A role of fibrinolytic activity in angiogenesis. Quantitative assay using in vitro method

The functional role of the fibrinolytic system in capillary growth was investigated using bovine capillary endothelial cells (BCEs) cultured on a Type I collagen gel matrix, into which the cells migrated to form capillary-like tubular structures. The length of the tubes formed were measured morphometrically using an image analyzer in the absence and presence of fibrinolytic proteases, namely plasminogen, plasminogen activators (PAs) and PA inhibitor (PAI). The addition of plasminogen (25 micrograms/ml) to the gel matrix significantly increased the length of BCE tubes found on the 9th day of culture (p less than 0.01), with a dose-dependent tendency. The simultaneous addition of a basic fibroblast growth factor (bFGF, 10 ng/ml) enhanced this tube formation as early as the 3rd day of culture (p less than 0.01). Cultured BCEs secreted both tissue-type and urokinase-type PAs (tPA and uPA) and PAI-1 into the culture medium, and the secretion of both PAs was enhanced by the addition of bFGF. However, the secreted tPA was composed mostly of an inactive form of tPA.PAI-1 complex, and the PA activity was derived mostly from uPA. Inhibitors of plasmin suppressed the enhancing effect of plasminogen on angiogenesis. In addition, anti-uPA IgG markedly inhibited the enhancing effect of plasminogen on the 4th and 7th days of culture (p less than 0.01), whereas anti-tPA IgG showed an inhibitory tendency only on the 4th day of culture (p less than 0.05). These findings indicate that the plasminogen-PAs system, especially uPA synthesized and secreted by BCEs, plays an important role in regulating angiogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)