Hepatocyte growth factor releases epithelial and endothelial cells from growth arrest induced by transforming growth factor-beta1

Human lung fibroblasts and Mv1Lu mink lung epithelial cells were used as a model to study the role of extracellular matrix in epithelial-mesenchymal interactions. Extracellular matrices of fibroblasts were found to contain growth promoting activity that reduced the sensitivity of Mv1Lu cells to the growth inhibitory effects of transforming growth factor-beta (TGF-beta). The majority of the activity was identified as hepatocyte growth factor/scatter factor (HGF) by inhibition with specific antibodies and by reconstitution of the effect by recombinant HGF. HGF induced cell proliferation when contact-inhibited Mv1Lu cells were trypsinized and plated in the presence of TGF-beta1. The effect was valid also in assays where Madin-Darby canine kidney epithelial cells or bovine capillary endothelial cells were used. The multiplication of chronically TGF-beta1 inhibited Mv1Lu cells was also induced by HGF. In addition, HGF induced anchorage independent growth of Mv1Lu cells that was refractory to TGF-beta1 growth inhibition. Immunoprecipitation analysis indicated that HGF prevented the suppression of Cdk4 and Cdk2, but not the induction of p21, by TGF-beta1. Since both TGF-beta1 and HGF require proteolysis for activation, the results imply that proteolytic activity of epithelial and endothelial cells directs their responses to signals from mesenchymal-type extracellular matrices, and that during development, matrix-bound growth and invasion promoting and suppressing factors are activated in a coordinated manner.     

Glycemia and regulation of endothelial adhesion molecules

Hyperglycemia is one of the most prominent factors associated with diabetic vascular disease, the development of which is initated by functional and morphological alterations of the endothelium. The review summarizes several issues of glycemia and advanced glycation end products induced changes concerning soluble as well as cell bound endothelial adhesion molecules, and further addresses the aspect of apoptosis in the light of diabetic vascular complications.     

Sequential effects of high glucose on mesangial cell transforming growth factor-beta 1 and fibronectin synthesis

BACKGROUND: Transforming growth factor (TGF)-beta is recognized as the final common mediator of the principal lesions of diabetic nephropathy such as renal hypertrophy and mesangial expansion. To gain better understanding of the temporal relationships between high glucose (HG) and mesangial cell (MC) TGF-beta 1 synthesis and between TGF-beta 1 and extracellular matrix (ECM) synthesis, the present study examined early and sequential effects of HG on TGF-beta 1 and fibronectin (FN) mRNA expression and protein synthesis. METHODS: Confluent primary rat MC was stimulated with 5.6 (control) or 30 (high) mM glucose after synchronizing the growth by incubation with serum-free media for 48 hours. RESULTS: Mesangial cell TGF-beta 1 mRNA expression increased significantly in six hours and continued to increase until 48 hours in response to HG. The level of TGF-beta 1 mRNA was 1.5-fold higher than that of control glucose at six hours and 1.8-fold at 48 hours. TGF-beta activity in heat-activated conditioned media under HG increased 1.5- and 1.6-fold at 24 and 48 hours, respectively, compared to control glucose. FN mRNA increased significantly at 24 and 48 hours and 1.4-fold that of control glucose at both time points. FN protein also increased 1.5-fold that of control glucose at 48 hours. Anti-TGF-beta antibody completely abolished HG-induced FN synthesis. CONCLUSIONS: The present finding demonstrate that HG stimulates TGF-beta 1 very early and prior to FN production and that HG-induced FN production is mediated by TGF-beta. This finding is consistent with the view that TGF-beta mediates increased ECM accumulation by MC under high glucose conditions.     

Autoregulation of periodontal ligament cell phenotype and functions by transforming growth factor-beta1

During orthodontic tooth movement, mechanical forces acting on periodontal ligament (PDL) cells induce the synthesis of mediators which alter the growth, differentiation, and secretory functions of cells of the PDL. Since the cells of the PDL represent a heterogeneous population, we examined mechanically stress-induced cytokine profiles in three separate clones of human osteoblast-like PDL cells. Of the four pro-inflammatory cytokines investigated, only IL-6 and TGF-beta1 were up-regulated in response to mechanical stress. However, the expression of other pro-inflammatory cytokines such as IL-1 beta, TNF-alpha, or IL-8 was not observed. To understand the consequences of the increase in TGF-beta1 expression following mechanical stress, we examined the effect of TGF-beta1 on PDL cell phenotype and functions. TGF-beta1 was mitogenic to PDL cells at concentrations between 0.4 and 10 ng/mL. Furthermore, TGF-beta1 down-regulated the osteoblast-like phenotype of PDL cells, i.e., alkaline phosphatase activity, calcium phosphate nodule formation, expression of osteocalcin, and TGF-beta1, in a dose-dependent manner. Although initially TGF-beta1 induced expression of type I collagen mRNA, prolonged exposure to TGF-beta1 down-regulated the ability of PDL cells to express type I collagen mRNA. Our results further show that, within 4 hrs, exogenously applied TGF-beta1 down-regulated IL-6 expression in a dose-dependent manner, and this inhibition was sustained over a six-day period. In summary, the data suggest that mechanically stress-induced TGF-beta1 expression may be a physiological mechanism to induce mitogenesis in PDL cells while down-regulating its osteoblast-like features and simultaneously reducing the IL-6-induced bone resorption.     

Differential regulation of endothelial cell adhesion molecule expression by nitric oxide donors and antioxidants

Although nitric oxide (NO) and antioxidants inhibit adhesion molecule expression, their inhibitory effects on nuclear factor kappaB (NF-kappaB) activation may differ. The NO donors, but not 8-bromo-cGMP, decreased tumor necrosis factor alpha (TNF-alpha)-induced VCAM-1, ICAM-1, and E-selectin expression by 11-70%. In contrast, NAC completely abolished VCAM-1 and E-selectin expression and decreased ICAM-1 expression by 56%. Gel shift assays demonstrate that NF-kappaB activation was inhibited by both NO and antioxidants. The activation of NF-kappaB involves the phosphorylation and degradation of its cytoplasmic inhibitor IkappaB-alpha by 26S proteasomes. The 26S proteasome inhibitor MG132 prevented the degradation of phosphorylated IkappaB-alpha. NAC inhibited IkappaB kinase (IKK) activity and prevented IkappaB-alpha phosphorylation and degradation. In contrast, NO did not inhibit IKK activity, IkappaB-alpha phosphorylation, or IkappaB-alpha degradation. However, NO, but not antioxidants, induced IkappaB-alpha promoter activity. The inhibitory effects of NO on adhesion molecule expression, therefore, differs from that of antioxidants in terms of the mechanism by which NF-kappaB is inactivated.     

Modulation of transforming growth factor-beta 1 effects by cytokines

The purpose of the present study was to determine the effects of human recombinant transforming growth factor-beta 1 (TGF-beta 1) on the proliferation of normal cell and cancer cell lines and to evaluate the mechanism of TGF-beta-induced immunosuppression. Murine H238 fibrosarcoma and human UC-11 glioblastoma cells showed no proliferative change in the presence of TGF-beta, whereas the growth of human LS174T colon adenocarcinoma cells was significantly enhanced at the lower concentrations of TGF-beta. In contrast, Mono/Mac-6, a human monocyte cell line, human peripheral blood mononuclear (PBMN) cells, and BALB/c mouse spleen cells were significantly suppressed by 2.5 to 250 ng/ml of TGF-beta. In order to investigate the mode of action, TGF-beta and other cytokines were added 0, 1, and 2 days after initiation of the culture. Mono/Mac-6 cells showed that 2 days are needed for TGF-beta-induced suppression. Simultaneous addition of TGF-beta and tumor necrosis-alpha (TNF-alpha; 600 units/ml) to Mono/Mac-6 cells resulted in nearly complete suppression by day 3. IL-2, and to a lesser extent IL-4, was able to counteract the suppressive effects of TGF-beta on mitogen-stimulated spleen cells. However, our results indicate that IL-2 is not as effective in restoring responsiveness once T cell activation is well underway. IL-1 and interferon-gamma had no effects on TGF-beta-mediated immunosuppression. Since TGF-beta depressed normal cell growth and since IL-2 could effectively counteract the suppression, we assayed for IL-2 production. When normal spleen cells were treated with 2.5 ng of TGF-beta/ml, a 3.4-fold decrease in IL-2 production was observed. This is a potential mechanism for TGF-beta-mediated immunosuppression.     

Expression of cell adhesion molecules and vascular endothelial growth factor in experimental choroidal neovascularisation in the rat

AIMS: To investigate the longevity and reproducibility of choroidal neovascularisation (CNV) induced by krypton laser photocoagulation in the rat. The presence of cell adhesion molecules (CAMs) and vascular endothelial growth factor (VEGF) during the development of CNV was also studied. METHODS: 67 pigmented rats underwent retinal photocoagulation by krypton laser. The eyes were examined by either single or serial fluorescein angiography at 3 days, 1, 2-3, 4-5, 7-8, and 12 weeks post photocoagulation. The expression of CAMs (ICAM-1, E-selectin, and CD44) and VEGF post photocoagulation was studied by immunohistochemistry. RESULTS: CNV related fluorescein leakage appeared in 46.4% of 766 laser spots delivered to the 58 eyes that were tested at 2-3 weeks post treatment. The ratio of hyperfluorescent laser sites did not change significantly at 8 weeks post laser. The number of leaky spots was independent of the total number of lesions delivered to each eye (at 2-3 weeks post laser 10-15 spots/eye: 44% and 25-30 spots/eye: 49%; t = 0.7673; p = 0.3903). Nine eyes were followed by serial angiography between 2 and 12 weeks. The laser spots with fluorescein leakage at 2 weeks (51.5%) remained leaky at 12 weeks (51.5%). Histopathologically, macrophage accumulation peaked at 5 days and CNV was firstly observed at 1 week post photocoagulation. ICAM-1, E-selectin, CD44, and VEGF were maximally induced at 3-5 days post laser photocoagulation, and were localised to RPE, choroidal vascular endothelial, and inflammatory cells. VEGF was also detected in intravascular leucocytes at the sites of laser lesions. CONCLUSIONS: These studies demonstrated that krypton laser photocoagulation can be successfully used to produce lesions similar to those of human CNV. The response induced remained present for an extended period of time (12 weeks), thus offering a potential model to screen candidate CNV inhibitory agents. In addition, it is proposed that the expression of ICAM-1, E-selectin, CD44, and VEGF before new vessel formation might be linked to the initiation of CNV.     

Modulation of skin cell functions by transforming growth factor-beta1 and ACTH after ultraviolet irradiation

A new method was developed for binding poly-(ethylene oxide) (PEO) to polymer surfaces that involves the use of electron beam irradiation in two steps. In the first, methacrylic acid was grafted and polymerized to a polymer surface, changing it from hydrophobic to hydrophilic. Exposure of this surface to aqueous PEO solutions resulted in strong hydrogen bonding of the PEO, which was covalently grafted in a second radiation step. The PEO grafts were stable; they could not be removed with extensive washing with water, soaking in basic solution, or gentle mechanical scraping. Both monolayers and multilayers of PEO were formed. The density of the monolayers were found to have little dependence on the molecular weight or concentration of the PEO solution; multilayers could be controlled by varying the viscosity of the PEO solution and the method of application. The PEO-grafted monolayers were tested for their ability to prevent protein adsorption of cytochrome-c, albumin, and fibronectin. Monolayers of star PEO were the most effective, at best showing a 60% decrease in adsorption from untreated controls. One million molecular wight linear PEO monolayers were almost as effective as star monolayers, and 35,000 g/mol linear PEO was bound too closely to the surface, owing to its small size, to have much impact in preventing protein adsorption. The reason for the continued protein adsorption was believed to be due to a close grafting of the PEO to the surface, as well as the grafted methacrylic acid chains being long enough to extend through the PEO monolayer, thus being accessible on the surface.     

Differential transforming growth factor-beta secretion in adenocarcinoma and squamous cell carcinoma of the uterine cervix

Tumor cells from eight freshly isolated cervical cancers (i.e., four adenocarcinomas and four squamous carcinomas) were analyzed for their production of the immune-inhibitory cytokine transforming growth factor-beta (TGF-beta) in vitro. All fresh adenocarcinomas secreted significant levels of TGF-beta (mean 397, range between 207 and 782 pg/ml/10(5) cells/48 hr). In contrast, no detectable TGF-beta was present in the supernatants from the four fresh squamous carcinoma cultures (P < 0.001). These data suggest that major differences in the secretion of the immunoinhibitory cytokine TGF-beta exist between squamous cell carcinomas and adenocarcinomas of the uterine cervix. Furthermore, these findings suggest that at least some of the differences in the natural biologic behavior, as well as in the response to radiation treatment, between these two histologic types of cervical cancer could be related to differences in secretion of this immune-inhibitory cytokine.     

Growth-inhibitory effects of transforming growth factor-beta 1 on myeloid leukemia cell lines

Transforming growth factor-beta1 is a pleiotropic cytokine involved in a variety of biological processes in both transformed and normal cells, including regulation of cellular proliferation and differentiation; its predominant action on hematopoietic cells is to inhibit cell growth. We used growth factor-dependent cell lines to assess TGF-beta1 effects on human myeloid leukemia cell growth. While four lines were completely or predominantly resistant, TGF-beta1 inhibited effectively, albeit to various extents, the growth of 12 other cell lines. This effect was dose dependent and specific, because a neutralizing anti-TGF-beta1 antibody prevented TGF-beta1-induced growth suppression. In the present system, basic fibroblast growth factor, known as an antagonist of TGF-beta1 counteracting its inhibitory effects, did not abrogate the suppressive effects of TGF-beta1. Other growth-stimulatory cytokines negated the TGF-beta1-induced inhibition in several cell lines, again to various extents. When proliferation was enhanced by growth-promoting cytokines (e.g. granulocyte-macrophage colony-stimulating factor, GM-CSF, stem cell factor, SCF, or PIXY-321), some previously TGF-beta1-sensitive cell lines acquired cellular resistance toward TGF-beta1-mediated growth suppression, whereas four other cell lines remained susceptible to TGF-beta1 growth inhibition despite possible counteraction by other cytokines. Thus, three growth response patterns to TGF-beta1 were seen: (1) constitutive resistance; (2) factor-dependent relative resistance; and (3) sensitivity to growth inhibition indifferent to counteracting cytokines. In the latter case, TGF-beta1 did not downregulate expression of one specific growth factor receptor. These studies indicate that human myeloid leukemia cells, represented here by leukemia cell lines as model systems, exhibit heterogeneous growth responses to TGF-beta1; its inhibitory effects can be modulated or completely alleviated by positive antagonistic cytokines. The availability of TGF-beta1-susceptible and -refractory cell lines allows for detailed investigations on the mechanisms of these regulatory pathways, the nature of TGF-beta1-resistance, and the possible contribution of acquired TGF-beta1-resistance to disease progression.     

Immunolocalization of transforming growth factor-beta 1 and transforming growth factor-beta 2 in the mouse ovary during gonadotrophin-induced follicular maturation

An immunohistochemical approach was utilized to evaluate the cellular distribution of transforming growth factor-beta 1 (TGF beta 1) and transforming growth factor beta 2 (TGF beta 2) at different stages of follicle development in the prepubertal mouse ovary under the following conditions: (i) after pregnant mare's serum gonadotrophin (PMSG) treatment; (ii) after PMSG and human chorionic gonadotrophin (HCG) treatment; (iii) after PMSG and HCG treatment plus mating. In the immature ovary, TGF beta 1 and TGF beta 2 immunoreactivities are localized in theca and granulosa cells and in oocytes. After PMSG treatment, TGF beta 1 and TGF beta 2 immunoreactivities are localized in granulosa cells; in addition, TGF beta 2 staining is noted in the matrix surrounding antral cells. Staining for both TGR beta 1 and TGF beta 2 drops in the theca but persists in the oocyte. PMSG plus HCG treatment results in a significant increase in TGF beta 1 and TGF beta 2 immunoreactivity in the theca and in the maintenance of TGF beta 1 staining in both basal granulosa cells and cumulus cells whereas TGF beta 2 immunoreactivity is essentially localized in the matrix surrounding cumulus cells. Staining for TGF beta 1 and TGF beta 2 persists in the oocyte. Following PMSG plus HCG treatment and mating, TGF beta 1 immunoreactivity is localized in the luteal cells of corpora lutea and TGF beta 2 shows a similar localization pattern. This study provides evidence that TGF beta 1 and TGF beta 2 peptides are expressed in specific cell types during induced follicular maturation in the mouse ovary.     

Characterization and regulation of the latent transforming growth factor-beta complex secreted by vascular pericytes

Transforming growth factor-beta (TGF-beta) stimulates the accumulation of extracellular matrix in renal and hepatic disease. Kidney glomerular mesangial cells (GMC) and liver fat-storing cells (FSC) produce latent of inactive TGF-beta. In this study, we characterized the latent TGF-beta complexes secreted by these cells. Human FSC produce a single latent TGF-beta complex, predominantly of the TGF-beta 1 isoform, whereas GMC secrete multiple complexes of latent TGF-beta, containing beta 1 and beta 2 isoforms. At least four forms were identified in GMC using ion exchange chromatography, including a peak not previously described in other cell types which eluted at 0.12 M NaCl, and predominantly of the beta 2 isoform. Both cell types secrete the latent TGF-beta 1 binding protein of 190 kDa, as part of a high molecular weight TGF-beta complex. Epidermal growth factor stimulates the secretion of latent TGF-beta and latent TGF-beta binding protein in both cell types. Secretion of latent TGF-beta in both cell types was found to be associated with secretion of decorin. This study shows that vascular pericytes from the kidney and the liver have distinctly different profiles of latent TGF-beta complexes, with GMC secreting a unique form of latent TGF-beta 2. The regulatory effect of epidermal growth factor and platelet-derived growth factor has potential implication for the pathophysiology of liver regeneration and chronic liver and kidney diseases.     

Expression of transforming growth factor-beta 1 messenger ribonucleic acid and the modulation of deoxyribonucleic acid synthesis by transforming growth factor-beta 1 in human endometrial cells

OBJECTIVE: The purpose of this study was (1) to evaluate the potential sites of transforming growth factor-beta 1 synthesis in human endometrium by analyzing separated endometrial glands and stromal cells for transforming growth factor-beta 1 messenger ribonucleic acid by Northern analysis of total ribonucleic acid and (2) to investigate the effects of transforming growth factor-beta 1 on deoxyribonucleic acid synthesis in endometrial epithelial and stromal cells in culture. STUDY DESIGN: Endometrial glands and stroma from proliferative and secretory endometrium were isolated after collagenase treatment of endometrial tissue minces and were analyzed for transforming growth factor-beta 1 messenger ribonucleic acid by Northern analysis. We studied the effects of estradiol-17 beta and transforming growth factor-beta 1 on deoxyribonucleic acid synthesis in endometrial epithelium and transforming growth factor-beta 1 on stromal cells in culture by evaluating tritiated thymidine incorporation into trichloroacetic acid-precipitable material. RESULTS: Transforming growth factor-beta 1 messenger ribonucleic acid was detected for Northern analysis in separated endometrial stromal cells in levels that were greatest during the secretory phase and in greater levels than in epithelial cells from that same tissue. Transforming growth factor-beta 1 messenger ribonucleic acid in glandular epithelium in culture was not increased to detectable levels by treatment with transforming growth factor-beta 1. Deoxyribonucleic acid synthesis in endometrial glandular epithelium was inhibited by transforming growth factor-beta 1, but transforming growth factor-beta 1 stimulated deoxyribonucleic acid synthesis in endometrial stromal cells in culture. After treatment for 5 days with estradiol-17 beta (10(-8) mol/L), deoxyribonucleic acid synthesis in endometrial glands in culture was decreased by 40%. Transforming growth factor-beta 1 (1 ng/ml) did not alter this effect of estradiol-17 beta on deoxyribonucleic acid synthesis. CONCLUSIONS: Transforming growth factor-beta 1 acts to decrease deoxyribonucleic acid synthesis in epithelial cells and to increase it in stromal cells isolated from human endometrium and maintained in monolayer culture. Transforming growth factor-beta 1, potentially of stromal cell origin, could participate in the regulation of endometrial cell proliferation and differentiation in vivo.     

Granulocyte-macrophage colony-stimulating factor abrogates transforming growth factor-beta 1-mediated cell cycle arrest by up-regulating cyclin D2/Cdk6

Beh?et's disease (BD) is a chronic multisystemic inflammatory disorder characterized mainly by recurrent oral and genital aphthous ulcerations and uveitis. Etiology and pathogenesis of BD remain unknown. T cell receptor (TCR) V alpha/V beta gene product expression as well as Jbeta gene segment expression in peripheral blood of BD patients were analysed to investigate the possible role of T lymphocytes in the etiopathogenesis of BD. Flow cytometry with 12 TCR V-specific MoAbs was used for TCRV analyses. Jbeta gene segment usage by T cell populations expressing certain V betas was determined by polymerase chain reaction (PCR) technique with V beta- and C beta-specific primers, Southern blotting of PCR products, and subsequent hybridization with radiolabelled Jbeta gene segment-specific probes. Although 13 of the 23 BD patients exhibited increases in expression of one or more TCR V-gene products, only expansions among the CD4+ T cell subset were significantly more frequent in BD patients (7/23) compared with healthy controls (0/15) (P = 0.019). Six out of eight cases followed for up to 20 months had at least one expansion correlated with disease activity. A strict preference for particular Jbeta gene segments implicating clonality was apparent in all analysed T cell expansions and correlated well with disease activity. These results suggest a possible involvement of antigen-specific T lymphocytes in the pathogenesis of BD.     

Collagen type XVI expression is modulated by basic fibroblast growth factor and transforming growth factor-beta

A new instrument for laparoscopic access consists of a trocarless, reusable, visual-access cannula with an external thread that ends in a blunt tip. The device has no sharp ends or moving parts. The cannula does not transect but radially stretches and elevates vessels, fascia, and muscle fibers, preserving the fascia's natural gridiron shutter mechanism at the access site. The outer thread stabilizes the cannula, and no fascial suture is necessary. In a prospective clinical trial between 1994 and 1997, the instrument was used in 203 patients requiring 234 access ports for diagnostic and operative laparoscopies. No device-related complications or failed attempts were recorded. The cannula caused less tissue trauma at access sites, and may decrease the frequency of hernias and postoperative access site pain.     

Differential regulation of eosinophil adhesion and transmigration by pulmonary microvascular endothelial cells

In bronchial asthma, eosinophils (EOS) adhere to, and migrate across, the lung microvasculature to exert their effector functions in the airways. This study was conducted to determine the effect of cytokines on adhesion molecule expression on human pulmonary microvascular endothelial cells (HPMEC) and the influence of these molecules on EOS adhesion and transmigration in vitro. Unlike ICAM-1 expression (>80% positive cytokine-treated HPMEC by flow cytometry), VCAM-1 expression varied with the cytokine(s) pretreatment; the order of potency was: TNF-alpha + IL-4 (82.2 +/- 4.2% positive cells) > TNF-alpha (41.8 +/- 5.1%) > IL-1beta (20.8 +/- 4.7%). IL-4 alone had no effect on either ICAM-1 or VCAM-1 expression. EOS adhesion to cytokine-treated HPMEC followed the same order as that observed for VCAM-1 expression. Interestingly, EOS migration across cytokine-treated HPMEC varied inversely with VCAM-1 expression on, and EOS adhesion to, HPMEC; IL-1beta (21.2 +/- 1.4% migration) > TNF-alpha (12.6 +/- 2.6%) > TNF-alpha + IL-4 (9.1 +/- 2.0%). EOS adhesion was greatest with TNF-alpha + IL-4-treated HPMEC, was dependent on VCAM-1, and inhibited with anti-alpha4 integrin mAb (67.7 +/- 7.5% inhibition, p < 0.0005). In contrast, the highest EOS migration occurred across IL-1beta-treated HPMEC and was inhibited by anti-beta2 integrin mAb (40.4 +/- 2.5% inhibition, p < 0.005). Viable HPMEC were required for EOS migration but not adhesion. Our results suggest that EOS adhesion and transmigration are differentially regulated by VCAM-1 and ICAM-1 expression and the interaction of these adhesion proteins with their respective counterligands, i.e., alpha4 and beta2 integrins on EOS.