Extracellular collagen modulates the regulation of chondrocytes by transforming growth factor-β1

This article describes the modulation, by extracellular collagen, of DNA and proteoglycan synthesis in articular chondrocytes stimulated with transforming growth factor-β1, Type-I and type-II collagen, heat denatured type-II collagen, and bovine serum albumin were each incorporated into alginate in increasing concentrations. Bovine articular chondrocytes were isolated and were resuspended in the alginate, yielding alginate beads with final extracellular protein concentrations of 0-1.5% (wt/vol) for the collagens and 0-2.5% (wt/vol) for bovine serum albumin. Cultures of beads were maintained for 7 days in basal Dulbecco's modified Eagle medium or in medium supplemented with 10 ng/ml transforming growth factor-β1. Subsequently, the synthesis of DNA and proteoglycan was measured by radiolabel-incorporation methods with [³⁵S]sulfate and [³H]thymidine, and the values were normalized to the DNA content. Transforming growth factor-β1 stimulated the synthesis of both DNA and proteoglycan in a bimodal fashion. The presence of extracellular type-II collagen increased the rate of DNA and proteoglycan synthesis in a dose-dependent fashion in cultures stimulated by transforming growth factor-β1, whereas heat-inactivated type-II collagen abrogated the effects observed with type-II collagen for synthesis of both DNA and proteoglycan. In contrast, the presence of extracellular type-I collagen caused a dose-dependent inhibition of synthesis of both DNA and proteoglycan in cultures stimulated with transforming growth factor-β1. Extracellular bovine serum albumin brought about a limited increase in synthesis rates, presumably by blocking nonspecific cytokine binding. These results suggest that type-II collagen has a specific role in chondrocyte regulation and serves to mediate the response of chondrocytes to transforming growth factor-β1.     

Autocrine transforming growth factor-β1 activity and glycosaminoglycan synthesis by human cutaneous scar fibroblasts

Transforming growth factor-beta(1) is a well-known and potent biological response modifier that plays an important role in tissue repair and fibrosis. Among the extracellular constituents known to accumulate in fibrotic tissues, glycosaminoglycans are prominent. In this study we examined transforming growth factor-beta(1) synthesis by human dermal fibroblasts derived from both normal and fibrotic cutaneous tissues. We studied the influence of transforming growth factor-beta(1) on glycosaminoglycan synthesis and explored the role of transforming growth factor-beta(1) as an autocrine mediator of its own expression. These investigations are directed at understanding the persistence of the fibrotic phenotype in scarred skin. Transforming growth factor-beta(1) activity was measured by means of a mink lung epithelium growth inhibitory assay. Replicate explants (n = 3) of fibroblasts each derived from normal skin, normal scar, or hypertrophic scar were studied by adding exogenous transforming growth factor-beta(1) at a concentration range of 0 to 10 ng/ml. The resulting conditioned media were removed and assayed for transforming growth factor-beta(1) activity, then the cells were pulsed for an additional 24 hours with radiolabeled glycosaminoglycan precursor, [(3)H]-glucosamine, to evaluate glycosaminoglycan production. Cell-free glycosaminoglycan synthetic profiles were also developed. Transforming growth factor-beta(1) was found to cause a dose-dependent increase in glycosaminoglycan synthesis in hypertrophic scar and normal skin but not in normal scar fibroblasts in cell-mediated glycosaminoglycan synthesis; the reverse was observed in cell-free glycosaminoglycan synthesis, where transforming growth factor-beta(1) increased glycosaminoglycan synthesis in normal scar but not in normal skin or hypertrophic scar. Most endogenous transforming growth factor-beta(1) existed in latent form for normal skin cells but in active form for normal scar and hypertrophic scar fibroblasts.     

Coordinate upregulation of cartilage matrix synthesis in fibrin cultures supplemented with exogenous insulin-like growth factor-I.

The addition of insulin-like growth factor-I to cartilage cultures is known to stimulate the synthesis of proteoglycan and type-II collagen in explant and monolayer studies. The purpose of this study was to determine the effects of long-term supplementation with insulin-like growth factor-I in chondrocytes cultured in fibrin discs as a preliminary investigation to in vivo application of chondrocyte/insulin-like growth factor-I/fibrin grafts to articular-cartilage repair procedures. Chondrocyte-fibrin cultures were maintained for 14 days, with insulin-like growth factor-I added at varying concentrations of 0, 10, 50, or 100 ng/ml medium. Cultures supplemented with 50 or 100 ng of growth factor/ml had increased levels of aggrecan and type-IIB procollagen mRNA, and translation to aggrecan and type-IIB collagen was confirmed by dye-binding assay of total proteoglycan, type-II collagen immunohistochemistry, and determination of collagen content by high-performance liquid chromatography. Maintenance of the chondrocyte phenotype during the 14 days of culture was confirmed by round cell morphology on routine staining, expression of type-II procollagen mRNA on in situ hybridization, evidence of production of pericellular type-II collagen on immunocytochemistry, synthesis of large-molecular-size aggrecan monomer on CL-2B column chromatography, and lack of appreciable message expression for type I or IIA collagen on Northern blot hybridization. Dose-response effects of insulin-like growth factor-I on the expression of chondrocyte matrix constituents were most pronounced at 50 and 100 ng of growth factor per milliliter of medium. These data confirm that (a) culture of chondrocytes for extended periods in three-dimensional cultures of fibrin maintains the chondrocyte phenotype and (b) supplementation with increasing concentrations of insulin-like growth factor-I enhances chondrocyte matrix synthesis and may provide a means to enhance chondrocyte phenotypic stability and function during transplantation grafting procedures.     

Effects of transforming growth factor-β1 and fibroblast growth factor on DNA synthesis in growth plate chondrocytes are enhanced by insulin-like growth factor-I

The local tissue metabolism is controlled through the complex interaction between systemic and local growth factors. In recent years, an increasing number of autocrine or paracrine growth regulators have been identified in physeal cartilage. While these factors act to alter chondrocytes phenotypically and presumably are important mediators in the process of endochondral ossification, the manner in which they interact with the systemically regulated growth factor insulin-like growth factor-I is unknown. In the present study, the interactive effects of insulin-like growth factor-I with transforming growth factor-β1 or basic fibroblast growth factor were examined in short-term monolayer cultures of chick growth plate chondrocytes. [³H]thymidine incorporation was maximally stimulated 11-fold by fibroblast growth factor (10 ng/ml) and 3.5-fold by transforming growth factor-β1 following a 24-hour exposure in serum-containing cultures. The effects of transforming growth factor-β1 and fibroblast growth factor at both high and low concentrations were enhanced in a dose-dependent manner by insulin-like growth factor-I, with a 40–50% increase in DNA synthesis in the presence of 100 ng/ml of insulin-like growth factor-I. Since insulin-like growth factor-I increased [³H]thymidine incorporation after 48 hours (50% increase) but not after 24 hours of exposure, these observations represent a synergistic interaction. Total DNA in cultures treated for 5 days confirmed the modulating effect of insulin-like growth factor-I with transforming growth factor-β1 and fibroblast growth factor. The growth factors were further examined for their effects on markers of chondrocyte differentiation. While all three caused a dose-dependent inhibition of alkaline phosphatase activity, the effects of insulin-like growth factor-I were additive only to those of transforming growth factor-β1 and fibroblast growth factor. Similarly, insulin-like growth factor-I did not affect the sulfate incorporation stimulated by fibroblast growth factor or transforming growth factor-β1. Insulin-like growth factor-I had no effect on total protein synthesis after 24 hours and, although type-II collagen mRNA levels were stimulated, it had no effect on type-X collagen mRNA, as determined by quantitative in situ hybridization. Finally, insulin-like growth factor-I did not alter the dose-dependent stimulation of noncollagen protein synthesis and the inhibition of collagen synthesis caused by fibroblast growth factor and transforming growth factor-β1 in 24-hour cultures. Thus, the data suggest that insulin-like growth factor-I may have a role in augmenting the effects of other growth factors found in cartilage. Since insulin-like growth factor-I is under systemic control by growth hormones, this permits an endocrine regulation of transforming growth factor-β1 and fibroblast growth factor activity and may bring local growth factor effects under systemic control.     

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.     

Modulation of α-smooth muscle actin expression in fibroblasts by transforming growth factor-β isoforms: an in vivo and in vitro study

Myofibroblasts are granulation tissue fibroblasts bearing ultrastructural and biochemical features of smooth muscle cells, such as cytoplasmic microfilaments and alpha-smooth muscle actin expression. They appear transiently during wound healing and more permanently during several pathologic situations such as fibrotic diseases. Transforming growth factor-beta1 has been suggested to be an important promoter of the myofibroblastic phenotype. Here we show that (1) transforming growth factor-beta2, like transforming growth factor-beta1, induces myofibroblast formation in vivo and in vitro; (2) transforming growth factor-beta3 acts as a negative regulator of the myofibroblastic phenotype in vivo but not in vitro; and (3) in vitro, the three different transforming growth factor-beta isoforms are equally able to induce alpha-smooth muscle actin messenger RNA and protein expression in growing and quiescent cultured human and rat subcutaneous tissue fibroblasts. These data confirm that in vitro the behavior of the three different transforming growth factor-beta isoforms is similar, whereas in vivo transforming growth factor-beta isoforms possibly play different but complementary roles in myofibroblast modulation during wound repair.     

Recombinant human bone morphogenetic protein-2 maintains the articular chondrocyte phenotype in long-term culture

Bone morphogenetic proteins have been shown to increase matrix synthesis by articular chondrocytes in short-term cultures. Members of this family of proteins have also been shown to induce endochondral ossification in vivo. The present study was performed to determine if the addition of human recombinant bone morphogenetic protein-2 to a long-term monolayer articular chondrocyte cell culture system affected the ability of the chondrocytes to divide in vitro, whether the cytokine altered expression of the articular chondrocyte phenotype and synthesis of matrix proteoglycans, and whether the cytokine was capable of inducing differentiation to a hypertrophic chondrocyte. Human recombinant bone morphogenetic protein-2 did not alter cell proliferation. It caused 3.5–6.2 times more proteoglycan synthesis by articular chondrocytes during each of the time points tested after 4 days in culture. Total proteoglycan accumulation in the extracellular matrix after 28 days in culture was 6.7 times as great in the treated cultures as in the control. Treatment with human recombinant bone morphogenetic protein-2 maintained the articular chondrocyte phenotype of cells in culture as demonstrated by Northern blot analysis: the expression of type-I collagen genes was increased and that of type-II collagen and aggrecan mRNA was lost in untreated chondrocyte cultures after 14–21 days in culture. In contrast, exposure to 100 ng/ml human recombinant bone morphogenetic protein-2 maintained expression of type-II collagen and increased expression of aggrecan compared with controls during the 28-day culture period. Northern blot analysis of the expression of type-X collagen and osteocalcin by chondrocytes treated with human recombinant bone morphogenetic protein-2 showed a lack of expression of these genes, indicating no alteration in phenotype. These experiments demonstrated the ability of human recombinant bone morphogenetic protein-2 to promote the articular chondrocyte phenotype and matrix synthesis in long-term culture. Characteristics of cell growth were not affected, and the cytokine did not induce differentiation to a hypertrophic chondrocyte.     

αv integrins play an important role in myofibroblast differentiation

Transforming growth factor-beta1 is a potent mediator of the differentiation of fibroblasts into myofibroblasts, which is characterized by the appearance of the cytoskeletal protein alpha-smooth muscle actin. The aim of this study was to investigate the role of integrin extracellular matrix receptors in transforming growth factor-beta1-induced myofibroblast differentiation. We show that blockade of the alphav and/or beta1 integrins prevents the transforming growth factor-beta1-induced myofibroblast differentiation, seen by the increased expression of alpha-smooth muscle actin and enhanced collagen gel contraction in three human fibroblast cell lines (from the mouth, skin, and kidney). Further, blockade of alphav specific integrins alphavbeta5 and alphavbeta3 suppressed myofibroblast differentiation in fibroblasts from the mouth and skin; however, in the kidney cells, the prevention of differentiation was seen only with blockade of alphavbeta5 integrin but not alphavbeta3. A possible reason for this result may be different degrees of responsiveness to transforming growth factor-beta1 treatment seen from different anatomical origins of the cell lines. These data indicate a novel role for alphav integrins in the differentiation of human fibroblasts from the mouth, skin, and kidney into myofibroblasts and suggest that there is a common differentiation pathway.     

Transforming growth factor-beta1 regulates cell growth and causes downregulation of SMemb/non-muscle myosin heavy chain B mRNA in human prostate stromal cells

OBJECTIVES: SMemb/non-muscle myosin heavy chain B (SMemb/NMMHC-B) is most abundantly expressed in proliferating smooth muscle cells and correlates with phenotypic changes from a contractive to a proliferative type. The stromal cells of the prostate play a crucial role in the regulation of prostatic growth and function. The aim of this study was to investigate the effects of the multifunctional cytokine transforming growth factor-beta1 (TGF-beta1) on SMemb/NMMHC-B mRNA expression and stromal cell growth. The expression of the SM2 isoform of smooth muscle myosin heavy chain (SMMHC) mRNA was also examined. MATERIAL AND METHODS: Primary cultures of prostate stromal cells were established by means of an explant method from eight normal prostates. The effects of TGF-beta1 on stromal cell growth were determined by means of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide conversion assay. SMemb/NMMHC-B and SM2 mRNA expression were analyzed quantitatively by means of real-time polymerase chain reaction. RESULTS: In the absence of TGF-beta1, cells expressed alpha-smooth muscle actin and vimentin. After TGF-beta1 treatment, the expression of alpha-smooth muscle actin increased and cells also expressed desmin. TGF-beta1 at concentrations of 1.0, 5.0 and 10 ng/ml suppressed cell growth by 72%, 62% and 56%, respectively, downregulated SMemb/NMMHC-B mRNA expression by 71%, 52% and 38%, respectively and upregulated SM2 mRNA expression 2.1-, 3.0- and 5.3-fold, respectively. CONCLUSIONS: These results demonstrate that TGF-beta1 modulates the smooth muscle cell phenotype from a proliferative to a contractile type and that the inhibitory effects of TGF-beta1 on stromal cell growth correlate with downregulation of the SMemb/NMMHC-B gene.     

Mechanisms of TGF-β action in connective tissue repair of rat mesenteric wounds

We have recently reported that transforming growth factor-beta stimulates genuine connective tissue repair in the perforated rat mesentery and that this stimulation is not caused by increased macrophage chemotaxis. To further characterize the effect of transforming growth factor-beta(1) on the enhanced rate of wound closure, we performed a series of morphometric analyses with determination of mitotic index, fibroblast labeling index, cellular density, neovascularization, and scar tissue formation. Actin expression close to the wound margin was also evaluated morphologically. Fibroblast cell proliferation was not stimulated by transforming growth factor-beta(1) in either wounded or unwounded tissue. Transforming growth factor-beta(1) did, however, significantly increase the formation of healing tissue postoperative days 5 to 10 (p < 0.05) and angiogenesis was significantly stimulated by transforming growth factor-beta(1) postoperative days 7 and 10 (p < 0.005). The mean cellular density was significantly increased in unperforated, transforming growth factor-beta(1)-treated membranes from days 3 to 10, and increased expression of actin with time was observed close to the wound margin. Transforming growth factor-beta(1) was thus shown to be a potent stimulator of angiogenesis and healing tissue formation in connective tissue repair, but this stimulation mainly occurred after closure of perforations. The increased cellular density in the absence of stimulated proliferation and increased actin expression in wound cells indicate that contraction may be an important mechanism of connective tissue repair in the perforated rat mesentery.     

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.     

Regulating effects of transforming growth factor-beta (TGF-beta) on gene expression of collagen type II in human intervertebral discs

OBJECTIVE: To assess the regulating effects of TGF-beta on gene expression of collagen type II in the human intervertebral discs. METHODS: In situ hybridization was used to investigate the effect of TGF-beta1 on collagen mRNA in confluent primary and passaged monolayer cell cultures of annulus fibrosus (AF) as well as nucleus pulposus (NP). The mean photodensitometry of cell smears as semi-quantitative analysis was evaluated by VIDAS software. RESULTS: In primary cultures, 1 ng/ml and 10 ng/ml TGF-beta1 inhibited the collagen type II mRNA levels by 74.6% and 60.2% respectively in AF; they also inhibited the mRNA levels by 69.6% and 55.5% respectively in NP. In passaged cultures in which the notochordal cells and chondrocytes were in dedifferentiation status, 1 ng/ml and 10 ng/ml TGF-beta1 increased the collagen type II mRNA levels by 151% and 166% respectively in AF and also increased the mRNA levels by 145% and 198% respectively in NP. CONCLUSIONS: The regulation effect of TGF-beta on collagen type II gene expression is dependent on whether the cells are fully differentiated or undergoing phenotype loss, and TGF-beta may play an important role in the repair process during early disc degeneration, especially in nucleus pulposus.