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• W1984216075 abstract "Transforming growth factor β1 (TGFβ1) activation leads to tissue fibrosis. Here, we report on the role of LOX-1, a lectin-like 52-kDa receptor for oxidized low density lipoprotein, in TGFβ1-mediated collagen expression and underlying signaling in mouse cardiac fibroblasts. TGFβ1 was overexpressed in wild-type (WT) and LOX-1 knock-out mouse cardiac fibroblasts by transfection with adeno-associated virus type 2 vector carrying the active TGFβ1 moiety (AAV/TGFβ ACT1). Transfection of WT mouse cardiac fibroblasts with AAV/TGFβ ACT1 markedly enhanced the expression of NADPH oxidases (p22phox, p47phox, and gp91phox subunits) and LOX-1, formation of reactive oxygen species, and collagen synthesis, concomitant with an increase in the activation of p38 and p44/42 mitogen-activated protein kinases (MAPK). The TGFβ1-mediated increase in collagen synthesis was markedly attenuated in the LOX-1 knock-out mouse cardiac fibroblasts as well as in WT mouse cardiac fibroblasts treated with a specific anti-LOX-1 antibody. Treatment with anti-LOX-1 antibody also reduced NADPH oxidase expression and MAPK activation. The NADPH oxidase inhibitors and gp91phox small interfering RNA reduced LOX-1 expression, MAPK activation, and collagen formation. The p38 MAPK inhibitors as well as the p44/42 MAPK inhibitors reduced collagen formation without affecting LOX-1 expression in cardiac fibroblasts. These observations suggest that collagen synthesis in cardiac fibroblasts involves a facilitative interaction between TGFβ1-NADPH oxidase and LOX-1. Further, the activation of MAPK pathway appears to be downstream of TGFβ1-reactive oxygen species-LOX-1 cascade. Transforming growth factor β1 (TGFβ1) activation leads to tissue fibrosis. Here, we report on the role of LOX-1, a lectin-like 52-kDa receptor for oxidized low density lipoprotein, in TGFβ1-mediated collagen expression and underlying signaling in mouse cardiac fibroblasts. TGFβ1 was overexpressed in wild-type (WT) and LOX-1 knock-out mouse cardiac fibroblasts by transfection with adeno-associated virus type 2 vector carrying the active TGFβ1 moiety (AAV/TGFβ ACT1). Transfection of WT mouse cardiac fibroblasts with AAV/TGFβ ACT1 markedly enhanced the expression of NADPH oxidases (p22phox, p47phox, and gp91phox subunits) and LOX-1, formation of reactive oxygen species, and collagen synthesis, concomitant with an increase in the activation of p38 and p44/42 mitogen-activated protein kinases (MAPK). The TGFβ1-mediated increase in collagen synthesis was markedly attenuated in the LOX-1 knock-out mouse cardiac fibroblasts as well as in WT mouse cardiac fibroblasts treated with a specific anti-LOX-1 antibody. Treatment with anti-LOX-1 antibody also reduced NADPH oxidase expression and MAPK activation. The NADPH oxidase inhibitors and gp91phox small interfering RNA reduced LOX-1 expression, MAPK activation, and collagen formation. The p38 MAPK inhibitors as well as the p44/42 MAPK inhibitors reduced collagen formation without affecting LOX-1 expression in cardiac fibroblasts. These observations suggest that collagen synthesis in cardiac fibroblasts involves a facilitative interaction between TGFβ1-NADPH oxidase and LOX-1. Further, the activation of MAPK pathway appears to be downstream of TGFβ1-reactive oxygen species-LOX-1 cascade. Cardiac fibrosis, characterized by accumulation of matrix proteins in the extracellular space in the perivascular region, is closely associated with the development of heart failure. Among the extracellular matrix proteins, up to 85% are collagens, of which type 1 (collagen I) and type 3 (collagen III) constitute two-thirds of total collagens in most tissues (1Heeneman S. Cleutjens J.P. Faber B.C. Creemers E.E. van Suylen R.J. Lutgens E. Cleutjens K.B. Daemen M.J. J. Pathol. 2003; 200: 516-525Crossref PubMed Scopus (121) Google Scholar). Transforming growth factor (TGF) 3The abbreviations used are: TGF, transforming growth factor; rTGF, recombinant TGF; siRNA, small interfering RNA; WT, wild-type; KO, knock-out; m.o.i., multiplicity of infection; GFP, green fluorescent protein; ROS, reactive oxygen species; MAPK, mitogen-activated protein kinase; DCF, dihydrofluorescein. β1 is one of the most pleiotropic and multifunctional peptides known (2Mehta J.L. Attramadal H. Cardiovasc. Res. 2007; 74: 181-183Crossref PubMed Scopus (18) Google Scholar). It exerts potent effects on many different cell types and is involved in a wide variety of biological processes (2Mehta J.L. Attramadal H. Cardiovasc. Res. 2007; 74: 181-183Crossref PubMed Scopus (18) Google Scholar). The cellular actions of TGFβ1 are dependent not only on the cell type but also on its state of differentiation and the cytokine milieu (3Letterio J.J. Roberts A.B. Annu. Rev. Immunol. 1998; 16: 137-161Crossref PubMed Scopus (1674) Google Scholar). Although there is evidence that TGFβ1 stimulates fibroblast growth, enhances collagen synthesis, and suppresses collagen degradation (2Mehta J.L. Attramadal H. Cardiovasc. Res. 2007; 74: 181-183Crossref PubMed Scopus (18) Google Scholar), the specific effect of TGFβ1 on the cardiac remodeling process remains unclear. TGFβ1 is synthesized in cells as a precursor molecule, TGFβ Latent1. Conversion from cysteine (Cys223/225) into serine (Ser223/225) in the TGFβ Latent1 molecule is associated with the formation of TGFβ ACT1 (active TGFβ1) (4Blobe G. Schiemann W. Lodish H. N. Eng. J. Med. 2000; 342: 1350-1358Crossref PubMed Scopus (2189) Google Scholar). It is the TGFβ ACT1 that appears to be functionally relevant in the process of ischemia-reperfusion (5Yang B.C. Zander D. Mehta J.L. J. Pharmacol. Exp. Ther. 1999; 291: 733-738PubMed Google Scholar). LOX-1 is a lectin-like receptor for oxidized low density lipoprotein (6Mehta J.L. Chen J. Hermonat P.L. Romeo F. Novelli G. Cardiovasc. Res. 2006; 69: 36-45Crossref PubMed Scopus (388) Google Scholar). It is also up-regulated in response to oxidative stress (7Hu C.P. Dandapat A. Chen J. Fujita Y. Inoue N. Kawase Y. Jishage K.I. Suzuki H. Sawamura T. Mehta J.L. Cardiovasc. Res. 2007; 76: 292-302Crossref PubMed Scopus (59) Google Scholar). Activation of LOX-1 enhances the growth of cardiac fibroblasts and promotes collagen synthesis (8Chen K. Chen J. Liu Y. Xie J. Li D. Sawamura T. Hermonat P.L. Mehta J.L. Hypertension. 2005; 46: 622-627Crossref PubMed Scopus (37) Google Scholar, 9Joseph J. Ranganathan S. Mehta J.L. J. Cardiovas. Pharmacol. Ther. 2003; 8: 161-166Crossref PubMed Scopus (5) Google Scholar). It has been reported that TGFβ1 can regulate LOX-1 expression in vascular endothelial cells, smooth muscle cells, and monocytes/macrophages (10Draude G. Lorenz R.L. Am. J. Physiol. 2000; 278: H1042-H1048Crossref PubMed Google Scholar, 11Minami M. Kume N. Kataoka H. Morimoto M. Hayashida K. Sawamura T. Masaki T. Kita T. Biochem. Biophys. Res. Commun. 2000; 272: 357-361Crossref PubMed Scopus (94) Google Scholar). Another study showed that LOX-1 blockade reduces TGFβ1 protein expression in endothelial cells (12Chen H. Li D. Saldeen T. Mehta J.L. Circ. Res. 2001; 89: 1155-1160Crossref PubMed Scopus (59) Google Scholar). The present study was conducted to examine whether and how LOX-1 mediates TGFβ1-mediated collagen production. Materials and Reagents—Monoclonal antibody against mouse LOX-1 raised in rat with mouse Fc portion of IgG has been reported earlier to block the effect of LOX-1 (13Mehta J.L. Sanada N. Hu C.P. Chen J. Dandapat A. Sugawara F. Satoh H. Inoue K. Kawase Y. Jishage K. Suzuki H. Takeya M. Schnackenberg L. Beger R. Hermonat P.L. Thomas M. Sawamura T. Circ. Res. 2007; 100: 1634-1642Crossref PubMed Scopus (375) Google Scholar). The following reagents and antibodies were purchased: p38 MAPK inhibitors SB203580 (Sigma) and SB202190 (Calbiochem); p44/42 MAPK inhibitors U0126 (Sigma) and PD98059 (Calbiochem); NADPH oxidase inhibitors apocynin (Aldrich) and diphenyliodonium (Sigma); GeneSilencer® siRNA transfection reagent (Gene Therapy Systems); siCONTROL® non-targeting siRNA and siGENOME SMARTpool® NADPH oxidase gp91phox subunit siRNA (Dharmacon); human recombinant TGFβ1 (rTGFβ1) and mouse nonspecific IgG (Sigma); all primary antibodies for Western blot analysis except anti-LOX-1 antibody (Santa Cruz Biotechnology). 2′, 7′-dichlorodihydrofluorescein diacetate was purchased from Cayman. U0126, PD98059, SB203580, SB202190, diphenyliodonium, and apocynin were dissolved in Me2SO. The final concentration of Me2SO was <0.1% in these experiments. Animals—C57BL/6 mice (also referred to as WT mice) were obtained from Jackson Laboratories. The homozygous LOX-1 knock-out (KO) mice were developed as described recently (13Mehta J.L. Sanada N. Hu C.P. Chen J. Dandapat A. Sugawara F. Satoh H. Inoue K. Kawase Y. Jishage K. Suzuki H. Takeya M. Schnackenberg L. Beger R. Hermonat P.L. Thomas M. Sawamura T. Circ. Res. 2007; 100: 1634-1642Crossref PubMed Scopus (375) Google Scholar) and backcrossed eight times with the C57BL/6 strain to replace the genetic background. Male C57BL/6 and LOX-1 KO mice weighing 22–26 g were utilized at 8–10 weeks of age. All animals received humane care in compliance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals published by the National Institutes of Health. Construction of AAV/TGFβ ACT1 and Generation of Recombinant AAV Stocks—The TGFβ1 mutant TGFβ ACT1 cDNA was generated, sequenced, and ligated into an adeno-associated virus (AAV) vector, dl6–95, as described recently (14Hu C.P. Dandapat A. Liu Y. Hermonat P.L. Mehta J.L. Am. J. Physiol. 2007; 293: H1833-H1838Crossref PubMed Scopus (28) Google Scholar). Recombinant AAV vector hereafter will be referred to as AAV/TGFβ ACT1. The generation of AAV/Neo virus has been described earlier (15Li D. Liu Y. Chen J. Velchala N. Amani F. Nemarkommula A. Chen K. Rayaz H. Zhang D. Liu H. Sinha A.K. Romeo F. Hermonat P.L. Mehta J.L. Biochem. Biophys. Res. Commun. 2006; 344: 701-707Crossref PubMed Scopus (38) Google Scholar). Virus stocks were generated as described previously (15Li D. Liu Y. Chen J. Velchala N. Amani F. Nemarkommula A. Chen K. Rayaz H. Zhang D. Liu H. Sinha A.K. Romeo F. Hermonat P.L. Mehta J.L. Biochem. Biophys. Res. Commun. 2006; 344: 701-707Crossref PubMed Scopus (38) Google Scholar). The titer of purified virus, in encapsidated genomes per milliliter (eg/ml), was calculated by dot-blot hybridization and determined to be ∼1011 eg/ml. Cell Culture and AAV Vector Infection—Mouse (WT and LOX-1 KO) cardiac fibroblasts were isolated and cultured as described earlier (16Chen J. Mehta J.L. Am. J. Physiol. 2006; 291: H1738-H1745Crossref PubMed Scopus (82) Google Scholar). Cells cultured to fifth passage were used in all experiments. To transfect the cultured cells, AAV vectors were added to cell culture dishes at multiplicity of infection (m.o.i.) of 103 and incubated with the cells for 72 h at 37 °C in 5% CO2/95% air. The infection efficiency was evaluated by AAV/GFP expression using fluorescent microscopy. AAV/GFP was obtained from the University of North Carolina Gene Therapy Center, and the titer was 8 × 1012 virus particles/ml (3.1 × 1010 infectious units). Experimental Protocols—Cardiac fibroblasts were transfected with AAV vector (or only culture medium) for 72 h in the absence or presence of anti-LOX-1 antibody (10 μg/ml), nonspecific IgG (10 μg/ml), apocynin (600 μm), diphenyliodonium (10 μm), U0126 (10 μm), PD98059 (10 μm), SB203580 (10 μm), SB202190 (10 μm), gp91phox siRNA (50 nm), or non-targeting siRNA (50 nm), or Me2SO (as vehicle control). These concentrations were chosen on the basis of published data (13Mehta J.L. Sanada N. Hu C.P. Chen J. Dandapat A. Sugawara F. Satoh H. Inoue K. Kawase Y. Jishage K. Suzuki H. Takeya M. Schnackenberg L. Beger R. Hermonat P.L. Thomas M. Sawamura T. Circ. Res. 2007; 100: 1634-1642Crossref PubMed Scopus (375) Google Scholar, 17Usatyuk P.V. Romer L.H. He D. Parinandi N.L. Kleinberg M.E. Zhan S. Jacaobson J.R. Dudek S. Pendyala S. Garcia J.G. Natarajan V. J. Biol. Chem. 2007; 282: 23284-23295Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 18Lee O.H. Kim Y.M. Lee Y.M. Moon E.J. Lee D.J. Kim J.H. Kim K.W. Kwon Y.G. Biochem. Biophys. Res. Commun. 1999; 264: 743-750Crossref PubMed Scopus (335) Google Scholar, 19Chen J.X. Zeng H. Tuo Q.H. Yu H. Meyrick B. Aschner J.L. Am. J. Physiol. 2007; 292: H1664-H1674Crossref PubMed Scopus (84) Google Scholar, 20Dandapat A. Hu C.P. Sun L.Q. Mehta J.L. Arterioscler. Thromb. Vasc. Biol. 2007; 27: 2435-2442Crossref PubMed Scopus (128) Google Scholar) and modified in accordance with the results of pilot experiments. In other experiments, cardiac fibroblasts were treated with rTGFβ1 (2 ng/ml) for 24 h. At the end of the experiments, culture medium was collected for the determination of soluble collagen production and fibroblasts were examined for measurement of reactive oxygen species (ROS) release and expression of specific proteins. Evaluation of Cell Growth—Cell growth was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma). Meanwhile, cell number was determined in triplicate using a hemocytometer. Results were normalized to the conversion of MTT and cell number in control cultures as 100%, respectively. Soluble Collagen Assay—Soluble collagen production was determined by the Sircol assay (Biocolor) as described earlier (21Huang M. Sharma S. Zhu L.X. Keane M.P. Luo J. Zhang L. Burdick M.D. Lin Y.Q. Dohadwala M. Gardner B. Batra R.K. Strieter R.M. Dubinett S.M. J. Clin. Investig. 2002; 109: 931-937Crossref PubMed Google Scholar) and expressed as μg/106 cells. Measurement of ROS in Cardiac Fibroblasts—Intracellular ROS generation was measured with the use of 2′, 7′-dichlorodihydrofluorescein diacetate (10 μm) fluorescent signal, a cell-permeant indicator for ROS, as described previously (14Hu C.P. Dandapat A. Liu Y. Hermonat P.L. Mehta J.L. Am. J. Physiol. 2007; 293: H1833-H1838Crossref PubMed Scopus (28) Google Scholar). Results were displayed in a ratiometric fashion normalized for the control conditions. Western Blot Analysis—The expression of TGFβ1, collagen type I, collagen type III, LOX-1, p38, and p44/42 MAPK isoforms, NADPH oxidase subunits (p22phox, p47phox, and gp91phox), or β-actin was assessed by Western analysis using standard methodologies (14Hu C.P. Dandapat A. Liu Y. Hermonat P.L. Mehta J.L. Am. J. Physiol. 2007; 293: H1833-H1838Crossref PubMed Scopus (28) Google Scholar). Densities of protein bands relative to β-actin were analyzed. Statistical Analysis—Data, based on at least four separate experiments, are expressed as means ± S.E. All values were analyzed by using one-way analysis of variance and the Newman-Keuls-Student t test. The significance level was chosen as p < 0.05. AAV Facilitates Transfection of Fibroblasts—In pilot experiments, AAV/GFP vectors were added to cell culture dishes at an m.o.i. of 0–104 and incubated with the cells for 72 h at 37°Cin5%CO2/95% air. More than 90% of the cells showed GFP expression at m.o.i. of 103. There was no difference in GFP expression in cardiac fibroblasts from WT and LOX-1 KO mice (Fig. 1A). Because the most optimal transfection was observed at m.o.i. of 103, transfection in subsequent experiments with AAV vectors was performed at m.o.i. of 103. Increase in TGFβ1 Expression— After 72 h of AAV infection, fibroblasts were harvested for Western blotting. As shown in Fig. 1B, total TGFβ1 expression was increased in AAV/TGFβ ACT1-transfected cells, indicating successful delivery of the transgene. AAV/Neo alone had no effect on TGFβ1 expression. Note that there was no difference in TGFβ1 expression in cardiac fibroblasts from WT and LOX-1 KO mice. AAV/TGFβ ACT1 Transfection Facilitates Cell Growth— AAV/TGFβ ACT1 transfection had a stimulatory effect on fibroblast growth, and the number of fibroblasts increased by ∼50% (versus fibroblasts kept under control conditions). This effect was much less evident in fibroblasts from LOX-1 KO mice (p < 0.01 versus AAV/TGFβ ACT1-transfected fibroblasts from wild-type mice) (Fig. 2, A and B). AAV/Neo alone had no effect on cell growth. Interaction between TGFβ ACT1, LOX-1 Expression, and Collagen Synthesis in Fibroblasts—AAV/TGFβ ACT1 transfection increased the expression of collagen type I and III (p < 0.01 versus fibroblasts kept under control conditions) in WT mouse cardiac fibroblasts, but this effect was much less pronounced in fibroblasts from LOX-1 KO mice (p < 0.01 versus AAV/TGFβ ACT1-transfected fibroblasts from WT mice) (Fig. 3A). Note that AAV/Neo alone had no effect on collagen expression. We also measured soluble collagen in the medium bathing the fibroblasts, and in keeping with collagen I and III expression data, soluble collagen increased in the supernates of AAV/TGFβ ACT1-transfected cardiac fibroblasts (Fig. 3B). To further confirm the effect of AAV/TGFβ ACT1 on collagen expression, we treated cells with rTGFβ1 directly and found that the effects of rTGFβ1 were consistent with that of transfection with AAV/TGFβ ACT1 (Fig. 3C). These observations suggest that LOX-1 plays an important role in TGFβ1-induced collagen production. Because the observations in LOX-1 KO mouse cardiac fibroblasts may reflect effects of genes other than LOX-1 in modulation of TGFβ1-mediated collagen synthesis, the role of LOX-1 in the effects of TGFβ ACT1 was confirmed by use of a specific anti-LOX-1 antibody. As shown in Fig. 4A, the expression of collagens was less in cells treated with anti-LOX-1 antibody than in fibroblasts kept under control conditions (p < 0.01). Note that nonspecific IgG had no effect on AAV/TGFβ ACT1-induced collagen expression (Fig. 4B). Interaction between TGFβ1, NADPH Oxidase, and LOX-1— Oxidant stress up-regulates the expression and activation of LOX-1 (7Hu C.P. Dandapat A. Chen J. Fujita Y. Inoue N. Kawase Y. Jishage K.I. Suzuki H. Sawamura T. Mehta J.L. Cardiovasc. Res. 2007; 76: 292-302Crossref PubMed Scopus (59) Google Scholar), and activation of LOX-1 itself can stimulate the formation of ROS (22Cominacini L. Rigoni A. Pasini A.F. Garbin U. Davoli A. Campagnola M. Pastorino A.M. Lo Cascio V. Sawamura T. J. Biol. Chem. 2001; 276: 13750-13755Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 23Chen X.P. Xun K.L. Wu Q. Zhang T.T. Shi J.S. Du G.H. Vascul. Pharmacol. 2007; 47: 1-9Crossref PubMed Scopus (101) Google Scholar). NADPH oxidase activation is a major source of ROS in cardiac fibroblasts (19Chen J.X. Zeng H. Tuo Q.H. Yu H. Meyrick B. Aschner J.L. Am. J. Physiol. 2007; 292: H1664-H1674Crossref PubMed Scopus (84) Google Scholar). We, therefore, measured intracellular ROS generation and NADPH oxidase expression in fibroblasts transfected with AAV/TGFβ ACT1. Indeed, AAV/TGFβ ACT1 transfection enhanced the expression of NADPH oxidase (p22phox, p47phox, and gp91phox subunits) as well as dihydrofluorescein (DCF) fluorescence reflecting intracellular ROS generation. The treatment of cells with anti-LOX-1 antibody reduced the expression of NADPH oxidase (all three subunits) (Fig. 5A). In addition, anti-LOX-1 antibody treatment reduced TGFβ ACT1-induced increase in DCF fluorescence (Fig. 5C), reflecting a reduction in intracellular ROS generation, concomitant with suppression of collagen production. Nonspecific IgG had no effect on TGFβ ACT1-induced expression of NADPH oxidase and ROS generation (Fig. 5, A and C). Further, treatment of fibroblasts with apocynin and diphenyliodonium, the NADPH oxidase inhibitors, attenuated the expression of LOX-1 as well as collagen expression despite forced up-regulation of TGFβ ACT1 (Fig. 4, A and B). To confirm the role of NADPH oxidase, we conducted experiments using gp91phox subunit knockdown methodology. As shown in Figs. 5B and 4C, gp91phox siRNA markedly inhibited the expression of gp91phox, LOX-1, and collagen induced by TGFβ ACT1. The non-targeting siRNA had no effect. Signaling of TGFβ1-LOX-1-mediated Collagen Formation—To study the intracellular signaling mechanism of LOX-1-TGFβ1 interaction, the AAV/TGFβ ACT1-transfected cardiac fibroblasts from WT mice were treated with a variety of inhibitors. As shown in Fig. 4, A and B, treatment of cells with the p44/42 MAPK inhibitors (U0126 and PD98059) or the p38 MAPK inhibitors (SB203580 and SB202190) reduced the expression of collagens (type I and type III) in response to AAV/TGFβ ACT1 transfection. Importantly, TGFβ ACT1-mediated up-regulation of LOX-1 was inhibited by the NADPH oxidase inhibitors, but not by the MAPK inhibitors, indicating that NADPH oxidase activation plays a significant role in TGFβ ACT1-mediated LOX-1 expression and that NADPH oxidase activation is upstream of MAPKs. Next, we measured the expression of redox-sensitive p38 and p44/42 MAPKs. As shown in Fig. 6, A and B, the expression of p38 or p44/42 MAPKs was not altered by TGFβ ACT1 up-regulation. However, TGFβ1 up-regulation markedly increased the phosphorylation of both p38 and p44/42 MAPKs (p < 0.01), which was inhibited by anti-LOX-1 antibody, the NADPH oxidase inhibitors, and the MAPK inhibitors. AAV/Neo transfection or nonspecific IgG had no effect. In this study, we set out to examine the hypothesis that LOX-1 plays an important role in collagen generation in mouse cardiac fibroblasts in response to TGFβ1. Toward that goal, we transfected WT and LOX-1 KO mouse cardiac fibroblasts with TGFβ ACT1 using AAV as the delivery vector. In previous studies, AAV has been shown to transfect smooth muscle cells, cardiomyocytes, and fibroblasts very efficiently (14Hu C.P. Dandapat A. Liu Y. Hermonat P.L. Mehta J.L. Am. J. Physiol. 2007; 293: H1833-H1838Crossref PubMed Scopus (28) Google Scholar, 24Nykanen A.I. Pajusola K. Krebs R. Keranen M.A. Raisky O. Koskinen P.K. Alitalo K. Lemstrom K.B. Circ. Res. 2006; 98: 1373-1380Crossref PubMed Scopus (32) Google Scholar, 25Zhao W. Zhong L. Wu J. Chen L. Qing K. Weigel-Kelley K.A. Larsen S.H. Shou W. Warrington Jr., K.H. Srivastava A. Virology. 2006; 353: 283-293Crossref PubMed Scopus (51) Google Scholar). Indeed, we observed that there was >90% AAV/TGFβ ACT1 transfection efficiency at an m.o.i. of 103. In keeping with the well established role of TGFβ1 in the genesis of fibrosis, the forced up-regulation of TGFβ ACT1 in WT mouse cardiac fibroblasts dramatically enhanced the expression of collagens (types I and III) and increased the level of soluble collagen in the fibroblast supernates. We used TGFβ ACT1 to up-regulate TGFβ1 because previous studies (5Yang B.C. Zander D. Mehta J.L. J. Pharmacol. Exp. Ther. 1999; 291: 733-738PubMed Google Scholar, 14Hu C.P. Dandapat A. Liu Y. Hermonat P.L. Mehta J.L. Am. J. Physiol. 2007; 293: H1833-H1838Crossref PubMed Scopus (28) Google Scholar) have shown that the active moiety of TGFβ1 results in significantly greater biological effects than the latent form of TGFβ ACT1. The suggestion of a role of LOX-1 in the biological effects of TGFβ1 comes from studies that indicate that TGFβ1 influences the pro-inflammatory effect of oxidized low density lipoprotein (12Chen H. Li D. Saldeen T. Mehta J.L. Circ. Res. 2001; 89: 1155-1160Crossref PubMed Scopus (59) Google Scholar). In these studies, pretreatment of endothelial cells with anti-LOX-1 antibody attenuated the synthesis of TGFβ1 in response to oxidized low density lipoprotein. Minami et al. (11Minami M. Kume N. Kataoka H. Morimoto M. Hayashida K. Sawamura T. Masaki T. Kita T. Biochem. Biophys. Res. Commun. 2000; 272: 357-361Crossref PubMed Scopus (94) Google Scholar) provided evidence that TGFβ1 (0.1–10 ng/ml) induces LOX-1 expression in a transcriptional manner in both bovine aortic endothelial and smooth muscle cells in a dose- and time-dependent fashion. Draude and Lorenz (10Draude G. Lorenz R.L. Am. J. Physiol. 2000; 278: H1042-H1048Crossref PubMed Google Scholar) described TGFβ1-mediated stimulation of oxidized low density lipoprotein uptake in freshly isolated and cultured human monocytes with simultaneous severalfold increase in LOX-1 mRNA. All these observations suggest a link between LOX-1 and TGFβ1 in the biology of a variety of cell types. This study provides conclusive evidence that TGFβ1-mediated collagen formation in fibroblasts involves a critical role of LOX-1. Forced overexpression of TGFβ1 in WT mouse cardiac fibroblasts induced a 3–4-fold increase in LOX-1 expression. The evidence for the critical role of LOX-1 in TGFβ1-mediated collagen formation was obtained by two different approaches: first, the use of fibroblasts from LOX-1 KO mouse hearts and second, the use of a specific monoclonal anti-LOX-1 antibody. Both these approaches resulted in a marked inhibition of TGFβ1-mediated collagen formation. It is noteworthy that TGFβ1-mediated fibroblast growth was also blocked in fibroblasts from LOX-1 KO mice. In other experiments, we observed that NADPH oxidase expression was greatly increased in WT mouse cardiac fibroblasts transfected with AAV/TGFβ ACT1. The activity of TGFβ1 has been shown to be associated with generation of ROS (26Sturrock A. Cahill B. Norman K. Huecksteadt T.P. Hill K. Sanders K. Karwande S.V. Stringham J.C. Bull D.A. Gleich M. Kennedy T.P. Hoidal J.R. Am. J. Physiol. 2006; 290: L661-L673Crossref PubMed Scopus (348) Google Scholar, 27Rhyu D.Y. Yang Y. Ha H. Lee G.T. Song J.S. Uh S.T. Lee H.B. J. Am. Soc. Nephrol. 2005; 16: 667-675Crossref PubMed Scopus (455) Google Scholar). This was confirmed in the present studies by examination of NADPH oxidase (p22phox, p47phox, and gp91phox subunits) expression and direct measurement of intracellular ROS as DCF fluorescence. Further, ROS have been shown to activate LOX-1 (7Hu C.P. Dandapat A. Chen J. Fujita Y. Inoue N. Kawase Y. Jishage K.I. Suzuki H. Sawamura T. Mehta J.L. Cardiovasc. Res. 2007; 76: 292-302Crossref PubMed Scopus (59) Google Scholar) and LOX-1 activation itself leads to ROS generation (22Cominacini L. Rigoni A. Pasini A.F. Garbin U. Davoli A. Campagnola M. Pastorino A.M. Lo Cascio V. Sawamura T. J. Biol. Chem. 2001; 276: 13750-13755Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 23Chen X.P. Xun K.L. Wu Q. Zhang T.T. Shi J.S. Du G.H. Vascul. Pharmacol. 2007; 47: 1-9Crossref PubMed Scopus (101) Google Scholar). We observed that the inhibition of NADPH oxidase by apocynin, diphenyliodonium, or gp91phox siRNA reduced LOX-1 expression and collagen formation in WT mouse cardiac fibroblasts despite forced up-regulation of TGFβ ACT1. Interestingly, treatment of fibroblasts with the anti-LOX-1 antibody itself reduced the up-regulation of NADPH oxidase (p22phox, p47phox, and gp91phox subunits) despite forced up-regulation of TGFβ1. These observations provide compelling evidence for a positive feedback between TGFβ1, NADPH oxidase-mediated ROS generation, and LOX-1 (summarized in Fig. 7). Next, we examined the role of redox-sensitive MAPKs in TGFβ1-mediated collagen formation. The AAV/TGFβ ACT1-transfected WT mouse cardiac fibroblasts did not exhibit any change in p38 or p44/42 MAPK protein levels, but their phosphorylation was increased 2–3-fold. MAPK phosphorylation was blocked by the treatment of cells with NADPH oxidase inhibitors as well as anti-LOX-1 antibody. On the other hand, treatment of fibroblasts with the MAPK inhibitors significantly reduced the expression of collagens in fibroblasts transfected with AAV/TGFβ ACT1 without effect on LOX-1 expression (Fig. 4), suggesting that MAPK activation is downstream of ROS generation in response to TGFβ1-NADPH oxidase-LOX-1 cascade (Fig. 7). In essence, this study provides the missing link between TGFβ1 activation and collagen formation in the heart during pro-oxidant states such as hypertension and ischemia-reperfusion injury. Identification of LOX-1 as an important molecule in TGFβ1-mediated collagen synthesis provides a new target for therapy of disease states characterized by excessive tissue remodeling." @default.
• W1984216075 created "2016-06-24" @default.
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• W1984216075 creator A5005269170 @default.
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• W1984216075 date "2008-04-01" @default.
• W1984216075 modified "2023-10-06" @default.
• W1984216075 title "Regulation of TGFβ1-mediated Collagen Formation by LOX-1" @default.
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