The phenotypic plasticity of mature vascular smooth muscle cells (VSMCs) facilitates angiogenesis and wound healing, but VSCM dedifferentiation also contributes to vascular pathologies such as intimal hyperplasia. Insulin/insulin-like growth factor I (IGF-I) is unique among growth factors in promoting VSMC differentiation via preferential activation of phosphatidylinositol 3-kinase (PI3K) and Akt. We have previously reported that rapamycin promotes VSMC differentiation by inhibiting the mammalian target of rapamycin (mTOR) target S6K1. Here, we show that rapamycin activates Akt and induces contractile protein expression in human VSMC in an insulin-like growth factor I-dependent manner, by relieving S6K1-dependent negative regulation of insulin receptor substrate-1 (IRS-1). In skeletal muscle and adipocytes, rapamycin relieves mTOR/S6K1-dependent inhibitory phosphorylation of IRS-1, thus preventing IRS-1 degradation and enhancing PI3K activation. We report that this mechanism is functional in VSMCs and crucial for rapamycin-induced differentiation. Rapamycin inhibits S6K1-dependent IRS-1 serine phosphorylation, increases IRS-1 protein levels, and promotes association of tyrosine-phosphorylated IRS-1 with PI3K. A rapamycin-resistant S6K1 mutant prevents rapamycin-induced Akt activation and VSMC differentiation. Notably, we find that rapamycin selectively activates only the Akt2 isoform and that Akt2, but not Akt1, is sufficient to induce contractile protein expression. Akt2 is required for rapamycin-induced VSMC differentiation, whereas Akt1 appears to oppose contractile protein expression. The anti-restenotic effect of rapamycin in patients may be attributable to this unique pattern of PI3K effector regulation wherein anti-differentiation signals from S6K1 are inhibited, but pro-differentiation Akt2 activity is promoted through an IRS-1 feedback signaling mechanism. Vascular smooth muscle cells (VSMCs)3 maintain a phenotypic plasticity that is important in physiological processes such as arteriogenesis, and in pathological responses, including atherosclerosis, intimal hyperplasia, and restenosis. Mature VSMCs are quiescent and exhibit a differentiated, contractile phenotype. Differentiation status in vitro can be measured by expression of smooth muscle-specific contractile proteins, including calponin, caldesmon, and smooth muscle myosin heavy chain (SM-MHC) (1). In response to injury, or upon in vitro culture, VSMCs re-enter the cell cycle, proliferate, migrate toward attractants, down-regulate expression of contractile proteins, and up-regulate protein synthesis, particularly of the extracellular matrix. This de-differentiated phenotype is referred to as "synthetic" because of this property (1).VSMC de-differentiation and resultant intimal hyperplasia in response to vessel injury are common problems following vascular interventions such as angioplasty, stent placement, and bypass grafts. Since receiving FDA approval in 2003, the use of the mTOR inhibitor rapamycin on drug-eluting stents has had a profound impac...

Vascular smooth muscle cells (VSMC) in mature, normal blood vessels exhibit a differentiated, quiescent, contractile morphology, but injury induces a phenotypic modulation toward a proliferative, dedifferentiated, migratory phenotype with upregulated extracellular matrix protein synthesis (synthetic phenotype), which contributes to intimal hyperplasia. The mTOR (the mammalian target of rapamycin) pathway inhibitor rapamycin inhibits intimal hyperplasia in animal models and in human clinical trials. We report that rapamycin treatment induces differentiation in cultured synthetic phenotype VSMC from multiple species. VSMC treated with rapamycin assumed a contractile morphology, quantitatively reflected by a 67% decrease in cell area. Total protein and collagen synthesis were also inhibited by rapamycin. Rapamycin induced expression of the VSMC differentiation marker contractile proteins smooth muscle (SM) alpha-actin, calponin, and SM myosin heavy chain (SM-MHC), as observed by immunoblotting and immunohistochemistry. Notably, we detected a striking rapamycin induction of calponin and SM-MHC mRNA, suggesting a role for mTOR in transcriptional control of VSMC gene expression. Rapamycin also induced expression of the cyclin-dependent kinase inhibitors p21(cip) and p27(kip), consistent with cell cycle withdrawal. Rapamycin inhibits mTOR, a signaling protein that regulates protein synthesis effectors, including p70 S6K1. Overexpression of p70 S6K1 inhibited rapamycin-induced contractile protein and p21(cip) expression, suggesting that this kinase opposes VSMC differentiation. In conclusion, we report that regulation of VSMC differentiation is a novel function of the rapamycin-sensitive mTOR signaling pathway.

The human prostacyclin receptor is a seven-transmembrane ␣-helical G-protein coupled receptor, which plays important roles in both vascular smooth muscle relaxation as well as prevention of blood coagulation. The position of the native ligand-binding pocket for prostacyclin as well as other derivatives of the 20-carbon eicosanoid, arachidonic acid, has yet to be determined. Through the use of prostanoid receptor sequence alignments, site-directed mutagenesis, and the 2.8-Å x-ray crystallographic structure of bovine rhodopsin, we have developed a three-dimensional model of the agonistbinding pocket within the seven-transmembrane (TM) domains of the human prostacyclin receptor. Upon mutation to alanine, 11 of 29 candidate residues within TM domains II, III, IV, V, and VII exhibited a marked decrease in agonist binding. Of this group, four amino acids, Arg-279 (TMVII), Phe-278 (TMVII), Tyr-75 (TMII), and Phe-95 (TMIII), were identified (via receptor amino acid sequence alignment, ligand structural comparison, and computer-assisted homology modeling) as having direct molecular interactions with ligand side-chain constituents. This binding pocket is distinct from that of the biogenic amine receptors and rhodopsin where the native ligands (also composed of a carbon ring and a carbon chain) are accommodated in an opposing direction. These findings should assist in the development of novel and highly specific ligands including selective antagonists for further molecular pharmacogenetic studies of the human prostacyclin receptor.Vascular smooth muscle relaxation and inhibition of platelet aggregation are two key physiological processes mediated by human prostacyclin. Dysfunctional prostacyclin activity has been implicated in the development of a number of cardiovascular diseases including thrombosis, myocardial infarction, stroke, myocardial ischemia, atherosclerosis, and systemic and pulmonary hypertension (1). In contrast to other members of the rhodopsin-like G-protein coupled receptor (GPCR) 1 subfamily such as the adrenergic receptors or other members of the prostanoid family, there are currently no high affinity selective antagonists for the prostacyclin receptor. This finding suggests that the prostacyclin receptor may possess a unique ligandbinding pocket.Receptor activation is contingent upon ligand binding interactions, which initiate a conformational change in protein structure that is subsequently transmitted to the G-protein.Determining the exact nature and location of receptor-ligand binding interactions at the molecular level is essential for understanding the functions of prostanoid receptor physiology. Moreover, such insights would lend to the development of novel and highly specific modes of treatment for prostanoid-related disorders. Based upon the position of the chromophore (covalently bound 11-cis-retinal) within the binding pocket of rhodopsin along with the location of other ligands within similar rhodopsin-type GPCRs (2), the putative binding pocket for GPCRs with small nonpeptide ligands is beli...

Interactions between endothelial cells (ECs) and smooth muscle cells (SMCs) are fundamental in diverse cardiovascular processes such as arteriogenesis, collateral blood vessel development, atherosclerosis, and restenosis. Alterations in SMC phenotype occur in each of these processes. Endothelial denudation has been suggested to contribute to the SMC proliferative response to vessel injury by angioplasty or other catheterization procedures. We have employed a co-culture approach to dissect the molecular signals that are dependent on the spatial relationship between ECs and SMCs, and have identified the importance of the PI3K/Akt pathway in EC-induced SMC differentiation. This pathway may suggest targets for therapeutic interventions for intimal hyperplasia and restenosis.