Transforming Growth Factor-β Regulates the Growth of Valve Interstitial Cells in Vitro
2011; Elsevier BV; Volume: 179; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2011.06.007
ISSN1525-2191
Autores Tópico(s)Periodontal Regeneration and Treatments
ResumoAlthough 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)-β promotes VIC proliferation in early-stage wound repair; thus, herein, we tested the hypothesis that TGF-β 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 α-smooth muscle actin and vinculin. TGF-β–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-β–treated VICs adhered better to substratum and had more vinculin plaques and α-smooth muscle actin stress fibers than did controls. Thus, the regulation of VIC growth by TGF-β is context dependent. TGF-β 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. 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)-β promotes VIC proliferation in early-stage wound repair; thus, herein, we tested the hypothesis that TGF-β 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 α-smooth muscle actin and vinculin. TGF-β–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-β–treated VICs adhered better to substratum and had more vinculin plaques and α-smooth muscle actin stress fibers than did controls. Thus, the regulation of VIC growth by TGF-β is context dependent. TGF-β 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. Valve interstitial cells (VICs) are present in all three layers1Lester W. Rosenthal A. Granton B. Gotlieb A.I. Porcine mitral valve interstitial cells in culture.Lab Invest. 1988; 59: 710-719PubMed Google Scholar, 2Liu A.C. Joag V.R. Gotlieb A.I. The emerging role of valve interstitial cell phenotypes in regulating heart valve pathobiology.Am J Pathol. 2007; 171: 1407-1418Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar of the adult heart valve and are the most prevalent cell type present.3Mulholland D.L. Gotlieb A.I. Cell biology of valvular interstitial cells.Can J Cardiol. 1996; 12: 231-236PubMed Google Scholar, 4Filip D.A. Radu A. Simionescu M. Interstitial cells of the heart valves possess characteristics similar to smooth muscle cells.Circ Res. 1986; 59: 310-320Crossref PubMed Scopus (208) Google Scholar, 5Taylor P.M. Batten P. Brand N.J. Thomas P.S. Yacoub M.H. The cardiac valve interstitial cell.Int J Biochem Cell Biol. 2003; 35: 113-118Crossref PubMed Scopus (245) Google Scholar They are situated underneath the surface endocardium1Lester W. Rosenthal A. Granton B. Gotlieb A.I. Porcine mitral valve interstitial cells in culture.Lab Invest. 1988; 59: 710-719PubMed Google Scholar and embedded in an extracellular matrix (ECM) that they secrete and actively remodel.5Taylor P.M. Batten P. Brand N.J. Thomas P.S. Yacoub M.H. The cardiac valve interstitial cell.Int J Biochem Cell Biol. 2003; 35: 113-118Crossref PubMed Scopus (245) Google Scholar VICs are the master cells within the valve because they regulate both physiological and pathological processes.2Liu A.C. Joag V.R. Gotlieb A.I. The emerging role of valve interstitial cell phenotypes in regulating heart valve pathobiology.Am J Pathol. 2007; 171: 1407-1418Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar, 6Durbin A.D. Gotlieb A.I. Advances towards understanding heart valve response to injury.Cardiovasc Pathol. 2002; 11: 69-77Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar In their quiescent state, VICs show characteristics of fibroblasts and are critical for the maintenance of normal valve structure and function.2Liu A.C. Joag V.R. Gotlieb A.I. The emerging role of valve interstitial cell phenotypes in regulating heart valve pathobiology.Am J Pathol. 2007; 171: 1407-1418Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar, 5Taylor P.M. Batten P. Brand N.J. Thomas P.S. Yacoub M.H. The cardiac valve interstitial cell.Int J Biochem Cell Biol. 2003; 35: 113-118Crossref PubMed Scopus (245) Google Scholar During development or in response to injury and disturbed hemodynamic flow, VICs become activated2Liu A.C. Joag V.R. Gotlieb A.I. The emerging role of valve interstitial cell phenotypes in regulating heart valve pathobiology.Am J Pathol. 2007; 171: 1407-1418Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar, 6Durbin A.D. Gotlieb A.I. Advances towards understanding heart valve response to injury.Cardiovasc Pathol. 2002; 11: 69-77Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 7Rabkin-Aikawa E. Farber M. Aikawa M. Schoen F.J. Dynamic and reversible changes of interstitial cell phenotype during remodeling of cardiac valves.J Heart Valve Dis. 2004; 13: 841-847PubMed Google Scholar and take on features of myofibroblasts.2Liu A.C. Joag V.R. Gotlieb A.I. The emerging role of valve interstitial cell phenotypes in regulating heart valve pathobiology.Am J Pathol. 2007; 171: 1407-1418Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar, 5Taylor P.M. Batten P. Brand N.J. Thomas P.S. Yacoub M.H. The cardiac valve interstitial cell.Int J Biochem Cell Biol. 2003; 35: 113-118Crossref PubMed Scopus (245) Google Scholar, 6Durbin A.D. Gotlieb A.I. Advances towards understanding heart valve response to injury.Cardiovasc Pathol. 2002; 11: 69-77Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 7Rabkin-Aikawa E. Farber M. Aikawa M. Schoen F.J. Dynamic and reversible changes of interstitial cell phenotype during remodeling of cardiac valves.J Heart Valve Dis. 2004; 13: 841-847PubMed Google Scholar Activated VICs are characterized by increased α-smooth muscle actin (α-SMA) expression, stress fiber formation, contractility, and proteolytic enzyme secretion.2Liu A.C. Joag V.R. Gotlieb A.I. The emerging role of valve interstitial cell phenotypes in regulating heart valve pathobiology.Am J Pathol. 2007; 171: 1407-1418Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar, 4Filip D.A. Radu A. Simionescu M. Interstitial cells of the heart valves possess characteristics similar to smooth muscle cells.Circ Res. 1986; 59: 310-320Crossref PubMed Scopus (208) Google Scholar, 6Durbin A.D. Gotlieb A.I. Advances towards understanding heart valve response to injury.Cardiovasc Pathol. 2002; 11: 69-77Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 7Rabkin-Aikawa E. Farber M. Aikawa M. Schoen F.J. Dynamic and reversible changes of interstitial cell phenotype during remodeling of cardiac valves.J Heart Valve Dis. 2004; 13: 841-847PubMed Google Scholar, 8Liu A.C. Gotlieb A.I. Transforming growth factor-beta regulates in vitro heart valve repair by activated valve interstitial cells.Am J Pathol. 2008; 173: 1275-1285Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar Like other myofibroblasts,9Gabbiani G. The myofibroblast in wound healing and fibrocontractive diseases.J Pathol. 2003; 200: 500-503Crossref PubMed Scopus (1248) Google Scholar VICs regulate wound repair, as demonstrated by the increased number and enhanced production of ECM components, both in diseased valves and in in vitro wound models.10Fayet C. Bendeck M.P. Gotlieb A.I. Cardiac valve interstitial cells secrete fibronectin and form fibrillar adhesions in response to injury.Cardiovasc Pathol. 2007; 16: 203-211Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 11Lester W.M. Damji A.A. Tanaka M. Gedeon I. Bovine mitral valve organ culture: role of interstitial cells in repair of valvular injury.J Mol Cell Cardiol. 1992; 24: 43-53Abstract Full Text PDF PubMed Scopus (35) Google Scholar Thus, understanding the regulation of the biological and pathobiological features of VICs is essential for understanding the pathogenesis of heart valve diseases. Quiescent VICs are activated by several cytokines and growth factors, one of them being transforming growth factor (TGF)-β,2Liu A.C. Joag V.R. Gotlieb A.I. The emerging role of valve interstitial cell phenotypes in regulating heart valve pathobiology.Am J Pathol. 2007; 171: 1407-1418Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar, 12Werner S. Grose R. Regulation of wound healing by growth factors and cytokines.Physiol Rev. 2003; 83: 835-870Crossref PubMed Scopus (2606) Google Scholar a 25-kDa protein of the TGF-β superfamily.13Roberts A.B. Anzano M.A. Wakefield L.M. Roche N.S. Stern D.F. Sporn M.B. Type beta transforming growth factor: a bifunctional regulator of cellular growth.Proc Natl Acad Sci U S A. 1985; 82: 119-123Crossref PubMed Scopus (969) Google Scholar The TGF-β ligand binds and brings together TGF-β receptor I (TGF-βRI) and TGF-βRII Ser-Thr kinases.14Shi Y. Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus.Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4795) Google Scholar TGF-βRII phosphorylates TGF-βRI (also known as ALK5) and propagates the signal through phosphorylation of Smad proteins or via non-Smad mechanisms, such as mitogen-activated protein kinase, ρ-like Rho-GTPase, and phosphatidylinositol 3-kinase (PI3K)–AKT pathways.14Shi Y. Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus.Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4795) Google Scholar, 15Zhang Y.E. Non-Smad pathways in TGF-beta signaling.Cell Res. 2009; 19: 128-139Crossref PubMed Scopus (1309) Google Scholar TGF-β regulates a wide range of cellular processes, including cell proliferation, apoptosis, differentiation, migration, and ECM remodeling.8Liu A.C. Gotlieb A.I. Transforming growth factor-beta regulates in vitro heart valve repair by activated valve interstitial cells.Am J Pathol. 2008; 173: 1275-1285Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 12Werner S. Grose R. Regulation of wound healing by growth factors and cytokines.Physiol Rev. 2003; 83: 835-870Crossref PubMed Scopus (2606) Google Scholar, 14Shi Y. Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus.Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4795) Google Scholar, 16Heldin C.H. Landstrom M. Moustakas A. Mechanism of TGF-beta signaling to growth arrest, apoptosis, and epithelial-mesenchymal transition.Curr Opin Cell Biol. 2009; 21: 166-176Crossref PubMed Scopus (524) Google Scholar This multifunctionality allows TGF-β to participate in wound repair in multiple tissues and organs of the body.12Werner S. Grose R. Regulation of wound healing by growth factors and cytokines.Physiol Rev. 2003; 83: 835-870Crossref PubMed Scopus (2606) Google Scholar, 17Border W.A. Ruoslahti E. Transforming growth factor-beta in disease: the dark side of tissue repair.J Clin Invest. 1992; 90: 1-7Crossref PubMed Scopus (1045) Google Scholar Overexpression of TGF-β is often observed in in vivo wound sites.12Werner S. Grose R. Regulation of wound healing by growth factors and cytokines.Physiol Rev. 2003; 83: 835-870Crossref PubMed Scopus (2606) Google Scholar It is also present in several heart valve–related diseases (eg, calcific aortic stenosis,18Hinton Jr, R.B. Lincoln J. Deutsch G.H. Osinska H. Manning P.B. Benson D.W. Yutzey K.E. Extracellular matrix remodeling and organization in developing and diseased aortic valves.Circ Res. 2006; 98: 1431-1438Crossref PubMed Scopus (323) Google Scholar, 19Jian B. Narula N. Li Q.Y. Mohler 3rd, E.R. Levy R.J. Progression of aortic valve stenosis: TGF-beta1 is present in calcified aortic valve cusps and promotes aortic valve interstitial cell calcification via apoptosis.Ann Thorac Surg. 2003; 75 (discussion 465–466): 457-465Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar mitral valve prolapse,20Ng C.M. Cheng A. Myers L.A. Martinez-Murillo F. Jie C. Bedja D. Gabrielson K.L. Hausladen J.M. Mecham R.P. Judge D.P. Dietz H.C. TGF-beta-dependent pathogenesis of mitral valve prolapse in a mouse model of Marfan syndrome.J Clin Invest. 2004; 114: 1586-1592Crossref PubMed Scopus (473) Google Scholar, 21Rabkin E. Aikawa M. Stone J.R. Fukumoto Y. Libby P. Schoen F.J. Activated interstitial myofibroblasts express catabolic enzymes and mediate matrix remodeling in myxomatous heart valves.Circulation. 2001; 104: 2525-2532Crossref PubMed Scopus (490) Google Scholar and Marfan syndrome).22Matt P. Schoenhoff F. Habashi J. Holm T. Van Erp C. Loch D. Carlson O.D. Griswold B.F. Fu Q. De Backer J. Loeys B. Huso D.L. McDonnell N.B. Van Eyk J.E. Dietz H.C. Circulating transforming growth factor-beta in Marfan syndrome.Circulation. 2009; 120: 526-532Crossref PubMed Scopus (216) Google Scholar By using a well-characterized wound model,11Lester W.M. Damji A.A. Tanaka M. Gedeon I. Bovine mitral valve organ culture: role of interstitial cells in repair of valvular injury.J Mol Cell Cardiol. 1992; 24: 43-53Abstract Full Text PDF PubMed Scopus (35) Google Scholar we also reported an up-regulation of TGF-β at the in vitro wound edge. The exogenous addition of TGF-β further activates the VICs along the wound edge and stimulates their proliferation to enhance wound closure within the first 24 hours after wounding.8Liu A.C. Gotlieb A.I. Transforming growth factor-beta regulates in vitro heart valve repair by activated valve interstitial cells.Am J Pathol. 2008; 173: 1275-1285Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar Although the beneficial role of TGF-β in wound repair has been studied, its effects on cell growth remain controversial, owing to its context-dependent nature. Depending on TGF-β concentration,23Battegay E.J. Raines E.W. Seifert R.A. Bowen-Pope D.F. Ross R. TGF-beta induces bimodal proliferation of connective tissue cells via complex control of an autocrine PDGF loop.Cell. 1990; 63: 515-524Abstract Full Text PDF PubMed Scopus (666) Google Scholar, 24Zhou W. Park I. Pins M. Kozlowski J.M. Jovanovic B. Zhang J. Lee C. Ilio K. Dual regulation of proliferation and growth arrest in prostatic stromal cells by transforming growth factor-beta1.Endocrinology. 2003; 144: 4280-4284Crossref PubMed Scopus (32) Google Scholar cell type of interest,25Lee K.Y. Bae S.C. TGF-beta-dependent cell growth arrest and apoptosis.J Biochem Mol Biol. 2002; 35: 47-53Crossref PubMed Google Scholar and degree of cell differentiation,26Heredia A. Villena J. Romaris M. Molist A. Bassols A. The effect of TGF-beta 1 on cell proliferation and proteoglycan production in human melanoma cells depends on the degree of cell differentiation.Cancer Lett. 1996; 109: 39-47Abstract Full Text PDF PubMed Scopus (11) Google Scholar varying responses in proliferation and apoptosis have been reported. Because VIC growth in vitro is poorly understood, we investigated the regulation of VIC proliferation by TGF-β. VICs were harvested from porcine heart valve explants, as previously described.1Lester W. Rosenthal A. Granton B. Gotlieb A.I. Porcine mitral valve interstitial cells in culture.Lab Invest. 1988; 59: 710-719PubMed Google Scholar VICs at passages 3 to 5 were plated in triplicate 35-mm tissue culture dishes (Falcon; BD Biosciences, San Jose, CA) at a density of 2000 cells/cm2 and cultured in 2 mL of 5% fetal bovine serum containing media. Porcine TGF-β1 (R&D Systems, Minneapolis, MN) was reconstituted in a vehicle solution of 4 mmol/L HCl and 0.1% bovine serum albumin. Media containing the vehicle solution or active porcine TGF-β1 (0.1, 0.5, or 5 ng/mL) were first added to cultures 18 hours after plating and every 2 days thereafter. Cell counts were performed in triplicate during a 10-day period using the Countess Automated Cell Counter (Invitrogen, Eugene, OR) at 18, 42, and 66 hours after plating and then every 2 days. In an attempt to rescue the TGF-β–mediated reduction in cell number, both a neutralizing antibody and a potent receptor inhibitor were used. TGF-β–neutralizing antibody (R&D Systems) was reconstituted in sterile PBS to a 10-mg/mL stock solution. At 18 hours after plating and every 2 days thereafter, media containing 15-μg/mL neutralizing antibody was added to the culture. SB431542 is a potent inhibitor of TGF-βRI.27Inman G.J. Nicolás F.J. Callahan J.F. Harling J.D. Gaster L.M. Reith A.D. Laping N.J. Hill C.S. SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7.Mol Pharmacol. 2002; 62: 65-74Crossref PubMed Scopus (1332) Google Scholar, 28Watabe T. Nishihara A. Mishima K. Yamashita J. Shimizu K. Miyazawa K. Nishikawa S. Miyazono K. TGF-beta receptor kinase inhibitor enhances growth and integrity of embryonic stem cell-derived endothelial cells.J Cell Biol. 2003; 163: 1303-1311Crossref PubMed Scopus (149) Google Scholar, 29Halder S.K. Beauchamp R.D. Datta P.K. A specific inhibitor of TGF-beta receptor kinase, SB-431542, as a potent antitumor agent for human cancers.Neoplasia. 2005; 7: 509-521Abstract Full Text PDF PubMed Scopus (228) Google Scholar Solid anhydrous SB431542 (Sigma, St Louis, MO) was dissolved in sterile dimethyl sulfoxide (DMSO) and prepared into a 10 mmol/L stock solution. A specific amount of the stock solution was added to the culture media to achieve desired further dilutions. In experiments involving SB431542 treatment alone, media containing DMSO or 1 to approximately 10 μmol/L of SB431542 were added to the cell cultures, as previously described. For the experiments involving both SB431542 and TGF-β1, VICs were pretreated with the inhibitor 2 hours before the addition of TGF-β1. Cell counts were performed in triplicate during a 10-day period using a Coulter counter (Coulter Electronics Inc., Hialeah, FL) at the time points previously specified. To confirm the results of SB431542 inhibition, SD208 (Tocris Bioscience, Bristol, UK) was used. SD208 is also a potent and selective inhibitor of TGF-βRI.30Uhl M. Aulwurm S. Wischhusen J. Weiler M. Ma J.Y. Almirez R. Mangadu R. Liu Y.W. Platten M. Herrlinger U. Murphy A. Wong D.H. Wick W. Higgins L.S. Weller M. SD-208, a novel transforming growth factor beta receptor I kinase inhibitor, inhibits growth and invasiveness and enhances immunogenicity of murine and human glioma cells in vitro and in vivo.Cancer Res. 2004; 64: 7954-7961Crossref PubMed Scopus (356) Google Scholar Preparation and treatment with SD208 follows the previously mentioned procedure for SB431542. VICs were plated on 22 × 22-mm square glass coverslips (Fisher Scientific, Pittsburgh, PA) in 35-mm tissue culture dishes at a density of 2000 cells/cm2 and cultured under the same conditions as previously described. Vehicle and TGF-β–treated monolayers were immunofluorescently stained 4 or 6 days after first addition of TGF-β. Briefly, VICs on glass coverslips were fixed with methanol-acetone (1:1) at 4°C (in the case of α-SMA) or 4% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA) [in the case of vinculin and phosphorylated Smad (p-Smad)], rinsed three times with PBS for 5 minutes in tissue culture dishes, permeabilized with 0.1% Triton X-100 (Sigma) in PBS for 3.5 minutes (in the case of α-SMA and p-Smad) or with 0.2% Triton X-100 in PBS for 5 minutes (in the case of vinculin), and rinsed again three times in PBS at 5-minute intervals. The coverslips were incubated with mouse anti-α-SMA (1:400; Sigma), mouse anti-vinculin (1:50; Sigma), and rabbit anti-p-Smad2/3 (Ser423/425) (1:50; Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies at room temperature for 1 hour and were then washed three times with PBS at 5-minute intervals. Secondary antibodies were goat anti-mouse and goat anti-rabbit Alexa 488 (1:200; Molecular Probes, Invitrogen, Eugene, OR). Hoechst 33342 (1:2000; Lonza, Basel, Switzerland) was used to counterstain nuclei. After a 20-minute incubation with secondary antibody, coverslips were dipped in deionized water, mounted with Prolong Gold antifade reagent (Molecular Probes, Invitrogen), and stored at 4°C. Coverslips were examined using ×10 and ×60 objectives of an Olympus FluoView 1000 Laser Scanning Confocal Microscope (Olympus, Markham, ON, Canada) equipped with lasers providing exciting wavelengths of 405 and 488 nm, respectively. Serial optical sections were obtained at 0.1-μm intervals for a total of 4.5 to 8.5 μm. Images were captured at randomly selected regions using FV10-ASW 1.6 software (Olympus) to avoid saturation. TUNEL was performed using the TACS TdT-fluorescein in situ apoptosis detection kit (R&D Systems) to identify apoptotic VICs. Total nuclei were counterstained with Hoechst. A positive and a negative control were used for comparison. Bromodeoxyuridine (BrdU) labeling reagent (Amersham Sciences, Buckinghamshire, UK) was added in 1:1000 dilution to vehicle and TGF-β–treated cultures 2 and 4 days after first treatment at the time of media change and incubated for 6 hours. The coverslips were then washed in PBS, fixed in ethanol-acetic acid (95:5) for 20 minutes at 4°C in a coplin jar, and denatured with 2N HCl for 30 minutes in an incubator at 37°C. Incubation for 1 hour with mouse anti-BrdU (1:1000; Sigma) primary antibodies at 37°C was followed by secondary antibody, as previously described. VIC nuclei were counterstained with propidium iodide (1:1000; Sigma). Images were captured using the ×20 objective of a Nikon TE300 (Nikon, Melville, NY) inverted fluorescence microscope, starting two fields away from the left top corner, taking every other field until the whole coverslip was covered. A total of 15 fields were captured for each coverslip, with approximately 200 to 500 cells and 30 to 100 cells per fields for vehicle and TGF-β–treated monolayers, respectively. The total number of nuclei was counted using Simple PCI 6.5 (Hamamatsu, Shizuoka Prefecture, Japan), and the number of BrdU-stained nuclei was counted manually. Proliferation was quantitated as the percentage of labeled cells in the total number of cells counted. By using a previously characterized trypsin adhesion assay31Zacks S. Rosenthal A. Granton B. Havenith M. Opas M. Gotlieb A.I. Characterization of Cobblestone mitral valve interstitial cells.Arch Pathol Lab Med. 1991; 115: 774-779PubMed Google Scholar with slight modification, the degree of adhesiveness to the substratum was compared. Briefly, vehicle or TGF-β–treated VICs were incubated in 1.0 mL of 1:10 diluted trypsin-EDTA (Invitrogen) for 0.5 or 1.0 minutes, followed by immediate neutralization with 0.5 mL of media containing 5% fetal bovine serum. The number of VICs that were detached during the incubation period was counted using a Coulter counter. The remaining VICs in the monolayer were washed three times with PBS, detached using full trypsin-EDTA, and counted. The percentages of vehicle or TGF-β–treated VICs detached during the incubation period were compared. To harvest enough cells for protein extraction, TGF-β– or SB431542-treated VICs and their respective controls were cultured in 100-mm tissue culture dishes (Falcon) until they reached confluency. Whole cell protein extracts were obtained by lysing the VICs in ice-cold radioimmunoprecipitation assay buffer (50 mmol/L Tris, pH7.4; 150 mmol/L NaCl; 1% Triton; 0.25% Na-deoxycholate; and 1 mmol/L EDTA) supplemented with protease inhibitor (Roche Applied Science, Indianapolis, IN), 1 mmol/L sodium orthovanadate, 1 mmol/L phenylmethylsulfonyl fluoride, and 1 mmol/L sodium fluoride (Sigma). Nuclear and cytosolic protein extracts were obtained using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Rockford, IL), supplemented with protease inhibitor (Roche Applied Science), 1 mmol/L sodium orthovanadate, 1 mmol/L phenylmethylsulfonyl fluoride, and1 mmol/L sodium fluoride, following the extraction protocols specified by the kit. The protein concentration was determined by Bradford analysis, and 10 to 40 μg of protein per lane was resolved by SDS-PAGE. Transfer and blotting were performed on an iBlot gel transfer device (Invitrogen) and a SNAP i.d. protein detection system (Millipore, Danvers, MA), following the instructions specified. The blots were reacted with rabbit anti-phosphorylated retinoblastoma protein (pRb) (Ser807/811) (1:200), mouse anti-α-SMA (1:5000), anti-cyclin D1 (1:100), anti-CDK4 (1:200), and anti-p27 (1:100) (Santa Cruz Biotechnology). Mouse anti-β-tubulin (1:5000; Sigma) and anti-histone H1 (1:5000; Millipore) antibodies were used to confirm equal protein loading in the whole cell/cytosolic and nuclear portion, respectively. Goat anti-mouse and goat anti-rabbit secondary antibodies (horseradish peroxidase conjugated), at a dilution of 1:5000, were used. The immune complexes were detected using Luminata Western HRP Substrates (Millipore). The intensities of protein bands were quantified and compared using Image J 1.43 (US NIH, Bethesda, MA). The densitometric data were normalized such that the group that has a higher expression level is set at 100. The Student's t-test was used to compare vehicle with TGF-β1–treated groups. P < 0.05 was considered significant. All statistical analyses were performed using GraphPad Prism version 5 software (GraphPad Software Inc., San Diego, CA). Staining for the p-Smad2/3 complex, a key complex involved in TGF-β signaling, is used to confirm activation of the pathway. At both 4 and 6 days after treatment, TGF-β–treated VICs (Figure 1, B and D) showed significantly more prominent p-Smad nuclear localization (Figure 1E) compared with the control, in which nuclei are only faintly stained (Figure 1, A and C). Under phase-contrast microscopy, the vehicle and TGF-β–treated VICs initially showed no observable difference in cell density or morphological features (Figure 2, A–D). After 4 days, treatment with TGF-β resulted in a significant reduction in VIC density (Figure 2, E–J). Although vehicle-treated VICs became superconfluent 8 days after treatment, TGF-β–treated VICs failed to reach confluency, even when they were cultured for up to 12 days. Instead, prolonged treatment caused VICs to cluster and form nodules. The growth of TGF-β–treated VICs leveled off after 4 days of treatment, whereas that of the vehicle-treated cells remained in the log phase. The inhibitory effect is concentration dependent, as shown in Figure 3A. Maximal inhibition was observed with 0.5 ng/mL TGF-β treatment because a higher concentration (5 ng/mL) resulted in the same extent of inhibition (data not shown).Figure 3Growth curves of control and TGF-β–treated VICs. A: VIC growth in 0.1 or 0.5 ng/mL TGF-β and corresponding vehicle-treated subconfluent monolayers. TGF-β–treated VICs showed significantly delayed growth after 4 days of treatment. The growth-inhibitory effect is concentration dependent because 0.5 ng/mL treatment resulted in a significantly greater extent of inhibition than 0.1 ng/mL treatment. (Only the statistical significance between the two treatment groups is shown.) B: VIC growth in two independent groups. In the control group, DMSO was added to the culture 2 hours before the addition of TGF-β vehicle solution (4 mmol/L HCl and 0.1% bovine serum albumin). In the treatment group, 2 μmol/L SB431542 was added to the culture 2 hours before the addition of 5 ng/mL TGF-β. The TGF-β–mediated growth inhibition is completely abolished in the presence of SB431542, and the two growth curves overlap. C: VIC growth in DMSO and 1 μmol/L SB431542 treated subconfluent monolayers. The inhibitor alone inhibits VIC growth. D: VIC growth in vehicle- and TGF-β–neutralizing antibody (Ab)–treated monolayers. No significant change in cell number was observed. In all experiments, cell counts were performed in triplicate. Error bars denote SEM. Statistical significance between groups: *P < 0.05, **P < 0.01, and ***P < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As confirmed by intracellular labeling of the p-Smad2/3 complex (data not shown), TGF-β signaling via the Smad pathway was effectively blocked by the inhibitor, SB431542. Because SB431542 functions by disrupting the kinase activity of TGF-βRI, we incubated the cells with the inhibitor for 2 hours before adding any exogenous TGF-β in an effort to block any of its downstream signaling. The inhibitory effect of TGF-β was abolished in the presence of the inhibitor (Figure 3B)
Referência(s)