Upregulation of neuropilin-1 by basic fibroblast growth factor enhances vascular smooth muscle cell migration in response to VEGF

Neuropilin-1 (NRP-1) is a co-receptor for vascular endothelial growth factor (VEGF). During neovascularization, vascular smooth muscle cells (VSMCs) and pericytes modulate the function of endothelial cells. Factors that mediate NRP-1 in human VSMCs (hVSMCs) remain to be elucidated. We studied various angiogenic cytokines to identify factors that increase NRP-1 expression in hVSMCs. Treatment of hVSMCs with basic fibroblast growth factor (b-FGF) induced expressions of NRP-1 mRNA and protein whereas epidermal growth factor, insulin-like growth factor-1, and interleukin-1beta did not. b-FGF induced phosphorylation of Erk-1/2 and JNK. MEK1/2 and nuclear factor kappa B (NF-kappaB) inhibitors (U0126 and TLCK, respectively) blocked the ability of b-FGF to induce NRP-1 mRNA expression, but inhibition of JNK (SP600125) or PI3-kinase activity (wortmannin) did not. Further, the increase in NRP-1 expression by b-FGF enhanced hVSMCs migration in response to VEGF(165). This effect was dependent on the binding of VEGF(165) to VEGFR-2, as blocking antibodies to VEGFR-2, but not VEGFR-1, inhibited VEGF(165)-induced migration. In conclusion, b-FGF increased NRP-1 expression in hVSMCs that in turn enhance the effect of VEGF(165) on cell migration. The enhanced migration of hVSMCs was mediated through binding of VEGF(165) to both NRP-1 and VEGFR-2, as inhibition of VEGFR-2 on these cells blocked the effect of VEGF-mediated cell migration.     

The basic fibroblast growth factor-saporin mitotoxin acts through the basic fibroblast growth factor receptor.

We have confirmed the hypothesis that a mitotoxin resulting from the conjugation of basic fibroblast growth factor and saporin exerts its cytotoxic effect through specific interaction with the basic fibroblast growth factor (FGF) receptor. Accordingly, the mitotoxin stimulates tyrosine phosphorylation of the 90 kD substrate that characterizes the initial cellular response to basic FGF. Cross-linking experiments show that radio-labeled basic fibroblast growth factor-saporin (FGF-SAP) binds to the receptor. Suramin, an inhibitor of growth factor receptor binding, inhibits the cytotoxicity of basic FGF-SAP. In a study of 4 different cell types, there is a decrease in the ED50 of the mitotoxin as the receptor number per cell increases. We have verified the cytotoxicity of the mitotoxin in 3 different assay systems. As expected, it is effective in the inhibition of protein synthesis and DNA synthesis, as well as of cell count. Binding of basic FGF-SAP which will result in cytotoxicity occurs very rapidly; 5 minutes of incubation of 10 nM basic FGF-SAP with cells results in 80% inhibition of cell count. The in vitro data indicate that the basic FGF-SAP is a receptor specific and potent suicide antagonist of basic FGF. Its potential as an anti-FGF for therapeutic and research uses in vivo is discussed.     

Modulation of the epidermal growth factor receptor by basic fibroblast growth factor

Treatment of Swiss 3T3 fibroblasts with basic fibroblast growth factor (bFGF) lead to a rapid reduction in epidermal growth factor (EGF) binding and a slower inhibition of EGF receptor autophosphorylation. The reduction in binding was due to a complete loss of the highest affinity EGF binding sites and a reduction in the lower affinity binding sites. Neither the inhibition of EGF binding nor the inhibition of EGF receptor autophosphorylation required protein kinase C. Treatment of cells with bFGF stimulated the phosphorylation of the EGF receptor, which persisted for several hours. The inhibition of EGF receptor autophosphorylation by bFGF was reduced in the presence of cycloheximide. However, cycloheximide had no effect on the reduction of EGF binding by bFGF. In contrast to these results with Swiss 3T3 fibroblasts, treatment of PC12 cells with bFGF lead to a reduction in EGF binding but no inhibition of EGF receptor autophosphorylation. Thus inhibited of EGF receptor autophosphorylation and inhibition of EGF binding can be uncoupled. © 1993 Wiley-Liss, Inc.     

Induction of vascular smooth muscle cell growth by selective activation of the thrombin receptor. Effect of heparin.

The synthetic peptide, SFLLRNPNDKYEPF, has been recently described as a peptide mimicking the new amino-terminus created by cleavage of the thrombin receptor, therefore acting as an agonist of the thrombin receptor. This peptide was a potent mitogen for rabbit arterial smooth muscle cells (SMC) and exhibited the same activity as that of native alpha-thrombin. Both compounds stimulated the proliferation of growth-arrested SMCs with half-maximum mitogenic responses at 1 nM. NAPAP, a synthetic inhibitor of the enzymatic activity of thrombin, specifically inhibited thrombin-induced SMC growth (IC50 = 0.35 +/- 0.04 microM) but was without effect on the mitogenic effect of the agonist peptide. These results therefore demonstrate that the mitogenic effect of alpha-thrombin for SMCs is intimately linked to its esterolytic activity. Heparin, which inhibited fetal calf serum-induced SMC growth, was without effect on thrombin-induced SMC growth but strongly reduced the mitogenic effect of the agonist peptide (IC50 = 32 +/- 5 micrograms/ml). This effect was not related to the anti-coagulant activity of heparin but was highly dependent on molecular mass and on the global charge of the molecule and was also observed for other sulphated polysaccharides such as pentosan polysulphate.     

Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor or vasculotropin, is a recently characterized endothelial-specific mitogen which is angiogenic in vivo. Here we demonstrate that VEGF is angiogenic in vitro: when added to microvascular endothelial cells grown on the surface of three-dimensional collagen gels, VEGF induces the cells to invade the underlying matrix and to form capillary-like tubules, with an optimal effect at approximately 2.2nM (100ng/ml). When compared to basic fibroblast growth factor (bFGF) at equimolar (0.5nM) concentrations, VEGF was about half as potent. The most striking effect was seen in combination with bFGF: when added simultaneously, VEGF and bFGF induced an in vitro angiogenic response which was far greater than additive, and which occurred with greater rapidity than the response to either cytokine alone. These results demonstrate that like bFGF, VEGF induces an angiogenic response via a direct effect on endothelial cells, and that by acting in concert, these two cytokines have a potent synergistic effect on the induction of angiogenesis in vitro. We suggest that the synergism between VEGF and bFGF plays an important role in the control of angiogenesis in vivo.     

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.     

Characterization of the receptor for keratinocyte growth factor. Evidence for multiple fibroblast growth factor receptors

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family. KGF exhibits potent mitogenic activity for a variety of epithelial cell types but is distinct from other known FGFs in that it is not mitogenic for fibroblasts or endothelial cells. We report saturable specific binding of 125I-KGF to surface receptors on intact Balb/MK mouse epidermal keratinocytes. 125I-KGF binding was completed efficiently by acidic FGF (aFGF) but with 20-fold lower efficiency by basic FGF (bFGF). The pattern of 125I-acidic FGF binding and competition on Balb/MK keratinocytes and NIH/3T3 fibroblasts suggests that these cell types possess related but distinct FGF receptors. Scatchard analysis of 125I-KGF binding suggested major and minor high affinity receptor components (KD = 400 and 25 pM, respectively) as well as a third high capacity/low affinity heparin-like component. Covalent affinity cross-linking of 125I-KGF to its receptor on Balb/MK cells revealed two species of 115 and 140 kDa. KGF also stimulated the rapid tyrosine phosphorylation of a 90-kDa protein in Balb/MK cells but not in NIH/3T3 fibroblasts. Together these results indicate that Balb/MK keratinocytes possess high affinity KGF receptors to which the FGFs may also bind. However, these receptors are distinct from the receptor(s) for aFGF and bFGF on NIH/3T3 fibroblasts, which fail to interact with KGF.     

Epidermal growth factor receptor transactivation is regulated by glucose in vascular smooth muscle cells

We hypothesized that glucose-mediated alterations in vascular smooth muscle cell signal transduction contribute to diabetic complications. We found enhanced AngII activation of Akt and extracellular ERK1/2 in vascular smooth muscle cells incubated with high glucose (27.5 mM) compared with low glucose (5.5 mM). Because AngII-mediated transactivation of the epidermal growth factor receptor (EGFR) is important in Akt and ERK1/2 activation, we studied the effects of glucose on EGFR function. The EGFR in cells cultured for 48 h in low glucose was smaller (145 kDa) than the EGFR in cells cultured with high glucose (170 kDa). The shift from the 170-kDa isoform to the 145-kDa isoform was reversible and dependent upon glucose concentration with EC50 approximately 1 mM. N-Glycosylation was responsible because peptide N-glycosidase F treatment of isolated 170-kDa EGFR yielded a single band at 145 kDa. Cell surface biotinylation showed that the 145-kDa EGFR was present on plasma membrane. AngII and other G-protein-coupled receptor ligands known to transactivate EGFR phosphorylated the 170-kDa EGFR but not the 145-kDa EGFR, whereas EGF, heparin-binding EGF-like growth factor, and transforming growth factor-alpha phosphorylated both receptors. Subcellular fractionation showed that the 145-kDa receptor localized to a different plasma membrane domain than the 170-kDa receptor. These results establish a novel mechanism by which glucose-dependent EGFR N-glycosylation modulates AngII signal transduction and suggest a potential mechanism for pathogenic effects of AngII in diabetic vasculopathy.     

Culture-induced increase in acidic and basic fibroblast growth factor activities and their association with the nuclei of vascular endothelial and smooth muscle cells.

The activity of acidic and basic fibroblast growth factor-like mitogens (aFGF, bFGF) extracted from cultured bovine aortic endothelial (BAEC) and rat aortic smooth muscle cells (SMC) was compared with that of freshly isolated cells from the same tissues. Extracts of subendothelial extracellular matrix (ECM) and cell lysates of cultured BAEC contained 4-fold more bFGF-like activity than the extracts of fresh cells. ECM and cell lysates of SMC yielded 10-fold more bFGF-like activity than the fresh cell lysates. We consistently find aFGF-like activity in both cell types. In the case of BAEC, cultured cells and ECM contained 3-fold more aFGF-like activity when compared with freshly isolated cells, whereas in cultured SMC, aFGF-like activity in cell and ECM extracts was 8-fold higher than in fresh cell extracts. The mitogens extracted from cell lysates and from the ECM are closely related to aFGF or bFGF by the criteria that they bind to heparin-sepharose and elute at 1.1 M (aFGF) or 1.5 M (bFGF) NaCl, have molecular weights of about 18,000, and react with anti-aFGF (1.1 M), or anti-bFGF (1.5 M) antibodies when analyzed by Western blots and by radioimmunoassay specific for aFGF and bFGF. This mitogenic activity is inhibited by neutralizing antibodies to aFGF and bFGF. In addition, the column fractions are potent mitogens for Balb/c 3T3 fibroblasts. Acidic and basic FGF-like mitogenic activity could also be extracted from the cell nuclei. The subcellular localization of both FGFs was visualized in both nuclei and cytoplasm with immunoperoxidase. Compared with primary SMC, secondary SMC had an increased capacity to bind 125IaFGF to high affinity receptors, while binding to freshly isolated BAEC and SMC was negligible. We conclude that FGFs are present at low levels in freshly isolated cells and that propagation in cell culture provides a stimulus for production of these mitogens.     

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.     

Basic fibroblast growth factor modulates the mitogenic potency of the platelet-derived growth factor (PDGF) isoforms by specific upregulation of the PDGF alpha receptor in vascular smooth muscle cells.

Platelet-derived growth factor AA (PDGF AA), in contrast to PDGF AB and BB, is a poor mitogen for smooth muscle cells (SMC). However, together with basic fibroblast growth factor (bFGF) it acts synergistically on DNA synthesis of these cells. Northern blot analysis revealed that bFGF selectively increases the PDGF-receptor alpha subtype (PDGF-R alpha) mRNA level without a significant effect on the PDGF-R beta mRNA level. The amount of PDGF-R alpha protein is also selectively increased after stimulating SMC with bFGF as shown by immunoprecipitation of lysates from SMC with anti-PDGF-R alpha antibodies. The number of binding sites for 125I-PDGF AA is more than doubled after bFGF-treatment, whereas the specific binding for PDGF AB and BB increased only by approximately 30 and 20%, respectively. The increase in the number of PDGF-R alpha renders the SMC responsive for PDGF AA as demonstrated by the induction of the proto-oncogene c-fos as well as by an increased cell proliferation. The enhanced PDGF binding after bFGF treatment may in fact explain the observed synergistic behavior. These data are discussed with regard to a possible role of growth factor-induced transmodulation of receptor expression during atherogenesis.     

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.     

Platelet-derived growth factor induces c-fms and scavenger receptor genes in vascular smooth muscle cells.

Vascular smooth muscle cells proliferate and transform to foam cells in the process of atherosclerosis. In the present study, we demonstrated that platelet-derived growth factor (PDGF)-BB induced expression of proto-oncogene c-fms in vascular smooth muscle cells, which normally do not express c-fms, isolated from either human umbilical artery or rabbit aorta. No effect of the protein kinase C activator, phorbol ester, was demonstrated on mRNA expression of c-fms. In contrast, the scavenger receptor activity was induced by both PDGF-BB and phorbol ester. These results indicate that two characteristic genes of monocyte-macrophages were induced by PDGF-BB via the different pathways, and suggest that PDGF-BB plays an important role in initiating phenotypic conversion of smooth muscle cells to macrophage-like cells.     

Mechanical stretch stimulates growth of vascular smooth muscle cells via epidermal growth factor receptor

We have studied whether activation of epidermal growth factor receptor (EGFR) is involved in stretch-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation and protein synthesis in cultured rat vascular smooth muscle cells (VSMC). Cyclic stretch (1 Hz) induced a rapid (within 5 min) phosphorylation of ERK1/2, an effect that was time and strength dependent and inhibited by an EGFR kinase inhibitor (AG-1478) but not by a platelet-derived growth factor receptor kinase inhibitor (AG-1296). The stretch rapidly (within 2 min) induced tyrosine phosphorylation of several proteins, among which 180-kDa protein was shown to be EGFR as revealed by blockade with AG-1478 as well as immunoprecipitation with anti-EGFR antibody coupled with immunoblotting with anti-phosphotyrosine antibody. The stretch rapidly (within 2 min) induced association of tyrosine-phosphorylated EGFR with adaptor proteins (Shc/Grb2) as revealed by coprecipitation with glutathione-S-transferase-Grb2 fusion protein coupled with immunoblotting with anti-phosphotyrosine, anti-EGFR, and anti-Shc antibodies. Transfection of a dominant-negative mutant of H-Ras also inhibited stretch-induced ERK1/2 activation. Treatment with a stretch-activated ion channel blocker (Gd(3+)) and an intracellular Ca(2+) antagonist (TMB-8) inhibited stretch-induced phosphorylation of EGFR and ERK1/2. Treatment with AG-1478 and a mitogen-activated protein kinase kinase inhibitor (PD-98059), but not AG-1296, blocked [(3)H]leucine uptake stimulated by a high level of stretch. These data suggest that ERK1/2 activation by mechanical stretch requires Ca(2+)-sensitive EGFR activation mainly via stretch-activated ion channels, thereby leading to VSMC growth.     

Nicotine-induced vascular endothelial growth factor release via the EGFR-ERK pathway in rat vascular smooth muscle cells

Cigarette smoke has been firmly established as an independent risk factor for atherosclerosis and other vascular diseases. The proliferation and migration of vascular smooth muscle cells (VSMC) induced by growth factors have been proposed to play an important role in the progression of atherosclerosis. In the present study, we investigated the effects of nicotine, which is one of the important constituents of cigarette smoke, on vascular endothelial growth factor (VEGF) release, in rat VSMC. The stimulation of cells with nicotine resulted in a time- and concentration-dependent release of VEGF. Hexamethonium, an antagonist of nicotinic acetylcholine receptor (nAChR), inhibited nicotine-induced VEGF release. We next investigated the mechanisms by which nicotine induces VEGF release in the cells. The nicotine-induced VEGF release was inhibited by treatment with U0126, a selective inhibitor of MEK, which attenuated the nicotine-induced ERK phosphorylation. Nicotine induced a transient phosphorylation of ERK. Furthermore, AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) kinase, inhibited nicotine-induced ERK phosphorylation and VEGF release. These data suggest that nicotine releases VEGF through nAChR in VSMC. Moreover, VEGF release induced by nicotine is mediated by an EGFR-ERK pathway in VSMC. VEGF may contribute to the risk of cardiovascular diseases in cigarette smokers.     

Evidence for prostacyclin and cAMP upregulation by bradykinin and insulin-like growth factor 1 in vascular smooth muscle cells

Although bradykinin (BK) and insulin like growth factor-1 (IGF-1) have been shown to modulate the functional and structural integrity of the arterial wall, the cellular mechanisms through which this regulation occurs is still undefined. The present study examined the role of second messenger molecules generated by BK and IGF-1 that could ultimately result in proliferative or antiproliferative signals in vascular smooth muscle cells (VSMC).

Activation of BK or IGF-1 receptors stimulated the synthesis and release of prostacyclin (PGI₂) leading to increased production of cAMP in VSMC. Inhibition of p42/p44^mapk or src kinases prevented the increase in PGI₂ and cAMP observed in response to BK or IGF-1, indicating a role for these kinases in the regulation of cPLA₂ activity in the VSMC. Inhibition of PKC failed to alter production of PGI₂ in response to BK, but further increased both p42/p44^mapk activation and the synthesis of PGI₂ produced in response to IGF-1. In addition, both BK and IGF-1 significantly induced the expression of c-fos mRNA levels in VSMC, and this effect of BK was accentuated in the presence a cPLA₂ inhibitor. Finally, inhibition of cPLA₂ activity and/or cyclooxygenase activity enhanced the expression of collagen I mRNA levels in response to BK and IGF-1 stimulation.

These findings indicate that the effect of BK or IGF-1 to stimulate VSMC growth is an integrated response to the activation of multiple signaling pathways. Thus, the excessive cell growth that occurs in certain forms of vascular disease could reflect dysfunction in one or more of these pathways.