HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Membrane-type matrix metalloproteinase-mediated angiogenesis in a fibrin-collagen matrix

Adult angiogenesis, associated with pathologic conditions, is often accompanied by the formation of a fibrinous exudate. This temporary matrix consists mainly of fibrin but is intermingled with plasma proteins and collagen fibers. The formation of capillary structures in a fibrinous matrix in vivo was mimicked by an in vitro model, in which human microvascular endothelial cells (hMVECs) seeded on top of a fibrin-10% collagen matrix form capillarylike tubular structures after stimulation with basic fibroblast growth factor/tumor necrosis factor (bFGF/TNF) or vascular endothelial growth factor (VEGF)/TNF. In the fibrincollagen matrix the metalloproteinase inhibitor BB94 inhibited tubule formation by 70% to 80%. Simultaneous inhibition of plasmin and metalloproteinases by aprotinin and BB94 caused a nearly complete inhibition of tubule formation. Adenoviral transduction of tissue inhibitor of metalloproteinases 1 (TIMP-1) and TIMP-3 into endothelial cells revealed that TIMP-3 markedly inhibited angiogenesis, whereas TIMP-1 had only a minor effect. Immunohistochemical analysis showed the presence of matrix metalloproteinase 1 (MMP-1), MMP-2, and membrane-type 1 (MT1)–MMP, whereas MMP-9 was absent. The endothelial production of these MMPs was confirmed by antigen assays and real-time polymerase chain reaction (PCR). MT1-MMP mRNA was markedly increased in endothelial cells under conditions that induced tubular structures. The presence of MMP-1, MMP-2, and MT1MMP was also demonstrated in vivo in the newly formed vessels of a recanalized arterial mural thrombus. These data suggest that MMPs, in particular MT-MMPs, play a pivotal role in the formation of capillarylike tubular structures in a collagen-containing fibrin matrix in vitro and may be involved in angiogenesis in a fibrinous exudate in vivo. (Blood. 2003; 101:1810-1817)