Attenuation of advanced atherosclerosis and aortic aneurysm in hph-1/apoE DKO mice by folic acid and DHFR overexpression.

Introduction: Atherosclerosis and aortic aneurysms often co-exist, attributed to common risk factors predisposing to development of these severe vascular disorders. The apoE null mice represent a classical model of atherosclerosis. It has been shown that endothelial nitric oxide synthase (eNOS) is modestly uncoupled in apoE null mice at baseline to contribute to oxidative stress. Methods: Notably, we have previously demonstrated marked activation of eNOS in its uncoupled state in angiotensin II (Ang II) infused hph-1 mice, which triggers formation of abdominal aortic aneurysm (AAA), representing the first evidence documenting a direct causal role of eNOS uncoupling in AAA formation that can be reversed by recoupling of eNOS with oral administration of folic acid (FA) or plasmid-mediated overexpression of dihydrofolate reductase (DHFR). In the present study, hph-1(hyperphenylalaninemia-1)/apoE double mutant mice were created in-house, and exposed to Ang II infusion (0.7 mg/kg/day) for 2 weeks alongside apoE null mice, during which formation of AAA was monitored by echo determination of abdominal aortic size on weekly basis and post-mortem inspection. Results: Indeed, although AAA formation was not significantly different between apoE and hph-1/apoE mice, there were significantly more atherosclerotic lesions formed in hph-1/apoE double mutant mice. These data indicate that AAA forms consequent to a certain extent of eNOS uncoupling activity. By contrast, worsened eNOS uncoupling activity is still associated with deteriorated atherogenesis in hph-1/apoE double mutant mice. In addition, oral FA administration or overexpression of DHFR substantially alleviated excessive atherosclerosis and AAA formation in apoE null mice and hph-1/apoE double mutant mice. Discussion: These findings seem to establish that targeting DHFR to fully restore eNOS function or to fully recouple eNOS into its native coupled state, can be employed as a novel and robust therapeutic strategy to alleviate advanced atherosclerosis.