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The endothelium maintains and regulates vascular homeostasis mainly by balancing interplay between vasorelaxation and vasoconstriction via regulating Nitric Oxide (NO) availability. Endothelial nitric oxide synthase (eNOS) is one of three NOS isoforms that catalyses the synthesis of NO to regulate endothelial function. However, eNOS's role in the regulation of endothelial function, such as cell proliferation and migration remain unclear. To gain a better understanding, we genetically knocked down eNOS in cultured endothelial cells using sieNOS and evaluated cell proliferation, migration and also tube forming potential in vitro. To our surprise, loss of eNOS significantly induced endothelial cell proliferation, which was associated with significant downregulation of both cell cycle inhibitor p21 and cell proliferation antigen Ki-67. Knockdown of eNOS induced cell migration but inhibited formation of tube-like structures in vitro. Mechanistically, loss of eNOS was associated with activation of MAPK/ERK and inhibition of PI3-K/AKT signaling pathway. On the contrary, pharmacologic inhibition of eNOS by inhibitors L-NAME or L-NMMA, inhibited cell proliferation. Genetic and pharmacologic inhibition of eNOS, both promoted endothelial cell migration but inhibited tube-forming potential. Our findings confirm that eNOS regulate endothelial function by inversely controlling endothelial cell proliferation and migration, and by directly regulating its tube-forming potential. Differential results obtained following pharmacologic versus genetic inhibition of eNOS indicates a more complex mechanism behind eNOS regulation and activity in endothelial cells, warranting further investigation. [ABSTRACT FROM AUTHOR]

Under different pathophysiological conditions, endothelial cells lose endothelial phenotype and gain mesenchymal cell-like phenotype via a process known as endothelial-to-mesenchymal transition (EndMT). At the molecular level, endothelial cells lose the expression of endothelial cell-specific markers such as CD31/platelet-endothelial cell adhesion molecule, von Willebrand factor, and vascular-endothelial cadherin and gain the expression of mesenchymal cell markers such as α-smooth muscle actin, N-cadherin, vimentin, fibroblast specific protein-1, and collagens. EndMT is induced by numerous different pathways triggered and modulated by multiple different and often redundant mechanisms in a context-dependent manner depending on the pathophysiological status of the cell. EndMT plays an essential role in embryonic development, particularly in atrioventricular valve development; however, EndMT is also implicated in the pathogenesis of several genetically determined and acquired diseases, including malignant, cardiovascular, inflammatory, and fibrotic disorders. Among cardiovascular diseases, aberrant EndMT is reported in atherosclerosis, pulmonary hypertension, valvular disease, fibroelastosis, and cardiac fibrosis. Accordingly, understanding the mechanisms behind the cause and/or effect of EndMT to eventually target EndMT appears to be a promising strategy for treating aberrant EndMT-associated diseases. However, this approach is limited by a lack of precise functional and molecular pathways, causes and/or effects, and a lack of robust animal models and human data about EndMT in different diseases. Here, we review different mechanisms in EndMT and the role of EndMT in various cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Endothelial autophagy plays an important role in the regulation of endothelial function. The inhibition of endothelial autophagy is associated with the reduced expression of protein disulfide isomerase 4 (PDIA-4); however, its role in endothelial cells is not known. Here, we report that endothelial cell-specific loss of PDIA-4 leads to impaired autophagic flux accompanied by loss of endothelial function and apoptosis. Endothelial cell-specific loss of PDIA-4 also induced marked changes in endothelial cell architecture, accompanied by the loss of endothelial markers and the gain of mesenchymal markers consistent with endothelial-to-mesenchymal transition (EndMT). The loss of PDIA-4 activated TGFβ-signaling, and inhibition of TGFβ-signaling suppressed EndMT in PDIA-4-silenced endothelial cells in vitro. Our findings help elucidate the role of PDIA-4 in endothelial autophagy and endothelial function and provide a potential target to modulate endothelial function and/or limit autophagy and EndMT in (patho-)physiological conditions. [ABSTRACT FROM AUTHOR]