Published online before print December 23, 2008, doi: 10.1161/CIRCRESAHA.108.181131
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© 2009 American Heart Association, Inc.
Molecular Medicine |
G2A Deficiency in Mice Promotes Macrophage Activation and Atherosclerosis
From the Cardiovascular Research Center (D.T.B., M.D.S., C.C.H.), Department of Pharmacology (L.E.J., C.C.H.), Department of Microbiology (K.S.R.), Department of Molecular Physiology and Biological Physics (C.C.H.), University of Charlottesville, Charlottesville, Virginia and Cardiovascular Research Center (D.H., A.D.), University of Kentucky; and Department of Physiology (S.-C.K.), Kwandong University College of Medicine, South Korea.
Correspondence to Catherine C. Hedrick, PhD, Cardiovascular Research Center, University of Virginia, PO Box 801394, 415 Lane Rd, MR5 Rm G123, Charlottesville, VA 22908. E-mail cch6n{at}virginia.edu
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Abstract |
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G2A is a stress-inducible G protein–coupled receptor that is expressed on several cell types within atherosclerotic lesions. We demonstrated previously that G2A deficiency in mice increased aortic monocyte recruitment and increased monocyte:endothelial interactions. To investigate the impact of G2A deficiency in macrophages, we isolated peritoneal macrophages from G2A+/+ApoE–/– and G2A–/–ApoE–/– mice. G2A–/–ApoE–/– macrophages had significantly lower apoptosis than control macrophages. The prosurvival genes BCL-2, BCL-xL, and cFLIP were increased in G2A–/–ApoE–/– macrophages. Macrophages from G2A–/–ApoE–/– mice also had increased proinflammatory status that was indicative of a M1 macrophage phenotype. This was indicated by significantly increased nuclear translocation of nuclear factor
B, as well as production of interleukin-12p40, tumor necrosis factor 
, and interleukin-6, and reduced expression of arginase-I. Moreover, G2A–/–ApoE–/– macrophages had reduced ability to engulf apoptotic cells in vitro. We examined atherosclerosis in mice fed a Western diet for 10 weeks and found that G2A deficiency increased lesion size in the aortic root by 50%. Plasma lipid levels were not changed in G2A–/–ApoE–/– mice. However, we found that absence of G2A increased the number of aortic macrophages and attenuated apoptosis in this cell type. Moreover, bone marrow transplantation studies indicated that deficiency of G2A in marrow-derived cells significantly contributed to atherosclerosis development. In the absence of G2A, increased macrophage activation and decreased apoptosis is associated with accumulation of macrophages in the aorta and increased atherosclerosis.
Key Words: apoptosis • macrophages • vascular inflammation • atherosclerosis
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Introduction |
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Macrophages and T lymphocytes play critical roles in the initiation and development of atherosclerosis.1 Activated endothelium recruits monocytes by secretion of chemoattractants, after which monocytes bind to and subsequently transmigrate though the endothelial layer.2–6 In addition to macrophages, CD4+ lymphocytes are detected in early atherogenesis and late-stage unstable atherosclerotic lesions, consistent with a role for acquired immunity in lesion development.7–9 Lesional lymphocytes secrete the inflammatory cytokine interferon-
, which further activate macrophages and vascular cells.10–13 The G protein–coupled receptor G2A is a stress-inducible receptor. Overexpression in fibroblasts causes cell cycle arrest at the G2 phase of mitosis, thus the name G2A for G2 Accumulation.14 G2A expression attenuates Bcr-Abl oncogene-mediated cell proliferation, whereas mice lacking G2A have an increased mortality rate in an oncogene-induced model of leukemia.15 The endogenous ligand for G2A is unknown, although putative ligands include lysophosphatidylcholine,16,17 9(S)HODE,18 and possibly other free fatty acids.18 G2A and other receptors within the OGR1 family (TDAG8, GPR4, and OGR1) respond to changes in extracellular pH.19 However G2A is less responsive to pH changes compared to other receptor family members.20
G2A is highly expressed on macrophages and lymphocytes, with lower expression found on macrovascular endothelium.21,22 G2A has been localized to atherosclerotic lesions in mice, consistent with a contributory role in the disease process.23 We have reported that G2A deficiency increases monocyte:endothelial interactions in vivo, resulting in increased monocyte accumulation in aorta.22 In this prior study, we demonstrated a critical role of G2A in endothelium; however, the impact of G2A deficiency on the macrophage was not investigated.
In the present study, we hypothesize that G2A deficiency in macrophages would result in a proinflammatory macrophage phenotype. Our data are consistent with this hypothesis, because macrophages from G2A–/–ApoE–/– show increased cytokine secretion, nuclear factor (NF)B activation, and associated increases in survival gene expression. G2A–/–ApoE–/– mice fed a diet high in saturated fat for 10 weeks develop increased aortic root atherosclerosis compared to G2A+/+ApoE–/–. These data demonstrate that G2A deficiency results in a proinflammatory macrophage M1 phenotype that is associated with increased atherosclerosis.
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Materials and Methods |
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Detailed methods can be found in the online data supplement at http://circres.ahajournals.org. For the present studies, we fed G2A+/+ApoE–/– and G2A–/–ApoE–/– double knockout mice a Western (Harlan Teklad 88137) for 10 weeks. We measured atherosclerosis using aortic root and en face techniques. In some studies, thioglycollate-elicited peritoneal macrophages were obtained from mice for measurements of apoptosis using flow cytometry and real-time PCR and macrophage inflammatory phenotype using ELISA and real-time PCR. We also performed atherosclerosis measurements in ApoE–/– and Ldlr–/– recipient mice fed a Western diet for 10 weeks that had received ApoE–/–, G2A–/–ApoE–/–, Ldlr–/–, or G2A–/–Ldlr–/– bone marrow cells in a series of bone marrow transplantation studies.
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Results |
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Activation of NF
B in G2A-Deficient MacrophagesOn activation, NF
B is translocated to the nucleus where it acts as a transcription factor. We found that NFB p65 levels were significantly increased in macrophage nuclear extracts from G2A–/–ApoE–/– mice (Figure 1A). Phosphorylation of nuclear NFB p65, another measure of NFB activation, was also increased in G2A–/–ApoE–/– macrophages (data not shown). AKT is involved in NFB signaling,24 and AKT phosphorylation activates the BCL family of antiapoptotic factors. Peritoneal macrophages from G2A–/–ApoE–/– mice showed increased AKT phosphorylation compared to G2A+/+ ApoE–/– control (Figure 1B). Figure 1C represents densitometry of 6 mice per group.
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G2A–/–ApoE–/– Macrophages Have Reduced Apoptosis
We examined expression of genes involved in inflammation and apoptosis. Several survival gene targets of NFB were upregulated in G2A–/–ApoE–/– macrophages compared to control, including BCL-2, BCL-xL, and cFLIP (Figure 2A). In addition, IAP2 was increased 1.8-fold, and expression of p73 and p53, 2 proapoptotic genes, was reduced 2.7-fold (Figure 2A). Interestingly though, we observed significant upregulation of several other proapoptotic genes, including FasL, PAK7, and caspase-12 (Figure 2A), suggesting that G2A expression in the macrophage must regulate apoptotic/survival pathways, and in the absence of G2A, these pathways become dysregulated.
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To examine whether the macrophages exhibited a functionally proapoptotic or prosurvival phenotype, we performed several assays. First, we measured Annexin V staining on freshly isolated peritoneal macrophages from G2A–/– ApoE–/– and G2A+/+ApoE–/– mice using flow cytometry (Figure 2B). G2A–/–ApoE–/– macrophages showed significantly less Annexin V staining, as measured by flow cytometry (Figure 2B). Alternatively, peritoneal macrophages were plated overnight on chamber slides and stained for TUNEL or cleaved caspase-3. G2A–/–ApoE–/– macrophages showed significantly less TUNEL staining, (P<0.001; Figure 2C), and less cleaved caspase-3 than G2A+/+ApoE–/– (Figure 2C). Taken together, these results indicate that G2A deficiency in macrophages activates prosurvival signaling pathways thereby preventing apoptosis, even though several proapoptotic pathways are activated in these macrophages.
G2A–/–ApoE–/– Macrophages Are Proinflammatory and Have Impaired Apoptotic Cell Engulfment
We next examined the inflammatory phenotype of G2A–/– ApoE–/– and G2A+/+ApoE–/– macrophages. IL-12p70, tumor necrosis factor (TNF), monocyte chemoattractant protein (MCP)-1, IL-10, and IL-6 secretion into culture media were all significantly higher in G2A–/–ApoE–/– macrophages compared to control (Figure 3A). Interferon- levels were unchanged. Additionally, quantitative real-time RT-PCR was performed on RNA isolated from peritoneal macrophages from G2A–/–ApoE–/– and G2A+/+ ApoE–/– mice, and we observed similar inductions of cytokine mRNA expression (Figure 3B). In addition, we found significant reductions in Arginase-I and transforming growth factor β expression in G2A–/–ApoE–/– macrophages (Figure 3B), both of which have been linked to a M2 alternative, antiinflammatory macrophage phenotype.25 We also found similar changes in gene expression in macrophages isolated from whole aorta (data not shown). Taken together, these data suggest that G2A deficiency promotes an inflammatory M1-like macrophage phenotype.
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Next, we measured the ability of the G2A–/–ApoE–/– macrophages to engulf apoptotic cells. Efficient clearance of apoptotic cells by phagocytic macrophages is associated with promotion of an antiinflammatory, M2-like phenotype. As shown in Figure 3C, G2A–/–ApoE–/– macrophages showed reduced ability to engulf apoptotic cells in vitro. Because defective apoptotic cell clearance from the vessel wall is associated with increased atherosclerosis,26,27 these data would suggest that G2A deficiency in macrophages would contribute to atherosclerosis development.
Apoptosis and Antiapoptotic Gene Expression After Treatment With Oxidized Low-Density Lipoprotein
Peritoneal macrophages from 6 each of G2A+/+ApoE–/– and G2A–/–ApoE–/– mice fed chow were isolated and stimulated with the treatment of with oxidized low-density lipoprotein (LDL) (50 µg/mL) for 18 hours. After treatment, cells were collected for measurement of apoptosis and survival gene expression. Apoptosis, as measured by Annexin V staining, was significantly reduced in G2A–/–ApoE–/– macrophages compared to G2A+/+ApoE–/– control (P<0.002). Treatment with oxLDL increased apoptosis in G2A+/+ ApoE–/– by 50% (P<0.01) but did not significantly increase apoptosis in G2A–/–ApoE–/– macrophages. Figure 4A shows representative dot plots from each group. The survival genes BCL-2, BCL-xL, and cFLIP, as well as inducible NOS expression, were all significantly increased in G2A–/– ApoE–/– macrophages compared to control (P<0.001), whereas treatment with oxLDL further increased survival gene and inducible NOS expression in G2A–/–ApoE–/– macrophages (P<0.002) (Figure 4B). Treatment of G2A+/+ApoE–/– macrophages with oxLDL reduced expression of the survival genes BCL-2, BCL-xL, and cFLIP (P<0.005). Arginase I expression was significantly reduced in untreated G2A–/– ApoE–/– compared to G2A+/+ApoE–/– (P<0.02) but was restored to control levels on stimulation with oxLDL (Figure 4B).
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Atherosclerosis Lesion Analysis and Characterization
We next examined atherosclerosis development in G2A-deficient mice after 10 weeks of Western diet feeding. We found that fasting total cholesterol concentrations were elevated in both groups. There were no significant differences between G2A+/+ApoE–/– and G2A–/–ApoE–/– mice in blood glucose levels (306±36.2 versus 282.7±48.1 mg/dL), total cholesterol (1084±107.4 versus 972.5±161.5 mg/dL), high-density lipoprotein (HDL) (16.1±2.1 versus 16.4±3.1 mg/dL), LDL (1052±124.9 versus 943.3±157 mg/dL), or triglycerides (128.7±28.6 versus 132.4±28.9 mg/dL), respectively. Body weight at the end of the experiment also did not differ between groups (22.2 g±2.8 g for G2A+/+ApoE–/– vs 21.6±3.6 g for G2A–/–ApoE–/–).
G2A deficiency did lead to significantly increased concentrations of MCP-1 and IL-6 in plasma, however (P<0.005; Figure 5). Plasma concentrations of interferon-, IL-12p70, IL-10, and TNF were below the detection sensitivity of the assay in all groups (data not shown).
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Aortic root sections from G2A+/+ApoE–/– and G2A–/– ApoE–/– mice were collected, and lesion area was measured in both the sinus and the ascending aortas as indicated in Figure 6A, with the "0" point indicating the transition between these areas. After 10 weeks of Western diet feeding, G2A–/–ApoE–/– mice showed increased atherosclerosis development within –240 to +240 µm from the transition (P<0.001; Figure 6A). Movat pentachrome and picrosirius red staining of 5-µm sections from aortic roots (taken at +160 µm from the transition) demonstrated that G2A deficiency led to greater collagen content in the aortic root, suggesting formation of complex atherosclerotic plaques (Figure 6B). Using Movat stain, collagen content is apparent by dark purple to black coloration.28 Using picrosirius red staining, collagen content of the lesion is visible by red staining under normal light. Under polarized light, red, orange, yellow, and green colors are apparent (the colors of collagen fibers in order of decreasing thickness) (Figure 6C).29
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Absence of G2A in Marrow-Derived Cells Contributes to Early Atherosclerosis Development
Bone marrow transplantation studies were performed in ApoE–/– recipients using ApoE–/– and G2A–/–ApoE–/– bone marrow and in Ldlr–/– recipients using Ldlr–/– and G2A–/–Ldlr–/– bone marrow. After 6 weeks of reconstitution, mice were placed on a Western diet for 10 weeks. Atherosclerosis was measured using en face analysis. First, as expected, the percentage plaque area was greater in ApoE–/– recipients receiving apoE–/– marrow compared to Ldlr–/– recipients receiving Ldlr–/– marrow (Figure 7). In the apoE–/– recipients, we observed a trend toward increased plaque area in the mice that received G2A–/–ApoE–/– bone marrow, but the data did not reach statistical significance. However, in the Ldlr–/– recipients, absence of G2A in marrow-derived cells significantly increased aortic plaque area, P<0.003 (Figure 7). Taken together, these data suggest that G2A deficiency in macrophages contributes significantly to atherosclerosis development yet does not rule out some contribution of G2A deficiency in non–marrow-derived cells.
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Higher Macrophage Content in Aortic Wall of G2A–/– Mice
Finally, we examined macrophage numbers in the aortic wall using a novel flow cytometric method developed by Ley and colleagues.30 Macrophage content in vivo in the aortic wall of G2A–/–ApoE–/– mice was significantly higher than that of ApoE–/– control (P<0.002), as measured by dual staining for CD45 and F4/80 (Figure 8A). However, total monocyte and lymphocyte counts in blood were similar between the 2 experimental groups (data not shown). Thus, the increase in aortic wall macrophage accumulation does not occur as a result of increased leukocyte numbers in blood of the G2A–/– ApoE–/– mice. We anticipate that the increased aortic wall content is attributable to increased monocyte:endothelial cell interactions in the arterial wall in the absence of G2A, which we have previously reported,22 as well as increased macrophage survival. Indeed, G2A–/–ApoE–/– aortic root cross-sections, whereas having a greater plaque area and macrophage content had significantly less TUNEL+/DAPI+ costaining (Figure 8B). The regions of positive TUNEL staining in G2A–/–ApoE–/– lesions showed very little costaining for either DAPI nuclei or MAC-2 macrophage, indicating these areas are necrotic. We found an 
5% increase in the percentage necrotic core/total plaque area ratio in the aortic roots of G2A–/–ApoE–/– mice compared to G2A+/+ApoE–/– mice. Aortic root sections from ApoE–/– mice had more macrophage staining, as measured by MAC-2+ staining, as well as greater TUNEL+/DAPI+ staining. These data combined with our in vitro data on macrophage function suggest that the reduced macrophage apoptosis observed in G2A–/–ApoE–/– mice contributes to aortic macrophage accumulation and atherosclerosis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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G2A is a G protein–coupled receptor that is highly expressed in macrophages and lymphocytes.31 The endogenous ligand for G2A remains unclear16,18,32,33; however, there is some evidence that G2A is involved in atherosclerotic lesion development in animal models.21,23 We have recently found that absence of G2A in mice contributes to cholesterol gallstone disease,34 although G2A has not, to date, been defined as a Lith gene. The gene in humans that corresponds to G2A is GPR132. Currently, there are no known reported associations of polymorphisms in GPR132 with clinical disease in humans. Recently, however, we have discovered single-nucleotide polymorphisms within GPR132 that are associated with internal intimal media thickness of the carotid artery in patients (data not shown). Thus, GPR132 (G2A) may indeed represent a clinically relevant gene for lipid metabolism and atherosclerosis.
In the present study, we investigated the impact of G2A deficiency on macrophage function and atherosclerosis in ApoE–/– mice. G2A–/–ApoE–/– mice fed a fat-enriched diet for 10 weeks had significantly greater aortic sinus lesion area compared to G2A+/+ApoE–/– mice. G2A–/–ApoE–/– mice additionally had significantly higher numbers of macrophages present in the aortic wall compared to G2A+/+ ApoE–/–. Although there was no difference in circulating lipid levels, G2A–/–ApoE–/– mice had significantly greater plasma IL-6 and MCP-1 levels. Moreover, macrophages isolated from these mice had increased production of proinflammatory cytokines and reduced expression of antiinflammatory genes. These data suggest that G2A deficiency promotes a proinflammatory M1 macrophage phenotype that contributes to atherosclerotic lesion development.
In related studies of G2A function, Parks et al examined G2A-deficient mice on a LDLR–/– background and noted increased macrophage content and decreased macrophage apoptosis, with no effect on atherosclerotic lesion size after either 6 or 12 weeks of Western diet feeding.21 A second study from the same group demonstrated that G2A deficiency decreased atherosclerosis in G2A–/–Ldlr–/– mice at later times points of diet feeding.35 In the second study, these investigators reported significant elevations in plasma HDL levels in the G2A–/–Ldlr–/– mice when fed a Western diet for both 9 and 20 weeks. They observed a significant decrease in atherosclerosis in G2A–/–Ldlr–/– mice at both time points of feeding that could possibly be attributed to increased plasma HDL concentrations. Our studies were performed in ApoE–/– mice for 10 weeks, and we did not observe changes in HDL. Moreover, we performed bone marrow transplantation studies in both genetic backgrounds and found that deficiency of G2A in bone marrow–derived cells significantly contributed to atherosclerosis (Figure 7). In the apoE–/– background, this trend did not reach statistical significance, most likely because of the small number of animals available for study. However, the effect was quite dramatic in the Ldlr–/– mice, and our findings are opposite to those of Parks and colleagues.21,35 HDL levels were similar among groups in our bone marrow transplant studies (data not shown), which is different from the studies by Parks and colleagues. However, taken together, the collective results of our studies and those of Parks et al suggest that G2A expression in multiple cell types influences atherosclerosis. Indeed, absence of G2A in hepatocytes, lymphocytes, and endothelium clearly influences inflammatory and immune processes related to atherosclerosis.22,41,34 Development of floxed mice for cell-specific studies of G2A deficiency is needed to dissect the important contributions of G2A in each cell type on atherosclerosis.
G2A expression has been shown to influence apoptosis in leukocytes.16,21,36 Recent studies suggest an important role of apoptosis in atherosclerotic plaque formation.37,38 Our previous study demonstrated that G2A deficiency resulted in increased NFB activation in murine aortic endothelial cells.22 Because NFB activation targets survival gene expression resulting in decreased apoptosis,39 we investigated whether this was relevant in macrophages in the present study. Indeed, G2A–/–ApoE–/– macrophages showed significantly higher levels of NFB p65 expression in the nucleus that corresponded with increased survival gene expression. Expression of NFB-controlled genes that are important for cell survival, including bcl-2, bcl-xL, TRAF, and cFLIP was elevated in macrophages from G2A–/– mice. These G2A-deficient macrophages also showed significantly lower TUNEL, cleaved caspase-3, and Annexin V staining. Furthermore, we observed downregulation of the proapoptotic genes, p53 and p73, which have been shown to be downregulated by NFB.40 Concomitantly with the increase in prosurvival genes, we observed increased numbers of macrophages in the aortic wall of G2A–/– mice in vivo, suggesting that G2A deficiency causes macrophage accumulation in the aortic wall through promoting macrophage survival. We speculate from our data that NFB activation is the primary regulator of macrophage survival in the absence of G2A. Because we have observed activation of NFB in endothelium as well, we anticipate that G2A expression somehow serves to inhibit NFB. The mechanisms for this are unknown but are presently being studied in the laboratory. In preliminary studies, we have not observed significant changes in IB expression (data not shown), but we have observed increased AKT activity in the G2A–/–ApoE–/– macrophages, which has been shown to increase NFB.41 However, we cannot rule out contributions of other survival pathway genes in regulating apoptosis in the G2A–/– macrophages. Moreover, we found upregulation of a few proapoptotic genes, including FasL, and caspase-12 in G2A–/– macrophages, which suggest that the macrophages in G2A–/– mice have become dysregulated, most likely impacting their inflammatory phenotype as well. Indeed, caspase-12 is induced by endoplasmic reticulum stress, which often occurs as a result of free cholesterol loading in macrophages.42 Endoplasmic reticulum stress can contribute to a proinflammatory macrophage phenotype.38
Under normal conditions, macrophages act to maintain homeostasis in the aortic wall. Classically activated (M1-type) macrophages exhibit strong microbicidal properties, thereby promoting IL-12 and TNF-mediated Th1 responses.43 In contrast, alternatively activated (M2-type) macrophages secrete antiinflammatory cytokines such as transforming growth factor β, ingest and clear cell debris, and are rapidly cleared from the wall, thereby contributing to the resolution of inflammation. In normal tissue and during early atherosclerosis, M2-type macrophages help to stabilize the environment of the vessel wall by promoting effective efferocytosis of dying cells.44 Chronic activation of M1-like macrophages promotes an unstable vessel environment, reducing efferocytosis and triggering secondary necrosis of vascular wall cells, thereby contributing to the advanced atherosclerotic plaque formation.38 G2A has recently been identified as a phagocyte receptor on macrophages, in which G2A recognizes "find me" signals such as lysophosphatidylcholine metabolites that are secreted by dying cells. The increased collagen content observed in aortic roots of G2A-deficient mice (Figure 6) suggests that there is increased secondary necrosis of vascular wall cells that contribute to plaque complexity. This finding is consistent with the notion of Peter et al that G2A may serve as a phagocytic receptor.45 Thus, the absence of G2A on macrophages may contribute to atherosclerosis through impairing efferocytosis pathways, in which various find me signals secreted by dying cells are no longer recognized by the macrophage in the absence of G2A. We did find reductions in apoptotic cell engulfment in G2A–/– macrophages in vitro, supporting this hypothesis (Figure 3). Moreover, defective apoptotic cell clearance has been linked to atherosclerosis in mice.26 Future studies to determine the specific roles of the G2A receptor in apoptotic cell clearance and its subsequent impact on atherosclerosis will be needed to fully address this hypothesis.
In conclusion, we demonstrate that G2A deficiency caused increased atherosclerosis in the aortic sinus of ApoE–/– mice fed a Western diet. We propose this is attributable to increased numbers of lesional macrophages and decreased macrophage apoptosis in the aortic wall. These findings demonstrate that G2A deficiency triggers an abnormal inflammatory macrophage phenotype that contributes to atherosclerosis development, suggesting a critical role of G2A in macrophage homeostasis.
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After submission of this manuscript, Frasch et al reported that antibody blockade of the G2A receptor reduces apoptotic cell uptake by macrophages, further confirming our engulfment data in G2A-deficient macrophages.
Frasch SC, Berry KZ, Fernandez-Boyanapalli R, Jin HS, Leslie C, Henson PM, Murphy RC, Bratton DL. NADPH oxidase-dependent generation of lysophosphatidylserine enhances clearance of activated and dying neutrophils via G2A. J Biol Chem. 2008;28283:33736–33749.
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Acknowledgments |
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We thank Nathan Linden for assistance with real-time RT-PCR, and Angela Whetzel for helpful technical assistance. We thank Dr Owen Witte (University of California, Los Angeles) for the gift of the G2A–/– mice.
Sources of Funding
These studies were funded by NIH R01 HL071141 (to C.C.H.).
Disclosures
None.
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Footnotes |
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Original received June 9, 2008; revision received November 6, 2008; accepted December 11, 2008.
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References |
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