A possible mechanism of basic fibroblast growth factor-promoted scarless wound healing: the induction of myofibroblast apoptosis

ARTICLE

ejd.2011.1582

Auteur(s) : Masatoshi Abe masaabe@med.gunma-u.ac.jp, Yoko Yokoyama, Osamu Ishikawa

Department of Dermatology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi 371-8511, Japan

Reprints: M. Abe

Human recombinant basic fibroblast growth factor (bFGF) is been available for the treatment of non-healing skin ulcers. bFGF is very effective for promoting scarless wound healing, as well as for accelerating both dermal and epidermal wound healing as confirmed in daily clinical practice. However, there still remain unknown biological effects of bFGF on wound healing, especially on dermal myofibroblasts [1, 2].

The motile mechanisms, by which fibroblasts remodel the extracellular matrices during the morphogenetic processes, have been previously studied using cultured cells in three dimensional collagen matrices [3, 4]. As fibroblasts exert a force on and move collagen fibrils of the matrix, the collagen concentration can be measured as a decrease in the diameter of the free matrices or as a decrease in the height of the restrained matrices. During the contraction of the restrained matrices named as stress matrix contraction (SMC), the collagen fibrils become oriented in the same plane as the restraint, and thereafter mechanical loading develops. In contrast, the floating matrix contraction (FMC) occurs without a particular orientation of collagen fibrils, and the matrix remains mechanically unloaded [3-6].

The signaling mechanisms in fibroblasts, which direct them to regulate collagen matrix contraction, depend on the mechanical loading status at the time the contraction is initiated, as well as on the growth factors added to initiate the contraction. For example, stimulation of the fibroblasts with lysophosphatidic acid (LPA), generates a robust force in the restrained matrices [1, 7], whereas both LPA and bFGF equally stimulate the FMC [8, 9]. FMC is an enigma since the bFGF stimulation of fibroblasts in the floating matrices causes the activation of small G protein Rho (GTP loading) [1]. The current in vitro study was conducted to elucidate the mechanisms how bFGF promotes scarless wound healing via the myofibroblast-collagen matrix.

Materials and methods

Reagents

Dulbecco's modified Eagle's medium (DMEM) and trypsin solution were obtained from Nihon Seiyaku (Tokyo, Japan). Bovine serum albumin (BSA) and LPA were obtained from Sigma, Steinheim (St. Louis, MO). Fetal bovine serum (FBS) was obtained from Cytosystems (Castle Hill, NSW, Australia). bFGF was obtained from Kaken Pham. Co. Ltd. (Tokyo, Japan). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG H+L and goat anti-rabbit IgG H+L antibodies were obtained from ICN Biomedicals, Inc. (Aurora, OH). The enhanced chemiluminescence (ECL) Western blotting reagent was obtained from Amersham Pharmacia Biotechnologies (Piscataway, NJ). Vitrogen “100” collagen was obtained from Cohesion (Palo Alto, CA). The G-LISA^TM RhoA and Rac 1 activation assay biochemical kits were obtained from Cytoskeleton (Denver, CO). Lipofectin was obtained from Invitrogen (Carlsbad, CA). C3 exotransferase, Akt inhibitor VI, Y27632, and LY294002 were obtained from Calbiochem-Novabiochem Corp. (La Jolla, CA). The mouse anti-actin, mouse anti-α-smooth muscle actin (αSMA) and mouse anti-myosin regulatory light chain (20K) antibodies were obtained from Sigma Chemicals. Rabbit anti-diphosphorylated myosin light chain (di-MLC) (Thr 18/Ser 19) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). RITC-conjugated phalloidin and FITC-conjugated goat anti-mouse IgG H+L antibodies were obtained from Molecular Probes, Inc. (Eugene, OR). The polyvinylidene difluoride (PVDF) membranes were obtained from Millipore Corp. (Bedford, MA). The western blotting reagents were obtained from Amersham Pharmacia Biotechnologies (Piscataway, NJ). Fluoromount G was obtained from Southern Biotechnology Associates (Birmingham, AL). The ApoStrand Enzyme-Linked Immunosorbent Assay (ELISA) Apoptosis Detection Kit was obtained from Biomol International (Plymouth Meeting, PA).

Monolayer and collagen matrix culture

The study protocol conforms to the principles of the Helsinki Declaration and the institutional ethics review board approved it. After informed consent was obtained, dermal fibroblasts were cultivated from five healthy volunteers. The skin specimens were cut into small pieces, and outgrown fibroblasts were trypsinized and grown in DMEM supplemented with 10% FBS at 37̊C in 5% CO₂ and 95% humidified air incubator. The culture medium was changed every 3 days. The cells were used within the fifth passage. The cells were harvested using 0.25% trypsin for 1 minute, followed by treatment with DMEM supplemented with 10% FBS. For the monolayer culture experiments, harvested cells were seeded at a density of 4 × 10⁴ cells on 22-mm sq. glass coverslips (Fisher Scientific, Chicago, IL), which were coated with collagen (50 μg/mL for 30 minutes), and then incubated in DMEM containing 5 mg/mL BSA and the respective growth factors and inhibitors, as indicated. Transforming growth factor-β₁ (TGFβ₁) has been reported to be required to render fibroblasts to express the myofibroblast phenotype in vitro, i.e., an increased expression of αSMA. In the present study, fibroblasts were incubated for 3 to 6 days in 10% FBS/DMEM containing 10 ng/mL TGFβ₁, and the medium was changed every 2 days.

The collagen matrix cultures were prepared using Vitrogen “100” collagen as previously described [10, 11]. Briefly, neutralized collagen solution (1.5 mg/mL) containing the harvested myofibroblasts (5×10⁵ cells/mL) was prewarmed to 37̊C for 4 minutes, and then aliquots (200 μL) were placed on an area outlined by a 12-mm-diameter circular score within a well of a 24-well culture plate (Greiner Bio-one, Frickenhausen, Germany) and were allowed to polymerize for 1 hour at 37̊C in a 5% CO₂ humidified incubator. In order to initiate the FMC, the matrices were gently released from the culture dish with a spatula into 0.8-1.0 mL of DMEM containing 5 mg/mL BSA (DMEM/BSA), and the respective growth factors and inhibitors, as indicated. For the stressed matrix contraction (SMC), the polymerized matrices were incubated for 24 hour before release, in 1.0 mL of DMEM/10% FBS containing 50 μg/mL ascorbic acid.

The matrix contraction was carried out for the indicated times. Thereafter, the samples were fixed for 10 minutes at room temperature with 3% paraformaldehyde in phosphate-buffered saline (PBS) (150 mM NaCl, 3 mM KCl, 1 mM KH₂PO₄, 6 mM Na₂HPO₄, pH 7.2). In order to quantify the contraction, the fixed matrices were washed, placed on a flat surface, and the diameters were measured. The contraction data were presented as the change in the diameter (starting minus final) in millimeters. All the experiments were carried out in duplicate, and each experiment was repeated three or more times. The data points and error bars in the figures represent the averages and the standard deviations. In the data points where the error bars are not visible, the data points overlapped.

In order to load fibroblasts with C3 exotransferase, Lipofectin was used as a delivery system. Lipofectin/C3 was prepared in 120 μL of DMEM and diluted with additional DMEM after 1 hour at 22̊C to yield a final concentration of 10 μg/mL Lipofectin and 5 μg/mL C3. Subsequently, the cells were incubated with Lipofectin/C3 mixture or with the identically prepared Lipofectin without C3 for 30 minutes at 37̊C. Following the treatment with Lipofectin/C3 or Lipofectin alone, the cells were rinsed and further incubated with DMEM and 10% FBS for 60 minutes at 37̊C before harvesting.

SDS-PAGE and immunoblotting

SDS-PAGE and immunoblotting were performed as previously described [11]. Briefly, the cells in extraction buffer (150 mM NaCl, 6 mM Na₂HPO₄, 4 mM NaH₂PO₄, 2 mM EDTA, 1% NaDOC, 1% NP-40, 0.1% SDS, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 mM AEBSF, 50 mM NaF, 1 mM Na₃VO₄ and 1 mM (NH₄)₂MoO₄, pH 7.0) (150 μl per 35-mm tissue-culture dish) were homogenized using 100 strokes in a Dounce homogenizer (pestle B; Wheaton Scientific, Millville, NJ). The samples were clarified by centrifugation at 14,000 rpm (Beckman Microfuge II) for 10 minutes at 4̊C and the supernatants were dissolved in 4× reducing sample buffer (250 mM Tris, 8% SDS, 40% glycerol, 20% mercaptoethanol, 0.04% bromophenol blue) and then boiled for 5 minutes. Equal amounts of extract (protein concentration was determined by the LDH assay) were subjected to SDS-PAGE using 12% acrylamide minislab gels. The transfer to PVDF membranes was carried out at 100 V for 1 hour. The membranes were blocked with 5% milk in TTBS (0.1% Tween 20, 150 mM NaCl, 20 mM Tris, pH 7.5), and then incubated with anti-actin antibody (1:1000), anti-αSMA (1:500), anti-MLC antibody (1:1000) or anti-di-MLC (1:400) in blocking solution at 4̊C for 12 hr. After washing with TTBS, the membranes were incubated with either HRP conjugated goat anti-mouse IgG in 5% milk in TTBS (for anti-actin antibody and anti-α smooth muscle actin antibody) or in 3% BSA in TTBS (for anti-MLC kinase antibody and anti-MLC antibody) or HRP-conjugated goat anti-rabbit IgG in 3% BSA in TTBS (for anti-phosphoMLC) for 1 hour. After washing with TTBS, the membranes were visualized by the ECL system.

RhoA activation assay

RhoA activation was assessed using the G-LISA^TM RhoA activation assay biochemical kit. The luminescence assay was performed according to the manufacturer's instructions. The raw data were recorded at 100 gain, 100 millisecond integration time on a luminometer. In each experiment, the luminescence was calculated based on the values of triplicate wells.

Rac1 activation assay

Rac1 activation was assessed using the G-LISA^TM Rac activation assay biochem kit. ELISA was performed according to the manufacturer's instructions. The raw data were recorded at an optical density of 405 nm. In each experiment, the luminescence was calculated from the values of triplicate wells.

Apoptosis detection assay

The detection of apoptosis was assessed using the ApoStrand ELISA Apoptosis Detection Kit according to the manufacturer's instructions. The data were recorded at an optical density of 405 nm. In each experiment, the luminescence was calculated from the values of triplicate wells.

Immunofluorescence microscopy

The cells in matrices were fixed for 10 minutes with 3% paraformaldehyde in PBS at room temperature, blocked with 2% glycine/1% BSA in DPBS (150 mM NaCl, 3 mM KCl, 1 mM KH₂PO₄, 6 mM Na₂HPO4, 0.5 mM MgCl_2, 1mM CaCl₂, pH 7.2) for 30 minutes, and then permeabilized for 15 minutes with 0.5% triton X-100 in DPBS. Subsequently, the samples were washed with DPBS and treated for 10 minutes with 1% BSA in DPBS. RITC-conjugated phalloidin was diluted in 1% BSA in DPBS (8 units/ml) and was then added to the matrices for 30 minutes at 37̊C. After washing with DPBS, the samples were mounted on glass slides with Fluoromount G. The observed images were collected with an Olympus DP70 camera and an Olympus DP Controller system.

Statistical analysis

Data were expressed as the mean±SD. The statistical analyses for the effects of bFGF on the RhoA activation were performed with the Statview software program (version 4.0; Abacus Concepts, Berkeley, CA, USA). The group data were subjected to analysis of variance testing to determine the overall impact of sample treatments within an experiment, with additional post hoc testing via the Fisher Protected Least Significant Difference (PLSD) test to determine the statistical significance of the individual sample treatments on the parameters in question. The analysis of variance results was reported as significant only if both the analysis of variance and the Fisher PLSD tests yielded a probability (P) value of 0.05 or lower.

Results

TGFβ-activated expression of the myofibroblast phenotype

Previous studies reported that 3 to 10 days of TGFβ₁ treatment were required to render fibroblasts to express the myofibroblast phenotype in vitro, i.e., increased expression of αSMA [12]. We obtained the similar results in our early passaged human fibroblast cultures. Figure 1 shows a representative result of the western blot analysis of the αSMA expression in TGFβ-treated fibroblasts in the presence or absence of bFGF. The levels of cellular αSMA increased, albeit variably, after 2 to 4 days of 10 ng/mL TGFβ1 treatment alone and were consistently elevated after 5 days. In contrast, levels of cellular αSMA increased 4 to 6 days after the co-stimulation of bFGF and TGFβ1.

bFGF and collagen matrix contraction

Figure 2 shows the representative result of the stressed collagen matrices 1 hour after the myofibroblast promoting-contraction in the presence or absence of bFGF. The studies were performed in order to elucidate the mechanisms of the FMC using four different kinase inhibitors: PI3K inhibitor, LY294002; Akt inhibitor VI; Rho inhibitor, C3 exotransferase; and Rho kinase (ROCK) inhibitor, Y27632. The diameter of the matrices was 10.5 mm at the time the contraction was initiated. Myofibroblasts, which increased the αSMA actin expression induced by TGFβ1, exhibited marked SMC. In contrast, SMC was suppressed when the myofibroblasts were stimulated with bFGF. This result suggests that bFGF may block the myofibroblast promoting-matrix contraction. Among the four different inhibitors, only 10μM Y27632 and 5μM C3 exotransferase were able to block the matrix relaxation promoted by bFGF. On the other hand, bFGF did not promote floating matrix contraction at any concentration (data not shown).

Effect of bFGF on myofibroblast dendritic extensions in collagen matrixes

Figure 3 shows the appearance of myofibroblasts in the collagen matrices after 4 hours. The myofibroblasts in the basal medium with or without 10 ng/mL of bFGF form dendritic extensions. No significant differences were observed in cell spreading in the myofibroblast-collagen gel even in the presence of the four inhibitors. However, the cell density in matrix apparently decreased when they were stimulated with bFGF alone or with LY294002 and the Akt inhibitor (figure 3 B-D).

bFGF and Rho family activation on myofibroblasts

It is possible that, in SMC, the Rac signaling pathway may reside in the upstream of Rho kinase. In the subsequent studies, we focused on Rac, a member of the Rho family, as well as on Rho. G-LISA^TM to detect GTP-loaded Rac 1 and Rho A was carried out to confirm whether or not Rac 1 and Rho A activation was involved in fibroblasts as well as in myofibroblasts. bFGF stimulation (10 ng/mL) caused transient Rho A activation in the fibroblasts, and sustained Rho A activation in the myofibroblasts. In contrast, bFGF stimulation (10 ng/mL) caused significant Rac 1 activation both in myofibroblasts and in fibroblasts (figure 4).

Comparative studies of bFGF induced MLC phosphorylation between fibroblasts and myofibroblasts

Further experiments were performed in order to investigate the effects of bFGF on MLC phosphorylation in the fibroblast and the myofibroblast collagen matrices. MLC phosphorylation in the fibroblasts of the collagen matrices was assessed using antibodies specific to di-MLC, with total MLC as a loading control. We also confirmed the expression of the myofibroblast phenotype by immunoblotting with anti-αSMA antibodies; in this experiment, actin was used as a loading control. Diphosphorylated MLC, as well as the monophosphorylated form, has been implicated in cell contractility. The levels of di-MLC were highest in the fibroblasts 30 minutes after the bFGF stimulation. In contrast, the basal level of di-MLC was elevated in the myofibroblasts without bFGF stimulation. After bFGF stimulation, the levels of di-MLC were further elevated up to 60 minutes (figure 5).

Apoptosis induction by bFGF on myofibroblasts

Since the cell numbers decreased when they were stimulated with bFGF in the myofibroblast-collagen gel, experiments were performed to elucidate the mechanisms of relaxation of the bFGF-stimulated SMC, with a focus on apoptosis. bFGF did not promote apoptosis in the fibroblasts, even in the presence of four different inhibitors. In contrast, bFGF significantly promoted apoptosis in the myofibroblasts. The two different inhibitors, Y27632 and C3 exotransferase were able to block the myofibroblast apoptosis promoted by bFGF. It is of note that the degree of apoptosis in the presence of the other inhibitors, LY294002 and Akt inhibitor, was less than that of bFGF alone (figure 6).

Discussion

Various types of cells are involved in skin wound healing, which goes through phases of bleeding and coagulation, inflammation, proliferation, and remodeling, by the interactions of cells involving the activities of cytokines or growth factors [13, 14]. bFGF is a potent mitogen and chemoattractant for endothelial cells and fibroblasts [15-18]. The topical administration of recombinant bFGF to skin wounds has been shown to accelerate both dermal and epidermal wound healing. Previously, the effect of bFGF on sutured incised wounds of full-thickness skin prepared in animals was studied, and it was found that the width of the resultant scar was narrower and flatter after a single treatment with bFGF compared with controls [19]. However, little is known about the effects of topically applied bFGF on the remodeling phase of wound healing. In the present study, myofibroblasts stimulated with bFGF exhibited a transient Rac and Rho activation similar to that of fibroblasts. This result suggests that bFGF plays a specific role in not only the proliferation phase but also in the remodeling phase of wound healing.

It has been recognized that myofibroblasts play a key role in promoting hypertrophic scars in the remodeling phase of skin wound healing. The studies of myofibroblasts in three-dimensional matrices also suggest that reciprocal and adaptive mechanical interactions play a pivotal role in the regulation of morphogenesis. Therefore, fibroblasts cultured in collagen matrices have been used as a model system to study the organization of cells into connective tissue. It was confirmed that myofibroblasts isolated from wound tissue or hypertrophic scars contract the matrices more than fibroblasts from unwounded skin [20, 21]. TGFβ can activate fibroblasts to differentiate into myofibroblasts [22, 23]; αSMA-expressing cells that have been implicated in wound contraction [24, 25] and wound contractures [26]. TGFβ can potentially activate fibroblasts to differentiate into myofibroblasts, even if fibroblasts are mechanically loaded. It was shown that TGFβ activation of fibroblasts to differentiate into myofibroblasts occurred when the cells were in stressed collagen matrices but not the in floating collagen matrices [27, 28]. In the present study, we found that the levels of αSMA expression of fibroblasts increased after 2 to 4 days of TGFβ1 treatment alone and increased 4 to 6 days after co-stimulation with bFGF and TGFβ1. The results indicated that bFGF may delay the expression of αSMA by fibroblasts.

Spontaneous contraction was observed in the stressed myofibroblast-collagen matrix, and bFGF cancelled the gel contraction. However, in the presence of C3 exotransferase or Y27632, bFGF allowed the contraction. Since the Rho kinase has been shown to induce apoptosis [29], our results suggest that the Rho/Rho kinese signaling pathway is involved in bFGF-promoted myofibroblast apoptosis. Our observations support the proposed role of bFGF for myofibroblasts in wound contraction [24, 25] and may provide a model to study the cycle of cell contraction and relaxation that mediates wound contracture in situ [30]. In particular, there is only limited information available with respect to the mechanism of cellular loss and apoptosis during tissue repair following the administration of bFGF to acute incisional skin wounds [19]. Recent studies suggest that apoptosis is the main cause of reduced cellularity during the various phases of normal wound healing [31] and also involved in the process of remodeling [32-34]. In a rat wound model, the resolution of inflammatory changes in injured tissue and the decrease in tissue cellularity during the healing process were considered to be associated with apoptosis [31]. In a study of impaired wound healing in diabetic mice (C57BL/KsJ-db/db), a significant delay in the appearance of apoptosis was reported [35]. However, the topical administration of insulin-like growth factor II (IGF-II) and platelet-derived growth factor (PDGF) to the wound markedly promoted apoptosis and improved wound healing [35]. In a porcine wound model, the inhibitory effect on wound contraction positively correlates with the number of fibroblasts in the collagen sponge [36]. Our results showed that bFGF significantly increased apoptosis in myofibroblasts, consistent with the previous report that showed increased apoptosis in incisional wounds after treatment with bFGF. In addition, both LY294002 and Akt inhibitor in the presence of bFGF also markedly induced apoptosis in the myofibroblasts. Akt has been recognized to promote cell survival by apoptosis inhibition [37]. Considering the stressed myofibroblast-collagen matrix experiment, the present study suggests that the PI3K/Akt as well as the Rho/Rho kinese signaling pathway is partially involved in bFGF-promoted myofibroblast apoptosis.

We suggest that bFGF can control the role of myofibroblast functions through apoptosis and can promote scarless wound repair. Acidic FGF (aFGF) induces the apoptosis of human lung myofibroblasts by prior treatment with TGFβ1 in vitro, in which aFGF reduced the growth rate of the fibroblasts, despite the induction of DNA synthesis in these cells [38]. Such dual effects may be implicated in the significant heterogeneity of the cultured fibroblast population that contains fibroblasts, proto-myofibroblasts, and myofibroblasts, and the differential ability of these cell types to induce apoptosis.

In addition to its agonist effect, bFGF induced MLC diphosphorylation in the fibroblast but not in the myofibroblast whose MLC had diphosphorylated spontaneously. These findings suggest that relaxation of the myofibroblast collagen matrix with bFGF was independent of MLC phosphorylation.

bFGF is reported to be a potent inhibitor of mesodermal differentiation, whereas PDFG AB favours myofibroblast formation and up-regulates expression of TGFβ receptors I and II [39]. The latest study indicated that gene-modified artificial skin including human fibroblasts expressing bFGF resulted in a reduced wound contraction and a well-organized human epidermis and better formed dermis [40]. These techniques should be a promising approach in the future treatment of chronic wounds.

In conclusion, the present study suggests that down regulation of PI3K/Akt signaling as well as up regulation of Rho/Rho kinese signaling is involved in bFGF-promoted myofibroblast apoptosis. These results suggest that bFGF can promote scarless wound healing upon the induction of apoptosis in myofibroblasts. Our preliminary study may help in the prevention and treatment of tissue fibrosis and in particular in the eradication of hypertrophic and keloid scars.

Disclosure

Financial support: none. Conflicts of interest: none.

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