PCSK9 Modulates Macrophage Polarization-Mediated Ventricular Remodeling after Myocardial Infarction - PubMed (original) (raw)

PCSK9 Modulates Macrophage Polarization-Mediated Ventricular Remodeling after Myocardial Infarction

Feifei Wang et al. J Immunol Res. 2022.

Abstract

Background and aims: An increasing number of high-risk patients with coronary heart disease (similar to acute myocardial infarction (AMI)) are using PCSK9 inhibitors. However, whether PCSK9 affects myocardial repair and the molecular mechanism of PCSK9 modulation of immune inflammation after AMI are not known. The present research investigated the role of PCSK9 in the immunomodulation of macrophages after AMI and provided evidence for the clinical application of PCSK9 inhibitors after AMI to improve cardiac repair.

Methods and results: Wild-type C57BL6/J (WT) and PCSK9-/- mouse hearts were subjected to left anterior descending (LAD) coronary artery occlusion to establish an AMI model. Correlation analysis showed that higher PCSK9 expression indicated worse cardiac function after AMI, and PCSK9 knockout reduced infarct size, improved cardiac function, and attenuated inflammatory cell infiltration compared to WT mice. Notably, the curative effects of PCSK9 inhibition were abolished after the systemic depletion of macrophages using clodronate liposomes. PCSK9 showed a regulatory effect on macrophage polarization in vivo and in vitro. Our studies also revealed that activation of the TLR4/MyD88/NF-_κ_B axis was a possible mechanism of PCSK9 regulation of macrophage polarization.

Conclusion: Our data suggested that PCSK9 modulated macrophage polarization-mediated ventricular remodeling after myocardial infarction.

Copyright © 2022 Feifei Wang et al.

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Conflict of interest statement

The authors report no conflicts of interest.

Figures

Figure 1

Figure 1

High expression of PCSK9 after acute myocardial infarction and the relationship between cardiac function. (a, b) Compared with WT control and WT sham group, the mice after AMI have a high level of PCSK9 protein and mRNA. (c) LVEF% and LVIDd measured by echocardiography 7 days after AMI, n = 6. (d) The correlation between the level of PCSK9 protein and cardiac function EF%, LVIDs in the mice after AMI, n = 10. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ns: not significant.

Figure 2

Figure 2

Inhibition of highly expressed PCSK9 reduced infarct size, inflammation, and myocardial fibrosis and improved cardiac function after 7 days of AMI. (a–c) Cardiac function measured by echocardiography, n = 5. (d) TTC staining showed the infarct size and quantitative analysis by ImageJ in each group, n = 5. (e) Masson staining for infarct size and myocardial fibrosis. Scale bar = 1 μ_m, n = 5. (f) Quantitative analysis by ImageJ for collagen volume fraction and percentage infarct size of hearts in (e). (g) HE staining for the infract regions in hearts. Scale bar = 50 μ_m, n = 5, and quantification of inflammatory cell infiltration. ∗_P < 0.05; ∗∗_P < 0.01; ∗∗∗P < 0.001; ns: not significant.

Figure 3

Figure 3

Systemic depletion of macrophages reduced the benefits of PCSK9 knockout in cardiac repair after myocardial infarction. (a) After being adaptively fed for 6 days, Cl2MDP or PBS were injected into the tail vein to systemically deplete macrophages. (b) Immunohistochemical staining for F4/80 expression in mouse hearts from Cl2MDP- and PBS-treated mice after myocardial infarction. Scale bar = 50 μ_m, n = 5. Quantitative analysis by ImageJ for F4/80+ cells of myocardium in (b). (c, d) Cardiac function measured by echocardiography after Cl2MDP and PBS treatment in the WT ischemia group, n = 4. (e, f) Cardiac function measured by echocardiography after Cl2MDP and PBS treatment in the PCSK9−/− ischemia group, n = 4. ∗_P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ns: not significant.

Figure 4

Figure 4

PCSK9 knockout inhibited M1 polarization and promoted M2 polarization in myocardial macrophages after infarction. (a) Representative immunofluorescence staining showing the percentages of M1 (F4/80+iNOS+CD206−) and M2 (F4/80+iNOS−CD206+) in WT/PCSK9−/− mouse myocardium after ischemia or sham. Nuclei were counterstained with DAPI. Scale bar = 50 _μ_m, n = 5. Quantitative analysis of the percentage of M1 and M2 macrophages of (a). (b, c) q-PCR analysis of IL-6, iNOS, TGF-β, and CD206 mRNA expression in WT/PCSK9−/− mouse myocardium after ischemia or sham, n = 3. (d) Representative images of Western blots for PCSK9, IL6, iNOS, and TGF-β in WT/PCSK9−/− mouse myocardium after ischemia or sham, n = 3.

Figure 5

Figure 5

In vitro, the exogenous PCSK9 protein induced inflammatory macrophages to acquire the M1 phenotype. The morphologic changes in macrophages stimulated by LPS/IL4. Cell shape changed from round to fusiform in LPS-stimulated RAW264.7 cells to ellipse in IL4-stimulated RAW264.7 cells. Scale bar = 50 μ_m. (b) 0.5 μ_g/mL PCSK9 protein significantly induced IL6 expression in RAW264.7 and have no effect on cell viability. (c) Representative flow cytometry plots showing the percentages of M1 (F4/80+/iNOS+/CD206−) and M2 (F4/80+/iNOS−/CD206+) phenotype in LPS/IL4-stimulated RAW264.7 cells after cocultivation with PCSK9 protein for 24 h, n = 3. Pooled flow cytometry data from (c). (e, f) q-PCR analysis of IL-6, iNOS, TGF-β, and CD206 mRNA expression in LPS/IL4-stimulated RAW264.7 cells after cocultivation with PCSK9 protein for 24 h, n = 3. (g) Representative images of Western blots for IL6, iNOS, and TGF-β in LPS/IL4-stimulated RAW264.7 cells after cocultivation with PCSK9 protein for 24 h, n = 3. Protein levels of IL6, iNOS, and TGF-β of (g). ∗_P < 0.05; ∗∗_P < 0.01; ∗∗∗P < 0.001; ns: not significant.

Figure 6

Figure 6

PCSK9 regulated M1 macrophage polarization by targeting TLR4. Representative images of Western blots for TLR4 and downstream MyD88/NF-_κ_B in WT/PCSK9−/− mouse myocardium after ischemia or sham, n = 3. (b) Protein levels of TLR4 and downstream MyD88/NF-_κ_B of (a). (c) Representative images of Western blots for TLR4 and downstream MyD88/NF-κ_B in LPS/IL4-stimulated RAW264.7 cells after cocultivation with PCSK9 protein for 24 h, n = 3. (d) Protein levels of TLR4 and downstream MyD88/NF-κ_B of (c). (e)TLR4 inhibitor (TAK242) was used to analyze whether TLR4 is involved in PCSK9-regulated macrophage polarization; (f) protein levels of IL6, iNOS, TLR4, and downstream MyD88/NF-κ_B of (e). ∗_P < 0.05; ∗∗_P < 0.01; ∗∗∗_P < 0.001; ns: not significant.

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