TGF-β through Smad3 signaling stimulates vascular smooth muscle cell proliferation and neointimal formation (original) (raw)
Related papers
Cardiovascular Research, 2009
Time for primary review: 36 days Aims Although transforming growth factor-beta (TGF-b) is believed to stimulate intimal hyperplasia after arterial injury, its role in remodelling remains unclear. We investigate whether Smad3, a TGF-b signalling protein, might facilitate its effect on remodelling. Methods and results Using the rat carotid angioplasty model, we assess Smad3 expression following arterial injury. We then test the effect of arterial Smad3 overexpression on the response to injury, and use a conditioned media experimental design to confirm an Smad3-dependent soluble factor that mediates this response. We use small interfering RNA (siRNA) to identify this factor as connective tissue growth factor (CTGF). Finally, we attempt to replicate the effect of medial Smad3 overexpression through adventitial application of recombinant CTGF. Injury induced medial expression of Smad3; overexpression of Smad3 caused neointimal thickening and luminal expansion, suggesting adaptive remodelling. Smad3 overexpression, though exclusively medial, caused adventitial changes: myofibroblast transformation, proliferation, and collagen production, all of which are associated with adaptive remodelling. Supporting the hypothesis that Smad3 initiated remodelling and these adventitial changes via a secreted product of medial smooth muscle cells (SMCs), we found that media conditioned by Smad3expressing recombinant adenoviral vector (AdSmad3)-infected SMCs stimulated adventitial fibroblast transformation, proliferation, and collagen production in vitro. This effect was attenuated by pre-treatment of SMCs with siRNA specific for CTGF, abundantly produced by AdSmad3-infected SMCs, and significantly up-regulated in Smad3-overexpressing arteries. Moreover, periadventitial administration of CTGF replicated the effect of medial Smad3 overexpression on adaptive remodelling and neointimal hyperplasia. Conclusion Medial gene transfer of Smad3 promotes adaptive remodelling by indirectly influencing the behaviour of adventitial fibroblasts. This arterial cell-cell communication is likely to be mediated by Smad3-dependent production of CTGF.
Arteriosclerosis, Thrombosis, and Vascular Biology, 2006
Objective— The purpose of these studies is to investigate the mechanism by which transforming growth factor (TGF)β1 regulates the synthesis of the extracellular matrix protein fibronectin (FN). Methods and Results— TGFβ1 elicited a time-dependent induction of FN protein and mRNA in A10 rat aortic smooth muscle cells (SMCs). Ectopic expression of Smad3 in A10 cells stimulated both basal and TGFβ1-induced FN expression, whereas expression of Smad7 eliminated the TGFβ response. Because TGFβ activated PKCδ in SMCs, we tested the role of PKCδ in regulation of FN expression. Inhibition of PKCδ activity by rottlerin or dominant-negative adenovirus (AdPKCδ DN) blocked TGFβ1’s induction of FN, whereas overexpression of PKCδ enhanced TGFβ’s effect. Moreover, aortic SMCs isolated from PKCδ −/− mice exhibited diminished FN induction in response to TGFβ. Furthermore, we found that Smad3 protein and mRNA were markedly reduced in AdPKCδ DN-treated A10 cells and in PKCδ null cells. Finally, restori...
Cardiovascular Research, 2009
Aims Although transforming growth factor-beta (TGF-b) is believed to stimulate intimal hyperplasia after arterial injury, its role in remodelling remains unclear. We investigate whether Smad3, a TGF-b signalling protein, might facilitate its effect on remodelling. Methods and results Using the rat carotid angioplasty model, we assess Smad3 expression following arterial injury. We then test the effect of arterial Smad3 overexpression on the response to injury, and use a conditioned media experimental design to confirm an Smad3-dependent soluble factor that mediates this response. We use small interfering RNA (siRNA) to identify this factor as connective tissue growth factor (CTGF). Finally, we attempt to replicate the effect of medial Smad3 overexpression through adventitial application of recombinant CTGF. Injury induced medial expression of Smad3; overexpression of Smad3 caused neointimal thickening and luminal expansion, suggesting adaptive remodelling. Smad3 overexpression, though exclusively medial, caused adventitial changes: myofibroblast transformation, proliferation, and collagen production, all of which are associated with adaptive remodelling. Supporting the hypothesis that Smad3 initiated remodelling and these adventitial changes via a secreted product of medial smooth muscle cells (SMCs), we found that media conditioned by Smad3expressing recombinant adenoviral vector (AdSmad3)-infected SMCs stimulated adventitial fibroblast transformation, proliferation, and collagen production in vitro. This effect was attenuated by pre-treatment of SMCs with siRNA specific for CTGF, abundantly produced by AdSmad3-infected SMCs, and significantly up-regulated in Smad3-overexpressing arteries. Moreover, periadventitial administration of CTGF replicated the effect of medial Smad3 overexpression on adaptive remodelling and neointimal hyperplasia. Conclusion Medial gene transfer of Smad3 promotes adaptive remodelling by indirectly influencing the behaviour of adventitial fibroblasts. This arterial cell-cell communication is likely to be mediated by Smad3-dependent production of CTGF.
American Journal of Physiology - Heart and Circulatory Physiology, 2015
Dysfunctional vascular growth is a major contributor to cardiovascular disease, the leading cause of morbidity and mortality worldwide. Growth factor-induced activation of vascular smooth muscle cells (VSMCs) results in a phenotypic switch from a quiescent, contractile state to a proliferative state foundational to vessel pathology. Transforming growth factor-β (TGF-β) is a multifunctional signaling protein capable of growth stimulation via Smad signaling. Although Smad signaling is well characterized in many tissues, its role in VSM growth disorders remains controversial. Recent data from our lab and others implicate the metabolic regulator AMP-activated protein kinase (AMPK) in VSM growth inhibition. We hypothesized that AMPK inhibits VSMC proliferation by reducing TGF-β-mediated growth in a Smad-dependent fashion. Treatment of rat VSMCs with the AMPK agonist AICAR significantly decreased TGF-β-mediated activation of synthetic Smad2 and Smad3 and increased inhibitory Smad7. Flow c...
2015
12 13 Dysfunctional vascular growth is a major contributor to cardiovascular disease, the 14 leading cause of morbidity and mortality worldwide. Growth factor-induced activation of 15 vascular smooth muscle cells (VSMCs) results in a phenotypic switch from a quiescent, 16 contractile state to a proliferative state foundational to vessel pathology. Transforming 17 growth factor-beta (TGFβ) is a multifunctional signaling protein capable of growth 18 stimulation via Smad signaling. Although Smad signaling is well characterized in many 19 tissues, its role in VSM growth disorders remains controversial. Recent data from our 20 lab and others implicate the metabolic regulator AMP-activated protein kinase (AMPK) 21 in VSM growth inhibition. We hypothesized that AMPK inhibits VSMC proliferation by 22 reducing TGFβ-mediated growth in Smad-dependent fashion. Treatment of rat VSMCs 23 with the AMPK agonist AICAR significantly decreased TGFβ-mediated activation of 24 synthetic Smad2 and Smad3 a...
PLoS ONE, 2014
Atherosclerotic-associated diseases are the leading cause of death in the United States. Despite recent progress, interventional treatments for atherosclerosis can be complicated by restenosis resulting from neo-intimal hyperplasia. We have previously demonstrated that TGF-b and its downstream signaling protein Smad3:1) are up-regulated following vascular injury, 2) together drive smooth muscle cell (SMC) proliferation and migration and 3) enhance the development of intimal hyperplasia. In order to determine a mechanism through which TGF-b/Smad3 promote these effects, Affymetrix gene expression arrays were performed on primary rat SMCs infected with Smad3 and stimulated with TGF-b or infected with GFP alone. More than 200 genes were differentially expressed (.2.0 fold change, p,0.05) in TGF-b/Smad3 stimulated SMCs. We then performed GO term enrichment analysis using the DAVID bioinformatics database and found that TGF-b/ Smad3 activated the expression of multiple genes related to either development or cell differentiation, several of which have been shown to be associated with multipotent stem or progenitor cells. Quantitative real-time PCR confirmed upregulation of several developmental genes including FGF1, NGF, and Wnt11 (by 2.5, 6 and 7 fold, respectively) as well as stem/progenitor cell associated genes CD34 and CXCR4 (by 10 and 45 fold, respectively). In addition, up-regulation of these factors at protein levels were also confirmed by Western blotting, or by immunocytochemistry (performed for CXCR4 and NGF). Finally, TGF-b/Smad3 down regulated transcription of SMC contractile genes as well as protein production of smooth muscle alpha actin, calponin, and smooth muscle myosin heavy chain. These combined results suggest that TGF-b/Smad3 stimulation drives SMCs to a phenotypically altered state of de-differentiation through the up-regulation of developmental related genes.
Mechanisms of TGF-β-Induced Differentiation in Human Vascular Smooth Muscle Cells
Journal of Vascular Research, 2011
Background: Transforming growth factor- (TGF- ) plays an important role in vascular homeostasis through effects on vascular smooth muscle cells (SMC). Fine-tuning of TGF- signaling occurs at the level of ALK receptors or Smads, and is regulated with cell type specificity. Methods: Our goal was to understand TGF- signaling in regulating SMC differentiation marker expression in human SMC. Activation of Smads was characterized, and loss-and gain-of-function reagents used to define ALK pathways. In addition, Smad-independent mechanisms were determined. Results: TGF- type I receptors, ALK1 and ALK5, are expressed in human SMC, and TGF- 1 phosphorylates Smad1/5/8 and Smad2/3 in a timeand dosage-dependent pattern. ALK5 activity, not bone morphogenetic protein type I receptors, is required for Smad phosphorylation. Endoglin, a TGF- type III receptor, is a TGF- 1 target in SMC, yet endoglin does not modify TGF- 1 responsiveness. ALK5, not ALK1, is required for TGF- 1-induction of SMC differentiation markers, and ALK5 signals through an ALK5/Smad3-and MAP kinase-dependent pathway. Conclusion: The definition of the specific signaling downstream of TGF- regulating SMC differentiation markers will contribute to a better understanding of vascular disorders involving changes in SMC phenotype.
Essential Role of Smad3 in Angiotensin II-Induced Vascular Fibrosis
Circulation Research, 2006
Angiotensin II (Ang II) plays a pivotal role in vascular fibrosis, which leads to serious complications in hypertension and diabetes. However, the underlying signaling mechanisms are largely unclear. In hypertensive patients, we found that arteriosclerosis was associated with the activation of Smad2/3. This observation was further investigated in vitro by stimulating mouse primary aorta vascular smooth muscle cells (VSMCs) with Ang II. There were several novel findings. First, Ang II was able to activate an early Smad signaling pathway directly at 15 to 30 minutes. This was extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) dependent but transforming growth factor- (TGF-) independent because Ang II-induced Smad signaling was blocked by addition of ERK1/2 inhibitor and by dominant-negative (DN) ERK1/2 but not by DN-TGF- receptor II (TRII) or conditional deletion of TRII. Second, Ang II was also able to activate the late Smad2/3 signaling pathway at 24 hours, which was TGF- dependent because it was blocked by the anti-TGF- antibody and DN-TRII. Finally, activation of Smad3 but not Smad2 was a key and necessary mechanism of Ang II-induced vascular fibrosis because Ang II induced Smad3/4 promoter activities and collagen matrix expression was abolished in VSMCs null for Smad3 but not Smad2. Thus, we concluded that Ang II induces vascular fibrosis via both TGF--dependent and ERK1/2 MAPK-dependent Smad signaling pathways. Activation of Smad3 but not Smad2 is a key mechanism by which Ang II mediates arteriosclerosis.