In the past three and a half years of this MERIT award we have made significant progress in dissecting the mechanisms of caveolin-1 (Cav-1) regulation of cardiovascular functions using state of the art technological approaches. We have shown that endothelial caveolin-1 controls the extent of atherosclerosis since the loss of Cav-1 reduces atherosclerosis, while over-expression of Cav-1 in the endothelium enhances atherogenesis. The differences in atherosclerosis cannot be explained based on changes in lipoprotein metabolism since the loss of Cav-1 paradoxically increases plasma triglyceride and cholesterol levels, but reduces atherogenesis. Mechanistically, the loss of Cav-1 in endothelium enhances NO release and reduces endothelial expression of cytokine inducible adhesion molecules. Interestingly, the loss of Cav-1 reduces the uptake of Di-labeled LDL into the vessel wall suggesting that Cav-1 regulates the entry/exit of LDL into the vessel wall. We have also documented the loss of Cav-1 reduces the size ofthe fenestration in the sinusoidal endothelium of the liver which may explain the enhanced levels of plasma VLDL, triglycerides/cholesterol found in these mice. In the next project, we sought to understand the nature of the protein partners of Cav-1 in vivo. To this end, we have identified a series of proteins that interact with Cav-1 in the lung. Quantitative proteomics on proteins enriched/de-enriched in lipid raft domains were isolated from lung lysates prepared from WT and Cav- 1 KO mice. Interestingly, the genetic loss of Cav-1 only changed the levels of 10 proteins, including Cav-2, cavins-1, -2 and -4. Cavins are recently identified regulators of caveolae assembly that form a stable complex with Cav-1 in a variety of cells. siRNA mediated knockdown of Cavin-1, reduces the levels of Cav-1 and Cav-2, enhances NO release and reduces endothelial cell growth and migration. Presently we are characterizing the cardiovascular phenotypes of Cavin-1 knockout mice. Finally, we have developed a potential allosteric activator of eNOS based on Cav-1 It is well appreciated that caveolin-1 (Cav-1) is the major structural protein that is essential to the formation of the caveolae in endothelial cells. The aberrant regulation of endothelial nitric oxide synthase (eNOS) and associated NO, release is directly associated with a range of vascular diseases. Caveolin-1 (Cav-1), the main coat protein of caveolae, is highly expressed in endothelial cells'(EC), and its scaffolding domain serves as an endogenous negative regulator of eNOS function. Structure-function analysis of Cav-1 has shown that phenylalanine 92 (F92) is critical forthe inhibitory actions of Cav-I towards eNOS. In a paper published in 2011, we showed that F92A Cav-1 and a mutant cell permeable scaffolding domain peptide, called Cavnoxin, increased basal NO release in eNOS-expressing cells. Moreover, Cavnoxin reduced vascular tone ex vivo and lowered blood pressure in normal mice. In contrast, similar experiments performed using eNOS- or Cav-1- deficient mice showed that Cavnoxin's vasodilatory effect was abolished in the absence of these gene products, indicating a high level of eNOS/Cav-1 specificity. Mechanistically, biochemical assays indicated that non-inhibitory F92A Cav-1 and Cavnoxin specifically disrupted the inhibitory actions of endogenous Cav-1 towards eNOS, enhancing basal NO release. Collectively, these data raise the possibility of studying the inhibitory influence of Cav-1 on eNOS without interfering with endogenous Cav-1's other actions and suggest a therapeutic application for regulated eNOS/Cav-1 interaction in disease characterized by decreased NO release. These studies as well as several other published papers derived from the data and concepts of this MERIT award have made a significant impact in the field and are worth pursuing in the extended grant application.

Public Health Relevance

This research is relevant to public health since endothelial dysfunction is a common manifestafion of most cardiovascular diseases. Our research has discovered the major mechanisms of how the endothelium control blood flow and inflammation. Research supported by this grant may help idenfify new drugs that reduce heart disease and improve the quality of life of people suffering with cardiovascular disease.

Agency
National Institute of Health (NIH)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
4R37HL061371-16
Application #
8517273
Study Section
No Study Section (in-house review) (NSS)
Program Officer
Charette, Marc F
Project Start
Project End
Budget Start
Budget End
Support Year
16
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Yale University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
City
New Haven
State
CT
Country
United States
Zip Code
06510
Park, Eon Joo; Grabi?ska, Kariona A; Guan, Ziqiang et al. (2014) Mutation of Nogo-B receptor, a subunit of cis-prenyltransferase, causes a congenital disorder of glycosylation. Cell Metab 20:448-57
Goodwin, Julie E; Feng, Yan; Velazquez, Heino et al. (2014) Loss of the endothelial glucocorticoid receptor prevents the therapeutic protection afforded by dexamethasone after LPS. PLoS One 9:e108126
Goodwin, Julie E; Feng, Yan; Velazquez, Heino et al. (2013) Endothelial glucocorticoid receptor is required for protection against sepsis. Proc Natl Acad Sci U S A 110:306-11
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Zhang, Pei; Huang, Angela; Ferruzzi, Jacopo et al. (2012) Inhibition of microRNA-29 enhances elastin levels in cells haploinsufficient for elastin and in bioengineered vessels--brief report. Arterioscler Thromb Vasc Biol 32:756-9
Briand, Nolwenn; Le Lay, Soazig; Sessa, William C et al. (2011) Distinct roles of endothelial and adipocyte caveolin-1 in macrophage infiltration and adipose tissue metabolic activity. Diabetes 60:448-53
Bernatchez, Pascal; Sharma, Arpeeta; Bauer, Philip M et al. (2011) A noninhibitory mutant of the caveolin-1 scaffolding domain enhances eNOS-derived NO synthesis and vasodilation in mice. J Clin Invest 121:3747-55
Davalos, Alberto; Fernandez-Hernando, Carlos; Sowa, Grzegorz et al. (2010) Quantitative proteomics of caveolin-1-regulated proteins: characterization of polymerase i and transcript release factor/CAVIN-1 IN endothelial cells. Mol Cell Proteomics 9:2109-24
Albinsson, Sebastian; Suarez, Yajaira; Skoura, Athanasia et al. (2010) MicroRNAs are necessary for vascular smooth muscle growth, differentiation, and function. Arterioscler Thromb Vasc Biol 30:1118-26

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