The Fibrin-derived Peptide FX06 Protects Human Pulmonary Endothelial Cells Against the COVID- 19-Triggered Cytokine Storm

Objectives: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been a major health emergency since 2019. Endothelial dysfunction is a hallmark of COVID-19, which leads to severe illness, i.e. multi-organ failure, coagulopathy, and death (1). FX06, a fibrin-derived peptide naturally occurring in the human body, formerly known as Bβ15-42, protects the vasculature in myocardial ischemia-reperfusion in animal models (2). Therefore, it is a promising therapeutic candidate for endothelial complications like capillary leak in COVID-19 and other forms of acute respiratory disorders. The aim of this project is to investigate whether FX06 can attenuate COVID-19 cytokine-triggered inflammatory processes in vitro. Methods: To mimic the inflammatory status of COVID-19, cells from a human pulmonary endothelial cell line (ECs) were treated with the so-called severe cytokine cocktail comprised of ten different cytokines or chemokines at concentrations found in serum profiles of COVID-19 patients with severe illness (3–7). ECs were cultured under both static and shear stress/laminar flow conditions and treated with the severe cytokine cocktail for 24 h, in the absence or presence of FX06 for the last 2 h. Results: Under both static and shear stress conditions, the COVID-19-triggered cytokine cocktail increased the adhesion and migration of Peripheral Blood Mononuclear Cells (PBMCs) through the endothelial monolayer. This deleterious effect was significantly reduced by FX06. FX06 was also shown to protect ECs against the cytotoxic activity of allogeneic CD8+ T cells, which increased upon the severe cytokine cocktail treatment. Interestingly, FX06 did not protect ECs from the severe cytokine cocktail-triggered apoptosis and necrosis, suggesting that restoration of vascular integrity was caused by cytoskeletal reorganisation rather than cellular rescue. In fact, FX06 reversed morphology changes caused by the severe cytokine cocktail, i.e.FX06 shortened the F-actin fibre length. Furthermore, FX06 reduced the pro-inflammatory angiogenic activity enhanced by the severe cytokine cocktail. Additionally, FX06 restored continuous VE-cadherin/CD144 distribution on the EC surface after disruption by the severe cytokine cocktail. FX06 might also assist in maintaining the normal vascular barrier function of ECs by reducing the surface expression of Syndecan-1 (SDC1/CD138). From whole proteomics and phosphoproteomics analyses, FX06 increased the activity of tight and gap junctions and reduced the abundance of proteins associated with immune cell infiltration. Notably, FX06 downregulated Rho GTPase which is, again, enhanced by the severe cytokine cocktail. It was confirmed that the severe cytokine cocktail promoted Ras homolog family member A (RhoA) expression, while FX06 reversed this. FX06 also downregulated the downstream protein of RhoA signalling, phosphorylated Rho-associated Coiled-coil Containing Protein Kinase 1 (ROCK1). Conclusions: FX06 shows great potential in reducing vascular leakage to protect the endothelium against the proinflammatory effect of COVID-19-triggered cytokines. The investigation of immunological markers in ECs will further advance our understanding of the mechanism of action (MOA) of FX06 and its potential to counteract systemic endothelial inflammation and disease progression in COVID-19 and other forms of acute respiratory disease syndrome (ARDS).