The expression of intron-containing messages has been shown to occur in a variety of diseases including lactic acidosis, Cowden Syndrome, and several cancers. However, it is unknown whether these intron-containing messages result in protein production in vivo. Indeed, intron-containing RNAs are typically retained in the nucleus, targeted for degradation, or are repressed translationally. Here, we show that during vascular lesion formation in rats, an alternative isoform of the helix-loop-helix transcription factor Id3 (Id3a) generated by intron retention is abundantly expressed. We demonstrate that Id3 is expressed early in lesion formation when the proliferative index of the neointima is highest and that Id3 promotes smooth muscle cell (SMC) proliferation and S-phase entry and inhibits transcription of the cell-cycle inhibitor p21 Cip1 . Using an Id3a-specific antibody developed by our laboratory, we show that Id3a protein is induced during vascular lesion formation and that Id3a expression peaks late when the proliferative index is low or declining and extensive apoptosis is observed. Furthermore, Id3a fails to promote SMC growth and S-phase entry or to inhibit p21 Cip1 promoter transactivation. In contrast, Id3a stimulates SMC apoptosis and inhibits endogenous Id3 production. Adenoviral delivery of Id3a inhibited lesion formation in balloon-injured rat carotid arteries in vivo. These data describe a novel feedback loop whereby intron retention generates an Id3 isoform that acts to limit SMC growth during vascular lesion formation, providing the first evidence that regulated intron retention can modulate a pathologic process in vivo.

Key Words: helix-loop-helix motifs Ⅲ muscle, smooth, vascular Ⅲ proliferation Ⅲ differentiation U nderstanding VSMC biology is important for understanding the vasculoproliferative disorders (such as in-stent restenosis, vein graft failure, and transplant arteriopathy) and atherosclerosis. In the vasculoproliferative disorders, vessel wall injury results in the release of cytokines and mitogens, such as IL-1, thrombin, PDGF, FGF, Ang II, and transforming growth factor-␤ from platelets, inflammatory cells, and cells within the vessel wall. [1][2][3][4][5] These potent factors can induce normally quiescent VSMC in the vessel wall to modulate to a less differentiated phenotype, re-enter the cell cycle, proliferate, migrate, and secrete extracellular matrix, contributing to neointimal formation. 6 In atherosclerosis, lipid deposition with subsequent macrophage infiltration initiates a similar, although less accelerated, cascade of events. Accumulation of phenotypically modulated VSMC within fatty streaks over time leads to progression to fibrous plaques that may become occlusive to the vascular lumen. 7,8 However, matrix secretion by VSMC late in atherosclerotic plaque development appears to increase plaque stability and thus protect against rupture and thrombosis-the cause of most acute coronary syndromes-so that VSMC may have both deleterious and protective roles in atherosclerosis. 7,9,10 Therefore, a further understanding of the mechanisms regulating the phenotypic modulation and growth of VSMC may provide important insights that could lead to the development of strategies to limit the VSMC accumulation leading to neointimal formation and enhance VSMC proliferation and matrix production to stabilize advanced atherosclerotic plaques.Regulated gene expression is essential for the maintenance of normal vessel structure and function and, not surprisingly, the expression of multiple genes is altered in the vasculoproliferative disorders and atherosclerosis. Recent comprehensive reviews broadly summarize regulation of gene transcription in the vessel wall and in VSMC and suggest that transcriptional modulation of genes involved in cholesterol and fatty acid metabolism, inflammation, and cell cycle progression may hold promise as favorable therapeutic approaches to limit atherosclerosis and the vascular response to injury. 11,12 Transcription factors implicated in regulating these processes (such as the sterol responsive element-binding protein [SREBP], nuclear factor kappa B, E2F, and others) and their target genes (low-density lipoprotein receptor, adhesion molecules, cytokines, and cell cycle proteins) are known. 11 However, the transcription factors and mechanisms that link phenotypic modulation and growth regulation in VSMC are poorly understood. Several classes of transcription factors are known or likely to be involved in the regulation of VSMC differentiation and, in some instances, proliferation. 13 Notably, the HLH class of transcription factors is able to modulate both the expression of VSMC-specific and cell...

Understanding the mechanisms that regulate cell cycle progression in vascular smooth muscle cells (VSMCs) is key to understanding and modulating vascular lesion formation.Results of the present study provide the first evidence that phosphorylation of the helix-loop-helix factor Id3 in VSMCs occurs in vitro and in vivo and provides a regulatory switch controlling Id3-induced regulation of p21Cip1 and VSMC growth.T argeting cell cycle progression has emerged as a promising strategy for limiting vascular smooth muscle cell (VSMC) proliferation in vivo. 1,2 The cyclin-dependent kinase inhibitor p21Cip1 regulates VSMC cell cycle progression and proliferation both in vitro and in vivo. 3,4 The promoter region of p21Cip1 contains 8 E-box elements, consensus sites for basic helix-loop-helix (bHLH) transcription factors, such as E47, and bHLH expression can promote p21Cip1 transcription. 5 The Id family of HLH proteins function at least in part as dominant-negative inhibitors of bHLH-mediated transcription. Id3 has recently emerged as an important regulator of VSMC growth. Antisense inhibition of Id3 can block VSMC proliferation and S-phase entry while increasing p21Cip1 protein levels. 6 In addition, Id3 expression is upregulated in VSMCs in vivo after vascular injury in mice and rats. 7,8 Id3 contains an N-terminal cdk2 consensus site where it can be phosphorylated at a serine residue (Ser5) by cyclin-cdk2 complexes. 9 Ser5 phosphorylation can alter the ability of Id3 to prevent bHLH dimers from binding DNA in vitro as well as the ability of Id3 to promote S-phase entry in cultured fibroblasts. 9 However, the specific mechanism(s) whereby cdk2 phosphorylation of Id3 regulates cell cycle progression remain unknown. Materials and MethodsProduction of antisera specific for Ser5 phosphorylated Id3 is described in detail in the expanded Materials and Methods section of the online data supplement available at http://circres.ahajournals.org.Western blot analysis, transient transfections, luciferase and CAT assays, immunohistochemistry, and FACS analysis were performed as previously described 8 and in the online data supplement.

Hyperlipemia and oxidized LDL (ox-LDL) are important independent cardiovascular risk factors. Ox-LDL has been shown to stimulate vascular smooth muscle cell (VSMC) proliferation. However, the effects of hyperlipemia and the molecular mechanisms mediating hyperlipemia and ox-LDL effects on VSMC growth are poorly understood. The helix-loop-helix (HLH) transcription factor, Id3, is a redox-sensitive gene expressed in VSMC in response to mitogen stimulation and vascular injury. Accordingly, we hypothesize that Id3 is an important mediator of ox-LDL and hyperlipemia-induced VSMC growth. Aortas harvested from hyperlipemic pigs demonstrated significantly more Id3 than normolipemic controls. Primary VSMC were stimulated with ox-LDL, native LDL, sera from hyperlipemic pigs, or normolipemic pigs. VSMC exposed to hyperlipemic sera demonstrated increased Id3 expression, VSMC growth and S-phase entry and decreased p21^cip1 expression and transcription. Cells stimulated with ox-LDL demonstrated similar findings of increased growth and Id3 expression and decreased p21^cip1 expression. Moreover, the effects of ox-LDL on growth were abolished in cells devoid of the Id3 gene. Results provide evidence that the HLH factor Id3 mediates the mitogenic effect of hyperlipemic sera and ox-LDL in VSMC via inhibition of p21^cip1 expression, subsequently increasing DNA synthesis and proliferation.

Objective-12/15-Lipoxygenase (12/15-LO) has been implicated in the pathogenesis of vascular disease. Vascular smooth muscle cell (VSMC) proliferation is a key component of the response to injury in vascular disease. -LO), highly homologous to human 15-lipoxygenase, and its products have been shown to be increased in animal models of native atherosclerosis and injury-induced restenosis. Further, pharmacological inhibition and knockout (KO) of the 12/15-LO gene in atherosclerosis and injury models result in significantly less vascular lesion formation. [1][2][3][4][5][6][7] There are several theories as to how 12/15-LO induces lesion formation. It has been shown that lipoxygenases have the ability to oxidize low-density lipoprotein (LDL), a step important in lesion initiation and progression, and KO of the lipoxygenase gene decreases oxidant stress. 6,8,9 It has also been demonstrated that 12/15-LO and its products have the ability to mediate the effects of angiotensin II (Ang II), platelet-derived growth factor, and cytokines, all important mediators of vascular injury. 10 -14 12/15-LO is expressed in macrophages, fibroblasts, endothelial cells, and vascular smooth muscle cells (VSMCs), all important contributors to vascular lesion formation and progression. 9,12,[15][16][17] Recently, its role in the VSMC has begun to be defined. Indeed, the 12/15-LO products of arachidonic and linoleic acids 12S-hydroxy-eicosatetraenoic acid (12S-HETE), 15S-HETE, and 13S-hydroperoxyoctadecadienoic acid (13S-HPODE) are produced in VSMCs. Lipoxygenase activity has been shown to be increased by and to mediate the hypertrophic effects of Ang II, the mitogenic effects of cytokines, and the chemotactic effects of platelet-derived growth factor in VSMCs, whereas 12/15-LO inhibition blocks these effects. 10,11,13,14 12/15-LO products have direct hypertrophic effects in VSMCs. 10,18 12/15-LO appears to also affect VSMC proliferation because 12/15-LO increases dramatically with arterial injury in VSMCs, and inhibition of 12/15-LO decreases smooth muscle cell proliferation and neointimal formation after balloon injury. 4 Recent studies show that VSMCs from 12/15-LO KO mice display decreased S-phase entry and growth-related responses relative to those from wildtype mice. 19 However, although much is known about the effects of 12/15-LO and its products, less is known about the specific mechanisms by which 12/15-LO regulates key nuclear transcription factors related to VSMC growth.