Adenovirus-mediated expression of tissue factor pathway inhibitor-2 inhibits endothelial cell migration and angiogenesis.

OBJECTIVE Extracellular matrix (ECM) remodeling during angiogenesis is accomplished through plasmin-dependent pericellular proteolysis and through the action of matrix metalloproteinases (MMPs). Because tissue factor pathway inhibitor-2 (TFPI-2), a Kunitz-type protease inhibitor with prominent ECM localization, inhibits plasmin and MMPs activity, we investigated the role of TFPI-2 in endothelial cell (EC) migration and angiogenesis. METHODS AND RESULTS Real-time polymerase chain reaction and immunostaining showed that the expression of TFPI-2 mRNA and protein was upregulated in migrating ECs. The effect of TFPI-2 on angiogenesis was studied in mouse models of Matrigel and polyvinylalcohol sponge implants by overexpressing TFPI-2 through infection with a replication-deficient adenovirus (AdTFPI-2). Using (immuno)fluorescence and confocal microscopy we observed that TFPI-2 reduced neovascularization and promoted ECM deposition. Lateral cell migration and capillary tube formation in vitro also were impaired by TFPI-2, a process reversed by anti-TFPI-2 antibodies. Increased apoptosis occurred both in AdTFPI-2-treated ECs and in the mouse implants. Zymography and assays in the absence of plasminogen confirmed plasmin inhibition as a main mechanism through which TFPI-2 inhibits EC migration. CONCLUSIONS Our data suggest that TFPI-2 may be an important regulator of aberrant angiogenesis associated with tumor growth/metastasis, cardiovascular diseases, chronic inflammation, or diabetes.