Caveolin 1 is critical for abdominal aortic aneurysm formation induced by angiotensin II and inhibition of lysyl oxidase

Abstract

Although AngII (angiotensin II) and its receptor AT1R (AngII type 1 receptor) have been implicated in AAA (abdominal aortic aneurysm) formation, the proximal signalling events primarily responsible for AAA formation remain uncertain. Caveolae are cholesterol-rich membrane microdomains that serve as a signalling platform to facilitate the temporal and spatial localization of signal transduction events, including those stimulated by AngII. Cav1 (caveolin 1)-enriched caveolae in vascular smooth muscle cells mediate ADAM17 (a disintegrin and metalloproteinase 17)-dependent EGFR (epidermal growth factor receptor) transactivation, which is linked to vascular remodelling induced by AngII. In the present study, we have tested our hypothesis that Cav1 plays a critical role for the development of AAA at least in part via its specific alteration of AngII signalling within caveolae. Cav1-/- mice and the control wild-type mice were co-infused with AngII and β-aminopropionitrile to induce AAA. We found that Cav1-/- mice with the co-infusion did not develop AAA compared with control mice in spite of hypertension. We found an increased expression of ADAM17 and enhanced phosphorylation of EGFR in AAA. These events were markedly attenuated in Cav1-/- aortas with the co-infusion. Furthermore, aortas from Cav1-/- mice with the co-infusion showed less endoplasmic reticulum stress, oxidative stress and inflammatory responses compared with aortas from control mice. Cav1 silencing in cultured vascular smooth muscle cells prevented AngII-induced ADAM17 induction and activation. In conclusion, Cav1 appears to play a critical role in the formation of AAA and associated endoplasmic reticulum/oxidative stress, presumably through the regulation of caveolae compartmentalized signals induced by AngII.

Figure 1. Increased mortality and fatal aortic rupture were prevented in Cav1 deficient mice co-infused with Ang II and BAPN

8 week old Cav1−/− mice and the control Cav1+/+ (C57Bl/6) mice were infused with Ang II (1 μg/kg/min for 4 weeks) and BAPN (150 mg/kg/day for the first 2 weeks). Percentage survival curve with black and grey circles represent Cav1−/− and Cav1+/+ mice, respectively (n=survived number/total number) (A). Summary data showing percentages of animals with fatal rupture or unspecified death following treatment with Ang II plus BAPN (B).

Figure 2. Prevention of Ang II-dependent AAA development in Cav1 deficient mice

8 week old Cav1−/− mice and the control Cav1+/+ (C57Bl/6) mice were infused with Ang II (1 μg/kg/min for 4 weeks) and BAPN (150 mg/kg/day for the first 2 weeks) or saline for 4 weeks. Incidence of AAA in surviving mice after Ang II plus BAPN infusion for 4 weeks (A). Representative aorta after Ang II plus BAPN infusion, arrows indicate AAA (B). Measurements of maximal external width of abdominal aortas after the 4 week infusion (C).

Figure 3. Histological assessment of abdominal aortas

8 week old Cav1−/− mice and the control Cav1+/+ (C57Bl/6) mice were infused with Ang II (1 μg/kg/min 4 weeks) and BAPN (150 mg/kg/day 2 weeks) or saline for 4 weeks. Cross sections of abdominal aortas were stained with Masson’s trichrome (MT) or hematoxylin and eosin (HE) protocol. Representative staining was shown from n=4 for each.

Figure 4. ADAM17 induction was prevented by Cav1 silencing

8 week old Cav1−/− mice and the control Cav1+/+ (C57Bl/6) mice were infused with Ang II+BAPN or saline for 4 weeks as in Fig 1. qPCR analysis of ADAM17 mRNA expression in abdominal aorta (n=4) (A). Rat VSMC were infected with adenoviruses (50 moi) encoding HB-EGF-AP and miRNA targeting rat Cav1 or non-targeting control miRNA (100 moi) for 72 h and HB-EGF-AP shedding assay was performed with or without 100 nM Ang II stimulation (means ± SEM from three separate experiments). The combined cell lysates from each experimental condition were analyzed by immunoblotting as indicated (B). Rat VSMC cotransfected with ADAM17 promoter-Luc construct and miR ADAM17 or the control miR construct were stimulated with or without 100 nM Ang II for 24 hours (means ± SEM from three separate experiments) (C). *p<0.05