Macrophage NLRP3-dependent IL-1β production contributes to aortic fibrosis in heart failure with preserved ejection fraction

Figures

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  Figure 1 HFpEF mouse model generated by HFD and ALD treatment exhibits aortic fibrosis (A) Schematic representation of the HFpEF mouse model. (B) Percentage of the left ventricular ejection fraction. n = 5 mice per group. (C,D) Representative pulsed-wave Doppler (top) and tissue Doppler (bottom) tracings and the ratio between the mitral E wave and e′ wave (E/e′). n = 5 mice per group. (E,F) Representative images of the M mode for the abdominal aorta monitored by ultrasound and analysis of circumferential cyclic strain. n = 5 mice per group. (G) SBP. n = 5 mice per group (31 recordings in the sham group and 27 recordings in the HFD + ALD group). (H‒J) Representative images of Masson and Sirius Red staining of the aortas and quantification of the collagen content. Scale bar: 25 μm. n = 5 mice per group. (K,L) Protein levels of MMP2 and MMP9 in aortas detected by western blot analysis. n = 5 mice per group. (M,N) mRNA levels of MMP2 (M) and MMP9 (N) in aortas detected by real-time PCR. n = 5 mice per group. Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 2 Macrophage markers are upregulated in the aortas from the HFD and ALD treatment groups (A,B) Protein levels of CCL2 in the aortas were detected by western blot analysis. n = 5 mice per group. (C) mRNA levels of CCL2 in aortas detected by RT-PCR. n = 4 mice per group. (D,E) Protein levels of ICAM-1 in aortas were detected by western blot analysis. n = 5 mice per group. (F) mRNA levels of ICAM-1 in aortas detected by RT-PCR. n = 4 mice per group. (G) Representative immunofluorescent staining images of ICAM-1-positive aortic endothelial cells. Scale bar: 20 μm. n = 4 mice per group. (H,I) Protein levels of CD68 in the aortas were detected by western blot analysis. n = 5 mice per group. (J‒M) Representative images of immunofluorescence staining and quantification of CD68, CD4, and CD20 in the aortas. Scale bar: 20 μm. n = 5 mice per group. Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 3 Macrophage NLRP3 inflammasome activation results in IL-1β overproduction in the aortas of mice treated with HFD and ALD (A,B) mRNA levels of pro-IL-1β (A) and NLRP3 (B) in the aortas detected by RT-PCR. n = 4 mice per group. (C,D) Protein levels of NLRP3, cleaved caspase-1, pro-IL-1β, and IL-1β in the aortas were detected by western blot analysis. n = 5 mice per group. (E) Bone marrow transplant (BMT) strategy. (F,G) Representative images and analysis of the percentage of green fluorescence in blood cells detected by flow cytometry for evaluating BMT efficiency. n = 5 mice per group. (H,I) Protein levels of NLRP3, cleaved caspase-1, pro-IL-1β, and IL-1β in the aortas were detected by western blot analysis. n = 5 mice per group. Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 4 NLRP3 deficiency in macrophages attenuates aortic fibrosis and improves cardiac diastolic dysfunction (A,B) Representative images of the M mode for the abdominal aorta monitored by ultrasound and analysis of circumferential cyclic strain. n = 5 mice per group. (C) SBP. n = 5 mice per group (38 recordings in the H11-G/R BM group and 32 recordings in the NLRP3–/– BM group). (D‒F) Representative images of Masson and Sirius Red staining of the aorta and quantification of the collagen content. Scale bar: 25 μm. n = 5 mice per group. (G‒J) Protein levels of MMP2 (G‒H) and ICAM-1 (I,J) in the aortas detected by western blot analysis. n = 5 mice per group. (K) Percentage of the left ventricular ejection fraction. n = 5 mice per group. (L,M) Representative pulsed-wave Doppler (top) and tissue Doppler (bottom) tracings and ratios between the mitral E wave and e′ wave (E/e′). n = 5 mice per group. Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01.