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).     

The opposing effects of basic fibroblast growth factor and transforming growth factor beta on the regulation of plasminogen activator activity in capillary endothelial cells

Basic fibroblast growth factor (bFGF), a potent inducer of angiogenesis in vivo, stimulates the production of both urokinase- and tissue-type plasminogen activators (PAs) in cultured bovine capillary endothelial cells. The observed increase in proteolytic activity induced by bFGF was effectively diminished by picogram amounts of transforming growth factor beta (TGF beta), but could not be abolished by increasing the amount of TGF beta. However, the inhibition by TGF beta was greatly enhanced if the cells were pretreated with TGF beta before addition of bFGF. After prolonged incubation of cultures treated simultaneously with bFGF and TGF beta, the inhibitory effect of TGF beta diminished and the stimulatory effect of the added bFGF dominated as assayed by PA levels. TGF beta did not alter the receptor binding of labeled bFGF, nor did a 6-h pretreatment with TGF beta reduce the amount of bFGF bound. The major difference between the effects of bFGF and TGF beta was that while bFGF effectively enhanced PA activity expressed by the cells, TGF beta decreased the amounts of both cell-associated and secreted PA activity by decreasing enzyme production. Both bFGF and TGF beta increased the secretion of the endothelial-type plasminogen activator inhibitor.     

A distinct basic fibroblast growth factor (FGF-2)/FGF receptor interaction distinguishes urokinase-type plasminogen activator induction from mitogenicity in endothelial cells.

Basic fibroblast growth factor (FGF-2) induces cell proliferation and urokinase-type plasminogen activator (uPA) production in fetal bovine aortic endothelial GM 7373 cells. In the present paper we investigated the role of the interaction of FGF-2 with tyrosine-kinase (TK) FGF receptors (FGFRs) in mediating uPA up-regulation in these cells. The results show that FGF-2 antagonists suramin, protamine, heparin, the synthetic peptide FGF-2(112-155), and a soluble form of FGFR-1 do not inhibit FGF-2-mediated uPA up-regulation at concentrations that affect growth factor binding to cell surface receptors and mitogenic activity. In contrast, tyrosine phosphorylation inhibitors and overexpression of a dominant negative TK- mutant of FGFR-1 abolish the uPA-inducing activity of FGF-2, indicating that FGFR and its TK activity are essential in mediating uPA induction. Accordingly, FGF-2 induces uPA up-regulation in Chinese hamster ovary cells transfected with wild-type FGFR-1, -2, -3, or -4 but not with TK- FGFR-1 mutant. Small unilamellar phosphatidyl choline:cholesterol vesicles loaded with FGF-2 increased uPA production in GM 7373 cells in the absence of a mitogenic response. Liposome-encapsulated FGF-2 showed a limited but significant capacity, relative to free FGF-2, to interact with FGFR both at 4 degrees C and 37 degrees C and to be internalized within the cell. uPA up-regulation by liposome-encapsulated FGF-2 was quenched by neutralizing anti-FGF-2 antibodies, indicating that the activity of liposome-delivered FGF-2 is mediated by an extracellular action of the growth factor. Taken together, the data indicate that a distinct interaction of FGF-2 with FGFR, quantitatively and/or qualitatively different from the one that leads to mitogenicity, is responsible for the uPA-inducing activity of the growth factor.     

Basic fibroblast growth factor-induced activation of latent transforming growth factor beta in endothelial cells: regulation of plasminogen activator activity

Plasmodium falciparum malaria parasites invade human erythrocytes by means of a parasite receptor for erythrocytes, the 175-kD erythrocyte binding antigen (EBA-175). Similar to invasion efficiency, binding requires N-acetylneuraminic acid (Neu5Ac) on human erythrocytes, specifically the glycophorins. EBA-175 bound to erythrocytes with receptor-like specificity and was saturable. The specificity of EBA-175 binding was studied to determine if its binding is influenced either by simple electrostatic interaction with the negatively charged Neu5Ac (on the erythrocyte surface); or if Neu5Ac indirectly affected the conformation of an unknown ligand, or if Neu5Ac itself in specific linkage and carbohydrate composition was the primary ligand for EBA-175 as demonstrated for hemagglutinins of influenza viruses. Most Neu5Ac on human erythrocytes is linked to galactose by alpha 2-3 and alpha 2-6 linkages on glycophorin A. Soluble Neu5Ac by itself in solution did not competitively inhibit the binding of EBA-175 to erythrocytes, suggesting that linkage to an underlying sugar is required for binding in contrast to charge alone. Binding was competitively inhibited only by Neu5Ac(alpha 2-3)Gal-containing oligosaccharides. Similar oligosaccharides containing Neu5Ac(alpha 2-6)Gal-linkages had only slight inhibitory effects. Binding inhibition assays with modified sialic acids and other saccharides confirmed that oligosaccharide composition and linkage were primary factors for efficient binding. EBA-175 bound tightly enough to glycophorin A that the complex could be precipitated with an anti-glycophorin A monoclonal antibody. Selective cleavage of O-linked tetrasaccharides clustered at the NH2 terminus of glycophorin A markedly reduced binding in inhibition studies. We conclude that the Neu5Ac(a2,3)-Gal- determinant on O-linked tetrasaccharides of glycophorin A appear to be the preferential erythrocyte ligand for EBA-175.     

Autocrine activities of basic fibroblast growth factor: regulation of endothelial cell movement, plasminogen activator synthesis, and DNA synthesis

We have found that the spontaneous migration of bovine aortic endothelial cells from the edge of a denuded area in a confluent monolayer is dependent upon the release of endogenous basic fibroblast growth factor (bFGF). Cell movement is blocked by purified polyclonal rabbit IgG to bFGF as well as affinity purified anti-bFGF IgG and anti-bFGF F(ab')2 fragments. The inhibitory effect of the immunoglobulins is dependent upon antibody concentration, is reversible, is overcome by the addition of recombinant bFGF, and is removed by affinity chromatography of the antiserum through a column of bFGF-Sepharose. Cell movement is also reversibly inhibited by the addition of protamine sulfate and suramin; two agents reported to block bFGF binding to its receptor. The addition of recombinant bFGF to wounded monolayers accelerates the movement of cells into the denuded area. Transforming growth factor beta which has been shown to antagonize several other effects of bFGF also inhibits cell movement. The anti-bFGF IgG prevents the movement of bovine capillary endothelial cells, BHK-21, NIH 3T3, and human skin fibroblasts into a denuded area. Antibodies to bFGF, as well as suramin and protamine sulfate also suppress the basal levels of plasminogen activator and DNA synthesis in bovine aortic endothelial cells.     

Interaction of single-chain urokinase-type plasminogen activator with human endothelial cells

The interaction of urokinase-type plasminogen activators with receptors on the surface of endothelial cells may play an important role in the regulation of fibrinolysis and cell migration. Therefore, we investigated whether human umbilical vein endothelial cells (HUVEC) express receptors for single-chain urokinase (scu-PA) on the cell surface and examined the effect of such binding on plasminogen activator activity. Binding of 125I-labeled scu-PA to HUVEC, performed at 4 degrees C, was saturable, reversible, and specific (k+1 4 +/- 1 X 10(6) min-1 M-1, k-1 6.2 +/- 1.4 X 10(-3) min-1, Kd 2.8 +/- 0.1 nM; Bmax 2.2 +/- 0.1 X 10(5) sites/cell; mean +/- S.E.). Binding of radiolabeled scu-PA was inhibited by both natural and recombinant wild-type scu-PA, high molecular weight two-chain u-PA (tcu-PA), catalytic site-inactivated tcu-PA, an amino-terminal fragment of u-PA (amino acids 1-143), and a smaller peptide (amino acids 4-42) corresponding primarily to the epidermal growth factor-like domain. Binding was not inhibited by low molecular weight urokinase or by a recombinant scu-PA missing amino acids 9-45. Cell-bound scu-PA migrated at its native molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of plasminogen, scu-PA bound to endothelial cells generated greater plasmin activity than did scu-PA in the absence of cells. In contrast, when tcu-PA was added directly to HUVEC, sodium dodecyl sulfate-stable complexes formed with cell or matrix-associated plasminogen activator inhibitors with a loss of plasminogen activator activity. These studies suggest that endothelial cells in culture express high affinity binding sites for the epidermal growth factor domain of scu-PA. Interaction of scu-PA with these receptors may permit plasminogen activator activity to be expressed at discrete sites on the endothelial cell membrane.     

The expression and localization of urokinase-type plasminogen activator and its type 1 inhibitor are regulated by retinoic acid and fibroblast growth factor in human teratocarcinoma cells.

Human Tera 2 embryonal carcinoma cells switch gradually from rapidly growing undifferentiated cells to almost nonproliferating cells during retinoic acid (RA)-induced neuronal differentiation. This process is associated with the increased expression of type 1 plasminogen activator inhibitor (PAI 1) mRNA, and the secreted inhibitor is immobilized to the pericellular area. Furthermore, the differentiation is accompanied by a decrease in the amount of both the secreted tissue-type PA (tPA) and the mainly cell-associated urokinase-type PA (uPA) activity. In RA-differentiated cells, uPA becomes localized at the vinculin-rich cell-substratum adhesion sites. Fibroblast growth factor activity has been associated with various events during embryonal growth and with the regulation of proteolytic enzymes. A short-term treatment of the undifferentiated Tera 2 cells with basic fibroblast growth factor (bFGF) increases uPA mRNA levels and the cell-associated uPA activity, whereas the secretory tPA activity decreases. bFGF induces PAI 1 mRNA expression in the undifferentiated cells, but unlike PAI 1 protein after RA-treatment, the inhibitor does not accumulate around the cells but is released in the medium. A similar exposure to bFGF has less effect on the RA-differentiated Tera 2 cells. Under these conditions bFGF treatment leads to an increase in the amounts of PAI 1 and uPA mRNAs, but no changes in the localization of these components can be seen. Differentiation of human embryonal carcinoma cells is thus connected with an altered response to bFGF.     

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.     

The inactivation of single-chain urokinase-type plasminogen activator by thrombin on cultured human endothelial cells

Single-chain urokinase-type plasminogen activator (scu-PA) is cleaved by thrombin, resulting in an inactive molecule called thrombin-cleaved two-chain urokinase-type plasminogen activator (tcu-PA/T). There is no knowledge about cell-mediated inactivation of scu-PA. We have studied whether scu-PA bound to cultured human umbilical vein endothelial cells (HUVEC) could be inactivated by thrombin. High molecular weight scu-PA was bound to HUVEC and incubated with increasing amounts of thrombin for 30 min at 37 degrees C. Cell-bound urokinase-type plasminogen activator (u-PA) was released and levels of scu-PA, tcu-PA/T and active two-chain u-PA were measured using sensitive bioimmunoassays. Cell-bound scu-PA was efficiently inactivated by thrombin. Fifty percent inactivation of scu-PA occurred at about 0.2 nM thrombin. In the presence of monoclonal anti-urokinase receptor IgG, at least 50% of the binding of scu-PA to HUVEC was inhibited. The relative amount of tcu-PA/T that was generated by thrombin was not affected by the monoclonal antibody. These results indicated that scu-PA bound to HUVEC via the urokinase receptor can be inactivated by thrombin. The efficient inactivation of cell-bound scu-PA suggests that a cofactor for thrombin may be involved, like thrombomodulin or glycosaminoglycans. It is concluded that scu-PA bound to the urokinase receptor on a cell surface can be inactivated by thrombin, which may have profound effects on u-PA-mediated local fibrinolysis and extracellular proteolysis during processes in which thrombin is also involved.     

Metabolism of receptor-bound and matrix-bound basic fibroblast growth factor by bovine capillary endothelial cells

Bovine capillary endothelial (BCE) cells were incubated at 4 degrees C with 5 ng/ml 125I-basic fibroblast growth factor (bFGF) to equilibrate 125I-bFGF with high affinity cell surface receptors and low affinity matrix binding sites. 67% of the added 125I-bFGF bound to the matrix and 7% bound to receptors. The fate of bound bFGF was followed after cells were incubated in bFGF-free medium and were shifted to 37 degrees C to restore cell metabolism. 125I-bFGF bound to receptors decreased rapidly while the amount of 125I-bFGF bound to matrix was reduced more slowly. The rapid decrease in receptor-bound 125I-bFGF appeared to be due to a down-regulation of bFGF receptors; cells that had been treated for 5 h with bFGF had 60% fewer high affinity receptors than untreated cells. Despite the initial high level of 125I-bFGF binding to matrix, most of this 125I-bFGF was mobilized and metabolized by the cells. 125I-bFGF was internalized by the cells at 37 degrees C, leading to a constant accumulation of 125I-bFGF within the cell. Internalized bFGF was rapidly cleaved from an 18-kD form to a 16-kD form. The 16-kD form was more slowly degraded with a half-life of approximately 8 h. Degradation of internalized 125I-bFGF was inhibited by chloroquine, suggesting that the digestion occurred in a lysosomal compartment. The role of matrix binding sites in the internalization process was investigated. Binding to matrix sites seemed not to be directly involved in the internalization process, since addition of heparin at a concentration that blocked 95% of the binding to matrix had no effect on the initial rate of internalization of bFGF. BCE cells also released a substance that competed for the binding of bFGF to matrix but not to receptors. This substance bound to DEAE-cellulose and was sensitive to heparinase treatment, suggesting that it was a heparinlike molecule. Thus, heparinlike molecules produced by BCE cells can modulate the cellular interaction with bFGF. Matrix-associated heparinlike molecules bind bFGF which can later be metabolized by the cell, and secreted heparinlike molecules release bFGF from matrices.     

Activation mechanisms of the urokinase-type plasminogen activator promoter by hepatocyte growth factor/scatter factor.

Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic effector inducing invasion and metastasis of tumor cells that express the Met tyrosine kinase receptor. One of the effectors of HGF/SF is the urokinase-type plasminogen activator, a serine protease that facilitates tumor progression and metastasis by controlling the synthesis of the extracellular matrix degrading plasmin. Stimulation of NIH 3T3 cells that were stably transfected with the human Met receptor (NIH 3T3-Methum) with HGF/SF induced a trans-activation of the urokinase promoter and urokinase secretion. Induction of the urokinase promoter by HGF/SF via the Met receptor was blocked by co-expression of a dominant-negative Grb2 and Sos1 expression construct. Further, the expression of the catalytically inactive mutants of Ha-Ras, RhoA, c-Raf, and Erk2 or addition of the Mek1-specific inhibitor PD 098059 abrogated the stimulation of the urokinase promoter by HGF/SF. A sequence residing between -2109 and -1870 base pairs (bp) was critical for stimulation of the urokinase gene by HGF/SF. Mobility shift assays with oligonucleotides spanning an AP-1 site at -1880 bp or a combined PEA3/AP-1 site at -1967 bp showed binding of nuclear factors from NIH 3T3-Methum cells. Expression of an expression plasmid that inhibits DNA binding of AP-1 proteins (A-Fos) abrogated inducible and basal activation of the urokinase promoter. Nuclear extract from unstimulated NIH 3T3-Methum cells contained more JunD and showed a stronger JunD supershift with the AP-1 oligonucleotides, compared with HGF/SF-stimulated cells. Consistent with the levels of JunD expression being functionally important for basal expression of the urokinase promoter, we found that overexpression of wild type JunD inhibited the induction of the urokinase promoter by HGF/SF. These data suggest that the induction of urokinase by HGF/SF is regulated by a Grb2/Sos1/Ha-Ras/c-Raf/RhoA/Mek1/Erk2/c-++ +Jun-dependent mitogen-activated protein kinase pathway.     

Release of basic fibroblast growth factor-heparan sulfate complexes from endothelial cells by plasminogen activator-mediated proteolytic activity

Cultured bovine capillary endothelial (BCE) cells synthesize heparan sulfate proteoglycans (HSPG), which are both secreted into the culture medium and deposited in the cell layer. The nonsoluble HSPGs can be isolated as two predominant species: a larger 800-kD HSPG, which is recovered from preparations of extracellular matrix, and a 250-kD HSPG, which is solubilized by nonionic detergent extraction of the cells. Both HSPG species bind bFGF. 125I-bFGF bound to BCE cell cultures is readily released by either heparinase or plasmin. When released by plasmin, the growth factor is recovered from the incubation medium as a complex with the partly degraded high molecular mass HSPG. Endogenous bFGF activity is released by a proteolytic treatment of cultured BCE cells. The bFGF-binding HSPGs are also released when cultures are incubated with the inactive proenzyme plasminogen. Under such experimental conditions, the release of the extracellular proteoglycans can be enhanced by treating the cells either with bFGF, which increases the plasminogen activating activity expressed by the cells, or decreased by treating the cells with transforming growth factor beta, which decreases the plasminogen activating activity of the cells. Specific immune antibodies raised against bovine urokinase also block the release of HSPG from BCE cell cultures. We propose that this plasminogen activator-mediated proteolysis provides a mechanism for the release of biologically active bFGF-HSPG complexes from the extracellular matrix and that bFGF release can be regulated by the balance between factors affecting the pericellular proteolytic activity.