Alleviation of accelerated diabetic atherogenesis in STZ-treated apoE/NOX1 DKO mice, apoE−/−/tg-EC-DHFR mice, and by folic acid

We and others have previously shown that uncoupling of endothelial nitric oxide synthase (eNOS) induces oxidative stress in diabetes, contributing to endothelial dysfunction. Activation of NADPH oxidase (NOX) isoform NOX1 by angiotensin II (Ang II) triggers eNOS uncoupling via deficiency in dihydrofolate reductase (DHFR) in streptozotocin (STZ)-induced type 1 diabetic mice. Presently, we investigated whether accelerated atherosclerosis is attenuated in apoE/NOX1 double knockout, and whether mice overexpressing DHFR in the endothelium (tg-EC-DHFR, generated in house) recouples eNOS to alleviate diabetic atherogenesis. At baseline, endothelial-specific DHFR overexpression recoupled eNOS and improved vasorelaxation in the aortas and mesenteric arteries of STZ-induced diabetic mice. Accelerated atherogenesis in STZ/high-fat diet (HFD) treated apoE^−/− mice was markedly abrogated in tg-EC-DHFR background, establishing an important role of endothelial DHFR in maintaining vascular function and protecting from diabetic atherogenesis. Moreover, by crossing apoE^−/− with NOX1 null mice (NOX1^-/y), we found that NOX1 deletion markedly diminished atherosclerotic lesion formation in HFD + STZ-treated apoE^−/−/NOX1^-/y mice, indicating that NOX1 lies upstream of eNOS uncoupling in facilitating diabetic atherogenesis. Oral administration with folic acid (FA), shown to upregulate DHFR, robustly attenuated atherosclerotic lesion formation in HFD + STZ-treated apoE^−/− mice similarly to NOX1 deletion. Taken together, our data for the first time demonstrate that endothelial DHFR plays an important role in the preservation of endothelial function and inhibition of atherosclerosis in diabetes, deficiency of which consequent to NOX1 activation mediates eNOS uncoupling driven lesion formation. Strategies targeting uncoupled eNOS prove to be robust treatment options for diabetic endothelial dysfunction and atherogenesis.