Inflammatory, Thrombotic, and Diabetic Responses in Human Arterial Fibroblasts and Endothelial Cells are Regulated by SARS‐CoV‐2 Structural Proteins

Freda, Christopher; Yin, Wei; Ghebrehiwet, Berhane; Rubenstein, David

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is responsible for many pathological processes, such as altered vascular disease development, dysfunctional thrombosis, and a heightened inflammatory state. Although many clinical reports support these observations, there has been limited experimental work to determine the underlying mechanisms and cellular processes induced by exposure to SARS‐CoV‐2 structural proteins. Importantly, these structural proteins are conserved amongst all current known SARS‐CoVs. Our objective was to investigate the effects of the Spike (S), Nucleocapsid (N), and Membrane‐Envelope (M) SARS‐CoV‐2 structural proteins on inflammation, thrombosis, and diabetic disease markers of human aortic adventitial fibroblasts and human umbilical vein endothelial cells. We hypothesized that short‐term exposure to SARS‐CoV‐2 structural proteins would result in increased expression of inflammatory, thrombotic, and diabetic proteins in both cell types, which would support a mechanism for altered vascular disease progression. To test this, the cells were incubated independently with the three SARS‐CoV‐2 proteins for one hour, after which we analyzed the expression of gC1qR, ICAM‐1, tissue factor, RAGE, GLUT‐4, thrombomodulin, PECAM‐1, and Connexin‐43 (in their respective cell types), using an ELISA approach. All cells were monitored for maintenance of typical culture parameters using a live/dead cell cytotoxicity assay and the MTT assay (for metabolic activity). We observed that each of these markers were significantly up‐regulated after exposure to SARS‐CoV‐2 structural proteins as compared to fibroblasts or endothelial cells that were not exposed to these proteins. Interestingly, the extent of the expression of these markers was sometimes significantly different for each of the SARS‐CoV‐2 structural proteins. This suggests that each of the SARS‐CoV‐2 proteins interacts with these cells through different mechanisms, however, more work will need to be undertaken to determine the mechanisms by which these proteins interact with cells. Thus, these results indicate that the cellular response of vascular cells towards SARS‐CoV‐2 structural proteins promotes inflammatory, thrombotic, and vascular dysfunction. However, these interactions are regulated by complex and possibly different cellular receptors/signal transduction pathways that should be explored further.