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 #
7R01HL097111-04
Application #
8383468
Study Section
Electrical Signaling, Ion Transport, and Arrhythmias Study Section (ESTA)
Program Officer
OH, Youngsuk
Project Start
2010-01-19
Project End
2014-11-30
Budget Start
2012-12-01
Budget End
2013-11-30
Support Year
4
Fiscal Year
2013
Total Cost
$359,124
Indirect Cost
$123,504
Name
State University of New York at Albany
Department
None
Type
Schools of Engineering
DUNS #
152652822
City
Albany
State
NY
Country
United States
Zip Code
12222
Kassan, Modar; Ait-Aissa, Karima; Radwan, Eman et al. (2016) Essential Role of Smooth Muscle STIM1 in Hypertension and Cardiovascular Dysfunction. Arterioscler Thromb Vasc Biol 36:1900-9
Stolwijk, Judith A; Zhang, Xuexin; Gueguinou, Maxime et al. (2016) Calcium Signaling Is Dispensable for Receptor Regulation of Endothelial Barrier Function. J Biol Chem 291:22894-22912
Shakerley, Nicole L; Chandrasekaran, Akshaya; Trebak, Mohamed et al. (2016) Francisella tularensis Catalase Restricts Immune Function by Impairing TRPM2 Channel Activity. J Biol Chem 291:3871-81
Wang, Huiyun; Zhang, Xuexin; Xue, Li et al. (2016) Low-Voltage-Activated CaV3.1 Calcium Channels Shape T Helper Cell Cytokine Profiles. Immunity 44:782-94
Beardslee, Luke A; Stolwijk, Judith; Khaladj, Dimitrius A et al. (2016) A sacrificial process for fabrication of biodegradable polymer membranes with submicron thickness. J Biomed Mater Res B Appl Biomater 104:1192-201
Vashisht, Ayushi; Trebak, Mohamed; Motiani, Rajender K (2015) STIM and Orai proteins as novel targets for cancer therapy. A Review in the Theme: Cell and Molecular Processes in Cancer Metastasis. Am J Physiol Cell Physiol 309:C457-69
Stolwijk, Judith A; Matrougui, Khalid; Renken, Christian W et al. (2015) Impedance analysis of GPCR-mediated changes in endothelial barrier function: overview and fundamental considerations for stable and reproducible measurements. Pflugers Arch 467:2193-218
Zhang, Wei; Zhang, Xuexin; González-Cobos, José C et al. (2015) Leukotriene-C4 synthase, a critical enzyme in the activation of store-independent Orai1/Orai3 channels, is required for neointimal hyperplasia. J Biol Chem 290:5015-27
Kassan, Modar; Choi, Soo-Kyoung; Galán, Maria et al. (2015) Nuclear factor kappa B inhibition improves conductance artery function in type 2 diabetic mice. Diabetes Metab Res Rev 31:39-49
Spinelli, Amy M; Liu, Yongfeng; Sun, Li-Yan et al. (2015) Smooth muscle CaMKIIδ promotes allergen-induced airway hyperresponsiveness and inflammation. Pflugers Arch 467:2541-54

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