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. 2025 Sep 25;26(19):9349.
doi: 10.3390/ijms26199349.

Identification of miR136, miR155, and miR183 in Vascular Calcification in Human Peripheral Arteries

Tom Le Corvec  1   2 , Mathilde Burgaud  3 , Marja Steenman  2 , Robel A Tesfaye  4 , Yann Gouëffic  5 , Blandine Maurel  1   2 , Thibaut Quillard  2
Affiliations

Identification of miR136, miR155, and miR183 in Vascular Calcification in Human Peripheral Arteries

Tom Le Corvec et al. Int J Mol Sci. .

Abstract

Vascular calcification (V) is an independent risk factor for all-cause and cardiovascular mortality. Vascular smooth muscle cells (VSMCs) play a major role in VC as they can acquire mineralizing properties when exposed to osteogenic conditions. Despite its clinical impact, there are still no dedicated therapeutic strategies targeting VC. To address this issue, we used human calcified and non-calcified atherosclerotic arteries (ECLAGEN Biocollection) to screen and identify microRNA (miR) associated with VC. We combined non-biased miRNomic (microfluidic arrays) and transcriptomic analysis to select miR candidates and their putative target genes with expression associated with VC and ossification. We further validated miR functional regulation and function in relation to cell mineralization using primary human VSMCs. Our study identified 12 miRs associated with VC in carotid and femoral arteries. Among those, we showed that miR136, miR155, and miR183 expression were regulated during VSMC mineralization and that overexpression of these miRs promoted VSMC mineralization. Cross-analysis of this miRNomic and a transcriptomic analysis led to the identification of CD73 and Smad3 pathways as putative target genes responsible for mediating the miR155 pro-mineralizing function. These results highlight the potential benefit of miR155 inhibition in limiting VC development in peripheral atherosclerotic arteries.

Keywords: atherosclerosis; miRNAs; vascular calcification; vascular smooth muscle cells.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
miRs associated with VC and ossification. Volcano plot graphs and tables representing fold changes of miRs differentially expressed between calcified and non-calcified pathological carotid arteries (CA) (n = 20 patients) (A) and between ossified and non-ossified femoral arteries (FA) (n = 20 patients) (B). CA = Carotid Arteries; FA = Femoral Arteries; OM = Osteoid Metaplasia.
Figure 2
Figure 2
Expression and regulation of miRs during VSMC mineralization in vitro. miR expression levels during VSMC mineralization induced by inorganic phosphate (3 mM) (A) and osteogenic medium (B) for each miR candidate: 127, 136, 155, and 183. Bars represent means ± SEMs (* p < 0.05). Pi = Inorganic Phosphate; OB = Osteogenic Medium; NT = Non-Treated.
Figure 3
Figure 3
Functional impact of miR candidates on human VSMC mineralization. (A) miR136, -155, and -183 overexpression in VSMCs 7 days after transfection of individual miR mimics. (B) Representative images of Alizarin red staining for mineralization (in red) of VSMCs 7 days after inducing miR overexpression (negative control, mir136, miR155, and miR183 mimics) in inorganic phosphate-enriched medium (3 mM) and quantification of the mineralized area percentage (stained area/total area) between conditions. (C) Transcriptional regulation of osteoblastic (in green) and contractile-associated genes (in red) 7 days after inducing miR overexpression compared to a mimic negative control. Bars represent means ± SEMs (* p < 0.05). (D) Representative Western blotting for contractile-associated proteins 7 days after miR mimic transfection. NT = Non-Treated.
Figure 4
Figure 4
Genes associated with VC. Volcano plot graphs representing fold changes of genes differentially expressed between calcified and non calcified pathological carotid arteries (A) and between ossified and non-ossified pathological femoral arteries (n= 20) (B). (C) REVIGO scatterplot of their gene ontology annotations (biological functions) highlighting their contributing roles in the biomineralization process. CA = Carotid Arteries; FA = Femoral Arteries; OM = Osteoid Metaplasia.
Figure 4
Figure 4
Genes associated with VC. Volcano plot graphs representing fold changes of genes differentially expressed between calcified and non calcified pathological carotid arteries (A) and between ossified and non-ossified pathological femoral arteries (n= 20) (B). (C) REVIGO scatterplot of their gene ontology annotations (biological functions) highlighting their contributing roles in the biomineralization process. CA = Carotid Arteries; FA = Femoral Arteries; OM = Osteoid Metaplasia.
Figure 5
Figure 5
Putative targets for miR155. (A) Putative interaction sites between miR155 and Smad3 (NCBI gene ID: 4088) and CD73 (NCBI gene ID: 5167). (B) Smad3 and CD73 protein and mRNA levels 2 days following overexpression of indicated miR mimics. Bars represent means ± SEMs (* p < 0.05).
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