Inflammatory processes are characterized by an increase in microvascular permeability (hypermeability) to macromolecules. Signaling interactions between vascular wall and blood cells provide a unique way of communicating, coordinating, and integrating an appropriate physiologic response to the changing tissue environment in vivo. There is a paucity of information on permeability-related signaling mechanisms in the complex cellular environment of the in vivo microcirculation. Knowledge of these important interactions in vivo is fundamental to understand the integrated regulation of microvascular transport and its functional alterations in vascular disease. In particular, the role of nitric oxide (NO) in the control of microvascular permeability remains controversial. We propose to test in vivo three major hypotheses: 1) an eNOS-assoctated signaling cascade regulates microvascular transport. 2) molecular movement (translocation, trafficking) of eNOS from membrane to other cellular compartments is a functionally and differentially important step in the endothelium-mediated regulatory mechanisms. 3) eNOS signaling mechanisms regulate microvascular permeability responses to ischemia-reperfusion (I-R). To test these hypotheses, we will A) determine whether or not a cause-effect relationship exists between eNOS activity and hyperpermeability in vivo in eNOS -/- mice and their wild-type eNOS +/+ control; B) test translocation of eNOS from membrane to cytosol after stimulation with a pure vasodilator (ACh) and a non-vasodilator that causes hyperpermeability (PAF); C) determine whether or not differences in microvascular permeability responses exist in eNOS +/+ and eNOS -/- mice and the subcellular location of eNOS under baseline and after I-R. In addition, we will determine whether or not changes in eNOS phosphorylation occur in response ACh, PAF and to I-R, and assess the significance of PKB and MAP kinases in these processes. Methods of immunoprecipitation, western blotting, intravital and confocal microscopy, as well as NO measurements and computer-assisted digital image analysis will be applied to evaluate the regulation of microvascular permeability in vivo.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL070634-01
Application #
6506567
Study Section
Cardiovascular and Renal Study Section (CVB)
Program Officer
Goldman, Stephen
Project Start
2002-07-01
Project End
2007-06-30
Budget Start
2002-07-01
Budget End
2003-06-30
Support Year
1
Fiscal Year
2002
Total Cost
$380,508
Indirect Cost
Name
University of Medicine & Dentistry of NJ
Department
Pharmacology
Type
Schools of Medicine
DUNS #
605799469
City
Newark
State
NJ
Country
United States
Zip Code
07107
Durán, Walter N; Sánchez, Fabiola A (2016) New Member of Endothelial Arsenal Against Inflammation. Circ Res 119:178-80
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Sanchez, Fabiola A; Rana, Roshniben; Gonzalez, Francisco G et al. (2011) Functional significance of cytosolic endothelial nitric-oxide synthase (eNOS): regulation of hyperpermeability. J Biol Chem 286:30409-14
Duran, Walter N; Breslin, Jerome W; Sanchez, Fabiola A (2010) The NO cascade, eNOS location, and microvascular permeability. Cardiovasc Res 87:254-61
Kim, David D; Kleinman, David M; Kanetaka, Takehito et al. (2010) Rapamycin inhibits VEGF-induced microvascular hyperpermeability in vivo. Microcirculation 17:128-36
Kim, David D; Kanetaka, Takehito; Durán, Ricardo G et al. (2009) Independent regulation of periarteriolar and perivenular nitric oxide mechanisms in the in vivo hamster cheek pouch microvasculature. Microcirculation 16:323-30
Sanchez, Fabiola A; Rana, Roshniben; Kim, David D et al. (2009) Internalization of eNOS and NO delivery to subcellular targets determine agonist-induced hyperpermeability. Proc Natl Acad Sci U S A 106:6849-53

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