UC Riverside

Despite significant advances in medicine, atherosclerosis and its complications remain the leading causes of morbidity and mortality in most developed countries. Endothelium, lining the first layer of blood vessels, serves as a dynamic interface in maintaining vascular homeostasis. Endothelial dysfunction is a hallmark of atherosclerosis development, signified by impaired nitric oxide (NO) bioavailability derived from endothelial nitric oxide synthase (eNOS). To investigated the molecular mechanisms by which eNOS-NO is regulated under physiological, pathophysiological and pharmacological contexts, I examined the role of two metabolic master molecules, AMP-activated protein kinase (AMPK) and sirtuin 1 (silent information regulator 2 homolog, SIRT1), in eNOS regulation and NO bioavailability.

Primarily, I demonstrated that AMPK directly phosphorylates eNOS Ser-633, in addition to Ser-1177, leading to endothelial NO production, in response to hormonal (adiponectin), physical (shear stress), and pharmacological (atorvastatin) stimuli. Further, I found reduced eNOS phosphorylation in aortas from mice deficient in AMPKa2 (AMPKa2^-/-), confirming the AMPK-mediated eNOS regulation in vivo. In addition, with the application of Nano-liquid chromatography/tandem mass spectrometry (LC/MS/MS), I proved that eNOS Ser-633 was able to compete with acetyl-CoA carboxylase (ACC) Ser-79 for AMPK phosphorylation. These findings suggest that AMPK phosphorylation of eNOS Ser-633 is a functional signaling event for NO bioavailability in endothelial cells (ECs).

In the second part, I focused on SIRT1, a NAD⁺-dependent deacetylase and its modulation on eNOS. First, I found that SIRT1 responds to laminar shear stress with increased protein level and activity in ECs. In parallel to the observation that SIRT1 was significantly higher in ECs exposed to physiological pulsatile flow than pathophysiological oscillatory flow, SIRT1 was shown to be higher in the mouse thoracic aorta exposed to athero-protective flow than in the aortic arch under athero-prone flow. Next, I revealed the functional consequences of the shear stress-enhanced SIRT1 in ECs, including mitochondrial biogenesis and eNOS deacetylation. Subsequently, I studied the interplay of AMPK phosphorylation and SIRT1 deacetylation on eNOS. By using AMPK inhibitor and eNOS Ser-633 and -1177 mutants, I demonstrated that AMPK phosphorylation of eNOS may prime SIRT1 deacetylation of eNOS with attendant NO production. To verify this finding in vivo, I compared eNOS acetylation status in thoracic aortas from AMPKa2^-/- mice and their wild-type littermates and found that AMPKa2^-/- mice had a higher eNOS acetylation. Collectively, my study suggests that AMPK and SIRT1 could synergistically enhance eNOS-derived NO under physiological cues, such as athero-protective flow.