Project Summary Vascular endothelial cell (EC) metabolism is essential for functional endothelium, and maladapted energy use severely affects EC health. AMP-activated protein kinase (AMPK) is a key regulator of cellular energy status and homeostatic function. Our preliminary studies showed that energy stress results in spatially defined AMPK activity at cellular organelles, which indicates that AMPK activity is compartmentalized in the cell. An emerging AMPK substrate in the vascular endothelium is angiotensin-converting enzyme 2 (ACE2), and we have found that the AMPK–ACE2 axis enhances EC function and is atheroprotective. SARS-CoV viruses invade the host cells by binding the viral spike protein (S protein) to ACE2, which leads to decreased membrane ACE2 levels, increased extracellular soluble ACE2, and increased glycolysis, thus resulting in host cell damage. In preliminary studies, we have also found that the SARS-CoV-2 S protein deactivates the AMPK–ACE2 axis and impairs EC function in vitro and in vivo. This impairment is likely to constitute a risk factor for the long-term effects of SARS- CoV-2 infection or post-acute sequelae of SARS-CoV-2 infection (PASC). These preliminary findings lead to the hypothesis that EC homeostasis is maintained via the spatiotemporal regulation of the AMPK–ACE2 axis. In contrast, S protein entry disrupts cellular energetics in ECs, leading to dysregulated AMPK and the ensuing ACE2 hypo-phosphorylation, which critically contributes to the COVID-19–associated EC dysfunction and PASC. The three specific aims proposed to test this novel hypothesis are as follows: Aim 1. To investigate the spatiotemporal regulation of AMPK in ECs under physiological [e.g., pulsatile shear stress (PS)], pharmacological (e.g., metformin), and pathophysiological (e.g., S protein) conditions; Aim 2. To decipher the mechanisms by which physiological, pharmacological, and pathophysiological stimuli modulate the AMPK– ACE2 axis in ECs; Aim 3. To investigate the role of impaired AMPK–ACE2 axis in S protein-accelerated atherosclerosis in the context of PASC. In the proposed research, we will use live cell imaging, in vitro EC biology, and in vivo animal models to determine the role of the AMPK–ACE2 axis in endothelial health and disease. These findings will result in otherwise missing insights into the pathophysiology of PASC, which will continue to be a long-term consequence of SARS-CoV-2 infection.