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• W2079658573 abstract "Bone marrow-derived progenitor cells have recently been shown to be involved in the development of intimal hyperplasia after vascular injury. Transforming growth factor-β (TGF-β) has profound stimulatory effects on intimal hyperplasia, but it is unknown whether these effects involve progenitor cell recruitment. In this study we found that although TGF-β had no direct effect on progenitor cell recruitment, conditioned media derived from vascular smooth muscle cells (VSMC) stimulated with TGF-β induced migration of both total bone marrow (BM) cells and BM-mesenchymal stem cells (MSC) and also induced MSC differentiation into smooth muscle like cells. Furthermore, overexpression of the signaling molecule Smad3 in VSMC via adenovirus-mediated gene transfer (AdSmad3) enhanced the TGF-β's chemotactic effect. Microarray analysis of VSMC stimulated by TGF-β/AdSmad3 revealed monocyte chemoattractant protein-1 (MCP-1) as a likely factor responsible for progenitor cell recruitment. We then demonstrated that TGF-β through Smad3 phosphorylation induced a robust expression of MCP-1 in VSMC. Recombinant MCP-1 mimicked the stimulatory effect of conditioned media on BM and MSC migration. In the rat carotid injury model, Smad3 overexpression significantly increased MCP-1 expression after vascular injury, consistent with our in vitro results. Interestingly, TGF-β/Smad3-induced MCP-1 was completely blocked by both Ro-32-0432 and rotterlin, suggesting protein kinase C-δ (PKCδ) may play a role in TGF-β/Smad3-induced MCP-1 expression. In summary, our data demonstrate that TGF-β, through Smad3 and PKCδ, stimulates VSMC production of MCP-1, which is a chemoattractant for bone marrow-derived cells, specifically MSC. Manipulation of this signaling system may provide a novel approach to inhibition of intimal hyperplasia. Bone marrow-derived progenitor cells have recently been shown to be involved in the development of intimal hyperplasia after vascular injury. Transforming growth factor-β (TGF-β) has profound stimulatory effects on intimal hyperplasia, but it is unknown whether these effects involve progenitor cell recruitment. In this study we found that although TGF-β had no direct effect on progenitor cell recruitment, conditioned media derived from vascular smooth muscle cells (VSMC) stimulated with TGF-β induced migration of both total bone marrow (BM) cells and BM-mesenchymal stem cells (MSC) and also induced MSC differentiation into smooth muscle like cells. Furthermore, overexpression of the signaling molecule Smad3 in VSMC via adenovirus-mediated gene transfer (AdSmad3) enhanced the TGF-β's chemotactic effect. Microarray analysis of VSMC stimulated by TGF-β/AdSmad3 revealed monocyte chemoattractant protein-1 (MCP-1) as a likely factor responsible for progenitor cell recruitment. We then demonstrated that TGF-β through Smad3 phosphorylation induced a robust expression of MCP-1 in VSMC. Recombinant MCP-1 mimicked the stimulatory effect of conditioned media on BM and MSC migration. In the rat carotid injury model, Smad3 overexpression significantly increased MCP-1 expression after vascular injury, consistent with our in vitro results. Interestingly, TGF-β/Smad3-induced MCP-1 was completely blocked by both Ro-32-0432 and rotterlin, suggesting protein kinase C-δ (PKCδ) may play a role in TGF-β/Smad3-induced MCP-1 expression. In summary, our data demonstrate that TGF-β, through Smad3 and PKCδ, stimulates VSMC production of MCP-1, which is a chemoattractant for bone marrow-derived cells, specifically MSC. Manipulation of this signaling system may provide a novel approach to inhibition of intimal hyperplasia. Intimal hyperplasia after vascular injury is a complex process involving vascular smooth muscle cell proliferation, migration, and extracellular matrix deposition. It is believed that vascular injuries transform vascular smooth muscle cells (VSMC) 3The abbreviations used are: VSMCvascular smooth muscle cell(s)SMCsmooth muscle cell(s)BMPCbone marrow (BM)-derived progenitor cell(s)RSMCrat SMC(s)MSCmesenchymal stem cellAdadenovirusSDF-1αstromal-derived factor-1αMCPmonocyte chemoattractant proteinTGF-βtransforming growth factor-βDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumELISAenzyme-linked immunosorbent assayPKCprotein kinase CGFPgreen fluorescent proteinsiRNAsmall interfering RNARTreverse transcriptionα-SMAα-smooth muscle actin. from a quiescent to a synthetic and proliferative phenotype (1Newby A.C. Zaltsman A.B. J. Pathol. 2000; 190: 300-309Crossref PubMed Scopus (518) Google Scholar). These “activated” VSMC migrate to the subintima space where they continue to proliferate and, thus, form the neointimal lesion (2Ryer E.J. Hom R.P. Sakakibara K. Nakayama K.I. Nakayama K. Faries P.L. Liu B. Kent K.C. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 780-786Crossref PubMed Scopus (46) Google Scholar). Recent evidence, however, suggests that bone marrow-derived progenitor cells (BMPC) are recruited to sites of vascular injury and may also play an essential role in the development of intimal hyperplasia (3Zernecke A. Schober A. Bot I. von Hundelshausen P. Liehn E.A. Möpps B. Mericskay M. Gierschik P. Biessen E.A. Weber C. Circ. Res. 2005; 96: 784-791Crossref PubMed Scopus (325) Google Scholar, 4Massberg S. Konrad I. Schürzinger K. Lorenz M. Schneider S. Zohlnhoefer D. Hoppe K. Schiemann M. Kennerknecht E. Sauer S. Schulz C. Kerstan S. Rudelius M. Seidl S. Sorge F. Langer H. Peluso M. Goyal P. Vestweber D. Emambokus N.R. Busch D.H. Frampton J. Gawaz M. J. Exp. Med. 2006; 203: 1221-1233Crossref PubMed Scopus (369) Google Scholar, 5Wang C.H. Anderson N. Li S.H. Szmitko P.E. Cherng W.J. Fedak P.W. Fazel S. Li R.K. Yau T.M. Weisel R.D. Stanford W.L. Verma S. Circ. Res. 2006; 99: 617-625Crossref PubMed Scopus (67) Google Scholar, 6Sata M. Saiura A. Kunisato A. Tojo A. Okada S. Tokuhisa T. Hirai H. Makuuchi M. Hirata Y. Nagai R. Nat. Med. 2002; 8: 403-409Crossref PubMed Scopus (1064) Google Scholar, 7Tanaka K. Sata M. Hirata Y. Nagai R. Circ. Res. 2003; 93: 783-790Crossref PubMed Scopus (259) Google Scholar). vascular smooth muscle cell(s) smooth muscle cell(s) bone marrow (BM)-derived progenitor cell(s) rat SMC(s) mesenchymal stem cell adenovirus stromal-derived factor-1α monocyte chemoattractant protein transforming growth factor-β Dulbecco's modified Eagle's medium fetal bovine serum enzyme-linked immunosorbent assay protein kinase C green fluorescent protein small interfering RNA reverse transcription α-smooth muscle actin. The mechanisms underlying BMPC recruitment to the site of arterial injury are now being elucidated. The following three steps are believed to occur with BMPC in response to arterial injury; 1) mobilization of cells from the bone marrow, 2) recruitment of these cells to the site of injury, and 3) differentiation of progenitor cells into cells of the arterial wall (8Tsai S. Butler J. Rafii S. Liu B. Kent K.C. J. Vasc. Surg. 2009; 49: 502-510Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). A number of chemokines are responsible for the second phase of this process, and proteins that have been implicated include but are not limited to stromal-derived factor-1α (SDF-1α) (3Zernecke A. Schober A. Bot I. von Hundelshausen P. Liehn E.A. Möpps B. Mericskay M. Gierschik P. Biessen E.A. Weber C. Circ. Res. 2005; 96: 784-791Crossref PubMed Scopus (325) Google Scholar, 4Massberg S. Konrad I. Schürzinger K. Lorenz M. Schneider S. Zohlnhoefer D. Hoppe K. Schiemann M. Kennerknecht E. Sauer S. Schulz C. Kerstan S. Rudelius M. Seidl S. Sorge F. Langer H. Peluso M. Goyal P. Vestweber D. Emambokus N.R. Busch D.H. Frampton J. Gawaz M. J. Exp. Med. 2006; 203: 1221-1233Crossref PubMed Scopus (369) Google Scholar), c-kit (5Wang C.H. Anderson N. Li S.H. Szmitko P.E. Cherng W.J. Fedak P.W. Fazel S. Li R.K. Yau T.M. Weisel R.D. Stanford W.L. Verma S. Circ. Res. 2006; 99: 617-625Crossref PubMed Scopus (67) Google Scholar), and monocyte chemoattractant protein 3 (MCP-3) (9Schenk S. Mal N. Finan A. Zhang M. Kiedrowski M. Popovic Z. McCarthy P.M. Penn M.S. Stem Cells. 2007; 25: 245-251Crossref PubMed Scopus (233) Google Scholar, 10Wang L. Li Y. Chen J. Gautam S.C. Zhang Z. Lu M. Chopp M. Exp. Hematol. 2002; 30: 831-836Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). We and others have recently found that some of these chemoattractants can be released by VSMC at the time of arterial injury, although the signals that stimulate their release have not been elucidated. Mesenchymal stem cells (MSC) are present in bone-marrow stroma and capable of differentiating into multiple cell types, including smooth muscle cells, endothelial cells, and cardiomyoblast progenitors. MSC are believed to contribute to the repair of injured myocardial and nerve tissue (9Schenk S. Mal N. Finan A. Zhang M. Kiedrowski M. Popovic Z. McCarthy P.M. Penn M.S. Stem Cells. 2007; 25: 245-251Crossref PubMed Scopus (233) Google Scholar, 10Wang L. Li Y. Chen J. Gautam S.C. Zhang Z. Lu M. Chopp M. Exp. Hematol. 2002; 30: 831-836Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Furthermore, it has recently been shown in a mouse femoral artery wire injury model that MSC are recruited to the neointimal lesion (11Wang C.H. Cherng W.J. Yang N.I. Kuo L.T. Hsu C.M. Yeh H.I. Lan Y.J. Yeh C.H. Stanford W.L. Arterioscler. Thromb. Vasc. Biol. 2008; 28: 54-60Crossref PubMed Scopus (116) Google Scholar). Transforming growth factor-β (TGF-β) has been repeatedly demonstrated to be a provocative factor in the development of intimal hyperplasia. Studies have shown that TGF-β mRNA and protein levels are elevated during progressive neointimal thickening (12Nikol S. Isner J.M. Pickering J.G. Kearney M. Leclerc G. Weir L. J. Clin. Invest. 1992; 90: 1582-1592Crossref PubMed Scopus (356) Google Scholar, 13Ryan S.T. Koteliansky V.E. Gotwals P.J. Lindner V. J. Vasc. Res. 2003; 40: 37-46Crossref PubMed Scopus (90) Google Scholar). Furthermore, administration of recombinant TGF-β after injury has been shown to enhance intimal hyperplasia (14Kanzaki T. Tamura K. Takahashi K. Saito Y. Akikusa B. Oohashi H. Kasayuki N. Ueda M. Morisaki N. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1951-1957Crossref PubMed Scopus (115) Google Scholar), whereas blockade of TGF-β signaling reduces this process (15Wolf Y.G. Rasmussen L.M. Ruoslahti E. J. Clin. Invest. 1994; 93: 1172-1178Crossref PubMed Scopus (290) Google Scholar). Active TGF-β signals by binding to a specific serine/threonine kinase type II receptor, which then recruits and phosphorylates the type I receptor. Receptor activation by TGF-β leads to phosphorylation of Smads 2 and 3, both which heterodimerize with Smad4. The resulting complex enters the cell nucleus to directly regulate transcriptional activation of target genes (16Derynck R. Zhang Y.E. Nature. 2003; 425: 577-584Crossref PubMed Scopus (4323) Google Scholar, 17ten Dijke P. Hill C.S. Trends Biochem. Sci. 2004; 29: 265-273Abstract Full Text Full Text PDF PubMed Scopus (1052) Google Scholar). We have previously shown that TGF-β through Smad3 signaling increases the expression of fibronectin in VSMC, an extracellular matrix protein that has been implicated in intimal hyperplasia (2Ryer E.J. Hom R.P. Sakakibara K. Nakayama K.I. Nakayama K. Faries P.L. Liu B. Kent K.C. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 780-786Crossref PubMed Scopus (46) Google Scholar). Monocyte chemoattractant protein-1 (MCP-1) is a member of the C-C chemokine family, which is classically known to play a major role in inducing monocyte migration to sites of inflammation and injury. Recently it has been reported that MCP-1, acting through its receptor CCR2, plays a key role in the recruitment of monocytes from the peripheral circulation into early atherosclerotic and restenotic lesions (18Charo I.F. Taubman M.B. Circ. Res. 2004; 95: 858-866Crossref PubMed Scopus (646) Google Scholar). Furthermore, suppression of MCP-1 by administration of a neutralizing MCP-1 antibody or gene transfer of mutant MCP-1 inhibited neointimal hyperplasia in injured rat carotid arteries (19Furukawa Y. Matsumori A. Ohashi N. Shioi T. Ono K. Harada A. Matsushima K. Sasayama S. Circ. Res. 1999; 84: 306-314Crossref PubMed Scopus (218) Google Scholar, 20Egashira K. Zhao Q. Kataoka C. Ohtani K. Usui M. Charo I.F. Nishida K. Inoue S. Katoh M. Ichiki T. Takeshita A. Circ. Res. 2002; 90: 1167-1172Crossref PubMed Scopus (157) Google Scholar). Although these studies of MCP-1 have focused on the recruitment of monocytes and macrophages to the arterial wall, the possibility that MCP-1 might also enhance intimal hyperplasia through the recruitment of stem or progenitor cells has not been addressed. Suggesting this as a possibility is recent evidence that mesenchymal stem cells express the MCP-1 receptor, CCR2 (21Simper D. Stalboerger P.G. Panetta C.J. Wang S. Caplice N.M. Circulation. 2002; 106: 1199-1204Crossref PubMed Scopus (472) Google Scholar, 22Zhao Y. Glesne D. Huberman E. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 2426-2431Crossref PubMed Scopus (446) Google Scholar). Additionally, the related cytokine MCP-3 has been shown to play an essential role in recruiting mesenchymal stem cells to injured myocardial tissue (9Schenk S. Mal N. Finan A. Zhang M. Kiedrowski M. Popovic Z. McCarthy P.M. Penn M.S. Stem Cells. 2007; 25: 245-251Crossref PubMed Scopus (233) Google Scholar). Together, these findings suggest that MCP-1 may be involved in the pathogenesis of intimal hyperplasia by inducing progenitor cell recruitment in a manner similar to its known effects on mononuclear cells. In the present study, we hypothesize that TGF-β through Smad3 signaling induces VSMC secretion of MCP-1, which acts as a bone marrow-derived progenitor cell or mesenchymal stem cell chemoattractant. These findings raise the possibility that the stimulatory effect of TGF-β on intimal hyperplasia includes at least in part, the recruitment of bone marrow progenitor cells to the neointima. Recombinant TGF-β1 and rat MCP-1 were obtained from R&D Systems. Dulbecco's modified Eagle's medium (DMEM) and cell culture reagents were from Invitrogen. Other reagents, if not specified, were purchased from Sigma. Rat aortic SMC were isolated from the thoracic aorta of male Sprague-Dawley rats based on a protocol described by Clowes et al. (23Clowes M.M. Lynch C.M. Miller A.D. Miller D.G. Osborne W.R. Clowes A.W. J. Clin. Invest. 1994; 93: 644-651Crossref PubMed Scopus (103) Google Scholar) and maintained in DMEM containing 10% fetal FBS at 37 °C with 5% CO2. Cell viability was assayed by the trypan blue exclusion method, which indicated that <5% of the cells took up the dye both before and after the infection of adenoviral vectors or treatment with recombinant TGF-β1 or inhibitors. For experiments using conditioned media, RSMC (2 × 105 cells/well in 6-well plates) were infected with adenovirus (30,000 particles/cell in DMEM containing 2% FBS for 4 h at 37 °C) followed by starvation in DMEM containing 0.5% FBS for 48 h. The cells were then treated with recombinant TGF-β1 (5 ng/ml) or solvent (4 mm HCl with 2% bovine serum albumin) for 6 h. Media were collected and used for ELISA or chemotaxis assays as described below. Rat MSC isolated from rat bone marrow were obtained from Cell Applications Inc. (San Diego, CA). The cells were maintained through the use of mesenchymal stem cell growth medium kit (Cell Applications Inc.). MSC populations were passaged 1–4 times. Cells were harvested from plates using a solution containing 0.25% trypsin and 1 mm EDTA (Invitrogen). Confirmation of MSC was made through the presence of cell surface markers such as CD90, CD44, and CD54 and the absence of CD31 and CD45. For cell differentiation experiments, MSC up to passage 4 were plated in MSC growth medium (non-differentiating). Bone marrow from Sprague-Dawley rats was isolated by flushing the long bones with Dulbecco's phosphate-buffered saline containing 2% bovine serum albumin, and heparin (1000 units/ml). Cells from individual animals were pooled. Bone marrow was dissociated by aspiration with a Pasteur pipette. Red blood cells were excluded using Ficoll-Hypaque (GE Healthcare) density centrifugation. The remaining cells were then rinsed twice in phosphate-buffered saline followed by centrifugation. Adenoviral vectors expressing Smad3 (AdSmad3), PKCδ (AdPKCδ), and GFP (AdGFP) were constructed as previously described (2Ryer E.J. Hom R.P. Sakakibara K. Nakayama K.I. Nakayama K. Faries P.L. Liu B. Kent K.C. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 780-786Crossref PubMed Scopus (46) Google Scholar). 2 × 105 bone marrow cells or 2 × 105 MSC were placed in the upper chamber of Costar 24-well transwell plates with 5-μm pore filters (Corning, Inc., Corning, NY). Cultured conditioned medium was placed into the lower chambers or wells. After incubating plates for 6 h at 37 °C, migrated cells were collected from the lower chambers and counted. RSMC were plated at 50–60% confluence in DMEM culture medium in 6-well plates and incubated for 24 h. Cells were then transfected in Opti-MEM I medium with 20 nmol of siRNA for rat MCP-1 (Santa Cruz Biotechnology), 100 pmol of siRNA for Smad3 (Invitrogen), or control siRNA using RNAiMax transfection reagent (Invitrogen), as described by the manufacturer's protocol. After 6 h, the Opti-MEM I medium was replaced by DMEM containing 0.5% FBS, and cells were starved for 48 h. Cells that were infected with adenovirus were infected the day after transfection, then starved for the remaining 48 h. The cells were then stimulated with recombinant TGF-β1 (5 ng/ml) or solvent for 6 h, after which conditioned media was collected for chemotaxis assays and MCP-1 ELISA, and cells were lysed for protein used in Western blotting. Cells were lysed in RIPA buffer (50 mm Tris, 150 mm NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, and 10 μg/ml aprotinin). Fifty micrograms of protein from each sample were separated on 10% SDS-PAGE gels. The protein samples were then transferred to nitrocellulose membranes. Protein expression was confirmed by immunoblotting with the following antibodies: rabbit anti-Smad3 (AbCam), rabbit anti-pSmad3 (AbCam), and mouse anti-β-actin (Sigma). After incubation with the appropriate primary and horseradish peroxidase-conjugated secondary antibodies, the membranes were developed with enhanced chemiluminescence reagent (Amersham Biosciences). RSMC were infected with AdSmad3 or AdGFP followed by starvation for 48 h in DMEM containing 0.5% FBS, then treated with recombinant TGF-β1 (5 ng/ml) or solvent for 6 h. Cells were harvested, and total RNA was extracted by RNeasy Mini Kit (Qiagen, Valencia, CA). This RNA was then processed according to the protocol recommended by Affymetrix using the Superscript Choice kit (Invitrogen) for double-stranded cDNA synthesis and the Enzo Bioarray kit for in vitro transcription and labeling of cRNA. 15-μg samples of fragmented cRNA were hybridized for 16 h at 45 °C to rat 230.2.0 arrays (Affymetrix). Data analysis was performed using GeneSpringTM software 5.1 (Silicon Genetics) for normalization, -fold change calculations, and clustering. Expression of the mRNA of MCP-1 was detected by RT-PCR. RNA was isolated from rat SMC using a kit from Qiagen. A segment of the cDNA was detected using RT-PCR for MCP-1 using MCP-1 primers, which were obtained from Santa Cruz (Santa Cruz Biotechnology). ELISA for MCP-1-ELISA to detect MCP-1 secreted by RSMC was performed using rat MCP-1 ELISA kit (BD Biosciences). RSMC were cultured at a density of 1 × 105/ml in 1 ml of complete medium in the presence or absence of different stimuli in 6-well plates (Costar). After incubation for specified periods of time at 37 °C, cell-free culture supernatants were obtained. The concentrations of MCP-1 were then measured according to the manufacturer's instructions. Male Sprague-Dawley rats (450–500 g) underwent balloon injury of the left common carotid artery as described elsewhere in accordance with institutional guidelines and approval (24Hollenbeck S.T. Sakakibara K. Faries P.L. Workhu B. Liu B. Kent K.C. J. Surg. Res. 2004; 120: 288-294Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 25Clowes A.W. Reidy M.A. Clowes M.M. Lab. Invest. 1983; 49: 208-215PubMed Google Scholar). Briefly, after induction of anesthesia with isoflurane, a 2-French balloon catheter was inserted through the left external carotid artery into the common carotid and insufflated with 1 ml of air three times. Recombinant adenoviral vectors were constructed to express Smad3 as previously described; an empty viral vector was used as a control (26Ryer E.J. Sakakibara K. Wang C. Sarkar D. Fisher P.B. Faries P.L. Kent K.C. Liu B. J. Biol. Chem. 2005; 280: 35310-35317Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). After injury, animals received intraluminal administration of adenoviral vectors as indicated (2.5 × 109 plaque-forming unit in 200 μl of serum-free media over 20 min). The external carotid artery was then ligated, and flow was re-established through the common carotid and internal carotid arteries. Rats were sacrificed 5 days after injury, and perfusion was fixed with 4% paraformaldehyde diluted in phosphate-buffered saline at a pressure of 100 mm Hg for 15 min. The left and right carotid arteries were then harvested and fixed in 4% paraformaldehyde for an additional 30 min. The arteries were then paraffin-embedded and cut into 7-μm sections for histological analysis. Bone marrow cells were extracted from rat long bones and labeled with fluorescent dyes using a PKH26 red fluorescent cell linker kit (Sigma-Aldrich). After left carotid balloon angioplasty was performed, a 24-gauge angiocath was inserted into the external carotid arteriotomy and secured. 5 × 107 BM suspended in 1 ml phosphate-buffered saline were injected systemically through the arteriotomy. The external carotid artery was then ligated, and flow was re-established from the common carotid artery to the internal carotid artery. Rats were sacrificed 48 h after BM cell injection, and the tissue was frozen into blocks with optimal cutting temperature medium (Sakura Finetek Inc.). Immunostaining for MCP-1 using monoclonal anti-MCP-1 antibody (Santa Cruz) was performed as described previously (24Hollenbeck S.T. Sakakibara K. Faries P.L. Workhu B. Liu B. Kent K.C. J. Surg. Res. 2004; 120: 288-294Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Slides were visualized with a Nikon Eclipse E800 upright microscope. Digital images were acquired using a RetigaEXi CCD digital camera and processed and analyzed using IPLab software. Semiquantitative immunohistochemical analysis was performed using Adobe Photoshop 7.0 and NIH imaging software (ImageJ) (24Hollenbeck S.T. Sakakibara K. Faries P.L. Workhu B. Liu B. Kent K.C. J. Surg. Res. 2004; 120: 288-294Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Data are expressed as the mean ± S.E. Unpaired Student's t test was used to evaluate the statistical differences between control and treated groups. In cases of multiple groups, differences were first analyzed with one-way analysis of variance. Values of p < 0.05 were considered significant. All experiments were repeated at least in triplicate. To begin testing our hypothesis that TGF-β stimulates intimal hyperplasia through the recruitment of BMPC, we first established a bone marrow cell chemotaxis assay using SDF-1α as a positive control (Fig. 1A). We then evaluated whether recombinant TGF-β could directly stimulate either total bone marrow cell or MSC migration. TGF-β alone had no direct effect on progenitor cell migration (Fig. 1, A and B). We then asked whether TGF-β might stimulate VSMC to produce progenitor cell chemoattractants as it is well established that TGF-β levels are elevated after vascular injury. As shown in Fig. 1, C and D, conditioned media derived from rat aortic VSMC treated with TGF-β significantly increased both total bone marrow cell as well as isolated rat MSC chemotaxis. These findings suggest that TGF-β activates VSMC, which in turn produce a secreted factor that is responsible for recruitment or chemotaxis of bone marrow progenitor cells. Smad3 is a major signaling molecule for TGF-β that we and others have shown to be important in mediating many of the TGF-β effects on VSMC. We, therefore, hypothesized that Smad3 might mediate the effect of TGF-β on progenitor cell recruitment. To examine whether Smad3 plays a role in TGF-β-induced secretion of BMPC chemoattractants by VSMC, we infected rat aortic VSMC with adenoviral vectors expressing Smad3 (AdSmad3) and then stimulated cells with recombinant TGF-β1, thus enhancing TGF-β/Smad3 signaling. As shown in Fig. 2, conditioned media from VSMC treated with AdSmad3 and TGF-β significantly increased both total bone marrow cell as well as isolated rat MSC migration, compared with conditioned media from VSMC treated with TGF-β alone. To further study the role of Smad3 in this process, we examined the effect of inhibition of Smad3 signaling on BMPC recruitment. VMSC were pretreated with SB431542 (10 μg/ml), a TGF-β receptor kinase inhibitor that, at this concentration, is specific for Smad2/3 phosphorylation. As shown in Fig. 3A, treatment with SB431542 effectively inhibited TFG-β-induced phosphorylation of both endogenous and adenovirus-derived Smad3 but did not affect total expression of Smad3. VSMC treated with TGF-β or TGF-β/AdSmad3 were pretreated with SB431542, and the effect of this conditioned media on bone marrow cell and MSC migration was evaluated. As shown in Fig. 3, B and C, pretreatment of VSMC with SB431542 followed by stimulation with TGF-β or TGF-β/AdSmad3 resulted in a significant decrease in the chemotaxis of both bone marrow cells and MSC in response to conditioned media derived from VSMC. These data confirm that phosphorylation of Smad3 is an essential step in TGF-β mediated recruitment of progenitor cells by vascular SMC. To more specifically inhibit Smad3 function we developed a siRNA to rat Smad3. As shown in Fig. 3D, transient transfection of VSMC with Smad3 siRNA effectively down-regulated Smad3 expression. Furthermore, conditioned media derived from VSMC transiently transfected with Smad3 siRNA followed by stimulation with TGF-β produced significantly decreased total BM and MSC migration compared with conditioned media from VSMC transfected with scrambled siRNA (Fig. 3, E and F). Taken together, these results demonstrate that Smad3 is the signaling protein through which TGF-β mediates its effect on progenitor cell recruitment. Our next goal was to determine the identity of the progenitor cell chemoattractant released by VSMC in response to TGF-β. To explore the possibilities, we performed a microarray analysis of TGF-β/AdSmad3-stimulated VSMC. Treatment with TGF-β/AdSmad3 increased by more than 4-fold the expression of 566 genes and decreased expression by at least 4-fold of 284 genes. The selected genes were clustered into groups that influence cell differentiation or cell growth using GeneSpringTM software. We were able to identify a number of known progenitor cell chemoattractants stimulated by TGF-β in Smad3-overexpressing cells, as summarized in Table 1. One of the most highly induced genes was that of MCP-1, with a greater than 80-fold induction. Because MCP-1 has been shown to attract neural progenitors cells to areas of tissue injury (27Belmadani A. Tran P.B. Ren D. Miller R.J. J. Neurosci. 2006; 26: 3182-3191Crossref PubMed Scopus (272) Google Scholar), we hypothesized that MCP-1 may be the critical factor that allows TGF-β-stimulated vascular SMC to function as a chemoattractant of bone marrow progenitor cells to sites of arterial injury.TABLE 1Potential progenitor cell chemoattractants (-fold change)Growth factorsChemokinesOther moleculesVEGF (7.7)MCP-1 (83)Sphingosine kinase (3.2)VEGF receptor (5.5)MCP-3 (37)Sphingosine-1 phosphate (1.4)Platelet-derived growth factor (4.7)Interleukin-6 (8.5)Matrix metalloprotein-9 (1.4)Fibroblast growth factor-2 (4.6)Interleukin-1 (4.4)Insulin-like growth factor-1 (4)RANTES (3)Stem cell factor (3.3) Open table in a new tab As shown in Fig. 4A, TGF-β stimulated the production of MCP-1 in RSMC in a dose-dependent manner. Smad3 overexpression via adenovirus-mediated gene transfer also increased the production of MCP-1, with further enhancement when Smad3-overexpressing cells were stimulated with TGF-β (Fig. 4B). To demonstrate that the increase in protein level was secondary to an increase in gene transcription, RT-PCR confirmed that TGF-β and Smad3 overexpression both increased expression of MCP-1 mRNA in VSMC (Fig. 4C). We next investigated the impact of inhibiting Smad3 signaling on TGF-β-induced MCP-1. As shown in Fig. 4D, inhibition of Smad2/3 phosphorylation using SB431542 (10 μg/ml) significantly blocked TGF-β-induced MCP-1 in both control RSMC and RSMC overexpressing Smad3. Furthermore, Smad3 siRNA also significantly decreased TGF-β-induced MCP-1 expression in RSMC (Fig. 4E). To determine the role of MCP-1 in the recruitment of bone marrow cells and MSC, we first tested the effect of recombinant MCP-1 on total bone marrow cell and MSC migration. Fig. 5, A and B, show that recombinant MCP-1 markedly induced both total bone marrow cell and MSC chemotaxis. To further examine whether MCP-1 is necessary for the recruitment of bone marrow cells by VSMC treated with TGF-β and AdSmad3, we used a siRNA to silence MCP-1 expression in these cells. As shown in Fig. 5C, MCP-1-specific siRNA significantly reduced the amount of MCP-1 released by SMC treated with TGF-β, whereas scrambled siRNA had no significant effect. Inhibition of MCP-1 gene expression by the siRNA was also confirmed by RT-PCR (Fig. 5D). Transfection of MCP-1 siRNA in" @default.
• W2079658573 created "2016-06-24" @default.
• W2079658573 creator A5006328463 @default.
• W2079658573 creator A5012140656 @default.
• W2079658573 creator A5028140419 @default.
• W2079658573 creator A5033818458 @default.
• W2079658573 creator A5050738541 @default.
• W2079658573 creator A5053447705 @default.
• W2079658573 creator A5090799132 @default.
• W2079658573 creator A5090815103 @default.
• W2079658573 date "2009-06-01" @default.
• W2079658573 modified "2023-10-15" @default.
• W2079658573 title "Transforming Growth Factor-β Promotes Recruitment of Bone Marrow Cells and Bone Marrow-derived Mesenchymal Stem Cells through Stimulation of MCP-1 Production in Vascular Smooth Muscle Cells" @default.
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