Abstract
The fibrinous exudate of a wound or tumor stroma facilitates angiogenesis. We studied the involvement of RGD-binding integrins during tube formation in human plasma-derived fibrin clots and human purified fibrin matrices. Capillary-like tube formation by human microvascular endothelial cells in a 3D plasma-derived fibrinous matrix was induced by FGF-2 and TNF-alpha and depended largely on cell-bound u-PA and plasmin activities. While tube formation was minimally affected by the addition of either the alphavbeta3-integrin inhibiting mAb LM609 or the alpha5-integrin inhibiting mAb IIA1, the general RGD-antagonist echistatin completely inhibited this process. Remarkably, when alphavbeta3- and alpha5beta1-integrins were inhibited simultaneously, tube formation was reduced by 78%. It was accompanied by a 44% reduction of u-PA antigen accumulation and 41% less production of fibrin degradation products. alphavbeta5-integrin-blocking antibodies further enhanced the inhibition by mAb LM609 and mAb IIA1 to 94%, but had no effect by themselves. alphav-specific cRGD only inhibited angiogenesis when alpha5beta1-integrin was simultaneously blocked. Endostatin mimicked the effect of alpha5beta1-integrin and inhibited tube formation only in the presence of LM609 or cRGD (73 and 80%, respectively). Comparable results were obtained when purified fibrin matrices were used instead of the plasma-derived fibrinous matrices. These data show that blocking of tube formation in a fibrinous exudate requires the simultaneous inhibition of alphavbeta3- and alpha5beta1-integrins. This may bear impact on attempts to influence angiogenesis in a fibrinous environment.