Korean J Physiol Pharmacol.  2018 Mar;22(2):127-134. 10.4196/kjpp.2018.22.2.127.

Silencing MR-1 attenuates atherosclerosis in ApoE(−/−) mice induced by angiotensin II through FAK-Akt–mTOR-NF-kappaB signaling pathway

• Chen Y
• Cao J
• Zhao Q
• Luo H
• Wang Y
• Dai W

• KMID: 2410102
• DOI: http://doi.org/10.4196/kjpp.2018.22.2.127

Abstract

Myofibrillogenesis regulator-1 (MR-1) is a novel protein involved in cellular proliferation, migration, inflammatory reaction and signal transduction. However, little information is available on the relationship between MR-1 expression and the progression of atherosclerosis. Here we report atheroprotective effects of silencing MR-1 in a model of Ang II-accelerated atherosclerosis, characterized by suppression focal adhesion kinase (FAK) and nuclear factor kappaB (NF-κB) signaling pathway, and atherosclerotic lesion macrophage content. In this model, administration of the siRNA-MR-1 substantially attenuated Ang II-accelerated atherosclerosis with stabilization of atherosclerotic plaques and inhibited FAK, Akt, mammalian target of rapamycin (mTOR) and NF-kB activation, which was associated with suppression of inflammatory factor and atherogenic gene expression in the artery. In vitro studies demonstrated similar changes in Ang II-treated vascular smooth muscle cells (VSMCs) and macrophages: siRNA-MR-1 inhibited the expression levels of proinflammatory factor. These studies uncover crucial proinflammatory mechanisms of Ang II and highlight actions of silencing MR-1 to inhibit Ang II signaling, which is atheroprotective.

Keyword

Angiotensin II; Apolipoprotein E; Atherosclerosis; Myofibrillogenesis regulator-1; NF-κB; siRNA

MeSH Terms

Angiotensin II*
Angiotensins*
Animals
Arteries
Atherosclerosis*
Cell Proliferation
Focal Adhesion Protein-Tyrosine Kinases
Gene Expression
In Vitro Techniques
Macrophages
Mice*
Muscle Development
Muscle, Smooth, Vascular
NF-kappa B
Plaque, Atherosclerotic
RNA, Small Interfering
Signal Transduction
Sirolimus
Angiotensin II
Angiotensins
Focal Adhesion Protein-Tyrosine Kinases
NF-kappa B
RNA, Small Interfering
Sirolimus

Figure

• 

  Fig. 1 Upregulation of MR-1 expression in atherosclerotic aortas of ApoE−/− mice and VSMCs and macrophages. (A) Aortic MR-1 mRNA expression after 4 weeks of Ang II+siRNA or Ang II+siRNA-control treatment. Data are mean±SEM (n=6 per group). (B) Aortic MR-1 protein expression after 4 weeks of Ang II+siRNA or Ang II+siRNA-control treatment. (C) Analysis of MR-1 protein levels of VSMCs and macrophages. (D) MR-1 colocalization with VSMC and macrophages in atherosclerotic plaques (double immunohistochemistry/ Immunofluorescence).

• 

  Fig. 2 siRNA-MR-1 exhibited a significant suppression of atheroma formation in the vascular induced by Ang II. H&E staining quantitative analysis of (A) plaque area, (B) fibrous cap thickness, (C) cap-to-core ratio and (D) picrosirius red staining analysis of collagen area in three groups of mice. At least three independent experiments were performed and the data are presented as the mean±SEM. p<0.05 vs. siRNA-control denotes statistically significant difference. (E) The effect of siRNA-MR-1 on the expression of two macrophage marker genes, CD68 and F4/80 induced by Ang II in the vascular tissues.

• 

  Fig. 3 siRNA-MR-1 inhibited vascular and cellular inflammatory gene expression and matrix metalloproteinase MMP-2 induced by Ang II. (A) Western blot analysis of TNF-α, MCP-1 and MMP-2 protein expression in aortas (n=6). (B) Western blot analysis of TNF-α, MCP-1 and MMP-2 protein expression in VSMCs (n=6). Top, representative blots; bottom, quantitative results. Values are means±SEM. *p<0.05 vs. siRNA-control injected groups and untreated groups induced by Ang II. Experiments were repeated at least three times.

• 

  Fig. 4 Silencing MR-1 inhibites inflammatory signaling pathways in lesions of ApoE−/− mice induced by Ang II. (A) siRNA-MR-1 markedly reduced the levels of p-FAK, p-Akt, p-mTOR and p-p65 in artery wall induced by Ang II compared with the siRNA-control group. (B) Rapamycin inhibited p65 phosphorylation and nuclear translocation in VSMCs. Western blot analysis of p65 translocation and phosphorylation in cytoplasmic extracts (CE) and nuclear extracts (NE) of 3 groups of VSMCs. (C) The phosphorylation of Akt was not affected by rapamycin. Each assay was done in triplicate.

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