Phosphorylation of eNOS on serine 1179 by Akt is important for eNOS activation in response to agonists such as vascular endothelial growth factor (VEGF), estrogen or flow. In this competitive renewal, we provide data showing that the genetic loss of eNOS severely impairs ischemia-induced arteriogenesis in mice, a phenotype rescued by adenoviral transduction of the ischemic limb with S1179D eNOS, a constitutively active form of eNOS, using a new, more efficient approach for in vivo gene transfer. More importantly, we have observed an identical phenotype of critical limb ischemia in mice deficient in the gene for Akt1, but not for Akt2, suggesting that the Akt-eNOS axis may be critical for arteriogenesis following tissue ischemia. In addition, utilizing caveolin-1 (-/-) mice and new strategies aimed at the hsp90/Akt interaction (dominant negative hsp90 and cell permeable peptides that antagonize the docking of Akt/eNOS to hsp90), we will dissect the importance of caveolin-1 and hsp90 in regulating Akt-dependent eNOS phosphorylation. We hypothesize that a major physiological role of Akt in the cardiovascular system is to modulate eNOS, thus preserve blood flow and promote arterial remodeling and arteriogenesis. As a corollary to this hypothesis, we predict that the scaffolding function of hsp90 is crucial to coupling signal transduction via Akt to eNOS activation. We will: 1. determine the importance of eNOS vs. Akt in a model of ischemia-induced arteriogenesis; 2. dissect the importance of Akt1/Akt 2 in mediating angiogenic phenotypes in vitro; and 3. examine the molecular regulation of the eNOS phosphorylation (via the hsp90/Akt signaling module) in blood vessels, endothelial cells isolated from mice deficient in the negative regulator of eNOS, caveolin-1. We will also manipulate the levels and activity of hsp90 in endothelial cells using new methodologies (dominant negative mutations and cell permeable peptides) to assess the importance of hsp90 in regulating the functions of Akt and eNOS. Collectively, this work will facilitate our understanding of the importance of Akt/eNOS axis in arteriogenesis and vascular remodeling and provide insights into the molecular machinery required for eNOS regulation by protein phosphorylation using a multi-disciplinary approach.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL064793-07
Application #
7010681
Study Section
Pathology A Study Section (PTHA)
Program Officer
Goldman, Stephen
Project Start
2000-03-07
Project End
2008-02-29
Budget Start
2006-03-01
Budget End
2007-02-28
Support Year
7
Fiscal Year
2006
Total Cost
$359,231
Indirect Cost
Name
Yale University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Luciano, Amelia K; Zhou, Wenping; Santana, Jeans M et al. (2018) CLOCK phosphorylation by AKT regulates its nuclear accumulation and circadian gene expression in peripheral tissues. J Biol Chem 293:9126-9136
Kuo, Andrew; Lee, Monica Y; Yang, Kui et al. (2018) Caveolin-1 regulates lipid droplet metabolism in endothelial cells via autocrine prostacyclin-stimulated, cAMP-mediated lipolysis. J Biol Chem 293:973-983
Luciano, Amelia K; Santana, Jeans M; Velazquez, Heino et al. (2017) Akt1 Controls the Timing and Amplitude of Vascular Circadian Gene Expression. J Biol Rhythms 32:212-221
Grabi?ska, Kariona A; Edani, Ban H; Park, Eon Joo et al. (2017) A conserved C-terminal RXG motif in the NgBR subunit of cis-prenyltransferase is critical for prenyltransferase activity. J Biol Chem 292:17351-17361
Hoffmann, Reuben; Grabi?ska, Kariona; Guan, Ziqiang et al. (2017) Long-Chain Polyprenols Promote Spore Wall Formation in Saccharomyces cerevisiae. Genetics 207:1371-1386
Kuo, Andrew; Lee, Monica Y; Sessa, William C (2017) Lipid Droplet Biogenesis and Function in the Endothelium. Circ Res 120:1289-1297
Kraehling, Jan R; Sessa, William C (2017) Contemporary Approaches to Modulating the Nitric Oxide-cGMP Pathway in Cardiovascular Disease. Circ Res 120:1174-1182
Grabi?ska, Kariona A; Park, Eon Joo; Sessa, William C (2016) cis-Prenyltransferase: New Insights into Protein Glycosylation, Rubber Synthesis, and Human Diseases. J Biol Chem 291:18582-90
Chamorro-Jorganes, Aránzazu; Lee, Monica Y; Araldi, Elisa et al. (2016) VEGF-Induced Expression of miR-17-92 Cluster in Endothelial Cells Is Mediated by ERK/ELK1 Activation and Regulates Angiogenesis. Circ Res 118:38-47
Kraehling, Jan R; Chidlow, John H; Rajagopal, Chitra et al. (2016) Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells. Nat Commun 7:13516

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