Low Concentrations Of TGF-beta Research Articles

Serum derived from multiple trauma patients promotes the differentiation of endothelial progenitor cells in vitro: possible role of transforming growth factor-beta1 and vascular endothelial growth factor165.

Ischemia in various organs and tissues takes place during and as a direct result of multiple trauma (MT). Bone marrow-derived endothelial progenitor cells (EPCs) are involved in neovascularization after ischemic incidences. Here, we report that serum derived from patients with MT stimulates differentiation of EPCs in vitro from peripheral blood mononuclear cells (PBMCs). EPCs were identified by DiL-Acetyl-LDL-uptake with concomitant UEA-I-lectin binding. A significant increase in EPC numbers was noted when PBMCs were cultivated for 72 h with the serum of MT patients (n = 25) obtained at 5 days. Furthermore, serum from MT patients enhanced the functional acting of EPCs to form prevascular structures in matrigel. Reverse transcription polymerase chain reaction analysis revealed gene expression of transforming growth factor (TGF)-beta1- and vascular endothelial growth factor (VEGF) receptors 1 and 2. Reverse transcription polymerase chain reaction analysis was based on further cultivated cell preparations, which contained at least 80% EPCs. Moreover, the addition of recombinant VEGF or low concentrations of TGF-beta increased EPC differentiation. In addition, neutralization of TGF-beta1 and of VEGF165 in MT serum using specific antibodies resulted in a significant decrease in EPC differentiation. Our data indicate that TGF-beta1 and VEGF165 play a pivotal role for EPC differentiation induced by serum of polytrauma patients.

Indomethacin suppresses the anti-proliferative effects of transforming growth factor-beta isoforms on fibroblast cell cultures.

The transforming growth factor-beta (TGFbeta) family of mediators consists of five closely related isoforms, of which three are present in mammals. TGFbeta1 has been shown to exert a biphasic effect on the proliferation of several cell types, including fibroblasts, with stimulation at low concentrations and inhibition at higher concentrations. The stimulatory effects are well characterized, but the mechanisms by which TGFbeta1 inhibits cell proliferation are incompletely understood. In the present study we have compared the effects of all three mammalian TGFbeta isoforms on human lung fibroblast proliferation, and have elucidated the role of the TGFbeta-induced synthesis of prostaglandin E2 (PGE2) in mediating their actions. All three isoforms stimulated fibroblast proliferation with maximal effects at 5 pg/ml (0.2 pM) and an order of potency of TGFbeta3 > TGFbeta2 > TGFbeta1. At higher concentrations, proliferation declined, and at 40 pg/ml and above all isoforms inhibited fibroblast proliferation. Again TGFbeta3 was the most potent, but there were no significant differences between the inhibitory effects of TGFbeta1 and TGFbeta2. Addition of indomethacin, an inhibitor of PGE2 synthesis, did not alter the proliferative activity of any of the TGFbeta isoforms, but completely overcame their inhibitory effects, restoring the stimulatory actions observed at lower TGFbeta concentrations. All TGFbeta isoforms stimulated PGE2 synthesis; TGFbeta3 was approximately twice as potent as TGFbeta1 and TGFbeta2, each of which had similar effects. These data suggest that the inhibition of fibroblast proliferation at higher concentrations of TGFbeta isoforms may be mediated by autocrine stimulation of PGE2 synthesis.

Rapid Flux in Transforming Growth Factor-β Receptors on Bone Cells

The proportion of transforming growth factor-beta (TGF-beta) binding among conventional membrane receptors on bone cells can vary with hormone or growth factor treatment or with the state of osteoblast-like activity and appears to determine the nature of its biological effects. Therefore, functional TGF-beta receptor stability could be an important aspect of regulation. Suppression of protein synthesis reduced TGF-beta binding to types I and II receptors with t1/2 of 2 h and to betaglycan with t1/2 of 6 h. In contrast, suppression of mRNA transcription reduced TGF-beta binding at least 3-fold more slowly at each receptor site. Preexposure to TGF-beta decreased its binding at all three sites within 4 h in osteoblast-enriched cultures. This effect was transient with lower TGF-beta concentrations, where the receptor profile was nearly fully restored within 24-48 h. In contrast, less differentiated bone cells were less sensitive to ligand-dependent receptor down-regulation. Agents that alter protein kinase and phosphatase activity also modified the TGF-beta binding profile in specific ways. Together, these results indicate that cell surface TGF-beta receptors turn over rapidly by ligand-independent and ligand-dependent mechanisms, demonstrate that the binding capacity of TGF-beta receptors is less stable than their mRNAs, and that functional receptor levels may be determined in part by post-transcriptional events.

Characteristics and expression of transforming growth factor-beta receptor subtypes on vascular smooth muscle cells from spontaneously hypertensive rats.

To investigate the characteristics and expression of transforming growth factor (TGF)-beta receptor subtypes on vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. The effects of TGF-beta 1 on DNA synthesis were evaluated by [3H]-thymidine incorporation into quiescent VSMC plated at high (5 x 10(4) cells/cm2) or low (5 x 10(3) cells/cm2) cell density. Specific binding of TGF-beta to VSMC was assessed by incubation of the cells with [125I]-TGF-beta 1. Affinity labelling of receptor subtypes was achieved by exposure of the cells to [125I]-TGF-beta 1 and cross-linking with disuccimidyl suberate. VSMC from SHR displayed a biphasic DNA synthesis response to TGF-beta 1 at high cell density, with DNA synthesis stimulated by low concentrations of TGF-beta 1 but not by high concentrations, whereas at low cell density there was a small increase in DNA synthesis in response to TGF-beta 1. TGF-beta 1 inhibited DNA synthesis in VSMC from WKY rats at both high and low cell densities. Binding assays revealed that VSMC from SHR had a larger number of TGF-beta receptors and a higher affinity for TGF-beta at high and at low cell densities. The affinity labelling with [125I]-TGF-beta 1 revealed the presence of receptor subtypes with relative molecular masses of 280-300, 85, 70, 60 and 50 x 10(3) on vascular smooth muscle cells from both rat strains at high cell density. The abundance of the 85 x 10(3) molecular mass receptor subtype was greater in VSMC from SHR. The 85 x 10(3) molecular mass receptor subtype was not detected on VSMC from either strain at low cell density. The present results suggest a different expression of TGF-beta receptor subtypes on VSMC from SHR and WKY rats. These differences may account for the exaggerated proliferative response of VSMC from SHR to TGF-beta.

Transforming growth factor-beta 1 is required for secretion of IgG of all subclasses by LPS-activated murine B cells in vitro.

Addition of transforming growth factor-beta 1 (TGF-beta 1) to in vitro cultures of murine B cells activated with bacterial LPS selectively stimulates IgG2b and IgA class switching and decreases cellular proliferation. To assess a possible role for endogenous TGF-beta in modulating the Ig isotypes produced by LPS-activated cells, we utilized a neutralizing anti-TGF-beta mAb to abrogate endogenous TGF-beta activity. Anti-TGF-beta antibody, over a range of relatively low cell densities, strikingly inhibited both IgG3 and IgG2b production in response to LPS, with little or no change in the concentrations of secreted IgM. This effect of anti-TGF-beta antibody was specific, since it did not occur with an isotype-matched control mAb and was completely reversed with exogenous TGF-beta 1. Optimal IgG3 secretion occurred at concentrations of TGF-beta that were approximately eightfold lower than that necessary for maximal synthesis of IgG2b. Neutralization of endogenous TGF-beta in LPS-activated cultures was associated with an approximately twofold increase in proliferation and viable cell yields, a modest decrease in the percentage of membrane (m)IgG2b+ cells, and a modest increase in the percentage of mIgG3+ cells. This latter finding indicated that TGF-beta was not required for IgG3 class switching, but for maturation of mIgG3+ cells into Ig secretors. Highly purified B cells, obtained by electronic cell sorting, released active TGF-beta in response to LPS and showed a similar marked reduction in LPS-mediated IgG3 and IgG2b secretion in the presence of anti-TGF-beta antibody. Abrogation of endogenous TGF-beta activity in LPS-activated cultures also resulted in a striking reduction in IFN-gamma-mediated IgG2a production, and a more modest decrease in the synthesis of IgG1 and IgE in the presence of IL-4. These data indicate that relatively low concentrations of TGF-beta are essential for stimulating optimal IgG secretion by LPS-activated B cells, in an Ig isotype-nonspecific manner, and may regulate these responses in an autocrine fashion.

Effects of transforming growth factor beta on cells derived from bone and callus of patients with osteogenesis imperfecta

We studied the influence of transforming growth factor beta (TGF-beta) on cultured bone cells derived from two patients with osteogenesis imperfecta (OI) and from human controls. Additionally, cells from a hyperplastic callus that had developed spontaneously at the femur of the patient in Case 1 and cells from a normal fracture callus were included in the study. TGF-beta increased the synthesis of total protein and collagen of all cells without changing the pattern of interstitial collagens. Proliferation was stimulated by TGF-beta in the OI bone cells from Case 1, in cells from the central part of the hyperplastic callus, and in cells from the fracture callus. In Case 2, proliferation of bone cells was decreased by low concentrations of TGF-beta. Alkaline phosphatase (AP) activity was enhanced by TGF-beta in normal human bone cells, not affected in bone cells from the patient in Case 2 or in cells from the central part of the hyperplastic callus, and inhibited in bone cells and cells from the peripheral part of the hyperplastic callus of Case 1 and in cells from the fracture callus. We conclude that TGF-beta has common and specific effects on cultured human cells derived from different types of skeletal tissues. Simultaneous stimulation of collagen synthesis and AP activity by TGF-beta was restricted to normal human bone cells and might reflect their mature state of osteoblastic differentiation. Cells derived from bone of both patients with OI, from the hyperplastic callus, and from the fracture callus showed a different response pattern to TGF-beta.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor(TGF)‐β1 induces leukemic cell‐growth‐promoting activity in fibroblast cells.

The production of the leukemic cell-growth-promoting factor (LGF) in TGF-beta 1-treated fibroblast cells was studied. BALB/c3T3 mouse fibroblast (3T3) cells cultured in Eagle's medium containing a low concentration of TGF-beta 1 (0.04-1 ng/ml) secreted 3-5 times more LGF than the cells cultured in the absence of TGF-beta 1. The amount of LGF secretion was dose-dependent on the concentration of post-cultured medium and time-dependent after the addition of TGF-beta 1. Similar findings were obtained in human diploid fibroblasts, WI-38 cells. LGF is a 18KD glycoprotein that is acid-stable but heat-unstable.

Selective upregulation of platelet-derived growth factor alpha receptors by transforming growth factor beta in scleroderma fibroblasts.

Transforming growth factor beta (TGF-beta), a multifunctional cytokine, is an indirect mitogen for human fibroblasts through platelet-derived growth factor (PDGF), particularly the A ligand-alpha receptor arm of that system. TGF-beta effects on PDGF alpha receptor expression were studied in vitro using ligand binding techniques in three human dermal fibroblast strains: newborn foreskin, adult skin, and scleroderma (systemic sclerosis, SSc). Each cell strain responded differently to TGF-beta. In newborn foreskin fibroblasts, PDGF alpha receptor number decreased in a dose-dependent manner after exposure to low concentrations of TGF-beta (0.1-1 ng/ml). Responses of normal skin fibroblasts were varied, and mean net receptor number was unchanged. Increases in PDGF alpha receptor number by TGF-beta occurred consistently with SSc fibroblasts and low concentrations of TGF-beta (0.1-1 ng/ml) were particularly stimulatory. Increased surface expression of alpha receptor subunit by TGF-beta in SSc fibroblasts correlated with increased new PDGF alpha receptor synthesis as demonstrated by radioimmunoprecipitation analysis of metabolically labeled cells and with increased steady-state levels of corresponding mRNAs. In normal adult skin fibroblasts, TGF-beta had no effect on either synthesis or mRNA expression of alpha receptor subunits. Proliferative responses to PDGF-AA after pretreatment with TGF-beta correlated positively with effects of TGF-beta on expression of alpha receptor subunit. Decreased mitogenic responses to PDGF-AA were observed in foreskin fibroblasts, small changes in responses in adult fibroblasts, and significant increases in SSc fibroblasts. Thus, costimulation with PDGF-AA and TGF-beta selectively enhanced proliferation of fibroblasts with the SSc phenotype. Immunohistochemical examination of SSc and control skin biopsies revealed the presence of PDGF-AA in SSc skin. Data obtained by ligand binding, immunoprecipitation, mRNA, and mitogenic techniques are consistent with the hypothesis that activation of the PDGF-AA ligand/alpha receptor pathway is a characteristic of the SSc fibroblast and may contribute to the expansion of fibroblasts in SSc.

Loss of transforming growth factor beta 1 receptors and its effects on the growth of EBV-transformed human B cells.

Transforming growth factor-beta (TGF-beta) is a potent negative regulator of normal human B cell growth mediated by exogenous signals, including IL-2 and low m.w. B cell growth factor 12 kDa (BCGF-12 kDa). In the present study, we investigated the regulatory linkage between viral or nonviral transformation of human B cells and the growth inhibitory effects of TGF-beta 1. A panel of EBV+ and EBV- B cell lines, derived either by in vitro EBV B cell transformation, or from cases of lymphoma was used to quantitate the negative growth effects of TGF-beta 1. The proliferative response of three EBV- B cell lines to rBCGF-12 kDa or serum was inhibited by low concentrations of TGF-beta 1 (0.2-0.5 ng/ml for 50% maximal effect), as measured by tritiated thymidine uptake and viable cellular recovery. In contrast, rBCGF-12 kDa or serum mediated proliferation of three EBV+ B cell lines was refractory to the growth inhibitory effects of TGF-beta 1. In an attempt to understand the mechanism(s) for this differential growth control in EBV+ and EBV- B cells, we studied the expression of TGF-beta 1, c-myc, and TGF-beta 1 receptors. No correlation was observed between the expression of TGF-beta 1 or c-myc gene and growth inhibition by TGF-beta 1 in the cell lines studied. Our results indicate that sensitivity or resistance to TGF-beta 1 correlated with the presence or absence (loss) of high affinity receptors for TGF-beta 1. EBV- B cell lines expressed levels of high affinity receptors similar to those found on activated normal B or T cells. In contrast, EBV+ B cell lines showed no detectable high affinity receptors. Chemical cross-linking studies with a bifunctional reagent, dissuccinimidyl suberate revealed a normal expression of type I (65-70 kDa), type II (85-90 kDa), and type III (280-300 kDa) TGF-beta 1 high affinity receptors on EBV- B cell lines. In contrast, EBV+ B cell lines did not express type I and type II receptors, whereas type III receptors were expressed but could not be inhibited by unlabeled TGF-beta 1.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of transforming growth factor-beta 1 on human arterial smooth muscle cells in vitro.

Control of the thickness of the arterial wall is critical, as excessive overgrowth of constituent smooth muscle cells (SMCs) may interfere with blood flow. Effects on SMCs in vitro of several growth factors that are present in blood and/or that are produced endogenously in the arterial wall under certain conditions suggest that influences of endocrine, paracrine, and autocrine nature from stimulating and inhibiting factors may control the smooth muscle tissue mass in the artery. This possibility was explored further by investigating the degree of myodifferentiation in terms of the presence of differentiation-specific filamentous alpha-smooth muscle actin and growth, as measured by the synthesis of DNA and cell number, of SMCs as influenced by their exposure to the mitogens, platelet-derived growth factor and epidermal growth factor, and the bifunctional growth factor, transforming growth factor-beta 1 (TGF-beta 1). Exposure to TGF-beta 1 markedly enhanced differentiation-specific filamentous alpha-smooth muscle actin. This effect did not require arrest of growth, which speaks against a direct causal relation between loss of myodifferentiation (modulation) and multiplication. When quiescent cultures were exposed to TGF-beta 1, alpha-smooth muscle actin was further increased, indicating a more specific differentiation-promoting effect by TGF-beta 1 than mere inhibition of growth. Exposure to TGF-beta 1 also increased spreading, which occurred in parallel with increased filamentous alpha-smooth muscle actin and appearance of stress fibers. Exposure to platelet-derived growth factor under serum-free conditions and to epidermal growth factor in cultures exposed to serum markedly decreased the number of alpha-actin-positive SMCs, indicating a dedifferentiating effect by these mitogens. Exposure of SMCs to TGF-beta 1 under serum-free conditions had pronounced effects on growth, with a concentration-dependent inhibition of platelet-derived growth factor-induced DNA synthesis and cell multiplication. The basal synthesis of DNA in the absence of added growth factors was also greatly inhibited. With serum-free cultures, some loss of cells occurred even with very low concentrations of TGF-beta 1 (5 pg/ml), against which platelet-derived growth factor or a dense cultural state had a protective effect. Enhancement of cell multiplication was not detected for cultivated human SMCs exposed to TGF-beta 1, irrespective of culture density, in contrast to that reported for dense cultures of rat SMCs. TGF-beta 1 is present in and may be released from platelets in situations that promote platelet adherence such as endothelial injury; TGF-beta 1 may also be released from activated macrophages and T lymphocytes either during an immune reaction or inflammation or from the endothelium.(ABSTRACT TRUNCATED AT 400 WORDS)

Antagonism of the Inhibitory Effects of Transforming Growth Factor‐β on Colony Formation of Mouse Bone Marrow Cells in Vitro by Increasing Concentrations of Growth Factorsa

Low concentrations of TGF-beta inhibited the clonal proliferation of three different preparations of mouse bone marrow progenitor cells in vitro. Normal marrow cells responsive to CSF-1; two-day post-fluorouracil cells responsive to CSF-1 plus IL-3 and IL-1 alpha; and regenerating seven-day post-fluorouracil cells responsive to CSF-1 plus IL-3, IL-1 alpha and IL-6 were all severely inhibited by TGF-beta 1; and the inhibitory effects were antagonized by increasing the concentrations of the relevant growth factors.

Stimulation of fibronectin production by TGF‐β1 is independent of effects on cell proliferation: The example of bovine adrenocortical cells

We reported previously that transforming growth factor beta (TGF-beta) does not influence the proliferation of bovine adrenocortical cells but is a very potent inhibitor of their steroidogenic functions (Feige et al.: Journal of Biological Chemistry 262:13491-13495, 1987). In the present study, we addressed the question of whether these cells modify the synthesis of their extracellular matrix (in particular of fibronectin) in response to TGF-beta 1, similarly to the changes observed in cell types whose growth is modified by this factor (e.g., fibroblasts). Immunofluorescence studies using anti-fibronectin antibodies revealed that TGF-beta 1 treatment in serum-free medium induced the formation of fibronectin-containing fibrils associated with adrenocortical cells. Metabolic labeling of adrenocortical cells with [35S]-methionine showed that fibronectin synthesis and secretion were highly stimulated by low concentrations of TGF-beta 1. Half-maximal stimulation was observed for TGF-beta 1 concentrations in the range of 0.1 to 0.5 ng/ml and maximal stimulation reached 35-fold over control at the concentration of 2 ng/ml. The earlier detectable effect was observed after 8 h of treatment (6-fold stimulation) and the maximal increase was reached after 24 h of treatment. Stimulation of adrenocortical fibronectin synthesis by TGF-beta 1 appeared to imply a transcriptional event since it was no longer observed in the presence of DRB, a potent inhibitor of RNA polymerases, and because the level of fibronectin mRNA was stimulated under TGF-beta 1 treatment. Taken together, these results indicate that the increased expression of fibronectin is not closely related to growth-regulatory effects of TGF-beta 1 since it is also observed in adrenocortical cells, whose proliferation is unaffected by TGF-beta 1.

Osteoinductive factor inhibits formation of human osteoclast-like cells.

Osteoinductive factor (OIF) is a glycoprotein in bone that induces ectopic bone formation. Implantation of OIF plus transforming growth factor beta (TGF-beta) type 1 or 2 into subcutaneous tissues of rats induces formation of bone at the implantation site. Since TGF-beta is also present in bone matrix and inhibits formation of multinucleated cells that express an osteoclast phenotype in long-term human marrow cultures, we tested the effects of OIF on formation of these osteoclast-like cells to determine the effects of OIF on cells in the osteoclast lineage. We found that OIF inhibited total multinucleated cell (MNC) formation in a dose-dependent fashion and preferentially inhibited formation of MNCs that react with monoclonal antibody 23c6 (23c6-positive MNCs), an antibody that identifies osteoclasts. In addition, low concentrations of OIF in combination with low concentrations of TGF-beta acted synergistically to inhibit 23c6-positive MNC formation. The inhibition of 23c6-positive MNC formation by OIF was not mediated by prostaglandin synthesis. These data suggest that regulatory growth factors, such as OIF or TGF-beta, that are stored within the bone matrix and released when bone is resorbed can serve as natural inhibitors of osteoclast activity by inhibiting osteoclast formation.

Transforming growth factor-beta regulates growth as well as collagen and fibronectin synthesis of human marrow fibroblasts.

Three growth factors present in platelets, namely platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF), have been implicated in the pathogenesis of bone marrow fibrosis frequently associated with myeloproliferative disorders. In this study, regulation of the proliferation, as well as collagen and fibronectin synthesis from marrow fibroblasts by TGF-beta was investigated. TGF-beta alone at high plating density stimulated the proliferation of cells at low concentrations, but rather showed inhibition at high concentrations in both MPD patients and control subjects. In the presence of PDGF, which has been confirmed to be a main growth factor for marrow fibroblasts, low concentration of TGF-beta inhibited the proliferation at low cell density, but there was no inhibition at high cell density. The synthesis of both type I and type III procollagen was enhanced by high concentrations of TGF-beta in both MPD patients and control subjects, while PDGF or EGF showed no effect. The fibronectin synthesis was also enhanced by TGF-beta, but not by PDGF or EGF. These results suggest that growth and stromal protein synthesis of fibroblasts causing marrow fibrosis are regulated by TGF-beta as well as PDGF and EGF, when these factors are released or leaked from platelets or megakaryocytes into marrow environment in MPD patients.