Abstract 329: Microparticles Induce Antiangiogenic Signaling in Microvascular Endothelial Cells by Activating a CD36-Dependent Signaling Pathway Involving Src Kinases and Reactive Oxygen Species
Cell-derived microparticles (MP), ≤1μm vesicles shed from vascular and blood cells during activation or apoptosis, circulate in plasma and are biomarkers of cardiovascular disease. The type 2 scavenger receptor CD36 binds MP via phosphatidylserine exposed on their surface. On microvascular endothelial cells (MVEC) CD36 induces anti-angiogenic signals via its cognate protein ligands, such as thrombospondin-1. We hypothesized that CD36-MP interactions on MVEC would inhibit angiogenesis. MP generated in vitro from THP1 cells dose dependently inhibited MVEC tube formation in in vitro matrigel angiogenesis assays (>50% inhibition of number of tubes formed, branch points, and segments; p<0.05). MP derived from human umbilical vein endothelial cells (HUVEC), MVEC and erythrocytes also inhibited tube formation showing that the anti-angiogenic effect of MP is independent of their cell of origin. THP1 MP also inhibited MVEC migration in a transwell assay by 80% (p<0.001). Early passage MVEC with high CD36 expression were inhibited more than late passage MVEC with low CD36 expression (80% vs 33%; p<0.001) establishing the specificity of CD36 in this process. Further, in HUVEC that do not express CD36, inhibition of migration by THP1 MP was only 44% (p<0.001) but lentiviral-mediated heterologous expression of murine CD36 in HUVEC restored inhibition of migration to levels similar to that of MVEC (82%; p<0.001), corroborating the specificity of CD36 in MP mediated inhibition of migration. Since CD36 dependent generation of reactive oxygen species (ROS) is necessary to inhibit cell migration in macrophages, we tested if MP can induce ROS in MVEC. THP1 MP dose dependently induced ROS in MVEC by two fold compared to control at 1hr (p<0.05). NADPH oxidase inhibitor, apocynin, blocked MP induced ROS induction by >95% and partially (33%) reversed the inhibition of tube formation in MVEC by THP1 MP (p<0.05). Western blots of THP1 MP treated MVEC showed time-dependent activation of Fyn kinase, and PP2, a Src family kinase inhibitor, blocked MP induced ROS induction by >95%. These studies identify a novel CD36 dependent mechanistic pathway through which cell-derived MP inhibit MVEC angiogenic activities through ROS generation involving NADPH oxidase and Src kinase Fyn.
Author Disclosures: D. Ramakrishnan: None. R.L. Silverstein: None.
- © 2014 by American Heart Association, Inc.