Cardiovascular disease is the leading cause of death in patients with diabetes. Among coronary vascular defects, obstructive coronary artery disease (CAD) is the primary cause of cardiac mortality. However, there is growing evidence that patients with coronary microvascular disease (CMD, also known as non-obstructive coronary disease) also experience chest pain and occasional heart attack. Furthermore, recent studies show that diabetes is the risk factor of CMD. However, the molecular mechanisms by which diabetes develops CMD is not fully understood. MicroRNAs (miRNAs) are a non-coding small RNA that regulates gene expression at the post-transcriptional level, and the change of miRNA profile is implicated in many diseases. In this study, we identified the core miRNAs which are involved in the development of coronary endothelial dysfunction in diabetes. We used an inducible type 2 diabetic (T2D) mouse model generated by high-fat diet and a single injection of low-dose streptozotocin. Our T2D mice did not show detectable atherosclerotic plaque, but developed CMD evidenced by reduced coronary flow velocity reserve. We conducted a miRNA array using mouse coronary endothelial cells (MCECs) isolated from diabetic and control mice, and selected seven miRNAs based on the result. Next, real-time PCR assays were carried out with seven miRNAs and found that miR210 and miR342-5p were downregulated, and miR378 was upregulated in MCECs of diabetic mice compared to the control. To identify the target genes of miR342-5p and miR378, we inhibited miR342-5p or overexpressed miR378 in MCECs. After testing 93 genes that are involved in endothelial functions, we found that inhibition of miR342-5p decreased the levels of Atp2a3 , Opa1 , Sod3 , and Vegfb , whereas miR378 overexpression significantly decreased Aggf1, HK1, Mapk3, Pak1, Panx1, Stim1, Stim2, Vcam1, and Vegfb and increased Casp2 and Gja1 . Western blotting data revealed that the expression levels of Affg1, Opa1, and Pak1 were significantly decreased in the MCECs from diabetic mice compared to the control. In summary, these data suggest that miR342-5p and miR378 could be a potential therapeutic target for CMD in diabetic patients, and Affg1 Opa1 and Pak1 are the downstream mRNAs of miR342-5p or miR378.
Diabetes mellitus is a prominent risk factor of coronary microvascular disease (CMD) and the comorbidity of CMD in diabetic patients leads to an increasing occurrence of cardiac mortality. Increased protein O -linked β-N-acetylglucosaminylation ( O -GlcNAcylation), a post-translational modification of proteins, is implicated in the development of vascular complications in diabetes. Therefore, we investigated the molecular mechanisms in which excess protein O -GlcNAcylation advances the progression of CMD in diabetes through coronary endothelial cell dysfunction. We hypothesize that excess protein O -GlcNAcylation will up-regulate p53 which will induce the development of CMD. We had conducted in vivo and in vitro experiments in control mice, TALLYHO/Jng (TH) mice, a polygenic type 2 diabetic mouse model, and endothelial cell-specific O -GlcNAcase (OGA, a regulatory enzyme that catalyzes the removal of O- GlcNAc from proteins)-overexpressing TH mice. We found that compared to control mice, TH mice exhibited a decrease in coronary flow velocity reserve (CFVR, an indicator of coronary microvascular function), reduced capillary density, and increased endothelial cell apoptosis in the left ventricle. In addition, TH mice showed a significant increase of p53 protein expression in mouse coronary endothelial cells (MCECs) compared to control mice. Moreover, overexpression of OGA lowered protein O -GlcNAcylation and decreased p53 levels in MCECs. p53 inhibition with pifithrin-α restoratively increased CFVR and cardiac contractility in TH mice. From this data, we conclude OGA overexpression lowers p53 expression levels and thus attenuates endothelial cell apoptosis and restores CFVR and cardiac function in diabetes. The downregulation of p53 through OGA overexpression could be a potential therapeutic target for CMD in diabetic patients.
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