Abstract-We previously showed that changes in vascular smooth muscle cell (SMC) PTEN/Akt signaling following vascular injury are associated with increased SMC proliferation and neointima formation. In this report, we used a genetic model to deplete PTEN specifically in SMCs by crossing PTEN LoxP/LoxP mice to mice expressing Cre recombinase under the control of the SM22␣ promoter. PTEN was downregulated with increases in phosphorylated Akt in major vessels, hearts, and lungs of mutant mice. SMC PTEN depletion promoted widespread medial SMC hyperplasia, vascular remodeling, and histopathology consistent with pulmonary hypertension. Increased vascular deposition of the chemokine stromal cell-derived factor (SDF)-1␣ and medial and intimal cells coexpressing SM-␣-actin and CXCR4, the SDF-1␣ receptor, was detected in SMC PTEN-depleted mice. PTEN deficiency in cultured aortic SMCs induced autocrine growth through increased production of SDF-1␣. Blocking SDF-1␣ attenuated autocrine growth and blocked growth of control SMCs induced by conditioned media from PTEN-deficient SMCs. In addition, SMC PTEN deficiency enhanced progenitor cell migration toward SMCs through increased SDF-1␣ production. SDF-1␣ production by other cell types is regulated by the transcription factor hypoxia-inducible factor (HIF)-1␣. We found SMC nuclear HIF-1␣ expression in PTEN-depleted mice and increased nuclear HIF-1␣ in PTEN-deficient SMCs. Key Words: smooth muscle cell Ⅲ PTEN Ⅲ neointima Ⅲ autocrine growth Ⅲ conditional knockout mouse S mooth muscle cell (SMC) accumulation in the arterial intima is a key event in the pathogenesis of atherosclerosis, postangioplasty/in-stent restenosis, and graft arteriosclerosis, 1 with changes in the biological function and phenotype of SMCs contributing to the pathology. 2 These conditions are characterized, to varying degrees, by dedifferentiation, migration, and proliferation of medial-derived SMCs to form the neointima. Recent data suggest that bone marrow-derived, circulating, and/or resident progenitor/ proinflammatory cells are recruited to the injured vessel, differentiate down a SMC lineage, and proliferate, thereby contributing to neointimal lesion formation. [3][4][5][6] Compelling evidence supports the contribution of both processes to intimal hyperplasia, and major advances have identified numerous factors involved in this complex pathobiology. However, the underlying mechanism(s) initiating lesion formation are not clearly defined. Increased SMC production of chemokines, such as stromal cell-derived factor (SDF)-1␣ (CXCL12), has been shown to be centrally involved in progenitor cell recruitment, 7 although their role in inducing an autocrine growth pathway within the artery wall itself is unknown. We focused on the hypothesis that SMCs are central mediators of the injury response. Perturbations in SMC signaling, result in the production of soluble factors that regulate significant SMC hyperplasia and progenitor/proinflammatory cell recruitment through an autocrine/paracrine mechanism.Under physio...

Platelet-derived growth factor (PDGF) inhibits expression of smooth muscle (SM) genes in vascular smooth muscle cells and blocks induction by arginine vasopressin (AVP). We have previously demonstrated that suppression of SM-␣-actin by PDGF-BB is mediated in part through a Ras-dependent pathway. This study examined the role of phosphatidylinositol 3-kinase (PI3K)y and its downstream effector, Akt, in regulating SM gene expression. PDGF caused a rapid sustained activation of Akt, whereas AVP caused only a small transient increase. PDGF selectively caused a sustained stimulation of p85/p110␣ PI3K. In contrast, p85/110␤ PI3K activity was not altered by either PDGF or AVP, whereas both agents caused a delayed activation of Class IB p101/110␥ PI3K. Expression of a gain-of-function PI3K or myristoylated Akt (myr-Akt) mimicked the inhibitory effect of PDGF on SM-␣-actin and SM22␣ expression. Pretreatment with LY 294002 reversed the inhibitory effect of PDGF. Expression of myr-Akt selectively inhibited AVP-induced activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinases, which we have shown are critical for induction of these genes. Nuclear extracts from PDGF-stimulated or myr-Akt expressing cells showed reduced serum response factor binding to SM-specific CArG elements. This was associated with appearance of serum response factor in the cytoplasm. These data indicate that activation of p85/p110␣/Akt mediates suppression of SM gene expression by PDGF.

In vascular smooth muscle cells (VSMC), platelet-derived growth factor (PDGF) suppresses expression of multiple smooth muscle contractile proteins, useful markers of differentiation. Conversely, hypertrophic agents induce expression of these genes. The goal of this study was to employ genomic approaches to identify classes of genes differentially regulated by PDGF and hypertrophic stimuli. Changes in gene expression were determined using Affymetrix RAE-230 GeneChips in rat aortic VSMC stimulated with PDGF. For comparison with a model hypertrophic stimulus, a microarray was performed with VSMC stably expressing constitutively active G␣ 16 , which strongly induces smooth muscle marker expression. We identified 75 genes whose expression was increased by exposure to PDGF and decreased by expression of G␣ 16 and 97 genes whose expression was decreased by PDGF and increased by G␣ 16 . These genes included many smooth muscle-specific proteins; several extracellular matrix, cytoskeletal, and chemotaxis-related proteins; cell signaling molecules; and transcription factors. Changes in gene expression for many of these were confirmed by PCR or immunoblotting. The contribution of signaling pathways activated by PDGF to the gene expression profile was examined in VSMC stably expressing gain-of-function H-Ras or myristoylated Akt. Among the genes that were confirmed to be differentially regulated were CCAAT/enhancer-binding protein ␦, versican, and nexilin. All of these genes also had altered expression in injured aortas, consistent with a role for PDGF in the response of injured VSMC. These data indicate that genes that are differentially regulated by PDGF and hypertrophic stimuli may represent families of genes and potentially be biomarkers for vascular injury.

Abstract-Promoters of many smooth muscle-specific genes (SM-genes) contain multiple CArG boxes, which represent a binding site for serum response factor (SRF). Transciptional control through these regions involves interactions with SRF and specific coactivators such as myocardin. We have previously reported that suppression of SM-gene expression by platelet derived growth factor (PDGF) is associated with redistribution of SRF, leading to lower intra-nuclear levels, and a reduction in SRF transactivation. To further assess the role of SRF depletion on VSMC phenotype, the current study used RNA interference (RNAi). Two SRF-specific sequences constructed as hairpins were stably expressed in rat VSMC. Clones expressing SRF RNAi had no detectable SRF expression by immunoblotting, and showed diminished levels of SM ␣-actin protein and promoter activity. Unexpectedly, depletion of VSMC resulted in increased rates of proliferation and migration. Several genes whose expression is increased by PDGF stimulation, including c-Jun, were similarly induced in cells lacking SRF. Key Words: vascular smooth muscle cells Ⅲ serum response factor Ⅲ RNA interference Ⅲ platelet derived growth factor P henotypic modulation of vascular smooth muscle cells (VSMC 1 ) is critical during development and in the onset of diseases such as atherosclerosis and hypertension. Atherosclerosis is a leading cause of heart disease and stroke and causes Ϸ50% of all deaths. 2,3 Therefore, understanding the physiology of large vessels will help to identify the cause of a primary cardiovascular disease. VSMC in mature animals express multiple contractile proteins to maintain vascular tone. These proteins are markers for the differentiated contractile phenotype. Unlike myocardial and skeletal muscle cells, VSMC are highly plastic and retain the ability to modulate their phenotype. 4 -6 Environmental factors can alter the expression levels of these contractile proteins, leading to a switch from a differentiated phenotype, characterized by high expression, to a proliferative, dedifferentiated phenotype characterized by low expression. This occurs in pathophysiologic states of large arteries such as atherosclerosis, where migration and proliferation of neointimal VSMC lead to vascular remodeling. 7,8 In adult cultured VSMC, vasoconstrictors, such as angiotensin II or arginine vasopressin (AVP), increase expression of smooth muscle (SM) markers such as SMA and SM22␣, whereas growth factors such as platelet derived growth factor (PDGF) decrease protein and mRNA levels of these proteins. 1,9 -11 We have shown that these effects are mediated through transcriptional regulation of SM specific promoter activity, and have defined specific signal transduction pathways that regulate SM specific gene expression by AVP or PDGF. AVP-induced increases in gene expression require c-jun amino terminal kinase and p38 MAP kinases, whereas PDGF-induced suppression is mediated by Ras, and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. 1,[11][12][13] The promoters of these SM...

This process involves coordinated changes in expression of multiple SM-specific genes. In cultured VSMC, arginine vasopressin (AVP) increases and PDGF decreases expression of SM ␣-actin (SMA), the earliest marker of SM cells (SMC). However, it is unknown whether these agents regulate other SM genes in a similar fashion. SM22␣ appears secondary to SMA during development and is also a marker for SMC. This study examined the regulation of SM22␣ expression by AVP and PDGF in cultured VSMC. Levels of SM22␣ mRNA and protein were increased by AVP and suppressed by PDGF. Consistent with these changes, AVP increased SM22␣ promoter activity, whereas PDGF inhibited basal promoter activity and blocked AVP-induced increase. Activation of both JNK and p38 MAPK pathways was necessary for AVP-mediated induction of SM22␣ promoter. Expression of constitutively active Ras produced similar suppressions on SM22␣ promoter activity as PDGF. Signaling relayed from PDGF/Ras activation involved Raf, or a protein that competes for this site, Ral-GDS, and phosphatidylinositol 3-kinase activation. Truncational analysis showed that the proximal location of three CArG boxes in the promoter was sufficient for AVP stimulation. Mutations in this CArG box reduced basal and AVP-stimulated promoter activity without effecting PDGF suppression. Overexpression of serum response factor enhanced basal and AVP-stimulated promoter activity but had no effect on PDGF-BB-induced suppression. These data indicate that AVP and PDGF initiate specific signaling pathways that control expression of multiple SM genes leading to phenotypic modulation.