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Id Subject Object Predicate Lexical cue
T1 0-77 Sentence denotes Injury to the Endothelial Glycocalyx in Critically Ill Patients with COVID-19
T2 79-93 Sentence denotes To the Editor:
T3 94-377 Sentence denotes Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the so-called coronavirus disease (COVID-19), which is characterized by a broad spectrum of clinical presentations ranging from asymptomatic patients to critically ill individuals with a high case fatality rate (1).
T4 378-521 Sentence denotes The critical care community has increasingly recognized that cardiovascular and thrombotic complications are relatively common in COVID-19 (2).
T5 522-658 Sentence denotes Indeed, direct involvement of the vascular endothelium was recently reported in a series of patients suffering from severe COVID-19 (3).
T6 659-927 Sentence denotes The endothelial glycocalyx (eGC), which covers the luminal surface of endothelial cells, contributes to the maintenance of vascular homeostasis, whereas disruption of the eGC is observed early in critically ill patients and is associated with inferior outcomes (4, 5).
T7 928-1107 Sentence denotes Here, we investigated in translational human and cellular studies whether injury to the eGC can be found in critically ill patients with COVID-19 early after admission to the ICU.
T8 1108-1353 Sentence denotes We collected plasma and serum from 19 adult individuals within 24 hours after invasive ventilation for acute respiratory distress syndrome and from 10 healthy human donors after written informed consent of patients or their legal representative.
T9 1354-1454 Sentence denotes The first patient was admitted on March 19, 2020, and the observation period was until May 17, 2020.
T10 1455-1529 Sentence denotes The median (interquartile range) observation duration was 47 (40–54) days.
T11 1530-1664 Sentence denotes Baseline patient characteristics at study inclusion as well as a description of the further clinical course are summarized in Table 1.
T12 1665-1807 Sentence denotes Organ failure was not restricted to the lungs, and multiorgan dysfunction was common both at inclusion and during the further clinical course.
T13 1808-1901 Sentence denotes Surprisingly, global markers of endothelial injury such as Angpt-1 (angiopoietin-1) (control:
T14 1902-1936 Sentence denotes 29 [26.2–30.9] ng/ml vs. COVID-19:
T15 1937-1992 Sentence denotes 27.8 [23.4–36.2] ng/ml; P = 0.79) and Angpt-2 (control:
T16 1993-2032 Sentence denotes 0.655 [0.336–1.113] ng/ml vs. COVID-19:
T17 2033-2110 Sentence denotes 0.434 [0.035–1.338] ng/ml; P = 0.6) were unchanged in patients with COVID-19.
T18 2111-2353 Sentence denotes In contrast, marked increases in the soluble form of the sTie2 (Tie2 receptor) (Figure 1A) and in syndecan-1 (Figure 1B)—indicating pathological shedding of transmembrane proteins involved in glycocalyx structure and processing—were observed.
T19 2354-2478 Sentence denotes The key eGC sheddase Hpa-1 (heparanase-1) and its enzymatic activity were both not significantly increased (data not shown).
T20 2479-2622 Sentence denotes To the contrary, the Hpa-1 counterpart, the protective Hpa-2 (heparanase-2), was pertinently reduced in all patients with COVID-19 (Figure 1C).
T21 2623-2752 Sentence denotes Driven by this acquired Hpa-2 deficiency, the Hpa-1:Hpa-2 ratio was higher in patients with COVID-19 (P = 0.012; data not shown).
T22 2753-2975 Sentence denotes Together, this indicates that critically ill patients with COVID-19 suffer from an acquired Hpa-2 deficiency that can contribute to the degradation of the eGC, maybe even before classical endothelial activation and injury.
T23 2976-3076 Sentence denotes Next, eGC structure was analyzed in humans, employing sublingual sidestream darkfield (SDF) imaging.
T24 3077-3305 Sentence denotes We quantified the size of the individual patients’ eGC using an indirect surrogate termed the perfused boundary region and found a decrease of perfused boundary region, indicating reduced eGC thickness in patients with COVID-19.
T25 3306-3556 Sentence denotes To demonstrate that the deficiency of Hpa-2 is mechanistically involved in the degradation of the eGC, we used a microfluidic chamber with cultured endothelial cells (ECs) under flow that synthesize an intact and stable eGC under in vitro conditions.
T26 3557-3711 Sentence denotes After stimulation with COVID-19 or control serum, the eGC was visualized by confocal microscopy followed by computerized three-dimensional reconstruction.
T27 3712-3798 Sentence denotes Its thickness was then quantified by analyzing the heparan sulfate (HS)-positive area.
T28 3799-3893 Sentence denotes We found that stimulation with COVID-19 was sufficient to severely damage the eGC (Figure 1E).
T29 3894-3943 Sentence denotes The HS-positive area was reduced by 34% (control:
T30 3944-3968 Sentence denotes 6.1 ± 0.9% vs. COVID-19:
T31 3969-3989 Sentence denotes 4 ± 0.4% P < 0.001).
T32 3990-4199 Sentence denotes Consistent with our observation in patients, we found that the transcription of Hpa-2 in COVID-19–stimulated ECs was significantly reduced after 6 hours (0.63 ± 0.02 relative expression to control; P = 0.003).
T33 4200-4464 Sentence denotes Of note, transgenic overexpression of Hpa-2 in a lentivirus-transduced EC line was sufficient to reverse this phenotype, as the HS area in COVID-19 serum–treated lenti-control cells was 1.9 ± 0.6% but was 4.2 ± 1.2% in lenti-Hpa-2–overexpressing cells (P < 0.001).
T34 4465-4574 Sentence denotes In other words, if ECs overexpress Hpa-2, the serum of patients with COVID-19 cannot degrade the eGC anymore.
T35 4575-4583 Sentence denotes Table 1.
T36 4585-4655 Sentence denotes Patient Characteristics at Study Inclusion and Further Clinical Course
T37 4656-4679 Sentence denotes Characteristic Results
T38 4680-4702 Sentence denotes Number of patients 19
T39 4703-4752 Sentence denotes Age, yr, median (interquartile range) 57 (45–69)
T40 4753-4766 Sentence denotes Sex, n (%)  
T41 4767-4778 Sentence denotes  M 18 (95)
T42 4779-4788 Sentence denotes  F 1 (5)
T43 4789-4847 Sentence denotes BMI, kg/m2, median (interquartile range) 27.8 (26.2–33.5)
T44 4848-4871 Sentence denotes Comorbidities, n (%)  
T45 4872-4886 Sentence denotes  Asthma 1 (5)
T46 4887-4900 Sentence denotes  HTN 10 (53)
T47 4901-4913 Sentence denotes  CHF 2 (11)
T48 4914-4925 Sentence denotes  CAD 1 (5)
T49 4926-4943 Sentence denotes  Diabetes 5 (26)
T50 4944-4955 Sentence denotes  CKD 1 (5)
T51 4956-4972 Sentence denotes  Obesity 7 (37)
T52 4973-4992 Sentence denotes  Malignancy 2 (11)
T53 4993-5019 Sentence denotes  Immunosuppression 2 (11)
T54 5020-5057 Sentence denotes Invasive ventilation, n (%) 19 (100)
T55 5058-5135 Sentence denotes Time from hospital to ICU admission, d, median (interquartile range) 0 (0–2)
T56 5136-5225 Sentence denotes Time from ICU admission to invasive ventilation, d, median (interquartile range) 0 (0–0)
T57 5226-5280 Sentence denotes Respirator parameters, median (interquartile range)  
T58 5281-5301 Sentence denotes  FiO2, % 50 (30–60)
T59 5302-5325 Sentence denotes  PEEP, mbar 12 (12–15)
T60 5326-5350 Sentence denotes  Pplat, mbar 27 (23–30)
T61 5351-5432 Sentence denotes Oxygenation index (PaO2/FiO2), mm Hg, median (interquartile range) 173 (152–260)
T62 5433-5487 Sentence denotes PaCO2, mm Hg, median (interquartile range) 46 (40–51)
T63 5488-5538 Sentence denotes pH, median (interquartile range) 7.36 (7.32–7.41)
T64 5539-5599 Sentence denotes Lactate, mmol/L, median (interquartile range) 1.6 (1.5–2.9)
T65 5600-5629 Sentence denotes Vasopressor, n (%) 16 (84.2)
T66 5630-5711 Sentence denotes Norepinephrine dose, μg/kg/min, median (interquartile range) 0.083 (0.019–0.182)
T67 5712-5752 Sentence denotes Renal replacement therapy, n (%) 3 (16)
T68 5753-5783 Sentence denotes vv-ECMO support, n (%) 2 (11)
T69 5784-5843 Sentence denotes SOFA score, points, median (interquartile range) 11 (9–14)
T70 5844-5877 Sentence denotes Organ-specific failures, n (%)  
T71 5878-5918 Sentence denotes  Respiratory (PaO2/FiO2 < 300) 19 (100)
T72 5919-5959 Sentence denotes  Coagulation (thrombocytes < 100) 1 (5)
T73 5960-5997 Sentence denotes  Liver (bilirubin > 33 μmol/L) 1 (5)
T74 5998-6053 Sentence denotes  Cardiovascular (dobutamine or norepinephrine) 16 (84)
T75 6054-6088 Sentence denotes  Neurological (GCS < 13) 19 (100)
T76 6089-6129 Sentence denotes  Renal (creatinine > 170 μmol/L) 4 (21)
T77 6130-6166 Sentence denotes Lab, median (interquartile range)  
T78 6167-6205 Sentence denotes  CRP (normal: <5), mg/L 174 (121–203)
T79 6206-6246 Sentence denotes  PCT normal: <0.5), μg/L 3.8 (0.5–10.9)
T80 6247-6286 Sentence denotes  IL-6 (normal: <7), ng/L 272 (142–541)
T81 6287-6326 Sentence denotes  LDH (normal: <248), U/L 548 (384–657)
T82 6327-6374 Sentence denotes  D-Dimer (normal: <0.5), mg/L 3.56 (0.84–8.88)
T83 6375-6418 Sentence denotes  Troponin T (normal: <14), ng/L 17 (11–23)
T84 6419-6464 Sentence denotes  NT-proBNP (normal: <86), ng/L 260 (82–1108)
T85 6465-6498 Sentence denotes Further clinical course, n (%)  
T86 6499-6524 Sentence denotes  Prone position 19 (100)
T87 6525-6544 Sentence denotes  Inhaled NO 4 (21)
T88 6545-6561 Sentence denotes  vv-ECMO 7 (37)
T89 6562-6596 Sentence denotes  Renal replacement therapy 8 (42)
T90 6597-6619 Sentence denotes  Septic shock 10 (53)
T91 6620-6662 Sentence denotes  Died by end of observation period 3 (16)
T92 6663-6704 Sentence denotes  Still dependent on critical care 6 (32)
T93 6705-6755 Sentence denotes  Still dependent on mechanical ventilation 2 (11)
T94 6756-7259 Sentence denotes Definition of abbreviations: BMI = body mass index; CAD = coronary artery disease; CHF = congestive heart failure; CKD = chronic kidney disease; CRP = C-reactive protein; ECMO = extracorporeal membrane oxygenation; GCS = Glasgow Coma Scale; HTN = hypertension; LDH = lactate dehydrogenase; NT-proBNP = N-terminal pro–B-type natriuretic peptide; PCT = procalcitonin; PEEP = positive end-expiratory pressure; Pplat = plateau pressure; SOFA = Sequential Organ Failure Assessment; vv-ECMO = venovenous ECMO.
T95 7260-7429 Sentence denotes Demographic and clinical characteristics are given at the time of study inclusion as well as a description of the further clinical outcome within the observation period.
T96 7430-7530 Sentence denotes Values are presented as median (interquartile range) or, if categorical, as numbers and percentages.
T97 7531-7784 Sentence denotes Demographic characteristics for the control patients were as follows: age 32 (28–33) years and 9/10 male sex for laboratory investigation control patients; 39 (36–44) years and 5/10 male sex for sidestream darkfield imaging measurement control patients.
T98 7785-7862 Sentence denotes All individuals of the control cohorts had no relevant medical preconditions.
T99 7863-7872 Sentence denotes Figure 1.
T100 7874-7952 Sentence denotes Injury to the endothelial glycocalyx in severe coronavirus disease (COVID-19).
T101 7953-8091 Sentence denotes Scatter dot plots (median [interquartile range]) show (A) sTie-2 and (B) syndecan-1 concentrations for control and patients with COVID-19.
T102 8092-8136 Sentence denotes Concentrations for both syndecan-1 (control:
T103 8137-8174 Sentence denotes 41.5 [32.6–105.4] ng/ml vs. COVID-19:
T104 8175-8222 Sentence denotes 336.5 [196.7–377.1] ng/ml) and sTie-2 (control:
T105 8223-8256 Sentence denotes 18.4 [16–21.3] ng/ml vs COVID-19:
T106 8257-8543 Sentence denotes 22.3 [19.7–27.3] ng/ml) were increased in patients with COVID-19. (C) Although both Hpa-1 (heparanase-1) concentration and activity were not significantly altered in comparison with control subjects, patients with COVID-19 showed an acquired deficiency in Hpa-2 (heparanase-2) (control:
T107 8544-8579 Sentence denotes 18.7 [10.6–31.1] U/ml vs. COVID-19:
T108 8580-8600 Sentence denotes 4.7 [2.6–5.1] U/ml).
T109 8601-8686 Sentence denotes Consequentially, the Hpa-1:Hpa-2 ratio was higher in patients with COVID-19 (control:
T110 8687-8722 Sentence denotes 0.08 [0.05–0.17] ng/U vs. COVID-19:
T111 8723-8960 Sentence denotes 0.35 [0.27–0.66] ng/U). (D) Sidestream darkfield imaging in patients allows the quantification of endothelial glycocalyx thickness as indicated by increased perfused boundary region; this was increased in patients with COVID-19 (control:
T112 8961-8991 Sentence denotes 1.9 [1.8–1.9] μm vs. COVID-19:
T113 8992-9063 Sentence denotes 2.1 [1.8–2.2] μm), indicating reduced endothelial glycocalyx thickness.
T114 9064-9743 Sentence denotes For comparison of groups, first, the normal distribution (D’Agostino-Pearson omnibus and Shapiro-Wilk normality test) of variables was tested, and then (A and D) two-sided unpaired t tests and (B and C) Mann-Whitney tests were used accordingly. (E) Exemplary three-dimensional reconstruction of the heparan sulfate (HS) layer images of naive endothelial cells in a microfluidic chip (HS in red; DAPI nuclei staining in blue) after perfusion with serum of a patient with COVID-19 (middle row) compared with a healthy control subject (upper row) in both front and isometric view angles demonstrates the diffuse loss of HS-rich glycocalyx layer in cells treated with COVID-19 serum.
T115 9744-9971 Sentence denotes In Hpa-2–overexpressing endothelial cells (lower row), the HS surface layer is protected from injurious effects by perfusion with COVID-19 serum. overexp = overexpressing; PBR = perfused boundary region; sTie-2 = soluble Tie-2.
T116 9972-10093 Sentence denotes This exploratory study has obvious limitations, most importantly, its small sample size and hypothesis-generating nature.
T117 10094-10215 Sentence denotes Injury to the eGC is not a finding specific for COVID-19 but can be found in a wide range of critically ill patients (5).
T118 10216-10463 Sentence denotes Because only a small selection of molecules that may participate in endothelial injury have been investigated in this study, we cannot exclude that further mediators may play a critical role in endothelial and eGC injury in patients with COVID-19.
T119 10464-10699 Sentence denotes In addition, because of concerns of viral transmission, SDF imaging values could not be obtained from the same control patients from whom blood analysis was performed but were obtained from a separate historic in-center control cohort.
T120 10700-10876 Sentence denotes Both control cohorts were not matched to the individual patients in terms of age, but the control group that blood was collected from was matched in terms of male predominance.
T121 10877-11046 Sentence denotes In summary, we found injury of the eGC and speculate that this might represent a potentially critical hallmark of later widespread endothelial injury in severe COVID-19.
T122 11047-11140 Sentence denotes Reduced eGC thickness was visualized in vivo by employing sublingual SDF imaging in patients.
T123 11141-11392 Sentence denotes At the same time, increased syndecan-1 and sTie-2 concentrations in the blood of these patients indicated shedding of important endothelial transmembrane proteins responsible for building (5) and maintaining (6) the structure of the eGC, respectively.
T124 11393-11530 Sentence denotes Interestingly, eGC shedding could be reproduced when patient blood was transferred ex vivo into an endothelial microcapillary chip model.
T125 11531-11763 Sentence denotes Although Hpa-1 and its enzymatic activity (primarily responsible for HS degradation) (7) was found to be normal, Hpa-2, a protein that has been described as a protective antagonist of Hpa-1 (8, 9), was severely depleted in COVID-19.
T126 11764-11884 Sentence denotes Importantly, degradation of the eGC after perfusion with COVID-19 serum could be attenuated in ECs overexpressing Hpa-2.
T127 11885-12076 Sentence denotes We therefore postulate that acquired Hpa-2 deficiency might represent a potential mechanism of injury to the eGC, which could later progress to widespread endothelial dysfunction in COVID-19.
T128 12077-12274 Sentence denotes In conclusion, our data suggest that in critically ill patients with COVID-19, endothelial injury involves glycocalyx integrity, and acquired Hpa-2 deficiency might be a potential causative factor.
T129 12276-12290 Sentence denotes Acknowledgment
T130 12291-12479 Sentence denotes The authors thank Yvonne Nicolai for excellent technical support and Dr. Markus Busch and Dr. Olaf Wiesner and the nursing staff for their infrastructural support in conducting this study.
T131 12480-12586 Sentence denotes S.D. is supported by the German Research Foundation (DA 1209/4-3) and the German Center for Lung Research.
T132 12587-12687 Sentence denotes Authors Contributions: K.S., P.A.G., B.S., T.W., H.H., M.M.H., and S.D. obtained retrospective data.
T133 12688-12769 Sentence denotes K.S., P.A.G., A.B., and S.D. performed sidestream darkfield imaging measurements.
T134 12770-12866 Sentence denotes P.A.G. and T.P. performed ELISA measurements, and Y.K. performed and analyzed flow measurements.
T135 12867-12975 Sentence denotes K.S., P.A.G., H.H., T.W., M.M.H., and S.D. analyzed and discussed the data and generated figures and tables.
T136 12976-13058 Sentence denotes K.S., P.A.G., and S.D. wrote the manuscript; all authors proofread the manuscript.
T137 13059-13096 Sentence denotes Originally Published in Press as DOI:
T138 13097-13142 Sentence denotes 10.1164/rccm.202007-2676LE on August 24, 2020
T139 13143-13228 Sentence denotes Author disclosures are available with the text of this letter at www.atsjournals.org.