Am J Physiol Heart Circ Physiol 306: H789–H796, 2014. First published January 24, 2014; doi:10.1152/ajpheart.00559.2013.
Vascular endothelial growth factor-C: its unrevealed role in fibrogenesis Tieqiang Zhao,1 Wenyuan Zhao,1 Weixin Meng,2 Chang Liu,2 Yuanjian Chen,1 and Yao Sun1 1
Division of Cardiovascular Diseases, Department of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee; and 2Division of Cardiac Surgery, Department of Surgery, First Affiliate Hospital of Harbin Medical University, Harbin, China Submitted 24 July 2013; accepted in final form 21 January 2014
Zhao T, Zhao W, Meng W, Liu C, Chen Y, Sun Y. Vascular endothelial growth factor-C: its unrevealed role in fibrogenesis. Am J Physiol Heart Circ Physiol 306: H789 –H796, 2014. First published January 24, 2014; doi:10.1152/ajpheart.00559.2013.—Vascular endothelial growth factor (VEGF)-C is a key mediator of lymphangiogenesis. Our recent study shows that VEGF-C/VEGF receptors (VEGFR)3 are significantly increased in the infarcted rat myocardium, where VEGFR-3 is expressed not only in lymph ducts but also in myofibroblasts, indicating that VEGF-C has an unrevealed role in fibrogenesis during cardiac repair. The current study is to explore the regulation and molecular mechanisms of VEGF-C in fibrogenesis. The potential regulation of VEGF-C on myofibroblast differentiation/growth/migration, collagen degradation/synthesis, and transforming growth factor (TGF)- and ERK pathways was detected in cultured cardiac myofibroblasts. Our results showed that VEGF-C significantly increased myofibroblast proliferation, migration, and type I/III collagen production. Matrix metalloproteinase (MMP)-2 and -9 were significantly elevated in the medium of VEGF-C-treated cells, coincident with increased tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. Furthermore, VEGF-C activated the TGF-1 pathway and ERK phosphorylation, which was significantly suppressed by TGF- or ERK blockade. This is the first study indicating that in addition to lymphangiogenesis, VEGF-C is also involved in fibrogenesis through stimulation of myofibroblast proliferation, migration, and collagen synthesis, via activation of the TGF-1 and ERK pathways. myofibroblasts; VEGF-C; collagen synthesis; collagen degradation; TGF-1; ERK VEGF IS A SUBFAMILY OF GROWTH factors, composed of five different isoforms: VEGF-A, -B, -C, -D, and placenta growth factor. VEGF initiates cellular responses by interacting with tyrosine kinase receptors on the cell surface. There are three main VEGF receptor subtypes, VEGFR-1, -2, and -3. VEGF-A binds to VEGFR-1 and -2; VEGF-B binds to VEGFR-1; and VEGF-C and -D bind to VEGFR-2 and -3. VEGF-A and -B are the key mediators of angiogenesis, while VEGF-C and -D are recognized to stimulate lymphangiogenesis in physiological and pathological conditions (14, 25). Ventricular dysfunction appears most commonly in patients with previous myocardial infarction (MI). Cardiac repair occurring at the site of myocyte loss preserves the structural integrity of the heart and is essential to its recovery. Fibrogenesis (scar formation) is central to cardiac repair. Myofibroblasts, which are phenotypically transformed fibroblasts, are responsible for the production of collagen and scar contraction in the infarcted myocardium (6, 20). Fibroblasts are the major source of myofibroblasts. Stimulated by TGF- and mechani-
Address for reprint requests and other correspondence: Y. Sun, Div. of Cardiovascular Diseases, Dept. of Medicine, Univ. of Tennessee Health Science Center, 956 Court Ave, Rm. B324, Memphis, TN 38163 (e-mail: [email protected]). http://www.ajpheart.org
cal stretch, fibroblasts differentiate into myofibroblasts in the repairing tissue and, possessing the features of both fibroblasts and smooth muscle cells, are dynamic in collagen synthesis and fibrous tissue contraction (3). Maintaining the extracellular matrix in the infarcted myocardium is essential in preventing the heart from dilatation. Local factors regulating cardiac repair/remodeling have drawn great attention. Our recent study has shown that VEGF-C expression is significantly increased in the infarcted myocardium (31). Our study has further shown that VEGFR-3 is significantly increased in the infarcted myocardium in both early and late stages of MI. In addition to lymphatic vessels, VEGFR-3 is highly expressed in myofibroblasts of the infarcted myocardium, suggesting that VEGF-C plays a role in cardiac fibrous tissue formation in an autocrine/paracrine manner. These observations imply that, in addition to lymphangiogenesis, VEGF-C has unidentified functions in fibrogenesis during cardiac repair post-MI. The molecular basis of VEGF-C on cardiac fibrogenic response, however, remains unknown and is investigated in the current study. Primary cultures of rat cardiac fibroblasts automatically transform to myofibroblasts in the first or second passage (P1 or P2) (18). Both in vitro and in vivo cardiac myofibroblasts are of a similar phenotype. With the use of cultured myofibroblasts, the potential influence of VEGF-C on myofibroblast growth, migration, proliferation, and collagen turnover was explored in the study. The TGF- and MAPK/ERK signaling cascade are the major pathways controlling cellular processes associated with fibrogenesis, including growth, proliferation, and survival. Activation of the TGF- and MAPK/ERK pathways is detected in various fibrotic diseases (24, 28). The current study also determined whether the regulation of VEGF-C on fibrogenesis is through TGF- and/or ERK pathways. MATERIAL AND METHODS
Cell culture. Cardiac fibroblasts were isolated from 8-wk-old male Sprague-Dawley rats (14). Briefly, rat was anesthetized with isoflurane inhalation, and the heart was excised, washed in PBS, and cut into 1-mm3 pieces. The tissue was digested at 37°C in digestion medium containing a mixture of collagenase B (115 mg/100 ml; Worthington, Lakewood, NJ) and trypsin (50 mg/100 ml; Sigma, St. Louis, MO) for 10 min with constant shaking. Cells from the third to tenth digestions were pooled and pelleted down. The pellet was resuspended in 5 ml DMEM 10% FBS, seeded into 60-mm dishes, and kept at 37°C in CO2 incubator for a preplating period of 150 min. Unattached cells were discarded, and attached cells were washed and grown in the plating medium. Cultures were maintained at 37°C in 95% humidified air and 5% CO2 atmosphere. Fibroblasts in culture automatically differentiate into myofibroblasts at P1 and P2 (18). Myofibroblasts were confirmed by immunohistochemical ␣-smooth muscle actin (SMA) staining. The dose (0, 10, 50, and 200 ng/ml
0363-6135/14 Copyright © 2014 the American Physiological Society
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medium) and time (6, 12 and 24 h) response of VEGF-C on fibrous tissue formation were determined. This study was approved by the University of Tennessee Health Science Center Animal Care and Use Committee. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Cell proliferation assay. Myofibroblast proliferation was assessed using bromodeoxyuridine (BrdU) cell proliferation assay kit (Cell Signaling, Danvers, MA). The assay was performed according to the manufacturer’s instructions. Myofibroblasts were seeded in 96-well plates. After being quiescient in serum-deprived medium for 24 h, cells were exposed to VEGF-C (200 ng/ml) for 20 h. BrdU (10 M) was then added, and cells were further incubated for 4 h. At the endpoint of the incubation, medium was withdrawn, 100 l/well of fixing solution were added, and the plates were incubated at room temperature for 30 min. Afterwards, the solution was aspirated and the wells were rinsed three times with a washing solution and eventually dried on a paper towel. Anti-BrdU monoclonal antibodies were then added (100 l/well), and the wells were incubated for 1 h at room temperature. Medium was removed, and wells rinsed three times with a washing solution. Horseradish peroxidase-conjugated anti-mouse IgG was added, and the plate was incubated at room temperature for 30 min. After three washes, 100 l TMB substrate were added to each well, followed by 100 l of stop solution for 30 min. The absorbance was read at 450 nm in a microplate reader (4). Eight different wells were counted in each group. Cell migration assay. Myofibroblast migration was detected with a modified Boyden’s chamber assay. The cell culture inserts, which contain membranes with 8.0-m pore size, were placed in a 24-well tissue culture plate (Millipore ECM508, Billerica, MA). Myofibroblasts were quiesced for 24 h, then trypsinized, resuspended in DMEM, and seeded into the upper chamber at 1⫻ 105 cells/well. The lower chamber contains DMEM with VEGF-C (200 ng/ml) as a chemoattractant. After incubation for 6 h at 37°C, cells/media were removed from the top side of the insert by pipetting out the remaining cell suspension. The inserts were placed in clean wells containing 400 l of cell stain for 20 min.
The inserts were then rinsed in water, and cotton-tipped swabs were used to remove nonmigratory cells from the interior of the insert. The inserts were transferred to a clean well containing 200 l of extraction buffer for 15 min. The dye mixture (100 l) was transferred to a 96-well plate, and optical density was measured at 560 nm (15). Six different samples were tested in each group. Western blotting. The effects of TGF-1 (10 ng/ml) on type I collagen and matrix metalloproteinase (MMP)-2 and MMP-9 levels and VEGF-C on expression of type I and III collagen, MMP-2, MMP-9, inhibitor of metalloproteinase (TIMP)-1 and -2, and TGF-1 proteins in culture medium and ␣-SMA, Smad2, and phosphorylated Smad2 in myofibroblasts were assessed by Western blot. The cells were plated in sixwell plates, grown to subconfluence, and then quiesced for 24 h. Cells were then incubated with or without VEGF-C for 24 h. Medium was concentrated by centrifugation for 30 min in Amicon ultra centrifugal filters (Millipore, Billerica, MA). Proteins from cells and medium were loaded on the gel, subjected to SDSPAGE (10% polyacrylamide gel), and transferred onto nitrocellulose membranes using a Bio-Rad Mini Trans Blot electrophoretic transfer unit. Membranes were blocked for nonspecific protein with 5% nonfat dry milk in TBS and then probed overnight at 4°C with primary antibodies against type I and III collagen, MMP-2, MMP-9, TIMP-1, TIMP-2, TGF-1, ␣-SMA, Smad2, and phosphorylated Smad2 (Millipore and Sigma). Membranes were then washed three times (10 min per wash) with TBS with 0.05% Tween-20 to remove unbound antibodies, and further incubated with appropriate horseradish peroxidase-conjugated secondary antibody (1:2,000). Membranes were developed by a chemiluminescence reagent kit (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s protocol. The amount of protein detected was assessed by means of quantitative densitometry analysis with a computer image analyzing system (29). Six different samples were tested in each group. ELISA. The effect of VEGF-C on TGF-1 production in cardiac myofibroblasts was determined by ELISA. Supernatants were collected for TGF-1 detection with a commercial ELISA kit (R&D Systems, Minneapolis, MN). Briefly, TGF-1 standards, positive control, and 50-ul activated samples were added to a 96-well plate
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Fig. 1. Effect of VEGF-C on myofibroblast differentiation, proliferation, and migration. Cultured cardiac fibroblasts automatically differentiated into myofibroblasts at P1 and expressed ␣-smooth muscle actin (␣-SMA; A, left, arrows). Compared with vehicletreated myofibroblasts, VEGF-C did not affect myofibroblast differentiation (B), significantly increased myofibroblast proliferation (C) and migration (D). A, right: negative control of ␣-SMA labeling. *P ⬍ 0.05 vs. controls (CTL); n ⫽ 8/group.
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coated with TGF-1 antibody. The plate was incubated for 2 h at room temperature and then washed by wash buffer (400 l) four times. TGF-1 conjugate (100 l) was then added to the plate. After a 2-h incubation, the plate was washed again four times as above. Substrate solution (100 l) was added to the plate, followed by stop solution (100 l). The optical density of each well was measured within 30 min using a microplate reader set to 450 nm with a correction at 540 nm. Six samples were included in each group. TGF-1 blockade. TGF-1 siRNA and neutralizing antibody were used to detect whether the regulation of VEGF-C on fibrogenesis is via the TGF-1 pathway. At confluence of 50%, myofibroblasts were quiesced for 24 h. TGF-1 siRNA (final concentration 30 nM; control siRNA sequence: sense: 5=-UAACGACGCGACGACGUAATT-3=, antisense: 5=-UUACGUCGUCGCGUCGUUATT-3=; TGF-b1 siRNA sequence: sense: 5=-GGAGAGCCCUGGAUACCAATT-3=, antisense: 5=-UUGGUAUCCAGGGCUCUCCGG-3=) was introduced to cells through Lipofectamine RNAiMAX reagent (Invitrogen, Grand Island, NY) (9, 30). The transfection was performed as described by the manufacturer’s manual. At 6 h after the transfection, VEGF-C was administered to the cells with final concentration of 200 ng/ml. The neutralizing antibody of TGF- 1 (10 ug/ml; R&D Systems) was applied to cells, followed by VEGF-C (200 ng/ml) for 1 h. The medium and cell lysate were collected at 24 h. Myofibroblast proliferation and collagen synthesis were analyzed as described above. ERK activation and blockade. To detect ERK1/2 activation, myofibroblasts were quiesed for 24 h and VEGF-C (200 ng/ml) and then administered to the cells for 10 min. Cell lysate was collected in modified RIPA buffer. ERK phosphorylation was detected by Western blot using antibodies against ERK1/2 and phospho-ERK1/2 (Cell Signaling). To determine whether the role of VEGF-C on fibrogenesis is through the ERK pathway, U0126 (ERK inhibitor, 10 uM) was added to the plates for 1 h, followed by VEGF-C treatment. Myofibroblast proliferation and collagen production were analyzed as described above. Statistical analysis. Statistical analysis of cell differentiation, proliferation, migration, Western blot, and ELIZA data among the groups was performed using Student t-test or ANOVA. Values are expressed as means ⫾ SE with P ⬍ 0.05 considered significant. Multiple group comparisons among controls and each group were made by Scheffé’s F-test.
VEGF-C promotes type I and III collagen synthesis. Collagen production is the key function of myofibroblasts. Collagen is a secreted protein released from cells into the interstitial space. In cultured myofibroblasts, collagen is secreted into the culture medium. Type I and III collagen are the major collagen isoforms. Via Western blot, we observed low levels of type I collagen in the medium of the control cells. VEGF-C treatment significantly increased type I collagen contents in the culture medium compared with that of the control group. Our study further shows that VEGF-C stimulated type I collagen synthesis in a time- and dose-dependent manner (Fig. 2, A and B, respectively). Further-
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Fibroblast-myofibroblast differentiation. The hallmark of myofibroblasts is their expression of ␣-SMA, which is not expressed by fibroblasts. Fibroblasts spontaneously transform into myofibroblasts in culture. By immunohistochemical ␣-SMA staining, results showed that cultured cardiac fibroblasts began to express ␣-SMA at P1 (Fig. 1A). Western blotting detection shows that VEGF-C treatment did not alter ␣-SMA expression in cultured cells (Fig. 1B). VEGF-C increases myofibroblast proliferation. The potential role of VEGF-C on myofibroblast proliferation was examined by the BrdU cell proliferation assay. We found that VEGF-C treatment significantly increased myofibroblast proliferation compared with vehicle-treated control cells (Fig. 1C). VEGF-C stimulates myofibroblast migration. ␣-SMA is a cytoskeletal protein involved in cell contraction and migration. In the early phase of MI, migration enables the rapid recruitment of myofibroblasts to the infarct site. The potential role of VEGF-C on myofibroblast migration was examined by the Boyden’s chamber assay. We found that myofibroblast migration was significantly increased in cells receiving VEGF-C compared with vehicle-treated cells (Fig. 1D).
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Fig. 2. VEGF-C promotes type I and III collagen synthesis in myofibroblasts. Compared with controls, VEGF-C treatment significantly stimulated type I collagen secretion in a time (A)- and dose-dependent (B) manner. VEGF-C also promoted type III collagen production (C). *P ⬍ 0.05 vs. controls (CTL); n ⫽ 6/group.
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more, VEGF-C also significantly elevated type III collagen release in myofibroblasts (Fig. 2C). VEGF-C upregulates MMP-2 and MMP-9 expression. MMPs are also secreted proteins. Western blot detection revealed that MMP-2 and MMP-9 levels were barely detectable in the medium of the control cells. VEGF-C treatment was found to increase MMP-2 and MMP-9 secretion by cultured myofibroblasts (Fig. 3, A and B, respectively). VEGF-C promotes TIMP-1 and TIMP-2 expression. TIMPs are glycoprotein peptidases involved in inhibition of extracellular matrix degradation. TIMPs are also secreted proteins that are released into the interstitial space and culture medium. Western blot detection showed that myofibroblasts in the control group produced extremely low levels of TIMP-1 and TIMP-2. VEGF-C treatment significantly increased TIMP-1 and TIMP-2 levels in the medium compared with that of the control group (Fig. 3, C and D, respectively). VEGF-C activates TGF-1 pathway. TGF-1 is a profibrogenic mediator produced by several types of cells, including myofibroblasts, and is a secreted protein. TGF-1 protein levels in the culture medium were measured by Western blot and ELISA (Fig. 4, A and B). TGF-1 was barely detectable in the medium of the control cells. In VEGF-C-treated cells, TGF-1 protein level was significantly elevated in the medium compared with that of the controls. TGF- signaling is initiated by phosphorylation of the cytoplasmic signaling molecules Smad2/3. The current study shows that VEGF-C treatment enhanced Smad2 phosphorylation in myofibroblasts, which started at 10 min, peaked at 30 min, and then declined at 1 h (Fig. 4C). TGF-1 stimulates type I collagen, MMP-2, and MMP-9 release into the medium (Fig. 5A). To determine whether the
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role of VEGF-C on fibrogenesis is via activation of the TGF- pathway, we blocked TGF-1 synthesis with TGF-1 siRNA or the neutralizing antibody. Both TGF-1 siRNA and neutralizing treatments significantly suppressed VEGF-C-induced myofibroblast proliferation (Fig. 5A) and type I collagen synthesis (Fig. 5, B and C). VEGF-C activates ERK pathway. The ERK signal pathway functions in cellular proliferation, differentiation, and survival. To determine whether the role of VEGF-C on fibrogenesis is via activation of the ERK pathway, we examined the effect of VEGF-C on ERK phosphorylation. Compared with vehicletreated cells, we observed significantly increased ERK phosphorylation in myofibroblasts after 10 min of VEGF-C treatment compared with controls (Fig. 6A). To determine whether VEGF-C-induced fibrogenesis is through the ERK pathway, we blocked the ERK pathway with an ERK inhibitor (U0126). We found that U0126 treatment significantly diminished VEGF-C-induced myofibroblast proliferation (Fig. 6B) and type I collagen synthesis (Fig. 6C). Interaction between TGF-1 and ERK pathways. To study whether the TGF-1 and ERK pathways cross talk in myofibroblast-induced fibrogenesis, we cotreated myofibroblasts with a VEGF-C and ERK inhibitor (U0126). We found that U-126 did not suppress VEGF-C-induced TGF- synthesis (Fig. 7). DISCUSSION
VEGF-C and VEGFR-3 are coexpressed in myofibroblasts in the infarcted myocardium, suggesting a potential role of VEGF-C in myofibroblast growth and function. The current
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Fig. 3. VEGF-C increases matrix metalloproteinase (MMP)-2 (A) and -9 (B) and tissue inhibitor of metalloproteinase (TIMP)-1 (C) and -2 (D) synthesis in myofibroblasts. MMP-2, MMP-9, TIMP-1 and TIMP-2 were not detectable in the medium of vehicle-treated myofibroblasts. VEGF-C treatment induced MMP-2, MMP-9, TIMP-1, and TIMP-2 release; n ⫽ 6/group.
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Fig. 4. VEGF-C upregulates TGF-1 production and Smad2 phosphorylation. Detected by Western blot (A) and ELISA (B), TGF-1 was not detectable in the medium of vehicle-treated cells, while VEGF-C treatment significantly enhanced TGF-1 levels in the medium. C: VEGF-C treatment significantly enhanced Smad2 phosphorylation in myofibroblasts. *P ⬍ 0.05 vs. controls (CTL); n ⫽ 6/group.
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Fig. 5. TGF- blockade abolishes the role of VEGF-C on fibrogenesis. Compared with control cells, TGF-1 neutralizing antibody significantly suppressed VEGF-C-induced myofibroblast proliferation (B) and type I collagen synthesis (A). TGF-1 siRNA treatment significantly suppressed VEGF-C-induced type I collagen production (C and D). *P ⬍ 0.05 vs. controls; #P ⬍ 0.05 vs. VEGFC treatment; n ⫽ 6/group.
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broblast growth factor, etc., have been shown to regulate myofibroblast proliferation (5, 19, 30). The current study has revealed that VEGF-C treatment has no effect on myofibroblast differentiation but markedly increases myofibrobalst proliferation. Previously, VEGF-C has been reported to stimulate lymph endothelial cell replication (2, 27). Anti-VEGFR-3 antibody has been shown to inhibit VEGF-C-induced tumor lymphangiogenesis and metastasis by suppressing lymph endothelial cell proliferation (2). Our observation indicates that in addition to lymph endothelial cells, VEGF-C also promotes the proliferation of other cell lines, including myofibroblasts, and is involved in other cellular actions, such as fibrogenesis during tissue repair. Myofibroblasts are mobile and contractile. Following MI, myofibroblasts migrate into the infarct area. Their contractile properties, combined with their synthesis of extracellular matrix proteins, serve the purpose of preventing infarct expansion. The process of migration of myofibroblasts in the infarct area is not completely elucidated. Myofibroblasts are highly responsive to certain growth factors. Platelet-derived growth factor, interstitial growth factor, and endothelial growth factor and TGF-1 stimulate the migration of myofibroblasts (10). The result of the present study shows that VEGF-C serves as another mediator of myofibroblast migration. Previously, VEGF-C has been reported to play an autocrine role in metastasis by promoting tumor cell motility (23). Depletion of endogenous VEGF-C by treatments with a VEGF-C siRNA inhibited tumor cell migration (17). Thus VEGF-C is involved in cell migration in various pathological conditions. Next, we detected the potential regulation of VEGF-C on the function of myofibroblasts. The key role of myofibroblasts is to produce extracellular matrix. Collagen is the major component of extracellular matrix. So far, 29 types of collagen have been identified and described. Among all collagen subtypes, type I and III collagens are the main elements of cardiac fibrous tissue (7, 21). Myofibroblast-induced collagen synthesis is known to
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study explored the regulation of VEGF-C on myofibroblastinduced fibrogenesis and its underlying molecular mechanisms. First, we tested whether VEGF-C regulated cardiac myofibroblast differentiation, proliferation, and migration. Myofibroblasts are primarily differentiated from cardiac interstitial fibroblasts in the infarcted heart. Following MI, they first appear at the border zone (the region between the infarcted and noninfarcted myocardium) and rapidly proliferate and accumulate in the infarcted myocardium. Locally released factors and cytokines, such as TGF-, platelet-derived growth factor, fi-
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Fig. 6. VEGF-C stimulates ERK phosphorylation in myofibroblasts. Compared with controls, VEGF-C elevated ERK phosphorylation (p-ERK1/2), which was blocked by the ERK inhibitor U0126 (A). VEGF-C-induced myofibroblast proliferation (B) and type I collagen synthesis (C) were significantly reduced by U0126; n ⫽ 6/group. *P ⬍ 0.05 vs. controls; #P ⬍ 0.05 vs. VEGFC treatment.
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Fig. 7. Effect of the ERK inhibitor on VEGF-C-induced TGF-1 production in myofibroblasts. VEGF-C enhanced TGF-1 production by myofibroblasts, which was not diminished by U0126; n ⫽ 6/group.
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be primarily stimulated by TGF- (8, 12). The results from the present study reveal that VEGF-C serves as a stimulatory factor in the function of myofibroblasts and promotes type I and III collagen production. Fibrous tissue accumulation relies on the balance between collagen synthesis and degradation with MMPs playing a central role in collagen degradation. MMPs are a large family of proteases with ⬎20 members. MMPs are secreted proteins produced by various types of cells, including myofibroblasts (13). After secretion into the interstitial space, MMP activity is controlled by TIMPs. TIMPs function as an important regulatory brake on MMP activity by inhibiting the active species, thereby suppressing collagen degradation (11). TIMPs comprise a family of four subtypes and are produced in various cells, including myofibroblasts (16). Our data have shown that VEGF-C treatment significantly increased MMP-2 and MMP-9 release and is coincident with upregulated TIMP-1 and TIMP-2 expression in myofibroblasts. These observations suggest that even though MMPs are elevated, coexpression of TIMPs counteract the activity of MMPs and limit collagen degradation. Finally, we investigated whether the role of VEGF-C on fibrogenesis is via activating TGF-1 and/or ERK pathways. TGF-1 is a central profibrotic factor, which promotes fibroblast proliferation and extracellular matrix production in both physiological and pathological situations, including in the infarcted heart (1, 22). The current study has shown that VEGF-C treatment significantly elevates TGF-1 release from myofibroblasts. Smads are intracellular proteins that transduce extracellular signals from TGF-1 ligands to the nucleus where they activate downstream gene transcription. In response to TGF- signal, Smad2 is phosphorylated by the TGF- receptors and activates TGF- pathway. The current study shows that VEGF-C treatment stimulates Smad2 phosphorylation. Our data further reveal that TGF-1 siRNA or neutralizing antibody abolishes VEGF-C-induced fibrogenic effects. These observations indicate that regulation of VEGF-C on myofibroblast growth and function is mediated through activating the TGF- pathway. The MAPK/ERK pathway is involved in the regulation of a variety of growth and differentiation pathways through several phosphorylation cascades. The signaling cascade is activated by a number of receptors, which then transduce the signal to adaptors that eventually activate Raf, MEK1/2, and ERK, the core components of the pathway (26). ERK regulates transcription factors via phosphorylation. We further examined whether the stimulatory role of VEGF-C on fibrogenesis was also through the ERK pathway. Our data indicate that VEGF-C stimulates ERK phosphorylation, and VEGF-C-induced myofibroblast proliferation and fibrogenesis are markedly suppressed by the ERK inhibitor. These observations demonstrate that, in addition to TGF- pathway, VEGF-C also promotes fibrogenesis via activating the ERK pathway. We then examined whether TGF- and ERK pathways interacted in myofibroblast-induced fibrogenesis. Our data reveal that TGF- synthesis is not blocked by ERK inhibitor treatment in myofibroblasts. The result indicates that VEGFC-induced ERK phosphorylation in myofibroblasts is not via TGF- pathway. In summary, VEGF-C controls multiple levels of the fibrogenic process. It upregulates myofibroblast proliferation, mi-
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gration, and collagen synthesis. Unlike TGF-, which directly induces fibrogenesis, VEGF-C stimulates the growth and function of myofibroblasts through stimulating TGF-1 production and ERK phosphorylation. GRANTS This work was supported by National Heart, Lung, and Blood Institute Grant 1RO1-HL-096503 (to Y. Sun). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: T.Z., W.Z., W.M., C.L., and Y.C. performed experiments; T.Z. and Y.S. analyzed data; T.Z. and Y.S. interpreted results of experiments; T.Z. and Y.S. prepared figures; T.Z., W.Z., W.M., C.L., Y.C., and Y.S. approved final version of manuscript; Y.S. conception and design of research; Y.S. drafted manuscript. REFERENCES 1. Bujak M, Frangogiannis NG. The role of TGF-beta signaling in myocardial infarction and cardiac remodeling. Cardiovasc Res 74: 184 –195, 2007. 2. Chaudary N, Milosevic M, Hill RP. Suppression of vascular endothelial growth factor receptor 3 (VEGFR3) and vascular endothelial growth factor C (VEGFC) inhibits hypoxia-induced lymph node metastases in cervix cancer. Gynecol Oncol 123: 393–400, 2011. 3. Chen W, Frangogiannis NG. Fibroblasts in post-infarction inflammation and cardiac repair. Biochim Biophys Acta 1833: 945–953, 2013. 4. Cohen S, Efraim AN, Levi-Schaffer F, Eliashar R. The effect of hypoxia and cycloxygenase inhibitors on nasal polyp derived fibroblasts. Am J Otolaryngol 32: 564 –573, 2011. 5. Daskalopoulos EP, Hermans KC, Blankesteijn WM. Cardiac (myo)fibroblast: novel strategies for its targeting following myocardial infarction. Curr Pharm Des 2013 Jun 18 [Epub ahead of print]. 6. Daskalopoulos EP, Janssen BJ, Blankesteijn WM. Myofibroblasts in the infarct area: concepts and challenges. Microsc Microanal 18: 35–49, 2012. 7. Deten A, Holzl A, Leicht M, Barth W, Zimmer HG. Changes in extracellular matrix and in transforming growth factor beta isoforms after coronary artery ligation in rats. J Mol Cell Cardiol 33: 1191–1207, 2001. 8. Dobaczewski M, Bujak M, Li N, Gonzalez-Quesada C, Mendoza LH, Wang XF, Frangogiannis NG. Smad3 signaling critically regulates fibroblast phenotype and function in healing myocardial infarction. Circ Res 107: 418 –428, 2010. 9. Hwang M, Kim HJ, Noh HJ, Chang YC, Chae YM, Kim KH, Jeon JP, Lee TS, Oh HK, Lee YS, Park KK. TGF-beta1 siRNA suppresses the tubulointerstitial fibrosis in the kidney of ureteral obstruction. Exp Mol Pathol 81: 48 –54, 2006. 10. Leeb SN, Vogl D, Falk W, Scholmerich J, Rogler G, Gelbmann CM. Regulation of migration of human colonic myofibroblasts. Growth Factors 20: 81–91, 2002. 11. Li YY, McTiernan CF, Feldman AM. Interplay of matrix metalloproteinases, tissue inhibitors of metalloproteinases and their regulators in cardiac matrix remodeling. Cardiovasc Res 46: 214 –224, 2000. 12. Lian R, Chen Y, Xu Z, Zhang X. Soluble transforming growth factorbeta1 receptor II might inhibit transforming growth factor-beta-induced myofibroblast differentiation and improve ischemic cardiac function after myocardial infarction in rats. Coron Artery Dis 21: 369 –377, 2010. 13. Lindsey ML, Zamilpa R. Temporal and spatial expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases following myocardial infarction. Cardiovasc Ther 30: 31–41, 2012. 14. Lohela M, Bry M, Tammela T, Alitalo K. VEGFs and receptors involved in angiogenesis versus lymphangiogenesis. Curr Opin Cell Biol 21: 154 –165, 2009. 15. Manso AM, Kang SM, Plotnikov SV, Thievessen I, Oh J, Beggs HE, Ross RS. Cardiac fibroblasts require focal adhesion kinase for normal proliferation and migration. Am J Physiol Heart Circ Physiol 296: H627– H638, 2009. 16. McKaig BC, McWilliams D, Watson SA, Mahida YR. Expression and regulation of tissue inhibitor of metalloproteinase-1 and matrix metallo-
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proteinases by intestinal myofibroblasts in inflammatory bowel disease. Am J Pathol 162: 1355–1360, 2003. Ochi N, Matsuo Y, Sawai H, Yasuda A, Takahashi H, Sato M, Funahashi H, Okada Y, Manabe T. Vascular endothelial growth factor-C secreted by pancreatic cancer cell line promotes lymphatic endothelial cell migration in an in vitro model of tumor lymphangiogenesis. Pancreas 34: 444 –451, 2007. Santiago JJ, Dangerfield AL, Rattan SG, Bathe KL, Cunnington RH, Raizman JE, Bedosky KM, Freed DH, Kardami E, Dixon IM. Cardiac fibroblast to myofibroblast differentiation in vivo and in vitro: expression of focal adhesion components in neonatal and adult rat ventricular myofibroblasts. Dev Dyn 239: 1573–1584, 2010. Strutz F, Zeisberg M, Hemmerlein B, Sattler B, Hummel K, Becker V, Muller GA. Basic fibroblast growth factor expression is increased in human renal fibrogenesis and may mediate autocrine fibroblast proliferation. Kidney Int 57: 1521–1538, 2000. Sun Y, Weber KT. Angiotensin converting enzyme and myofibroblasts during tissue repair in the rat heart. J Mol Cell Cardiol 28: 851–858, 1996. Sun Y, Zhang JQ, Zhang J, Lamparter S. Cardiac remodeling by fibrous tissue after infarction in rats. J Lab Clin Med 135: 316 –323, 2000. Sun Y, Zhang JQ, Zhang J, Ramires FJ. Angiotensin II, transforming growth factor-beta1 and repair in the infarcted heart. J Mol Cell Cardiol 30: 1559 –1569, 1998. Timoshenko AV, Rastogi S, Lala PK. Migration-promoting role of VEGF-C and VEGF-C binding receptors in human breast cancer cells. Br J Cancer 97: 1090 –1098, 2007.
24. Xiao L, Du Y, Shen Y, He Y, Zhao H, Li Z. TGF-beta 1 induced fibroblast proliferation is mediated by the FGF-2/ERK pathway. Front Biosci 17: 2667–2674, 2012. 25. Yamazaki Y, Morita T. Molecular and functional diversity of vascular endothelial growth factors. Mol Divers 10: 515–527, 2006. 26. Yeh CC, Li H, Malhotra D, Turcato S, Nicholas S, Tu R, Zhu BQ, Cha J, Swigart PM, Myagmar BE, Baker AJ, Simpson PC, Mann MJ. Distinctive ERK and p38 signaling in remote and infarcted myocardium during post-MI remodeling in the mouse. J Cell Biochem 109: 1185–1191, 2010. 27. Yonemura Y, Endo Y, Fujita H, Fushida S, Ninomiya I, Bandou E, Taniguchi K, Miwa K, Ohoyama S, Sugiyama K, Sasaki T. Role of vascular endothelial growth factor C expression in the development of lymph node metastasis in gastric cancer. Clin Cancer Res 5: 1823–1829, 1999. 28. Zeisberg M, Maeshima Y, Mosterman B, Kalluri R. Renal fibrosis. Extracellular matrix microenvironment regulates migratory behavior of activated tubular epithelial cells. Am J Pathol 160: 2001–2008, 2002. 29. Zhao T, Zhao W, Chen Y, Ahokas RA, Sun Y. Vascular endothelial growth factor (VEGF)-A: role on cardiac angiogenesis following myocardial infarction. Microvasc Res 80: 188 –194, 2010. 30. Zhao T, Zhao W, Chen Y, Li VS, Meng W, Sun Y. Platelet-derived growth factor-D promotes fibrogenesis of cardiac fibroblasts. Am J Physiol Heart Circ Physiol 304: H1719 –H1726, 2013. 31. Zhao T, Zhao W, Chen Y, Liu L, Ahokas RA, Sun Y. Differential expression of vascular endothelial growth factor isoforms and receptor subtypes in the infarcted heart. Int J Cardiol 167: 2638 –2645, 2013.
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Vascular endothelial growth factor-C: its unrevealed role in fibrogenesis Tieqiang Zhao,1 Wenyuan Zhao,1 Weixin Meng,2 Chang Liu,2 Yuanjian Chen,1 and Yao Sun1 1
Division of Cardiovascular Diseases, Department of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee; and 2Division of Cardiac Surgery, Department of Surgery, First Affiliate Hospital of Harbin Medical University, Harbin, China Submitted 24 July 2013; accepted in final form 21 January 2014
Zhao T, Zhao W, Meng W, Liu C, Chen Y, Sun Y. Vascular endothelial growth factor-C: its unrevealed role in fibrogenesis. Am J Physiol Heart Circ Physiol 306: H789 –H796, 2014. First published January 24, 2014; doi:10.1152/ajpheart.00559.2013.—Vascular endothelial growth factor (VEGF)-C is a key mediator of lymphangiogenesis. Our recent study shows that VEGF-C/VEGF receptors (VEGFR)3 are significantly increased in the infarcted rat myocardium, where VEGFR-3 is expressed not only in lymph ducts but also in myofibroblasts, indicating that VEGF-C has an unrevealed role in fibrogenesis during cardiac repair. The current study is to explore the regulation and molecular mechanisms of VEGF-C in fibrogenesis. The potential regulation of VEGF-C on myofibroblast differentiation/growth/migration, collagen degradation/synthesis, and transforming growth factor (TGF)- and ERK pathways was detected in cultured cardiac myofibroblasts. Our results showed that VEGF-C significantly increased myofibroblast proliferation, migration, and type I/III collagen production. Matrix metalloproteinase (MMP)-2 and -9 were significantly elevated in the medium of VEGF-C-treated cells, coincident with increased tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. Furthermore, VEGF-C activated the TGF-1 pathway and ERK phosphorylation, which was significantly suppressed by TGF- or ERK blockade. This is the first study indicating that in addition to lymphangiogenesis, VEGF-C is also involved in fibrogenesis through stimulation of myofibroblast proliferation, migration, and collagen synthesis, via activation of the TGF-1 and ERK pathways. myofibroblasts; VEGF-C; collagen synthesis; collagen degradation; TGF-1; ERK VEGF IS A SUBFAMILY OF GROWTH factors, composed of five different isoforms: VEGF-A, -B, -C, -D, and placenta growth factor. VEGF initiates cellular responses by interacting with tyrosine kinase receptors on the cell surface. There are three main VEGF receptor subtypes, VEGFR-1, -2, and -3. VEGF-A binds to VEGFR-1 and -2; VEGF-B binds to VEGFR-1; and VEGF-C and -D bind to VEGFR-2 and -3. VEGF-A and -B are the key mediators of angiogenesis, while VEGF-C and -D are recognized to stimulate lymphangiogenesis in physiological and pathological conditions (14, 25). Ventricular dysfunction appears most commonly in patients with previous myocardial infarction (MI). Cardiac repair occurring at the site of myocyte loss preserves the structural integrity of the heart and is essential to its recovery. Fibrogenesis (scar formation) is central to cardiac repair. Myofibroblasts, which are phenotypically transformed fibroblasts, are responsible for the production of collagen and scar contraction in the infarcted myocardium (6, 20). Fibroblasts are the major source of myofibroblasts. Stimulated by TGF- and mechani-
Address for reprint requests and other correspondence: Y. Sun, Div. of Cardiovascular Diseases, Dept. of Medicine, Univ. of Tennessee Health Science Center, 956 Court Ave, Rm. B324, Memphis, TN 38163 (e-mail: [email protected]). http://www.ajpheart.org
cal stretch, fibroblasts differentiate into myofibroblasts in the repairing tissue and, possessing the features of both fibroblasts and smooth muscle cells, are dynamic in collagen synthesis and fibrous tissue contraction (3). Maintaining the extracellular matrix in the infarcted myocardium is essential in preventing the heart from dilatation. Local factors regulating cardiac repair/remodeling have drawn great attention. Our recent study has shown that VEGF-C expression is significantly increased in the infarcted myocardium (31). Our study has further shown that VEGFR-3 is significantly increased in the infarcted myocardium in both early and late stages of MI. In addition to lymphatic vessels, VEGFR-3 is highly expressed in myofibroblasts of the infarcted myocardium, suggesting that VEGF-C plays a role in cardiac fibrous tissue formation in an autocrine/paracrine manner. These observations imply that, in addition to lymphangiogenesis, VEGF-C has unidentified functions in fibrogenesis during cardiac repair post-MI. The molecular basis of VEGF-C on cardiac fibrogenic response, however, remains unknown and is investigated in the current study. Primary cultures of rat cardiac fibroblasts automatically transform to myofibroblasts in the first or second passage (P1 or P2) (18). Both in vitro and in vivo cardiac myofibroblasts are of a similar phenotype. With the use of cultured myofibroblasts, the potential influence of VEGF-C on myofibroblast growth, migration, proliferation, and collagen turnover was explored in the study. The TGF- and MAPK/ERK signaling cascade are the major pathways controlling cellular processes associated with fibrogenesis, including growth, proliferation, and survival. Activation of the TGF- and MAPK/ERK pathways is detected in various fibrotic diseases (24, 28). The current study also determined whether the regulation of VEGF-C on fibrogenesis is through TGF- and/or ERK pathways. MATERIAL AND METHODS
Cell culture. Cardiac fibroblasts were isolated from 8-wk-old male Sprague-Dawley rats (14). Briefly, rat was anesthetized with isoflurane inhalation, and the heart was excised, washed in PBS, and cut into 1-mm3 pieces. The tissue was digested at 37°C in digestion medium containing a mixture of collagenase B (115 mg/100 ml; Worthington, Lakewood, NJ) and trypsin (50 mg/100 ml; Sigma, St. Louis, MO) for 10 min with constant shaking. Cells from the third to tenth digestions were pooled and pelleted down. The pellet was resuspended in 5 ml DMEM 10% FBS, seeded into 60-mm dishes, and kept at 37°C in CO2 incubator for a preplating period of 150 min. Unattached cells were discarded, and attached cells were washed and grown in the plating medium. Cultures were maintained at 37°C in 95% humidified air and 5% CO2 atmosphere. Fibroblasts in culture automatically differentiate into myofibroblasts at P1 and P2 (18). Myofibroblasts were confirmed by immunohistochemical ␣-smooth muscle actin (SMA) staining. The dose (0, 10, 50, and 200 ng/ml
0363-6135/14 Copyright © 2014 the American Physiological Society
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medium) and time (6, 12 and 24 h) response of VEGF-C on fibrous tissue formation were determined. This study was approved by the University of Tennessee Health Science Center Animal Care and Use Committee. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Cell proliferation assay. Myofibroblast proliferation was assessed using bromodeoxyuridine (BrdU) cell proliferation assay kit (Cell Signaling, Danvers, MA). The assay was performed according to the manufacturer’s instructions. Myofibroblasts were seeded in 96-well plates. After being quiescient in serum-deprived medium for 24 h, cells were exposed to VEGF-C (200 ng/ml) for 20 h. BrdU (10 M) was then added, and cells were further incubated for 4 h. At the endpoint of the incubation, medium was withdrawn, 100 l/well of fixing solution were added, and the plates were incubated at room temperature for 30 min. Afterwards, the solution was aspirated and the wells were rinsed three times with a washing solution and eventually dried on a paper towel. Anti-BrdU monoclonal antibodies were then added (100 l/well), and the wells were incubated for 1 h at room temperature. Medium was removed, and wells rinsed three times with a washing solution. Horseradish peroxidase-conjugated anti-mouse IgG was added, and the plate was incubated at room temperature for 30 min. After three washes, 100 l TMB substrate were added to each well, followed by 100 l of stop solution for 30 min. The absorbance was read at 450 nm in a microplate reader (4). Eight different wells were counted in each group. Cell migration assay. Myofibroblast migration was detected with a modified Boyden’s chamber assay. The cell culture inserts, which contain membranes with 8.0-m pore size, were placed in a 24-well tissue culture plate (Millipore ECM508, Billerica, MA). Myofibroblasts were quiesced for 24 h, then trypsinized, resuspended in DMEM, and seeded into the upper chamber at 1⫻ 105 cells/well. The lower chamber contains DMEM with VEGF-C (200 ng/ml) as a chemoattractant. After incubation for 6 h at 37°C, cells/media were removed from the top side of the insert by pipetting out the remaining cell suspension. The inserts were placed in clean wells containing 400 l of cell stain for 20 min.
The inserts were then rinsed in water, and cotton-tipped swabs were used to remove nonmigratory cells from the interior of the insert. The inserts were transferred to a clean well containing 200 l of extraction buffer for 15 min. The dye mixture (100 l) was transferred to a 96-well plate, and optical density was measured at 560 nm (15). Six different samples were tested in each group. Western blotting. The effects of TGF-1 (10 ng/ml) on type I collagen and matrix metalloproteinase (MMP)-2 and MMP-9 levels and VEGF-C on expression of type I and III collagen, MMP-2, MMP-9, inhibitor of metalloproteinase (TIMP)-1 and -2, and TGF-1 proteins in culture medium and ␣-SMA, Smad2, and phosphorylated Smad2 in myofibroblasts were assessed by Western blot. The cells were plated in sixwell plates, grown to subconfluence, and then quiesced for 24 h. Cells were then incubated with or without VEGF-C for 24 h. Medium was concentrated by centrifugation for 30 min in Amicon ultra centrifugal filters (Millipore, Billerica, MA). Proteins from cells and medium were loaded on the gel, subjected to SDSPAGE (10% polyacrylamide gel), and transferred onto nitrocellulose membranes using a Bio-Rad Mini Trans Blot electrophoretic transfer unit. Membranes were blocked for nonspecific protein with 5% nonfat dry milk in TBS and then probed overnight at 4°C with primary antibodies against type I and III collagen, MMP-2, MMP-9, TIMP-1, TIMP-2, TGF-1, ␣-SMA, Smad2, and phosphorylated Smad2 (Millipore and Sigma). Membranes were then washed three times (10 min per wash) with TBS with 0.05% Tween-20 to remove unbound antibodies, and further incubated with appropriate horseradish peroxidase-conjugated secondary antibody (1:2,000). Membranes were developed by a chemiluminescence reagent kit (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s protocol. The amount of protein detected was assessed by means of quantitative densitometry analysis with a computer image analyzing system (29). Six different samples were tested in each group. ELISA. The effect of VEGF-C on TGF-1 production in cardiac myofibroblasts was determined by ELISA. Supernatants were collected for TGF-1 detection with a commercial ELISA kit (R&D Systems, Minneapolis, MN). Briefly, TGF-1 standards, positive control, and 50-ul activated samples were added to a 96-well plate
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Fig. 1. Effect of VEGF-C on myofibroblast differentiation, proliferation, and migration. Cultured cardiac fibroblasts automatically differentiated into myofibroblasts at P1 and expressed ␣-smooth muscle actin (␣-SMA; A, left, arrows). Compared with vehicletreated myofibroblasts, VEGF-C did not affect myofibroblast differentiation (B), significantly increased myofibroblast proliferation (C) and migration (D). A, right: negative control of ␣-SMA labeling. *P ⬍ 0.05 vs. controls (CTL); n ⫽ 8/group.
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coated with TGF-1 antibody. The plate was incubated for 2 h at room temperature and then washed by wash buffer (400 l) four times. TGF-1 conjugate (100 l) was then added to the plate. After a 2-h incubation, the plate was washed again four times as above. Substrate solution (100 l) was added to the plate, followed by stop solution (100 l). The optical density of each well was measured within 30 min using a microplate reader set to 450 nm with a correction at 540 nm. Six samples were included in each group. TGF-1 blockade. TGF-1 siRNA and neutralizing antibody were used to detect whether the regulation of VEGF-C on fibrogenesis is via the TGF-1 pathway. At confluence of 50%, myofibroblasts were quiesced for 24 h. TGF-1 siRNA (final concentration 30 nM; control siRNA sequence: sense: 5=-UAACGACGCGACGACGUAATT-3=, antisense: 5=-UUACGUCGUCGCGUCGUUATT-3=; TGF-b1 siRNA sequence: sense: 5=-GGAGAGCCCUGGAUACCAATT-3=, antisense: 5=-UUGGUAUCCAGGGCUCUCCGG-3=) was introduced to cells through Lipofectamine RNAiMAX reagent (Invitrogen, Grand Island, NY) (9, 30). The transfection was performed as described by the manufacturer’s manual. At 6 h after the transfection, VEGF-C was administered to the cells with final concentration of 200 ng/ml. The neutralizing antibody of TGF- 1 (10 ug/ml; R&D Systems) was applied to cells, followed by VEGF-C (200 ng/ml) for 1 h. The medium and cell lysate were collected at 24 h. Myofibroblast proliferation and collagen synthesis were analyzed as described above. ERK activation and blockade. To detect ERK1/2 activation, myofibroblasts were quiesed for 24 h and VEGF-C (200 ng/ml) and then administered to the cells for 10 min. Cell lysate was collected in modified RIPA buffer. ERK phosphorylation was detected by Western blot using antibodies against ERK1/2 and phospho-ERK1/2 (Cell Signaling). To determine whether the role of VEGF-C on fibrogenesis is through the ERK pathway, U0126 (ERK inhibitor, 10 uM) was added to the plates for 1 h, followed by VEGF-C treatment. Myofibroblast proliferation and collagen production were analyzed as described above. Statistical analysis. Statistical analysis of cell differentiation, proliferation, migration, Western blot, and ELIZA data among the groups was performed using Student t-test or ANOVA. Values are expressed as means ⫾ SE with P ⬍ 0.05 considered significant. Multiple group comparisons among controls and each group were made by Scheffé’s F-test.
VEGF-C promotes type I and III collagen synthesis. Collagen production is the key function of myofibroblasts. Collagen is a secreted protein released from cells into the interstitial space. In cultured myofibroblasts, collagen is secreted into the culture medium. Type I and III collagen are the major collagen isoforms. Via Western blot, we observed low levels of type I collagen in the medium of the control cells. VEGF-C treatment significantly increased type I collagen contents in the culture medium compared with that of the control group. Our study further shows that VEGF-C stimulated type I collagen synthesis in a time- and dose-dependent manner (Fig. 2, A and B, respectively). Further-
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Fibroblast-myofibroblast differentiation. The hallmark of myofibroblasts is their expression of ␣-SMA, which is not expressed by fibroblasts. Fibroblasts spontaneously transform into myofibroblasts in culture. By immunohistochemical ␣-SMA staining, results showed that cultured cardiac fibroblasts began to express ␣-SMA at P1 (Fig. 1A). Western blotting detection shows that VEGF-C treatment did not alter ␣-SMA expression in cultured cells (Fig. 1B). VEGF-C increases myofibroblast proliferation. The potential role of VEGF-C on myofibroblast proliferation was examined by the BrdU cell proliferation assay. We found that VEGF-C treatment significantly increased myofibroblast proliferation compared with vehicle-treated control cells (Fig. 1C). VEGF-C stimulates myofibroblast migration. ␣-SMA is a cytoskeletal protein involved in cell contraction and migration. In the early phase of MI, migration enables the rapid recruitment of myofibroblasts to the infarct site. The potential role of VEGF-C on myofibroblast migration was examined by the Boyden’s chamber assay. We found that myofibroblast migration was significantly increased in cells receiving VEGF-C compared with vehicle-treated cells (Fig. 1D).
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Fig. 2. VEGF-C promotes type I and III collagen synthesis in myofibroblasts. Compared with controls, VEGF-C treatment significantly stimulated type I collagen secretion in a time (A)- and dose-dependent (B) manner. VEGF-C also promoted type III collagen production (C). *P ⬍ 0.05 vs. controls (CTL); n ⫽ 6/group.
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more, VEGF-C also significantly elevated type III collagen release in myofibroblasts (Fig. 2C). VEGF-C upregulates MMP-2 and MMP-9 expression. MMPs are also secreted proteins. Western blot detection revealed that MMP-2 and MMP-9 levels were barely detectable in the medium of the control cells. VEGF-C treatment was found to increase MMP-2 and MMP-9 secretion by cultured myofibroblasts (Fig. 3, A and B, respectively). VEGF-C promotes TIMP-1 and TIMP-2 expression. TIMPs are glycoprotein peptidases involved in inhibition of extracellular matrix degradation. TIMPs are also secreted proteins that are released into the interstitial space and culture medium. Western blot detection showed that myofibroblasts in the control group produced extremely low levels of TIMP-1 and TIMP-2. VEGF-C treatment significantly increased TIMP-1 and TIMP-2 levels in the medium compared with that of the control group (Fig. 3, C and D, respectively). VEGF-C activates TGF-1 pathway. TGF-1 is a profibrogenic mediator produced by several types of cells, including myofibroblasts, and is a secreted protein. TGF-1 protein levels in the culture medium were measured by Western blot and ELISA (Fig. 4, A and B). TGF-1 was barely detectable in the medium of the control cells. In VEGF-C-treated cells, TGF-1 protein level was significantly elevated in the medium compared with that of the controls. TGF- signaling is initiated by phosphorylation of the cytoplasmic signaling molecules Smad2/3. The current study shows that VEGF-C treatment enhanced Smad2 phosphorylation in myofibroblasts, which started at 10 min, peaked at 30 min, and then declined at 1 h (Fig. 4C). TGF-1 stimulates type I collagen, MMP-2, and MMP-9 release into the medium (Fig. 5A). To determine whether the
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role of VEGF-C on fibrogenesis is via activation of the TGF- pathway, we blocked TGF-1 synthesis with TGF-1 siRNA or the neutralizing antibody. Both TGF-1 siRNA and neutralizing treatments significantly suppressed VEGF-C-induced myofibroblast proliferation (Fig. 5A) and type I collagen synthesis (Fig. 5, B and C). VEGF-C activates ERK pathway. The ERK signal pathway functions in cellular proliferation, differentiation, and survival. To determine whether the role of VEGF-C on fibrogenesis is via activation of the ERK pathway, we examined the effect of VEGF-C on ERK phosphorylation. Compared with vehicletreated cells, we observed significantly increased ERK phosphorylation in myofibroblasts after 10 min of VEGF-C treatment compared with controls (Fig. 6A). To determine whether VEGF-C-induced fibrogenesis is through the ERK pathway, we blocked the ERK pathway with an ERK inhibitor (U0126). We found that U0126 treatment significantly diminished VEGF-C-induced myofibroblast proliferation (Fig. 6B) and type I collagen synthesis (Fig. 6C). Interaction between TGF-1 and ERK pathways. To study whether the TGF-1 and ERK pathways cross talk in myofibroblast-induced fibrogenesis, we cotreated myofibroblasts with a VEGF-C and ERK inhibitor (U0126). We found that U-126 did not suppress VEGF-C-induced TGF- synthesis (Fig. 7). DISCUSSION
VEGF-C and VEGFR-3 are coexpressed in myofibroblasts in the infarcted myocardium, suggesting a potential role of VEGF-C in myofibroblast growth and function. The current
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Fig. 3. VEGF-C increases matrix metalloproteinase (MMP)-2 (A) and -9 (B) and tissue inhibitor of metalloproteinase (TIMP)-1 (C) and -2 (D) synthesis in myofibroblasts. MMP-2, MMP-9, TIMP-1 and TIMP-2 were not detectable in the medium of vehicle-treated myofibroblasts. VEGF-C treatment induced MMP-2, MMP-9, TIMP-1, and TIMP-2 release; n ⫽ 6/group.
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Fig. 4. VEGF-C upregulates TGF-1 production and Smad2 phosphorylation. Detected by Western blot (A) and ELISA (B), TGF-1 was not detectable in the medium of vehicle-treated cells, while VEGF-C treatment significantly enhanced TGF-1 levels in the medium. C: VEGF-C treatment significantly enhanced Smad2 phosphorylation in myofibroblasts. *P ⬍ 0.05 vs. controls (CTL); n ⫽ 6/group.
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2.5 2.0 1.5 1.0 .5 0.0
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Fig. 5. TGF- blockade abolishes the role of VEGF-C on fibrogenesis. Compared with control cells, TGF-1 neutralizing antibody significantly suppressed VEGF-C-induced myofibroblast proliferation (B) and type I collagen synthesis (A). TGF-1 siRNA treatment significantly suppressed VEGF-C-induced type I collagen production (C and D). *P ⬍ 0.05 vs. controls; #P ⬍ 0.05 vs. VEGFC treatment; n ⫽ 6/group.
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broblast growth factor, etc., have been shown to regulate myofibroblast proliferation (5, 19, 30). The current study has revealed that VEGF-C treatment has no effect on myofibroblast differentiation but markedly increases myofibrobalst proliferation. Previously, VEGF-C has been reported to stimulate lymph endothelial cell replication (2, 27). Anti-VEGFR-3 antibody has been shown to inhibit VEGF-C-induced tumor lymphangiogenesis and metastasis by suppressing lymph endothelial cell proliferation (2). Our observation indicates that in addition to lymph endothelial cells, VEGF-C also promotes the proliferation of other cell lines, including myofibroblasts, and is involved in other cellular actions, such as fibrogenesis during tissue repair. Myofibroblasts are mobile and contractile. Following MI, myofibroblasts migrate into the infarct area. Their contractile properties, combined with their synthesis of extracellular matrix proteins, serve the purpose of preventing infarct expansion. The process of migration of myofibroblasts in the infarct area is not completely elucidated. Myofibroblasts are highly responsive to certain growth factors. Platelet-derived growth factor, interstitial growth factor, and endothelial growth factor and TGF-1 stimulate the migration of myofibroblasts (10). The result of the present study shows that VEGF-C serves as another mediator of myofibroblast migration. Previously, VEGF-C has been reported to play an autocrine role in metastasis by promoting tumor cell motility (23). Depletion of endogenous VEGF-C by treatments with a VEGF-C siRNA inhibited tumor cell migration (17). Thus VEGF-C is involved in cell migration in various pathological conditions. Next, we detected the potential regulation of VEGF-C on the function of myofibroblasts. The key role of myofibroblasts is to produce extracellular matrix. Collagen is the major component of extracellular matrix. So far, 29 types of collagen have been identified and described. Among all collagen subtypes, type I and III collagens are the main elements of cardiac fibrous tissue (7, 21). Myofibroblast-induced collagen synthesis is known to
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study explored the regulation of VEGF-C on myofibroblastinduced fibrogenesis and its underlying molecular mechanisms. First, we tested whether VEGF-C regulated cardiac myofibroblast differentiation, proliferation, and migration. Myofibroblasts are primarily differentiated from cardiac interstitial fibroblasts in the infarcted heart. Following MI, they first appear at the border zone (the region between the infarcted and noninfarcted myocardium) and rapidly proliferate and accumulate in the infarcted myocardium. Locally released factors and cytokines, such as TGF-, platelet-derived growth factor, fi-
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Fig. 6. VEGF-C stimulates ERK phosphorylation in myofibroblasts. Compared with controls, VEGF-C elevated ERK phosphorylation (p-ERK1/2), which was blocked by the ERK inhibitor U0126 (A). VEGF-C-induced myofibroblast proliferation (B) and type I collagen synthesis (C) were significantly reduced by U0126; n ⫽ 6/group. *P ⬍ 0.05 vs. controls; #P ⬍ 0.05 vs. VEGFC treatment.
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Fig. 7. Effect of the ERK inhibitor on VEGF-C-induced TGF-1 production in myofibroblasts. VEGF-C enhanced TGF-1 production by myofibroblasts, which was not diminished by U0126; n ⫽ 6/group.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00559.2013 • www.ajpheart.org
VEGF-C AND FIBROGENESIS
be primarily stimulated by TGF- (8, 12). The results from the present study reveal that VEGF-C serves as a stimulatory factor in the function of myofibroblasts and promotes type I and III collagen production. Fibrous tissue accumulation relies on the balance between collagen synthesis and degradation with MMPs playing a central role in collagen degradation. MMPs are a large family of proteases with ⬎20 members. MMPs are secreted proteins produced by various types of cells, including myofibroblasts (13). After secretion into the interstitial space, MMP activity is controlled by TIMPs. TIMPs function as an important regulatory brake on MMP activity by inhibiting the active species, thereby suppressing collagen degradation (11). TIMPs comprise a family of four subtypes and are produced in various cells, including myofibroblasts (16). Our data have shown that VEGF-C treatment significantly increased MMP-2 and MMP-9 release and is coincident with upregulated TIMP-1 and TIMP-2 expression in myofibroblasts. These observations suggest that even though MMPs are elevated, coexpression of TIMPs counteract the activity of MMPs and limit collagen degradation. Finally, we investigated whether the role of VEGF-C on fibrogenesis is via activating TGF-1 and/or ERK pathways. TGF-1 is a central profibrotic factor, which promotes fibroblast proliferation and extracellular matrix production in both physiological and pathological situations, including in the infarcted heart (1, 22). The current study has shown that VEGF-C treatment significantly elevates TGF-1 release from myofibroblasts. Smads are intracellular proteins that transduce extracellular signals from TGF-1 ligands to the nucleus where they activate downstream gene transcription. In response to TGF- signal, Smad2 is phosphorylated by the TGF- receptors and activates TGF- pathway. The current study shows that VEGF-C treatment stimulates Smad2 phosphorylation. Our data further reveal that TGF-1 siRNA or neutralizing antibody abolishes VEGF-C-induced fibrogenic effects. These observations indicate that regulation of VEGF-C on myofibroblast growth and function is mediated through activating the TGF- pathway. The MAPK/ERK pathway is involved in the regulation of a variety of growth and differentiation pathways through several phosphorylation cascades. The signaling cascade is activated by a number of receptors, which then transduce the signal to adaptors that eventually activate Raf, MEK1/2, and ERK, the core components of the pathway (26). ERK regulates transcription factors via phosphorylation. We further examined whether the stimulatory role of VEGF-C on fibrogenesis was also through the ERK pathway. Our data indicate that VEGF-C stimulates ERK phosphorylation, and VEGF-C-induced myofibroblast proliferation and fibrogenesis are markedly suppressed by the ERK inhibitor. These observations demonstrate that, in addition to TGF- pathway, VEGF-C also promotes fibrogenesis via activating the ERK pathway. We then examined whether TGF- and ERK pathways interacted in myofibroblast-induced fibrogenesis. Our data reveal that TGF- synthesis is not blocked by ERK inhibitor treatment in myofibroblasts. The result indicates that VEGFC-induced ERK phosphorylation in myofibroblasts is not via TGF- pathway. In summary, VEGF-C controls multiple levels of the fibrogenic process. It upregulates myofibroblast proliferation, mi-
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gration, and collagen synthesis. Unlike TGF-, which directly induces fibrogenesis, VEGF-C stimulates the growth and function of myofibroblasts through stimulating TGF-1 production and ERK phosphorylation. GRANTS This work was supported by National Heart, Lung, and Blood Institute Grant 1RO1-HL-096503 (to Y. Sun). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: T.Z., W.Z., W.M., C.L., and Y.C. performed experiments; T.Z. and Y.S. analyzed data; T.Z. and Y.S. interpreted results of experiments; T.Z. and Y.S. prepared figures; T.Z., W.Z., W.M., C.L., Y.C., and Y.S. approved final version of manuscript; Y.S. conception and design of research; Y.S. drafted manuscript. REFERENCES 1. Bujak M, Frangogiannis NG. The role of TGF-beta signaling in myocardial infarction and cardiac remodeling. Cardiovasc Res 74: 184 –195, 2007. 2. Chaudary N, Milosevic M, Hill RP. Suppression of vascular endothelial growth factor receptor 3 (VEGFR3) and vascular endothelial growth factor C (VEGFC) inhibits hypoxia-induced lymph node metastases in cervix cancer. Gynecol Oncol 123: 393–400, 2011. 3. Chen W, Frangogiannis NG. Fibroblasts in post-infarction inflammation and cardiac repair. Biochim Biophys Acta 1833: 945–953, 2013. 4. Cohen S, Efraim AN, Levi-Schaffer F, Eliashar R. The effect of hypoxia and cycloxygenase inhibitors on nasal polyp derived fibroblasts. Am J Otolaryngol 32: 564 –573, 2011. 5. Daskalopoulos EP, Hermans KC, Blankesteijn WM. Cardiac (myo)fibroblast: novel strategies for its targeting following myocardial infarction. Curr Pharm Des 2013 Jun 18 [Epub ahead of print]. 6. Daskalopoulos EP, Janssen BJ, Blankesteijn WM. Myofibroblasts in the infarct area: concepts and challenges. Microsc Microanal 18: 35–49, 2012. 7. Deten A, Holzl A, Leicht M, Barth W, Zimmer HG. Changes in extracellular matrix and in transforming growth factor beta isoforms after coronary artery ligation in rats. J Mol Cell Cardiol 33: 1191–1207, 2001. 8. Dobaczewski M, Bujak M, Li N, Gonzalez-Quesada C, Mendoza LH, Wang XF, Frangogiannis NG. Smad3 signaling critically regulates fibroblast phenotype and function in healing myocardial infarction. Circ Res 107: 418 –428, 2010. 9. Hwang M, Kim HJ, Noh HJ, Chang YC, Chae YM, Kim KH, Jeon JP, Lee TS, Oh HK, Lee YS, Park KK. TGF-beta1 siRNA suppresses the tubulointerstitial fibrosis in the kidney of ureteral obstruction. Exp Mol Pathol 81: 48 –54, 2006. 10. Leeb SN, Vogl D, Falk W, Scholmerich J, Rogler G, Gelbmann CM. Regulation of migration of human colonic myofibroblasts. Growth Factors 20: 81–91, 2002. 11. Li YY, McTiernan CF, Feldman AM. Interplay of matrix metalloproteinases, tissue inhibitors of metalloproteinases and their regulators in cardiac matrix remodeling. Cardiovasc Res 46: 214 –224, 2000. 12. Lian R, Chen Y, Xu Z, Zhang X. Soluble transforming growth factorbeta1 receptor II might inhibit transforming growth factor-beta-induced myofibroblast differentiation and improve ischemic cardiac function after myocardial infarction in rats. Coron Artery Dis 21: 369 –377, 2010. 13. Lindsey ML, Zamilpa R. Temporal and spatial expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases following myocardial infarction. Cardiovasc Ther 30: 31–41, 2012. 14. Lohela M, Bry M, Tammela T, Alitalo K. VEGFs and receptors involved in angiogenesis versus lymphangiogenesis. Curr Opin Cell Biol 21: 154 –165, 2009. 15. Manso AM, Kang SM, Plotnikov SV, Thievessen I, Oh J, Beggs HE, Ross RS. Cardiac fibroblasts require focal adhesion kinase for normal proliferation and migration. Am J Physiol Heart Circ Physiol 296: H627– H638, 2009. 16. McKaig BC, McWilliams D, Watson SA, Mahida YR. Expression and regulation of tissue inhibitor of metalloproteinase-1 and matrix metallo-
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