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 angiostatic 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.

Basic fibroblast growth factor (bFGF) is a potent mitogen for human bone marrow stromal cells. Normally, large numbers of human bone marrow stromal cells are difficult to obtain. However, nanogram/ml concentrations of bFGF stimulate the growth of passaged bone marrow stromal cells both in media formulated for optimal growth of stromal cells and in a simple mixture of RPMI-1640 and 10% fetal calf serum facilitating the successive expansion of stromal cells through multiple passages. bFGF also greatly accelerates the formation of a primary stromal cell layer following inoculation of newly harvested bone marrow cells into dishes. In the presence of bFGF, the stromal cells attain high densities, lose their contact inhibition and grow in multilayered sheets. Heparin greatly potentiates the stimulatory effect of low concentrations of bFGF. The effects of bFGF are fully reversible: cells cultured in the presence of this factor for multiple passages revert to normal growth rates following trypsinization and subculture. A short (4 h) exposure of the cells to bFGF elicits profound growth stimulation. This supports the hypothesis that this factor binds to glycosaminoglycans in the cell matrix which act as a storage reservoir for this cytokine.