Molecular processes mediating hyperhomocysteinemia-induced metabolic reprogramming, redox regulation and growth inhibition in endothelial cells

Hyperhomocysteinemia (HHcy) is an established and potent independent risk factor for degenerative diseases, including cardiovascular disease (CVD), Alzheimer disease, type II diabetes mellitus, and chronic kidney disease. HHcy has been shown to inhibit proliferation and promote inflammatory responses in endothelial cells (EC), and impair endothelial function, a hallmark for vascular injury. However, metabolic processes and molecular mechanisms mediating HHcy-induced endothelial injury remains to be elucidated. This study examined the effects of HHcy on the expression of microRNA (miRNA) and mRNA in human aortic EC treated with a pathophysiologically relevant concentration of homocysteine (Hcy 500 μM). We performed a set of extensive bioinformatics analyses to identify HHcy-altered metabolic and molecular processes. The global functional implications and molecular network were determined by Gene Set Enrichment Analysis (GSEA) followed by Cytoscape analysis. We identified 244 significantly differentially expressed (SDE) mRNA, their relevant functional pathways, and 45 SDE miRNA. HHcy-altered SDE inversely correlated miRNA-mRNA pairs (45 induced/14 reduced mRNA) were discovered and applied to network construction using an experimentally verified database. We established a hypothetical model to describe the biochemical and molecular network with these specified miRNA/mRNA axes, finding: 1) HHcy causes metabolic reprogramming by increasing glucose uptake and oxidation, by glycogen debranching and NAD+/CoA synthesis, and by stimulating mitochondrial reactive oxygen species production via NNT/IDH2 suppression-induced NAD+/NADP-NADPH/NADP+ metabolism disruption; 2) HHcy activates inflammatory responses by activating inflammasome-pyroptosis mainly through ↓miR193b→↑CASP-9 signaling and by inducing IL-1β and adhesion molecules through the ↓miR29c→↑NEDD9 and the ↓miR1256→↑ICAM-1 axes, as well as GPCR and interferon α/β signaling; 3) HHcy promotes cell degradation by the activation of lysosome autophagy and ubiquitin proteasome systems; 4) HHcy causes cell cycle arrest at G1/S and S/G2 transitions, suppresses spindle checkpoint complex and cytokinetic abscission, and suppresses proliferation through ↓miRNA335/↑VASH1 and other axes. These findings are in accordance with our previous studies and add a wealth of heretofore-unexplored molecular and metabolic mechanisms underlying HHcy-induced endothelial injury. This is the first study to consider the effects of HHcy on both global mRNA and miRNA expression changes for mechanism identification. Molecular axes and biochemical processes identified in this study are useful not only for the understanding of mechanisms underlying HHcy-induced endothelial injury, but also for discovering therapeutic targets for CVD in general.
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Highlights • Identified multiple HHcy-altered metabolic and molecular processes potentially responsible for HHcy-induced endothelial injury via examining global mRNA/miRNA expression changes in Hcy-treated EC and performing comprehensive bioinformatic studies. • HHcy may activate glucose uptake signaling via the ↓miR148b→↑SLC2A axis. • HHcy may induce glucose oxidation signaling by switching pyruvate metabolism from lactate synthesis to mitochondrial oxidation via expression changes of ↑MPC1 & ↓LDHB. • HHcy may disrupt redox homeostasis mostly by suppressing NNT/IDH2-related mt-NADPH/mt-NAD+ signaling. • HHcy may increase FA β-oxidation, glutamine, TCA cycle and OXPHOS signaling. • HHcy may activate inflammatory signaling via the ↓miR29c→↑NEDD9 and the ↓miR1256→↑ICAM-1 axes. • HHcy may activate inflammasome/pyroptosis-related signaling by the ↓miR137→↑TLR3, the ↓miR574→↑TRAF5, and the ↓miR193b→↑CASP-9 axes, and induce IL1α/β and CASP-10/7. • HHcy may induce inflammation signaling via GPCR activation through the ↓miRNA335→↑CXCR4/↑GNA14 axes. • HHcy may activate molecular degradation process signaling through the ↓miRNA335→↑ASAH1/↑ABCB9 axes. • HHcy may suppress cell cycle and proliferation through the miR491→↓HMGA2→↓CCNA2/CCNB2, the ↓miR335→↑VASH1, the ↓miR181a→↑PHLDA1, the miR6045→↓CENPH, the miR22→↓PRR11/↓BRCA2, and the miR605/miR497/miR514a→CEP55 axes