It is well known that local hemodynamic forces/stresses modulate the phenotype of vascular endothelial cells (ECs), and this phenotypic modulation contributes to the focal nature of atherosclerotic disease. ECs in athero- prone arterial regions, which are exposed to oscillatory shear stress (OS), experience higher levels of reactive oxygen species (ROS) and exhibit inflammation and increased sensitization to apoptosis compared to ECs in atheroprotective regions, which are exposed to pulsatile shear stress (PS). Our group has made seminal discov- eries on the structure/function of the Mitochondrial Calcium (Ca2+) Uniporter (MCU) complex, an inner mitochon- drial membrane channel responsible for mitochondrial Ca2+ ([Ca2+]m) uptake. It consists of a pore-forming protein, also called MCU, and auxiliary subunits. We showed that EC MCU expression is regulated by the redox-sensitive transcription factor CREB. MCU is activated following oxidative modification by mitochondrial ROS (mROS), and persistent activation results in [Ca2+]m overload and cell death. We recently showed that MCU knockdown inhibits the intracellular Ca2+ ([Ca2+]i) oscillations in cultured ECs exposed to steady laminar shear stress suggesting that the MCU activity ([Ca2+]m uptake) is critical for shear-induced [Ca2+]i signaling and EC function. Since MCU expression/activity are redox regulated and OS-exposed ECs encounter oxidative stress, we hy- pothesized that EC MCU expression/activity will be enhanced in atheroprone (OS) regions compared to athero- protective (PS) ones, and the increased EC [Ca2+]m uptake in OS-exposed regions will be responsible for mito- chondrial and cell dysfunction, and for initiation of atherosclerosis. Preliminary data showed significantly higher EC MCU protein expression and activity in OS-exposed, compared to PS-exposed, regions in mouse aortas. We propose to: (a) Determine the differential effects of PS vs. OS on EC MCU gene/protein expression and activity in vivo and in vitro. To establish the causative role of OS in EC MCU expression in vivo, a mouse partial carotid artery ligation model will be employed. (b) Define the signaling events upstream and downstream of MCU in cultured ECs, from different species/vascular beds, exposed to PS vs. OS. The effects of either MCU knock- down, overexpression, or persistent activation on the basal and PS/OS-induced [Ca2+]m, [Ca2+]i, mROS, cytosolic ROS, mitochondrial (bioenergetics, respiration) and EC function will be determined. (c) Assess whether targeting the EC MCU will confer protection from atherosclerotic disease. The MCU role in OS-induced EC inflammation will be examined in EC-specific MCU conditional knockout (MCU?EC), CRISPR/Cas9 knock-in (gain-of-function mutant), and control mice. To assess the MCU as a therapeutic target, double transgenic MCU?EC+ApoE-/- mice will be generated and the role of MCU ablation in post-ligation atherosclerosis will be examined in ApoE-/- mice. Completion of this collaborative project will provide new insights into the MCU-mediated Ca2+ signaling and its role in EC mechanotransduction and atherosclerotic disease development. Furthermore, the discovery of novel molecular mechanisms of atherosclerosis will undoubtedly lead to the design of new therapeutic interventions.

Public Health Relevance

Modulation of the vascular endothelial cell (EC) phenotype by hemodynamic forces is known to contribute to the localized nature of atherosclerotic disease. Intracellular Ca2+ dynamics in ECs exposed to fluid shear stress were found to depend on mitochondrial Ca2+ uptake, which is mediated by the Mitochondrial Ca2+ Uniporter (MCU). This project will investigate the hemodynamically-induced spatio-temporal regulation of MCU expression/activity and its role in potentiating EC mitochondrial Ca2+ uptake/overload, EC dysfunction and atherosclerosis in athero- prone arterial regions, and, thus, it will uncover novel therapeutic targets in atherosclerosis.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Atherosclerosis and Inflammation of the Cardiovascular System Study Section (AICS)
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Chen, Jue
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State University of New York at Buffalo
Biomedical Engineering
Biomed Engr/Col Engr/Engr Sta
United States
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