In vitro evidence for differential involvement of CTGF, TGFß, and PDGF-BB in mesangial response to injury

Ingrid E. Blom1, Anette J. van Dijk1, Lotte Wieten1, Karen Duran1, Yasuhiko Ito2,3, Livio Kleij4, Mark deNichilo5, Ton J. Rabelink4, Jan J. Weening2, Jan Aten2 and Roel Goldschmeding1,

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



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Connective tissue growth factor (CTGF) is a profibrotic growth factor, which is upregulated in wound healing and renal fibrosis, including anti-Thy-1.1 nephritis. The kinetics of CTGF mRNA expression in anti-Thy-1.1 nephritis suggested that CTGF regulation might contribute to glomerular response to injury downstream of transforming growth factor-ß (TGFß). In anti-Thy-1.1 nephritis the initial damage is followed by mesangial repair and limited sclerosis, which involves mesangial cell (MC) activation ({alpha}-smooth-muscle actin ({alpha}SMA) expression), proliferation, migration, and extracellular matrix production. The present in vitro study addresses the possible role of CTGF in these different aspects of mesangial response to injury, and how CTGF activity might relate to effects of TGFß and platelet-derived growth factor-BB (PDGF-BB).

Methods and Results. Immunostaining and ELISA showed that {alpha}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



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Connective tissue growth factor (CTGF) is a 38-kDa cysteine-rich protein and is a member of the CCN (CTGF/Fisp 12, Cyr 61/CEF-10, Nov) immediate early gene family of proteins. CTGF was originally cloned from human umbilical vein endothelial cells (HUVEC) and identified by its platelet-derived growth factor-like (PDGF) activity for normal rat kidney (NRK) fibroblasts [1]. CTGF expression is upregulated by transforming growth factor ß (TGFß), but not by PDGF in NRK, human foreskin fibroblasts, NIH3T3 cells, glomerular visceral epithelial cells (GVEC) and mesangial cells (MC) [26]. We have previously shown that renal CTGF expression is highly upregulated in a subset of human kidney diseases and that it may be a common factor in the development of renal fibrosis, as a downstream mediator of TGFß [3]. CTGF is thought to be essential for the mediation of some of the actions of TGFß [4,5,7]. Full length CTGF (38 kDa) has been shown to induce mitogenic activity for NIH 3T3 cells, whereas the 10-kDa form is mitogenic for Balb/c 3T3 cells and vascular smooth-muscle cells [1]. Furthermore, CTGF is involved in regulation of endothelial cell migration [8], matrix production by human foreskin fibroblasts and mesangial cells in vitro and in granulation tissue formation [911].

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 {alpha} smooth-muscle actin ({alpha}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 {alpha}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.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Growth factors and antibodies
Recombinant human (rh) CTGF, pre-immune chicken IgY (pCIgY13) and neutralizing chicken anti-CTGF antibody (pIgY13) were provided by FibroGen Inc. (South San Francisco, USA). rhCTGF was generated using a baculovirus expression system and purified by heparin–Sepharose affinity chromatography as described previously [9]. Peak fractions containing rhCTGF were determined by immunoblotting and Coomassie staining of SDS–polyacrylamide gels. Neutralizing anti-CTGF antibody was raised in chicken by immunization with purified baculovirus-derived full-length rhCTGF protein as previously described [4], and was subsequently affinity purified through a rhCTGF–Sepharose column. rhTGFß1, rhPDGF-BB, and neutralizing rabbit pan-specific TGFß antibody (panTGFß) were purchased from R&D Systems (Abingdon, United Kingdom).

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 heparin–Sepharose 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% SDS–polyacrylamide 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-{alpha}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.

{alpha}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-{alpha}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.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
TGFß induction of CTGF protein in rat mesangial cells
Western blot analysis was performed on culture supernatants from mesangial cells that were serum-starved for 24 h and subsequently stimulated with TGFß1 (5 ng/ml). As indicated in Figure 1Go, conditioned medium showed an increase in CTGF protein expression in TGFß1-stimulated cultures compared to control cultures (lanes 2 and 3) of the same molecular weight (36–38 kDa) as the recombinant protein (lane 1). Supernatants of heparin–Sepharose treated conditioned medium did not show any reactivity with the anti-CTGF antibody (Ab) (lanes 4 and 5). In contrast, when heparin-binding proteins were resolved from heparin–Sepharose beads by boiling in SDS sample buffer, a strong CTGF protein signal was observed in TGFß1-stimulated cultures (lanes 6 and 7).



View larger version (48K):
[in this window]
[in a new window]
 
Fig. 1. Induction of CTGF protein expression by TGFß1 in cultured rat mesangial cells. Cells were serum starved and stimulated with TGFß1 (5 ng/ml) for 24 h. Heparin-binding proteins in the conditioned medium (CM) were precipitated using heparin–Sepharose (HS) CL 6B beads. Lane 1, rhCTGF (10 ng) was run as a standard; lane 2, control CM; lane 3, TGFß1-stimulated CM; lane 4, supernatant of control HS-treated cultures; lane 5, supernatant of TGFß1 HS-treated cultures; lane 6, SDS-eluted heparin-binding proteins from HS-treated control cultures; lane 7, SDS-eluted heparin binding proteins from HS-treated TGFß1-stimulated cultures.

 

Induction of morphological changes and {alpha}SMA expression by TGFß1, but not by PDGF-BB or CTGF
Upregulation of {alpha}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 {alpha}SMA expression was observed after the elevation of CTGF mRNA [6]. Therefore we examined scrape-wounded cultures for changes in morphology and {alpha}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-{alpha}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 {alpha}SMA-positive cells appeared hypertrophic and polygonal, and resembled myofibroblasts (Figure 2aGo). The morphology and the {alpha}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 2bGo,cGo, 7Go). In contrast, cells in TGFß1-stimulated cultures developed a uniformly spindle-shaped and fibroblast-like phenotype throughout the monolayer. Immunostaining of these cultures for {alpha}SMA was positive in over 95% of the cells throughout the monolayer (Figure 2dGo).



View larger version (127K):
[in this window]
[in a new window]
 
Fig. 2. Scraped rat mesangial cells were stained for {alpha}SMA. (A) Control, (B) rhCTGF (20 ng/ml) and (C) PDGF-BB (10 ng/ml) stimulated cultures showed positive staining of a minority of cells with a hypertrophic appearance scattered throughout the monolayer. (D) TGFß1-stimulated cells (5 ng/ml) were more spindle-shaped and fibroblast-like and {alpha}SMA staining was positive throughout the monolayer.

 


View larger version (129K):
[in this window]
[in a new window]
 
Fig. 7. (A) Control cells and cells exposed to (B) CTGF (20 ng/ml) or (D) TGFß1 (5 ng/ml) did not express Ki67 antigen as judged by immunostaining. However, (C) a large fraction (about 58%) of mesangial cells stimulated with PDGF-BB (10 ng/ml) showed strong staining for Ki67.

 
To measure {alpha}SMA expression, an ELISA was performed on methanol-fixed rat mesangial cell monolayers. As shown in Figure 3Go, 3.1- and 2.4-fold inductions of {alpha}SMA expression were observed after incubation with TGFß1 for 24 and 48 h respectively. In contrast, {alpha}SMA expression in CTGF- and PDGF-BB-stimulated cultures did not significantly differ from control levels. {alpha}SMA expression induced by TGFß1 was not influenced by addition of neutralizing anti-CTGF antibody, suggesting that TGFß1-induced expression of {alpha}SMA was mediated by a CTGF-independent pathway.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3. {alpha}SMA expression was measured semi-quantitatively in an ELISA on mesangial cells, stimulated for 24 and 48 h with CTGF (20 ng/ml), TGFß1 (5 ng/ml), PDGF-BB (10 ng/ml) or TGFß1 (5 ng/ml) in the presence of anti-CTGF neutralizing antibody (pIgY13; 0.4 µg/ml). Significant increase in {alpha}SMA expression was observed only in TGFß1-stimulated cultures (P<0.05) after both 24 and 48 h of stimulation. Co-addition of CTGF neutralizing antibody to TGFß1-stimulated cultures did not influence TGFß1-induced {alpha}SMA expression (P<0.05).

 

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 4Go). rhCTGF enhanced wound closure in a dose-dependent manner (Figure 5aGo). 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 5bGo). 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).



View larger version (112K):
[in this window]
[in a new window]
 
Fig. 4. Time-lapse photography of a rat mesangial cell monolayer, stimulated with rhCTGF (20 ng/ml), at 0, 24, 48 and 72 h after scraping. At 72 h after scraping 16±27.3% of the original distance between the wound edges remained. Magnification x200.

 


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 5. (a) CTGF-induced wound closure of scraped rat mesangial cell monolayers in a dose-dependent manner. (b) CTGF-induced wound closure of scraped rat mesangial cell monolayers is not affected by neutralizing anti-TGFß antibodies. Wound closure was measured as percentage of starting distance. For better comparison, data are presented after correction for ‘spontaneous’ wound closure as observed in control cultures. CTGF (20 ng/ml)-induced wound closure was completely blocked by addition of a chicken neutralizing anti-CTGF antibody (pIgY13; 0.4 µg/ml). Significant differences in wound closure were observed at 48 and 72 h after scraping (P<0.05; control vs CTGF; control vs CTGF+panTGFß at 48 h. Control vs CTGF, control vs CTGF+pCIgY13, control vs CTGF+ panTGFß, CTGF vs CTGF+pIgY13, CTGF+pIgY13 vs CTGF+ pCIgY13, CTGF+pIgY13 vs CTGF+panTGFß at 72 h). Co-addition of control chicken IgY (pCIgY13; 0.4 µg/ml) did not affect CTGF- induced wound healing. Co-addition of a neutralizing pan-TGFß antibody (1 µg/ml) did not influence CTGF-induced wound closure, suggesting that CTGF was capable of induction of wound healing in a TGFß-independent manner.

 

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 6aGo).



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 6. (a) TGFß-induced wound closure of scraped rat mesangial cell monolayers is independent of CTGF. Wound closure is presented as percentage of starting distance and corrected for background values of control cultures. TGFß1 (5 ng/ml) induces significant wound closure of rat mesangial cell monolayers at 72 h after scraping (P<0.05; control vs TGFß, control vs TGFß+pIgY13, TGFß vs TGFß+panTGFß). Effects of TGFß1 on wound closure could be blocked completely by co-addition of neutralizing pan-specific TGFß antibody (1 µg/ml) (P<0.05) but not by neutralizing anti-CTGF antibody (pIgY13; 0.4 µg/ml). (b) PDGF-BB-induced wound healing of scraped rat mesangial cell monolayers is independent of CTGF. Wound closure is presented as percentage of starting distance and corrected for background values of control cultures. PDGF-BB (10 ng/ml)-induced wound closure of mesangial cell monolayers is already significant 24 h after scraping (P<0.05; control vs PDGF-BB, control vs PDGF-BB+pIgY13, control vs PDGF-BB+panTGFß). PDGF-BB effects were not affected by the addition of neutralizing anti-CTGF antibody (pIgY13; 0.4 µg/ml), nor by the addition of a neutralizing panTGFß antibody (1 µg/ml).

 
In PDGF-BB-stimulated cultures, a much quicker closure of the wounds was observed, as compared to cultures stimulated with CTGF or TGFß1. Addition of neutralizing anti-CTGF antibody or neutralizing pan-specific TGFß antibody did not influence wound closure of PDGF-BB-stimulated cultures (Figure 6bGo).

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 7Go). In agreement with this, only PDGF-BB and not CTGF and TGFß increased [3H]thymidine incorporation (10 times that of control cultures, Figure 8Go). 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).



View larger version (9K):
[in this window]
[in a new window]
 
Fig. 8. No induction of [3H]thymidine incorporation was observed in control, CTGF or TGFß1-stimulated cultures. However, a 10-fold increase in proliferation was measured in PDGF-BB-stimulated cultures as compared to controls (P<0.05).

 

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 9Go).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 9. Dot blot analysis of fibronectin expression in conditioned medium of rat mesangial cell cultures. Both rhCTGF (4.3±0.2-fold 20 ng/ml) and TGFß1 (7.8±1-fold 5 ng/ml) induced expression of fibronectin. CTGF stimulated fibronectin expression was completely blocked by neutralizing anti-CTGF Ab (pIgY13, 0.4 µg/ml) but not by control IgY (pCIgY13, 0.4 µg/ml). TGFß1 induced fibronectin expression was significantly blocked by anti-CTGF Ab. PDGF-BB (10 ng/ml) did not show induction of fibronectin protein expression.

 



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Tissue repair is a highly regulated process of obvious importance throughout life. To study tissue repair in response to glomerular injury, anti-Thy-1.1 nephritis has been used extensively as an in vivo model for mesangial proliferative glomerulonephritis.

Recently we observed early transient upregulation of CTGF mRNA in this model, which preceded the increase in {alpha}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 10Go).



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 10. Different pathways by which TGFß mediates its action on cells, independent and dependent of CTGF. In italic: effects of TGFß and CTGF observed in this study (modified from Kothapalli et al. 1997 [4]).

 
Conversely, CTGF is known to have mitogenic effects on proliferation, migration, matrix production, angiogenesis, adherence and granulation tissue formation [1,4,5,810]. However, it has not been firmly established whether these actions of CTGF require TGFß activity.

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 {alpha}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 {alpha}SMA. The morphology and number of {alpha}SMA-positive cells and the level of {alpha}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, {alpha}SMA expression was upregulated in all cells throughout the monolayer in these cultures and semi-quantitative analysis by an indirect ELISA for {alpha}SMA on mesangial cells showed up to a threefold increase of the {alpha}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 {alpha}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 {alpha}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 {alpha}SMA expression and proliferation, morphology or cellular hypertrophy seems contradictory. Stephenson et al. [23] reported a strong correlation of {alpha}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 {alpha}SMA is expressed mainly in proliferating Ki67-positive mesangial cells. We observed no increase in {alpha}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 {alpha}SMA. As far as the in vivo correlation of {alpha}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 {alpha}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.



   Acknowledgments
 
The authors wish to thank Dick van Wichen and Roel Broekhuizen (Department of Pathology, University Medical Center Utrecht) and Wim Verrijp (Audio-visual Service, University Medical Center Utrecht) for help with photographic work and Drs C. L. Kruitwagen (Center for Biostatistics) for advice on statistical analysis. George Martin (FibroGen Inc.) is acknowledged for critical reading of the manuscript.



   Notes
 
Correspondence and offprint requests to: R. Goldschmeding MD, Department of Pathology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Brigstock DR. The connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN) family. Endocr Rev1990; 20: 189–206[Abstract/Free Full Text]
  2. Igarashi A, Okochi H, Bradham DM, Grotendorst GR. Regulation of connective tissue growth factor gene expression in human skin fibroblasts and during wound repair. Mol Biol Cell1993; 4: 637–645[Abstract]
  3. Ito Y, Aten J, Bende RJ et al. Expression of connective tissue growth factor in human renal fibrosis. Kidney Int1998; 53: 853–861[ISI][Medline]
  4. Kothapalli D, Frazier KS, Welpy A, Segarini PR, Grotendorst GR. Transforming growth factor ß induces anchorage-independent growth of NRK fibroblasts via a connective tissue growth factor-dependent signaling pathway. Cell Growth Differ1997; 8: 61–68[Abstract]
  5. Kothapalli D, Hayashi N, Grotendorst GR. Inhibition of TGFß-stimulated CTGF gene expression and anchorage-independent growth by cAMP identifies a CTGF-dependent restriction point in the cell cycle. FASEB J1998; 12: 1151–1161[Abstract/Free Full Text]
  6. Ito Y, Kleij L, Goldschmeding R et al. Kinetics of connective tissue growth factor expression during experimental proliferative glomerulonephritis. J Am Soc Nephrol2001; 12: 472–484[Abstract/Free Full Text]
  7. Duncan MR, Frazier KS, Abramson S et al. Connective tissue growth factor mediates transforming growth factor ß-induced collagen synthesis: downregulation by cAMP. FASEB J1999; 13: 1774–1786[Abstract/Free Full Text]
  8. Shimo T, Nakanashi T, Kimura Y et al. Inhibition of endogenous expression of connective tissue growth factor by its antisense oligonucleotide and antisense RNA suppresses proliferation and migration of vascular endothelial cells. Biochem1998; 124: 130–140
  9. Frazier K, Williams S, Kothapalli D, Klapper H, Grotendorst GR. Stimulation of fibroblast cell growth, matrix production, and granulation tissue formation by connective tissue growth factor. J Invest Dermatol1996; 107: 404–411[Abstract]
  10. Murphy M, Godson C, Cannon S et al. Suppression subtractive hybridisation identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells. J Biol Chem1999; 274: 5830–5834[Abstract/Free Full Text]
  11. Riser BL, DeNichilo M, Cortes P et al. Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis. J Am Soc Nephrol2000; 11: 25–38[Abstract/Free Full Text]
  12. Hugo C, Shankland SJ, Bowen-Pope DF, Couser WG, Johnson RJ. Extraglomerular origin of the mesangial cell after injury, a new role of the juxtaglomerular apparatus. J Clin Invest1997; 100: 786–794[Abstract/Free Full Text]
  13. Abboud HE. Role of platelet-derived growth factor in renal injury. Annu Rev Physiol1995; 57: 297–309[ISI][Medline]
  14. Border WA, Okuda S, Languino LR, Sporn MB, Ruoslahti E. Suppression of experimental glomerulonephritis by antiserum against transforming growth factor ß1. Nature1990; 346: 371–374[ISI][Medline]
  15. Floege J, Johnson RJ. Multiple roles for platelet-derived growth factor in renal disease. Miner Electrolyte Metab1995; 21: 271–282[ISI][Medline]
  16. Johnson RJ, Raines EW, Floege J et al. Inhibition of mesangial cell proliferation and matrix expansion in glomerulonephritis in the rat by antibody to platelet-derived growth factor. J Exp Med1992; 175: 1413–1416[Abstract]
  17. Wolthuis A, Boes A, Rodemann HP, Grond J. Vasoactive agents affect growth and protein synthesis of cultured rat mesangial cells. Kidney Int1992; 41: 124–131[ISI][Medline]
  18. Johnson RJ, Iida H, Alpers CE et al. Expression of smooth muscle cell phenotype by rat mesangial cells in immune complex nephritis. Alpha-smooth muscle actin is a marker of mesangial cell proliferation. J Clin Invest1991; 87: 847–858[ISI][Medline]
  19. Roberts AB, Flanders KC, Heine UI et al. Transforming growth factor-ß: Multifunctional regulator of differentiation and development. Phil Trans R Soc Lond B1990; 327: 145–154[ISI][Medline]
  20. Grotendorst GR. Connective tissue growth factor: a mediator of TGF-ß action on fibroblasts. Cytokine Growth Factor Rev1997; 8: 171–179[Medline]
  21. Babic AM, Chen CC, Lau LF. Fisp12/Mouse connective tissue growth factor mediates endothelial cell adhesion and migration through integrin {alpha}vß3, promotes endothelial cell survival and induces angiogenesis in vivo. Mol Cell Biol1999; 19: 2958–2966[Abstract/Free Full Text]
  22. Pawar S, Kartha S, Toback FG. Differential gene expression in migrating renal epithelial cells after wounding. J Cell Physiol1995; 165: 556–565[ISI][Medline]
  23. Stephenson LA, Haney LB, Hussaini IM, Karns LR, Glass WF. Regulation of smooth muscle {alpha}-actin expression and hypertrophy in cultured mesangial cells. Kidney Int1998; 54: 1175–1187[ISI][Medline]
  24. Floege J, Eng E, Young BA et al. Infusion of platelet-derived growth factor or basic fibroblast growth factor induces selective glomerular mesangial cell proliferation and matrix accumulation in rats. J Clin Invest1993; 92: 2952–2962[ISI][Medline]
Received for publication: 8. 9.00
Revision received 15.12.00.