PubAnnotation

Id	Subject	Object	Predicate	Lexical cue
T1	0-38	Sentence	denotes	The Yin and Yang of ACE/ACE2 Pathways:
T2	39-124	Sentence	denotes	The Rationale for the Use of Renin-Angiotensin System Inhibitors in COVID-19 Patients
T3	126-134	Sentence	denotes	Abstract
T4	135-342	Sentence	denotes	The article describes the rationale for inhibition of the renin-angiotensin system (RAS) pathways as specific targets in patients infected by SARS-CoV-2 in order to prevent positive feedback-loop mechanisms.
T5	343-620	Sentence	denotes	Based purely on experimental studies in which RAS pathway inhibitors were administered in vivo to humans/rodents, a reasonable hypothesis of using inhibitors that block both ACE and ACE2 zinc metalloproteases and their downstream pathways in COVID-19 patients will be proposed.
T6	621-983	Sentence	denotes	In particular, metal (zinc) chelators and renin inhibitors may work alone or in combination to inhibit the positive feedback loops (initially triggered by SARS-CoV-2 and subsequently sustained by hypoxia independently on viral trigger) as both arms of renin-angiotensin system are upregulated, leading to critical, advanced and untreatable stages of the disease.
T7	985-987	Sentence	denotes	1.
T8	988-1000	Sentence	denotes	Introduction
T9	1001-1178	Sentence	denotes	The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is the cause of the COVID-19 pandemic, for which there is currently no specific pharmacotherapy.
T10	1179-1421	Sentence	denotes	In the intensive care unit, depending on the severity of the COVID-19 infection, patients might receive supportive care such as oxygen supply, interferons, glucocorticoids, anti-virals, chloroquine, and macrolides given as deemed appropriate.
T11	1422-1624	Sentence	denotes	The SARS-CoV-2 infection produces either non-severe or severe symptoms and, in the latter case, it may precipitate into severe acute respiratory syndrome (SARS) and ultimately death [1,2,3,4,5,6,7,8,9].
T12	1625-1705	Sentence	denotes	Aging and different comorbidities are associated with patients’ critical status.
T13	1706-1892	Sentence	denotes	Most patients display lymphopaenia and eosinopaenia, and critical ones show extremely low levels of eosinophils, suggesting that eosinopaenia may be a potential diagnostic biomarker [1].
T14	1893-2148	Sentence	denotes	Most serious cases of SARS are closely associated with cardiac dysfunction/injury, respiratory distress/illness, coagulopathy, hypoproteinaemia, acidosis, hypoxia and a cytokine storm mainly involving IL-1β, IL-6, IL-8, IL-10 and TNFα [1,2,3,4,5,6,7,8,9].
T15	2149-2622	Sentence	denotes	For instance, hypertension, an age-related disease, is the most common comorbidity (15–31% up to 24–58% in severe disease cases according to clinical reports), while only five cases of asthma (0.9%) were identified in one report of 548 infected patients in Wuhan [6], but no other asthmatic or allergic patients were described in other reports [1,2,3,4,5,7,8,9], considering that the overall prevalence of asthma is estimated to be 6.4% in Wuhan [6] and ~ 4% in China [10].
T16	2623-2788	Sentence	denotes	Viral infections usually increase the risk of allergic disease exacerbation [1]; however, the reported data seem to suggest that, in this case, the opposite is true.
T17	2789-3255	Sentence	denotes	Indeed, in more than 72.000 patient cohorts examined, the number of patients defined as asthmatic was low (5), which suggests that asthmatics might be protected from virus-induced SARS, whereas pre-existing hypertensive disease and/or pre-existing antihypertensive treatments may represent a risk factor for increased virus infection, considering that the overall prevalence of hypertension in Chinese adult population ≥18 years of age is estimated to be ~ 23% [11].
T18	3256-3558	Sentence	denotes	Therefore, according to the clinical picture of infected patients, Th2-mediated allergic diseases (usually with high eosinophil counts) may play a protective role against SARS (usually with low eosinophil counts), while yet unknown mechanisms related to hypertensive conditions may exacerbate symptoms.
T19	3559-3875	Sentence	denotes	To complete the picture, COVID-19 hospitalized patients suffering from chronic renal disease were usually low (0-3%) [1,2,3,4,5,6,7,8,9] if compared with the prevalence of the disease in China (10.2-11.3%) [12], suggesting that chronic kidney disease patients might be protected from COVID-19 as for asthma patients.
T20	3877-3879	Sentence	denotes	2.
T21	3880-3928	Sentence	denotes	ACE2-mediated SARS-CoV and SARS-CoV-2 Infections
T22	3929-4092	Sentence	denotes	Angiotensin-converting enzyme 2 (ACE2) was identified as a receptor for the spike (S) protein of SARS-CoV, finally facilitating viral entry into target cells [13].
T23	4093-4286	Sentence	denotes	ACE2 is abundantly expressed in airway epithelial cells and vascular endothelial cells, and it is believed to play a crucial role in the mechanism of acute lung injury induced by SARS-CoV [14].
T24	4287-4540	Sentence	denotes	The ability of spike-Fc protein treatment (3h) to downregulate ACE2 protein expression has been shown in an in vitro system (cell lines) and also in vivo in lung cells of mice [14,15], suggesting that ACE2 pathway may be down-modulated during infection.
T25	4541-4683	Sentence	denotes	However, ACE2 is constitutively expressed and released from the apical cell surface of human airway epithelia into airway surface liquid [16].
T26	4684-4925	Sentence	denotes	Of note, its surface down-modulation upon spike protein challenge has been shown to be due to ACE2 shedding mediated by activation of extracellular ADAM17/TACE metalloprotease, which concomitantly induces shedding/production of TNFα [17,18].
T27	4926-5157	Sentence	denotes	Interestingly, ACE2 shedding is enhanced not only by binding with spike protein [17,18], but also by IL-1β and TNFα inflammatory cytokines [16], cytokines that are secreted at relatively high concentration in COVID-19 patients [2].
T28	5158-5442	Sentence	denotes	Moreover, soluble (s)ACE2 (induced or not by virus binding) released from human airway epithelia has been demonstrated to retain both its enzymatic activity and its binding ability for spike viral protein, finally reducing spike protein-mediated viral entry into target cells [16,17].
T29	5443-5607	Sentence	denotes	Therefore, the interaction of ACE2 with spike protein of SARS-CoV induces a cellular “protective” ACE2 shedding feedback response that initially limits viral entry.
T30	5608-5795	Sentence	denotes	Nevertheless, ADAM17/TACE-mediated ACE2 shedding or ACE2 enzymatic activity have been shown to intriguingly correlate positively with viral infection and disease complications [17,19,20].
T31	5796-5987	Sentence	denotes	In contrast, HNL63-CoV, which similarly binds to ACE2 through its spike protein, infects ACE2-bearing cells and mainly induces the common cold, leading to neither ACE2 shedding nor SARS [17].
T32	5988-6250	Sentence	denotes	Moreover, catalytically inactive forms of sACE2 can potently inhibit SARS-CoV infection [19,21], suggesting that events downstream of ACE2 shedding and/or its enzymatic activity may indirectly and subsequently favour viral infection and/or disease complications.
T33	6251-6522	Sentence	denotes	To this regard, sACE2 was also associated with myocardial pathological conditions [22] and cardiovascular complications including hypotension (known to enhance both renin and angiotensin I, the substrate of ACE/ACE2) and tachycardia were common in SARS-CoV patients [23].
T34	6523-6791	Sentence	denotes	Since spike protein has been shown to not inhibit ACE2 enzymatic activity that is retained by sACE2-spike protein complex [16,17,24] and, in general, sACE2 maintains its enzymatic activity, we cannot consider its higher circulating expression a mere disease biomarker.
T35	6792-6907	Sentence	denotes	Indeed, ACE2 shedding might repress its local function but it certainly enhances its circulating/systemic activity.
T36	6908-7236	Sentence	denotes	Interestingly, a recent integrative bioinformatics analysis shows that the gene expression of ACE2 in human bronchial cells infected with SARS-CoV is dramatically increased 24h after infection and remained at a high level for at least 2 days, suggesting that ACE2 may be involved in a positive feedback loop post-infection [25].
T37	7237-7539	Sentence	denotes	In the same report, it has been shown that ACE2 expression level in bronchial epithelium obtained by brushing from asthmatic and normal subjects was similar, suggesting that respiratory epithelial cells of healthy subjects and asthmatic patients have similar ability to bind to SARS-CoV-2 through ACE2.
T38	7540-7637	Sentence	denotes	Of note, ACE2 was also identified as the receptor for the novel spike protein of SARS-CoV-2 [13].
T39	7638-7801	Sentence	denotes	Although the role of ACE2 in the pathogenesis of SARS-CoV-2 and in inducing lung injury is still unknown, ACE2 behaves similarly to original SARS-CoV [13] (Box 1).
T40	7802-7988	Sentence	denotes	Box 1 The formation of spike-ACE2 protein complexes may initiate ACE2 systemic upregulation and may impair autologous antibody generation/recognition of anti-S1-Receptor Binding Domain.
T41	7989-8258	Sentence	denotes	The ACE2 interacts with the spike (S) protein, thereby serves as an entry receptor for SARS-CoVs, and this event is likely followed by conformational changes, cleavages and fusion of both spike viral and ACE2 proteins at the level of infected host cell plasma-membrane.
T42	8259-8444	Sentence	denotes	The SARS coronavirus surface spike glycoproteins consist of trimers formed by two spike, capsid-distal S1 and –proximal S2, protein regions, located on the outer envelope of the virion.
T43	8445-8701	Sentence	denotes	Spike proteins are clove-shaped trimers with three S1 heads and a trimeric S2 stalk that can bind the cellular ACE2 receptor when one S1 (possessing the receptor-binding domain) in the trimer adopts an “up” conformation (see Figure later in the Section 4).
T44	8702-9113	Sentence	denotes	The subsequent binding of ACE2 receptor to the spike glycoprotein triggers both the ADAM17-mediated ACE2 shedding and dissociation of S1 fragments by exogenous (furin-related or other) proteases from the spike trimer, which leads on one hand to one S1-ACE2 complex and two S1 free fragments, and on the other hand to fusion of S2 viral trimers and cellular membrane structures, finally producing cell infection.
T45	9114-9337	Sentence	denotes	The molecular mechanism model for SARS-CoV recognition and infection was well described by Song and collaborators [26] and a similar molecular interactions between SARS-CoV-2 and ACE2 has been recently hypothesized [27,28].
T46	9338-9530	Sentence	denotes	To complete the picture, a recent structural work reveals that the full-length human ACE2 is assembled as a dimer associated or not with amino acid transporter B0AT1, which sandwich ACE2 [28].
T47	9531-9668	Sentence	denotes	Binding of the spike protein trimer onto the ACE2 dimer suggests simultaneous binding of two spike protein trimers to an ACE2 dimer [28].
T48	9669-9937	Sentence	denotes	Each monomer of the ACE2 homodimer is composed of a membrane-proximal collectrin-like domain mediating homodimerization and necessary to position the molecule to the cell membrane, and of a membrane-distal ACE2 peptidase domain responsible for ACE2 substrate cleavage.
T49	9938-10124	Sentence	denotes	Collectrin-like domain consists of an extracellular ferredoxin-like fold domain, also called neck domain (residues 616 to 726), a single transmembrane helix and an intracellular segment.
T50	10125-10309	Sentence	denotes	The ACE2 cleavage site mediated by ADAM17/TACE has been predicted between amino acids 716 and 741 [16], which corresponds to neck-transmembrane boundary of ACE2 collectrin-like domain.
T51	10310-10542	Sentence	denotes	It is tempting to speculate that, after ACE2 shedding, S2 viral trimers may fuse with the residual ACE2 collectrin-like fragments (complexed or not with B0AT1 transporters) which are eventually exposed on the cell surface membranes.
T52	10543-10647	Sentence	denotes	A model for SARS-CoV’s internalization by target cells that was already proposed several years ago [29].
T53	10648-10893	Sentence	denotes	Then, when the virus is inside the cell, a second proteolytic cleavage mediated by endosomal proteases (such as TMPRSS2) in the S2 region of the spike-ACE2 fusion protein might be necessary for intracellular virus “release” and active infection.
T54	10894-11051	Sentence	denotes	Several S1-sACE2 complexes, free S1 fragments or sACE2 are likely released in bloodstream of COVID-19 infected patients by spike and ACE2 receptor cleavages.
T55	11052-11527	Sentence	denotes	As already mentioned, circulating sACE2 protein shedding independent on virus contact has been also described either spontaneously when ACE2 transcription is upregulated or upon cytokine activation; it is therefore likely that in the bloodstreams ACE2 proteins are available to bind to both free S1 fragments and SARS-CoV-2 particles, finally leading to more and more S1-sACE2 and SARS-CoV-2-sACE2 complexes bearing enzymatic active sACE2 (see Figure later in the Section 4).
T56	11528-11803	Sentence	denotes	Of note, SARS-CoV-2-sACE2 complexes, sequestrating sACE2, can drive part of the sACE2 systemic activity and concentrate it locally where the organs possess cells expressing ACE2 on their surface membrane, i.e., following the viral tropism (see Figure later in the Section 4).
T57	11804-12098	Sentence	denotes	Indeed, although the viral load in sputum of adult SARS-CoV patients was usually higher than 104 copies/mL, reports indicate that plasma viral RNA concentrations are low, usually lower than 190 copies/mL and lymphocytes have much higher RNA-copy concentrations of SARS-CoV RNA than plasma [30].
T58	12099-12244	Sentence	denotes	Very low RNA concentrations with no difference between patients with mild or severe symptoms were detected in plasma from COVID-19 patients [30].
T59	12245-12376	Sentence	denotes	The viral load in nasal swabs of asymptomatic and symptomatic COVID-19-positive subjects was also not significantly different [31].
T60	12377-12661	Sentence	denotes	Of interest, SARS-CoV infected pediatric patients have more than double the amount of plasma RNA copies/mL when compared to adult patients [30], suggesting a different viral tropism between adults and children (different cellular ACE2 expression possibly leading to Kawasaki disease).
T61	12662-12789	Sentence	denotes	Notably, free S1 fragments may have a decoy function for autologous/exogenous anti-S1-receptor binding domain (RBD) antibodies.
T62	12790-13020	Sentence	denotes	On the other hand, sACE2-S1 complexes masking S1-RBD might also impair either generation of autologous high affinity antibodies against S1-RBD or recognition/inactivation of S1-RBD on SARS-CoV-2 by autologous/exogenous antibodies.
T63	13021-13393	Sentence	denotes	Therefore, antibodies that do not compete with ACE2 for the binding to S1-RBD might be more effective in neutralizing SARS-CoV-2 [32,33], knowing that the number of spike proteins per virion is estimated to be around 70 and that every spike protein bears three S1 receptor binding domains, which are masked to autologous antibody recognition when in a “down” conformation.
T64	13394-13589	Sentence	denotes	These highly organized molecular features clearly demonstrate that the coronaviruses are armed with sophisticated infection machinery able to evade immune responses induced or not by vaccination.
T65	13590-13772	Sentence	denotes	Nevertheless, passive immunotherapy by means of convalescent plasma from COVID-19 recovered donors is a promising option for prevention and treatment of COVID-19 [32,33,34,35,36,37].
T66	13774-13776	Sentence	denotes	3.
T67	13777-13826	Sentence	denotes	Pathological Effects of ACE2 Pathway Upregulation
T68	13827-13949	Sentence	denotes	ACE and ACE2 are two zinc metalloproteases involved in the biogenesis of the components of renin-angiotensin system (RAS).
T69	13950-14150	Sentence	denotes	ACE2 processes angiotensin (Ang) I and II into Ang (1–9) and Ang (1–7), respectively, and it also has other known peptide targets, such as des-Arg(9)-bradykinin, which mediates B1 receptor activation.
T70	14151-14497	Sentence	denotes	Ang (1–7) and Ang (1–9) peptides, opposing the effects of ACE/Ang II/Ang II type 1 receptor (AT1R) pathway, are known to mediate vasodilatative (hypotension), antiproliferative and apoptotic effects through Mas and AT2 receptors, respectively, both involving downstream nitric oxide synthase (NOS) pathway activation [38,39,40,41,42,43,44,45,46].
T71	14498-14653	Sentence	denotes	Physiology teaches us that there is a range of normality for every biological parameter, below (defect) and above (excess) of which there is a dysfunction.
T72	14654-14708	Sentence	denotes	This is likely to be true for ACE as well as for ACE2.
T73	14709-14923	Sentence	denotes	It is well known that excessive Ang II/AT1R pathway activation induces vasoconstriction, aldosterone, inflammation, excessive cell proliferation, thrombosis, cardiac dysfunction and lung alterations (see Figure 1).
T74	14924-15216	Sentence	denotes	Moreover, most of the experiments show that increased ACE2 activity leads to beneficial effects; however, the experiments were usually performed using models in which its “antagonist“ (ACE) pathway was upregulated or ACE2 itself was downregulated, therefore balancing an unbalanced situation.
T75	15217-15276	Sentence	denotes	What does it happen in models in which the opposite occurs?
T76	15277-15488	Sentence	denotes	(s)ACE2 or Ang (1–7) upregulation have been associated to some pathological conditions such as inflammation of the renal and gastrointestinal tract, cardiac dysfunction, human cirrhosis and lung injury/fibrosis.
T77	15489-15652	Sentence	denotes	Tissue/organ specific effects of excessive pathway activation that in some case resemble those produced by excessive Ang II/AT1R pathway activation (see Figure 1).
T78	15653-15792	Sentence	denotes	For example, systemic infusion of Ang (1–7) into mice had renal proinflammatory properties mediated by Mas receptor (MasR) activation [47].
T79	15793-15943	Sentence	denotes	Moreover, Ang (1–7) infusion was associated with increases in blood pressure, cardiac hypertrophy and fibrosis in rats with subtotal nephrectomy [48].
T80	15944-16124	Sentence	denotes	Similarly, elevated plasma sACE2 activity was associated both with greater severity of myocardial dysfunction and with an independent prediction of adverse clinical events [22,49].
T81	16125-16385	Sentence	denotes	Transgenic mice with increased cardiac ACE2 expression suffered from lethal ventricular arrhythmia (heart block, ventricular tachycardia and terminal ventricular fibrillation) consequent upon downregulation of connexins involved in gap junction formation [50].
T82	16386-16534	Sentence	denotes	In another model, ACE2 transgenic mice suffered from cardiac fibrosis with concomitant deficits in ejection fraction and fractional shortening [51].
T83	16535-16928	Sentence	denotes	Interestingly, in a rat model of myocardial infarction following coronary artery ligation, there is evidence that C16/MLN-4760 (a specific ACE2 inhibitor, see later) administration inhibits fibrosis and hypertrophy of non-infarcted myocardium and increases diastolic relaxation, raising the possibility that ACE2 activity may have some adverse effects on post-myocardial infarction heart [52].
T84	16929-17067	Sentence	denotes	The risk factors for cardiovascular disease include alterations in platelet function and coagulation with an increased risk of thrombosis.
T85	17068-17244	Sentence	denotes	Ang II and hypercholesterolemia are known to participate in microvascular thrombosis and enhanced thrombus formation in the microvasculature may contribute to microinfarctions.
T86	17245-17441	Sentence	denotes	To this end, in a rat model in which AT1R activation produce baseline thrombosis by platelet aggregation, Ang (1–9), known to bind AT2R [42], has been shown to enhance the thrombotic process [53].
T87	17442-17751	Sentence	denotes	Moreover, in a mouse model, AT2R activation (inhibited by AT2R antagonist, PD12319) mediated the onset of arteriolar microvascular thrombosis following chronic Ang II infusion [54], indicating both the recruitment of the AT2R pathway downstream of AT1R activation and its involvement in arteriolar thrombosis.
T88	17752-18061	Sentence	denotes	Of interest, disseminated intravascular coagulation is associated with hypoproteinaemia and deficit of coagulation and anticoagulation proteins, which can originate by their renal loss (and consequent proteinuria) and/or by their increased (pathological) consumption and/or by their reduced hepatic synthesis.
T89	18062-18270	Sentence	denotes	To this end, in healthy livers, ACE2 is limited to perivenular hepatocytes and endothelial cells; instead, in human hepatitis C cirrhosis, ACE2 protein expression is widespread in the hepatic parenchyma [55].
T90	18271-18462	Sentence	denotes	Notably, human hepatocytes cultured in hypoxic conditions upregulated ACE2 protein expression [55] and ACE2 mRNA, protein and activity were increased in response to hypoxia and by IL-1β [56].
T91	18463-18666	Sentence	denotes	In line with these observations, in peripheral blood human CD34+ cells, MasR expression and ACE2 expression, activity and shedding (of sACE2 catalytically active forms) were increased under hypoxia [57].
T92	18667-18924	Sentence	denotes	Moreover, under hypoxic conditions human pulmonary artery smooth muscle cells upregulated both (arms of the RAS) ACE and ACE2 mRNA and protein expression, and ACE2 was subsequently downregulated by (ACE-derived) Ang II through an AT1R-mediated process [58].
T93	18925-19110	Sentence	denotes	Similarly, both ACE and ACE2 expression were increased in vascular endothelium and smooth muscle of human left ventricle from patients with ischaemic (local hypoxia) heart disease [59].
T94	19111-19233	Sentence	denotes	Concomitant upregulation of both arms of the RAS that suggests a tight link between ACE and ACE2 under hypoxic conditions.
T95	19234-19372	Sentence	denotes	Indeed, several reports have shown a complex interplay of regulation between the two arms of the RAS independently on hypoxia (see Box 2).
T96	19373-19910	Sentence	denotes	Interestingly, chronic hypoxia induced activation of ACE2/Ang (1–7)/MasR axis and suppression of ACE/Ang II/AT1 receptor axis in lungs of pulmonary hypertensive Ren-2 transgenic rats (constructed by inserting the mouse Ren-2 renin gene) but not in normotensive transgene-negative control rats [60], suggesting that the baseline renin activity in hypertensive rats may be crucial to determine the differential response to hypoxia and that renin inhibition might be useful to inhibit the ACE to ACE2 pathway shift under hypoxic conditions.
T97	19911-20086	Sentence	denotes	Of interest, hypoxia alone or combined with hypercapnia has been shown to significantly increase both plasma renin expression or activity and plasma Ang II expression [61,62].
T98	20087-20582	Sentence	denotes	Moreover, hypercapnic acidosis (pH 6.8/6.9, a condition that could occur in SARS) induced in isolated rat lungs has been shown to induce a (compensatory) venular dilatation mediated by cyclooxygenase activation (inhibited by indomethacin) [63], an enzyme that has been shown to be induced downstream of Ang (1–7)/MasR pathway in isolated rat hearts (again inhibited by indomethacin) [64], suggesting the involvement of ACE2/Ang (1–7) pathway in mediating CO2-dependent lung venular vasodilation.
T99	20583-20978	Sentence	denotes	Altogether these observations indicate that there is a high probability that hypoxia/hypercapnia, a condition that occurs in SARS patients, might upregulate the activity of both arms of the RAS by suppling high amounts of renin product, Ang I, to ACE and ACE2, which, together, can produce high amounts of Ang II, Ang (1–9), Ang (1–7) and its (ACE-produced) metabolite, Ang (1–5) (see Figure 1).
T100	20979-21264	Sentence	denotes	Similarly to both Ang (1–7)/MasR and Ang (1–9)/AT2R pathways, Ang (1–5) has been shown to induce the secretion of atrial natriuretic peptide via MasR/NOS pathway in isolated perfused rat atria [65], indicating that Ang (1–5) is a heart active peptide (at least in Sprague-Dawley rats).
T101	21265-21431	Sentence	denotes	Interestingly, in the plasma of patients with inflammatory bowel disease, Ang (1–7) concentrations and (s)ACE2 activity were higher compared to healthy subjects [66].
T102	21432-21585	Sentence	denotes	Moreover, Ang (1–7) alone can either activate the Bax/Caspase–dependent apoptotic pathway or upregulate NF-kB signaling in lung fibroblast cultures [67].
T103	21586-21859	Sentence	denotes	Moreover, in vivo administration of Ang (1–7) alone (in Wistar rats) promoted morphological lung alterations, extracellular matrix accumulation and inflammatory cytokine production (including TNF-α and IL-6), characteristics of lung inflammation in pulmonary fibrosis [67].
T104	21860-21957	Sentence	denotes	Thereby, a condition that deteriorates respiratory compliance, could lead to hypoxia/hypercapnia.
T105	21958-22169	Sentence	denotes	Hypoxia, in turn, will induce ACE2 upregulation which possibly increases cardiac/lung detrimental effects mediated by Ang (1–7)/MasR pathway activation finally leading to further ACE2 cell membrane upregulation.
T106	22170-22405	Sentence	denotes	A condition that in COVID-19 can increase the probability of both SARS-CoV-2 entry and ACE2 shedding/systemic activity, finally generating positive feedback loops that might sustain SARS independently of viral infection (see Figure 2).
T107	22406-22723	Sentence	denotes	This hypothesis is supported by the observations that, in lung aspirates of acid- and/or spike-treated mice, Ang II and ACE2 are synergistically upregulated and cell surface downregulated (shed), respectively, suggesting their involvement in the increased lung microvascular permeability and pulmonary oedema [14,15].
T108	22724-22931	Sentence	denotes	Indeed, this condition might subsequently favour the diffusion, in neighbouring lung tissues and systemic circulation, of both (s)ACE2, Ang II and Ang (1–7), the Ang II-derived product of (s)ACE2 processing.
T109	22932-23121	Sentence	denotes	As already proposed, enhanced ACE2 shedding may locally reduce ACE2 activity in lung, however, it likely increases ACE2 systemic activity and subsequent production of circulating Ang (1–7).
T110	23122-23259	Sentence	denotes	Interestingly, Ang (1–7) has been also reported to promote eosinophil apoptosis in lungs and in bronco-alveolar lavage fluid (BALF) [40].
T111	23260-23531	Sentence	denotes	Moreover, Ang (1–7)/MasR axis inhibits allergic airway inflammation and eosinophil cell counts in the BALF of a murine model of asthma, indicating both that an impairment of ACE2 pathway may favour asthma and that ACE2 pathway activation can reduces asthma symptoms [68].
T112	23532-23763	Sentence	denotes	Moreover, a compound that mimics the Ang (1–7) actions has been shown to induce IL-10 upregulation via a MasR-dependent pathway in BALF [69] and IL-10 is thought to mediate anti-inflammatory effects of MasR pathway activation [70].
T113	23764-23884	Sentence	denotes	IL-10 secretion in mouse plasma was also induced by an AT2 receptor agonist and downstream nitric oxide signalling [71].
T114	23885-24078	Sentence	denotes	Increased IL-10 production (e.g., by T regulatory cells) is often associated with immune tolerance, which is the consequence of reduced number and function of specific immune compartments [72].
T115	24079-24439	Sentence	denotes	Indeed, IL-10 has been shown not only to suppress antigen-specific Th2-mediated immune responses including eosinophil expansion in allergic inflammation [72], but also to augment airway reactivity, suggesting that despite its potent anti-inflammatory activity, an excess of IL-10, as well as Ang (1–7), may contribute to the decline in pulmonary function [73].
T116	24440-24752	Sentence	denotes	Of note, IL-10 is one of the cytokines downstream ACE2 pathway [25] and is significantly upregulated in the most severe forms of COVID-19 [2,8,9], indicating an important correlation between ACE2/Ang (1–7) axis activation and IL-10 upregulation which might lead to eosinopaenia/lymphopaenia in COVID-19 patients.
T117	24753-24824	Sentence	denotes	Box 2 The reciprocal feedback-loop mechanisms of both arms of the RAS.
T118	24825-25157	Sentence	denotes	Similarly to hypoxic conditions, a strong correlation between the gene expression of ACE and that of ACE2 was also observed in human renal cortical biopsy specimen, suggesting a link between the two gene transcription, possibly related to the amount/excess of their substrates (Ang I and Ang II) and not exclusively to hypoxia [74].
T119	25158-25518	Sentence	denotes	A link that tends to maintain the balance of ACE/ACE2 ratio, but that may be disrupted by ACE inhibitors (ACEIs) or by Ang II type 1 receptor blockers (ARBs) which both have been shown to upregulate ACE2 mRNA expression in left ventricle of Lewis rats, possibly through two different mechanisms involving upregulation of Ang (1–7) or Ang II, respectively [75].
T120	25519-25784	Sentence	denotes	In this regard, in cardiac myocytes isolated from neonatal rats, Ang II significantly reduced ACE2 mRNA and its activity, effects blocked by ARBs, indicating that Ang II downregulates ACE2 expression/activity through an AT1R-dependent mechanism [76] (see Figure 1).
T121	25785-26139	Sentence	denotes	Moreover, in both cardiac myocytes and rat aortic vascular smooth muscle cells, Ang (1–7) prevented the Ang II-mediated reduction in ACE2 mRNA, an effect blocked by a selective MasR antagonist, D-Ala7-Ang-(1–7) (also known as A779), indicating that Ang (1–7) downregulates Ang II/AT1R signalling through a MasR-dependent mechanism [76,77] (see Figure 1).
T122	26140-26483	Sentence	denotes	Therefore, Ang (1–7) (as well as ACEIs and ARBs), preventing Ang II-mediated ACE2 downregulation, shift the Ang peptide balance in favour of Ang II metabolisation by ACE2, which, in turn, leads to further production of Ang (1–7), finally sustaining ACE2 transcription, membrane protein expression and eventually shedding and systemic activity.
T123	26484-26695	Sentence	denotes	Altogether these observations indicate a complex interplay of regulation between the two arms of the RAS in which feedback mechanisms of reciprocal (ACE/ACE2) pathway inhibition are involved at different levels:
T124	26696-26883	Sentence	denotes	Ang II/AT1 receptor mediates a negative feedback signal on the ACE2 expression/activity and Ang (1–7)/MasR mediated a negative feedback signal on the AT1 receptor activity (see Figure 1).
T125	26884-27114	Sentence	denotes	These reciprocal inhibitions in some cases (e.g., hypoxia/SARS-CoV-2 infection) can give rise to positive feedback loops that markedly shift the balance between Ang II/AT1R and the antagonist Ang (1–7)/MasR pathway (see Figure 1).
T126	27115-27412	Sentence	denotes	For example, under hypoxic conditions both arms of the RAS are upregulated and the presence of SARS-CoV-2 can affect Ang (1–7)/Ang II balance (which might be further influenced by ACEI/ARBs) by shifting it in favour of an increased ACE2 systemic activity, Ang (1–7) production and MasR activation.
T127	27413-27628	Sentence	denotes	This, in turn, can lead to further ACE2 cell membrane expression (increasing the probability of viral entry), which, after ACE2 shedding by binding to spike-SARS-CoV-2, ultimately establish a positive feedback loop.
T128	27630-27632	Sentence	denotes	4.
T129	27633-27709	Sentence	denotes	COVID-19 Can Induce RAS-mediated Positive Feedback Loops at Different Levels
T130	27710-27885	Sentence	denotes	Intriguingly, the binding affinity of ACE2 to SARS-CoV-2 binding domain has been reported to be either equal to or 10- to 20-fold higher than ACE2 binding to SARS-CoV [27,32].
T131	27886-27987	Sentence	denotes	The affinity of spike protein for ACE2 has been shown to correlate with the severity of disease [24].
T132	27988-28261	Sentence	denotes	Although SARS-CoV produces more severe respiratory symptoms than NL63-CoV does, both viral receptor binding domains bind to ACE2 with similar affinity [78], indicating that SARS development is not related to the strength of binding affinity and depends on other mechanisms.
T133	28262-28500	Sentence	denotes	In contrast to SARS-CoV, NL63-CoV did not induce ADAM17/TACE-mediated both ACE2 shedding and TNF-α secretion [17], suggesting that increased cleavability of ACE2 receptor and possibly of S1-S2 boundary may be crucial for disease severity.
T134	28501-28672	Sentence	denotes	Indeed, the spike glycoprotein of SARS-CoV-2 but not of SARS-CoV, contains a furin-like cleavage site at the S1-S2 boundary which indicates an increased cleavability [13].
T135	28673-28827	Sentence	denotes	Of note, TNF-α and IL-1β were shown to both be upregulated in SARS-CoV-2 [2] and induce viral-independent ACE2 shedding from epithelial airway cells [16].
T136	28828-29101	Sentence	denotes	Moreover, viral-independent ACE2 surface release from epithelial cells was not only inducible by cytokines (e.g., TNF-α and IL-1β) but also constitutively and spontaneously produced when ACE2 surface expression was upregulated [16], for example upon IL-1β stimulation [56].
T137	29102-29250	Sentence	denotes	This suggests that systemic release of proinflammatory cytokines such as TNF-α and IL-1β can mediate an increase of sACE2 and its systemic activity.
T138	29251-29391	Sentence	denotes	Of note, activation of ADAM17/TACE metalloprotease was induced by SARS-CoV and necessary for efficient infection (and TNF-α secretion) [17].
T139	29392-29653	Sentence	denotes	Intriguingly, both SARS-CoV infection and concomitant TNF-α secretion were significantly attenuated not only by knock-down of ADAM17/TACE expression by siRNA but also by deletion of the ACE2 cytoplasmic tail which is responsible for ADAM17/TACE activation [17].
T140	29654-29824	Sentence	denotes	Altogether these observations suggest the possibility that SARS-CoV may induce a positive feedback loop leading to surface expression and shedding of both ACE2 and TNF-α.
T141	29825-30108	Sentence	denotes	Indeed, upon spike protein binding to ACE2, downstream pathway activation can sustain positive feedback loops at different levels (Figure 2):(1) SARS-CoV can induce IL-1β and TNF-α systemic secretion that can mediate viral-independent surface membrane ACE2 upregulation and shedding.
T142	30109-30290	Sentence	denotes	Of note, ACE2 shedding, on one hand, protects from viral infection but, on the other hand, increases circulating/systemic active sACE2, leading to its downstream pathway activation.
T143	30291-30662	Sentence	denotes	(2) Hypoxia in combination or not with hypercapnia can upregulate the activity of both arms of the renin–angiotensin system by inducing renin, ACE and ACE2 synthesis, which can increase expression of Ang I, Ang II, Ang (1–7), Ang (1–9), Ang (1–5) and the inactive metabolite bradykinin (1–7), but also membrane bound ACE2, finally giving more chances to SARS-CoV-2 entry.
T144	30663-30739	Sentence	denotes	(3) ACE2 can induce vasodilatative hypotensive effects by Ang II catabolism.
T145	30740-30883	Sentence	denotes	Hypotension can induce again renin and ACE upregulation finally providing further Ang II, as a ACE2 substrate for further Ang (1–7) production.
T146	30884-31199	Sentence	denotes	(4) Ang (1–7) antiproliferative and apoptotic effects, possibly in part through IL-10, may mediate eosinopaenia and lymphopaenia that, on one hand, reduce inflammatory responses but, on the other hand, impair immune system ability to counter virus infection, finally predisposing the organism to further infections.
T147	31200-31363	Sentence	denotes	Ang (1–7) immunosuppressive activity, mediated or not by IL-10, may also support the reduced ability to generate an effective immunization to SARS-CoV-2 infection.
T148	31364-31581	Sentence	denotes	(5) Ang (1–7)/MasR pathway can sustain ACE2 synthesis even in the presence of elevated concentrations of Ang II (such as in hypoxia), by inhibiting Ang II/AT1R-mediated down-modulation of ACE2 activity (see Figure 1).
T149	31582-31755	Sentence	denotes	(6) Ang (1–7)/MasR pathway can produce cardiac dysfunction and lung alteration leading to systemic hypoxia, which, in turn, upregulates the activity of both arms of the RAS.
T150	31756-32113	Sentence	denotes	(7) Although the ACE2 catalytic efficiency is 400-fold lower with Ang I than with Ang II [79], high concentration of circulating ACE2 may be able to produce significant increase of Ang (1–9) that, by binding AT2 receptors, can produce arteriolar microvascular thrombosis and local hypoxic conditions finally inducing local upregulation both arms of the RAS.
T151	32114-32461	Sentence	denotes	Finally, ACE2-loaded SARS-CoV-2 virions and S1-ACE2 complexes may impair both sACE2 proteolytic degradation by blood proteases and its renal excretion, therefore blunting the removal of its systemic enzymatic activity and indicating that circulating viral particles are dangerous even when they are not able to entry into the cells (see Figure 2).
T152	32462-32665	Sentence	denotes	On the one hand, ACE2 hypertranscription and consequent increase of membrane (m)ACE2 exposure induced by hypoxia/hypotension may facilitate SARS-CoV-2 entry and its lifecycle into mACE2 expressing cells.
T153	32666-32900	Sentence	denotes	On the other hand, the release of ACE2 from the cell membranes and its subsequent activity in the bloodstream and in local (lung/cardiac) extracellular fluids are likely critical steps in contributing to systemic disease pathogenesis.
T154	32901-33075	Sentence	denotes	Nevertheless, the same aetiological agent, SARS-CoV-2, can produce a variety of clinical syndromes, which involves several organs in relation to the subject’s predisposition.
T155	33076-33463	Sentence	denotes	Although in the present work I have supposed that most of the COVID-19 symptoms might derive from Ang (1–7), Ang II and Ang (1–9) peptide upregulation, the involvement of B1 receptor pathway suppression mediated by ACE2 metabolisation of des-Arg(9)-bradykinin to inactive bradykinin (1–7) or other ACE2-downstream peptides such as apelins, casomorphins and dynorphins cannot be rule out.
T156	33464-33646	Sentence	denotes	Based on the above observations, in order to block the RAS-induced positive feedback loops, different pharmacological targets can be hypothesized and pursued alone or in combination.
T157	33647-33700	Sentence	denotes	They will be discussed in the next section and Box 3.
T158	33701-33804	Sentence	denotes	Box 3 Possible targets of therapeutic intervention to inhibit the RAS-induced positive feedback loops.
T159	33805-34078	Sentence	denotes	Pharmacological inhibition of enzymes involved in both viral entry, such as TMPRSS2 and ADAM17, and the RAS function such as renin, ACE and ACE2 enzymatic activity or their downstream pathways are expected to stop positive feedback loops and their detrimental consequences.
T160	34079-34137	Sentence	denotes	Inhibition of TMPRSS2 and ADAM17 metalloprotease activity.
T161	34138-34433	Sentence	denotes	Inhibition of the serine protease TMPRSS2 (necessary for SARS-CoV-2 entry) by a clinically proven protease inhibitor has been recently suggested by Hoffmann and colleagues [13] and inhibition of ADAM17 enzymatic activity has been already proposed about ten years ago by Haga and colleagues [20].
T162	34434-34788	Sentence	denotes	Indeed, inhibition of ADAM17-mediated ACE2 shedding is expected to increase membrane ACE2 expression and therefore the probability of viral entry; nevertheless, in the early phases of the disease, inhibition of ACE2 circulating activity might be sufficient to inhibit the systemic RAS pathway upregulation and the development of severe forms of COVID-19.
T163	34789-35163	Sentence	denotes	It is, in fact, possible that maintenance/recovery of correct organismal immune responses, by preventing ACE2-mediated immune suppression, in concert with cellular adaptive immune responses mediated by apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) systems [80] may anyway work to induce both an effective “immunization” and the viral eradication.
T164	35164-35199	Sentence	denotes	Inhibition of both arms of the RAS.
T165	35200-35397	Sentence	denotes	Among the inhibitors of the RAS pathways, different strategy can be pursued involving either ACE2 enzymatic activity or its upstream renin and ACE enzymatic activity or its downstream MasR pathway.
T166	35398-35624	Sentence	denotes	Inhibition of ACE2, ACE and renin enzymatic activities and their involvement in SARS-CoVs will be extensively discussed in the next sections, instead a brief description of MasR inhibition will be presented in the present Box.
T167	35625-35707	Sentence	denotes	Mas receptor pathway inhibition and side effects of using Mas receptor inhibitors.
T168	35708-35871	Sentence	denotes	A779 also known as D-Ala7-Ang-(1–7) and D-Pro7-Ang-(1–7) are two distinct MasR antagonists able to prevent Ang-(1–7)-mediated downstream activation in human cells.
T169	35872-36245	Sentence	denotes	The existence of several MasR subtypes has been suggested based on the differential capacity of the two MasR blockers to fully inhibit some biological actions of Ang-(1–7) [and perhaps of Ang (1–5), see Figure 1] [39,70]; therefore, differently from ACE2 enzymatic inhibitors, MasR antagonists should be administered in combinations, in order to inhibit ACE2 hyperactivity.
T170	36246-36436	Sentence	denotes	In human aortic smooth muscle cells, they have been shown to restore NADPH oxidase/NF-kB/iNOS inflammatory pathway induced by Ang II when it is inhibited by Ang (1–7) co-administration [81].
T171	36437-36815	Sentence	denotes	In mice studies a MasR blocker (A779) administered alone was not associated with systolic blood pressure alterations, and the hypotensive effect produced by rACE2 co-infused with Ang II was unaffected by A779 co-administration, indicating that the hypotensive activity of rACE2 mainly depended on Ang II degradation rather than on increase of Ang (1–7) and MasR activation [82].
T172	36816-37009	Sentence	denotes	In another report, spontaneously hypertensive rats (SHRs) that received A-779 alone for a total of two weeks did not significantly alter basal blood pressure and urinary protein excretion [83].
T173	37010-37282	Sentence	denotes	Moreover, in SHRs treated with A-779 in combination with Ang II, renal injury and interstitial infiltration of macrophages and T cells were surprisingly reduced as compared with SHRs treated with Ang II alone, suggesting a safe use of A-779 drug in in vivo infusions [83].
T174	37283-37439	Sentence	denotes	Another report showed that infusion of A-779 alone for 7 days did not produce a significant effect neither on blood pressure nor on heart rate in SHRs [84].
T175	37440-37656	Sentence	denotes	In a rat model of cardiac arrhythmia, administration of A-779 alone did not cause any significant alteration in the number of arrhythmic events, confirming that A-779 can be safely delivered to rodents in vivo. [85].
T176	37657-38007	Sentence	denotes	Although MasR antagonists has been shown to be safe in acute and chronic in vivo studies either with mice or rats, there are no data on administration in humans and the existence of different MasR subtypes in the vasculature require combinations of MasR antagonists to inhibit an excess of ACE2 activity as for example may occur in COVID-19 patients.
T177	38009-38011	Sentence	denotes	5.
T178	38012-38097	Sentence	denotes	Mechanism of Action and Potential Risk of Using RAS Pathway Inhibitors Targeting ACE2
T179	38099-38103	Sentence	denotes	5.1.
T180	38104-38133	Sentence	denotes	ACE2 (and ACE) Hyperactivity:
T181	38134-38164	Sentence	denotes	Is It a Matter of (Free) Zinc?
T182	38165-38260	Sentence	denotes	ACE2 and ACE are two zinc metalloprotease that function differently despite their similarities.
T183	38261-38451	Sentence	denotes	ACE2 is a monocarboxypeptidase (cleaves a C-terminal single amino acid from its substrate), whereas ACE is a dipeptidylpeptidase (releases a C-terminal dipeptide from its substrate) [86,87].
T184	38452-38758	Sentence	denotes	ACE2 has a substrate preference for hydrolysis between proline and a hydrophobic or basic C-terminal residue [79,86,87] and acts not only on the RAS and bradykinin peptides but also on the C-terminus of the apelin, casomorphin dynorphin peptides [79], possibly inhibiting Apelin-13 hypotensive action [44].
T185	38759-38875	Sentence	denotes	It is expressed in the heart, kidneys, liver, colon, small intestine, lung, brain and testes [39,44,45,46,76,77,87].
T186	38876-39085	Sentence	denotes	Among the tissues expressing ACE2 there are cardiomyocytes, endothelium, alveolar type II cells, ciliated airway cells, vascular smooth muscle cells and testicular Leydig cells [14,39,44,45,46,76,77,87,88,89].
T187	39086-39382	Sentence	denotes	ACE2 is present on the apical surface of epithelial cells of renal tubules, lung alveoli and vascular endothelia, in these last cells the production of Ang (1–7) induces autocrine activation of MasR axis leading to NOS-mediated vasodilation and consequently hypotension [14,44,45,46,86,87,88,89].
T188	39383-39568	Sentence	denotes	ACE2, as well as ACE, is a zinc metalloprotease that can be inhibited by zinc chelating EDTA and activated by high concentrations of chloride or fluoride anions and zinc cation [79,90].
T189	39569-39633	Sentence	denotes	Indeed, both enzymes have chloride- and zinc-binding sites [90].
T190	39634-39758	Sentence	denotes	It is conceivable that these sites are responsible for the anion and zinc activation effect in both homologous enzymes [90].
T191	39759-40017	Sentence	denotes	However, the presence of chloride ions increases Ang I cleavage by ACE and ACE2 (increasing both Ang II and Ang (1–9)) and decreases Ang II cleavage by ACE2, suggesting that chloride induces specific ACE2 conformational changes in its active site (see [44]).
T192	40018-40188	Sentence	denotes	To this regard, in plasma samples of COVID–19 patients, chloride range is usually normal or slightly higher (99.6–107.0 mmol/L) than normal values (96.–106.0 mmol/L) [5].
T193	40189-40275	Sentence	denotes	Unfortunately, I was not able to find plasma zinc concentrations in COVID-19 patients.
T194	40276-40395	Sentence	denotes	Zinc is involved in several cellular activities and its systemic concentration is tightly regulated by several factors.
T195	40396-40996	Sentence	denotes	Of interest, the major transporter/reservoir of zinc in plasma is albumin [91], a protein that was detected at significantly low concentrations in SARS-CoV-2 as well as severe adult respiratory distress syndrome (ARDS, a COVID-19-like disease) (usually < 3.5 g/dL, normal range 3.5–5.4) and inflammatory bowel disease (IBD, a disease with increased ACE2 activity) [6,8,66,92] Since most of zinc in plasma is sequestered by albumin, the total amount of plasma zinc mostly depends on albumin concentration and even small changes in albumin’s capacity for zinc binding may have significant consequences.
T196	40997-41219	Sentence	denotes	To this regard, reduced concentrations of plasma albumin is believed to increase plasma levels of free zinc, while reducing the total plasma zinc concentrations [91], a condition that was observed in ARDS patients [93,94].
T197	41220-41362	Sentence	denotes	Indeed, albumin can potentially deplete free bioavailable forms of zinc; vice versa, hypoalbuminaemia may increase free zinc levels in plasma.
T198	41363-41498	Sentence	denotes	Therefore, some ARDS patients with low levels of both total zinc and albumin might have rather higher than lower free zinc levels [93].
T199	41499-41713	Sentence	denotes	The free zinc concentration, which describes the fraction of zinc that is loosely bound and easily exchangeable, represents the highly bioavailable (and toxic) part of plasma zinc but it is not clinically detected.
T200	41714-41794	Sentence	denotes	To that end, zinc/albumin ratio might be a surrogate marker of free zinc levels.
T201	41795-41934	Sentence	denotes	Indeed, only recently it was described a fluorescence-based method for determining the free zinc concentration in human serum samples [95].
T202	41935-42058	Sentence	denotes	With this method it was shown that free zinc concentration in sera from females was significantly lower than in males [95].
T203	42059-42208	Sentence	denotes	Moreover, free zinc concentration did correlate neither with total serum concentrations of zinc (or other metal ions such as iron) nor with age [95].
T204	42209-42432	Sentence	denotes	Notably, increased plasma zinc bioavailability consequent to reduced albumin levels induces zinc import into cells and likely increases the cellular functions associated to cellular zinc concentrations such as ACE and ACE2.
T205	42433-42619	Sentence	denotes	Indeed, enzymatic activity of both enzymes seems significantly upregulated in ARDS patients as detected by analysis of Ang peptide concentrations in plasma (see Box later in ARDS topic).
T206	42620-42858	Sentence	denotes	In addition, serum albumin levels in COVID-19 were usually very low (in severe forms is usually < 3.1 g/dL) [6,8], raising the possibility of a negative correlation between albumin concentration in plasma and increase of the RAS activity.
T207	42859-43181	Sentence	denotes	In line with these hypotheses, both asthmatic/allergic disease patients and chronic kidney disease patients without a history of cardiovascular disease, which are protected from developing COVID-19, showed a significant decrease in both circulating zinc levels and ACE2 activity as compared to normal values [96,97,98,99].
T208	43182-43488	Sentence	denotes	Interestingly, chronic renal disease patients with low zinc levels had normal concentrations of albumin but higher urinary zinc excretion than healthy controls [97], suggesting that low ACE2 activity in chronic renal diseases (and protection from SARS) might derive from a higher free Zn2+ renal excretion.
T209	43489-43626	Sentence	denotes	A similar mechanism mediated by a reduction of extracellular levels of free Zn2+ might be hypothesised for asthma patients, as well [98].
T210	43627-43824	Sentence	denotes	Unfortunately, I could not find information on plasma albumin concentrations for asthma patients; nevertheless, we might suppose that in these patients the albumin concentration is in normal range.
T211	43825-44058	Sentence	denotes	Intriguingly, inhalation exposure to ZnCl2/ZnO/hexachloroethane (the main ingredients in smoke bombs) induces both elevated plasma levels of zinc and ARDS with clinical pictures that strongly resemble those of COVID-19 [100,101,102].
T212	44059-44294	Sentence	denotes	Since both ACE2 and ACE enzymes are activated by high concentrations of chloride and zinc ions [79,90], it is possible to hypothesise an activation of both arms of the RAS in ARDS induced by smoke bombs as well as in COVID-19 patients.
T213	44295-44546	Sentence	denotes	In addition, metal fume fever, a flu-like syndrome caused by inhalation of welding fumes (mainly containing zinc) can be a predictor for the development of respiratory symptoms and welders have an increased pneumonia and cardiovascular risk [103,104].
T214	44547-44819	Sentence	denotes	In this regard, metal fume fever has been shown to produce fever, fatigue, muscle ache, cough, dyspnea, and an increase of several cytokines including IL-1β, IL-6, IL-8 and TNF-α [103], symptoms and cytokines that have already been described for COVID-19 patients [2,8,9].
T215	44820-45005	Sentence	denotes	Notably, TNF-α was upregulated early while IL-6 and IL-8 increased later, suggesting a subsequent involvement of these latter cytokines in response to inhalation of welding fumes [103].
T216	45006-45174	Sentence	denotes	Moreover, elevated levels of zinc have been shown not only to stimulate pro-inflammatory cytokine secretion by monocytes, but also inhibit T cell functions (see [102]).
T217	45175-45537	Sentence	denotes	Zinc supplementation that has also been proposed for COVID-19 treatment [105,106,107], is able to suppress allogeneic immune response (see [102]) and incubation of blood cells with welding fume particles resulted in a considerable increase not only of pro-inflammatory cytokines (including IL-1β, IL-6, IL-8, TNF-α) but also the tolerogenic cytokine IL-10 [104].
T218	45538-45866	Sentence	denotes	Similarly, in pulmonary arterial hypertension (PAH) as well as SARS-CoV, both arms of the inflammatory system (namely IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 and TNF-α) have been shown to be upregulated [108,109,110], suggesting a common systemic pathway of activation in welding syndrome, ARDS, PAH and SARS-CoV infections.
T219	45867-46036	Sentence	denotes	Finally, the correlations between zinc exposure (metal fume) and cytokine storm [103,104] suggests a possible link between free zinc increase and these type of diseases.
T220	46037-46217	Sentence	denotes	More information on systemic zinc homeostasis and multiorgan effects of elevated levels of extracellular free zinc (such as in subjects inhaling ZnCl2 smoke) is available in Box 4.
T221	46218-46320	Sentence	denotes	Box 4 Systemic zinc homeostasis and multiorgan effects of elevated levels of extracellular free zinc.
T222	46321-46562	Sentence	denotes	Zinc absorption occurs throughout the gastrointestinal tract into enterocytes by zinc transporters of the Zrt- and Irt-like protein (ZIP) family that take up Zn2+, but also Cu2+ and Fe2+ ions, both of which can inhibit zinc absorption [111].
T223	46563-46860	Sentence	denotes	The bioavailability of zinc is affected by several factors among them the amount of zinc in food, its chemical form (from very low availability, e.g., zinc oxide, to comparatively high, e.g., zinc chloride salt) and its binding to other (intestinal absorbed or non-absorbed) food components [111].
T224	46861-47065	Sentence	denotes	For example, phytates (components present in foods like rice, cereals and corn) and folic acid can form insoluble complexes with zinc that decrease zinc absorption and increase fecal zinc excretion [111].
T225	47066-47302	Sentence	denotes	On the other hand, Zn2+ can form complexes with most natural amino acids (AA) that for example can come from digested proteins; each Zn2+ can be coordinated with the amine groups and carboxylate anions of two AAs (a molar ratio of 1:2).
T226	47303-47577	Sentence	denotes	Interestingly, in an in vitro model, the formulation of either ZnCl2 salt or Zn-AA complexes were absorbed by enterocytes and zinc was released on the opposite side of the enterocyte membrane, thus mimicking the intestinal absorption and release into the blood stream [111].
T227	47578-47709	Sentence	denotes	It is therefore conceivable that zinc chelates of AA, as well as zinc salts, may participate in bioavailability and uptake of zinc.
T228	47710-47996	Sentence	denotes	The uptake of zinc-AA complexes by AA transporters of human enterocytes suggests that B0AT1 neutral AA transporter associated with ACE2 [28] might also be involved in zinc-AA uptake (a similar mechanism might be supposable for insulin biosynthesis/secretion in pancreatic β cells [91]).
T229	47997-48148	Sentence	denotes	Indeed, a recycling of zinc-AAs, possibly supplied by membrane bound ACE2 monocarboxypeptidase cleavage, for ACE2 synthesis could be also hypothesised.
T230	48149-48366	Sentence	denotes	Plasma zinc concentration is mainly regulated through a balance between intestinal absorption and renal excretion involving specific mechanisms sensitive to both dietary zinc availability and its cellular utilization.
T231	48367-48493	Sentence	denotes	The majority of Zn2+ in blood binds to serum albumin, which represents the major zinc transporter/reservoir protein in plasma.
T232	48494-48753	Sentence	denotes	On the other hand, labile-bound pool of Zn2+ labile complexes (e.g., of AA) and free Zn2+ are the biologically available forms of zinc for cellular internalization and/or enzymatic activities (0.1–1% of the total zinc in blood plasma under normal conditions).
T233	48754-48937	Sentence	denotes	Free Zn2+ is subsequently accumulated by endothelial cells and zinc uptake and release by endothelial cells is the crucial step for redistribution of Zn2+ from plasma to tissues [91].
T234	48938-49117	Sentence	denotes	Zinc is considered a relatively nontoxic metal; however, several data report that excessive free extracellular zinc is toxic and induce apoptosis in different cell types [91,102].
T235	49118-49451	Sentence	denotes	Plasma excess of free Zn2+ is uptaken by ZIP transporters on the cell surface of each cells in contact with plasma (including immune cells, endothelial cells, neurons and cardiomyocytes), leading to increase of intracellular zinc that can modulate zinc associated cellular functions in both physiological and pathological conditions.
T236	49452-49882	Sentence	denotes	Indeed, zinc is an important mediator/messenger involved in several cellular activities and the shift of zinc from plasma to cells may be significant for both normal physiological processes including energy metabolism, blood coagulation, and zinc signalling, and a range of disease states, including neurodegenerative and cardiovascular diseases, metabolic syndrome, inflammation, endothelial stress, diabetes and thrombosis [91].
T237	49883-50109	Sentence	denotes	For example, excess zinc intake has been shown to contribute to both amyloid beta-peptide plaque formation in Alzheimer’s disease and cardiac dysfunction, indicating that free zinc ion may be much more toxic than thought [91].
T238	50110-50386	Sentence	denotes	It has been reported that Alzheimer and other neurodegenerative disease patients show elevated levels not only of Zn2+ but also of other divalent metals such as Fe2+ and Cu2+ [112], suggesting a possible link between the increase metal ions and saturation of ZIP transporters.
T239	50387-50600	Sentence	denotes	On the other hand, increased plasma ferritin levels (such as in COVID-19 [2,3,6]) might reduce free Fe2+ and its “competition” with Zn2+ for ZIP transporters, finally increasing cellular zinc uptake and functions.
T240	50601-50740	Sentence	denotes	To this regard, EDTA metal chelator has been shown to protect from neural cell death and it was tested in neurodegenerative diseases [102].
T241	50741-50923	Sentence	denotes	Of interest, plasma albumin acts as a transport protein for both metal ions such as zinc and hydrophobic molecules such as free fatty acid (FFA) to allow their systemic distribution.
T242	50924-51105	Sentence	denotes	Indeed, albumin is well beyond a mere carrier of metal ions and hydrophobic molecules, and by binding of these different molecules mediates their crosstalk and bioavailability [91].
T243	51106-51218	Sentence	denotes	To this regard, one particularly intriguing allosteric link exists between FFA and zinc binding to albumin [91].
T244	51219-51424	Sentence	denotes	When FFAs with ten or more carbon atoms bind to albumin causes the release of Zn2+, suggesting a possible link between dyslipidemia and toxic effects mediated by consequent increase of free zinc in plasma.
T245	51425-51613	Sentence	denotes	For example, endothelial stress and activation and pro-inflammatory action of FFAs may be mediated by their effects on free zinc release from albumin and its consequent efflux from plasma.
T246	51614-51784	Sentence	denotes	Different studies have suggested a role of albumin to inhibit interaction of free Zn2+ with amyloid beta-peptides and combat plaque formation in Alzheimer’s disease [91].
T247	51785-51898	Sentence	denotes	Zn2+ has also been shown to promote clot stability by binding to fibrinogen and inhibiting heparin activity [91].
T248	51899-52020	Sentence	denotes	An evidence that has suggested a possible positive correlation between increased levels of FFAs and thrombotic risk [91].
T249	52021-52250	Sentence	denotes	In addition, physiological conditions associated with elevated plasma FFAs, such as physical exercise, can also produce an increased plasma level of free zinc and its downstream physiological events (e.g., activation of the RAS).
T250	52251-52419	Sentence	denotes	On the other hand, reduced concentrations of plasma albumin (such as IBD, ARDS and COVID-19) will induce elevated plasma levels of both free FFAs and bioavailable zinc.
T251	52420-52582	Sentence	denotes	Intriguingly, ARDS is induced after inhalation exposure to ZnCl2/ZnO/hexachloroethane, the main ingredients in smoke bombs used for crowd dispersal [100,101,102].
T252	52583-52700	Sentence	denotes	To this regard, soldiers that breathed smoke-bomb fumes have been shown to quickly or slowly develope ARDS [100,101].
T253	52701-52883	Sentence	denotes	Interestingly, a patient who had the highest level of serum zinc rapidly developed ARDS that leads to death from multiorgan failure (including respiratory and hepatic failure) [100].
T254	52884-53171	Sentence	denotes	Instead, a slow progressive clinical course was associated with a significant increase of plasma zinc concentration that negatively correlated with pulmonary and liver function [100], finally leading to death for severe respiratory failure three to five weeks after inhalation [100,101].
T255	53172-53272	Sentence	denotes	At autopsy diffuse microvascular obliteration and marked endothelial cell injury was apparent [101].
T256	53273-53465	Sentence	denotes	Widespread occlusion of the pulmonary arteries may contribute to the development of acute pulmonary hypertension and extensive interstitial and intra-alveolar fibrosis was also observed [101].
T257	53466-53678	Sentence	denotes	Other soldiers wearing gas masks, immediately developed severe coughing and dyspnea, and slowly improved their lung function (only twelve months after exposure their lung function tests were nearly normal) [101].
T258	53679-53763	Sentence	denotes	All clinical conditions that strongly resemble those described in COVID-19 patients.
T259	53765-53769	Sentence	denotes	5.2.
T260	53770-53836	Sentence	denotes	Safety and Efficacy Concerns of MLN4760 and Dx600 ACE2 Inhibitors.
T261	53837-53976	Sentence	denotes	It is known that the catalytic cleft of ACE2 consists of two peptidase subdomains: one membrane-distal and the other one membrane-proximal.
T262	53977-54097	Sentence	denotes	Their weak interactions are consistent with the ability to transition from open to the closed ACE2 conformation [28,90].
T263	54098-54373	Sentence	denotes	Indeed, the two subdomains undergo a large hinge-bending motion in which membrane-proximal subdomain remains almost unchanged, while membrane-distal subdomain moves to close the distance between the two subdomains, mimicking the opening/closing movement of a clam shell [90].
T264	54374-54664	Sentence	denotes	ACE2 open conformation likely reflects free state of the enzyme available to catch substrates (or inhibitors), then, when the ACE2 receptor binds to a substrate, the membrane-distal subdomain closes around the substrate (or the inhibitor), finally performing the enzymatic activity [24,90].
T265	54665-54845	Sentence	denotes	Interestingly, in cryo–electron microscopy structures of full-length human ACE2, only the closed/substrate-bound conformation of ACE2 was observed in the spike-ACE2 complexes [28].
T266	54846-55090	Sentence	denotes	Since human ACE2 is assembled on cell surface as a homodimer [28], binding of the spike protein trimer onto ACE2 dimer suggests simultaneous binding of two spike protein trimers to substrate-bound conformer of ACE2 homodimer on plasma membrane.
T267	55091-55347	Sentence	denotes	The spike binding sites on ACE2 homodimer are localized above the membrane-distal peptidase subdomain of each ACE2 monomer, nevertheless neither ACE2 shedding nor ACE2 binding to spike proteins have been shown to inhibit ACE2 enzymatic activity [16,17,24].
T268	55348-55615	Sentence	denotes	On the other hand, the S-protein-binding region of membrane-distal ACE2 subdomain is not significantly perturbed by the receptor conformational changes and maintains the ability to associate with soluble spike proteins independently on open/closed conformations [24].
T269	55616-55818	Sentence	denotes	Interestingly, an ACE2 specific inhibitor (MLN-4760) has been shown to induce the closed (inhibitor-bound) ACE2 structure [90] and to retain its inhibitory effects on sACE2 bound to spike proteins [24].
T270	55819-56019	Sentence	denotes	MLN4760 is a potent and selective human ACE2 inhibitor (IC50 = 0.44 nM against soluble human ACE2) whose synthesis produces a racemic mixture of two diastereomers that showed 75:25 ratio for Isomer A:
T271	56020-56129	Sentence	denotes	Isomer B [113,114] and the purified isomer B is the isomer commercially available from Merck Millipore [114].
T272	56130-56315	Sentence	denotes	Testing MLN-4760 racemic mixture and its isomers, it was observed a concentration-dependent inhibition of recombinant human (rh)ACE or rhACE2 activities with all three inhibitors [114].
T273	56316-56666	Sentence	denotes	The isomer B was less selective (near minimal-maximal rhACE inhibition range 10−6M-10−4M) than the racemate or the isomer A (near minimal-maximal rhACE inhibition range 10−6M-10−2M) and less effective (near maximal rhACE2 inhibition at 10−7M) than the racemate or the isomer A (near maximal rhACE2 inhibition at 10−8 M) for rhACE2 versus rhACE [114].
T274	56667-57033	Sentence	denotes	Moreover, all three inhibitors exerted a significantly higher inhibitory activity against soluble than membrane-bound forms of (m)ACE2 (near maximal mACE2 inhibition at 10−6M), being the isomer B more selective and effective than the racemate or the isomer A for mACE2 vs. mACE expressed on the surface of mononuclear cells (or CD34 hematopoietic progenitors) [114].
T275	57034-57133	Sentence	denotes	A second ACE2 inhibitor, Dx600, produced similar results to those obtained with the isomer B [114].
T276	57134-57300	Sentence	denotes	Another study evaluated the inhibitory activity of both MLN-4760 and DX600 (either the linear conformational form or the disulfide bridged cyclic variant) inhibitors.
T277	57301-57512	Sentence	denotes	In this report, the experiments were performed at pH 6.5 [115], a pH at which rhACE2 proteolysis operates at maximal activity [79] and a condition that resembles hypercapnic acidosis which may occur during SARS.
T278	57513-57849	Sentence	denotes	MLN-4760 was still able to strongly and specifically inhibit rhACE2 activity (near maximal inhibition at 10−8 M), preventing rhACE2-driven Ang II degradation into Ang (1–7), whereas DX600 (either linear or cyclic variant) inhibits rhACE2 at relatively higher concentration (near maximal inhibition at 10−6 M–10−7 M, respectively) [115].
T279	57850-57994	Sentence	denotes	Altogether these data indicate that the racemate or the isomer A are more effective in inhibiting soluble forms of ACE2 than isomer B and Dx600.
T280	57995-58348	Sentence	denotes	Therefore, these inhibitors at opportune (low) concentrations are expected to preferentially reduce systemic sACE2 activity, while preserving the ACE2 activity of (local) membrane-associated forms of ACE2, knowing that catalytic activity of circulating ACE2 was undetectable in human plasma of healthy subjects due to an endogenous ACE2 inhibitor [116].
T281	58349-58704	Sentence	denotes	Since MLN-4760 inhibitor promotes the closed ACE2 conformation [90] which is the preferential conformer for virus binding [28], MLN-4760 is expected to not prevent viral entry; nevertheless, its inhibitory function on ACE2 pathway might work on the positive feedback loops (above described) that ultimately favour ACE2 membrane expression and viral entry.
T282	58705-58822	Sentence	denotes	A potential risk factor of inhibiting the Ang II metabolization into Ang 1–7 could be the increase of blood pressure.
T283	58823-59332	Sentence	denotes	Although ACE2 pathway inhibition might lead to hypertensive effects, treatment with MLN-4760 for 4-5 weeks had no effect on blood pressure when administered 10 mg/kg/day in drinking water in wild type mice [117] nor in male (mRen2)27 transgenic hypertensive rats (administered 30 mg/kg/day subcutaneously via mini-osmotic pumps) [118], suggesting that hypertensive activity mediated by ACE2 inhibition is promptly balanced by compensatory mechanisms either in normal or hypertensive blood pressure conditions.
T284	59333-59538	Sentence	denotes	In addition, injections of MLN4760 into the nucleus tractus solitarii has been shown to reduce the baroreceptor reflex in rats, suggesting a role for ACE2 in controlling a reflex bradycardia (see [39,44]).
T285	59539-59773	Sentence	denotes	Finally, chronic inhibition of ACE2 with MLN-4760 led to increase of ACE, albuminuria and glomerular injury in streptozotocin-induced diabetic mice, indicating a possible adverse effect of the inhibitor in a diabetic background [119].
T286	59774-59947	Sentence	denotes	Extensive experiments have been also performed with DX600, a specific peptide ACE2 inhibitor that exhibited a mixed competitive and non-competitive type of inhibition [120].
T287	59948-60045	Sentence	denotes	Actually, several reports describing Dx600 inhibitor administration in mice suggest its safe use.
T288	60046-60326	Sentence	denotes	Of interest, a report described its (safe) use alone (1 mg/kg per day) by nasal inhalation for 3 days in a mouse model of endotoxin-induced lung inflammation [121]; however, this inhibitor is less efficacious than MLN-4760 in inhibiting the soluble forms of human rACE2 [114,115].
T289	60327-60523	Sentence	denotes	Altogether these reports on administration of ACE2 inhibitors do not reveal significant adverse impacts or mortality in experimental animals, which suggests their safety in chronic administration.
T290	60524-60623	Sentence	denotes	This was also confirmed in human clinical trials with MLN-4760 (clinical name: ORE1001, see later).
T291	60624-61143	Sentence	denotes	Of interest, a report showed that on day 28 post-myocardial infarction, adult male Sprague-Dawley rats that had received MLN-4760 (also called C16) 25 mg/mL/day by daily intraperitoneal injection (as a solution of 42 mg/mL in distilled water) tended to have lower left ventricular pulse pressure, mean arterial pressures and left ventricular relaxation time constant-Tau compared to untreated group (see table 2 of the paper) [52], suggesting a possible protective role of ACE2 inhibition in post-myocardial infarction.
T292	61144-61424	Sentence	denotes	Therefore, MLN-4760 might be helpful not only for COVID-19 but also in targeted therapies for pathologies correlated with an excessive increase of ACE2 activity that may involve heart, lung, liver, colon or other tissues/organs expressing ACE2 such as blood and endothelial cells.
T293	61425-61651	Sentence	denotes	As an example, GL1001 (old name of MLN-4760) showed to produce an anti-inflammatory activity in a mouse model of colitis [122], highlighting the importance of the yin-yang balance of ACE/ACE2 pathways (“in medio stat virtus”).
T294	61653-61657	Sentence	denotes	5.3.
T295	61658-61706	Sentence	denotes	Safety and Efficacy Concerns of Chelating Agents
T296	61707-61924	Sentence	denotes	Based on ACE2 mechanism of action, there are alternative ways to inhibit ACE2 (and ACE), for example concentration of cation/anion might also targets of intervention to inhibit the zinc metalloprotease ACE2 (and ACE).
T297	61925-62022	Sentence	denotes	In this regard, EDTA, a cation chelator, has been shown to be able to inhibit ACE2 activity [79].
T298	62023-62317	Sentence	denotes	Of interest, EDTA binds with 106- and 102-fold higher affinity to Zn2+ than to Ca2+ and Fe2+, respectively [123], and in plasma, free Ca2+ concentration (1.05–1.30 mmol/L) is “only” about 103-fold higher than free Zn2+ (0.1–2.0 µmol/L), while majority of iron in plasma is bound to transferrin.
T299	62318-62430	Sentence	denotes	As a consequence, the iron concentration in plasma of healthy subjects is very low on the order of 10−18M [112].
T300	62431-62738	Sentence	denotes	Among the essential metal ions present in the organisms, the specific affinity of EDTA for systemic Zn2+ in physiologic conditions is highlighted by the evidence that prolonged treatment with CaNa2EDTA results in specific zinc depletion that is believed to mediate the teratogenic effects of the drug [123].
T301	62739-63068	Sentence	denotes	As already mentioned, zinc is an important mediator/messenger involved in several cellular activities and excess of free zinc has been shown to be toxic (see also Box 4); however, zinc deficiency has also detrimental effects on growth, neuronal development, and immunity, and in severe cases its consequences can be deadly [102].
T302	63069-63261	Sentence	denotes	For these reasons, treatments with CaNa2EDTA require a careful monitoring of the patient for the therapeutic effects as well as possible complications, so as to titrate the appropriate dosage.
T303	63262-63367	Sentence	denotes	CaNa2EDTA was approved by FDA in chelation therapy for lowering blood lead levels a long time ago (1953).
T304	63368-63501	Sentence	denotes	Then, different (commercially available) iron chelating agents, expected to work in chelating zinc ion as well, were approved by FDA.
T305	63502-63635	Sentence	denotes	Chelation therapy comprises intravenous or oral administration of chelating agents that remove metal ions such as zinc from the body.
T306	63636-63733	Sentence	denotes	Cells synthesising high amounts of ACE or ACE2 needs of high amounts of bioavailable (free) zinc.
T307	63734-63876	Sentence	denotes	Therefore, it is conceivable that plasma levels of free zinc may influence not only ACE and ACE2 activities but also their cellular synthesis.
T308	63877-64008	Sentence	denotes	Indeed, both ACE and ACE2 synthesis might be particularly sensitive to reduction of free zinc levels in case of their upregulation.
T309	64009-64201	Sentence	denotes	In addition, metal chelating agents, by limiting the availability of free zinc to cells, might have effects on both ACE2/ACE synthesis and conformation (when assembled on the plasma membrane).
T310	64202-64499	Sentence	denotes	Indeed, the closed conformer of ACE2 homodimer that is the preferential conformation for virus binding [28], needs the presence of both zinc and substrate/inhibitor in the catalytic site [90], suggesting that zinc chelation (differently from MLN-4760) might also inhibit ACE2-mediated viral entry.
T311	64500-64746	Sentence	denotes	Intriguingly, chloroquine has been shown to specifically enhance zinc uptake and its accumulation/sequestration in the lysosomes [124], raising the possibility that it might work on COVID-19 patients by reducing zinc recycling and zinc functions.
T312	64747-64956	Sentence	denotes	As already mentioned, free Zn2+ promotes clot stability by binding to fibrinogen (see Box 4 and [91]) and chloroquine has been shown to have an anti-thrombotic activity by inhibiting platelet activation [125].
T313	64957-65337	Sentence	denotes	To this regard, upon platelet activation the release of Zn2+ store from their secretory granules has been shown to participate to the pro-coagulant activity in platelet-dependent fibrin formation [126], suggesting that an elevated free Zn2+ concentration might occur and contribute to thrombotic predisposition in COVID-19 patients, a phenomenon possibly countered by chloroquine.
T314	65338-65579	Sentence	denotes	For all of the above reasons, cation chelating agents, administered alone or in combination with other therapies, might be effective to counter COVID-19 infection, in particular when, induced by hypoxia, both arms of the RAS are upregulated.
T315	65580-65627	Sentence	denotes	A scenario that would deserve an investigation.
T316	65628-65990	Sentence	denotes	However, some formulations of metal chelating agents carry a black box warning because they may cause serious and fatal renal toxicity and failure, hepatic toxicity and failure, gastrointestinal haemorrhage, arrhythmias, tetany, hypocalcaemia, hypotension, convulsions, respiratory arrest, and agranulocytosis that can lead to serious infections and death [123].
T317	65991-66158	Sentence	denotes	For the sake of completeness, besides renal toxicity linked to the route of drug excretion, EDTA has been shown to be effective in chronic renal artery diseases [123].
T318	66159-66249	Sentence	denotes	Finally, for its teratogenic effects, the drug is also contraindicated in pregnancy [123].
T319	66250-66472	Sentence	denotes	As a result, treatments with metal chelating agents require close patient monitoring, including laboratory tests of renal and hepatic function, and absolute neutrophil count should be monitored before and during treatment.
T320	66473-66544	Sentence	denotes	Alternative ways of RAS pathway inhibition are also described in Box 5.
T321	66545-66595	Sentence	denotes	Box 5 Alternative ways of RAS pathway inhibition.
T322	66596-66703	Sentence	denotes	Of interest, a small cationic inhibitor of ACE2 (but not of ACE) has been detected in plasma samples [116].
T323	66704-66958	Sentence	denotes	The endogenous inhibitor might play a compensatory fine-control (within a threshold limit) for normal fluctuation of ACE2 protein in plasma and it has been hypothesized to be a basic AA or a small basic peptide able to compete with ACE2 substrates [116].
T324	66959-67066	Sentence	denotes	I have already mentioned that AA can chelate zinc forming labile zinc complexes of amino acids (see Box 4).
T325	67067-67252	Sentence	denotes	It is therefore possible that a basic AA or a small basic peptide capable to specifically accommodate into the catalytic site of ACE2, but into that of ACE, could inhibit ACE2 activity.
T326	67253-67417	Sentence	denotes	Indeed, despite the similarities, ACE and ACE2 possess different substrate specificity that depends on the smaller ACE2 binding pocket compared to that of ACE [90].
T327	67418-67594	Sentence	denotes	This specific aspect might determine the specificity for a specific basic amino acid or molecule that is able to accommodate into the catalytic pocket of ACE2 and bind to zinc.
T328	67595-67755	Sentence	denotes	Among the possible endogenous ACE2 inhibitors, agmatine, decarboxylated arginine, has a chemical structure that resembles that of an ACE2 inhibitor, NAAE [127].
T329	67756-67932	Sentence	denotes	NAAE is a small molecule that had demonstrated an anti-SARS-CoV activity, by acting on both ACE2 catalytic activity and ACE2 binding domain for spike protein of SARS-CoV [127].
T330	67933-67981	Sentence	denotes	Unfortunately, NAAE has never been used in vivo.
T331	67982-68272	Sentence	denotes	Indeed, NAAE is a weak ACE2 inhibitor, it is in fact more than a thousand-fold less potent than MLN-4760; however, if agmatine will be proven to have ACE2 inhibitory activity, it might be helpful to prevent the trigger of the positive feedback loops in the first mild phases of the disease.
T332	68273-68395	Sentence	denotes	Indeed, it has an important role in down-regulating NO synthesis reducing NO overproduction by different mechanisms [128].
T333	68396-68557	Sentence	denotes	Of note, NOS pathway has been shown to be upregulated by both Ang (1–7)/MasR and Ang (1–9)/AT2 receptor pathways that are downstream ACE2 activity [38,39,41,43].
T334	68558-68749	Sentence	denotes	Moreover, agmatine has a regulated plasma concentration in the range of 20-80 ng/mL and the use of dietary agmatine has been shown to be safe and effective in reducing neuropathic pain [129].
T335	68750-68825	Sentence	denotes	Moreover, agmatine sulfate is regularly taken as a bodybuilding supplement.
T336	68826-69036	Sentence	denotes	Among natural metal-chelating agents, phytates and folic acid are two chelating agents from vegetables that might reduce zinc intestinal absorption and possibly its systemic concentration (see Box 4 and [111]).
T337	69037-69189	Sentence	denotes	Similarly, nicotianamine that is extracted from plants (soybean) is a low-molecular weight metal chelator with high affinity for divalent metal cations.
T338	69190-69293	Sentence	denotes	Nicotianamine has been shown to inhibit the activity of both zinc metalloproteases, ACE2 and ACE [130].
T339	69294-69375	Sentence	denotes	In addition, zeolites might also be effective in reducing free zinc availability.
T340	69376-69504	Sentence	denotes	Zeolites are a group of aluminosilicate minerals with crystalline microporous structure that are originated from volcanic rocks.
T341	69505-69676	Sentence	denotes	Their molecular structure generates cavities that allow high absorbency capacity for a wide range of charged elements such as water, heavy metals, cations and many toxins.
T342	69677-69777	Sentence	denotes	Clinoptilolite is one of the zeolites that has been widely studied in veterinary and human medicine.
T343	69778-69964	Sentence	denotes	The increased usage of clinoptilolite-based products in vivo has stimulated several investigations on its safety and its positive medical effects related to human health (see [131,132]).
T344	69965-70102	Sentence	denotes	Finally, soluble and catalytically inactive forms of ACE2 have been shown to be potent inhibitors of SARS-CoV infection products [19,21].
T345	70103-70203	Sentence	denotes	An approach that could be pursued (in combination with other therapies) to inhibit SARS-CoV-2 entry.
T346	70204-70420	Sentence	denotes	Indeed, soluble forms of ACE2 are expected to protect from viral infection and a similar strategy using a recombinant form of human ACE2 has been proposed not only in COVID-19, but also in ARDS and PAH [108,133,134].
T347	70421-70551	Sentence	denotes	However, it is possible that catalytic active form of ACE2 might favour adverse effects in these specific pathological conditions.
T348	70552-70862	Sentence	denotes	To this regard, a clinical trial using recombinant hACE2 protein has been recently started (ClinicalTrials.gov number, NCT04287686) in COVID-19 patients and pilot clinical trials of rhACE2 in ARDS and PAH started in 2017 and 2018, respectively [108,134]; unfortunately, no conclusive results are available yet.
T349	70864-70866	Sentence	denotes	6.
T350	70867-70980	Sentence	denotes	Correlation of Pre-existing Circulating ACE2 Activity and Increased Potential to Develop Severe Forms of COVID-19
T351	70981-71124	Sentence	denotes	Severe symptoms of COVID-19 have been described to correlate with pre-existing hypertension, diabetes, age and male gender [1,2,3,4,5,6,7,8,9].
T352	71125-71502	Sentence	denotes	It is not still clear whether it depends on constitutive hypertensive conditions and/or on anti-hypertensive treatments or on other age-related conditions, considering that the prevalence of hypertension in Chinese adults is ~ 23% and that only about 41% take prescribed antihypertensive medications, being calcium channel blockers the most commonly used (~ 50%) in China [11].
T353	71503-71741	Sentence	denotes	To this regard, there is an interesting report describing sACE2 activity in plasma samples of Spanish healthy subjects and patients, in which a total of 2572 subjects from a multicenter study (NEFRONA project, 2009–2011) was studied [96].
T354	71742-71860	Sentence	denotes	The report shows that male and advanced age were identified as independent predictors of enhanced sACE2 activity [96].
T355	71861-72052	Sentence	denotes	Furthermore, subjects with hypertension, diabetes, dyslipidemia, or plaques also had significantly increased circulating ACE2 activity when compared with those without these pathologies [96].
T356	72053-72268	Sentence	denotes	Notably, hypertensive (the most frequent comorbidity with COVID-19) and diabetic patients are often treated with ACE inhibitors (ACEIs) and/or with ARBs, suggesting a possible positive correlation [135,136,137,138].
T357	72269-72554	Sentence	denotes	Interestingly, circulating ACE2 activity is significantly increased in subjects on therapy with ARBs or taking oral antidiabetic agents as compared with non-treated patients, while treatment with ACEIs and cholecalciferol had no significant influence on circulating ACE2 activity [96].
T358	72555-72710	Sentence	denotes	In line with this observations, losartan (an ARB), but not lisinopril (an ACEI), was able to upregulate ACE2 activity in left ventricle of Lewis rats [75].
T359	72711-73020	Sentence	denotes	Nevertheless, plasma concentration of Ang (1–7) was significantly increased with both treatments when compared to vehicle-treated rats and it was significantly higher in ACEI-treated than ARB-treated rats [75], suggesting that ACE inhibition is able to induce an activation of the ACE2/Ang (1–7)/MasR pathway.
T360	73021-73192	Sentence	denotes	Although more evidence is needed in humans, the above observations suggest that ARBs should be precautionarily avoided to reduce possible ACE2-mediated viral consequences.
T361	73193-73366	Sentence	denotes	Indeed, under hypoxic conditions, either an ACEI or an ARB have been shown to upregulate membrane ACE2 protein expression on human pulmonary artery smooth muscle cells [58].
T362	73367-73518	Sentence	denotes	ACEI and ARB have been shown to work by reducing the concentration of Ang II and by inhibiting its AT1-mediated ACE2 downregulation, respectively [58].
T363	73519-73774	Sentence	denotes	ACEIs and ARBs might therefore play a role in the upregulation of membrane ACE2 expression under hypoxic conditions such as COVID-19, knowing that the increase of membrane bound ACE2 (before its shedding) will also increase the probability of viral entry.
T364	73775-73877	Sentence	denotes	Of note, ACEI and ARB antihypertensive medications are more commonly used in Europe/USA than in China.
T365	73878-74102	Sentence	denotes	Nevertheless, recent reports show that the use of ACEI/ARB medications in patients with COVID-19 is safe and ACEI/ARB exposure was not associated with a higher risk of having severe forms of COVID-19 [6,139,140,141,142,143].
T366	74103-74345	Sentence	denotes	Moreover, some reports show that ACEI/ARB treated patients may even be protected from COVID-19 and ACEI/ARB exposure was associated with a lower risk of mortality compared to those on non-ACEI/ARB antihypertensive drugs [144,145,146,147,148].
T367	74346-74546	Sentence	denotes	Among these reports, one indicates that the risk of severe symptoms of COVID-19 was significantly decreased in patients who took ARB drugs (but not ACEIs) compared to patients who took no drugs [144].
T368	74547-74773	Sentence	denotes	Differently, another report suggests that the use of ACEIs (but not ARBs) reduced risk of death and/or critical disease [146], suggesting that some of these reports did not adjust for confounders and further analyses are need.
T369	74774-74972	Sentence	denotes	Nevertheless, clinical trials of losartan as a treatment for COVID-19, are actually underway among patients who have not previously been treated with ARBs and/or ACEIs (NCT04312009 and NCT04311177).
T370	74973-75222	Sentence	denotes	However, most of hypertensive/diabetic patients are ACEI- or ARB-“pretreated”, nevertheless they have an increased risk to develop SARS, therefore ACEIs and ARBs, if not detrimental, are not expected to face the disease in non-hypertensive patients.
T371	75223-75554	Sentence	denotes	A possible beneficial effect of ACEIs in COVID-19 patients could indirectly come by reducing ACE2 substrate, Ang II, and finally limiting Ang (1–7) [but not Ang (1–9)] production and its (detrimental) effects; however, this is only a hypothetical possibility since, in this case, the ACE2/ACE pathway might be even more unbalanced.
T372	75555-75747	Sentence	denotes	To complete the picture, smokers and subjects on therapy with insulin tend to have an increased (although not significantly) circulating ACE2 activity when compared with control subjects [96].
T373	75748-76009	Sentence	denotes	On the other hand, increased ACE2 protein expression was reported in plasma and/or urine of physically active men after acute aerobic training or in renal cortices of spontaneous hypertensive, but not normotensive, rats after chronic aerobic training [149,150].
T374	76010-76239	Sentence	denotes	In addition, recent works reveal that asthma and other allergic diseases, which protect from developing COVID-19, were associated with significant reductions in levels of both zinc in plasma and ACE2 mRNA in airway cells [98,99].
T375	76240-76498	Sentence	denotes	On the other hand, as already mentioned, some patients with cardiovascular diseases (and inflammatory bowel disease) have an increased circulating ACE2 [22,49,151,152], which might explain the higher probability of elderly heart patients to develop COVID-19.
T376	76499-76728	Sentence	denotes	Interestingly, in a report on atrial fibrillation, increased plasma ACE2 activity was significantly associated not only with cardiac dysfunction, increasing age, male gender and hypertension, but also with vascular disease [152].
T377	76729-76970	Sentence	denotes	Altogether the data suggest a strong correlation between circulating ACE2 activity and the predisposition to develop the most severe symptoms of COVID-19, suggesting that circulating sACE2 might be a predictive biomarker of SARS development.
T378	76971-77206	Sentence	denotes	The surprising aspect is that circulating (differently from membrane bound) ACE2 is expected to protect from viral infection and clinical trials using recombinant ACE2 protein are being pursued (ClinicalTrials.gov number, NCT04287686).
T379	77207-77410	Sentence	denotes	However, even if it might not be detrimental, recombinant ACE2 is not expected to protect from COVID-19, as circulating ACE2 upregulation correlates with the most common comorbidities in severe COVID-19.
T380	77411-77707	Sentence	denotes	The correlation is extremely impressive and striking if we also consider the low prevalence of chronic renal disease in COVID-19 hospitalized patients (0–3%) [1,2,3,4,5,6,7,8,9] (prevalence of the disease in China ~11% [12]) which might be protected by a higher sACE2 and/or Zn2+ renal excretion.
T381	77708-77926	Sentence	denotes	Indeed, in chronic kidney disease patients without a history of cardiovascular disease, there was a significant decrease in circulating ACE2 activity and zinc levels when compared with healthy control subjects [96,97].
T382	77927-78236	Sentence	denotes	Since proteinuria was associated with lower blood levels of sACE2 protein [153] and chronic kidney disease patients had higher urinary zinc excretion than healthy controls [97], low sACE2 activity in chronic renal diseases and protection from SARS might derive from a higher sACE2 and/or Zn2+ renal excretion.
T383	78237-78483	Sentence	denotes	Indeed, sACE2 is detectable in urine of healthy subjects and urinary sACE2 protein levels are elevated in patients with chronic renal diseases and in hypertensive patients treated with the Ang II type 1 receptor blocker (ARB) olmesartan [41,154].
T384	78484-78709	Sentence	denotes	It can, therefore, be supposed that a basal hyperactivity of ACE2 and consequent ACE/ACE2 pathway disequilibrium in blood might predispose to the development of more severe COVID-19 symptoms that involve both arms of the RAS.
T385	78710-79086	Sentence	denotes	In this regard, different organism predisposition to infections including SARS-CoV-2 or to pathologies associated with an increased risk to develop severe forms of SARS-CoV-2 infection might depend not only on genetic ACE2 polymorphisms and organ-specific ACE2 gene/protein expression [138,155,156,157,158,159,160,161,162,163] but also on anatomical and environmental factors.
T386	79087-79263	Sentence	denotes	For example, ACE2 gene variants have been associated with risk for hypertension, dyslipidemia, type 2 diabetes and cardiovascular dysfunction [138,155,156,157,158,159,160,161].
T387	79264-79508	Sentence	denotes	Most of these variants are intronic and located in splice-site junctions or in enhancer regions while the others are placed in the 3’ UTR promoter region, suggesting their involvement in the structure/function and expression of ACE2 gene [138].
T388	79509-79661	Sentence	denotes	On the other hand, COVID-19 has similar risk factors of obstructive sleep apnea (OSA), suggesting a possible association between OSA and COVID-19 [164].
T389	79662-79828	Sentence	denotes	Indeed, high prevalence of pre-existing OSA in COVID-19 patients has been reported [165,166], indicating that OSA could be a risk factor for severe forms of Covid-19.
T390	79829-80055	Sentence	denotes	OSA is characterised by repetitive airway collapse with apnea/hypopnea and intermittent hypoxaemia during sleep and it is associated with hypertension, cardiovascular disease, diabetes, obesity and systemic inflammation [167].
T391	80056-80312	Sentence	denotes	Most of these diseases are known to predispose to increased levels of circulating ACE2 and, as already mentioned, hypoxia has been shown to upregulate both arms of the RAS; therefore, OSA might predispose to the deleterious effects of SARS-CoV-2 infection.
T392	80313-80553	Sentence	denotes	In OSA patients, upper airway collapsibility under passive conditions (critical closing pressure) is higher in males than in females due to anatomical factors, i.e., longer upper airways, as upper airway length correlates with OSA severity.
T393	80554-80794	Sentence	denotes	Indeed, upper airways in females are less collapsible and more stable during sleep than in males [167], suggesting that an anatomical factor might contribute to and result in different gender susceptibility to COVID-19 and disease severity.
T394	80795-80976	Sentence	denotes	Moreover, diet (e.g., zinc assumption), physical exercise and external temperature and UV radiation are all “environmental” aspects that can seasonally influence the RAS activities.
T395	80977-81090	Sentence	denotes	Seasonal differences of the RAS activities leading, for example, to differences in blood pressure are well known.
T396	81091-81213	Sentence	denotes	Of interest, the majority of coronaviruses cause cold-like illnesses and have a relatively low mutational frequency [168].
T397	81214-81287	Sentence	denotes	Similarly SARS-CoV-2 seems to mutate much slower than seasonal flu [169].
T398	81288-81522	Sentence	denotes	Nevertheless, like influenza virus, they are responsible for seasonal episodes of common cold in humans worldwide [168], suggesting that coronaviruses may possess an ability to evade immune control that is different from seasonal flu.
T399	81523-81615	Sentence	denotes	For example, they might favour a relatively short-term activation/memory of immune response.
T400	81616-81752	Sentence	denotes	Indeed, as for SARS-CoV and MERS-CoV, a short duration of immunity after SARS-CoV-2 infection has been recently suggested [170,171,172].
T401	81753-81933	Sentence	denotes	Seasonal cold is an annually recurring time period characterized by the prevalence of outbreaks of cold and the season occurs during the cold period of the year in each hemisphere.
T402	81934-82304	Sentence	denotes	Altogether these observations suggest that seasonal predisposition to some virus infections (including SARS-CoV-2) might not depend only on environmental features that directly inactivate the viruses “unprotected” outside the host (e.g., higher ultraviolet rays and temperatures), but also on seasonal indirect effects produced in the organisms by environmental changes.
T403	82306-82308	Sentence	denotes	7.
T404	82309-82418	Sentence	denotes	Monitoring ACE/ACE2 Activity in COVID-19 in Order to Determine a Rationale Use of the Specific RAS Inhibitors
T405	82419-82526	Sentence	denotes	There is currently no specific pharmacotherapy to combat SARS-CoV-2 infection and its pathological effects.
T406	82527-82922	Sentence	denotes	Actually, the use of plasma from convalescent COVID-19 patients or of SARS-CoV-2-specific neutralizing antibodies has been shown to be a promising option for the treatment of critically COVID-19 patients [32,33,34,35,36,37,172,173]; however, it has some limitations that might be overcome by vaccine strategies or by the production of specific anti SARS-Co-V-2 therapeutic monoclonal antibodies.
T407	82923-83099	Sentence	denotes	Despite the promising data that demonstrate vaccine protection against SARS-CoV-2 in nonhuman primates [174], we cannot be sure to get a vaccine against SARS-CoV-2 (see [175]).
T408	83100-83350	Sentence	denotes	Indeed, animals immunized with inactivated SARS-CoV vaccines developed a severe (asthma-like) lung eosinophilic immunopathology when challenged with SARS-CoV, further indicating a central role of eosinophil “balanced numbers” in this pathology [176].
T409	83351-83534	Sentence	denotes	Vaccines might generate antibodies against viral ligand/ACE2 complex that finally blocks ACE2 activity during SARS-CoV infection and consequent downstream asthma-like events/symptoms.
T410	83535-83846	Sentence	denotes	On the other hand, fatal outcomes in SARS-CoV infection correlated with a cytokine storm involving elevated Th2 serum cytokines (including IL-4, IL-5 and IL-10), suggesting that an increase of Th2 cytokines possibly mediated by virus-specific CD4 T cells might be crucial in the severe forms of infection [110].
T411	83847-84261	Sentence	denotes	In addition to cytokine profile (such as IL-10), eosinopaenia, tachycardia, normo/hypotension (although COVID-19 and hypoxia increase Ang II and many patients are “hypertensive” and/or receiving anti-hypertensive medications) and hypoxia in SARS-CoV-2 patients are compatible with downstream events stemming from both an excessive ACE2 pathway upregulation and activation of positive feedback loops (see Figure 2).
T412	84262-84458	Sentence	denotes	ACE2 and (anti-inflammatory) IL-10 hyperproduction may ultimately trigger subsequent compensatory (and deleterious) responses, leading to both renin/ACE and pro-inflammatory cytokine upregulation.
T413	84459-84578	Sentence	denotes	In line with this hypothesis, a longitudinal study showed that IL-6 increases late during the COVID-19 progression [3].
T414	84579-84875	Sentence	denotes	Altogether these data imply not only that the ACE2 is the “vehicle” of viral entry into the host cells, but also that virus-dependent and virus-independent mechanisms may sustain activation of both arms of the RAS and of the inflammatory system, finally promoting SARS-induced multi-organ injury.
T415	84876-85186	Sentence	denotes	Unfortunately, complete analysis of circulating levels of ACE/ACE2 proteins and activities, together with ACE and ACE2 substrates and products i.e., Ang I, Ang 1–9, Ang II, Ang (1–7) and Ang (1–5), des-Arg(9)-bradykinin and the inactive metabolite bradykinin (1–7) is lacking to address the current hypothesis.
T416	85187-85370	Sentence	denotes	The required data could be obtained from blood sample analyses, however, it is important to consider that:(1) Organ-specific local microenvironment might not reflect the systemic one;
T417	85371-85607	Sentence	denotes	(2) S1-sACE2 and SARS-CoV-2-sACE2 complexes, formed by viral-induced ACE2 shedding or by subsequent binding of sACE2 with viral particles or S1 fragments in the circulation, might not be detectable by some anti-ACE2 antibodies in ELISA.
T418	85608-85831	Sentence	denotes	Therefore, since the complexes might prevent/mask sACE2 antibody recognition but not the enzymatic activity, sACE2 detection (and its real concentration) by ELISA in blood samples of COVID-19 patients might not be reliable;
T419	85832-86128	Sentence	denotes	(3) In order to evaluate circulating ACE/ACE2 activity, we can determine it by adding fluorogenic (RAS) substrates to plasma samples without (thus considering also the endogenous ACE2 inhibitor present in plasma [116]) or with adding ZnCl2, which is able to induce ACE/ACE2 maximal activity [96];
T420	86129-86280	Sentence	denotes	(4) circulating concentrations ACE/ACE2 substrates/products likely depend on the:(a) level of ACE/ACE2 pathway activity and availability of substrates;
T421	86281-86414	Sentence	denotes	(b) level of expression of the respective receptors (AT1R, AT2R and MasR) that bind and remove ligand products away from circulation.
T422	86415-86637	Sentence	denotes	For example, Ang (1–7) concentration in blood by ACE2 pathway upregulation is dependent both on the increase of the (antagonistic) Ang II (and its precursor, Ang I) and on MasR expression on surface of cells in the organs.
T423	86638-86879	Sentence	denotes	To this regard, the detection of inactive metabolites, such as bradykinin (1–7), in COVID-19 patients and comparison with its concentration in healthy subjects could be the most indicative and reliable markers of ACE2 activity in the plasma.
T424	86880-87283	Sentence	denotes	Of interest, a report that described the ex vivo effect of human rACE2 on the levels of several endogenous Ang peptides in plasma samples, thus mimicking an acute increase of systemic ACE2 activity in plasma [115] showed a strong reduction of Ang II (as expected), a moderate reduction of Ang I and a significant (more than five-fold) increase of Ang (1–9), Ang (1–7) and (ACE-produced) Ang (1–5) [115].
T425	87284-87406	Sentence	denotes	Therefore, Ang (1–5) may also be an interesting surrogate marker of ACE-ACE2 combined activity in COVID-19 patients [115].
T426	87407-87927	Sentence	denotes	At the present time, it has been reported that plasma levels of Ang II in SARS-CoV-2 infected patients are markedly elevated as compared with healthy subjects (interquartile range between 260–360 pg/mL and 65–120 pg/mL, respectively) and linearly associated to viral load and lung injury [177]; unfortunately, nobody has tested the levels of Ang (1–7), Ang (1–9), Ang (1–5) and bradykinin (1–7), some of which might be at high levels as well, thus justifying both normo/hypotension and eosinopaenia in COVID-19 patients.
T427	87928-88111	Sentence	denotes	More information on the RAS peptides in plasma of Acute Respiratory distress syndrome, a COVID-19-like disease, (and pulmonary arterial hypertension) is instead available (see Box 6).
T428	88112-88195	Sentence	denotes	Box 6 Acute Respiratory distress syndrome and hyperactivation of the RAS pathways.
T429	88196-88322	Sentence	denotes	The main clinical feature of ARDS patients is the progressive deterioration of lung function leading to progressive hypoxemia.
T430	88323-88509	Sentence	denotes	ARDS can be induced by different causes such as ZnCl2 aspiration, sepsis, trauma, acute pancreatitis, or pneumonias following virus infections such as SARS-CoVs or human influenza virus.
T431	88510-88646	Sentence	denotes	ACE insertion/deletion polymorphisms correlated with severity of ARDS in humans, suggesting that the RAS could have a role in ARDS [44].
T432	88647-88825	Sentence	denotes	In animal models, ACE2 protected from acute lung by inhibiting Ang II/AT1R activity [15], for this reason the use of recombinant ACE2 in ARDS has been proposed and pursued [134].
T433	88826-89156	Sentence	denotes	Clinical trials with recombinant human ACE2 started in healthy volunteers in 2009 (ClinicalTrials.gov number, NCT00886353 [178]), then it was tested in ARDS (ClinicalTrials.gov number, NCT01597635 [134]), PAH (ClinicalTrials.gov number, NCT01884051 [108]) and finally in COVID-19 patients (ClinicalTrials.gov number, NCT04287686).
T434	89157-89255	Sentence	denotes	Despite its safe use, no conclusive evidence in support of its use in these diseases was reported.
T435	89256-89572	Sentence	denotes	Of interest, in the plasma samples from ARDS patients, levels of Ang I have been shown to be significantly increased in non-survivors (interquartile range 1990–16950 pg/mL) compared to survivors (interquartile range 730–5660 pg/mL) [92], suggesting a marked increase of renin activity in non-surviving ARDS patients.
T436	89573-90027	Sentence	denotes	Unfortunately, no comparisons of the two ARDS groups with healthy subjects (interquartile range 35–66 pg/mL, markedly lower than both patient groups [179]) were reported; this is important to consider since the plasma concentrations of the RAS peptides may significantly differ depending on method of detection (ELISA versus vs. mass spectrometry) and, in particular, on protocol of determination (endogenous versus equilibrium Ang peptide levels [179]).
T437	90028-90161	Sentence	denotes	Endogenous RAS peptide concentrations can be evaluated collecting and stabilizing blood samples with a RAS enzyme inhibitor cocktail.
T438	90162-90374	Sentence	denotes	Differently, heparinized plasma samples can be incubated at 37 °C to induce ex vivo equilibrium, which unveils the presence of renin, ACE and/or ACE2 activity in the plasma (equilibrium Ang peptide levels) [179].
T439	90375-90462	Sentence	denotes	This last protocol usually gives significantly higher levels of the RAS peptides [179].
T440	90463-90990	Sentence	denotes	For example, in healthy subjects, the normal ranges (interquartile ranges) of endogenous RAS peptides, Ang I, Ang II, Ang (1–7), Ang (1–9) and Ang (1–5) were 12–24 pg/mL, 1–12 pg/mL, <2 pg/mL, <4 pg/mL and < 1 pg/mL, respectively, differently the normal range after ex vivo equilibrium of the same peptides were 35–66 pg/mL, 110–200 pg/mL, < 2 pg/mL, < 4 pg/mL and < 1 pg/mL, respectively [179], indicating the presence of renin and ACE (but no ACE2) activity (as highlighted by Ang peptide increases upon ex vivo equilibrium).
T441	90991-91193	Sentence	denotes	Similarly, another report that evaluated Ang II and Ang (1–7) in healthy males showed normal endogenous ranges ~ 14.5 pg/mL and 0.9 pg/mL, respectively, and an Ang (1–7)/Ang II ratio ~ 0.06 ratio [180].
T442	91194-91456	Sentence	denotes	Moreover, Ang (1–7), Ang I, Ang (1–9) and Ang (1–7)/Ang II ratio were significantly higher in ACEI-treated hypertensive patients compared to healthy subjects [180], suggesting that inhibition of ACE predisposes to upregulation of the ACE2/Ang (1–7)/MasR pathway.
T443	91457-91678	Sentence	denotes	Notably, two different reports that evaluated the RAS peptides in ARDS patients using the similar method of detection (high performance liquid chromatography plus mass spectrometry) showed significantly different results.
T444	91679-91811	Sentence	denotes	One report showed that Ang II ranged (interquartile range) between 120–630 pg/mL (non-survivors) and 70–2220 pg/mL (survivors) [92].
T445	91812-92096	Sentence	denotes	A second report showed significantly lower values of Ang II, (interquartile range) between 40–150 ng/mL (non-survivors with placebo) and 5–20 pg/mL (survivors with placebo) (see Supplementary Data [134]), suggesting that they used different protocols of the RAS peptide determination.
T446	92097-92250	Sentence	denotes	Nevertheless, both Ang (1–7) and Ang (1–7)/Ang II ratios found in ARDS patients of both reports [92,134] were significantly higher than healthy subjects.
T447	92251-92443	Sentence	denotes	One report showed that Ang (1–7) ranged (interquartile range) between 2.5–10 pg/mL (see Figure 2B of [134]), Ang II between 5–20 pg/mL (see Figure 2 of [134]) and Ang (1–7)/Ang II ratio ~ 0.5.
T448	92444-92612	Sentence	denotes	Moreover, in the report in which the elevated concentrations of Ang I and Ang II indicate an ex vivo equilibrium of the RAS peptides, the increase was even more marked.
T449	92613-92931	Sentence	denotes	In that report, Ang (1–7) (< 1pg/mL in healthy subjects) ranged between 80–1070 pg/mL and Ang (1–7)/Ang II ratio (~ 0.06 in healthy subjects) ranged between 0.24–1.82 [92], suggesting that ACE2 activity (and ACE2 presence in plasma samples) was significantly increased in ARDS patients as compared to healthy subjects.
T450	92932-93113	Sentence	denotes	In line with this hypothesis, there was the determination of Ang (1–9), whose production from Ang I exclusively depends on ACE2 (which also compete with ACE for the same substrate).
T451	93114-93515	Sentence	denotes	In normal healthy subjects, it was reproducibly below the limit of quantification [179,180], differently, both in ex vivo treatments of plasma samples with human rACE2 [115,181] and in ARDS patients, it was markedly increased (Ang (1–9) ranged between 100–3080 pg/mL) [92], suggesting both that the dominating activity of ACE in plasma of healthy subjects is overcome by that of ACE2 in ARDS patients.
T452	93516-93593	Sentence	denotes	Therefore, Ang (1–9) may represent a good surrogate marker for ACE2 activity.
T453	93594-93802	Sentence	denotes	Similarly to Ang (1–9), there was the determination of Ang (1–5), whose production depend on both ACE2 [that produces Ang (1–7) from Ang II] and ACE [that produces Ang II from I and Ang (1–5) from Ang (1–7)].
T454	93803-94068	Sentence	denotes	Ang (1–5) was undetectable in healthy subjects [178,179]; however, it was significantly increased in ARDS patients (interquartile range between 50–730 pg/mL [92]), suggesting that both ACE2 and ACE activity were likely upregulated as compared with healthy subjects.
T455	94069-94192	Sentence	denotes	The activation for both arms of the RAS needs of the contemporary activation renin, the rate-determining enzyme of the RAS.
T456	94193-94523	Sentence	denotes	Indeed, analysis of plasma samples (from a healthy subjects), treated ex vivo with recombinant renin, showed an increase of Ang I, Ang II, Ang (1–7) and Ang (1–5), but not Ang (1–9) [115,179,181], further suggesting both the ACE2 specific activity in producing Ang (1–9) and an activation of both arms of the RAS in ARDS patients.
T457	94524-94825	Sentence	denotes	Finally, in vivo or ex vivo administration of human rACE2 in plasma samples produced an increase not only of Ang (1–9) but also of Ang (1–5) and Ang (1–7) in both healthy subjects [178] and ARDS patients [134], indicating that all three Ang peptides might represent surrogate markers of ACE2 activity.
T458	94826-95127	Sentence	denotes	Of interest, the mean arterial pressure of ARDS patients was markedly low and significantly lower in non–survivors (mean value 65 mmHg and more likely need vasopressor support) compared to survivors (mean value 71 mmHg) [92], which may likely be a consequence of the above described ACE2 upregulation.
T459	95128-95215	Sentence	denotes	Interestingly, similar features were also detected in PAH and coronary atherosclerosis.
T460	95216-95444	Sentence	denotes	In particular, a significant increase of both Ang II and Ang (1–7) peptides was observed in plasma of these patients [108,182] and plasma levels of TNF-α were significantly elevated in the critical coronary artery disease [182].
T461	95445-95664	Sentence	denotes	Moreover, despite a significant increase of Ang II/Ang (1–7) ratio (that suggested a therapeutic use of recombinant hACE2 in PAH patients) systolic and diastolic blood pressures were in the range of normality [108,182].
T462	95665-95875	Sentence	denotes	Altogether these observations suggest that Ang peptide ratios are not reliable markers for disease status in these pathologies; rather, increased plasma amounts of each RAS peptide should be careful considered.
T463	95877-95879	Sentence	denotes	8.
T464	95880-95933	Sentence	denotes	Hypothesizing Pharmacological Treatments for COVID-19
T465	95934-96044	Sentence	denotes	Based on the above described hypothesis, inhibition of ACE2 pathway might be beneficial for COVID-19 patients.
T466	96045-96305	Sentence	denotes	Indeed, the clinical picture as a whole is consistent with an ACE2 gain of function (possibly due to both circulating active forms of S1-sACE2 complexes and local forms of SARS-CoV-2-sACE2 complexes) rather than an ACE2 loss of function, as initially supposed.
T467	96306-96500	Sentence	denotes	Therefore, inhibition of ACE2/Ang (1–7)/MasR axis or other ACE2 pathways to restore ACE/ACE2 balance might be needed, at least in the first phases of the disease when hypoxia is not yet induced.
T468	96501-96642	Sentence	denotes	Different strategies could be pursued through ACE2 pathway inhibitors and/or MasR antagonists and/or renin inhibitors and/or metal chelators.
T469	96643-96848	Sentence	denotes	Several different molecules have been designed to specifically inhibit human ACE2 enzyme (both membrane bound and soluble forms) or human MasR signal transduction, but only a few have been studied in vivo.
T470	96849-96995	Sentence	denotes	Some ACE2 pathway inhibitors have been widely used in mouse/rat models, as control of human and mouse ACE2 activity or in mouse models of colitis.
T471	96996-97288	Sentence	denotes	Thanks to mouse/rat in vivo experiments and clinical trial results in human participants it has been possible to infer toxicity/efficacy for some human ACE2 inhibitory molecules (MLN-4760/C16/GL1001/ORE1001, Dx600 and A779) that may be exploited to face this exceptionally dramatic situation.
T472	97289-97539	Sentence	denotes	Unfortunately, to my knowledge, only one (MLN-4760/C16/GL1001/ORE1001) of the ACE2-specific inhibitors has been tested in vivo in humans in a Phase I clinical trial long time ago (http://oreholdings.com/wp-content/uploads/2013/06/09.02.09-S-4-A.pdf).
T473	97540-97654	Sentence	denotes	Here, I have focused my attention on inhibitors of human ACE2 pathway that were consistently administered in vivo.
T474	97655-98031	Sentence	denotes	Making use of published reports in which human/rodent ACE2 pathway inhibitors were administered in vivo, I have hypothesized a possible therapeutic pharmacological intervention through an inhibition strategy of the RAS pathways for COVID-19 in patients experiencing both mild and critical, advanced and untreatable stages of the disease (the most problematic cases to manage).
T475	98033-98037	Sentence	denotes	8.1.
T476	98038-98066	Sentence	denotes	Inhibition of ACE2: MLN-4760
T477	98067-98738	Sentence	denotes	Briefly, one of the best candidates to treat COVID-19 patients, but not to prevent viral entry, is the small synthetic molecule MLN-4760 (specific ACE2 inhibitor, also known as C16, GL1001 or ORE1001, [113]) for the following reasons:(1) It has been shown to bind/inhibit ACE2 enzymatic activity even at low/acidic pH (pH 6.5, [115]) typical of hypercapnia (as it might occur in lungs of COVID-19 patients) when human ACE2 activity is maximal [79]; nevertheless, it retains its inhibitory effects on soluble ACE2 bound to spike proteins [24], indicating that it is able to bind and inhibit ACE2 activity regardless ACE2 binding to SARS-CoV-2 particles or to S1 fragments.
T478	98739-99143	Sentence	denotes	(2) No significant adverse effects were described upon its chronic administration neither alone nor in combination with ACE2 activators (while inhibiting their activating effects) nor after inducing functional impairment of ACE2 activity in rodent experiments in vivo [52,117,118,122,183,184] nor in a clinical Phase I trial in humans (http://oreholdings.com/wp-content/uploads/2013/06/09.10.09-425.pdf);
T479	99144-99226	Sentence	denotes	(3) Its administration by different route is well described in rodents and humans.
T480	99227-99577	Sentence	denotes	In particular:(a) Chronic administration (about 4 weeks) of C-16/DLM-4760 in combination with ACE2 activating treatments was performed by daily intraperitoneal injection at a dose of 25mg/kg in distilled water (as a solution of 42 mg/mL) or 0.9% sterile saline (as a solution of 84 mg/mL using a 0.5-mL insulin syringe) freshly prepared [52,183,184].
T481	99578-99909	Sentence	denotes	(b) Alternatively, chronic administration (about 8 days) of GL1001/DLM-4760 disodium salt in combination with an ACE2 activating treatment was performed by subcutaneous injection (5mL/kg) containing up to a dose of 300 mg/kg, twice a day, formulated in a vehicle solution [15% 2-hydroxypropyl-β-cyclodextrin (HPBDC)/85% H2O] [122].
T482	99910-99995	Sentence	denotes	Subchronic doses of GL1001 indicate no adverse effects up to 1,000 mg/kg (see [122]).
T483	99996-100085	Sentence	denotes	(c) In humans ORE1001/GL1001/MLN-4760 was already proposed and tested in clinical trials.
T484	100086-100254	Sentence	denotes	Its pharmaceutical indication was for digestive tract inflammations (Inflammatory bowel disease, gastritis and colitis) that are correlated with overexpression of ACE2.
T485	100255-100331	Sentence	denotes	In a Phase I clinical testing up 14 days dosing, ORE1001 was well tolerated.
T486	100332-100416	Sentence	denotes	Subjects received drug (dosing up to 2100 mg) with no side adverse effects reported.
T487	100417-100554	Sentence	denotes	In particular, 47 subjects received single-dose from 2.1 to 2100 mg and 24 subjects received 14 day multiple doses from 50 mg to 1800 mg.
T488	100555-100644	Sentence	denotes	All doses were well tolerated, with no significant side effects including blood pressure.
T489	100645-100802	Sentence	denotes	Pharmacokinetics of orally administered capsules was consistent with once-daily dosing. (http://oreholdings.com/wp-content/uploads/2013/06/09.10.09-425.pdf).
T490	100803-100961	Sentence	denotes	300 mg (active drug) oral capsules were used in a Phase Ib/IIa clinical trial that was, however, abandoned. (https://clinicaltrials.gov/ct2/show/NCT01039597).
T491	100962-101111	Sentence	denotes	(d) Finally, MLN-4760 was also administered (2.5 mg/kg per day) by nasal inhalation for 2–3 days in lung-infected mice by Pseudomonas bacteria [185].
T492	101112-101253	Sentence	denotes	Interestingly, the report underscores the role played by local concentration of molecules (ACE2) in modulating lung inflammation and disease.
T493	101254-101487	Sentence	denotes	For these reasons, in diseases involving respiratory tract, like SARS, inhalation treatment is preferable, even for the lower concentration (and hopefully lower toxicity) of MLN-4760 needed for this route of treatment administration.
T494	101488-101603	Sentence	denotes	Different routes of MLN-4760 treatment administration can be pursued depending on the hospital condition/expertise.
T495	101604-101766	Sentence	denotes	In this exceptionally critical situation, it could be delivered to critical untreatable patients as a controlled “compassionate use”, in particular by inhalation.
T496	101767-101966	Sentence	denotes	However, when, under hypoxic conditions, both arms of the RAS are upregulated, it might have a limited action and, by shifting the balance of ACE/ACE2 ratio in favour of ACE, might be even dangerous.
T497	101967-102178	Sentence	denotes	Instead, specific inhibition of ACE2 enzymatic activity might effectively work in preventing the establishment of positive feedback loops in COVID-19 patients who are suffering from mild symptoms of the disease.
T498	102179-102342	Sentence	denotes	MLN-4760 is sold by different companies, that, in case it works, could be encouraged to manufacture the molecule, actively contributing to face this global threat.
T499	102343-102490	Sentence	denotes	On the other hand, the drug could be also synthesized in University chemistry labs because, to my knowledge, is no longer under patent restriction.
T500	102491-102922	Sentence	denotes	MLN-4760, whose clinical development was abandoned after phase I trials (clinical name, ORE1001), is actually an interesting compound that could be useful for all patients in which a specific ACE2 upregulation is proven and associated to different (heart/lung/liver/intestine/kidney/immune system/blood/coagulation, etc.) pathological conditions related to ACE2 pathway downstream events, as it seems to occur in COVID-19 patients.

T1

0-38

Sentence

denotes

The Yin and Yang of ACE/ACE2 Pathways:

T2

39-124

Sentence

denotes

The Rationale for the Use of Renin-Angiotensin System Inhibitors in COVID-19 Patients

T3

126-134

Sentence

denotes

Abstract

T4

135-342

Sentence

denotes

The article describes the rationale for inhibition of the renin-angiotensin system (RAS) pathways as specific targets in patients infected by SARS-CoV-2 in order to prevent positive feedback-loop mechanisms.

T5

343-620

Sentence

denotes

Based purely on experimental studies in which RAS pathway inhibitors were administered in vivo to humans/rodents, a reasonable hypothesis of using inhibitors that block both ACE and ACE2 zinc metalloproteases and their downstream pathways in COVID-19 patients will be proposed.

T6

621-983

Sentence

denotes

In particular, metal (zinc) chelators and renin inhibitors may work alone or in combination to inhibit the positive feedback loops (initially triggered by SARS-CoV-2 and subsequently sustained by hypoxia independently on viral trigger) as both arms of renin-angiotensin system are upregulated, leading to critical, advanced and untreatable stages of the disease.

T7

985-987

Sentence

denotes

1.

T8

988-1000

Sentence

denotes

Introduction

T9

1001-1178

Sentence

denotes

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is the cause of the COVID-19 pandemic, for which there is currently no specific pharmacotherapy.

T10

1179-1421

Sentence

denotes

In the intensive care unit, depending on the severity of the COVID-19 infection, patients might receive supportive care such as oxygen supply, interferons, glucocorticoids, anti-virals, chloroquine, and macrolides given as deemed appropriate.

T11

1422-1624

Sentence

denotes

The SARS-CoV-2 infection produces either non-severe or severe symptoms and, in the latter case, it may precipitate into severe acute respiratory syndrome (SARS) and ultimately death [1,2,3,4,5,6,7,8,9].

T12

1625-1705

Sentence

denotes

Aging and different comorbidities are associated with patients’ critical status.

T13

1706-1892

Sentence

denotes

Most patients display lymphopaenia and eosinopaenia, and critical ones show extremely low levels of eosinophils, suggesting that eosinopaenia may be a potential diagnostic biomarker [1].

T14

1893-2148

Sentence

denotes

Most serious cases of SARS are closely associated with cardiac dysfunction/injury, respiratory distress/illness, coagulopathy, hypoproteinaemia, acidosis, hypoxia and a cytokine storm mainly involving IL-1β, IL-6, IL-8, IL-10 and TNFα [1,2,3,4,5,6,7,8,9].

T15

2149-2622

Sentence

denotes

For instance, hypertension, an age-related disease, is the most common comorbidity (15–31% up to 24–58% in severe disease cases according to clinical reports), while only five cases of asthma (0.9%) were identified in one report of 548 infected patients in Wuhan [6], but no other asthmatic or allergic patients were described in other reports [1,2,3,4,5,7,8,9], considering that the overall prevalence of asthma is estimated to be 6.4% in Wuhan [6] and ~ 4% in China [10].

T16

2623-2788

Sentence

denotes

Viral infections usually increase the risk of allergic disease exacerbation [1]; however, the reported data seem to suggest that, in this case, the opposite is true.

T17

2789-3255

Sentence

denotes

Indeed, in more than 72.000 patient cohorts examined, the number of patients defined as asthmatic was low (5), which suggests that asthmatics might be protected from virus-induced SARS, whereas pre-existing hypertensive disease and/or pre-existing antihypertensive treatments may represent a risk factor for increased virus infection, considering that the overall prevalence of hypertension in Chinese adult population ≥18 years of age is estimated to be ~ 23% [11].

T18

3256-3558

Sentence

denotes

Therefore, according to the clinical picture of infected patients, Th2-mediated allergic diseases (usually with high eosinophil counts) may play a protective role against SARS (usually with low eosinophil counts), while yet unknown mechanisms related to hypertensive conditions may exacerbate symptoms.

T19

3559-3875

Sentence

denotes

To complete the picture, COVID-19 hospitalized patients suffering from chronic renal disease were usually low (0-3%) [1,2,3,4,5,6,7,8,9] if compared with the prevalence of the disease in China (10.2-11.3%) [12], suggesting that chronic kidney disease patients might be protected from COVID-19 as for asthma patients.

T20

3877-3879

Sentence

denotes

2.

T21

3880-3928

Sentence

denotes

ACE2-mediated SARS-CoV and SARS-CoV-2 Infections

T22

3929-4092

Sentence

denotes

Angiotensin-converting enzyme 2 (ACE2) was identified as a receptor for the spike (S) protein of SARS-CoV, finally facilitating viral entry into target cells [13].

T23

4093-4286

Sentence

denotes

ACE2 is abundantly expressed in airway epithelial cells and vascular endothelial cells, and it is believed to play a crucial role in the mechanism of acute lung injury induced by SARS-CoV [14].

T24

4287-4540

Sentence

denotes

The ability of spike-Fc protein treatment (3h) to downregulate ACE2 protein expression has been shown in an in vitro system (cell lines) and also in vivo in lung cells of mice [14,15], suggesting that ACE2 pathway may be down-modulated during infection.

T25

4541-4683

Sentence

denotes

However, ACE2 is constitutively expressed and released from the apical cell surface of human airway epithelia into airway surface liquid [16].

T26

4684-4925

Sentence

denotes

Of note, its surface down-modulation upon spike protein challenge has been shown to be due to ACE2 shedding mediated by activation of extracellular ADAM17/TACE metalloprotease, which concomitantly induces shedding/production of TNFα [17,18].

T27

4926-5157

Sentence

denotes

Interestingly, ACE2 shedding is enhanced not only by binding with spike protein [17,18], but also by IL-1β and TNFα inflammatory cytokines [16], cytokines that are secreted at relatively high concentration in COVID-19 patients [2].

T28

5158-5442

Sentence

denotes

Moreover, soluble (s)ACE2 (induced or not by virus binding) released from human airway epithelia has been demonstrated to retain both its enzymatic activity and its binding ability for spike viral protein, finally reducing spike protein-mediated viral entry into target cells [16,17].

T29

5443-5607

Sentence

denotes

Therefore, the interaction of ACE2 with spike protein of SARS-CoV induces a cellular “protective” ACE2 shedding feedback response that initially limits viral entry.

T30

5608-5795

Sentence

denotes

Nevertheless, ADAM17/TACE-mediated ACE2 shedding or ACE2 enzymatic activity have been shown to intriguingly correlate positively with viral infection and disease complications [17,19,20].

T31

5796-5987

Sentence

denotes

In contrast, HNL63-CoV, which similarly binds to ACE2 through its spike protein, infects ACE2-bearing cells and mainly induces the common cold, leading to neither ACE2 shedding nor SARS [17].

T32

5988-6250

Sentence

denotes

Moreover, catalytically inactive forms of sACE2 can potently inhibit SARS-CoV infection [19,21], suggesting that events downstream of ACE2 shedding and/or its enzymatic activity may indirectly and subsequently favour viral infection and/or disease complications.

T33

6251-6522

Sentence

denotes

To this regard, sACE2 was also associated with myocardial pathological conditions [22] and cardiovascular complications including hypotension (known to enhance both renin and angiotensin I, the substrate of ACE/ACE2) and tachycardia were common in SARS-CoV patients [23].

T34

6523-6791

Sentence

denotes

Since spike protein has been shown to not inhibit ACE2 enzymatic activity that is retained by sACE2-spike protein complex [16,17,24] and, in general, sACE2 maintains its enzymatic activity, we cannot consider its higher circulating expression a mere disease biomarker.

T35

6792-6907

Sentence

denotes

Indeed, ACE2 shedding might repress its local function but it certainly enhances its circulating/systemic activity.

T36

6908-7236

Sentence

denotes

Interestingly, a recent integrative bioinformatics analysis shows that the gene expression of ACE2 in human bronchial cells infected with SARS-CoV is dramatically increased 24h after infection and remained at a high level for at least 2 days, suggesting that ACE2 may be involved in a positive feedback loop post-infection [25].

T37

7237-7539

Sentence

denotes

In the same report, it has been shown that ACE2 expression level in bronchial epithelium obtained by brushing from asthmatic and normal subjects was similar, suggesting that respiratory epithelial cells of healthy subjects and asthmatic patients have similar ability to bind to SARS-CoV-2 through ACE2.

T38

7540-7637

Sentence

denotes

Of note, ACE2 was also identified as the receptor for the novel spike protein of SARS-CoV-2 [13].

T39

7638-7801

Sentence

denotes

Although the role of ACE2 in the pathogenesis of SARS-CoV-2 and in inducing lung injury is still unknown, ACE2 behaves similarly to original SARS-CoV [13] (Box 1).

T40

7802-7988

Sentence

denotes

Box 1 The formation of spike-ACE2 protein complexes may initiate ACE2 systemic upregulation and may impair autologous antibody generation/recognition of anti-S1-Receptor Binding Domain.

T41

7989-8258

Sentence

denotes

The ACE2 interacts with the spike (S) protein, thereby serves as an entry receptor for SARS-CoVs, and this event is likely followed by conformational changes, cleavages and fusion of both spike viral and ACE2 proteins at the level of infected host cell plasma-membrane.

T42

8259-8444

Sentence

denotes

The SARS coronavirus surface spike glycoproteins consist of trimers formed by two spike, capsid-distal S1 and –proximal S2, protein regions, located on the outer envelope of the virion.

T43

8445-8701

Sentence

denotes

Spike proteins are clove-shaped trimers with three S1 heads and a trimeric S2 stalk that can bind the cellular ACE2 receptor when one S1 (possessing the receptor-binding domain) in the trimer adopts an “up” conformation (see Figure later in the Section 4).

T44

8702-9113

Sentence

denotes

The subsequent binding of ACE2 receptor to the spike glycoprotein triggers both the ADAM17-mediated ACE2 shedding and dissociation of S1 fragments by exogenous (furin-related or other) proteases from the spike trimer, which leads on one hand to one S1-ACE2 complex and two S1 free fragments, and on the other hand to fusion of S2 viral trimers and cellular membrane structures, finally producing cell infection.

T45

9114-9337

Sentence

denotes

The molecular mechanism model for SARS-CoV recognition and infection was well described by Song and collaborators [26] and a similar molecular interactions between SARS-CoV-2 and ACE2 has been recently hypothesized [27,28].

T46

9338-9530

Sentence

denotes

To complete the picture, a recent structural work reveals that the full-length human ACE2 is assembled as a dimer associated or not with amino acid transporter B0AT1, which sandwich ACE2 [28].

T47

9531-9668

Sentence

denotes

Binding of the spike protein trimer onto the ACE2 dimer suggests simultaneous binding of two spike protein trimers to an ACE2 dimer [28].

T48

9669-9937

Sentence

denotes

Each monomer of the ACE2 homodimer is composed of a membrane-proximal collectrin-like domain mediating homodimerization and necessary to position the molecule to the cell membrane, and of a membrane-distal ACE2 peptidase domain responsible for ACE2 substrate cleavage.

T49

9938-10124

Sentence

denotes

Collectrin-like domain consists of an extracellular ferredoxin-like fold domain, also called neck domain (residues 616 to 726), a single transmembrane helix and an intracellular segment.

T50

10125-10309

Sentence

denotes

The ACE2 cleavage site mediated by ADAM17/TACE has been predicted between amino acids 716 and 741 [16], which corresponds to neck-transmembrane boundary of ACE2 collectrin-like domain.

T51

10310-10542

Sentence

denotes

It is tempting to speculate that, after ACE2 shedding, S2 viral trimers may fuse with the residual ACE2 collectrin-like fragments (complexed or not with B0AT1 transporters) which are eventually exposed on the cell surface membranes.

T52

10543-10647

Sentence

denotes

A model for SARS-CoV’s internalization by target cells that was already proposed several years ago [29].

T53

10648-10893

Sentence

denotes

Then, when the virus is inside the cell, a second proteolytic cleavage mediated by endosomal proteases (such as TMPRSS2) in the S2 region of the spike-ACE2 fusion protein might be necessary for intracellular virus “release” and active infection.

T54

10894-11051

Sentence

denotes

Several S1-sACE2 complexes, free S1 fragments or sACE2 are likely released in bloodstream of COVID-19 infected patients by spike and ACE2 receptor cleavages.

T55

11052-11527

Sentence

denotes

As already mentioned, circulating sACE2 protein shedding independent on virus contact has been also described either spontaneously when ACE2 transcription is upregulated or upon cytokine activation; it is therefore likely that in the bloodstreams ACE2 proteins are available to bind to both free S1 fragments and SARS-CoV-2 particles, finally leading to more and more S1-sACE2 and SARS-CoV-2-sACE2 complexes bearing enzymatic active sACE2 (see Figure later in the Section 4).

T56

11528-11803

Sentence

denotes

Of note, SARS-CoV-2-sACE2 complexes, sequestrating sACE2, can drive part of the sACE2 systemic activity and concentrate it locally where the organs possess cells expressing ACE2 on their surface membrane, i.e., following the viral tropism (see Figure later in the Section 4).

T57

11804-12098

Sentence

denotes

Indeed, although the viral load in sputum of adult SARS-CoV patients was usually higher than 104 copies/mL, reports indicate that plasma viral RNA concentrations are low, usually lower than 190 copies/mL and lymphocytes have much higher RNA-copy concentrations of SARS-CoV RNA than plasma [30].

T58

12099-12244

Sentence

denotes

Very low RNA concentrations with no difference between patients with mild or severe symptoms were detected in plasma from COVID-19 patients [30].

T59

12245-12376

Sentence

denotes

The viral load in nasal swabs of asymptomatic and symptomatic COVID-19-positive subjects was also not significantly different [31].

T60

12377-12661

Sentence

denotes

Of interest, SARS-CoV infected pediatric patients have more than double the amount of plasma RNA copies/mL when compared to adult patients [30], suggesting a different viral tropism between adults and children (different cellular ACE2 expression possibly leading to Kawasaki disease).

T61

12662-12789

Sentence

denotes

Notably, free S1 fragments may have a decoy function for autologous/exogenous anti-S1-receptor binding domain (RBD) antibodies.

T62

12790-13020

Sentence

denotes

On the other hand, sACE2-S1 complexes masking S1-RBD might also impair either generation of autologous high affinity antibodies against S1-RBD or recognition/inactivation of S1-RBD on SARS-CoV-2 by autologous/exogenous antibodies.

T63

13021-13393

Sentence

denotes

Therefore, antibodies that do not compete with ACE2 for the binding to S1-RBD might be more effective in neutralizing SARS-CoV-2 [32,33], knowing that the number of spike proteins per virion is estimated to be around 70 and that every spike protein bears three S1 receptor binding domains, which are masked to autologous antibody recognition when in a “down” conformation.

T64

13394-13589

Sentence

denotes

These highly organized molecular features clearly demonstrate that the coronaviruses are armed with sophisticated infection machinery able to evade immune responses induced or not by vaccination.

T65

13590-13772

Sentence

denotes

Nevertheless, passive immunotherapy by means of convalescent plasma from COVID-19 recovered donors is a promising option for prevention and treatment of COVID-19 [32,33,34,35,36,37].

T66

13774-13776

Sentence

denotes

3.

T67

13777-13826

Sentence

denotes

Pathological Effects of ACE2 Pathway Upregulation

T68

13827-13949

Sentence

denotes

ACE and ACE2 are two zinc metalloproteases involved in the biogenesis of the components of renin-angiotensin system (RAS).

T69

13950-14150

Sentence

denotes

ACE2 processes angiotensin (Ang) I and II into Ang (1–9) and Ang (1–7), respectively, and it also has other known peptide targets, such as des-Arg(9)-bradykinin, which mediates B1 receptor activation.

T70

14151-14497

Sentence

denotes

Ang (1–7) and Ang (1–9) peptides, opposing the effects of ACE/Ang II/Ang II type 1 receptor (AT1R) pathway, are known to mediate vasodilatative (hypotension), antiproliferative and apoptotic effects through Mas and AT2 receptors, respectively, both involving downstream nitric oxide synthase (NOS) pathway activation [38,39,40,41,42,43,44,45,46].

T71

14498-14653

Sentence

denotes

Physiology teaches us that there is a range of normality for every biological parameter, below (defect) and above (excess) of which there is a dysfunction.

T72

14654-14708

Sentence

denotes

This is likely to be true for ACE as well as for ACE2.

T73

14709-14923

Sentence

denotes

It is well known that excessive Ang II/AT1R pathway activation induces vasoconstriction, aldosterone, inflammation, excessive cell proliferation, thrombosis, cardiac dysfunction and lung alterations (see Figure 1).

T74

14924-15216

Sentence

denotes

Moreover, most of the experiments show that increased ACE2 activity leads to beneficial effects; however, the experiments were usually performed using models in which its “antagonist“ (ACE) pathway was upregulated or ACE2 itself was downregulated, therefore balancing an unbalanced situation.

T75

15217-15276

Sentence

denotes

What does it happen in models in which the opposite occurs?

T76

15277-15488

Sentence

denotes

(s)ACE2 or Ang (1–7) upregulation have been associated to some pathological conditions such as inflammation of the renal and gastrointestinal tract, cardiac dysfunction, human cirrhosis and lung injury/fibrosis.

T77

15489-15652

Sentence

denotes

Tissue/organ specific effects of excessive pathway activation that in some case resemble those produced by excessive Ang II/AT1R pathway activation (see Figure 1).

T78

15653-15792

Sentence

denotes

For example, systemic infusion of Ang (1–7) into mice had renal proinflammatory properties mediated by Mas receptor (MasR) activation [47].

T79

15793-15943

Sentence

denotes

Moreover, Ang (1–7) infusion was associated with increases in blood pressure, cardiac hypertrophy and fibrosis in rats with subtotal nephrectomy [48].

T80

15944-16124

Sentence

denotes

Similarly, elevated plasma sACE2 activity was associated both with greater severity of myocardial dysfunction and with an independent prediction of adverse clinical events [22,49].

T81

16125-16385

Sentence

denotes

Transgenic mice with increased cardiac ACE2 expression suffered from lethal ventricular arrhythmia (heart block, ventricular tachycardia and terminal ventricular fibrillation) consequent upon downregulation of connexins involved in gap junction formation [50].

T82

16386-16534

Sentence

denotes

In another model, ACE2 transgenic mice suffered from cardiac fibrosis with concomitant deficits in ejection fraction and fractional shortening [51].

T83

16535-16928

Sentence

denotes

Interestingly, in a rat model of myocardial infarction following coronary artery ligation, there is evidence that C16/MLN-4760 (a specific ACE2 inhibitor, see later) administration inhibits fibrosis and hypertrophy of non-infarcted myocardium and increases diastolic relaxation, raising the possibility that ACE2 activity may have some adverse effects on post-myocardial infarction heart [52].

T84

16929-17067

Sentence

denotes

The risk factors for cardiovascular disease include alterations in platelet function and coagulation with an increased risk of thrombosis.

T85

17068-17244

Sentence

denotes

Ang II and hypercholesterolemia are known to participate in microvascular thrombosis and enhanced thrombus formation in the microvasculature may contribute to microinfarctions.

T86

17245-17441

Sentence

denotes

To this end, in a rat model in which AT1R activation produce baseline thrombosis by platelet aggregation, Ang (1–9), known to bind AT2R [42], has been shown to enhance the thrombotic process [53].

T87

17442-17751

Sentence

denotes

Moreover, in a mouse model, AT2R activation (inhibited by AT2R antagonist, PD12319) mediated the onset of arteriolar microvascular thrombosis following chronic Ang II infusion [54], indicating both the recruitment of the AT2R pathway downstream of AT1R activation and its involvement in arteriolar thrombosis.

T88

17752-18061

Sentence

denotes

Of interest, disseminated intravascular coagulation is associated with hypoproteinaemia and deficit of coagulation and anticoagulation proteins, which can originate by their renal loss (and consequent proteinuria) and/or by their increased (pathological) consumption and/or by their reduced hepatic synthesis.

T89

18062-18270

Sentence

denotes

To this end, in healthy livers, ACE2 is limited to perivenular hepatocytes and endothelial cells; instead, in human hepatitis C cirrhosis, ACE2 protein expression is widespread in the hepatic parenchyma [55].

T90

18271-18462

Sentence

denotes

Notably, human hepatocytes cultured in hypoxic conditions upregulated ACE2 protein expression [55] and ACE2 mRNA, protein and activity were increased in response to hypoxia and by IL-1β [56].

T91

18463-18666

Sentence

denotes

In line with these observations, in peripheral blood human CD34+ cells, MasR expression and ACE2 expression, activity and shedding (of sACE2 catalytically active forms) were increased under hypoxia [57].

T92

18667-18924

Sentence

denotes

Moreover, under hypoxic conditions human pulmonary artery smooth muscle cells upregulated both (arms of the RAS) ACE and ACE2 mRNA and protein expression, and ACE2 was subsequently downregulated by (ACE-derived) Ang II through an AT1R-mediated process [58].

T93

18925-19110

Sentence

denotes

Similarly, both ACE and ACE2 expression were increased in vascular endothelium and smooth muscle of human left ventricle from patients with ischaemic (local hypoxia) heart disease [59].

T94

19111-19233

Sentence

denotes

Concomitant upregulation of both arms of the RAS that suggests a tight link between ACE and ACE2 under hypoxic conditions.

T95

19234-19372

Sentence

denotes

Indeed, several reports have shown a complex interplay of regulation between the two arms of the RAS independently on hypoxia (see Box 2).

T96

19373-19910

Sentence

denotes

Interestingly, chronic hypoxia induced activation of ACE2/Ang (1–7)/MasR axis and suppression of ACE/Ang II/AT1 receptor axis in lungs of pulmonary hypertensive Ren-2 transgenic rats (constructed by inserting the mouse Ren-2 renin gene) but not in normotensive transgene-negative control rats [60], suggesting that the baseline renin activity in hypertensive rats may be crucial to determine the differential response to hypoxia and that renin inhibition might be useful to inhibit the ACE to ACE2 pathway shift under hypoxic conditions.

T97

19911-20086

Sentence

denotes

Of interest, hypoxia alone or combined with hypercapnia has been shown to significantly increase both plasma renin expression or activity and plasma Ang II expression [61,62].

T98

20087-20582

Sentence

denotes

Moreover, hypercapnic acidosis (pH 6.8/6.9, a condition that could occur in SARS) induced in isolated rat lungs has been shown to induce a (compensatory) venular dilatation mediated by cyclooxygenase activation (inhibited by indomethacin) [63], an enzyme that has been shown to be induced downstream of Ang (1–7)/MasR pathway in isolated rat hearts (again inhibited by indomethacin) [64], suggesting the involvement of ACE2/Ang (1–7) pathway in mediating CO2-dependent lung venular vasodilation.

T99

20583-20978

Sentence

denotes

Altogether these observations indicate that there is a high probability that hypoxia/hypercapnia, a condition that occurs in SARS patients, might upregulate the activity of both arms of the RAS by suppling high amounts of renin product, Ang I, to ACE and ACE2, which, together, can produce high amounts of Ang II, Ang (1–9), Ang (1–7) and its (ACE-produced) metabolite, Ang (1–5) (see Figure 1).

T100

20979-21264

Sentence

denotes

Similarly to both Ang (1–7)/MasR and Ang (1–9)/AT2R pathways, Ang (1–5) has been shown to induce the secretion of atrial natriuretic peptide via MasR/NOS pathway in isolated perfused rat atria [65], indicating that Ang (1–5) is a heart active peptide (at least in Sprague-Dawley rats).

T101

21265-21431

Sentence

denotes

Interestingly, in the plasma of patients with inflammatory bowel disease, Ang (1–7) concentrations and (s)ACE2 activity were higher compared to healthy subjects [66].

T102

21432-21585

Sentence

denotes

Moreover, Ang (1–7) alone can either activate the Bax/Caspase–dependent apoptotic pathway or upregulate NF-kB signaling in lung fibroblast cultures [67].

T103

21586-21859

Sentence

denotes

Moreover, in vivo administration of Ang (1–7) alone (in Wistar rats) promoted morphological lung alterations, extracellular matrix accumulation and inflammatory cytokine production (including TNF-α and IL-6), characteristics of lung inflammation in pulmonary fibrosis [67].

T104

21860-21957

Sentence

denotes

Thereby, a condition that deteriorates respiratory compliance, could lead to hypoxia/hypercapnia.

T105

21958-22169

Sentence

denotes

Hypoxia, in turn, will induce ACE2 upregulation which possibly increases cardiac/lung detrimental effects mediated by Ang (1–7)/MasR pathway activation finally leading to further ACE2 cell membrane upregulation.

T106

22170-22405

Sentence

denotes

A condition that in COVID-19 can increase the probability of both SARS-CoV-2 entry and ACE2 shedding/systemic activity, finally generating positive feedback loops that might sustain SARS independently of viral infection (see Figure 2).

T107

22406-22723

Sentence

denotes

This hypothesis is supported by the observations that, in lung aspirates of acid- and/or spike-treated mice, Ang II and ACE2 are synergistically upregulated and cell surface downregulated (shed), respectively, suggesting their involvement in the increased lung microvascular permeability and pulmonary oedema [14,15].

T108

22724-22931

Sentence

denotes

Indeed, this condition might subsequently favour the diffusion, in neighbouring lung tissues and systemic circulation, of both (s)ACE2, Ang II and Ang (1–7), the Ang II-derived product of (s)ACE2 processing.

T109

22932-23121

Sentence

denotes

As already proposed, enhanced ACE2 shedding may locally reduce ACE2 activity in lung, however, it likely increases ACE2 systemic activity and subsequent production of circulating Ang (1–7).

T110

23122-23259

Sentence

denotes

Interestingly, Ang (1–7) has been also reported to promote eosinophil apoptosis in lungs and in bronco-alveolar lavage fluid (BALF) [40].

T111

23260-23531

Sentence

denotes

Moreover, Ang (1–7)/MasR axis inhibits allergic airway inflammation and eosinophil cell counts in the BALF of a murine model of asthma, indicating both that an impairment of ACE2 pathway may favour asthma and that ACE2 pathway activation can reduces asthma symptoms [68].

T112

23532-23763

Sentence

denotes

Moreover, a compound that mimics the Ang (1–7) actions has been shown to induce IL-10 upregulation via a MasR-dependent pathway in BALF [69] and IL-10 is thought to mediate anti-inflammatory effects of MasR pathway activation [70].

T113

23764-23884

Sentence

denotes

IL-10 secretion in mouse plasma was also induced by an AT2 receptor agonist and downstream nitric oxide signalling [71].

T114

23885-24078

Sentence

denotes

Increased IL-10 production (e.g., by T regulatory cells) is often associated with immune tolerance, which is the consequence of reduced number and function of specific immune compartments [72].

T115

24079-24439

Sentence

denotes

Indeed, IL-10 has been shown not only to suppress antigen-specific Th2-mediated immune responses including eosinophil expansion in allergic inflammation [72], but also to augment airway reactivity, suggesting that despite its potent anti-inflammatory activity, an excess of IL-10, as well as Ang (1–7), may contribute to the decline in pulmonary function [73].

T116

24440-24752

Sentence

denotes

Of note, IL-10 is one of the cytokines downstream ACE2 pathway [25] and is significantly upregulated in the most severe forms of COVID-19 [2,8,9], indicating an important correlation between ACE2/Ang (1–7) axis activation and IL-10 upregulation which might lead to eosinopaenia/lymphopaenia in COVID-19 patients.

T117

24753-24824

Sentence

denotes

Box 2 The reciprocal feedback-loop mechanisms of both arms of the RAS.

T118

24825-25157

Sentence

denotes

Similarly to hypoxic conditions, a strong correlation between the gene expression of ACE and that of ACE2 was also observed in human renal cortical biopsy specimen, suggesting a link between the two gene transcription, possibly related to the amount/excess of their substrates (Ang I and Ang II) and not exclusively to hypoxia [74].

T119

25158-25518

Sentence

denotes

A link that tends to maintain the balance of ACE/ACE2 ratio, but that may be disrupted by ACE inhibitors (ACEIs) or by Ang II type 1 receptor blockers (ARBs) which both have been shown to upregulate ACE2 mRNA expression in left ventricle of Lewis rats, possibly through two different mechanisms involving upregulation of Ang (1–7) or Ang II, respectively [75].

T120

25519-25784

Sentence

denotes

In this regard, in cardiac myocytes isolated from neonatal rats, Ang II significantly reduced ACE2 mRNA and its activity, effects blocked by ARBs, indicating that Ang II downregulates ACE2 expression/activity through an AT1R-dependent mechanism [76] (see Figure 1).

T121

25785-26139

Sentence

denotes

Moreover, in both cardiac myocytes and rat aortic vascular smooth muscle cells, Ang (1–7) prevented the Ang II-mediated reduction in ACE2 mRNA, an effect blocked by a selective MasR antagonist, D-Ala7-Ang-(1–7) (also known as A779), indicating that Ang (1–7) downregulates Ang II/AT1R signalling through a MasR-dependent mechanism [76,77] (see Figure 1).

T122

26140-26483

Sentence

denotes

Therefore, Ang (1–7) (as well as ACEIs and ARBs), preventing Ang II-mediated ACE2 downregulation, shift the Ang peptide balance in favour of Ang II metabolisation by ACE2, which, in turn, leads to further production of Ang (1–7), finally sustaining ACE2 transcription, membrane protein expression and eventually shedding and systemic activity.

T123

26484-26695

Sentence

denotes

Altogether these observations indicate a complex interplay of regulation between the two arms of the RAS in which feedback mechanisms of reciprocal (ACE/ACE2) pathway inhibition are involved at different levels:

T124

26696-26883

Sentence

denotes

Ang II/AT1 receptor mediates a negative feedback signal on the ACE2 expression/activity and Ang (1–7)/MasR mediated a negative feedback signal on the AT1 receptor activity (see Figure 1).

T125

26884-27114

Sentence

denotes

These reciprocal inhibitions in some cases (e.g., hypoxia/SARS-CoV-2 infection) can give rise to positive feedback loops that markedly shift the balance between Ang II/AT1R and the antagonist Ang (1–7)/MasR pathway (see Figure 1).

T126

27115-27412

Sentence

denotes

For example, under hypoxic conditions both arms of the RAS are upregulated and the presence of SARS-CoV-2 can affect Ang (1–7)/Ang II balance (which might be further influenced by ACEI/ARBs) by shifting it in favour of an increased ACE2 systemic activity, Ang (1–7) production and MasR activation.

T127

27413-27628

Sentence

denotes

This, in turn, can lead to further ACE2 cell membrane expression (increasing the probability of viral entry), which, after ACE2 shedding by binding to spike-SARS-CoV-2, ultimately establish a positive feedback loop.

T128

27630-27632

Sentence

denotes

4.

T129

27633-27709

Sentence

denotes

COVID-19 Can Induce RAS-mediated Positive Feedback Loops at Different Levels

T130

27710-27885

Sentence

denotes

Intriguingly, the binding affinity of ACE2 to SARS-CoV-2 binding domain has been reported to be either equal to or 10- to 20-fold higher than ACE2 binding to SARS-CoV [27,32].

T131

27886-27987

Sentence

denotes

The affinity of spike protein for ACE2 has been shown to correlate with the severity of disease [24].

T132

27988-28261

Sentence

denotes

Although SARS-CoV produces more severe respiratory symptoms than NL63-CoV does, both viral receptor binding domains bind to ACE2 with similar affinity [78], indicating that SARS development is not related to the strength of binding affinity and depends on other mechanisms.

T133

28262-28500

Sentence

denotes

In contrast to SARS-CoV, NL63-CoV did not induce ADAM17/TACE-mediated both ACE2 shedding and TNF-α secretion [17], suggesting that increased cleavability of ACE2 receptor and possibly of S1-S2 boundary may be crucial for disease severity.

T134

28501-28672

Sentence

denotes

Indeed, the spike glycoprotein of SARS-CoV-2 but not of SARS-CoV, contains a furin-like cleavage site at the S1-S2 boundary which indicates an increased cleavability [13].

T135

28673-28827

Sentence

denotes

Of note, TNF-α and IL-1β were shown to both be upregulated in SARS-CoV-2 [2] and induce viral-independent ACE2 shedding from epithelial airway cells [16].

T136

28828-29101

Sentence

denotes

Moreover, viral-independent ACE2 surface release from epithelial cells was not only inducible by cytokines (e.g., TNF-α and IL-1β) but also constitutively and spontaneously produced when ACE2 surface expression was upregulated [16], for example upon IL-1β stimulation [56].

T137

29102-29250

Sentence

denotes

This suggests that systemic release of proinflammatory cytokines such as TNF-α and IL-1β can mediate an increase of sACE2 and its systemic activity.

T138

29251-29391

Sentence

denotes

Of note, activation of ADAM17/TACE metalloprotease was induced by SARS-CoV and necessary for efficient infection (and TNF-α secretion) [17].

T139

29392-29653

Sentence

denotes

Intriguingly, both SARS-CoV infection and concomitant TNF-α secretion were significantly attenuated not only by knock-down of ADAM17/TACE expression by siRNA but also by deletion of the ACE2 cytoplasmic tail which is responsible for ADAM17/TACE activation [17].

T140

29654-29824

Sentence

denotes

Altogether these observations suggest the possibility that SARS-CoV may induce a positive feedback loop leading to surface expression and shedding of both ACE2 and TNF-α.

T141

29825-30108

Sentence

denotes

Indeed, upon spike protein binding to ACE2, downstream pathway activation can sustain positive feedback loops at different levels (Figure 2):(1) SARS-CoV can induce IL-1β and TNF-α systemic secretion that can mediate viral-independent surface membrane ACE2 upregulation and shedding.

T142

30109-30290

Sentence

denotes

Of note, ACE2 shedding, on one hand, protects from viral infection but, on the other hand, increases circulating/systemic active sACE2, leading to its downstream pathway activation.

T143

30291-30662

Sentence

denotes

(2) Hypoxia in combination or not with hypercapnia can upregulate the activity of both arms of the renin–angiotensin system by inducing renin, ACE and ACE2 synthesis, which can increase expression of Ang I, Ang II, Ang (1–7), Ang (1–9), Ang (1–5) and the inactive metabolite bradykinin (1–7), but also membrane bound ACE2, finally giving more chances to SARS-CoV-2 entry.

T144

30663-30739

Sentence

denotes

(3) ACE2 can induce vasodilatative hypotensive effects by Ang II catabolism.

T145

30740-30883

Sentence

denotes

Hypotension can induce again renin and ACE upregulation finally providing further Ang II, as a ACE2 substrate for further Ang (1–7) production.

T146

30884-31199

Sentence

denotes

(4) Ang (1–7) antiproliferative and apoptotic effects, possibly in part through IL-10, may mediate eosinopaenia and lymphopaenia that, on one hand, reduce inflammatory responses but, on the other hand, impair immune system ability to counter virus infection, finally predisposing the organism to further infections.

T147

31200-31363

Sentence

denotes

Ang (1–7) immunosuppressive activity, mediated or not by IL-10, may also support the reduced ability to generate an effective immunization to SARS-CoV-2 infection.

T148

31364-31581

Sentence

denotes

(5) Ang (1–7)/MasR pathway can sustain ACE2 synthesis even in the presence of elevated concentrations of Ang II (such as in hypoxia), by inhibiting Ang II/AT1R-mediated down-modulation of ACE2 activity (see Figure 1).

T149

31582-31755

Sentence

denotes

(6) Ang (1–7)/MasR pathway can produce cardiac dysfunction and lung alteration leading to systemic hypoxia, which, in turn, upregulates the activity of both arms of the RAS.

T150

31756-32113

Sentence

denotes

(7) Although the ACE2 catalytic efficiency is 400-fold lower with Ang I than with Ang II [79], high concentration of circulating ACE2 may be able to produce significant increase of Ang (1–9) that, by binding AT2 receptors, can produce arteriolar microvascular thrombosis and local hypoxic conditions finally inducing local upregulation both arms of the RAS.

T151

32114-32461

Sentence

denotes

Finally, ACE2-loaded SARS-CoV-2 virions and S1-ACE2 complexes may impair both sACE2 proteolytic degradation by blood proteases and its renal excretion, therefore blunting the removal of its systemic enzymatic activity and indicating that circulating viral particles are dangerous even when they are not able to entry into the cells (see Figure 2).

T152

32462-32665

Sentence

denotes

On the one hand, ACE2 hypertranscription and consequent increase of membrane (m)ACE2 exposure induced by hypoxia/hypotension may facilitate SARS-CoV-2 entry and its lifecycle into mACE2 expressing cells.

T153

32666-32900

Sentence

denotes

On the other hand, the release of ACE2 from the cell membranes and its subsequent activity in the bloodstream and in local (lung/cardiac) extracellular fluids are likely critical steps in contributing to systemic disease pathogenesis.

T154

32901-33075

Sentence

denotes

Nevertheless, the same aetiological agent, SARS-CoV-2, can produce a variety of clinical syndromes, which involves several organs in relation to the subject’s predisposition.

T155

33076-33463

Sentence

denotes

Although in the present work I have supposed that most of the COVID-19 symptoms might derive from Ang (1–7), Ang II and Ang (1–9) peptide upregulation, the involvement of B1 receptor pathway suppression mediated by ACE2 metabolisation of des-Arg(9)-bradykinin to inactive bradykinin (1–7) or other ACE2-downstream peptides such as apelins, casomorphins and dynorphins cannot be rule out.

T156

33464-33646

Sentence

denotes

Based on the above observations, in order to block the RAS-induced positive feedback loops, different pharmacological targets can be hypothesized and pursued alone or in combination.

T157

33647-33700

Sentence

denotes

They will be discussed in the next section and Box 3.

T158

33701-33804

Sentence

denotes

Box 3 Possible targets of therapeutic intervention to inhibit the RAS-induced positive feedback loops.

T159

33805-34078

Sentence

denotes

Pharmacological inhibition of enzymes involved in both viral entry, such as TMPRSS2 and ADAM17, and the RAS function such as renin, ACE and ACE2 enzymatic activity or their downstream pathways are expected to stop positive feedback loops and their detrimental consequences.

T160

34079-34137

Sentence

denotes

Inhibition of TMPRSS2 and ADAM17 metalloprotease activity.

T161

34138-34433

Sentence

denotes

Inhibition of the serine protease TMPRSS2 (necessary for SARS-CoV-2 entry) by a clinically proven protease inhibitor has been recently suggested by Hoffmann and colleagues [13] and inhibition of ADAM17 enzymatic activity has been already proposed about ten years ago by Haga and colleagues [20].

T162

34434-34788

Sentence

denotes

Indeed, inhibition of ADAM17-mediated ACE2 shedding is expected to increase membrane ACE2 expression and therefore the probability of viral entry; nevertheless, in the early phases of the disease, inhibition of ACE2 circulating activity might be sufficient to inhibit the systemic RAS pathway upregulation and the development of severe forms of COVID-19.

T163

34789-35163

Sentence

denotes

It is, in fact, possible that maintenance/recovery of correct organismal immune responses, by preventing ACE2-mediated immune suppression, in concert with cellular adaptive immune responses mediated by apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) systems [80] may anyway work to induce both an effective “immunization” and the viral eradication.

T164

35164-35199

Sentence

denotes

Inhibition of both arms of the RAS.

T165

35200-35397

Sentence

denotes

Among the inhibitors of the RAS pathways, different strategy can be pursued involving either ACE2 enzymatic activity or its upstream renin and ACE enzymatic activity or its downstream MasR pathway.

T166

35398-35624

Sentence

denotes

Inhibition of ACE2, ACE and renin enzymatic activities and their involvement in SARS-CoVs will be extensively discussed in the next sections, instead a brief description of MasR inhibition will be presented in the present Box.

T167

35625-35707

Sentence

denotes

Mas receptor pathway inhibition and side effects of using Mas receptor inhibitors.

T168

35708-35871

Sentence

denotes

A779 also known as D-Ala7-Ang-(1–7) and D-Pro7-Ang-(1–7) are two distinct MasR antagonists able to prevent Ang-(1–7)-mediated downstream activation in human cells.

T169

35872-36245

Sentence

denotes

The existence of several MasR subtypes has been suggested based on the differential capacity of the two MasR blockers to fully inhibit some biological actions of Ang-(1–7) [and perhaps of Ang (1–5), see Figure 1] [39,70]; therefore, differently from ACE2 enzymatic inhibitors, MasR antagonists should be administered in combinations, in order to inhibit ACE2 hyperactivity.

T170

36246-36436

Sentence

denotes

In human aortic smooth muscle cells, they have been shown to restore NADPH oxidase/NF-kB/iNOS inflammatory pathway induced by Ang II when it is inhibited by Ang (1–7) co-administration [81].

T171

36437-36815

Sentence

denotes

In mice studies a MasR blocker (A779) administered alone was not associated with systolic blood pressure alterations, and the hypotensive effect produced by rACE2 co-infused with Ang II was unaffected by A779 co-administration, indicating that the hypotensive activity of rACE2 mainly depended on Ang II degradation rather than on increase of Ang (1–7) and MasR activation [82].

T172

36816-37009

Sentence

denotes

In another report, spontaneously hypertensive rats (SHRs) that received A-779 alone for a total of two weeks did not significantly alter basal blood pressure and urinary protein excretion [83].

T173

37010-37282

Sentence

denotes

Moreover, in SHRs treated with A-779 in combination with Ang II, renal injury and interstitial infiltration of macrophages and T cells were surprisingly reduced as compared with SHRs treated with Ang II alone, suggesting a safe use of A-779 drug in in vivo infusions [83].

T174

37283-37439

Sentence

denotes

Another report showed that infusion of A-779 alone for 7 days did not produce a significant effect neither on blood pressure nor on heart rate in SHRs [84].

T175

37440-37656

Sentence

denotes

In a rat model of cardiac arrhythmia, administration of A-779 alone did not cause any significant alteration in the number of arrhythmic events, confirming that A-779 can be safely delivered to rodents in vivo. [85].

T176

37657-38007

Sentence

denotes

Although MasR antagonists has been shown to be safe in acute and chronic in vivo studies either with mice or rats, there are no data on administration in humans and the existence of different MasR subtypes in the vasculature require combinations of MasR antagonists to inhibit an excess of ACE2 activity as for example may occur in COVID-19 patients.

T177

38009-38011

Sentence

denotes

5.

T178

38012-38097

Sentence

denotes

Mechanism of Action and Potential Risk of Using RAS Pathway Inhibitors Targeting ACE2

T179

38099-38103

Sentence

denotes

5.1.

T180

38104-38133

Sentence

denotes

ACE2 (and ACE) Hyperactivity:

T181

38134-38164

Sentence

denotes

Is It a Matter of (Free) Zinc?

T182

38165-38260

Sentence

denotes

ACE2 and ACE are two zinc metalloprotease that function differently despite their similarities.

T183

38261-38451

Sentence

denotes

ACE2 is a monocarboxypeptidase (cleaves a C-terminal single amino acid from its substrate), whereas ACE is a dipeptidylpeptidase (releases a C-terminal dipeptide from its substrate) [86,87].

T184

38452-38758

Sentence

denotes

ACE2 has a substrate preference for hydrolysis between proline and a hydrophobic or basic C-terminal residue [79,86,87] and acts not only on the RAS and bradykinin peptides but also on the C-terminus of the apelin, casomorphin dynorphin peptides [79], possibly inhibiting Apelin-13 hypotensive action [44].

T185

38759-38875

Sentence

denotes

It is expressed in the heart, kidneys, liver, colon, small intestine, lung, brain and testes [39,44,45,46,76,77,87].

T186

38876-39085

Sentence

denotes

Among the tissues expressing ACE2 there are cardiomyocytes, endothelium, alveolar type II cells, ciliated airway cells, vascular smooth muscle cells and testicular Leydig cells [14,39,44,45,46,76,77,87,88,89].

T187

39086-39382

Sentence

denotes

ACE2 is present on the apical surface of epithelial cells of renal tubules, lung alveoli and vascular endothelia, in these last cells the production of Ang (1–7) induces autocrine activation of MasR axis leading to NOS-mediated vasodilation and consequently hypotension [14,44,45,46,86,87,88,89].

T188

39383-39568

Sentence

denotes

ACE2, as well as ACE, is a zinc metalloprotease that can be inhibited by zinc chelating EDTA and activated by high concentrations of chloride or fluoride anions and zinc cation [79,90].

T189

39569-39633

Sentence

denotes

Indeed, both enzymes have chloride- and zinc-binding sites [90].

T190

39634-39758

Sentence

denotes

It is conceivable that these sites are responsible for the anion and zinc activation effect in both homologous enzymes [90].

T191

39759-40017

Sentence

denotes

However, the presence of chloride ions increases Ang I cleavage by ACE and ACE2 (increasing both Ang II and Ang (1–9)) and decreases Ang II cleavage by ACE2, suggesting that chloride induces specific ACE2 conformational changes in its active site (see [44]).

T192

40018-40188

Sentence

denotes

To this regard, in plasma samples of COVID–19 patients, chloride range is usually normal or slightly higher (99.6–107.0 mmol/L) than normal values (96.–106.0 mmol/L) [5].

T193

40189-40275

Sentence

denotes

Unfortunately, I was not able to find plasma zinc concentrations in COVID-19 patients.

T194

40276-40395

Sentence

denotes

Zinc is involved in several cellular activities and its systemic concentration is tightly regulated by several factors.

T195

40396-40996

Sentence

denotes

Of interest, the major transporter/reservoir of zinc in plasma is albumin [91], a protein that was detected at significantly low concentrations in SARS-CoV-2 as well as severe adult respiratory distress syndrome (ARDS, a COVID-19-like disease) (usually < 3.5 g/dL, normal range 3.5–5.4) and inflammatory bowel disease (IBD, a disease with increased ACE2 activity) [6,8,66,92] Since most of zinc in plasma is sequestered by albumin, the total amount of plasma zinc mostly depends on albumin concentration and even small changes in albumin’s capacity for zinc binding may have significant consequences.

T196

40997-41219

Sentence

denotes

To this regard, reduced concentrations of plasma albumin is believed to increase plasma levels of free zinc, while reducing the total plasma zinc concentrations [91], a condition that was observed in ARDS patients [93,94].

T197

41220-41362

Sentence

denotes

Indeed, albumin can potentially deplete free bioavailable forms of zinc; vice versa, hypoalbuminaemia may increase free zinc levels in plasma.

T198

41363-41498

Sentence

denotes

Therefore, some ARDS patients with low levels of both total zinc and albumin might have rather higher than lower free zinc levels [93].

T199

41499-41713

Sentence

denotes

The free zinc concentration, which describes the fraction of zinc that is loosely bound and easily exchangeable, represents the highly bioavailable (and toxic) part of plasma zinc but it is not clinically detected.

T200

41714-41794

Sentence

denotes

To that end, zinc/albumin ratio might be a surrogate marker of free zinc levels.

T201

41795-41934

Sentence

denotes

Indeed, only recently it was described a fluorescence-based method for determining the free zinc concentration in human serum samples [95].

T202

41935-42058

Sentence

denotes

With this method it was shown that free zinc concentration in sera from females was significantly lower than in males [95].

T203

42059-42208

Sentence

denotes

Moreover, free zinc concentration did correlate neither with total serum concentrations of zinc (or other metal ions such as iron) nor with age [95].

T204

42209-42432

Sentence

denotes

Notably, increased plasma zinc bioavailability consequent to reduced albumin levels induces zinc import into cells and likely increases the cellular functions associated to cellular zinc concentrations such as ACE and ACE2.

T205

42433-42619

Sentence

denotes

Indeed, enzymatic activity of both enzymes seems significantly upregulated in ARDS patients as detected by analysis of Ang peptide concentrations in plasma (see Box later in ARDS topic).

T206

42620-42858

Sentence

denotes

In addition, serum albumin levels in COVID-19 were usually very low (in severe forms is usually < 3.1 g/dL) [6,8], raising the possibility of a negative correlation between albumin concentration in plasma and increase of the RAS activity.

T207

42859-43181

Sentence

denotes

In line with these hypotheses, both asthmatic/allergic disease patients and chronic kidney disease patients without a history of cardiovascular disease, which are protected from developing COVID-19, showed a significant decrease in both circulating zinc levels and ACE2 activity as compared to normal values [96,97,98,99].

T208

43182-43488

Sentence

denotes

Interestingly, chronic renal disease patients with low zinc levels had normal concentrations of albumin but higher urinary zinc excretion than healthy controls [97], suggesting that low ACE2 activity in chronic renal diseases (and protection from SARS) might derive from a higher free Zn2+ renal excretion.

T209

43489-43626

Sentence

denotes

A similar mechanism mediated by a reduction of extracellular levels of free Zn2+ might be hypothesised for asthma patients, as well [98].

T210

43627-43824

Sentence

denotes

Unfortunately, I could not find information on plasma albumin concentrations for asthma patients; nevertheless, we might suppose that in these patients the albumin concentration is in normal range.

T211

43825-44058

Sentence

denotes

Intriguingly, inhalation exposure to ZnCl2/ZnO/hexachloroethane (the main ingredients in smoke bombs) induces both elevated plasma levels of zinc and ARDS with clinical pictures that strongly resemble those of COVID-19 [100,101,102].

T212

44059-44294

Sentence

denotes

Since both ACE2 and ACE enzymes are activated by high concentrations of chloride and zinc ions [79,90], it is possible to hypothesise an activation of both arms of the RAS in ARDS induced by smoke bombs as well as in COVID-19 patients.

T213

44295-44546

Sentence

denotes

In addition, metal fume fever, a flu-like syndrome caused by inhalation of welding fumes (mainly containing zinc) can be a predictor for the development of respiratory symptoms and welders have an increased pneumonia and cardiovascular risk [103,104].

T214

44547-44819

Sentence

denotes

In this regard, metal fume fever has been shown to produce fever, fatigue, muscle ache, cough, dyspnea, and an increase of several cytokines including IL-1β, IL-6, IL-8 and TNF-α [103], symptoms and cytokines that have already been described for COVID-19 patients [2,8,9].

T215

44820-45005

Sentence

denotes

Notably, TNF-α was upregulated early while IL-6 and IL-8 increased later, suggesting a subsequent involvement of these latter cytokines in response to inhalation of welding fumes [103].

T216

45006-45174

Sentence

denotes

Moreover, elevated levels of zinc have been shown not only to stimulate pro-inflammatory cytokine secretion by monocytes, but also inhibit T cell functions (see [102]).

T217

45175-45537

Sentence

denotes

Zinc supplementation that has also been proposed for COVID-19 treatment [105,106,107], is able to suppress allogeneic immune response (see [102]) and incubation of blood cells with welding fume particles resulted in a considerable increase not only of pro-inflammatory cytokines (including IL-1β, IL-6, IL-8, TNF-α) but also the tolerogenic cytokine IL-10 [104].

T218

45538-45866

Sentence

denotes

Similarly, in pulmonary arterial hypertension (PAH) as well as SARS-CoV, both arms of the inflammatory system (namely IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 and TNF-α) have been shown to be upregulated [108,109,110], suggesting a common systemic pathway of activation in welding syndrome, ARDS, PAH and SARS-CoV infections.

T219

45867-46036

Sentence

denotes

Finally, the correlations between zinc exposure (metal fume) and cytokine storm [103,104] suggests a possible link between free zinc increase and these type of diseases.

T220

46037-46217

Sentence

denotes

More information on systemic zinc homeostasis and multiorgan effects of elevated levels of extracellular free zinc (such as in subjects inhaling ZnCl2 smoke) is available in Box 4.

T221

46218-46320

Sentence

denotes

Box 4 Systemic zinc homeostasis and multiorgan effects of elevated levels of extracellular free zinc.

T222

46321-46562

Sentence

denotes

Zinc absorption occurs throughout the gastrointestinal tract into enterocytes by zinc transporters of the Zrt- and Irt-like protein (ZIP) family that take up Zn2+, but also Cu2+ and Fe2+ ions, both of which can inhibit zinc absorption [111].

T223

46563-46860

Sentence

denotes

The bioavailability of zinc is affected by several factors among them the amount of zinc in food, its chemical form (from very low availability, e.g., zinc oxide, to comparatively high, e.g., zinc chloride salt) and its binding to other (intestinal absorbed or non-absorbed) food components [111].

T224

46861-47065

Sentence

denotes

For example, phytates (components present in foods like rice, cereals and corn) and folic acid can form insoluble complexes with zinc that decrease zinc absorption and increase fecal zinc excretion [111].

T225

47066-47302

Sentence

denotes

On the other hand, Zn2+ can form complexes with most natural amino acids (AA) that for example can come from digested proteins; each Zn2+ can be coordinated with the amine groups and carboxylate anions of two AAs (a molar ratio of 1:2).

T226

47303-47577

Sentence

denotes

Interestingly, in an in vitro model, the formulation of either ZnCl2 salt or Zn-AA complexes were absorbed by enterocytes and zinc was released on the opposite side of the enterocyte membrane, thus mimicking the intestinal absorption and release into the blood stream [111].

T227

47578-47709

Sentence

denotes

It is therefore conceivable that zinc chelates of AA, as well as zinc salts, may participate in bioavailability and uptake of zinc.

T228

47710-47996

Sentence

denotes

The uptake of zinc-AA complexes by AA transporters of human enterocytes suggests that B0AT1 neutral AA transporter associated with ACE2 [28] might also be involved in zinc-AA uptake (a similar mechanism might be supposable for insulin biosynthesis/secretion in pancreatic β cells [91]).

T229

47997-48148

Sentence

denotes

Indeed, a recycling of zinc-AAs, possibly supplied by membrane bound ACE2 monocarboxypeptidase cleavage, for ACE2 synthesis could be also hypothesised.

T230

48149-48366

Sentence

denotes

Plasma zinc concentration is mainly regulated through a balance between intestinal absorption and renal excretion involving specific mechanisms sensitive to both dietary zinc availability and its cellular utilization.

T231

48367-48493

Sentence

denotes

The majority of Zn2+ in blood binds to serum albumin, which represents the major zinc transporter/reservoir protein in plasma.

T232

48494-48753

Sentence

denotes

On the other hand, labile-bound pool of Zn2+ labile complexes (e.g., of AA) and free Zn2+ are the biologically available forms of zinc for cellular internalization and/or enzymatic activities (0.1–1% of the total zinc in blood plasma under normal conditions).

T233

48754-48937

Sentence

denotes

Free Zn2+ is subsequently accumulated by endothelial cells and zinc uptake and release by endothelial cells is the crucial step for redistribution of Zn2+ from plasma to tissues [91].

T234

48938-49117

Sentence

denotes

Zinc is considered a relatively nontoxic metal; however, several data report that excessive free extracellular zinc is toxic and induce apoptosis in different cell types [91,102].

T235

49118-49451

Sentence

denotes

Plasma excess of free Zn2+ is uptaken by ZIP transporters on the cell surface of each cells in contact with plasma (including immune cells, endothelial cells, neurons and cardiomyocytes), leading to increase of intracellular zinc that can modulate zinc associated cellular functions in both physiological and pathological conditions.

T236

49452-49882

Sentence

denotes

Indeed, zinc is an important mediator/messenger involved in several cellular activities and the shift of zinc from plasma to cells may be significant for both normal physiological processes including energy metabolism, blood coagulation, and zinc signalling, and a range of disease states, including neurodegenerative and cardiovascular diseases, metabolic syndrome, inflammation, endothelial stress, diabetes and thrombosis [91].

T237

49883-50109

Sentence

denotes

For example, excess zinc intake has been shown to contribute to both amyloid beta-peptide plaque formation in Alzheimer’s disease and cardiac dysfunction, indicating that free zinc ion may be much more toxic than thought [91].

T238

50110-50386

Sentence

denotes

It has been reported that Alzheimer and other neurodegenerative disease patients show elevated levels not only of Zn2+ but also of other divalent metals such as Fe2+ and Cu2+ [112], suggesting a possible link between the increase metal ions and saturation of ZIP transporters.

T239

50387-50600

Sentence

denotes

On the other hand, increased plasma ferritin levels (such as in COVID-19 [2,3,6]) might reduce free Fe2+ and its “competition” with Zn2+ for ZIP transporters, finally increasing cellular zinc uptake and functions.

T240

50601-50740

Sentence

denotes

To this regard, EDTA metal chelator has been shown to protect from neural cell death and it was tested in neurodegenerative diseases [102].

T241

50741-50923

Sentence

denotes

Of interest, plasma albumin acts as a transport protein for both metal ions such as zinc and hydrophobic molecules such as free fatty acid (FFA) to allow their systemic distribution.

T242

50924-51105

Sentence

denotes

Indeed, albumin is well beyond a mere carrier of metal ions and hydrophobic molecules, and by binding of these different molecules mediates their crosstalk and bioavailability [91].

T243

51106-51218

Sentence

denotes

To this regard, one particularly intriguing allosteric link exists between FFA and zinc binding to albumin [91].

T244

51219-51424

Sentence

denotes

When FFAs with ten or more carbon atoms bind to albumin causes the release of Zn2+, suggesting a possible link between dyslipidemia and toxic effects mediated by consequent increase of free zinc in plasma.

T245

51425-51613

Sentence

denotes

For example, endothelial stress and activation and pro-inflammatory action of FFAs may be mediated by their effects on free zinc release from albumin and its consequent efflux from plasma.

T246

51614-51784

Sentence

denotes

Different studies have suggested a role of albumin to inhibit interaction of free Zn2+ with amyloid beta-peptides and combat plaque formation in Alzheimer’s disease [91].

T247

51785-51898

Sentence

denotes

Zn2+ has also been shown to promote clot stability by binding to fibrinogen and inhibiting heparin activity [91].

T248

51899-52020

Sentence

denotes

An evidence that has suggested a possible positive correlation between increased levels of FFAs and thrombotic risk [91].

T249

52021-52250

Sentence

denotes

In addition, physiological conditions associated with elevated plasma FFAs, such as physical exercise, can also produce an increased plasma level of free zinc and its downstream physiological events (e.g., activation of the RAS).

T250

52251-52419

Sentence

denotes

On the other hand, reduced concentrations of plasma albumin (such as IBD, ARDS and COVID-19) will induce elevated plasma levels of both free FFAs and bioavailable zinc.

T251

52420-52582

Sentence

denotes

Intriguingly, ARDS is induced after inhalation exposure to ZnCl2/ZnO/hexachloroethane, the main ingredients in smoke bombs used for crowd dispersal [100,101,102].

T252

52583-52700

Sentence

denotes

To this regard, soldiers that breathed smoke-bomb fumes have been shown to quickly or slowly develope ARDS [100,101].

T253

52701-52883

Sentence

denotes

Interestingly, a patient who had the highest level of serum zinc rapidly developed ARDS that leads to death from multiorgan failure (including respiratory and hepatic failure) [100].

T254

52884-53171

Sentence

denotes

Instead, a slow progressive clinical course was associated with a significant increase of plasma zinc concentration that negatively correlated with pulmonary and liver function [100], finally leading to death for severe respiratory failure three to five weeks after inhalation [100,101].

T255

53172-53272

Sentence

denotes

At autopsy diffuse microvascular obliteration and marked endothelial cell injury was apparent [101].

T256

53273-53465

Sentence

denotes

Widespread occlusion of the pulmonary arteries may contribute to the development of acute pulmonary hypertension and extensive interstitial and intra-alveolar fibrosis was also observed [101].

T257

53466-53678

Sentence

denotes

Other soldiers wearing gas masks, immediately developed severe coughing and dyspnea, and slowly improved their lung function (only twelve months after exposure their lung function tests were nearly normal) [101].

T258

53679-53763

Sentence

denotes

All clinical conditions that strongly resemble those described in COVID-19 patients.

T259

53765-53769

Sentence

denotes

5.2.

T260

53770-53836

Sentence

denotes

Safety and Efficacy Concerns of MLN4760 and Dx600 ACE2 Inhibitors.

T261

53837-53976

Sentence

denotes

It is known that the catalytic cleft of ACE2 consists of two peptidase subdomains: one membrane-distal and the other one membrane-proximal.

T262

53977-54097

Sentence

denotes

Their weak interactions are consistent with the ability to transition from open to the closed ACE2 conformation [28,90].

T263

54098-54373

Sentence

denotes

Indeed, the two subdomains undergo a large hinge-bending motion in which membrane-proximal subdomain remains almost unchanged, while membrane-distal subdomain moves to close the distance between the two subdomains, mimicking the opening/closing movement of a clam shell [90].

T264

54374-54664

Sentence

denotes

ACE2 open conformation likely reflects free state of the enzyme available to catch substrates (or inhibitors), then, when the ACE2 receptor binds to a substrate, the membrane-distal subdomain closes around the substrate (or the inhibitor), finally performing the enzymatic activity [24,90].

T265

54665-54845

Sentence

denotes

Interestingly, in cryo–electron microscopy structures of full-length human ACE2, only the closed/substrate-bound conformation of ACE2 was observed in the spike-ACE2 complexes [28].

T266

54846-55090

Sentence

denotes

Since human ACE2 is assembled on cell surface as a homodimer [28], binding of the spike protein trimer onto ACE2 dimer suggests simultaneous binding of two spike protein trimers to substrate-bound conformer of ACE2 homodimer on plasma membrane.

T267

55091-55347

Sentence

denotes

The spike binding sites on ACE2 homodimer are localized above the membrane-distal peptidase subdomain of each ACE2 monomer, nevertheless neither ACE2 shedding nor ACE2 binding to spike proteins have been shown to inhibit ACE2 enzymatic activity [16,17,24].

T268

55348-55615

Sentence

denotes

On the other hand, the S-protein-binding region of membrane-distal ACE2 subdomain is not significantly perturbed by the receptor conformational changes and maintains the ability to associate with soluble spike proteins independently on open/closed conformations [24].

T269

55616-55818

Sentence

denotes

Interestingly, an ACE2 specific inhibitor (MLN-4760) has been shown to induce the closed (inhibitor-bound) ACE2 structure [90] and to retain its inhibitory effects on sACE2 bound to spike proteins [24].

T270

55819-56019

Sentence

denotes

MLN4760 is a potent and selective human ACE2 inhibitor (IC50 = 0.44 nM against soluble human ACE2) whose synthesis produces a racemic mixture of two diastereomers that showed 75:25 ratio for Isomer A:

T271

56020-56129

Sentence

denotes

Isomer B [113,114] and the purified isomer B is the isomer commercially available from Merck Millipore [114].

T272

56130-56315

Sentence

denotes

Testing MLN-4760 racemic mixture and its isomers, it was observed a concentration-dependent inhibition of recombinant human (rh)ACE or rhACE2 activities with all three inhibitors [114].

T273

56316-56666

Sentence

denotes

The isomer B was less selective (near minimal-maximal rhACE inhibition range 10−6M-10−4M) than the racemate or the isomer A (near minimal-maximal rhACE inhibition range 10−6M-10−2M) and less effective (near maximal rhACE2 inhibition at 10−7M) than the racemate or the isomer A (near maximal rhACE2 inhibition at 10−8 M) for rhACE2 versus rhACE [114].

T274

56667-57033

Sentence

denotes

Moreover, all three inhibitors exerted a significantly higher inhibitory activity against soluble than membrane-bound forms of (m)ACE2 (near maximal mACE2 inhibition at 10−6M), being the isomer B more selective and effective than the racemate or the isomer A for mACE2 vs. mACE expressed on the surface of mononuclear cells (or CD34 hematopoietic progenitors) [114].

T275

57034-57133

Sentence

denotes

A second ACE2 inhibitor, Dx600, produced similar results to those obtained with the isomer B [114].

T276

57134-57300

Sentence

denotes

Another study evaluated the inhibitory activity of both MLN-4760 and DX600 (either the linear conformational form or the disulfide bridged cyclic variant) inhibitors.

T277

57301-57512

Sentence

denotes

In this report, the experiments were performed at pH 6.5 [115], a pH at which rhACE2 proteolysis operates at maximal activity [79] and a condition that resembles hypercapnic acidosis which may occur during SARS.

T278

57513-57849

Sentence

denotes

MLN-4760 was still able to strongly and specifically inhibit rhACE2 activity (near maximal inhibition at 10−8 M), preventing rhACE2-driven Ang II degradation into Ang (1–7), whereas DX600 (either linear or cyclic variant) inhibits rhACE2 at relatively higher concentration (near maximal inhibition at 10−6 M–10−7 M, respectively) [115].

T279

57850-57994

Sentence

denotes

Altogether these data indicate that the racemate or the isomer A are more effective in inhibiting soluble forms of ACE2 than isomer B and Dx600.

T280

57995-58348

Sentence

denotes

Therefore, these inhibitors at opportune (low) concentrations are expected to preferentially reduce systemic sACE2 activity, while preserving the ACE2 activity of (local) membrane-associated forms of ACE2, knowing that catalytic activity of circulating ACE2 was undetectable in human plasma of healthy subjects due to an endogenous ACE2 inhibitor [116].

T281

58349-58704

Sentence

denotes

Since MLN-4760 inhibitor promotes the closed ACE2 conformation [90] which is the preferential conformer for virus binding [28], MLN-4760 is expected to not prevent viral entry; nevertheless, its inhibitory function on ACE2 pathway might work on the positive feedback loops (above described) that ultimately favour ACE2 membrane expression and viral entry.

T282

58705-58822

Sentence

denotes

A potential risk factor of inhibiting the Ang II metabolization into Ang 1–7 could be the increase of blood pressure.

T283

58823-59332

Sentence

denotes

Although ACE2 pathway inhibition might lead to hypertensive effects, treatment with MLN-4760 for 4-5 weeks had no effect on blood pressure when administered 10 mg/kg/day in drinking water in wild type mice [117] nor in male (mRen2)27 transgenic hypertensive rats (administered 30 mg/kg/day subcutaneously via mini-osmotic pumps) [118], suggesting that hypertensive activity mediated by ACE2 inhibition is promptly balanced by compensatory mechanisms either in normal or hypertensive blood pressure conditions.

T284

59333-59538

Sentence

denotes

In addition, injections of MLN4760 into the nucleus tractus solitarii has been shown to reduce the baroreceptor reflex in rats, suggesting a role for ACE2 in controlling a reflex bradycardia (see [39,44]).

T285

59539-59773

Sentence

denotes

Finally, chronic inhibition of ACE2 with MLN-4760 led to increase of ACE, albuminuria and glomerular injury in streptozotocin-induced diabetic mice, indicating a possible adverse effect of the inhibitor in a diabetic background [119].

T286

59774-59947

Sentence

denotes

Extensive experiments have been also performed with DX600, a specific peptide ACE2 inhibitor that exhibited a mixed competitive and non-competitive type of inhibition [120].

T287

59948-60045

Sentence

denotes

Actually, several reports describing Dx600 inhibitor administration in mice suggest its safe use.

T288

60046-60326

Sentence

denotes

Of interest, a report described its (safe) use alone (1 mg/kg per day) by nasal inhalation for 3 days in a mouse model of endotoxin-induced lung inflammation [121]; however, this inhibitor is less efficacious than MLN-4760 in inhibiting the soluble forms of human rACE2 [114,115].

T289

60327-60523

Sentence

denotes

Altogether these reports on administration of ACE2 inhibitors do not reveal significant adverse impacts or mortality in experimental animals, which suggests their safety in chronic administration.

T290

60524-60623

Sentence

denotes

This was also confirmed in human clinical trials with MLN-4760 (clinical name: ORE1001, see later).

T291

60624-61143

Sentence

denotes

Of interest, a report showed that on day 28 post-myocardial infarction, adult male Sprague-Dawley rats that had received MLN-4760 (also called C16) 25 mg/mL/day by daily intraperitoneal injection (as a solution of 42 mg/mL in distilled water) tended to have lower left ventricular pulse pressure, mean arterial pressures and left ventricular relaxation time constant-Tau compared to untreated group (see table 2 of the paper) [52], suggesting a possible protective role of ACE2 inhibition in post-myocardial infarction.

T292

61144-61424

Sentence

denotes

Therefore, MLN-4760 might be helpful not only for COVID-19 but also in targeted therapies for pathologies correlated with an excessive increase of ACE2 activity that may involve heart, lung, liver, colon or other tissues/organs expressing ACE2 such as blood and endothelial cells.

T293

61425-61651

Sentence

denotes

As an example, GL1001 (old name of MLN-4760) showed to produce an anti-inflammatory activity in a mouse model of colitis [122], highlighting the importance of the yin-yang balance of ACE/ACE2 pathways (“in medio stat virtus”).

T294

61653-61657

Sentence

denotes

5.3.

T295

61658-61706

Sentence

denotes

Safety and Efficacy Concerns of Chelating Agents

T296

61707-61924

Sentence

denotes

Based on ACE2 mechanism of action, there are alternative ways to inhibit ACE2 (and ACE), for example concentration of cation/anion might also targets of intervention to inhibit the zinc metalloprotease ACE2 (and ACE).

T297

61925-62022

Sentence

denotes

In this regard, EDTA, a cation chelator, has been shown to be able to inhibit ACE2 activity [79].

T298

62023-62317

Sentence

denotes

Of interest, EDTA binds with 106- and 102-fold higher affinity to Zn2+ than to Ca2+ and Fe2+, respectively [123], and in plasma, free Ca2+ concentration (1.05–1.30 mmol/L) is “only” about 103-fold higher than free Zn2+ (0.1–2.0 µmol/L), while majority of iron in plasma is bound to transferrin.

T299

62318-62430

Sentence

denotes

As a consequence, the iron concentration in plasma of healthy subjects is very low on the order of 10−18M [112].

T300

62431-62738

Sentence

denotes

Among the essential metal ions present in the organisms, the specific affinity of EDTA for systemic Zn2+ in physiologic conditions is highlighted by the evidence that prolonged treatment with CaNa2EDTA results in specific zinc depletion that is believed to mediate the teratogenic effects of the drug [123].

T301

62739-63068

Sentence

denotes

As already mentioned, zinc is an important mediator/messenger involved in several cellular activities and excess of free zinc has been shown to be toxic (see also Box 4); however, zinc deficiency has also detrimental effects on growth, neuronal development, and immunity, and in severe cases its consequences can be deadly [102].

T302

63069-63261

Sentence

denotes

For these reasons, treatments with CaNa2EDTA require a careful monitoring of the patient for the therapeutic effects as well as possible complications, so as to titrate the appropriate dosage.

T303

63262-63367

Sentence

denotes

CaNa2EDTA was approved by FDA in chelation therapy for lowering blood lead levels a long time ago (1953).

T304

63368-63501

Sentence

denotes

Then, different (commercially available) iron chelating agents, expected to work in chelating zinc ion as well, were approved by FDA.

T305

63502-63635

Sentence

denotes

Chelation therapy comprises intravenous or oral administration of chelating agents that remove metal ions such as zinc from the body.

T306

63636-63733

Sentence

denotes

Cells synthesising high amounts of ACE or ACE2 needs of high amounts of bioavailable (free) zinc.

T307

63734-63876

Sentence

denotes

Therefore, it is conceivable that plasma levels of free zinc may influence not only ACE and ACE2 activities but also their cellular synthesis.

T308

63877-64008

Sentence

denotes

Indeed, both ACE and ACE2 synthesis might be particularly sensitive to reduction of free zinc levels in case of their upregulation.

T309

64009-64201

Sentence

denotes

In addition, metal chelating agents, by limiting the availability of free zinc to cells, might have effects on both ACE2/ACE synthesis and conformation (when assembled on the plasma membrane).

T310

64202-64499

Sentence

denotes

Indeed, the closed conformer of ACE2 homodimer that is the preferential conformation for virus binding [28], needs the presence of both zinc and substrate/inhibitor in the catalytic site [90], suggesting that zinc chelation (differently from MLN-4760) might also inhibit ACE2-mediated viral entry.

T311

64500-64746

Sentence

denotes

Intriguingly, chloroquine has been shown to specifically enhance zinc uptake and its accumulation/sequestration in the lysosomes [124], raising the possibility that it might work on COVID-19 patients by reducing zinc recycling and zinc functions.

T312

64747-64956

Sentence

denotes

As already mentioned, free Zn2+ promotes clot stability by binding to fibrinogen (see Box 4 and [91]) and chloroquine has been shown to have an anti-thrombotic activity by inhibiting platelet activation [125].

T313

64957-65337

Sentence

denotes

To this regard, upon platelet activation the release of Zn2+ store from their secretory granules has been shown to participate to the pro-coagulant activity in platelet-dependent fibrin formation [126], suggesting that an elevated free Zn2+ concentration might occur and contribute to thrombotic predisposition in COVID-19 patients, a phenomenon possibly countered by chloroquine.

T314

65338-65579

Sentence

denotes

For all of the above reasons, cation chelating agents, administered alone or in combination with other therapies, might be effective to counter COVID-19 infection, in particular when, induced by hypoxia, both arms of the RAS are upregulated.

T315

65580-65627

Sentence

denotes

A scenario that would deserve an investigation.

T316

65628-65990

Sentence

denotes

However, some formulations of metal chelating agents carry a black box warning because they may cause serious and fatal renal toxicity and failure, hepatic toxicity and failure, gastrointestinal haemorrhage, arrhythmias, tetany, hypocalcaemia, hypotension, convulsions, respiratory arrest, and agranulocytosis that can lead to serious infections and death [123].

T317

65991-66158

Sentence

denotes

For the sake of completeness, besides renal toxicity linked to the route of drug excretion, EDTA has been shown to be effective in chronic renal artery diseases [123].

T318

66159-66249

Sentence

denotes

Finally, for its teratogenic effects, the drug is also contraindicated in pregnancy [123].

T319

66250-66472

Sentence

denotes

As a result, treatments with metal chelating agents require close patient monitoring, including laboratory tests of renal and hepatic function, and absolute neutrophil count should be monitored before and during treatment.

T320

66473-66544

Sentence

denotes

Alternative ways of RAS pathway inhibition are also described in Box 5.

T321

66545-66595

Sentence

denotes

Box 5 Alternative ways of RAS pathway inhibition.

T322

66596-66703

Sentence

denotes

Of interest, a small cationic inhibitor of ACE2 (but not of ACE) has been detected in plasma samples [116].

T323

66704-66958

Sentence

denotes

The endogenous inhibitor might play a compensatory fine-control (within a threshold limit) for normal fluctuation of ACE2 protein in plasma and it has been hypothesized to be a basic AA or a small basic peptide able to compete with ACE2 substrates [116].

T324

66959-67066

Sentence

denotes

I have already mentioned that AA can chelate zinc forming labile zinc complexes of amino acids (see Box 4).

T325

67067-67252

Sentence

denotes

It is therefore possible that a basic AA or a small basic peptide capable to specifically accommodate into the catalytic site of ACE2, but into that of ACE, could inhibit ACE2 activity.

T326

67253-67417

Sentence

denotes

Indeed, despite the similarities, ACE and ACE2 possess different substrate specificity that depends on the smaller ACE2 binding pocket compared to that of ACE [90].

T327

67418-67594

Sentence

denotes

This specific aspect might determine the specificity for a specific basic amino acid or molecule that is able to accommodate into the catalytic pocket of ACE2 and bind to zinc.

T328

67595-67755

Sentence

denotes

Among the possible endogenous ACE2 inhibitors, agmatine, decarboxylated arginine, has a chemical structure that resembles that of an ACE2 inhibitor, NAAE [127].

T329

67756-67932

Sentence

denotes

NAAE is a small molecule that had demonstrated an anti-SARS-CoV activity, by acting on both ACE2 catalytic activity and ACE2 binding domain for spike protein of SARS-CoV [127].

T330

67933-67981

Sentence

denotes

Unfortunately, NAAE has never been used in vivo.

T331

67982-68272

Sentence

denotes

Indeed, NAAE is a weak ACE2 inhibitor, it is in fact more than a thousand-fold less potent than MLN-4760; however, if agmatine will be proven to have ACE2 inhibitory activity, it might be helpful to prevent the trigger of the positive feedback loops in the first mild phases of the disease.

T332

68273-68395

Sentence

denotes

Indeed, it has an important role in down-regulating NO synthesis reducing NO overproduction by different mechanisms [128].

T333

68396-68557

Sentence

denotes

Of note, NOS pathway has been shown to be upregulated by both Ang (1–7)/MasR and Ang (1–9)/AT2 receptor pathways that are downstream ACE2 activity [38,39,41,43].

T334

68558-68749

Sentence

denotes

Moreover, agmatine has a regulated plasma concentration in the range of 20-80 ng/mL and the use of dietary agmatine has been shown to be safe and effective in reducing neuropathic pain [129].

T335

68750-68825

Sentence

denotes

Moreover, agmatine sulfate is regularly taken as a bodybuilding supplement.

T336

68826-69036

Sentence

denotes

Among natural metal-chelating agents, phytates and folic acid are two chelating agents from vegetables that might reduce zinc intestinal absorption and possibly its systemic concentration (see Box 4 and [111]).

T337

69037-69189

Sentence

denotes

Similarly, nicotianamine that is extracted from plants (soybean) is a low-molecular weight metal chelator with high affinity for divalent metal cations.

T338

69190-69293

Sentence

denotes

Nicotianamine has been shown to inhibit the activity of both zinc metalloproteases, ACE2 and ACE [130].

T339

69294-69375

Sentence

denotes

In addition, zeolites might also be effective in reducing free zinc availability.

T340

69376-69504

Sentence

denotes

Zeolites are a group of aluminosilicate minerals with crystalline microporous structure that are originated from volcanic rocks.

T341

69505-69676

Sentence

denotes

Their molecular structure generates cavities that allow high absorbency capacity for a wide range of charged elements such as water, heavy metals, cations and many toxins.

T342

69677-69777

Sentence

denotes

Clinoptilolite is one of the zeolites that has been widely studied in veterinary and human medicine.

T343

69778-69964

Sentence

denotes

The increased usage of clinoptilolite-based products in vivo has stimulated several investigations on its safety and its positive medical effects related to human health (see [131,132]).

T344

69965-70102

Sentence

denotes

Finally, soluble and catalytically inactive forms of ACE2 have been shown to be potent inhibitors of SARS-CoV infection products [19,21].

T345

70103-70203

Sentence

denotes

An approach that could be pursued (in combination with other therapies) to inhibit SARS-CoV-2 entry.

T346

70204-70420

Sentence

denotes

Indeed, soluble forms of ACE2 are expected to protect from viral infection and a similar strategy using a recombinant form of human ACE2 has been proposed not only in COVID-19, but also in ARDS and PAH [108,133,134].

T347

70421-70551

Sentence

denotes

However, it is possible that catalytic active form of ACE2 might favour adverse effects in these specific pathological conditions.

T348

70552-70862

Sentence

denotes

To this regard, a clinical trial using recombinant hACE2 protein has been recently started (ClinicalTrials.gov number, NCT04287686) in COVID-19 patients and pilot clinical trials of rhACE2 in ARDS and PAH started in 2017 and 2018, respectively [108,134]; unfortunately, no conclusive results are available yet.

T349

70864-70866

Sentence

denotes

6.

T350

70867-70980

Sentence

denotes

Correlation of Pre-existing Circulating ACE2 Activity and Increased Potential to Develop Severe Forms of COVID-19

T351

70981-71124

Sentence

denotes

Severe symptoms of COVID-19 have been described to correlate with pre-existing hypertension, diabetes, age and male gender [1,2,3,4,5,6,7,8,9].

T352

71125-71502

Sentence

denotes

It is not still clear whether it depends on constitutive hypertensive conditions and/or on anti-hypertensive treatments or on other age-related conditions, considering that the prevalence of hypertension in Chinese adults is ~ 23% and that only about 41% take prescribed antihypertensive medications, being calcium channel blockers the most commonly used (~ 50%) in China [11].

T353

71503-71741

Sentence

denotes

To this regard, there is an interesting report describing sACE2 activity in plasma samples of Spanish healthy subjects and patients, in which a total of 2572 subjects from a multicenter study (NEFRONA project, 2009–2011) was studied [96].

T354

71742-71860

Sentence

denotes

The report shows that male and advanced age were identified as independent predictors of enhanced sACE2 activity [96].

T355

71861-72052

Sentence

denotes

Furthermore, subjects with hypertension, diabetes, dyslipidemia, or plaques also had significantly increased circulating ACE2 activity when compared with those without these pathologies [96].

T356

72053-72268

Sentence

denotes

Notably, hypertensive (the most frequent comorbidity with COVID-19) and diabetic patients are often treated with ACE inhibitors (ACEIs) and/or with ARBs, suggesting a possible positive correlation [135,136,137,138].

T357

72269-72554

Sentence

denotes

Interestingly, circulating ACE2 activity is significantly increased in subjects on therapy with ARBs or taking oral antidiabetic agents as compared with non-treated patients, while treatment with ACEIs and cholecalciferol had no significant influence on circulating ACE2 activity [96].

T358

72555-72710

Sentence

denotes

In line with this observations, losartan (an ARB), but not lisinopril (an ACEI), was able to upregulate ACE2 activity in left ventricle of Lewis rats [75].

T359

72711-73020

Sentence

denotes

Nevertheless, plasma concentration of Ang (1–7) was significantly increased with both treatments when compared to vehicle-treated rats and it was significantly higher in ACEI-treated than ARB-treated rats [75], suggesting that ACE inhibition is able to induce an activation of the ACE2/Ang (1–7)/MasR pathway.

T360

73021-73192

Sentence

denotes

Although more evidence is needed in humans, the above observations suggest that ARBs should be precautionarily avoided to reduce possible ACE2-mediated viral consequences.

T361

73193-73366

Sentence

denotes

Indeed, under hypoxic conditions, either an ACEI or an ARB have been shown to upregulate membrane ACE2 protein expression on human pulmonary artery smooth muscle cells [58].

T362

73367-73518

Sentence

denotes

ACEI and ARB have been shown to work by reducing the concentration of Ang II and by inhibiting its AT1-mediated ACE2 downregulation, respectively [58].

T363

73519-73774

Sentence

denotes

ACEIs and ARBs might therefore play a role in the upregulation of membrane ACE2 expression under hypoxic conditions such as COVID-19, knowing that the increase of membrane bound ACE2 (before its shedding) will also increase the probability of viral entry.

T364

73775-73877

Sentence

denotes

Of note, ACEI and ARB antihypertensive medications are more commonly used in Europe/USA than in China.

T365

73878-74102

Sentence

denotes

Nevertheless, recent reports show that the use of ACEI/ARB medications in patients with COVID-19 is safe and ACEI/ARB exposure was not associated with a higher risk of having severe forms of COVID-19 [6,139,140,141,142,143].

T366

74103-74345

Sentence

denotes

Moreover, some reports show that ACEI/ARB treated patients may even be protected from COVID-19 and ACEI/ARB exposure was associated with a lower risk of mortality compared to those on non-ACEI/ARB antihypertensive drugs [144,145,146,147,148].

T367

74346-74546

Sentence

denotes

Among these reports, one indicates that the risk of severe symptoms of COVID-19 was significantly decreased in patients who took ARB drugs (but not ACEIs) compared to patients who took no drugs [144].

T368

74547-74773

Sentence

denotes

Differently, another report suggests that the use of ACEIs (but not ARBs) reduced risk of death and/or critical disease [146], suggesting that some of these reports did not adjust for confounders and further analyses are need.

T369

74774-74972

Sentence

denotes

Nevertheless, clinical trials of losartan as a treatment for COVID-19, are actually underway among patients who have not previously been treated with ARBs and/or ACEIs (NCT04312009 and NCT04311177).

T370

74973-75222

Sentence

denotes

However, most of hypertensive/diabetic patients are ACEI- or ARB-“pretreated”, nevertheless they have an increased risk to develop SARS, therefore ACEIs and ARBs, if not detrimental, are not expected to face the disease in non-hypertensive patients.

T371

75223-75554

Sentence

denotes

A possible beneficial effect of ACEIs in COVID-19 patients could indirectly come by reducing ACE2 substrate, Ang II, and finally limiting Ang (1–7) [but not Ang (1–9)] production and its (detrimental) effects; however, this is only a hypothetical possibility since, in this case, the ACE2/ACE pathway might be even more unbalanced.

T372

75555-75747

Sentence

denotes

To complete the picture, smokers and subjects on therapy with insulin tend to have an increased (although not significantly) circulating ACE2 activity when compared with control subjects [96].

T373

75748-76009

Sentence

denotes

On the other hand, increased ACE2 protein expression was reported in plasma and/or urine of physically active men after acute aerobic training or in renal cortices of spontaneous hypertensive, but not normotensive, rats after chronic aerobic training [149,150].

T374

76010-76239

Sentence

denotes

In addition, recent works reveal that asthma and other allergic diseases, which protect from developing COVID-19, were associated with significant reductions in levels of both zinc in plasma and ACE2 mRNA in airway cells [98,99].

T375

76240-76498

Sentence

denotes

On the other hand, as already mentioned, some patients with cardiovascular diseases (and inflammatory bowel disease) have an increased circulating ACE2 [22,49,151,152], which might explain the higher probability of elderly heart patients to develop COVID-19.

T376

76499-76728

Sentence

denotes

Interestingly, in a report on atrial fibrillation, increased plasma ACE2 activity was significantly associated not only with cardiac dysfunction, increasing age, male gender and hypertension, but also with vascular disease [152].

T377

76729-76970

Sentence

denotes

Altogether the data suggest a strong correlation between circulating ACE2 activity and the predisposition to develop the most severe symptoms of COVID-19, suggesting that circulating sACE2 might be a predictive biomarker of SARS development.

T378

76971-77206

Sentence

denotes

The surprising aspect is that circulating (differently from membrane bound) ACE2 is expected to protect from viral infection and clinical trials using recombinant ACE2 protein are being pursued (ClinicalTrials.gov number, NCT04287686).

T379

77207-77410

Sentence

denotes

However, even if it might not be detrimental, recombinant ACE2 is not expected to protect from COVID-19, as circulating ACE2 upregulation correlates with the most common comorbidities in severe COVID-19.

T380

77411-77707

Sentence

denotes

The correlation is extremely impressive and striking if we also consider the low prevalence of chronic renal disease in COVID-19 hospitalized patients (0–3%) [1,2,3,4,5,6,7,8,9] (prevalence of the disease in China ~11% [12]) which might be protected by a higher sACE2 and/or Zn2+ renal excretion.

T381

77708-77926

Sentence

denotes

Indeed, in chronic kidney disease patients without a history of cardiovascular disease, there was a significant decrease in circulating ACE2 activity and zinc levels when compared with healthy control subjects [96,97].

T382

77927-78236

Sentence

denotes

Since proteinuria was associated with lower blood levels of sACE2 protein [153] and chronic kidney disease patients had higher urinary zinc excretion than healthy controls [97], low sACE2 activity in chronic renal diseases and protection from SARS might derive from a higher sACE2 and/or Zn2+ renal excretion.

T383

78237-78483

Sentence

denotes

Indeed, sACE2 is detectable in urine of healthy subjects and urinary sACE2 protein levels are elevated in patients with chronic renal diseases and in hypertensive patients treated with the Ang II type 1 receptor blocker (ARB) olmesartan [41,154].

T384

78484-78709

Sentence

denotes

It can, therefore, be supposed that a basal hyperactivity of ACE2 and consequent ACE/ACE2 pathway disequilibrium in blood might predispose to the development of more severe COVID-19 symptoms that involve both arms of the RAS.

T385

78710-79086

Sentence

denotes

In this regard, different organism predisposition to infections including SARS-CoV-2 or to pathologies associated with an increased risk to develop severe forms of SARS-CoV-2 infection might depend not only on genetic ACE2 polymorphisms and organ-specific ACE2 gene/protein expression [138,155,156,157,158,159,160,161,162,163] but also on anatomical and environmental factors.

T386

79087-79263

Sentence

denotes

For example, ACE2 gene variants have been associated with risk for hypertension, dyslipidemia, type 2 diabetes and cardiovascular dysfunction [138,155,156,157,158,159,160,161].

T387

79264-79508

Sentence

denotes

Most of these variants are intronic and located in splice-site junctions or in enhancer regions while the others are placed in the 3’ UTR promoter region, suggesting their involvement in the structure/function and expression of ACE2 gene [138].

T388

79509-79661

Sentence

denotes

On the other hand, COVID-19 has similar risk factors of obstructive sleep apnea (OSA), suggesting a possible association between OSA and COVID-19 [164].

T389

79662-79828

Sentence

denotes

Indeed, high prevalence of pre-existing OSA in COVID-19 patients has been reported [165,166], indicating that OSA could be a risk factor for severe forms of Covid-19.

T390

79829-80055

Sentence

denotes

OSA is characterised by repetitive airway collapse with apnea/hypopnea and intermittent hypoxaemia during sleep and it is associated with hypertension, cardiovascular disease, diabetes, obesity and systemic inflammation [167].

T391

80056-80312

Sentence

denotes

Most of these diseases are known to predispose to increased levels of circulating ACE2 and, as already mentioned, hypoxia has been shown to upregulate both arms of the RAS; therefore, OSA might predispose to the deleterious effects of SARS-CoV-2 infection.

T392

80313-80553

Sentence

denotes

In OSA patients, upper airway collapsibility under passive conditions (critical closing pressure) is higher in males than in females due to anatomical factors, i.e., longer upper airways, as upper airway length correlates with OSA severity.

T393

80554-80794

Sentence

denotes

Indeed, upper airways in females are less collapsible and more stable during sleep than in males [167], suggesting that an anatomical factor might contribute to and result in different gender susceptibility to COVID-19 and disease severity.

T394

80795-80976

Sentence

denotes

Moreover, diet (e.g., zinc assumption), physical exercise and external temperature and UV radiation are all “environmental” aspects that can seasonally influence the RAS activities.

T395

80977-81090

Sentence

denotes

Seasonal differences of the RAS activities leading, for example, to differences in blood pressure are well known.

T396

81091-81213

Sentence

denotes

Of interest, the majority of coronaviruses cause cold-like illnesses and have a relatively low mutational frequency [168].

T397

81214-81287

Sentence

denotes

Similarly SARS-CoV-2 seems to mutate much slower than seasonal flu [169].

T398

81288-81522

Sentence

denotes

Nevertheless, like influenza virus, they are responsible for seasonal episodes of common cold in humans worldwide [168], suggesting that coronaviruses may possess an ability to evade immune control that is different from seasonal flu.

T399

81523-81615

Sentence

denotes

For example, they might favour a relatively short-term activation/memory of immune response.

T400

81616-81752

Sentence

denotes

Indeed, as for SARS-CoV and MERS-CoV, a short duration of immunity after SARS-CoV-2 infection has been recently suggested [170,171,172].

T401

81753-81933

Sentence

denotes

Seasonal cold is an annually recurring time period characterized by the prevalence of outbreaks of cold and the season occurs during the cold period of the year in each hemisphere.

T402

81934-82304

Sentence

denotes

Altogether these observations suggest that seasonal predisposition to some virus infections (including SARS-CoV-2) might not depend only on environmental features that directly inactivate the viruses “unprotected” outside the host (e.g., higher ultraviolet rays and temperatures), but also on seasonal indirect effects produced in the organisms by environmental changes.

T403

82306-82308

Sentence

denotes

7.

T404

82309-82418

Sentence

denotes

Monitoring ACE/ACE2 Activity in COVID-19 in Order to Determine a Rationale Use of the Specific RAS Inhibitors

T405

82419-82526

Sentence

denotes

There is currently no specific pharmacotherapy to combat SARS-CoV-2 infection and its pathological effects.

T406

82527-82922

Sentence

denotes

Actually, the use of plasma from convalescent COVID-19 patients or of SARS-CoV-2-specific neutralizing antibodies has been shown to be a promising option for the treatment of critically COVID-19 patients [32,33,34,35,36,37,172,173]; however, it has some limitations that might be overcome by vaccine strategies or by the production of specific anti SARS-Co-V-2 therapeutic monoclonal antibodies.

T407

82923-83099

Sentence

denotes

Despite the promising data that demonstrate vaccine protection against SARS-CoV-2 in nonhuman primates [174], we cannot be sure to get a vaccine against SARS-CoV-2 (see [175]).

T408

83100-83350

Sentence

denotes

Indeed, animals immunized with inactivated SARS-CoV vaccines developed a severe (asthma-like) lung eosinophilic immunopathology when challenged with SARS-CoV, further indicating a central role of eosinophil “balanced numbers” in this pathology [176].

T409

83351-83534

Sentence

denotes

Vaccines might generate antibodies against viral ligand/ACE2 complex that finally blocks ACE2 activity during SARS-CoV infection and consequent downstream asthma-like events/symptoms.

T410

83535-83846

Sentence

denotes

On the other hand, fatal outcomes in SARS-CoV infection correlated with a cytokine storm involving elevated Th2 serum cytokines (including IL-4, IL-5 and IL-10), suggesting that an increase of Th2 cytokines possibly mediated by virus-specific CD4 T cells might be crucial in the severe forms of infection [110].

T411

83847-84261

Sentence

denotes

In addition to cytokine profile (such as IL-10), eosinopaenia, tachycardia, normo/hypotension (although COVID-19 and hypoxia increase Ang II and many patients are “hypertensive” and/or receiving anti-hypertensive medications) and hypoxia in SARS-CoV-2 patients are compatible with downstream events stemming from both an excessive ACE2 pathway upregulation and activation of positive feedback loops (see Figure 2).

T412

84262-84458

Sentence

denotes

ACE2 and (anti-inflammatory) IL-10 hyperproduction may ultimately trigger subsequent compensatory (and deleterious) responses, leading to both renin/ACE and pro-inflammatory cytokine upregulation.

T413

84459-84578

Sentence

denotes

In line with this hypothesis, a longitudinal study showed that IL-6 increases late during the COVID-19 progression [3].

T414

84579-84875

Sentence

denotes

Altogether these data imply not only that the ACE2 is the “vehicle” of viral entry into the host cells, but also that virus-dependent and virus-independent mechanisms may sustain activation of both arms of the RAS and of the inflammatory system, finally promoting SARS-induced multi-organ injury.

T415

84876-85186

Sentence

denotes

Unfortunately, complete analysis of circulating levels of ACE/ACE2 proteins and activities, together with ACE and ACE2 substrates and products i.e., Ang I, Ang 1–9, Ang II, Ang (1–7) and Ang (1–5), des-Arg(9)-bradykinin and the inactive metabolite bradykinin (1–7) is lacking to address the current hypothesis.

T416

85187-85370

Sentence

denotes

The required data could be obtained from blood sample analyses, however, it is important to consider that:(1) Organ-specific local microenvironment might not reflect the systemic one;

T417

85371-85607

Sentence

denotes

(2) S1-sACE2 and SARS-CoV-2-sACE2 complexes, formed by viral-induced ACE2 shedding or by subsequent binding of sACE2 with viral particles or S1 fragments in the circulation, might not be detectable by some anti-ACE2 antibodies in ELISA.

T418

85608-85831

Sentence

denotes

Therefore, since the complexes might prevent/mask sACE2 antibody recognition but not the enzymatic activity, sACE2 detection (and its real concentration) by ELISA in blood samples of COVID-19 patients might not be reliable;

T419

85832-86128

Sentence

denotes

(3) In order to evaluate circulating ACE/ACE2 activity, we can determine it by adding fluorogenic (RAS) substrates to plasma samples without (thus considering also the endogenous ACE2 inhibitor present in plasma [116]) or with adding ZnCl2, which is able to induce ACE/ACE2 maximal activity [96];

T420

86129-86280

Sentence

denotes

(4) circulating concentrations ACE/ACE2 substrates/products likely depend on the:(a) level of ACE/ACE2 pathway activity and availability of substrates;

T421

86281-86414

Sentence

denotes

(b) level of expression of the respective receptors (AT1R, AT2R and MasR) that bind and remove ligand products away from circulation.

T422

86415-86637

Sentence

denotes

For example, Ang (1–7) concentration in blood by ACE2 pathway upregulation is dependent both on the increase of the (antagonistic) Ang II (and its precursor, Ang I) and on MasR expression on surface of cells in the organs.

T423

86638-86879

Sentence

denotes

To this regard, the detection of inactive metabolites, such as bradykinin (1–7), in COVID-19 patients and comparison with its concentration in healthy subjects could be the most indicative and reliable markers of ACE2 activity in the plasma.

T424

86880-87283

Sentence

denotes

Of interest, a report that described the ex vivo effect of human rACE2 on the levels of several endogenous Ang peptides in plasma samples, thus mimicking an acute increase of systemic ACE2 activity in plasma [115] showed a strong reduction of Ang II (as expected), a moderate reduction of Ang I and a significant (more than five-fold) increase of Ang (1–9), Ang (1–7) and (ACE-produced) Ang (1–5) [115].

T425

87284-87406

Sentence

denotes

Therefore, Ang (1–5) may also be an interesting surrogate marker of ACE-ACE2 combined activity in COVID-19 patients [115].

T426

87407-87927

Sentence

denotes

At the present time, it has been reported that plasma levels of Ang II in SARS-CoV-2 infected patients are markedly elevated as compared with healthy subjects (interquartile range between 260–360 pg/mL and 65–120 pg/mL, respectively) and linearly associated to viral load and lung injury [177]; unfortunately, nobody has tested the levels of Ang (1–7), Ang (1–9), Ang (1–5) and bradykinin (1–7), some of which might be at high levels as well, thus justifying both normo/hypotension and eosinopaenia in COVID-19 patients.

T427

87928-88111

Sentence

denotes

More information on the RAS peptides in plasma of Acute Respiratory distress syndrome, a COVID-19-like disease, (and pulmonary arterial hypertension) is instead available (see Box 6).

T428

88112-88195

Sentence

denotes

Box 6 Acute Respiratory distress syndrome and hyperactivation of the RAS pathways.

T429

88196-88322

Sentence

denotes

The main clinical feature of ARDS patients is the progressive deterioration of lung function leading to progressive hypoxemia.

T430

88323-88509

Sentence

denotes

ARDS can be induced by different causes such as ZnCl2 aspiration, sepsis, trauma, acute pancreatitis, or pneumonias following virus infections such as SARS-CoVs or human influenza virus.

T431

88510-88646

Sentence

denotes

ACE insertion/deletion polymorphisms correlated with severity of ARDS in humans, suggesting that the RAS could have a role in ARDS [44].

T432

88647-88825

Sentence

denotes

In animal models, ACE2 protected from acute lung by inhibiting Ang II/AT1R activity [15], for this reason the use of recombinant ACE2 in ARDS has been proposed and pursued [134].

T433

88826-89156

Sentence

denotes

Clinical trials with recombinant human ACE2 started in healthy volunteers in 2009 (ClinicalTrials.gov number, NCT00886353 [178]), then it was tested in ARDS (ClinicalTrials.gov number, NCT01597635 [134]), PAH (ClinicalTrials.gov number, NCT01884051 [108]) and finally in COVID-19 patients (ClinicalTrials.gov number, NCT04287686).

T434

89157-89255

Sentence

denotes

Despite its safe use, no conclusive evidence in support of its use in these diseases was reported.

T435

89256-89572

Sentence

denotes

Of interest, in the plasma samples from ARDS patients, levels of Ang I have been shown to be significantly increased in non-survivors (interquartile range 1990–16950 pg/mL) compared to survivors (interquartile range 730–5660 pg/mL) [92], suggesting a marked increase of renin activity in non-surviving ARDS patients.

T436

89573-90027

Sentence

denotes

Unfortunately, no comparisons of the two ARDS groups with healthy subjects (interquartile range 35–66 pg/mL, markedly lower than both patient groups [179]) were reported; this is important to consider since the plasma concentrations of the RAS peptides may significantly differ depending on method of detection (ELISA versus vs. mass spectrometry) and, in particular, on protocol of determination (endogenous versus equilibrium Ang peptide levels [179]).

T437

90028-90161

Sentence

denotes

Endogenous RAS peptide concentrations can be evaluated collecting and stabilizing blood samples with a RAS enzyme inhibitor cocktail.

T438

90162-90374

Sentence

denotes

Differently, heparinized plasma samples can be incubated at 37 °C to induce ex vivo equilibrium, which unveils the presence of renin, ACE and/or ACE2 activity in the plasma (equilibrium Ang peptide levels) [179].

T439

90375-90462

Sentence

denotes

This last protocol usually gives significantly higher levels of the RAS peptides [179].

T440

90463-90990

Sentence

denotes

For example, in healthy subjects, the normal ranges (interquartile ranges) of endogenous RAS peptides, Ang I, Ang II, Ang (1–7), Ang (1–9) and Ang (1–5) were 12–24 pg/mL, 1–12 pg/mL, <2 pg/mL, <4 pg/mL and < 1 pg/mL, respectively, differently the normal range after ex vivo equilibrium of the same peptides were 35–66 pg/mL, 110–200 pg/mL, < 2 pg/mL, < 4 pg/mL and < 1 pg/mL, respectively [179], indicating the presence of renin and ACE (but no ACE2) activity (as highlighted by Ang peptide increases upon ex vivo equilibrium).

T441

90991-91193

Sentence

denotes

Similarly, another report that evaluated Ang II and Ang (1–7) in healthy males showed normal endogenous ranges ~ 14.5 pg/mL and 0.9 pg/mL, respectively, and an Ang (1–7)/Ang II ratio ~ 0.06 ratio [180].

T442

91194-91456

Sentence

denotes

Moreover, Ang (1–7), Ang I, Ang (1–9) and Ang (1–7)/Ang II ratio were significantly higher in ACEI-treated hypertensive patients compared to healthy subjects [180], suggesting that inhibition of ACE predisposes to upregulation of the ACE2/Ang (1–7)/MasR pathway.

T443

91457-91678

Sentence

denotes

Notably, two different reports that evaluated the RAS peptides in ARDS patients using the similar method of detection (high performance liquid chromatography plus mass spectrometry) showed significantly different results.

T444

91679-91811

Sentence

denotes

One report showed that Ang II ranged (interquartile range) between 120–630 pg/mL (non-survivors) and 70–2220 pg/mL (survivors) [92].

T445

91812-92096

Sentence

denotes

A second report showed significantly lower values of Ang II, (interquartile range) between 40–150 ng/mL (non-survivors with placebo) and 5–20 pg/mL (survivors with placebo) (see Supplementary Data [134]), suggesting that they used different protocols of the RAS peptide determination.

T446

92097-92250

Sentence

denotes

Nevertheless, both Ang (1–7) and Ang (1–7)/Ang II ratios found in ARDS patients of both reports [92,134] were significantly higher than healthy subjects.

T447

92251-92443

Sentence

denotes

One report showed that Ang (1–7) ranged (interquartile range) between 2.5–10 pg/mL (see Figure 2B of [134]), Ang II between 5–20 pg/mL (see Figure 2 of [134]) and Ang (1–7)/Ang II ratio ~ 0.5.

T448

92444-92612

Sentence

denotes

Moreover, in the report in which the elevated concentrations of Ang I and Ang II indicate an ex vivo equilibrium of the RAS peptides, the increase was even more marked.

T449

92613-92931

Sentence

denotes

In that report, Ang (1–7) (< 1pg/mL in healthy subjects) ranged between 80–1070 pg/mL and Ang (1–7)/Ang II ratio (~ 0.06 in healthy subjects) ranged between 0.24–1.82 [92], suggesting that ACE2 activity (and ACE2 presence in plasma samples) was significantly increased in ARDS patients as compared to healthy subjects.

T450

92932-93113

Sentence

denotes

In line with this hypothesis, there was the determination of Ang (1–9), whose production from Ang I exclusively depends on ACE2 (which also compete with ACE for the same substrate).

T451

93114-93515

Sentence

denotes

In normal healthy subjects, it was reproducibly below the limit of quantification [179,180], differently, both in ex vivo treatments of plasma samples with human rACE2 [115,181] and in ARDS patients, it was markedly increased (Ang (1–9) ranged between 100–3080 pg/mL) [92], suggesting both that the dominating activity of ACE in plasma of healthy subjects is overcome by that of ACE2 in ARDS patients.

T452

93516-93593

Sentence

denotes

Therefore, Ang (1–9) may represent a good surrogate marker for ACE2 activity.

T453

93594-93802

Sentence

denotes

Similarly to Ang (1–9), there was the determination of Ang (1–5), whose production depend on both ACE2 [that produces Ang (1–7) from Ang II] and ACE [that produces Ang II from I and Ang (1–5) from Ang (1–7)].

T454

93803-94068

Sentence

denotes

Ang (1–5) was undetectable in healthy subjects [178,179]; however, it was significantly increased in ARDS patients (interquartile range between 50–730 pg/mL [92]), suggesting that both ACE2 and ACE activity were likely upregulated as compared with healthy subjects.

T455

94069-94192

Sentence

denotes

The activation for both arms of the RAS needs of the contemporary activation renin, the rate-determining enzyme of the RAS.

T456

94193-94523

Sentence

denotes

Indeed, analysis of plasma samples (from a healthy subjects), treated ex vivo with recombinant renin, showed an increase of Ang I, Ang II, Ang (1–7) and Ang (1–5), but not Ang (1–9) [115,179,181], further suggesting both the ACE2 specific activity in producing Ang (1–9) and an activation of both arms of the RAS in ARDS patients.

T457

94524-94825

Sentence

denotes

Finally, in vivo or ex vivo administration of human rACE2 in plasma samples produced an increase not only of Ang (1–9) but also of Ang (1–5) and Ang (1–7) in both healthy subjects [178] and ARDS patients [134], indicating that all three Ang peptides might represent surrogate markers of ACE2 activity.

T458

94826-95127

Sentence

denotes

Of interest, the mean arterial pressure of ARDS patients was markedly low and significantly lower in non–survivors (mean value 65 mmHg and more likely need vasopressor support) compared to survivors (mean value 71 mmHg) [92], which may likely be a consequence of the above described ACE2 upregulation.

T459

95128-95215

Sentence

denotes

Interestingly, similar features were also detected in PAH and coronary atherosclerosis.

T460

95216-95444

Sentence

denotes

In particular, a significant increase of both Ang II and Ang (1–7) peptides was observed in plasma of these patients [108,182] and plasma levels of TNF-α were significantly elevated in the critical coronary artery disease [182].

T461

95445-95664

Sentence

denotes

Moreover, despite a significant increase of Ang II/Ang (1–7) ratio (that suggested a therapeutic use of recombinant hACE2 in PAH patients) systolic and diastolic blood pressures were in the range of normality [108,182].

T462

95665-95875

Sentence

denotes

Altogether these observations suggest that Ang peptide ratios are not reliable markers for disease status in these pathologies; rather, increased plasma amounts of each RAS peptide should be careful considered.

T463

95877-95879

Sentence

denotes

8.

T464

95880-95933

Sentence

denotes

Hypothesizing Pharmacological Treatments for COVID-19

T465

95934-96044

Sentence

denotes

Based on the above described hypothesis, inhibition of ACE2 pathway might be beneficial for COVID-19 patients.

T466

96045-96305

Sentence

denotes

Indeed, the clinical picture as a whole is consistent with an ACE2 gain of function (possibly due to both circulating active forms of S1-sACE2 complexes and local forms of SARS-CoV-2-sACE2 complexes) rather than an ACE2 loss of function, as initially supposed.

T467

96306-96500

Sentence

denotes

Therefore, inhibition of ACE2/Ang (1–7)/MasR axis or other ACE2 pathways to restore ACE/ACE2 balance might be needed, at least in the first phases of the disease when hypoxia is not yet induced.

T468

96501-96642

Sentence

denotes

Different strategies could be pursued through ACE2 pathway inhibitors and/or MasR antagonists and/or renin inhibitors and/or metal chelators.

T469

96643-96848

Sentence

denotes

Several different molecules have been designed to specifically inhibit human ACE2 enzyme (both membrane bound and soluble forms) or human MasR signal transduction, but only a few have been studied in vivo.

T470

96849-96995

Sentence

denotes

Some ACE2 pathway inhibitors have been widely used in mouse/rat models, as control of human and mouse ACE2 activity or in mouse models of colitis.

T471

96996-97288

Sentence

denotes

Thanks to mouse/rat in vivo experiments and clinical trial results in human participants it has been possible to infer toxicity/efficacy for some human ACE2 inhibitory molecules (MLN-4760/C16/GL1001/ORE1001, Dx600 and A779) that may be exploited to face this exceptionally dramatic situation.

T472

97289-97539

Sentence

denotes

Unfortunately, to my knowledge, only one (MLN-4760/C16/GL1001/ORE1001) of the ACE2-specific inhibitors has been tested in vivo in humans in a Phase I clinical trial long time ago (http://oreholdings.com/wp-content/uploads/2013/06/09.02.09-S-4-A.pdf).

T473

97540-97654

Sentence

denotes

Here, I have focused my attention on inhibitors of human ACE2 pathway that were consistently administered in vivo.

T474

97655-98031

Sentence

denotes

Making use of published reports in which human/rodent ACE2 pathway inhibitors were administered in vivo, I have hypothesized a possible therapeutic pharmacological intervention through an inhibition strategy of the RAS pathways for COVID-19 in patients experiencing both mild and critical, advanced and untreatable stages of the disease (the most problematic cases to manage).

T475

98033-98037

Sentence

denotes

8.1.

T476

98038-98066

Sentence

denotes

Inhibition of ACE2: MLN-4760

T477

98067-98738

Sentence

denotes

Briefly, one of the best candidates to treat COVID-19 patients, but not to prevent viral entry, is the small synthetic molecule MLN-4760 (specific ACE2 inhibitor, also known as C16, GL1001 or ORE1001, [113]) for the following reasons:(1) It has been shown to bind/inhibit ACE2 enzymatic activity even at low/acidic pH (pH 6.5, [115]) typical of hypercapnia (as it might occur in lungs of COVID-19 patients) when human ACE2 activity is maximal [79]; nevertheless, it retains its inhibitory effects on soluble ACE2 bound to spike proteins [24], indicating that it is able to bind and inhibit ACE2 activity regardless ACE2 binding to SARS-CoV-2 particles or to S1 fragments.

T478

98739-99143

Sentence

denotes

(2) No significant adverse effects were described upon its chronic administration neither alone nor in combination with ACE2 activators (while inhibiting their activating effects) nor after inducing functional impairment of ACE2 activity in rodent experiments in vivo [52,117,118,122,183,184] nor in a clinical Phase I trial in humans (http://oreholdings.com/wp-content/uploads/2013/06/09.10.09-425.pdf);

T479

99144-99226

Sentence

denotes

(3) Its administration by different route is well described in rodents and humans.

T480

99227-99577

Sentence

denotes

In particular:(a) Chronic administration (about 4 weeks) of C-16/DLM-4760 in combination with ACE2 activating treatments was performed by daily intraperitoneal injection at a dose of 25mg/kg in distilled water (as a solution of 42 mg/mL) or 0.9% sterile saline (as a solution of 84 mg/mL using a 0.5-mL insulin syringe) freshly prepared [52,183,184].

T481

99578-99909

Sentence

denotes

(b) Alternatively, chronic administration (about 8 days) of GL1001/DLM-4760 disodium salt in combination with an ACE2 activating treatment was performed by subcutaneous injection (5mL/kg) containing up to a dose of 300 mg/kg, twice a day, formulated in a vehicle solution [15% 2-hydroxypropyl-β-cyclodextrin (HPBDC)/85% H2O] [122].

T482

99910-99995

Sentence

denotes

Subchronic doses of GL1001 indicate no adverse effects up to 1,000 mg/kg (see [122]).

T483

99996-100085

Sentence

denotes

(c) In humans ORE1001/GL1001/MLN-4760 was already proposed and tested in clinical trials.

T484

100086-100254

Sentence

denotes

Its pharmaceutical indication was for digestive tract inflammations (Inflammatory bowel disease, gastritis and colitis) that are correlated with overexpression of ACE2.

T485

100255-100331

Sentence

denotes

In a Phase I clinical testing up 14 days dosing, ORE1001 was well tolerated.

T486

100332-100416

Sentence

denotes

Subjects received drug (dosing up to 2100 mg) with no side adverse effects reported.

T487

100417-100554

Sentence

denotes

In particular, 47 subjects received single-dose from 2.1 to 2100 mg and 24 subjects received 14 day multiple doses from 50 mg to 1800 mg.

T488

100555-100644

Sentence

denotes

All doses were well tolerated, with no significant side effects including blood pressure.

T489

100645-100802

Sentence

denotes

Pharmacokinetics of orally administered capsules was consistent with once-daily dosing. (http://oreholdings.com/wp-content/uploads/2013/06/09.10.09-425.pdf).

T490

100803-100961

Sentence

denotes

300 mg (active drug) oral capsules were used in a Phase Ib/IIa clinical trial that was, however, abandoned. (https://clinicaltrials.gov/ct2/show/NCT01039597).

T491

100962-101111

Sentence

denotes

(d) Finally, MLN-4760 was also administered (2.5 mg/kg per day) by nasal inhalation for 2–3 days in lung-infected mice by Pseudomonas bacteria [185].

T492

101112-101253

Sentence

denotes

Interestingly, the report underscores the role played by local concentration of molecules (ACE2) in modulating lung inflammation and disease.

T493

101254-101487

Sentence

denotes

For these reasons, in diseases involving respiratory tract, like SARS, inhalation treatment is preferable, even for the lower concentration (and hopefully lower toxicity) of MLN-4760 needed for this route of treatment administration.

T494

101488-101603

Sentence

denotes

Different routes of MLN-4760 treatment administration can be pursued depending on the hospital condition/expertise.

T495

101604-101766

Sentence

denotes

In this exceptionally critical situation, it could be delivered to critical untreatable patients as a controlled “compassionate use”, in particular by inhalation.

T496

101767-101966

Sentence

denotes

However, when, under hypoxic conditions, both arms of the RAS are upregulated, it might have a limited action and, by shifting the balance of ACE/ACE2 ratio in favour of ACE, might be even dangerous.

T497

101967-102178

Sentence

denotes

Instead, specific inhibition of ACE2 enzymatic activity might effectively work in preventing the establishment of positive feedback loops in COVID-19 patients who are suffering from mild symptoms of the disease.

T498

102179-102342

Sentence

denotes

MLN-4760 is sold by different companies, that, in case it works, could be encouraged to manufacture the molecule, actively contributing to face this global threat.

T499

102343-102490

Sentence

denotes

On the other hand, the drug could be also synthesized in University chemistry labs because, to my knowledge, is no longer under patent restriction.

T500

102491-102922

Sentence

denotes

MLN-4760, whose clinical development was abandoned after phase I trials (clinical name, ORE1001), is actually an interesting compound that could be useful for all patients in which a specific ACE2 upregulation is proven and associated to different (heart/lung/liver/intestine/kidney/immune system/blood/coagulation, etc.) pathological conditions related to ACE2 pathway downstream events, as it seems to occur in COVID-19 patients.

T501

102923-103319

Sentence

denotes

Although in blood of normal subjects sACE2 activity is undetectable because its catalytic activity in human plasma is masked by an endogenous inhibitor [116] and chronic ACE2 activation may be deleterious, some patients might need of sACE2 activity to better face an acute pathological condition, therefore a careful monitoring of clinical parameters should be performed during patient treatment.

T502

103321-103325

Sentence

denotes

8.2.

T503

103326-103355

Sentence

denotes

Inhibition of Renin Activity:

T504

103356-103365

Sentence

denotes

Aliskiren

T505

103366-103473

Sentence

denotes

An alternative approach is to inhibit the RAS pathway at its origin by inhibiting renin enzymatic activity.

T506

103474-103687

Sentence

denotes

Regarding renin inhibitors, aliskiren is the sole compound in this class of drugs that was approved by the US Food and Drug Administration in March 2007 and commercialized for the management of hypertension [186].

T507

103688-103824

Sentence

denotes

Aliskiren may be used alone or in combination with other drugs to face COVID-19, in particular the severe forms of SARS-CoV-2 infection.

T508

103825-103930

Sentence

denotes

Indeed, in patients with severe symptoms of COVID-19 hypoxia might have upregulated both arms of the RAS.

T509

103931-104093

Sentence

denotes

Therefore, aliskiren might be a useful “tool” to reduce production of Ang I, the necessary fuel for both ACE and ACE2 hyperactivity and their detrimental effects.

T510

104094-104310

Sentence

denotes

Aliskiren treatment in rats has been shown to upregulate both AT1R and MasR expression probably as consequence of a compensation mechanism when both Ang II and Ang (1–7) ligands are reduced by renin inhibition [187].

T511

104311-104531

Sentence

denotes

Of particular interest in the context of SARS-CoV-2 infection, the treatment has been shown to reduce expression of both AT2R and ACE2, thus possibly performing a multiple action in inhibiting both arms of the RAS [187].

T512

104532-104648

Sentence

denotes

Similarly, cation chelating agents such as CaNa2EDTA or nicotianamine might also work, although at different levels.

T513

104650-104654

Sentence

denotes

8.3.

T514

104655-104672

Sentence

denotes

Chelating Agents:

T515

104673-104682

Sentence

denotes

CaNa2EDTA

T516

104683-104961

Sentence

denotes

As already mentioned, chelating agents, by limiting zinc cellular availability, might influence hyperactivity, synthesis and conformation of both ACE2 and ACE, which may impair not only ACE2 (and ACE) enzymatic activity but also its availability on cell surface for viral entry.

T517

104962-105144

Sentence

denotes

For these reasons, chelating agents might be effective to counter SARS-CoV-2 infection, and in particular when (e.g., induced by hypoxia) both arms of the RAS are likely upregulated.

T518

105145-105336

Sentence

denotes

Differently from renin inhibitors, more chelating agents are commercially available and both classes of drugs might be administered, alone or in combination with other therapies for COVID-19.

T519

105337-105627

Sentence

denotes

Of note, these classes of drugs might be beneficial not only for SARS-CoV-2 infection but also for other disease in which both arms of the RAS (and of the inflammatory system) are upregulated, as for example ARDS, PAH and other pathologies associated to chronic hypoxia and cardiac failure.

T520

105628-105924

Sentence

denotes

Indeed, both Ang-II and Ang-(1–7) upregulation and a cytokine storm are common aspects in patients not only with ARDS and PAH but also with critical coronary artery disease [182], suggesting an upregulation of both arms of the RAS and of the inflammatory system in several different dysfunctions.

T521

105925-106028

Sentence

denotes

Notably, a number of clinical trials have indicated the utility of CaNa2EDTA in coronary heart disease.

T522

106029-106148

Sentence

denotes

It was believed that chelation therapy might alter plaque morphology and volume, or improve endothelial function [123].

T523

106149-106196

Sentence

denotes

However, the evidence was not convincing [123].

T524

106197-106302

Sentence

denotes

Future clinical trials on selected patients with both ACE and ACE2 upregulation might be more convincing.

T525

106303-106432

Sentence

denotes

It is clear that similar pathways of activation can lead to different outcomes depending on individual predisposition/background.

T526

106433-106651

Sentence

denotes

Therefore, future efforts to understand the genetic, morpho-physiological, environmental and lifestyle factors that individually affect the predisposition to a specific disease with a precision medicine must be pursed.

T527

106652-106823

Sentence

denotes

To avoid disrupting extracellular calcium levels, CaNa2EDTA formulation is typically employed as an antidote for lead poisoning or as an extracellular zinc chelator [123].

T528

106824-106965

Sentence

denotes

CaNa2EDTA is poorly absorbed in the gastrointestinal tract therefore can be preferentially administered by parenteral route or by inhalation.

T529

106966-107207

Sentence

denotes

Nevertheless, oral tablets (e.g., 250–500 mg/tablet) or suppositories of CaNa2EDTA might also work in reducing plasma zinc levels by inhibiting intestinal zinc absorption (the only physiological way of zinc uptake by the organism) [188,189].

T530

107208-107340

Sentence

denotes

In this regard, eight men showing elevated lead concentrations were given a seven-day course of CaNa2EDTA, 4 g/day in divided doses.

T531

107341-107476

Sentence

denotes

Oral CaNa2EDTA caused a rise in lead excretion within few hours, suggesting that orally administered drug was partially absorbed [188].

T532

107477-107621

Sentence

denotes

Importantly, the trial in the eight lead workers demonstrated that oral administration of CaNa2EDTA was safe and produced no side-effects [188].

T533

107622-107892

Sentence

denotes

However, the FDA-approved method for delivery of CaNa2EDTA in treatment of lead poisoning is intravenously or intramuscularly and the recommended dose for asymptomatic adults and pediatric patients with elevated blood lead levels is 1000 g/m2/day (~0,05 g/kg/day) [190].

T534

107893-108019

Sentence

denotes

CaNa2EDTA was usually administered for 5–10 days followed by an interval of at least 5–10 days before a second administration.

T535

108020-108078

Sentence

denotes

Unfortunately, intravenous administration is very painful.

T536

108079-108362

Sentence

denotes

Administration of CaNa2EDTA via nebulizer has also been pursued for inhalation therapy in pulmonary metal poisonings to protect lung tissue and reduce its systemic uptaken [123]; moreover, nebulized solutions containing EDTA have been proven safe, not inducing adverse effects [191].

T537

108363-108588

Sentence

denotes

For the sake of completeness, the unconventional methods for administering chelating agents such as rectal or inhalatory administration are not FDA approved methods for delivery and they have not been studied in detail [192].

T538

108589-108739

Sentence

denotes

CaNa2EDTA diffuses mainly in the extracellular fluids, where it is not significantly metabolized, and it is excreted rapidly by glomerular filtration.

T539

108740-108838

Sentence

denotes

The chelator has a half-life of 1.4 to 3 h in adults and is completely excreted within 24 h [123].

T540

108839-108920

Sentence

denotes

For all of the above reasons, CaNa2EDTA might be employed in therapy of COVID-19.

T541

108921-109175

Sentence

denotes

However, before beginning any therapy with medication, particularly in the case of new diseases (such as COVID-19) or new treatments, a careful patient selection for these new treatments is essential to prevent unnecessary toxicity (“first do not harm”).

T542

109177-109181

Sentence

denotes

8.4.

T543

109182-109210

Sentence

denotes

Safety and Efficacy Concerns

T544

109211-109595

Sentence

denotes

Unfortunately, diseases of different aetiologies may present similar final dysfunction/alterations (e.g., cardiac dysfunction/lung alterations/thrombosis are induced by excessive activation of either AT1R or MasR/AT2R pathways, see Figure 1), while diseases with same aetiologies may present different final dysfunction/alterations depending on patients (COVID-19 is a clear example).

T545

109596-109808

Sentence

denotes

This confounding aspect of diseases can lead to erroneous interpretations, and only defining the correct and early molecular origin of diseases is possible to reach a successful outcome if a therapy is available.

T546

109809-110001

Sentence

denotes

In order to decide who to treat, and when to start treatment, it is important to define the entity of dysfunctions, probability of treatment success and probability to develop adverse effects.

T547

110002-110115

Sentence

denotes

To promptly intervene in order to prevent severe forms of COVID-19, we need early markers of disease progression.

T548

110116-110512

Sentence

denotes

To this regard, if plasma (free) Zn2+ (or total zinc/albumin molar ratio), albumin, IL-10, Ang (1–9), Ang (1–7), Ang (1–5) and/or bradykinin (1–7) will be proven to be reliable surrogate markers for ACE and/or ACE2 enzymatic activity in vivo, they could be exploited, together with circulating ACE and ACE2 activity evaluation, in order to decide who and when to start treating COVID-19 patients.

T549

110513-110611

Sentence

denotes

Alternatively, eosinopaenia and hypotension may be also exploited as signs of disease progression.

T550

110612-111038

Sentence

denotes

In any case, since the conditions of some patients can be critical, it is highly recommended to administer the minimal effective dose in order to reduce possible side effects, starting with low and increasing doses, while at the same time monitoring patient status and early signs of disease progression (e.g., blood pressure, eosinophil counts, molecular concentrations of the RAS peptides and other markers of inflammation).

T551

111039-111439

Sentence

denotes

The aim of the project was not necessarily to stop viral entry (the epithelial cells take already care of it shedding ACE2) but to block positive feedback loops that follow the cellular response to SARS-CoV-2 infection, which are likely the main cause of the severe symptoms associated with COVID-19, doing so, the disease might hopefully become like a simple flu, a spontaneously eradicable disease.

T552

111440-111570

Sentence

denotes

At the moment, I am alone in proposing these new and non-conventional therapeutic approaches that may or may not work on COVID-19.

T553

111571-111756

Sentence

denotes

In doing so, I have not only described a rationale/strategy to counter COVID-19, but also provided practical information to optimize the RAS inhibitors based on known experimental data.

T554

111757-111946

Sentence

denotes

Before any in vivo drug administration (e.g., ACE2 inhibitor), it has to be checked the biological activity (e.g., ACE2) that is affected by the drug, considering the possible side effects.

T555

111947-112125

Sentence

denotes

However, this is not always the case and, in some cases, patients are treated without a careful evaluation of the biological parameter that is modulated by the administered drug.

T556

112126-112338

Sentence

denotes

In this regard, I have tried to address all aspects of the RAS inhibition in detail because my main concern is that the use of these approaches in a wrong way may lead to a failure or even to deleterious effects.

T557

112339-112582

Sentence

denotes

That is why I have meticulously described all the steps before possible administration and provided the guideline suggestions and specific recommendations in order to increase the probability of success and to minimize the deleterious effects.

T558

112583-112683

Sentence

denotes

The readers are, however, expected and encouraged to debate and make their own optimized guidelines.

T559

112685-112700

Sentence

denotes

Acknowledgments

T560

112701-113042

Sentence

denotes

I have to thank Josè Enrique O’Connor, Kishore Wary, Claudio Sorio, Nadir Mario Maraldi, Patrizia Pignatti, John Byrnes, Reza Abdi, Genny del Zotto, Gregary Marhefka, Claudio Ortolani, Renato Zambello, Claudio Pioli, Rita Monticelli, Alessandro Minelli, Piero Sestili, Moreno Zamai and Giuliana Benvenuti for their support and encouragement.

T561

113043-113110

Sentence

denotes

Time was precious, as the virus works 24 h a day and 7 days a week.

T562

113111-113293

Sentence

denotes

I really hoped that the scientific efforts could become decisive in order to face this dramatic situation and to save people as early and as much as possible but it was not possible.

T563

113294-113399

Sentence

denotes

With my work, I hope to reach the future generations and tell them that we cannot play with human health.

T564

113400-113509

Sentence

denotes

I would also like to apologize to those whose relevant contributions could not be cited because of my limits.

T565

113511-113531

Sentence

denotes

Author Contributions

T566

113532-113574

Sentence

denotes

L.Z. is sole author and sole investigator.

T567

113575-113624

Sentence

denotes

The author conceived of the article and wrote it.

T568

113625-113669

Sentence

denotes

L.Z. read and approved the final manuscript.

T569

113671-113678

Sentence

denotes

Funding

T570

113679-113722

Sentence

denotes

This research received no external funding.

T571

113724-113745

Sentence

denotes

Conflicts of Interest

T572

113746-113800

Sentence

denotes

The author declares that he has no competing interest.

T573

113802-113984

Sentence

denotes

Figure 1 Interplay of regulation between the two arms of the renin-angiotensin system and pathophysiological consequences of excessive systemic activation of different RAS pathways.

T574

113985-114149

Sentence

denotes

Reciprocal (ACE/ACE2) pathway inhibition, RAS inhibitor sites of action and influence of hypoxia/SARS-CoV-2 on the RAS are indicated. (for reference, see the text).

T575

114150-114347

Sentence

denotes

Figure 2 SARS-CoV-2 induced positive feedback loops mediated by RAS activation and pathophysiological consequences of the systemic excess of circulating ACE2 enzyme. (for reference, see the text).

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