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. 2015 May 22;116(11):1744-52.
doi: 10.1161/CIRCRESAHA.116.305895. Epub 2015 Apr 13.

Genetic Evidence Supports a Major Role for Akt1 in VSMCs During Atherogenesis

Noemi Rotllan  1 , Amarylis C Wanschel  1 , Ana Fernández-Hernando  1 , Alessandro G Salerno  1 , Stefan Offermanns  1 , William C Sessa  1 , Carlos Fernández-Hernando  2
Affiliations

Genetic Evidence Supports a Major Role for Akt1 in VSMCs During Atherogenesis

Noemi Rotllan et al. Circ Res. .

Abstract

Rationale: Coronary artery disease, the direct result of atherosclerosis, is the most common cause of death in Western societies. Vascular smooth muscle cell (VSMC) apoptosis occurs during the progression of atherosclerosis and in advanced lesions and promotes plaque necrosis, a common feature of high-risk/vulnerable atherosclerotic plaques. Akt1, a serine/threonine protein kinase, regulates several key endothelial cell and VSMC functions including cell growth, migration, survival, and vascular tone. Although global deficiency of Akt1 results in impaired angiogenesis and massive atherosclerosis, the specific contribution of VSMC Akt1 remains poorly characterized.

Objective: To investigate the contribution of VSMC Akt1 during atherogenesis and in established atherosclerotic plaques.

Methods and results: We generated 2 mouse models in which Akt1 expression can be suppressed specifically in VSCMs before (Apoe(-/-)Akt1(fl/fl)Sm22α(CRE)) and after (Apoe(-/-)Akt1(fl/fl)SM-MHC-CreER(T2E)) the formation of atherosclerotic plaques. This approach allows us to interrogate the role of Akt1 during the initial and late steps of atherogenesis. The absence of Akt1 in VSMCs during the progression of atherosclerosis results in larger atherosclerotic plaques characterized by bigger necrotic core areas, enhanced VSMC apoptosis, and reduced fibrous cap and collagen content. In contrast, VSMC Akt1 inhibition in established atherosclerotic plaques does not influence lesion size but markedly reduces the relative fibrous cap area in plaques and increases VSMC apoptosis.

Conclusions: Akt1 expression in VSMCs influences early and late stages of atherosclerosis. The absence of Akt1 in VSMCs induces features of plaque vulnerability including fibrous cap thinning and extensive necrotic core areas. These observations suggest that interventions enhancing Akt1 expression specifically in VSMCs may lessen plaque progression.

Keywords: atherogenesis; atherosclerosis; proto-oncogene proteins c-akt.

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Figures

Figure 1
Figure 1. Prolonged Akt1 silencing in VSMCs results in moderately increased atherosclerosis
A) Experimental outline of the atherosclerosis study of Apoe−/−Akt1fl/fl and Apoe−/−Akt1fl/flSm22αCRE mice fed a WD during 3 or 5 months. B) Representative Western blot analysis of Akt1, Akt2, total Akt and downstream Akt targets from aortic lysates of male Apoe−/−Akt1fl/fl and Apoe−/−Akt1fl/flSm22αCRE mice fed a WD during 3 months (n=6). Hsp90 was used as a loading control. Band densitometry is shown in the right panels. *Indicates p<p 0.05 compared with Apoe−/−Akt1fl/fl mice. C–E) Body weight (C), total cholesterol (D) and triglyceride levels (E) at the starting point (0 month) and after 1.5, 3 and 5 months on WD. All of the data represent the mean ± SEM; (Apoe−/−Akt1fl/fl n=14 and Apoe−/−Akt1fl/flSm22αCRE n=10). F) Representative histological analysis of cross-sections from the aortic sinus stained with hematoxylin and eosin (H&E). The right panel shows the quantification of the lesion area. All of the data represents the mean ± SEM; (3 months: Apoe−/−Akt1fl/fl n=8, Apoe−/−Akt1fl/flSm22αCRE n=13; 5 months: Apoe−/−Akt1fl/fl n=14, Apoe−/−Akt1fl/flSm22αCRE n=10). *Indicates p< 0.05 compared to Apoe−/−Akt1fl/fl mice.
Figure 2
Figure 2. Absence of Akt1 in VSMCs during the progression of atherosclerosis results in larger atherosclerotic plaques characterized by bigger necrotic core areas, reduced VSMCs and collagen content and thinner fibrous caps
A–C) Representative histological analysis of cross-sections from the aortic sinus stained with hematoxylin and eosin (H&E) (A), Picrosirus red (B), and CD68 (green) and SMA (red) staining (C). Quantification of the fibrous cap, necrotic core, collagen, macrophage and VSMC content are represented in the right panels. All of the data represent the mean ± SEM; (3 months: Apoe−/−Akt1fl/fl n=8, Apoe−/−Akt1fl/flSm22αCRE n=13; 5 months: Apoe−/−Akt1fl/fl n=14, Apoe−/−Akt1fl/flSm22αCRE n=10). The dashed lines demarcate necrotic core areas in panel A and the intima of the atherosclerotic lesions in panel C.
Figure 3
Figure 3. Loss of Akt1 in VSMCs results in marked VSMC and macrophage apoptosis in atherosclerotic plaques
A and B) Representative immunofluorescence analysis of atherosclerotic plaques isolated from Apoe−/−Akt1fl/fl and Apoe−/−Akt1fl/flSm22αCRE mice stained with smooth muscle cell alpha actin (SMA) and TUNEL (A) or with CD68 and TUNEL (B). Quantification is shown in the right panels. Data represent the mean ± SEM; (3 months: Apoe−/−Akt1fl/fl n=8 Apoe−/−Akt1fl/flSm22αCRE n=13; 5 months: Apoe−/−Akt1fl/fl n=14, Apoe−/−Akt1fl/flSm22αCRE n=10). Arrows indicate cells that are positive for SMA or CD68 and TUNEL labeling. C) qRT-PCR analysis of Mcp1, Il-6, Il-1β, Mmp9 and Mmp2 in whole aortic samples isolated from Apoe−/−Akt1fl/fl and Apoe−/−Akt1fl/flSm22αCRE mice fed a WD for 3 and 5 months. Data are expressed as relative expression and correspond to mean ± SEM (n=5 each group).
Figure 4
Figure 4. Silencing Akt1 in VSMCs in established lesions promotes fibrous cap thinning
A) Representative Western blot analysis of Akt1 expression after Tamoxifen (TMX) treatment from aortic lysates of Apoe−/−Akt1fl/flSM-MHC-CreERT2E mice. Hsp90 was used as a loading control. B) Experimental outline of the atherosclerosis study and Tamoxifen (TMX) administration in Apoe−/−Akt1fl/flSM-MHC-CreERT2 mice fed a WD for 5 months. C) Representative Western blot analysis of Akt1, Akt2, total Akt and downstream Akt targets from aortic lysates of male Apoe−/−Akt1fl/flSM-MHC-CreERT2 mice 3 weeks post-tamoxifen treatment. Hsp90 was used as a loading control. Band densitometry is shown in the right panels. *Indicates p<0.05 compared to non-treated Apoe−/−Akt1fl/flSM-MHC-CreERT2 mice. D–F) Representative histological analysis of cross-sections from the aortic sinus stained with hematoxylin and eosin (H&E) (D), Picrosirus red (E), and CD68 (green) and SMC (red) staining (F). Quantification of the lesion area, necrotic core, fibrous cap, collagen, macrophage and VSMC content are shown in the right panels. All of the data represent the mean ± SEM; (TMX− n=10, TMX+ n=8). *Indicates p< 0.05 compared to control untreated mice. The dashed white lines demarcate the intima of the atherosclerotic lesions.
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References

    1. Lusis AJ. Atherosclerosis. Nature. 2000;407:233–241. - PMC - PubMed
    1. Glass CK, Witztum JL. Atherosclerosis. the road ahead. Cell. 2001;104:503–516. - PubMed
    1. Libby P. Inflammation in atherosclerosis. Nature. 2002;420:868–874. - PubMed
    1. Clarke MC, Figg N, Maguire JJ, Davenport AP, Goddard M, Littlewood TD, Bennett MR. Apoptosis of vascular smooth muscle cells induces features of plaque vulnerability in atherosclerosis. Nature medicine. 2006;12:1075–1080. - PubMed
    1. Allard D, Figg N, Bennett MR, Littlewood TD. Akt regulates the survival of vascular smooth muscle cells via inhibition of FoxO3a and GSK3. J Biol Chem. 2008;283:19739–19747. - PubMed

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