Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using modeling of heparin-fibroblast growth factor interactions, we replaced four basic residues of basic fibroblast growth factor (FGF-2) with neutral glutamine residues by site-specific mutagenesis to give the mutants K128Q, K138Q, K128Q-K138Q, R129Q, K134Q, and R129Q-K134Q. The FGF mutants were characterized for their receptor and heparin binding affinities, mitogenic and cell proliferation activities, and their ability to induce plasminogen activator (PA) production and in vitro angiogenesis by cultured endothelial cells. Heparin binding properties and biological activities of the three mutants involving R129 and K134 remained essentially unchanged; however, significant changes for three mutants involving K128 and K138 were found. The KD values for heparin binding for K128Q and K138Q mutants were increased about 10-fold, and that for the K128Q-K138Q double mutant was increased by about 100-fold. The mutant K128Q-K138Q required a 10-fold higher concentration of heparin to promote binding to heparan sulfate proteoglycan (HSPG)-deficient CHO cells transfected with fibroblast growth factor receptor-1 (FGFR1) or to induce DNA synthesis in HSPG-deficient myeloid cells transfected with FGFR1. Binding affinities of the mutants to cell surface receptors on BHK-21 cells, however, were similar to that of wild-type FGF-2. In endothelial cell proliferation assays the activities of K128Q and K128Q-K138Q were about 10-fold lower than that of the wild-type protein, whereas the K138Q mutant exhibited wild-type activity. In addition, the K128Q-K138Q mutant displayed a markedly lowered capacity to induce PA activity in cultured endothelial cells and to form capillary-like structures in an in vitro angiogenesis model.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Diminished heparin binding of a basic fibroblast growth factor mutant is associated with reduced receptor binding, mitogenesis, plasminogen activator induction, and in vitro angiogenesis. 752 51

Basic fibroblast growth factor (bFGF; FGF-2) lacks a signal sequence and thus is not secreted by classical pathways. It has been speculated that one mode of bFGF release may be injury, either sublethal or lethal; and, transient disruption of the plasma membrane has been shown to release bFGF [Muthukrihnan et al. (1991): J Cell Physiol 148:1-16]. This observation has led to the concept of bFGF as a "wound hormone," involved in tissue integrity and repair. Findings of elevated bFGF following injury in vivo support this concept. Using an in vitro model, we have examined the regulation of bFGF gene expression following its release by sublethal injury. Analysis of bFGF protein by ELISA revealed that scraping subconfluent bovine aortic EC (BAE) released up to 80% of their bFGF. Following scraping, there was a 4- to 10-fold increase in the steady state level of bFGF mRNA, which reached a maximum at 2-3 h. There was a parallel increase in protein so that by 6 h after the scrape-induced release, bFGF levels were restored to those measured prior to scraping. Since bFGF has been reported to induce its own expression, we hypothesized that the released bFGF might be responsible for the increase in bFGF mRNA. However, inclusion of neutralizing antibodies against bFGF had a negligible effect on the scrape-induced increase in bFGF mRNA levels. Because of the important role of transforming growth factor type-beta 1 (TGF-beta 1), the plasminogen/plasminogen activator system, and thrombin in wound healing, we investigated their potential contributions to the increase in bFGF expression. Addition of anti-TGF-beta 1 antibodies, plasminogen activator inhibitor-1 (PAI-1), or the thrombin inhibitory combination of heparin and anti-thrombin III (AT III) to the cells at the time of scraping blocked about 50% of the increase in bFGF mRNA; the effects of these agents were not additive. The suppression of bFGF mRNA was associated with a proportional reduction in bFGF protein. Inclusion of the antagonists for 2 h at the time of scraping led to reduced cell proliferation, suggesting that cell-associated bFGF may be required for recovery and growth. Finally, studies to characterize the molecular mechanisms underlying the increased bFGF mRNA following sublethal injury revealed an increase in the transcriptional activation of bFGF gene. These results indicate that in spite of the fact that bFGF is not a secreted protein, levels of bFGF in the cell are tightly regulated. Furthermore, these findings suggest a role for bFGF in recovery from cell injury.
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PMID:Regulation of basic fibroblast growth factor (bFGF) gene and protein expression following its release from sublethally injured endothelial cells. 759 55

The roles of heparan sulfate proteoglycans and tyrosine kinase fibroblast growth factor (FGF) receptors in mediating the induction of plasminogen activator (PA) by FGF-2 were investigated using L6 myoblast cells that normally do not express detectable FGF receptors. PA was induced by FGF-2 in a dose-dependent manner in L6 cells expressing transfected FGF receptor-1 but not in nontransfected cells or cells transfected with the vector alone. The PA produced in these cells was characterized as urokinase-type PA (uPA). Thus, expression of a tyrosine kinase FGF receptor was required for induction of uPA. Internalization of FGF through heparan sulfates does not seem to be involved in this response as soluble heparin and suramin at concentrations which inhibited FGF-2 binding to heparan sulfates but not receptors did not affect the induction of uPA by FGF-2. Mutant receptors in which the tyrosine kinase was inactivated were not able to respond to FGF-2. In contrast, mutation of the site of phospholipase Cgamma1 (PLCgamma) binding in the receptor, which causes loss of PLCgamma activation, had no effect on uPA induction by FGF-2. These results suggest that PLCgamma activation is not required for induction of uPA by FGF-2.
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PMID:Induction of urokinase-type plasminogen activator by fibroblast growth factor (FGF)-2 is dependent on expression of FGF receptors and does not require activation of phospholipase Cgamma1. 894 Jan 13

Heparin-binding growth factors have been implicated in central nervous system development, regeneration and pathology. To assess the expression pattern and possible function in multiple sclerosis, the heparin-binding growth factors pleiotrophin (PTN), midkine (MK), basic fibroblast growth factor (FGF-2) and one of its receptors (FGFR1/flg) mRNA and protein levels were examined in an experimental autoimmune encephalomyelitis (EAE) model in the Lewis rat. We assessed the time course of expression of PTN, MK and FGF-2 during EAE and determined the cellular origin of FGF-2 and FGFR1 in normal spinal cord and during inflammatory demyelination. Basal expression of PTN and MK mRNAs in normal spinal cords was significantly upregulated after induction of EAE. MK expression was upregulated two to threefold correlating with disease progression, whereas PTN expression reached peak levels threefold above basal levels during the clinical recovery period. FGF-2 mRNA expression was low in normal spinal cord and dramatically increased in correlation with progressive demyelination. FGF-2 was confined to neurons in normal tissue and shifted dramatically to microglia, paralleling their activation during EAE. Double immunohistochemistry revealed colocalization of FGF-2 to activated microglia/macrophages with strongest expression in the macrophage-rich perivascular core area and microglial expression at the edges of white and gray matter perivascular regions. FGFR1, like its ligand, was induced in activated macrophages/microglia. Growth factor expression in demyelinating diseases could serve several functions, e.g., to modulate the activity of microglia/macrophage in an autocrine fashion, to induce the expression of other factors like insulin-like growth factor 1 or plasminogen activator, which can effect regeneration or degeneration, respectively, and finally to stimulate directly localized proliferation and/or regeneration of oligodendrocytes within the lesion area.
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PMID:Basic FGF and FGF receptor 1 are expressed in microglia during experimental autoimmune encephalomyelitis: temporally distinct expression of midkine and pleiotrophin. 981 19

Pancreatic tumors overexpress FGF-2 and t-PA, but the implication of the growth factor in t-PA synthesis and t-PA-dependent tumor invasion remains unknown. FGF-2 is present in different isoforms: The 18 kDa FGF-2 is secreted, while the 22.5 kDa one is nuclearized and exerts intracrine regulations bypassing cell-surface FGF receptors. Rat pancreatic carcinoma AR4-2J cells producing either the 18 or the 22.5 kDa FGF-2 after transfection with FGF-2 cDNAs have been used to analyze the role of FGF-2 in t-PA expression and t-PA-related cell spreading. The 22.5 kDa FGF-2 reduced t-PA and PAI-1 synthesis 2-fold. Addition of recombinant 18 kDa FGF-2 (rFGF-2) to cell cultures resulted in increased t-PA and decreased PAI-1 expression. By contrast, rFGF-2 did not significantly modify t-PA synthesis in cells producing the 22.5 kDa FGF-2. Cell spreading was t-PA-dependent. Furthermore, cells producing the 22.5 kDa FGF-2 migrated less than control cells and cells producing the 18 kDa FGF-2. Overall, our data show that secretory FGF-2 is involved in t-PA synthesis by pancreatic cancer cells and facilitates cell spreading. The 22.5 kDa FGF-2 exerts opposite effects by decreasing t-PA expression in basal conditions and during rFGF-2 stimulation. Since the expression of the 22.5 kDa FGF-2 is under specific controls, its up-regulation might have the potential to reduce spreading of pancreatic cancer cells.
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PMID:FGF-2 isoforms of 18 and 22.5 kDa differentially modulate t-PA and PAI-1 expressions on the pancreatic carcinoma cells AR4-2J: consequences on cell spreading and invasion. 1069 30

Angiogenesis, the formation of new capillary blood vessels, occurs almost exclusively in the microcirculation. This process is controlled by the interaction between factors with positive and negative regulatory activity. In this study, we have compared the effect of two well described positive regulators, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2) on bovine adrenal cortex-derived microvascular endothelial (BME) and bovine aortic endothelial (BAE) cells. The parameters we assessed included (a) cellular reorganization and lumen formation following exposure of the apical cell surface to a three-dimensional collagen gel; (b) organization of the actin cytoskeleton; (c) expression of thrombospondin-1 (TSP-1), an endogenous negative regulator of angiogenesis; and (d) extracellular proteolytic activity mediated by the plasminogen activator (PA)/plasmin system. We found that (a) collagen gel overlay induces rapid reorganization and lumen formation in BME but not BAE cells; (b) FGF-2 but not VEGF induced dramatic reorganization of actin microfilaments in BME cells, with neither cytokine affecting BAE cells; (c) FGF-2 decreased TSP-1 protein and mRNA expression in BME cells, an effect which was specific for FGF-2 and BME cells, since TSP-1 protein levels were unaffected by VEGF in BME cells, or by FGF-2 or VEGF in BAE cells; (d) FGF-2 induced urokinase-type PA (uPA) in BME and BAE cells, while VEGF induced uPA and tissue-type PA in BME cells with no effect on BAE cells. Taken together, these findings reveal endothelial cell-type specific responses to FGF-2 and VEGF, and point to the greater specificity of these cytokines for endothelial cells of the microvasculature than for large vessel (aortic) endothelial cells. Furthermore, when viewed in the context of our previous observation on the synergistic interaction between VEGF and FGF-2, our present findings provide evidence for complementary mechanisms which, when acting in concert, might account for the synergistic effect.
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PMID:Response of bovine endothelial cells to FGF-2 and VEGF is dependent on their site of origin: Relevance to the regulation of angiogenesis. 1150 Sep 40

After wounding, the corneal endothelium heals primarily by migration of adjacent cells into the denuded wound area. In this study, it has been attempted to identify elements of the intracellular signaling pathway activated through basic Fibroblast Growth Factor (FGF-2)- and Protein Kinase C (PKC)-modulated migration, using specific inhibitors and stimulators of second messengers in a cell culture model. Bovine corneal endothelial cells (BCEC) were grown to confluency and experiments performed with first passage cells under serum-free conditions. A central circular 'wound' was made with a specially designed trephine. In different experiments, cells were incubated with either FGF-2 (10 ng ml(-1)), pertussis toxin (PTX; 1-50 ng ml(-1)), phorbol 12-myristate 13-acetate (PMA; 50 ng ml(-1)), 2,4'-di-bromoacetophenone (DAP; 5 microM), 1-(5-iosquinolinesulphonyl)-2-methyl-piperazine dihydrochloride (H7; 10 microM), indomethacin (5 ng ml(-1)), nordihydroguaiaretic acid (NDGA; 10 ng ml(-1)), 2-(4-morpholinyl)-8-pheny-4H-1-benzopyran-4-one (LY294002; 10 microM) or different combinations of these agents. Unsupplemented cultures served as controls. Migration was quantitated by counting the cells inside the denuded area in one randomly chosen section from the wound edge 72 hr after wounding. Cell toxicity was determined with the trypan blue exclusion test. Results were statistically analysed by Student's t-test. FGF-2 and PMA (a protein kinase C activator) both stimulated migration of endothelial cells at 2.2- and 3.1-fold, respectively. The PLA(2) inhibitor DAP and the PKC inhibitor H7 both significantly reduced PMA-stimulated migration to control levels but had no effect (DAP) or even stimulated (H7) FGF-2-modulated migration. PTX did not affect FGF-2-stimulated migration. The phosphoinositol (3)-kinase inhibitor LY294002 significantly reduced FGF-2-mediated stimulation of endothelial migration similar to the rate of control cultures. LY294002 had no effect when applied together with PMA. The cyclooxygenase inhibitor indomethacin did not influence migration rates of the cells added either alone or in combination with PMA and FGF-2, respectively. The lipoxygenase inhibitor NDGA significantly reduced the number of migrating cells in cultures with no other supplements, or of those supplemented with either PMA or FGF-2. FGF-2-induced endothelial migration in vitro is not dependent on PKC/PLA(2) or pertussis-toxin sensitive G-protein pathways but rather requires activation of a phosphoinositol (3)-kinase-like enzyme and/or arachidonic acid release with subsequent liberation of lipoxygenase products. Independent of FGF-2, PKC is a major intracellular effector of corneal endothelial migration activity after wounding and stimulates migration via the PLA(2)-dependent generation of lipoxygenase metabolites.
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PMID:Intracellular signaling pathway of FGF-2-modulated corneal endothelial cell migration during wound healing in vitro. 1174 64

An in vitro angiogenesis system was designed for screening angiogenic agonists and antagonists. In order to obtain large quantities of cells and reproducibility, human endothelial cells with extended life spans were developed by retroviral transfection. The resulting cells grown in a serum-free medium containing endothelial cell growth supplement (ECGS) have a telomerase activity, extended life spans of at least 21 passages, and an endothelial cell phenotype (diI-acetylated-LDL upake, factor VIII-related antigen, VEGFR-1 and R-2, and tissue-type plasminogen activator (tPA)) that resembled that of unaltered primary endothelial cells. Exceptions were (i) a higher expression of tPA, and (ii) a non-significant growth response to FGF-2 or VEGF stimulation. Within three-dimensional fibrin gels, specific cell clones rapidly formed tubular structures in a more reproducible manner than those observed with low-passage primary cells. Tube formation by primary endothelial cells and those with extended life spans was dependent upon FGF-2 and ECGS, respectively. Both cell types produced FGF-2 and VEGF cytokines. Increasing doses of suramin significantly decreased the size of microvessels formed by both cell lines. These functional results indicate that a vascular matrix system containing human cells with extended life spans can be successfully utilized as an in vitro assay for antiangiogenic compounds.
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PMID:Human vascular endothelial cells with extended life spans: in vitro cell response, protein expression, and angiogenesis. 1254 57

Trophoblast invasion, accompanied by degradation of extracellular matrix, is crucial to normal pregnancy development, whereas shallow placental invasion and implantation likely plays a role in the subsequent development of pre-eclampsia. The growth factors vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and fibroblast growth factor (FGF) are placental growth factors that activate degradation of extracellular matrix. We determined the effect of VEGF, EGF, FGF-2, FGF-4 and FGF-10 on the plasminogen activator system of first trimester cytotrophoblasts cultured in vitro. We studied the activity of urokinase plasminogen activator (uPA), its inhibitor plasminogen activator inhibitor-1 (PAI-1), and 92 kDa gelatinase-B (matrix metalloproteinase-9, MMP-9), using protein gel and reversed gel zymography. The expression pattern of FGF-4 and FGF-10 in human placental sections was determined by immunohistochemistry. FGF-4 was expressed in first trimester villi stroma, primarily in endothelial cells. FGF-10 expression was localized to first trimester extravillous trophoblasts. VEGF, EGF, FGF-4 and FGF-10, but not FGF-2, stimulate the activity of trophoblast uPA, PAI-1 and MMP-9. These results support the hypothesis that specific growth factors modulate the invasive potential of trophoblasts, and therefore may play an important role in early placental development. Our findings may contribute to the understanding of the pathophysiology of diseases associated with shallow placentation, such as pre-eclampsia.
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PMID:Vascular endothelial growth factor, epidermal growth factor and fibroblast growth factor-4 and -10 stimulate trophoblast plasminogen activator system and metalloproteinase-9. 1499 96

Primary cultures of bovine microvascular endothelial cells (BME) isolated from the adrenal cortex, are commonly used to study vascular endothelium, but have a limited life span. To circumvent these limitations, we have immortalized BME cells with either simian virus 40 (SV40) or with a retrovirus containing the coding region of human telomerase reverse transcriptase (hTERT), and have investigated whether the clonal populations obtained, maintain differentiated properties characteristic of microvascular endothelium. Immortalized cells were characterized for maintenance of typical endothelial morphology, marker expression, and functional characteristics including uptake of Acetylated low-density lipoprotein (Ac-LDL), capillary-like tube formation in three-dimensional collagen gels, as well as metalloproteinase (MMP) and plasminogen activator (PA)-mediated extracellular proteolysis. Whilst immortalization of BME cells with SV40 was associated with loss of endothelial-specific properties, hTERT-BME exhibited an endothelial phenotype similar to that of wild-type endothelial cells. Specifically, they showed a typical cobblestone morphology, were contact-inhibited, expressed endothelial cell-specific markers (e.g., CD31, vWF) and both fibroblast growth factor receptor 1 (FGFR-1) and vascular endothelial growth factor receptor-2 (VEGFR-2). In addition, they expressed receptors for LDL. Importantly, when grown on collagen gels, hTERT-BME cells underwent MMP-dependent tube-like structure formation in response to VEGFR-2 activation. In a collagen gel sandwich assay, hTERT-BME formed tubular structures in the absence of exogenously added angiogenic cytokines. Sustained tube formation was induced by VEGF-A alone or in combination with FGF-2. From 17 sub-clones that displayed a non-transformed phenotype, a high proliferative capacity and tubulogenic properties in three-dimensional collagen gels, we isolated two distinct subpopulations that display a highly specific response to VEGF-A or to FGF-2. We have generated hTERT-BME cells that maintain endothelial-specific properties and function and have isolated clones that respond differentially to VEGF-A or FGF-2. These immortalized cell lines will facilitate the study of endothelial cell biology.
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PMID:Bovine microvascular endothelial cells immortalized with human telomerase. 1640 75


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