Apoptosis in human cultured trophoblasts is enhanced by hypoxia and diminished by epidermal growth factor

Preeclampsia and fetal growthrestriction are associated with placental hypoperfusion and villoushypoxia. The villous response to this environment includes diminishedtrophoblast differentiation and enhanced apoptosis. We tested thehypothesis that hypoxia induces apoptosis in cultured trophoblasts, andthat epidermal growth factor (EGF), an enhancer of trophoblastdifferentiation, diminishes hypoxia-induced apoptosis. Trophoblastsisolated from placentas of term-uncomplicated human pregnancies werecultured up to 72 h in standard (PO₂ = 120 mmHg) or hypoxic (PO₂ < 15 mmHg) conditions. Exposure to hypoxia for 24 h markedly enhanced trophoblast apoptosis as determined by DNA laddering, internucleosomal in situ DNA fragmentation, and histomorphology, as well as by thereversibility of the apoptotic process with a caspase inhibitor. Apoptosis was accompanied by increased expression of p53 and Bax anddecreased expression of Bcl-2. Addition of EGF to cultured trophoblastsor exposure of more differentiated trophoblasts to hypoxiasignificantly lowered the level of apoptosis. We conclude that hypoxiaenhances apoptosis in cultured trophoblasts by a mechanism thatinvolves an increase in p53 and Bax expression. EGF and enhancement ofcell differentiation protect against hypoxic-induced apoptosis.

     

Monosomy 6 in human cultured fibroblast-like cells permanently stimulated by fibroblast growth factor 1: evidence for selection.

The appearance of cells with monosomy 6 (mono6 cells) in cultures of human fibroblast-like cells during long-term stimulation with acidic fibroblast growth factor (FGF1) was confirmed in five of the six lines newly investigated. Aneugenic pretreatment at the start of the cultures accelerated the emergence of mono6 cells, as would be expected if selection, rather than induction, is the main mechanism involved. This could be confirmed by using an incidental rearrangement, der(8)t(6p;8p), that emerged in one of the lines by monitoring the proliferation of the mono6 cells (here monosomic for 6p22.1-->qter) in mixtures with normal cells. During growth in the presence of FGF1, the proportion of mono6 cells increased six fold, whereas in the absence of FGF1, it declined to background levels. Selection rather than induction of the mono6 cells is further supported by their clonal origin, as ascertained on the basis of X-inactivation patterns in three informative cases. In addition, colonies grown in the presence of FGF1 from single cells did not reveal higher proportions of mono6 cells by fluorescence in situ hybridization analysis than those grown without the growth factor. During permanent stimulation with FGF1, the growth of mono6 cells did not become dependent on FGF1, nor did these cells lose their responsiveness to FGF1. Although evidence in favor of selection of preexistent mono6 cells by FGF1 is provided in this study, the contribution of a primary inducing mechanism cannot be entirely excluded.     

Expression of the urokinase receptor in vascular endothelial cells is stimulated by basic fibroblast growth factor

Basic fibroblast growth factor, a potent angiogenesis inducer, stimulates urokinase (uPA) production by vascular endothelial cells. In both basic fibroblast growth factor-stimulated and -nonstimulated bovine capillary endothelial and human umbilical vein endothelial cells single-chain uPA binding is mediated by a membrane protein with a Mr of 42,000. Exposure of bovine capillary or endothelial human umbilical vein endothelial cells to pmolar concentrations of basic fibroblast growth factor results in a dose-dependent, protein synthesis-dependent increase in the number of membrane receptors for uPA (19,500-187,000) and in a parallel decrease in their affinity (KD = 0.144-0.790 nM). With both cells, single-chain uPA binding is competed by synthetic peptides whose sequence corresponds to the receptor-binding sequence in the NH2-terminal domain of uPA. Exposure of bovine capillary endothelial cells to transforming growth factor beta 1, which inhibits uPA production and upregulates type 1 plasminogen activator inhibitor, the major endothelial cell plasminogen activator inhibitor, has no effect on uPA receptor levels. These results show that basic fibroblast growth factor, besides stimulating uPA production by vascular endothelial cells, also increases the production of receptors, which modulates their capacity to focalize this enzyme on the cell surface. This effect may be important in the degradative processes that occur during angiogenesis.     

Expression of fibroblast growth factor receptor 3 by fibroblast growth factor 2 in cultured chick embryo chondrocytes

Although fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptor 3 (FGFR3) both inhibit longitudinal bone growth, little is known about the relationship between FGF2 and FGFR3. Accordingly, the current study examined the expression of FGFR3 mRNA after the administration of FGF2 using cultured chondrocytes from day 17 chick embryos to evaluate the relationship between FGF2 and FGFR3. The chondrocytes were isolated from the caudal one-third portion (LS) of sterna, peripheral regions (USP) and central core regions (USC) of the cephalic portion of the sterna, and lower portion of the proximal tibial growth plate (Ti) of day 17 chick embryo. The expression of FGFR1, FGFR3, and type II and X collagen mRNA in the chondrocytes from the LS, USP, USC, and Ti was determined. FGFR1 was not expressed in the LS and USP chondrocytes, yet strongly expressed in the USC and Ti chondrocytes. With a treatment of FGF2, the expression of FGFR1 slightly increased in the USC chondrocytes and was not related with the concentration of FGF2 in the Ti chondrocytes. FGFR3 was expressed in all the chondrocyte types, yet strongly increased in the LS, USC, USP, and Ti in that order according to the concentration of FGF2. For the LS and USP chondrocytes, the expression of FGFR3 with FGF2 increased in a 4-day culture, yet decreased in a 6-day culture, whereas for the USC chondrocytes, the expression of FGFR3 mRNA with FGF2 increased in a 2-day culture, yet decreased in a 4-day culture, suggesting that the hypertrophic chondrocytes were more numerous and sensitive compared to the proliferative chondrocytes. For all the chondrocyte types, FGF2 appeared to be up-regulated to FGFR3, as the expression of FGFR3 mRNA increased with a higher concentration of FGF2 until a peak level. In conclusion, FGF2 was found to up-regulate to FGFR3 until the peak level of FGFR3 mRNA expression, while in hypertrophic chondrocytes, FGFR3 appeared to cause the differentiaton of chondrocytes, resulting in the inhibition of longitudinal bone growth after the peak level of FGFR3 mRNA expression.     

The effect of recombinant human basic fibroblast growth factor rhfgf-2 on human osteoblast in growth and phenotype expression

This paper describes the studies of human recombinant basic fibroblast growth factor (rhFGF-2) for its effects on human osteoblast growth and phenotype expression. During a 24-h period of treatment, rhFGF-2 highly stimulated DNA synthesis in a dose-related fashion with a maximum stimulation of 150% for 1 ng/ml. On the other hand, rhFGF-2 decreases alkaline phosphatase activity, synthesis of type I collagen, and cumulative amount of osteocalcin. Moreover, rhFGF-2 provoked a threefold increase in the amount of intracellular cAMP. Scatchard plots show the presence of two classes of [¹²⁵I] rhFGF-2 receptors. This data suggests that rhFGF-2 which stimulate cell replication may act indirectly as an anabolic agent and stimulate some of the phenotypic expression markers.     

Hepatocyte growth factor and other fibroblast secretions modulate the phenotype of human bronchial epithelial cells

The luminal airway surface is lined with epithelial cells that provide a protective barrier from the external environment and clear inhaled pathogens from the lung. To accomplish this important function, human bronchial epithelial (HBE) cells must be able to rapidly regenerate a mucociliary layer of cells following epithelial injury. Whereas epithelial-fibroblast interactions are known to modulate the airway architecture during lung development and repair, little is known about how these two cells interact. Using a primary HBE and lung fibroblast coculture system, we demonstrate that 1) subepithelial fibroblasts provide a suitable environment for differentiation of HBE cells into a polarized ciliated phenotype despite being cultured in media that induces terminal squamous differentiation and growth arrest in the absence of fibroblasts, 2) HBE cells cocultured with subepithelial fibroblasts exhibit augmented ciliogenesis, accelerated wound repair, and diminished polarized ion transport compared with cells grown in control conditions, and 3) hepatocyte growth factor (HGF) is important for subepithelial fibroblast modulation of HBE cell differentiation. These results provide a model to study fibroblast modulation of epithelial phenotype and indicate that HGF secreted by subepithelial fibroblasts contributes to HBE cell differentiation.     

Growth of normal human keratinocytes and fibroblasts in serum-free medium is stimulated by acidic and basic fibroblast growth factor

Keratinocytes and fibroblasts isolated from human neonatal foreskin can be plated and grown through multiple rounds of division in vitro under defined serum-free conditions. We utilized these growth conditions to examine the mitogenic potential of acidic and basic fibroblast growth factor (aFGF and bFGF) on these cells. Our results demonstrate that both aFGF and bFGF can stimulate the proliferation of keratinocytes and fibroblasts. aFGF is a more potent mitogen than bFGF for keratinocytes. In contrast, bFGF appears to be more potent than aFGF in stimulating the growth of fibroblast cultures. Heparin sulfate (10 micrograms/ml) dramatically inhibited the ability of bFGF to stimulate the proliferation of keratinocytes. In comparison, heparin slightly inhibited the stimulatory effect of aFGF and had no effect on epidermal growth factor (EGF) stimulation in keratinocyte cultures. In fibroblast cultures the addition of heparin enhanced the mitogenic effect of aFGF, had a minimal stimulatory effect on the mitogenic activity of bFGF, and had no effect on EGF-stimulated growth. Our results demonstrate that the proliferation in vitro of two normal cell types found in the skin can be influenced by aFGF and bFGF and demonstrate cell-type specific differences in the responsiveness of fibroblasts and keratinocytes to these growth factors and heparin.     

Regulation of retinal capillary cells by basic fibroblast growth factor, vascular endothelial growth factor, and hypoxia

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) feature prominently in retinal neovascular diseases. Although the role of VEGF in retinal angiogenesis is well established, the importance of bFGF in this process requires further clarification. This study was undertaken to investigate the responses of retinal capillary cells (endothelial cells and pericytes) to bFGF under hypoxic conditions, as well as the potentially synergistic effects of bFGF and VEGF on the proliferation and cord formation of retinal endothelial cells. Cell proliferation was determined by cell number and by 3H-thymidine incorporation. Cord formation was assessed in three-dimensional gels of collagen type I. VEGF and bFGF increased 3H-thymidine incorporation by both cell types, an effect that was more pronounced in a hypoxic environment. Moreover, the proliferation of pericytes was stimulated to a greater extent by bFGF relative to VEGF. Endothelial migration in collagen gels, however, was induced more effectively by VEGF than by bFGF. A synergistic effect of VEGF and bFGF on cell invasion was observed in the collagen gel assay. VEGF and bFGF each augment proliferation of these cells, especially under hypoxia. We thus propose that these two cytokines have a synergistic effect at several stages of angiogenesis in the retina.     

Synthesis and secretion of an epidermal growth factor (EGF) by human fibroblast cells in culture

Human fibroblast (WS-1) cells in culture synthesized and secreted an epidermal growth factor which cross-reacted with human epidermal growth factor (hEGF) purified from human urine. hEGF secreted by the cells (designated as WS-1 EGF or fibroblast EGF) and hEGF isolated from urine (designated as urine EGF) were immunologically indistinguishable. The molecular weight of fibroblast EGF estimated by gel filtration was identical with that of hEGF from urine. On chromatofocusing chromatography, fibroblast EGF was eluted mainly at pH 4.26 as a sharp symmetric peak with a minor peak at pH 4.62, like urine EGF. These results suggested that EGF synthesized and secreted by human fibroblast cells is an identical molecule to that of hEGF in human urine.     

Basic fibroblast growth factor stimulates hepatocyte growth factor/scatter factor secretion by human mesenchymal cells

Basic fibroblast growth factor (bFGF) together with other pleiotropic factors plays an important role in many complex physiological processes such as embryonic development, angiogenesis, and wound repair. Among these factors, hepatocyte growth factor/scatter factor (HGF/SF) which is secreted by cells of mesodermal origin exerts its mito- and motogenic activities on cells of epithelial and endothelial origin. Knowledge of the regulatory mechanisms of HGF/SF may contribute to the understanding of its role in physio-pathological processes. We observed that the secretion of HGF/SF by MRC-5 cells and by other fibroblast-derived cell cultures in conditioned media was enhanced by exposure to bFGF. HGF/SF was measured by the scatter assay, a bioassay for cell motility, and was further characterized by Western blot analysis with anti-HGF/SF antibodies. Exposure of MRC-5 cultures to 10 ng/ml of bFGF resulted already 6 h posttreatment in a threefold higher amount of scatter factor secreted into the medium as compared to untreated cultures. HGF/SF secretion was sustained after bFGF treatment for the following 72 h when increased amounts of HGF/SF were detected both in conditioned media as well as associated to the extracellular matrix. The secretion of HGF/SF in cell supernatants increased dose dependently upon treatment with bFGF starting from basal levels of 6 U/ml and reaching 27 U/ml at 30 ng/ml bFGF, plateauing thereafter. Upregulation of HGF/SF by IL-1, already described by others, was confirmed in this study. Based on our findings an articulated interaction can be speculated for bFGF, HGF/SF, and IL-1, e.g., in tissue regeneration during inflammatory processes or in wound healing. © 1996 Wiley-Liss, Inc.     

Interaction of platelet factor 4 with fibroblast growth factor 2 is stabilised by heparan sulphate

The CXC chemokine platelet factor 4 (PF4) appears to inhibit tumour growth through its modulation of the activity of angiogenic growth factors. We investigated the heparan sulphate-dependent mechanism of PF4 inhibition of fibroblast growth factor 2 (FGF-2). The ability of PF4 to bind simultaneously to both FGF-2 and HS was assessed using affinity gel chromatography. Thirty-three to forty-two percent more HS bound to the FGF-2 affinity gel in the presence of PF4 than with HS alone. Protection assays showed that PF4 and FGF-2 bound to adjacent or overlapping sites together covering a 12 kDa stretch of HS. This study suggests that the three components may form a ternary complex. PF4 released at sites of angiogenesis may bind to angiogenic growth factors attached to endothelial cell surface HS to disrupt or prevent them from interacting with their signalling receptors. Manipulation of this mechanism may prove useful for clinical intervention of angiogenesis.     

Induction of synaptic development in cultured muscle cells by basic fibroblast growth factor.

The role of basic fibroblast growth factor (bFGF) in signaling the development of the neuromuscular junction was examined. Beads coated with bFGF induced the formation of acetylcholine receptor (AChR) clusters in cultured Xenopus myotomal muscle cells. Tyrphostin, a tyrosine kinase inhibitor, abolished AChR clustering induced by bFGF beads, suggesting a role of tyrosine kinase activation in AChR clustering. Using specific antibodies, we demonstrated the presence of both bFGF and its receptor in the myotomal muscle in vivo during the period of neuromuscular connection. However, similar tissue from older animals with mature neuromuscular junctions showed an apparently truncated form of the bFGF receptor. These data suggest that bFGF may play a role in signaling synaptogenesis in skeletal muscle.     

Fibroblast growth factor release by bovine endothelial cells and human astrocytoma cells in culture is density dependent

Basic fibroblast growth factor (bFGF) is a potent endothelial cell mitogen whose actions are mediated by binding to specific cell surface receptors on a variety of cell types. However, the amino acid sequence of bFGF does not contain a classical signal peptide sequence and the extent to which cellular stores of this mitogen are released is still a matter of some controversy. In the present study we examined the release of immunoreactive bFGF into serum-free conditioned medium of bovine corneal endothelial cells (BCE) and a human astrocytoma cell line, U87-MG. Western blotting analysis of BCE conditioned medium using N-terminal specific anti-bFGF serum revealed a single immunoreactive band of 32 kilodaltons, which was reduced to 18 kilodaltons in the presence of 8 M urea. Using a sensitive two-site immunoradiometric assay we were able to quantify the release of immunoreactive bFGF into the culture medium by BCE cells and by the human astrocytoma cell line U87-MG. In each case the release of bFGF was cell density dependent, but under all conditions the level of bFGF released was significantly greater in the transformed astrocytoma line, ranging from 15- to 50-fold higher than in the BCE cultures under various conditions. At 30% confluence the concentration of immunoreactive bFGF in the medium was maintained at a constant level for up to 24 h. However, the level of immunoreactive bFGF declined rapidly in confluent cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

BCL-2 is upregulated in human SH-SY5Y neuroblastoma cells differentiated by overexpression of fibroblast growth factor 1.

Fibroblast growth factor 1 (FGF1) is a multipotent factor in the development and differentiation of the central nervous system. Recent studies in PC12 cells attribute these effects to high endogenous FGF1 expression. To examine the differentiation mechanisms induced by FGF1, we performed studies in SH-SY5Y human neuroblastoma cells. We monitored the impact of FGF1 overexpression in SH-SY5Y either after addition of exogenous FGF1 and heparin or after stable transfection with the FGF1 eukaryotic expression vector. Under both conditions, the FGF1 endogenous rise caused SH-SY5Y cell differentiation with morphological changes (appearance of neuritic extensions), increased GAP-43 gene expression, decreased of N-myc gene expression, and prolonged long-term survival in serum-free media. These modifications were correlated with Bcl-2 upregulation. These results suggest that there is a link between the endogenous FGF1 signaling pathway and Bcl-2 in neuronal survival modulation.