Structural and functional analysis of vascular permeability
Vascular permeability and its regulation are indispensable for normal circulatory function and tissue homeostasis. When unregulated, vascular permeability contributes significantly to blood vessel related pathologies, such as cancer and diabetic retinopathy. Interposed between blood and tissues, endothelial cells become structurally and functionally differentiated, to mediate controlled permeability. The aim of this thesis work was to develop suitable systems to study two different aspects of vascular permeability, endothelial fenestrae formation and tyrosine phophatase-based regulation of paracellular permeability. Transcellular permeability describes the passage of water and macromolecules across endothelial cells. A route for transcellular permeability is provided by fenestrae, the 60 nm circular pores that span the entire thickness of highly attenuated endothelia encountered in endocrine or filtrating organs. In an attempt to gain insight into the structure and biogenesis of fenestrae I helped set up an in vitro culture system where fenestrae could be rapidly induced in cell biological and biochemical quantities. To understand the molecular composition of fenestrae I established a biochemical method for their enrichment. Subtractive proteomic analysis performed on a subcellular fraction enriched in plasma membranes revealed proteins with roles in actin filament disassembly, plasma membrane remodeling, endocytosis, and membrane to cytoskeleton linkage. One particular candidate, the membrane-cytoskeleton linker moesin, was validated as a component of fenestrae by immunocytochemical means. Paracellular permeability occurs through tight or adherens junctions that join endothelial cells. In order to understand the regulation of paracellular permeability through adherens junctions I focused on Vascular Endothelial Protein Tyrosine Phosphatase (VE-PTP), a demonstrated phosphatase modulator of the junctional protein VE-cadherin. To this end I established transgenic mice with inducible expression of wildtype or mutant VE-PTP in endothelial cells. A correlation between VE-PTP expression at the RNA level and embryonic defects suggests the potential utility of the mice as models of adherens junction dysfunction.