Effect of transforming growth factor beta on cell proliferation and glycosaminoglycan synthesis by rabbit growth-plate chondrocytes in culture

The effects of the transforming growth factor beta (TGF-beta) on the growth and glycosaminoglycan synthesis of rabbit growth plate-chondrocytes in culture were studied. In serum-free medium, TGF-beta caused dose-dependent inhibition of DNA synthesis by chondrocytes, measured as [3H]thymidine incorporation (ED50 = 0.1-0.3 ng/ml). The inhibitory effect was maximal at a dose of 1 ng/ml, and extended for a duration of 16-42 h. In contrast, TGF-beta potentiated the synthesis of DNA stimulated by fetal calf serum (FCS). Addition of TGF-beta (1 ng/ml) to cultures containing 10% FCS increased [3H]thymidine incorporation to 1.6-times that in cultures with 10% FCS alone. Consistent with this finding, TGF-beta potentiated DNA synthesis stimulated by the purified growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and fibroblast growth factor (FGF). The maximal stimulation of DNA synthesis by FGF (0.4 ng/ml) was further potentiated dose dependently by TGF-beta (ED50 = 0.1 ng/ml, maximum at 1 ng/ml). When the cultures were treated with the optimal concentrations of TGF-beta (1 ng/ml) and FGF (0.4 ng/ml), [3H]thymidine incorporation was 3-times higher than that of cultures treated with FGF alone. This TGF-beta-induced potentiation of DNA synthesis was associated with replication of chondrocytes, as shown by a marked increase in the amount of DNA during treatment of sparse cultures of the cells with the growth factors for 5 days. In contrast, TGF-beta caused dose-dependent stimulation of glycosaminoglycan synthesis in confluent cultures of growth-plate chondrocytes (ED50 = 0.3 ng/ml, maximum at 1 ng/ml). This stimulatory effect of TGF-beta was greater than that of insulin-like growth factor I (IGF-I) or PDGF. Furthermore, TGF-beta stimulated glycosaminoglycan synthesis additively with IGF-I or PDGF. Recently, it has been suggested that bone and articular cartilage are rich sources of TGF-beta, whereas epiphyseal growth cartilage is not. Thus, the present data indicate that TGF-beta may be important in bone formation by modulating growth and phenotypic expression of chondrocytes in the growth plate, possibly via a paracrine mechanism.     

Regulation of rat pulmonary artery endothelial cell transforming growth factor-beta production by IL-1 beta and tumor necrosis factor-alpha.

Recent studies suggest that transforming growth factor-beta (TGF-beta) production is up-regulated at sites of tissue injury, inflammation and repair, or fibrosis. Endothelial cells represent a potentially important in vivo source of TGF-beta; however, the identity of endogenous modulators of TGF-beta production by these cells remains unclear. To address this issue, the effects of the cytokines, IL-1 beta, and TNF-alpha on TGF-beta production by rat pulmonary artery endothelial cells were examined. Conditioned media from cells treated with 0 to 20 ng/ml IL-1 beta and/or TNF-alpha were assayed for TGF-beta activity using a mink lung epithelial cell line. The results show that rat pulmonary artery endothelial cells secreted undetectable amounts of active TGF-beta in the absence of cytokines. However, upon acidification of the conditioned media before assay, a time-dependent increase in TGF-beta activity was noted in media from both untreated and cytokine-treated cells. However, both IL-1 beta and TNF-alpha treatment caused the secretion of significantly greater amounts of TGF-beta activity than control cells, in a dose-dependent manner, with maximal response obtained at cytokine doses of greater than 10 ng/ml. At equivalent doses of cytokine tested, the magnitude of the response was significantly greater with IL-1 beta. These responses were paralleled by increases in steady state mRNA levels for TGF-beta 1. Addition of both cytokines resulted in a synergistic response. Synergism with IL-1 beta was also noted with the fibrogenic agent bleomycin. Kinetic studies indicated that a minimum of 4 h of treatment with either IL-1 beta or TNF-alpha was required for detection of significant increases in either secreted TGF-beta activity or steady state TGF-beta 1 mRNA levels. Thus, endothelial cells could play a role in various TGF-beta-dependent processes in vivo, in situations wherein IL-1 beta and/or TNF-alpha may be present at comparable concentrations.     

Bi-functional action of transforming growth factor-beta on DNA synthesis in early passage human fetal fibroblasts

We investigated the influence of transforming growth factor-beta (TGF-beta) on DNA synthesis in human fetal fibroblasts, as measured by the incorporation of [3H]thymidine and cell replication. In serum-free medium, without additional peptide growth factors, TGF-beta had no action on thymidine incorporation. However, in the presence of 0.1% v/v fetal calf serum, TGF-beta exhibited a bi-functional action on the cells. A dose-dependent stimulation of [3H]thymidine incorporation, and an increase in cell number, occurred with fibroblasts established from fetuses under 50 g body weight, with a maximum stimulation seen at 1.25 ng/ml. For fibroblasts from fetuses of 100 g or greater body weight, TGF-beta caused a dose-related decrease in thymidine uptake with a maximal inhibition at 2.5 ng/ml, and a small decrease in cell number. When DNA synthesis was stimulated by the addition of somatomedin-C/insulin-like growth factor I, epidermal growth factor, or platelet-derived growth factor, their actions were potentiated by the presence of TGF-beta on cells derived from fetuses under 50 g body weight, but inhibited on cells obtained from the larger fetuses weighing more than 100 g. Similar results were found for changes in cell number in response to TGF-beta when stimulated by SM-C/IGF I. The ability of TGF-beta to modulate [3H] thymidine incorporation did not involve a change in the time required for growth-restricted cells to enter the S phase of the replication cycle. These data suggest that TGF-beta may exert either a growth-promoting or growth-inhibiting action on human fetal connective tissues in the presence of other peptide growth factors, which is dependent on fetal age and development.     

Effects of transforming growth factor-beta and IL-1 alpha on prostaglandin synthesis in serum-deprived osteoblastic cells.

We investigated the effects that the combination of IL-1 alpha and transforming growth factor-beta (TGF-beta) had on PGE2 production in a murine clonal osteoblastic cell line MC3T3-E1 and primary rat calvarial osteoblast-like cells. In serum-supplemented medium, IL-1 alpha was a potent stimulator of PGE2 production in MC3T3-E1 cells (50-fold increase with 0.1 ng/ml). TGF-beta (10 ng/ml) had only a small effect alone and no additional effect on IL-1 alpha-induced responses. In serum-deprived MC3T3-E1 cells, PGE2 responses to IL-1 alpha were either absent or markedly reduced. TGF-beta alone had small effects. However, simultaneous addition of TGF-beta with IL-1 alpha to MC3T3-E1 cells partially restored the ability of IL-1 alpha to generate a PGE2 response (10-fold increase in PGE2 with 0.1 ng/ml of both IL-1 alpha and TGF-beta). As with MC3T3-E1 cells, serum-deprived primary fetal rat calvarial osteoblastic cells also did not respond to IL-1 alpha, unless TGF-beta was present in the medium (sixfold increase in PGE2 with 0.1 ng/ml IL-1 alpha and 10 ng/ml TGF-beta). The synergistic effect of TGF-beta and IL-1 alpha was specific for PGE2 responses, because these factors did not synergistically affect cell proliferation, collagen and noncollagen protein synthesis, or alkaline phosphatase activity. The observed synergy was not associated with changes in the steady state cyclooxygenase (PGH synthase) mRNA levels. However, it did correlate with increased release of [3H]arachidonic acid from prelabeled serum-depleted MC3T3-E1 cells. Hence, the synergistic interactions of IL-1 alpha and TGF-beta on PGE2 appear to occur through an increase in the release of arachidonic acid substrate from phospholipid pools. These effects may be important for both normal bone turnover and the responses of bone to inflammatory and immune stimuli.     

Can transforming growth factor-beta1 and retinoids modify the activity of estradiol and antiestrogens in MCF-7 breast cancer cells?

Retinoic acid and transforming growth factor-beta (TGF-beta) affect differentiation, proliferation and carcinogenesis of epithelial cells. The effect of both compounds on the proliferation of cells of the hormone sensitive human breast cancer cell line (ER+) MCF-7 was assessed in the presence of estradiol and tamoxifen. The assay was based on [3H]thymidine incorporation and the proliferative activity of PCNA- and Ki 67-positive cells. The apoptotic index and expression of the Bcl-2 and p53 antigens in MCF-7 cells were also determined. Exogenous TGF-beta1 added to the cell culture showed antiproliferative activity within the concentration range of 0.003-30 ng/ml. Irrespective of TGF-beta1 concentrations, a marked reduction in the stimulatory action of estradiol (10(-9) and 10(-8) M) was observed whereas in combination with tamoxifen (10(-7) and 10(-6) M) only 30 ng/ml TGF-beta1 caused a statistically significant reduction to approximately 30% of the proliferative cells. In further experiments we examined the effect of exposure of breast cancer cells to retinoids in combination with TGF-beta1. The incorporation of [3H]thymidine into MCF-7 cells was inhibited to 52 +/- 19% (control =100%) by 3 ng/ml TGF-beta1, and this dose was used throughout. It was found that addition of TGF-beta1 and isotretinoin to the culture did not decrease proliferation, while TGF-beta1 and tretinoin at low concentrations (3 x 10(-8) and 3 x 10(-7) M) reduced the percentage of proliferating cells by approximately 30% (67+/-8% and 67+/-5%, P<0.05 compared to values in the tretinoin group). Both retinoids also led to a statistically significant decrease in the stimulatory effect of 10(-9) M estradiol, attenuated by TGF-beta1. In addition, the retinoids in combination with TGF-beta1 and tamoxifen (10(-6) M) caused a further reduction in the percentage of proliferating cells. Immunocytochemical analysis showed that all the examined compounds gave a statistically significant reduction in the percentage of cells with a positive reaction to PCNA and Ki 67 antigen. TGF-beta1, isotretinoin and tretinoin added to the culture resulted in the lowest percentage of PCNA positive cells. However, the lowest fraction of Ki 67 positive cells was observed after addition of isotretinoin. The obtained results also confirm the fact that the well-known regulatory proteins Bcl-2 and p53 play an important role in the regulation of apoptosis in the MCF-7 cell line, with lowered Bcl-2 expression accompanying easier apoptotic induction. The majority of the examined compounds act via the p53 pathway although some bypass this important proapoptotic factor.     

Endoglin expression regulates basal and TGF-beta1-induced extracellular matrix synthesis in cultured L6E9 myoblasts.

BACKGROUND/AIMS: TGF-beta1 plays a major role in extracellular matrix (ECM) accumulation in tissue fibrosis. Connective tissue growth factor appears to play a critical role in this effect. Endoglin is a component of the transforming growth factor b (TGF-beta) receptor complex. Endoglin is upregulated by TGF-beta1, but its functional role in ECM regulation is unknown. Using rat myoblasts as a model system, we have assessed the role of endoglin on regulating CTGF expression and ECM synthesis and accumulation in the presence or absence of TGF-beta1. METHODS: L6E9 myoblast cell line was transfected with human endoglin, and collagen, fibronectin and CTGF production was assessed by Western blot and by proline incorporation to collagen proteins. RESULTS: Northern blot analysis revealed that parental rat myoblasts L6E9 do not express endogenous endoglin. Upon endoglin transfection, endoglin-expressing cells displayed a decreased CTGF expression and decreased collagen and fibronectin accumulation respect to mock transfectants. Northern blot analysis also revealed a decreased alpha2 (I) procollagen mRNA expression in endoglin transfectants. TGF-beta1 treatment induced an increase in CTGF expression and collagen synthesis and accumulation in L6E9 myoblasts. This effect was significantly lower in endoglin-transfected than in mock-transfected cells. CONCLUSION: These results demonstrate that endoglin expression negatively regulates basal and TGF-beta1-induced CTGF and collagen expression and synthesis.     

Human skeletal growth factor stimulates collagen synthesis and inhibits proliferation in a clonal osteoblast cell line (MC3T3-E1)

Human skeletal growth factor (hSGF), an 11-kD polypeptide purified from human bone, has been proposed to be a local regulator of bone formation. To investigate the underlying cellular mechanisms in an in vitro model system, we examined the effects of hSGF on proliferation and collagen synthesis in cells of the clonal osteoblast cell line MC3T3-E1. This line was isolated from newborn mouse calvarial cells and retains many characteristics of mature osteoblasts (Sudo, H., et al., (1984) J. Cell Biol. 96:191). A 14-hr treatment with hSGF increased noncollagenous protein synthesis to 215% of unstimulated controls and increased collagen synthesis to 630% of controls as determined by [3H]proline incorporation and high-pressure liquid chromatographic separation of [3H]proline and [3H]hydroxyproline in acid hydrolysates of trichloroacetic acid-insoluble protein. HSGF did not increase cell number over a 48-hr period and caused a reversible inhibition of DNA synthesis. Half-maximal hSGF concentration for stimulation of [3H]proline incorporation and inhibition of [3H]thymidine incorporation was 100 ng/ml. HSGF also inhibited DNA synthesis in cells stimulated by serum. In contrast, hSGF stimulated both collagen synthesis and DNA synthesis in primary cultures of chick embryo bone cells, which may be developmentally less mature than MC3T3-E1 cells. The results suggest that hSGF directly stimulated mature osteoblast matrix synthetic activity and that hSGF has differential effects on proliferation of osteoblast progenitor cells and mature osteoblasts.     

Negative regulation of hepatocyte growth factor gene expression in human lung fibroblasts and leukemic cells by transforming growth factor-beta 1 and glucocorticoids.

Hepatocyte growth factor (HGF), a mesenchymal-derived factor which regulates growth, motility, and morphogenesis of epithelial and endothelial cells, functions as a hepatotrophic and renotrophic factor for regeneration of the liver and kidney. We have now obtained evidence that transforming growth factor-beta 1 (TGF-beta 1) and glucocorticoids are negative regulators for HGF gene expression. When TGF-beta 1 or dexamethasone was added to cultures of MRC-5 human embryonic lung fibroblasts and HL-60 human promyelocytic leukemic cells, the amount of HGF secreted into the culture medium was inhibited to 30-40% of that of control cultures by 10 ng/ml TGF-beta 1 and to 40-50% by 10(-6) M dexamethasone. The inhibitory effect of TGF-beta 1 and dexamethasone on HGF synthesis in MRC-5 cells was additive, thereby suggesting that TGF-beta 1 and dexamethasone exert effects through distinct mechanisms. Hydrocortisone also inhibited HGF synthesis with the same potency as dexamethasone; however, testosterone, estriol, and beta-estradiol had no effect. The rate of HGF synthesis in MRC-5 cells, as measured by pulse labeling with [35S]methionine and subsequent immunoprecipitation, was suppressed to 30-40% of the control with 10 ng/ml TGF-beta 1, and to 30-45% by 10(-6) M dexamethasone. HGF mRNA levels in MRC-5 cells and HL-60 cells were dose-dependently suppressed by TGF-beta 1 and dexamethasone; 10 ng/ml TGF-beta 1 suppressed HGF mRNA levels to 32% and 35% of control culture, respectively, in MRC-5 cells and HL-60 cells, and 10(-6) M dexamethasone suppressed to 43% and 38%, respectively. Thus, TGF-beta 1 and glucocorticoids seem to inhibit HGF synthesis by suppressing the expression of the HGF gene. We propose that a negative regulation of HGF gene expression by TGF-beta 1 or glucocorticoids may be involved in physiological or pathological processes during tissue regeneration.     

Regulation of rat proximal tubule epithelial cell growth by fibroblast growth factors, insulin-like growth factor-1 and transforming growth factor-beta, and analysis of fibroblast growth factors in rat kidney.

Growth factors may play an important role in regulating the growth of the proximal tubule epithelium. To determine which growth factors could be involved, we have investigated the mitogenicity of various purified factors in rat kidney proximal tubule epithelial (RPTE) cells cultured in defined medium. Fibroblast growth factors, aFGF (acidic FGF) and bFGF (basic FGF), stimulate DNA synthesis in a dose-dependent manner, with ED50 values of 4.5 and 3.2 ng/ml, respectively; their effects are not additive. With cholera toxin in the medium, both aFGF and bFGF can replace insulin or epidermal growth factor (EGF) to attain the maximum level of cell growth, but they cannot replace cholera toxin. Cholera toxin specifically potentiates the effects of FGFs on DNA synthesis. At high cell density, both insulin and insulin-like growth factor 1 (IGF-1) induce DNA synthesis more effectively than EGF, FGFs and cholera toxin. The high concentration (0.2-1.0 microgram/ml) of insulin required for cell growth can be replaced by a low concentration of IGF-1 (10-20 ng/ml), indicating that insulin probably acts through a low affinity interaction with the IGF-1 receptor. Transforming growth factor-beta 1 (TGF-beta 1) inhibits DNA synthesis induced by individual factors and combinations of factors in a concentration-dependent manner. Northern blot analysis shows that mRNA for TGF-beta 1, IGF-1, and aFGF, but not bFGF are present in rat kidney. Western blot analysis and bioassay data confirmed that the majority of FGF-like protein in rat kidney is aFGF. The data suggest that in addition to EGF, IGFs, and TGF-beta, FGFs may also be important kidney-derived regulators of proximal tubule epithelial cell growth in vivo and in vitro.     

Synergistic actions of cytokines and growth factors in enhancing porcine granulosa cell growth.

In our studies of the growth-promoting effect of a cytokine, interleukin-1 (IL-1), on cultured porcine granulosa cells, we found that the potency of IL-1 action correlated with the serum concentration in the culture medium and that IL-1 acted synergistically with insulin to increase the number of cells in the presence of low serum concentrations (0.1-1%). With granulosa cells maintained in a quiescent state under serum-free conditions, we therefore examined the effects of combined treatment with IL-1 and peptide growth factors, including insulin, on [3H]thymidine incorporation by these cells. IL-1 by itself enhanced [3H]thymidine incorporation in a concentration-dependent manner. Moreover, IL-1 acted synergistically with insulin, epidermal growth factor (EGF), or fibroblast growth factor (FGF) to enhance [3H]thymidine incorporation. Combinations of maximally effective concentrations of insulin (1 micrograms/ml), EGF (1 ng/ml), or FGF (50 ng/ml) with the maximally effective concentration of IL-1 (10 ng/ml) increased the levels of [3H]thymidine incorporation to 10-, 22-, and 20-fold, respectively, over the control values. Whereas IL-2 (0.1-100 ng/ml) did not affect [3H]thymidine incorporation, tumor necrosis factor alpha (TNF alpha) stimulated [3H]thymidine incorporation by itself and reproduced the actions of IL-1 to act synergistically with insulin, EGF, or FGF. When IL-1 and TNF alpha were added together in relatively low concentrations (1 ng/ml each), the combination had synergistic effects in enhancing [3H]thymidine incorporation. The present study demonstrates that cytokines and peptide growth factors act synergistically to markedly enhance porcine granulosa cell growth in vitro.     

Mechanisms by which extracellular ATP and UTP stimulate the release of prostacyclin from bovine pulmonary artery endothelial cells.

Extracellular ATP and UTP caused increases in the concentration of cytoplasmic free calcium ([Ca2+]i) and the intracellular level of inositol 1,4,5-trisphosphate (IP3), a second messenger for calcium mobilization, prior to the release of prostacyclin (PGI2) from cultured bovine pulmonary artery endothelial (BPAE) cells. The agonist specificity and dose-dependence were similar for nucleotide-mediated increases in IP3 levels, [Ca2+]i and PGI2 release. An increase in [Ca2+]; and PGI2 release was observed after addition of ionomycin, a calcium ionophore, to BPAE cells incubated in a calcium-free medium. The addition of ATP to the ionomycin-treated cells caused no further increase in [Ca2+]i or PGI2 release. The inability of ATP to cause an increase in [Ca2+]i or PGI2 release in ionomycin-treated cells was apparently due to the ionomycin-dependent depletion of intracellular calcium stores since the subsequent addition of extracellular calcium caused a significant increase in both [Ca2+]i and PGI2 release. Introduction of BAPTA, a calcium buffer, into BPAE cells inhibited ATP-mediated increases in [Ca2+]i and PGI2 release, further evidence that PGI2 release is dependent upon an increase in [Ca2+]i. The increase in [Ca2+]i elicited by ATP apparently caused the activation of a calmodulin-dependent phospholipase A2 since trifluoperazine, an inhibitor of calmodulin, and quinacrine, an inhibitor of phospholipase A2, prevented the stimulation of PGI2 release by ATP. Furthermore, ATP caused the specific hydrolysis of [14C]arachidonyl-labeled phosphatidylcholine and the generation of free arachidonic acid, the rate-limiting substrate for PGI2 synthesis, prior to the release of PGI2 from BPAE cells. These findings suggest that the increase in PGI2 release elicited by ATP and UTP is at least partially dependent upon a phospholipase C-mediated increase in [Ca2+]i and the subsequent activation of a phosphatidylcholine-specific phospholipase A2. ATP analogs modified in the adenine base or phosphate moiety caused PGI2 release with a rank order of agonist potency of adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) greater than 2-methylthioATP (2-MeSATP) greater than ATP, whereas alpha, beta methyleneATP and beta, gamma methyleneATP had no effect on PGI2 release.     

Phytolacca americana inhibits the high glucose-induced mesangial proliferation via suppressing extracellular matrix accumulation and TGF-beta production.

This study describes a potential of Phytolaccaceae (Phytolacca americana var.) as an inhibitor of high glucose-stimulated production of extracellular matrix (ECM) proteins and TGF-beta in cultured glomerular mesangial cells (GMCs). Raising the ambient glucose concentration for 24 hrs caused a dose-dependent increase in [3H]thymidine incorporation of GMCs, and the maximal response was achieved at 20 mM. Phytolaccaceae extracts (2.5-20 microg/ml) inhibited the high glucose-induced [3H]thymidine incorporation in a dose-dependent manner, and the concentrations tested here did not affect to the cell viability. Exposure of the GMCs to 20 mM glucose caused both ECM (collagen and fibronectin) accumulation and TGF-beta secretion, and these changes were significantly diminished by treatment of GMCs with Phytolaccaceae (10 microg/ml). Taken together, these results indicate that Phytolaccaceae inhibits the high glucose-induced GMCs proliferation partially through suppressing accumulation of ECM components and TGF-beta production, suggesting that Phytolaccaceae may be a promising agent for treating the development and progression of diabetic glomerulopathy.     

Triacylglycerol synthesis in isolated fat cells. An effect of insulin on microsomal fatty acid coenzyme A ligase activity.

Fatty acid CoA ligase (AMP) (EC 6.2.1.3) specific activity was increased approximately 2-fold in microsomes prepared from isolated rat fat cells incubated with 400 microunits of insulin/ml (2.9 nM) for 45 to 60 min compared to paired controls using an assay based on the conversion of [3H]oleic acid to [3H]oleoyl-CoA. Similar insulin-dependent increases in microsomal fatty acid CoA ligase specific activities were observed using an assay based on the conversion of [3H]CoA to fatty acyl-[3H]CoA. Fatty acid CoA ligase activity was predominately (about 80%) associated with the microsomal fraction. The insulin-dependent increase in microsomal fatty acid CoA ligase specific activity was maximal in 2 to 5 min at 400 microunits/ml. At 10 min, 80 to 100 microunits of insulin/ml caused a maximal increase in fatty acid CoA ligase specific activity. Similar apparent Km values for ATP, CoA, and fatty acid were observed for fatty acid CoA ligase activity in microsomal preparations from control and insulin-exposed cells. These data suggest that fatty acid CoA ligase activity is regulated in adipose tissue by insulin. Such regulation may serve to promote the capture of fatty acid and thereby, triacylglycerol synthesis in adipose tissue.     

Hepatocyte growth factor is a paracrine factor for renal epithelial cells: stimulation of DNA synthesis and NA,K-ATPase activity.

The expressions of mRNAs of hepatocyte growth factor (HGF) and its receptor (c-met) and its effects were examined in cultured renal epithelial cell lines (OK, LLCPK1, and MDCK cells) and rat mesangial cells in primary culture. Northern blot analysis revealed the presence of HGF mRNA in mesangial cells, but not in epithelial cells. c-met mRNA was detected in epithelial cells, but not in mesangial cells. HGF stimulated [3H]-thymidine incorporation (DNA synthesis) dose-dependently in OK and LLCPK1 cells, but not in MDCK and mesangial cells. Ouabaine sensitive rubidium uptake (Na,K-ATPase activity) was stimulated by 63% with HGF (10 ng/ml) treatment for 16hr in MDCK cells. The results suggest that HGF is produced in the kidney, at least in mesangial cells and works on epithelial cells to stimulate the proliferation and/or to modify cell functions in a paracrine manner.     

Heparan mimetic regulates collagen expression and TGF-beta1 distribution in gamma-irradiated human intestinal smooth muscle cells.

Radiation-induced intestinal fibrosis is characterized by collagen accumulation, a process in which TGF-beta1 plays a key role. We analyzed the effects of gamma radiation on collagen expression and TGF-beta1 distribution in human intestinal smooth muscle cells (HISM). We investigated the activity of a carboxymethylated and sulfated dextran (RG-1503), exhibiting antifibrotic properties and promoting in vivo intestinal tissue repair, on irradiated HISM. After (60)Co irradiation (10 Gy), HISM were labeled with [(3)H] proline (+/-RG-1503). Radiolabeled collagen I, III, and V were quantified by SDS-PAGE. TGF-beta1 was quantified by ELISA in culture medium, pericellular and intracellular compartments. Irradiation induced a specific 2.85-fold increase in collagen III production by HISM. Collagen V decreased by 80% 72 h after irradiation. Pericellular TGF-beta1 was increased (up to twofold) in irradiated HISM. RG-1503 added before or after irradiation reversed both mRNA and protein levels of collagen III and V to control values. RG-1503 decreased the amount of TGF-beta1 in the cell layer below the control values. Irradiation of HISM induced the development of a fibrotic phenotype in terms of collagen production and TGF-beta1 distribution. The antifibrotic RG-1503 restored HISM physiological characteristics and may represent a promising therapeutic approach for radiation-induced intestinal fibrosis.     

Transforming growth factor-beta 1 induces angiogenesis in vitro via VEGF production in human airway smooth muscle cells

Increase in size and number of bronchial blood vessels as well as hyperaemia are factors that contribute to airway wall remodelling in patients with chronic airway diseases, such as asthma and chronic obstructive pulmonary diseases (COPD). Expression of transforming growth factor beta 1 (TGF-beta 1), a multifunctional cytokine as well as vascular endothelial growth factor (VEGF), a key angiogenic molecule, has been shown in the inflammed airways in patients with chronic airway diseases. TGF-beta 1 has been implicated in the regulation of extracellular matrix, leading to airway remodelling in patients with chronic airway diseases. However, the role of TGF-beta 1 in regulating VEGF expression in patients with chronic airway diseases, as well as the underlying mechanisms are not yet well established. We investigated whether TGF-beta 1 stimulates VEGF expression in vitro and hence could influence vascular remodelling. Cultured human airway smooth muscle cells (HASMC) were serum deprived for 60 h before incubation with 5ng/ml of TGF-beta 1 for different time points. Control cells received serum-free culture medium. TGF-beta 1 treatment resulted in time dependent HASMC cell proliferation with maximal values for DNA biosynthesis at 24 h and cell number at 48 h. Northern blot analysis of VEGF mRNA expression showed increased levels in cells treated with TGF-beta 1 for 4 to 8 h. TGF-beta 1 also induced a time-dependent release of VEGF proteins in the conditioned medium after 48 h of treatment. Furthermore, the ability of HASMC-released VEGF proteins to induce human umbilical vein endothelial cells proliferation was inhibited by VEGF receptor antagonist, confirming that TGF-beta 1 induced VEGF was biologically active. We conclude that TGF-beta 1 in addition to an extracellular matrix regulator also could play a key role in bronchial angiogenesis and vascular remodelling via VEGF pathway in asthma.