Flow Effects on Endothelial Cell Glucose Metabolism: Glycolytic Flux and O-GlcNAcylation in Health and Disease

Abstract

Cardiovascular disease, the leading global cause of death, is precipitated by endothelial cell (EC) dysfunction. When EC are exposed to unidirectional steady laminar flow (>12 dynes/cm2 shear stress), the cells assume a healthy, quiescent phenotype in which EC phosphorylate endothelial nitric oxide synthase (eNOS) to produce NO, a vasodilator that is also important in preventing vascular disease. However, when EC fail to adapt to flow, for example in areas of oscillating disturbed flow, they have impaired NO production. Areas of disturbed flow are linked to EC dysfunction and diseases such as atherosclerosis. Endothelial metabolism has recently emerged as a powerful tool to regulate the vasculature. Both cancer cells and ECs generate the majority of their energy through glycolysis even in the presence of oxygen (the Warburg effect). When EC glucose metabolism was decreased by PFKFB3 blockade, EC proliferation, angiogenic sprouting, and cancer metastasis were inhibited both in vitro and in vivo. Endothelial metabolism also decreases in chronic laminar flow, which put EC in a quiescent state and decreased angiogenesis. Little is known about how different blood flow regimes regulate EC metabolism and how this could impact macrovascular disease. The goal of this research was to elucidate how hemodynamics regulate EC glucose metabolism, and how EC glucose metabolism can be modulated to restore EC function in disturbed flow. We hypothesized that steady laminar flow (shear stress of 20 dynes/cm2) but not oscillating disturbed flow (shear stress of 4±6 dynes/cm2) reduces glycolytic flux and eNOS O-GlcNAcylation to promote a healthy endothelial phenotype. First, I adapted human EC to steady laminar and oscillating disturbed flow in a cone-and-plate device in vitro and quantified changes in (1) endothelial glycolytic activity and (2) eNOS O-GlcNAcylation. Stable isotope mass spectrometry and YSI Bioanalysis revealed an overall decrease in glucose uptake and glycolytic activity in EC exposed to steady laminar but not oscillating disturbed flow. HUVEC exposed to oscillating disturbed flow had over two times more glucose consumption compared to cells exposed to steady laminar flow. Glycolytic intermediate total pool sizes such as fructose-1,6-bisphosphate, 1,3-bisphosphoglyercate, and phosphoenolpyruvate were 60-70% lower in HUVEC exposed to steady laminar flow compared to cells exposed to oscillating disturbed flow. Next, I showed that eNOS O-GlcNAcylation was abolished in EC exposed to steady laminar (~75% lower) but not oscillating disturbed flow. Interestingly, there was no change in protein level, localization, or activity of key O-GlcNAcylation enzymes (OGT, OGA, or GFAT). Instead, glycolysis inhibition via 2-deoxy-2-glucose (2-DG) in cells exposed to disturbed flow efficiently decreased eNOS O-GlcNAcylation by 60%, thereby increasing eNOS phosphorylation by 20% and NO production by 65%. Finally, I investigated altered glucose metabolism in pulmonary arterial hypertension (PAH). Human pulmonary artery EC from PAH patients showed three times as much glucose uptake as healthy patients. Additionally, glycolytic intermediates such as fructose-1-6, bisphosphate and 3-phosphoglycerate showed 50-75% higher total pool levels in PAH patients compared to healthy patients. Furthermore, PAH patients showed reduced eNOS O-GlcNAcylation and NO bioavailability. My data demonstrate that steady laminar but not oscillating disturbed flow decreases glycolytic activity as well as HBP activity. Specifically, glycolytic flux controls eNOS O-GlcNAcylation and UDP-GlcNAc substrate availability, thus impacting eNOS phosphorylation and NO production. This research shows for the first time that O-GlcNAcylation is regulated by mechanical stimuli; relates flow-induced glycolytic flux changes to macrovascular disease; and highlights O-GlcNAcylation as a novel therapeutic target to restore eNOS activity and to prevent EC dysfunction in cardiovascular disease.Ph.D., Biomedical Engineering -- Drexel University, 201