Abstract Body (Do not enter title and authors here): BACKGROUND. Diabetes mellitus (DM) and hyperglycemia are known to drive endothelial impairment and coronary microvascular dysfunction. However, currently no established biomarkers of endothelial dysfunction in patients with "Ischemia with non-obstructive coronary artery" (INOCA). We evaluated the expression of circulating microRNAs (miRs) involved in the regulation of endothelial function in individuals with confirmed INOCA, comparing patients with and without DM.

METHODS. We evaluated consecutive INOCA patients referred for percutaneous coronary intervention (PCI) in a multicenter study. DM was defined according to the American Diabetes Association (ADA) guidelines. INOCA was defined based on symptoms of myocardial ischemia, non-obstructive coronary artery stenosis (<50% diameter reduction or fractional flow reserve >0.80), objective evidence of myocardial ischemia, and impaired coronary microvascular function (coronary flow reserve ≤2.0, abnormal coronary microvascular resistance indices, coronary microvascular spasm, endothelial dysfunction with ≥20% luminal constriction during acetylcholine infusion, or coronary slow flow phenomenon). Circulating miRs were isolated from plasma samples using EDTA-containing tubes and the miRNeasy Serum/Plasma kit. Quality control included using the Agilent Small RNA Kit [33] and the RNA Spike-in kit (Qiagen) with synthetic RNA spike-ins (UniSp2, UniSp4, UniSp5) and UniSp6 spike-in and cel-miR-39-3p for cDNA synthesis control. Relative gene expression was determined using the 2-ΔΔCT method, with miRs miR-125a-5p and miR-19a-5p selected for normalization using bioinformatic algorithms. Hemolysis was assessed by measuring absorbance at 414 nm and ΔCq of miR-16-5p, miR-451a, and miR-23a-3p.

RESULTS. 42 patients completed the study (28 without DM, 14 with DM). We measured the expression levels of validated miRs and found that in INOCA patients with DM, there was increased expression of miRs associated with endothelial dysfunction and reduced expression of miRs protective for endothelial function. Specifically, miR-363-5p was significantly downregulated (P<0.001), and miR-92 was significantly upregulated (P<0.001) in individuals with DM.

CONCLUSIONS. Our results revealed significant dysregulation of miR-363-5p and miR-92a-3p in INOCA patients with DM compared to those without DM, supporting their role as biomarkers for endothelial dysfunction in DM and INOCA.