6. Correlation of Pre-existing Circulating ACE2 Activity and Increased Potential to Develop Severe Forms of COVID-19
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]. 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]. 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]. The report shows that male and advanced age were identified as independent predictors of enhanced sACE2 activity [96]. Furthermore, subjects with hypertension, diabetes, dyslipidemia, or plaques also had significantly increased circulating ACE2 activity when compared with those without these pathologies [96]. 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]. 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]. 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]. 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. Although more evidence is needed in humans, the above observations suggest that ARBs should be precautionarily avoided to reduce possible ACE2-mediated viral consequences. 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]. 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]. 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. Of note, ACEI and ARB antihypertensive medications are more commonly used in Europe/USA than in China. 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]. 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]. 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]. 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. 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). 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. 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.
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]. 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]. 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]. 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. 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]. 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. 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). 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. 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. 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]. 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. 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]. 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. 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. 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]. 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]. 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]. 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. 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]. 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. 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. 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. 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. Seasonal differences of the RAS activities leading, for example, to differences in blood pressure are well known. Of interest, the majority of coronaviruses cause cold-like illnesses and have a relatively low mutational frequency [168]. Similarly SARS-CoV-2 seems to mutate much slower than seasonal flu [169]. 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. For example, they might favour a relatively short-term activation/memory of immune response. 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]. 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. 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.