The activation of platelets is the final common pathway for most ischemic strokes. Acute thrombus formation in the setting of vascular dysfunction and inflammation initiates a cascade of events that culminates in necrotic death of neurons and injury to their supportive structures in the neurovascular unit. However, the signaling pathways that link these events are not well understood. The Rho/Rho-associated coiled-coil forming kinases (ROCK1 and ROCK2) are important regulators of the actin cytoskeleton. Because changes in the actin cytoskeleton underlie platelet aggregation, vascular contractility, and inflammatory cell recruitment, it is likely that the Rho/ROCK pathway will play a central role in ischemic strokes. Accordingly, the overall aim of this proposal is to investigate the role of ROCK isoforms in platelets and to determine how they might contribute to thromboembolic strokes. To achieve this goal, we will target ROCK deletion in platelets using knockout (KO), bone marrow transplantation (BMT), and Cre/loxP technology and will investigate the subsequent loss-of-function of platelet ROCKs in thrombus formation, clot propagation, and focal cerebral ischemia. The results of these proposed studies will hopefully lead to the development of isoform-specific ROCK inhibitors as novel therapies for patients with ischemic strokes.
Specific aim 1 will determine the mechanisms by which ROCKs contribute to platelet function and arterial thrombosis. We will test the hypothesis that ROCKs play differential roles in regulating the assembly of the platelet cytoskeleton and mediating platelet function. To determine and compare the effect of ROCK1 and ROCK2 on thrombosis, platelets derived from ROCK1-/- and ROCK2-/- bone marrow transplanted (BMT) mice will be studied for aggregation, adhesion, hetero- and homo-typic aggregate formation, and by direct visualization with electron microscopy after activation with various platelet agonists. Furthermore, we will investigate the potential downstream signaling pathways of ROCKs that regulates platelet actin cytoskeleton and function.
Specific aim 2 will determine the pathophysiological consequences of platelet ROCK deletion on thrombus formation and propagation in a clot embolic model of stroke. We will test the hypothesis that ROCKs are critically important for platelet function in vivo, and that platelet deletion of ROCKs confers stroke protection in a mouse model of thrombosis-mediated focal cerebral ischemia. To do this, we will develop platelet-specific ROCK KO mice (ROCK1Plt-/- and ROCK2Plt-/- mice) and utilize (1) a carotid artery injury model for measurement of arterial occlusive thrombosis, (2) an agonist-dependent platelet consumptive model to study micro thrombi formation, and (3) a clot-embolic stroke model using preformed thrombi to determine the ability of a clot to form and adhere to the vasculature, mediate vascular occlusion, and cause cerebral ischemia and infarction.

Public Health Relevance

Stroke is the 3rd leading cause of death and a major cause of disability in the Western society. The final common pathway of most strokes is platelet activation and aggregation. This research application proposes to investigate the role of an emerging signaling pathway, Rho kinase (ROCK), in platelets as a potential therapeutic target for preventing and treating ischemic strokes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
7R01NS070001-04
Application #
8415552
Study Section
Acute Neural Injury and Epilepsy Study Section (ANIE)
Program Officer
Bosetti, Francesca
Project Start
2010-02-01
Project End
2015-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
4
Fiscal Year
2013
Total Cost
$326,858
Indirect Cost
$119,986
Name
University of Chicago
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
005421136
City
Chicago
State
IL
Country
United States
Zip Code
60637
Zee, Robert Y L; Wang, Qing-Mei; Chasman, Daniel I et al. (2014) Gene variations of ROCKs and risk of ischaemic stroke: the Women's Genome Health Study. Clin Sci (Lond) 126:829-35
Sawada, Naoki; Liao, James K (2014) Rho/Rho-associated coiled-coil forming kinase pathway as therapeutic targets for statins in atherosclerosis. Antioxid Redox Signal 20:1251-67
Miao, Yanying; Liao, James K (2014) Potential serum biomarkers in the pathophysiological processes of stroke. Expert Rev Neurother 14:173-85
Kajikawa, Masato; Noma, Kensuke; Maruhashi, Tatsuya et al. (2014) Rho-associated kinase activity is a predictor of cardiovascular outcomes. Hypertension 63:856-64
Toque, Haroldo A; Nunes, Kenia P; Yao, Lin et al. (2013) Activated Rho kinase mediates diabetes-induced elevation of vascular arginase activation and contributes to impaired corpora cavernosa relaxation: possible involvement of p38 MAPK activation. J Sex Med 10:1502-15
Dong, Ming; Jiang, Xin; Liao, James K et al. (2013) Elevated rho-kinase activity as a marker indicating atherosclerosis and inflammation burden in polyvascular disease patients with concomitant coronary and peripheral arterial disease. Clin Cardiol 36:347-51
Liao, James K (2013) Linking endothelial dysfunction with endothelial cell activation. J Clin Invest 123:540-1
Tanaka, Shin-ichi; Fukumoto, Yoshihiro; Nochioka, Kotaro et al. (2013) Statins exert the pleiotropic effects through small GTP-binding protein dissociation stimulator upregulation with a resultant Rac1 degradation. Arterioscler Thromb Vasc Biol 33:1591-600
Okamoto, Ryuji; Li, Yuxin; Noma, Kensuke et al. (2013) FHL2 prevents cardiac hypertrophy in mice with cardiac-specific deletion of ROCK2. FASEB J 27:1439-49
Wang, Chao-Yung; Liao, James K (2012) A mouse model of diet-induced obesity and insulin resistance. Methods Mol Biol 821:421-33

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