Recoupling of eNOS with Folic Acid Prevents Abdominal Aortic Aneurysm Formation in Angiotensin II-Infused Apolipoprotein E Null Mice

Abstract

We have previously shown that eNOS uncoupling mediates abdominal aortic aneurysm (AAA) formation in hph-1 mice. In the present study we examined whether recoupling of eNOS prevents AAA formation in a well-established model of Angiotensin II-infused apolipoprotein E (apoE) null mice by targeting some common pathologies of AAA. Infusion of Ang II resulted in a 92% incidence rate of AAA in the apoE null animals. In a separate group, animals were treated orally with folic acid (FA), which is known to recouple eNOS through augmentation of dihydrofolate reductase (DHFR) function. This resulted in a reduction of AAA rate to 19.5%. Imaging with ultrasound showed that FA markedly inhibited expansion of abdominal aorta. FA also abolished elastin breakdown and macrophage infiltration in the AAA animals. The eNOS uncoupling activity, assessed by L-NAME-sensitive superoxide production, was minimal at baseline but greatly exaggerated with Ang II infusion, which was completely attenuated by FA. This was accompanied by markedly improved tetrahydrobiopterin and nitric oxide bioavailability. Furthermore, the expression and activity of DHFR was decreased in Ang II-infused apoE null mice specifically in the endothelial cells, while FA administration resulted in its recovery. Taken together, these data further establish a significant role of uncoupled eNOS in mediating AAA formation, and a universal efficacy of FA in preventing AAA formation via restoration of DHFR to restore eNOS function.

Circulating tetrahydrobiopterin as a novel biomarker for abdominal aortic aneurysm

“…Previous studies have shown that in these mice, infusion of ANG II for 4 wk results in ϳ90% incidence of AAA (6,29). Our group has also shown that aortic H 4 B levels were reduced in these animals at 4 wk of ANG II infusion (29).…”

“…For plasma, equal volumes of plasma and H 4B lysis buffer were mixed and incubated on ice for 20 min in the dark and then centrifuged at 12,000 g for 3 min at 4°C in the dark. The supernatant for both the aorta and plasma was subjected to oxidation in acidic (0.2 M trichloroacetic acid with 2.5% I 2 and 10% KI) and alkalytic solutions (0.1 M NaOH with 0.9% I 2 and 1.5% KI) as described previously (10,24,29,35). After centrifugation, the supernatant was injected into a fluorescent detector-equipped HPLC system (Shimadzu) set at 350-nm excitation and 450-nm emission wavelengths.…”

“…Recent studies from our laboratory have identified that uncoupling of endothelial nitric oxide synthase (eNOS), in which the normally nitric oxide (NO)-producing eNOS produces superoxide to induce oxidative stress, plays a causal role in the formation of AAA (10,29). This uncoupling of eNOS is caused by a reduced bioavailability of tetrahydrobiopterin (H 4 B), which is the essential cofactor for proper eNOS coupling activity (4,16,17,24).…”

“…This response of depressed H 4 B levels remained until the end of the experimental protocol for both strains of animals. Oral administration of folic acid (FA), which has been shown to reduce AAA development in these two animal models (10,29), increased both circulating and tissue levels of H 4 B starting at week 1, with H 4 B levels remaining enhanced until the end of the experiment. Linear correlation was found between tissue and plasma levels of H 4 B under both H 4 B reduced and enhanced conditions, suggesting that plasma levels of H 4 B accurately predicts its tissue levels.…”

Arrested Hematopoiesis and Vascular Relaxation Defects in Mice with a Mutation in Dhfr

“…DHFR expression levels are reduced in the aortas of diabetic mice (45,46), angiotensin II-infused wild-type mice (47), BH4-deficient hph-1 mice, (48), ApoE gene knockout mice (49), and aged hypercholesterolemic LDL receptor gene knockout mice (50), which all correlates with reduced vascular BH4 levels and NO bioactivity. Where examined, restoration of DHFR levels via endothelium-targeted overexpression of the DHFR gene or folic acid supplements prevents eNOS uncoupling and inhibits the degree of hypertension or aortic aneurysm in Ang II-infused hph-1 (48) or ApoE gene knockout (49) mice and improves endothelial function in diabetic mice (46).…”