Recent studies demonstrate that oxidative inactivation of tetrahydrobiopterin (H4B) may cause uncoupling of endothelial nitric oxide synthase (eNOS) to produce superoxide (O 2 •؊ ). H4B was found recyclable from its oxidized form by dihydrofolate reductase (DHFR) in several cell types. Functionality of the endothelial DHFR, however, remains completely unknown. Here we present findings that specific inhibition of endothelial DHFR by RNA interference markedly reduced endothelial H4B and nitric oxide (NO • ) bioavailability. Furthermore, angiotensin II (100 nmol͞liter for 24 h) caused a H4B deficiency that was mediated by H2O2-dependent downregulation of DHFR. This response was associated with a significant increase in endothelial O 2•؊ production, which was abolished by eNOS inhibitor N-nitro-L-arginine-methyl ester or H2O2 scavenger polyethylene glycol-conjugated catalase, strongly suggesting H2O2-dependent eNOS uncoupling. Rapid and transient activation of endothelial NAD(P)H oxidases was responsible for the initial burst production of O 2 • (Rac1 inhibitor NSC 23766 but not an N-nitro-L-arginine-methyl ester-attenuated ESR O 2•؊ signal at 30 min) in response to angiotensin II, preceding a second peak in O 2•؊ production at 24 h that predominantly depended on uncoupled eNOS. Overexpression of DHFR restored NO • production and diminished eNOS production of O 2•؊ in angiotensin II-stimulated cells. In conclusion, these data represent evidence that DHFR is critical for H4B and NO • bioavailability in the endothelium. Endothelial NAD(P)H oxidase-derived H2O2 down-regulates DHFR expression in response to angiotensin II, resulting in H4B deficiency and uncoupling of eNOS. This signaling cascade may represent a universal mechanism underlying eNOS dysfunction under pathophysiological conditions associated with oxidant stress.hydrogen peroxide ͉ tetrahydrobiopterin ͉ superoxide

It has been shown that eNOS uncoupling occurs in hypertension and atherosclerosis. However its causal role in vascular pathogenesis has not been previously characterized. Here, we challenged eNOS pre-uncoupled hph-1 mice (deficient in eNOS cofactor tetrahydrobiopterin biosynthetic enzyme GTPCHI) with Ang II (0.7 mg/kg/day, 14 days). Both wild-type (WT) and hph-1 groups developed hypertension similarly up to day 6 to 7. Thereafter approximately 14% of Ang II-infused (0.7 mg/kg/day) hph-1 mice (n=72) started to die suddenly of ruptured AAA. Among the survivors, 65% developed AAA, resulting in a total morbidity rate of 79%. In contrast, none of the Ang II-infused WT mice died or developed AAA. Ang II progressively deteriorated eNOS uncoupling in hph-1 mice, while augmenting H4B and nitric oxide (NO•) deficiencies. The abundance of the H4B salvage enzyme dihydrofolate reductase (DHFR) in the endothelium was decreased in hph-1 mice and further diminished by Ang II infusion. Intriguingly, restoration of DHFR expression by oral administration of folic acid (FA), or overexpression of DHFR, completely prevented AAA formation in Ang II-infused hph-1 mice while attenuating progressive uncoupling of eNOS. Folic acid also attenuated vascular remodelling and inflammation characterized by medial elastin break down, augmented MMP2 activity and activation of MMP9, as well as macrophage infiltration. In conclusion, these data innovatively suggest a causal role of eNOS uncoupling/H4B deficiency in AAA formation. Therefore oral FA administration, endothelium targeted DHFR gene therapy, and perhaps other countermeasures directed against eNOS uncoupling, could be used as new therapeutics for AAA.

Background-Folate supplementation improves endothelial function in patients with hyperhomocysteinemia. Mechanistic insights into potential benefits of folate on vascular function in general population however, remain mysterious.