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.     

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.

     

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.

     

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.

     

Alteration of articular cartilage frictional properties by transforming growth factor β, interleukin‐1β, and oncostatin M

Objective

To evaluate the functional effects of transforming growth factor β1 (TGFβ1), interleukin‐1β (IL‐1β), and oncostatin M (OSM) on the frictional properties of articular cartilage and to determine the role of cytokine‐mediated changes in cartilage frictional properties by extracting and redepositing lubricin on the surface of cartilage explants.

Methods

Neonatal bovine cartilage explants were cultured in the presence or absence of 10 ng/ml of TGFβ1, IL‐1β, or OSM over 48 hours. Boundary lubrication tests were conducted to determine the effects of endogenously produced surface localized lubricin and of exogenous lubricin at the tissue surface and in the lubricant solution. The initial friction coefficient (μ₀), equilibrium friction coefficient (μ_eq), and Young's modulus (E_Y) were determined from the temporal load data.

Results

IL‐1β and OSM decreased tissue glycosaminoglycan (GAG) content by ∼20% over 48 hours and decreased E_Y to a similar extent (11–17%), but TGFβ did not alter GAG content or E_Y. Alterations in proteoglycan content corresponded to changes in μ₀, but endogenous lubricin decreased boundary mode μ_eq. The addition of exogenous lubricin, either localized at the tissue surface or in the lubricating solution, did not modulate μ₀, but it did lower μ_eq in cytokine‐treated cartilage.

Conclusion

This study provides new insight into the functional consequences of cytokine‐mediated changes in friction coefficient. In combination with established pathways of cytokine‐mediated lubricin metabolism, these data provide evidence of distinct biochemical origins of boundary and biphasic pressure‐mediated lubrication mechanisms in cartilage, with boundary lubrication regulated by surface accumulation of lubricants and biphasic lubrication controlled by factors such as GAG content that affect water movement through the tissue.

     

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.