Loss of eNOS in Endothelial Cells Promotes Proliferation

Background Endothelial cells help maintain vascular homeostasis mainly by balancing interplay between vasorelaxation and vasoconstriction via regulating availability of Nitric Oxide (NO). Endothelial nitric oxide synthase (eNOS) is one of three NOS isoforms that catalyses the synthesis of NO to regulate endothelial function. To date, however, eNOS’s role in endothelial cell proliferation, remains unclear. Methods and Results To gain a better understanding about eNOS and cell proliferation, we genetically inhibited eNOS via silencing by transfecting cultured human umbilical vein endothelial cells with sieNOS or scrambled control. Successful eNOS silencing was confirmed at transcript and protein levels, and then endothelial cell proliferation was evaluated. Surprisingly, loss of eNOS significantly induced endothelial cell proliferation, concomitant with significant downregulation of both cell cycle inhibitor p21 and cell proliferation antigen Ki‐67. To confirm the specificity of eNOS silencer, we used another sieNOS molecule, which also showed the similar results as endothelial proliferation was significantly induced in eNOS‐silenced endothelial cells. Later, to demonstrate that the induced endothelial cell proliferation is due to eNOS inhibition, we pharmacologically inhibited eNOS by treating endothelial cells with L‐NAME. Contrastingly, L‐NAME‐treatment significantly inhibited endothelial cell proliferation in a dose‐dependent manner. Conclusion Our findings, for the first time, indicate that eNOS regulate endothelial function by directly controlling endothelial cell proliferation. Observed inhibition of endothelial cell proliferation by L‐NAME, may be due to its non‐specific effects. The findings also indicate that eNOS and L‐NAME might employ different pathways regulating endothelial cell proliferation warranting further investigation.