Nifedipine attenuation of abdominal aortic aneurysm in hypertensive and non-hypertensive mice: Mechanisms and implications

Abstract

Rupture of abdominal aortic aneurysm (AAA) is a lethal event. No oral medicine has been available to prevent or treat AAA. We have recently identified a novel mechanism of eNOS uncoupling by which AAA develops, in Angiotensin II (Ang II) infused hyperphenylalaninemia 1 (hph-1) mice. Using this unique model we investigated effects on AAA formation of the L-type calcium channel blocker nifedipine, in view of the unclear relationship between hypertension and AAA, and unclear mechanisms of aneurysm protective effects of some blood pressure lowering drugs. Six-month old hph-1 mice were infused with Ang II (0.7 mg/kg/day) for 2 weeks, and fed nifedipine chow at two different doses (5 and 20 mg/kg/day). While the high dose of nifedipine reduced blood pressure, the lower dose had no effect. Interestingly, the incidence rate of AAA dropped from 71% to 7 and 12.5% for low and high dose nifedipine, respectively. Expansion of abdominal aorta, determined by ultrasound imaging, was abolished by both doses of nifedipine, which recoupled eNOS completely to improve NO bioavailability. Both also abrogated aortic superoxide production. Of note, Ang II activation of NADPH oxidase in vascular smooth muscle cells and endothelial cells, known to uncouple eNOS, was also attenuated by nifedipine. Although low dose was a sub-pressor while the high dose reduced blood pressure via inhibition of calcium channels, both doses were highly effective in preventing AAA by preserving eNOS coupling activity to eliminate sustained oxidative stress from uncoupled eNOS. These data demonstrate that oral treatment of nifedipine is highly effective in preserving eNOS function to attenuate AAA formation. Nifedipine may be used for AAA prevention either at low dose to AAA risk group, or at high dose to patients with co-existing hypertension.

NOX isoforms in the development of abdominal aortic aneurysm

“…Our previous studies have established a novel causal role of eNOS uncoupling in AAA formation [[4], [5], [6], [23], [24]]. In addition, there are evidences that Ang II transiently activates NOXs, resulting in uncoupling of eNOS in endothelial cells [7], [8].…”

“…The PCR amplification was performed using the conditions of 95 °C for 3 min, followed by 30 cycles of 94 °C for 60 s, 50 °C for 60 s and 72 °C for 30 s. For the hph-1, the primers used were: 5′-AGGCTGCCCATAAAAGGG-3′ and 5′-GTTTGTGCTAATGTTCTCATCTGG-3′. The PCR amplification was performed using the conditions of 95 °C for 15 min, followed by 35 cycles of 94 °C for 20 s, 58 °C for 60 s and 72 °C for 45 s [5], [6] .…”

“…Aortic superoxide was measured by ESR as previously described [4], [5], [23], [6], [24]. Briefly, freshly isolated aortas were homogenized on ice in lysis buffer containing 1:100 protease inhibitor cocktail, and centrifuged at 12,000 g for 15 min.…”

“…Aortic NO production was also measured using ESR as previous described [4], [5], [23], [6], [24]. Briefly, freshly isolated aortas were cut into 2 mm rings, and then incubated in freshly prepared NO specific spin trap trap Fe 2+ (DETC) 2 (0.5 mmol/L) in nitrogen bubbled, modified Krebs/HEPES buffer (as described above) at 37°C for 60 min, in the presence of calcium ionophore A23187 (10 μmol/L).…”

“…Aortic H 4 B and its oxidized species were measured using HPLC as previously described [4], [5], [23], [6], [24]. Briefly, freshly isolated aortas were lysed in H 4 B lysis buffer (0.1 mol/L phosphoric acid, 1 mmol/L EDTA, 10 mmol/L DL-Dithiothreitol), centrifuged at 12,000 g for 3 min, and the lysates were subjected to differential oxidation in acidic (0.2 mol/L trichloroacetic acid with 2.5% I2 and 10% KI) and alkalytic (0.1 mol/L NaOH with 0.9% I2 and 1.5% KI) solutions.…”