Recombinant ACE2 Expression Is Required for SARS-CoV-2 To Infect Primary Human Endothelial Cells and Induce Inflammatory and Procoagulative Responses

Abstract

SARS-CoV-2 causes COVID-19, an acute respiratory distress syndrome (ARDS) characterized by pulmonary edema, viral pneumonia, multiorgan dysfunction, coagulopathy, and inflammation. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) receptors to infect and damage ciliated epithelial cells in the upper respiratory tract. In alveoli, gas exchange occurs across an epithelial-endothelial barrier that ties respiration to endothelial cell (EC) regulation of edema, coagulation, and inflammation. How SARS-CoV-2 dysregulates vascular functions to cause ARDS in COVID-19 patients remains an enigma focused on dysregulated EC responses. Whether SARS-CoV-2 directly or indirectly affects functions of the endothelium remains to be resolved and is critical to understanding SARS-CoV-2 pathogenesis and therapeutic targets. We demonstrate that primary human ECs lack ACE2 receptors at protein and RNA levels and that SARS-CoV-2 is incapable of directly infecting ECs derived from pulmonary, cardiac, brain, umbilical vein, or kidney tissues. In contrast, pulmonary ECs transduced with recombinant ACE2 receptors are infected by SARS-CoV-2 and result in high viral titers (∼1 × 107/ml), multinucleate syncytia, and EC lysis. SARS-CoV-2 infection of ACE2-expressing ECs elicits procoagulative and inflammatory responses observed in COVID-19 patients. The inability of SARS-CoV-2 to directly infect and lyse ECs without ACE2 expression explains the lack of vascular hemorrhage in COVID-19 patients and indicates that the endothelium is not a primary target of SARS-CoV-2 infection. These findings are consistent with SARS-CoV-2 indirectly activating EC programs that regulate thrombosis and endotheliitis in COVID-19 patients and focus strategies on therapeutically targeting epithelial and inflammatory responses that activate the endothelium or initiate limited ACE2-independent EC infection. IMPORTANCE SARS-CoV-2 infects pulmonary epithelial cells through ACE2 receptors and causes ARDS. COVID-19 causes progressive respiratory failure resulting from diffuse alveolar damage and systemic coagulopathy, thrombosis, and capillary inflammation that tie alveolar responses to EC dysfunction. This has prompted theories that SARS-CoV-2 directly infects ECs through ACE2 receptors, yet SARS-CoV-2 antigen has not been colocalized with ECs and prior studies indicate that ACE2 colocalizes with alveolar epithelial cells and vascular smooth muscle cells, not ECs. Here, we demonstrate that primary human ECs derived from lung, kidney, heart, brain, and umbilical veins require expression of recombinant ACE2 receptors in order to be infected by SARS-CoV-2. However, SARS-CoV-2 lytically infects ACE2-ECs and elicits procoagulative and inflammatory responses observed in COVID-19 patients. These findings suggest a novel mechanism of COVID-19 pathogenesis resulting from indirect EC activation, or infection of a small subset of ECs by an ACE2-independent mechanism, that transforms rationales and targets for therapeutic intervention.

Increased susceptibility of human endothelial cells to infections by SARS-CoV-2 variants

“…HCMVEC, HLMVEC, HUVEC and HPAEC did not express ACE2 and were not permissive for SARS-CoV-2. However, transducing endothelial cells with recombinant ACE2 may enable SARS-CoV-2 infection as shown recently [ 20 ], suggesting that the lack of sufficient ACE2 expression might be a limiting factor. Subsequent steps in the viral life cycle appear to be blocked in HCAECs.…”

COVID-19 Vasculopathy: Mounting Evidence for an Indirect Mechanism of Endothelial Injury

“…This gap has now been filled by recently published work by different groups in which isolated human endothelial cells were cultured in the presence of the SARS-CoV-2 virus. Using immunohistochemistry for the N or S protein and a viral progeny detection assay, Nascimento Conde et al showed that SARS-CoV-2 is incapable of infecting primary human endothelial cells from lung, brain, and renal glomeruli as well as HUVEC 50 . Ajmetaj-Shala and coworkers demonstrated that blood outgrowth, lung and aortic primary human endothelial cells were not susceptible to infection by SARS-CoV-2 or SARS-CoV-2 pseudovirus but could be infected with Ebola and Vesicular Stomatitis Virus 49 .…”

“…These abortive infections obtained with high SARS-CoV-2 titers in endothelial cells that do not express the ACE2 receptor or express it weakly have been attributed to engagement by the virus of noncanonical receptors and lack of surface enzymes needed to cleave the S protein and mediate effective viral-host cell membrane fusion 43 , 45 , 47 . Notably, endothelial cells became permissive to SARS-CoV-2 only following transduction with recombinant ACE2 receptor 50 . Thus, in vitro studies with human endothelial cells isolated from different organs have consistently shown that the vascular endothelium is resistant to SARS-CoV-2 infection.…”

Mechanisms of SARS‐CoV‐2‐induced lung vascular disease: potential role of complement

“…Even in the presence of inflammation, where an endothelial cell was pretreated with IL-1, the cells remained refractory to SARS-CoV-2 infection. A separate study demonstrated that primary human endothelial cells lack ACE2 receptors at the RNA and protein levels, and that SARS-CoV-2 is incapable of directly infecting endothelial cells from pulmonary, cardiac, kidney, or brain tissues, even after stimulation with a variety of factors (e.g., IL-1β, TNF-α, IL-6) 41 . In contrast, pulmonary endothelial cells transfected with ACE2 receptors can be infected, indicating that endothelial cells are permissive for SARS-CoV-2 replication 41 .…”

“… 27 In contrast, pulmonary endothelial cells transfected with ACE2 receptors can be infected, indicating that endothelial cells are permissive for SARS-CoV-2 replication. 27 …”