Shangxi Liu

CCN2 is required for bleomycin‐induced skin fibrosis in mice

OBJECTIVE No therapy for fibrotic disease is available. The proadhesive matricellular protein connective tissue growth factor CCN2 is a marker of fibrotic cells; however, the specific role of CCN2 in connective tissue biology in general and in fibrogenesis in particular is unclear. The aim of this study was to assess whether adult mice bearing a smooth muscle cell/fibroblast-specific deletion of CCN2 are resistant to bleomycin-induced skin scleroderma. METHODS Cutaneous fibrosis was induced in mice by subcutaneous injection of bleomycin. Untreated control groups were injected with phosphate buffered saline. Mice bearing a fibroblast/smooth muscle cell-specific deletion of CCN2 were investigated for changes in dermal thickness, collagen content, and the number of α-smooth muscle actin (α-SMA)-positive cells. Dermal fibroblasts were isolated to assess whether the induction of collagen and α-SMA messenger RNA in response to transforming growth factor β (TGFβ) was impaired. RESULTS The loss of CCN2 resulted in resistance to bleomycin-induced skin fibrosis. In response to bleomycin, wild-type mice possessed, but CCN2-deficient mice lacked, abundant α-SMA-expressing myofibroblasts within fibrotic lesions. Fibroblast responses to TGFβ, a potent inducer of myofibroblast differentiation, were not affected. Collectively, these results indicate that CCN2 is essential for bleomycin-induced skin fibrosis, likely due to a defect in myofibroblast recruitment. CONCLUSION These data indicate that therapeutic strategies that involve blocking CCN2 in vivo may be of benefit in combating fibrotic skin disease.

Expression of integrin β1 by fibroblasts is required for tissue repair in vivo

In tissue repair, fibroblasts migrate into the wound to produce and remodel extracellular matrix (ECM). Integrins are believed to be crucial for tissue repair, but their tissue-specific role in this process is poorly understood. Here, we show that mice containing a fibroblast-specific deletion of integrin β1 exhibit delayed cutaneous wound closure and less granulation tissue formation, including reduced production of new ECM and reduced expression of α-smooth muscle actin (α-SMA). Integrin-β1-deficient fibroblasts showed reduced expression of type I collagen and connective tissue growth factor, and failed to differentiate into myofibroblasts as a result of reduced α-SMA stress fiber formation. Loss of integrin β1 in adult fibroblasts reduced their ability to adhere to, to spread on and to contract ECM. Within stressed collagen matrices, integrin-β1-deficient fibroblasts showed reduced activation of latent TGFβ. Addition of active TGFβ alleviated the phenotype of integrin-β1-deficient mice. Thus integrin β1 is essential for normal wound healing, where it acts, at least in part, through a TGFβ-dependent mechanism in vivo.

Inhibition of focal adhesion kinase prevents experimental lung fibrosis and myofibroblast formation

OBJECTIVE Enhanced adhesive signaling, including activation of focal adhesion kinase (FAK), is a hallmark of fibroblasts from lung fibrosis patients, and FAK has therefore been hypothesized to be a key mediator of this disease. This study was undertaken to characterize the contribution of FAK to the development of pulmonary fibrosis both in vivo and in vitro. METHODS FAK expression and activity were analyzed in lung tissue samples from lung fibrosis patients by immunohistochemistry. Mice orally treated with the FAK inhibitor PF-562,271, or with small interfering RNA (siRNA)-mediated silencing of FAK were exposed to intratracheally instilled bleomycin to induce lung fibrosis, and lungs were harvested for histologic and biochemical analysis. Using endothelin 1 (ET-1) as a stimulus, cell adhesion and contraction, as well as profibrotic gene expression, were studied in fibroblasts isolated from wild-type and FAK-deficient mouse embryos. ET-1-mediated FAK activation and gene expression were studied in primary mouse lung fibroblasts, as well as in wild-type and β1 integrin-deficient mouse fibroblasts. RESULTS FAK expression and activity were up-regulated in fibroblast foci and remodeled vessels from lung fibrosis patients. Pharmacologic or siRNA-mediated targeting of FAK resulted in marked abrogation of bleomycin-induced lung fibrosis in mice. Loss of FAK impaired the acquisition of a profibrotic phenotype in response to ET-1. Profibrotic gene expression leading to myofibroblast differentiation required cell adhesion, and was driven by JNK activation through β1 integrin/FAK signaling. CONCLUSION These results implicate FAK as a central mediator of fibrogenesis, and highlight this kinase as a potential therapeutic target in fibrotic diseases.

Loss of peroxisome proliferator–activated receptor γ in mouse fibroblasts results in increased susceptibility to bleomycin-induced skin fibrosis

OBJECTIVE There is increasing evidence that the transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) plays an important role in controlling cell differentiation, and that PPARgamma ligands can modify inflammatory and fibrotic responses. The aim of the present study was to examine the role of PPARgamma in a mouse model of skin scleroderma, in which mice bearing a fibroblast-specific deletion of PPARgamma were used. METHODS Cutaneous sclerosis was induced by subcutaneous injection of bleomycin, while untreated control groups were injected with phosphate buffered saline. Mice bearing a fibroblast-specific deletion of PPARgamma were investigated for changes in dermal thickness, inflammation, collagen content, and the number of alpha-smooth muscle actin-positive cells. The quantity of the collagen-specific amino acid hydroxyproline was also measured. In addition, the effect of PPARgamma deletion on transforming growth factor beta1 (TGFbeta1) signaling in the fibroblasts was investigated. RESULTS Bleomycin treatment induced marked cutaneous thickening and fibrosis in all treated mice. Deletion of PPARgamma resulted in enhanced susceptibility to bleomycin-induced skin fibrosis, as indicated by increases in all measures of skin fibrosis and enhanced sensitivity of fibroblasts to TGFbeta1 in PPAR-deficient mice. CONCLUSION These results indicate that PPARgamma suppresses fibrogenesis. Specific agonists of PPARgamma may therefore alleviate the extent of the development of cutaneous sclerosis.

Loss of β1 Integrin in Mouse Fibroblasts Results in Resistance to Skin Scleroderma in a Mouse Model

OBJECTIVE Activated adhesive signaling is a hallmark of fibroblasts isolated from the scars of scleroderma (systemic sclerosis) lesions. Beta-1 integrin plays a key role in adhesive signaling. The aim of the present study was to examine the role of beta1 integrin in a mouse model of skin scleroderma using mice bearing a fibroblast-specific deletion of beta1 integrin. METHODS Cutaneous sclerosis was induced by subcutaneous injection of bleomycin. Control groups were treated with phosphate buffered saline. Mice bearing a fibroblast-specific deletion of beta1 integrin and control mice were investigated. Dermal thickness, collagen production, and the number of alpha-smooth muscle actin-positive cells were determined. The quantity of the collagen-specific amino acid hydroxyproline was also measured. RESULTS Bleomycin treatment induced marked cutaneous thickening and fibrosis in control mice. Conversely, the deletion of beta1 integrin resulted in resistance to bleomycin-induced fibrosis. CONCLUSION Expression of beta1 integrin by fibroblasts is required for fibrogenesis. Inhibition of beta1 integrin may be a viable method to alleviate the development of cutaneous sclerosis.

GSK-3β in mouse fibroblasts controls wound healing and fibrosis through an endothelin-1–dependent mechanism

Glycogen synthase kinase-3 (GSK-3) is a widely expressed and highly conserved serine/threonine protein kinase encoded by 2 genes, GSK3A and GSK3B. GSK-3 is thought to be involved in tissue repair and fibrogenesis, but its role in these processes is currently unknown. To investigate the function of GSK-3beta in fibroblasts, we generated mice harboring a fibroblast-specific deletion of Gsk3b and evaluated their wound-healing and fibrogenic responses. We have shown that Gsk3b-conditional-KO mice (Gsk3b-CKO mice) exhibited accelerated wound closure, increased fibrogenesis, and excessive scarring compared with control mice. In addition, Gsk3b-CKO mice showed elevated collagen production, decreased cell apoptosis, elevated levels of profibrotic alpha-SMA, and increased myofibroblast formation during wound healing. In cultured Gsk3b-CKO fibroblasts, adhesion, spreading, migration, and contraction were enhanced. Both Gsk3b-CKO mice and fibroblasts showed elevated expression and production of endothelin-1 (ET-1) compared with control mice and cells. Antagonizing ET-1 reversed the phenotype of Gsk3b-CKO fibroblasts and mice. Thus, GSK-3beta appears to control the progression of wound healing and fibrosis by modulating ET-1 levels. These results suggest that targeting the GSK-3beta pathway or ET-1 may be of benefit in controlling tissue repair and fibrogenic responses in vivo.

Focal Adhesion Kinase/Src Suppresses Early Chondrogenesis CENTRAL ROLE OF CCN2

Adhesive signaling plays a key role in cellular differentiation, including in chondrogenesis. Herein, we probe the contribution to early chondrogenesis of two key modulators of adhesion, namely focal adhesion kinase (FAK)/Src and CCN2 (connective tissue growth factor, CTGF). We use the micromass model of chondrogenesis to show that FAK/Src signaling, which mediates cell/matrix attachment, suppresses early chondrogenesis, including the induction of Ccn2, Agc, and Sox6. The FAK/Src inhibitor PP2 elevates Ccn2, Agc, and Sox6 expression in wild-type mesenchymal cells in micromass culture, but not in cells lacking CCN2. Our results suggest a reduction in FAK/Src signaling is a critical feature permitting chondrogenic differentiation and that CCN2 operates downstream of this loss to promote chondrogenesis.

Rac1 Expression by Fibroblasts Is Required for Tissue Repair in Vivo

Tissue repair requires that fibroblasts migrate into the wound to produce and remodel extracellular matrix, a process that requires adhesion. Failure to suppress the tissue repair program results in fibrotic disorders that are characterized by excessive adhesive signaling. The role of specific components of adhesive signaling in fibrogenic responses is unclear, but may involve small GTPases such as Rac1. To address the functions of Rac1 in fibroblasts, we generated mice containing a fibroblast-specific deletion of Rac1. These mice show delayed cutaneous wound closure, including reduced collagen production and myofibroblast formation. In cultured Rac1-deficient fibroblasts, adhesion, spreading, and migration were significantly inhibited. Rac1-deficient fibroblasts possessed impaired myofibroblast formation and function as visualized by reduced alpha-smooth muscle actin expression as well as matrix contraction. Both in vivo and in vitro, Rac1- deficient fibroblasts showed a reduced generation of reactive oxygen species; in vitro, hydrogen peroxide alleviated the phenotype of Rac1-deficient fibroblasts. Thus, Rac1 is an essential signaling integrator that is required for normal wound healing and dermal homeostasis.

Connective Tissue Growth Factor Promoter Activity in Normal and Wounded Skin

In skin, connective tissue growth factor (CTGF/CCN2) is induced during tissue repair. However, what the exact cell types are that express CTGF in normal and wounded skin remain controversial. In this report, we use transgenic knock-in mice in which the Pacific jellyfish Aequorea victoria enhanced green fluorescent protein (E-GFP) gene has been inserted between the endogenous CTGF promoter and gene. Unwounded (day 0) and wounded (days 3 and 7) skin was examined for GFP to detect cells in which the CTGF promoter was active, α-smooth muscle actin (α-SMA) to detect myofibroblasts, and NG2 expression to detect pericytes. In unwounded mice, CTGF expression was absent in epidermis and was present in a few cells in the dermis. Upon wounding, CTGF expression was induced in the dermis. Double immunolabeling revealed that CTGF-expressing cells also expressed α-SMA, indicating the CTGF was expressed in myofibroblasts. A subset (~30%) of myofibroblasts were also NG2 positive, indicating that pericytes significantly contributed to the number of myofibroblasts in the wound. Pericytes also expressed CTGF. Collectively, these results indicate that CTGF expression in skin correlates with myofibroblast induction, and that CTGF-expressing pericytes are significant contributors to myofibroblast activity during cutaneous tissue repair.

Connective tissue growth factor is induced in bleomycin-induced skin scleroderma

The origin of fibrotic cells within connective tissue is unclear. For example, the extent to which microvascular pericytes contribute to the number of myofibroblasts present in dermal fibrosis in uncertain. Connective tissue growth factor (CTGF/CCN2) is a marker and mediator of fibrosis. In this report, we use an antibody recognizing CCN2 to assess the cell types in mouse dermis which express CCN2 in the bleomycin model of skin scleroderma. Control (PBS injected) and fibrotic (bleomycin-injected) dermis was examined for CCN2, α-smooth muscle actin (α-SMA) (to detect myofibroblasts), and NG2 (to detect pericytes) expression. Consistent with previously published data, CCN2 expression was largely absent in the dermis of control mice. However, upon exposure to bleomycin, CCN2 was observed in the dermis. Cells that expressed CCN2 were α−SMA-expressing myofibroblasts. Approximately 85% of myofibroblasts were NG2-positive, CCN2-expressing pericytes, indicating that pericytes significantly contributed to the presence of myofibroblasts in sclerotic dermis. Thus CCN2 is induced in fibrotic skin, correlating with the induction of myofibroblast induction. Moreover, CCN2-expressing pericytes significantly contribute to the appearance of myofibroblasts in bleomycin-induced skin scleroderma.