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OS 02-03 EFFECT OF HIGH GLUCOSE EXPOSURE ON ENDOTHELIAL MICROPARTICLE FORMATION AND COMPOSITION.

OBJECTIVE
Diabetes and hypertension are cognate diseases that often coexist, potentiating one's risk for cardiovascular complications. Both diseases are typified by the development of endothelial dysfunction and are accompanied by increased circulating endothelial microparticles (eMPs). Microparticles are small membrane-derived vesicles that are secreted ubiquitously following cell stress. We have previously shown that eMPs are sensitive markers of vascular injury in hypertension and identified eMP-mediated signaling pathways that lead to endothelial injury. However, whether eMPs differ based on stimuli for their formation is unclear. To investigate the effects of HG on the molecular composition and biological actions of eMPs.


DESIGN AND METHOD
Cultured endothelial cells (ECs) were treated with normal D-glucose (NG, 5.6 mM), 25 mM D-glucose (HG), or L-glucose (osmotic control). eMPs were then isolated from the collected media via differential centrifugation. eMP levels were quantified using nanoparticle tracking analysis (NTA), and their effects on endothelial cell oxidative stress were assessed by dihydroethidium-HPLC. Protein composition was assessed via liquid chromatography-tandem mass spectrometry (LC-MS/MS) and relative differences were assessed by label-free spectral counting. Candidate proteins were verified by Western blot analysis.


RESULTS
eMP formation increased following HG treatment relative to the NG (3-fold, P < 0.05). eMPs formed under high glucose were more potent inducers of oxidative stress than NG eMPs (∼4-fold). LC-MS identified 14,631 unique peptides of which resulted in 220 independent proteins with at least 2 peptides per protein and an average sequence coverage of 16%. Both LC-MS and Western Blot analysis of HG eMPs showed a 4-fold increase in thrombospondin-1 and a 5-11-fold increase in its membrane-anchored binding partners (Versican, Agrin, Perlecan).


CONCLUSIONS
HG increases eMP formation and leads to molecular changes, including enrichment in thrombospondin-1 and its glycocalyx binding partners. Such alterations may contribute to the development of vascular injury in diabetes.