Abstract
Apoptosis of vascular smooth muscle cells (VSMCs) may lead to atherosclerotic plaque instability and rupture, resulting in myocardial infarction, stroke, and sudden death. However, the molecular mechanisms mediating survival of VSMCs in atherosclerotic plaques remain unknown. Although plaque VSMCs exhibit increased susceptibility to apoptosis and reduced expression of the IGF1 receptor (IGF1R) when compared with normal VSMCs, a causative effect has not been established. Here we show that increased expression of the IGF1R can rescue plaque VSMCs from oxidative stress-induced apoptosis, demonstrating that IGF-1 signaling is a critical regulator of VSMC survival. Akt mediates the majority of the IGF1R survival signaling, and ectopic activation of Akt was sufficient to protect VSMCs in vitro. Both IGF1R and phospho-Akt expression were reduced in human plaque (intimal) VSMCs when compared with medial VSMCs, suggesting that Akt mediates survival signaling in atherosclerosis. Importantly, downstream targets of Akt were identified that mediate its protective effect as inhibition of FoxO3a or GSK3 by Akt-dependent phosphorylation protected VSMCs in vitro. We conclude that Akt and its downstream targets FoxO3a and GSK3 regulate a survival pathway in VSMCs and that their deregulation due to a reduction of IGF1R signaling may promote apoptosis in atherosclerosis.
Highlights
Atherosclerotic plaque underlie the majority of myocardial infarctions, stroke, and sudden death [2]
IGF1 receptor (IGF1R) Mediates Survival of vascular smooth muscle cells (VSMCs)—Previously, we demonstrated that VSMCs derived from human plaques express lower levels of IGF1R when compared with VSMCs derived from normal human aortas [4, 7]. pVSMCs show reduced proliferation and increased sensitivity to apoptosis [3, 4]
When compared with plaque VSMCs microinjected with empty vector or IGF1R-YF, those injected with wild-type IGF1R showed increased protection against H2O2induced apoptosis in response to IGF1 (Fig. 1A)
Summary
Cell Culture—Human VSMCs were isolated from aortas of cardiac transplant patients or from atherosclerotic plaques following carotid endarterectomy with informed consent and approval of the Local Ethics Committee. Expression plasmids were transfected into rat VSMCs using SuperFect (Qiagen). Infectious replication-deficient retrovirus was harvested from Bosc packaging cells and used to infect rat VSMCs in the presence of 8 g/ml Polybrene (hexadimethrine bromide, Sigma). Human plaque-derived VSMCs were microinjected at 150 hectopascals for 0.1 s with expression plasmids at 1 mg/ml using Eppendorf Femtotips II and an Eppendorf Femtojet/Injectman 2. IGF1R wild type and YF mutant [18] were cloned into pBMN IRES puro [17]. Dominant negative Akt (DN-Akt, T308A, S473A [19]) was cloned into pCDNA3, and the wild type and the A3 mutant of FoxO3a [20] were cloned into pCDNA3.1. Antibodies—Antibodies to the following proteins were used: Akt (Cell Signaling antibodies 9272 and 4691), phospho-Akt Ser-473 (Cell Signaling antibodies 9271 and 4060), GSK3␣/ (Upstate Biotechnology antibody 05-412), GSK3␣ (AbCam antibody 28833), phosphoGSK3␣/ Ser-21/Ser-9 (Cell Signaling antibody 9331), phosphoGSK3␣ Ser-21 (Cell Signaling antibody 9316), FoxO1 (Cell Signaling antibody 9462 and AbCam antibody 39670), FoxO3a (Santa Cruz Biotechnology antibody sc-11351 and Cell Signaling antibody 9467), phospho-FoxO1/phospho-FoxO3a
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