Role of Uncoupled Endothelial Nitric Oxide Synthase in Abdominal Aortic Aneurysm Formation

Abstract

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.

Protein Phosphotyrosine Phosphatase 1B (PTP1B) in Calpain-dependent Feedback Regulation of Vascular Endothelial Growth Factor Receptor (VEGFR2) in Endothelial Cells

“…Next, a mouse aortic disc angiogenesis assay was carried out. In vivo siRNA transfection was achieved by lipid-based transfection kit as previously described (12)(13)(14). siRNA targeting mouse PTP1B was found highly effective in attenuating PTP1B expression in mouse aortas (Fig.…”

Circulating tetrahydrobiopterin as a novel biomarker for abdominal aortic aneurysm

“…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). Accordingly, the Orana heterozygote mouse represents a promising model to further define the in vivo role of DHFR in cardiovascular disease, similar to the hph-1 mouse also generated by ENU mutagenesis as a model of BH4 deficiency due to 90% constitutive reduction in GTPCH-1 activity (51).…”