Suppression of the production of extracellular matrix and α-smooth muscle actin induced by transforming growth factor-β1 in fibroblasts of the flexor tendon sheath by hepatocyte growth factor

We examined the effectiveness of hepatocyte growth factor (HGF) in blocking production of transforming growth factor (TGF)-β1-induced collagen I, fibronectin, and α-smooth muscle actin (α-SMA) in the flexor tendon sheath of rabbits in vitro. Fibroblasts were obtained from the sheaths. Cell culture was supplemented with TGF-β1 5 ng/ml and increasing doses of HGF (10-40 ng/ml). The production of collagen I and fibronectin in supernatants culture were examined using an enzyme-linked immunosorbent assay (ELISA). α-SMA expression was assessed by western blot. TGF-β1 stimulated production of collagen I, fibronectin, and α-SMA greatly, while HGF significantly (p<0.05) reduced production of all components induced by TGF-β1 in a dose-dependent manner. This suggests that HGF effectively antagonises the action of TGF-β1 in cultured fibroblasts from flexor tendon sheaths. The results provide a cellular and molecular basis for HGF acting as a therapeutic agent for adhesions in flexor tendons.     

HGF拮抗TGF-β1诱导腱鞘成纤维细胞α-SMA及细胞外基质合成

目的 研究HGF能否阻抑TGF-β1诱导的腱鞘成纤维细胞α-SMA及细胞外基质过度合成.方法 选取成年新西兰大白兔7只,体重3.75～4.00 kg,无菌切取前肢中趾趾深屈肌腱,进行腱鞘成纤维细胞的分离和培养,待细胞生长成单层后,以胰蛋白酶消化传代.取第3代成纤维细胞用于实验,当细胞达到70%融合时,培养液中加入TGF-β1(5 ng/ml)及HGF(10～40 ng/ml).培养72 h后,利用Westernblot检测α-SMA表达;ELISA测定细胞Ⅰ型胶原及纤维结合素的表达.结果 TGF-β1能显著诱导α-SMA表达,半定量分析提示,TGF-β1作用后的成纤维细胞α-SMA表达量是对照组的1.8倍.随HGF的同时加入,α-SMA的表达则明显受抑制(P＜0.05),且随HGF浓度的升高其阻抑作用呈逐渐增强趋势.TGF-β1同样能诱导Ⅰ型胶原及纤维结合素的表达(P＜0.01),而HGF则可以有效地阻抑其表达,其效应呈剂量依赖性(P＜0.05).结论 HGF可以有效阻抑TGF-β1诱导的腱鞘成纤维细胞α-SMA、Ⅰ型胶原及纤维结合素的表达,这为利用HGF预防和治疗屈指肌腱损伤后粘连及瘢痕在细胞和分子水平提供了依据.     

Flexor tendon healing in vitro: effects of TGF-beta on tendon cell collagen production

Flexor tendon healing is complicated by adhesions to the surrounding sheath. Transforming growth factor beta (TGF-beta) is a cytokine with numerous activities related to wound healing. We examined the effects of TGF-beta-1, -2 and -3 on tendon cell proliferation and collagen production. Three separate cell lines--sheath fibroblasts, epitenon and endotenon tenocytes--were isolated from rabbit flexor tendons and cultured separately. Cell culture media was supplemented with 1 or 5 ng/mL of TGF-beta-1, -2, or -3. Cell number and collagen I and III production were measured and compared with unsupplemented control cultures. The addition of TGF-beta to cell culture media resulted in a decrease in cell number in all 3 lines that did not reach statistical significance. There was a significant increase (p <.05) in collagen I and III production with the addition of all 3 TGF-beta isoforms. Modulation of TGF-beta production may provide a mechanism to modulate adhesion formation clinically.     

Inhibition of TGF-beta-induced collagen production in rabbit flexor tendons

PURPOSE: Postoperative adhesions frequently compromise the success of flexor tendon repair. Manipulation of growth factors responsible for scar formation may be a method of decreasing adhesion formation. Transforming growth factor beta (TGF-beta) is a key cytokine in the pathogenesis of tissue fibrosis. The purpose of this study was to examine the effectiveness of TGF-beta neutralizing antibody in blocking TGF-beta-induced collagen I production in rabbit flexor tendons in vitro. METHODS: Sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were obtained from rabbit flexor tendons. Each cell culture was supplemented with 1 ng/mL of TGF-beta along with increasing doses of TGF-beta neutralizing antibody (0.1-2.0 microg/mL). Collagen I production was measured by enzyme-linked immunoabsorbent assay and TGF-beta bioactivity was measured by the luciferase assay. Results were compared with TGF-beta alone and unsupplemented controls. RESULTS: The addition of neutralizing antibody significantly reduced TGF-beta-induced collagen I production in a dose-dependent manner in all 3 cell cultures. TGF-beta bioactivity was also reduced by its neutralizing antibody. CONCLUSIONS: This study shows that TGF-beta inhibition through its neutralizing antibody was effective in cultured flexor tendon cells. The results encourage further experiments that use such agents to modulate flexor tendon wound healing in in vivo models in the hope of eventually blocking the effect of TGF-beta on flexor tendons clinically.     

Phenotypic differences between oral and skin fibroblasts in wound contraction and growth factor expression

Wounds of the oral mucosa heal in an accelerated fashion with reduced scarring compared with cutaneous wounds. The differences in healing outcome between oral mucosa and skin could be because of phenotypic differences between the respective fibroblast populations. This study compared paired mucosal and dermal fibroblasts in terms of collagen gel contraction, alpha-smooth muscle actin expression (alpha-SMA), and production of the epithelial growth factors: keratinocyte growth factor (KGF) and hepatocyte growth factor/scatter factor (HGF). The effects of transforming growth factor -beta1 and -beta3 on each parameter were also determined. Gel contraction in floating collagen lattices was determined over a 7-day period. alpha-SMA expression by fibroblasts was determined by Western blotting. KGF and HGF expression were determined by an enzyme-linked immunosorbent assay. Oral fibroblasts induced accelerated collagen gel contraction, yet surprisingly expressed lower levels of alpha-SMA. Oral cells also produced significantly greater levels of both KGF and HGF than their dermal counterparts. Transforming growth factor-beta1 and -beta3, over the concentration range of 0.1-10 ng/mL, had similar effects on cell function, stimulating both gel contraction and alpha-SMA production, but inhibiting KGF and HGF production by both cell types. These data indicate phenotypic differences between oral and dermal fibroblasts that may well contribute to the differences in healing outcome between these two tissues.     

Transforming growth factor-beta regulates tubular epithelial-myofibroblast transdifferentiation in vitro.

BACKGROUND: We recently found evidence of tubular epithelial-myofibroblast transdifferentiation (TEMT) during the development of tubulointerstitial fibrosis in the rat remnant kidney. This study investigated the mechanisms that induce TEMT in vitro. METHODS: The normal rat kidney tubular epithelial cell line (NRK52E) was cultured for six days on plastic or collagen type I-coated plates in the presence or absence of recombinant transforming growth factor-beta1 (TGF-beta1). Transdifferentiation of tubular cells into myofibroblasts was assessed by electron microscopy and by expression of alpha-smooth muscle actin (alpha-SMA) and E-cadherin. RESULTS: NRK52E cells cultured on plastic or collagen-coated plates showed a classic cobblestone morphology. Culture in 1 ng/ml TGF-beta caused only very minor changes in morphology, but culture in 10 or 50 ng/ml TGF-beta1 caused profound changes. This involved hypertrophy, a loss of apical-basal polarity and microvilli, with cells becoming elongated and invasive, the formation of a new front-end back-end polarity, and the appearance of actin microfilaments and dense bodies. These morphological changes were accompanied by phenotypic changes. Double immunohistochemistry staining showed that the addition of TGF-beta1 to confluent cell cultures caused a loss of the epithelial marker E-cadherin and de novo expression of alpha-SMA. An intermediate stage in transdifferentiation could be seen with hypertrophic cells expressing both E-cadherin and alpha-SMA. De novo alpha-SMA expression was confirmed by Northern blotting, Western blotting, and flow cytometry. In particular, cells with a transformed morphology showed strong alpha-SMA immunostaining of characteristic microfilament structures along the cell axis. There was a dose-dependent increase in the percentage of cells expressing alpha-SMA with increasing concentrations of TGF-beta1, which was completely inhibited by the addition of a neutralizing anti-TGF-beta1 antibody. Compared with growth on plastic, cell culture on collagen-coated plates showed a threefold increase in the percentage of cells expressing alpha-SMA in response to TGF-beta1. CONCLUSION: TGF-beta1 is a key mediator that regulates, in a dose-dependent fashion, transdifferentiation of tubular epithelial cells into alpha-SMA+ myofibroblasts. This transdifferentiation is markedly enhanced by growth on collagen type I. These findings have identified a novel pathway that may contribute to renal fibrosis associated with overexpression of TGF-beta1 within the diseased kidney.     

Hepatocyte growth factor (HGF) modulates matrix turnover in human glomeruli

BACKGROUND: The imbalance between synthesis and degradation of mesangial matrix causes glomerulosclerosis and leads to renal failure. Hepatocyte growth factor (HGF) has been shown to reduce the progression in murine models of chronic renal failure. The present study evaluated the effect of HGF on the extracellular matrix turnover and on c-met receptor in human glomeruli. METHODS: Human glomeruli microdissected from donor kidney biopsies before transplantation were incubated with culture media containing HGF (50 ng/mL). After 24 and 48 hours, the expression of c-met, (alpha2) IV collagen, transforming growth factor-beta (TGF-beta), metalloprotease (MMP) 2 and 9 and of the inhibitor of MMP-2, tissue inhibitors of metalloprotease-1 (TIMP-1), was evaluated by polymerase chain reaction (PCR). beta-actin was used as housekeeping gene. The production of collagen type IV and TGF-beta was evaluated by enzyme-linked immunosorbent assay (ELISA) and Western blotting and the activity of MMP by zymography. RESULTS: (alpha2) IV collagen, TGF-beta, and TIMP-1 mRNA levels were markedly decreased in glomeruli treated with HGF at 24 and 48 hours. The expression of c-met was up-regulated by HGF treatment. HGF reduced the production of collagen type IV and TGF-beta. MMP-2 but not MMP-9 mRNA level was increased in HGF-treated glomeruli, although the gelatinolytic activity of the supernatant was not changed. By light microscopic examination kidney biopsies neither showed glomerular hypercellularity nor mesangial expansion. CONCLUSION: HGF reduced expression and synthesis of TGF-beta and collagen type IV and increased MMP-2 mRNA level in normal human glomeruli. These results suggest an antifibrotic effect of HGF on glomerular cells and may explain its beneficial role in glomerulosclerosis.     

Role of hepatocyte growth factor in invasion of prostate cancer cell lines through tumor-stromal interaction

We examined how prostate stromal cell-derived hepatocyte growth factor (HGF) affects invasion of prostate cancer cells through tumor-stromal interaction. The effects of HGF, various growth factors [transforming growth factor (TGF)-alpha, TGF-beta 1, basic fibroblast growth factor, keratinocyte growth factor, and platelet-derived growth factor], and conditioned medium (CM) from prostate stromal cells (PrSC) on prostate cancer cells (LNCaP, PC-3 and DU145) were determined by collagen gel invesion assay. DU145 cells and PrSC were co-cultured for matrigel invasion chamber assay. LNCaP and PC-3 cells did not respond to any of the factors examined. Invasion of DU145 cells into the collagen gel matrix was induced by HGF and TGF-beta 1, but not by any of the other factors tested. When DU145 cells were cultured in CM from PrSC or co-cultured with PrSC, the cells acquired invasive potential, and this invasion was inhibited by an antibody against HGF, but not against TGF-beta 1. Induction activity of CM from cancer cells to stimulate HGF production by PrSC was studied by ELISA method and Western blotting. Native type HGF production in PrSC was enhanced by some unknown inducer(s) produced by cancer cells. In summary, PrSC-derived HGF enhanced invasive activity of the prostate cancer cell line DU145 through tumor-stromal interaction wherein DU145 cells secreted some HGF-inducer(s) for PrSC.     

Temporal expression of growth factors and matrix molecules in healing tendon lesions.

Overuse tendon injuries are common among elite and recreational athletes. Tendon healing may be enhanced at the cellular level through the use of exogenous growth factors; however, little is known about the endogenous expression of growth factors in healing tendon. This study describes the temporal expression of insulin-like growth factor-I (IGF-I), transforming growth factor-beta1 (TGF-beta1), and collagen types I and III in healing tendon lesions. Collagenase-induced lesions were created in the tensile region of the flexor digitorum superficialis tendon of both forelimbs of 14 horses. Tendons were harvested from euthanatized horses 1, 2, 4, 8 or 24 weeks following injury. Gene expression was evaluated using Northern blot analysis (collagen types I and III), real time PCR (IGF-I and TGF-beta1), and in situ hybridization. Protein content was assayed by dye-binding assay (collagen types I and III), radioimmunoassay (IGF-I), ELISA (TGF-beta1), and immunohistochemistry. Samples were also processed for differential collagen typing, DNA and glycosaminoglycan content, and routine H&E staining. Microscopically, lesions progressed from an amorphous, acellular lesion soon after injury to scar tissue filled with collagen fibers and mature fibroblasts organized along lines of tension. Early lesions were characterized by immediate increases in expression of growth factors and collagen. Message levels for TGF-beta1 peaked early in the wound healing process (1 week), while IGF-I peaked later (4 weeks), as the regenerative phase of healing was progressing. In the first 2 weeks after lesion induction, tissue levels of IGF-I protein actually decreased approximately 40% compared to normal tendon. By 4 weeks, these levels had exceeded those of normal tendon and remained elevated through 8 weeks. Message expression for collagen types I and III increased by 1 week following injury and remained elevated throughout the course of the study. Collagen type I represented the major type of collagen in healing tendon at all time points of the study. Based on these results, IGF-I, administered exogenously during the first 2 weeks following injury, may provide a therapeutic advantage by bolstering low endogenous tissue levels and enhancing the metabolic response of individual tendon fibroblasts.     

Prostate stromal cell-derived hepatocyte growth factor induces invasion of prostate cancer cell line DU145 through tumor-stromal interaction.

BACKGROUND: In prostate cancer, several growth factors derived from stromal cells regulate tumor cell growth. Hepatocyte growth factor (HGF) possesses biological activities that promote cancer proliferation and invasion through tumor-stromal interaction. We examined how prostate stromal cell-derived HGF affects invasion of prostate cancer cells through this interaction. METHODS: The effects of HGF, various growth factors (transforming growth factor (TGF)-alpha, TGF-beta1, basic fibroblast growth factor, keratinocyte growth factor, and platelet-derived growth factor), and conditioned medium (CM) from prostate stromal cells (PrSC) on prostate cancer cells (LNCaP, PC-3, and DU145) were determined by collagen gel invasion assay. DU145 cells and PrSC were cocultured for Matrigel invasion chamber assay. Induction activity of CM from cancer cells to stimulate HGF production by PrSC was studied by the ELISA method and Western blotting. RESULTS: LNCaP and PC-3 cells did not respond to any of the factors examined. Invasion of DU145 cells into the collagen gel matrix was induced by HGF and TGF-beta1, but not by any of the other factors tested. When DU145 cells were cultured in CM from PrSC or cocultured with PrSC, the cells acquired invasive potential, and this invasion was inhibited by an antibody against HGF, but not against TGF-beta1. Native-type HGF production in PrSC was enhanced by some unknown inducer(s) produced by cancer cells. CONCLUSIONS: PrSC-derived HGF enhanced invasive activity of the prostate cancer cell line DU145 through tumor-stromal interaction, wherein DU145 cells secreted some HGF-inducer(s) for PrSC.     

Relaxin down-regulates renal fibroblast function and promotes matrix remodelling in vitro.

BACKGROUND: Renal fibroblasts are important effector cells in tubulointerstitial fibrosis, with experimental antifibrotic strategies focusing on the functional down-regulation of these cells. Several experimental models of fibrosis have provided evidence for the effectiveness of the polypeptide hormone relaxin as a potential antifibrotic agent. This study was conducted to further elucidate the antifibrotic mechanisms of relaxin on renal fibroblasts in vitro. METHODS: Rat cortical fibroblasts were obtained from outgrowth culture of renal tissue isolated from kidneys 3 days post-unilateral ureteric obstruction and constituted 100% of cells studied. A relaxin radio-receptor assay was used to establish binding of relaxin to renal fibroblasts in vitro. Functional studies then examined the effects of H2 relaxin (0, 1, 10 and 100 ng/ml) on fibroblast kinetics, expression of alpha-smooth muscle actin (alpha-SMA), total collagen synthesis, collagenase production and collagen-I lattice contraction. CTGF mRNA expression was also measured by northern analysis. RESULTS: H2 relaxin bound with high affinity to rat renal fibroblasts, but receptor numbers were low. Consistent with its previously reported bimodal effect, transforming growth factor (TGF-beta 1) reduced fibroblast proliferation, an effect abrogated by H2 relaxin. Fibroblasts exposed to H2 relaxin (100 ng/ml) for 24 h demonstrated decreased immunostaining for alpha-SMA and reduced alpha-SMA protein expression compared with controls. There was a trend for a relaxin-mediated reduction in total collagen synthesis and alpha 1(I) mRNA expression with large dose-related increases in collagenase protein expression being observed. TGF-beta 1-stimulated collagen-I lattice contraction was significantly inhibited following co-incubation with 100 ng/ml relaxin. Incremental doses of H2 relaxin had no significant effect on CTGF mRNA expression. CONCLUSIONS: The findings of this study suggest that the antifibrotic effects of relaxin involve down-regulation of fibroblast activity, increase in collagenase synthesis and restructuring of collagen-I lattices, which are consistent with its known physiological role of matrix remodelling. Although there appears to be an interaction between TGF-beta 1 and H2 relaxin, this does not appear to involve a reduction in CTGF mRNA expression.     

Intravenous administration of hepatocyte growth factor gene ameliorates diabetic nephropathy in mice

Diabetic nephropathy is characterized by progressive loss of renal function, persistent proteinuria, and relentless accumulation of extracellular matrix leading to glomerulosclerosis and interstitial fibrosis. This study investigated the potential effects of long-term expression of exogenous hepatocyte growth factor (HGF) on normal and diabetic kidneys. Intravenous injection of human HGF gene via naked plasmid vector resulted in abundant HGF protein specifically localized in renal glomeruli, despite an extremely low level of transgene mRNA in the kidney. In uninephrectomized mice made diabetic with streptozotocin, delivery of exogenous HGF gene ameliorated the progression of diabetic nephropathy. HGF attenuated urine albumin and total protein excretion in diabetic mice. Exogenous HGF also mitigated glomerular mesangial expansion, reduced fibronectin and type I collagen deposition, and prevented interstitial myofibroblast activation. In addition, HGF prevented kidney cells from apoptotic death in the glomeruli and tubulointerstitium. Moreover, expression of HGF inhibited renal expression of TGF-beta1 and reduced urine level of TGF-beta1 protein. Therefore, despite the effects of HGF on diabetic nephropathy being controversial, these observations suggest that supplementation of HGF is beneficial in ameliorating diabetic renal insufficiency in mice.     

Chondrocytic differentiation of mesenchymal stem cells sequentially exposed to transforming growth factor-beta1 in monolayer and insulin-like growth factor-I in a three-dimensional matrix.

This study evaluated chondrogenesis of mesenchymal progenitor stem cells (MSCs) cultured initially under pre-confluent monolayer conditions exposed to transforming growth factor-beta1 (TGF-beta1), and subsequently in three-dimensional cultures containing insulin-like growth factor I (IGF-I). Bone marrow aspirates and chondrocytes were obtained from horses and cultured in monolayer with 0 or 5 ng of TGF-beta 1 per ml of medium for 6 days. TGF-beta 1 treated and untreated cultures were distributed to three-dimensional fibrin disks containing 0 or 100 ng of IGF-I per ml of medium to establish four treatment groups. After 13 days, cultures were assessed by toluidine blue staining, collagen types I and II in situ hybridization and immunohistochemistry, proteoglycan production by [35S]-sulfate incorporation, and disk DNA content by fluorometry. Mesenchymal cells in monolayer cultures treated with TGF-beta1 actively proliferated for the first 4 days, developed cellular rounding, and formed cell clusters. Treated MSC cultures had a two-fold increase in medium proteoglycan content. Pretreatment of MSCs with TGF-beta1 followed by exposure of cells to IGF-I in three-dimensional culture significantly increased the formation of markers of chondrocytic function including disk proteoglycan content and procollagen type II mRNA production. However, proteoglycan and procollagen type II production by MSC's remained lower than parallel chondrocyte cultures. MSC pretreatment with TGF-beta1 without sequential IGF-I was less effective in initiating expression of markers of chondrogenesis. This study indicates that although MSC differentiation was less than complete when compared to mature chondrocytes, chondrogenesis was observed in IGF-I supplemented cultures, particularly when used in concert with TGF-beta1 pretreatment.     

Transforming growth factor-beta promotes pro-fibrotic behavior by serosal fibroblasts via PKC and ERK1/2 mitogen activated protein kinase cell signaling

OBJECTIVE: To assess the role of fibroblasts, transforming growth factor (TGF)-beta, and cell signal pathways in promoting fibrosis in Crohn's disease (CD). SUMMARY BACKGROUND DATA: Intestinal strictures are a major source of morbidity in CD. Fibroblasts found at sites of stricture promote fibrogenesis. The mechanisms underlying this pro-fibrotic behavior remain elusive. METHODS: Fibroblasts were isolated from strictured and macroscopically normal serosa in patients with CD and from normal serosa in patients with colorectal cancer. Whole cell connective tissue growth factor (CTGF) and fibronectin expression were determined by Western blot analysis. Fibroblast type I collagen expression was evaluated by real-time PCR, while fibroblast contractile activity was measured using fibroblast populated collagen lattices. Cells were stimulated with TGF-beta1 and inhibitors of the protein kinase C (PKC) and ERK 1/2 mitogen activated protein (MAP) kinase cell signaling pathways. RESULTS: Stricture fibroblasts displayed enhanced constitutive expression of fibronectin. TGF-beta promoted fibroblast CTGF, fibronectin, and type I collagen expression and enhanced fibroblast contractile activity. Inhibition of PKC reduced basal collagen expression and contractile activity in Crohn's fibroblasts and attenuated the effect of TGF-beta on fibroblast CTGF, fibronectin, and collagen I expression as well as fibroblast contractility. ERK 1/2 inhibition had a similar effect on TGF-beta-induced CTGF and fibronectin expression. CONCLUSIONS: TGF-beta is a critical pro-fibrotic growth factor in CD, and its effects are mediated via PKC and ERK 1/2 MAP kinase cell signaling. These pathways may represent novel therapeutic targets for patients with CD characterized by recurrent intestinal stricture formation.     

Expression of mRNA for vascular endothelial growth factor at the repair site of healing canine flexor tendon.

Neovascularization is an important and prominent feature of tendon healing that contributes to wound repair and potentially to adhesion formation. To define the location of cell populations that recruit and organize the angiogenic response during early healing of flexor tendon, we examined the gene expression pattern of the prototypic angiogenic factor, vascular endothelial growth factor, at and around the tenorrhaphy site in a canine model of flexor tendon repair. In situ hybridization with radiolabeled antisense riboprobes was used to identify tendon cell populations that contribute to the neovascularization process by expressing vascular endothelial growth factor and to relate this cell population to the previously described cell populations that participate in matrix synthesis (express type alpha1(I) collagen) and mitotic renewal (express histone H4). The majority of cells (approximately 67%) within the repair site itself express vascular endothelial growth factor mRNA; however, minimal levels accumulate within cells of the epitenon (approximately 10% of cells; p < 0.0002). By contrast, expression of type alpha1(I) collagen and histone H4 does not differ significantly between the epitenon and the repair site (uniformly approximately 30% of cells). Thus, a gradient of cell populations expressing vascular endothelial growth factor exists in the repairing tendon. These data suggest a potential contribution of cells within the repair site to the organization of angiogenesis during the early postoperative phase of tendon healing.