Reversal of hyperlipidemia with a genetic switch favorably affects the content and inflammatory state of macrophages in atherosclerotic plaques

Abstract

Background: We previously showed that the progression of atherosclerosis in the Reversa mouse (Ldlr(-/-Apob100/100Mttpfl/fl) Mx1Cre(+/+)) was arrested when the hyperlipidemia was normalized by inactivating the gene for microsomal triglyceride transfer protein. Here, we tested whether atherosclerosis would regress if the lipid levels were reduced after advanced plaques formed.

Methods and results: Reversa mice were fed an atherogenic diet for 16 weeks. Plasma lipid levels were then reduced. Within 2 weeks, this reduction led to decreased monocyte-derived (CD68(+)) cells in atherosclerotic plaques and was associated with emigration of these cells out of plaques. In addition, the fall in lipid levels was accompanied by lower plaque lipid content and by reduced expression in plaque CD68(+) cells of inflammatory genes and higher expression of genes for markers of antiinflammatory M2 macrophages. Plaque composition was affected more than plaque size, with the decreased content of lipid and CD68(+) cells balanced by a higher content of collagen. When the reduced lipid level was combined with the administration of pioglitazone to simulate the clinical aggressive lipid management and proliferator-activated receptor-γ agonist treatment, the rate of depletion of plaque CD68(+) cells was unaffected, but there was a further increase in their expression of antiinflammatory macrophage markers.

Conclusion: The Reversa mouse is a new model of atherosclerosis regression. After lipid lowering, favorable changes in plaque composition were independent of changes in size. In addition, plaque CD68(+) cells became less inflammatory, an effect enhanced by treatment with pioglitazone.

Figure 1. Correcting hyperlipidemia in Reversa mice reduces the content of CD68+ cells in plaques

(A) CD68+ cell content in plaques of Reversa mice after plasma lipid normalization. Reversa mice (n=13/group) were fed a western diet for 16 weeks, at which point their high plasma lipid levels were either maintained (saline group) or normalized (pIpC group), as described in Methods. CD68+ cell content, quantified by morphometric techniques, was significantly lower 14 days after plasma lipids stabilized at the reduced levels. Statistical analysis within each treatment group was by ANOVA followed by Dunnett’s test to compare results at each time point to those on day 0. ⁺p < 0.03 or *p <0.0001 compared to day 0. (B) Immunohistochemical staining of CD68. By 56 days, CD68+ cells (red) in pIpC-treated mice were virtually undetectable.

Figure 2. Transplantation of aortic segments from hyperlipidemic Reversa mice into normolipidemic Reversa mice leads to CD68+ cell depletion

(A) Male hyperlipidemic Reversa mice were fed western diet for 16 weeks and aortic segments transplanted into normolipidemic Reversa (i.e., treated with pIpC and used 2 weeks thereafter). The transplanted aortic segments were then harvested 5 days later and morphometric analysis of plaque CD68 content was performed. *p=0.0045 compared to baseline by unpaired two-tailed t-test; n = 6/group. (B) Representative sections demonstrating a decrease in CD68+ cells as assessed by immunohistochemistry.

Figure 3. Correcting hyperlipidemia in Reversa mice lowers neutral lipid content in aortic atherosclerotic plaques

(A) Immunohistochemistry and co-localization studies were performed in the Baseline and pIpC-treated Reversa mice after 16 weeks of western diet feeding and 14 days after reduction of plasma lipids, respectively. The pictures are representative images showing Oil-red-O staining (neutral lipid, presumably cholesteryl ester). Note that although the staining is less for lipid and CD68+ cells in the pIpC-treated mouse, in both cases, there is co-localization of the 2 plaque components as assessed by confocal immunofluorescence microscopy. (B) Oil-red-O staining of the aortae of Reversa mice at baseline and 14 days after reduction of the plasma lipids (by pIpC injection). Reversa mice treated with pIpC had visibly reduced amounts of Oil-red-O staining. (C) Morphometric analysis of Oil-red-O staining. *p < 0.05 compared to baseline. n = 8/group. “L” indicates the lumen of the artery.

Figure 4. Effects of the correction of hyperlipidemia on CCR7 expression levels in plaque CD68+ cells and on the emigration from plaques of macrophages derived from Ly6C^hi and Ly6C^lo circulating monocytes

(A) Mice were treated as in Figure 1. CCR7 mRNA levels in plaque CD68+ cells laser captured 14 days after pIpC or saline treatments were assessed by qRT-PCR. (B) CCR7 protein expression in aortic plaques under the conditions in panel A, as judged by immunohistochemistry with a CCR7-specific antibody. (C, D) Quantification of the relative levels in plaques of macrophages derived from circulating Ly6C^hi (“CCR2+”) and Ly6C^lo (“CX3CR1+”) monocytes. One week before the first pIpC or saline injection, the two major subsets types of circulating monocytes were labeled in vivo with fluorescent beads (Methods). Fourteen days after the baseline time point, the number of fluorescent beads in plaques was counted. Fewer beads were present in sections from pIpC-treated mice, indicating emigration of the macrophages containing them. *p < 0.05; n >10/group in panel A; n = 5/group for panels C and D. “L” indicates the lumen of the artery.

Figure 5. Correction of the hyperlipidemia in Reversa mice has little effect on the size of plaques, but increases their collagen content

(A) Plaque areas at baseline (day 0) and at various time points after treating the mice with pIpC or normal saline, as measured by morphometric analysis of hematoxylin and eosin–stained sections. Statistical analysis of the means in the pIpC group was based on N=13 for each time point; * indicates p<0.01 for the comparison between day 0 and any other time point; ns indicates no statistical difference among the other time points. (B) Quantification of collagen content in plaques at baseline (day 0) or 14 days after saline or pIpC treatment, as judged by morphometric analysis of Sirius Red–stained sections examined under polarizing light. Statistical analysis of the means was based on N=11 for each group; * indicates p<0.0001 for the comparison of the pIpC group to either the baseline (day 0) or saline treatment group. (C) Representative section showing a diffuse intimal mesh and a sub-endothelial collection of collagen under regression conditions from a pIpC-treated mouse used for the analysis shown in panel B. “L” and “M” indicate the lumen and the medial layer of the artery, respectively.

Figure 6. Correction of the hyperlipidemia in Reversa mice is associated with reduced expression of inflammatory markers in plaque CD68+ cells

CD68+ cells in plaques were obtained by laser-capture microdissetion 14 days after baseline. Levels of gene expression were compared in CD68+ cells from pIpC- and saline–treated Reversa mice. Total RNA was isolated, and transcript levels of VCAM-1, ICAM-1, MCP-1, and TNFα were measured by qRT-PCR. Data were from two pools of RNA, each from three mice, and are expressed as fold change compared with samples obtained from mice at baseline. *p < 0.05.

Figure 7. M2 macrophage markers are enriched in CD68+ cells in the plaques of Reversa mice under regression conditions, with further enhancement by PPARγ activation

Mice were treated as in Figure 1 and CD68+ cells were harvested by laser-capture microdissection and their RNA isolated as in Figure 6. qRT-PCR was then used to measure the levels of the indicated M2 markers. Note that in pIpC-treated mice, the expression of these markers were higher than in baseline mice, with further enhancement found with pIpC and PPARγ activator pioglitazone co-treatment. *p < 0.05 compared with baseline; **p < 0.05 compared with pIpC. Data are from three pools of RNA, each from three mice, and are expressed as fold change compared with samples obtained from mice at baseline.

Figure 8. PPARγ activation does not increase the rate of CD68+ cell depletion, but further reduces PAI-1 expression, in Reversa mouse plaques after hyperlipidemia was corrected

(A) Reversa mice were treated with pIpC to reverse the hyperlipidemia as in Figure 1; one-half of the mice received pioglitazone in their diet. Plaque CD68+ cell content was measured by morphometric analysis of sections stained by immunohistochemistry. n = 8/group. (B) Reversa mice were treated as in Figure 1 and CD68+ cells were obtained by laser-capture microdissection. The level of PAI-1 transcripts was measured by qRT-PCR and normalized to cyclophilin A. *p < 0.05 compared to baseline mice. **p < 0.05 compared with the mice treated with pIpC alone. Data are from three pools of RNA, each from three mice.