Work done over the last funding period has demonstrated that ceramide production and clustering of membrane rafts (MR) (formerly lipid rafts) drive aggregation of NADPH oxidase (Nox) subunits together with other regulatory molecules in the membrane of endothelial cells (ECs) forming a MR redox signaling platform. This signaling platform produces superoxide to regulate coronary endothelial function. This competitive renewal now seeks to elucidate the molecular mechanisms mediating the formation and function of such MR signaling platforms. The hypothesis being tested is that a rapid fusion of membrane proximal lysosomes to plasma membrane via the SNARE-centered exocytic machinery mediates translocation of ASMase, local ceramide production, and consequent formation of MR redox signaling platforms in coronary arterial endothelial cells. This lysosome-MR redox signalosome contributes to endothelial dysfunction in response to activation of death receptors and other danger signals.
Specific Aim 1 will determine whether the formation of MR redox signaling platforms are mediated by a rapid fusion of membrane proximal lysosomes to plasma membrane via a SNARE- centered exocytic machinery in coronary ECs.
Specific Aim 2 will identify the topographic characteristics of lysosome-MR signalosomes in coronary ECs using scanning and immunogold electron microscopy and determine possible interactions of different molecules.
Specific Aim 3 will determine the functionality of the lysosome MR signalosomes in coronary ECs with a focus on interactions of ASMase activity and Nox activation.
Specific Aim 4 will determine whether the formation of such lysosome MR signalosomes via the SNARE-centered exocytic machinery contributes to endothelial dysfunction in the intact coronary arterial endothelium. These proposed studies will elucidate the molecular mechanisms mediating the trafficking of intracellular organelles and molecules to cell plasma membrane for transmembrane signaling. The results will reveal a novel lysosomal function serving as a signaling organelle in ECs, which may importantly contribute to the formation of MR redox signaling platform and thereby to the regulation of endothelial function under physiological and pathological conditions.

Public Health Relevance

This grant proposal seeks to study a novel signaling mechanism by which a membrane signaling complex is formed, which is referred to as membrane raft redox signaling platform. This signaling platform will regulate the function of heart arteries and blood flow. The major focus of the proposed studies is to determine whether or how a small functional body-lysosome within endothelial cells of the heart arteries moves to cell membrane to help form signaling platforms. The findings will importantly contribute to our understanding of the mechanisms responsible for contraction of the heart arteries and therefore may help develop new therapeutic strategies for coronary arterial disease to prevent heart attack in the future.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL075316-07
Application #
8266398
Study Section
Vascular Cell and Molecular Biology Study Section (VCMB)
Program Officer
Fleg, Jerome
Project Start
2004-03-15
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
7
Fiscal Year
2012
Total Cost
$400,330
Indirect Cost
$118,944
Name
Virginia Commonwealth University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
105300446
City
Richmond
State
VA
Country
United States
Zip Code
23298
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