Plasminogen Activator and Collagenase by Cultured Capillary Endothelial Cells Production

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10 -~ to 10 -8 M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyI-TPA and 4c~-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolytic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel endothelial cells. Capillary proliferation in vivo is marked by fragmentation of the capillary basal lamina and subsequent migration and proliferation of distinct endothelial cell populations in response to stimuli (2). New blood vessel formation, or angiogenesis, can be studied in a number of systems (9, 10), and is promoted by angiogenic factors derived from both normal tissues (4, 11), and tumors (2, 9). Since the formation of capillaries is marked by both the destruction of the basal lamina and the invasion of cells through interstitial tissue, capillary formation may require the elaboration of proteases to degrade the proteins of the basal lamina and interstitial stroma. Therefore, we have proposed (20) that angiogenesis requires the secretion of proteases and have initiated experiments to characterize the pro974 teases produced by endothelial cells in response to various stimuli. Previous work has shown that endothelial ceils derived from large vessels of several tissues and species synthesize at least two extracellular proteases: the serine protease plasminogen activator (PA) and vertebrate interstitial collagenase (19). Endothelial cells isolated from bovine aorta (13, 14), rabbit vena cava (17), and human umbilical vein (14) secrete variable amounts of PA, an enzyme which converts plasminogen to plasmin, a neutral protease with broad substrate specificity. The amount of PA activity is dependent upon the species and source of endothelium (14), the level of cellular inhibitors (17), and on the exact culture conditions (15). However, significantly THE IOURNAL Of CELL BIOLOGY • VOLUME 95 DECEMBER 1982 974-981 © The Rockefeller University Press • 0021-9525/82/12/0C~0/00 $t,00 on D ecem er 5, 2017 jcb.rress.org D ow nladed fom higher levels of PA production can be induced in many endothelial cells by the inclusion of the tumor promoter 12-0tetradecanoyl phorbol-13-acetate (TPA) in the growth media (14). In contrast, the basal level of collagenase production by endothelial cells is quite low (19). However, in the presence of TPA, human umbilical vein endothelial cells produce significant amounts of a latent collagenase, which, upon activation, is capable of degrading types I, II, and III collagens (19). Bovine vein endothelial ceils also have been reported to secrete low levels of a type IV collagenase (16). However, no single endothelial cell type has been shown to synthesize significantly increased amounts of both collagenase and PA in response to TPA stimulation. The endothelial cells involved in neovascularization and therefore of greatest interest in terms of their protease production are the capillary endothelial ceils. Thus, we examined the synthesis of PA and collagenase by cultured bovine capillary endothelial (BCE) ceils. To determine if collagenase and PA synthesis could be stimulated in BCE cells, we also investigated the modulation of these two enzymes in response to TPA, a known inducer of PA in endothelial cells (17). Since the responses of endothelial cells derived from large vessels and capillaries may be quite different, the levels of PA and collagenase activities produced by both BCE and bovine aortic endothelial (BAE) cells were compared. MATERIALS AND METHODS