Involvement of αvβ5 integrin–mediated activation of latent transforming growth factor β1 in autocrine transforming growth factor β signaling in systemic sclerosis fibroblasts

Objective

To confirm the involvement of αvβ5 in the self‐activation system in systemic sclerosis (SSc) fibroblasts.

Methods

Levels of αvβ5 expression were analyzed by immunoprecipitation. The promoter activity of the human α2(I) collagen gene was determined by transient transfection assay. Phosphorylation levels and DNA binding ability of Smad3 were investigated by immunoprecipitation and DNA affinity precipitation, respectively. The localization of active transforming growth factor β (TGFβ) was determined by coculture assay using TMLC cells (mink lung epithelial reporter cells that stably express a portion of the plasminogen activator inhibitor 1 promoter). The morphologic features of cells were determined by immunofluorescence analysis.

Results

Levels of αvβ5 expression were significantly elevated in SSc fibroblasts compared with normal fibroblasts. Treatment with anti‐αvβ5 antibody or β5 antisense oligonucleotide significantly reduced human α2(I) collagen gene promoter activity in SSc fibroblasts. In SSc fibroblasts pretreated with TGFβ1 antisense oligonucleotide, the exogenous latent TGFβ1 stimulation significantly increased human α2(I) collagen gene promoter activity; this effect was significantly reduced in the presence of anti‐αvβ5 antibody. Phosphorylation levels and DNA binding ability of Smad3 in SSc fibroblasts were significantly reduced by treatment with β5 antisense oligonucleotide. The luciferase activity of TMLC cells cocultured with SSc fibroblasts was significantly elevated compared with that of TMLC cells cocultured with normal fibroblasts and was significantly reduced in the presence of anti‐αvβ5 antibody. Anti‐αvβ5 antibody reversed the myofibroblastic features of SSc fibroblasts.

Conclusion

Up‐regulated expression of αvβ5 contributes to the establishment of autocrine TGFβ signaling in SSc fibroblasts through activation of endogenous latent TGFβ1.

     

Mast cells are a source of transforming growth factor β in systemic sclerosis

Objective

To describe the cellular source of transforming growth factor β (TGFβ) in the dermis of patients with systemic sclerosis (SSc).

Methods

We performed electron microscopy (EM) with immunogold labeling on skin biopsy specimens from 7 patients with SSc and 3 healthy control subjects. For TGFβ quantification, the numbers of gold particles per square micron were calculated. The origin of mast cells was confirmed and quantified by toluidine blue staining and light microscopy. Degranulation was assessed on toluidine blue–stained sections and on EM images.

Results

In all patients, active TGFβ was observed uniquely in mast cell vesicles, some of which were released into the extracellular space. Patients with progressive SSc and a more recent onset of non–Raynaud's phenomenon symptoms had higher numbers of mast cells and gold particles per mast cell. Mast cells from healthy control subjects also contained active TGFβ but, in contrast to SSc samples, showed a resting character with no or low‐level degranulation and uniformly dense osmiophilic vesicles.

Conclusion

Degranulation of skin mast cells can be an important mechanism of TGFβ secretion in SSc.

     

Morphologic and mitogenic responses of rabbit synovial fibroblasts to transforming growth factor β require transforming growth factor α or epidermal growth factor

We tested the hypothesis that normal synovial fibroblasts might proliferate in response to transforming growth factors (TGFs), peptides that are extracted with acid-ethanol from bovine kidney or salivary gland and that cause anchorage-independent growth of normal cells. A 72-hour exposure of confluent monolayers of rabbit synovial fibroblasts in 10% fetal calf serum to partially purified TGF-β in the presence of TGF-β gave a 2- to 5-fold increase in incorporation of ³H-thymidine, protein content, and cell number. Similar results were obtained with high pressure liquid chromatography-purified TGF-β in the presence of epidermal growth factor (EGF) (a type of TGF-β). By itself, purified TGF-β was not mitogenic; it depended absolutely on EGF. However, only TGF-β along with EGF, and not EGF alone, induced a marked morphologic change: a piling up of cells into foci resembling those commonly seen in primary cultures of rheumatoid synovial cells. Mitogenic responses induced by the TGF-β-EGF combination were prevented by all-trans-retinoic acid but not by indomethacin or dexamethasone. The data indicate that TGF-β, a peptide extracted from normal cells, can act in concert with EGF to cause proliferation and piling up of synovial cells and raise the possibility that these factors may play a role in rheumatoid arthritis and other proliferative but nonmalignant diseases as well.     

Hepatocyte growth factor and transforming growth factor β1 ratio at baseline can predict early response to cyclophosphamide in systemic lupus erythematosus nephritis

Objective

To determine whether the ratio of hepatocyte growth factor (HGF) to transforming growth factor β1 (TGFβ1) in systemic lupus erythematosus (SLE) nephritis could be a prognostic factor for response to therapy with cyclophosphamide (CYC) and steroids at 6 months, and to examine whether the molecular ratio of HGF to TGFβ1 correlates with the activity index (AI) and chronicity index (CI) and has predictive value for remission at the sixth month.

Methods

Thirty‐six SLE patients with new‐onset nephritis, 25 of whom were treated with CYC and steroids, entered into a prospective observational cohort trial at a tertiary university referral center. Renal biopsy findings and clinical parameters were recorded for all patients. Histopathologic, clinical, and immunohistochemical data at baseline served to define the predictive value for the outcome at 6 months.

Results

AI and CI at baseline did not distinguish patients who had achieved remission from those who had not achieved remission after receiving CYC plus steroids. HGF and TGFβ1 were expressed in the tubuli, not in the glomeruli. The CI correlated directly with the TGFβ1 extension score (TGFβ1‐ES) (r = 0.43, P = 0.008), but correlated indirectly with the HGF intensity score (HGF‐IS) (r = −0.39, P = 0.02) and the HGF‐ES (r = −0.45, P = 0.006). An HGF‐ES:TGFβ1‐ES ratio of ≥1 at baseline distinguished patients who had achieved remission from those who had not achieved remission, with a predictive value of 94%.

Conclusion

These findings indicate that baseline expression of renal HGF and TGFβ1 predicts short‐term renal outcome after therapy with CYC and steroids.

     

JAK‐2 as a novel mediator of the profibrotic effects of transforming growth factor β in systemic sclerosis

Objective

To investigate whether JAK‐2 contributes to the pathologic activation of fibroblasts in patients with systemic sclerosis (SSc) and to evaluate the antifibrotic potential of JAK‐2 inhibition for the treatment of SSc.

Methods

Activation of JAK‐2 in human skin and in experimental fibrosis was determined by immunohistochemical analysis. JAK‐2 signaling was inhibited by the selective JAK‐2 inhibitor TG101209 or by small interfering RNA. Bleomycin‐induced dermal fibrosis in mice and TSK‐1 mice were used to evaluate the antifibrotic potential of specific JAK‐2 inhibition in vivo.

Results

Increased activation of JAK‐2 was detected in the skin of patients with SSc, particularly in fibroblasts. The activation of JAK‐2 was dependent on transforming growth factor β (TGFβ) and persisted in cultured SSc fibroblasts. Inhibition of JAK‐2 reduced basal collagen synthesis selectively in SSc fibroblasts but not in resting healthy dermal fibroblasts. Moreover, inhibition of JAK‐2 prevented the stimulatory effects of TGFβ on fibroblasts. Treatment with TG101209 not only prevented bleomycin‐induced fibrosis but also effectively reduced skin fibrosis in TSK‐1 mice.

Conclusion

We demonstrated that JAK‐2 is activated in a TGFβ‐dependent manner in SSc. Considering the potent antifibrotic effects of JAK‐2 inhibition, our study might have direct translational implications, because inhibitors of JAK‐2 are currently being evaluated in clinical trials for myeloproliferative disorders and would also be available for evaluation in patients with SSc.

     

原发性胆汁性肝硬化患者肝组织转化生长因子β-1表达水平变化

目的 探讨原发性胆汁性肝硬化（PBC）患者肝组织转化生长因子-β1（TGF-β1）表达的变化。 方法 52例PBC患者接受超声引导下肝穿刺活检,选择病理科留取的正常肝组织20份作为对照,另选择20例健康人采集静脉血。采用免疫组化法检测肝组织TGFβ-1表达,采用ELISA法检测血清肿瘤坏死因子α（TNF-α）和白介素6（IL-6）水平。 结果 正常肝组织不表达或仅有少量TGF-β1表达,而PBC患者肝实质细胞胞浆内呈TGF-β1高表达;PBC患者血清TNF-α和IL-6水平分别为（28.71±13.54） pg/ml和（21.3±9.4） pg/ml,显著高于健康人【分别为（21.3±15.4） pg/ml和（2.1±1.6） pg/ml,P<0.01】;7例PBC肝组织肝纤维化S0期患者肝组织TGF-β1表达及血清TNF-α和IL-6水平分别为（0.5±0.2）10-2、（7.1±4.1） pg/ml和（5.1±1.0） pg/ml,显著低于12例S1期【（4.2±1.3） 10-2、（18.6±6.2） pg/ml、（11.5±3.6） pg/ml,P<0.01】、18例S2期【（6.9±1.2） 10-2、（27.3±9.9） pg/ml、（19.4±4.1） pg/ml,P<0.01】、9例S3期【（13.3±15.1） 10-2、（39.7±15.18） pg/ml、（27.3±8.1） pg/ml,P<0.01】和6例S4期【（21.2±17.1） 10-2、（53.4±17.3） pg/ml、（47.8±11.0） pg/ml,P<0.01】;15例Child-Pugh A级患者肝组织TGF-β1表达及血清TNF-α和IL-6水平分别为（1.9±1.6） 10-2、（12.2±3.1） pg/ml和（7.3±2.5） pg/ml,显著低于25例Child-Pugh B级【（15.9±13.6） 10-2、（32.9±8.6） pg/ml、（21.8±6.3） pg/ml,P<0.01】或12例C级患者【（22.6±18.5） 10-2、（49.1±19.3） pg/ml、（45.5±12.7） pg/ml,P<0.01】。 结论 PBC患者肝组织TGFβ-1表达及血清TNF-α和IL-6水平显著升高,可能与肝组织纤维化增生和/或肝功能受损有关。     

Dialysis‐associated systemic fibrosis (nephrogenic fibrosing dermopathy): Study of inflammatory cells and transforming growth factor β1 expression in affected skin

Objective

Nephrogenic fibrosing dermopathy (NFD) is a newly recognized cutaneous fibrotic disorder occurring in individuals with end‐stage renal disease (ESRD). The aim of the present study was to describe the clinical and histopathologic features of 9 new cases and to characterize the inflammatory cells and expression of transforming growth factor β1 (TGFβ1) in affected skin.

Methods

Clinical and laboratory assessments, including serology and pulmonary function studies, were performed in 9 patients undergoing long‐term dialysis (8 hemodialysis; 1 peritoneal dialysis) for ESRD of diverse etiologies. Skin, fascia, striated muscles, lungs, and heart were examined by histopathology. Inflammatory cells were characterized by immunophenotyping using specific monoclonal antibodies. TGFβ1 expression was determined by in situ hybridization.

Results

All patients displayed cutaneous features resembling both systemic sclerosis and diffuse fasciitis, with severe loss of motion and flexion contractures in multiple joints. Six patients displayed woody induration of the muscles of the legs, thighs, and forearms. Five of the 6 patients with lung involvement had a reduced diffusion capacity for carbon monoxide on pulmonary function testing. Marked elevations of the erythrocyte sedimentation rate and/or C‐reactive protein level were found in 6 patients. Antinuclear antibodies were present at low titers in 4 patients. Histopathologic studies indicated that in addition to the dermis, the fibrotic process affected the subcutaneous tissue, fascia, striated muscles, lungs, and myocardium. Large numbers of CD68+/factor XIIIa+ dendritic cells and increased expression of TGFβ1 were found in affected skin and muscle.

Conclusion

Our findings indicate that the fibrotic process of NFD affects not only the dermis, but also the subcutaneous tissues, fascia, and other organs, including striated muscles, heart, and lungs. We therefore believe this is a systemic fibrosing process, and we suggest that dialysis‐associated systemic fibrosis would be a better term for the condition.

     

A comparison of the effect of transforming growth factor β1 on pyrophosphate elaboration from various articular tissues

Objective. The purpose of this study was to determine the effect of transforming growth factor β1 (TGFβ1) on inorganic pyrophosphate (PPi) elaboration from articular tissues to better understand the pathophysiology of calcium pyrophosphate dihydrate (CPPD) crystal deposition in the joint. Methods. PPi was measured in the media of adult porcine articular tissue in organ culture and monolayer cultures. Results. TGF/β1 strongly stimulated PPi elaboration by porcine fibrocartilage and hyaline cartilage. It modestly increased PPi elaboration by ligament, and had no effect on PPi elaborated by synovium. Of all cell types tested in cell culture, only chondrocytes responded to TGF/β1 by significantly increasing PPi elaboration. Conclusion. TGF/β1 stimulates PPi elaboration from hyaline cartilage, fibrocartilage, and ligament, indicating that there is in situ CPPD crystal formation in these tissues. The ability of tissues to respond to TGFβ1 by increasing PPi elaboration correlates with the prevalence of CPPD crystal deposition found clinically. The unique response of chondrocyte monolayers to TGF/β1 reinforces the key role of the chondrocyte in PPi elaboration in the joint. These findings support an etiologic role for responsiveness to TGFβ1 in CPPD disease.     

Diminished transforming growth factor β2 production leads to increased expression of a profibrotic procollagen α2 type I messenger RNA variant in embryonic fibroblasts of UCD‐200 chickens,a model for systemic sclerosis

Objective

A procollagen α2(I) messenger RNA (mRNA) variant, with a 115‐bp band and an expected band of 180 bp, was found to be increased during early, acute scleroderma‐like disease in UCD‐200 chickens. The present study investigated the influence of cytokines on the expression of these 2 proα2(I) mRNA variants.

Methods

Embryonic fibroblasts of UCD‐200 chickens (UCD‐200‐CEF) and normal white leghorns (NWL‐CEF) were grown in 3‐dimensional collagen gels. Procollagen mRNA expression was analyzed by RNase protection assay, and proliferation was determined by ³H‐thymidine incorporation. Transforming growth factor β1 (TGFβ1) and TGFβ2 were measured in culture supernatants by enzyme‐linked immunosorbent assay.

Results

Compared with NWL‐CEF, UCD‐200‐CEF expressed 7.2 times more of the smaller profibrotic proα2(I) mRNA variant. TGFβ1 stimulated the proliferation of UCD‐200‐CEF, but not NWL‐CEF. The 115 bp:180 bp ratio was increased by TGFβ1 in both NWL‐CEF and UCD‐CEF. TGFβ2 and TGFβ3 reduced the expression of the profibrotic proα2(I) mRNA in UCD‐200‐CEF to the same levels observed in healthy control NWL‐CEF. In culture supernatants, NWL‐CEF produced 4.1 times more TGFβ2 than that produced by UCD‐CEF. Inhibition of endogenous TGFβ2 in NWL‐CEF resulted in the same 115 bp:180 bp ratio as seen in untreated UCD‐CEF.

Conclusion

TGFβ2 reduces the expression of a profibrotic proα2(I) mRNA variant in UCD‐200‐CEF. The constitutive overproduction of this proα2(I) mRNA variant and the diminished synthesis of TGFβ2 in untreated UCD‐200‐CEF suggest that TGFβ2 can act as an antifibrotic cytokine and might be a key player during fibrosis onset. These results shed light on the contradictory observations regarding the role of TGFβ2 in human systemic sclerosis.

     

Fibroblast growth factor 2 and transforming growth factor β1 induce precocious maturation of articular cartilage

Objective

We have discovered that a combination of fibroblast growth factor 2 and transforming growth factor β1 induce profound morphologic changes in immature articular cartilage. The purpose of this study was to test the hypothesis that these changes represent accelerated postnatal maturation.

Methods

Histochemical and biochemical assays were used to confirm the nature of the morphologic changes that accompany growth factor stimulation of immature bovine articular cartilage explants in serum‐free culture medium. Growth factor–induced apoptosis, cellular proliferation, and changes in the collagen network were also quantitatively analyzed.

Results

Growth factor stimulation resulted in rapid resorption from the basal aspect of immature cartilage explants that was simultaneously opposed by cellular proliferation from the apical aspect driven from a pool of chondroprogenitor cells we have previously described. Maturation‐dependent changes in tissue stiffness, collagen crosslinking, and collagen fibril architecture as well as differentiation of the extracellular matrix into distinct pericellular, territorial, and interterritorial domains were all present in growth factor–stimulated cartilage samples and absent in control samples.

Conclusion

Our data demonstrate that it is possible to significantly enhance the maturation of cartilage tissue using specific growth factor stimulation. This may have applications in transplantation therapy or in the treatment of diseased cartilage, through phenotype modulation of osteoarthritic chondrocytes in order to stimulate growth and maturation of cartilage repair tissue.