Cooperative effect of TNF alpha, bFGF, and VEGF on the formation of tubular structures of human microvascular endothelial cells in a fibrin matrix. Role of urokinase activity

In angiogenesis associated with tissue repair and disease, fibrin and inflammatory mediators are often involved. We have used three-dimensional fibrin matrices to investigate the humoral requirements of human microvascular endothelial cells (hMVEC) to form capillary-like tubular structures. bFGF and VEGF(165) were unable to induce tubular structures by themselves. Simultaneous addition of one or both of these factors with TNF alpha. induced outgrowth of tubules, the effect being the strongest when bFGF, VEGF(165), and TNF alpha were added simultaneously, Exogenously added u-PA, but not its nonproteolytic amino-terminal fragment, could replace TNF alpha, suggesting that TNF alpha-induced u-PA synthesis was involved. Soluble u-PA receptor (u-PAR) or antibodies that inhibited u-PA activity prevented the formation of tubular structures by 59-99%. epsilon-ACA and trasylol which inhibit the formation and activity of plasmin reduced the extent of tube formation by 71-95%. TNF alpha or u-PA did not induce tubular structures without additional growth factors. bFGF and VEGF(165) enhanced of the u-PAR by 72 and 46%, but TNF alpha itself also increased u-PAR in hMVEC by 30%. Induction of mitogenesis was not the major contribution of bFGF and VEGF(165) because the cell number did not change significantly in the presence of TNF alpha, and tyrphostin A47, which inhibited mitosis completely, reduced the formation of tubular structures only by 28-36%. These data show that induction of cell-bound u-PA activity by the cytokine TNF alpha is required in addition to the angiogenic factors VEGF(165) and/or bFGF to induce in vitro formation of capillary-like structures by hMVEC in fibrin matrices. These data may provide insight in the mechanism of angiogenesis as occurs in pathological conditions.