Clinical data suggest an association between elevated levels of Hcy, also known as hyperhomocysteine-mia (HHcy) and stroke. HHcy is generated due to increase in de-methylation of methionine by S-adenosine- homocysteine hydrolase (SAHH) and a decrease in methyltetrahydrofolate reductase (MTHFR) and cystathionine-y-lyase (CSE, an enzyme responsible for Hcy metabolism to H2S, a most potent vasodilator, antioxidant and anti-hypertensive agent) contribute to mitochondria dysfunction (mitophagy) and ischemic stroke. Cytochrome-C transports electrons and facilitates mitochondrial bioenergetics. Interestingly, during HHcy, cytochrome-C becomes homocysteinylated (N-Hcy-cyt-c). However, it's consequence to mitophagy and stroke is unclear. The long-term goal of this project is to understand the mechanism of mitophagy, mitochondrial repair and permeability in brain vasculature during I/R injury. Our preliminary studies suggest that during I/R ,total Hcy levels increases, causes N-Hcy-cyt-C , increases mitochondrial matrix metalloproteinase-9 (mtMMP-9), in-part degradation of mt-matrix (connexin and tight junction protein, TJP) which led to mitophagy and permeability in brain vasculature. Interestingly, THC decreases Hcy level and mitigates brain damage. Tetra hydro-curcumin (THC), a major herbal antioxidant and anti-inflammatory agent, has shown to protect brain against I/R injury. The central hypothesis of this proposal is that HHcy contributes to mitophagy mediated brain damage through N-Hcy-cyt-C in part, by increasing oxidative stress, mtMMP-9, degrades connexin-43 and TJP (Figure 1). The treatment with THC, CSE gene and SAHH shRNA gene transfer attenuates mitophagy and permeability. We will test this hypothesis by following three specific aims:
Specific Aim #1 : To determine whether the Hcy contributes to mitophagy, in part by inducing oxidative stress, exacerbating homocysteinylation of cytochrome-c in ischemia reperfusion and if THC, CSE and SAHH shRNA gene therapy mitigates these changes.
Specific Aim #2 : To determine whether the homocysteinylation of cytochrome-c activates mt-MMP-9, disruption of collagen/elastin ratio, mtCxn43 and mt-tight junction proteins in ischemia reperfusion and if THC, CSE and SAHH shRNA gene therapy ameliorate.
Specific Aim #3 : To determine whether Hcy alters mitochondrial (mt) bioenergetics and cerebro-vascular remodeling in ischemia reperfusion and if THC, CSE and SAHH shRNA gene therapy alleviate. These studies will demonstrate the novel mechanism of cerebrovascular remodeling and have therapeutic ramifications for mitochondrial repair in cerebral ischemic stroke.

Public Health Relevance

These studies will delineate the mechanisms of mitophagy and cerebrovascular remodeling in brain vasculature. The treatment with dietary herbal antioxidant tetrahydrocurcumin, naked CSE DNA and SAHH shRNA gene therapy mediated stabilization of mitochondrial membrane potential by inhibition of mitochondrial DNA methylation;oxidant and proteolysis stress via novel mechanistic pathways will be powerful therapeutic strategy for preventing ischemic stroke.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL107640-03
Application #
8625825
Study Section
Special Emphasis Panel (ZRG1-VH-J (02))
Program Officer
Reid, Diane M
Project Start
2012-03-01
Project End
2017-02-28
Budget Start
2014-03-01
Budget End
2015-02-28
Support Year
3
Fiscal Year
2014
Total Cost
$705,000
Indirect Cost
$235,000
Name
University of Louisville
Department
Physiology
Type
Schools of Medicine
DUNS #
057588857
City
Louisville
State
KY
Country
United States
Zip Code
40292
Kamat, Pradip K; Kalani, Anuradha; Rai, Shivika et al. (2016) Mechanism of Oxidative Stress and Synapse Dysfunction in the Pathogenesis of Alzheimer's Disease: Understanding the Therapeutics Strategies. Mol Neurobiol 53:648-61
Kamat, Pradip K; Kyles, Philip; Kalani, Anuradha et al. (2016) Hydrogen Sulfide Ameliorates Homocysteine-Induced Alzheimer's Disease-Like Pathology, Blood-Brain Barrier Disruption, and Synaptic Disorder. Mol Neurobiol 53:2451-67
Kalani, Anuradha; Pushpakumar, Sathnur B; Vacek, Jonathan C et al. (2016) Inhibition of MMP-9 attenuates hypertensive cerebrovascular dysfunction in Dahl salt-sensitive rats. Mol Cell Biochem 413:25-35
Kalani, Anuradha; Chaturvedi, Pankaj; Kamat, Pradip K et al. (2016) Curcumin-loaded embryonic stem cell exosomes restored neurovascular unit following ischemia-reperfusion injury. Int J Biochem Cell Biol 79:360-369
Kamat, Pradip K; Mallonee, Carissa J; George, Akash K et al. (2016) Homocysteine, Alcoholism, and Its Potential Epigenetic Mechanism. Alcohol Clin Exp Res 40:2474-2481
Kamat, Pradip K; Kalani, Anuradha; Tyagi, Suresh C et al. (2015) Hydrogen Sulfide Epigenetically Attenuates Homocysteine-Induced Mitochondrial Toxicity Mediated Through NMDA Receptor in Mouse Brain Endothelial (bEnd3) Cells. J Cell Physiol 230:378-94
Kamat, Pradip Kumar; Kalani, Anuradha; Tyagi, Neetu (2015) Role of hydrogen sulfide in brain synaptic remodeling. Methods Enzymol 555:207-29
Kamat, Pradip Kumar; Kalani, Anuradha; Metreveli, Naira et al. (2015) A possible molecular mechanism of hearing loss during cerebral ischemia in mice. Can J Physiol Pharmacol 93:505-16
Kalani, Anuradha; Kamat, Pradip K; Kalani, Komal et al. (2015) Epigenetic impact of curcumin on stroke prevention. Metab Brain Dis 30:427-35
Kamat, P K; Vacek, J C; Kalani, A et al. (2015) Homocysteine Induced Cerebrovascular Dysfunction: A Link to Alzheimer's Disease Etiology. Open Neurol J 9:9-14

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