Shear stress Regulation of Endothelial Glycolysis via METTL3-mediated RNA m6A Modification

Project Summary Shear stresses resulting from distinct blood flow patterns impact endothelial cell (EC) functions. ECs under atheroprone flow exhibit increased proliferation, inflammation, and glycolysis, compared to those under atheroprotective flow. Our recent studies have uncovered a novel role of epitranscriptional regulation (i.e., functional changes to RNAs without altered nucleotide sequence) in EC mechanobiology. Thus, atheroprone flow induces METTL3, a methyltransferase that catalyzes N6-methyladenosine (m6A) to result in the most abundant RNA modification found in eukaryotes. Our newly conducted preliminary studies indicate that METTL3 inhibition in ECs suppresses the atheroprone flow-induced pro-inflammatory response and glycolysis. Through transcriptome and epitranscriptome mapping, we found that METTL3 caused hypermethylation of the mRNAs encoding the key enzymes involved in glycolysis and pentose phosphate pathway (PPP, a branch of glycolysis). These findings led to the guiding hypothesis that atheroprone flow upregulates METTL3 to modulate m6A epitranscriptomes and hence promote glycolysis and PPP in ECs, thus contributing to EC dysfunction and atherosclerosis. The four specific aims proposed to test this hypothesis are: Aim 1. To delineate the dynamics of METTL3-regulated epitranscriptomes in ECs under atheroprone vs. atheroprotective flow patterns; Aim 2. To identify the METTL3-modulated m6A RNA targets that drive the atheroprone flow enhancement of EC glycolysis and PPP; Aim 3. To elucidate the effects of the flow-regulated EC m6A epitranscriptomes on the phenotypic changes of co-cultured SMCs and EC glycolysis by SRS imaging; Aim 4. To validate flow regulation of METTL3 and m6A epitranscriptomes in mouse atherosclerosis models. This interdisciplinary research will unveil novel epitranscriptional mechanisms underlying EC mechanobiology and atherosclerotic diseases.