Role of endothelial dysfunction in the thrombotic complications of COVID-19 patients
Authors: Emanuela Falcinelli, Eleonora Petito, Cecilia Becattini, Edoardo De Robertis, Ugo Paliani, Manuela Sebastiano, Gaetano Vaudo, Giuseppe Guglielmini, Francesco Paciullo, Vittorio Cerotto, Marco Malvestiti, Fabio Gori, Loredana Bury, Teseo Lazzarini, Paolo Gresele
Journal: Journal of Infection (2020)

We note with interest the review of Kunutsor SK. et al.1Kunutsor S.K. Laukkanen J.A. Cardiovascular complications in COVID-19: a systematic review and meta-analysis.J Infect. 2020; 81 (Epub 2020 Jun 3PMID:32504747): e139-e141https://doi.org/10.1016/j.jinf.2020.05.068Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar on cardiovascular implications of coronavirus disease 2019 (COVID-19). Indeed, Sars-Cov-2 infection begins in lungs but moves rapidly to the vascular system with platelet alterations and blood clotting abnormalities, and associates with a high incidence of cardiovascular events and venous thromboembolism (VTE), especially in critically ill patients (10–34%).2Nopp S. Moik F. Jilma B. Pabinger I. Ay C. Risk of venous thromboembolism in patients with COVID-19: a systematic review and meta-analysis.Res Pract Thromb Haemost. 2020; 4: 1178-1191Crossref PubMed Scopus (312) Google Scholar Based on autopsy findings, endothelial injury has been hypothesized to play a crucial role in the Sars-Cov-2 associated pro-coagulant condition.3López Castro J. COVID-19 and thrombosis: beyond a casual association.Med Clin (Engl Ed). 2020; 155: 44PubMed Google Scholar Very few studies, however, have assessed circulating biomarkers of endothelial damage in COVID-19 patients. Among these particularly interesting are circulating endothelial cells (CECs), circulating endothelial progenitor cells (EPCs), endothelial extracellular vesicles (EEVs) and soluble forms of endothelial adhesive proteins (CAM) which are known to be altered in conditions associated with enhanced cardiovascular risk and to be predictive of vascular complications in various conditions, including infectious diseases.4Page A.V. Liles W.C. Biomarkers of endothelial activation/dysfunction in infectious diseases.Virulence. 2013; 4: 507-516https://doi.org/10.4161/viru.24530Crossref PubMed Scopus (210) Google Scholar For these parameters no data are available, to our knowledge, in Sars-Cov-2 infection. Aim of our study was to assess the role of cellular and soluble circulating endothelial derangement parameters as markers of endothelial damage in COVID-19 patients and to unravel if they may identify patients developing VTE or adverse outcome. Fifty-six COVID-19 patients and 36 healthy, age- and sex-matched controls were enrolled in a multicenter study in the Umbria Region, Italy. Peripheral blood was collected for EEVs, CECs and EPCs by flow cytometry and for sVCAM and sICAM by ELISA.5Gresele P. Migliacci R. Procacci A. De Monte P. Bonizzoni E. Prevention by NCX 4016, a nitric oxide-donating aspirin, but not by aspirin, of the acute endothelial dysfunction induced by exercise in patients with intermittent claudication.Thromb Haemost. 2007; 97: 444-450Crossref PubMed Scopus (44) Google Scholar Four of the enrolled patients were reassessed after disease recovery confirmed by 2 negative nasopharyngeal swabs (mean 72.7 days, 95% CI 32.8–112.6, after the second). Statistical analyses were performed using GraphPad Prism 8.4 for Windows software. Data not normally distributed were analyzed with the Mann Whitney test; otherwise with the two-tailed unpaired Student's t-test. P<0.05 was considered statistically significant. The study was approved by the local Ethics Committees (CEAS Umbria n. 3656/20, University of Perugia Bioethics Committee n. 2020–36,346). Demographics, clinical and main laboratory features of the study population are summarized in Table 1. All patients were hospitalized (median hospitalization 26 days) and were studied on average 4.2 ± 0.5 days (95%CI 3.2–5.2) from the last positive nasopharyngeal swab. Sixteen patients (28.5%) were admitted into the intensive care unit (ICU) while the others into non-ICU COVID-19 wards. Five ICU and 3 non-ICU patients died during hospitalization. Of the 56 patients, 19 had a partial pressure of oxygen/fraction of inspiration oxygen ratio (PaO2/FiO2) lower than 300 and 31 required mechanical ventilation (15 invasive and 16 non invasive). Ten (17.8%) developed 11 thrombotic events (one suffered two thrombotic events) during or immediately after hospitalization (median 9.5 days) confirmed by computed tomography pulmonary angiography or compression ultrasonography (6 pulmonary embolism, 4 deep vein thrombosis, 1 cava vein thrombosis): of these, six were under prophylactic low molecular weight heparin (LMWH) (n = 3 standard-dose, n = 3 intermediate-dose) and four under therapeutic-dose LMWH (one for atrial fibrillation and one for a previous pulmonary embolism).Table 1Demographic and clinical characteristics of the study population.COVID-19 patients n = 56Healthy subjects n = 36p value Age (years)72.1 ± 1.868.0 ± 3.0ns Sex (% M)57.1%40.1%ns Leukocytes (x 103/μL)7.0 ± 0.75.7 ± 0.8ns Neutrophil-to-lymphocyte ratio (NLR)8.0 ± 0.92.0 ± 0.2<0.005 Platelets (x 103/μL)211.1 ± 17.0208 ± 17.2ns D-dimer (ng/ml)1663 ± 299.0180.6 ± 21.7<0.0001 Fibrinogen (mg/dL)403.1 ± 27.0323.2 ± 26ns VWF: Ag (%)273.8 ± 26.4104.0 ± 7.0<0.0001 VWF: RCo (%)298.0 ± 28.090.0 ± 4.9<0.0001 Procalcitonin (ng/ml)1.2 ± 0.5N.A. CRP (mg/dL)4.8 ± 1.5N.A. LDH (U/L)247.2 ± 33.3N.A. PaO2/FiO2241.3 ± 27.1N.A. SOFA score (total)6.0 ± 0.4N.A. Days from positive swab4.2 ± 0.5N.A. Thrombotic events (n)11N.A.Comorbidities Hypertension (n)323<0.01 Type 2 Diabetes Mellitus (n)111<0.05 Obesity (n)121<0.05 Smoker (n)63ns Atrial Fibrillation (n)70ns Cirrhosis (n)10ns Kidney failure (n)70ns Stroke (n)40ns Peripheral artery disease (n)70ns COPD (n)40nsDrugsAntihypertensive agents (n)111<0.05Statins (n)110<0.05Antiplatelet treatments: Aspirin (n)90<0.05 Anti P2Y12 (n)30nsAnticoagulant treatments: LMWH (n)450<0.0001 -standard32 -incremented8 -therapeutic5Apixaban (n)60nsCOVID-19 TreatmentsHydroxycloroquine (n)5N.A.Darunavir/Cobicistat (n)2N.A.Tolicizumab (n)1N.A.Results are reported as mean±SEM if not differently indicated. N.A. not applicable; SOFA: sequential organ failure assessment. Open table in a new tab Results are reported as mean±SEM if not differently indicated. N.A. not applicable; SOFA: sequential organ failure assessment. COVID-19 patients had significantly higher CECs and EEVs in comparison with healthy subjects (21.5 ± 2.2 vs 8.1 ± 1.4/μl, p<0.01 and 286.5 ± 38 vs 127.6 ± 21/μl, p<0.05 respectively). CECs correlated with C-reactive protein levels (r = 0.49, p<0.05), neutrophil-to-lymphocyte ratio (r = 0.40, p<0.01) and d-Dimer (r = 0.45, p<0.05), biomarkers of inflammation and hypercoagulability, but did not differ between patients who developed a thrombotic event and those who did not. Three distinct populations of circulating EPCs (CD34+ and CD309+) were detected based on their CD45 expression. CD45 negative (CD45neg), which express the regenerative potential of EPCs against vascular damage, were significantly lower in COVID-19 patients compared to controls (Fig. 1A), while a significant increase of CD45 positive intermediate (CD45+int) (Fig. 1B) and CD45 positive high (CD45+high) was observed, suggesting that these EPCs with high phagocytic capability may represent a reactive mechanism to limit viral proliferation.6Wang Q.R. Wang B.H. Zhu W.B. Huang Y.H. Li Y. Yan Q. An in vitro study of differentiation of hematopoietic cells to endothelial cells.Bone Marrow Res. 2011; 2011846096Crossref PubMed Google Scholar COVID-19 patients also had higher plasma levels of soluble markers of EC disturbance, sVCAM-1 (3122 ± 324 vs 1135 ± 82 ng/ml, p<0.001) and sICAM-1 (Fig. 1C) and VWF:Ag and VWF:RCo (Table 1), as compared with controls. Notably, sICAM-1 was significantly more elevated in COVID-19 patients admitted into ICU compared to those not in ICU (Fig. 1D) and in patients with reduced PaO2/FiO2 ratio compared to those with normal PaO2/FiO2 (Fig. 1E), suggesting that severe respiratory syndrome and hypoxemia are associated with endothelial damage. A significant correlation was also found between sICAM-1 and the SOFA score (r = 0.65, p<0.01), suggesting that elevated sICAM-1 may represent a marker of severe disease evolution in Sars-Cov-2 infection. D-dimer, VWF:Ag (not shown) and sICAM-1 (Fig. 1F) were significantly higher in patients who developed VTE than in patients who did not. ROC curve analysis showed that sICAM-1 >519.06 ng/ml discriminates COVID-19 patients with VTE from those without with moderate accuracy (AUC= 0.83, p<0.01) (Suppl. Fig. 1). Most patients were under standard- (n = 32) or incremented-dose (n = 8) prophylactic LMWH (40/56, 71%) but no differences between treated and untreated patients were found for any of the circulating endothelial dysfunction markers assessed. In patients who had recovered from COVID-19, CECs, EMPs, EPCs, VWF:Ag, VWF:RCo, sICAM-1 and sVCAM-1 returned to levels close to those of healthy controls, suggesting that endothelial damage is strictly dependent on active COVID-19 infection (Suppl. Fig. 2). Our results show that COVID-19 patients have increased circulating CECs, EMPs and phagocytic EPCs and increased plasma levels of sICAM-1, sVCAM-1, VWF:Ag and VWF:RCo, with concomitant decrease of angiogenic EPCs, proving that circulating parameters of endothelial derangement are strongly altered in COVID-19 patients. In particular, plasma levels of sICAM-1 and of sVCAM-1 were more than threefold increased probably reflecting the enhanced adhesiveness of microvascular endothelium mediating the strong leukocyte extravasation in tissue, in particular in lungs. Moreover, the endothelial activation triggered by SARS-CoV-2 probably contributes to the strong in vivo platelet activation found in COVID-19 patients and to platelet adhesion to lung endothelium leading to lung injury. Elevated sICAM-1 predicts cardiovascular events in apparently healthy men and in patients with cardiovascular disease and is associated with recurrent VTE,7Dzikowska-Diduch O. Domienik-Karłowicz J. Górska E. Demkow U. Pruszczyk P. Kostrubiec M. E-selectin and sICAM-1, biomarkers of endothelial function, predict recurrence of venous thromboembolism.Thromb Res. 2017; 157: 173-180Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar and in our study strongly associated with VTE incidence and disease severity, therefore this marker warrants more extensive investigation for prognostic prediction in COVID-19 patients. In our study prophylactic-dose LMWH did not affect biomarkers of endothelial dysfunction, in agreement with low clinical efficacy in preventing VTE in COVID-19 patients.8Al-Ani F. Chehade S. Lazo-Langner A. Thrombosis risk associated with COVID-19 infection. A scoping review.Thromb Res. 2020; 192: 152-160Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar A recent study evaluated the impact of therapeutic-dose anticoagulation given to COVID-19 patients prior to hospitalization on endothelial damage, measured by CECs, suggesting that early treatment may prevent COVID-19-associated endothelial lesion.9Khider L. Gendron N. Goudot G. Chocron R. Hauw-Berlemont C. Cheng C. Rivet N. Pere H. Roffe A. Clerc S. Lebeaux D. Debuc B. Veyer D. Rance B. Gaussem P. Bertil S. Badoual C. Juvin P. Planquette B. Messas E. Sanchez O. Hulot J.S. Diehl J.L. Mirault T. Smadja D.M. Curative anticoagulation prevents endothelial lesion in COVID-19 patients.J Thromb Haemost. 2020 Jun 18; (Epub ahead of print. PMID:32558198PMCID: PMC7323356)https://doi.org/10.1111/jth.14968Crossref Scopus (65) Google Scholar Thus sICAM-1 might be used as an indicator to switch to therapeutic dose heparin in high-risk patients. Finally, our data, strongly confirming that COVID-19 is an endothelial disease, provide the rationale for the search of novel therapeutic strategies targeting inflammatory mediators and/or promoting endothelial protection/repair to prevent the thrombotic and systemic complications of COVID-19. The authors declare no conflict of interest. Laura Franco(a), Luca Saccarelli(b), Maria Lapenna(c), Marco D'Abbondanza(d), Stefano Cristallini(f). The contribution of Luisa Golia (Section of Anesthesia, Intensive Care, and Pain Medicine, Azienda Ospedaliera-Universitaria Santa Maria della Misericordia, Perugia, Italy), Valentina Bubba (Division of Internal Medicine, ASL 1 Umbria, Città di Castello, Italy), Pierluigi Piergentili (Section of Anesthesia and Intensive Care, Presidio Alto Chiascio, USL Umbria 1, Gubbio, Italy) with patient enrollment is kindly acknowledged. Stago s.r.l. (Italy) kindly gave some of the reagents used for the study. This work was supported by a fellowship from Fondazione Umberto Veronesi to L. Bury and E. Falcinelli.