Endothelium Infection and Dysregulation by SARS-CoV-2: Evidence and Caveats in COVID-19

Abstract

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by the acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) poses a persistent threat to global public health. Although primarily a respiratory illness, extrapulmonary manifestations of COVID-19 include gastrointestinal, cardiovascular, renal and neurological diseases. Recent studies suggest that dysfunction of the endothelium during COVID-19 may exacerbate these deleterious events by inciting inflammatory and microvascular thrombotic processes. Although controversial, there is evidence that SARS-CoV-2 may infect endothelial cells by binding to the angiotensin-converting enzyme 2 (ACE2) cellular receptor using the viral Spike protein. In this review, we explore current insights into the relationship between SARS-CoV-2 infection, endothelial dysfunction due to ACE2 downregulation, and deleterious pulmonary and extra-pulmonary immunothrombotic complications in severe COVID-19. We also discuss preclinical and clinical development of therapeutic agents targeting SARS-CoV-2-mediated endothelial dysfunction. Finally, we present evidence of SARS-CoV-2 replication in primary human lung and cardiac microvascular endothelial cells. Accordingly, in striving to understand the parameters that lead to severe disease in COVID-19 patients, it is important to consider how direct infection of endothelial cells by SARS-CoV-2 may contribute to this process.

Keywords: ACE2; ADAM17; COVID-19; RAAS; SARS-CoV-2; bradykinin–kallikrein pathway; endothelial dysfunction; immunothrombosis; pericyte; therapeutics.

Figure 1

Architecture of healthy and dysfunctional vascular endothelium. (1) In homeostatic conditions, a healthy endothelium is a thin monolayer of endothelial cells at the interface between the circulation and tissue. A basement membrane separates the endothelium from smooth muscle and underlying connective tissue in arterioles. Smooth muscle cells regulate vascular tone, promoting vasodilation in conjunction with endothelial cells. As the vessel diameter decreases in capillaries, the vessel walls consist uniquely of the endothelial monolayer, basement membrane, and pericytes within the basement membrane wrapping around the abluminal surface of endothelial cells. Functionally, pericyte–endothelial cell cross-talk is essential to maintain normal endothelial cell shape and function. Together, endothelial cells, smooth muscle cells, and pericytes promote vasodilation, limit vascular permeability, promote an anti-thrombotic state, and reduce hyperinflammation in healthy patients. (2) However, disruption of the endothelial barrier and endothelial cell function leads to deleterious vasoconstriction, increased vascular permeability, thrombosis, and hyperinflammation. Dysfunction of the endothelium is indicated by a variety of biomarkers.

Figure 2

SARS-CoV-2 infects human endothelial cells. (A) Human primary lung and cardiac microvascular cells were infected with SARS-CoV-2 (MOI = 3) for 48 h followed by cell lysates collection for RNA isolation and qRT-PCR using SYBR Green (Quanta BioSciences). Viral spike protein expression was quantitated using a Bio-Rad CFX96 Touch System. Values are expressed as the mean of three independent experiments. Error bars represent standard errors of the mean. (B) Representative confocal imaging (20×) of human primary lung microvascular cells infected with SARS-CoV-2 (MOI = 3) for 48 h. After fixation, coronavirus-infected cells were stained using mouse monoclonal antibody to SARS spike protein (GeneTex) and Alexa Fluor 488 donkey anti-mouse (Invitrogen). Nuclei were stained with DAPI. Confocal images were acquired using Olympus 1 × 81 spinning disk confocal microscope with Volocity 6.2.1 software.

Figure 3

SARS-CoV-2-mediated cellular ACE2 downregulation leads to RAAS dysfunctions. (1) In normal conditions, the liver releases angiotensinogen, which is hydrolyzed into Ang I by renin (angiotensinogenase) from renal juxtaglomerular cells. ACE produced in the kidneys and lungs further converts Ang I into Ang II, which are then converted by ACE2 into Ang 1-9 and Ang 1-7 respectively. Ang 1-7 binds to MasR to mediate vasoprotective effects. (2) During SARS-CoV-2 infection, viral internalization results in cellular ACE2 downregulation and increased ACE. Consequently, Ang II accumulates and binds to AT1R, mediating detrimental vascular effects. (3) ADAM17 proteolytically sheds sACE2, which exacerbates inflammation, may further amplify RAAS dysfunction by decreasing cellular ACE2, and blocks SARS-CoV-2 entry.

Figure 4

SARS-CoV-2-mediated cellular ACE2 downregulation leads to bradykinin–kallikrein pathway dysfunction. (1) In normal conditions, kallikrein converts kininogen to bradykinin, which can then bind to B2R on endothelial cells. B2R stimulation increase tPA/uPA to mediate protective vasodilation and fibrinolysis. (2) During SARS-CoV-2 infection, increased activity of kallikrein increase bradykinin. However, ACE2 downregulation increases Ang II. Ang II can convert bradykinin to its inactive peptides through aldosterone-induced ACE expression and increase PAI-1, which inhibits the protective effects of tPA/uPA. Furthermore, ACE2 downregulation prevents the inactivation of des-Arg(9)-BK and Lys-des-Arg(9)-BK. These can bind to endothelial B1R to promote detrimental vascular effects, which together with Ang II, upregulate B1R. In all, inhibition of B2R and stimulation of B1R results in hypofibrinolysis and adverse vascular consequences.

Figure 5

Endothelial dysfunction leads to immunothrombosis by inducing a pro-thrombotic state, hyperinflammation, and increasing platelets. Deleterious thrombosis is mediated by hypercoagulation and hypofibrinolysis. (1) The intrinsic coagulation pathway is stimulated by vascular endothelial damage and NET formation by PMNs. The extrinsic pathway is stimulated by COVID-19-mediated TF upregulation and TFPI inhibition. These pathways converge on the common pathway, promoting hypercoagulation through excessive fibrin formation. (2) Fibrinolysis is inhibited by the Ang II-mediated increased PAI-1:tPA/uPA ratio. Together, accumulation of fibrin, platelets, and hyperinflammation due to endothelial dysfunction contribute to the formation of immunothrombotic clots.