Plasminogen activator and collagenase production by cultured capillary endothelial cells

Abstract

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 pro-974 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 collage...

Purification of a factor from human placenta that stimulates capillary endothelial cell protease production, DNA synthesis, and migration.

“…All collagenase was in a latent form and was activated with trypsin to detect activity. The cell layers from these same cultures were washed twice with cold phosphate-buffered saline, pH 7.5, and were extracted with 0.5% Triton X-100 in 0.1 M sodium phosphate, pH 8.1, and the cell extracts were assayed for PA activity as described (15). Previous experiments have demonstrated that the amount of PA in cell extracts is proportional to the amount found in conditioned medium (15).…”

“…Bovine capillary endothelial (BCE) cells were isolated from the adrenal cortex of recently slaughtered yearling cattle by the method of Folkman et al (14). Cells were grown to confluence in a minimal essential medium (aMEM) containing 10% (vol/vol) calf serum and supplemented with medium conditioned by mouse sarcoma 180 cells as described (15). When cultures reached confluence, the medium was changed to aMEM containing 5% (vol/vol) calf serum and no conditioning factors.…”

“…The cell layers from these same cultures were washed twice with cold phosphate-buffered saline, pH 7.5, and were extracted with 0.5% Triton X-100 in 0.1 M sodium phosphate, pH 8.1, and the cell extracts were assayed for PA activity as described (15). Previous experiments have demonstrated that the amount of PA in cell extracts is proportional to the amount found in conditioned medium (15). One unit of protease-inducing activity was defined as the amount necessary to give half the maximal stimulation of PA and collagenase production.…”

“…The most obvious was the striking effect of the factor on the morphology of cultured BCE cells. Incubation of BCE cells for 24 hr with concentrations of the protease-inducing factor in the range that induced PA and collagenase altered the morphology of the cells from their typical cobblestone appearance to a more elongated, spindle-shaped appearance, as described for another protease-inducing molecule (15).…”

Correlation between basic fibroblast growth factor immunostaining of stromal cells and stromelysin-3 mRNA expression in human breast carcinoma

“…A number of studies have suggested that the ability of specific factors to induce angiogenic responses correlates with their stimulation of matrix protease synthesis in endothelial cells (Gross et al, 1982;Montesano and Orci, 1985). For example, antibodies to interstitial collagenase inhibit endothelial cell invasion (Montesano and Orci, 1985), and metalloprotease inhibitors, such as TIMP, inhibit the angiogenic process (Johnson et al, 1994).…”