Abstract 16379: The Role of Endothelial Microparticles in the Regulation of Pulmonary Endothelial Function in vitro
Rationale: Microparticles (MPs) are small membrane vesicles (<100 nm) released in extracellular space upon cell activation or apopotosis. Increased levels of endothelium-derived MPs in human plasma are associated with the severity of several cardiovascular disorders, including pulmonary hypertension (PH). Small GTPase RhoA and its effector, Rho kinase are activated in pulmonary vasculature in all forms of PH, independent of etiology, and their activation is associated with endothelial dysfunction. Interestingly, RhoA has been shown to stimulate MP generation. We hypothesized that RhoA-induced generation of microparticles helps to propagate pro-inflammatory and pro-angiogenic signals in human pulmonary endothelium, exacerbating endothelial dysfunction.
Methods: Conditioned medium was collected from human pulmonary artery endothelial cells (HPAECs), untreated or overexpressing active from of RhoA. MPs were isolated by serial centrifugation. Isolated MPs were analysed by flow cytometry and electron scanning microscopy. The effects of isolated MPs on endothelial cell function were examined after 24-48 hour co-incubation.
Results and Conclusion: Activation of RhoA in HPAECs increased microparticle generation by 50%. In electron scanning microscopy, MPs could be seen clustering and docking on the cell membrane of HPAECs. When seeded on to HPAECs, isolated MPs stimulated endothelial cell proliferation (20% increase in MTS reduction assay, P<0.05), tube formation on Matrigel (25% increase in total tube length, P<0.05), and inflammatory response (50% increase in NFkB expression, P<0.01), but had no effect on trans-endothelial permeability. MPs stimulated phosphorylation of the focal adhesion kinase protein (FAK), which could be the mechanism leading to increased tube formation. Equal quantities of MPs from unstimulated or RhoA stimulated cells appear to have similar effects. These results suggest that endothelial MPs are likely to induce abnormal endothelial cell function in the pulmonary vasculature. This study provides a useful model for studying microparticle-induced endothelial dysfunction in vitro.
- © 2012 by American Heart Association, Inc.