key: cord-0935112-m82735kf
authors: Andrianto; Al-Farabi, Makhyan Jibril; Nugraha, Ricardo Adrian; Marsudi, Bagas Adhimurda; Azmi, Yusuf
title: Biomarkers of endothelial dysfunction and outcomes in coronavirus disease 2019 (COVID-19) patients: A systematic review and meta-analysis
date: 2021-07-15
journal: Microvasc Res
DOI: 10.1016/j.mvr.2021.104224
sha: b21b31fd22383c63e39fb2963374add809dc7dbf
doc_id: 935112
cord_uid: m82735kf

BACKGROUND: Several studies have reported that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can directly infect endothelial cells, and endothelial dysfunction is often found in severe cases of coronavirus disease 2019 (COVID-19). To better understand the prognostic values of endothelial dysfunction in COVID-19-associated coagulopathy, we conducted a systematic review and meta-analysis to assess biomarkers of endothelial cells in patients with COVID-19. METHODS: A literature search was conducted on online databases for observational studies evaluating biomarkers of endothelial dysfunction and composite poor outcomes in COVID-19 patients. RESULTS: A total of 1187 patients from 17 studies were included in this analysis. The estimated pooled means for von Willebrand Factor (VWF) antigen levels in COVID-19 patients was higher compared to healthy control (306.42 [95% confidence interval (CI) 291.37–321.48], p < 0.001; I(2):86%), with the highest VWF antigen levels was found in deceased COVID-19 patients (448.57 [95% CI 407.20–489.93], p < 0.001; I(2):0%). Meta-analysis showed that higher plasma levels of VWF antigen, tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1 antigen (PAI-1) antigen, and soluble thrombomodulin (sTM) were associated with composite poor outcome in COVID-19 patients ([standardized mean difference (SMD) 0.74 [0.33–1.16], p < 0.001; I(2):80.4%], [SMD 0.55 [0.19–0.92], p = 0.003; I(2):6.4%], [SMD 0.33 [0.04–0.62], p = 0.025; I(2):7.9%], and [SMD 0.55 [0.10–0.99], p = 0.015; I(2):23.6%], respectively). CONCLUSION: The estimated pooled means show increased levels of VWF antigen in COVID-19 patients. Several biomarkers of endothelial dysfunction, including VFW antigen, t-PA, PAI-1, and sTM, are significantly associated with increased composite poor outcomes in patients with COVID-19. PROSPERO REGISTRATION NUMBER: CRD42021228821.

Although initially recognized as a disease affecting the respiratory system, coronavirus disease 2019 often manifests as cardiovascular complications such as myocarditis, myocardial injuries, arrhythmias, and venous thromboembolism events (VTE) 1 . There are several possible mechanisms for these phenomena, one of which may be an unrestrained and unbalanced innate immune response, which in turn negates effective adaptive immunity, supporting the progression of COVID- 19 . Frequent laboratory abnormalities in patients with unfavorable progression of COVID-19, including abnormal cytokine profiles, led to the initial presumption that the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection involved a cytokine storm 2 .

However, recent evidence suggests that increased inflammatory cytokines including interleukin-6 (IL-6) in patients with severe and critical COVID-19 are significantly lower compared to patients with sepsis and acute respiratory distress syndrome (ARDS) not associated with COVID-19, thus doubting the role of a cytokine storm in COVID-19-related multiple organ damage 3 .

Several studies have reported that the SARS-CoV-2 can directly infect endothelial cells, and endothelial dysfunction is often found in severe cases of COVID-19 4 . Autopsy findings have also demonstrated endothelial dysfunction and microvascular thrombosis together with pulmonary embolism (PE) and deep-vein thrombosis in COVID-19 patients 5 . These findings suggest that endothelial injury, endotheliitis, and microcirculatory dysfunction in different vascular beds contribute significantly to life-threatening complications of COVID-19, such as VTE and multiple organ involvement 6 . To better understand the prognostic values of endothelial dysfunction in COVID-19-associated coagulopathy, we conducted a systematic review and meta-analysis to assess biomarkers of endothelial cells in patients with COVID-19.

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A systematic literature search of PubMed, PMC Europe, and the Cochrane Library Database from 1 January 2020 to 20 December 2020 was performed using the search strategy outlined in Supplementary Table S1 . Additional records were retrieved from Google Scholar. Duplicate articles were removed after the initial search. Two authors (MJA and YA) independently screened the title and abstract of the articles. Articles that passed the screening were assessed in full text based on the eligibility criteria. Disagreements were resolved by discussion with the senior author (A). This study was conducted following the Preferred Reporting Item for Systematic Reviews and Meta-Analysis (PRISMA) statement and registered with the International Prospective Register of Systematic Reviews (PROSPERO) database (registration number CRD42021228821).

We included all observational studies examining biomarkers of endothelial dysfunction and outcomes from patients who tested positive for SARS-CoV-2 using the reverse transcriptionpolymerase chain reaction (RT-PCR) test. The following types of articles were excluded from the analysis: case reports, review articles, non-English language articles, research articles on the pediatric population, animal or in-vitro studies, unpublished studies, and studies with irrelevant or nonextractable results.

Data extraction was carried out by two authors (MJA and YA) independently using piloted data extraction forms consisting of the author, year of publication, study design, number and characteristics of samples, levels of several biomarkers of endothelial dysfunction, and patient outcomes. The biomarkers of endothelial dysfunction analyzed in this study were von Willebrand Factor (VWF) antigen, tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1 antigen (PAI-1), and soluble thrombomodulin (sTM). The primary endpoint was composite poor J o u r n a l P r e -p r o o f outcomes consisting of ICU admission, severe illness, worsening of the respiratory status, the need for mechanical ventilation, and mortality. Moreover, if the included studies reported the data using median and quartile values, we used the formula developed by Wan et al. to estimate mean and standard deviation 7 . Disease severity was defined based on the WHO R&D Blueprint on COVID-19 8 .

The quality and risk of bias assessment of included studies were conducted using the Newcastle-Ottawa score (NOS) 9 by all authors independently, and discrepancies were resolved through discussion. This scoring system consists of three domains: the selection of sample cohorts, comparability of cohorts, and assessment of outcomes (Supplementary Table S2 ).

Stata software V.14.0 (College Station) was used for meta-analysis, and figure of estimated pooled means were produced using GraphPad Prism 9. We pooled multiple means and standard errors of the same population characteristic from different studies into a single group using the fixed-effect model of the meta-analysis algorithm. Pooled effect estimates of the outcomes were reported as standardized means differences (SMD). Fixed-effects and random-effects models were used for pooled analysis with low heterogeneity (I 2 statistic <50% or P-value <0.1) and high heterogeneity (I 2 statistic> 50% or P-value ≤ 0.1), respectively. Statistical significance was determined with P-value < 0.05. We performed a sensitivity analysis to test the robustness of the pooled effect estimates for VWF antigen levels by excluding each study sequentially and rerunning the meta-analysis. The funnel-plot analysis was used to assess the symmetrical distribution of effect sizes, and the regression-based Egger test was performed to assess publication bias on continuous endpoints.

We identified 697 articles from the database search, and 492 articles remained after the duplication was removed. Screening on titles and abstracts excluded 465 articles, and the remaining 27 full-text articles were assessed according to eligibility criteria. As a result, 17 studies 10-26 with a total of 1187 patients were subjected to qualitative analysis and meta-analysis ( Figure 1 and Table 1 ). Quality assessment with NOS showed that 13 studies 10, [13] [14] [15] [16] [17] [18] [19] [21] [22] [23] [24] 26 were of good quality with ≥ 7 NOS score and four studies 11, 12, 20, 25 were considered as moderate quality with six NOS score (Supplementary Table S2 ).

There was an increase in the VWF antigen levels in COVID- 19 

A single layer of healthy endothelial cells lines the entire vascular system and plays essential roles in maintaining laminar blood flow 27 . Conditions that cause endothelial activation, such as infection and inflammation, support proatherogenic mechanisms by stimulating thrombin formation, coagulation, and fibrin deposition in blood vessel walls 28 . Activated endothelial cells promote coagulation and induce thrombosis by favoring the expression of antifibrinolytics (e.g., PAI-1) and procoagulants (e.g., tissue factor) over the expression of profibrinolytic mediators (e.g., t-PA) and anticoagulants (e.g., heparin-like molecules and thrombomodulin) [28] [29] [30] . Taken together, these mechanisms contribute to massive platelet binding and formation of fibrin, leading to deposition of blood clots in the microvasculature and systemic thrombosis 2 . Since viral RNA of SARS-CoV-2 is rarely detected in the blood 31 , it suggests that rather than direct viral infection of endothelial cells, additional hostdependence factors may contribute to systemic endothelial dysfunction and vasculopathy in COVID-

Von Willebrand factor (VWF) is a large multidomain adhesive glycoprotein produced by megakaryocytes and endothelial cells 32 . Endothelial activation in COVID-19 infection and elevated VWF levels as an acute-phase protein released from endothelial cells are recognized as a response to inflammation, but high levels of VWF, in this case, indicate suspicion of endothelial disturbance 33 .

Expression of VWF and its release from the Weibel-Palade body of endothelial cells may also be stimulated by hypoxia. Hypoxia-induced upregulation of VWF is associated with thrombus in cardiac and pulmonary vessels that promote leukocyte recruitment 34 . Several studies have shown that COVID-19 patients have higher plasma levels of VWF antigen than healthy controls 10, 12, 18, 35 . Elevated

The procoagulant state resulting from endothelial activation can also be measured from changes in the balance between the levels of t-PA and PAI-1 39 . Although t-PA is recognized as a profibrinolytic mediator, t-PA is found to be higher in COVID-19 patients with poor outcomes. This paradox may be explained by a study showing that PAI-1, a major inhibitor of t-PA, facilitates the dissociation of t-PA from the surface of vascular endothelial cells (VES), which would therefore decrease cell surfaceassociated fibrinolytic potential 40 . Decreased amounts of t-PA on the VEC surface and increased concentration of t-PA in the plasma due to increased PAI-1 plasma level may be the possible mechanism of hypofibrinolysis that leads to poor outcomes in COVID-19 patients.

PAI-1 is one of the acute-phase proteins that is increased during inflammatory disorders where the concentrations are affected by the changes in its production by hepatocytes. 41 In patients with ARDS, increased levels of PAI-1 produced by endothelial cells and lung epithelium are associated with severe hypofibrinolytic state 42 . In addition to endothelial activation due to proinflammatory cytokines, binding of SARS-CoV-2 to the ACE2 receptor induces enhanced shredding of ACE2, thereby increasing Ang II levels which further stimulates PAI-1 expression in various cells including, endothelial cells, adipocytes, and smooth muscle cells 43 . In COVID-19 patients, plasma levels of PAI-1 were found to be 3.7 times higher than in healthy controls 35 . Furthermore, COVID-19 patients with severe respiratory dysfunction show significantly higher levels of PAI-1 compared to patients with burns, ARDS, and sepsis 44 .

Soluble thrombomodulin (sTM) is a product of proteolytic cleavage of the intact thrombomodulin protein from the surface of endothelial cells after endothelial injury and dysfunction 45 J o u r n a l P r e -p r o o f

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