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. 2015:2015:497280.
doi: 10.1155/2015/497280. Epub 2015 Jun 28.

miR-126 Is Involved in Vascular Remodeling under Laminar Shear Stress

Ana Mondadori dos Santos  1 , Laurent Metzinger  2 , Oualid Haddad  3 , Eléonore M'baya-Moutoula  4 , Fatiha Taïbi  4 , Nathalie Charnaux  3 , Ziad A Massy  5 , Hanna Hlawaty  3 , Valérie Metzinger-Le Meuth  6
Affiliations

miR-126 Is Involved in Vascular Remodeling under Laminar Shear Stress

Ana Mondadori dos Santos et al. Biomed Res Int. 2015.

Abstract

Morphology and changes in gene expression of vascular endothelium are mainly due to shear stress and inflammation. Cell phenotype modulation has been clearly demonstrated to be controlled by small noncoding micro-RNAs (miRNAs). This study focused on the effect of laminar shear stress (LSS) on human endothelial cells (HUVECs), with an emphasis on the role of miRNA-126 (miR-126). Exposure of HUVECs in vitro to LSS modified the shape of HUVECs and concomitantly regulated the expression of miR-126, vascular cell adhesion molecule 1 (VCAM-1), and syndecan-4 (SDC-4). A significant upregulation of miR-126 during long-term exposure to flow was shown. Interestingly, LSS enhanced SDC-4 expression on the HUVEC membranes. Overexpression of miR-126 in HUVECs decreased the levels of targets stromal cell-derived factor-1 SDF-1/CXCL12 and VCAM-1 but increased the expression of RGS16, CXCR4, and SDC-4. No significant difference in terms of cell proliferation and apoptosis was observed between scramble, anti-miR-126, and pre-miR-126 transfected HUVECs. In Apo-E KO/CKD mice aortas expressing a high level of miR-126, SDC-4 was concomitantly increased. In conclusion, our results suggest that miR-126 (i) is overexpressed by long-term LSS, (ii) has a role in up- and downregulation of genes involved in atherosclerosis, and (iii) affects SDC-4 expression.

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Figures

Figure 1
Figure 1
LSS alters the morphology of HUVEC cells. Representative phase contrast (A–C) or fluorescence (A1–C2) photographs of endothelial cells. HUVECs were grown under static conditions (A–A2) or LSS conditions for 10 min (B–B2) or 24 h (C–C2). Cells were then stained with Phalloidin (F-actin: red) and DAPI (Nucleus: blue). Global distribution of F-actin and cellular shape was analyzed for static conditions (A1), 10 min of LSS (B1) or 24 h of LSS (C1). High resolution zoom images of F-actin distribution in endothelial cells (A2-C2). Direction of flow is indicated by white arrows. For A–C scale bar = 40 μm, for A1–C1 scale bar = 20 μm, and for A2–C2 scale bar = 5 μm.
Figure 2
Figure 2
LSS induces SDC-1, SDC-4, and VCAM-1 expression in HUVECs. (a) Representative immunofluorescence staining of SDC-1, SDC-4, and VCAM-1 protein on endothelial cells cultured under static or LSS conditions for 10 min and 24 h. HUVEC cells were immunolabeled with anti-SDC-1 (A1–3), anti-SDC-4 (B1–3), and anti-VCAM-1 (C1–3) antibodies (green) or with their respective control isotypes. The nuclei were stained with DAPI (blue). High resolution zoom images of cells were added to each figure. Scale bar = 20 μm. (b) Immunofluorescence quantification of proteins normalized with the nuclei number (n = 3) was done and 5 different fields in HUVECs submitted to static conditions and LSS (10 min and 24 h) were counted and presented as fluorescence intensity/nuclei (AU: arbitrary units). A progressive increase of SDC-1 and SDC-4 expression up to 24 h and an increase at 10 min and then a decrease of VCAM-1 expression were observed. One-way Global ANOVA and Student's t-test, for SDC-1: ∗ P < 0.05 (10 min of LSS versus static), ∗∗ P < 0.001 (24 h of LSS versus static); for SDC-4: ∗ P < 0.05 (10 min of LSS versus static), ∗ P < 0.05 (24 h of LSS versus static); for VCAM-1: ∗∗ P < 0.001 (10 min of LSS versus static), ∗ P < 0.05 (24 h of LSS versus static).
Figure 3
Figure 3
LSS increases KLF-2 mRNA expression and regulates miRNA expression in HUVECs. HUVECs cells were exposed to various durations of laminar shear stress (10 min and 24 h). (a) LSS increased KLF-2 mRNA expression. Values are expressed as mean ± SD of 3 experiments. (b) 24 h of LSS increased miR-126 levels in HUVECs. Values are expressed as mean ± SD of 3 independent experiments (AU: arbitrary units). Student's t-test, for KLF-2: ∗∗ P < 0.001 (10 min of LSS versus static), ∗ P < 0.05 (24 h of LSS versus static); for miR-126: ∗ P < 0.05 (10 min of LSS versus static), ∗ P < 0.05 (24 h of LSS versus static).
Figure 4
Figure 4
Study of HUVEC transfection after 48 h with scramble, anti-miR-126, and pre-miR-126 under static conditions. (a) Confirmation of miR-126 overexpression and knockdown under the respective conditions. Values are expressed as mean ± SD of 3 independent experiments (AU: arbitrary units). Student's t-test, for miR-126: ∗∗∗ P < 0.0001 (scramble versus anti-miR-126), ∗∗∗ P < 0.0001 (scramble versus pre-miR-126). (b) HUVEC metabolic activity was measured using MTT test after HUVEC transfection. Representative flow cytometry histogram study of apoptosis from HUVECs. (c) Expression of various mRNA targets in HUVECs after 48 h of transfection with anti-miR-126 and pre-miR-126. Values are expressed as mean ± SD of 3 independent experiments (AU: arbitrary units). Student's t-test, for VCAM-1: ∗∗∗ P < 0.0001 (scramble versus anti-miR-126), ∗ P < 0.05 (scramble versus pre-miR-126); for RGS-16, SDC-4, CXCR4: ∗ P < 0.05 (scramble versus anti-miR-126), ∗ P < 0.05 (scramble versus pre-miR-126); for CXCL12: ∗ P < 0.05 (scramble versus anti-miR-126), ∗∗ P < 0.001 (scramble versus pre-miR-126).
Figure 5
Figure 5
miR-126, miR-126, and SDC-4 expression in control and atherosclerotic mice. Twenty-week-old mice aortas were isolated from wild-type (WT) and Apo-E KO mice for miRNA and mRNA studies at the indicated times. CKD mice were subjected to 10 weeks of uremia. (a) miR-126 expression expressed as RQ normalized to U6. (b) SDC-4 expression normalized to GAPDH. Values are expressed as mean ± SD of 3 independent experiments (AU: arbitrary units). Student's t-test, for miR-126: ∗ P < 0.05 (WT sham versus Apo-E CKD); for SDC-4: ∗ P < 0.05 (WT sham versus WT CKD), ∗ P < 0.05 (WT sham versus Apo-E CKD).
Figure 6
Figure 6
Representative network of miR-126 in HUVECs submitted to LSS. LSS induces at short-term the expression of transcription factor, KLF-2, and at long-term miR-126 expression. miR-126 downregulates the endothelial adhesion molecule VCAM-1 and chemokine SDF-1/CXCL12. Loss of VCAM-1 and SDF-1/CXCL12 can be associated with decrease of leukocyte homing over the endothelium. miR-126 overexpression enhanced the SDC-4 which can induce the transduction pathway leading to remodeling of F-actin cytoskeleton and favors cell adhesion and spreading. VCAM-1: vascular cell adhesion molecule-1; miR-126: small noncoding micro-RNA-126; SDF-1/CXCL12: stromal cell-derived factor-1; KLF-2: Krüppel-like factor-2; SDC-4: syndecan-4.
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