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Published on: September 26, 2018
PEP-1-MsrA ameliorates inflammation and reduces atherosclerosis in apolipoprotein E deficient mice
Yao Wu1, Guanghui Xie2, Yanyong Xu3
1Department of Biochemistry and Molecular Biology, Hubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan University School of Basic Medical Sciences, 185 Donghu Road, Bldg. 2, 2-209, Wuhan, 430071, Hubei, China. isabel_wuyao@whu.edu.cn.
Background:
Methionine sulfoxide reductase A (MsrA) is a potent intracellular oxidoreductase and serves as an essential factor that protects cells against oxidative damage. However, therapeutic use of exogenous MsrA in oxidative stress-induced diseases is limited, because it cannot enter the cells. The aim of this study is to investigate whether MsrA with PEP-1, a cell penetrating peptide, fused to its N-terminus can protect against oxidative stress in macrophages and can attenuate atherosclerosis in apolipoprotein E deficient (apoE(-/-)) mice.
Methods:
MsrA and the fusion protein PEP-1-MsrA were expressed and purified using a pET28a expression system. Transduction of the fusion protein into macrophages was confirmed by Western blot and immunofluorescence staining. Intracellular reactive oxygen species (ROS) and apoptosis levels were measured by flow cytometry. In in vivo study, MsrA or PEP-1-MsrA proteins were intraperitoneally injected into apoE(-/-) mice fed a Western diet for 12 weeks. Plasma lipids levels, inflammatory gene expression, and paraoxonase-1 (PON1) and superoxide dismutase (SOD) activities were assessed. Atherosclerotic lesions were analyzed by Oil Red O staining and immunohistochemistry.
Results:
PEP-1-MsrA could penetrate the cells and significantly reduced intracellular ROS levels and apoptosis in H2O2-treated macrophages. It also decreased TNFα and IL-1β mRNA levels and increased the IL-10 mRNA level in lipopolysaccharide-treated macrophages. In in vivo study, PEP-1-MsrA injection significantly increased plasma PON1 and SOD activities and decreased plasma monocyte chemoattractant protein 1 (MCP-1) level compared to the injection of vehicle control or MsrA. In PEP-1-MsrA injected mice, hepatic PON1 levels were increased, while the expression of TNFα and IL-6 mRNA in the liver was suppressed. Although plasma total cholesterol and triglyceride levels did not change, the aortic atherosclerosis in PEP-1-MsrA treated mice was significantly reduced. This was accompanied by a reduction of total and apoptotic macrophages in the lesions.
Conclusion:
Our study provides evidence that PEP-1-MsrA may be a potential therapeutic agent for atherosclerosis-related cardiovascular diseases.
Insights
This study shows that PEP-1-MsrA, a fusion protein, effectively penetrates cells and reduces oxidative stress and inflammation. It significantly attenuates atherosclerosis in mice, suggesting its potential as a therapeutic agent for cardiovascular diseases.
Area of Science:
- Biochemistry
- Molecular Biology
- Cardiovascular Research
Background:
- Methionine sulfoxide reductase A (MsrA) protects cells from oxidative damage.
- Exogenous MsrA is limited in therapeutic use due to poor cell penetration.
- PEP-1 peptide enhances cell penetration for therapeutic applications.
Purpose of the Study:
- To investigate the cell-penetrating ability of PEP-1-MsrA.
- To evaluate the protective effects of PEP-1-MsrA against oxidative stress in macrophages.
- To assess the therapeutic potential of PEP-1-MsrA in attenuating atherosclerosis in apoE(-/-) mice.
Main Methods:
- PEP-1-MsrA fusion protein expression and purification.
- Confirmation of cellular transduction via Western blot and immunofluorescence.
- Measurement of intracellular reactive oxygen species (ROS) and apoptosis by flow cytometry.
- In vivo studies involving intraperitoneal injection of proteins into apoE(-/-) mice.
- Assessment of plasma lipids, inflammatory markers, antioxidant enzyme activities (PON1, SOD), and atherosclerotic lesions.
Main Results:
- PEP-1-MsrA demonstrated significant cell penetration, reducing ROS and apoptosis in macrophages.
- It modulated inflammatory cytokine mRNA levels (decreased TNFα, IL-1β; increased IL-10).
- In vivo, PEP-1-MsrA increased PON1 and SOD activities, reduced MCP-1, and suppressed hepatic TNFα and IL-6 mRNA.
- Aortic atherosclerosis was significantly reduced in PEP-1-MsrA treated mice, with decreased macrophage infiltration and apoptosis.
Conclusions:
- PEP-1-MsrA effectively penetrates cells and exerts antioxidant and anti-inflammatory effects.
- The fusion protein shows significant therapeutic potential in reducing atherosclerosis.
- PEP-1-MsrA is a promising candidate for treating atherosclerosis-related cardiovascular diseases.
