Platelet-derived growth factor stimulates tyrosine-specific protein kinase activity in Swiss mouse 3T3 cell membranes.

Platelet-derived growth factor (PDGF) stimulates the incorporation of 32P from [gamma-32P]ATP into a Mr approximately 170,000 protein by an endogenous tyrosine-specific protein kinase in membrane preparations of Swiss mouse 3T3 cells. Epidermal growth factor (EGF), but not fibroblast growth factor (FGF) or insulin, stimulates limited incorporation of 32P into a protein of similar molecular weight. The ligand concentration required for half-maximal activity (S0.5) for PDGF stimulation of phosphorylation is 50 ng/ml; saturation is achieved at 300 ng/ml. The S0.5 for ATP is 15 microM. Mg2+ or Mn2+ is required for protein kinase activity. Stimulation of PDGF results in the preferential phosphorylation of tyrosine residues in this Mr approximately 170,000 membrane protein. The Mr approximately 170,000 protein can be resolved into Mr approximately 180,000 and 160,000 components in 4% NaDodSO4 gels. PDGF stimulates 32P incorporation preferentially into the Mr approximately 180,000 and less extensively into the Mr approximately 160,000 protein. EGF stimulates 32P incorporation predominantly into a protein of Mr approximately 160,000. The similarity of PDGF and EGF in stimulating phosphotyrosine-specific protein kinase activity and the stimulation of a similar activity by viral transformation (src) genes suggest that a common mechanism may exist for the phenotypic expression of increased DNA synthesis and cell growth stimulated by these separate factors.     

The effect of sodium selenite on chondrocytes in monolayer culture

The effect of sodium selenite on DNA and sulfated proteoglycan synthesis by cultured rabbit articular and growth plate chondrocytes was studied as an in vitro model for Kashin-Beck disease. The selenium content of a defined medium (DMEM, fibroblast growth factor, insulin, and dexamethasone) was below the limit of detection by isotope dilution mass spectrometry. The chondrocytes were viable in the Se-free basal medium. Selenite over a range of 5 X 10(-9) M to 5 X 10(-7) M had no stimulatory effect on DNA or sulfated proteoglycan synthesis by either type of chondrocyte or skin fibroblasts. Proliferation of bovine endothelial cells was enhanced by 5 X 10(-7) M Se. At Se concentrations of greater than or equal to 10(-6) M, there was progressive inhibition of cell growth and radiosulfate incorporation of the connective tissue cells; bovine endothelial cells were more resistant. Twice equimolar concentrations of vitamins C and E exerted no protective effect against the cytotoxicity of higher concentrations of Se. Se supplementation also failed to stimulate growth of human infant chondrocytes. The model enabled simulation of conditions of hyposelenosis below those encountered in nature. The data provide no evidence that chondrocytes have idiosyncratic requirements for Se, and do not support the hypothesis that Se deficiency is a major etiologic factor in Kashin-Beck disease.     

Responses of normal and rheumatic human articular chondrocytes cultured under various experimental conditions in agarose

The purpose of the present study was to test if agarose could support the maintenance of normal and arthritic human chondrocytes in culture, and under which experimental conditions they could be successfully grown. Cultures of chondrocytes isolated from articular cartilage from patients with rheumatoid arthritis (RA), juvenile rheumatoid arthritis (JRA), and healthy controls were assessed by light microscopy, alcian blue staining, formazan uptake and incorporation of radiosulfate into the extracellular matrix. The results showed that both normal and arthritic chondrocytes proliferated, and synthesized proteoglycan (PG) in agarose in short term and long term culture. Proliferation and PG synthesis occurred at a slower rate in chondrocytes from adult rheumatic patients than from healthy controls. Supplements to the medium influenced chondrocyte proliferation, PG synthesis and release into the medium. Serum from RA patients stimulated chondrocyte responses more than normal human serum (NHS), and NHS promoted PG synthesis more than fetal calf serum (FCS). Exposure to inflammatory synovial fluid (SF) enhanced PG synthesis of healthy chondrocytes, but suppressed it in arthritic chondrocytes. We conclude that species-specific serum is optimal for chondrocyte cultures, and that disease related culture conditions change the chondrocyte response. As metabolic responses of human chondrocytes are maintained in agarose, this culture system appears as a suitable in vitro tool for further studies of human joint disease.     

Maintenance of the synthesis of large proteoglycans in anatomically intact murine articular cartilage by steroids and insulin-like growth factor I.

OBJECTIVES--The exact regulation of the synthesis of cartilage specific molecules, such as collagen type II and aggrecan, by articular chondrocytes is unknown, but growth factors and hormones probably play an important part. The effects of glucocorticosteroids (prednisolone and triamcinolone), in combination with insulin-like growth factor I (IGF-I), on the synthesis and hydrodynamic volume of proteoglycans from murine patellar cartilage were investigated. METHODS--The in vitro effect of IGF-I and steroids on proteoglycan synthesis in murine patellar cartilage was evaluated by [35S]sulphate incorporation in combination with dissociative gel chromatography using a Sephacryl S-1000 column. The impact of in vivo prednisolone (0-5 mg/kg) on proteoglycan synthesis in murine patellar cartilage was analysed by [35S]sulphate incorporation immediately after dissection from the knee joint. RESULTS--Prednisolone stimulated proteoglycan synthesis in murine patellar cartilage from normal knees and in cartilage from knees injected with papain in vitro in the absence and presence of IGF-I. Moreover, oral administration of prednisolone for seven days to C57Bl10 mice resulted in enhanced proteoglycan synthesis in patellar cartilage. The incubation of patellar cartilage for 48 hours without serum or growth factors led to the synthesis of proteoglycans with a smaller hydrodynamic volume than those synthesised immediately after dissection of the patellae. This could either be circumvented by the addition of IGF-I or by the addition of glucocorticosteroids (prednisolone or triamcinolone) to the culture medium. CONCLUSIONS--These results show that in a dose range of 0.0003-0.3 mmol/l, glucocorticosteroids, like IGF-I, stimulate proteoglycan synthesis and maintain the synthesis of hydrodynamically large proteoglycans by chondrocytes from murine articular cartilage. This indicates that glucocorticosteroids might play a part in the preservation of matrix integrity in articular cartilage.     

Presence of epidermal growth factor, platelet-derived growth factor, and their receptors in human myometrial tissue and smooth muscle cells: their action in smooth muscle cells in vitro.

Immunohistochemical observations indicate that human myometrial smooth muscle cells express epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)-AB and contain EGF and PDGF-beta receptors with no variation in intensity with phases of the menstrual cycle. Furthermore, immunofluorescent microscopic studies revealed that primary myometrial smooth muscle cell cultures also express EGF, PDGF-AB, and contain EGF and PDGF-beta, but not alpha-receptor. Incubation of subconfluent smooth muscle cells in serum-free medium leads to quiescence within 48 h as demonstrated by 3H-thymidine incorporation and labeling index. Exposure of quiescent cells to 10% fetal bovine serum stimulates resumption of DNA synthesis and proliferation in a time-dependent manner with a doubling time of 41.6 h. EGF (1.5-50 ng/ml) and PDGF-AB (1-10 ng/ml) in a dose- and time-dependent manner significantly stimulated 3H-thymidine incorporation by quiescent myometrial smooth muscle cells (P less than 0.05). Combinations of EGF (15 ng/ml) and PDGF-AB (10 ng/ml) significantly increased 3H-thymidine incorporation induced by either growth factor alone (P less than 0.05). PDGF-BB at 10 ng/ml also stimulated 3H-thymidine incorporation and its effect was similar to that induced by PDGF-AB at the same concentration. 17 beta-Estradiol (E2) at 1 microM inhibited 3H-thymidine incorporation by the smooth muscle cells (P less than 0.05). E2 also reduced the stimulatory effect of EGF (15 ng/ml) and PDGF (3 ng/ml). Progesterone at 1 microM either alone or in combination with E2 did not have any effect on 3H-thymidine incorporation or alter the mitogenic action of EGF and PDGF. The effect of EGF and PDGF on cell growth and 3H-thymidine incorporation by myometrial smooth muscle cells was independent of phases of the menstrual cycle. In summary, the results of present studies indicate that human myometrial tissue and myometrial smooth muscle cells in primary culture locally produce EGF and PDGF-AB and contain EGF and PDGF-beta, but not alpha-receptors. Moreover, the myometrial smooth muscle cells in culture respond to the mitogenic action of EGF and PDGF.     

Effect of osmolarity on glycosaminoglycan production and cell metabolism of articular chondrocyte under three-dimensional culture system

OBJECTIVES:This study examined how physiological levels of extracellular osmolarity influence proteoglycan accumulation in articular chondrocytes in a three-dimensional culture system.METHODS:Cells were obtained from metacarpal phalangeal joints of 18-24 month bovine. They were cultured for 6 days in alginate beads at 4 million cells/ml in DMEM containing 6% FBS under 21% O2. Medium osmolarity was altered by NaCl addition over the range 270-570 mOsm and monitored using a freezing point osmometer. Profiles across intact beads were determined by manual counting using fluorescent probes and transmission electron microscope. Lactate production was measured enzymatically and glycosaminoglycan (GAG) accumulation was measured using a modified dimethylmethylene blue assay. Rate of sulfate GAG synthesis was measured using a standard 35S-sulfate radioactive method.RESULTS:The cell viability was similar for the high and low osmolarity cultures. However, confocal microscopy showed that the cells were the largest under 270 mOsm and became smaller with increasing osmotic pressure. GAG production was largest in the 370mOsm, and the capacity for GAG production and cell metabolism (lactate production) was low under hypo-osmolarity and hyper-osmolarity, and cell deaths were often observed on electron microscopy.CONCLUSIONS:In our model the prevailing osmolarity was a powerful regulator of GAG accumulation by cultured chondrocytes. These results thus indicate GAG synthesis rates are regulated by GAG concentration, with implications both for the aetiology of osteoarthritis and for tissue engineering.     

Effects of tetrandrine on growth factor-induced DNA synthesis and proliferative response of rat pulmonary artery smooth muscle cells

The objective of the present study was to investigate the effect of tetrandrine (a plant alkaloid isolated from Stephenia tetrandra) on growth factor-induced DNA synthesis and proliferative responses of rat pulmonary artery smooth muscle cells. Male rat and bovine pulmonary artery smooth muscle cells (PASMC) were cultured in Medium 199 containing FBS (10%). DNA synthesis was monitored from [(3)H]-thymidine uptake and cell proliferation by direct cell counting. In the present study FBS (1% v/v) caused a small increase in DNA synthesis above basal levels in rat and bovine PASMC (6% and 11% respectively). Platelet-derived growth factor (PDGF, 50 ng/ml), fibroblast growth factor (FGF, 50 ng/ml) or interleukin-1alpha (IL-1alpha, 100 pg/ml) alone increased rat PASMC proliferation (69-85%) and DNA synthesis above basal levels (76-92%). The addition of these growth factors in combination with FBS (1%) resulted in higher increases in DNA synthesis above basal levels (rat PASMC:PDGF, 465%; FGF, 421%; IL-1alpha, 406%; bovine PASMC:PDGF, 279%). Tetrandrine (10(-5) M) inhibited FBS (10%)-induced rat PASMC proliferation (90.5%) and DNA synthesis (89.0%). Tetrandrine significantly inhibited cell proliferation (86.5-98.5%) and DNA synthesis (79.9-89.0%) induced by FBS (1%) in combination with one of the following mitogens; PDGF (50 ng/ml), FGF (50 ng/ml), IL-1alpha (100 pg/ml). The inhibitory effects of tetrandrine were observed between 10(-6) and 10(-5)M and PASMC viability was not affected by tetrandrine below 3x10(-5) m. In summary, these results suggest that tetrandrine can exert anti-proliferative effects against a range of mitogenic stimuli for vascular smooth muscle cells in vitro. Such effects may contribute to the inhibitory effect of tetrandrine on pulmonary vascular remodelling associated with pulmonary hypertension.     

Recombinant human osteogenic protein 1 is a potent stimulator of the synthesis of cartilage proteoglycans and collagens by human articular chondrocytes

Objective. To study the effects of recombinant human osteogenic protein-1 (rHuOP-1; bone morphogenetic protein-7) on proteoglycan and collagen synthesis by human articular chondrocytes. Methods. Articular chondrocytes from fetal, adolescent, and adult human donors were cultured in alginate beads for 4 days in a mixture of Ham's F-12, Dulbecco's modified Eagle's medium, 10% fetal bovine serum (FBS), then for an additional 3-10 days in the presence and absence of rHuOP-1, with and without FBS. Chondrocyte synthetic activity was measured as the amount of incorporation of ³⁵S-sulfate into proteoglycans and ³H-proline into hydroxyproline. Sieve chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were performed to identify specific proteoglycans and collagens. Results. Recombinant human OP-1 markedly stimulated the synthesis of proteoglycans (mostly aggrecan) and collagens (predominantly type II) by all chondrocyte preparations. This did not require the presence of FBS and was associated with continued expression of the chondrocyte phenotype. Conclusion. Recombinant human OP-1 is a more potent stimulator of the synthesis of cartilage-specific molecules by human articular chondrocytes than are other factors tested for comparison, including TGFβ1 and activin A.     

Responsiveness of articular cartilage from normal and inflamed mouse knee joints to various growth factors.

OBJECTIVE--Disturbed anabolic signalling might contribute to the decreased chondrocyte proteoglycan (PG) synthesis during joint inflammation. Articular cartilage obtained from mouse knee joints with experimentally-induced arthritis exhibits a state of nonresponsiveness towards stimulation of chondrocyte PG synthesis by insulin-like growth factor-1 (IGF-1). Investigations were carried out on the role of other growth factors apart from IGF-1 on regulation of chondrocyte PG synthesis under pathological conditions, that is, during repair after IL-1 exposure as well as during early and later arthritis. METHODS--Mouse patellae were obtained from normal knee joints and joints injected with IL-1 or zymosan. The patellae were cultured with basic fibroblast growth factor [bFGF], platelet-derived growth factor [PDGF], epidermal growth factor [EGF] or transforming growth factor beta [TGF beta] for 24 hours in the presence or absence of IGF-1. Chondrocyte PG synthesis was measured by 35S-sulphate incorporation. RESULTS--In normal cartilage none of the tested growth factors elicited stimulatory effects on the chondrocyte PG synthesis as caused by IGF-1. EGF and TGF beta even caused significant inhibition of chondrocyte PG synthesis. Combination of bFGF or PDGF with IGF-1 exerted significant additional stimulation of the 35S-sulphate incorporation. IL-1 exposed cartilage displayed reactivity to IGF-1 as well as to the other growth factors similar to control cartilage. Cartilage obtained from joints with experimentally-induced arthritis exhibited a state of nonresponsiveness towards all individually tested growth factors as well as growth factor combinations. CONCLUSION--Arthritis causes nonresponsiveness to stimulation of chondrocyte PG synthesis by the tested growth factors, which might be caused by a general receptor function defect.     

Effect of purified growth factors on rabbit articular chondrocytes in monolayer culture. I. Dna synthesis

The ability of purified growth factors, insulin, ascorbate, and several other compounds to stimulate DNA synthesis by rabbit articular chondrocytes was studied in monolayer culture. Platelet-derived growth factor (1 U/ml), pituitary fibroblast growth factor (1-100 ng/ml), and epidermal growth factor (1-50 ng/ml) were stimulatory in a basal medium supplemented with 1% heat-inactivated fetal bovine serum. Insulin, 1-50 micrograms/ml, has small growth-promoting effects but acted synergistically with platelet-derived, pituitary fibroblast, and epidermal growth factors. Increasing concentrations of serum up to 10% enhanced the growth-promoting action of the purified factors, but not of insulin. There were indications of cooperation between insulin and bovine serum albumin and dexamethasone. Ascorbate (0.2 mM) reduced or had little growth-promoting action in the basal medium. At 5 and 10% serum concentrations, however, ascorbate promoted DNA synthesis as effectively as the purified growth factors. No significant stimulatory effect was shown by transferrin, thrombin, L-glutamine, putrescine, selenous acid, dexamethasone, 7S nerve growth factor, or multiplication-stimulating activity.     

Interaction of receptors for insulin-like growth factor I, platelet-derived growth factor, and fibroblast growth factor in rat aortic cells

Serum contains various growth factors which regulate the proliferation of cells. We investigated the growth of cultured arterial smooth muscle cells under the influence of insulin-like growth factor I (IGF I), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF), and examined the effect of these growth factors on the binding of [125I] IGF I and on the binding of [125I]PDGF to these cells. IGF I, FGF, and PDGF stimulated [6-3H]thymidine incorporation into DNA of confluent cultures of cells which were incubated in modified Dulbecco's modified Eagle medium. However, the effect of these growth factors on DNA synthesis was much more potent in Dulbecco's modified Eagle medium with 1% fetal calf serum. FGF and PDGF potentiated the growth-promoting effect of IGF I. The binding of [125I]IGF I to the cells was increased after a preincubation with FGF and PDGF. The binding was potently increased by FGF (100 ng/ml) after a preincubation time of 30 min. There was an increase in binding during the first 3 h of preincubation followed by a decrease after 4-5 h. PDGF (10-1000 ng/ml) stimulated [125I]IGF I binding only after 2 h of preincubation. The stimulation was dose dependent. Maximal stimulation of the binding was observed after 3 h of preincubation followed by a decrease after 4-5 h of preincubation. Specific binding sites for PDGF on smooth muscle cells could be demonstrated too. A preincubation of confluent cells with IGF I caused a dose-dependent increase in [125I]PDGF binding. These results support the hypothesis that the regulation of the binding of a specific growth factor by a second growth factor is important for the control of cell growth.     

Differential effect of transforming growth factor beta on freshly isolated and cultured articular chondrocytes.

Transforming growth factor beta (TGF beta) is a multipotent regulator of cell proliferation and extracellular matrix synthesis. In our study we show that the effect of TGF beta on proliferation and proteoglycan synthesis of articular cartilage chondrocytes appears to be dependent on the period in culture of these chondrocytes. TGF beta inhibits DNA and proteoglycan synthesis of freshly isolated chondrocytes while the DNA and proteoglycan synthesis of chondrocytes cultured in monolayer was stimulated by TGF beta. Since TGF beta is present in high concentrations in synovial fluid of patients with osteoarthritis, TGF beta might play a role in the elevated proteoglycan synthesis and cell proliferation in osteoarthritic cartilage.     

Inhibition of proteoglycan synthesis by transforming growth factor beta in anatomically intact articular cartilage of murine patellae.

The effect of transforming growth factor beta (TGF beta) on proteoglycan synthesis and degradation in anatomically intact articular cartilage of murine patellae was studied. Exogenously added TGF beta up to a concentration of 200 pmol/l had no effect on proteoglycan synthesis in intact articular cartilage. Neutralisation of endogenously produced TGF beta with a specific monoclonal antibody to TGF beta, however, led to stimulation of proteoglycan synthesis, indicating that TGF beta itself inhibits proteoglycan synthesis in anatomically intact cartilage. Transforming growth factor beta decreased the degradation of proteoglycans in intact cartilage in the absence of fetal calf serum or insulin-like growth factor 1. In the presence of fetal calf serum or insulin-like growth factor 1, TGF beta had no additional effect on proteoglycan breakdown.     

Dissecting the hematopoietic microenvironment. VI. The effects of several growth factors on the in vitro growth of murine bone marrow CFU-F

The effects of several growth factors on the proliferation of fibroblastic colony-forming units (CFU-F) were studied. In the present study CFU-F colonies were found to consist of fibroblasts, macrophages, and endothelial cells. Growth factors, including interleukin 3 (IL-3), interleukin 1 alpha (IL-1 alpha), epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and buffalo rat liver cell-conditioned medium (BRL-CM) were tested for stimulation of the proliferation of CFU-F in a standard culture in both 2% and 15% serum. Overall, the colony numbers produced in 15% serum were much higher than in 2% serum with or without growth factors. However, the influence of several growth factors on CFU-F cultured in 2% serum was relatively greater than in 15% serum when compared to controls. The stimulation of CFU-F by FGF only occurred in culture with 15% serum, and the stimulation by PDGF only occurred with 2% serum. Overall, the strongest stimulations were produced by PDGF, IL-3, and BRL-CM. Combining the other growth factors with IL-3, PDGF, or IL-1 alpha enhanced their effects only modestly. The stimulation by growth factors included increases of the cell numbers between and within colonies as well as an increase in the number of colonies. The study produced results that suggest a complex interaction mediated by growth factors between fibroblasts and other stromal cells within the CFU-F colonies and within the bone marrow itself.     

Growth factor-specific regulation of insulin receptor substrate-1 expression in MCF-7 breast carcinoma cells: effects on the insulin-like growth factor signaling pathway

IGFs are potent mitogens that play a crucial role in cell proliferation and/or differentiation and tumorigenesis. Insulin receptor substrate-1 (IRS-1) is a key protein in the IGF signaling pathway in the estrogen-dependent MCF-7 breast carcinoma cell line. In this study, three growth factors [fibroblast growth factor (FGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF)] were tested for their ability to modulate IRS-1 protein expression and the IGF-I signaling pathway. FGF and, to a lesser extent, EGF were found to increase IRS-1 protein, whereas PDGF had no effect. This indicates that growth factors can specifically modulate IRS-1 protein content. The increases provoked by EGF and FGF were dependent on the MAPK signaling pathway but independent of phosphatidylinositol 3-kinase (PI 3-kinase) signaling and required de novo protein synthesis. We noted that the kinetics of MAPK activation was continuous in response to FGF but transient in response to EGF. In addition, transfection of cells with a constitutively active form of MAPK kinase, which results in continuous MAPK activity, increased IRS-1 expression. Taken together, these results suggest that stimulation of IRS-1 expression was therefore stronger when MAPK activity was sustained. Pretreatment of cells with EGF, FGF, or PDGF for 24 h reduced IGF-I-induced tyrosine phosphorylation per molecule of IRS-1. However, IGF-I-induced PI 3-kinase activity was decreased by 24 h of pretreatment with EGF or PDGF but not with FGF. Our results therefore demonstrate that different growth factors are capable of specifically modulating the IGF-I signaling via IRS-1. They further suggest that the FGF-induced increase in IRS-1 counterbalances the inhibition of IRS-1 tyrosine phosphorylation to allow normal stimulation of IGF-I-induced PI 3-kinase activity.     

Genetic enhancement of matrix synthesis by articular chondrocytes: comparison of different growth factor genes in the presence and absence of interleukin-1

OBJECTIVE: To determine whether articular chondrocytes express growth factor genes delivered by adenoviral vectors and whether expression of these genes influences matrix synthesis in the presence and absence of interleukin-1 (IL-1). METHODS: Monolayer cultures of rabbit articular chondrocytes were infected with recombinant adenovirus carrying genes encoding the following growth factors: insulin-like growth factor 1 (IGF-1), transforming growth factor beta1 (TGFbeta1), and bone morphogenetic protein 2 (BMP-2). As a control, cells were transduced with the lac Z gene. Cultures were also treated with each growth factor supplied as a protein. Levels of gene expression were noted, and the synthesis of proteoglycan, collagen, and noncollagenous proteins was measured by radiolabeling. Collagen was typed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The effects of growth factor gene transfer on proteoglycan synthesis in the presence of IL-1 were also measured. RESULTS: The expression of all transgenes was high following adenoviral transduction. Proteoglycan synthesis was stimulated approximately 8-fold by the BMP-2 gene and 2-3-fold by the IGF-1 gene. The effects of BMP-2 and IGF-1 genes were additive upon cotransduction. Synthesis of collagen and noncollagenous proteins, in contrast, was most strongly stimulated by the IGF-1 gene. In each case, collagen typing confirmed the synthesis of type II collagen. IL-1 suppressed proteoglycan synthesis by 50-60%. IGF-1 and TGFbeta genes restored proteoglycan synthesis to control levels in the presence of IL-1. The BMP-2 gene, in contrast, elevated proteoglycan synthesis beyond control levels in the presence of IL-1. CONCLUSION: Transfer of growth factor genes to articular chondrocytes can greatly increase matrix synthesis in vitro, even in the presence of the inflammatory cytokine IL-1. This result encourages the further development of gene therapy for the repair of damaged cartilage.     

Mitogenic action of gastrin-releasing polypeptide on isolated epiphyseal growth plate chondrocytes from the ovine fetus.

Gastrin-releasing polypeptide (GRP) has been implicated in the development of the human fetal lung. To determine whether GRP has a wider role in fetal development, its actions on DNA synthesis and cell replication by isolated epiphyseal growth plate chondrocytes obtained from ovine fetuses between 35 days gestation and near term (145 days) were examined. Chondrocytes were isolated using collagenase from the proximal tibia and cultured in monolayer. Synthesis of DNA was assessed from the incorporation of [3H]thymidine into previously growth-restricted cells after incubation in medium supplemented with GRP1-27 (40-1280 nM). Increase in cell number was assessed after incubation with test medium for 1 week. GRP caused a dose-dependent increase in both cell number and DNA synthetic rate compared to control incubations. Cell number was increased by 50% in the presence of a maximally effective 160 nM GRP and DNA synthesis by up to 800% utilizing chondrocytes obtained from animals of 75-80 days gestation. The mean (+/- SEM) half-maximal concentration of GRP for the stimulation of DNA synthesis was 97 +/- 12 nM (5 separate fetuses). Concentrations of GRP in excess of 160 nM caused a sharp reduction in both cell replication and DNA synthesis. To determine where within the cell cycle GRP exerted its mitogenic action, synchronized chondrocytes were transiently exposed to fetal bovine serum and cultured with GRP for increasing periods of time before pulse labeling with [3H]thymidine during S phase. GRP was as effective in stimulating DNA synthesis when present for the initial 4 h of G1 as when present for the entire G1 period. Since isolated fetal growth plate chondrocytes release insulin-like growth factor II (IGF II) and basic fibroblast growth factor (basic FGF) the possible mediation of GRP action by the release of these peptides or synergistic interactions were examined. Specific antibodies shown to negate the mitogenic actions of exogenous IGFs or basic FGF on chondrocytes did not alter GRP-stimulated DNA synthesis. The release of radioimmunoassayable IGF II by chondrocytes was not altered in the presence of GRP. Coincubation of GRP with submaximal concentrations of IGF I or basic FGF showed additive effects on DNA synthesis. When the actions of galanin were examined it was found to inhibit basal DNA synthesis by chondrocytes at a concentration of 167 nM. However, 66 nM or greater galanin was able to render 160 nM GRP inactive as a mitogen. These results suggest that GRP may potentially influence skeletal development in the ovine fetus and may interact with locally released peptide growth factors or other neuropeptides.     

Basic fibroblast growth factor inhibits the anabolic activity of insulin-like growth factor 1 and osteogenic protein 1 in adult human articular chondrocytes

OBJECTIVE: To determine the effects of basic fibroblast growth factor (bFGF) on the chondrocyte anabolic activity promoted by insulin-like growth factor 1 (IGF-1) and osteogenic protein 1 (OP-1). METHODS: Human articular chondrocytes were cultured in alginate beads or as cartilage explants in serum-free medium with or without IGF-1 (100 ng/ml), OP-1 (100 ng/ml), or bFGF (0-100 ng/ml). Cell survival, proliferation, proteoglycan synthesis, and total proteoglycan accumulation were measured after 21 days of culture in alginate beads, and proteoglycan synthesis was measured in explants. RESULTS: Cell survival was not altered by bFGF at any dose, and chondrocyte proliferation was stimulated only at doses above 1 ng/ml. When combined with IGF-1, 1 ng/ml of bFGF stimulated proliferation to 170% of control, but when combined with IGF-1 and OP-1, proliferation increased to 373% of control. Doses of bFGF of 100 ng/ml decreased total proteoglycan levels accumulated per cell by 60% compared with control and also inhibited the ability of IGF-1 or OP-1 to increase proteoglycan production. Likewise, sulfate incorporation in response to IGF-1 and OP-1 alone or together was completely inhibited by 50 ng/ml bFGF in both alginate and explant cultures. CONCLUSION: The anabolic activity of IGF-1 and OP-1, alone and in combination, is significantly inhibited by bFGF. The results suggest that excessive release of bFGF from the cartilage matrix during injury, with loading, or in arthritis could contribute to increased proliferation and reduced anabolic activity in articular cartilage.     

Isolation and cultivation of human articular chondrocytes

Adult human articular chondrocytes were isolated from surgically excised articular cartilage from 15 patients suffering from trauma or disease of the knee. Cells were isolated with an enzymatic digestion method and cultured with F12 medium supplemented with serum. The effects of serum and basic fibroblast growth factor (bFGF) on the growth of cultured chondrocytes were studied. Serum stimulated the growth of chondrocytes at concentrations from 1-30%. bFGF significantly stimulated the growth of chondrocytes in a dose-dependent manner at concentrations from 1-100 ng/ml. Chondrocytes grew well in F12 medium supplemented with 10% fetal bovine serum (FBS). These cultured chondrocytes could be passaged for many generations and revealed an average of 2,702-fold increase of cell numbers during 2-6 months period (cumulative population doublings = 11 times). Cumulative population doublings increased to 24 times in cell cultured with medium supplemented with bFGF (10 ng/ml). Immunocytochemical study using anti-S-100 antibodies demonstrated that these cultures were pure cell cultures of chondrocytes. We have demonstrated that cell cultures of adult human articular chondrocytes can be established with these methods. Cultured chondrocytes provide an in vitro model system for studying the physiology and pathology of human articular chondrocytes and may be used for autologous transplantation of chondrocytes to treat articular cartilage defects.