Transforming growth factor beta and basic fibroblast growth factor synergistically promote early bovine embryo development during the fourth cell cycle.

Developmentally competent bovine blastocysts were produced by adding transforming growth factor beta (TGF beta) and basic fibroblast growth factor (bFGF) to serum-free cultures of in vitro produced, 2-cell bovine embryos. The effects of TGF beta were evaluated because this growth factor signals synthesis and secretion of the extracellular matrix component fibronectin and its receptor. Previous investigations have demonstrated that fibronectin promotes early bovine embryo development in vitro. The effects of TGF beta can be potentiated by bFGF; bFGF itself is an effector of protein synthesis and a potent mitogen. A positive interaction between the 2 growth factors resulted in 38.8% of fertilized oocytes maturing beyond the 16-cell stage; of these, 24.6% formed blastocysts. Transfer of early blastocysts produced using serum-free medium supplemented with growth factors resulted in pregnancy in 3 of 9 recipients. These results support the hypothesis that TGF beta and bFGF act synergistically to promote development of bovine embryos beyond the "8-cell block" observed in vitro.     

Soluble production and function of vascular endothelial growth factor/basic fibroblast growth factor complex peptide

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are important proangiogenic factors in tumor procession. The autocrine and paracrine bFGF and the VEGF in tumor tissue can promote tumor angiogenesis, tumor growth, and metastasis. A VEGF/bFGF Complex Peptide (VBP3) was designed on the basis of epitope peptides from both VEGF and bFGF to elicit in vivo production of anti‐bFGF and anti‐VEGF antibodies. In this study, we reported on the production of recombinant VBP3 using high cell density fermentation. Fed‐batch fermentation for recombinant VBP3 production was conducted, and the production procedure was optimized in a 10‐L fermentor. The fraction of soluble VBP3 protein obtained reached 78% of total recombinant protein output under fed‐batch fermentation. Purified recombinant VBP3 could inhibit tumor cell proliferation in vitro and stimulate C57BL/6 mice to produce high titer anti‐VEGF and anti‐bFGF antibodies in vivo. A melanoma‐grafted mouse model and an immunohistochemistry assay showed that tumor growth and tumor angiogenesis were significantly inhibited in VBP3‐vaccinated mice. These results demonstrated that soluble recombinant VBP3 could be produced by large‐scale fermentation, and the product, with good immunogenicity, elicited production of high‐titer anti‐bFGF and anti‐VEGF antibodies, which could be used as a therapeutic tumor vaccine to inhibit tumor angiogenesis and tumor growth. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:194–203, 2015     

Transcriptional regulation of basic fibroblast growth factor gene expression in capillary endothelial cells.

The growth of capillary endothelial cells (BCE) is an important regulatory step in the formation of capillary blood vessels. In vivo, the proliferation of these cells is stringently controlled. In vitro they can be stimulated by polypeptide growth factors, such as acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF). Since bFGF is synthesized and stored by vascular endothelial cells, this mitogen may play an important role in an autocrine growth regulation during angiogenesis. Here, evidence is presented for induction of the mRNA of bFGF by bFGF itself. A similar increase of bFGF mRNA was observed in response to thrombin and after treatment with phorbol ester. These results suggest that an autocrine loop may exist that may serve to modulate the mitogenic response in BCE under various physiological conditions, (e.g., wound healing and new capillary formation).     

Growth factor regulation of cell growth and proliferation in the nervous system

This article discusses a novel intracrine mechanism of growth-factor action in the nervous system whereby fibroblast growth factor-2 (FGF-2) and its receptor accumulate in the cell nucleus and act as mediators in the control of cell growth and proliferation. In human and rat brain the levels and subcellular localization of FGF-2 differ between quiescent and reactive astrocytes. Quiescent cells express a low level of FGF-2, which is located predominantly within the cytoplasm. In reactive astrocytes, the expression of FGF-2 increases and the proteins are found in both the cytoplasm and nucleus. In glioma tumors, FGF-2 is overexpressed in the nuclei of neoplastic cells. Similar changes in FGF-2 expression and localization are found in vitro. The nuclear accumulation of FGF-2 reflects a transient activation of the FGF-2 gene by potentially novel transactivating factors interacting with an upstream regulatory promoter region. In parallel with FGF-2, the nuclei of astrocytes contain the high-affinity FGF-2 receptor, FGFR1. Nuclear FGFR1 is full length, retains kinase activity, and is localized within the nuclear interior in association with the nuclear matrix. Transfection of either FGF-2 or FGFR1 into cells that do not normally express these proteins results in their nuclear accumulation and concomitant increases in cell proliferation. A similar regulation of nuclear FGF-2 and FGFR1 is observed in neural crest-derived adrenal medullary cells and of FGF-2 in the nuclei of cerebellar neurons. Thus, the regulation of the nuclear content of FGF-2 and FGFR1 could serve as a novel mechanism controlling growth and proliferation of glial and neuronal cells.     

Effect of local neutralization of basic fibroblast growth factor or vascular endothelial growth factor by a specific antibody on the development of the corpus luteum in the cow

Active angiogenesis and progesterone (P) synthesis occur in parallel during development of the corpus luteum (CL). Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) are known to stimulate angiogenesis and P synthesis in vitro. The aim of the present study was to investigate the impact of bFGF or VEGF on the CL development in the cow by using a specific antibody against bFGF or VEGF. bFGF antibody, VEGF antibody, or saline as a control (n = 4 cows/treatment) were injected directly into the CL immediately after ovulation (Day 1), and the treatment was continued for 3 times/day over 7 days. Luteal biopsies were applied on Day 8 of the estrous cycle to determine the expression of genes associated with P synthesis and angiogenesis. Intraluteal injections with the bFGF antibody or the VEGF antibody markedly decreased the CL volume, plasma P concentration and StAR mRNA expression. bFGF antibody treatment decreased the mRNA expression of bFGF, FGF receptor-1, VEGF120, and angiopoietin (ANPT)-1, and increased ANPT-2/ANPT-1 ratio. However, VEGF antibody treatment decreased ANPT-2 mRNA expression and ANPT-2/ANPT-1 ratio. These results indicate that local neutralization of bFGF or VEGF changes genes regulating angiogenesis and P synthesis, and remarkably suppresses the CL size and P secretion during the development of CL in the cow, supporting the concept that bFGF and VEGF control the CL formation and function.     

Effects of basic fibroblast growth factor on angiogenin expression and cell proliferation in H7402 human hepatoma cells

Hepatocellular carcinoma （HCC） is one of the most common cancers worldwide. Basic fibroblast growth factor （bFGF）, which is highly expressed in developing tissues and malignant cells, regulates cell growth, differentiation, and migration. Its expression is essential for the progression and metastasis of HCC. This study aims to investigate the effects of bFGF on the expression of angiogenin, another growth factor, which plays an important role in tumor angiogenesis, and on cell proliferation in H7402 human hepatoma cells. The bFGF sense cDNA or antisense cDNA was stably transfected into H7402 cells. Genomic DNA PCR analysis demonstrated that human bFGF sense cDNA or antisense cDNA was inserted into the genome. Furthermore, the expression of bFGF and angiogenin was examined by RT-PCR and Western blot assays. MTT and colony formation assays were employed to determine cell proliferation. Stable bFGF over-expressing and under-expressing transfectants were successfully established. Expression of angiogenin was decreased in the over-expressing bFGF cells （sense transfectants） and was increased in the under-expressing bFGF cells （antisense transfectants）. Cell proliferation increased in the bFGF sense transfectants and decreased in the bFGF antisense transfectants. These results demonstrated that the endogenous bFGF may not only negatively regulate the angiogenin expression but also contribute to the overall cell proliferation in H7402 human hepatoma cells. This study may be helpful in finding a potential therapeutic approach to HCC.     

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.     

Correlation of cell migration, cell invasion, receptor number, proteinase production, and basic fibroblast growth factor levels in endothelial cells

The levels of endogenous basic fibroblast growth factor (bFGF) in seven clones of cultured bovine capillary endothelial (BCE) cells were assayed, and their relation to cell morphology, bFGF receptor number, cell migration, amniotic membrane invasivity, and proteinase levels were studied. Immunoblotting experiments with anti-bFGF IgG demonstrated that cells from these clones contained different amounts of bFGF. The cells containing high levels of bFGF had a spindle or elongated appearance at confluence and a low number of high affinity receptors for bFGF. The cells containing low levels of bFGF had a cobblestone-like appearance and a higher number of high affinity receptors. When exposed to 10 ng/ml bFGF, cells containing a low level of bFGF took on an elongated appearance with a crisscross pattern similar to that seen with the high producer bFGF cells. The endogenous bFGF levels of the BCE cell clones correlated with the extent of cell migration after wounding of a monolayer and the degree of invasion of the human amniotic membrane. Cells from the clone with the highest endogenous bFGF level migrated well, invaded the amnion membrane without the addition of exogenous bFGF, and were relatively unaffected by the addition of bFGF. Cells from the clone containing the lowest level of bFGF did not migrate or invade under normal conditions. However, the addition of bFGF to the culture medium strongly enhanced both of these processes. The inclusion of anti-bFGF IgG in the media suppressed cell migration and invasion. The plasminogen activator (PA) activities of cell lysates of the clones, assayed by the 125I-fibrin plate technique, indicated that the PA levels did not correlate with the bFGF levels. Metalloproteinase activities in the conditioned medium, assayed by gelatin zymography, correlated with the endogenous bFGF levels, suggesting that the degree of expression of metalloproteinases might be critical for cell migration and invasion. These data suggest that endogenous bFGF may have an important role for migration and invasion of BCE cells during neovascularization via the induction and/or activation of specific metalloproteinases.     

Binding, internalization, and degradation of basic fibroblast growth factor in human microvascular endothelial cells

The binding, internalization, and degradation of basic fibroblast growth factor (bFGF) in human omental microvascular endothelial cells (HOME cells) were investigated. Binding studies of bFGF in human endothelial cells have not yet been reported. Basic FGF bound to HOME cells (KD of 42.0 +/- 3.8 pM and 70,526 +/- 6121 binding sites/cell for the high-affinity sites, KD of 0.933 +/- 0.27 nM and 630,252 +/- 172,459 sites/cell for low-affinity binding sites). The number of low-affinity binding sites was found to be variable. Washing the cells with 2 M phosphate-buffered saline removed completely 125I-bFGF bound to low-affinity binding sites but decreased also the high-affinity binding. The majority of the surface-bound 125I-bFGF was removed by washing the cells with acetic acid buffer at pH 3. At 37 degrees C, 30% of the cell-associated 125I-bFGF became resistant to the acidic wash after 90 min, suggesting that this fraction of bound 125I-bFGF was internalized. At this temperature, degradation of the internalized ligand was followed after 1 h by the appearance of three major bands of 15,000, 10,000, and 8,000 Da and was inhibited by chloroquine. These results demonstrated two classes of binding sites for bFGF in HOME cells; the number of high-affinity binding sites being larger than the number reported for bovine capillary endothelial cells. The intracellular processing of bFGF in HOME cells seems to be different from that of heparin binding growth factor-1 in murine lung capillary endothelial cells and of eye-derived growth factor-1 in Chinese hamster fibroblasts.     

Involvement of interleukin-8, vascular endothelial growth factor, and basic fibroblast growth factor in tumor necrosis factor alpha-dependent angiogenesis.

Tumor necrosis factor alpha (TNF-alpha) is a macrophage/monocyte-derived polypeptide which modulates the expression of various genes in vascular endothelial cells and induces angiogenesis. However, the underlying mechanism by which TNF-alpha mediates angiogenesis is not completely understood. In this study, we assessed whether TNF-alpha-induced angiogenesis is mediated through TNF-alpha itself or indirectly through other TNF-alpha-induced angiogenesis-promoting factors. Cellular mRNA levels of interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and their receptors were increased after the treatment of human microvascular endothelial cells with TNF-alpha (100 U/ml). TNF-alpha-dependent tubular morphogenesis in vascular endothelial cells was inhibited by the administration of anti-IL-8, anti-VEGF, and anti-bFGF antibodies, and coadministration of all three antibodies almost completely abrogated tubular formation. Moreover, treatment with Sp1, NF-kappaB, and c-Jun antisense oligonucleotides inhibited TNF-alpha-dependent tubular morphogenesis by microvascular endothelial cells. Administration of a NF-kappaB antisense oligonucleotide almost completely inhibited TNF-alpha-dependent IL-8 production and partially abrogated TNF-alpha-dependent VEGF production, and an Sp1 antisense sequence partially inhibited TNF-alpha-dependent production of VEGF. A c-Jun antisense oligonucleotide significantly inhibited TNF-alpha-dependent bFGF production but did not affect the production of IL-8 and VEGF. Administration of an anti-IL-8 or anti-VEGF antibody also blocked TNF-alpha-induced neovascularization in the rabbit cornea in vivo. Thus, angiogenesis by TNF-alpha appears to be modulated through various angiogenic factors, both in vitro and in vivo, and this pathway is controlled through paracrine and/or autocrine mechanisms.     

Nuclear matrix proteins in well and poorly differentiated human breast cancer cell lines

The nuclear matrix, besides providing the structural support of the nucleus, is involved in various cellular functions of the nucleus. Nuclear matrix proteins (NMPs), which are both tissue- and cell type–specific, are altered with transformation and state of differentiation. Furthermore, NMPs have been identified as informative markers of disease states. Here, the NMP profiles from human breast cancer cell lines and breast tumours were analyzed using two-dimension gel electrophoresis. We identified NMPs that are associated with well and poorly differentiated human breast cancer cells in vitro and in vivo. Five NMPs (NMBC 1–5) were found to be exclusive for well-differentiated human breast cancer cells, while one NMP (NMBC-6) was found to be present only in poorly differentiated human breast cancer cells. The identification of these proteins suggests the potential use of nuclear matrix proteins as prognostic indicators. J. Cell. Biochem. 66:9–15, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Autonomous migration of human fetal skin fibroblasts into a denuded area in a cell monolayer is mediated by basic fibroblast growth factor and collagen

Summary Human fetal skin fibroblasts (TIG-3S) were found to migrate into a denuded area in a cell monolayer when cultured in both serum-depleted and serum-supplemented media, unlike adult-donor skin fibroblasts which migrated well only when cultured in serum-supplemented medium. Therefore, a series of experiments was carried out to determine whether autocrine factors are involved in their migration. The migration of TIG-3S cells in serum-depleted medium was suppressed by the addition of suramin, a factor with growth factor antagonist properties, which suggests that growth factors are important for cell migration. The suramin-induced inhibition was reversed completely by adding excess basic fibroblast growth factor (bFGF) to the culture medium and partially by platelet-derived growth factor (PDGF). Treatment with neutralizing anti-PDGF antibody did not suppress TIG-3S cell migration, whereas neutralizing anti-bFGF antibody did, which indicates that bFGF is an autocrine and PDGF a paracrine factor involved in cell migration. Next, an experiment was performed to ascertain whether the extracellular matrix is involved in TIG-3S cell migration. Monensin, an inhibitor of extracellular matrix secretion, inhibited cell migration, which was reversed by adding excess type I collagen, but not excess plasma fibronectin. In addition, further evidence for the involvement of collagen was provided by the observation that ethyl-3,4-dihydroxybenzoate, a specific inhibitor of collagen synthesis, suppressed cell migration. These results suggest that the autonomous migration of TIG-3S human fetal skin fibroblasts is mediated by bFGF and type I collagen, which they produce and secrete.     

Isolation and partial characterization of an endothelial cell growth factor from the bovine kidney: homology with basic fibroblast growth factor

Two kidney-derived mitogens have been isolated by ion exchange, heparin-Sepharose and reverse-phase high-performance liquid chromatography on the basis of their capacity to stimulate the proliferation of bovine vascular endothelial cells in vitro. Gas phase sequence analysis identified the amino terminal sequences His-Phe-Lys-Asp-Pro-Lys-Arg-Leu-Tyr-X-Lys-Asn-Gly-Gly-Phe-Phe-Leu and His-Phe-Lys-Asp-Pro-Lys-Arg-Leu, respectively. The sequences are identical to residues 16-32 and 16-23 of bovine basic pituitary Fibroblast Growth Factor (FGF). The possibility that these kidney-derived mitogens are related, if not identical, to pituitary basic FGF is supported by the observations that they have similar molecular weights (15-16 kDa), similar retention behavior on all steps of chromatography and similar amino acid compositions, and they share at least some structural homology. Moreover, the kidney-derived growth factors, like basic FGF, are potent stimulators of capillary endothelial cells, granulosa cells, adrenocortical cells and vascular smooth-muscle cells (ED50 = 50 pg). The results demonstrate the existence of a kidney-derived FGF and suggest that at least some of the mitogenic, angiogenic and neovascularising activities described to be present in the kidney are due to the presence of an FGF-like molecule in this tissue.     

Localisation of acidic and basic fibroblast growth factors during mouse palate development and their effects on mouse palate mesenchyme cells in vitro

Summary The distribution of acidic and basic fibroblast growth factors (aFGF, bFGF) was mapped during mouse embryonic palate development. Generally, they localised most intensely in the basement membrane and epithelia rather than the mesenchyme. Localisation was predominantly restricted to the palatal nasal, and medial edge epithelia. Staining was particularly intense in the medial edge epithelia at the time of mid-line epithelial seam formation. Intense staining persisted in the epithelia of the degenerating seam and later in the oral and nasal epithelial triangles. Mouse embryonic palate mesenchyme (MEPM) cells cultured in vitro on a variety of substrata (on plastic, on the surface of a collagen gel and within a collagen gel) responded to treatment with aFGF or bFGF. These responses were modulated by the culture substratum. The FGFs stimulated MEPM cell proliferation on plastic and on collagen, but inhibited cell growth in collagen. The FGFs had little effect on protein production when cells were cultured on plastic, but caused a large reduction in on-collagen and incollagen cultures. This reduction was greater in collagenous than non-collagenous proteins. Generally, treatment with FGFs stimulated the production of glycosaminoglycans (GAGs), particularly hyaluronan (HA) and dermatan sulphate (DS). In addition, the size class of HA was shifted to a higher molecular weight form. These data indicate that aFGF and bFGF may play a role in modulating mesenchymal cell matrix biosynthesis, so facilitating palatal epithelial seam degeneration. Correspondence to: M.W.J. Ferguson