Effects of integrin ανβ3 on differentiation and collagen synthesis induced by connective tissue growth factor in human hypertrophic scar fibroblasts

  • Authors:
    • Xiaolong Hu
    • Na Li
    • Ke Tao
    • Xiaobing Fang
    • Jiaqi Liu
    • Yaojun Wang
    • Hongtao Wang
    • Jihong Shi
    • Yunchuan Wang
    • Peng Ji
    • Weixia Cai
    • Xiaozhi Bai
    • Xiongxiang Zhu
    • Juntao Han
    • Dahai Hu
  • View Affiliations

  • Published online on: August 22, 2014     https://doi.org/10.3892/ijmm.2014.1912
  • Pages: 1323-1334
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Abstract

CCN2 is a matricellular protein that appears to be important in scar formation. CCN2 mediates the pro-fibrotic effects in hypertrophic scars (HTSs) through an unknown mechanism. However, many activities of CCN2 protein are known to be mediated by direct binding to integrin receptors. In this study, we investigated the role of integrin ανβ3 in the differentiation of hypertrophic scar fibroblasts (HTSFs) induced by CCN2. The levels of integrin ανβ3 between normal skin and hypertrophic scar (HTS) tissues were compared, and integrin ανβ3 was found to be upregulated in HTS. CCN2 was shown to induce HTSF differentiation and collagen (COL) synthesis at the mRNA and protein levels. Based on these results, the expression of integrin ανβ3 was upregulated by CCN2 stimulation during HTSF differentiation. Blockade of integrin ανβ3 prevented CCN2-induced HTSF differentiation and COL synthesis. Furthermore, the CCN2-induced increase in contractility of the HTSF in COL lattices was inhibited by integrin ανβ3 blocking antibodies. HTSs were established in a rabbit ear model, and the inhibitor of integrin ανβ3 significantly improved the architecture of the rabbit ear scar. Results of the present study showed that integrin ανβ3 contributes to pro-fibrotic CCN2 signaling. Blocking this pathway may therefore be beneficial for the treatment of HTS.

Introduction

Over 70% of patients with burn injuries develop hypertrophic scarring (HTS), which may manifest as tissue disfigurement or disruption of normal physiologic and/or physical function. There is currently no effective and safe strategy for the prevention or inhibition of HTS formation (1). Therefore, investigations on how to reduce HTS occurrence during the skin wound-healing process is of great clinical value.

CCN2, also known as connective tissue growth factor, plays an important role in promoting fibrosis and scarring in numerous tissues (2,3). CCN2 has been characterized as a cysteine-rich protein that stimulates a broad repertoire of cell responses, including proliferation, migration, adhesion, matrix production and differentiation (4). Overexpression of CCN2 has been shown to promote fibrosis and scar formation in skin, kidney, liver, brain, lung, vasculature and pancreas (3,5,6).

Previous studies have provided strong evidence of a role for CCN2 in HTS, especially as a cofactor of transforming growth factor β1 (TGF-β1) function (7,8). Several cell types have been shown to produce CCN2 in HTS (911), with attention being focused on hypertrophic scar fibroblasts (HTSFs). Baseline CCN2 expression is increased in unstimulated HTSF, as compared to that in normal fibroblasts, and HTSF production is increased by the CCN2-stimulated activation of TGF-β1 (9). CCN2 also plays a critical role in mediating many of the important fibroproliferative effects of TGF-β1 (12). Furthermore, antisense oligonucleotides targeting CCN2 have no measurable effect on early wound closure events but significantly decrease the transcription of TIMP-1 and collagen (COL) types I and III in rabbit models (13). Therefore, understanding the regulatory mechanisms of CCN2 may benefit therapeutic efforts to inhibit HTS.

In recent years, progress has been made in determining the mechanisms that underlie the diverse effect of CCN2 proteins on cell function. However, the specific mechanisms involved in the CCN2-dependent fibrogenic response remain to be clarified. In general, the intracellular signaling pathways activated by CCN2 remain unknown because the bona fide CCN2 receptor has yet to be identified. Findings of a recent study have shown that many activities of CCN2 proteins are mediated by directly binding to integrin receptors (2).

Integrin ανβ3 cluster with the TGF-β type II receptor and mediate TGF-β-induced proliferation of human lung fibroblasts (14). Moreover, integrin ανβ3 appear to mediate the profibrotic effects of TGF-β in scleroderma fibroblasts (15). Previous studies have shown that integrin ανβ3 is crucial in several processes related to tissue remodeling, such as the binding and activation of matrix metalloproteinases and growth factors, as well as cell proliferation, migration and differentiation (1619). However, the potential for integrin ανβ3 to interact with CCN2 in HTS remains unclear.

Based on the previous findings in the literature, we hypothesized that integrin ανβ3 mediates COL synthesis and HTSF differentiation in HTS. In this study, we investigated whether CCN2 induced HTSF differentiation and COL synthesis. The expression levels of integrin ανβ3 in normal, undamaged skin was compared to that in HTS. An in vitro and in vivo HTS model was used to investigate the effect of integrin ανβ3 on COL synthesis and HTSF differentiation.

Materials and methods

Reagents

Recombinant human CCN2 (rhCCN2) was obtained from PeproTech (London, UK). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibco (Grand Island, NY, USA). Blocking antibody targeting integrin ανβ3 were obtained from Millipore Biotechnology (Temecula, CA, USA). Murine polyclonal antibody against COL type IA2 and goat polyclonal antibody against COL type IIIA1 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Murine monoclonal antibody against α-smooth muscle actin (SMA) and rabbit polyclonal antibody against β-actin were obtained from Boster Biological Technology Co. (Wuhan, China). Cy3-conjugated goat anti-mouse IgG antibodies were purchased from Cwbio (Beijing, China). Inhibitor of activin receptor-like kinase (ALK)5 (the TGF-β type I receptor) was purchased from Selleck (SB431542). Polyvinylidene fluoride (PVDF) membranes and Immobilon Western chemiluminescent horseradish peroxidase (HRP) substrate were purchased from Millipore Co. (Bedford, MA, USA).

Cell culture and treatment

Normal skin and hypertrophic scar tissues were obtained from patients who underwent plastic surgery at the Xijing Hospital (Xi’an, China). Each patient demonstrated extensive areas of HTS, which manifested as raised, erythematous, pruritic and thickened scars restricted to the injury site. The demographic data of the patients enrolled in the present study are shown in Table I. Specimens were obtained from the Human Subjects Committee of the local institution. Dermal portions were minced and incubated in a solution of collagenase type I (0.1 mg/ml) at 37°C for 3 h to separate the fibroblasts. The fibroblasts were pelleted and grown in DMEM supplemented with 10% fetal calf serum (Gibco), 100 U/ml penicillin, and 100 U/ml streptomycin at 37°C in a 5% (v/v) CO2 humidified atmosphere. Cells between the third and sixth sub-passages were used for subsequent experiments.

Table I

Demographic data of patient samples used in the present study.

Table I

Demographic data of patient samples used in the present study.

Patient no.DiagnosisAge (years)GenderDuration (months)Ethnicity
1Hypertrophic scar35Female3Chinese
2Hypertrophic scar40Male10Chinese
3Hypertrophic scar20Male6Chinese
4Hypertrophic scar29Female8Chinese
5Hypertrophic scar41Male5Chinese
6Hypertrophic scar36Male12Chinese

The fibroblasts were seeded in 60-mm culture dishes and grown to 80–90% confluence. Prior to treatment, the cells were cultured in serum-free DMEM for 12 h. The cells were then treated in the absence or presence of integrin ανβ3 antibody (LM609, 10 μg/ml) or ALK5 inhibitor (SB431542, 10 μM) for 1 h. CCN2 (5–80 ng/ml) and/or TGF-β1 (10 ng/ml) were subsequently added to the culture for a time period determined by the specific experiment. The cells of the control group were added to an equal volume of serum-free medium. The culture media and cells were harvested and analyzed at time points indicated in each experiment to measure RNA and protein levels.

Rabbit ear model of hypertrophic scar tissue and treatment

A model for hypertrophic scar in rabbit was used as previously described (7). New Zealand white rabbits were anaesthetized by the intravenous administration of sodium pentobarbital (30 mg/kg). Six 10-mm dermal wounds were created on the ventral surface of each ear down to bare cartilage after removal of the perichondrium. On postoperative day 29 and afterwards, scars were randomly classified into five groups (n=18 scars per group): one control group, one PBS group and three treatment groups. CCN2 (40 ng/ml), integrin ανβ3 antibody (LM609, 10 μg/ml) and CCN2 (40 ng/ml) + integrin ανβ3 antibody (LM609, 10 μg/ml), respectively, were applied once daily to the scars.

RNA isolation and quantitative PCR

Quantitative PCR (qPCR) was performed as previously reported (20). Briefly, total RNA was isolated with the TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Total RNA was reverse transcribed using the PrimeScript RT kit (Takara, Dalian, China) according to the manufacturer’s instructions. qPCR was carried out with the Bio-Rad IQ5 Real-Time System (Bio-Rad, Hercules, CA, USA) using the SYBR® Premix ExTaq™ II kit (Takara). Primer sequences were: α-SMA, forward 5′-GAC AATGGCTCTGGGCTCTGTAA-3′ and reverse 5′-TGTGCT TCGTCACCCACGTA-3′; COL A2, forward 5′-GAGGGCAAC AGCAGGTTCACTTA-3′ and reverse 5′-TCAGCACCACCG ATGTCCA-3′; COL IIIA1, forward 5′-CCACGGAAACAC TGGTGGAC-3′ and reverse 5′-GCCAGCTGCACATCAAG GAC-3′; integrin αν, forward 5′-AGCTGAGCTCATCGTTTC CATTC-3′ and reverse 5′-CCTTCATTGGGTTTCCAA GGTC-3′; integrin β3, forward 5′-GAGGTCATCCCT GGCCTCAA-3′ and reverse 5′-CTGGCAGGCACAGTC ACAATC-3′; CCN2, forward 5′-CTTGCGAAGCTGACC TGGAA-3′ and reverse 5′-AGCTCAAACTTGATAGGCTTG GAGA-3′; GAPDH, forward 5′-GCACCGTCAAGGCTG AGAAC-3′ and reverse 5′-TGGTGAAGACGCCAGTGGA-3′. qPCRs were performed in triplicate. Gene expression levels were normalized against the expression level of the housekeeping gene GAPDH.

Western blot analysis

Western blotting was performed as previously described (21). Briefly, 50 μg protein samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a PVDF membrane. The membranes were blocked with 5% non-fat milk, and incubated with antibodies for murine anti-human COL type IA2 (1:200), goat anti-human COL type IIIA1 (1:200), rabbit anti-human β-catenin (1:700) or murine anti-human α-SMA (1:350) overnight at 4°C. HRP-conjugated goat anti-rabbit IgG (1:3,000), goat anti-mouse IgG (1:3,000) or rabbit anti-goat IgG (1:3,000) were used as secondary antibodies. Proteins were visualized by an enhanced chemiluminescence system using FluorChem FC (Alpha Innotech, San Leandro, CA, USA).

Immunocytochemistry

Immunocytochemistry was performed as previously reported (7). Cell samples were fixed with 90% ethanol and blocked with 3% bovine serum albumin. The samples were incubated with murine anti-α-SMA antibodies at a dilution of 1:100. After being washed with PBS, the samples were incubated with Cy3-conjugated goat anti-murine IgG antibody at a dilution of 1:50. The samples were subsequently stained with 4′,6′-diamidino-2-phenylindole (DAPI). The coverslips were subsequently mounted onto glass slides and viewed with Olympus Box-Type Photofluorography Unit Model FSX100. DAPI-positive and α-SMA-positive cells were counted on the slide at three randomly selected regions. Fluorescence was analyzed by Image-Pro Plus system.

Fibroblast-populated COL lattices contraction and improvement assays

Type I COL was extracted from rat-tail tendons as described by Bell et al (22). The HTSF-populated COL lattices contractility assays were performed in 24-well plates. A 0.5 ml suspension containing 1×105 cells, 1.4 mg/ml COL, CCN2 and integrin ανβ3 antibody (LM609) was added to the triplicate wells and incubated at 37°C for 1 h to allow the mixture to gel. DMEM (1 ml) supplemented with either CCN2 (40 ng/ml), LM609 (10 μg/ml), or both was added and stirred gently. The gels were incubated at 37°C for 24 h, followed by mechanical detachment from the side of the wells. Images were captured at 0 and 48 h after the gels were released. The surface areas of the gels at each time point were measured using Image ProPlus 6.0 software, and were normalized by the surface area and then measured immediately after detachment.

After detachment from the side of the wells at 48 h, fibroblast-populated COL lattices were fixed with 4% paraformaldehyde in PBS overnight. The sections were stained with hematoxylin and eosin (H&E) and analyzed using the framework of FPCL by FSX100.

Masson’s trichrome staining for COL fibers

Masson’s trichrome staining analysis of COL fibers was performed as previously described (7). Briefly, the samples were rehydrated through gradient ethanol immersion. The tissues were subsequently stained by placing them in Weigert’s iron hematoxylin working solution. The slides were then stained in Biebrich scarlet-acid fuchsin solution, and placed in 1% phosphomolybdic-phosphotungstic acid solution. The sections were directly transferred into aniline blue solution and rinsed in 1% acetic acid solution. The tissue samples were then dehydrated, cleared, and mounted. A bluestain indicated the presence of COL fibers. The histologic characteristics and the COL fibers in the samples were observed under FSX100.

Statistical analysis

Results are presented as mean ± standard error of three independent experiments. Statistical analysis was performed by analysis of variance (ANOVA). The paired-samples t-test was used for comparison of gene expression levels between scar and normal skin samples. P<0.05 was considered to indicate a statistically significant result.

Results

Expression levels of CCN2, COL IA2 and COL IIIA1 mRNA in normal skin and HTS

As an initial experiment, we compared the transcript levels of CCN2, COL IA2 and COL IIIA1 between normal and HTS skin in vivo. Total RNA was extracted from the scar and normal skin tissues derived from six patients with HTS, and qPCR was performed. The relative transcript levels of CCN2, COL IA2 and COL IIIA1 were significantly higher in HTS skin than those in the control skin (Fig. 1).

Expression levels of CCN2, COL IA2 and COL IIIA1 mRNA in cultured normal and HTSF

Cultured human dermal fibroblasts obtained from normal and HTS skin were incubated under the same conditions, and their mRNA expression was analyzed. The expression of CCN2, COL IA2 and COL IIIA1 mRNA was upregulated in HTSF (Fig. 2), which was consistent with the in vivo results.

Expression levels of integrin αν and β3 mRNA in normal skin and HTS

Subsequently, we compared the transcript levels of integrin αν and β3 between normal and HTS skin in vivo. Total RNA was extracted from the scar and normal skin tissues derived from six patients with HTS, and qPCR was performed. The relative transcript levels of integrin αν and β3 were significantly lower in the normal skin samples. By contrast, the integrin αν and β3 expression levels were significantly higher in the HTS skin than those in the control skin (Fig. 3).

Expression levels of integrin αν and β3 mRNA in cultured normal and HTSF

Cultured human dermal fibroblasts obtained from normal and HTS skin were incubated under the same conditions, and their mRNA expression was analyzed. The expression of integrin αν and β3 mRNA was also upregulated in HTSF (Fig. 4), which was consistent with the in vivo results.

CCN2 induces HTSF differentiation and COL synthesis

Myofibroblasts play a key role in the wound-healing process, promoting wound closure and matrix deposition. The differentiation of fibroblasts into myofibroblasts can be identified by the expression of α-SMA, which is organized into stress fibers. The regulation of myofibroblast differentiation is crucial because an excessive number of myofibroblasts is associated with increased pathologic scar formation (23). CCN2 has an established role in regulating the extracellular matrix (ECM). Then, we evaluated the effects of CCN2 on the differentiation and COL synthesis of HTSF. The mRNA levels of α-SMA, COL IIIA1 and COL IA2 increased gradually after rCCN2 stimulation in a dose-dependent manner and peaked with 40 ng/ml (Figs. 5 and 6). Similarly, the protein levels of α-SMA, COL IA2 and COL IIIA1 increased after rCCN2 stimulation and peaked with 40 ng/ml.

CCN2 increases integrin αν and β3 mRNA expression in HTSF

To determine whether CCN2 altered the cellular surface expression of integrin αν and β3 on HTSF, HTSFs were treated with CCN2 and integrin αν or β3 mRNA expression was measured. The cells were treated with 5–80 ng/ml CCN2 for 24 h. CCN2 gradually increased the transcription of integrin αν and β3 mRNA, which peaked at CCN2 (40 ng/ml) (Fig. 7).

Functional blockade of integrin ανβ3 blocks CCN2-induced HTSF differentiation and COL synthesis

To investigate whether integrin ανβ3 was required for CCN2-induced α-SMA and COL I expression, HTSFs were treated with or without LM609. α-SMA, COL IA2 mRNA and protein expression was then measured. Treatment with LM609 significantly inhibited α-SMA and COL IA2 mRNA expression (Fig. 8). Similarly, the expression of α-SMA COL IA2 protein was significantly decreased in the presence of LM609 (Fig. 9). Immunocytochemistry also showed that the expression of α-SMA by CCN2 was significantly decreased after the presence of LM609 (Fig. 10).

HTSF is controlled by blockage of the intergrin receptor following TGF-β1 stimulation

PCR and WB for α-SMA, COL I and COL III in HTSFs treated with different receptor inhibitors after TGF-β1 stimulation were conducted. Compared with the control, TGF-β1 markedly stimulated α-SMA COL I and COL III expression, while treatment with the ALK5 (SB-431542) and ανβ3 integrin-specific (LM609) inhibitor suppressed the TGF-β1-induced expression of α-SMA COL I and COL III. Moreover, SB431542 significantly inhibited the expression of α-SMA COL I and COL III compared with LM609 (Figs. 11 and 12).

LM609-mediated blockade of CCN2 inhibited the contractility of HTSF

To determine whether the CCN2-induced changes in α-SMA protein and their inhibition by LM609 had functional consequences, the fibroblast-populated COL lattices (FPCLs) contraction assay was performed. As a model of a three-dimensional wound environment, HTSFs were embedded in COL matrices attached to the culture plate to generate FPCL in the presence of CCN2. HTSFs in mechanically stretched microenvironments have their cytoskeletons remodeled and exhibit a contractive phenotype (20). In our study model, CCN2 treatment (40 ng/ml) induced a significant (p<0.05) increase in contraction compared to the control untreated cells after 48 h. When LM609 was added to the gels at the same time as CCN2, CCN2-induced gel contraction was blocked (Fig. 13). To evaluate the whether the change of the contractility was associated with α-SMA expression, the total RNA of the HTSF in the COL lattices was extracted and qPCR was conducted. Similar to the results above, the fibroblasts cultured in the three-dimensional environment showed an increased expression of α-SMA mRNA after CCN2 stimulation, which was inhibited by LM609 (Fig. 13C). Additionally, H&E staining results showed the architecture of FPCL. Of note, in the CCN2 + LM609 treatment groups, the architecture of FPCL was significantly improved compared with the CCN2 activation group (Fig. 14).

Effects of CCN2 and ανβ3 integrin-specific inhibitor on HTS in the rabbit ear scar model

On day 56 post-wounding, significant hypertrophic scarring was evident in the control, PBS and CCN2 groups. Overproduction of COL is a major characteristic of HTSs. Thus, Masson’s trichrome staining of scar tissue was performed on day 56 post-wounding. Light microscopy revealed more features typical of COL fibers in scar tissue (Fig. 15B–D) in the control group compared with unwounded dermal tissue (Fig. 15A). The COL bundles were more abundant, denser, thicker and disorganized. By contrast, the COL fibers in the groups treated with CCN2 + LM609 and LM609 were fewer, thinner and more regularly arranged (Fig. 15E and F).

Discussion

Hypertrophic scarring is a common complication of wound healing. It is associated with an abnormal proliferation of fibroblasts and an overproduction of COL and other ECM elements. Fibroblast proliferation and matrix synthesis induced by growth factors have been considered to be involved in initiating and maintaining fibrosis (24). Although the underlying mechanisms involved are complex, different effects of CCN2 mediate many harmful aspects of hypertrophic scarring.

CCN2 has been involved in many fibrotic disorders of the lung, liver, kidney and pancreas. Treatment with antisense oligonucleotides or antibodies to CCN2 in cell culture or animal models decreased ECM synthesis and reduced scarring (13,2527). Moreover, CCN2 mediates the fibroproliferative effects of TGF-β1. Levels of CCN2 are correlated with an increased expression of ECM, such as COL I, integrins and fibronectin. Therefore, it is important to define the signaling pathway through CCN2 (28). Constitutive CCN2 expression is a hallmark of fibrosis (29). In adults, CCN2 is induced during tissue repair (30). Simultaneous co-injection of CCN2 and TGF-β causes sustained fibrotic responses in vivo, in contrast to the application of TGF-β alone, which causes only a transient fibrotic response that depends on the constant injection of ligand (31). In general, the biological effect of cytokines is mainly determined by the occurrence of cytokine-receptor interaction, which is modulated by the concentration and activity of cytokines and/or their receptors. For example, in scleroderma fibroblasts, the upregulated expression of TGF-β receptors contributes to the increased biological effect of autocrine TGF-β, resulting in excessive ECM deposition (32). Integrin ανβ3 plays an important role in a variety of physiological and patho-physiological processes, including tumor angiogenesis, rheumatoid arthritis and a number of inflammatory and repair processes (33). Integrin ανβ3 has been shown to bind CCN2 proteins. Previously, it was shown that integrin ανβ3 function as co-receptors with heparan sulfate proteoglycans (HSPGs) for CCN2-mediated hepatic stellate cell adhesion (34).

In the present study, we found that the mRNA levels of integrin αν and β3 increased in HTS. In HTSF, the expression of integrin αν and β3 mRNA was also significantly increased (Fig. 3). CCN2 stimulation of HTSF resulted in upregulation of the cell expression of α-SMA, COL IA2, and COL IIIA1, which are important components of the ECM in skin tissue (Figs. 5 and 6). In addition, we detected increased expression levels of integrin αν and β3 subunits during the transition (Fig. 7).

Integrin αν is involved in differentiation in a variety of cell types, including osteoclasts, oligodendrocytes, keratinocytes and myoblasts (3538). In scleroderma fibroblasts, integrin ανβ5 is upregulated and was shown to be capable of interacting with TGF-β receptors. Furthermore, the blockade of integrin ανβ5 reversed the myofibroblastic phenotype in scleroderma fibroblasts (39). In our experiments, treatment of HTSF cells with the ανβ3 integrin-specific inhibitor (LM609) significantly reduced the cell expression of α-SMA and COL IA2, while immunocytochemistry demonstrated that LM609 led to suppression of the CCN2-induced expression of α-SMA (Figs. 810).

HTSF can be controlled by blockage of the integrin receptor following TGF-β1 stimulation because it has been demonstrated that an increased TGF-β1 occurs in HTS in vitro and in vivo. To confirm this, we detected the expression level of α-SMA COL I and COL III in HTS fibroblasts treated with different receptor inhibitors following TGF-β1 stimulation. The data showed that compared with the control, TGF-β1 markedly stimulated α-SMA COL I and COL III expression, while the treatment with ALK5 inhibitor (SB431542) and ανβ3 integrin-specific inhibitor (LM609) suppressed the TGF-β1-induced expression of α-SMA, COL I and COL III. Moreover, SB431542 significantly inhibited the expression of α-SMA COL I and COL III compared with LM609 (Figs. 11 and 12).

To validate our hypothesis, fibroblast-populated COL lattices contraction assays and a rabbit ear HTS model was established, and the role of integrin ανβ3 was investigated. In the FPCLs model, the integrin ανβ3-specific inhibitor (LM609) blocked the CCN2-induced increase in contractility of the HTSFs, suggesting that LM609 may negatively regulate the contractility of HTSFs. Furthermore, the fibroblasts cultured in the three-dimensional environment showed an increased expression of α-SMA mRNA after CCN2 stimulation, which was inhibited by LM609. Additionally, the H&E staining results showed that the architecture of FPCL in the CCN2 + LM609 treatment groups was significantly improved compared with that of the CCN2 activation group (Figs. 13 and 14). In a rabbit ear HTS model, Masson staining revealed that the COL bundles were more abundant, denser, thicker, and disorganized in the control group. By contrast, there were fewer, thinner, and more organized COL fibers in the groups treated with CCN2 + LM609 and LM609 (Fig. 15). The results of this study suggest that ανβ3 integrin inhibition was able to significantly inhibit post-injury scarring of the model rabbit ears. These findings suggest that integrin ανβ3 is a key receptor for modulating the signals by which CCN2 promotes α-SMA and collagen expression in HTSF.

In summary, we have demonstrated that the upregulated expression of integrin αν and β3 induce myofibroblastic differentiation of HTSF. This finding may provide insights into the regulatory mechanisms of fibrotic disorders, such as HTS.

Acknowledgements

This study was supported by the Grant-in-Aid for scientific research from the Xijing hospital assist (XJZT13M16), and the National Natural Science Foundation of China (grant no. 81171811).

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November-2014
Volume 34 Issue 5

Print ISSN: 1107-3756
Online ISSN:1791-244X

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Copy and paste a formatted citation
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Spandidos Publications style
Hu X, Li N, Tao K, Fang X, Liu J, Wang Y, Wang H, Shi J, Wang Y, Ji P, Ji P, et al: Effects of integrin ανβ3 on differentiation and collagen synthesis induced by connective tissue growth factor in human hypertrophic scar fibroblasts. Int J Mol Med 34: 1323-1334, 2014
APA
Hu, X., Li, N., Tao, K., Fang, X., Liu, J., Wang, Y. ... Hu, D. (2014). Effects of integrin ανβ3 on differentiation and collagen synthesis induced by connective tissue growth factor in human hypertrophic scar fibroblasts. International Journal of Molecular Medicine, 34, 1323-1334. https://doi.org/10.3892/ijmm.2014.1912
MLA
Hu, X., Li, N., Tao, K., Fang, X., Liu, J., Wang, Y., Wang, H., Shi, J., Wang, Y., Ji, P., Cai, W., Bai, X., Zhu, X., Han, J., Hu, D."Effects of integrin ανβ3 on differentiation and collagen synthesis induced by connective tissue growth factor in human hypertrophic scar fibroblasts". International Journal of Molecular Medicine 34.5 (2014): 1323-1334.
Chicago
Hu, X., Li, N., Tao, K., Fang, X., Liu, J., Wang, Y., Wang, H., Shi, J., Wang, Y., Ji, P., Cai, W., Bai, X., Zhu, X., Han, J., Hu, D."Effects of integrin ανβ3 on differentiation and collagen synthesis induced by connective tissue growth factor in human hypertrophic scar fibroblasts". International Journal of Molecular Medicine 34, no. 5 (2014): 1323-1334. https://doi.org/10.3892/ijmm.2014.1912