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.     

Inhibition of endothelial cell movement by pericytes and smooth muscle cells: activation of a latent transforming growth factor-beta 1-like molecule by plasmin during co-culture

When a confluent monolayer of bovine aortic endothelial (BAE) cells is wounded with a razor blade, endothelial cells (ECs) spontaneously move into the denuded area. If bovine pericytes or smooth muscle cells (SMCs) are plated into the denuded area at low density, they block the movement of the ECs. This effect is dependent upon the number of cells plated into the wound area and contact between ECs and the plated cells. Antibodies to transforming growth factor-beta 1 (TGF-beta 1) abrogate the inhibition of BAE cell movement by pericytes or SMCs. TGF-beta 1, if added to wounded BAE cell monolayers, also inhibits cell movement. When cultured separately, BAE cells, pericytes, and SMCs each produce an inactive TGF-beta 1-like molecule which is activated in BAE cell-pericyte or BAE cell-SMC co-cultures. The activation appears to be mediated by plasmin as the inhibitory effect on cell movement in co-cultures of BAE cells and pericytes is blocked by the inclusion of inhibitors of plasmin in the culture medium.     

Plasminogen activator inhibitor-1 is induced in migrating endothelial cells.

It has been proposed that a finely tuned protease-anti-protease equilibrium must be maintained during processes of cell migration in order to limit extracellular proteolysis to the close proximity of the cell surface, and thereby to prevent excessive extracellular matrix degradation. We have previously shown that urokinase-type plasminogen activator (u-PA) activity is induced in microvascular endothelial cells migrating from the edges of a wounded monolayer in vitro (Pepper et al., J. Cell Biol., 105:2535-2541, 1987). By Northern analysis, we now demonstrate that plasminogen activator inhibitor 1 (PAI-1) mRNA is increased in multiple-wounded monolayers of bovine microvascular (BME) or aortic (BAE) endothelial cells, with a maximal 7- and 9-fold increase 4 h after wounding, respectively. By in situ hybridization, we demonstrate that the increase in PAI-1 mRNA is localized to cells at the edge of a wounded BME or BAE cell monolayer. The increase in PAI-1 mRNA observed in BME cells is independent of cell division and is inhibited by antibodies to basic fibroblast growth factor (bFGF), suggesting that PAI-1 induction in migrating cells is mediated by the autocrine activity of bFGF. Taken together with our previous observations on the induction of u-PA, these results support the hypothesis that the proteolytic balance in the pericellular environment of migrating cells is regulated through the concomitant production of proteases and protease inhibitors.     

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.     

Centrosome reorientation in regenerating endothelial monolayers requires bFGF

Monolayers of endothelial cells respond to physical denudation with a characteristic sequence of lamellipodia extrusion, cell migration, and cell proliferation. Basic fibroblast growth factor (bFGF) has been implicated as a necessary component of this process: addition of exogenous bFGF enhances monolayer regeneration both in vitro and in vivo, and monolayer regeneration can be inhibited in vitro by treatment with neutralizing antibodies raised against bFGF. Centrosome reorientation from a random location to one preferentially situated between the nucleus and the denudation edge has been postulated as a mechanism essential for cell polarization and subsequent migration. This present study examined the effects of a polyclonal antibody to bFGF and suramin on monolayer regeneration, actin microfilament staining, and centrosome orientation at the wound edge of partially denuded bovine large vessel endothelial monolayers. Treatment with anti-bFGF or suramin abolished monolayer repair in these cultures. Cells at the denudation edge showed altered actin staining patterns and reduced lamellipodia extrusion, and there was complete inhibition of centrosome reorientation in treated cultures. Monolayer repair and centrosome reorientation could be restored by addition of exogenous bFGF in antibody but not suramin treated cultures. Recent evidence suggests that preferential centrosome location in migrating cells may be a consequence of lamellipodia protrusion and cell spreading, rather than an indication of cell polarization. However, these results indicate that agents which interfere with bFGF availability prevent endothelial monolayer regeneration via mechanisms involving cell spreading and/or centrosome reorientation.     

Urokinase-type plasminogen activator is induced in migrating capillary endothelial cells

Cellular migration is an essential component of invasive biological processes, many of which have been correlated with an increase in plasminogen activator production. Endothelial cell migration occurs in vivo during repair of vascular lesions and angiogenesis, and can be induced in vitro by wounding a confluent monolayer of cells. By combining the wounded monolayer model with a substrate overlay technique, we show that cells migrating from the edges of an experimental wound display an increase in urokinase-type plasminogen activator (uPA) activity, and that this activity reverts to background levels upon cessation of movement, when the wound has closed. Our results demonstrate a direct temporal relationship between endothelial cell migration and uPA activity, and suggest that induction of uPA activity is a component of the migratory process.     

Endothelial cell confluence regulates cyclooxygenase-2 and prostaglandin E2 production that modulate motility

Endothelial cells line the vasculature and, after mechanical denudation during invasive procedures or cellular loss from natural causes, migrate to reestablish a confluent monolayer. We find confluent monolayers of human umbilical vein endothelial cells were quiescent and expressed low levels of cyclooxygenase-2, but expressed cyclooxygenase-2 at levels comparable with cytokine-stimulated cells when present in a subconfluent culture. Mechanically wounding endothelial cell monolayers stimulated rapid cyclooxygenase-2 expression that increased with the level of wounding. Cyclooxygenase-2 re-expression occurred throughout the culture, suggesting signaling from cells proximal to the wound to distal cells. Media from wounded monolayers stimulated cyclooxygenase-2 expression in confluent monolayers, which correlated with the level of wounding of the donor monolayer. Wounded monolayers and cells in subconfluent cultures secreted enhanced levels of prostaglandin (PG) E(2) that depended on cyclooxygenase-2 activity, and PGE(2) stimulated cyclooxygenase-2 expression in confluent endothelial cell monolayers. Cells from subconfluent monolayers migrated through filters more readily than those from confluent monolayers, and the cyclooxygenase-2-selective inhibitor NS-398 suppressed migration. Adding PGE(2) to NS-398-treated cells augmented migration. Endothelial cells also migrated into mechanically denuded areas of confluent monolayers, and this too was suppressed by NS-398. We conclude that endothelial cells not in contact with neighboring cells express cyclooxygenase-2 that results in enhanced release of PGE(2), and that this autocrine and paracrine loop enhances endothelial cell migration to cover denuded areas of the endothelium.     

Basic fibroblast growth factor and transforming growth factor beta-1: Synergistic mediators of angiogenesis in vitro

We investigated the relative roles of basic fibroblast growth factor (bFGF) and transforming growth factor beta-1 (TGF-b) on bovine aortic endothelial cell mitogenesis and morphogenesis using two-dimensional Petri dish cultures and a threedimensional hydrated collagen gel. bFGF alone stimulated endothelial cell proliferation with an EC₅₀ of 0.5 ng/ml. At bFGF levels greater than 2.5 ng/ml, morphologic alterations in confluent monolayers predominated; cells changed from a cobblestone morphology to an elongated cell pattern and showed enhanced migration into a denuded area of a Petri dish. In the three-dimensional model, exposure of endothelial cell monolayers to high bFGF levels stimulated minor cell migration directly under the monolayer but no invasion into the gel matrix. In combination with bFGF, heparin potentiated morphogenic changes, but not mitogenesis. bFGF, modification of the antiproliferative effect of TGF-b in confluent cultures was evidenced by induction of endothelial cell sprouting in response to 0.5 ng/ml TGF-b and 10–20 ng/ml bFGF in two-dimensional cultures. On collagen gels, endothelial cells migrated into the deep layers of the gel in a dose-dependent manner: invasion was maximal at 0.3–0.7 ng/ml TGF-b with decreased invasion at higher concentrations. The optimal collagen concentration that supported cell invasion was 0.075% collagen with the number of invading cells decreasing with increasing collagen gel density. By scanning electron microscopy, invading endothelial cells assumed a fibroblast-like appearance with slender cell extensions. We concluded that bFGF and TGF-b had independent effects on endothelial cell morphology and mitogenesis in culture. In combination at specific doses, these agents stimulated sprouting in the two-dimensional model and cell invasion in a collagen gel model. Morphogenic changes may be the primary event in determining angiogenesis. © 1993 Wiley-Liss, Inc.     

Heparin and heparan sulfate increase the radius of diffusion and action of basic fibroblast growth factor

The radius of diffusion of basic FGF (bFGF) in the presence and in the absence of the glycosaminoglycans heparin and heparan sulfate was measured. Iodinated 125I-bFGF diffuses further in agarose, fibrin, and on a monolayer of bovine aortic endothelial (BAE) cells in the presence of heparin than in its absence. Heparan sulfates affected the diffusion of 125I-bFGF in a manner similar to, though less pronounced than, heparin. When applied at the center of a monolayer of BAE cells, bFGF plus heparin stimulated morphological changes at a 10-fold greater radius than bFGF alone. These results suggest that bFGF-heparin and/or heparan sulfate complexes may be more effective than bFGF alone in stimulating cells located away from the bFGF source because the bFGF-glycosaminoglycan complex partitions into the soluble phase rather than binding to insoluble glycosaminoglycans in the extracellular matrix. Thus, the complex of bFGF and glycosaminoglycan may represent one of the active forms of bFGF in vivo.     

Correlation of cell migration, cell invasion, receptor number, proteinase production, and basic fibroblast growth factor levels in endothelial cells

The levels of endogenous basic fibroblast growth factor (bFGF) in seven clones of cultured bovine capillary endothelial (BCE) cells were assayed, and their relation to cell morphology, bFGF receptor number, cell migration, amniotic membrane invasivity, and proteinase levels were studied. Immunoblotting experiments with anti-bFGF IgG demonstrated that cells from these clones contained different amounts of bFGF. The cells containing high levels of bFGF had a spindle or elongated appearance at confluence and a low number of high affinity receptors for bFGF. The cells containing low levels of bFGF had a cobblestone-like appearance and a higher number of high affinity receptors. When exposed to 10 ng/ml bFGF, cells containing a low level of bFGF took on an elongated appearance with a crisscross pattern similar to that seen with the high producer bFGF cells. The endogenous bFGF levels of the BCE cell clones correlated with the extent of cell migration after wounding of a monolayer and the degree of invasion of the human amniotic membrane. Cells from the clone with the highest endogenous bFGF level migrated well, invaded the amnion membrane without the addition of exogenous bFGF, and were relatively unaffected by the addition of bFGF. Cells from the clone containing the lowest level of bFGF did not migrate or invade under normal conditions. However, the addition of bFGF to the culture medium strongly enhanced both of these processes. The inclusion of anti-bFGF IgG in the media suppressed cell migration and invasion. The plasminogen activator (PA) activities of cell lysates of the clones, assayed by the 125I-fibrin plate technique, indicated that the PA levels did not correlate with the bFGF levels. Metalloproteinase activities in the conditioned medium, assayed by gelatin zymography, correlated with the endogenous bFGF levels, suggesting that the degree of expression of metalloproteinases might be critical for cell migration and invasion. These data suggest that endogenous bFGF may have an important role for migration and invasion of BCE cells during neovascularization via the induction and/or activation of specific metalloproteinases.     

Response of bovine endothelial cells to FGF-2 and VEGF is dependent on their site of origin: Relevance to the regulation of angiogenesis

Angiogenesis, the formation of new capillary blood vessels, occurs almost exclusively in the microcirculation. This process is controlled by the interaction between factors with positive and negative regulatory activity. In this study, we have compared the effect of two well described positive regulators, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2) on bovine adrenal cortex-derived microvascular endothelial (BME) and bovine aortic endothelial (BAE) cells. The parameters we assessed included (a) cellular reorganization and lumen formation following exposure of the apical cell surface to a three-dimensional collagen gel; (b) organization of the actin cytoskeleton; (c) expression of thrombospondin-1 (TSP-1), an endogenous negative regulator of angiogenesis; and (d) extracellular proteolytic activity mediated by the plasminogen activator (PA)/plasmin system. We found that (a) collagen gel overlay induces rapid reorganization and lumen formation in BME but not BAE cells; (b) FGF-2 but not VEGF induced dramatic reorganization of actin microfilaments in BME cells, with neither cytokine affecting BAE cells; (c) FGF-2 decreased TSP-1 protein and mRNA expression in BME cells, an effect which was specific for FGF-2 and BME cells, since TSP-1 protein levels were unaffected by VEGF in BME cells, or by FGF-2 or VEGF in BAE cells; (d) FGF-2 induced urokinase-type PA (uPA) in BME and BAE cells, while VEGF induced uPA and tissue-type PA in BME cells with no effect on BAE cells. Taken together, these findings reveal endothelial cell-type specific responses to FGF-2 and VEGF, and point to the greater specificity of these cytokines for endothelial cells of the microvasculature than for large vessel (aortic) endothelial cells. Furthermore, when viewed in the context of our previous observation on the synergistic interaction between VEGF and FGF-2, our present findings provide evidence for complementary mechanisms which, when acting in concert, might account for the synergistic effect.     

Repair of wounded monolayers of cultured bovine aortic endothelial cells is inhibited by calcium spirulan,a novel sulfated polysaccharide isolated from Spirulina platensis

Calcium spirulan (Ca-SP) is a novel sulfated polysaccharide isolated from a blue-green alga Spirulina platensis. Ca-SP inhibits thrombin by activation of heparin cofactor II. Therefore, it could serve as an origin of anti-atherogenic medicines. Since maintenance of vascular endothelial cell monolayers is important for prevention of vascular lesions such as atherosclerosis, the effect of Ca-SP at 20 microg/ml or less on the repair of wounded bovine aortic endothelial cell monolayers in culture was investigated in the present study. When the monolayers were wounded and cultured in the presence of Ca-SP, the polysaccharide inhibited the appearance of the cells in the wounded area. The inhibition was also observed even when the repair was promoted by excess basic fibroblast growth factor, which is one of the autocrine growth factors that are involved in the endothelial cell monolayer maintenance. On the other hand, Ca-SP inhibited the cell growth and the incorporation of [3H]thymidine into the acid-insoluble fraction of proliferating endothelial cells, suggesting that Ca-SP inhibits endothelial cell proliferation. From these results, it is concluded that Ca-SP may retard the repair process of damaged vascular endothelium through inhibition of vascular endothelial cell proliferation by induction of a lower ability to respond to stimulation by endogenous basic fibroblast growth factor.     

A high-affinity receptor for urokinase plasminogen activator on human keratinocytes: characterization and potential modulation during migration.

Low passage cultures of normal human keratinocytes produce several components of the plasminogen activator/plasmin proteolytic cascade, including urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), and two specific inhibitors. Studies here presented demonstrate that these cells also contain a high-affinity (Kd = 3 x 10(-10) M) plasma membrane-binding site for uPA. High molecular weight uPA, either as the single-chain precursor or two-chain activated form, bound to the receptor; however, low molecular weight (33 kD) uPA, tPA, or epidermal growth factor did not compete for binding, demonstrating specificity. Acid treatment, which removed endogenous uPA from the receptor, was required to detect maximal binding (45,000 sites per cell). To investigate the possibility that the uPA receptor on keratinocytes may be involved in epithelial migration during wound repair, cultures were wounded and allowed to migrate into the wounded site. Binding sites for uPA were localized by autoradiographic analysis of 125I-uPA binding as well as by immunocytochemical studies using anti-uPA IgG. With both techniques uPA binding sites were detected selectively on the plasma membrane of cells at the leading edge of the migrating epithelial sheet. This localization pattern suggests that uPA receptor expression on keratinocytes may be coupled to cell migration during cutaneous wounding.     

Apolipoprotein E: A potent inhibitor of endothelial and tumor cell proliferation

Recombinant human apolipoprotein E3 (apoE), purified from E. coli, inhibited the proliferation of several cell types, including endothelial cells and tumor cells in a dose- and time-dependent manner. ApoE inhibited both de novo DNA synthesis and proliferation as assessed by an increase in cell number. Maximal inhibition of cell growth by apoE was achieved under conditions where proliferation was dependent on heparin-binding growth factors. Thus, at low serum concentrations (0–2.5%) basic fibroblast growth factor (bFGF) stimulated the proliferation of bovine aortic endothelial (BAE) cells severalfold. The bFGF-dependent proliferation was dramatically inhibited by apoE with an IC₅₀ ≈ 50 nM. Under conditions where cell proliferation was mainly serum-dependent, apoE also suppressed growth but required higher concentrations to be effective (IC₅₀ ≈ 500 nM). ApoE also inhibited growth of bovine corneal endothelial cells, human melanoma cells, and human breast carcinoma cells. The IC₅₀ values obtained with these cells were generally 3–5 times higher than with BAE cells. Inhibition of cell proliferation by apoE was reversible and dependent on the time of apoE addition to the culture. In addition, apoE inhibited the chemotactic response of endothelial cells that were induced to migrate by a gradient of soluble bFGF. Inhibition of cell proliferation by apoE may be mediated both by competition for growth factor binding to proteoglycans and by an antiadhesive activity of apoE. The present results demonstrate that apoE is a potent inhibitor of proliferation of several cell types and suggest that apoE may be effective in modulating angiogenesis, tumor cell growth, and metastasis.     

Modulation of sulfated proteoglycan synthesis by bovine aortic endothelial cells during migration

The rates of 35S-sulfate incorporation into proteoglycan were compared in multi-scratch wounded and confluent cultures of bovine aortic endothelial cells to determine whether proteoglycan synthesis is altered as cells are stimulated to migrate and proliferate. Incorporation was found to be stimulated in a time-dependent manner, reaching maximal levels 44-50 h after wounding, as cells migrated into wounded areas of the culture dish. Quantitative autoradiography of 35S-sulfate-labeled single-scratch wounded cultures demonstrated a 2-4-fold increase in the number of silver grains over migrating cells near the wound edge when compared to cells remote from the wound edge. Furthermore, when cell proliferation was blocked by inhibition of DNA synthesis, the increase in 35S-sulfate incorporation into proteoglycan after wounding was unaffected. These data indicate that cell division is not required for the modulation of proteoglycan synthesis to occur after wounding. Characterization of the newly synthesized proteoglycan by ion-exchange and molecular sieve chromatography demonstrated that heparan sulfate proteoglycan constitutes approximately 80% of the labeled proteoglycan in postconfluent cultures, while after wounding, chondroitin sulfate proteoglycan and/or dermatan sulfate proteoglycan (CS/DSPG) increases to as much as 60% of the total labeled proteoglycan. These results suggest that CS/DSPG synthesis is stimulated concomitant with the stimulation of endothelial cell migration after wounding.     

Upregulation of urokinase receptor expression on migrating endothelial cells

One of the phenotypic hallmarks of migrating endothelial cells, both in vivo and in vitro, is expression of the urokinase-type plasminogen activator (u-PA), a key mediator of extracellular proteolysis. In the study reported here, we have used an in vitro model of endothelial cell migration to explore the mechanism of this phenomenon. We have found that wounding of an endothelial cell monolayer triggers a marked, rapid and sustained increase in expression of a specific high-affinity receptor for u-PA (u-PAr) on the surface of migrating cells. Migrating cells displayed an increase in the levels of u-PA and u-PAr mRNAs, and this increase was mediated by endogenous basic fibroblast growth factor (bFGF). We also show that the increase in u-PA activity on migrating cells can be accounted for by an increase in receptor-bound u-PA, and that the increase in activity is also dependent on endogenous bFGF. These results demonstrate that the expression of plasmin-mediated proteolytic activity by migrating endothelial cells is a consequence of increased production of both u-PA and its receptor, and that this in turn is mediated by endogenous bFGF. This suggests that u-PA, produced at increased levels by migrating cells, binds to u-PAr whose expression is upregulated on the same cells. These observations are in accord with the postulated role of u-PAr in mediating efficient and spatially restricted extracellular proteolysis, particularly in the context of cell migration.     

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.     

Cytoskeleton in TFG-beta- and bFGF-modulated endothelial monolayer repair

Endothelial cell proliferation and migration in vitro is depressed by transforming growth factor beta (TFG-beta) and enhanced by basic fibroblast growth factor (bFGF) treatment. This study examines interactions between cytoskeletal changes and cell proliferation in regenerating endothelial monolayers treated with bFGF, TFG-beta, and both factors. As previously described by others, monolayer regeneration is enhanced by bFGF and reduced by TFG-beta. Endothelial cell morphology is altered by TFG-beta treatment. Cells lose their cobblestone appearance and assume a pleomorphic shape. Actin microfilament staining is modified in both intact and regenerating TFG-beta-treated monolayers as well. There is a loss of dense peripheral band staining and an enhancement in staining intensity of cytoplasmic stress fibers. No such alterations are seen in bFGF-treated cultures. Cell proliferation at the wound edge, as indicated by bromodeoxyuridine incorporation, is inhibited by TGF-beta. Although monolayer repair is modulated by growth factor treatment, centrosome reorientation and microtubule staining patterns are not altered by either factor. Thus these factors appear to have effects on a mechanism(s) other than centrosome reorientation which may be involved in repair of denuded endothelial monolayers.