1 Department of Pathology, University Medical Center Utrecht, 2 Department of Pathology, Academic Medical Center, University of Amsterdam, The Netherlands, 3 Department of Internal Medicine, Chubu Rousai Hospital, Nagoya, Japan, 4 Department of Vascular Medicine, University Medical Center Utrecht, The Netherlands and 5 FibroGen Inc., South San Francisco, USA
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Abstract |
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Methods and Results. Immunostaining and ELISA showed that SMA expression and transformation of MC into myofibroblast-like cells was induced by TGFß, but not affected by PDGF-BB, CTGF, or neutralizing anti-CTGF antibodies. [3H]thymidine incorporation and Ki67 staining demonstrated that, unlike PDGF-BB, neither CTGF nor TGFß induced the proliferation of MC. In contrast, both CTGF and TGFß induced MC migration, as evidenced by approximation of wound edges in scrape-wounded, non-proliferating rat MC monolayers. In addition, fibronectin expression was upregulated by both CTGF and TGFß, as measured by dot-blot analysis. Anti-CTGF completely blocked the effect of added CTGF. Moreover, anti-CTGF significantly reduced TGFß-induced increase in fibronectin.
Conclusion. It thus appears that CTGF is specifically involved in a subset of the adaptive changes of MC involved in mesangial repair and sclerosis, which makes it an interesting candidate target for future intervention strategies.
Keywords: CTGF; extracellular matrix; mesangial; migration; TGFß; wound healing
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Introduction |
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In experimental mesangial proliferative glomerulonephritis, induced by a single injection with anti- Thy-1.1 antibody, complement-mediated lysis of the mesangium can be largely restored in 2 weeks by repopulation of the mesangial area by the activation, proliferation and migration of extraglomerular mesangial cells, as well as by remodulation of the mesangial matrix, including transient increase of extracellular matrix proteins [6,12]. Both PDGF and TGFß have been implicated as essential factors in the pathogenesis of anti-Thy-1.1 nephritis. We have observed in this model, that CTGF mRNA is highly upregulated in MC and GVEC, and that this precedes the increase in expression of smooth-muscle actin (
SMA) by periglomerular and mesangial cells, which is indicative of activation and transformation into myofibroblast-like cells. In vitro, all TGFß isoforms are equally capable of inducing transient CTGF mRNA upregulation in rat mesangial cells, and more sustained upregulation of CTGF mRNA in GVEC [6]. It is not known what aspects of the mesangial response to glomerular injury involve CTGF, and how this would relate to the role of TGFß and PDGF-BB in anti-Thy-1.1 nephritis and other processes of tissue repair [1316].
To study these questions, we compared the effects of CTGF with those of TGFß and PDGF-BB on rat mesangial cells in an in vitro model for wound healing. For this, a scrape-wounding assay was developed using mesangial cell monolayers, in which SMA expression, cell proliferation, and cell migration could be addressed. Expression of fibronectin was assessed in conditioned medium of cultured mesangial cells after stimulation with CTGF, TGFß, or PDGF-BB.
CTGF, TGFß1 and PDGF-BB may each contribute to different aspects of the mesangial response to injury.
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Subjects and methods |
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Culture of rat mesangial cells
An established rat mesangial cell line [17] was used and cultured in complete medium consisting of DMEM (Gibco BRL, Breda, The Netherlands), supplemented with 20% fetal calf serum (Gibco BRL), 5 µg/ml insulin (Sigma, Zwijndrecht, The Netherlands), penicillin (Gibco BRL) and streptomycin (Gibco BRL) in a humidified 37°C, 5% CO2, 95% air incubator.
Western blot analysis
Rat mesangial cells were cultured for 24 h with and without TGFß1 (5 ng/ml) in the absence of fetal calf serum (i.e. under serum-free conditions), and conditioned medium was harvested to which protease inhibitors (PMSF 1 mmol/l, aprotinin 2 µg/ml, leupeptin 5 µg/ml, pepstatin 0.7 µg/ml (Roche, Almere, The Netherlands)) were added. Heparin-binding proteins were extracted from conditioned medium with heparinSepharose CL-6B beads (Amersham Pharmacia, Roosendaal, The Netherlands) for 2 h at 4°C. Bound proteins were eluted by boiling in 2x SDS sample buffer and resolved on a 8% SDSpolyacrylamide gel and subsequently transferred to a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). Membranes were blocked in PBS/0.05% Tween 20/5% BSA and incubated with pIgY13 or control chicken Ab (pCIgY13) at 0.5 µg/ml in blocking buffer, followed by HRPO-conjugated rabbit anti-chicken Ab (Zymed, South San Francisco, USA) in blocking buffer. Immobilized antibodies were detected with the enhanced chemiluminescence system (ECL, NEN Lifesciences, Zaventem, Belgium) according to the manufacturer's instructions and exposure to X-Omat blue XB-1 films (Kodak, NEN Lifesciences).
Scrape wounding assay
Cells (1.5x105/ml) were plated in 12-well plates (Costar, Badhoevedorp, The Netherlands), in complete medium. After overnight culturing, cells were kept in serum free media for 24 h and two wounds per well were made by scraping the surface of the dish with a plastic pipette tip. Subsequently, plates were washed with Dulbecco's PBS (Gibco BRL) and DPBS was replaced by fresh serum free medium, to which various factors were added. In each experiment, wound closure was measured at several time points in triplicate wells for each condition, at six marked sites per well (i.e. 3x6=18 observations per time point/condition/experiment), by inverse light microscopy with a grid reticule in the eyepiece of the microscope. Wound closure was quantified as percentage of the starting distance of the wound edges. All experiments were repeated three times, on separate days for each condition. Data are presented as the mean±standard deviation of three independent experiments, performed on separate days. For statistical evaluation, a repeated measurement test followed by a Duncan's post hoc test was applied.
Cell proliferation assay
[3H]Thymidine incorporation was measured to assess DNA synthesis in mesangial cells. Cells were plated in 24-well (Costar) tissue culture plates at a concentration of 2.5x104 cells/well. Cells were serum-starved for 24 h, followed by a 48-h incubation with growth factors before addition of [3H]thymidine (5 µCi/well) during the last 2 h of the assay. Cells were washed with Dulbecco's PBS (Gibco BRL) and fixed in methanol, followed by solubilization with 0.2 N NaOH and quantification of [3H]thymidine in a liquid scintillation counter. Experiments were performed in triplicate and data are expressed as means±standard error. For statistical evaluation, a LSD test with Bonferroni correction was applied.
Immunocytochemistry
Scrape-wounded mesangial cell monolayers were fixed in methanol and washed in PBS/0.05% Tween 20, followed by incubation with a monoclonal anti-SMA antibody (Sigma, clone 1A4, Zwijndrecht, The Netherlands) or a monoclonal anti-Ki67 antibody (Dako, Glostrup, Denmark). Immobilized mouse antibodies were detected by a two-step immunoperoxidase technique using HRPO-conjugated rabbit anti-mouse immunoglobulin (Dako), followed by HRPO-conjugated swine anti-rabbit immunoglobulin (Dako). Enzyme activity of HRPO was detected using 3,3'-diaminobenzidine (Sigma). Specificity was checked by omission of the primary antibodies and the use of isotype-matched non-immune IgG as negative controls.
SMA ELISA
Rat mesangial cells were plated at 5000 cells/well in a 96-well tissue culture plate (Costar). Cells were serum-starved for 24 h after reaching approximately a 70% confluence level, after which growth factors were added in fresh serum-free medium in triplicate wells. After another 24 or 48 h of incubation, cells were fixed in methanol at 4°C and non-specific protein binding sites were blocked for 1 h by PBS/1% BSA. Cells were incubated with a monoclonal anti-SMA antibody in PBS/1% BSA followed by a 2-step detection with HRPO-conjugated rabbit anti-mouse IgG (Dako) and HRPO-conjugated swine anti-rabbit IgG (Dako). Finally, o-phenylenediamine dihydrochloride (Sigma) was added as substrate for HRPO. Absorbance was measured at 490 nm in an ELISA Titertek reader. Experiments were repeated at least 3 times per condition and evaluated by a LSD test with Bonferroni correction.
Fibronectin dot-blot analysis
Rat mesangial cells were cultured and subsequently serum-starved for 24 h after reaching a 70% confluence level. Conditioned medium of cells exposed for 24 h to various factors in the absence or presence of neutralizing antibodies were harvested and protease inhibitors (PMSF 1 mmol/l, aprotinin 2 µg/ml, leupeptin 5 µg/ml, pepstatin 0.7 µg/ml (Roche, Almere, The Netherlands)) were added. Conditioned media were blotted in dilution series onto a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) with a Biorad dot-blot apparatus (Biorad, Utrecht, The Netherlands). Blots were blocked with PBS/0.05% Tween 20/5% non-fat milk and incubated with a polyclonal rabbit anti-fibronectin antibody (Dako, Glostrup, Denmark), followed by a HRPO-conjugated swine anti-rabbit antibody (Dako). Immobilized antibodies were detected with an enhanced chemiluminescence detection system (NEN Lifesciences), followed by exposure to a X-Omat blue film (Kodak, NEN Lifesciences). Specificity was checked by omission of the primary antibody and use of isotype-matched IgG as negative control. Experiments were repeated at least three times per condition and statistically evaluated by a LSD test with Bonferroni correction.
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Results |
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Induction of morphological changes and SMA expression by TGFß1, but not by PDGF-BB or CTGF
Upregulation of SMA in vivo is a marker for transformation of mesangial cells into myofibroblast-like cells [18]. In the anti-Thy-1.1 glomerulonephritis model, increased
SMA expression was observed after the elevation of CTGF mRNA [6]. Therefore we examined scrape-wounded cultures for changes in morphology and
SMA expression in the presence or absence of added growth factors. Phase-contrast microscopy revealed that scrape-wounded cultures exhibited a seemingly uniform monolayer of slightly elongated, flattened cells with multiple slender extensions. However, immunostaining with monoclonal anti-
SMA antibody revealed heterogeneity, with intense positive staining of a small minority of cells (<14%), which were scattered throughout the monolayer either as single cells or grouped in small clusters between the large majority of negative cells throughout the monolayer. These
SMA-positive cells appeared hypertrophic and polygonal, and resembled myofibroblasts (Figure 2a
). The morphology and the
SMA staining pattern of mesangial cells were not different in CTGF- or PDGF-BB-stimulated cultures, except that the latter stood out by the appearance of many mitotic figures (Figure 2b
,c
, 7
). In contrast, cells in TGFß1-stimulated cultures developed a uniformly spindle-shaped and fibroblast-like phenotype throughout the monolayer. Immunostaining of these cultures for
SMA was positive in over 95% of the cells throughout the monolayer (Figure 2d
).
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Induction of wound closure by CTGF
Wound closure in the absence of added growth factor occurred only very slowly, leaving a minimum of 88±15% of the original width of the wound at 72 h after scraping. At 72 h after scraping, wound closure induced by rhCTGF at a concentration of 20 ng/ml was almost complete (Figure 4). rhCTGF enhanced wound closure in a dose-dependent manner (Figure 5a
). In the presence of neutralizing chicken anti-CTGF antibody (pIgY13) at 0.4 µg/ml no significant wound closure was measured as compared to controls. Addition of control chicken IgY (pCIgY13) (0.4 µg/ml) or neutralizing pan-specific TGFß antibody (1 µg/ml) did not influence CTGF-induced wound healing (Figure 5b
). Short term exposure (4 h) of scrape-wounded cells to rhCTGF did not result in significant wound closure (data not shown). So continuous presence of rhCTGF in the culture medium was required to induce wound healing of mesangial cell monolayers. Since previous studies have shown that heparin can significantly increase the proliferative response of NRK fibroblasts to CTGF, the influence of heparin on CTGF-induced wound closure was examined [9]. Heparin (10 µg/ml), added in the presence or absence of exogenous growth factors, did not alter the rate of wound closure induced by CTGF in our assays (data not shown).
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Induction of wound closure by TGFß1 and PDGF-BB
When PDGF-BB (10 ng/ml) or TGFß1 (5 ng/ml) was added to cultures, significantly increased wound closure was measured after 48 and 72 h post scraping, leaving 24±1.4 and 41±6.4% respectively of the width of the wound. The kinetics of wound healing in TGFß1-stimulated cultures were similar to those of CTGF-stimulated cultures. Addition of neutralizing anti-CTGF antibody to TGFß1-stimulated cultures did not influence TGFß1-induced wound healing, but neutralizing pan-specific TGFß antibody completely blocked TGFß1-induced wound closure (Figure 6a).
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Induction of proliferation by PDGF-BB, but not by TGFß1 or CTGF
To get an impression of the relative contribution of proliferation to wound closure, immunostaining was performed with a monoclonal anti-Ki67 antibody. Only the PDGF-BB-stimulated cultures, but not the CTGF- or TGFß1-stimulated cells, displayed a high fraction of Ki67-positive cells. These were located mainly at the wound edges of the mesangial cell monolayer (Figure 7). In agreement with this, only PDGF-BB and not CTGF and TGFß increased [3H]thymidine incorporation (10 times that of control cultures, Figure 8
). Addition of heparin (10 µg/ml) in the presence or absence of added growth factors did not influence the proliferation rate of mesangial cells (data not shown).
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Induction of fibronectin expression by CTGF and TGFß
An important aspect of the mesangial response to injury is growth factor-induced expression of extracellular matrix proteins, which is essential for normal wound healing. A strong accumulation of fibronectin, among other matrix proteins, is typically seen in anti-Thy-1.1 nephritis. Therefore, we tested the ability of CTGF to influence fibronectin expression in mesangial cells and investigate the possible role of CTGF in the previously reported induction of fibronectin synthesis by TGFß.
In conditioned medium of CTGF-stimulated cells a 4.3±0.2-fold upregulation of fibronectin protein was measured compared to control cultures. This CTGF-induced fibronectin synthesis could be completely blocked by neutralizing anti-CTGF antibodies (pIgY13, 0.4 µg/ml) but not by pre-immune control chicken IgY (pCIgY13, 0.4 µg/ml). TGFß-stimulated fibronectin expression was significantly reduced in the presence of neutralizing anti-CTGF antibodies (3.0±0.5-fold, as compared to 7.8±1.0-fold over baseline). PDGF-BB (0.9±0.1) did not significantly change the basal fibronectin expression in this rat mesangial cell line (Figure 9).
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Discussion |
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Recently we observed early transient upregulation of CTGF mRNA in this model, which preceded the increase in SMA [6]. As far as growth factor involvement in the anti-Thy-1.1 model is concerned, TGFß and PDGF-BB have been implicated as key factors in the repair process. Effects of PDGF-BB include proliferation, chemotaxis, and extracellular matrix synthesis, and its expression is strongly correlated with the degree of glomerular injury in glomerulonephritis [13,15,16].
TGFß1 is critically involved in wound healing and fibrosis, but also in tumour suppression and control of the immune response and inflammation [9,14,19,20]. Some of the effects of TGFß1, such as anchorage-independent growth of NRK fibroblasts and collagen synthesis by these cells, have been demonstrated to be mediated via a CTGF-dependent pathway [4,5,7,11]. However, CTGF without active TGFß was not able to induce anchorage-independent growth. The question remains concerning which other actions of TGFß might be mediated via CTGF-dependent and -independent pathways (Figure 10).
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The present in vitro studies were performed to investigate whether the increased expression of CTGF in the healing phase of the anti-Thy-1.1 single injection model might contribute to the various aspects of mesangial repair and how this would relate to effects of TGFß and PDGF-BB. Therefore we assessed the in vitro response of mesangial cells to these growth factors in terms of changes in morphology and SMA expression, migration, proliferation, and fibronectin induction.
To assess the effect of CTGF, TGFß, and PDGF-BB on activation of mesangial cells and on their transformation into myofibroblast-like cells, scrape-wounded cultures were stained for SMA. The morphology and number of
SMA-positive cells and the level of
SMA expression were not affected by CTGF and PDGF-BB. In contrast, TGFß1-stimulated cells were more spindle-shaped and arranged in typical fibroblast-like bundles. Moreover,
SMA expression was upregulated in all cells throughout the monolayer in these cultures and semi-quantitative analysis by an indirect ELISA for
SMA on mesangial cells showed up to a threefold increase of the
SMA expression level. Neutralizing anti-CTGF antibodies did not influence these effects of TGFß on mesangial cells, which suggests that the effect of TGFß on
SMA expression is not dependent on CTGF.
Migration and proliferation of mesangial cells, two other aspects of mesangial repair in anti-Thy-1.1 nephritis, were investigated by measuring the closure of wound margins by the scraped mesangial cell monolayers. We found that CTGF stimulates wound closure of mesangial cell monolayers in a dose-dependent manner. This effect of CTGF was completely blocked by the addition of neutralizing chicken anti-CTGF antibody but not by neutralizing pan-specific TGFß antibody. From this it seems that CTGF-induced wound healing in this in vitro model is TGFß-independent although we cannot exclude some role for autocrine TGFß stimulation, which might have escaped TGFß blocking antibodies in the culture medium. To stimulate significant wound closure, short-term exposure to CTGF was not sufficient. When scrape-wounded cultures were stimulated with TGFß, similar kinetics of wound closure were observed as in the presence of CTGF. TGFß-induced wound healing was completely blocked by a neutralizing pan-specific TGFß antibody, but neutralizing anti-CTGF antibody had no detectable effect on TGFß-induced wound healing. It thus appears that CTGF- and TGFß-induced wound healing share various characteristics but are mutually independent.
To investigate the possible involvement of proliferation in CTGF- and TGFß-induced wound closure, cells were evaluated for the expression of the Ki67 antigen and for [3H]thymidine incorporation. Although CTGF is a mitogen for NRK fibroblasts and for vascular smooth-muscle cells [9,22] and while TGFß is known to be mitogenic for some mesenchymal cells (e.g. NRK fibroblasts), neither CTGF nor TGFß induced mesangial-cell proliferation in our assays. This suggests that mitogenic responses to CTGF and TGFß may be cell-type dependent.
In PDGF-BB-stimulated cultures wound closure was more rapid than in cultures exposed to CTGF or TGFß, complete at 24 h as compared to the 72 h needed in CTGF- and TGFß1-stimulated cultures. PDGF-BB-induced wound closure was characterized by strongly increased proliferation, as shown by [3H]thymidine incorporation and anti-Ki67 staining.
Since proliferation appeared not to be necessary for CTGF- and TGFß-induced wound closure, it seemed more likely that wound healing was the result of hypertrophy and/or migration of mesangial cells. Based on data from earlier studies [22], that CTGF-induced migration of vascular endothelial cells depends on expression of vß3 integrin, it might be suggested that this could also be the case for mesangial cells. However, involvement of integrins in CTGF-stimulated mesangial cells has not been studied thus far.
The literature on a possible relationship between SMA expression and proliferation, morphology or cellular hypertrophy seems contradictory. Stephenson et al. [23] reported a strong correlation of
SMA expression with hypertrophy and an inverse correlation with proliferation of mesangial cells in vitro, whereas Hugo et al. [12] showed in the in vivo anti-Thy-1.1 model that
SMA is expressed mainly in proliferating Ki67-positive mesangial cells. We observed no increase in
SMA expression in proliferative PDGF-BB-stimulated cultures, nor in non-proliferative CTGF-stimulated cultures. This observation indicates that in vitro neither the proliferation, nor the migration of rat mesangial cells requires the upregulation of
SMA. As far as the in vivo correlation of
SMA expression with proliferation and migration is concerned, this might reflect independent effects of regulation by multiple growth factors.
A further aspect of mesangial repair in anti-Thy-1.1 nephritis, upregulation of extracellular matrix production, was addressed by investigating the effects of CTGF and TGFß on fibronectin synthesis. Both CTGF and TGFß stimulated fibronectin expression by mesangial cells. Moreover, TGFß-induced fibronectin expression was significantly diminished in the presence of neutralizing anti-CTGF antibodies. This suggests that, at least in part, TGFß-induced fibronectin synthesis is regulated by a CTGF-dependent pathway. Whether this holds also true for regulation of expression of other extracellular matrix (ECM) components, and ECM-degrading enzymes, such as MMPs, remains to be elucidated. Although it has been reported [24], that PDGF-BB can induce fibronectin expression, we did not observe any effect on fibronectin expression by mesangial cells in our system. Differences in cell lines and culture conditions might be responsible for this apparent discrepancy.
In the complex process of wound repair, the levels of growth factors may determine the outcome of the repair process, i.e. hypertrophic scarring, vs true repair and restoration of functional tissue architecture. From in vitro as well as in vivo studies it is well known that TGFß is critically involved in various aspects of the repair process, but TGFß also plays a pivotal role as a suppressor of immune function and tumour growth. The fact that CTGF thus far seems to be involved more specifically in the tissue response to injury might make it a more attractive target for intervention strategies to prevent excess fibrosis, when CTGF is overexpressed.
The results of this study show that in the case of mesangial response to injury, TGFß effects may occur in part indirectly, through the effects of TGFß-induced CTGF expression on mesangial cells, and in part directly, through CTGF-independent pathways. It also has become apparent that the mesangial response to injury might also involve TGFß-like effects induced by CTGF overexpression in response to other stimuli, and independent of TGFß activity. However, it is still unclear whether increased expression of CTGF is primarily involved in temporary adaptation to the situation of acute damage and repair, or mainly in progressive scarring. Therefore the crucial question remains as to how therapeutic modulation of CTGF expression might affect the outcome of repair and scarring following glomerular injury. This will be investigated in the rat anti-Thy-1.1 model by analysis of histomorphological integrity and residual function after in vivo administration of neutralizing anti-CTGF antibodies during different phases of glomerulonephritis.
In conclusion, PDGF-BB, TGFß, and CTGF all induce in vitro wound healing of rat mesangial cell monolayers. In the case of PDGF-BB, wound closure appears to be independent of CTGF and to be mainly due to increased proliferation. TGFß-induced wound healing was in part independent of CTGF (migration and SMA expression), and in part regulated via a CTGF-dependent pathway (fibronectin synthesis). CTGF effects (migration and fibronectin production) appeared to be TGFß independent. These data add to the notion that mesangial response to injury involves the interaction of a number of growth factors with different regulatory and effector profiles. By manipulation of the balance between these factors it might become possible to influence the outcome of the repair process favourably, and to minimize (progressive) loss of function by excessive scarring. CTGF might be one of the more suitable targets for future intervention strategies.
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Acknowledgments |
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Notes |
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References |
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