Stimulation of motility in cultured bovine capillary endothelial cells by angiogenic preparations

Several angiogenic preparations that have been shown to stimulate plasminogen activator (PA) and collagenase production by cultured bovine capillary endothelial (BCE) cells were tested for their ability to stimulate BCE cell motility in the phagokinetic track assay. Bovine retinal extract, medium conditioned by 3T3-F442A differentiated mouse adipocytes, SK HEP-1 human hepatoma cell lysate, mouse sarcoma 180 cell lysate, and medium conditioned by mouse sarcoma 180 cells stimulated motility 68.7%, 48.5%, 140.9%, 56.5%, and 102.1%, respectively, relative to untreated cells. The motility-stimulating activity of these preparations was dose dependent and linear over the 16-h assay period. Several hormones and growth factors were tested for BCE cell motility-stimulating activity, including insulin, vasopressin, fibroblast growth factor, and a partially purified preparation of sarcoma growth factor, and were found to be ineffective. 12-0-tetradecanoyl-phorbol-acetate (TPA), a potent stimulator of both PA and collagenase activities in BCE cells, also did not stimulate motility, indicating that protease production is not sufficient to stimulate BCE cell motility in this assay. Neither SK HEP-1 hepatoma cell lysate nor TPA was effective in stimulating motility in bovine aortic endothelial (BAE) cells. The inability of SK HEP-1 hepatoma cell lysate to stimulate movement in BAE cells is consistent with the observation that angiogenesis occurs by sprouting of capillaries, not large vessels.     

Tetradecanoyl phorbol acetate stimulates latent collagenase production by cultured human endothelial cells

Angiogenesis is associated with the fragmentation of blood vessel basement membranes. Since collagen is a major constituent of basement membranes, cultured human endothelial cells derived from umbilical cord veins were assayed for their ability to produce collagenase. Unstimulated cultured human endothelial cells did not secrete detectable levels of active collagenase into the culture medium. However, if the post-culture medium was treated with trypsin or plasmin, low levels of collagenolytic activity were detected, indicating that endothelial cells secrete small amounts of latent collagenase. Addition of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) to the culture medium stimulated the secretion of collagenase by endothelial cells 5–30 fold. More than 90% of the collagenase was secreted in the latent form. Stimulation of collagenase production was detected at 10^?9 M TPA and was maximal at 10^?8 M TPA. An increase in the rate of collagenase production could be detected within 3 hr after the addition of TPA, and full induction occurred by 12 hr. Cycloheximide (3 μg/ml) or actinomycin D (0.1 μg/ml) inhibited both basal levels of collagenase production and the stimulation of collagenase production by TPA. Phorbol-12,13-didecanoate (PDD), a tumor-promoting analog of TPA, also stimulated collagenase production when administered at the same concentrations that were effective for TPA. However, 4-O-methyl TPA and 4-αPDD, two analogs of TPA which are not tumor promoters, did not stimulate collagenase production at concentrations up to 10^?7 M. The collagenase produced by endothelial cells was a typical vertebrate collagenase as judged by the following criteria: it cleaved collagen into only two fragments which were three quarters and one quarter of the length of the intact molecule; it was inhibited by EDTA and human serum; it was not inhibited by inhibitors of serine, thiol or aspartate proteases. Thus TPA causes an increase in the production of latent collagenase by cultured human endothelial cells.     

Both normal and tumor cells produce basic fibroblast growth factor

We have previously purified from human placenta a basic fibroblast growth factor (FGF)-like molecule which stimulates the production of plasminogen activator (PA) and collagenase, induces DNA synthesis, produces an increase in motility in cultured bovine capillary endothelial (BCE) cells, and induces angiogenesis in vivo. The ability of basic FGF to stimulate PA production in BCE cells was used as an assay for the presence of basic FGF-like molecules in extracts of both normal and tumor-derived cultured cells. The identity of the PA-stimulatory activity with basic FGF was confirmed by its high affinity for heparin and by its cross-reactivity with antibodies to human placental basic FGF. Basic FGF-like molecules were identified in eight of ten cell lines tested, and the amount of FGF-like activity present in these cells bore no relation to their origin from normal or tumor tissue. The test cells, BCE cells, had one of the highest levels of FGF-like activity, suggesting that it may have an autocrine role in these cells.     

Production of latent collagenase by human umbilical vein endothelial cells in response to angiogenic preparations

Three preparations known to be angiogenic in vivo and which stimulate production of latent collagenase by cultured bovine capillary endothelial (BCE) cells were tested for their ability to stimulate production of latent collagenase by cultured human umbilical vein endothelial (HUVE) cells. Bovine retinal extract and murine adipocyte-conditioned medium had no effect on production of latent collagenase by HUVE cells at concentrations that were effective in stimulating production of latent collagenase by BCE cells. However, with higher concentrations of bovine retinal extract, production of latent collagenase by HUVE cells was stimulated. Human hepatoma cell sonicate stimulated production of latent collagenase by HUVE cells in a dose-dependent manner. The concentration of human hepatoma cell sonicate which stimulated production of latent collagenase by HUVE cells was lower than the concentration that was effective for the stimulation of production of latent collagenase by BCE cells. Plasminogen activator production by HUVE cells was unaffected by human hepatoma cell sonicate. Varying the concentration of serum in HUVE cultures did not affect the stimulation of latent collagenase production by human hepatoma cell sonicate, suggesting that serum components neither block nor stimulate the action of the collagenase-inducing factor. Although human hepatoma cell sonicate is reported to stimulate endothelial cell multiplication, purified and partially purified endothelial cell mitogens had no effect on production of latent collagenase. Thus, at least two preparations which contain angiogenic activity will stimulate production of latent collagenase by HUVE cells.     

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.     

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.     

Fibroblast heterogeneity in collagenolytic response to cyclosporine.

To investigate the mechanism of cyclosporine (CS)-induced fibrotic gingival enlargement, the effect of CS on the collagenolytic activities of 14 different human gingival fibroblast strains derived from healthy individuals with non-inflammed gingiva was examined in vitro. There was marked heterogeneity among individuals in basal levels of collagenase activity, and there was also variation among the subpopulations derived from one strain. Fibroblasts from different individuals also varied markedly in their collagenolytic response to CS (0.1 to 0.75 micrograms/ml). In most strains, CS decreased collagenase activity, but in some, the drug caused no change or significantly increased activities. In most of the subpopulations CS significantly decreased collagenolytic activity. Two of the fibroblasts strains and the subpopulations described above were examined for the production of immunoreactive collagenase and tissue inhibitor of metalloproteinase (TIMP). The two strains made similar amounts of collagenase, but differed markedly in TIMP levels; CS affected their collagenase production differently but had similar effects on TIMP. Among the subpopulations there was variation in the production of collagenase, although none made detectable levels of TIMP; they also varied in the production of both proteins in response to CS. In two of the subpopulations and in both strains at some concentrations, the effect of CS on the relative levels of collagenase and TIMP could account for the decreased collagenase activity; i.e., the level of collagenase was unchanged or decreased, and TIMP production was unchanged or increased. This study demonstrates the variation among individuals as well as intrastrain heterogeneity of human gingival fibroblasts with regard to collagenase activity and the production of collagenase and TIMP. The heterogeneity of the collagenolytic response of different gingival fibroblast strains and their subpopulations to CS treatment may partly explain the susceptibility of only some individuals to CS-induced gingival enlargement.     

Characterization of the activation of latent TGF-beta by co-cultures of endothelial cells and pericytes or smooth muscle cells: a self- regulating system

The conversion of latent transforming growth factor beta (LTGF-beta) to the active species, transforming growth factor beta (TGF-beta), has been characterized in heterotypic cultures of bovine aortic endothelial (BAE) cells and bovine smooth muscle cells (SMCs). The formation of TGF-beta in co-cultures of BAE cells and SMCs was documented by a specific radioreceptor competition assay, while medium from homotypic cultures of BAE cells or SMCs contained no active TGF-beta as determined by this assay. The concentration of TGF-beta in the conditioned medium of heterotypic co-cultures was estimated to be 400-1,200 pg/ml using the inhibition of BAE cell migration as an assay. Northern blotting of poly A+ RNA extracted from both homotypic and heterotypic cultures of BAE cells and SMCs revealed that BAE cells produced both TGF-beta 1 and TGF-beta 2, while SMCs produced primarily TGF-beta 1. No change in the expression of these two forms of TGF-beta was apparent after 24 h in heterotypic cultures. Time course studies on the appearance of TGF-beta indicated that most of the active TGF-beta was generated within the first 12 h after the establishment of co-cultures. The generation of TGF-beta in co-cultures stimulated the production of the protease inhibitor plasminogen activator inhibitor-1 (PAI-1). The inclusion of neutralizing antibodies to TGF-beta in the co-culture medium blocked the observed increase in PAI-1 levels. The increased expression of PAI-1 subsequent to TGF-beta formation blocked the activation of the protease required for conversion of LTGF-beta to TGF-beta as the inclusion of neutralizing antibodies to PAI-1 in the co-culture medium resulted in prolonged production of TGF-beta. This effect was lost upon removal of the PAI-1 antibodies. Thus, the activation of LTGF-beta appears to be a self-regulating system.     

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.     

Cell density dependent effects of TGF-beta demonstrated by a plasminogen activator-based assay for TGF-beta.

Transforming growth factor-beta 1 (TGF-beta 1) induces a decrease in plasminogen activator (PA) expression in confluent cultures of bovine aortic endothelial (BAE) cells. We describe an assay using the suppression of PA expression in confluent BAE cells by TGF-beta 1 which detects concentrations of the growth factor ranging from 5 to 200 pg/ml and has an ED50 of 15-20 pg/ml. The assay can be performed in 96-well plates and requires a minimum of 35 ul of solution per sample, thereby limiting the amount of reagents required and allowing many samples to be tested in a single assay. Here we demonstrate that the effect of TGF-beta 1 on PA expression in BAE cells depends on the length of time the cells are exposed to the growth factor and the density at which the cells are plated. In cells plated at a high density (3.5 x 10(5) cells/cm2), both 4 h and 24 h exposures to TGF-beta 1 suppress PA expression. However, with cells plated sparsely (3.5 x 10(4) cells/cm2), a 4 h exposure to TGF-beta 1 increases PA expression 2-fold, whereas a 24 h exposure results in an 85% inhibition of basal PA expression. The paradoxical stimulation of PA expression in cells at a sparse density upon 4 h exposure to TGF-beta 1 occurs in a dose-dependent manner with an ED50 of 15-20 pg/ml. This bifunctional response of PA production in cells exposed to TGF-beta 1 may have implications with regard to the role of TGF-beta 1 in angiogenesis.     

Urokinase-type and tissue-type plasminogen activators have different distributions in cultured bovine capillary endothelial cells

The cell extracts and conditioned medium from cultured bovine capillary endothelial (BCE) cells were examined to determine the types of plasminogen activator (PA) present in each of these two fractions. The fractions were first analyzed by fibrin autography after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The cell extracts contained two species of PA of Mr 48,000 and 28,000. Multiple forms of PA were detected in the conditioned medium: variable amounts of the Mr 48,000 and 28,000 forms and a broad band of activity with Mr in the range of 67,000-93,000. The major fraction of the Mr 48,000 form was in the cell extract. Treatment of the cells with 12-0-tetradecanoyl phorbol-13-acetate or with a preparation containing angiogenic activity resulted in a proportionate increase in the levels of all forms. The Mr 48,000 form was demonstrated to be a urokinase-like PA, since it was immunoprecipitated with antibodies to urokinase. When conditioned medium or cell extracts from biosynthetically labelled BCE cells were incubated with antiserum to urokinase, the Mr 48,000 form was immunoprecipitated only from the cell extract. The Mr 67,000-93,000 forms were demonstrated to be tissue-type PAs, since they were immunoprecipitated with antibodies to tissue PA. When the same conditioned medium or cell extracts were incubated with antiserum to tissue-type PA, the Mr 67,000-93,000 forms were immunoprecipitated only from the conditioned medium. Therefore, BCE cells are able to produce both tissue-type PA, which is primarily secreted, and urokinase-type PA, which remains primarily cell associated.     

Characterization of extracellular alkaline proteases and collagenase induction in Vibrio alginolyticus

The number and approximate molecular weights of extracellular alkaline proteases produced by Vibrio alginolyticus were determined by gelatin-PAGE. Three major bands of protease activity with apparent molecular weights of approximately 28 000, 22 500 and 19 500 (proteases 1, 2 and 3, respectively) and two minor bands of protease activity with apparent molecular weights of approximately 15 500 and 14 500 (proteases 4 and 5, respectively) were obtained after gelatin-PAGE. The activities of the five proteases were inhibited by serine protease inhibitors but their activities were not affected by inhibitors of trypsin-like enzymes. Histidine, which inhibited V. alginolyticus collagenase, did not inhibit the activities of the alkaline serine proteases. The production of protease 1, however, was enhanced by histidine. Protease 1 production was also affected by temperature and production was depressed at 37 degrees C. Gelatin-PAGE of a commercial V. alginolyticus collagenase preparation revealed four bands of activity which were identified as collagenases with apparent molecular weights of approximately 45 000, 38 500, 33 500 and 31 000. The collagenase preparation was contaminated with two serine proteases. The release of [3H]proline from collagen matrices produced by smooth muscle cells was shown to be a sensitive assay for bacterial collagenases and was used to show that V. alginolyticus produced a basal constitutive level of extracellular collagenase. The constitutive levels of collagenase were affected by aeration.     

Tumor-promoting phorbol esters and cell proliferation stimulate secretion of basement membrane (type IV) collagen-degrading metalloproteinase by human fibroblasts

The secretion of a type IV collagen-specific proteinase is stimulated in cultured human skin fibroblasts by the phorbol ester tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) and during cell proliferation. Exposure of the cells at the late log phase of growth to 10(-9) to 10(-6) M TPA resulted in the secretion of type IV collagenase activity to the medium, this effect being reversible. Incubation of intact type IV procollagen with TPA-induced fibroblast medium protein produced six peptides, four of which corresponded in size to the fragments produced by a type IV collagen-specific collagenase (Fessler, L., Duncan, K., Fessler, J., Salo, T., and Tryggvason (1984) J. Biol. Chem. 259, 9783-9789). The TPA-induced type IV collagen-degrading enzyme could be activated by trypsin, was inhibited by EDTA, but was not affected by soybean trypsin inhibitor, N-ethylmaleimide, aprotinin, or cysteine. Therefore, in human skin fibroblasts, TPA can induce a type IV collagen-specific, metal-dependent collagenase as was previously described in some invasive tumor cells. Furthermore, another metalloprotease is apparently secreted under the same conditions of TPA exposure. The production of metal-dependent, type IV collagen-degrading activity was also studied at different stages of cellular proliferation. In early log phase, a significant amount of enzyme activity was observed in the control cell medium; this activity disappeared during both late log and stationary growth phases. This activity could be markedly increased by the addition of 10(-8) M TPA to the culture medium. The production of matrix-degrading proteinases is therefore likely to be associated with rapid cell proliferation in both transformed and untransformed cells.     

The identification, purification and characterisation of an inhibitor of collagenase (20K) produced by neoplastic epithelial cells

A rat carcinoma cell line (T2/H7) constitutively synthesised interstitial collagenase. When these cells were incubated with 12-O-tetradecanoylphorbol 13-acetate (TPA) they secreted an inhibitor of collagenase, which resulted in a net decrease of collagenolytic activity being detected in conditioned medium. Using reverse zymography, the Mr of the inhibitor was found to be 20,000 which suggests that it may be the rat homologue of inhibitor of metalloproteinase 2 (IMP2; TIMP-2), as it inhibited both the gelatinolytic and collagenolytic activities of rat collagenase. The inhibitor was separated from collagenase by filtration through a YM30 membrane. The inhibitor was purified further by sequential chromatography on heparin-Sepharose and Con A-Sepharose. It bound to heparin-Sepharose in 75 mM NaCl and was eluted with 300 mM NaCl. It did not bind to Con A-Sepharose, suggesting that it was a non-glycosylated molecule. The inhibitor was resistant to treatment with either trypsin, APMA or heat.     

Production of procollagenase by cultured human keratinocytes

Using a collagen film assay utilizing 14C-labeled type I collagen, we demonstrated that cultured human keratinocytes produced a procollagenase after treatment with the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Production of collagenase paralleled alterations in cellular morphology induced by TPA. When procollagenase was immunoprecipitated with antibody to human fibroblast collagenase and analyzed on sodium dodecyl sulfate-polyacrylamide gels, the zymogen was revealed as a 56- and 51-kDa doublet. The keratinocyte-derived collagenase was a neutral metalloprotease, required activation with trypsin for detection in the collagenase assay and produced the characteristic three-quarter and one-quarter length collagen cleavage products when incubated with type I collagen at 25 degrees C. The enzyme was inhibited by serum and cysteine and was largely unaffected by serine, thiol, and carboxyl protease inhibitors. Cycloheximide inhibited the TPA-induced production of collagenase, suggesting that the procollagenase was not stored preformed in the keratinocytes. Keratinocytes treated with a tumor-promoting analogue of TPA also produced collagenase, but cells treated with cytochalasin B, interleukin-1, or two non-tumor promoting phorbol esters did not. Keratinocyte-derived collagenase may play a role in wound healing and morphogenesis.     

Role of extracellular matrix in the action of basic fibroblast growth factor: Matrix as a source of growth factor for long-term stimulation of plasminogen activator production and DNA synthesis

When bovine capillary endothelial (BCE) cells were treated with 10 ng/ml of basic fibroblast growth factor (bFGF) for 10 or 30 minutes at 37°C, washed extensively with phosphate-buffered saline (PBS) and incubated in bFGF-free medium, plasminogen activator (PA) production was stimulated to the same extent as in cells exposed continuously to bFGF. Three methods of removing bFGF from heparin-like binding sites in the extracellular matrix, but not from bFGF receptors, abolished this long-term effect of a brief exposure to bFGF. First, BCE cells exposed to bFGF for 30 minutes were washed with 2M NaCI and incubated in bFGF-free medium. Second, BCE cells were incubated with bFGF for 10 minutes in the presence of heparin, and cells were washed with PBS and incubated in bFGF-free medium. Third, BCE cell cultures were treated with heparinase and exposed to bFGF. Each of these treatments abolished the long-term (24-48 hours) stimulation of PA production normally observed after brief exposure to bFGF. In each of these experiments, incubation of cells in bFGF-containing medium after the treatments resulted in normal stimulation of PA production, demonstrating that the treatments did not harm the cells. Stimulation of DNA synthesis was observed when cells were exposed to bFGF for 2 hours at 4°C, incubated in bFGF-free medium for 24 hours at 37°C, and assayed for ³H-thymidine incorporation. However, no stimulation was observed if the 2 hours incubation at 4°C was carried out in the presence of heparin. Thus, long-term stimulation of PA activity and DNA synthesis after a brief exposure to bFGF seems to be a consequence of bFGF binding to the extracellular matrix. The extracellular matrixmay act as a physiologic buffer, binding bFGF when concentrations are high and releasing it later for interaction with its receptor. This interaction with matrix may be required for the in vivo action of bFGF.     

Nonspecific proteases: a new approach to the isolation of adult cardiocytes

The potent collagenolytic activity of nonspecific proteinases suggested their use as enzymatic agents for the dissociation of single adult heart cells. This was assessed in guinea pig hearts perfused for 1 min with solutions containing hyaluronidase (100-10 000 U/mL), trypsin (100-10 000 U/mL), crude collagenase (100-500 U/mL), or nonspecific protease (0.1-100 U/mL). No rod-shaped cells were observed among the cells isolated with these concentrations of hyaluronidase, trypsin, or crude collagenase. By contrast, 45-80% of the cells released with nonspecific protease (5-10 U/mL) were rod shaped and Ca2+ tolerant. Resting and action potentials recorded from cells dispersed with nonspecific protease were similar to those recorded from cells isolated after prolonged collagenase exposure.     

The mitogenic signaling pathway but not the plasminogen activator- inducing pathway of basic fibroblast growth factor is mediated through protein kinase C in fetal bovine aortic endothelial cells

Basic fibroblast growth factor (bFGF) induces cell proliferation and plasminogen activator (PA) activity in transformed fetal bovine aortic endothelial (FBAE) GM 7373 cells. A similar response is observed after treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In these cells, bFGF and TPA cause activation of protein kinase C (PKC), as demonstrated by the induction of the phosphorylation of an 87-kD PKC substrate in intact cells and by the increase in membrane-associated PKC activity. Activation of PKC by bFGF or TPA is inhibited in cells made PKC-deficient by pretreatment with high concentrations of TPA. The mitogenic activity of bFGF or of TPA is completely inhibited in PKC- deficient cells or in naive cells treated with the PKC inhibitor H-7. However, these cells proliferate in response to serum, epidermal growth factor, and dibutyryl cyclic AMP. Similar results are obtained in normal FBAE AG 7680 cells. These data indicate that activation of PKC is responsible for the mitogenic activity of bFGF in FBAE cells. On the contrary, the PA-inducing activity of bFGF is unaffected by down- regulation of PKC or by treatment with the PKC inhibitor H-7 in both transformed GM 7373 and normal AG 7680 cells. bFGF induces a rapid 45Ca influx in naive and in PKC-deprived GM 7373 cells. In these cells, addition of EGTA to the incubation medium prevents both the 45Ca influx and the increase in PA activity induced by bFGF, without affecting its mitogenic activity. Even though the involvement of PKC in the increase of cell-associated PA activity induced by bFGF can not be completely dismissed, the present results suggest a role of calcium entry in the modulation of the PA-inducing activity of bFGF.     

In vitro angiogenesis on the human amniotic membrane: requirement for basic fibroblast growth factor-induced proteinases

The role of basic fibroblast growth factor-(bFGF) induced proteinases in basement membrane (BM) invasion by bovine capillary endothelial (BCE) cells was studied using a quantitative in vitro assay previously described (Mignatti et al., 1986). 125I-iododeoxyuridine-labeled BCE cells were grown for 72 h on the human amnion BM, and cell invasion was determined by measuring the radioactivity associated with the tissue after removal of the noninvasive cell layer. BCE cells were noninvasive under normal conditions. Addition of human bFGF to either the BM or to the stromal aspect of the amnion induced BCE cell invasion with a dose-dependent response. This effect was maximal in the presence of 70 ng/ml bFGF, and was inhibited by anti-FGF antibody. Transforming growth factor beta, as well as plasmin inhibitors and anti-tissue type plasminogen activator antibody inhibited BCE cell invasion. The tissue inhibitor of metalloproteinases, 1-10 phenanthroline, anti-type IV and anti-interstitial collagenase antibodies had the same effect. On the contrary, anti-stromelysin antibody and Eglin, an inhibitor of elastase, were ineffective. The results obtained show that both the plasminogen activator-plasmin system and specific collagenases are involved in the invasive process occurring during angiogenesis.     

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.