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Transforming growth factor-β increases vascular smooth muscle cell proliferation through the Smad3 and extracellular signal-regulated kinase mitogen-activated.

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Presentation on theme: "Transforming growth factor-β increases vascular smooth muscle cell proliferation through the Smad3 and extracellular signal-regulated kinase mitogen-activated."— Presentation transcript:

1 Transforming growth factor-β increases vascular smooth muscle cell proliferation through the Smad3 and extracellular signal-regulated kinase mitogen-activated protein kinases pathways  Pasithorn A. Suwanabol, MD, Stephen M. Seedial, BS, Xudong Shi, MD, PhD, Fan Zhang, MD, PhD, Dai Yamanouchi, MD, PhD, Drew Roenneburg, MS, Bo Liu, PhD, K. Craig Kent, MD  Journal of Vascular Surgery  Volume 56, Issue 2, Pages e1 (August 2012) DOI: /j.jvs Copyright © 2012 Society for Vascular Surgery Terms and Conditions

2 Fig 1 Transforming growth factor-β (TGF-β) induces extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase phosphorylation in vascular smooth muscle cells (VSMCs). A, VSMCs were treated with TGF-β for 1 hour at the indicated concentrations. B, VSMCs were treated with TGF-β (5 ng/mL) at the indicated time points. Protein lysates were analyzed by Western blotting using antibodies against phospho-ERK (p-ERK), total ERK (t-ERK), or β-actin. Data are shown with the standard error (range bars) and are representative of three independent experiments. *P < .05. Journal of Vascular Surgery  , e1DOI: ( /j.jvs ) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

3 Fig 2 Smad3 is a signaling intermediate in transforming growth factor-β (TGF-β)–induced phosphorylation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase. A, Vascular smooth muscle cells (VSMCs) were infected with control virus adenovirus expressing green fluorescent protein (AdGFP) or Smad3 (AdSmad3), followed by stimulation with TGF-β (5 ng/mL) for 1 hour. B, VSMCs were transfected with 100 pmol of control small interfering RNA (siRNA; scramble) or with 100 pmol of Smad3 siRNA for 24 hours then stimulated with TGF-β for 1 hour. Protein lysates were analyzed by Western blotting using antibodies against phospho-ERK (p-ERK), total ERK (t-ERK), or β-actin. Data are shown with the standard error (range bars) and are representative of three independent experiments. *P < .05. Journal of Vascular Surgery  , e1DOI: ( /j.jvs ) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

4 Fig 3 Western blotting (WB) and immunoprecipitation (IP) show a direct protein–protein interaction between Smad3 and phosphorylated extracellular signal-regulated kinase (p-ERK) mitogen-activated protein kinase. Vascular smooth muscle cells (VSMCs) were infected with control adenovirus expressing green fluorescent protein (AdGFP) or Smad3 (AdSmad3), followed by stimulation with transforming growth factor-β (TGF-β; 5 ng/mL) for 1 hour. Cell lysates were immunoprecipitated with anti-Smad3 antibody (Ab) or immunoglobulin G and immunoblotted for p-ERK or phosphorylated Smad3 (p-Smad3). Data shown are representative of three independent experiments. Journal of Vascular Surgery  , e1DOI: ( /j.jvs ) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

5 Fig 4 Extracellular signal-regulated kinase inhibition decreases transforming growth factor-β (TGF-β)/Smad3-induced vascular smooth muscle cell (VSMC) proliferation. VSMCs were infected with adenovirus expressing green fluorescent protein (AdGFP; control) or Smad3 (AdSmad3). These cells were then pretreated for 30 minutes with the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase inhibitor PD98059 (10 μm/mL) or control, followed by stimulation with TGF-β (5 ng/mL) or solvent for 96 hours. Proliferation was evaluated by 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay (n = 3). *P < .05 comparison of Smad3 or Smad3 + TGF-β vs GFP alone; **P < .05 comparison of Smad3 or Smad3 + TGF-β with or without PD Data are shown with the standard error (range bars). Journal of Vascular Surgery  , e1DOI: ( /j.jvs ) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

6 Fig 5 Overexpression of Smad3 in vivo increases phosphorylation of extracellular signal-regulated kinase (pERK) mitogen-activated protein kinase and cell proliferation in the injured artery wall. Rat carotid arteries were balloon-injured, followed by infusion with adenovirus expressing Smad3 for 20 minutes (AdSmad3, 2.5 × 109 plaque-forming units) or adenovirus expressing green fluorescent protein (AdGFP, 2.5 × 109 plaque-forming units). Animals were euthanized at day 3, and arteries were subjected to paraffin embedding and sectioning. Immunohistochemistry was performed with immunoglobulin G (IgG), Smad3, pERK or proliferating cell nuclear antigen (PCNA) antibodies. The percentage of immunopositive cells was calculated. Left, Immunohistochemistry for IgG, Smad3, p-ERK, and PCNA. Right, Bar graphs show quantitative results for p-ERK MAPK (AdGFP 32.4 ±2.65 vs AdSmad ± 6.5) and PCNA (AdGFP 43.1 ±2.4 vs AdSmad ±4.65). Data shown are representative of three animals per group (**P < .01). Journal of Vascular Surgery  , e1DOI: ( /j.jvs ) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

7 Supplementary Fig 1 Transforming growth factor-β (TGF-β) induces phosphorylation of Smad3 in vascular smooth muscle cells (VSMCs). A, VSMCs were treated with TGF-β for 1 hour at the indicated concentrations. B, VSMCs overexpressing Smad3 were treated with TGF-β (5 ng/mL) at the indicated time points. Protein lysates were analyzed by Western blotting using antibodies against phospho-Smad3 (p-Smad3) or β-actin. Data shown are representative of three independent experiments. Journal of Vascular Surgery  , e1DOI: ( /j.jvs ) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

8 Supplementary Fig 2 Smad3 expression is upregulated after infection with adenovirus expressing Smad3 (AdSmad3), and decreased after incubation with small interfering RNA (siRNA) to Smad3. A, Vascular smooth muscle cells (VSMCs) were infected with control adenovirus expressing green fluorescent protein (AdGFP) or AdSmad3, followed by stimulation with transforming growth factor-β (TGF-β, 5 ng/mL) for 1 hour. B, VSMCs were transfected with 100 pmol of scramble or Smad3 siRNA for 24 hours. Protein lysates were analyzed by Western blotting using antibodies for Smad3 or β-actin. Data shown are representative of three independent experiments. Journal of Vascular Surgery  , e1DOI: ( /j.jvs ) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

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