Vascular smooth muscle cell (VSMC) proliferation and migration are essential for vascular development, angiogenesis and repair;their dysfunction contributes to vascular diseases such as atherosclerosis, hypertensive microvessel remodeling and Leiomyosarcomas. In these diseases VSMC switch from a quiescent differentiated and contractile phenotype to a synthetic proliferative and migratory phenotype, a condition that can be recapitulated in culture. There is clear evidence that this VSMC phenotypic modulation is of paramount clinical importance in atherosclerosis and other vascular occlusive diseases, yet the molecular mechanisms of this modulation remain incompletely understood. VSMC phenotypic modulation is accompanied by a change in ion channel expression: synthetic VSMC downregulate the expression of L-type Ca2+ channels and upregulate that of canonical transient receptor potential (TRPC) channels. Our preliminary studies have demonstrated an increase in TRPC6 and the newly discovered calcium sensor STIM1, and calcium channels Orai1 and Orai3 expression in synthetic cultured rat aortic VSMC, as compared to quiescent freshly isolated cells. We hypothesize that STIM1 is a master regulator of Ca2+ signaling in VSMC required for Orai1, Orai3 and TRPC6 channel function and that increased Ca2+ entry as a result of STIM1, Orai and TRPC upregulation contribute to VSMC proliferation and migration in disease. In support of this hypothesis we found that STIM1 knockdown using silencing RNA (siRNA) inhibited VSMC proliferation in culture and we revealed agonist-specific activation of distinct Ca2+ channels and remarkable STIM1 versatility in regulating these channels. Indeed, knockdown of STIM1 in synthetic VSMC abrogated the function of: i) PDGF-activated Orai1 channels;ii) thrombin-activated channels contributed by heteromultimeric Orai1/3 and iii) Diacylglycerol (DAG)-activated TRPC6 channels.
In Aim1 we will biophysically characterize thrombin-activated Ca2+ entry pathways using whole cell patch clamp and determine the role of STIM1 oligomerization, cellular localization and interaction with Orai1 and Orai3 in the activation of this Ca2+ entry pathway.
In Aim2 we will determine whether sarcoplasmic reticulum- or plasma membrane- associated STIM1 is involved in TRPC6 activation by DAG, examine the role of STIM1 oligomerization and the interaction of STIM1 and TRPC6 by FRET microscopy and the interaction of their native counterparts using co-immunoprecipitations.
In Aim3 we will test the hypothesis that Orai1, Orai3 and TRPC6 upregulation also occurs in vivo in a rat model of vascular injury and determine the effect on neointimal formation of in vivo silencing of these proteins using adenovirus encoding siRNA. The results from this proposal will generate a better understanding of VSMC physiology and unveil novel targets for drug therapy aimed at controlling VSMC proliferation and migration that occur during vascular diseases such as atherosclerosis and hypertension.

Public Health Relevance

The results from this proposal will generate a better understanding of VSMC physiology and unveil novel targets for drug therapy aimed at controlling VSMC proliferation and migration that occur during vascular diseases such as atherosclerosis and hypertension.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL097111-03
Application #
8197794
Study Section
Electrical Signaling, Ion Transport, and Arrhythmias Study Section (ESTA)
Program Officer
OH, Youngsuk
Project Start
2010-01-19
Project End
2012-11-08
Budget Start
2011-12-01
Budget End
2012-11-08
Support Year
3
Fiscal Year
2012
Total Cost
$432,868
Indirect Cost
$156,021
Name
Albany Medical College
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
190592162
City
Albany
State
NY
Country
United States
Zip Code
12208
Pierro, Cristina; Zhang, Xuexin; Kankeu, Cynthia et al. (2018) Oncogenic KRAS suppresses store-operated Ca2+ entry and ICRAC through ERK pathway-dependent remodelling of STIM expression in colorectal cancer cell lines. Cell Calcium 72:70-80
Pathak, Trayambak; Trebak, Mohamed (2018) Mitochondrial Ca2+ signaling. Pharmacol Ther 192:112-123
Zhou, Yandong; Nwokonko, Robert M; Cai, Xiangyu et al. (2018) Cross-linking of Orai1 channels by STIM proteins. Proc Natl Acad Sci U S A 115:E3398-E3407
Cai, Xiangyu; Nwokonko, Robert M; Loktionova, Natalia A et al. (2018) Pore properties of Orai1 calcium channel dimers and their activation by the STIM1 ER calcium sensor. J Biol Chem 293:12962-12974
Zhang, Xuexin; Spinelli, Amy M; Zhang, Wei et al. (2018) Study of the Endogenous CRAC Channel Using shRNA-Mediated Gene Silencing. Methods Mol Biol 1843:137-145
Verkhratsky, Alexei; Trebak, Mohamed; Perocchi, Fabiana et al. (2018) Crosslink between calcium and sodium signalling. Exp Physiol 103:157-169
Johnson, Martin; Trebak, Mohamed (2018) Slow Traffic Makes for Bad Circulation. Hypertension 72:585-587
Tanwar, Jyoti; Trebak, Mohamed; Motiani, Rajender K (2017) Cardiovascular and Hemostatic Disorders: Role of STIM and Orai Proteins in Vascular Disorders. Adv Exp Med Biol 993:425-452
Ben-Kasus Nissim, Tsipi; Zhang, Xuexin; Elazar, Assaf et al. (2017) Mitochondria control store-operated Ca2+ entry through Na+ and redox signals. EMBO J 36:797-815
Hempel, Nadine; Trebak, Mohamed (2017) Crosstalk between calcium and reactive oxygen species signaling in cancer. Cell Calcium 63:70-96

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