Expression of decorin by sprouting bovine aortic endothelial cells exhibiting angiogenesis in vitro.

In our recent studies, we have demonstrated that monolayer cultures of bovine aortic endothelial (BAE) cells that do not express type I collagen also fail to express and synthesize decorin, a small chondroitin/dermatan sulfate proteoglycan that interacts with type I collagen and regulates collagen fibrillogenesis in vitro. However, BAE cells exhibiting a spontaneous sprouting phenotype and a predisposition toward the formation of cords and tube-like structures (an in vitro model for angiogenesis) initiate the synthesis of type I collagen during their morphological transition from a polygonal monolayer to an angiogenic phenotype. In the present study, we examined whether BAE cells also initiate the synthesis of the proteoglycan decorin during this morphological transition. We show by Northern blot analysis and by immunochemical methods that BAE cell cultures containing sprouting cells and cords, but not monolayer cultures of these cells, express and synthesize decorin (M(r) approximately 100,000). We also show that type I collagen expression by BAE cell cultures is initiated concomitantly. However, the localization of decorin and type I collagen in cord and tube-forming BAE cell cultures is not completely identical. Type I collagen is detected only in sprouting BAE cells and in endothelial cords, whereas decorin is also apparent in BAE cells surrounding the cords and tubes. Our results indicate that the synthesis of decorin as well as type I collagen is associated with endothelial cord and tube formation in vitro.     

Vascular endothelial growth factor (VEGF)-C synergizes with basic fibroblast growth factor and VEGF in the induction of angiogenesis in vitro and alters endothelial cell extracellular proteolytic activity

Vascular endothelial growth factor-C (VEGF-C) is a recently characterized member of the VEGF family of angiogenic polypeptides. We demonstrate here that VEGF-C is angiogenic in vitro when added to bovine aortic or lymphatic endothelial (BAE and BLE) cells but has little or no effect on bovine microvascular endothelial (BME) cells. As reported previously for VEGF, VEGF-C and basic fibroblast growth factor (bFGF) induced a synergistic in vitro angiogenic response in all three cells lines. Unexpectedly, VEGF and VEGF-C also synergized in the in vitro angiogenic response when assessed on BAE cells. Characterization of VEGF receptor (VEGFR) expression revealed that BME, BAE, and BLE cell lines express VEGFR-1 and -2, whereas of the three cell lines assessed, only BAE cells express VEGFR-3. We also demonstrate that VEGF-C increases plasminogen activator (PA) activity in the three bovine endothelial cell lines and that this is accompanied by a concomitant increase in PA inhibitor-1. Addition of α₂-antiplasmin to BME cells co-treated with bFGF and VEGF-C partially inhibited collagen gel invasion. These results demonstrate, first, that by acting in concert with bFGF or VEGF, VEGF-C has a potent synergistic effect on the induction of angiogenesis in vitro and, second, that like VEGF and bFGF, VEGF-C is capable of altering endothelial cell extracellular proteolytic activity. These observations also highlight the notion of context, i.e., that the activity of an angiogenesis-regulating cytokine depends on the presence and concentration of other cytokines in the pericellular environment of the responding endothelial cell. J. Cell. Physiol. 177:439–452, 1998. © 1998 Wiley-Liss, Inc.     

Goniodomin A, an antifungal polyether macrolide, exhibits antiangiogenic activities via inhibition of actin reorganization in endothelial cells.

Goniodomin A (GDA) is an antifungal polyether macrolide isolated from the dinoflagellate Goniodoma pseudogoniaulax. Previous studies revealed that GDA profoundly affected cytoskeletal reorganization. We examined the effect of GDA on the angiogenic properties of vascular endothelial cells. GDA itself did not affect proliferation of, migration of, and tube formation in type I collagen gels by, bovine aortic endothelial cells (BAECs). Proliferation of BAECs stimulated by bFGF was not affected by GDA at concentrations of up to 10 nM. However, at similar concentrations, GDA significantly inhibited bFGF-induced migration and tube formation in type I collagen gels by BAECs. Actin reorganization is required for cell migration. GDA caused the perinuclear aggregation of filamentous actin and inhibited stress fiber formation in bFGF- or VEGF-stimulated BAECs and lysophosphatidic acid-stimulated HeLa cells. However, GDA did not affect stress fiber structures already formed through Gbetagamma expression or in constitutively active RhoA mutant HeLa cells. Finally, GDA inhibited forming of vasucular system in a chorioallantoic membrane. Our results indicated that GDA suppressed angiogenic properties of ECs at least in part through the inhibition of actin reorganization and inhibited angiogenesis in vivo.     

Organized type I collagen influences endothelial patterns during “spontaneous angiogenesis in vitro”: Planar cultures as models of vascular development

Summary Selected strains of vascular endothelial cells, grown as confluent monolayers on tissue culture plastic, generate flat networks of cellular cords that resemble beds of capillaries—a phenomenon referred to as “spontaneous angiogenesis in vitro”. We have studied spontaneous angiogenic activity by a clonal population (clone A) of bovine aortic endothelial cells to indentify processes that mediate the development of cellular networks. Confluent cultures of clone A endothelial cells synthesized type I collagen, a portion of which was incorporated into narrow, extracellular cables that formed a planar network beneath the cellular monolayer. The collagenous cables acted as a template for the development of cellular networks: flattened, polygonal cells of the monolayer that were in direct contact with the cables acquired spindle shapes, associated to form cellular cords, and became elevated above the monolayer. Networks of cables and cellular cords did not form in a strain of bovine aortic endothelial cells that did not synthesize type I collagen, or when traction forces generated by clone A endothelial cells were inhibited with cytochalasin D. In a model of cable development, tension applied by a confluent monolayer of endothelial cells reorganized a sheetlike substrate of malleable type I collagen into a network of cables via the formation and radial enlargement of perforations through the collagen sheet. Our results point to a general involvement of extracellular matrix templates in two-dimensional (planar) models of vascular development in vitro. For several reasons, planar models simulate invasive angiogenesis poorly. In contrast, planar models might offer insights into the growth and development of planar vascular systems in vivo.     

Docosapentaenoic acid (22:5, n-3) suppressed tube-forming activity in endothelial cells induced by vascular endothelial growth factor

It is generally accepted that n-3 polyunsaturated fatty acids have beneficial effects on vascular homeostasis. Among the several functions of endothelial cells, angiogenesis contributes to tumor growth, inflammation, and microangiopathy. We have already demonstrated that eicosapentaenoic acid (EPA, 20:5, n-3) suppressed angiogenesis. In this paper, we examined the effect of docosapentaenoic acid (DPA, 22:5, n-3), an elongated metabolite of EPA, on tube-forming activity in bovine aortic endothelial cells (BAE cells) incubated between type I collagen gels. The pretreatment of BAE cells with DPA suppressed tube-forming activity induced by vascular endothelial growth factor (VEGF). The effect of DPA was stronger than those of EPA and docosahexaenoic acid (22:6, n-3). The migrating activity of endothelial cells stimulated with VEGF was also suppressed by DPA pretreatment. The treatment of BAE cells with DPA caused the suppression of VEGF receptor-2 (VEGFR-2, the kinase insert domain-containing receptor, KDR) expression in both plastic dish and collagen gel cultures. These data indicate that DPA has a potent inhibitory effect on angiogenesis through the suppression of VEGFR-2 expression.     

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.     

The relative magnitudes of endothelial force generation and matrix stiffness modulate capillary morphogenesis in vitro

When suspended in collagen gels, endothelial cells elongate and form capillary-like networks containing lumens. Human blood outgrowth endothelial cells (HBOEC) suspended in relatively rigid 3 mg/ml floating collagen gels, formed in vivo-like, thin, branched multi-cellular structures with small, thick-walled lumens, while human umbilical vein endothelial cells (HUVEC) formed fewer multi-cellular structures, had a spread appearance, and had larger lumens. HBOEC exert more traction on collagen gels than HUVEC as evidenced by greater contraction of floating gels. When the stiffness of floating gels was decreased by decreasing the collagen concentration from 3 to 1.5 mg/ml, HUVEC contracted gels more and formed thin, multi-cellular structures with small lumens, similar in appearance to HBOEC in floating 3 mg/ml gels. In contrast to floating gels, traction forces exerted by cells in mechanically constrained gels encounter considerable resistance. In constrained collagen gels (3 mg/ml), both cell types appeared spread, formed structures with fewer cells, had larger, thinner-walled lumens than in floating gels, and showed prominent actin stress fibers, not seen in floating gels. These results suggest that the relative magnitudes of cellular force generation and apparent matrix stiffness modulate capillary morphogenesis in vitro and that this balance may play a role in regulating angiogenesis in vivo.     

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.     

Mechanism of action of angiostatic steroids: Suppression of plasminogen activator activity via stimulation of plasminogen activator inhibitor synthesis

Recently, a novel class of angiostatic steroids which block angiogenesis in several systems has been described. Since the elaboration of proteases is believed to be an important component of angiogenesis, we tested whether these steroids blocked the fibrinolytic response of endothelial cells to the angiogenic protein, basic fibroblast growth factor [bFGF]). Cultured bovine aortic endothelial (BAE) cells were incubated with bFGF and/or medroxyprogesterone acetate (MPA), an angio-static steroid which has been shown to inhibit vascularization, collagenolysis, and tumor growth. When bFGF (3 ng/ml) was added to confluent monolayers of BAE cells, plasminogen activator (PA) activity in the medium was increased threefold. In contrast, MPA at 10^?6 M, 10^?7 M, 10^?8 M, and 10^?9 M decreased PA levels in the medium by 83%, 83%, 75%, and 39%, respectively. The stimulation of PA levels in BAE cells by bFGF (3 ng/ml) was abrogated by the presence of 10^?6 M MPA. This decrease in PA activity was found to be mediated by a significant increase in plasminogen activator inhibitor type-1 (PAI-1) production. MPA, therefore, negated one of the important enzymatic activities associated with the angiogenic process. In contrast to the decreased levels of secreted PA in cultures exposed simultaneously to MPA and bFGF, cell-associated PA levels remained high, consistent with earlier observations indicating that PAI-1 does not inhibit cell-associated PA. Thus, angiostatic steroids may exert their inhibitory effects on angiogenesis by increasing the synthesis of PAI-1. This, in turn, inhibits PA activity and, therefore, plasmin generation, which is essential for the invasive aspect of angiogenesis. © 1993 Wiley-Liss, Inc.     

Suramin inhibits C6 glioma-induced angiogenesis in vitro

Aspects of tumor-induced angiogenesis in vitro were examined using an assay involving collagen gel invasion by a surface monolayer of bovine endothelial cells under the influence of serum free conditioned medium produced by C6 cells, an experimentally derived rat glial tumor cell line. The effects of the polyanionic compound suramin, known to interfere with growth factor/cell signaling on this process were evaluated. Collagen gel invasion was quantified by adding C6 conditioned medium with or without various doses of suramin to monolayers of bovine aortic endothelial cells grown on type I collagen gels in transwell inserts. Cultures were monitored with phase-contrast microscopy. After various periods of incubation collagen gels were fixed, embedded in epoxy resin, and 1-μm thick sections were stained with toluidine blue. Additional cultures were used to evaluate the effects of C6 conditioned medium and suramin on endothelial cell proliferation, and on chemotaxis through 8-μm pores. C6 glioma cell conditioned medium induced large vessel endothelial cells to sprout into the underlying collagen matrix and subsequently from networks of capillary like tubes. Conditioned medium was also chemotactic and mitogenic for these cells. The addition of suramin to C6 glioma conditioned medium prevents tube formation in collagen gels, and inhibits both endothelial cell proliferation and chemotaxis in a dose dependent manner. These results suggest that glial tumor cell conditioned medium induces angiongenesis in large vessel endothelial cells in vitro via mechanisms which are disrupted by suramin, most likely involving tumor-derived growth factor release and/or endothelium-mediated matrix proteolysis.     

The biosynthesis of extracellular-matrix components by bovine retinal endothelial cells displaying distinctive morphological phenotypes.

Previous studies have indicated that the morphology and behaviour of bovine retinal microvessel endothelial cells are influenced by culture conditions in vitro. Data are presented here concerning the biosynthesis of matrix macromolecules by bovine retinal endothelial cells cultured under conditions in which the cells display either the 'cobblestone' or the 'sprouting' phenotype. Newly synthesized matrix proteins were identified by their characteristic electrophoretic mobilities, immunoprecipitation with specific antibodies, susceptibilities to enzymic digestions and chromatographic behaviour. Type IV procollagen was the major collagenous species synthesized by early-passage cells forming a 'cobblestone' monolayer. In contrast, cells displaying the 'sprouting' morphology switched to the predominant synthesis of interstitial fibrillar collagens (types I and III). Fibronectin was synthesized by retinal endothelial cells under all the experimental conditions studied. A non-collagenous glycoprotein of Mr approx. 47,000 was also a major biosynthetic product of these cells. The synthesis of thrombospondin was very much dependent on the nature of the substratum on which the cells were cultured. This glycoprotein was synthesized in large amounts by 'cobblestone' endothelial cells cultured on gelatin-coated dishes, whereas its synthesis was markedly decreased by culturing the cells on collagen gels, and the protein appeared to be absent when the cells were plated within collagen gels ('sprouting' cells). Late-passage retinal cells synthesized predominantly type I procollagen, variable amounts of type III procollagen and only traces of type IV procollagen, irrespective of whether the cells displayed a 'cobblestone' or 'sprouting' morphology.     

In Vitro Angiogenic and Proteolytic Properties of Bovine Lymphatic Endothelial Cells

The aim of the present study was to determine whether angiogenic cytokines, which induce neovascularization in the blood vascular system, might also be operative in the lymphatic system. In an assay of spontaneous in vitro angiogenesis, endothelial cells isolated from bovine lymphatic vessels retained their histotypic morphogenetic properties by forming capillary-like tubes. In a second assay, in which endothelial cells could be induced to invade a three-dimensional collagen gel within which they formed tube-like structures, lymphatic endothelial cells responded to basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in a manner similar to what has previously been observed with endothelial cells derived from the blood vascular system. Finally, since angiogenesis is believed to require extracellular proteolytic activity, we investigated the effects of bFGF and VEGF on lymphatic endothelial cell proteolytic properties by focussing on the plasminogen activator (PA) system. bFGF and VEGF increased urokinase, urokinase receptor, and tissue-type PA expression. This was accompanied by an increase in PA inhibitor-l, which is thought to play an important permissive role in angiogenesis by protecting the extracellular matrix against excessive proteolytic degradation. Taken together, these results demonstrate that with respect to in vitro morphogenetic and proteolytic properties, lymphatic endothelial cells respond to the previously described angiogenic factors, bFGF and VEGF, in a manner very similar to what has been described for endothelial cells derived from the blood vascular system.     

Thrombospondin-1 Modulates Angiogenesisin Vitroby Up-Regulation of Matrix Metalloproteinase-9 in Endothelial Cells

Evidence suggests that thrombospondin-1 (TSP-1), a 450-kDa glycoprotein in platelets and extracellular matrix, is involved in angiogenesis. However, the mechanisms by which TSP-1 regulates angiogenesis are unknown, and the exact role of TSP-1 in angiogenesis has been controversial: both stimulatory and inhibitory effects of TSP-1 have been reported. In this study, we evaluated the effect of TSP-1 on the capacity of bovine aortic endothelial (BAE) cells to both invade and form microvessel-like tubes in collagen gels. BAE cell tube formation was enhanced by exogenous TSP-1 at relatively low concentrations (1–10 μg/ml) but inhibited at higher concentrations of TSP-1 (>15 μg/ml). In addition, we correlated this biphasic effect on tube formation with the capacity of TSP-1 to stimulate the activity of a matrix metalloproteinase-9 (MMP-9) in BAE cell collagen gel cultures. The TSP-1-mediated stimulation of MMP-9 activity was specific and dose- and time-dependent. Furthermore, TSP-1-stimulated BAE cell invasion and tube formation were reversed by antibodies against both TSP-1 and MMP-9, suggesting that TSP-1 modulates endothelial cell invasion and morphogenesisin vitroby a mechanism involving the regulation of MMP-9 activity. These findings support the conclusion that TSP-1 is a multifunctional modulator of angiogenesis and are consistent with the dynamic presence of TSP-1 in remodeling tissues in which matrix degradation is required.     

Influence of the extracellular matrix on the proliferative response of human skin fibroblasts to serum and purified platelet-derived growth factor

The culture of adult human skin fibroblasts on reconstituted bovine type 1 fibrillar collagen gels, ranging in concentration from 2.5-35.0 mg/ml, results in a reduction in proliferation rate by 40%-60% as measured by (3H) thymidine incorporation. The suppressive effect was noted when cells were cultured in both human and bovine serum. Drying the gels into thin films abolishes the suppressive effect of the fibrillar collagen on cell proliferation. Cell attachment studies showed that differences in the proliferation rate of cells on the various substrata were not simply due to differences in initial attachment. Studies with purified platelet-derived growth factor (PDGF) demonstrated that the reduced responsiveness of cells to this factor, when cultured on collagen gels as compared to plastic, was largely responsible for the reduced proliferative activity of the cells when cultured in the presence of serum. The reduced proliferative activity of fibroblasts in response to PDGF, when cultured on collagen gels, was confirmed by total DNA determination. It was shown that the reduced responsiveness of cells to PDGF was not simply because the factor bound to the fibrillar collagen gel or was inaccessible to the cells. The data indicate that the reduced proliferation rate of fibroblasts cultured on collagen gels is a direct result of the influence of the extracellular matrix on the cells' ability to respond to a soluble mitogenic mediator.     

8-prenylnaringenin, a novel phytoestrogen, inhibits angiogenesis in vitro and in vivo

8-Prenylnaringenin is a recently discovered phytoestrogen. Using an in vitro model of angiogenesis in which endothelial cells can be induced to invade a three-dimensional collagen gel within which they form capillary-like tubes, we demonstrate that 8-prenylnaringenin inhibits angiogenesis induced by basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), or the synergistic effect of the two cytokines in combination, with an IC(50) of between 3 and 10 microM. This effect was seen with bovine microvascular endothelial cells derived from the adrenal cortex (BME cells) and with endothelial cells from the bovine thoracic aorta (BAE cells). The inhibitory effects of 8-prenylnaringenin were found to be roughly equipotent to those of genistein that has previously been shown to inhibit angiogenesis in vitro. Early chorioallantoic membrane (CAM) assay results showed reductions in both vessel lengths and vein diameters, with similar potency in the 8-prenylnaringenin and genistein groups. Similar effects on the CAM vessels were seen when the two substances were co-added. These findings suggest that 8-prenylnaringenin has potential therapeutic applications for diseases in which angiogenesis is an important component.     

Regulation of Vascular Endothelial Growth Factor Receptor-2 (Flk-1) Expression in Vascular Endothelial Cells

We have previously reported the existence of a synergistic interaction between vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in the induction of angiogenesisin vitro.Here we demonstrate that bFGF increases VEGF receptor-2 (VEGFR-2/Flk-1) expression: mRNA levels were increased by 4.5- to 8.0-fold and total protein by 2.0- to 3.5-fold, in bovine microvascular endothelial (BME), aortic endothelial (BAE), and transformed fetal aortic (GM7373) endothelial cells. VEGF itself did not affect VEGFR-2 expression, and neither bFGF nor VEGF altered expression of FGF receptor-1. We also show that synergism occurs at the level of proliferation when this is measured in a three-dimensional but not in a conventional two-dimensional assay. Differences in the level of VEGFR-2 expression were also observed when cells were grown on or within collagen gels under different conditions: mRNA levels were lowest under sparse conditions, increased 20- to 26-fold at confluence, and increased even further (57-fold) when cells were cultured in suspension in three-dimensional collagen gels. Finally, a synergistic increase was seen in the level of expression of urokinase and urokinase receptor mRNAs when cells were exposed to bFGF and VEGF for 4 days. These findings demonstrate that the level of VEGFR-2 expression can be modulated by environmental factors including cytokines and the geometry of the culture conditions and provide some insight into the mechanisms of synergism between bFGF and VEGF in the induction of angiogenesisin vitro.     

Angiostatin-induced inhibition of endothelial cell proliferation/apoptosis is associated with the down-regulation of cell cycle regulatory protein cdk5

Endothelial cells (ECs) are quiescent in normal blood vessels, but undergo rapid bursts of proliferation after vascular injury, hypoxia or induced by powerful angiogenic cytokines like fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). Deregulated proliferation of ECs facilitates angiogenic processes and promotes tumor growth. In dividing cells, cell cycle-associated protein kinases, which are referred as cyclin-dependent kinases (cdks), regulate proliferation, differentiation, senescence, and apoptosis. Cyclin-dependent kinase-5 (cdk5) is expressed in neuronal cells and plays an important role in neurite outgrowth, of neuronal migration and neurogenesis, its functions in non-neuronal cells are unclear. Here, we show for the first time that the cdk5 is expressed at high levels in proliferating bovine aortic endothelial (BAE) cells, by contrast insignificant low levels of cdk5 expression in quiescent BAE cells. In addition, bFGF up-regulates cdk5 expression in a dose-dependent fashion. Interestingly, temporal expression data suggests that cdk5 expression is very low between 24-48 h, but high level of cdk5 expression was detected during 60-72 h. This later time corresponds to the time of completion of one cell cycle (doubling of cell population) of BAE cell culture. Angiostatin (AS), a powerful inhibitor of angiogenesis inhibits ECs proliferation in dose-dependent manner with concomitant down-regulation of cdk5 expression. The role of cdk5 in ECs, proliferation and apoptosis was confirmed by selective inhibition of cdk5 expression by the purine derivative roscovitine, which inhibits bFGF-stimulated BAE cells proliferation and induces apoptosis in dose-specific manner. By contrast, the roscovitine analog olomoucine, which is a specific inhibitor of cdk4, but not of cdk5 failed to affect ECs proliferation and apoptosis. These data suggest for the first time that neuron specific protein cdk5 may have significant role in the regulation of ECs proliferation, apoptosis, and angiogenesis and extends beyond its role in neurogenesis.     

Induction of angiogenesis in vitro by vanadate, an inhibitor of phosphotyrosine phosphatases

We have previously shown that capillary endothelial cells grown on the surface of three-dimensional collagen gels can be induced to invade the underlying fibrillar matrix and to form capillary-like tubular structures in response to tumor-promoting phorbol esters or the angiogenic agent fibroblast growth factor (FGF). Since both phorbol esters and FGF stimulate phosphorylation of tyrosine residues, we treated endothelial cells with vanadate, an inhibitor of phosphotyrosine-specific phosphatases, to determine whether this agent could induce the expression of an angiogenic phenotype in these cells. We show here that vanadate stimulates endothelial cells to invade collagen matrices and to organize into characteristic tubules resembling those induced by FGF or phorbol esters. We have further observed that vanadate concomitantly stimulates endothelial cells to produce plasminogen activators (PAs), proteolytic enzymes which are induced by phorbol esters and FGF, and which have been implicated in the neovascular response; this stimulation can be accounted for by an increase in the levels of urokinase-type PA and tissue type PA mRNA. These results suggest a role for tyrosine phosphorylation in the regulation of the angiogenic phenotype in capillary endothelial cells.     

Injury-induced release of basic fibroblast growth factor from bovine aortic endothelium

Although the basic fibroblast growth factor (bFGF) gene lacks a traditional consensus signal peptide domain indicative for secretion, many cell types have receptors for bFGF. Since endothelium is a rich source of cell-associated bFGF, we asked under what conditions could bFGF be released or secreted from confluent cultures of bovine aortic endothelial (BAE) cells. The level of bFGF in BAE cell lysates was compared with the level of heparin-releasable bFGF in intact BAE cell monolayers, intact cells with exposed extracellular matrix (nonlytic matrices), and extracellular matrices prepared by cell lysis (lytic matrices). Less than 10% of total cell-associated bFGF was released from intact cell monolayers and nonlytic matrices. In contrast, the levels of bFGF released from lytic matrices depended upon the conditions used to prepare the matrices. Cell lysis at neutral pH generated matrices that released the highest bFGF levels (approximately 50% of total cell-associated bFGF). These matrices were heavily contaminated by histones, indicating the cellular release and adsorption of intracellular proteins to the matrix. Matrices prepared by BAE cell exposure to basic pH (100 mM NH4OH) contained low bFGF content and minor histone contamination. These latter matrices were chosen to study bFGF sequestration, under physiological conditions, into the extracellular matrix of confluent BAE cell cultures. Incubation with endotoxin, an agent acutely toxic to BAE cells, resulted in cellular release and adsorption of endogenous bFGF to cells and matrices, accompanied by histone deposition in the matrices. These results suggested that one mechanism for bFGF release from BAE cell monolayers was passive release induced by severe cell injury and/or cell lysis with secondary adsorption to the matrix.