Effect of hyperglycemia on epigenetics of target genes in vascular cells -

Abstract

Background: Epigenetic alterations in vascular cells have been found to be implicated in microvascular and macrovascular complications in Diabetes Mellitus (DM). Metabolic memory is the phenomenon in which diabetic individuals who are chronically exposed to high blood glucose, exhibit diabetic complications even after achieving normoglycemia. This memory results partially from mitotically heritable changes in DNA methylation patterns on key genes involved in pathways that affect vascular remodeling. Aims: To assess epigenetic control of expression of key genes involved in diabetic vascular complications through performing quantitative DNA methylation profiling in 5’ promoter regions. To simulate the metabolic memory phenomenon by comparing DNA methylation signatures between control, diabetic, and insulin-treated diabetic ratsMethods: Type-1 Diabetes Mellitus (T1DM) was induced by streptozotocin injection in Male Sprague Dawley rats. After 6 weeks, the aortas of T1DM rats, control rats and insulin-treated rats were harvested for isolation of genomic DNA (gDNA), mRNA, and proteins from smooth muscle cells. High glucose-treated and high mannitol-treated rat aortic smooth muscle cells for 4 weeks and their controls were also collected for isolation of gDNA and mRNA. Real-time qPCR (RTqPCR) followed by High-Resolution Melt PCR and global proteomics analysis were done to assess gene expression, differential DNA methylation profiles, and protein synthesis and activity, respectively. Genes of interest included Bradykinin Receptor B1 (BDKRB1), B2 (BDKRB2), Connective Tissue Growth Factor (CTGF), Fibronectin1 (FN1), C-Reactive Protein (CRP), Interleukin 1 (IL1), Interleukin 6 (IL6), Interleukin 10 (IL10), Transforming Growth Factor beta 1 (TGF-1), Transforming growth factor beta 2 (TGF-2), Tumor Necrosis Factor alpha (TNF), NADPH oxidase 1 (Nox1), and NADPH oxidase 4 (Nox4). Results: RTqPCR on cDNA from in vitro and in vivo samples showed that high glucose treatment induces express