Abstract
Testisin (encoded by PRSS21) is a membrane anchored serine protease, which is tethered to the cell surface via a glycosylphosphatidylinositol (GPI)-anchor. While testisin is found in abundance in spermatozoa, it is also expressed in microvascular endothelial cells where its function is unknown. Here we identify 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 basement membranes. 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 and 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.
Highlights
The endothelium plays a critical role in regulating vascular wall functions, such as modulating vascular tone, controlling the exchange of fluids and cells, regulating local cellular growth and extracellular matrix deposition, and controlling homeostatic as well as inflammatory responses [1]
The cell-cell interactions between endothelial cells are mediated by the adherens junctions (AJ) protein, vascular endothelial (VE)-cadherin, which is responsible for endothelial AJ assembly and barrier architecture [8], and dictates the levels of expression and localization of other junctional molecules including claudin-5 and N-cadherin [9, 10]
While testisin shows limited expression or is not expressed in most other tissues [13, 14, 20,21,22,23], we showed that testisin mRNA is expressed by human microvascular endothelial cells (HMVECs) undergoing reorganization and tube-like formation in Matrigel angiogenesis assays in vitro and during pre-capillary morphogenesis on 3-D fibrillar type I collagen [12]
Summary
The endothelium plays a critical role in regulating vascular wall functions, such as modulating vascular tone, controlling the exchange of fluids and cells, regulating local cellular growth and extracellular matrix deposition, and controlling homeostatic as well as inflammatory responses [1]. The endothelium is the site of angiogenesis, the multistep process of vascular remodeling involving coordinated migration, proliferation, and junction formation of vascular endothelial cells to form new vessel branches in response to growth stimuli [2]. Intercellular junctions between endothelial cells mediate barrier integrity and control barrier permeability [5]. The cell-cell interactions between endothelial cells are mediated by the AJ protein, vascular endothelial (VE)-cadherin, which is responsible for endothelial AJ assembly and barrier architecture [8], and dictates the levels of expression and localization of other junctional molecules including claudin-5 and N-cadherin [9, 10]. Several lines of evidence demonstrate that VE-cadherin plays a pivotal role in angiogenesis and controls vascular permeability [11]
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