Active and tissue inhibitor of matrix metalloproteinase-free gelatinase B accumulates within human microvascular endothelial vesicles

Human gelatinase B is involved in tissue remodeling and angiogenesis, It is thought to be synthesized and rapidly secreted as an inactive precursor. In this report, we have shown that human endothelial cells accumulate active forms of gelatinase B in the cytosol. Microvascular but not macrovascular endothelial cells dramatically increased the expression of cytosolic gelatinase B in response to phorbol myristate acetate, Western blotting showed that tissue inhibitor of metalloproteinase-l (TIMP1) was also present in the cytosol, Whereas gelatinase B was complexed with TIMP1 in the conditioned medium, it existed as a free enzyme in the cytosol, suggesting that the formation of gelatinase B and TIMP1 complex occurs after their secretion, Immunogold electron microscopy revealed that gelatinase B was localized in secretory vesicles which were especially prominent in invading pseudopodia. In contrast, TIMP1 was found throughout the cytoplasm but was not present in the gelatinase vesicles, The accumulation of intracellular activated gelatinase B, ready for rapid release, may facilitate the migration of microvascular endothelial cells during angiogenesis.