Macrophage-Specific IGF-1 Overexpression Reduces CXCL12 Chemokine Levels and Suppresses Atherosclerotic Burden in Apoe-Deficient Mice

Abstract

Objective: IGF-1 (insulin-like growth factor 1) exerts pleiotropic effects including promotion of cellular growth, differentiation, survival, and anabolism. We have shown that systemic IGF-1 administration reduced atherosclerosis in Apoe^-/- (apolipoprotein E deficient) mice, and this effect was associated with a reduction in lesional macrophages and a decreased number of foam cells in the plaque. Almost all cell types secrete IGF-1, but the effect of macrophage-derived IGF-1 on the pathogenesis of atherosclerosis is poorly understood. We hypothesized that macrophage-derived IGF-1 will reduce atherosclerosis. Approach and Results: We created macrophage-specific IGF-1 overexpressing mice on an Apoe^-/- background. Macrophage-specific IGF-1 overexpression reduced plaque macrophages, foam cells, and atherosclerotic burden and promoted features of stable atherosclerotic plaque. Macrophage-specific IGF1 mice had a reduction in monocyte infiltration into plaque, decreased expression of CXCL12 (CXC chemokine ligand 12), and upregulation of ABCA1 (ATP-binding cassette transporter 1), a cholesterol efflux regulator, in atherosclerotic plaque and in peritoneal macrophages. IGF-1 prevented oxidized lipid-induced CXCL12 upregulation and foam cell formation in cultured THP-1 macrophages and increased lipid efflux. We also found an increase in cholesterol efflux in macrophage-specific IGF1-derived peritoneal macrophages.

Conclusions: Macrophage IGF-1 overexpression reduced atherosclerotic burden and increased features of plaque stability, likely via a reduction in CXCL12-mediated monocyte recruitment and an increase in ABCA1-dependent macrophage lipid efflux.

Keywords: atherosclerosis; cytokines; inflammation; intercellular signaling peptides and proteins; lipids.

Figure 1:. Macrophage-specific IGF-1 overexpressing mice.

A. IGF-1 protein isolated from mice with macrophage-specific IGF-1 overexpression (#12 and #17 mice) and control (Apoe^−/− mice) (N=4– 5 mice per group) showing no significant difference in any tissue. B. Serum IGF-1 levels measured by ELISA (N=4–5 mice per group). C. IGF-1 levels in conditioned media of peritoneal macrophages (N=22–25 mice per group). IGF-1 levels were normalized to total protein levels. D. IGF-1 mRNA levels in peritoneal macrophages in #17 (MF-IGF1) and control mice after 24 hours in SFM (N=12–14 mice per group). E. IGF-1 mRNA levels in plaque samples isolated by LCM in #17 (MF-IGF1) and control mice (N=3–6 mice per group). F. IGF-1 mRNA levels in circulating CD11b⁺/F4/80⁺ monocytes (N=5–6 mice per group). A, B, C used one-way ANOVA with a Tukey’s post-hoc test. All other statistical tests are Student’s two-tailed t-test.

Figure 2:. Macrophage-specific IGF-1 overexpression reduced atherosclerosis.

Macrophage-specific IGF-1 overexpressing (MF-IGF1 mice) and control mice (Con) were fed with a high-fat diet. A, C. Enface analysis of atherosclerotic burden (N=30–37 mice per group). B, D. H&E stained cross-sectional aortic valve sections to assess lesional area (N=16–29 mice per group). B Insert: magnified view of lesions showing that plaque in MF-IGF1 mice is less cellular. Scale bar, 100 μm. E. Necrotic core area in atherosclerotic plaque (N=14–15 mice per group). All statistical tests are Student’s two-tailed t-test.

Figure 3:. Macrophage-specific IGF-1 overexpression changed atherosclerotic plaque toward a more stable plaque phenotype.

A, E Serial aortic valve cross-sections were stained with Mac3 antibody, (N=16–29 mice per group). B, G. Aortic valve section were stained with trichrome (N=16–29 mice per group). C, H. Snap-frozen aortic valve sections were stained with Oil Red O (N=6–7 mice per group). D, I. TUNEL assay co-stained with Mac3 antibody (N=5–6 mice per group). Data is normalized to 1000 Mac3⁺ cells. Insert showing TUNEL⁺/Mac3⁺-positive plaque cells. F. Mac3/DAPI double positive cell number was normalized to total number of DAPI positive cells in the plaque. (N=13 mice per group). Scale bar, 100 μm. All statistical tests are Student’s two-tailed t-test, except (G), which has a Welch’s correction to account for a difference in SDs. L=Lumen

Figure 4:. Macrophage-specific IGF-1 overexpression reduced monocyte recruitment into atherosclerotic plaque and decreased CXCL12 chemokine expression.

A, B. Monocyte recruitment was measured by quantification of plaque levels of red beads after normalization to plaque size. Arrows, plaque red spots (N=4–7 mice per group). C. Circulating levels of CXCL12 measured by ELISA (N=8–11 mice per group). D, F. CXCL12 positive area was normalized to plaque area (N=10 mice per group). E,G. CXCL12/Mac3 positive area was normalized to cell number (N=8–10 mice per group). H. CXCL12 mRNA levels in LCM plaque isolates (N=3–5 mice per group). I. CXCL12 levels in peritoneal macrophages were quantified by immunoblotting. Predicted weight 7–14kDa. (N=8–9 mice per group). Scale bar, 100 μm. All statistical tests are Student’s two-tailed t-test, except B, which had a Welch’s correction due to differences in SDs. AP=Atherosclerotic Plaque, L=lumen

Figure 5:. Macrophage-specific IGF-1 overexpression upregulated ABCA1 expression.

A. ABCA1 mRNA levels in LCM plaque isolates (N=3–5 mice per group). B. ABCA1 protein levels in peritoneal macrophages were quantified by immunoblotting. Cells were incubated in SFM for 48 hours. Predicted weight 254 kDa. (N=10–11 mice per group). C. Peritoneal macrophages were pretreated with PPP and ABCA1 protein levels were measured (N=3–4 wells per group in 2 independent experiments). D. Cholesterol efflux assay with ApoAI as an acceptor (N=3 wells per animal, 3 mice in each group) or with HDL as an acceptor. (N=1 well per animal, 3 mice per group). All statistical tests are Student’s two-tailed t-test.

Figure 6:. IGF-1 reduced formation of THP-1 macrophages-derived foam cell formation.

THP-1-derived macrophages were pretreated with IGF-1 and then treated with oxLDL or ntLDL. A. CXCL12 mRNA levels in THP-1 macrophages (N=3 wells per group per experiment, 3 independent experiments). B.10μg/ml ApoA1 was used as a cholesterol acceptor and cells were pretreated with IGF-1 then treated with oxLDL. Cholesterol efflux capacity was normalized to 0ng/ml IGF-1 treatment (Con). (N=4–7 wells per group per experiment, 3 independent experiments). C. 200μg/ml HDL was used as a cholesterol acceptor and cells were pretreated with IGF-1 then treated with oxLDL. Cholesterol efflux capacity was normalized to 0ng/ml IGF-1 treatment (Con). (N=3 wells per group per experiment, 2 independent experiments). D. Representative images of Oil Red O-stained macrophages. E. Quantitative data. (N=3 wells per group per experiment, 3 independent experiments). F. Cells were treated with IGF-1 and/or CXCL12 and then Oil Red O staining was used to quantify neutral lipids. (N=3 wells per group, 3 independent experiments). All statistics are one-way ANOVA except in D and E, which used a Student’s two-tail t-test. B used a Tukey’s post-hoc test, and C used Dunnett’s post -hoc test.