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

Abstract

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 VEGF165 were unable to induce tubular structures by themselves. Simultaneous addition of one or both of these factors with TNFα induced outgrowth of tubules, the effect being the strongest when bFGF, VEGF165, and TNFα were added simultaneously. Exogenously added u-PA, but not its nonproteolytic amino-terminal fragment, could replace TNFα, suggesting that TNFα-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%. ε-ACA and trasylol which inhibit the formation and activity of plasmin reduced file extent of tube formation by 71-95%. TNFα or u-PA did not induce tubular structures without additional growth factors. bFGF and VEGF165 enhanced of the u-PAR by 72 and 46%, but TNFα itself also increased u-PAR in hMVEC by 30%. Induction of mitogenesis was not the major contribution of bFGF and VEGF165 because the cell number did not change significantly in the presence of TNFα, 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 TNFα is required in addition to the angiogenic factors VEGF165 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.