Different histological types of active intraplaque calcification underlie alternative miRNA-mRNA axes in carotid atherosclerotic disease

Arterial calcification is an actively regulated process, with different morphological manifestations. Micro-RNAs emerged as potential regulators of vascular calcification; they may become novel diagnostic tools and be used for a finest staging of the carotid plaque progression. The present study aimed at characterizing the different miRNA-mRNA axes in carotid plaques according to their histological patterns of calcification. Histopathological analysis was performed on 124 retrospective carotid plaques, with clinical data and preoperatory angio-CT. miRNA analysis was carried out with microfluidic cards. Real-time PCR was performed for selected miRNAs validation and for RUNX-2 and SOX-9 mRNA levels. CD31, CD68, SMA, and SOX-9 were analyzed by immunohistochemistry. miRNA levels on HUVEC cells were analyzed for confirming results under in vitro osteogenic conditions. Histopathological analysis revealed two main calcification subtypes of plaques: calcific cores (CC) and protruding nodules (PN). miRNA array and PCR validation of miR-1275, miR-30a-5p, and miR-30d indicated a significant upregulation of miR-30a-5p and miR-30d in the PN plaques. Likewise, the miRNA targets RUNX-2 and SOX-9 resulted poorly expressed in PN plaques. The inverse correlation between miRNA and RUNX-2 levels was confirmed on osteogenic- differentiated HUVEC. miR-30a-5p and miR-30d directly correlated with calcification extension and thickness at angio-CT imaging. Our study demonstrated the presence of two distinct morphological subtypes of calcification in carotid atheromatous plaques, supported by different miRNA signatures, and by different angio-CT features. These results shed the light on the use of miRNA as novel diagnostic markers, suggestive of plaque evolution.