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Abstract
Objective—Angiotensin II is a major determinant of atherosclerosis. Although macrophages are the most abundant cells in atherosclerotic plaques and express angiotensin II type 1 receptor (AT1), the pathophysiologic role of macrophage AT1 in atherogenesis remains uncertain. We examined the contribution of macrophage AT1 to accelerated atherosclerosis in an angiotensin II-responsive setting induced by uninephrectomy (UNx).
Methods and Results—AT1−/− or AT1+/+ marrow from apolipoprotein E deficient (apoE−/−) mice was transplanted into recipient apoE−/− mice with subsequent UNx or sham operation: apoE−/−/AT1+/+→apoE−/−+sham; apoE−/−/AT1+/+ →apoE−/−+UNx; apoE−/−/AT1−/−→apoE−/−+sham; apoE−/−/AT1−/−→apoE−/−+UNx. No differences in body weight, blood pressure, lipid profile, and serum creatinine were observed between the 2 UNx groups. ApoE−/−/AT1+/+ →apoE−/−+UNx had significantly more atherosclerosis (16907±21473 versus 116071±8180 μm2, P<0.05). By contrast, loss of macrophage AT1 which reduced local AT1 expression, prevented any effect of UNx on atherosclerosis (77174±9947 versus 75714±11333 μm2, P=NS). Although UNx did not affect total macrophage content in the atheroma, lesions in apoE−/−/AT1−/−→apoE−/−+UNx had fewer classically activated macrophage phenotype (M1) and more alternatively activated phenotype (M2). Further, UNx did not affect plaque necrosis or apoptosis in apoE−/−/AT1−/−→apoE−/− whereas it significantly increased both (by 2- and 6-fold, respectively) in apoE−/−/AT1+/+ →apoE−/− mice. Instead, apoE−/−/AT1−/−→apoE−/− had 5-fold–increase in macrophage-associated apoptotic bodies, indicating enhanced efferocytosis. In vitro studies confirmed blunted susceptibility to apoptosis, especially in M2 macrophages, and a more efficient phagocytic function of AT1−/− macrophages versus AT1+/+.
Conclusion—AT1 receptor of bone marrow-derived macrophages worsens the extent and complexity of renal injury-induced atherosclerosis by shifting the macrophage phenotype to more M1 and less M2 through mechanisms that include increased apoptosis and impaired efferocytosis.
Introduction
Experimental studies have established the proatherogenic effects of angiotensin II (AII).1–3 Conversely, ample experimental and clinical evidence suggests that antagonism of AII actions by angiotensin-converting enzyme inhibitors or angiotensin receptor blockers (ARB) provides antiatherogenic benefit.3–7 The proatherogenic effects of AII and the antiatherogenic effects of angiotensin-converting enzyme inhibitors and ARB have been linked to modulation of macrophage functions, including chemotaxis, intimal recruitment, inflammation, neovascularization, and proteolysis.3,4,8,9 AII can modulate macrophage-specific functions, as supported by observations that monocyte/macrophages express components of the angiotensin system, including the AII type 1 receptor (AT1), and that cellular exposure to AII promotes lipid accumulation, migration, and cytokine production.4,8,9 Nonetheless, the specific contribution in vivo of the macrophage AT1 receptor to atherogenesis has been controversial, ranging from little influence observed in some studies10 to significant proatherogenic effects reported by others, especially in the setting of infusion of exogenous AII.11,12
The variable impact of macrophage AT1 on atherogenesis may reflect heterogeneity in macrophages, with multiple functions differently expressed during progression of atherosclerosis.13 The classically activated M1 macrophage phenotype, stimulated by lipopolysaccaride and interferon-γ, enhances proinflammatory cytokines, including tumor necrosis factor-α, C-C chemokine receptor type 7 (CCR7), inducible nitric oxide synthases (iNOS), interleukin (IL)-1β, and IL-6. The alternatively activated M2 macrophage phenotype, stimulated by IL-4 and IL-13, is linked to inflammation resolution, tissue repair, and endocytic clearance with activation of antiinflammatory cytokines, including arginase-1 and Ym-1. Such antiinflammatory and endocytic functions contribute to efferocytosis, the clearance of apoptotic cells by phagocytes, which is an essential mechanism for vascular repair and remodeling.14 Modulation of macrophage phenotype transformation by AII was recently documented in a model of antiglomerular basement membrane glomerulonephritis.15 In this study, macrophages infiltrating glomeruli were predominantly of M1 phenotype in untreated rats and of M2 phenotype in rats receiving ARB-treatment, which also caused attenuation of renal injury. We also found that ARB treatment skewed the intrarenal macrophage population from M1 to M2 phenotype in an obesity-related kidney injury model.16 Previously, we showed that reduction in renal mass dramatically exacerbates atherosclerosis via a mechanism that is clearly responsive to ARB.4 Because absolute and relative abundance of M1 and M2 macrophages determines extent and composition of the atherosclerotic plaque, we examined the impact of modulating the macrophage AT1 on renal injury-enhanced atherogenesis, focusing on the macrophage phenotype and the mechanisms that influence plaque architecture.
Methods
Experimental Groups
Female apoE−/− mice on a C57BL/6 background (Jackson Laboratories, Bar Harbor, ME) maintained on normal mouse chow (RP5001; PMI Feeds, St. Louis, MO) served as recipients in bone marrow transplantation studies. AII type 1a receptor (AT1a) deficient mice on a C57BL/6 background previously generated in our laboratory17 have been backcrossed more than 10 times with apoE−/− mice to produce mice deficient in both apoE and AT1a. These double knock-outs (apoE−/−/AT1a−/−) as well as apoE−/− mice with intact AT1a (apoE−/−/AT1a+/+) were used as bone marrow donors. Rodents have 2 AT1 forms, a dominant AT1a receptor and a less expressed AII type 1b receptor (AT1b) subtype.12,18 Studies in systemic AT1a-deficient mice suggest compensatory effects of AT1b on renal damage in murine lupus nephritis model and in obesity-induced renal injury.16,19 Real time-PCR amplification performed on peritoneal macrophages used inventories TaqMan assay ID Mm01701115-m1 for AT1b and Hs99999901-s1 for 18s rRNA using an ABI prism 7700 sequence detection system (Applied Biosystems Inc., Foster City, CA) as previously described.16,20 Similar to previously published reports of results obtained from bone marrow-derived macrophages of AT1a−/− mice,12 there was no detectable expression of AT1b receptor in macrophages of either AT1a+/+ or AT1a−/− mice.
At 8 weeks of age, recipients were lethally irradiated with 9 Gy using a cesium gamma source and then transplanted with 5×106 bone marrow cells harvested from femurs of apoE−/−/AT1+/+ or apoE−/−/AT1−/− donors as previously described.17,21 Six weeks later, each recipient underwent sham operation (Sham) or uninephrectomy (UNx). Mice were subdivided into 4 groups: apoE−/− recipients reconstituted with apoE−/−/AT1+/+ marrow and sham operation (apoE−/−/AT1+/+→apoE−/−+sham, n=10); apoE−/− recipients reconstituted with apoE−/−/AT1+/+ marrow and UNx (apoE−/−/AT1+/+→apoE−/−+UNx, n=15); apoE−/− recipients reconstituted with apoE−/−/AT1−/− marrow and sham operation (apoE−/−/AT1−/−→apoE−/−+sham, n=10); and apoE−/− recipients reconstituted with apoE−/−/AT1−/− marrow and UNx (apoE−/−/AT1−/−→apoE−/−+UNx, n=15). Mice were euthanized at 25 weeks of age. Care and experimental procedures were in accordance with National Institutes of Health guidelines and approved by the Vanderbilt University Institutional Animal Care and Usage Committee.
Systemic Parameters
Systolic blood pressure (BP) was measured in conscious mice midway through the study and prior to sacrifice using the Bp-2000 BP analysis system automated tail cuff (Visitech Systems Inc, Apex, NC).22–24 Animals were first acclimated to the procedure, and mean values were based on an average of 10 stable readings. Body weight was assessed weekly. Serum total cholesterol and triglycerides levels were determined at the end of the experiment, according to standard methods.1,2,25 Serum creatinine was measured by HPLC.23,26
Assessment of Atherosclerosis and Necrotic Areas Within Atherosclerotic Lesions
Mice were euthanized under phenobarbital anesthesia and perfused with PBS through the left ventricle.1,2,25 The heart with the proximal aorta was embedded in optical cutting temperature and snap-frozen in liquid nitrogen. Ten-micrometer thick cryosections were cut from the proximal aorta beginning at the end of the aortic sinus with modifications specific for computer analysis.1,2,25 Cryosections were stained with Oil-Red-O to assess lipid deposition. Quantitative analysis of lesions was performed using Imaging System KS300 (Release 2.0; Kontron Elektronik GmbH, Poway, CA). To assess necrotic areas, cryosections were stained with Harris H&E (Sigma, St. Louis, MO). Both stained and acellular/anuclear areas in intimal lesions were included in quantitation of the total atherosclerotic lesions.23,27,28 Necrotic area was calculated as the ratio between a nuclear area and total lesion area as previously described.23,27,28 Quantitative analysis of lesions was performed using Imaging System KS300. The assessment was done without awareness of group assignment.
Assessment of Macrophage Content and Phenotype, AT1 Receptor Expression in Atherosclerotic Lesions
Serial, 5-μm thick cryosections of proximal aorta were fixed in acetone and incubated with monoclonal rat antibody to mouse macrophages (MOMA-2; Serotec, Raleigh, NC) to measure macrophage-positive area within atherosclerotic lesions as previously described.4,23 Rat anti-mouse CD68 (AbD Serotec, UK) and nuclear DAPI were used to stain macrophages. Rabbit anti-mouse CCR7 and iNOS (BD bioscience) were used to stain for M1 macrophage pheonotype,29–31 whereas rabbit anti-mouse Ym-1 (Stemcell Technologies) or arginase 1 (BD bioscience) were used to stain for M2 macrophage phenotype.32,33 The percentage of M1 or M2 subtypes was determined as the ratio of positive cells for each phenotype marker to total CD68 positive cells. To assess expression of AT1 receptor, immunofluorescence was performed on optical cutting temperature-embedded aorta sections (Supplement, available online at http://atvb.ahajournals.org).34
Assessment of Macrophage Apoptosis and Efferocytosis in Atherosclerotic Lesions
Apoptotic cells in lesions were detected by TUNEL (Tdt-mediated dUTP nick end labeling) using the in situ cell death detection kit TMR red (Roche, Indianapolis, IN). Nuclei were counterstained with the Hoechst 33342 stain (Invitrogen, CA), and macrophages were stained with rabbit antimacrophage cytoplasm antibody (AIA31240; Accurate Chemical and Scientific), goat anti-rabbit biotinylated conjugated secondary antibody, and Alexa Fluor 488 (Molecular Probes).27,35 Macrophage efferocytosis was evaluated by colocalization of apoptotic cells with intact macrophages.27,35 In situ quantitation of free versus macrophage-associated apoptotic cells in individual lesion sections was performed as described by us and others.27,35 Increased ratio of free to macrophage-associated apoptotic cells within the lesion represents inefficient efferocytosis.
Assessment of Apoptosis, Efferocytosis, and Biomediator Activity In Vitro
Thioglycollate-elicited peritoneal macrophages from apoE−/−/AT1−/− and apoE−/−/AT1+/+ mice were seeded at 1.0×106 cells/well in 2-well chamber slides. After overnight incubation in DMEM with 1% fetal bovine serum, macrophages were reacted with 50 ng/mL lipopolysaccaride for 24 hours. Apoptotic cells were detected by TUNEL using the in situ cell death detection kit (Roche) according to the manufacturer's instruction.27 TUNEL positive cells versus total cells were quantitated in triplicate chamber slide wells assessing at least 10 fields per well. Pro and antiapoptotic markers were assessed in apoE−/−/AT1−/− and apoE−/−/AT1+/+ macrophages seeded at 0.5×106 cells/well and exposed to lipopolysaccaride (50 ng/mL) or IL-13 (10 ng/mL) for 6 hours. Macrophage total RNA was extracted and gene expression was assessed by real time PCR with probes for bcl-2 (Mm00477631_m1), caspase-3 (Mm01195085_m1) and 18S rRNA obtained from Applied Biosystems.
To assess efferocytosis, peritoneal macrophages were seeded in 100-mm plates at 20×106 in DMEM with 10% FBS. The plated macrophages were labeled with 5 μmol/L carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) cell tracer (Molecular Probes, Eugene, OR) in DPBS for 30 minutes at 37°C. To induce apoptosis, the labeled macrophages were incubated with BAY11–7082×2 hours (20 μmol/L, Calbiochem, Germany), an agent that has been shown to be a powerful inducer of apoptosis of murine macrophages by inhibition of nuclear factor-κB (NF-κB),36 a key signaling pathway in macrophage function.8,27 The apoE−/−/AT1−/− and apoE−/−/AT1+/+ macrophages were seeded at 0.5×106 cells/well in 2-well chamber slides, washed with DPBS. 1.5×106 CFDA-SE cell tracer-labeled apoptotic cells in DMEM were added to the seeded macrophages. After 2 hours incubation, the seeded macrophages were vigorously washed with PBS to remove noningested apoptotic cells, fixed in 4% paraformaldehyde, and counterstained with DAPI for visualization of cell nuclei (Vector Labs, Burlingame, CA). Visualization of engulfed apoptotic cells was done with fluorescence microscopy. CFDA-SE cell tracer positive phagocytes versus total phagocytes were quantitated in triplicate chamber slide wells assessing at least 10 fields per well.
To assess biomediator activity, we assessed peritoneal macrophages with intact or deficient AT1. Macrophage total RNA was extracted and gene expression was assessed by real time PCR as previously described.23 Probes for IL-1β (Mm00434228_m1), iNOS (Mm01309900_m1), tumor necrosis factor-α (Mm99999068_m1), and 18S rRNA (Hs99999901_m1) were obtained from Applied Biosystems.
Statistical Analysis
Results are expressed as means±SEM. Statistical difference was assessed by a single-factor ANOVA followed by Tukey-Kramer's HSD. P<0.05 was considered to be significant.
Results
Systemic Parameters
Table 1 shows the systemic parameters at time of sacrifice. There were no differences in body weight or systolic BP among the groups. Uninephrectomy increased serum creatinine and total cholesterol levels in both apoE−/−/AT1+/+→ apoE−/− and apoE−/−/AT1−/−→apoE−/− mice. In agreement with previous reports,4,23 the intervention did not affect serum triglyceride levels.
Systemic Parameters
Atherosclerotic Lesions
Uninephrectomy significantly increased atherosclerosis in mice reconstituted with AT1+/+ marrow (Figure 1). The cross-sectional lesion area in the aortic sinus was 169071±21473 μm2 in apoE−/−/AT1+/+→apoE−/−+UNx mice and 116071±8180 μm2 in apoE−/−/AT1+/+→apoE−/− +sham mice (P<0.05). By contrast, UNx caused no increase in mice reconstituted with AT1−/− marrow (77174±9547 μm2 in apoE−/−/AT1−/− →apoE−/− +sham versus 75714±1133 μm2 in apoE−/−/AT1−/−→apoE−/−+UNx). Interestingly, reconstitution with AT1−/− cells reduced progression of atherosclerosis even in mice with intact kidneys. As shown in Figure 1, cross-sectional lesion area of apoE−/−/AT1−/−→apoE−/− +sham was 35% less than in apoE−/−/AT1+/+→apoE−/− +sham. AT1 immunostaining within the atherosclerotic plaque was reduced in atherosclerotic lesions of uninephrectomized mice repleted with AT1-deficient marrow (Supplemental Figure). Notably, AT1 expression in the vascular media was not reduced in mice repleted with AT1-deficient marrow.
Macrophages deficient in angiotensin type 1 receptor (AT1) prevent renal damage-induced increase in atherosclerosis. Representative Oil-Red-O-stained lesions of proximal aorta of mice with intact macrophage AT1 and sham operation (apolipoprotein E [apoE]−/−/AT1+/+→apoE−/− +sham, n=10) or uninephrectomy (apoE−/−/AT1+/+→apoE−/−+UNx, n=15) and mice with deficient macrophage AT1 and sham operation (apoE−/−/AT1−/−→apoE−/− +sham, n=10) or uninephrectomy (apoE−/−/AT1−/−→apoE−/−+UNx, n=15). Bars in photomicrographs correspond to 250 μm.
Macrophage Phenotype in Atherosclerotic Lesions
Macrophage-positive area was not significantly different among groups (apoE−/−/AT1+/+→apoE−/− +sham, 73999±7988 μm2; apoE−/−/AT1+/+→apoE−/−+UNx: 88644±6660 μm2; apoE−/−/AT1−/−→apoE−/− +sham: 66953±8079 μm2 apoE−/−/AT1−/−→apoE−/−+UNx: 78390±6032 μm2). Nevertheless, macrophage phenotypes were altered within the atherosclerotic lesions. In particular, in mice reconstituted with AT1+/+ bone marrow, UNx significantly increased macrophages with markers of the M1 phenotype, including CCR7 (63.6±2.8% versus 53.0±2.3, P<0.05, Figure 2A) and iNOS (80.3±1.7% versus 31.4±3.7, P<0.05, Figure 2B). Lesions of apoE−/−/AT1+/+→apoE−/− +UNx also had fewer cells with markers of the M2 phenotype, including Ym-1 (38.7±3.5 versus 64.0±4.2%, P<0.05, Figure 2C) and Arg1 (48.9±3.3 versus 65.6±4.2%, P<0.05, Figure 2D) compared with apoE−/−/AT1+/+→apoE−/− +sham. By contrast, in mice reconstituted with AT1−/− cells, UNx had no effect on CCR7 expression, and only modestly increased the number of iNOS positive cells (Figure 2B). Further, loss of kidney mass did not affect Ym-1 or Arg1 staining in lesions of apoE−/−/AT1−/−→apoE−/− (Figure 2C and D).
Macrophage angiotensin type 1 receptor (AT1) modulates renal damage-induced macrophage phenotypic change. Immunofluorescent staining for C-C chemokine receptor type 7 (CCR7) (A), inducible nitric oxide synthases (iNOS) (B), Ym-1 (C), and arginase 1 (D) as fractions of total macrophages stained with CD68 in atherosclerotic lesions of mice with intact macrophage AT1 and sham surgery (apolipoprotein E [apoE]−/−/AT1+/+→apoE−/−+sham, n=10) or uninephrectomy (apoE−/−/AT1+/+→apoE−/−+UNx, n=15) and mice with deficient macrophage AT1 (apoE−/−/AT1−/−→apoE−/− +sham, n=10) or uninephrectomy (apoE−/−/AT1−/−→apoE−/−+UNx, n=15). Bars in photomicrographs correspond to 100 μm.
Characteristics of Atherosclerotic Lesions
UNx dramatically increased apoptotic cells in lesions of apoE−/−/AT1+/+→apoE−/−+UNx (274.9±127.3 versus 1750.0±236.0, P<0.05, Figure 3A). By contrast, UNx in mice reconstituted with AT1−/− bone marrow showed no change in the number of apoptotic cells (195.6±114.2 versus 423.6±231.9, p=NS, Figure 3A). Free versus macrophage-associated apoptotic cells (an index of efferocytosis) were significantly increased in lesions of apoE−/−/AT1+/+→ apoE−/−+UNx versus apoE−/−/AT1+/+→apoE−/− +sham (1.71±0.4 versus 0.31±0.08, P<0.05, Figure 3B). By contrast, macrophage-associated apoptotic cell ratio was not different in lesions of apoE−/−/AT1−/−→apoE−/−+UNx versus apoE−/−/AT1−/−→apoE−/− +sham (0.56±0.16 versus 0.49±0.17, p=NS, Figure 3B).
Macrophages deficient in angiotensin type 1 receptor (AT1) lessen renal damage-induced increases in apoptosis and ineffective efferocytosis. A, Apoptosis (TUNEL staining). B, Efferocytosis (free-to-macrophage-associated ratio of apoptotic cells). Mice with intact macrophage AT1 and sham surgery (apolipoprotein E [apoE]−/−/AT1+/+→apoE−/−+sham, n=10) or uninephrectomy (apoE−/−/AT1+/+→apoE−/−+UNx, n=15) and mice with deficient macrophage AT1 and sham surgery (apoE−/−/ AT1−/−→apoE−/−+sham, n=10) or uninephrectomy (apoE−/−/AT1−/−→apoE−/−+UNx, n=15). Bars in photomicrographs correspond to 100 μm.
Expression of biomarkers was significantly increased in peritoneal macrophages from apoE−/−/AT1+/+→apoE−/− +UNx versus apoE−/−/AT1+/+→apoE−/− +sham (IL-1β; 0.99±0.03 versus 0.43±0.10, P<0.05, TNF-α; 1.57±0.17 versus 0.68±0.05, P<0.05, iNOS; 0.27±0.04 versus 0.16±0.02, P<0.05). By contrast, expression of inflammatory biomarkers was not different in peritoneal macrophages from apoE−/−/AT1−/−→apoE−/−+UNx versus apoE−/−/AT1−/−→apoE−/− +sham (IL-1β; 0.41±0.08 versus 0.12±0.04, p=NS, TNF-α; 1.02±0.12 versus 0.64±0.15, p=NS, iNOS; 0.12±0.04 versus 0.08±0.01, P=NS).
In vitro experiments paralleled the in vivo impact of macrophage AT1 on apoptosis and efferocytosis following UNx. Peritoneal macrophages lacking AT1 were significantly less susceptible to apoptotic stimuli and showed more efficient efferocytosis compared with AT1+/+ peritoneal macrophages (Figure 4A and B). In vitro studies confirmed both blunted susceptibility to apoptosis and a more efficient phagocytic function of AT1−/− macrophages. Thus, AT1−/− macrophages had 6.2±0.5% TUNEL-positive cells versus 12.4±1.0% in AT1+/+ macrophages (P<0.05, Figure 4A). AT1 also modulated gene expression of apoptotic markers. Macrophages lacking AT1 showed significantly less apoptosis and shifted the ratio of proapoptotic caspase 3 versus antiapoptotic bcl-2 (5.78±1.21 versus 9.24±2.43, P<0.05). Further, in AT1−/− macrophages showed 13.2±1.2% positive uptake for apoptotic cells versus 7.9±0.9% in AT1+/+ macrophages (P<0.05, Figure 4B).
Apoptosis and efferocytosis in macrophages with intact or deficient angiotensin type 1 receptor (AT1) (AT1+/+, n=3/4; AT1−/−, n=3/4, respectively). A, Apoptotic cells (TUNEL staining). B, Apoptotic cells identified with carboxy-fluorescein diacetate succinimidyl ester (CDFA-SE)/green label. DAPI (blue) staining identifies nuclei. CFDA-SE-positive phagocytes as percentage of total macrophages. Bars in photomicrographs correspond to 100 μm.
Figure 5A illustrates clustering of apoptotic cells (both free and macrophage-associated) around anuclear (presumably necrotic) areas within an atherosclerotic lesion of apoE−/−/AT1+/+→apoE−/−+UNx. These necrotic areas (Figure 5B) were significantly larger in apoE−/−/AT1+/+→apoE−/− +UNx versus apoE−/−/AT1+/+→apoE−/− +sham (20.18±2.29 versus 9.14±2.19%, P<0.05, Figure 5B). In contrast, no such increase occurred when UNx was done in apoE−/−/AT1−/−→apoE−/− mice (7.42±1.54 versus 8.31±1.31%, P=NS, Figure 5B).
A, Representative micrograph showing nuclei (Hoechst blue), TUNEL-positive apoptotic cells (red), and macrophages (green) surrounding acellular/anuclear areas in a lesion of apolipoprotein E [apoE]−/−/ angiotensin type 1 receptor [AT1]+/+→apoE−/−+uninephrectomy (UNx). B, Quantitation of necrotic areas assessed by the ratio of acellular/anuclear areas to total atherosclerotic lesion stained by Harris hematoxylin and eosin. Mice with intact macrophage AT1 and sham surgery (apoE−/−/AT1+/+→apoE−/−+sham, n=10) or UNx (apoE−/−/AT1+/+→apoE−/−+UNx, n=15) and mice with deficient macrophage AT1 and sham surgery (apoE−/−/AT1−/−→ apoE−/−+sham, n=10) or UNx (apoE−/−/AT1−/−→apoE−/−+UNx, n=15). Bars in photomicrographs correspond to 100 μm.
Discussion
Here we report the novel observation that macrophage AT1 plays a pivotal role in the accelerated progression of atherosclerosis induced by reduction in renal mass. The underlying mechanisms are independent of BP, serum lipids, or renal function. Instead, the data indicate that macrophage AT1 skews the distribution of the macrophage population within the atherosclerotic lesion toward the proinflammatory M1 phenotype.
Although macrophages are central in initiation and progression of atherosclerosis and possess components of the renin-angiotensin system, the specific role of macrophage AII receptors in the pathogenesis of atherosclerosis has not been defined. The current study shows a key role for macrophage AT1 receptor in both development and progression of atherosclerosis in the AII-responsive setting of renal damage. The worsening of atherosclerosis caused by uninephrectomy in AT1-replete mice was completely abrogated in mice lacking the AT1 receptor in macrophages apoE−/−/AT1−/− →apoE−/−+UNx (Figure 1). This benefit was independent of BP, lipid levels, or renal function, although it is possible that systemic, but not specifically BP, effects are perturbed by UNx which in turn influence the local angiotensin activity within the plaque.37 Although macrophages are central to development of atherosclerosis, bone marrow transplantation reconstitutes more than monocyte/macrophages. Therefore, it is possible that AT1 on other leukocytes also modulates plaque development and progression. AT1 receptor is also expressed on resident vascular cells, including endothelial and smooth muscle cells, which have important roles in atherogenesis and demonstrate antiatherogenic response to systemic receptor antagonism in vivo and in cell culture.38 Thus, although both the resident vascular and infiltrating cells participate to atherogenesis, the relative contribution of AT1 on each of these cell types remains to be determined.
The current data complement previous findings of accelerated atherosclerosis in response to a sustained infusion of AII in normal mice reconstituted with AT1+/+ bone marrow, but not AT1−/−.12,39 Interestingly, previous reports show that AII infusion into mice globally deficient in AT1 reconstituted with AT1+/+ marrow amplified atherosclerosis and caused decreased collagen content, consistent with a more unstable lesion.12 Our studies also fit with the finding that hypertensive humans express high levels of dimeric AT1 in monocytes, a parameter that correlates with greater AII-dependent adhesion to endothelial cells.40,41 Increased expression of angiotensin converting enzyme in monocytes of hemodialysis patients correlates with cardiovascular events and mortality42,43 and has been specifically linked to atherosclerotic vasculopathy.44 Taken together, these observations suggest that macrophage AT1 has an important role in advancing atherosclerosis caused by dyslipidemia and renal damage.
In addition, this study also reveals a direct role of macrophage AT1 in atherogenesis, even in the absence of administration of exogenous AII or reduced renal mass. In fact, even with intact kidneys, AT1 deficient mice had less atherosclerosis compared with AT1 replete mice (Figure 1). Previous studies concerning the impact of macrophage AT1 on atherogenesis reported variable effects.10,12,39 For example, reconstitution with AT1−/− bone marrow reduced atherogenesis in apoE−/− mice,12 but it had no significant effects in LDLR−/− mice.10,39 It is possible that these differences reflect the more subtle lesions prevailing in LDLR−/− mice (versus apoE−/− mice) and the substantially shorter duration of followup (18–20 weeks in LDLR−/− mice versus 32 weeks in apoE−/− mice). The current observation reveals a significant impact of macrophage AT1 on atherogenesis over the long term (24 weeks). The findings complement our previous studies that pharmacological antagonism of angiotensin receptor at a time of established atherosclerosis (24 weeks) lessens progression and stabilizes the plaque.3 The current data also suggest the importance of the AT1 receptor at the earliest stages of atherogenesis. Bone marrow transplantation was performed at 8 weeks, a time before substantial lipid deposition is seen. Thus, it appears that AT1 receptor modulates atherogenesis throughout the atherosclerotic process. These observations underscore the potential clinical benefit of angiotensin actions antagonism at different stages of atherosclerotic development.
The current study shows that renal injury modulates the M1/M2 phenotype and that the macrophage AT1 influences the phenotypic balance in the artery wall. The proinflammatory M1 is increased by UNx in AT1-replete mice, whereas the antiinflammatory M2 phenotype is decreased (Figure 2). On the other hand, in AT1 deficient mice, UNx had a markedly minor to no effect on M1 as determined by the iNOS and the CCR7 markers, respectively (Figure 2A and B). Moreover, UNx in AT1-deficient mice showed no decrease in the M2 phenotype (Figure 2C and D). These results suggest that both UNx and macrophage AT1 determine macrophage phenotype. Renal damage modulates M1, and in the face of macrophage AT1-deficiency, UNx did not completely preclude increasing the M1 phenotype. In contrast, macrophage AT1-deficiency completely prevented UNx-induced fall in the M2 macrophages, findings that appear pivotal to abrogating advancement of atherosclerosis. Indeed, gene expression of apoptotic factors in AT1-intact and -deficient peritoneal macrophages polarized to M1 or M2 phenotype revealed that cells deficient in AT1 had significantly greater expression of antiapoptotic bcl-2. Taken together, the results support the idea that reduced atherosclerosis in mice repleted with AT1-deficient marrow reflects not only decreased population of M1 and persistence of M2 macrophages in the atherosclerotic plaque, but also resistance of AT1-deficient M2 macrophages to apoptosis. In view of the central role of NF-κB in apoptosis, and the potent effect of lipopolysaccaride to activate this signaling pathway, it is possible that resistance of AT1-deficient macrophages to apoptotic stimuli reflects variable activation in NF-κB in these cells. Interestingly, systemic antagonism of AII with ARB was recently shown in a rat model of antiglomerular basement membrane glomerulonephritis model to suppress renal M1 macrophages and increase M2 macrophages together with suppression in M1-promoting proinflammatory mediators and significant inhibition in M1 cell generation.15 Interestingly some macrophages bore both M1 and M2 markers, suggesting the possibility of local switching of macrophage phenotypes from M1 to M2 within the ARB-treated glomeruli. Our data suggests this redistribution macrophage phenotype is modulated, at least in part, by susceptibility to apoptosis; although local switching of cellular phenotype is also feasible.
Previously, we showed that systemic inhibition of AII reduces macrophage infiltration and promotes compositional changes that stabilize the plaque.3,4 Current results support a key role for macrophage AT1 in such stabilization. Whereas apoptosis was significantly increased in AT1 replete mice with UNx, lesions of AT1−/− macrophage mice showed no such increase after UNx (Figure 3A). Our studies also find that renal damage impairs macrophage efferocytosis, evidenced by the observation of increased number of apoptotic cells not associated with macrophages (Figure 3B) that can further accelerate apoptosis and necrosis in the lesions in apoE−/−/AT1+/+→apoE−/−+UNx mice, but not apoE−/−/AT1−/−→apoE−/−+UNx. Furthermore, AT1−/− macrophages were less susceptible to in vitro apoptosis and showed more efficient capacity to engulf apoptotic cells (Figure 4). Together, these results suggest that macrophage AT1 is linked to impairment of the macrophage clean-up function induced by renal damage because efferocytosis in AT1−/− macrophages is maintained with in vivo UNx and in vitro.
Increased apoptosis and impairment in macrophage efferocytosis have been shown to promote necrosis in atherosclerotic lesions in LRP-1 deficient mice27 and in mice with selective deficiency in macrophage p38α MAPK, a cellular pathway activated by AII.28,35,45 Although renal damage accelerates atherosclerotic lesions rich in necrotic areas,23 whether this involves AII regulation of macrophage p38α MAPK or other cell signaling pathways known to affect AII actions, such as Akt or small G-proteins, remains to be determined. Previously, we showed that uninephrectomy-induced activation of NF-kB activity in peritoneal macrophages was regulated by AT1.8 NF-kB is a major regulator of inflammatory and immune responses and has a pivotal role in cholesterol trafficking in macrophages, including downregulation in ABCA1 expression. This suggests that the NF-kB's effect on inflammation and lipid handling can be regulated by AT1. In addition, the LXR agonist, GW3965, increases ABCA1 expression and enhances efferocytosis,46 whereas ABCA1/ABCG1 knockout macrophages have enhanced apoptosis in vitro.47 LXR activation lessens expression of AT1.48 These observations indicate that AT1 can regulate ABC transporters through both NF-κB and LXR pathways, which predict that upregulation of ABC transporters expected in AT1-deficient cells would enhance macrophage efferocytosis. In early atherosclerosis, efferocytosis is believed to be efficient and contribute to resolution of inflammatory reactions and inhibition in plaque progression whereas impaired efferocytosis is the hallmark of advanced atherosclerosis. Such processes are consistent with previous data that the predominant macrophage phenotype in early atherosclerotic lesion is the antiinflammatory M2, which changes to the predominance of the proinflammatory M1 phenotype in advanced lesions.13 The observations are also consistent with studies showing that M2 macrophages are much more efficient at efferocytosis compared to M1 macrophages and suggest the macrophage phenotypic change is crucial both in development and acceleration of atherosclerosis.49–51
In summary, our study shows that reduction in renal mass by UNx accelerates atherosclerosis in mice reconstituted with AT1+/+ bone marrow increases M1 and decreases M2 macrophage phenotypes. On the other hand, loss of macrophage AT1 prevents UNx-induced acceleration in atherosclerosis with blunted increase of M1 and abrogated decrease of M2 macrophages. Loss of macrophage AT1 lessens renal ablation-induced apoptosis and necrosis, and restores efficient efferocytosis. Thus, by shifting the M1/M2 phenotype, AT1 on macrophages causes impaired efferocytosis and enhanced necrosis, which are pivotal in renal injury-induced acceleration of atherosclerosis.
Sources of Funding
This work was supported in part by grants NIH HL087061, DK44757, HL65709, and HL57986, and the Lipid, Lipoprotein and Atherosclerosis Core of the Vanderbilt Mouse Metabolic Phenotyping Center (NIH DK59637–01).
Disclosures
None.
Acknowledgments
The authors acknowledge the expert technical assistance of Cathy Xu and Youmin Zhang.
- Received May 26, 2011.
- Accepted September 26, 2011.
- © 2011 American Heart Association, Inc.
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