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.
KeywordsAtheromatous disease Calcification Endothelial cells miRNA RUNX-2 SOX-9
Francesco Vasuri: study design, data analysis, histopathological analysis, paper drafting.
Carmen Ciavarella: study design, data analysis, performed experiments, paper drafting.
Silvia Fittipaldi: study design, performed experiments.
Rodolfo Pini: angio-CT, data analysis.
Andrea Vacirca: clinical data collection and analysis.
Mauro Gargiulo: clinical data collection, paper critical revision.
Gianluca Faggioli: angio-CT, paper critical revision.
Gianandrea Pasquinelli: study design, paper critical revision, paper drafting.
All authors gave substantial contributions to the conception or design of the work, or the acquisition, analysis, or interpretation of data for the work; drafted the work or revised it critically for important intellectual content; gave final approval of the final version to be published; agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Compliance with ethical standards
The present work was performed on archival tissue of human subjects, in compliance with Ethical standard. All patients are anonymous.
Conflict of interest
The authors declare that they have no conflict of interest.
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