The Role of Testisin in Endothelial Cells and Angiogenesis

Abstract

Testisin (PRSS21) is a membrane anchored serine protease, which is tethered to the cell surface via a glycosylphosphatidylinositol (GPI)-anchor. Testisin expression has been documented in eosinophiles, ovarian cancers, endothelial cells, and spermatozoa where its expression is highest. Although two substrates of testisin have so far been identified, protease activated receptor 2 (PAR2) and protein inhibitor C (PCI), little is known of the biological, physiological, and pathophysiologic characteristics of testisin. Thus far, there are no published data identifying an activator or inhibitor of testisin. To better characterize the biochemistry of testisin we produced the zymogen as inclusion bodies in E. coli and refolded using the insoluble cellular fraction. To better characterize the cellular functions of testisin, hybridomas producing anti-testisin monoclonal antibodies were acquired, antibodies purified, and then characterized. In an investigation of testisin’s function in endothelial cells we identified testisin as a novel regulator of physiological hormone-induced angiogenesis and microvascular endothelial permeability. Using a murine model of rapid physiological angiogenesis during corpus luteal development in the ovary, we found that mice genetically deficient in testisin (Prss21-/-) show a substantially increased incidence of hemorrhages which are significantly more severe than in littermate control Prss21+/+ mice. This phenotype was associated with increased vascular leakiness, demonstrated by a greater accumulation of extravasated Evans blue dye in Prss21-/- ovaries. Live cell imaging of in vitro cultured microvascular endothelial cells depleted of testisin by siRNA knockdown revealed that loss of testisin markedly impaired reorganization and tubule-like formation on Matrigel. Moreover, testisin siRNA knockdown increased the paracellular permeability to FITC-albumin across endothelial cell monolayers, which was associated with decreased expression of the adherens junction protein VE-cadherin and increased levels of phospho-(Tyr658)-VE-cadherin, without affecting the levels of the tight junction proteins occludin, claudin-5, or ZO-1. Decreased expression of VE-cadherin in the neovasculature of Prss21-/- ovaries was also observed without marked differences in endothelial cell content, vascular claudin-5 expression or pericyte recruitment. Together, these data identify testisin as a novel regulator of VE-cadherin adhesions during angiogenesis and indicate a potential new target for regulating neovascular integrity and associated pathologies.