Endothelium-derived microparticles (EMPs) are small vesicles released from endothelial cells in response to cell injury, apoptosis, or activation. Elevated concentrations of EMPs have been associated with many inflammatory and vascular diseases. EMPs also mediate long range signaling and alter downstream cell function. Unfortunately, the molecular and cellular basis of microparticle production and downstream cell function is poorly understood. We hypothesize that EMPs generated by different agonists will produce distinct populations of EMPs with unique protein compositions. To test this hypothesis, different EMP populations were generated from human umbilical vein endothelial cells by stimulation with plasminogen activator inhibitor type 1 (PAI-1) or tumor necrosis factor-alpha (TNF-α) and subjected to proteomic analysis by LC/MS. We identified 432 common proteins in all EMP populations studied. Also identified were 231 proteins unique to control EMPs, 104 proteins unique to PAI-1 EMPs and 70 proteins unique to TNF-α EMPs. Interestingly, variations in protein abundance were found among many of the common EMP proteins, suggesting that differences exist between EMPs on a relative scale. Finally, gene ontology (GO) and KEGG pathway analysis revealed many functional similarities and few differences between the EMP populations studied. In summary, our results clearly indicate that EMPs generated by PAI-1 and TNF-α produce EMPs with overlapping but distinct protein compositions. These observations provide fundamental insight into the mechanisms regulating the production of these particles and their physiological role in numerous diseases.

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Endothelial‐derived microparticles (EMPs) are produced by endothelial cells in response to cell injury, activation and apoptosis. Elevated plasma concentrations of EMPs are associated with a variety of vascular and inflammatory diseases, including acute lung injury (ALI). However, the mechanisms by which EMPs contribute to pulmonary disease are unclear. We hypothesize that EMPs contribute to ALI by inhibiting endothelium‐mediated vasodilation in the pulmonary vasculature. To test this hypothesis, acetylcholine (ACh)‐mediated vasodilation was quantified using isolated thoracic aorta (TA) and pulmonary artery (PA) rings of C57BL/6 mice in the presence or absence of EMPs (20 K or 100 K EMPs/ml). Concentration dependent inhibition of ACh‐mediated vasodilation was observed for both TA and PA rings; furthermore, PA rings are more sensitive to the inhibitory effects of EMPs. In conclusion, EMPs attenuate endothelium‐mediated vasodilation at pathophysiologically relevant concentrations. EMPs also act in a tissue specific manner affecting the pulmonary vasculature disproportionate to other vascular beds. These data suggest that EMP damage to pulmonary endothelial cells may contribute to ALI. Funding provided by the Children's Research Institute and MCW Department of Surgery.

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