Regulation of TGF 1-mediated Collagen Formation by LOX-1 STUDIES BASED ON FORCED OVEREXPRESSION OF TGF

Changping Hu, Abhijit Dandapat, Liuqin Sun, Junaid A. Khan, Yong Liu, Paul L. Hermonat, and Jawahar L. Mehta From Cardiovascular Medicine, University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, Arkansas 72205-7199, Department of Pharmacology, School of Pharmaceutical Sciences, Central South University, Changsha 410078 China, and Department of Ophthalmology, Heping Hospital, Changzhi Medical College, Changzhi 046000 China

forming growth factor (TGF) 3 ␤ 1 is one of the most pleiotropic and multifunctional peptides known (2). It exerts potent effects on many different cell types and is involved in a wide variety of biological processes (2). The cellular actions of TGF␤ 1 are dependent not only on the cell type but also on its state of differentiation and the cytokine milieu (3). Although there is evidence that TGF␤ 1 stimulates fibroblast growth, enhances collagen synthesis, and suppresses collagen degradation (2), the specific effect of TGF␤ 1 on the cardiac remodeling process remains unclear. TGF␤ 1 is synthesized in cells as a precursor molecule, TGF␤ 1 Latent . Conversion from cysteine (Cys 223/225 ) into serine (Ser 223/225 ) in the TGF␤ 1 Latent molecule is associated with the formation of TGF␤ 1 ACT (active TGF␤ 1 ) (4). It is the TGF␤ 1 ACT that appears to be functionally relevant in the process of ischemia-reperfusion (5).
LOX-1 is a lectin-like receptor for oxidized low density lipoprotein (6). It is also up-regulated in response to oxidative stress (7). Activation of LOX-1 enhances the growth of cardiac fibroblasts and promotes collagen synthesis (8,9). It has been reported that TGF␤ 1 can regulate LOX-1 expression in vascular endothelial cells, smooth muscle cells, and monocytes/macrophages (10,11). Another study showed that LOX-1 blockade reduces TGF␤ 1 protein expression in endothelial cells (12). The present study was conducted to examine whether and how LOX-1 mediates TGF␤ 1 -mediated collagen production.

EXPERIMENTAL PROCEDURES
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 (13). 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 gp91 phox subunit siRNA (Dharmacon); human recombinant TGF␤ 1 (rTGF␤ 1 ) and mouse nonspecific IgG (Sigma); all pri-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Both authors contributed equally to this work. 2  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 (13) 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/TGF␤ 1 ACT and Generation of Recombinant AAV Stocks-The TGF␤ 1 mutant TGF␤ 1 ACT cDNA was generated, sequenced, and ligated into an adeno-associated virus (AAV) vector, dl6 -95, as described recently (14). Recombinant AAV vector hereafter will be referred to as AAV/ TGF␤ 1 ACT . The generation of AAV/Neo virus has been described earlier (15). Virus stocks were generated as described previously (15). The titer of purified virus, in encapsidated genomes per milliliter (eg/ml), was calculated by dot-blot hybridization and determined to be ϳ10 11 eg/ml.
Cell Culture and AAV Vector Infection-Mouse (WT and LOX-1 KO) cardiac fibroblasts were isolated and cultured as described earlier (16). 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 10 3 and incubated with the cells for 72 h at 37°C in 5% CO 2 /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 ϫ 10 12 virus particles/ml (3.1 ϫ 10 10 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 g/ml), nonspecific IgG (10 g/ml), apocynin (600 M), diphenyliodonium   ACT had a stimulatory effect on fibroblast growth. This effect was much less evident in fibroblasts from LOX-1 KO mice. AAV/Neo alone had no effect on cell growth. Cell growth was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) (A) and cell number count (B). Error bars, Ϯ S.E. and modified in accordance with the results of pilot experiments. In other experiments, cardiac fibroblasts were treated with rTGF␤ 1 (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.
Soluble Collagen Assay-Soluble collagen production was determined by the Sircol assay (Biocolor) as described earlier (21) and expressed as g/10 6 cells.
Measurement of ROS in Cardiac Fibroblasts-Intracellular ROS generation was measured with the use of 2Ј,7Ј-dichlorodihydrofluorescein diacetate (10 M) fluorescent signal, a cellpermeant indicator for ROS, as described previously (14). Results were displayed in a ratiometric fashion normalized for the control conditions.
Statistical Analysis-Data, based on at least four separate experiments, are expressed as means Ϯ 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 Ͻ 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 -10 4 and incubated with the cells for 72 h at 37°C in 5% CO 2 /95% air. More than 90% of the cells showed GFP expression at m.o.i. of 10 3 . 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 10 3 , transfection in subsequent experiments with AAV vectors was performed at m.o.i. of 10 3 .
Increase in TGF␤ 1 Expression-After 72 h of AAV infection, fibroblasts were harvested for Western blotting. As shown in Fig. 1B, total TGF␤ 1 expression was increased in AAV/TGF␤ 1 ACT -transfected cells, indicating successful delivery of the transgene. AAV/Neo alone had no effect on TGF␤ 1 expression. Note that there was no difference in TGF␤ 1 expression in cardiac fibroblasts from WT and LOX-1 KO mice.
AAV/TGF␤ 1 ACT Transfection Facilitates Cell Growth-AAV/TGF␤ 1 ACT transfection had a stimulatory effect on fibroblast growth, and the number of fibroblasts increased by ϳ50% (versus fibroblasts kept under control conditions). This effect was much less evident in fibroblasts from LOX-1 KO mice (p Ͻ 0.01 versus AAV/TGF␤ 1 ACT -transfected fibroblasts from wildtype mice) (Fig. 2, A and B). AAV/Neo alone had no effect on cell growth.
Interaction between TGF␤ 1 ACT , LOX-1 Expression, and Collagen Synthesis in Fibroblasts-AAV/TGF␤ 1 ACT transfection increased the expression of collagen type I and III (p Ͻ 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 Ͻ 0.01 versus AAV/ TGF␤ 1 ACT -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/TGF␤ 1 ACT transfected cardiac fibroblasts (Fig. 3B). To further confirm the effect of AAV/TGF␤ 1 ACT on collagen expression, we treated cells with rTGF␤ 1 directly and found that the effects of rTGF␤ 1 were consistent with that of transfection with AAV/TGF␤ 1 ACT (Fig.  3C). These observations suggest that LOX-1 plays an important role in TGF␤ 1 -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 TGF␤ 1 -mediated collagen synthesis, the role of LOX-1 in the effects of TGF␤ 1 ACT was confirmed by use of a specific FIGURE 3. Effect of AAV/TGF␤ 1 ACT transfection or rTGF␤ 1 on collagen production. A and C, expression of collagen type I and III measured by Western blot analysis. B, soluble collagen levels measured by the Sircol assay. AAV/TGF␤ 1 ACT transfection or rTGF␤ 1 significantly increased the expression of collagen type I and III and soluble collagen level in WT mouse cardiac fibroblasts, which were much less pronounced in LOX-1 KO mouse fibroblasts. AAV/Neo alone had no effect on collagen production. Error bars, Ϯ S.E.
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 Ͻ 0.01). Note that nonspecific IgG had no effect on AAV/TGF␤ 1 ACTinduced collagen expression (Fig. 4B).
Interaction between TGF␤ 1 , NADPH Oxidase, and LOX-1-Oxidant stress up-regulates the expression and activation of LOX-1 (7), and activation of LOX-1 itself can stimulate the formation of ROS (22,23). NADPH oxidase activation is a major source of ROS in cardiac fibroblasts (19). We, therefore, measured intracellular ROS generation and NADPH oxidase expression in fibroblasts transfected with AAV/TGF␤ 1 ACT . Indeed, AAV/TGF␤ 1 ACT transfection enhanced the expression of NADPH oxidase (p22 phox , p47 phox , and gp91 phox 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 TGF␤ 1 ACT -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 TGF␤ 1 ACT -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 TGF␤ 1 ACT (Fig. 4, A and  B). To confirm the role of NADPH oxidase, we conducted experiments using gp91 phox subunit knockdown methodology. As shown in Figs. 5B and 4C, gp91 phox siRNA markedly inhibited the expression of gp91 phox , LOX-1, and collagen induced by TGF␤ 1 ACT . The nontargeting siRNA had no effect.
Signaling of TGF␤ 1 -LOX-1-mediated Collagen Formation-To study the intracellular signaling mechanism of LOX-1-TGF␤ 1 interaction, the AAV/TGF␤ 1 ACT -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/TGF␤ 1 ACT transfection. Importantly, TGF␤ 1 ACT -mediated up-regulation of LOX-1 was inhibited by the NADPH oxidase inhibitors, but not by the MAPK The expression of collagen type I and III was increased in AAV/TGF␤ 1 ACT -transfected fibroblasts, and this increase was inhibited by anti-LOX-1 antibody as well as the NADPH oxidase inhibitors apocynin and diphenyliodonium (DPI), the p38 MAPK inhibitors SB203580 and SB202190, the p44/42 MAPK inhibitors U0126 and PD98059 (A and B), and gp91phox siRNA (C). TGF␤ 1 ACT -mediated LOX-1 expression was inhibited by treatment of fibroblasts with anti-LOX-1 antibody, the NADPH oxidase inhibitors, and gp91phox siRNA, but not by MAPK inhibitors. Nonspecific IgG and non-targeting siRNA had no effect on collagen and LOX-1 expression. Error bars, Ϯ S.E.

FIGURE 5. Effect of AAV/TGF␤ 1 ACT transfection on NADPH oxidase expression and ROS generation. AAV/ TGF␤ 1
ACT transfection enhanced the expression of NADPH oxidase subunits p22 phox , p47 phox , and gp91phox (A and C) and ROS generation measured by 2Ј,7Ј-DCF fluorescence (B). The treatment of cells with anti-LOX-1 antibody and the NADPH oxidase inhibitors apocynin and diphenyliodonium (DPI) reduced the expression of NADPH oxidase (p22 phox , p47 phox , and gp91phox) and DCF fluorescence. AAV/Neo or nonspecific IgG alone had no effect on the expression of NADPH oxidase and DCF fluorescence. gp91phox siRNA, but not non-targeting siRNA, effectively inhibited the basal and AAV/TGF␤ 1 ACT -mediated expression of gp91phox. Error bars, Ϯ S.E.
inhibitors, indicating that NADPH oxidase activation plays a significant role in TGF␤ 1 ACT -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 TGF␤ 1 ACT up-regulation. However, TGF␤ 1 up-regulation markedly increased the phosphorylation of both p38 and p44/42 MAPKs (p Ͻ 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.

DISCUSSION
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 TGF␤ 1 . Toward that goal, we transfected WT and LOX-1 KO mouse cardiac fibroblasts with TGF␤ 1 ACT using AAV as the delivery vector. In previous studies, AAV has been shown to transfect smooth muscle cells, cardiomyocytes, and fibroblasts very efficiently (14,24,25). Indeed, we observed that there was Ͼ90% AAV/TGF␤ 1 ACT transfection efficiency at an m.o.i. of 10 3 . In keeping with the well established role of TGF␤ 1 in the genesis of fibrosis, the forced up-regulation of TGF␤ 1 ACT 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 TGF␤ 1 ACT to up-regulate TGF␤ 1 because previous studies (5,14) have shown that the active moiety of TGF␤ 1 results in significantly greater biological effects than the latent form of TGF␤ 1 ACT . The suggestion of a role of LOX-1 in the biological effects of TGF␤ 1 comes from studies that indicate that TGF␤ 1 influences the pro-inflammatory effect of oxidized low density lipoprotein (12). In these studies, pretreatment of endothelial cells with anti-LOX-1 antibody attenuated the synthesis of TGF␤ 1 in response to oxidized low density lipoprotein. Minami et al. (11) provided evidence that TGF␤ 1 (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 (10) described TGF␤ 1 -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 TGF␤ 1 in the biology of a variety of cell types.
This study provides conclusive evidence that TGF␤ 1 -mediated collagen formation in fibroblasts involves a critical role of LOX-1. Forced overexpression of TGF␤ 1 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 TGF␤ 1 -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 TGF␤ 1 -mediated collagen formation. It is noteworthy that TGF␤ 1 -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/TGF␤ 1 ACT . The activity of TGF␤ 1 has been shown to be associated with generation of ROS (26,27). This was confirmed in the present studies by examination of NADPH oxidase (p22 phox , p47 phox , and gp91 phox subunits) expression and direct measurement of intracellular ROS as DCF fluorescence. Further, ROS have been shown to activate LOX-1 (7) and LOX-1 activation itself leads to ROS generation ACT up-regulation. However, TGF␤ 1 up-regulation markedly increased the phosphorylation of both p38 and p44/42 MAPKs, which was inhibited by anti-LOX-1 antibody, apocynin, diphenyliodonium (DPI), and MAPK inhibitors. AAV/Neo transfection or nonspecific IgG had no effect. Error bars, Ϯ S.E. FIGURE 7. Hypothesized pathways of AAV/TGF␤ 1 ACT transfection-mediated up-regulation of collagen. TGF␤ 1 up-regulates the expression of LOX-1 and NADPH oxidase, and LOX-1 induces further ROS generation and TGF␤ 1 synthesis. These observations suggest a positive feedback between TGF␤ 1 , NADPH oxidase-mediated ROS generation, and LOX-1. ROS generation in response to TGF␤ 1 -NADPH oxidase-LOX-1 cascade causes activation of MAPKs followed by transcription of redox-sensitive transcription factor/s that induce the gene for collagens. (22,23). We observed that the inhibition of NADPH oxidase by apocynin, diphenyliodonium, or gp91 phox siRNA reduced LOX-1 expression and collagen formation in WT mouse cardiac fibroblasts despite forced up-regulation of TGF␤ 1 ACT . Interestingly, treatment of fibroblasts with the anti-LOX-1 antibody itself reduced the up-regulation of NADPH oxidase (p22 phox , p47 phox , and gp91 phox subunits) despite forced upregulation of TGF␤ 1 . These observations provide compelling evidence for a positive feedback between TGF␤ 1 , NADPH oxidase-mediated ROS generation, and LOX-1 (summarized in Fig. 7).
Next, we examined the role of redox-sensitive MAPKs in TGF␤ 1 -mediated collagen formation. The AAV/TGF␤ 1 ACTtransfected 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/TGF␤ 1 ACT without effect on LOX-1 expression (Fig.  4), suggesting that MAPK activation is downstream of ROS generation in response to TGF␤ 1 -NADPH oxidase-LOX-1 cascade (Fig. 7).
In essence, this study provides the missing link between TGF␤ 1 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 TGF␤ 1 -mediated collagen synthesis provides a new target for therapy of disease states characterized by excessive tissue remodeling.