This study determined that 1) deletion of 12/15-lipoxygenase (LOX) promotes the generation of epoxyeicosatrienoic acids, the cytochrome P-450-derived metabolites in postmyocardial infarction (post-MI) healing; 2) acute exposure of fatty acids to 12/15-LOX−/− mice drives leukocyte (neutrophils and macrophages) clearance post-MI; and 3) metabolic transformation of fats is the significant contributor in leukocyte clearance to drive either resolving or nonresolving inflammation post-MI.
Keywords: cardiac remodeling, fatty acids, inflammation, lipid mediators, leukocytes, myocardial infarction, neutrophils, polyunsaturated fatty acids, left ventricle, polyunsaturated fatty acids, lipoxygenase
Abstract
The metabolic transformation of fatty acids to form oxylipids using 12/15-lipoxygenase (LOX) can promote either resolving or nonresolving inflammation. However, the mechanism of how 12/15-LOX interacts with polyunsaturated fatty acids (PUFA) in postmyocardial infarction (post-MI) healing is unclear. Here, we reported the role of 12/15-LOX in post-MI cardiac remodeling in a PUFA [10% (wt/wt), 22 kcal]-enriched environment. Wild-type (WT; C57BL/6J) and 12/15-LOX-null (12/15-LOX−/−) male mice of 8–12 wk of age were fed a PUFA-enriched diet for 1 mo and subjected to permanent coronary artery ligation. Post-MI mice were monitored for day 1 or until day 5 along with standard diet-fed MI controls. No-MI surgery mice served as naïve controls. PUFA-fed WT and 12/15-LOX−/− mice improved ejection fraction and reduced lung edema greater than WT mice at day 5 post-MI (P < 0.05). Post-MI, neutrophil density was decreased in PUFA-fed WT and 12/15-LOX−/− mice at day 1 (P < 0.05). Deletion of 12/15-LOX in mice led to increased cytochrome P-450-derived bioactive lipid mediator epoxyeicosatrienoic acids (EETs), i.e., 11,12-EpETrE and 14,15-EpETrE, which were further enhanced by acute PUFA intake post-MI. Macrophage density was decreased in WT + PUFA and 12/15-LOX−/− mice compared with their respective standard diet-fed WT controls at day 5 post-MI. 12/15-LOX−/− + PUFA mice displayed an increased expression of chemokine (C-C motif) ligand 2 and reparative macrophages markers (Ym-1, Mrc-1, and Arg-1, all P < 0.05) in the infarcted area. Furthermore, 12/15-LOX−/− mice, with or without PUFA, showed reduced collagen deposition at day 5 post-MI compared with WT mice. In conclusion, deletion of 12/15-LOX and short-term exposure of PUFA promoted leukocyte clearance, thereby limiting cardiac remodeling and promoting an effective resolution of inflammation.
NEW & NOTEWORTHY This study determined that 1) deletion of 12/15-lipoxygenase (LOX) promotes the generation of epoxyeicosatrienoic acids, the cytochrome P-450-derived metabolites in postmyocardial infarction (post-MI) healing; 2) acute exposure of fatty acids to 12/15-LOX−/− mice drives leukocyte (neutrophils and macrophages) clearance post-MI; and 3) metabolic transformation of fats is the significant contributor in leukocyte clearance to drive either resolving or nonresolving inflammation post-MI.
chronic, low-grade inflammation is the primary factor influencing the severity of coronary artery disease due to fatty streak accumulation, which triggers myocardial infarction (MI). Post-MI, sustained ischemia of cardiac muscle activates the development of nonresolving inflammation that leads to progressive acute and chronic heart failure (2). Despite the clinical advancement of immediate ischemia-reperfusion therapies, 2–17% patients die within 1 yr post-MI due to the failure of resolving inflammation and >50% die within 5 yr. Post-MI, survivors may eventually progress to heart failure, resulting from adverse remodeling in the left ventricle (LV) (37). Although anti-inflammatory drug discovery and immune biology have advanced to a great extent in recent years, the emphasis of current heart failure treatments is focused on β-blockers, mineralocorticoid receptor antagonists, and angiotensin-converting enzyme inhibitors (4). Pathological remodeling is characterized by an uncontrolled inflammatory milieu and profound changes in size, shape, and function of the LV in post-MI patients. The ability to resolve the initial post-MI inflammatory response defines the extent of LV remodeling and post-MI survival.
Post-MI, the lipid-busting enzyme 12/15-lipoxygenase (LOX) catalyzes the incorporation of oxygen into polyunsaturated fatty acids (PUFA) to form differential lipid mediators that are involved in diabetes, atherogenesis, atheroprogression, advanced atherosclerosis, and other inflammatory diseases (8). Like 12/15-LOX, PUFA is also catabolized by two other enzymatic pathways that include cyclooxygenase (COX) to form prostaglandins and cytochrome P (CYP)-450 to form epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids. The lipid-busting 12/15-LOX enzyme catalyzes fatty acid substrate, and the subsequent fatty acid-derived mediators are widely known as major resolution factors in post-MI healing. It has been established that 12/15-LOX gene expression is upregulated during the progression of heart failure (27). Recent studies have confirmed that deletion of 12/15-LOX enzymes stimulates the effective resolution of inflammation post-MI compared with C57BL/6J wild-type (WT) mice (24). Compared with WT mice, effective resolution in 12/15-LOX-null mice was marked with the early clearance of neutrophils, limited fibrotic remodeling, and increased rates of survival, indicating that 12/15-LOX promotes the progression toward chronic heart failure (25). Our recent studies suggest that excess and long-term supply of n-6 PUFA leads to the development of the nonresolving chemokines milieu in the bone marrow, infarcted LV, and spleen, which promote inflammation (12, 15).
The next logical question was to test whether acute (1 mo) exposure to excess amounts of n-6 PUFA either stimulate or repress post-MI immune kinetics, thereby improving or impairing LV healing. Despite the essential nature of n-6 PUFA, the optimal intakes are the focus of much debate regarding their positive and negative effects in cardiovascular health. A recent epidemiological study revealed that linoleic acid, and not other n-6 PUFAs, was inversely associated with total and coronary heart disease (CHD) mortality in older adults (48). Many reports indicate that n-6 PUFA competes for LOX enzymes with n-3 PUFA [docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)], which have cardioprotective effects (29). Excess dietary intake of n-6 PUFA (particularly linoleic acid) increases collagen type I-to-III ratios, resulting in cardiac stiffening in mice (3). We selected the PUFA diet instead of saturated fat for several key reasons: 1) n-6 PUFA are predominant in a Western diet (28); 2) negative effects of dietary n-6 PUFA intake have been theorized to stem from their tendency to compete with n-3 PUFA for linoleic acid, thus blunting their cardiovascular benefits (1); and 3) the American Heart Association Science Advisory Committee suggests that increasing intakes of n-6 PUFA to at least 5–10% of energy consumption reduces the risk of CHD and that reducing n-6 PUFA intakes from current levels is more likely to increase risk of CHD than decrease (17). Conflicting reports of the effects of dietary n-6 PUFA intake on cardiovascular health highlight the need for mechanistic research in acute and chronic heart failure pathology. Using 12/15-LOX-null mice, we determined the interactions and acute effects of n-6 PUFA diet on post-MI leukocyte kinetics with a major emphasis on the resolution of inflammation (leukocytes clearance). Our quantitative results of the CYP-derived lipid metabolites that are generated in the absence of 12/15-LOX provided a novel outcome in post-MI heart failure pathology.
METHODS
Animals.
All animal surgery procedures and treatments were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th ed., 2011) and AVMA Guidelines for the Euthanasia of Animals (2013 ed.), and the animal protocol was approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham. For this study, 8- to 12-wk-old C57BL/6 (WT) and 12/15-LOX−/− mice of the C57BL/6 genetic background were obtained from The Jackson Laboratory (Bar Harbor, ME) and were maintained under constant temperature (19.8–22.2°C). Mice were given free access to water and standard chow diet before dietary intervention. The total numbers of mice used in this experiment were as follows: WT, 17; WT + PUFA, 14; 12/15-LOX−/−, 20; and 12/15-LOX−/−+PUFA, 15, for a total of n = 66 mice.
Acute PUFA diet exposure.
Before coronary artery ligation surgery, WT and 12/15-LOX−/− mice in the experimental PUFA groups were fed a 10% (wt/wt) safflower oil (SO; 22% kcal) diet, which is enriched with linoleic acid (n-6 PUFA), for a period of 4 wk. The customized SO-enriched diet was manufactured at Research Diets, and the diet was composed of approximately 15% (wt/wt) protein, 66% (wt/wt) carbohydrate, and 10% (wt/wt) SO ω-6 enriched diet. After completion of a 4-wk diet, mice were then subjected to permanent coronary ligation surgery, as presented in the study design (Fig. 1A) with respective outcomes of leukocyte, cytokines, and lipidomics parameters.
Fig. 1.
12/15-Lipoxygenase (LOX) deletion and polyunsaturated fatty acid (PUFA) intake increased neutrophils clearance postmyocardial infarction (post-MI). A: study design scheme and MI surgery plan for wild-type (WT) and 12/15-LOX−/− mice fed a PUFA or standard control diet for 1 mo. B: representative neutrophil immunohistochemical (IHC) images of left ventricular (LV) transverse sections of WT and 12/15-LOX−/− mice supplemented with standard or PUFA diet at day 1 (d1) and day 5 (d5) post-MI (magnification: ×40, scale = 50 µm). C: quantification of the neutrophil-stained area in the LV at day 1 and day 5 post-MI in WT and 12/15-LOX−/− mice supplemented with standard and PUFA diet. D: mRNA expression of chemokine (C-C motif) ligand (CCl)2 in no-MI control mice and day 1 and day 5 post-MI in WT and 12/15-LOX−/− mice supplemented with standard or PUFA diet. E: mRNA expression of arachidonate lipoxygenase (ALOX)5 in no-MI control mice and day 1 and day 5 post-MI in WT and 12/15-LOX−/− mice supplemented with standard or PUFA diet. Data are presented as means ± SE; n = 6–9 mice/group. *P < 0.05 vs. no-MI WT standard diet control; #P < 0.05 vs. WT mice with standard diet control at the respective time point.
Coronary ligation MI surgery.
To induce MI, WT and 12/15-LOX−/− were subjected to the surgical ligation of the left anterior descending coronary artery, as previously described (19, 49). Mice were anesthetized with 2% isoflurane using a tracheal intubation to control respiration through a MiniVent-type 845 ventilator (Hugo Sachs Elektronic), and the left anterior descending coronary artery was permanently ligated using nylon 8-0 sutures (ARO Surgical Instruments, Newport Beach, CA) in a minimally invasive surgery. We set the inclusion criteria based on the infarct area, and the infarct area above 45% is included from the study.
Necropsy.
Samples were collected at day 0 for each group as naïve controls, and post-MI samples were collected at day 1 or day 5 post-MI. No-MI control day (day 0), day 1, or day 5 post-MI mice were anesthetized with isoflurane briefly. Mice were then maintained under anesthesia using 2% isoflurane in 100% oxygen mix, and heparin (4 IU/g) was injected intraperitoneally. At 5 min after heparin injection, blood was collected from the carotid artery for plasma separation. The chest cavity was opened, and the LV was perfused with 2–3 ml of cardioplegic solution to arrest the heart in diastole; the heart, lung, and spleen were then removed. The lungs, LV, and right ventricle were extracted and weighed individually. The LV was divided into the apex, midcavity, and base under a microscope. All three LV sections and the right ventricle were stained with 1% 2,3,5-triphenyltetrazolium chloride (Sigma, St. Louis, MO) and photographed for the determination of infarct area using Adobe Photoshop CS5 (64 bit) software. LV infarct area and remote regions were separated under the microscope for precise segregation of infarcted and remote (noninfarcted) area. Samples were individually snap frozen and stored at −80°C. The midcavity section was fixed in 10% zinc-formalin and paraffin embedded for leukocyte (neutrophils and macrophages) immunohistochemistry. The lung mass and tibia were removed, and the wet and dry weights of lungs (24 h after necropsy) and tibia length were determined.
LV histology and immunohistochemistry.
For immunohistochemistry (IHC), LV midcavity sections were embedded in paraffin and cut in 5-µm sections. For assessment of neutrophils and macrophages, paraffin-embedded sections were deparaffinized in citrisolv and rehydrated through graded ethanol. Heat-mediated antigen retrieval was performed to expose antigen epitopes (Target Retrieval Solution, S1699, Dako, Glostrup, Denmark) using a pressure cooker (BioSB Tinto Retriever). Sections blocked with normal rabbit or goat serum as per antibody were incubated with rat anti-mouse neutrophils (CL 8993AP, clone 7, 1:50, Cedarlane, Burlington, ON, Canada) and rat anti-Mac-3 monoclonal antibody (CL 8943AP, clone M3/84, 1:100 dilution, Cedarlane). Neutrophil and macrophage staining was performed with the Vectastain Elite ABC kit (Vector). Slides were mounted using Permount and allowed to dry for image capture and quantification (14, 33).
Collagen staining using picrosirius red.
For picrosirius red (PSR) staining, paraffin-embedded LV tissue sections were processed, and collagen density was measured using image analysis software, as previously described (33).
Image analysis for IHC and PSR staining.
For each slide per mouse, five to seven images were captured using a microscope (BX43) with an attached camera (Olympus DP73), focusing on the infarct area and border zone for the LV and the whole section for the spleen. Images were captured using the cellSens Dimension program (Olympus version 1.9) and then analyzed for percent area stained using Image-Pro Premier 64-bit analyzer software. The percent area determined by the image-analysis software (Image-Pro Premier, Cybernetics) for the five to seven images of each sample was recorded and averaged to determine the percent area stained for neutrophils, macrophages, and collagen density in the infarcted area of the LV (14).
Measurements of mRNA levels using quantitative real-time PCR.
For quantitative RT-PCR, reverse transcription was performed with 2.5 μg total RNA using SuperScript VILO cDNA synthesis kit (Invitrogen, Carlsbad, CA). Quantitative RT-PCR for prostaglandin-endoperoxide synthase (Ptgs)-1, Ptgs-2, heme oxygenase (HO)-1, arachidonate 12-lipoxygenase (Alox)-12, Alox-15, Alox-5, Tnf-α, IL-6, chemokine (C-C motif) ligand (Ccl)2, IL-1β, Arg-1, Mrc-1, and Ym-1 genes was performed using TaqMan probes (Applied Biosystems, Foster City, CA) on master cycler ABI, 7900HT. Gene levels were normalized to hypoxanthine phosphoribosyltransferase as the housekeeping control. The results were reported as 2−ΔCt (ΔΔCt) values (where Ct is the threshold cycle). All experiments were performed in duplicate with n = 6–9 mice per group per time point.
Immunoblot analysis.
LV infarct tissue was homogenized in PBS with 1× protease inhibitor cocktail and centrifuged at 14,000 rpm for 5 min. Total 10 µg proteins were run on criterion XT bis-Tris 4–12% 18-gels (Bio-Rad) using MOPS buffer (Bio-Rad, Hercules, CA) and nitrocellulose membranes (Bio-Rad). Total protein stain was acquired using Pierce reversible protein stain and nitrocellulose membranes (Thermo Fisher Scientific, Waltham, MA). After being rinsed with water, the membrane was blocked for 1 h at room temperature using 5% nonfat milk powder (Bio-Rad) dissolved in PBS-Tween (PBST) and was probed overnight at 4°C with primary antibody (COX-2: 1:1,000, 5-LOX: 1:1,000, and HO-1 1:5,000) followed by the respective secondary antibody (Bio-Rad). Kaleidoscope precision plus standard (Bio-Rad) was used to determine the molecular weight of the protein. Proteins were detected using the Femto chemiluminescence detection system (Pierce Chemical, Rockford, IL). Densitometry was performed using ImageJ software (National Institutes of Health, Bethesda, MD).
Quantitative mass spectroscopy analyses of plasma lipid metabolites.
Targeted quantitative mass spectroscopy approach was used at day 5 post-MI to detect the lipid metabolites in PUFA-fed and laboratory chow diet-fed WT and 12/15-LOX−/− mice, as previously described (13, 31).
Post-MI lipidomics analysis using principal component and heat maps.
For the generation of the lipidome heat maps, the plasma-analytes concentration from WT, WT + PUFA, 12/15-LOX−/−, and 12/15-LOX−/− + PUFA groups at day 5 post-MI was normalized by the geometric mean for statistical significance. Normalized values were used for the generation of hierarchical heat maps using cluster 3.0 and Java TreeView software. Correlations between WT, WT + PUFA, 12/15-LOX−/−, and 12/15-LOX−/− + PUFA groups were observed using principal component analysis (PCA). The correlation matrixes were done using a Pearson (n) test to avoid inflating the impact of variables with high variances on the PCA (13).
Statistical analysis.
Data are expressed as means ± SE. Statistical analyses were performed using GraphPad Prism 5 (San Diego, CA). Analysis of variance (ANOVA) was followed by a Newman-Keuls post hoc test for multiple comparisons of real-time PCR and immunoblot data. Two-way ANOVA was performed on the lipid mediator data for statistical significance and interaction. For two-group comparisons, Student's t-test (unpaired) was applied. P < 0.05 was considered as statistically significant. All immunoblot densitometry data were normalized to total protein per lane. n = 4–6 samples were used for all groups/time points in all of the experiments except the immunoblot analysis, where n = 3 samples were used per time point.
RESULTS
Acute PUFA exposure attenuated LV remodeling, dysfunction, and the edema index in WT and 12/15-LOX−/− mice post-MI.
To evaluate the impact of an acute PUFA diet with deletion of 12/15-LOX (study design, Fig. 1A) in a post-MI setting, we measured LV function by echocardiography. As shown in Table 1, echocardiography data from the surviving 12/15-LOX−/− and acute PUFA-fed WT and 12/15-LOX−/− mice showed decreased end-systolic and diastolic volume at day 1 and day 5 post-MI, thereby improving ejection fraction, compared with standard diet-fed WT mice. The gravimetric analysis shown in Table 2 demonstrated that lung mass-to-body weight ratios were reduced in PUFA-fed WT and 12/15-LOX−/− mice compared with WT mice at day 5 post-MI. Thus, echocardiographic and gravimetric analyses indicated improved LV function, reduced pulmonary edema, and attenuated post-MI remodeling in PUFA-fed WT and 12/15-LOX−/− mice.
Table 1.
Echocardiographic measurements showing improved LV function in 12/15-LOX−/− and PUFA-fed mice
Control Day 0 |
MI Day 1 |
MI Day 5 |
||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Parameters | WT | WT + PUFA | 12/15-LOX−/− | 12/15-LOX−/− + PUFA | WT | WT + PUFA | 12/15-LOX−/− | 12/15-LOX−/− + PUFA | WT | WT + PUFA | 12/15-LOX−/− | 12/15-LOX−/− + PUFA |
n | 5 | 4 | 5 | 4 | 6 | 5 | 6 | 4 | 6 | 5 | 9 | 7 |
Heart rate, beats/min | 445 ± 16 | 465 ± 7 | 427 ± 11 | 418 ± 16 | 499 ± 16 | 475 ± 16 | 451 ± 18 | 462 ± 16 | 445 ± 17 | 484 ± 26 | 427 ± 8 | 436 ± 4 |
End-diastolic volume, µl | 53.3 ± 5 | 56.8 ± 5 | 45.0 ± 2 | 53.3 ± 4 | 96.6 ± 17* | 79.2 ± 6*† | 62.8 ± 4*† | 61.3 ± 4*† | 125.9 ± 17* | 102.2 ± 11* | 95.2 ± 5*† | 106.3 ± 20*† |
End-systolic volume, µl | 20.1 ± 3 | 24.8 ± 3 | 19.2 ± 2 | 22.0 ± 4 | 85.1 ± 16* | 68.0 ± 6*† | 52.0 ± 3*† | 52.3 ± 4*† | 113.2 ± 15* | 83.4 ± 138*† | 82.6 ± 4*† | 93.2 ± 19*† |
Posterior wall thickness at systole, mm | 1.1 ± 0.02 | 1.05 ± 0.06 | 1.05 ± 0.02 | 1.05 ± 0.04 | 0.54 ± 0.05* | 0.46 ± 0.02*† | 0.54 ± 0.03* | 0.47 ± 0.03*† | 0.50 ± 0.05* | 0.50 ± 0.02* | 0.51 ± 0.03* | 0.50 ± 0.05* |
Ejection fraction | 63 ± 4 | 62 ± 2 | 67 ± 2 | 64 ± 6 | 13 ± 1* | 14 ± 2* | 17 ± 1*† | 15 ± 1 | 10 ± 1* | 20 ± 5*† | 13 ± 0.7*† | 14 ± 3*† |
Values are expressed as means ± SE; n indicates sample size. No infarct served as the day 0 naïve control group. LV, left ventricular; LOX, lipoxygenase; PUFA, polyunsaturated fatty acid; MI, myocardial infarction.
P < 0.05 vs. day 0 wild-type (WT) control;
P < 0.05 vs. WT mice at the respective day time point.
Table 2.
Necropsy parameters indicating reduced LV remodeling and pulmonary edema in 12/15-LOX and PUFA-fed mice post-MI
Control Day 0 |
MI Day 1 |
MI Day 5 |
||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Necropsy Parameters | WT | WT + PUFA | 12/15-LOX−/− | 12/15-LOX−/− + PUFA | WT | WT + PUFA | 12/15-LOX−/− | 12/15-LOX−/− + PUFA | WT | WT + PUFA | 12/15-LOX−/− | 12/15-LOX−/− + PUFA |
n | 9 | 4 | 5 | 4 | 6 | 5 | 6 | 4 | 9 | 6 | 9 | 7 |
BW, g | 21 ± 2 | 25 ± 1 | 19 ± 1 | 20 ± 2 | 17 ± 2 | 25 ± 1 | 19 ± 1 | 19 ± 1 | 20 ± 1 | 25 ± 1 | 19 ± 0.5 | 20 ± 1 |
LV, mg | 65 ± 6 | 79 ± 1 | 64 ± 2 | 66 ± 7 | 75 ± 6 | 83 ± 2 | 73 ± 3 | 70 ± 3 | 96 ± 3* | 98 ± 4* | 88 ± 3*† | 90 ± 4*† |
LV/BW, mg/g | 3.1 ± 0.04 | 3.1 ± 0.1 | 3.3 ± 0.2 | 3.3 ± 0.1 | 4.4 ± 0.2* | 3.3 ± 0.1 | 3.8 ± 0.1*† | 3.7 ± 0.1*† | 4.9 ± 0.1* | 3.9 ± 0.2*† | 4.8 ± 0.2* | 4.6 ± 0.2* |
Right ventricle, mg | 17 ± 1 | 16 ± 1 | 15 ± 1 | 19 ± 4 | 16 ± 2 | 16 ± 1 | 17 ± 1 | 19 ± 1 | 18 ± 1 | 20 ± 1 | 16 ± 1 | 23 ± 3 |
Infarct area, % | ND | ND | ND | ND | 50 ± 2 | 50 ± 2 | 48 ± 2 | 50 ± 1 | 50 ± 1 | 51 ± 1 | 53 ± 1 | 50 ± 1 |
Lung mass/BW, mg/g | 6 ± 1 | 4 ± 1 | 6 ± 1 | 5 ± 1 | 8 ± 1* | 5 ± 1*† | 8 ± 1* | 7 ± 0.4* | 9 ± 1* | 5 ± 1*† | 9 ± 1* | 7 ± 1*† |
Values are expressed as means ± SE; n indicates sample size. BW, body weight; ND, no infarct in day 0 controls.
P < 0.05 vs. day 0 WT controls;
P < 0.05 vs. the WT group at the respective day time point.
Acute PUFA-expedited neutrophil clearance and decreased CCL2 in 12/15-LOX−/− post-MI.
First, we confirmed the deletion of 12/15-LOX, as shown by mRNA expression (Supplemental Fig. S1; Supplemental Material for this article is available at the American Journal of Physiology-Heart and Circulatory Physiology website). To determine the effect of acute PUFA intake and 12/15-LOX interactions on leukocyte infiltration, IHC was performed on LV sections compared with WT controls. In response to MI, neutrophil infiltration to infarcted area is the initial step (26). Thus, we determined the neutrophil density post-MI. Acute PUFA exposure reduced neutrophil density at day 1 post-MI in both WT and 12/15-LOX−/− mice compared with standard diet-fed WT and 12/15-LOX−/− mice. Acute PUFA exposure increased post-MI neutrophil clearance in both groups, as observed with the reduction of neutrophils density at day 5 using IHC. Time-dependent infarcted LV neutrophil clearance was prominent in WT + PUFA, 12/15-LOX−/−, and 12/15-LOX−/− + PUFA mice compared with WT mice (Fig. 1, B and C). Furthermore, mRNA expression of Ccl2 was determined because it directs leukocyte infiltration to the infarcted site. CCL2 was decreased in the infarcted area of PUFA-supplied WT and 12/15-LOX−/− mice compared with standard diet-fed mice (all P < 0.05; Fig. 1D). 12/15-LOX deletion was compensated with 5-LOX expression, and increased expression of 5-LOX mRNA in the infarcted LV indicated a compensatory metabolic redundant pathway. PUFA exposure further induced 5-LOX in 12/15-LOX−/− mice, and there was a robust increase in 5-LOX expression for 12/15-LOX−/− + PUFA mice compared with PUFA + WT mice at day 5 post-MI (Fig. 1E). Thus, acute PUFA exposure expedited neutrophil clearance with amplified expression of 5-LOX, suggesting that acute PUFA intake in 12/15-LOX−/− mice leads to an early induction of the resolving and reparative phase mediators in a post-MI setting.
Acute PUFA intake decreased proinflammatory mediators in 12/15-LOX−/− mice post-MI (decrease in arachidonic acid-derived HETEs).
Since the analyses of infarcted LVs showed an early initiation of resolving and reparative responses in 12/15-LOX−/− + PUFA mice compared with WT + PUFA mice, we quantitated PUFA-derived lipid mediators at day 5 post-MI. Short-term PUFA-fed and standard diet-fed plasma samples from day 5 post-MI mice were subjected to liquid chromotography-tandem mass spectroscopy (LC-MS/MS)-based comprehensive targeted lipidome analysis. In this, we analyzed 86 lipid analytes from PUFA- or chow-fed WT and 12/15-LOX−/− mice at day 5 post-MI. As expected, acute exposure of PUFA to WT and 12/15-LOX−/− mice resulted in elevated levels of arachidonic acid (AA)-derived lipid mediators in plasma at day 5 post-MI. This indicates bioavailability of fatty acids and the effective SO enrichment in the diet (Fig. 2). The PCAs for global changes indicated that WT and 12/15-LOX−/− cohorts on the standard diet were diversely separated post-MI (Supplemental Fig. S2). The WT + PUFA and 12/15-LOX−/− mice were observed to be closely associated with each other. Interestingly, the 12/15-LOX−/− + PUFA group showed differential metabolites compared with 12/15-LOX−/− and WT + PUFA groups, but all three groups were diverse for the enzymatic target (Fig. 2). Heat map analysis indicated that PUFA intake robustly increased the overall levels of AA-derived eicosanoids (Fig. 2A). WT + PUFA mice showed increased levels of proinflammatory 12-HETE and decreased levels of PGs compared with PUFA-fed 12/15-LOX−/− mice (Fig. 2, B and E). Interestingly, levels of CYP-450-mediated unstable 9 (10)-EpOME and its diol 9,10-DiHOME were higher in PUFA-fed WT mice compared with 12/15-LOX−/− + PUFA mice (Fig. 2D). Thus, PUFA exposure decreased proinflammatory mediators in 12/15-LOX−/− mice compared with WT mice, thus providing a suitable environment for infarcted LV healing and neutrophil clearance.
Fig. 2.
Acute PUFA intake expanded the arachidonic acid (AA) lipidome at day 5 post-MI. A: hierarchical cluster analysis of change in AA metabolites in WT and 12/15-LOX−/− mice supplemented with standard or PUFA diet at day 5 post-MI. B: bar graph representation of 12/15-LOX-mediated 12(S)-HETE and 15-HETE in WT and 12/15-LOX−/− mice supplemented with standard or PUFA diet at day 5 post-MI. C: bar graph representation of 5-LOX-mediated 5-oxoETE in WT and 12/15-LOX−/− mice supplemented with standard or PUFA diet at day 5 post-MI. D: bar graph representation of cytochrome P (CYP)-450-mediated 9,10-DiHOME and 9(10)-EpOME in WT and 12/15-LOX−/− mice supplemented with standard or PUFA diet at day 5 post-MI. E: bar graph representation of cyclooxygenase (COX)-2-mediated PGE2 and PGD2 in WT and 12/15-LOX−/− mice supplemented standard or PUFA diet at day 5 post-MI. Data are presented as means ± SE; n = 4 mice/group. $P < 0.05 vs. the respective genotype with standard diet-fed control analyzed by two-way ANOVA.
Acute PUFA supply enhanced reparative mediators in 12/15-LOX−/− mice post-MI (increase in EET metabolites).
Emerging evidence suggests that CYP450-derived EETs (i.e., 5,6-EpETrE, 8,9-EpETrE, 11,12-EpETrE, and 14,15-EpETrE) treatment is a viable strategy to control ischemic inflammation for cardioprotection and hypertension (20, 35). Compared with WT mice, 12/15-LOX deletion activated CYP2J2 (predominantly called CYP2J6 in the mouse) CYP450-derived metabolites 11,12-EpETrE and 14,15-EpETrE in plasma, which accelerated LV healing with the marked clearance of leukocytes after ligation-induced myocardial injury. Furthermore, PUFA exposure amplified 11,12-EpETrE and 14,15-EpETrE levels in 12/15-LOX−/− mice (Fig. 3A) post-MI. However, the levels of soluble, less active epoxides, dihydroxyeicosatrienoic acids (diHETrEs), i.e., 11,12-DiHETrE and 14,15-DiHETrE, remained unchanged in all four groups at day 5 post-MI (Fig. 3B). Immunoblot analysis showed that CYP2J2 was expressed at day 5 post-MI in 12/15-LOX−/− mice. Acute PUFA exposure increased CYP2J2 expression in 12/15-LOX−/− + PUFA mice (2.4 ± 0.04-fold) compared with PUFA-fed WT mice (Fig. 3C). Thus, comprehensive and quantitative lipid mediators analysis and estimation of CYP2J2 expression indicated that acute PUFA exposure to 12/15-LOX−/− mice resulted in an enzymatic shift of substrate utilization from 12/15-LOX to CYP450. Consequently, CYP450-derived endogenous lipid metabolites enhanced the reparative and resolving response in post-MI settings, thereby promoting early LV healing in 12/15-LOX−/−, PUFA-fed WT, and 12/15-LOX−/− mice.
Fig. 3.
Acute PUFA intake increased EpETrE in 12/15-LOX−/− mice. A: bar graph of CYP450-mediated EpETrE in WT and 12/15-LOX−/− mice supplemented with standard or PUFA diet at day 5 post-MI. B: bar graph of soluble epoxide DiHETrE in WT and 12/15-LOX−/− mice supplemented with standard or PUFA diet at day 5 post-MI. C: immunoblot representing CYP2J2 expression in LV infarct and controls. Densitometric analysis of CYP2J2 levels normalized to total protein are shown. n = 4–6 mice/group. *P < 0.05 vs. day 0 control. $P < 0.05 vs. MI-WT mice at the respective day. Data are presented as means ± SE; n = 3 or 4 mice/group. $P < 0.05 vs. the respective genotype with standard diet-fed control analyzed by two-way ANOVA.
Acute PUFA intake impacted the EPA series metabolome in LV healing post-MI.
Furthermore, we analyzed EPA-derived metabolites at day 5 post-MI in the plasma of both WT and 12/15-LOX−/− PUFA-fed and standard diet-fed mice. ω-6-enriched acute PUFA exposure globally decreased EPA-derived eicosanoids in WT and 12/15-LOX−/− mice (Fig. 4A, heat map). Short-term PUFA exposure decreased 12/15-LOX, CYP450-mediated, EPA-derived eicosanoids 15(S)-HEPE, 17,18-EpETE, and 5,6-DiHETE as well as nonenzymatic derived 18-HEPE and 12-HEPE (Fig. 4, B–D). Acute PUFA feeding also increased the levels of COX-mediated 15d-D12,14-PGJ3, and 11(R)-HEDE (Fig. 4E). Thus, ω-6-enriched acute PUFA exposure decreased EPA-derived metabolites in the post-MI setting, indicating an abundance of AA-derived metabolites and activation of the CYP pathway.
Fig. 4.
Acute PUFA intake reduced EPA metabolites at day 5 post-MI. A: hierarchical cluster analysis of change in EPA metabolites in WT and 12/15-LOX−/− mice supplemented with standard or PUFA diet at day 5 post-MI. B: bar graph of 12/15-LOX-mediated 15(S)-HEPE in WT and 12/15-LOX−/− mice with standard or PUFA diet at day 5 post-MI. C: bar graph of nonenzymatic 18-HEPE and 12-HEPE in WT and 12/15-LOX−/− mice supplemented with standard or PUFA diet at day 5 post-MI. D: bar graph of CYP450-mediated 17,18-EpETE and 5,6-DiHETE (EPA) in WT and 12/15-LOX−/− mice supplemented with standard or PUFA diet at day 5 post-MI. D: bar graph of COX2-mediated 15d-D12,14-PGJ3 and 15(R)-HEDE in WT and 12/15-LOX−/− mice supplemented with standard or PUFA diet at day 5 post-MI. Data are presented as means ± SE; n = 4 mice/group. $P < 0.05 vs. the respective genotype with standard diet-fed control analyzed by two-way ANOVA.
Acute PUFA intake decreased DHA metabolites in LV healing post-MI.
Analysis of the DHA metabolome after short-term PUFA exposure showed a decrease in overall DHA docosanoids in PUFA-fed WT and 12/15-LOX−/− mice compared with standard diet-fed mice. DHA docosanoids levels varied with diet and decreased after short-term PUFA exposure for both WT and 12/15-LOX−/− mice at day 5 post-MI (Fig. 5A). Autoxidation products of DHA 8-HDoHE, 13-HDoHE, 16-HDoHE, and 20-HDoHE (Fig. 5B) were significantly decreased in acute PUFA-fed WT and 12/15-LOX−/− mice at day 5 post-MI. Similarly, CYP450-mediated 19,20-DiHDoHE was decreased in standard diet-fed 12/15-LOX−/− mice compared with standard diet-fed WT controls. However, overall levels of 19,20-DiHDoHE were decreased after short-term PUFA feeding to both WT and 12/15-LOX−/− mice. Of note, PUFA feeding led to an increase in the levels of RvD2 in 12/15-LOX−/− mice compared with all three groups (Fig. 5C), indicating a complex feed-forward lipid mediator pathway. Thus, short-term PUFA exposure decreased DHA docosanoids in LV healing in 12/15-LOX−/− mice post-MI.
Fig. 5.
Acute PUFA intake in 12/15-LOX−/− mice decreased docosanoids at day 5 post-MI. A: hierarchical cluster analysis of change in DHA metabolites in WT and 12/15-LOX−/− mice supplemented with standard or PUFA diet at day 5 post-MI. B: bar graph of nonenzymatic HDoHE (8-HDoHE, 13-HDoHE, 16-HDoHE, and 20-HDoHE) in WT and 12/15-LOX−/− mice supplemented with standard or PUFA diet at day 5 post-MI. C: bar graph of CYP450-mediated 19,20-DiHDoHE in WT and 12/15-LOX−/− mice supplemented with standard or PUFA diet at day 5 post-MI. D: bar graph of 12/15-LOX mediated RvD2 in WT and 12/15-LOX−/− mice supplemented with standard or PUFA diet at day 5 post-MI. Data are presented as means ± SE; n = 3 or 4 mice/group. *P < 0.05 vs. WT mice within groups. $P < 0.05 vs. the respective genotype with standard diet-fed control analyzed by two-way ANOVA.
Acute PUFA intake increased proresolving cytokines in 12/15-LOX−/− mice post-MI.
To determine the impact of short-term PUFA intake post-MI on proinflammatory and proresolving markers, we quantified mRNA levels of IL-6, TNF-α, IL-1β, Mrc-1/CD206, Arg-1, and Ym-1. Short-term PUFA intake increased IL-6 expression (40-fold, P < 0.001) compared with standard diet-fed WT counterparts at day 1 post-MI. However, short-term PUFA intake did not impact TNF-α and IL-1β expression in WT mice. TNF-α levels were three-fold (P < 0.05) higher in standard diet-fed WT mice compared with WT + PUFA-fed mice at day 5 post-MI. Of note, PUFA feeding increased IL-6 levels in 12/15-LOX−/− mice at day 1 and day 5 post-MI compared with WT + PUFA-fed and standard diet-fed WT and 12/15-LOX−/− mice (Fig. 6A). PUFA intake did not alter the proresolving markers Mrc-1, Arg-1, and Ym-1 in WT + PUFA-fed mice compared with standard-diet WT mice. PUFA-fed 12/15-LOX−/− mice displayed an increase of Mrc-1 (25-fold, P < 0.05) compared with standard diet-fed 12/15-LOX−/− mice at day 5 post-MI (Fig. 6B). Both PUFA-fed and standard diet-fed 12/15-LOX−/− mice showed consistent increases in the mRNA expression of Ym-1 and Arg-1 compared with PUFA- and standard diet-fed WT mice. Thus, PUFA intake positively impacted proresolving markers in 12/15-LOX−/− mice, enhancing the LV reparative phase post-MI.
Fig. 6.
12/15-LOX−/− mice and PUFA feeding limited proinflammatory (M1) markers and stimulated proresolving (M2) markers post-MI. A and B: mRNA expression of IL-6, Tnf-α, and IL-1β (A) and Mrc-1, Ym-1, and Arg-1 (B) in the LV infarct of WT, 12/15-LOX−/−, and respective PUFA-fed groups. Data are presented as means ± SE; n = 4 mice/group for immunoblot analysis and n = 6 mice/group for mRNA. *P < 0.05 vs. WT mice within groups. #P < 0.05 vs. the respective genotype with standard diet-fed control analyzed by two-way ANOVA.
Acute PUFA intake decreased macrophages and collagen density in 12/15-LOX−/− mice post-MI.
In response to MI, macrophage density, residual time, and plasticity determine LV healing and, thereby, cardiac remodeling. Standard diet-fed WT and 12/15-LOX−/− mice displayed similar macrophages infiltration at day 5 post-MI. Short-term PUFA exposure reduced macrophage density in both groups (WT and 12/15-LOX−/−) at day 5 post-MI (Fig. 7, A and C). Post-MI macrophages expansion in the infarct area, scar maturation, and optimal matrix remodeling are the main factors of myocardial healing; therefore, to evaluate how short-term PUFA intake impacted matrix remodeling post-MI, collagen density was measured. Analysis of collagen-stained density area at day 5 post-MI revealed that collagen levels were lowered in standard diet-fed 12/15-LOX−/− mice compared with standard diet-fed WT mice. Collagen levels were relatively lower in PUFA-fed WT and 12/15-LOX−/− mice compared with their respective standard diet-fed groups (Fig. 6, B and C). Thus, acute PUFA intake decreased macrophage density with a marked reduction in collagen density in 12/15-LOX−/− mice during LV healing post-MI.
Fig. 7.
12/15-LOX deletion and PUFA supplementation attenuated collagen deposition by limiting fibrosis post-MI and decreased macrophage density. A: representative LV immunohistochemical images showing macrophages density in WT and 12/15-LOX−/− mice supplemented with standard or PUFA diet at day 5 post-MI. B: picrosirus red-stained infarcted LV collagen levels at day 5 post-MI in WT and 12/15-LOX−/− mice fed a standard or PUFA diet at day 5 post-MI. Magnification: ×40. scale bar = 50 µm. C and D: bar graphs depicting quantification of macrophages (C) and collagen levels (D). n = 6–9 mice/group. Data are presented as means ± SE. *P < 0.05 vs. WT mice within groups. $P < 0.05 vs. the respective genotype with standard diet-fed control analyzed by two-way ANOVA.
Acute PUFA intake increased reparative enzymes in 12/15-LOX−/− mice post-MI (increase in 5-LOX and HO-1 expression).
To confirm the increased LV reparative response post-MI in PUFA-fed 12/15-LOX−/− mice, we determined the expression levels of the resolving enzymes 5-LOX, COX-2, and HO-1. 12/15-LOX deletion increased expression of 5-LOX mRNA and protein in the infarcted LV. Acute PUFA intake further induced 5-LOX expression in both WT and 12/15-LOX−/− mice at day 5 post-MI (Fig. 8, A–B). Expression of 5-LOX was induced at day 1 and was amplified at day 5 post-MI with PUFA intake. COX-2 levels were elevated at post-MI day 1 and day 5 in WT mice compared with day 0. COX-2 levels in LV infarct tissue remained elevated at day 5 post-MI in WT and 12/15 LOX−/− mice and were associated with an influx of neutrophils. PUFA feeding further elevated COX-2 levels in both PUFA-fed WT and 12/15-LOX−/− mice. Inducible HO-1 levels were higher in 12/15-LOX−/− mice, which were further enhanced by acute PUFA feeding (Fig. 8, A and B). Thus, the amplified mRNA and protein expression of 5-LOX, HO-1, and COX-2 suggested that acute PUFA exposure leads to an early onset of the reparative phase in the post-MI setting.
Fig. 8.
12/15-LOX deletion and PUFA diet enhanced 5-LOX and heme oxygenase (HO)-1 post-MI. A and B: immunoblots representing 5-LOX (A) and HO-1 expression (B). Densitometric analyses of the respective blot and mRNA expression in the LV infarct of WT, 12/15-LOX−/−, and respective PUFA-fed groups is shown. Data are presented as means ± SE; n = 3 mice/group for immunoblot analysis and n = 6 mice/group for mRNA. *P < 0.05 vs. WT mice within groups. $P < 0.05 vs. the respective genotype with standard diet-fed control analyzed by two-way ANOVA. C: schematic diagram indicating fatty acid substrate and LOX interactions to form differential lipid meditors that impact on leukocyte clearance in WT and 12/15-LOX−/− mice to facilitate or impair resolution of inflammation post-MI.
DISCUSSION
Interactions of specific LOX enzymes to execute the metabolic transformation of fatty acids determine the class of lipid mediators in reparative and inflammation resolution. An essential component of the Western diet and a major substrate of n-6 fatty acid is AA, which is metabolized by three major pathways−COX, LOX, and CYP450−into biologically active products (8, 22, 39). Lipid mediators generated through LOX, COX, and CYP pathways regulate the inflammation-triggering and reparative pathway. The presented study defines the interaction of the lipid-busting enzyme 12/15-LOX with short-term dietary intake of PUFA (ω-6 essential fatty acid) in post-MI settings. We highlighted that acute exposure of PUFA in mice is beneficial during both the presence and absence of its metabolizing enzyme 12/15-LOX. The major outcomes observed were 1) increased neutrophil clearance from the infarcted area along with improved LV function, 2) elevated levels of 11,12-EpETrE and 14,15-EpETrE in 12/15-LOX−/− mice during the reparative phase post-MI, 3) increased reparative cytokine markers post-MI; and 4) decreased macrophages and collagen density at day 5 post-MI. Thus, our results indicate that inactivation of 12/15-LOX combined with acute PUFA intake can shift the substrate availability to the epoxygenase pathway to stimulate the reparative response in 12/15-LOX−/− mice (Fig. 8C).
Fat quality, quantity, and aging are the main variables of metabolism, and this contributes to the contextual reports that show both beneficial and detrimental effects of PUFA in cardiovascular disease (9, 17, 48). The ideal ratio of ω-6 and ω-3 fatty acids is 2:1, which is essential for maintaining cardiac homeostasis. The current Western diet ratio of ω-6 to ω-3 has been reported to be as high as 10:1, which is confounded due to decreased physical activity or lack of exercise (42). Since PUFA-derived eicosanoids possess both proinflammatory and anti-inflammatory properties, the optimal balance of lipid mediators coordinates the onset and turning off of inflammation to stimulate reparative mechanisms post-MI (23, 41). The innate inflammatory response has been well studied in different animal models with saturated fat exposure (32); however, it is important to understand how acute PUFA feeding regulates post-MI inflammation in both the presence and absence of the key metabolizing enzyme 12/15-LOX.
In the post-MI setting, innate responsive neutrophils are primed cells, which invade immediately post-MI to mediate collateral tissue damage by releasing the matrix-degrading enzyme in ischemia and reperfusion injury (5, 26). In a population-based study, post-MI patients with increased neutrophil counts showed signs of unresolved post-MI inflammation and proclivity to recurrent MI (10, 34). Post-MI, there is a robust increase in neutrophil trafficking and recruitment in the infarcted area, which is essential to clear the necrotic myocytes. However, the residual time of neutrophils in the infarcted area leads to unresolved inflammation, thus affecting the healing process (31). One-month PUFA diet exposure for both WT and 12/15-LOX−/− mice resulted in reduced neutrophils during the resolving phase of post-MI. This was further confirmed by a decrease in Ccl2, which mediates the firm adherence and subsequent transmigration of neutrophils in the infarcted LV post-MI (38). Thus, neutrophil clearance post-MI initiated the resolving phase, which is indicated by the improved LV function in 12/15-LOX−/− and PUFA-fed WT and 12/15-LOX−/− mice.
AA is extensively studied for the generation of PGs and leukotrienes in a wide variety of systems (in vitro, rodents, primates, and humans), with less emphasis on CYP-derived EETs. Eicosanoids, such as PGE2, thromboxane A2, and leukotriene B4, which are generated through COX and LOX, possess a diverse array of proinflammatory, vasoconstrictive, and/or proaggregatory properties (30). Other lipid mediators, such as lipoxin A4 and EETs, were generated through LOX and CYP pathway, respectively, and have anti-inflammatory/antiaggregatory properties. 12/15-LOX deletion increased levels of EETs, whereas acute exposure of PUFA further enhanced levels of 11,12-EpETrE and 14,15-EpETrE in plasma. CYPs are membrane-bound, heme-containing oxidase enzymes that oxidize numerous endogenous substances, including fatty acids. EETs are fatty acid epoxides produced from AA by CYP-450 epoxygenase (50). The predominant CYP isoforms in humans are CYP2C8, CYP2C9, and CYP2J2, which are expressed in the endothelium (50). The predominant rodent CYP isoforms include Cyp2j6, Cyp2c44, and Cyp2j5 in mice. CYP isoforms produce four regioisomeric EETs: 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET, which is a mixture of the S/R and R/S enantiomers. The activity of these regioisomeric EETs functionally varies (44). EETs possess important vasodilator properties via hyperpolarization and relaxation of vascular smooth muscle cells (40). EETs are known to significantly inhibit classical (M1) macrophage polarization and diminish proinflammatory cytokines at the transcriptional and posttranscriptional levels while retaining reparative (M2) macrophages (7). Thus, the upregulation of EETs promotes the reparative microenvironment to leukocytes post-MI (11).
Metabolic transformation of AA through CYP-450 produces EETs, which regulate insulin sensitivity and reduce the risk of coronary artery disease (6, 36). In contrast, dietary PUFA enrichment produces 5-HETE, 12-HETE, and 15-HETE, which aggravate insulin resistance, platelet aggregation, and cardiometabolic risk (46, 47). To increase the circulating concentration of CYP-derived EET, soluble epoxide hydrolases (sEH) have been developed, and they have been shown to reduce vascular resistance and cardioprotective effects (21, 43). Deletion of sEH offers cardioprotection; therefore, sEH inhibitors are effective in ischemic cardiomyopathy, pressure overload hypertrophy, and coronary reactive hyperemia (16, 43). Myocardial damage not only impacts myocytes but also regulates extracellular matrix remodeling. Post-MI, the LV undergoes a series of changes to form the reparative matrix scar, leading to the formation of myofibroblasts (45). In the present report, 12/15-LOX deletion reduced total collagen levels at day 5 post-MI compared with WT mice. PUFA interactions in the absence of 12/15-LOX in mice decreased collagen levels, suggesting limited remodeling and improving ventricular function. Leukocytes are the primary drivers of the post-MI wound healing process, as they modulate the cytokine and lipid mediator environment and thereby promote the resolution of inflammation. However, PUFA-fed 12/15-LOX−/− mice showed a relatively lower ejection fraction compared with PUFA-fed WT mice. This differential outcome could be due to PUFA-fed 12/15-LOX−/− mice having relatively higher levels of PGE2 and PGD2 compared with 12/15-LOX−/− mice fed a standard diet. Enrichment of AA-derived proinflammatory milieu is responsible for maintaining the differential neutrophils and macrophage phenotypes. The study highlights the interaction of fatty acids with lipid-busting enzymes as well as the subsequent generation of lipid mediators that direct the leukocyte phenotype and gene expression (Fig. 8C). In the homeostatic steady state, the myocardium primarily uses fatty acids (~60–70%) rather than carbohydrates (glucose, ~30%); however, in myocardial healing, whether differential metabolites serve as the energy source is the active area of research. Deletion of 12/15-LOX lowered Tnf-α, IL-1β, and IL-6 levels at day 5 post-MI with higher expression of Ym-1, Mrc-1/CD206, and Arg-1 at day 5 post-MI, suggesting that macrophages are diverging toward the M2 type. Thus, acute PUFA exposure resulted in a shift of leukocytes toward resolving macrophages, primarily in 12/15-LOX−/− mice, with significant changes in the lipid mediator milieu.
Study limitations.
A key limitation of this study was the mouse strain, age, and sex. Here, we presented data from 2- to 3-mo-old male C57BL/6J mice. Overall, the lipid regulation and dysregulation in post-MI cardiac remodeling are distinct in female mice, aging mice, or other strains of mice. Another limitation was that we studied initial acute (day 1) and reparative phases (day 5) as primary time points with minimal focus on long-term survival benefit post-MI. A study design with chronic heart failure and 12/15-LOX signaling is necessary in MI and heart failure patients.
Perspectives and Significance
The present study suggests that deletion of 12/15-LOX and acute PUFA intake amplifies reparative lipid mediators (EETs), thereby promoting neutrophil clearance and improved wound healing post-MI. Future studies with quantitative analysis of leukocytes are warranted to understand the degree of acute intake of PUFA needed to resolve or limit inflammatory cell trafficking. Future research studies are warranted to develop novel targets and therapy to support the onset of LV healing and to promote reparative inflammation in heart failure pathology.
GRANTS
This work was supported by National Institutes of Health Grants AT-006704 and HL-132989 (to G. V. Halade) and by 16POST31000008 (to V. Kain).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
G.V.H. conceived and designed research; G.V.H., V.K., and K.A.I. performed experiments; G.V.H., V.K., and K.A.I. analyzed data; G.V.H. interpreted results of experiments; G.V.H. and V.K. prepared figures; G.V.H. and V.K. drafted manuscript; G.V.H. and S.D.P. edited and revised manuscript; G.V.H. approved final version of manuscript.
Supplementary Material
REFERENCES
- 1.Ander BP, Dupasquier CM, Prociuk MA, Pierce GN. Polyunsaturated fatty acids and their effects on cardiovascular disease. Exp Clin Cardiol 8: 164–172, 2003. [PMC free article] [PubMed] [Google Scholar]
- 2.Anzai T. Post-infarction inflammation and left ventricular remodeling−a double-edged sword. Circ J 77: 580–587, 2013. doi: 10.1253/circj.CJ-13-0013. [DOI] [PubMed] [Google Scholar]
- 3.Beam J, Botta A, Ye J, Soliman H, Matier BJ, Forrest M, MacLeod KM, Ghosh S. Excess linoleic acid increases collagen I/III ratio and ‘stiffens’ the heart muscle following high fat diets. J Biol Chem 290: 23371–23384, 2015. doi: 10.1074/jbc.M115.682195. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Braunwald E. The management of heart failure: the past, the present, and the future. Circ Heart Fail 1: 58–62, 2008. doi: 10.1161/CIRCHEARTFAILURE.107.752162. [DOI] [PubMed] [Google Scholar]
- 5.Carbone F, Nencioni A, Mach F, Vuilleumier N, Montecucco F. Pathophysiological role of neutrophils in acute myocardial infarction. Thromb Haemost 110: 501–514, 2013. doi: 10.1160/TH13-03-0211. [DOI] [PubMed] [Google Scholar]
- 6.Chadderdon SM, Belcik JT, Bader L, Kievit P, Grove KL, Lindner JR. Vasoconstrictor eicosanoids and impaired microvascular function in inactive and insulin-resistant primates. Int J Obes 40: 1600–1603, 2016. doi: 10.1038/ijo.2016.117. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Dai M, Wu L, He Z, Zhang S, Chen C, Xu X, Wang P, Gruzdev A, Zeldin DC, Wang DW. Epoxyeicosatrienoic acids regulate macrophage polarization and prevent LPS-induced cardiac dysfunction. J Cell Physiol 230: 2108–2119, 2015. doi: 10.1002/jcp.24939. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Dobrian AD, Lieb DC, Cole BK, Taylor-Fishwick DA, Chakrabarti SK, Nadler JL. Functional and pathological roles of the 12- and 15-lipoxygenases. Prog Lipid Res 50: 115–131, 2011. doi: 10.1016/j.plipres.2010.10.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Farvid MS, Ding M, Pan A, Sun Q, Chiuve SE, Steffen LM, Willett WC, Hu FB. Dietary linoleic acid and risk of coronary heart disease: a systematic review and meta-analysis of prospective cohort studies. Circulation 130: 1568–1578, 2014. doi: 10.1161/CIRCULATIONAHA.114.010236. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Ghaffari S, Nadiri M, Pourafkari L, Sepehrvand N, Movasagpoor A, Rahmatvand N, Rezazadeh Saatloo M, Ahmadi M, Nader ND. The predictive value of total neutrophil count and neutrophil/lymphocyte ratio in predicting in-hospital mortality and complications after STEMI. J Cardiovasc Thorac Res 6: 35–41, 2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Gilroy DW, Edin ML, De Maeyer RP, Bystrom J, Newson J, Lih FB, Stables M, Zeldin DC, Bishop-Bailey D. CYP450-derived oxylipins mediate inflammatory resolution. Proc Natl Acad Sci USA 113: E3240–E3249, 2016. doi: 10.1073/pnas.1521453113. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Halade GV, El Jamali A, Williams PJ, Fajardo RJ, Fernandes G. Obesity-mediated inflammatory microenvironment stimulates osteoclastogenesis and bone loss in mice. Exp Gerontol 46: 43–52, 2011. doi: 10.1016/j.exger.2010.09.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Halade GV, Kain V, Black LM, Prabhu SD, Ingle KA. Aging dysregulates D- and E-series resolvins to modulate cardiosplenic and cardiorenal network following myocardial infarction. Aging (Albany NY) 8: 2611–2634, 2016. doi: 10.18632/aging.101077. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Halade GV, Ma Y, Ramirez TA, Zhang J, Dai Q, Hensler JG, Lopez EF, Ghasemi O, Jin YF, Lindsey ML. Reduced BDNF attenuates inflammation and angiogenesis to improve survival and cardiac function following myocardial infarction in mice. Am J Physiol Heart Circ Physiol 305: H1830–H1842, 2013. doi: 10.1152/ajpheart.00224.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Halade GV, Rahman MM, Bhattacharya A, Barnes JL, Chandrasekar B, Fernandes G. Docosahexaenoic acid-enriched fish oil attenuates kidney disease and prolongs median and maximal life span of autoimmune lupus-prone mice. J Immunol 184: 5280–5286, 2010. doi: 10.4049/jimmunol.0903282. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Hanif A, Edin ML, Zeldin DC, Morisseau C, Nayeem MA. Deletion of soluble epoxide hydrolase enhances coronary reactive hyperemia in isolated mouse heart: role of oxylipins and PPARγ. Am J Physiol Regul Integr Comp Physiol 311: R676–R688, 2016. doi: 10.1152/ajpregu.00237.2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Harris WS, Mozaffarian D, Rimm E, Kris-Etherton P, Rudel LL, Appel LJ, Engler MM, Engler MB, Sacks F. Omega-6 fatty acids and risk for cardiovascular disease: a science advisory from the American Heart Association Nutrition Subcommittee of the Council on Nutrition, Physical Activity, and Metabolism; Council on Cardiovascular Nursing; and Council on Epidemiology and Prevention. Circulation 119: 902–907, 2009. doi: 10.1161/CIRCULATIONAHA.108.191627. [DOI] [PubMed] [Google Scholar]
- 19.Hilgendorf I, Gerhardt LM, Tan TC, Winter C, Holderried TA, Chousterman BG, Iwamoto Y, Liao R, Zirlik A, Scherer-Crosbie M, Hedrick CC, Libby P, Nahrendorf M, Weissleder R, Swirski FK. Ly-6Chigh monocytes depend on Nr4a1 to balance both inflammatory and reparative phases in the infarcted myocardium. Circ Res 114: 1611–1622, 2014. doi: 10.1161/CIRCRESAHA.114.303204. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Imig JD. Epoxyeicosatrienoic acids, hypertension, and kidney injury. Hypertension 65: 476–482, 2015. doi: 10.1161/HYPERTENSIONAHA.114.03585. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Imig JD, Hammock BD. Soluble epoxide hydrolase as a therapeutic target for cardiovascular diseases. Nat Rev Drug Discov 8: 794–805, 2009. doi: 10.1038/nrd2875. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Jenkins CM, Cedars A, Gross RW. Eicosanoid signalling pathways in the heart. Cardiovasc Res 82: 240–249, 2009. doi: 10.1093/cvr/cvn346. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Kain V, Ingle KA, Colas RA, Dalli J, Prabhu SD, Serhan CN, Joshi M, Halade GV. Resolvin D1 activates the inflammation resolving response at splenic and ventricular site following myocardial infarction leading to improved ventricular function. J Mol Cell Cardiol 84: 24–35, 2015. doi: 10.1016/j.yjmcc.2015.04.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Kain V, Ingle KA, Kabarowski J, Prabhu SD, Halade GV. Deletion of 12/15 lipoxygenase improves left ventricle function and survival by resolving inflammation post myocardial infarction (Abstract). Circulation 130: A12648–A12648, 2014. [Google Scholar]
- 25.Kain V, Ingle KA, Prabhu SD, Halade GV. Genetic deficiency of 12/15 lipoxygenase activates EP4 receptor on proresolving macrophages to attenuate renal inflammation after myocardial infarction (Abstract). Circulation 134: A16952–A16952, 2016. [Google Scholar]
- 26.Kain V, Prabhu SD, Halade GV. Inflammation revisited: inflammation versus resolution of inflammation following myocardial infarction. Basic Res Cardiol 109: 444, 2014. doi: 10.1007/s00395-014-0444-7. [DOI] [PubMed] [Google Scholar]
- 27.Kayama Y, Minamino T, Toko H, Sakamoto M, Shimizu I, Takahashi H, Okada S, Tateno K, Moriya J, Yokoyama M, Nojima A, Yoshimura M, Egashira K, Aburatani H, Komuro I. Cardiac 12/15 lipoxygenase-induced inflammation is involved in heart failure. J Exp Med 206: 1565–1574, 2009. doi: 10.1084/jem.20082596. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Kris-Etherton PM, Taylor DS, Yu-Poth S, Huth P, Moriarty K, Fishell V, Hargrove RL, Zhao G, Etherton TD. Polyunsaturated fatty acids in the food chain in the United States. Am J Clin Nutr 71, Suppl: 179S–188S, 2000. [DOI] [PubMed] [Google Scholar]
- 29.Lands B. Historical perspectives on the impact of n-3 and n-6 nutrients on health. Prog Lipid Res 55: 17–29, 2014. doi: 10.1016/j.plipres.2014.04.002. [DOI] [PubMed] [Google Scholar]
- 30.Lianos EA. Eicosanoid biosynthesis and role in renal immune injury. Prostaglandins Leukot Essent Fatty Acids 41: 1–12, 1990. doi: 10.1016/0952-3278(90)90124-4. [DOI] [PubMed] [Google Scholar]
- 31.Lopez EF, Kabarowski JH, Ingle KA, Kain V, Barnes S, Crossman DK, Lindsey ML, Halade GV. Obesity superimposed on aging magnifies inflammation and delays the resolving response after myocardial infarction. Am J Physiol Heart Circ Physiol 308: H269–H280, 2015. doi: 10.1152/ajpheart.00604.2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Lumeng CN. Innate immune activation in obesity. Mol Aspects Med 34: 12–29, 2013. doi: 10.1016/j.mam.2012.10.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Ma Y, Halade GV, Zhang J, Ramirez TA, Levin D, Voorhees A, Jin YF, Han HC, Manicone AM, Lindsey ML. Matrix metalloproteinase-28 deletion exacerbates cardiac dysfunction and rupture after myocardial infarction in mice by inhibiting M2 macrophage activation. Circ Res 112: 675–688, 2013. doi: 10.1161/CIRCRESAHA.111.300502. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Meissner J, Irfan A, Twerenbold R, Mueller S, Reiter M, Haaf P, Reichlin T, Schaub N, Winkler K, Pfister O, Heinisch C, Mueller C. Use of neutrophil count in early diagnosis and risk stratification of AMI. Am J Med 124: 534–542, 2011. doi: 10.1016/j.amjmed.2010.10.023. [DOI] [PubMed] [Google Scholar]
- 35.Oni-Orisan A, Alsaleh N, Lee CR, Seubert JM. Epoxyeicosatrienoic acids and cardioprotection: the road to translation. J Mol Cell Cardiol 74: 199–208, 2014. doi: 10.1016/j.yjmcc.2014.05.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Oni-Orisan A, Edin ML, Lee JA, Wells MA, Christensen ES, Vendrov KC, Lih FB, Tomer KB, Bai X, Taylor JM, Stouffer GA, Zeldin DC, Lee CR. Cytochrome P450-derived epoxyeicosatrienoic acids and coronary artery disease in humans: a targeted metabolomics study. J Lipid Res 57: 109–119, 2016. doi: 10.1194/jlr.M061697. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Ponikowski P, Anker SD, AlHabib KF, Cowie MR, Force TL, Hu S, Jaarsma T, Krum H, Rastogi V, Rohde LE, Samal UC, Shimokawa H, Budi Siswanto B, Sliwa K, Filippatos G. Heart failure: preventing disease and death worldwide. ESC Heart Failure 1: 4–25, 2014. doi: 10.1002/ehf2.12005. [DOI] [PubMed] [Google Scholar]
- 38.Reichel CA, Rehberg M, Lerchenberger M, Berberich N, Bihari P, Khandoga AG, Zahler S, Krombach F. Ccl2 and Ccl3 mediate neutrophil recruitment via induction of protein synthesis and generation of lipid mediators. Arterioscler Thromb Vasc Biol 29: 1787–1793, 2009. doi: 10.1161/ATVBAHA.109.193268. [DOI] [PubMed] [Google Scholar]
- 39.Roman RJ. P-450 metabolites of arachidonic acid in the control of cardiovascular function. Physiol Rev 82: 131–185, 2002. doi: 10.1152/physrev.00021.2001. [DOI] [PubMed] [Google Scholar]
- 40.Sandoo A, van Zanten JJ, Metsios GS, Carroll D, Kitas GD. The endothelium and its role in regulating vascular tone. Open Cardiovasc Med J 4: 302–312, 2010. doi: 10.2174/1874192401004010302. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Serhan CN, Chiang N, Van Dyke TE. Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators. Nat Rev Immunol 8: 349–361, 2008. doi: 10.1038/nri2294. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Simopoulos AP. The importance of the ratio of omega-6/omega-3 essential fatty acids. Biomed Pharmacother 56: 365–379, 2002. doi: 10.1016/S0753-3322(02)00253-6. [DOI] [PubMed] [Google Scholar]
- 43.Sirish P, Li N, Liu JY, Lee KS, Hwang SH, Qiu H, Zhao C, Ma SM, López JE, Hammock BD, Chiamvimonvat N. Unique mechanistic insights into the beneficial effects of soluble epoxide hydrolase inhibitors in the prevention of cardiac fibrosis. Proc Natl Acad Sci USA 110: 5618–5623, 2013. doi: 10.1073/pnas.1221972110. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Sudhahar V, Shaw S, Imig JD. Epoxyeicosatrienoic acid analogs and vascular function. Curr Med Chem 17: 1181–1190, 2010. doi: 10.2174/092986710790827843. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45.Sullivan KE, Quinn KP, Tang KM, Georgakoudi I, Black LD III. Extracellular matrix remodeling following myocardial infarction influences the therapeutic potential of mesenchymal stem cells. Stem Cell Res Ther 5: 14, 2014. doi: 10.1186/scrt403. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 46.Taylor-Fishwick DA, Weaver J, Glenn L, Kuhn N, Rai G, Jadhav A, Simeonov A, Dudda A, Schmoll D, Holman TR, Maloney DJ, Nadler JL. Selective inhibition of 12-lipoxygenase protects islets and beta cells from inflammatory cytokine-mediated beta cell dysfunction. Diabetologia 58: 549−557, 2014. doi: 10.1007/s00125-014-3452-0. [DOI] [PubMed] [Google Scholar]
- 47.Tersey SA, Maier B, Nishiki Y, Maganti AV, Nadler JL, Mirmira RG. 12-Lipoxygenase promotes obesity-induced oxidative stress in pancreatic islets. Mol Cell Biol 34: 3735–3745, 2014. doi: 10.1128/MCB.00157-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 48.Wu JH, Lemaitre RN, King IB, Song X, Psaty BM, Siscovick DS, Mozaffarian D. Circulating omega-6 polyunsaturated fatty acids and total and cause-specific mortality: the Cardiovascular Health Study. Circulation 130: 1245–1253, 2014. doi: 10.1161/CIRCULATIONAHA.114.011590. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49.Zamilpa R, Lopez EF, Chiao YA, Dai Q, Escobar GP, Hakala K, Weintraub ST, Lindsey ML. Proteomic analysis identifies in vivo candidate matrix metalloproteinase-9 substrates in the left ventricle post-myocardial infarction. Proteomics 10: 2214–2223, 2010. doi: 10.1002/pmic.200900587. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50.Zeldin DC. Epoxygenase pathways of arachidonic acid metabolism. J Biol Chem 276: 36059–36062, 2001. doi: 10.1074/jbc.R100030200. [DOI] [PubMed] [Google Scholar]
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