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"Regulation of endothelial hyperpermeability deactivation by Epac-ERM protein interactions"

by
Patricio E. Mujica
Pharmacology & Physiology Program
B.Sc., 2009, Pontificia Universidad Católica de Chile
Santiago, Chile


Thesis Advisor: Walter N. Durán, Ph.D.
Professor
Department of Pharmacology & Physiology

Monday, May 5, 2014
11:00 A.M. , MSB H-609B


Abstract

Endothelial hyperpermeability is a hallmark of inflammation. Endothelial barrier function is restored after a period of hyperpermeability, but the mechanisms that deactivate hyperpermeability are largely unknown. Exchange protein activated by cAMP (Epac) is emerging as a possible deactivating/restorative factor in the endothelium. Epac localization, which can be modulated by Ezrin/Radixin/Moesin (ERM) proteins, may determine its function due to cellular compartmentalization of cAMP signaling. We hypothesize that ERM proteins facilitate deactivation of hyperpermeability by localizing Epac to the plasma membrane. Depletion of Radixin and Moesin by siRNA increased baseline permeability to macromolecules in human microvascular endothelial cell (HMVEC) monolayers, as well as PAF (platelet-activating factor)-induced permeability, whereas Ezrin knockdown did not affect permeability. We demonstrated association between Epac and ERM proteins in HMVEC by co-immunoprecipitation. Fluorescence microscopy showed that PAF induced co-localization of Epac with vascular endothelial (VE)-cadherin at the plasma membrane. Using in situ proximity ligation assay (PLA) we demonstrate that Epac and Radixin are associated in close proximity in endothelial cells and that this interaction is enhanced by PAF and VEGF. Inhibition of endothelial nitric oxide synthase decreased basal ERM phosphorylation, while inhibition of PKC abrogated PAF-induced ERM phosphorylation. We propose that deactivation of PAF-stimulated hyperpermeability is implemented in a time-orchestrated manner by PAF-induced activation of ERM proteins, which locates Epac to the plasma membrane promoting its interaction with endothelial junctional proteins.


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