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. 2022 Oct 15;206(8):961-972.
doi: 10.1164/rccm.202107-1774OC.

COVID-19-associated Lung Microvascular Endotheliopathy: A "From the Bench" Perspective

Jérémie Joffre  1 , Lauren Rodriguez  2   3 , Zachary A Matthay  4 , Elliot Lloyd  1 , Alexander T Fields  4 , Roland J Bainton  1 , Philip Kurien  1 , Anita Sil  2 , Carolyn S Calfee  5   6 , Prescott G Woodruff  5   6   7 , David J Erle  3   5   6   7 , Carolyn Hendrickson  5   6 , Matthew F Krummel  7   8 , Charles R Langelier  9   10 , Michael A Matthay  5   6 , Lucy Z Kornblith  4 , Judith Hellman  1 ; COVID-19 Multi-Phenotyping for Effective Therapies (COMET) Consortium; COVID-19 Associated Coagulopathy, Inflammation, and Thrombosis (Co-ACIT) Study Group
Collaborators, Affiliations

COVID-19-associated Lung Microvascular Endotheliopathy: A "From the Bench" Perspective

Jérémie Joffre et al. Am J Respir Crit Care Med. .

Abstract

Rationale: Autopsy and biomarker studies suggest that endotheliopathy contributes to coronavirus disease (COVID-19)-associated acute respiratory distress syndrome. However, the effects of COVID-19 on the lung endothelium are not well defined. We hypothesized that the lung endotheliopathy of COVID-19 is caused by circulating host factors and direct endothelial infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Objectives: We aimed to determine the effects of SARS-CoV-2 or sera from patients with COVID-19 on the permeability and inflammatory activation of lung microvascular endothelial cells. Methods: Human lung microvascular endothelial cells were treated with live SARS-CoV-2; inactivated viral particles; or sera from patients with COVID-19, patients without COVID-19, and healthy volunteers. Permeability was determined by measuring transendothelial resistance to electrical current flow, where decreased resistance signifies increased permeability. Inflammatory mediators were quantified in culture supernatants. Endothelial biomarkers were quantified in patient sera. Measurements and Main Results: Viral PCR confirmed that SARS-CoV-2 enters and replicates in endothelial cells. Live SARS-CoV-2, but not dead virus or spike protein, induces endothelial permeability and secretion of plasminogen activator inhibitor 1 and vascular endothelial growth factor. There was substantial variability in the effects of SARS-CoV-2 on endothelial cells from different donors. Sera from patients with COVID-19 induced endothelial permeability, which correlated with disease severity. Serum levels of endothelial activation and injury biomarkers were increased in patients with COVID-19 and correlated with severity of illness. Conclusions: SARS-CoV-2 infects and dysregulates endothelial cell functions. Circulating factors in patients with COVID-19 also induce endothelial cell dysfunction. Our data point to roles for both systemic factors acting on lung endothelial cells and viral infection of endothelial cells in COVID-19-associated endotheliopathy.

Keywords: COVID-19; acute respiratory distress syndrome; endothelial permeability; lung endothelial injury.

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Figures

Figure 1.
Figure 1.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) human lung microvascular endothelial cell (HMVEC) infection and endothelial permeability. (A) SARS-CoV-2 mRNA was detected and quantified in HMVEC lysates after 24 and 72 hours of exposure to live SARS-CoV-2 virus (multiplicity of infection [MOI] of 1; orange, strain nCoV/USA-WA1/2020) using quantitative PCR (TaqMan 2019-nCoV assay). This example shows cells from a single donor; dots are biological replicates. (B1–B6) Transendothelial resistance during treatment with medium (black line) or live virus (MOI of 1; orange) in six HMVEC donors. (B7) The corresponding area under the curve (AUC) for HMVECs on each of the six donors. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Mann-Whitney U test.
Figure 2.
Figure 2.
Pharmacological modulation of angiotensin-converting enzyme 2 receptor (ACE2R) pathway and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced human lung microvascular endothelial cell (HMVEC) permeability. (A1 and A2) Representative example of ACE2 agonist (Diminazene aceturate [DMZ] 20 μM; green) and ACE inhibitor (ACEi) effect (20 μM; claret red) on the transendothelial resistance and area under the curve (AUC) of HMVECs treated simultaneously with ACE2 agonists/antagonists and live SARS-CoV-2 (multiplicity of infection [MOI] of 1; orange) infection. (B) The stabilizing effects of DMZ on permeability induced by LPS (1 μg/ml). *P < 0.05, **P < 0.01 by two-tailed Mann-Whitney U test (AUC LPS vs. LPS + DMZ).
Figure 3.
Figure 3.
Effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on human lung microvascular endothelial cell (HMVEC) mediator secretion and proadhesive phenotype. (A) Concentrations of IL-6, IL-8, CCL-2, plasminogen activator inhibitor 1 (PAI-1), and vascular endothelial growth factor (VEGF) in supernatants of HMVECs from six different donors treated with medium or SARS-CoV-2 (multiplicity of infection [MOI] of 1; orange, strain nCoV/USA-WA1/2020). *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Mann-Whitney U test. (B1) Histograms for CD102 (ICAM-2), CD31 (platelet endothelial cell adhesion molecule 1 [PECAM-1]), CD106 (vascular cell adhesion molecule 1 [VCAM-1]), CD144 (VE-cadherin), and CD62P (P-selectin) expression on HMVECs from four different donors after 24 hours of treatment with medium (gray) or infection with SARS-CoV-2 (MOI of 1; orange, strain nCoV/USA-WA1/2020). (B2) A double-gradient heat map of the relative mean fluorescence intensity on endothelial cells from four different donors after 24 hours of treatment with medium versus virus.
Figure 4.
Figure 4.
Sera from patients with and without coronavirus disease (COVID-19) induce human lung microvascular endothelial cell (HMVEC) permeability proportionate to their clinical severity. (A1 and A2) Transendothelial resistance tracings of HMVECs during treatment with medium or with sera from healthy volunteers (n = 18); patients with COVID-19 with mild, moderate, or critical illness (n = 99; mild [n = 28], moderate [n = 41], critical [n = 30]); or patients without COVID-19 with mild, moderate, or critical illness (n = 43; mild [n = 14], moderate [n = 19], critical [n = 10]). Each curve represents the mean value of the group ± SD. (B) The corresponding electric cell substrate impedance sensing (ECIS) area under the curve (AUC) represents the intensity of sera-induced HMVEC permeability. *P < 0.05, ***P < 0.001 versus healthy donors by two-tailed Mann-Whitney U test.
Figure 5.
Figure 5.
Admission serum endothelial biomarkers in patients with versus without coronavirus disease (COVID-19). (A) Comparison of endothelium-related biomarkers in serum from patients with COVID-19 (n = 50) and patients without COVID-19 (n = 24). P values are based on two-sided Mann-Whitney U tests. (B) Comparison of endothelium-related biomarkers between patients with COVID-19 (n = 50) and patients without COVID-19 (n = 24), stratified according to the severity of illness. (C) Principal component analysis showing representation of COVID-19 and non-COVID-19 populations in the two dimensions (score plot). Scores and loadings are presented in a scatterplot of one principal component (PC) against another. The loadings are represented in a circle of correlations: The closer the arrow of a loading is to the circle (shown in blue), the more the variable is well represented in the space of the two plotted PCs and contributed to the building of these PCs. The first two PCs allowed the discrimination of patients with and without COVID-19, consolidating results of the univariate analysis. Patients with COVID-19 (dotted line ellipse) were characterized by increased cathepsin D, neural cell adhesion molecule (NCAM), soluble vascular cell adhesion molecule 1 (sVCAM-1), soluble intercellular adhesion molecule 1 (sICAM-1), plasminogen activator inhibitor 1 (PAI-1), and syndecan-1. Patients without COVID-19 (solid line ellipse) were characterized by increased brain-derived neurotrophic factor (BDNF), RANTES (regulated upon activation, normal T cell expressed and secreted), and angiotensin-converting enzyme (ACE).
Figure 6.
Figure 6.
Hypothetical model of coronavirus disease (COVID-19)-associated lung microvascular endotheliopathy. On the basis of our experimental results, we speculate that viral infection and secondary injury of the endothelium by inflammatory mediators and immune cells contribute to the lung endotheliopathy of COVID-19 and, in turn, COVID-19–associated acute respiratory distress syndrome (ARDS). (A) Endothelial cell exposure to live severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could occur early or later in the course of the disease if there is persistence of virus in the host, including in alveolar epithelial cells, which are in proximity to microvascular endothelial cells. In susceptible individuals, immune dysfunction could lead to increased viral load and release by alveolar epithelial cells. In this speculative model, SARS-CoV-2 infection of lung microvascular endothelial cells induces permeability, upregulates surface expression of leukocyte adhesion molecules and secretion of plasminogen activator inhibitor 1 (PAI-1) and vascular endothelial growth factor (VEGF) secretion, and induces low levels of secretion of cytokines and chemokines. In this context, infection of endothelial cells may promote and/or amplify endotheliopathy and ARDS. (B) Secondary injury to the lung endothelium is likely to be a principal determinant of COVID-19–induced endotheliopathy. Secondary injury may result from the actions of circulating inflammatory mediators and toxins and of activated pulmonary leukocytes and damaged alveolar epithelial cells. In susceptible individuals, impaired viral clearance and persistent viral replication in epithelial or endothelial cells may promote leukocyte recruitment to the lung and cell death and the release of damage-associated molecular patterns (DAMPs). These DAMPs could, analogously to their roles in sepsis and injury, further exacerbate the endotheliopathy, as well as acute lung injury and ARDS. RBC = red blood cells.
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Comment in

  • Insights into Endotheliopathy in COVID-19.
    Filbin MR. Filbin MR. Am J Respir Crit Care Med. 2022 Oct 15;206(8):926-928. doi: 10.1164/rccm.202207-1258ED. Am J Respir Crit Care Med. 2022. PMID: 35819867 Free PMC article. No abstract available.
  • SARS-CoV2 Endotheliopathy: Insights from Single Cell RNAseq.
    Khatun MS, Qin X, Pociask DA, Kolls JK. Khatun MS, et al. Am J Respir Crit Care Med. 2022 Nov 1;206(9):1178-1179. doi: 10.1164/rccm.202206-1105LE. Am J Respir Crit Care Med. 2022. PMID: 35839476 Free PMC article. No abstract available.

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