Autocrinological role of basic fibroblast growth factor on tube formation of vascular endothelial cells in vitro.

When bovine capillary endothelial (BCE) cells plated on type I collagen gel were covered with a second layer of collage gel, BCE cells reorganized into a network of capillary-like structures. In the presence of affinity purified anti-basic fibroblast growth factor (bFGF) antibody, this reorganization was inhibited. By using a computerized image analyzer, the formation of network structures and the effect of anti-bFGF antibody was quantitated. The inhibitory effect of anti-FGF antibody was dose-dependent and maximal inhibition was observed at 2.0 micrograms/ml of antibody. Exogenously added bFGF potentiated network formation of BCE cells and coadministration of bFGF abrogated the inhibitory effect of anti-bFGF antibody. Platelet factor 4, which blocks the binding of bFGF to its receptor, inhibited network formation. These results indicate that bFGF produced by endothelial cells regulates angiogenesis as an autocrine factor.     

Transforming growth factor beta-1 positively modulates the bioactivity of fibroblast growth factor on corneal endothelial cells

Transforming growth factor beta-1 (TGF beta-1), known as an inhibitor of vascular endothelial cell proliferation in vitro, stimulates bovine corneal endothelial cells (BCE) proliferation. It also positively modulates the response of BCE cells to fibroblast growth factor (FGF) and epidermal growth factor (EGF). This effect is concentration dependent within a physiological range of TGF beta-1, but it is blocked if cells are cultured on extracellular-matrix-coated dishes instead of plastic. TGF beta-1 does not modify the number or the affinity of bFGF receptors on BCE cell surface but increases the bFGF content of these cells. This suggests that TGF beta-1 might act through regulation of bFGF synthesis in BCE cells.     

In vitro angiogenesis on the human amniotic membrane: requirement for basic fibroblast growth factor-induced proteinases

The role of basic fibroblast growth factor-(bFGF) induced proteinases in basement membrane (BM) invasion by bovine capillary endothelial (BCE) cells was studied using a quantitative in vitro assay previously described (Mignatti et al., 1986). 125I-iododeoxyuridine-labeled BCE cells were grown for 72 h on the human amnion BM, and cell invasion was determined by measuring the radioactivity associated with the tissue after removal of the noninvasive cell layer. BCE cells were noninvasive under normal conditions. Addition of human bFGF to either the BM or to the stromal aspect of the amnion induced BCE cell invasion with a dose-dependent response. This effect was maximal in the presence of 70 ng/ml bFGF, and was inhibited by anti-FGF antibody. Transforming growth factor beta, as well as plasmin inhibitors and anti-tissue type plasminogen activator antibody inhibited BCE cell invasion. The tissue inhibitor of metalloproteinases, 1-10 phenanthroline, anti-type IV and anti-interstitial collagenase antibodies had the same effect. On the contrary, anti-stromelysin antibody and Eglin, an inhibitor of elastase, were ineffective. The results obtained show that both the plasminogen activator-plasmin system and specific collagenases are involved in the invasive process occurring during angiogenesis.     

Migratory and proliferative effect of platelet-derived growth factor in rabbit retinal endothelial cells: Evidence of an autocrine pathway of platelet-derived growth factor

Angiogenesis is a crucial event in the progression of diabetic retinopathy. Migration and proliferation of endothelial cells (EC) are important steps in angiogenesis and are caused by angiogenic factors such as basic fibroblast growth factor (bFGF). In this work, capillary EC were isolated from rabbit retinal tissues and rabbit retinal EC (RREC) were found to secrete a migration factor for RREC in conditioned medium (CM). The activity was inhibited by an anti-platelet-derived growth factor (PDGF) antibody, but not by an anti-bFGF antibody. We also found that RREC showed a migratory response to PDGF. The response was induced by PDGF-BB and PDGF-AB dose dependently, but not by PDGF-AA, indicating that it was mediated by PDGF-β receptor-dependent pathways, and that the PDGF-like factor was PDGF-BB or -AB. In addition, PDGF-BB induced the proliferation of RREC as well as bFGF. These data indicate that RREC have an autocrine pathway of PDGF by the secretion of and the response to PDGF. PDGF may play significant parts in angiogenesis in the progression of diabetic retinopathy. © 1994 Wiley-Liss, Inc.     

Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation

Cultured bovine capillary endothelial (BCE) cells were found to synthesize and secrete high molecular mass heparan sulfate proteoglycans and glycosaminoglycans, which bound basic fibroblast growth factor (bFGF). The secreted heparan sulfate molecules were purified by DEAE cellulose chromatography, followed by Sepharose 4B chromatography and affinity chromatography on immobilized bFGF. Most of the heparinase-sensitive sulfated molecules secreted into the medium by BCE cells bound to immobilized bFGF at low salt concentrations. However, elution from bFGF with increasing salt concentrations demonstrated varying affinities for bFGF among the secreted heparan sulfate molecules, with part of the heparan sulfate requiring NaCl concentrations between 1.0 and 1.5 M for elution. Cell extracts prepared from BCE cells also contained a bFGF-binding heparan sulfate proteoglycan, which could be released from the intact cells by a short proteinase treatment. The purified bFGF-binding heparan sulfate competed with 125I-bFGF for binding to low-affinity binding sites but not to high-affinity sites on the cells. Heparan sulfate did not interfere with bFGF stimulation of plasminogen activator activity in BCE cells in agreement with its lack of effect on binding of 125I-bFGF to high-affinity sites. Soluble bFGF was readily degraded by plasmin, whereas bFGF bound to heparan sulfate was protected from proteolytic degradation. Treatment of the heparan sulfate with heparinase before addition of plasmin abolished the protection and resulted in degradation of bFGF by the added proteinase. The results suggest that heparan sulfate released either directly by cells or through proteolytic degradation of their extracellular milieu may act as carrier for bFGF and facilitate the diffusion of locally produced growth factor by competing with its binding to surrounding matrix structures. Simultaneously, the secreted heparan sulfate glycosaminoglycans protect the growth factor from proteolytic degradation by extracellular proteinases, which are abundant at sites of neovascularization or cell invasion.     

Specific blockade of basic fibroblast growth factor gene expression in endothelial cells by antisense oligonucleotide.

The migration and proliferation of endothelial cells play a pivotal role in various vascular diseases. To elucidate the role of endogenous basic fibroblast growth factor (bFGF) produced within endothelial cells on cell growth, we introduced the antisense oligonucleotide complementary to bFGF mRNA into cultured bovine aortic endothelial cells by cationic liposome to block the production of autocrine bFGF. The treatment of the endothelial cells with the specific antisense oligomer efficiently inhibited the synthesis of bFGF with the concomitant suppression of endothelial proliferation, indicating the significant role of bFGF as an endothelial growth promotor. The neutralizing antibody against bFGF had no inhibition on basal DNA synthesis of the endothelial cells, in contrast to marked suppressive action of bFGF antisense oligomer. The results provide the new analytic and therapeutic implications in the use of the antisense methodology for the study of vascular biology.     

Both normal and tumor cells produce basic fibroblast growth factor

We have previously purified from human placenta a basic fibroblast growth factor (FGF)-like molecule which stimulates the production of plasminogen activator (PA) and collagenase, induces DNA synthesis, produces an increase in motility in cultured bovine capillary endothelial (BCE) cells, and induces angiogenesis in vivo. The ability of basic FGF to stimulate PA production in BCE cells was used as an assay for the presence of basic FGF-like molecules in extracts of both normal and tumor-derived cultured cells. The identity of the PA-stimulatory activity with basic FGF was confirmed by its high affinity for heparin and by its cross-reactivity with antibodies to human placental basic FGF. Basic FGF-like molecules were identified in eight of ten cell lines tested, and the amount of FGF-like activity present in these cells bore no relation to their origin from normal or tumor tissue. The test cells, BCE cells, had one of the highest levels of FGF-like activity, suggesting that it may have an autocrine role in these cells.     

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.     

Multiple forms of an angiogenesis factor: basic fibroblast growth factor

An angiogenesis factor has been isolated from human placenta and human hepatoma cells on the basis of its ability to stimulate protease production in cultured capillary endothelial cells. The purified angiogenesis factor also stimulated DNA synthesis and motility in capillary endothelial cells and induced angiogenesis in vivo. Amino acid sequence data revealed that the angiogenesis factor was human basic fibroblast growth factor (bFGF). Other angiogenesis factors isolated on the basis of their ability to stimulate endothelial cell proliferation have also been identified as bFGFs. The bFGFs that have been sequenced show variability in their N-termini. These different forms of bFGF may be naturally occurring processed forms or may be generated by proteases released during the isolation procedure. Recently a bFGF with a large N-terminal extension has been identified. This Mr 25,000 bFGF has the same biological activity and the same affinity for the bFGF receptor as the typical Mr 18,000 bFGFs. The Mr 25,000 bFGF can be converted into an Mr 18,000 form by treatment with low concentrations of trypsin, suggesting that it may be a precursor to the Mr 18,000 bFGF.     

Role of extracellular matrix in the action of basic fibroblast growth factor: Matrix as a source of growth factor for long-term stimulation of plasminogen activator production and DNA synthesis

When bovine capillary endothelial (BCE) cells were treated with 10 ng/ml of basic fibroblast growth factor (bFGF) for 10 or 30 minutes at 37°C, washed extensively with phosphate-buffered saline (PBS) and incubated in bFGF-free medium, plasminogen activator (PA) production was stimulated to the same extent as in cells exposed continuously to bFGF. Three methods of removing bFGF from heparin-like binding sites in the extracellular matrix, but not from bFGF receptors, abolished this long-term effect of a brief exposure to bFGF. First, BCE cells exposed to bFGF for 30 minutes were washed with 2M NaCI and incubated in bFGF-free medium. Second, BCE cells were incubated with bFGF for 10 minutes in the presence of heparin, and cells were washed with PBS and incubated in bFGF-free medium. Third, BCE cell cultures were treated with heparinase and exposed to bFGF. Each of these treatments abolished the long-term (24-48 hours) stimulation of PA production normally observed after brief exposure to bFGF. In each of these experiments, incubation of cells in bFGF-containing medium after the treatments resulted in normal stimulation of PA production, demonstrating that the treatments did not harm the cells. Stimulation of DNA synthesis was observed when cells were exposed to bFGF for 2 hours at 4°C, incubated in bFGF-free medium for 24 hours at 37°C, and assayed for ³H-thymidine incorporation. However, no stimulation was observed if the 2 hours incubation at 4°C was carried out in the presence of heparin. Thus, long-term stimulation of PA activity and DNA synthesis after a brief exposure to bFGF seems to be a consequence of bFGF binding to the extracellular matrix. The extracellular matrixmay act as a physiologic buffer, binding bFGF when concentrations are high and releasing it later for interaction with its receptor. This interaction with matrix may be required for the in vivo action of bFGF.     

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.     

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.     

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)     

Skeletal muscle capillarity and angiogenic mRNA levels after exercise training in normoxia and chronic hypoxia.

Gene expression of vascular endothelial growth factor (VEGF), and to a lesser extent of transforming growth factor-beta(1) (TGF-beta(1)) and basic fibroblast growth factor (bFGF), has been found to increase in rat skeletal muscle after a single exercise bout. In addition, acute hypoxia augments the VEGF mRNA response to exercise, which suggests that, if VEGF is important in muscle angiogenesis, hypoxic training might produce greater capillary growth than normoxic training. Therefore, we examined the effects of exercise training (treadmill running at the same absolute intensity) in normoxia and hypoxia (inspired O(2) fraction = 0.12) on rat skeletal muscle capillarity and on resting and postexercise gene expression of VEGF, its major receptors (flt-1 and flk-1), TGF-beta(1), and bFGF. Normoxic training did not alter basal or exercise-induced VEGF mRNA levels but produced a modest twofold increase in bFGF mRNA (P < 0.05). Rats trained in hypoxia exhibited an attenuated VEGF mRNA response to exercise (1.8-fold compared 3.4-fold with normoxic training; P < 0.05), absent TGF-beta(1) and flt-1 mRNA responses to exercise, and an approximately threefold (P < 0.05) decrease in bFGF mRNA levels. flk-1 mRNA levels were not significantly altered by either normoxic or hypoxic training. An increase in skeletal muscle capillarity was observed only in hypoxically trained rats. These data show that, whereas training in hypoxia potentiates the adaptive angiogenic response of skeletal muscle to a given absolute intensity of exercise, this was not evident in the gene expression of VEGF or its receptors when assessed at the end of training.     

Tumor necrosis factor inhibits the proliferation of cultured capillary endothelial cells

We have examined the effect of tumor necrosis factor (TNF) on the proliferation of capillary endothelial cells derived from brain or adrenal cortex. In both cell types, TNF inhibits basal as well as basic fibroblast growth factor (bFGF)-stimulated cell proliferation. TNF induces an additional cytotoxic effect in bFGF-stimulated, but not in unstimulated, capillary endothelial cells. These results suggest that TNF could act as a negative regulator of angiogenesis in vivo and further, that TNF might induce selective cytotoxicity of capillary endothelial cells stimulated by tumor-derived bFGF. These results could explain why TNF induces hemorrhagic necrosis of certain, solid tumors.