STAT6 Upregulation Promotes M2 Macrophage Polarization to Suppress Atherosclerosis - PubMed (original) (raw)
STAT6 Upregulation Promotes M2 Macrophage Polarization to Suppress Atherosclerosis
Min Gong et al. Med Sci Monit Basic Res. 2017.
Abstract
BACKGROUND Macrophages are highly heterogeneous and plastic cells that are involved in all stages of atherogenesis. They can undergo polarization by shifting between M1 and M2 functional phenotypes. However, the role of macrophage polarization and the molecular mechanism in modulating atherosclerotic plaque stability remain incompletely understood. Our study investigated the role of STAT6 in regulating macrophage phenotypes to affect atherosclerotic plaque stability. MATERIAL AND METHODS A murine atherosclerosis model with vulnerable plaques was induced with high-cholesterol diet and PCCP surgeries in ApoE-/- mice. Murine macrophages RAW264.7 treated with ox-LDL or IL-4 were used to simulate the in vitro process. pcDNA3.1(-)/STAT6-expressing vectors were transfected into RAW264.7 to evaluate its effect on cell polarization and the involved molecules. RESULTS Unstable plaques presented significantly increased M1 markers (CD86 and iNOS) and less M2 markers (Arg-1 and TGF-β) than the stable plaques. Moreover, we found that STAT6 and p-STAT6 were greatly decreased in the vulnerable plaques and ox-LDL-induced macrophages, while their expression was elevated after IL-4 stimulation. The overexpression of STAT6 substantially reversed the ox-LDL-stimulated macrophage apoptosis and lipid accumulation. STAT6 upregulation promoted the differentiation of macrophage to M2 subtype as reflected by the increased expression of Arg-1 and TGF-β. Furthermore, we found that STAT6 overexpression activated the Wnt-β-catenin signaling by enhancing the translocation of β-catenin, while β-catenin suppression inhibited STAT6 overexpression-induced M2 polarization. CONCLUSIONS STAT6 facilitated atherosclerotic plaque stabilization by promoting the polarization of macrophages to M2 subtype and antagonizing ox-LDL-induced cell apoptosis and lipid deposition in a Wnt-β-catenin-dependent manner.
Conflict of interest statement
Conflicts of interest
All authors declare that they have no conflicts of interest.
Figures
Figure 1
Macrophage polarization in atherosclerotic plaques of ApoE−/− mouse mice model. HCD-fed ApoE−/− mice underwent PCCP surgeries to rapidly induce the formation of vulnerable atherosclerotic plaques. (A) Right common carotid artery lesions were dissected and stained with HE (bar, 100 μm) or Oil Red O (bar, 50 μm) (n=6/group). Gross determination of plaque areas and Oil Red O-stained plaques in the 3 groups. (B) Serum levels of IL-6, TNF-α, total cholesterol, and ox-LDL in the animals. (C) RT-PCR determination of macrophage polarization markers in the plaques of each group. Data are presented as means ± SEM. * P<0.05, vs. control; # P<0.05, vs. HCD group.
Figure 2
Expression of STAT6 in AS plaques and ox-LDL-sensitized macrophages. (A) RT-PCR analysis of STAT6 mRNA levels in plaques of HCD-fed ApoE−/− mice underwent PCCP surgeries or not. (B) Western blot determination of STAT6 and p-STAT6 in the plaques of the animals. ** P<0.01 vs. HCD group; (C) Immunofluorescence staining and quantification for CD163 and STAT6 as well as their co-localization in atherosclerotic plaques of ApoE−/− mice. Bar=50 μm, n=5. (D) Relative STAT6 expression before and after ox-LDL stimulation for 24 h. (E) Western blot analysis of STAT6 and p-STAT6 in the ox-LDL-treated RAW264.7 cells. ** P<0.01 vs. control group.
Figure 3
STAT6 upregulation promoted macrophages polarization towards M2 subtype. (A, B) Effects of IL-4 on the expression of STAT6 and phosphorylated STAT6 determined using RT-PCR or Western blot. * P<0.05, ** P<0.01 vs. Control. (C) pcDNA3.1/STAT6 plasmid and the negative control vector were transfected to RAW264.7 cells for 48 h, the cell surface markers were labeled with anti-CD86 and anti-163 markers, and then were analyzed using flow cytometry. ** P<0.01, vs. STAT6 NC. (D) Representative images of immune labeling for CD163 and STAT6 and their localization in STAT6 siRNA/NC-transfected macrophages followed by treatment with IL-4 for 24 h. Bar=250 μm, ** P<0.01, vs. IL-4+siR-Ctrl.
Figure 4
STAT6 upregulation suppressed ox-LDL-induced foam cell formation and cell apoptosis. (A) The pcDNA3.1/STAT6 or its negative control transfected macrophages were treated with 50 nM ox-LDL, and the lipid accumulation in the cells was determined using Oil Red O staining after 48 h of incubation. (B) Cell apoptosis in response to the ox-LDL was detected by flow cytometry. ** P<0.01, vs. ox-LDL+STAT6 NC.
Figure 5
Wnt-β-catenin signaling was involved in STAT6-stimulated macrophages polarization. (A) Expression and quantification of total and nuclear β-catenin in stable and vulnerable plaques (n=5 for each group) and STAT6-overexpressing macrophages. ** P<0.01, compared with HCD or STAT6 vec. (B) Total and nuclear β-catenin levels in macrophages stimulated with IL-4 (10 ng/ml) for 24 h, * P<0.05, ** P<0.01 vs. control. (C) RT-PCR analysis of M2 macrophages markers in β-catenin silencing cells with STAT6 overexpression, ** P<0.01. (D) β-catenin siRNA or control transfected cells were exposed to IL-4 for 24 h, and the mRNA levels of Arg1 and TGF-β were examined using qRT-PCR, ** P<0.01 vs. control, ## P<0.01 vs. IL-4+NC siRNA.
References
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