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. 2008 Oct;22(10):3595-606.
doi: 10.1096/fj.08-112201. Epub 2008 Jun 17.

Atherosclerosis: evidence for impairment of resolution of vascular inflammation governed by specific lipid mediators

Aksam J Merched  1 , Kerry Ko, Katherine H Gotlinger, Charles N Serhan, Lawrence Chan
Affiliations

Atherosclerosis: evidence for impairment of resolution of vascular inflammation governed by specific lipid mediators

Aksam J Merched et al. FASEB J. 2008 Oct.

Abstract

Atherosclerosis is now recognized as an inflammatory disease involving the vascular wall. Recent results indicate that acute inflammation does not simply passively resolve as previously assumed but is actively terminated by a homeostatic process that is governed by specific lipid-derived mediators initiated by lipoxygenases. Experiments with animals and humans support a proinflammatory role for the 5-lipoxygenase system. In contrast, results from animal experiments show a range of responses with the 12/15-lipoxygenase pathways in atherosclerosis. To date, the only two clinical epidemiology human studies both support an antiatherogenic role for 12/15-lipoxygenase downstream actions. We tested the hypothesis that atherosclerosis results from a failure in the resolution of local inflammation by analyzing apolipoprotein E-deficient mice with 1) global leukocyte 12/15-lipoxygenase deficiency, 2) normal enzyme expression, or 3) macrophage-specific 12/15-lipoxygenase overexpression. Results from these indicate that 12/15-lipoxygenase expression protects mice against atherosclerosis via its role in the local biosynthesis of lipid mediators, including lipoxin A(4), resolvin D1, and protectin D1. These mediators exert potent agonist actions on macrophages and vascular endothelial cells that can control the magnitude of the local inflammatory response. Taken together, these findings suggest that a failure of local endogenous resolution mechanisms may underlie the unremitting inflammation that fuels atherosclerosis.

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Figures

Figure 1.
Figure 1.
Macrophage 12/15-LO expression delays atherosclerosis in apoE−/− mice. A) Characterization of the expression of 12/15-LO by real time qPCR in different tissues of transgenic mice showing high-level expression in macrophages and tissues rich in macrophages, e.g., lung. The primers were specific for the human 15-LO transgene transcripts and did not amplify mouse endogenous 12/15LO transcripts [see control macrophages (Mph-) and inset panels]. B) LXA4 production by activated macrophages. Adherent peritoneal macrophages were activated with ionophore A23187 and incubated with 100 mM of arachidonic acid. C) Aortic atherosclerotic lesion area in apoE−/−/15LOtg+/0 (tg) mice compared with apoE−/− littermate controls (+/+) by morphometric analysis of en face lesions performed at 35 and 43 wk on mice fed a regular chow. Bars represent means ± se (n=11, 15 and 7, 14, respectively; all females). D) Top: representative aortas displayed en face; atherosclerotic lesions are stained red by Oil Red O (35 w). Bottom: aortas taken from 90-wk-old tg and +/+ mice. The rectangular area from each aorta is viewed under higher magnification for a close-up view of the abdominal lesions. *P < 0.05; **P < 0.01.
Figure 2.
Figure 2.
Leukocyte 12/15-LO deficiency diminishes the progression of atheroscleosis. A) Graphs show cross-sectional analysis of atherosclerosis lesion involvement of the aortic sinus in 12/15-LO−/−/apoE−/− (−/−) and apoE−/− (+/+) mice at 22 wk (males: n=11 −/−, 11 +/+; females: n=17 −/−, 8 +/+). Bars represent means ± se. Images show collagen staining (blue) by Masson’s trichrome (left panels) in −/− (top panel) vs. +/+ (bottom panel) and macrophage staining (red-brown) with Mac-3 antibody (right panels). B) En face lesion analysis at 27 wk. C) LXA4 production by ionophore-activated macrophages incubated with 100 mM of arachidonic acid.. D) En face aortic lesion shows −/− mice that received bone marrow transplantation from two groups of donors; 12/15LO−/− apoE−/− (−/−) and 12/15LO+/+ apoE−/− (+/+). 12/15-LO expression (top bands, lanes 3 and 4) in macrophages was confirmed by RT-PCR using GAPDH (bottom bands) as an internal normal control. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 3.
Figure 3.
Plasma lipid and cytokine levels in 12/15-LO–/–/apoE−/− (−/−) ,apoE−/− (+/+) and apoE−/−/12/15-LOtg (tg) mice. A) Plasma cholesterol concentration in −/− and +/+ mice for both males and females. Values are means ± se (n=10 and 10 for males, and n=14 and 8 for females, respectively). B) Plasma lipoprotein profile by FPLC (males). C) Plasma triglycerides in male and female −/− and +/+ mice. D) Plasma cholesterol and triglycerides in +/+ and tg mice (all female). Values are means ± se (n=13 and 8). E) IL-17 and CCL5 were dowregulated in tg mice vs. +/+ controls. F) IL-12p40 and CCL5 were upregulated in −/− mice vs. +/+ controls. We measured 18 cytokines using Bioplex Protein Array system (Bio-Rad) as detailed in Material and Methods. Plasma from 4 to 6 mice was used for the determination of each cytokine. Values are means ± se; *P < 0.05, **P < 0.01.
Figure 4.
Figure 4.
Lipid mediators produced by LPS-activated macrophages from wild-type and 12/15-LOtg mice. A) Macrophages from three different mice were incubated with DHA (0.2 μg/ml) after exposure to 500 ng/ml of LPS. Samples were pooled and extracted for lipidomic study. Compared to those from wild-type (+/+) mice, macrophages isolated from 12/15LOtg mice produced markedly reduced PGE2, increased amounts of PD1 and 17HDHA. RvD1 was not identified in these conditions. B) Lipid mediators produced by macrophages incubated with 17HDHA (0.15 μg/ml) for 30 min. C–E) LC/MS/MS spectra of recovered D4-PGE2, PD1, and 17HDHA (respectively) identified in macrophages from 12/15-LOtg mice incubated with DHA. F) LC/MS/MS spectra of RvD1 produced by macrophages from wild-type mice incubated with 17HDHA.
Figure 5.
Figure 5.
LXA4, RvD1, and PD1 action on macrophages. A) Correlation of CCL5 plasma levels with macrophage (Mφ) CCL5 transcript expression in relation to 12/15LO gene dosage. CCL5 transcript levels in peritoneal macrophages by real-time RT-PCR. Each measurement is on RNA isolated from macrophages from 3 to 6 mice from each genotype. Error bars = sem. B) CCL5 secretion by macrophages treated with 50 nM of LXA4 shows down-regulation of CCL5. C) Macrophage global cytokine response to LXA4 treatment shows significant reduction of 10 inflammatory cytokines. D, E) PD1 and RvD1 actions on macrophages. Macrophages were activated with LPS for 4 h, then incubated with PD1 (D) or RvD1 (E). Cytokines produced under these conditions were measured by bioplex technology as described in Materials and Methods. Negative values indicate that the cytokine produced was reduced in the presence of PD1 or RvD1 compared to the amount produced in their absence. PD1 and RvD1 down-regulate LPS-induced production of the vast majority of the cytokines tested. n = 3 to 4. F) Actions of LXA4, PD1, and RvD1 (100 nM) on phagocytic activity of macrophages toward apoptotic thymocytes. Data are from 4 separate incubations; >500 cells counted in each assay. Values are means ± se; *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle. G) Representative views of macrophages (nuclei stained by DAPI, blue) that have taken up CellTracker-labeled apoptotic thymocytes (green).
Figure 6.
Figure 6.
Actions of LXA4, PD1, and RvD1 on the expression of chemokines, cytokines, and adhesion molecules by HAECs. A) RvD1 action on HAECs shows significant inhibition of IL8 and MCP-1 and increase of PDGF β as measured by bioplex technology. B) Effect of incubation with LO products on proteins involved in adhesions of leukocytes, evaluated by real time qPCR. Data are from 3 different cultures. Values are means ± se; *P < 0.05, **P < 0.01 vs. vehicle.
Figure 7.
Figure 7.
Model of atherosclerosis as a nonresolving form of vascular inflammation. The essential ω-6 PUFA arachidonic acid (AA) is released from phospholipids in cells by the action of cytosolic phospholipase A2. After specific enzymatic steps, AA is converted into different families of mediators: prostaglandins (PGs) and leukotrienes (LTs), which are mostly proinflammatory molecules, and lipoxins such as LXA4, a stop-inflammation mediator. The essential ω-3 PUFA DHA is converted to two novel mediators, RvD1 and PD1, that promote resolution. We propose a model in which absence of macrophage 12/15LO leads to a deficiency in proresolving end products, RvD1 and PD1, as well as LXA4, locally at the site of the ongoing inflammation, crippling multiple proresolving functions, leaving the proinflammatory milieu unabated, and fueling atherosclerosis progression. LOX, lipoxygenase.
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