Circulating miR-6821-5p levels and coronary calcification in asymptomatic familial hypercholesterolemia patients

Background and aims: Premature atherosclerotic cardiovascular disease (CVD) is a clinic characteristic of familial hypercholesterolemia (FH). Coronary calcium score (CCS) is a highly used imaging modality to evidence atherosclerotic plaque burden. microRNAs (miRNAs) are non-coding RNAs that epigenetically regulate gene expression. Here, we investigated whether CCS associates with a specific miRNA-signature in FH-patients. Methods: Patients with genetic diagnosis of FH (N = 86) from the nationwide SAFEHEART-cohort were investigated by computed tomography angiography imaging and classified depending on the presence of coronary calcification in FH-CCS (+) and FH-CCS (-) groups by the Agatston score. Differential miRNA profiling was performed in two stages: first by Affymetrix microarray technology (high-throughput differential profilingstudies) and second by RT-PCR using TaqMan-technology (analytical RT-qPCR study) in plasma of the two patient groups. Results: miR-193a-5p, miR-30e-5p and miR-6821-5p levels were significantly higher in FH-CCS (+) compared to FH-CCS (-). miR-6821-5p was the best miRNA to discriminate FH-patients CCS(+), according to receiver operating characteristic (ROC) analysis (AUC: 0.70 +/- 0.06, p = 0.006). High miR-6821-5p levels were associated with older age (p = 0.03) and high LDL-burden (p = 0.014) using a ROC-derived cut-off value. However, miR6821-5p did not correlate with age in either the CCS- or CCS + group. Genes involved in calcification processes were identified by in silico analysis. The relation of cell-calcification and expression levels of miR-6821-5p, BMP2 and SPP1 was validated experimentally in human vascular smooth muscle cell cultures. Conclusions: Up-regulated levels of miR-6821-5p are found in the plasma of asymptomatic FH-patients with coronary calcified atherosclerotic plaques, as well as in isolated human vascular smooth muscle cells expressing the pro-calcification genes BMP2 and SPP1. These findings highlight the impact of epigenetic regulation on the development of subclinical atherosclerosis.