Genetic deletion of 12/15 lipoxygenase promotes effective resolution of inflammation following myocardial infarction

Abstract

12/15 lipoxygenase (LOX) directs inflammation and lipid remodeling. However, the role of 12/15LOX in post-myocardial infarction (MI) left ventricular remodeling is unclear. To determine the role of 12/15LOX, 8-12 week-old C57BL/6 J wild-type (WT; n = 93) and 12/15LOX^-/- (n = 97) mice were subjected to permanent coronary artery ligation and monitored at day (d)1 and d5 post-operatively. Post-MI d28 survival was measured in male and female mice. No-MI surgery mice were maintained as d0 naïve controls. 12/15LOX^-/- mice exhibited higher survival rates with lower cardiac rupture and improved LV function as compared with WT post-MI. Compared to WT, neutrophils and macrophages in 12/15LOX^-/- mice were polarized towards N2 and M2 phenotypes, respectively, with increased of expression mrc-1, ym-1, and arg-1 post-MI. 12/15LOX^-/- mice exhibited lower levels of pro-inflammatory 12-(S)-hydroperoxyeicosatetraenoic acid (12(S)-HETE) and higher CYP2J-derived epoxyeicosatrienoic acids (EETs) levels. CYP2J-derived 5,6-, 8,9-, 11,12-, and 14,15-EETs activated macrophage-specific hemeoxygenase (HO)-1 marked with increases in F4/80⁺/Ly6C^low and F4/80⁺/CD206^high cells at d5 post-MI in 12/15LOX^-/- mice. In contrast, inhibition of HO-1 led to total mortality in 12/15LOX^-/- mice by post-MI d5. 12/15LOX^-/- mice exhibited reduced collagen density and lower α-smooth muscle actin (SMA) expression at d5 post-MI, indicating delayed or limited fibroblast-to-myofibroblast differentiation. In conclusion, genetic deletion of 12/15LOX reduces 12(S)-HETE and activates CYP2J-derived EETs to promote effective resolution of inflammation post-MI leading to reduced cardiac rupture, improved LV function, and better survival.

Keywords: Heart failure; Lipid mediators; Macrophages; Myocardial infarction; Neutrophils.

Figure 1. 12/15LOX^-/- deletion increased survival and reduced ruptures post-MI

(A) Survival rates post-MI in WT and 12/15LOX^-/- mice analyzed by log-lank test. *p<0.01 vs. WT, n=19 WT (male), n=19 WT (female), n=27 12/15LOX^-/- (male) and n=14 12/15LOX (female). (B) Bar graph representing post-MI ruptures and congestive heart failure in WT and 12/15LOX^-/- male and female mice. (C) Pathway scheme representing generation of lipid mediator from arachidonic acid. (D) Quantitative measurement of plasma 12(S)-HETE by mass spectrometry (LC-MS/MS). n=4; *p<0.05 vs no-MI, ^$p<0.05 vs WT post-MI. (E) Representative chromatograms of 12(S)-HETE at d1 and d5 post-MI. (F) Quantitative measurement of plasma 15(S)-HETE. n=4; *p<0.05 vs WT (G) Quantitative measurement of plasma 5(S)-HETE n=4; *p<0.05 vs WT.

Figure 2. 12/15LOX deletion upregulated cytochrome (450) subfamily CYP2J that promoted epoxyeicosatrienoic acids (EETs) biosynthesis in spleen

(A) Quantification of proinflammatory metabolites in spleens of WT and 12/15LOX^-/- mice at d5 post-MI. (B) Quantification of proresolving metabolites: EETs and EpDPE in WT and 12/15LOX^-/- mice at d5 post-MI. (C) Immunoblot representing CYP2J expression (upper panel) and in LV (lower panel) and spleen at d5 post-MI. n=2-3;*p<0.05 vs WT. (D) Densitometric analysis of CYP2J in LV and spleen normalized to total protein. n=2-3 mice; *p<0.05 vs MI-WT at d5 post-MI. (E) mRNA expression of Ephx2 in LV and spleen at d5 post-MI normalized to HPRT-1; n=4 mice; *p<0.05 vs MI-WT at d5 post-MI. (F) Quantification of HEPEs (5, 9 and 18) in spleens of WT and 12/15LOX-/- mice at d5 post-MI. n=4 mice; *p<0.05 vs MI-WT at d5 post-MI.

Figure 3. 12/15LOX^-/- deletion polarized neutrophils towards proresolving phenotype

(A) Representative neutrophil-stained LV images at 40× with their accompanying 1.25× sections at d1, and d5 post-MI; scale bar = 50 μm n=4. (B) Quantification of the neutrophils stained area in the LV at post-MI d1 and d5. n=4; *p<0.05 vs WT. (C) Venn diagram representing post-MI inflammatory gene expression in LVI regions of 12/15LOX^-/- mice at post-MI-d1. Post-MI-d1 gene expressions are normalized to respective no-MI. Red indicates ↑ed genes. Blue indicates ↓ed genes. Black indicates no change in genes. n=4; p<0.05 vs WT post-MI-d1. (D) Representative fluorescence-activated cell (FACS) sorting dot plots showing increased neutrophils by Ly6G^high strategy in LV mononuclear cells n=4-6 mice. (E) Quantification of CD11b⁺/F4/80^-, CD11b⁺/F4/80⁺, Ly6G⁺ and Ly6G⁺/Ly6C^high LV mononuclear cells post-MI-d1. n=4-6 mice; *p<0.05 vs WT. (F) mRNA expression of IL-6, tnf-α, from Ly6G⁺(N1) cells isolated from LV infarct. n=4mice; *p<0.05 vs WT ^$p<0.05 vs WT at d1 post-MI. (G) mRNA expression of Mrc-1/CD206, Arg-1 and Ym-1 from Ly6G⁺(N2) cells isolated from LV infarct. n=4-6 mice; *p<0.05 vs WT control, ^$p<0.05 vs WT at d1 post-MI.

Figure 4. 12/15LOX^-/- deletion promoted alternative macrophage polarization to resolve inflammation by d5 post-MI

(A) Representative IHC images of LV macrophages (Mac-3 positive) density at d5 post-MI. Representative images at 40× with accompanying 1.25× images scale bar = 50 μm (upper panel). (B) Quantification of % area of macrophages stained area in the LV infarcted zone at d5 post-MI (lower panel). n=4-6 mice; *p<0.05 vs WT. (C) Representative FACS sorting dot plots showing alternative macrophages polarization by F4/80⁺/Ly6C^low strategy in LV mononuclear cells (upper panel). FACs dot plots showing alternative macrophages polarization by F4/80⁺/CD206⁺ strategy in LV mononuclear cells (lower panel). n=4-6 mice. (D) Quantification of CD11b⁺/F4/80⁺, F4/80⁺/Ly6C^low, F4/80⁺/Ly6C^high F4/80⁺/CD206^high and F4/80⁺/CD206^low LV mononuclear cells at d5 post-MI. n=4 mice; *p<0.05 vs WT. (E-G) mRNA expression of Mrc-1/CD206, Arg-1, Ym-1 respectively from CD11b+ cells isolated from LV infarct at d5 post-MI. n=4 mice; *p<0.05 vs WT.

Figure 5. Macrophages derived HO-1 promoted effective resolution of inflammation in 12/15LOX^-/- mice

(A) Immunoblot representing 5-LOX and HO-1 expression in LVI. (B) Densitometric analysis showing increase 5LOX and HO-1; n=3 mice for no-MI; n=4 mice post-MI; *p<0.05 vs no-MI, ^$p<0.05 vs MI-WT at respective day. (C) mRNA expression of Alox-5 and Hmox-1 in LVI. n=4 mice; *p<0.05 vs No-MI, ^$p<0.05vs MI-WT at respective day. (D) Representative LV IHC images showing 5LOX and HO-1 expression at d5 post-MI. (E) Representative immunofluorescence co-localization images (yellow) of HO-1 (green) with Mac-3 (red) (Magnification 10×). (F) Gene expression of M1 and M2 markers. n=4 mice; *p<0.05 vs no-MI, ^$p<0.05vs MI-WT at respective day. (G) Survival curve presenting HO-1 inhibitor (tin protoporphyrin IX) increased mortality in 12/15LOX^-/- mice bylog-lank test *p<0.01.

Figure 6. 12/15LOX deletion attenuated extracellular matrix (ECM) deposition by limiting myofibroblast formation post-MI

(A) Picrosirus red (PSR) stained LV images (40× magnification; scale bar = 50 μm.) with complete LV acquired (1.25× magnification) at d5 post-MI, (B) Representative LV collagen deposition under plane polarized light (right panel). (C) Quantification of PSR stained collagen levels presented as % stained area in 5-6 images/mouse (left panel), n=4-6 mice; *p<0.05 vs WT. (D) Immunoblot representing α-SMA, MMP-9, TIMP-1 and collagen VI expression. (E) Densitometric analysis of α-SMA, MMP-9, TIMP-1 and collagen VI levels normalized to total protein. n=4-6 mice; *p<0.05 vs no-MI, ^$p<0.05 vs MI-WT at respective day. (F) Venn diagram representing post-MI ECM remodeling genes in LVI regions of 12/15LOX^-/- mice. Post-MI d5 gene expressions are normalized to respective no-MI. Red indicates ↑ed genes. Blue indicates ↓ed genes. Black indicates no change in genes. n=4; *p<0.05 vs WT at d5 post-MI. (G) Representative immunofluorescence images indicating reduced α-SMA (myofibroblast) and no change DDR2 (fibroblast) expression in LV of 12/15LOX^-/- at d5 post-MI.

Figure 7

Schematic design indicating lack of 12/15LOX promotes effective resolution of inflammation post-MI in heart failure pathology.