Vascular endothelial cell (VEC) permeability is largely dependent on the integrity of vascular endothelial cadherin (VEcadherin or VE-Cad)-based intercellular adhesions. Activators of protein kinase A (PKA) or of exchange protein activated by cAMP (EPAC) reduce VEC permeability largely by stabilizing VE-Cad-based intercellular adhesions. Currently, little is known concerning the nature and composition of the signaling complexes that allow PKA or EPAC to regulate VE-Cadbased structures and through these actions control permeability. Using pharmacological, biochemical, and cell biological approaches we identified and determined the composition and functionality of a signaling complex that coordinates cAMPmediated control of VE-Cad-based adhesions and VEC permeability. Thus, we report that PKA, EPAC1, and cyclic nucleotide phosphodiesterase 4D (PDE4D) enzymes integrate into VECad-based signaling complexes in human arterial endothelial cells. Importantly, we show that protein-protein interactions between EPAC1 and PDE4D serve to foster their integration into VE-Cad-based complexes and allow robust local regulation of EPAC1-based stabilization of VE-Cad-based adhesions. Of potential translational importance, we mapped the EPAC1 peptide motif involved in binding PDE4D and show that a cell-permeable variant of this peptide antagonizes EPAC1-PDE4D binding and directly alters VEC permeability. Collectively, our data indicate that PDE4D regulates both the activity and subcellular localization of EPAC1 and identify a novel mechanism for regulated EPAC1 signaling in these cells. Vascular endothelial cadherins (VE-Cad)3 localize at vascular endothelial cell-cell junctions and regulate vascular endothelial cell (VEC) permeability by forming Ca 2ϩ -dependent intercellular adhesions (1-3). VE-Cad-based adhesions are stabilized by recruitment of ␤-catenin, p120-catenin, and ␥-catenin and their subsequent interactions with the actin cytoskeleton (1-3). VE-Cad-based adhesion integrity is destabilized by vascular permeability-inducing factors including the vascular endothelial growth factor (VEGF) (4 -6). Indeed, VEGF reduces VE-Cad-based adhesion integrity and increases permeability by promoting phosphorylation of VE-Cad, or of components of the VE-Cad complex, and by inducing VE-Cad complex disassembly and internalization (5-10).By increasing the integrity of VE-Cad-based adhesions and promoting their interactions with the actin cytoskeleton, activators of cAMP signaling decrease basal VEC permeability and antagonize the effects of VEGF (11-15). These barrier-stabilizing effects of activators of cAMP signaling are coordinated through effects by either protein kinase A (PKA) or exchange protein activated by cAMP (EPAC). The selectivity of cAMPmediated cellular effects depends on selective anchoring of PKA, and perhaps EPAC, at defined intracellular sites (16 -18). In this context, recent evidence has established that the anchoring of cyclic nucleotide phosphodiesterases (PDEs) at these sites is equally critical to subcellular sele...

West Nile Virus (WNV) collected from 179 human blood donors in 25 US states and three Canadian provinces during the 2003 and 2004 epidemic seasons were genetically analyzed. The evolution of WNV during its Western spread was examined by envelope (E) gene sequencing of all 179 cases and full open reading frame sequencing of a subset of 20 WNV to determine if geographic and temporal segregation of distinct viral variants had occurred. Median joining network analysis was used to examine the genetic relationship between E gene variants and identified four large genetic clusters showing the gradual accumulation of mutations during the virus' western expansion. Two related WNV variants and their descendents, undetected in prior years, expanded in frequency. Apparent founder effects were observed in some regional outbreaks possibly due to local WNV colonization by a limited number of viruses. Amino acid mutations associated with newly expanding genetic variants reflect either selectively neutral mutational drift and/or mutations providing replicative advantages over the previously dominant forms of WNV.

Because both endothelin-1 (ET-1) and angiotensin II (AngII) are independent mediators of arterial remodeling, we sought to determine the role of ET receptor inhibition in AngII-accelerated atherosclerosis and aortic aneurysm formation. We administered saline or AngII and/or bosentan, an endothelin receptor antagonist (ERA) for 7, 14, or 28 days to 6-week- and 6-month-old apolipoprotein E-knockout mice. AngII treatment increased aortic atherosclerosis, which was reduced by ERA. ET-1 immunostaining was localized to macrophage-rich regions in aneurysmal vessels. ERA did not prevent AngII-induced aneurysm formation but instead may have increased aneurysm incidence. In AngII-treated animals with aneurysms, ERA had a profound effect on the non-aneurysmal thoracic aorta via increasing wall thickness, collagen/elastin ratio, wall stiffness, and viscous responses. These observations were confirmed in acute in vitro collagen sheet production models in which ERA inhibited AngII's dose-dependent effect on collagen type 1 α 1 (COL1A1) gene transcription. However, chronic treatment reduced matrix metalloproteinase 2 mRNA expression but enhanced COL3A1, tissue inhibitor of metalloproteinase 1 (TIMP-1), and TIMP-2 mRNA expressions. These data confirm a role for the ET system in AngII-accelerated atherosclerosis but suggest that ERA therapy is not protective against the formation of AngII-induced aneurysms and can paradoxically stimulate a chronic arterial matrix remodeling response.

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