Original Article

Insulin-Like Growth Factor-1 Receptor Deficiency in Macrophages Accelerates Atherosclerosis and Induces an Unstable Plaque Phenotype in Apolipoprotein E–Deficient MiceCLINICAL PERSPECTIVE

Yusuke Higashi, Sergiy Sukhanov, Shaw-Yung Shai, Svitlana Danchuk, Richard Tang, Patricia Snarski, Zhaohui Li, Patricia Lobelle-Rich, Meifang Wang, Derek Wang, Hong Yu, Ronald Korthuis, Patrice Delafontaine
https://doi.org/10.1161/CIRCULATIONAHA.116.021805
Circulation. 2016;133:2263-2278
Originally published May 6, 2016

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Abstract

Background—We have previously shown that systemic infusion of insulin-like growth factor-1 (IGF-1) exerts anti-inflammatory and antioxidant effects and reduces atherosclerotic burden in apolipoprotein E (Apoe)–deficient mice. Monocytes/macrophages express high levels of IGF-1 receptor (IGF1R) and play a pivotal role in atherogenesis, but the potential effects of IGF-1 on their function are unknown.

Methods and Results—To determine mechanisms whereby IGF-1 reduces atherosclerosis and to explore the potential involvement of monocytes/macrophages, we created monocyte/macrophage–specific IGF1R knockout (MΦ-IGF1R-KO) mice on an Apoe−/− background. We assessed atherosclerotic burden, plaque features of stability, and monocyte recruitment to atherosclerotic lesions. Phenotypic changes of IGF1R-deficient macrophages were investigated in culture. MΦ-IGF1R-KO significantly increased atherosclerotic lesion formation, as assessed by Oil Red O staining of en face aortas and aortic root cross-sections, and changed plaque composition to a less stable phenotype, characterized by increased macrophage and decreased α-smooth muscle actin–positive cell population, fibrous cap thinning, and decreased collagen content. Brachiocephalic artery lesions of MΦ-IGF1R-KO mice had histological features implying plaque vulnerability. Macrophages isolated from MΦ-IGF1R-KO mice showed enhanced proinflammatory responses on stimulation by interferon-γ and oxidized low-density lipoprotein and elevated antioxidant gene expression levels. Moreover, IGF1R-deficient macrophages had decreased expression of ABCA1 and ABCG1 and reduced lipid efflux.

Conclusions—Our data indicate that macrophage IGF1R signaling suppresses macrophage and foam cell accumulation in lesions and reduces plaque vulnerability, providing a novel mechanism whereby IGF-1 exerts antiatherogenic effects.

Introduction

Insulin-like growth factor -1 (IGF-1) is a pleiotropic factor that is produced and acts locally (ie, via autocrine or paracrine effects) or circulates in blood and exerts endocrine effects. Circulating IGF-1 levels reach a peak during the pubertal growth phase, eventually declining with the progression of age. Although its role in developmental and pubertal growth as the major mediator of the effects of growth hormone is well documented, the physiological roles of IGF-1 in aged subjects are not understood. Aging is a major independent risk factor for coronary heart disease; in fact, there is increased cardiovascular and coronary heart disease prevalence with age in both sexes. In light of the aging-dependent decline in circulating IGF-1 levels, a potential link between IGF-1 levels and the elevated prevalence of cardiovascular diseases has been suggested. Indeed, epidemiological data have suggested that low IGF-1 levels are an important predictor of coronary events in aged subjects.1–3 In an animal model of atherosclerosis, we have shown that low levels of circulating IGF-1 are associated with more atherosclerosis4 and, vice versa, that an increase in circulating IGF-1 decreases atherosclerotic burden.5

Clinical Perspective on p 2278

The pathogenesis of atherosclerosis is complicated, involving multiple cell types, including vascular endothelial cells, smooth muscle cells, and proinflammatory cells such as macrophages. To determine the potential target(s) whereby IGF-1 reduces atherosclerosis, we previously investigated whether smooth muscle–specific overexpression of IGF-1 alters atherosclerosis.6 Intriguingly, overexpression of IGF-1 in smooth muscle cells did not alter atherosclerotic burden6 but increased features of plaque stability,6,7 suggesting that other cellular components were a potential target for the antiatherogenic effects of IGF-1. Macrophages play a pivotal role in the pathogenesis of atherosclerosis by modulating inflammatory status and by scavenging and accumulating excess lipid to become foam cells. Thus, regulation of macrophage functions in terms of inflammation and phagocytosis is key to comprehending the disease process. Because macrophages have a predominant role in the inflammatory status of atherosclerotic lesions, it is critical to determine whether IGF-1 regulates macrophage function, particularly inflammatory activation and phagocytic activity. Thus far, there is very limited information on the potential link between IGF-1 effects and macrophage function, particularly in relation to vascular disease. IGF-1 has been reported to enhance chemotactic macrophage migration,8 to stimulate tumor necrosis factor-α (TNFα) expression,8 and to enhance low-density lipoprotein (LDL) uptake and cholesterol esterification.9 There are also reports from clinical investigations providing indirect evidence of anti-inflammatory effects of IGF-1. For example, there is an inverse relation between serum interleukin (IL)-6 and IGF-1 levels10; IGF-1/insulin-like growth factor–binding protein 3 administration to patients with severe burn injury induced an anti-inflammatory effect and reduced IL-6 and TNFα11,12; and low IGF-1 and high IL-6 and TNFα levels are associated with higher mortality in elderly patients.13,14 In this study, we used a well-established animal model of atherosclerosis to examine the role of macrophage IGF-1 signaling in atherosclerosis development and progression.

Methods

A detailed Materials and Methods section is available in the online-only Data Supplement.

Animals

All animal experiments were performed according to protocols approved by the institutional animal care and use committee. Monocyte/macrophage–targeted IGF-1 receptor–null mice on Apoe−/− background (MΦ-IGF1R-KO) and control mice (IGF1R-flox) were generated as described in the online-only Data Supplement. Eight-week old mice were fed a high-fat diet for 8 weeks before atherosclerosis was assessed.

Atherosclerotic Burden and Plaque Composition

Atherosclerotic burden was quantified with the use of en face preparations of whole aorta stained with Oil Red O and in cross sections of aortic root. Plaque composition was assessed in cross sections of aortic root by immunostaining for Mac-3 (macrophage) and α-smooth muscle actin (smooth muscle cell), and Masson’s Trichrome staining was used for collagen. Brachiocephalic artery lesions were analyzed by the Carstairs method as described by Gough et al.15

Tracing Recruitment of Circulating Monocytes

Circulating Ly6Chi or Ly6Clo monocytes were labeled in vivo with polychromatic red microspheres as described elsewhere,16 and numbers of red microsphere–positive cells were counted in a plaque and normalized to the efficiency of labeling in circulating monocytes (percent labeled/total number) and assessed by flow cytometry.

Intravital Fluorescence Microscopy

Intravital fluorescence microscopy was performed as described elsewhere.17

Macrophage Culture, Western Blot Analysis, and Quantitative Real-Time Reverse Transcription–Polymerase Chain Reaction

Thioglycolate-elicited macrophages were obtained from MΦ-IGF1R-KO and IGF1R-flox mice.18 Western blot analysis,19 total RNA extraction,20 and real-time reverse transcription–polymerase chain reaction20 were performed as previously described.

Statistical Analysis

All numeric data are expressed as mean±SEM. Statistical analyses were performed with GraphPad PRISM (version 6.07) software. Data sets were first assessed for residuals distribution with the D’Agostino-Pearson omnibus normality test and for equal variances with the Levene test for equality of variances. Differences in outcomes were determined by ANOVA and Bonferroni multiple-comparisons test, Kruskal– Wallis test, unpaired Student t test with or without the Welch correction, or Mann– Whitney U test, accordingly with the normality of residuals distribution. Differences were considered significant at P<0.05. The Fisher’s exact test was used to compare frequency of observed indexes of plaque vulnerability (Table).

View this table:
Table.

MΦ-IGF1R-KO-Induced Features of Ruptured Plaques in the Brachiocephalic Artery

Results

Generation of MΦ-IGF1R-KO/Apoe−/− Mice

MΦ-IGF1R-KO were generated on Apoe−/− background (LyzCre/+/Igf1rflox/flox/Apoe−/−: MΦ-IGF1R-KO) by crossing LyzCre/+ mice into Igf1rflox/flox/Apoe−/− (IGF1R-flox, served as a control). Genotype segregation in the offspring followed the expected mendelian frequency, and we did not recognize any developmental/morphological abnormalities. IGF1R deficiency was confirmed by lack of IGF1R protein detection by Western blot and IGF-1–dependent phosphorylation on Akt in peritoneal macrophages (Figure IA in the online-only Data Supplement). Consistent with myeloid-selective Cre expression,21–23 we found that the exon 3 of Igf1r was also excised in neutrophils (Figure IB in the online-only Data Supplement). However, we were unable to detect IGF1R protein in IGF1R-flox neutrophils, indicating extremely low levels of IGF1R protein expression in neutrophils (Figure IC in the online-only Data Supplement).

IGF1R and insulin receptor (InsR) can form a hybrid receptor (heterotetramer consisting of α+β subunits of IGF1R and α+β subunits of InsR),24–26 which binds IGF-1 with high affinity but not insulin.27,28 Depletion of IGF1R in endothelial cells is reported to allow InsR to form a holotetramer, thereby enhancing insulin sensitivity.29 Because insulin signaling in macrophages has significant effects on atherosclerosis (there are contradictory reports showing antiatherogenic30 or proatherogenic31 effects), we assessed insulin signaling activity in MΦ-IGF1R-KO macrophages. IGF1R deficiency did not alter InsR expression levels (Figure ID in the online-only Data Supplement). In IGF1R-flox macrophages, immunoprecipitation of InsR pulled down 100% of IGF1R (Figure IE, left, in the online-only Data Supplement), whereas anti-IGF1R immunoprecipitation pulled down 50% of InsR (Figure IE, right, in the online-only Data Supplement), suggesting that the ratio of IGF1R/InsR-hybrid receptors to InsR-holoreceptors was 2:1 without the presence of IGF1R-holoreceptors. IGF1R deficiency did not alter insulin-induced dose-dependent phosphorylation of Akt (Figure IF in the online-only Data Supplement).

Animals were assessed for circulating leukocyte counts (Table I in the online-only Data Supplement), cholesterol levels, and cytokine levels (Table II in the online-only Data Supplement). As has been reported,32 Western diet feeding for 8 weeks was associated with elevated monocyte count; however, IGF1R deficiency did not result in a significant difference in white blood cell count or circulating monocyte count (Table I in the online-only Data Supplement). A subpopulation of circulating monocytes as defined by CD11b+/CD90−/B220−/CD49b−/NK1.1−/Ly6G−/Ly6Chigh cells (Ly6Chi monocytes) has been reported to be proinflammatory and to be increased under atherogenic conditions such as hyperlipidemia.33 We did not observe a significant difference in Ly6Chi monocyte levels between MΦ-IGF1R-KO and IGF1R-flox mice (Table I in the online-only Data Supplement). MΦ-IGF1R-KO did not significantly alter circulating IGF-1, proinflammatory cytokine (IL-6, TNFα, monocyte chemoattractant protein-1 [MCP-1]), or cholesterol levels (Table II in the online-only Data Supplement).

Atherosclerosis Was Enhanced by MΦ-IGF1R-KO

Atherosclerotic lesion formation was assessed after 8 weeks of high-fat diet feeding. En face Oil Red O staining of aortas revealed a significant ≈64% increase in Oil Red O–positive lesion area, and there was a consistent ≈34% increase in plaque size at the aortic root in MΦ-IGF1R-KO mice (Figure 1). The effect was confirmed in each sex, indicating that there was no sex-specific effect of IGF1R deficiency (Figure II in the online-only Data Supplement). Plaque composition with regard to Mac3 (macrophage)-positive cells, α-smooth muscle actin–positive cells, and collagen content (Masson’s trichrome stain) was significantly altered by MΦ-IGF1R-KO (Figure 2); there was a 49% increase in Mac3 detection (Figure 2A) and a 31% decrease in α-smooth muscle actin detection (Figure 2B). Plaques in MΦ-IGF1R-KO mice had a thinner smooth muscle cap than IGF1R-flox mice (Figure 2B) and decreased collagen content (Figure 2C). Because we observed a decrease in collagen content in plaques from MΦ-IGF1R-KO mice, we hypothesized that this could be attributable to enhanced collagen degradation by matrix metalloproteinases (MMPs). Cultured peritoneal macrophages from MΦ-IGF1R-KO animals expressed higher levels of MMP-1, -2, -8, -9, -12, -13, and -14 than those from IGF1R-flox animals, suggesting enhanced MMP activity (Figure 2D). The MMP protein levels in tissue lysates of ascending aortas after 2 months of Western diet feeding (Figure IIIA and IIIB in the online-only Data Supplement) showed a significant increase in the expression of MMP-1, -2, -8, and -9 in MΦ-IGF1R-KO animals. These observations are consistent with enhanced lesion formation in MΦ-IGF1R-KO mice with a phenotypic shift toward increased features of plaque vulnerability.

Figure 1.
Figure 1.

Monocyte/macrophage insulin-like growth factor-1 receptor (IGF1R) deficiency aggravated atherosclerosis. Atherosclerotic lesion formation was assessed in monocyte/macrophage–specific (MΦ)–IGF1R knockout (KO) and IGF1R-flox mice (control) fed a high-fat diet for 2 months. A, Oil Red O staining of en face aortas; plaque covered area was determined and expressed as percent of total area (C; **P<0.0001 by the Mann–Whitney U test; n=25 for IGF1R-flox and n=23 for MΦ-IGF1R-KO). B, Plaque size was determined in hematoxylin and eosin–stained cross sections of aortic root; *P<0.05 by the Mann–Whitney U test; n=18 for IGF1R-flox and n=19 for MΦ-IGF1R-KO (D).

Figure 2.
Figure 2.

Monocyte/macrophage insulin-like growth factor-1 receptor (IGF1R) deficiency alters atherosclerotic plaque composition. Atherosclerotic plaque composition was assessed in cross sections of aortic root. A, Macrophage content was assessed by immunostaining against Mac3 antigen. *P<0.05 by the Student t test; n=18 for IGF1R-flox and n=17 for monocyte/macrophage–specific (MΦ)–IGF1R knockout (KO). B, Smooth muscle cell (SMC) content assessed as α-smooth muscle actin (αSMA)–positive cells by immunostaining (representative images shown in top left). *P<0.05 by the Student t test; n=16 for IGF1R-flox and n=18 for MΦ-IGF1R-KO. Thickness of SMC-positive fibrous cap was also determined by immunostaining against αSMA (representative images shown in bottom left). **P<0.001 by the Student t test; n=12 for IGF1R-flox and n=14 for MΦ-IGF1R-KO. C, Collagen content determined by Masson’s trichrome stain. **P<0.01 by the Mann–Whitney U test; n=17 for IGF1R-flox and n=16 for MΦ-IGF1R-KO. D, Western blot analysis of matrix metalloproteinases (MMPs) in cultured peritoneal macrophages isolated from IGF1R-flox (Flox) and MΦ-IGF1R-KO (Cre+) mice. Representative results of 3 independent experiments are shown.

Plaque Destabilization in MΦ-IGF1R-KO Mice

Because our findings suggested plaque destabilization in MΦ-IGF1R-KO mice, we assessed indicators of vulnerability in brachiocephalic artery plaques (Figure IV in the online-only Data Supplement) by staining cross sections of brachiocephalic artery with the Carstairs34 and Verhoeff–Van Gieson methods.35 Cross sections (5 μm thick) were made every 50 μm along the artery, for a total of 10 sections per artery. If 3 consecutive sections indicated a fibrous cap disruption, intraplaque hemorrhage, fibrin deposition, or medial elastin break, the artery was considered positive for signs of plaque vulnerability. After 8 weeks on a high fat diet, MΦ-IGF1R-KO animals had increased features of vulnerable plaques compared with IGF1R-flox animals, as determined by the presence of intraplaque hemorrhage (IGF1R-flox, 3 positive in 22 animals versus MΦ-IGF1R-KO, 8 positive in 17 animals; P=0.03; Table) and medial elastin breaks (IGF1R-flox, no positive in 22 animals versus MΦ-IGF1R-KO, 4 positive in 17 animals; P=0.03; Table), suggesting that plaques in MΦ-IGF1R-KO mice are more unstable than in IGF1R-flox mice.

Proinflammatory Responses Are Enhanced in IGF1R-Deficient Macrophages

Thioglycolate-elicited peritoneal macrophages from the MΦ-IGF1R-KO mice were assessed for proinflammatory cytokine and chemokine secretion (Figure 3). Among tested cytokines and chemokines, IL-1β production was below detectable levels, whereas IL-1α, IL-6, TNFα, MCP-1, and fractalkine were detected (Figure 3A–3F). IGF1R-deficient macrophages secreted significantly higher levels of all the tested cytokines and chemokines except fractalkine, which showed a strong trend toward an increase (P=0.0508). Interferon-γ (IFNγ) significantly enhanced TNFα (Figure 3C) and MCP-1 (Figure 3D) production in IGF1R-flox cells but not in MΦ-IGF1R-KO cells. Intriguingly, IFNγ did not enhance fractalkine production in IGF1R-flox macrophages, whereas in MΦ-IGF1R-KO cells, it decreased fractalkine production (Figure 3E). In a setting of exposure to IFNγ, MΦ-IGF1R-KO cells secreted higher levels of IL-1α, IL-6, and MCP-1 compared with IGF1R-flox. We tested nuclear factor-κB (NFκB) DNA binding activity in these cells (Figure 3F). There was significantly higher NFκB DNA binding activity in MΦ-IGF1R-KO, which was further enhanced by IFNγ, consistent with higher cytokine/chemokine production. To further assess NFκB involvement, we exposed cells to NFκB inhibitors (BMS-345541 and parthenolide, Figure 3G–3J). BMS-345541 completely abolished enhanced TNFα and IL-6 production in MΦ-IGF1R-KO cells (Figure 3G and 3H), and parthenolide recapitulated the BMS-345541 effect (Figure 3I and 3J), supporting the importance of NFκB-dependent cytokine production in MΦ-IGF1R-KO macrophages.

Figure 3.
Figure 3.

Proinflammatory cytokine and chemokine production by cultured macrophages isolated from insulin-like growth factor-1 receptor (IGF1R)–flox and monocyte/macrophage–specific (MΦ)–IGF1R knockout (KO) mice. Interleukin (IL)-1α (A), IL-6 (B), tumor necrosis factor-α (TNFα; C), monocyte chemoattractant protein-1 (MCP-1; D), and fractalkine (E) secretion by peritoneal macrophages was assessed by respective ELISA. Macrophages were isolated by peritoneal lavage from IGF1R-flox (open column) and MΦ-IGF1R-KO (closed column) mice and allowed to adhere on culture plates overnight. After removal of nonadhering cells, cells were primed by interferon-γ (IFNγ), and conditioned media were collected after 24 hours. *P<0.05, **P<0.01 vs IGF1R-flox; ##P<0.01 vs IGF1R-flox+IFNγ; $P<0.01 vs MΦ-IGF1R-KO. Statistical significance was assessed by 2-way ANOVA and subsequent post hoc analysis with the Bonferroni multiple-comparisons test; n=6. F, Nuclear factor-κB (NFκB) DNA binding activity was assessed (expressed as relative light units [RLU]) in cell lysates of IGF1R-flox and MΦ-IGF1R-KO macrophages with or without priming by IFNγ for 3 hours. *P<0.05 vs IGF1R-flox by 2-way ANOVA; n=3. G through J, Effects of NFκB inhibitors on TNFα (G and H) and IL-6 (I and J) secretion by peritoneal macrophages. IGF1R-flox (open column) and MΦ-IGF1R-KO (closed column) macrophages were exposed to the indicated dose of BMS-345541 or parthenolide for 1 hour before activation by IFNγ or lipopolysaccharide (LPS). Conditioned media were collected after 24 hours and assessed for cytokine concentration with the respective ELISA kit (R&D Systems). ##P<0.05 vs IGF1R-flox+IFNγ, #P<0.05 vs IGF1R-flox+LPS by the Mann–Whitney U test; n=4.

Macrophage Polarization

Because IGF1R-deficient macrophages manifested enhanced proinflammatory responses, we examined whether IGF1R deficiency influenced macrophage polarization. Classic activation (M1) was induced by exposure to IFNγ and subsequently to lipopolysaccharide (Figure 4A–4E). M1 marker gene (Tnf, Nos2, Il6, Ccl2, and Ccl5) expression levels were highly induced by exposure to IFNγ-LPS, and IGF1R deficiency further enhanced the expression, implying enhanced M1 activation in MΦ-IGF1R-KO macrophages. On the other hand, IL-4–induced gene expression levels (ie, M2 activation markers; Arg1, Mrc1, and Pparg) were not influenced by IGF1R deficiency (Figure 4F–4H). It has been reported that oxidatively modified LDL (oxLDL) and that oxidized lipid components of oxLDL alter macrophage activation status or induce a distinctive activation status.36–39 Hemeoxygenase-1 (Hmox1) and thioredoxin reductase-1 (Txnrd1) are signature genes that have been shown to be upregulated in macrophages exposed to oxidized phospholipid, leading to a polarization status of Mox.39 In fact, oxLDL exposure enhanced Hmox1 and Txnrd1 expression significantly (Figure 5A and 5B). Intriguingly, IGF1R deficiency did not influence Txnrd1 mRNA levels (Figure 5B) but significantly upregulated Hmox1 mRNA levels (Figure 5A). We further assessed whether IGF1R deficiency influences the effect of oxLDL on expression levels of macrophage activation marker genes (Figure 5). With regard to M1 activation markers, oxLDL by itself did not alter mRNA levels in IGF1R-flox cells (Tnf, Nos2, Il6, Ccl2, and Ccl5: Figure 5C–5G); however, in MΦ-IGF1R-KO cells, in which these mRNA levels were significantly elevated compared with IGF1R-flox cells, oxLDL significantly further upregulated Tnf, Nos2, Il6, and Ccl2 mRNA levels (Figure 5C–5F), whereas Ccl5 mRNA was suppressed (Figure 5G). On the other hand, of the M2 activation markers, Mrc1 mRNA levels were moderately upregulated by oxLDL (Figure 5I), whereas Arg1 and Pparg were not affected by oxLDL (Figure 5H and 5J). Although there were modestly lower Arg1 mRNA levels in IL-4/oxLDL–treated MΦ-IGF1R-KO cells than in IGF1R-flox cells, overall, oxLDL or IGF1R deficiency did not robustly alter M2 marker levels. These data suggested that IGF1R deficiency influenced macrophage polarization, namely enhancing the proinflammatory M1 phenotype as evoked by IFNγ and oxLDL stimulation; however, the effect is not entirely classic activation, as implicated by Ccl5 downregulation (Figure 5G). These results prompted us to examine whether Mox marker gene expression was altered by IGF1R deficiency in IFNγ-primed macrophages. IFNγ suppressed Hmox1 and Txnrd1 mRNA levels in IGF1R-flox macrophages (Figure 5A and 5B); however, in MΦ-IGF1R-KO cells, IFNγ exerted the opposite effect, leading to an upregulation of Hmox1 mRNA (Figure 5A). IFNγ suppressed Txnrd1 mRNA levels in IGF1R-deficient macrophages but to a lesser extent than in IGF1R-flox cells, resulting in higher expression levels compared with IGF1R-flox cells (Figure 5B).

Figure 4.
Figure 4.

mRNA expression levels representing classic activation (M1) are elevated in monocyte/macrophage–specific–insulin-like growth factor-1 receptor knockout (MΦ IGF1R-KO) macrophages. Classic activation marker (Tnf, A; Nos2, B; Il6, C; Ccl2, D; Ccl5, E) and alternative activation marker (Arg1, F; Mrc1, G; Pparg, H) mRNA levels were assessed by quantitative reverse transcription–polymerase chain reaction. After isolation and adhesion to culture plates, macrophages remained untreated (NA) or were exposed to interferon-γ (IFNγ) and lipopolysaccharide (LPS) to induce classic (ie, M1) activation or exposed to interleukin (IL)-4 to induce alternative (ie, M2) activation (IGF1R-flox, open column; MΦ-IGF1R-KO, closed column). Representative data from 4 independent experiments are shown. **P<0.01 or *P<0.05 vs IGF1R-flox by 2-way ANOVA and Bonferroni multiple-comparisons test, n=3.

Figure 5.
Figure 5.

Insulin-like growth factor-1 receptor (IGF1R) deficiency skews macrophage activation induced by oxidized low-density lipoprotein (oxLDL). Macrophages isolated from IGF1R-flox (open column) and monocyte/macrophage–specific (MΦ)–IGF1R knockout (KO; closed column) mice were primed with interferon-γ (IFNγ) for 6 hours and exposed to oxLDL for 18 hours. Total RNA was extracted and subjected to quantitative reverse transcription–polymerase chain reaction, testing oxidized lipid-induced activation (Mox) marker (Hmox1, A; Txnrd1, B), M1 activation marker (Tnf, C; Nos2, D; Il6, E; Ccl2, F; Ccl5, G), and M2 activation marker (Arg1, H; Mrc1, I; Pparg, J) mRNA levels. Representative data from 3 independent experiments are shown. ##P<0.01 vs respective condition without oxLDL, $P<0.01 vs IGF1R-flox of the respective condition, ¶P<0.01 vs respective condition without IFNγ, **P<0.01 vs IFNγ-treated MΦ-IGF1R-KO, #P<0.05 vs IL-4– and oxLDL-treated IGF1R-flox, *P<0.05 vs respective condition without oxLDL by 2-way ANOVA and Bonferroni multiple-comparisons test; n=3.

Efferocytosis, a process by which apoptotic cells are removed by phagocytosis, is considered a significant mechanism involved in the resolution of inflammation. We evaluated expression levels of genes involved in efferocytosis (Figure V in the online-only Data Supplement). M1 activation suppressed the expression of efferocytosis-related genes (Anxa1, Gas6, C1qa, and Mfge8), and M2 activation had no effect (Anxa1, C1qa, Mertk; Figure V in the online-only Data Supplement) or downregulated (Gas6, Mfge8) gene expression levels. IGF1R deficiency did not alter these mRNA levels in either activated or nonactivated cells. OxLDL by itself did not induce obvious effects except for an upregulation of Mertk mRNA levels (Figure VD in the online-only Data Supplement). IGF1R deficiency upregulated Anxa1 and Mfge8 in the presence of oxLDL (Figure VA and VE in the online-only Data Supplement), but it caused a trend to downregulation of C1qa (Figure VC in the online-only Data Supplement). Thus, IGF1R deficiency did not induce changes suggesting enhanced/reduced efferocytosis.

Lipid Internalization and Efflux

Macrophage internalization and accumulation of modified LDL lead to foam cell formation, which is a hallmark of atheroma formation. Macrophage lipid internalization was assessed by exposing cells to oxLDL and acetylated LDL (Figure 6A and 6B). Macrophages were exposed to IFNγ/lipopolysaccharide (M1 activation) or IL-4 (M2 activation) and then tested for lipid incorporation after exposure to modified LDL for 48 hours. M1 cells internalized far smaller amounts of lipid compared with the nonactivated cells or M2 cells (Figure 6A and 6B), whereas M2-activated cells incorporated more lipid than untreated cells when they were exposed to acetylated LDL (Figure 6A). IGF1R deficiency did not alter lipid incorporation (Figure 6A and 6B), regardless of activation status and exposure to acetylated LDL or oxLDL.

Figure 6.
Figure 6.

Cholesterol efflux from macrophages was adversely altered by insulin-like growth factor-1 receptor (IGF1R) deficiency. A and B, Peritoneal macrophages were incubated with interferon-γ+lipopolysaccharide (IFNγ+LPS) to induce M1 and interleukin (IL)-4 to induce M2 activation status, subsequently exposed to acetylated low-density lipoprotein (acLDL; A) or oxidized LDL (oxLDL; B) for 48 hours, and assessed for lipid uptake by Oil Red O stain. Fluorescent images of stained cells were assessed with ImagePro software. Oil Red O staining intensity was normalized to cell number and expressed as relative fluorescent unit (RFU)/ cell number. C and D, Peritoneal macrophages were not activated (NA) or were activated to M1 and M2 and then loaded with lipid by exposure to acLDL and 3H-cholesterol for 24 hours. Subsequently, apolipoprotein AI (ApoAI)–dependent (C) or high-density lipoprotein (HDL)–dependent (D) efflux of 3H-cholesterol for the subsequent 24 hours were assessed. IGF1R-flox, open column; monocyte/macrophage–specific (MΦ)–IGF1R knockout (KO), closed column. Statistical significance was assessed by Mann–Whitney U test; n=8.

Cholesterol efflux from cells to extracellular lipid acceptors also contributes to lipid accumulation and thus foam cell formation. Thus, activated and lipid-laden macrophages were tested for cholesterol efflux activity (Figure 6C and 6D). IGF1R deficiency markedly reduced apolipoprotein AI–dependent cholesterol efflux in M1-activated cells (Figure 6C). We measured HDL-dependent cholesterol efflux in nonactivated, M1-activated, and M2-activated cells (Figure 6D) and found that IGF1R deficiency caused a small but significant reduction in cholesterol efflux to HDL (Figure 6D). To gain insights into potential mechanisms, we assessed expression levels of ABCA1, ABCG1, and SRB1, which are major lipid transporters responsible for cholesterol efflux. They were differentially regulated by IGF1R deficiency (Figure 7); ABCG1 expression levels were downregulated by ≈50% in IGF1R-deficient macrophages, regardless of activation status (Figure 7B and 7D), whereas ABCA1 and SRB1 were not regulated. To investigate ABCG1 expression in MΦ-IGF1R-KO cells, we exposed the cells to ligands of LXR, which is a major regulator of ABCG1 gene expression.40 LXR agonist GW3965 or T0901317 induced ABCG1 and ABCA1 expression in IGF1R-flox macrophages but not in MΦ-IGF1R-KO cells (Figure 7E). Thus, LXR-dependent regulation of ABCG1 expression is compromised by IGF1R deficiency, potentially accounting for downregulation of ABCG1 in MΦ-IGF1R-KO cells.

Figure 7.
Figure 7.

Insulin-like growth factor-1 receptor (IGF1R) deficiency downregulated ABCG1 expression levels in acetylated low-density lipoprotein (acLDL)–loaded macrophages. Peritoneal macrophages were not activated (NA) or were activated to M1 and M2 and then exposed to acLDL for 24 hours. ABCA1 (A), ABCG1 (B), and SRB1 (C) expression levels were assessed by Western blot analysis (representative measurements and blots [D] from 3 independent experiments are shown). IGF1R-flox, open column; monocyte/macrophage–specific (MΦ)–IGF1R knockout (KO), closed column. *P<0.05 vs IGF1R-flox cells by 2-way ANOVA. E, Western blot analysis for ABCG1 expression, assessing effects of LXR agonists. AcLDL-loaded macrophages were exposed to LXR agonists (1 µmol/L GW3965, 1 µmol/L T0901317) for 48 hours and assessed for ABCG1 expression levels. Representative results from 3 independent experiments are shown. *P<0.05 vs IGF1R-flox, **P<0.01 vs IGF1R-flox by the Student t test; n=3.

Monocyte Recruitment, Macrophage Proliferation, and Apoptosis in MΦ-IGF1R-KO Mice

Because MΦ-IGF1R-KO caused an increase in Mac3 positivity within plaques, we evaluated monocyte recruitment to lesions, macrophage proliferation,41 and macrophage apoptosis, 3 major determinants of macrophage number in lesions, in plaques.42 To evaluate monocyte recruitment, we labeled circulating monocytes in vivo by intravenous administration of fluorescent microspheres.43 The microspheres are not capable of penetrating into tissue interstitial space; thus, red fluorescence–positive cells (identified by DAPI positivity) within plaque are considered recruited and infiltrated cells. We evaluated red microsphere labeling by flow cytometry. As described previously,16 intravenous injections of red microspheres labeled the Ly6Clo population among circulating monocytes (Figure VIA in the online-only Data Supplement), whereas clodronate administration 1 day before the injection of red microspheres introduced the label to Ly6Chi monocytes (Figure VIB in the online-only Data Supplement). As shown in Figure 8, we detected a significantly larger number of red fluorescence–positive cells in plaques from MΦ-IGF1R-KO mice both without (Figure 8C) and with (Figure 8D) clodronate administration. It is noteworthy that more red microsphere–positive cells are detected after clodronate administration, indicating that Ly6Chi monocytes are the dominant subpopulation to be recruited to lesions.33 To assess leukocyte adhesion and rolling on the luminal side of the endothelium in vivo, we performed intravital microscopy to detect CD11b-positive cells in the mesenteric circulation. MΦ-IGF1R-KO significantly increased CD11b+ leukocyte adhesion (Figure 8E) and rolling (Figure 8F) on the luminal surface of the endothelium, consistent with increased recruitment of CD11b+ cells, that is, monocytes and neutrophils. Because IGF1R expression was undetectable in neutrophils (Figure I in the online-only Data Supplement), it is unlikely that IGF1R gene deletion in neutrophils contributed significantly to these findings.

Figure 8.
Figure 8.

Monocyte/macrophage insulin-like growth factor-1 receptor (MΦ-IGF1R) deficiency increased monocyte recruitment and infiltration into atherosclerotic plaques. MΦ-IGF1R knockout (KO) and IGF1R-flox mice were fed a high-fat diet for 12 weeks. Red microspheres were administered via intravenous injection at 3 and 7 days before death (C) or clodronate liposomes were administered intravenously 1 day before the red microsphere administration to introduce label to the Ly6Chi monocyte population (D). Serial frozen sections were obtained at aortic root and stained with Oil Red O (A) or DAPI (B). Images (B) are magnified to view the corresponding serial sections of (A) at specified areas (rectangle). L indicates lumen; blue shows the DAPI-stained nuclei. Arrows indicate red microsphere–positive cells within the lesion. The number of red microsphere–positive cells was counted per plaque (C, without clodronate; D, with clodronate), and results are shown by box-and-whiskers plot in which a box extends from the 25th to 75th percentiles with the median indicated by a horizontal line and whiskers indicating the minimum and maximum. *P<0.05, **P<0.01 by Mann–Whitney U test; n=6 for IGF1R-flox and n=5 for MΦ-IGF1R-KO. E and F, Intravital microscopy assessment of CD11b+ cell adhesion (E) and rolling (F) on the luminal surface of the mesenteric circulation endothelium. Results are shown by box-and-whiskers plot as described above. *P<0.05 and **P<0.01 by the Mann–Whitney U test; n=4 for MΦ-IGF1R-KO and n=6 for IGF1R-flox.

To assess proliferation activity of macrophages in plaques, we assessed Ki67 and proliferating cell nuclear antigen gene expression levels44–46 (Figure VIC in the online-only Data Supplement). Macrophage Marker (SC-101447; a monoclonal antibody raised against isolated macrophages of mouse origin; Santa Cruz)47,48 –positive plaque area was laser dissected from aortic root for RNA isolation, followed by quantitative reverse transcription–polymerase chain reaction. Equal amplification of CD68 was confirmed between IGF1R-flox and MΦ-IGF1R-KO lesions. Macrophage-rich regions from MΦ-IGF1R-KO plaques expressed the same levels of Ki67 or proliferating cell nuclear antigen compared with IGF1R-flox (Figure VIC in the online-only Data Supplement), suggesting no difference in macrophage proliferation in plaques. Terminal deoxynucleotidyl transferase dUTP nick-end labeling/Mac3 double-positive cell numbers were not different (Figure VID in the online-only Data Supplement). Taken together, our data suggest that IGF1R deficiency enhances monocyte recruitment to lesions, thereby increasing the macrophage cell population.

OxLDL Downregulates IGF1R in Macrophages

OxLDL plays a critical role in atherogenesis, and we have previously shown that oxLDL downregulates IGF1R in vascular smooth muscle cells.49 To determine potential regulation of macrophage IGF1R expression by oxLDL, we used the human monocytic cell line THP-1, which was differentiated into macrophages and exposed to 100 µg/mL oxLDL (Figure VII in the online-only Data Supplement). OxLDL exposure for 24 hours downregulated IGF1R levels by 80%.

Discussion

IGF-1 production has been documented in macrophages8,9,50,51; however, precise effects of IGF-1 in macrophages in relation to the pathogenesis of atherosclerosis have not been elucidated. In this study, we generated macrophage/monocyte–specific IGF1R-deficient mice and discovered pivotal roles of IGF-1 in the regulation of inflammatory responses and lipid handling in macrophages, which are relevant to the antiatherogenic effects of IGF-1. Our data indicate that IGF1R deficiency in macrophages enhances proinflammatory activation (ie, M1 polarization), thereby promoting proinflammatory cytokine production (Figures 3 and 4), and enhances lipid accumulation as a result of reduced efflux (Figures 6 and 7). These phenotypic changes resulted in increased recruitment of macrophages to atherosclerotic plaques and increased atherosclerotic burden in MΦ-IGF1R-KO mice (Figures 1 and 2). Moreover, histological evaluation of brachiocephalic arteries indicated that the MΦ-IGF1R-KO induced features of unstable plaques (Table).

There is growing interest in the role of IGF-1 in cardiovascular disease. Low circulating IGF-1 levels have been associated with cardiovascular disease risk factors,52–56 and in particular, there is growing evidence for a role for IGF-1 deficiency in the pathogenesis of metabolic syndrome.57 Acromegaly (ie, excessive growth hormone and IGF-1) or otherwise growth hormone and IGF-1 deficiency have been linked to cardiovascular complications.58,59 However, epidemiological studies linking IGF-1 with cardiovascular disease report mixed results. Some cross-sectional and prospective studies60–65 suggest a positive association between IGF-1 (and in some cases insulin-like growth factor–binding protein 3) and atherosclerosis, but others have found that low IGF-1 is a predictor of ischemic heart disease and mortality, consistent with the potential anti-apoptotic, antioxidant, and plaque stabilization effects of IGF-1.1–3,13,66–72 Methodological constraints could explain these contradictions because measurement of total IGF-1 levels represents only a crude estimate of the biologically active IGF-1. Thus, an IGF-1–specific kinase receptor activation assay may better reflect IGF-1 bioactivity.73 In fact, it has been reported that higher IGF-1 bioactivity is associated with significantly longer survival in subjects with a high inflammatory risk profile or history of cardiovascular disease.74 Additionally, polymorphisms in the IGF-1 gene promoter region that influence circulating IGF-1 levels have been reported.75–77 The alleles that indicate lower circulating IGF-1 levels have been associated with increased risk for type 2 diabetes mellitus, myocardial infarction, left ventricular hypertrophy, higher carotid intima-media thickness, higher aortic pulse-wave velocity, and lower endothelium-dependent vasodilation.75–77 Intriguingly, IGF-1 resistance in the endothelium was reported in an animal model of obesity, thereby blunting the vasodilatory response to IGF-1 via attenuated endothelial nitric oxide synthase phosphorylation and nitric oxide production.78 Our present finding that reduced macrophage IGF-1 signaling is highly proinflammatory and increases atherosclerotic burden is consistent with the growing evidence that decreased IGF-1 action may be a significant contributor to the pathogenesis of atherosclerosis. Of note, we found that oxLDL downregulates IGF1R levels in human-derived THP1 macrophage (Figure VII in the online-only Data Supplement), which is consistent with our previous reports that both IGF-1 and IGF1R expression were significantly lower in human atherosclerotic plaque intimal regions with macrophage infiltration, where oxLDL was highly detected.79,80

We used MΦ-IGF1R-KO mice, which have 1 allele of Lys2 ablated; the IGF1R-flox mice (control) have both alleles intact. Previous studies in LysCre/+ mice with regard to potential alterations in monocyte/macrophage biology caused by the hemizygous deficiency of Lys2 showed no evidence of a heterozygous phenotype.21 In addition, complete ablation of both alleles of Lys2 does not influence atherosclerosis in Apoe−/− mice.81 Lys2-cre–mediated gene excision occurs in monocytes and neutrophils.21–23 We confirmed deletion of exon 3 of the Igf1r gene in both macrophages and neutrophils isolated from the MΦ-IGF1R-KO mouse, but we were unable to detect IGF1R protein even in Igf1r-normal neutrophils (ie, IGF1R-flox neutrophils), indicating extremely low expression levels. Thus, although contribution of neutrophils to the phenotype of MΦ-IGF1R-KO mice cannot be excluded, it appears much more likely that macrophages, which express significant levels of IGF1R, play the predominant role.

IGF1R and InsR are structurally similar and form a heteromeric hybrid receptor consisting of α+β subunits of IGF1R and α+β subunits of InsR. The hybrid receptor binds IGF-1 with high affinity but does not bind insulin at physiological ranges. To the best of our knowledge, this is the first study evaluating the expression ratio between IGF1R and InsR in macrophages, showing predominant expression of InsR. Thus, IGF1R-flox macrophages (expressing both IGF1R and InsR) responded to a physiological dose of insulin (Figure I in the online-only Data Supplement). Intriguingly, IGF1R deficiency in macrophages, although it should free up InsR hemidimers to form holoreceptors, did not increase insulin signaling. This finding is relevant because macrophage InsR deficiency has been shown to modulate atherosclerosis development (although results have been contradictory30,31). Because we found that MΦ-IGF1R-KO did not alter insulin signaling in macrophages, it is unlikely that changes in insulin action on macrophages plays a significant role in the phenotype of MΦ-IGF1R-KO mice.

Macrophages become activated as they infiltrate into a target tissue and are exposed to stimuli, expressing a highly proinflammatory phenotype or a less inflammatory but phagocytic and antigen-presenting phenotype. The former status was called classic activation or M1 activation; the latter was referred to as alternative activation or M2 activation. Recent investigations indicate, however, that macrophage activation status likely encompasses a broad spectrum in which M1 and M2 activation are likely 2 extremes.82–84 Recognizing that macrophage activation represents a continuum, we tested M1 and M2 activation, as well as Mox activation, which was described as a unique activation status found in plaque macrophages,39 to provide insights into the effects of IGF1R deficiency. Our results indicate that IGF1R deficiency resulted in macrophages acquiring a highly inflammatory (ie, M1) phenotype (Figure 3), whereas M2 marker gene expression levels were not altered (Figure 4F–4H). However, MΦ-IGF1R-KO macrophages are not simply skewed to a more inflammatory phenotype; these cells were also shifted to a phenotype induced by oxLDL, characterized by upregulation of the antioxidant genes Hmox1 and Txnrd1, described as Mox activation39 (Figure 5A and 5B). Reports of the effects of oxLDL and its specific lipid moieties on macrophage activation are variable and include enhancement of the inflammatory phenotype,36,38,85 induction of M2 activation,86 or a unique activation status.39 In summary, our results suggest that IGF1R deficiency skews macrophages to a unique activation state that can be characterized by enhanced proinflammatory response and elevated antioxidant system.

The effect of IGF1R deficiency on macrophage activation status prompted us to examine how IGF1R deficiency influences uptake of modified LDL, cholesterol efflux, and efferocytosis. It has been shown that M1-activated macrophages demonstrate reduced foam cell formation in response to oxLDL6,87 and that Mox-activated macrophages have attenuated phagocytosis and efferocytosis.39 It would thus have been reasonable to speculate that IGF1R deficiency, which enhances the proinflammatory phenotype and Mox marker expression levels, could lead to reduced foam cell formation. However, our results showed no evidence of altered acetylated LDL or oxLDL uptake by IGF1R-deficient macrophages (Figure 6A and 6B). We also assessed expression levels of genes that are functional in efferocytosis, but there was no apparent alteration caused by IGF1R deficiency (Figure V in the online-only Data Supplement). Thus, despite enhanced inflammatory responses and redox gene expression, IGF1R-deficient macrophages do not appear to have impairment in modified lipid uptake or efferocytosis. However, further assessment of lipid handling by IGF1R-deficient macrophages indicated that cholesterol efflux is impaired by IGF1R deficiency (Figure 6C and 6D), potentially promoting foam cell formation. Cholesterol efflux is mediated by the lipid transporters ABCA1, ABCG1, and SRB1.88 IGF1R deficiency caused lower expression levels of ABCG1 (Figure 7), which should, at least in part, account for lowered cholesterol efflux. Intriguingly, the LXR-dependent regulation of ABCG1 expression was compromised in MΦ-IGF1R-KO macrophages. LXR is a major regulator of ABCG1 expression.89,90 In fact, LXR drives ABCG1 expression on lipid loading by modified LDL (which causes accumulation of oxysterols).89,90 Thus, impaired LXR regulation of ABCG1 expression could be an important mechanism underlying lowered cholesterol efflux in MΦ-IGF1R-KO macrophages.

MΦ-IGF1R-KO increased atherosclerosis burden as assessed by Oil Red O staining of en face aortas and by histological analysis of aortic root cross sections (Figure 1). These mice had an increase in lesional macrophages and in recruitment of monocytes to atherosclerosis plaques (Figure 8). Consistent with the increased recruitment of monocytes, chemokine expression levels were upregulated in MΦ-IGF1R-KO macrophages (ie, MCP-1 and fractalkine; Figures 3–5). Moreover, MΦ-IGF1R-KO increased features of plaque vulnerability, as evidenced by histological features of intraplaque hemorrhage and medial elastin breaks in lesions from brachiocephalic artery (Figure IV in the online-only Data Supplement and Table). These findings were in accordance with the changes in plaque composition, notably the increased population of macrophages, the reduced population of smooth muscle cells particularly within the plaque cap, and decreased collagen (Figure 2). To obtain insights into the underlying mechanisms, we assessed MMP levels in peritoneal macrophages from MΦ-IGF1R-KO mice and showed significant upregulation of MMPs (Figure 2D), which have previously been shown to be relevant to atherogenesis and plaque vulnerability.91,92 In addition, we found significant upregulation of MMP-1, -2, -8, and -9 in lysates of ascending aorta from MΦ-IGF1R-KO animals, consistent with data obtained with peritoneal macrophages. A major source of collagen matrix synthesis and deposition in atherosclerotic plaques is smooth muscle cells, and thus, one can speculate that the enhanced inflammatory milieu induced by MΦ-IGF1R-KO altered smooth muscle cell homeostasis. In fact, it is noteworthy that MΦ-IGF1R-KO robustly enhanced macrophage production of proinflammatory cytokines such as IL-1α, TNFα, and IL-6 (Figure 3), and it has been reported that cytokines such as TNFα reduce IGF-1 and increase IGF binding protein-3 in vascular smooth muscle cells, leading to a reduction in bioactive IGF-1.93 IGF-1 positively regulates collagen synthesis by smooth muscle cells.94 Indeed, we have recently shown that increased IGF-1 signaling in vascular smooth muscle cells increases features of plaque stability, as determined by increased fibrous cap area, α-smooth muscle actin–positive smooth muscle cells, and collagen content, without affecting plaque burden,6 potentially mediated by IGF-1 induction of smooth muscle differentiation6 and collagen synthesis.94 Thus, it seems reasonable to speculate that increased production of inflammatory cytokines in MΦ-IGF1R-KO mice disrupts normal IGF-1 signaling in smooth muscle cells and reduces collagen deposition in plaques of MΦ-IGF1R-KO mice. Taken together with our previous results, the present study provides insights into potential interactions between macrophages and smooth muscle cells; that is, the enhanced inflammatory milieu by macrophages suppresses IGF-1 signaling in smooth muscle cells, leading to a reduction of plaque stability.

Our results are consistent with the growing body of experimental evidence that IGF1 has antiatherogenic effects. Infusion of IGF-1 in Apoe−/− mice reduces atherosclerotic burden,5 and overexpression of IGF-1 in vascular smooth muscle cells increases plaque collagen content and smooth muscle cell levels and reduces necrotic core size.6 IGF-1 has been shown to have antioxidant effects on the endothelium via upregulation of glutathione peroxidase levels.95 Our current findings demonstrate that IGF-1 signaling has a major effect on macrophage biology that is critical for atherogenesis. However, a limitation of experimental studies to date has been that they have been performed in murine models and that studies in larger animals phylogenetically closer to humans are lacking. Such studies will be important for the development of IGF-1–based therapeutic strategies.

Conclusions

We have shown that IGF1R deficiency in macrophages of Apoe−/− mice increases atherosclerotic burden and changes plaque composition to one of lowered smooth muscle cell and collagen content. Our data suggest that the loss of IGF-1 signaling skews macrophage activation to a proinflammatory status and promotes lipid accumulation in macrophages by lowering lipid efflux. There is increasing evidence linking low IGF-1 to multiple cardiovascular risk factors, including metabolic syndrome and aging.57,96 In fact, it has been reported that decreased IGF-1 bioavailability is an adverse prognostic factor for coronary heart disease (reviewed elsewhere97,98). Our findings herein suggesting that IGF-1 regulates macrophage inflammatory responses and lipid metabolism may provide the basis for a novel therapeutic approach for the treatment of atherosclerotic vascular disease development and progression.

Acknowledgments

We thank Chelsea Deroche, PhD (Biostatistics and Research Design Unit in the Office of Medical Research and Health Management and Informatics, University of Missouri School of Medicine), for her expert advice on statistical assessments.

Sources of Funding

This work was supported by National Institutes of Health grants R01-HL070241 (Dr Delafontaine), R01-HL080682 (Dr Delafontaine), R21-HL113705 (Dr Sukhanov), R01-HL059976 (Dr Korthuis), P01-HL095486 (Dr Korthuis), and R01-AA022108 (Dr Korthuis), as well as American Heart Association Grant-in-Aid 13GRNT17230069 (Dr Sukhanov).

Disclosures

None.

Footnotes

  • Received July 13, 2015.
  • Accepted April 27, 2016.

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CLINICAL PERSPECTIVE

Atherosclerosis is an inflammatory disease, and acute coronary events result largely from erosion or rupture of unstable plaques with increased inflammatory cells and a relative reduction in vascular smooth muscle cells. Macrophages play a major role in atherogenesis by scavenging and accumulating lipids to become lipid-laden foam cells. Furthermore, proinflammatory macrophages induce smooth muscle cell death by secreting cytokines and degrade extracellular matrix by producing enzymes such as matrix metalloproteinases, weakening the tensile strength of plaques and predisposing them to rupture. In the past decade, insulin-like growth factor-1 (IGF-1) has demonstrated antiatherogenic effects in experimental models, but the mechanisms are poorly elucidated. In this study, we investigated macrophage–IGF-1 receptor deficiency in a murine model of atherosclerosis. We found that IGF-1 receptor deficiency increased monocyte/macrophage recruitment to lesions, skewed macrophage activation to a proinflammatory status, promoted lipid accumulation in macrophages by lowering lipid efflux, and upregulated matrix metalloproteinase production, resulting in an increase in atherosclerotic burden and a decrease in features of plaque stability. Our findings are consistent with epidemiological studies suggesting that low circulating IGF-1 is a predictor of ischemic heart disease and mortality. There is also evidence linking low IGF-1 to multiple cardiovascular risk factors, including metabolic syndrome. Our findings suggest that IGF-1 regulation of macrophage inflammatory responses and lipid metabolism may be the basis for novel approaches to reduce atherosclerotic lesion progression and to promote plaque stability.

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  • Insulin-Like Growth Factor-1 Receptor Deficiency in Macrophages Accelerates Atherosclerosis and Induces an Unstable Plaque Phenotype in Apolipoprotein E–Deficient MiceCLINICAL PERSPECTIVE
    Yusuke Higashi, Sergiy Sukhanov, Shaw-Yung Shai, Svitlana Danchuk, Richard Tang, Patricia Snarski, Zhaohui Li, Patricia Lobelle-Rich, Meifang Wang, Derek Wang, Hong Yu, Ronald Korthuis and Patrice Delafontaine
    Circulation. 2016;133:2263-2278, originally published May 6, 2016
    https://doi.org/10.1161/CIRCULATIONAHA.116.021805
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