Involvement of miR-30a-5p and miR-30d in Endothelial to Mesenchymal Transition and Early Osteogenic Commitment under Inflammatory Stress in HUVEC.

Carmen Ciavarella,Francesco Vasuri,Silvia Fittipaldi,Gianandrea Pasquinelli,Mauro Gargiulo,Daniela Pollutri,Ilenia Motta,Sabrina Valente,Francesca Ricci

doi:10.3390/biom11020226

Carmen Ciavarella, Francesco Vasuri + Show 7 more

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https://doi.org/10.3390/biom11020226

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Abstract

The endothelial to mesenchymal transition (End–MT) can be associated with vascular calcification, by providing mesengenic progenitors. In this study, we investigated a link between End–MT and the osteogenic process and explored the involvement of miR-30a-5p and miR-30d as potential regulators of these processes. End–MT was induced in Human Umbilical Vein Endothelial Cells (HUVEC) through transforming growth factor-β1 (TGF-β1), TGFβ-3 and tumor necrosis factor-α (TNF-α), for 24 h and 6 days. End–MT mediators, mesenchymal and osteo/chondrogenic markers were analyzed through Real-Time PCR, immunofluorescence, flow cytometry and Western Blot. miR-30a-5p and miR-30d over-expression was carried out in HUVEC to explore their effects on End–MT and osteogenic differentiation. HUVEC at 24 h and 6 days gained mesenchymal morphology markers, including matrix metalloproteinase 9 (MMP-9), SLUG, VIMENTIN and α-smooth muscle actin (α-SMA), and a significant migratory potential, notably with TNF-α. After 6 days, the osteo/chondrogenic markers runt-related transcription factor 2 (RUNX-2) and SRY box transcription factor 9 (SOX-9) were upregulated. At this time point, miR-30a-5p and miR-30d decreased. Over-expression of miR-30a-5p and miR-30d affected End–MT mediators and the osteogenic potency in HUVEC, by reducing SLUG, VIMENTIN and RUNX-2. Our data suggest that End–MT represents a key link between inflammation and vascular calcification. Further, miR-30a-5p and miR-30d can regulate both the End–MT and the osteogenic processes, prompting future studies for exploring their potential use as therapeutic targets or biomarkers in vascular diseases.

Highlights

This article is an open access articleEndothelial cells (ECs) constitute the inner cell layer within the blood vessels and are primarily involved in vascular homeostasis, regulating blood flow, hemostasis, blood cells’ luminal adherence and vascular permeability [1]
In order to study the endothelial to mesenchymal transition (End–MT) occurring under pathological conditions in endothelial cells, we explored the effects of different soluble factors reproducing the atherosclerotic milieu in Human Umbilical Vein Endothelial Cells (HUVEC) for 24 h and 6 days
We analyzed the expression of the endothelial gene VE-cadherin that was reduced only in cells treated with transforming growth factor-β1 (TGF-β1) (0.28 ± 0.07, p = 0.0003) and transforming growth factor (TGF)-β3 (0.63 ± 0.3) (Figure 1C)

Summary

Introduction

This article is an open access articleEndothelial cells (ECs) constitute the inner cell layer within the blood vessels and are primarily involved in vascular homeostasis, regulating blood flow, hemostasis, blood cells’ luminal adherence and vascular permeability [1]. The endothelial dysfunction is considered the triggering event in the initiation of atherosclerotic disease [2], and it is associated with the loss of the EC barrier, allowing the recruitment of monocytes and the distributed under the terms and conditions of the Creative Commons. Biomolecules 2021, 11, 226 infiltration of low-density lipoproteins (LDLs) within the intima. Oxidation of LDLs is followed by foam cell generation, a powerful stimulus initiating the inflammatory cascade. Smooth muscle cells (SMCs) are induced to migrate and proliferate into the intima, forming the major component of the atherosclerotic plaque. Arterial calcification is commonly associated with vascular disease, and currently the coronary calcification score is used to define the burden of atherosclerotic disease

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