Targeted Disruption of TGF-β–Smad3 Signaling Leads to Enhanced Neointimal Hyperplasia With Diminished Matrix Deposition in Response to Vascular Injury

Kobayashi, Kazuki; Yokote, Koutaro; Fujimoto, M.; Yamashita, Kimihiro; Sakamoto, Akemi; Kitahara, Masaki; Kawamura, Hajime; Maezawa, Yoshiro; Asaumi, Sunao; Tokuhisa, Takeshi; Mori, Seijiro; Saitō, Yasushi

doi:10.1161/01.res.0000163980.55495.44

Cited by 68 publications

(57 citation statements)

References 61 publications

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“…9,10,12 However, the role of SMAD3 in the vasculature is somewhat less clear. As reported by Kobayashi et al, 49 Smad3 null mice showed enhanced neointimal hyperplasia with decreased matrix deposition in response to femoral artery injury.…”

Section: 43supporting

confidence: 64%

Functional Analysis of a Novel Genome-Wide Association Study Signal in SMAD3 That Confers Protection From Coronary Artery Disease

Turner

Martinuk

Silva

et al. 2016

ATVB

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Objective-A recent genome-wide association study meta-analysis identified an intronic single nucleotide polymorphism in SMAD3, rs56062135C>T, the minor allele (T) which associates with protection from coronary artery disease. Relevant to atherosclerosis, SMAD3 is a key contributor to transforming growth factor-β pathway signaling. Here, we seek to identify ≥1 causal coronary artery disease-associated single nucleotide polymorphisms at the SMAD3 locus and characterize mechanisms whereby the risk allele(s) contribute to coronary artery disease risk. Approach and Results-By genetic and epigenetic fine mapping, we identified a candidate causal single nucleotide polymorphism rs17293632C>T (D′, 0.97; r 2 , 0.94 with rs56062135) in intron 1 of SMAD3 with predicted functional effects. We show that the sequence encompassing rs17293632 acts as a strong enhancer in human arterial smooth muscle cells. The common allele (C) preserves an activator protein (AP)-1 site and enhancer function, whereas the protective (T) allele disrupts the AP-1 site and significantly reduces enhancer activity (P<0.001). Pharmacological inhibition of AP-1 activity upstream demonstrates that this allele-specific enhancer effect is AP-1 dependent (P<0.001). Chromatin immunoprecipitation experiments reveal binding of several AP-1 component proteins with preferential binding to the (C) allele. We show that rs17293632 is an expression quantitative trait locus for SMAD3 in blood and atherosclerotic plaque with reduced expression of SMAD3 in carriers of the protective allele. Finally, siRNA knockdown of SMAD3 in human arterial smooth muscle cells increases cell viability, consistent with an antiproliferative role. Phosphorylated SMAD3 then forms a complex with the common SMAD4 that subsequently translocates to the nucleus and regulates transcription. 6,7 Relevant to a role in atherosclerosis, in systems genetics analysis of multiple GWAS, we identified TGF-β signaling and SMAD transcriptional activities as enriched pathways for CAD association. 8 However, despite extensive data on the functions of TGF-β with respect to atherosclerosis, 9,10 the roles of SMAD proteins particularly SMAD3 and SMAD3 signaling are less well-understood. Conclusions-TheSMAD3 is expressed at low levels in healthy human aorta by immunohistochemistry and quantitative reverse transcription polymerase chain reaction (PCR).11 There is, however, a marked increase in SMAD3 and other SMAD proteins in fibrofatty lesions, with expression mostly limited to CD68-positive macrophages/macrophage-derived foam cells in these samples. Conversely, in fibrous atherosclerotic plaques, there are high levels of SMAD3 in vascular smooth muscle cells (SMCs), suggesting that the role of SMAD3 in atherosclerosis depends on cell type and stage of atherosclerosis.11 Higher SMAD3 expression in SMCs of the fibrous plaque coincides with TGF-β-mediated synthesis of collagen and other extracellular matrix proteins, which contribute to plaque stability.12 Rare SMAD3 mutations cause aneurysms-osteoarthritis syn...

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Section: 43supporting

confidence: 64%

Functional Analysis of a Novel Genome-Wide Association Study Signal in SMAD3 That Confers Protection From Coronary Artery Disease

Turner

Martinuk

Silva

et al. 2016

ATVB

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“…16 In the vascular system, TGF-␤ inhibits the proliferation of endothelial cells while it stimulates that of the adjacent smooth muscle cells, 33,34 despite their close proximity. Conversely, it has a pro-apoptotic effect on endothelial cells while being anti-apoptotic in smooth muscle cells.…”

Section: Discussionmentioning

confidence: 99%

Transforming Growth Factor-β Regulates the Growth of Valve Interstitial Cells in Vitro

Gotlieb

2011

The American Journal of Pathology

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Although valve interstitial cell (VIC) growth is an essential feature of injured and diseased valves, the regulation of VIC growth is poorly understood. Transforming growth factor (TGF)-␤ promotes VIC proliferation in early-stage wound repair; thus, herein, we tested the hypothesis that TGF-␤ regulates VIC proliferation under normal nonwound conditions using low-density porcine VIC monolayers. Cell numbers were counted during a 10-day period, whereas proliferation and apoptosis were quantified by bromodeoxyuridine staining and TUNEL, respectively. The extent of retinoblastoma protein phosphorylation and expression of cyclin D1, CDK 4, and p27 were compared using Western blot analysis. Adhesion was quantified using a trypsin adhesion assay, and morphological change was demonstrated by immunofluorescence localization of ␣-smooth muscle actin and vinculin. TGF-␤-treated VICs were rhomboid; significantly decreased in number, proliferation, and retinoblastoma protein phosphorylation; and concomitantly had decreased expression of cyclin D1/CDK4 and increased expression of p27. TGF-␤-treated VICs adhered better to substratum and had more vinculin plaques and ␣-smooth muscle actin stress fibers than did controls. Thus, the regulation of VIC growth by TGF-␤ is context dependent. TGF-␤ prevents excessive heart valve growth under normal physiological conditions while it promotes cell proliferation in the early stages of repair, when increased VICs are required.

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“…36 Interestingly, partial TGF-␤ pathway inhibition in Smad3-null mice enhanced SMC proliferation and intimal thickening in a mouse femoral artery injury model. 37 Although the biological response in the mouse femoral model differs somewhat from that of the rat in carotid injury, it may be that inhibition of the entire TGF-␤ signaling pathway via TGF-␤ ligand or receptor activity is preferred for preventing vascular fibrosis.…”

Section: Discussionmentioning

confidence: 99%

SM16, an Orally Active TGF-β Type I Receptor Inhibitor Prevents Myofibroblast Induction and Vascular Fibrosis in the Rat Carotid Injury Model

Corbley

Sun

et al. 2008

ATVB

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Objective-TGF-␤ plays a significant role in vascular injury-induced stenosis. This study evaluates the efficacy of a novel, small molecule inhibitor of ALK5/ALK4 kinase, in the rat carotid injury model of vascular fibrosis. Methods and Results-The small molecule, SM16, was shown to bind with high affinity to ALK5 kinase ATP binding site using a competitive binding assay and biacore analysis. SM16 blocked TGF-␤ and activin-induced Smad2/3 phosphorylation and TGF-␤-induced plasminogen activator inhibitor (PAI)-luciferase activity in cells. Good overall selectivity was demonstrated in a large panel of kinase assays, but SM16 also showed nanomolar inhibition of ALK4 and weak (micromolar) inhibition of Raf and p38. In the rat carotid injury model, SM16 dosed once daily orally at 15 or 30 mg/kg SM16 for 14 days caused significant inhibition of neointimal thickening and lumenal narrowing. SM16 also prevented induction of adventitial smooth muscle ␣-actin-positive myofibroblasts and the production of intimal collagen, but did not decrease the percentage of proliferative cells. Key Words: TGF beta Ⅲ restenosis Ⅲ fibrosis Ⅲ neointimal formation Ⅲ activin T GF-␤ promotes key cellular events in vascular fibrosis including the induction of adventitial myofibroblasts and activated smooth muscle cells as well as their proliferation, survival, contraction, and increased collagen deposition. 1 TGF-␤ is overexpressed in animal models of vascular fibrosis as well as human diseases such as restenosis, cardiac hypertrophy, primary pulmonary hypertension, and organ transplant vasculopathy. [1][2][3][4][5][6][7] Activin, another TGF-␤ superfamily member, is also upregulated during vascular remodeling after balloon injury of the rat carotid artery. 8,9 The association of overexpression of these 2 cytokines with inflammation and fibrotic responses to injury suggest that inhibition of either or both of these TGF-␤ superfamily pathways may prevent vascular fibrosis. [1][2][3][4][5][6][7][8][9][10] TGF-␤ and activin signal through binding to their respective type II receptors, TGF-␤RII and ActRII. The subsequent interaction of this complex with their respective type I receptors, ALK5 and ALK4, allows the interaction with and phosphorylation of type I receptor-associated signaling proteins, Smad2 and Smad3. 11,12 Blockade of TGF-␤ signaling with either the soluble TGF-␤ type II receptor extracellular domain-Fc fusion protein (sTGF-␤RII:Fc), 5,7 antibodies to TGF-␤1, 13 decorin, 14 TGF-␤ antisense oligonucleotides, 15-17 or adenovirus Smad7 18 was shown previously to inhibit vascular fibrosis in animal models. Here, we identified SM16, a potent, selective and orally active ALK5/ALK4 kinase inhibitor which potently inhibits TGF-␤-induced phosphorylation of Smad2 and Smad3 in cells and neointimal thickening and vascular remodeling in the rat carotid balloon injury model. This orally active, small molecule TGF-␤/activin signaling inhibitor prevents neointimal thickening and lumenal loss primarily through inhibition of myofibroblast induction a...

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Targeted Disruption of TGF-β–Smad3 Signaling Leads to Enhanced Neointimal Hyperplasia With Diminished Matrix Deposition in Response to Vascular Injury

Cited by 68 publications

References 61 publications

Functional Analysis of a Novel Genome-Wide Association Study Signal in SMAD3 That Confers Protection From Coronary Artery Disease

Functional Analysis of a Novel Genome-Wide Association Study Signal in SMAD3 That Confers Protection From Coronary Artery Disease

Transforming Growth Factor-β Regulates the Growth of Valve Interstitial Cells in Vitro

SM16, an Orally Active TGF-β Type I Receptor Inhibitor Prevents Myofibroblast Induction and Vascular Fibrosis in the Rat Carotid Injury Model

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