Human Coronary Artery Endothelial Cells Release Extracellular Vesicles that Have Angiostatic and Anti‐Proliferative Effects

Abstract

Endothelial cells (EC) are major contributors to the extracellular vesicles (EV) pool in systemic circulation. EVs released by vascular endothelium of different phenotypes mirror their molecular heterogeneity. Our laboratory showed that EVs released by human adipose tissue ECs exposed to chronic inflammation have angiostatic effects on naïve, proliferating ECs from adipose tissue in vitro. Whether the observed effect is specific to EVs from adipose tissue endothelium or is a more general feature of the endothelial EVs exposed to pro-inflammatory cues is currently unclear. Coronary circulation is critical for recovery of the ischemic myocardium and effective angiogenesis is key to support an adequate repair response. In this study, we compared the proliferative and angiogenic effects and cargo composition of EVs produced by ECs from coronary artery and adipose microvasculature exposed to prolonged treatment with pro-inflammatory cytokines (PIC) on naïve coronary artery ECs. To mimic the pro-inflammatory environment in vivo, ECs were treated for 6 days with a combination of TNFa, interferon g, and TGFb (5nM each). EVs were collected from conditioned media and separated using differential ultracentrifugation. Typically, vesicles produced by both adipose and coronary ECs exposed to PIC, produced ~2-fold more EVs/cell, and the size range for all EVs used in experiments was between 40-200nm. EVs from coronary EC had the most potent anti-proliferative effect that was time-dependent. After 24 hours of incubation with EVs produced by PIC-treated coronary ECs, proliferation of the naïve coronary ECs was reduced by ~80%, as assessed by BrdU incorporation. The reduced proliferation was not due to cellular senescence. Cells treated with EVs isolated from both adipose and coronary ECs showed reduced in vitro angiogenic responses, in the presence of VEGF, compared to untreated control cells. While all EV treatments were angiostatic, the magnitude of the effects was different for different EV preparations. Specifically, coronary cells treated with autologous EVs produced in response to PIC treatment displayed the most prominent reduction in the angiogenic response to VEGF, measured in vitro as tubule formation. Interestingly, the proteome of EVs produced by adipose ECs after prolonged cytokine treatment contained peptide signatures for all VEGF receptors, including VEGFR2. Immunogold EM showed VEGFR2 positivity on the membrane of some of the EVs produced following PIC treatment of the parent cells. We are currently investigating whether EVs may function as soluble decoy receptors that can exert their angiostatic effect via binding of circulating VEGF. The angiostatic effect of autologous EVs on coronary endothelium may be physiologically meaningful in post-ischemic myocardial events when such EVs may prevent or delay a robust angiogenic response needed for tissue re-vascularization.