Role and localization of urokinase receptor in the formation of new microvascular structures in fibrin matrices

Fibrin or a fibrinous exudate can facilitate angiogenesis in many pathological conditions. In vitro, the outgrowth of capillary-like structures in fibrin can be mimicked by exposing human microvascular endothelial cells (hMVECs) to an angiogenic growth factor and tumor necrosis factor (TNF)-alpha. Urokinase-type plasminogen activator (u-PA) and plasmin activities are required for this angiogenic process. This study focuses on the role and localization of the u-PA receptor (u-PAR) in newly formed microvascular structures. The u-PAR-blocking monoclonal antibody (MAb) H-2 completely inhibited the formation of capillary-like tubular structures induced by exposure of hMVECs to basic fibroblast growth factor and TNF-alpha. This was accompanied by a several-fold increase in u-PA accumulation in the conditioned medium. The effect of MAb H-2 was not caused by blocking cellular activation by u-PA/u-PAR interaction, as the aminoterminal fragment (ATF) of u-PA, which also activates u-PAR, prevented tube formation. In addition, the inhibition by MAb H-2 was not due to an effect of the antibody on u-PAR-vitronectin binding. These data show that inhibition of tube formation can be caused not only by inhibition of u-PA or plasmin activities but also by unavailability of the u-PAR for cell-bound proteolysis. Immunohistochemical analysis showed that in in vitro angiogenesis u-PAR and u-PA were localized on the invading, tube-forming hMVECs and not on the endothelial cells that are located on top of the fibrin matrix. u-PAR and u-PA were also prominently expressed on endothelial cells of neovessels present in an atherosclerotic plaque. These data may give more insight into the role of u-PAR in repair-associated angiogenesis.