It is known that local hemodynamic forces modulate the phenotype of vascular endothelial cells (ECs), and this phenotypic modulation contributes to the focal nature of atherosclerotic disease. ECs in atheroprone 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 atheroresistant regions, which are exposed to pulsatile shear stress (PS). Our group made seminal discoveries on the struc- ture/function of the Mitochondrial Calcium (Ca2+) Uniporter (MCU) complex, an inner mitochondrial membrane channel responsible for mitochondrial Ca2+ ([Ca2+]m) uptake. It consists of a pore-forming protein, also called MCU, and auxiliary subunits. MCU expression is regulated by the redox-sensitive transcription factor CREB. MCU is activated following oxidative modification by mitochondrial ROS (mROS), and persistent activation re- sults in [Ca2+]m overload and cell death. My lab showed that MCU knockdown inhibits intracellular Ca2+ ([Ca2+]i) oscillations in cultured ECs exposed to arterial-level steady laminar shear stress (an in vitro analog of PS) sug- gesting that MCU channel activity ([Ca2+]m uptake) is critical for shear-induced [Ca2+]i signaling and EC function. For this Supplement, the hypothesis is that aging may regulate both the basal MCU expression/activity and the MCU expression/activity following EC exposure to either atheroprone (OS) or atheroresistant (PS) flow. Since senescent ECs have increased ROS levels compared to young ones, it is anticipated that senescent ECs will express higher MCU basal levels/activity and will demonstrate differential changes in MCU expression/activity following exposure to OS or PS, compared to young ECs. Specifically, we propose to:
Aim 1 : Assess the MCU expression/activity levels in cultured young vs. senescent human umbilical vein ECs (HUVECs) and human carotid artery ECs (HCtAECs), prior to and following exposure to either atheroprone (OS) or atheroresistant shear stress (PS).
Aim 2 : Characterize the differences in mitochondrial and cell function, prior to and following exposure to OS or PS, between young and senescent HUVECs and HCtAECs, and assess the role of MCU in those differences. The effects of MCU knockdown on OS- and PS-induced changes in [Ca2+]m, [Ca2+]i, mROS, cytosolic ROS, and mitochondrial and EC function will be investigated in senescent vs. young ECs using the same methods as in the parent R01. By unveiling age-related changes in MCU expression and activity (hence, [Ca2+]m uptake) and its role in the EC's apoptotic threshold, this work will further advance our understanding of endothelial biology and may lead to development of drugs that will specifically target senescent ECs in atheroprone arterial regions and protect them from atherosclerosis.
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 potential role of MCU in mediating mitochondrial Ca2+ overload and cell dysfunc- tion in senescent, compared to young, ECs exposed to atheroprone flows, and it may lead to discovery of drugs/treatments that will protect senescent ECs in atheroprone arterial regions from atherosclerotic disease.
