The Apolipoprotein A-I Mimetic L-4F Attenuates Monocyte Activation and Adverse Cardiac Remodeling after Myocardial Infarction

Figures

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  Figure 1. L-4F attenuates left ventricular (LV) remodeling after reperfused myocardial infarction (MI). (A) Representative long axis B-Mode echocardiographic images at end-diastole and the corresponding group data. Serial images were acquired at 3- and 8-day post ischemia/reperfusion. Mice were either injected with PBS or 100 μg/day L-4F for 5 days starting at 3-day post-MI. The dotted line marks the LV cavity. (B) Echocardiographic group data depicting changes (Δ) in end-diastolic and end-systolic volume (EDV and ESV) and ejection fraction (EF) from 3- to 8-days post-MI or sham operation with treatment with either PBS or L-4F as in (A) n = 6–8/group, * p < 0.05, ** p < 0.005. NS – not significant.

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  Figure 2. L-4F suppresses blood pro-inflammatory monocyte levels. (A) Gating strategy for flow cytometric evaluation of peripheral blood monocytes. CD45+ cells within the lymphocyte/monocyte gate (P1) were further gated on the basis of CD11b and Ly6C expression. Pro-inflammatory monocytes were identified as CD45+CD11b+Ly6Chi cells while patrolling monocytes were identified as CD45+Cd11b+Ly6Clow cells. (B) Flow cytometric group data of peripheral blood monocytes after L-4F or PBS treatment post-reperfused MI. Data are depicted as percent CD45+ cells. n = 5–8/group, **** p < 0.0005. ns – not significant.

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  Figure 3. L-4F suppresses levels of pro-inflammatory splenic monocytes post-MI. (A) Representative gross images of spleens harvested from sham-operated or MI mice injected with either PBS or L-4F (100 μg/day) for 5 days starting at day 3 post-surgery, and the corresponding gravimetric quantitation. (B) Gating strategy and group data for flow cytometric quantitation of splenic monocytes following L-4F or PBS treatment. Splenic pro-inflammatory and patrolling monocytes were identified as CD45+CD11b+Ly6Chi and CD45+CD11b+Ly6Clow cells, respectively. Data are depicted as percent of CD45+ cells, n = 5–8/group. ** p < 0.005, **** p < 0.0005.

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  Figure 4. L-4F decreases myocardial pro-inflammatory macrophage expansion in infarcted hearts. Left panel: Gating strategy for flow cytometric evaluation of cardiac macrophages. Right panel: Quantitative group data for macrophage abundance in sham-operated and infarcted hearts after PBS or L-4F treatment for 5 days, starting at 3-day post-surgery. Statistical comparisons were performed after logarithmic data transformation to satisfy the normality assumption as described in the text. Data are presented as percent CD45+ cells by flow cytometry, n = 6–8/group, * p < 0.05. ns—not significant.

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  Figure 5. L-4F inhibits M1-macrophage activation and induces macrophage plasticity in vitro. (A) RT-PCR analysis of M1 and M2 macrophage-associated marker genes in mouse peritoneal macrophages polarized in vitro. (B) RT-PCR analysis of M1 and M2 macrophage-associated marker genes from polarized RAW 264.7 cells (upper panels) or polarized mouse peritoneal macrophages (lower panels) after overnight treatment with either PBS or L-4F (50 μg/mL). Data are presented as fold changes (mean ± SD) and compared to non-polarized M0 macrophages. * p < 0.05 vs. respective M0; $ p < 0.05 vs. PBS-treated M1 group, @ p < 0.05 vs. respective M1 and M2 groups, n = 3/group each done in triplicate for RAW 264.7 cells. Thioglycolate-elicited mouse peritoneal macrophages were isolated and pooled from 6 mice and also run in triplicate.

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  Figure 6. L-4F attenuates M1 macrophage-associated metabolic gene profile in polarized macrophages. RT-PCR analysis of M1-macrophage associated metabolic genes from polarized RAW 264.7 cells (upper panels) or polarized mouse peritoneal macrophages (lower panels) after overnight treatment with either PBS or L-4F (50 μg/mL). Data are presented as fold changes (mean ± SD) and compared to non-polarized M0 macrophages. * p < 0.05 vs. respective M0; $ p < 0.05 vs. PBS-treated M1 group, @ p < 0.05 vs. respective M0 and M2 groups, n = 3/group each done in triplicate as in Figure 5.