Modulation of Serine Proteinases and Metalloproteinases During Morphogenic Glial-Endothelial Interactions

Abstract: The regulation of microvessel formation and the expression of CNS-specific endothelial properties are attributed to perivascular astroglia. Specific proteolytic pathways mediate processes such as tissue remodeling, differentiation, invasion, and metastasis. We used a coculture system in which C6 glial cells induce CNS microvascular endothelial cells to form capillary-like structures to examine the role of plasminogen activators and collagenases in CNS microvessel morphogenesis. Fibrin zymography revealed the presence of high-molecular weight urokinase-type plasminogen activator (uPA), low-molecular weight uPA, and uPA/inhibitor complexes within endothelial cultures and cocultures. Gelatin zymography revealed the presence of 92-, 72-, and 62-kDa type IV collagenases within endothelial cultures and cocultures. uPA activity was confirmed by incubating the extracts with amiloride, an inhibitor of uPA. Collagenase activity was confirmed by incubating the gels with EDTA, an inhibitor of metalloproteinases. Quantitative densitometry showed a six- to eightfold decrease in coculture uPA during capillary-like structure formation. Substantially less change in type IV 72-kDa procollagenase activity was seen in cocultures during capillary-like structure formation, but active type IV 62-kDa collagenase activity was significantly increased during capillary-like structure formation. These findings establish that uPA and activated type IV collagenase activity specifically regulates morphogenic endothelial responses to glial interactions and suggest mechanisms by which microvessels respond within the CNS.     

Steroid Inhibition of Neural Micro vessel Morphogenesis In Vitro: Receptor Mediation and Astroglial Dependence

Steroid hormones alter several aspects of microvascular function within the CNS. Both microvessel formation and blood-brain barrier expression appear to be influenced by interactions between astrocytes and endothelial cells. To determine if steroids alter astrocyte-endothelial interactions, we studied their effects on astroglial-induced microvessel morphogenesis in vitro. C6 astroglial cells induce bovine retinal microvascular endothelial cells to differentiate into capillary-like structures. Dexamethasone, hydrocortisone, and progesterone at 10 nM inhibited C6-induced microvessel morphogenesis by 75, 35, and 30%, respectively. Inhibition by dexamethasone was both time and concentration dependent, reaching 80-100% at 1 microM. Tetrahydrocortisone and 17 alpha-hydroxyprogesterone had only marginal inhibitory effects. Cortexolone, a glucocorticoid receptor antagonist, blocked inhibition by dexamethasone. Progesterone receptors were expressed in C6 but not bovine retinal microvascular endothelial cells, identifying the astroglial cell as the likely effector of progesterone-mediated inhibition. Astroglial cells were further implicated as the effectors of steroid-mediated inhibition because none of the steroids inhibited astroglial-independent capillary-like structure formation in response to a reconstituted extracellular matrix, Matrigel. These findings are evidence that steroids modulate neural microvascular endothelial cell functions indirectly through perivascular astrocytes via a receptor-mediated mechanism.     

Astroglial-Induced In Vitro Angiogenesis: Requirements for RNA and Protein Synthesis

Astrocytes are believed to affect microvascular endothelial cell differentiation in brain and retina. Bovine retinal microvessel endothelial cells formed capillary-like structures when cocultured with C₆ astroglial cells or in the absence of C₆ cells in response to the reconstituted basement membrane protein Matrigel. Using quantitative computer-assisted image analysis, the requirements for RNA and protein synthesis in these two complementary models of in vitro microvessel morphogenesis were examined. Astroglial-dependent capillary-like structure formation was inhibited by up to 87% in a dose-dependent fashion by cycloheximide (0.01–0.1 μg/ml), puromycin (0.1–0.25 μg/ml), and actinomycin D (0.01– 0.025 μg/ml). In contrast, the astroglial-independent process in response to Matrigel was not affected by these metabolic inhibitors. These findings suggest that capillary-like structures form in response to astroglial cells in two distinct sequential stages. The first consists of inductive astroglial-endothelial interactions requiring both RNA and protein synthesis. This initiates endogenous endothelial morphogenic events that do not appear to require RNA or protein synthesis, consistent with posttranslational regulatory mechanisms. The first astroglial-dependent step is relevant to the regulation of microvessel formation in brain and retina, whereas the second may represent a morphogenic pathway common to micro-vessel formation in many tissues.     

Astrocytes induce neural microvascular endothelial cells to form capillary-like structures in vitro

Astrocytes maintain a unique association with the central nervous system microvasculature and are thought to play a role in neural microvessel formation and differentiation. We investigated the influence of astroglial cells on neural microvascular endothelial differentiation in vitro. Using an astroglial-endothelial coculture system, rat brain astrocytes and C6 cells of astroglial lineage are shown to induce bovine retinal microvascular endothelial (BRE) cells to form capillary-like structures. Light microscopic evidence for endothelial reorganization began within 48 hours and was complete 72-96 hours following the addition of BRE cells to 1-day-old astroglial cultures. The extent of BRE reorganization was quantitated by computer-assisted analysis and shown to be dependent upon the density of both the BRE and C6 cells within the cocultures. Coculture conditions in which BRE cells were separated from C6 cells by porous membranes failed to generate this endothelial cell change. Likewise, C6-conditioned media and C6-endothelial coculture conditioned media did not induce BRE cell reorganization. Extracellular laminin within the C6-endothelial cocultures, identified by indirect immunofluorescence, was concentrated at the endothelial-astroglial interface of capillary-like structures consistent with incipient basement membrane formation. Astroglial cells accumulated adjacent to capillary-like structures suggesting the presence of bidirectional influences between the reorganized endothelial cells and astroglia. This is the first demonstration of astroglial induction of angiogenesis in vitro and these findings support a functional role for perivascular astrocytes in the vascularization of neural tissue such as retina and brain.     

Cellular context–mediated Akt dynamics regulates MAP kinase signaling thresholds during angiogenesis

The formation of new blood vessels by sprouting angiogenesis is tightly regulated by contextual cues that affect angiogeneic growth factor signaling. Both constitutive activation and loss of Akt kinase activity in endothelial cells impair angiogenesis, suggesting that Akt dynamics mediates contextual microenvironmental regulation. We explored the temporal regulation of Akt in endothelial cells during formation of capillary-like networks induced by cell–cell contact with vascular smooth muscle cells (vSMCs) and vSMC-associated VEGF. Expression of constitutively active Akt1 strongly inhibited network formation, whereas hemiphosphorylated Akt1 epi-alleles with reduced kinase activity had an intermediate inhibitory effect. Conversely, inhibition of Akt signaling did not affect endothelial cell migration or morphogenesis in vSMC cocultures that generate capillary-like structures. We found that endothelial Akt activity is transiently blocked by proteasomal degradation in the presence of SMCs during the initial phase of capillary-like structure formation. Suppressed Akt activity corresponded to the increased endothelial MAP kinase signaling that was required for angiogenic endothelial morphogenesis. These results reveal a regulatory principle by which cellular context regulates Akt protein dynamics, which determines MAP kinase signaling thresholds necessary drive a morphogenetic program during angiogenesis.     

Plasminogen activators augment endothelial cell organization in vitro by two distinct pathways

Endothelial cell differentiation into capillary structures is a complex process that requires the concerted effects of several extracellular matrix proteases, including plasminogen activators. Here, the role of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) was evaluated in an in vitro model of endothelial morphogenesis involving organization of human umbilical vein endothelial cells into tubular structures when they are cultured on the basement membrane preparation, Matrigel. Both uPA and tPA were detected in HUVEC cultures on Matrigel, and inhibitors of plasminogen activators or of serine proteases decreased the extent of the tube network formed by the cells. The decrease resulting from serine protease inhibitors was additive to that from matrix metalloproteinase inhibitors which have previously been shown to decrease tube formation in this model, suggesting that the two classes of proteases modulate tube formation by distinct mechanisms. Plasminogen activator inhibitor (PAl)-1 decreased tube formation by 50% when added up to 4.5 h after the initiation of an 18 h assay and caused 25% inhibition when added 9.5 h after culture initiation, indicating that the effects of plasminogen activators are not limited to an early event in the differentiation process. Steady-state expression of mRNA for uPA increased during the first several hours of culture on Matrigel, further supporting a role for PA activity throughout the process of tube formation. These findings suggested that PAs may affect multiple events during tube-forming activity. A fucosylated peptide comprising the amino-terminal domain of uPA that binds to the uPA receptor (uPAR) but lacking proteolytic activity enhanced tube formation. In contrast, a defucosylated form of the same peptide had no effect. Since fucosylation of this fragment has been shown to be essential in other models of cell stimulation by uPA-uPAR interaction, these data support the hypothesis that uPA enhances endothelial morphogenesis both through proteolytic activity and via uPAR occupancy. Plasminogen activators could facilitate angiogenesis in vivo. © 1995 Wiley-Liss Inc.     

Autocrine EGF receptor activation mediates endothelial cell migration and vascular morphogenesis induced by VEGF under interstitial flow

We show here that autocrine ligand activation of epidermal growth factor (EGF) receptor in combination with interstitial flow is critically involved in the morphogenetic response of endothelial cells to VEGF stimulation. Human umbilical vein endothelial cell (HUVEC) monolayers cultured on a collagen gel and exposed to low interstitial flow in the absence of EGF and VEGF remained viable and mitotic but exhibited little evidence of vascular morphogenesis. Addition of VEGF produced a flow-dependent morphogenetic response within 48 to 72 h, characterized by branched capillary-like structures. The response was substantially abolished by inhibitors related to the autocrine EGF receptor pathway including Galardin, AG1478, PD98059, and an EGF receptor-blocking antibody, indicating that regulation of the morphogenetic process operates via autocrine EGF receptor activation. Moreover, we observed that in our system the EGF receptor was always activated independently of the interstitial flow, and, in addition, the EGF receptor inhibitors used above reduced the phosphorylation state of the receptor, correlating with inhibition of capillary morphogenesis. Finally, 5'bromo-2'-deoxyuridine (BrdU) labeling identified dividing cells at the monolayer but not in the extending capillary-like structures. EGF pathway inhibitors Galardin and AG1478 did not reduce BrdU incorporation in the monolayer, indicating that the EGF-receptor-mediated morphogenetic behavior is mainly due to cell migration rather than proliferation. Based on these results, we propose a two-step model for in vitro capillary morphogenesis in response to VEGF stimulation with interstitial fluid flow: monolayer maintenance by mitotic activity independent of EGF receptors and a migratory response mediated by autocrine EGF receptor activation wherein cells establish capillary-like structures.     

Caveolin-1 expression enhances endothelial capillary tubule formation.

The level of caveolin-1 expression closely correlates with the oncogenic transformation of NIH 3T3 cells, the proliferation of human cancer cells, and the differentiation of adipocytes and muscle cells. However, the role of caveolin-1 in endothelial cell proliferation and differentiation remains unknown. Here, we have shown that angiogenic growth factors that stimulate endothelial cell proliferation lead to dramatic reductions in caveolin-1 expression. In addition, using an in vitro Matrigel assay system, we studied the potential role of caveolin-1 in capillary-like tubule formation (i.e. endothelial cell differentiation) using human microvascular endothelial cells (HMEC-1). We showed that the level of endogenous caveolin-1 expression increased in a time-dependent manner when endothelial cells underwent differentiation and that the maximum level of caveolin-1 expression occurred just prior to the formation of capillary-like tubules. Interestingly, overexpression of caveolin-1, via an adenoviral gene delivery system, clearly accelerated endothelial cell differentiation/tubule formation and led to a dramatic approximately 3-fold increase in the number of capillary-like tubular structures. Conversely, down-regulation of caveolin-1 expression, via an antisense adenoviral approach, reduced the number of capillary-like tubules formed by >10-fold. Consistent with the unique function of caveolin-1 in interacting with key signaling molecules, delivery of the caveolin-1 scaffolding domain into the cytoplasm of living endothelial cells was also sufficient to enhance capillary-like tubule formation. Taken together, these results clearly demonstrate that caveolin-1 and the caveolin-1 scaffolding domain play an important positive role in the regulation of endothelial cell differentiation, a prerequisite step in the process of angiogenesis.     

Increased proteolytic activity is responsible for the aberrant morphogenetic behavior of endothelial cells expressing the middle T oncogene

Expression of the polyoma virus middle T (mT) oncogene in vivo is associated with a profound subversion of normal vascular development, which results in the formation of endothelial tumors (hemangiomas). In an attempt to understand the molecular mechanisms responsible for this phenomenon, we have investigated, in an in vitro system, the morphogenetic properties of endothelial cells expressing this oncogene. mT-expressing endothelioma (End) cells grown within fibrin gels formed large hemangioma-like cystic structures. All End cell lines examined expressed high levels of fibrinolytic activity resulting from increased production of urokinase-type plasminogen activator and decreased production of plasminogen activator inhibitors. Neutralization of excess proteolytic activity by exogenously added serine protease inhibitors corrected the aberrant in vitro behavior of End cells and allowed the formation of capillary-like tubules. These results suggest that tightly controlled proteolytic activity is essential for vascular morphogenesis and that physiological protease inhibitors play an important regulatory role in angiogenesis.     

FK506 induces endothelial dysfunction through attenuation of Akt and ERK1/2 independently of calcineurin inhibition and the caspase pathway

Calcineurin inhibitors such as cyclosporin A (CsA) and FK506 have been used in solid organ and hematopoietic stem cell transplantations to suppress immune function. However, these immunosuppresants are associated with severe endothelial dysfunction. We investigated whether CsA and FK506 induce endothelial dysfunction using a three-dimensional culture blood vessel model, in which human umbilical vein endothelial cells form and maintain capillary-like tube and lumen structures. We found that FK506, but not CsA, induced breakdown of the tube structures and endothelial cell death. FK506 inhibited calcineurin activity, but FK506-induced tube breakdown and cell death was not suppressed by RNA interference targeting calcineurin Aα. FK506 also induced caspase activation, but caspase inhibition by zVAD(OMe)-fmk failed to suppress FK506-induced tube breakdown and cell death. FK506 induced attenuation of Akt and extracellular-regulated kinase 1/2 (ERK1/2). Furthermore, Akt inhibition by LY294002 or ERK1/2 inhibition by PD98059 induced tube breakdown and cell death. Present results suggest that FK506 induces endothelial dysfunction through attenuation of Akt and ERK1/2 independently of calcineurin inhibition and the caspase pathway.     

Identification of a subpopulation of human renal microvascular endothelial cells with capacity to form capillary-like cord and tube structures

Summary Endothelial specialization is a prominent feature within distinct capillary beds of organs such as mammalian kidney, yet immunological markers for functionally distinct subpopulations of cultured endothelial cells from tissue sources such as kidney have not been available. We developed a simple and reproducible isolation and culture procedure to recover human renal microvascular endothelial cells (HRMEC) from the cortex of unused donor kidneys. This procedure yields highly purified preparations of cells that display endothelial markers that include Factor VIII antigen, acetyl-LDL receptors, and determinants that bind Ulex europaeus lectin. HRMEC assemble into capillary-like cord and tube structures when plated on the surface of basement membrane-like matrix (BMM) in media containing phorbol myristate acetate. To further define subpopulations of HRMEC, we generated a panel of monoclonal antibodies and screened for those recognizing cell surface determinants. One monoclonal antibody recovered from this screen recognized a cell surface protein expressed on a subpopulation of HRMEC that we have designated PEC-1 (pioneer endothelial cell antigen-1). Cells expressing PEC-1 extended long, interconnecting filopodial processes in response to phorbol myristate acetate and assembled into capillary-like structures when plated on BMM. Anti-PEC-1 immunoprecipitated proteins of 25 and 27 kDa. Magnetic bead separation of PEC-1 (+) cells selected cells that assemble into capillary-like cord and tube structures. The remaining PEC-1 (−) HRMEC population formed matrix adherent patches. In the kidney, the PEC-1 determinant is expressed on a small subpopulation of microvascular glomerular cells and is prominently expressed on the apical membrane of proximal tubule cells. The PEC-1 determinant discriminates among subpopulations of HRMEC, identifying a subpopulation that contributes to assembly of capillary-like structures.     

VEGF Stimulates RCAN1.4 Expression in Endothelial Cells via a Pathway Requiring Ca2+/Calcineurin and Protein Kinase C-δ

Background

Vascular endothelial growth factor (VEGF) has previously been shown to upregulate the expression of the endogenous calcineurin inhibitor, regulator of calcineurin 1, variant 4 (RCAN1.4). The aim of this study was to determine the role and regulation of VEGF-mediated RCAN1.4 expression, using human dermal microvascular endothelial cells (HDMECs) as a model system.

Methodology/Principal Findings

We show that VEGF is able to induce RCAN1.4 expression during cellular proliferation and differentiation, and that VEGF-mediated expression of RCAN1.4 was inhibited by the use of inhibitors to protein kinase C (PKC) and calcineurin. Further analysis revealed that siRNA silencing of PKC-delta expression partially inhibited VEGF-stimulated RCAN1.4 expression. Knockdown of RCAN1.4 with siRNA resulted in a decrease in cellular migration and disrupted tubular morphogenesis when HDMECs were either stimulated with VEGF in a collagen gel or in an endothelial/fibroblast co-culture model of angiogenesis. Analysis of intracellular signalling revealed that siRNA mediated silencing of RCAN1.4 resulted in increased expression of specific nuclear factor of activated T-cells (NFAT) regulated genes.

Conclusions/Significance

Our data suggests that RCAN1.4 expression is induced by VEGFR-2 activation in a Ca²⁺ and PKC-delta dependent manner and that RCAN1.4 acts to regulate calcineurin activity and gene expression facilitating endothelial cell migration and tubular morphogenesis.     

Human neutrophils promote angiogenesis by a paracrine feedforward mechanism involving endothelial interleukin-8

Neovascularization by sprouting angiogenesis is critical for inflammation-mediated tissue remodeling and wound healing. We report here that human polymorphonuclear neutrophils (PMN) stimulated for 1 h with 100 nM N-formyl-methionyl-leucyl-phenylalanine (fMLP) released a proangiogenic entity that induced sprouting of capillary-like structures in an in vitro angiogenesis assay. The effect was comparable to the response obtained on stimulation with 100 ng/ml basic FGF. The PMN-mediated response was inhibited by neutralizing antibodies against VEGF or IL-8. As measured by ELISA technique, we found that fMLP-activated PMN (5 x 10(6)/ml) released 78 pg/ml IL-8 and 39 pg/ml VEGF within 1 h after stimulation. IL-8 release was blocked by actinomycin D or cycloheximide, but the inhibitors had no effect on VEGF release, suggesting that IL-8 secretion required de novo synthesis whereas VEGF was secreted from preformed stores. Accordingly, RT-PCR analysis revealed that IL-8 mRNA was upregulated on PMN stimulation, whereas the expression of VEGF mRNA was not affected. Moreover, supernatant derived from activated PMN induced upregulation of endothelial IL-8 mRNA expression, suggesting that release of VEGF and IL-8 from activated PMN may activate a paracrine feedforward mechanism involving endothelial IL-8. Moreover, VEGF-induced upregulation of endothelial IL-8 expression as well as sprouting of capillary-like structures was inhibited by a neutralizing anti-IL-8 antibody. These findings suggest that bacteria-derived tripeptides stimulate human PMN to release VEGF and IL-8, which activate endothelial cells and induce angiogenesis by a paracrine feedforward mechanism involving endothelial IL-8 upregulation.     

Inhibition of p38 MAPK reduces tumor conditioned medium-induced angiogenesis in co-cultured human umbilical vein endothelial cells and fibroblasts

Tumor conditioned medium (CM) has been widely used to stimulate endothelial cells to form capillary-like structures in in vitro angiogenesis models. We report herein the effect of HT1080 and A549 CM after they were mixed with microvascular endothelial cells medium-2 (EGM-2) on angiogenesis in human umbilical vein endothelial cells (HUVECs). Both HT1080 and A549 CM decreased HUVEC proliferation, to different extents. While A549 CM significantly increased capillary-like structure formation in a co-culture system, no effect of HT1080 was apparent. Inhibition of p38 mitogen-activated protein kinase (MAPK) blocked both basal and A549 CM induced capillary-like structure formation, but inhibition of extracellular signal-regulated kinases (ERK) and that of c-Jun N-terminal protein kinases (JNK) MAPK had no such effect. Activation of ERK MAPK was inhibited by both CMs, whereas p38 MAPK was inactivated by HT1080 and activated by A549 CM and a control. Neither CM had an effect on JNK MAPK. The results suggest that p38 MAPK played a critical role in capillary-like structure formation in the co-culture, partly via promotion of apoptosis in HUVECs.     

Cooperative effect of TNFalpha, bFGF, and VEGF on the formation of tubular structures of human microvascular endothelial cells in a fibrin matrix. Role of urokinase activity

In angiogenesis associated with tissue repair and disease, fibrin and inflammatory mediators are often involved. We have used three- dimensional fibrin matrices to investigate the humoral requirements of human microvascular endothelial cells (hMVEC) to form capillary-like tubular structures. bFGF and VEGF165 were unable to induce tubular structures by themselves. Simultaneous addition of one or both of these factors with TNFalpha induced outgrowth of tubules, the effect being the strongest when bFGF, VEGF165, and TNFalpha were added simultaneously. Exogenously added u-PA, but not its nonproteolytic amino-terminal fragment, could replace TNFalpha, suggesting that TNFalpha-induced u-PA synthesis was involved. Soluble u-PA receptor (u- PAR) or antibodies that inhibited u-PA activity prevented the formation of tubular structures by 59-99%. epsilon-ACA and trasylol which inhibit the formation and activity of plasmin reduced the extent of tube formation by 71-95%. TNFalpha or u-PA did not induce tubular structures without additional growth factors. bFGF and VEGF165 enhanced of the u- PAR by 72 and 46%, but TNFalpha itself also increased u-PAR in hMVEC by 30%. Induction of mitogenesis was not the major contribution of bFGF and VEGF165 because the cell number did not change significantly in the presence of TNFalpha, and tyrphostin A47, which inhibited mitosis completely, reduced the formation of tubular structures only by 28-36%. These data show that induction of cell-bound u-PA activity by the cytokine TNFalpha is required in addition to the angiogenic factors VEGF165 and/or bFGF to induce in vitro formation of capillary-like structures by hMVEC in fibrin matrices. These data may provide insight in the mechanism of angiogenesis as occurs in pathological conditions.     

Inhibition of P38 MAPK Reduces Tumor Conditioned Medium-Induced Angiogenesis in Co-Cultured Human Umbilical Vein Endothelial Cells and Fibroblasts

Tumor conditioned medium (CM) has been widely used to stimulate endothelial cells to form capillary-like structures in in vitro angiogenesis models. We report herein the effect of HT1080 and A549 CM after they were mixed with microvascular endothelial cells medium-2 (EGM-2) on angiogenesis in human umbilical vein endothelial cells (HUVECs). Both HT1080 and A549 CM decreased HUVEC proliferation, to different extents. While A549 CM significantly increased capillary-like structure formation in a co-culture system, no effect of HT1080 was apparent. Inhibition of p38 mitogen-activated protein kinase (MAPK) blocked both basal and A549 CM induced capillary-like structure formation, but inhibition of extracellular signal-regulated kinases (ERK) and that of c-Jun N-terminal protein kinases (JNK) MAPK had no such effect. Activation of ERK MAPK was inhibited by both CMs, whereas p38 MAPK was inactivated by HT1080 and activated by A549 CM and a control. Neither CM had an effect on JNK MAPK. The results suggest that p38 MAPK played a critical role in capillary-like structure formation in the co-culture, partly via promotion of apoptosis in HUVECs.     

Downregulation of Fes inhibits VEGF-A-induced chemotaxis and capillary-like morphogenesis by cultured endothelial cells

The aim of this study was to determine whether the downregulation of endogenous Fes by siRNA in cultured endothelial cells affects vascular endothelial growth factor-A (VEGF-A)-induced chemotaxis and capillary-like morphogenesis, which are considered as angiogenic cellular responses in vitro. VEGF-A-treatment induced autophosphorylation of Fes in cultured endothelial cells. LY294002, a phosphoinositide 3-kinase inhibitor, significantly inhibited VEGF-A-induced chemotaxis and capillary-like morphogenesis. Downregulation of Fes attenuated these VEGF-A-induced cellular responses but LY294002 did not produce further inhibition of these responses. Downregulation of Fes neither affected VEGF-A-induced autophosphorylation of VEGF receptor 2 nor mitogen-activated protein kinase activation, but markedly decreased Akt activation. Taken together, our novel results indicate the involvement of Fes in VEGF-A-induced cellular responses by cultured endothelial cells.     

Urokinase-type plasminogen activator mediates basic fibroblast growth factor-induced bovine endothelial cell migration independent of its proteolytic activity.

The dependence of urokinase-type plasminogen activator (uPA) induction on endogenous basic fibroblast growth factor (bFGF) activity during endothelial cell migration was investigated utilizing a combination of wounded endothelial cell monolayers and substrate overlay techniques. Purified polyclonal rabbit immunoglobulin G (IgG) against bFGF blocked the appearance of uPA-dependent lytic activity normally observed at the edge of a wounded bovine aortic endothelial (BAE) cell monolayer. Additionally, the migration of cells into the denuded area was inhibited 30-50% by antibodies either to bFGF or to bovine uPA. Incubation of wounded monolayers with either purified bovine uPA or agents able to induce PA activity, such as phorbol myristate acetate (PMA), vanadate, or bFGF, resulted in enhanced migration of cells (28-50%). Anti-bovine uPA IgG blocked a significant fraction (25%) of BAE cell migration induced by exposure to exogenous bFGF. The role of uPA in migration of wounded BAE cells was not dependent on plasmin generation. Furthermore, the amino terminal fragment (ATF) of human recombinant (hr) uPA, which is enzymatically inactive, stimulated endothelial cell movement in the presence of anti-bFGF IgG. These results suggest that BAE cell migration from the edge of a wounded monolayer is dependent upon local increases of uPA mediated by endogenous bFGF. Moreover, the data support the conclusion that migration is stimulated via a signalling mechanism dependent upon occupancy of the uPA receptor but independent of uPA-mediated proteolysis.