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

Cardiovascular disease is the global leading cause of death, and atherosclerosis is the primary cause of cardiovascular disease.¹ Atherosclerosis is a chronic inflammatory disease² in which monocytes and macrophages are the predominant cell types mediating the inflammatory response and disease progression.^3,4 Oxidation of subendothelial LDLs (low-density lipoproteins) leads to an inflammatory cascade resulting in recruitment of monocytes and proliferation of macrophages that secrete chemokines⁵ that cause more monocytes to be recruited to the lesion,⁶ resulting in feed-forward inflammation that promotes plaque progression.⁷ Atherosclerotic plaque burden and the degree of macrophage infiltration are important contributors to plaque instability and the risk of plaque rupture, which underlies most acute coronary events.^8–10

Although IGF-1 (insulin-like growth factor 1) circulating levels peak at puberty, IGF-1 is continually and almost ubiquitously expressed throughout life.¹¹ It plays a critical role in normal growth and development.^12,13 In addition to the endocrine effects exerted by liver-derived circulating IGF-1, there are many IGF-1–mediated effects that result from autocrine and paracrine mechanisms. Most cells express both IGF-1 and its receptor IGF1R (IGF-1 receptor).¹⁴ There is growing evidence that circulating IGF-1 levels are inversely related to the risk of cardiovascular disease.^14–16 We have previously demonstrated that IGF-1 exerts antiatherosclerotic effects in Apoe^−/− (apolipoprotein E deficient) mice, a murine model of atherosclerosis.^17–21 Systemic IGF-1 administration reduced atherogenesis and decreased plaque macrophages and foam cell formation.²⁰ The effects of increased circulating IGF-1 could be mediated by many cell types that express IGF1R, notably monocyte/macrophages, endothelial cells, and smooth muscle cells (SMCs). All these cell types secrete IGF-1, and the role of locally produced IGF-1 in the pathogenesis of atherosclerosis is poorly understood. Deletion of monocyte/macrophage-specific IGF1R increased atherogenesis, promoted a proinflammatory macrophage phenotype, increased monocyte recruitment to the plaque, and IGF1R-deficient macrophages had reduced lipid efflux, suggesting that monocyte/macrophage IGF1R signaling had antiatherogenic effects.¹⁷

To determine the role of monocyte/macrophage-derived IGF-1 in atherogenesis, we generated an IGF-1 gain-of-function model specifically in macrophages and investigated potential mechanisms of IGF-1’s antiatherogenic effects. Our findings indicate that macrophage-derived IGF-1 downregulates expression of the chemokine CXCL12 (CXC chemokine ligand 12), thereby lowering monocyte recruitment to plaques and lipid accumulation within macrophages via elevated lipid efflux. Our results demonstrate a novel link between IGF-1 and CXCL12 suggesting that CXCL12 could play a major role in the antiatherosclerotic effects of macrophage-derived IGF-1.

Materials and Methods

The data that support the findings of this study are available from the corresponding author upon reasonable request. Detailed Materials and Methods are available in the Supplemental Material.

Animals

All animal experiments were approved by the Institutional Animal Care and Use Committees of the University of Missouri-Columbia and Tulane University. The rat IGF-1 gene (NC_005106.4) was cloned in a plasmid containing the human SRA (scavenger receptor-A) promoter cassette (plasmid kindly provided by Dr Glass, University of California-San Diego²²) and GFP cDNA to generate SRA-IGF-1-IRES-GFP vector. Southern blot²³ confirmed IGF-1 gene inserts in the genome of founders (Figure S1A). Transgenic lines #12 and #17 were crossed with Apoe^−/− mice (Jackson Laboratory #002052) to generate SRA-IGF-1/Apoe^−/− mice. Nontransgenic Apoe^−/− littermates were used as controls, and male and female mice were used equally.

Laser Capture Microdissection

Aortic valve sections were isolated and dissected as described previously.^24,25

Oil Red O Assay

Foam cell formation was assayed using human THP-1 mononuclear cells (ATCC TIB-202) as described previously.²⁶

Isolation of Peritoneal Macrophages

Peritoneal macrophages were isolated as described previously.¹⁷

Cholesterol Efflux Assay

THP-1 cells were treated with IGF-1 (100 ng/mL, 24 hours) and then IGF-1 and oxidized LDL (oxLDL; 50 µg/mL, 24 hours) in serum-free media. Efflux²⁷ was measured according to manufacturer’s instructions (Sigma-Aldrich; MAK192).

Isolation of Monocytes

Circulating and splenic monocytes were identified using antibodies from BioLegend as described previously.¹⁷ Cell identity of monocytes was confirmed by assessment of cell markers (Table 1).

Monocyte Recruitment Into Atherosclerotic Plaque

Mice were injected intravenously with fluorescent polychromatic red microspheres (Polyscience; 19507-5) at 7 and 3 days before sacrifice.^28,29

Circulating Cell Quantification

Circulating leukocytes and endothelial progenitor cells were quantified via FACs using modified previous gating strategies.^30–32

Atherosclerosis Quantification

After 12 weeks of high-fat diet, animals were sacrificed, and atherosclerosis was quantified as described previously.²¹

Atherosclerotic Plaque Composition

Plaque composition was assessed by immunostaining of aortic valve cross-sections for CD107b (Mac3), calponin, and αSMA (α-smooth muscle actin). Masson Trichrome staining was used to quantify collagen. All IgG controls for immunohistochemistry data can be found in Figure S9. Cell apoptosis was quantified with TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling)-Fluorescein kit (Roche; 11684795910) with costaining for Mac3 as described previously.²⁰

Immunoblotting Analysis

This was performed as described previously.¹⁷ Antibodies for immunohistochemistry and immunoblotting are listed in the major resources table.

Quantitative Real-Time Polymerase Chain Reaction

Total RNA extraction and real-time polymerase chain reaction were performed as described previously.¹⁷

Statistical Analysis

All numeric data are expressed as mean±SEM. Statistical analyses were performed with the GraphPad PRISM (version 8.0) software. Significant differences were determined by unpaired Student t test with or without the Welch correction or 1-way ANOVA with either a Dunnett or Tukey post hoc test accordingly with the normality of residuals distribution or sample size. Fisher exact test was used to compare frequency distribution differences between groups. The exact test used is mentioned in every figure legend. Differences were considered significant at P<0.05. We declare that the design, execution, and reporting of the current study adheres to the guidelines for experimental atherosclerosis studies described and recommended by the American Heart Association, and we also considered sex as a biological variable as explained by the ATVB Council.^33,34

Results

Generation of Macrophage-Specific IGF-1 Overexpressing Mice

We obtained 13 founder lines in which IGF-1 is overexpressed under the control of the SRA promoter (Figure S1A). Although we utilized internal ribosomal entry site (IRES)–mediated bicistronic gene expression to label rat IGF-1 expressing cells by GFP, we failed to observe any GFP-positive cells in macrophages from these animals both in vitro and in vivo (data not shown), likely due to the lower expression of a gene downstream of IRES as described previously.³⁵ We selected lines #12 and #17 because they were the two founder lines that had multiple tandem transgene copies and were successful breeders (Figure S1B). Because mature rat and mouse IGF-1 differ by only one amino acid,³⁶ we expected that IGF-1 signaling would be the same whether using rat or mouse IGF-1. Indeed, IGF-1 signaling (as measured by comparing pAKT/Akt levels in peritoneal macrophages) did not change when either mouse or rat recombinant IGF-1 (0–20 ng/mL) was added to cells (Figure S1C). We also confirmed that insertion of the IGF-1 transgene did not disrupt the CXCL12 gene by amplifying the entire CXCL12 sequence and then amplifying regions within that amplicon using nested polymerase chain reaction (Figure S1D).

Since mouse and rat mature IGF-1 protein are almost identical³⁶ and exert similar signaling, we measured total IGF-1 levels in all experiments. In almost all mouse models of IGF-1 overexpression, a rat IGF-1 construct was used,^18,37–40 and these models do not note any difference in signaling between endogenous and exogenous IGF-1. We found no difference in tissue and serum IGF-1 levels in the #12 and #17 transgenics versus control Apoe^−/− mice (Figure 1A and 1B). We also did not find a difference in SMC IGF-1 expression between groups (control, 3.0±1.4 pg/mL; macrophage-specific IGF1 [MF-IGF1], 1.4±0.8 pg/mL; P=NS). However, IGF-1 levels were significantly increased in the conditioned media of peritoneal macrophages isolated from the #17 line (1.8-fold increase) while there was no significant increase in IGF-1 levels in the #12 line (Figure 1C). After confirming IGF-1 gene overexpression and increased intracellular IGF-1 protein in peritoneal macrophage cell lysates (Figure 1D; Figure S2A), the #17 transgenic line was chosen for experiments outlined in the current report (henceforth referred to as MF-IGF1 mice). Starting at 8 weeks of age, animals were fed with a high-fat diet for 12 weeks to accelerate atherosclerosis development.

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To test whether atherosclerotic plaques in MF-IGF1 mice had increased IGF-1 gene expression, we isolated plaques by Laser Capture Microdissection (see Methods) and quantified IGF-1 mRNA levels using primers designed to detect both mouse and rat Igf1 genes.¹⁸ MF-IGF1 mice had an 18.4-fold increase in total IGF-1 gene expression in Laser Capture Microdissection plaque isolates (Figure 1E). We did not observe increased IGF-1 mRNA levels in circulating CD11b⁺/F4/80⁺ monocytes in MF-IGF1 mice, potentially due to low SRA promoter activity in this cell population (Figure 1F). Taken together, these results strongly suggest that MF-IGF1 mice have a selective increase in macrophage IGF-1 levels.

IGF-1 binds with high affinity to IGF1R and with lower affinity to InsR (insulin receptor), although both can mediate intracellular signaling.⁴¹ We found no difference in IGF1R or InsR expression in peritoneal macrophages isolated from MF-IGF1 mice compared with control mice (Figure S2B). oxLDL (50 µg/mL) treatment reduced IGF1R expression in peritoneal macrophages, but the effect was the same in control and MF-IGF1 mice (Figure S2C), which is consistent with the effect of oxLDL on IGF1R expression in SMCs⁴² and human-derived macrophages (THP-1 cells).¹⁷ There was also no difference in IGF1R mRNA levels in circulating monocytes between MF-IGF1 mice and controls (Figure S5A). Under normal laboratory diet (PicoLab Rodent Diet 5053) feeding, both sexes of MF-IGF1 mice showed a small trend of increase in BW compared with control (14.5% increase, P=NS) at 8 weeks of age. When fed with high-fat diet, MF-IGF1 mice showed higher body weight (average 24.1% increase) at weeks 10 to 18, whereas the difference was not significant at 20 weeks (Figure S2D). No difference was found in systolic blood pressure (Figure S2E), total cholesterol (control, 17.7±1.8 mmol/L; MF-IGF1, 20.2±1.2 mmol/L; P=NS), free cholesterol levels (control, 12.7±2.1 mmol/L; MF-IGF1, 15.4±1.1 mmol/L; P=NS), or triglyceride levels (control, 2.6±0.6 mmol/L; MF-IGF1, 2.4±0.9 mmol/L; P=NS) between control and MF-IGF1 mice after 12 weeks of high-fat diet. Neither cholesterol nor triglyceride levels differed between groups after 4 weeks of high-fat diet (free cholesterol: control, 9.2±0.9 mmol/L; MF-IGF1, 11.4±1.3 mmol/L; total cholesterol: control, 16.1±1.8 mmol/L; MF-IGF1, 19.0±1.8 mmol/L; triglycerides: control, 1.7±0.2 mmol/L; MF-IGF1, 2.5±0.4 mmol/L; P=NS for all).

Macrophage-Specific IGF-1 Overexpression Reduced Atherosclerotic Burden and Changed Atherosclerotic Plaque Composition Toward a Stable Plaque Phenotype

Macrophage-specific IGF-1 overexpression significantly reduced Oil Red O–positive plaque area by 29.7% (Figure 2A and 2C), as well as cross-sectional lesion area by 28.3% (Figure 2B and 2D). Total aortic area was not different between MF-IGF1 and control mice (control, 262.4±30.3 mm²; MF-IGF1, 268.1±32.6 mm²). Atherosclerotic plaques in MF-IGF1 mice had a significant 29.5% reduction in acellular/necrotic core area compared with control Apoe^−/− mice (Figure 2E). MF-IGF1 mice had a significant reduction in plaque macrophage levels (26.4% decrease in Mac3 immunopositivity; Figure 3A and 3E), and the percentage of cells that costained for DAPI (4′,6-diamidino-2-phenylindole) and Mac3 in the plaque was reduced by 30% in MF-IGF1 mice (Figure 3F). No change was detected in plaque SMC levels as was quantified by immunohistochemistry for 2 SMC contractile proteins^43,44: calponin and αSMA (Figure S4A through S4D). The ability of IGF-1 to reduce atherosclerotic burden, necrotic core area, and macrophage content was not different between sexes (Figure S3A through S3C). MF-IGF1 mice had a ≈2-fold increase in plaque collagen (trichrome staining; Figure 3B and 3G), but this increase was statistically significant only in female mice (Figure S3D). Oil Red O is widely used to stain neutral lipids, including those present in lipid-laden (foam) cells in vitro and in vivo.^45–47 Atherosclerotic plaques in MF-IGF1 mice had a marked reduction in Oil Red O–positive area compared with control (60% reduction; Figure 3C and 3H) indicating that macrophage-specific IGF-1 overexpression decreased lesional lipids. Furthermore, MF-IGF1 mice had reduced apoptosis specifically in plaque macrophages, as TUNEL⁺/Mac3⁺ cell numbers decreased by 55.4% (Figure 3D and 3I), whereas there was no difference in TUNEL⁺/Mac3⁻ cell number (Figure S4E). Thus, macrophage-specific IGF-1 overexpression reduced atherosclerotic burden, decreased plaque macrophages, reduced foam cells, and did not change plaque SMC levels. The decrease in plaque macrophage accumulation and necrotic core area, together with the increase in plaque collagen levels,¹⁰ suggested that macrophage IGF-1 overexpression promoted features of stable atherosclerotic plaque.

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Macrophage-Specific IGF-1 Overexpression Reduced Monocyte Recruitment Into Atherosclerotic Plaque and Decreased CXCL12 Chemokine Levels

As atherosclerosis develops, circulating monocytes infiltrate the plaque to become lesional macrophages, contributing to both formation of lipid-rich foam cells and to the physical bulk of developing plaques.⁷ To quantify monocyte recruitment and infiltration into the atherosclerotic plaque, we labeled circulating monocytes with red latex beads and tracked the persistence of this label in plaque.^48,49 Red latex microspheres undergo specific uptake only by mononuclear cells,²⁸ and this methodology has been validated for experiments with atherosclerotic mice by our group¹⁷ and others.^28,50 To assess the efficiency of monocyte labeling with red latex beads, we quantified the number of red bead-positive circulating and splenic monocytes in MF-IGF1 and control mice. We found that ≈20% of circulating monocytes were red bead positive and there was no difference in labeling efficiency of circulating and splenic monocytes in MF-IGF1 mice versus controls (Figure S6A through S6C). We detected a similar number of splenic and circulating monocytes as a subset of all leukocytes (Figure S6D and S6E), suggesting no change in monocytosis.⁵¹ MF-IGF1 mice had a significant reduction in the number of red bead-positive cells in plaques compared with control (74.2% decrease; Figure 4A and 4B) indicating that macrophage-specific IGF-1 overexpression reduced monocyte infiltration into atherosclerotic plaque. We also analyzed other leukocyte and endothelial progenitor cell populations in the circulation using flow cytometry. There was no change in natural killer, B-cell, or T-cell (lymphoid-derived cells) populations, nor in neutrophil, eosinophil, or monocyte (myeloid-derived cells) populations, nor in endothelial progenitor cell populations (Figure S5C through S5F).

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Leukocytes are trafficked via involvement of chemokines/chemokine receptors, where a concentration gradient of a chemokine will lead a leukocyte to the chemokine’s source.⁶ To investigate potential chemokine candidates mediating reduced monocyte infiltration into atherosclerotic plaque in MF-IGF1 mice and to determine potential changes in chemokines induced by macrophage-specific IGF-1 overexpression, we measured serum levels of 12 proatherogenic chemokines in MF-IGF1 and control mice via protein array (Table 2). Only CXCL12 levels were downregulated by macrophage-specific IGF-1 overexpression (P<0.0001). CXCL12—a heparin-binding chemokine⁵²—is highly expressed in macrophages, SMCs, and endothelial cells in atherosclerotic plaques but not in normal vessels,⁵³ and CXCL12 has been implicated in regulation of monocyte/macrophage differentiation,⁵⁴ macrophage-derived foam cell formation,⁵⁵ and in recruitment of macrophages to sites of injury,⁵⁶ and of monocytes to atherosclerotic plaque.⁵⁷

Downregulation of CXCL12 protein in serum of MF-IGF1 mice was confirmed by ELISA (26.7% reduction in MF-IGF1 mice compared with control; Figure 4C). We detected strong CXCL12 immunopositivity in aortic valve plaque lesions in control mice (Figure 4D and 4F) and CXCL12 signal overlapped with Mac3 positivity (Figure 4E and 4G), suggesting that macrophages were the predominant source of CXCL12 in murine plaque. There was a marked reduction in CXCL12 staining in plaque lesions in MF-IGF1 mice (Figure 4D through 4G) and a 5-fold decrease in CXCL12 mRNA levels in laser capture microdissection–isolated plaque samples from MF-IGF1 mice compared with control (Figure 4H). IGF-1 and CXCL12 mRNA levels in circulating monocytes in MF-IGF1 mice were not different from control (Figure 1F; Figure S5B), whereas CXCL12 protein levels in peritoneal macrophages from MF-IGF1 mice were decreased compared with control (Figure 4I). CXCL12 levels in conditioned media from peritoneal macrophages were not detectable (data not shown), likely because the majority of noncirculating CXCL12 is bound to the cellular membrane.^52,58 Thus, macrophage-specific IGF-1 overexpression reduced monocyte infiltration into atherosclerotic plaque and downregulated foam cells, and these effects were associated with decreased circulating, plaque, and macrophage CXCL12 expression.

Macrophage-Specific IGF-1 Overexpression Increased ABCA1 in Atherosclerotic Plaque and in Peritoneal Macrophages, With an Accompanying Increase in Cholesterol Efflux

Mechanisms mediating the proatherogenic effects of CXCL12 are complex and include promotion of monocyte differentiation, recruitment of macrophages to injured endothelium, and enhancement of foam cell formation.^59,60 Furthermore, CXCL12 is known to downregulate expression of ABCA1 (ATP-binding cassette transporter A1) in macrophages, thereby inhibiting cholesterol efflux and contributing to its proatherogenic effects.⁶¹ Because we found reduced macrophage CXCL12 expression and reduced foam cells in the plaques of MF-IGF1 mice, we hypothesized that ABCA1 is involved in macrophage-derived IGF-1’s effect on foam cells. ABCA1 gene expression was increased in laser capture microdissection plaque isolates in MF-IGF-1 mice compared with control (Figure 5A). ABCA1 protein expression was increased in peritoneal macrophages in MF-IGF1 mice compared with controls (Figure 5B). Furthermore, blocking IGF-1 signaling with picropodophyllin reduced ABCA1 protein levels to control levels in peritoneal macrophages from MF-IGF1 mice (Figure 5C). Cholesterol efflux in peritoneal macrophages from MF-IGF1 mice was increased by 2.4-fold compared with control when using ApoA1 (apolipoprotein A1) as the cholesterol acceptor and unchanged when using HDL (high-density lipoprotein) as the cholesterol acceptor (Figure 5D). Taken together, these results suggest that macrophage overexpression of IGF-1, in addition to reducing monocyte recruitment and infiltration into plaque, also upregulates ABCA1 and increases cholesterol efflux, contributing to macrophage-derived IGF-1’s antiatherogenic effect.

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IGF-1 Increased THP-1 Macrophage Cholesterol Efflux and Reduced Foam Cell Formation

To determine whether IGF-1 reduces macrophage-derived foam cell formation, we used human THP-1 monocyte-derived macrophages.²⁰ Treatment with oxLDL induced a significant increase in CXCL12 mRNA levels compared with native LDL treatment, and this increase was markedly and dose-dependently inhibited by IGF-1 pretreatment (Figure 6A; 68.9% decrease with 100 ng/mL IGF-1). Pretreatment with IGF-1 also reduced CXCL12 protein expression in oxLDL-treated THP-1 cells (Figure S7A; 56.2% decrease). IGF-1 (100 ng/mL) increased ApoA1-mediated cholesterol efflux capacity in THP-1 cells when exposed to oxLDL (28% increase) while there was no change in HDL-mediated efflux (Figure 6B and 6C; Figure S7C). THP-1 cells were pretreated with IGF-1 (0–100 ng/mL), and foam cell formation was induced by exposing cells to oxLDL or native LDL as control. OxLDL caused an almost 3-fold increase in Oil Red O staining, and this increase was almost completely blunted by IGF-1 (Figure 6D and 6E; 76.9% reduction in cells treated with 100 ng/mL IGF-1). Importantly, when recombinant CXCL12 pretreatment (80 µg/mL) was added to the cells, the IGF-1–mediated reduction in Oil Red O–positive area was completely abolished (Figure 6F).

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Discussion

Here, we report that macrophage-specific IGF-1 overexpression suppressed atherosclerotic burden and promoted features of stable atherosclerotic plaque in Apoe^−/− mice. MF-IGF1 transgenic animals had a reduction in total plaque burden in the aorta and in aortic root lesion size after 12 weeks on a high-fat diet. Plaques in MF-IGF1 mice had reduced macrophage and foam cell content and necrotic core size, no change in SMC content, and an increase in collagen levels. MF-IGF1 mice had a reduction of monocyte recruitment and infiltration into the plaque and decreased expression of CXCL12 in the plaque, the circulation, and in peritoneal macrophages. Importantly, these mice had increased levels of the cholesterol efflux regulator, ABCA1, in atherosclerotic plaques and in peritoneal macrophages and an increase in ApoA1-dependent macrophage cholesterol efflux. IGF-1 prevented oxLDL-induced increase in CXCL12 expression in cultured macrophages and IGF-1 increased ApoA1-dependent cholesterol efflux. IGF-1 also reduced neutral lipid accumulation in THP-1 macrophages, and this effect was blocked by exogenous CXCL12 treatment. Overall, our results indicate that macrophage IGF-1 overexpression downregulates macrophage CXCL12 expression and that IGF-1 potentially exerts its atheroprotective effect via this mechanism.

To our knowledge, ours is the first report of an animal model of macrophage-specific IGF-1 overexpression. MF-IGF1 mice showed a marked increase in IGF-1 mRNA levels in atherosclerotic plaque, and peritoneal macrophages isolated from these mice secreted 80% more IGF-1 in conditioned medium. As the expression of SRA is selectively confined to macrophages (including splenic, peritoneal, and other tissue macrophages^62–64) and only minor SRA expression has been reported for freshly isolated monocytes,^64,65 our observation of increased IGF-1 expression in both plaque and peritoneal macrophages but not in circulating monocytes in MF-IGF1 mice is consistent with the reported difference in SRA promoter activity in these cell types. SMCs do not express SRA basally but may express SRA upon dedifferentiation to a foam cell phenotype, although not to the same level as macrophages.^66–68 Of note, however, there was no increase in IGF-1 protein levels in aorta or other SMC-rich tissues like the bladder. Furthermore, we have previously shown that SMC-specific IGF-1 overexpression does not change atherosclerotic burden but does promote features of stable plaque.¹⁸ MF-IGF1 mice initially gained more weight compared with control mice, although the difference was not significant after 12 weeks of high-fat diet. One can speculate that since SRA expression is detected in bone marrow⁶⁹ and IGF-1 in bone marrow regulates bone density,^37,70–74 that perhaps MF-IGF1 mice have increased bone density, resulting in increased weight. Further studies will be required to address this hypothesis.

Macrophages are important for resolution of inflammation and for tissue repair in multiple tissues including the heart and skeletal muscle,^75–81 in some cases via IGF-1–dependent mechanisms. Indeed, macrophage-derived IGF-1 plays a role in regeneration of skeletal muscle⁸¹ and in protection against atrophy.⁸⁰ In atherosclerotic plaques, although macrophages may have anti-inflammatory effects, they are thought to play a critical role in plaque progression via secretion of proinflammatory cytokines, accumulation of lipoproteins to form foam cells, and via apoptosis and release of cellular debris promoting necrotic core formation.⁸² The role of macrophage IGF-1 signaling in these processes was virtually unknown until the recent demonstration by our group that deletion of monocyte/macrophage IGF1R in Apoe^−/− mice promoted a proinflammatory macrophage phenotype and increased atherosclerotic burden,¹⁷ suggesting that IGF-1 could exert beneficial anti-inflammatory effects on macrophages in atherosclerotic plaques. Many of the effects on atherosclerosis that resulted from monocyte/macrophage-specific knockout of the IGF1R are mirrored in our current findings in this novel model of macrophage-specific IGF-1 overexpression. These include effects on plaque burden, plaque stability, recruitment of monocytes, plaque macrophage content, and lipid efflux. This suggests that the ability of macrophage-derived IGF-1 to exert these effects is primarily via autocrine mechanisms, although additional effects on surrounding cells such as SMC or endothelial cells cannot be formally excluded. Furthermore, our current results uncover a novel mediator of these effects, CXCL12. Of note, in our prior model of monocyte/macrophage-specific IGF1R knockout, CXCL12 levels were not assessed.

In addition to an overall reduction of plaque burden, we found that MF-IGF1 mice have plaques that are characterized by features of increased stability, namely increased collagen content and smaller necrotic cores. There is evidence that features of unstable plaque (such as necrotic core size, cellular composition, and collagen levels) are important determinants of adverse cardiovascular events, including acute coronary syndromes.⁸³ In more stable plaques, collagen can confer a thicker fibrotic cap, lessening the chance of rupture.¹⁰ Our finding of increased collagen in the plaques of MF-IGF-1 mice could result from multiple mechanisms, including increased collagen synthesis or reduced collagen degradation by MMPs (matrix metalloproteinases), which are known to be highly expressed in plaque macrophages.⁸⁴ We have preliminary data that peritoneal macrophages in MF-IGF1 mice had a significant reduction in MMP8 and MMP14 expression (Figure S8), suggesting that reduced collagen breakdown could contribute to their increase in collagen content.

Macrophage levels in the plaque are regulated via several mechanisms such as changes in monocyte recruitment, macrophage efferocytosis, proliferation, and apoptosis.⁷ Plaque macrophage levels (as measured by Mac3 immunopositivity) were significantly reduced in MF-IGF1 mice. Mac3 has been used as a specific macrophage marker in atherosclerosis studies,⁸⁵ but if SMCs dedifferentiate into foam cells, they can also express the marker,^86–88 so it is possible that some cells we have considered as macrophages are actually phenotypically modified SMCs. We hypothesized that the change in macrophage number was due to a reduction of monocyte recruitment. It is of note that it has been reported that a reduction in monocyte recruitment to plaque results in reduced macrophage content.^89,90 We found a striking 70% reduction in the number of monocytes recruited to the plaques of MF-IGF1 mice. Chemotaxis of leukocytes is a key function of chemokines, and monocyte infiltration is regulated by chemokine secretion from multiple cell types.²⁸ Among the 12 circulating chemokines analyzed, only CXCL12 was reduced in MF-IGF1 mice. It is of note that 2 chemokines (T-cell attractants MDC [macrophage-derived chemokine] and MIG [monokine-induced by gamma interferon], respectively) were significantly, yet slightly, elevated so we cannot rule out a potential contribution of these chemokines to the phenotype of MF-IGF1 mice. CXCL12, originally discovered in the stromal cells of bone and to be responsible for homing and retention of hemopoietic stem and progenitor cells in the bone marrow,⁵⁶ has been recently implicated in atherosclerosis.^{59,61,91–93} Atherosclerotic plaque CXCL12 mRNA and protein expression were significantly reduced in MF-IGF1 mice. Importantly, MF-IGF1 peritoneal macrophages also showed a reduction of CXCL12 expression. These results suggest that decreased CXCL12 expression in the lesions of MF-IGF1 mice caused reduced monocyte recruitment, leading to a lower number of macrophages in the plaque.⁵⁴ It is of note that CXCL12 expression is increased within human plaque compared with its near absence in normal arteries.⁹² Most reports, but not all,⁹⁴ have indicated that CXCL12 is proatherogenic.^55,57,61 This study suggests that CXCL12 is a proatherogenic molecule when the source is macrophages.

Mishandled lipid metabolism in plaque macrophages results in lipid accumulation and ultimately macrophage apoptosis, resulting in a less stable plaque with a large necrotic core.⁸ CXCL12 reduces expression of ABCA1,⁶¹ the predominant lipid transporter mediating cholesterol efflux,⁵ resulting in increased macrophage lipid accumulation and foam cell formation. We found increased ABCA1 mRNA levels in MF-IGF1 mouse plaques and increased ABCA1 protein in MF-IGF1 peritoneal macrophages compared with control. Of note, HDL can bind to a number of receptors such as CD36 to mediate cholesterol efflux,⁵ whereas a component of HDL, ApoA1, specifically binds to ABCA1 to mediate efflux.⁹⁵ ApoA1/ABCA1 binding is considered the major cholesterol efflux pathway.⁹⁶ Our finding that IGF-1 increased ApoA1, but not HDL-dependent cholesterol efflux, is consistent with our data indicating that IGF-1 upregulates ABCA1 expression. Thus, our findings suggest that increased macrophage IGF-1 in MF-IGF1 mice increases ABCA1 expression in plaque, and this increase likely contributes to the reduction in lipid accumulation and foam cell formation.

It is important to note that IGF-1 and its receptor are downregulated in human atherosclerotic plaques and in SMCs exposed to oxLDL.^42,97 People with higher levels of bioavailable IGF-1 have a significant decrease in cardiovascular disease.^14–16,98 Results from the PRIME study (Prospective Epidemiological Study of Myocardial Infarction) showed that baseline IGF-1 is significantly lowered in patients who were developing an acute coronary syndrome.⁹⁸ A number of chemokines are involved in promoting atherogenesis,⁹⁹ including CXCL12.^{59,61,91–93} Studies have implicated CXCL12 as a biomarker of heart failure and all around mortality,¹⁰⁰ and a genome-wide association study identified CXCL12 as a potential therapeutic target in coronary disease.⁹¹ Increased plasma CXCL12 due to genetic diversity has been linked to increased risk of coronary disease.⁵⁷ It is of note that Döring et al⁵⁷ found that only endothelial cell–derived CXCL12 contributes to atherogenesis. However, that study used a model of CXCL12 deficiency in bone marrow–derived hematopoietic cells, which could have masked macrophage-specific CXCL12 effects.

Our finding that an increase in macrophage IGF-1 reduces levels of CXCL12 in atherosclerotic plaques, the circulation, and macrophages establishes a potentially important mechanism whereby macrophage-derived IGF-1 can reduce atherosclerotic plaque and promote features of plaque stability. To our knowledge, this is the first report of an animal model in which a local increase in plaque IGF-1 levels leads to a reduction in atherosclerosis. Our findings provide strong evidence that an increase in macrophage-derived IGF-1 may have significant antiatherosclerotic effects and provide potential new therapeutic targets.

Article Information

Acknowledgments

We would like to thank Dr Christopher Glass (University of California–San Diego) for the gift of SRA (scavenger receptor-A) promoter-containing plasmid. We also thank Daniel Jackson for cell sorting services and Alexander Jurkevich for laser capture microdissection training (University of Missouri at Columbia). We thank H. Alan Tucker for additional flow cytometry services (Tulane University). This work was supported by funding from the National Institutes of Health (R01HL070241, 3R01HL070241-16S1, P. Delafontaine, and R01HL142796, S. Sukhanov) and the American Heart Association (19TPA34850165 and 15SDG25240022, T. Yoshida).

Supplemental Material

Supplemental Materials and Methods

Supplemental Figures S1–S9

References ^{17,18,20,21,24–32}

Footnotes