Abstract 365: Microparticle-Induced Activation of the Vascular Endothelium Requires Caveolin-1/Caveolae

Abstract

Microparticles (MPs) are small membrane fragments shed from normal as well as activated, apoptotic or injured cells. Recent findings show a strong correlation between elevated levels of circulating MPs and several cardiovascular disease states including hypertension and atherosclerosis. Moreover, emerging evidence implicates MPs as a causal and/or contributing factor in altering normal vascular cell phenotype through initiation of proinflammatory signal transduction events and paracrine delivery of proteins, mRNA and miRNA. However, little is known regarding the mechanism by which MPs influence these events. Caveolae are important membrane microdomains, which function as centers of signal transduction and endocytosis. A number of known MP signaling events are associated with caveolae-mediated pathways, yet the relationship between caveolae and MPs has not been established. Here, we tested the concept that MP induced signaling in endothelial cells (ECs) depends on caveolae. MPs were generated from mouse lung ECs exposed to TNF-α for 24 hrs. MPs were isolated using low and high-speed centrifugation, resuspended and then exposed to ECs. Consistent with previous reports, MPs activated ECs as evidenced by upregulation of intracellular adhesion molecule (ICAM) expression. ICAM upregulation was associated with activation of NF-κB, Poly [ADP-ribose] polymerase 1 (PARP1) and epidermal growth factor receptor (EGFR). This response was absent in ECs lacking cavolin-1. To test whether caveolae-mediated endocytosis is a feature of the proinflammatory response, ECs were pretreated with dynasore, a dynamin2 inhibitor. Similar to observations in cells lacking caveolin-1, inhibition of endocytosis significantly attenuated MPs effects including activation of EGFR, NF-κB and PARP1 and ICAM expression. Last, we found that the EGFR localized in caveolae indicating that compartmentation of proximal signaling components in caveolae is essential for induction of an endothelial pro-inflammatory phenotype by MPs.