Abstract
Increased mortality in COVID-19 often associates with thrombotic and microvascular complications. We have recently shown that SARS-CoV-2 spike protein promotes inflammatory cytokine IL-6/IL-6R induced trans-signaling responses which modulate MCP-1 expression in human endothelial cells. MCP-1 is secreted as a major component of the senescence associated secretory phenotype (SASP). Virus infected or Spike transfected human pulmonary epithelial cells (A549) exhibited an increase in senescence related marker proteins. TMNK; as a representative human endothelial cell line, when exposed to cell culture supernatant derived from A549 cells expressing SARS-CoV-2 spike protein (Spike CM) exhibited a senescence phenotype with enhanced p16, p21, and SA-β-galactosidase expression. Inhibition of IL-6 trans-signaling by Tocilizumab, prior to exposure of supernatant to endothelial cells, inhibited p16 and p21 induction. Likewise, inhibition of receptor signaling by Zanabrutinib or Brd4 function by AZD5153 also led to limited induction of p16 expression. Senescence lead to an enhanced level of adhesion molecule, ICAM-1 and VCAM-1 in human endothelial cells, and TPH1 attachment by in vitro assay. Inhibition of senescence or SASP function prevented ICAM/VCAM expression and leukocyte attachment. We also observed an increase in oxidative stress in A549 spike transfected and endothelial cells exposed to Spike CM. ROS generation in TMNK was reduced after treatment with the IL-6 specific inhibitor Tociliximab, and with the specific inhibitors Zanabrutinib and AZD5153. Taken together, we identified that the exposure of human endothelial cells to cell culture supernatant derived from SARS-CoV-2 spike protein expression displayed cellular senescence markers leading to enhanced leukocyte adhesion with coronary blockade potential.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
The study was supported by seed grant funding from Saint Louis University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
T.P. and R.R. designed the experiments. K.M., T.P. and V.M. conducted the experiments. All authors drafted the manuscript, RR analyzed the results. All authors read and approved the final manuscript.
Abbreviations used in this article
- CM
- culture media
- ROS
- reactive oxygen species
- BRD4
- bromodomain-containing protein 4
- ICAM-1
- intercellular adhesion molecule 1
- VCAM-1
- vascular cell adhesion molecule 1
- SASP
- senescence associated secretory phenotype
- IL
- interleukin
- TNF
- tumor necrosis factor
- MCP-1
- monocyte chemoattractant protein-1
- SA-β-gal
- senescence associated beta-galactosidase
- DDR
- DNA damage response
- BTK
- Bruton’s tyrosine kinase