Proteinase inhibitors are of potential therapeutic importance as regulators of tumor cell mediated enzymatic degradation of the extracellular matrix. The present study focuses on the search for new endothelial proteinase inhibitors that may be part of the vascular defense mechanism against tumor invasion. Serine proteinase inhibitory activity was detected in serum-free culture supernatants of human umbilical vein endothelial cells (HUVEC) following phorbol 12-myristate 13-acetate (PMA) treatment. Three trypsin, chymotrypsin, elastase and plasmin resistant bands of 39 kDa, 35 kDa and 27 kDa were identified by reverse zymography. The PMA-mediated stimulation of the serine proteinase inhibitory activity was transcriptional and translational dependent, and was blocked by the protein kinase C (PKC) inhibitors, staurosporine, H7 and K252. Further evidence for PKC involvement was found through the use of a non-PKC activating phorbol ester (4-alpha-phorbol,12-13,-didecanoate), which was ineffective in stimulating the serine proteinase inhibitory activity. The three inhibitory bands were partially purified by trypsin affinity chromatography. The data obtained so far suggest that these may represent novel serine proteinase inhibitors.
