Role of uncoupled endothelial nitric oxide synthase in abdominal aortic aneurysm formation: treatment with folic acid

Abstract

It has been shown that endothelial NO synthase (eNOS) uncoupling occurs in hypertension and atherosclerosis. However, its causal role in vascular pathogenesis has not been characterized previously. Here, we challenged eNOS preuncoupled hyperphenylalaninemia (hph)-1 mice (deficient in eNOS cofactor tetrahydrobiopterin biosynthetic enzyme GTPCHI) with angiotensin II (Ang II; 0.7 mg/kg per day, 14 days). Both wild-type and hph-1 groups developed hypertension similarly up to day 6 to 7. Thereafter, ≈14% of Ang II-infused (0.7 mg/kg per day) hph-1 mice (n=72) started to die suddenly of ruptured abdominal aortic aneurysm (AAA). Among the survivors, 65% developed AAA, resulting in a total morbidity rate of 79%. In contrast, none of the Ang II-infused wild-type mice died or developed AAA. Ang II progressively deteriorated eNOS uncoupling in hph-1 mice while augmenting tetrahydrobiopterin and nitric oxide (NO(·)) deficiencies. The abundance of the tetrahydrobiopterin salvage enzyme dihydrofolate reductase in the endothelium was decreased in hph-1 mice and further diminished by Ang II infusion. Intriguingly, restoration of dihydrofolate reductase expression by oral administration of folic acid or overexpression of dihydrofolate reductase completely prevented AAA formation in Ang II-infused hph-1 mice while attenuating progressive uncoupling of eNOS. Folic acid also attenuated vascular remodeling and inflammation characterized by medial elastin breakdown and augmented matrix metalloproteinase 2 activity and activation of matrix metalloproteinase 9, as well as macrophage infiltration. In conclusion, these data innovatively suggest a causal role of eNOS uncoupling/tetrahydrobiopterin deficiency in AAA formation. Therefore, oral folic acid administration, endothelium-targeted dihydrofolate reductase gene therapy, and perhaps other countermeasures directed against eNOS uncoupling could be used as new therapeutics for AAA.

Figure 1. Ang II induces AAA formation in hph-1 mice

Wild-type and hph-1 mice were infused with Ang II (0.7 mg/kg/day) for 14 days. (A) Representative appearance of abdominal aortas in the different experimental groups of WT, WT/Ang II, hph-1 and hph-1/Ang II by day 14. Only Ang II-infused hph-1 mice developed AAA, with visible evidence of hemorrhage. H&E staining (arrows) of the AAA segment revealed thrombus formation. (B) AAA morbidity and mortality rates (no-AAA vs. non-lethal AAA vs. lethal AAA) in Ang II-infused hph-1 mice at 21%, 65% and 14% respectively. (C) Changes in mean blood pressure (MBP) in Ang II-infused hph-1 mice. The MBP was monitored by an intra-carotid telemetry method (Data Sciences International) continuously for 14 days.

Figure 2. Ang II infusion augments deficiency of H₄B and NO^•, and aggravates eNOS uncoupling, in hph-1 mice

Wild-type and hph-1 mice were infused with Ang II (0.7 mg/kg/day) for 14 days, after which aortas were harvested for: (A) aortic H₄B content; (B) aortic NO^• production; and (C) aortic O₂^•− production in the presence or absence of L-NAME. *p<0.05

Figure 3. Folic acid prevents eNOS uncoupling in Ang II-infused hph-1 mice via restoration of endothelial DHFR expression

Wild-type and hph-1 mice were started on oral administration of folic acid (FA, 15 mg/kg/day) 2 days prior to Ang II infusion (0.7 mg/kg/day), and treated throughout the study period of 14 days, after which aortas were harvested for: (A) endothelial and non-endothelial DHFR expression in aortic preparations; (B) aortic H₄B content; and (C) aortic eNOS uncoupling activity (indicated by L-NAME-sensitive O₂^•− production). *p<0.01

Figure 4. Folic acid prevents AAA formation and normalizes blood pressure in Ang II-infused hph-1 mice

Wild-type and hph-1 mice were started on oral administration of folic acid (FA, 15 mg/kg/day) 2 days prior to Ang II infusion (0.7 mg/kg/day), and treated throughout the study period of 14 days. At day 0 and day 14, abdominal ultrasound (Velvo 770 high-resolution echo system, Visualsonics) was performed to assess abdominal aorta (AA) dimensions (A-D) in Ang II-infused WT and hph-1 mice treated with or without FA. Aortic cross-sectional areas are depicted by blue circles, and calculated areas are listed below each image. MBP was assessed by telemetry during the course of the study (E-F), as described in Figure 1.

Figure 5. Folic acid prevents progressive uncoupling of eNOS in Ang II-infused hph-1 mice

Wild-type (WT) and hph-1 mice were started on oral administration of folic acid (FA, 15 mg/kg/day) 2 days prior to Ang II infusion (0.7 mg/kg/day), and treated throughout the study period of 14 days. Aortas were harvested on day 0, 4 or 8 days for analysis of O₂^•− production in the presence or absence of L-NAME in (A) WT, and (B) hph-1, mice. *p<0.05 vs L-NAME, #p<0.05 vs WT sham, +p<0.05 vs 4 days

Figure 6. Folic acid prevents vascular remodeling in Ang II-infused hph-1 mice

Wild-type and hph-1 mice were treated with our without folic acid (FA, 15 mg/kg/day) beginning 2 days prior to Ang II (0.7 mg/kg/day) or vehicle infusion, and treated throughout the study period of 14 days, after which aortas were harvested for (A) H&E staining (black arrows and red arrows indicating FA-induced changes in media and adventitia respectively); (B) VVG staining (black arrows showing elastin changes); (C) Macrophage staining indicating macrophage infiltraiton

Figure 7. Folic acid prevents Ang II-induced MMP2 and MMP9 activation in hph-1 mice

Wild-type and hph-1 mice were treated with our without folic acid (FA, 15 mg/kg/day) beginning 2 days prior to Ang II (0.7 mg/kg/day) or vehicle (sham) infusion. After the 14 day study period, aortas were harvested to assess MMP activity. (A) Representative zymogram showing MMP2 and MMP9 activities. (B) Quantitative data of MMP2 activity. (C) Quantitative data of MMP9 activity. *p<0.05 vs sham.

Fig. 8. DHFR overexpression recouples eNOS in Ang II-infused hph-1 mice

Wild-type and hph-1 mice were subjected to tail vein transfection of DHFR expression vector (pcDNA3.1-DHFR) in lipid-based reagent from Altogen Biosystems every other day for 14 days, starting 2 days prior to Ang II infusion. At the end of the 14 day infusion, aortas were harvested for: (A) endothelial DHFR expression in aortic preparations by western blotting; (B) aortic superoxide production in the presence or absence of L-NAME. *p<0.05 vs. WT sham, #p<0.05 vs. L-NAME, +p<0.05 vs. hph-1 sham