Published online before print August 14, 2006, doi: 10.1161/CIRCULATIONAHA.105.602359
(Circulation. 2006;114:807-819.)
© 2006 American Heart Association, Inc.
Molecular Cardiology |
Oxidized Lipid-Driven Chemokine Receptor Switch, CCR2 to CX3CR1, Mediates Adhesion of Human Macrophages to Coronary Artery Smooth Muscle Cells Through a Peroxisome Proliferator-Activated Receptor 
–Dependent Pathway
From the Molecular Signaling Section (J.B., Y.Z., P.M.M.) and Inflammation Biology Section (J.F.F.), Laboratory of Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md.
Correspondence to Philip M. Murphy, MD, 9000 Rockville Pike, Bldg 10, Room 11N113, NIH, Bethesda, MD 20892. E-mail pmm{at}nih.gov
Received November 18, 2005; revision received June 14, 2006; accepted June 19, 2006.
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Abstract |
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Background— Recent genetic data in mouse and humans suggest that the chemokine receptors CCR2 and CX3CR1 are involved in atherogenesis; however, detailed molecular and cellular mechanisms have not been fully delineated.
Methods and Results— Here, we show that oxidized linoleic acid metabolites, which are components of oxidized LDL found in large amounts in atherosclerotic plaque, were able to specifically induce differentiation of human monocytes to macrophages with decreased expression of CCR2, confirming a previous report, and increased expression of CX3CR1. These macrophages acquired the ability to adhere to coronary artery smooth muscle cells. The adhesion was mediated directly and predominantly by CX3CR1. Reciprocal effects of these lipids on CCR2 and CX3CR1 expression were mediated by the nuclear receptor peroxisome proliferator-activated receptor (PPAR) 
, and targeting the PPAR gene with sRNAi dramatically reduced macrophage adhesion to coronary artery smooth muscle cells.
Conclusions— These data suggest that in atherogenesis oxidized lipid-driven activation of macrophage PPAR in the intima may result in a proadhesive chemokine receptor switch–CCR2 off, CX3CR1 on–causing cessation of CCR2-dependent migration and activation of CX3CR1-dependent retention mechanisms, which together promote macrophage accumulation in vessel wall. Our results may explain at the molecular and cell biology levels the genetic link between CX3CR1 and atherosclerosis. Moreover, they identify macrophage binding to coronary artery smooth muscle cells as the first primary cell setting in which CX3CR1 functions as the major adhesion system.
Key Words: atherosclerosis • lipids • leukocytes • receptors • signal transduction
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Introduction |
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Atherosclerosis, a chronic inflammatory disease in the conductive arteries, is the leading cause of mortality in the Western world. Mechanisms of inflammation in atherosclerosis are poorly understood but are thought to involve coordinate action of platelets, leukocytes, oxidized lipoproteins, cytokines, and chemokines, among other factors. The monocyte-derived macrophage, the predominant leukocyte subtype found in atherosclerotic lesions, is localized in the intima.1 Mechanisms responsible for macrophage recruitment to and retention in the vessel wall are poorly understood; however, genetic data in humans and/or mouse have implicated the macrophage chemokine receptors CXCR2, CCR2, and CX3CR1 and their respective ligands CXCL8, CCL2, and CX3CL1, which may be induced in resident vascular cells by oxidized LDL and other factors.2–6
CX3CR1 is particularly noteworthy because both cx3cr1+/– and cx3cr1–/– mice have been reported to have reduced susceptibility to atherosclerosis4,6 and because human subjects heterozygous for the CX3CR1 M280 allele, which encodes a defective receptor, have reduced risk of atherosclerotic cardiovascular disease.7 CX3CR1 is an unusual chemokine receptor because, in addition to mediating leukocyte migration toward soluble CX3CL1, it is able to mediate direct adhesion of leukocytes to endothelial cells expressing a tethered form of CX3CL1 under both static and physiological flow conditions.8,9 These dual functions of CX3CR1 are due in part to the unusual structure of CX3CL1, a cleavable type I transmembrane protein with a chemokine domain extended from the cell surface on a mucin-like stalk.10–12
Clinical Perspective p 819
Ideas for how CX3CL1 and CX3CR1 might modulate risk of atherosclerosis at the molecular and cellular level have been confounded by the lack of clear evidence for CX3CL1 expression on coronary artery endothelial cells. Instead, CX3CL1 has been localized to macrophages and coronary artery smooth muscle cells (CASMCs) in human atherosclerotic vessels; no expression was detected in normal vessels.13,14 This suggests that accumulation and retention of monocytes/macrophages in plaque may occur by a CX3CR1/CASMC-dependent mechanism. Consistent with this, electron microscopy of early and advanced atherosclerotic lesions have shown that CASMCs are in contact with foamy macrophages,15 and CX3CL1 and CX3CR1 have been shown to colocalize in plaque.14 Here, we test this hypothesis in vitro using primary CASMCs and human peripheral blood monocyte-derived macrophages. To place this in a more relevant context, macrophages were first differentiated from monocytes by stimulation with 9-hydroxy-10E,12Z-octadecadienoic acid ester (9-HODE) and 13-hydroxy-9Z,11E-octadecadienoic acid ester (13-HODE), the 2 major oxidized linoleic acid metabolite components of oxidized LDL.16–19 These lipids are found at high concentrations in human atherosclerotic plaque16–19 and function as potent macrophage differentiation factors and potent and selective agonists for the transcription factor peroxisome proliferator-activated receptor (PPAR) .20,21 They have also been reported to downregulate monocyte/macrophage CCR2.22 Here, we show that they at the same time markedly upregulate the frequency of CX3CR1+ macrophages in a PPAR-dependent manner and promote macrophage-CASMC adhesion in a PPAR- and CX3CR1-dependent manner.
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Methods |
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Materials
LDL and oxidized LDL were purchased from Intracel (Frederick, Md). 9-HODE and 13-HODE, arachidonic acid–containing phospholipids PGPC (1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine) and POV-PC [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine], CAY 10410, and PPAR
-PAK (pioglitazone, rosiglitazone, troglitazone, and GW 9662) were from Cayman Chemical (Ann Arbor, Mich). Monoclonal antibodies (mAbs) included anti-CX3CR1 (MBL, Woburn, Mass); anti-CD36, anti-CD16, and anti-CD14 (BD Biosciences, San Diego, Calif); anti-HAM56 (Enzo, Farmingdale, NY); and anti-CCR2 (R&D, Minneapolis, Minn). Isotype-matched mAbs were from BD Biosciences. Anti-PPAR, anti-CX3CL1 rabbit polyclonal antiserum, and rabbit IgG were from Abcam (Cambridge, Mass). Human tumor necrosis factor-
, interferon-, and interleukin-1ß ELISAs were from R&D. Real-time polymerase chain reaction (PCR) reagents, including validated FAM-tagged human CX3CR1, CCR2, and CD36 primers and the GAPDH/JOE primer/probe set, were from Applied Biosystems (Foster City, Calif). Primary human CASMCs were from Cambrex (Rockville, Md). CASMC donors died as a result of suicide or accident and lacked history or pathological evidence of cardiovascular disease. RPMI 1640 and Vybrant cell adhesion assay kit were from Invitrogen (Carlsbad, Calif).
Cell Culture
Monocytes elutriated from peripheral blood of healthy donors were provided by the NIH Department of Transfusion Medicine. Cells were plated at 2.5x106 cells per well in a 6-well dish containing RPMI 1640 plus 20 vol% autologous serum, incubated at 37°C for 1 hour, and then stimulated with lipids for 24 hours.22 In experiments using peripheral blood mononuclear cells, 10 µg/mL of 9-HODE and 13-HODE induced maximal CX3CR1 upregulation and was the IC50 for inhibition of CCR2 expression, and therefore was used for macrophage experiments. After a 24-hour lipid stimulation, macrophage recovery was &88% and death was &8% by trypan blue exclusion. CASMCs were cultured in proprietary media with recommended supplements (Cambrex). Cells were passaged twice and grown to 90% confluence before analysis.
Flow Cytometry
For kinetic analysis, cells (106) were fixed (Cytofix buffer, BD Biosciences) and then stained with mAbs. Otherwise, cells were stained without fixation. Cells were stained with antibodies at 4°C for 30 minutes in labeling buffer (Hanks’ balanced salt solution with 0.1% BSA and 0.1% sodium azide) containing anti-Fc reagent (Miltenyi, Auburn, Calif). Flow cytometry was performed in duplicate with FacsCalibur and then analyzed with CellQuest (Becton-Dickinson, San Jose, Calif), correcting for nonspecific staining with isotype antibody controls.
mRNA Quantification
RNA was extracted by RNeasy (Qiagen, Valencia, Calif) and reverse transcribed with RETROscript (Ambion, Austin, Tex). cDNA was serially diluted and amplified in triplicate for standard curves for each primer/probe set. Relative target quantification was calculated with the 2–
CT method23 and normalized to GAPDH.
Chromatin Immunoprecipitation
Monocytes (5x106) fixed in 37% formaldehyde were homogenized in cold lysis buffer containing protease inhibitors from the Chromatin Immunoprecipitation kit (Active Motif, Carlsbad, Calif). DNA was sheared by sonication, and precleared chromatin ("input DNA," 500 ng) was immunoprecipitated with 5 µg anti-PPAR, control rabbit IgG, or polyclonal rabbit anti-CX3CR1. Chromatin was amplified using the following CX3CR1 promoter primer pairs (named by the 5'-most nucleotide relative to nucleotide 1 of codon 1): –13537 to –13764, –13760 to –13981, –13980 to –14243, –14189 to –14437, –14440 to –14565, and –14525 to –14690 (NCBI, GenBank accession number AY016370). PCR conditions were as follows: 95°C for 3 minutes, then 30 cycles of 20 seconds at 94°C, 30 seconds at 60°C, and 30 seconds at 72°C. Plateau was 38 cycles for immunoprecipitated DNA and 35 cycles for input DNA for each primer pair.
PPAR Knockdown
The PPAR Validated Stealth RNAi Duo Pack was from Invitrogen. PPAR-specific sRNAi were as follows: 5'-GCUUAUCUAUGAC-AGAUGUGAUCUU-3' (PPAR 1) and 5'-GCUUCAUGACAAG-GGAGUUUCUAAA-3' (PPAR 2). Control sRNAi had minimal sequence homology to any vertebrate transcript, and GC content matched the silencing sRNAi. Fluorescein-labeled dsRNA oligomer, used to assess transfection efficiency, had the same length, charge, and configuration as the sRNAi. Then, 5x106 monocytes were nucleofected (Amaxa, Cologne, Germany) with 100 nmol/L fluorescein-labeled dsRNA or with 100, 150, or 200 nmol/L negative control or PPAR-specific sRNAi, resuspended in 2 mL of RPMI 1640 prewarmed to 37°C and supplemented with 20 vol% of autologous serum, and then cultured with or without lipids for 24 hours.
Adhesion Assay
sRNAi-transfected or control monocytes (5x106/mL) were cultured with or without lipids for 24 hours, washed with prewarmed RPMI 1640, and loaded for 30 minutes with 5 µmol/L calcein AM at 37°C. Cells were resuspended at 0.5x106/100 µL and then incubated with CASMCs at 37°C for 60 minutes. Nonadherent cells were removed by washing 4 times, and end-point fluorescence (in units per milliliter) was measured with a fluorescein filter set (absorbance, 494 nm; emission, 517 nm) on a FlexStation (Molecular Devices, Sunnyvale, Calif). Autofluorescence was subtracted from peak fluorescence of each well.
Statistical Analysis
All conditions were performed in duplicate or triplicate, and each experiment was performed in 3 to 5 different monocyte and CASMC donors. Values for each condition were averaged, and data are presented as mean±SD in the figures. The 95% CIs are given in the text. The statistical significance of differences among matched groups was tested by the nonparametric Friedman 2-way ANOVA by ranks, followed by Dunn’s posttest, using the GraphPad Prism 3.0 program (GraphPad Software, San Diego, Calif). Values of P<0.05 were considered statistically significant.
The authors had full access to the data and take full responsibility for their integrity. All authors have read and agree to the manuscript as written.
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Results |
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Oxidized Linoleic Acid Metabolites Differentiate Monocytes to CX3CR1+ Macrophages
CD14+CD16– cells represented 78% (95% CI, 72 to 84) of total freshly elutriated monocytes (data not shown) and were 40% CCR2+ (95% CI, 29 to 47) and 70% CX3CR1+ (95% CI, 64 to 72) (Figure 1), confirming previous reports.24 CD14+CD16+ cells, which are increased in patients with hypercholesterolemia or coronary artery disease,25 represented only 8% (95% CI, 4.6 to 11.2) of freshly elutriated monocytes (data not shown), confirming previous reports,26 and were 3% CCR2+ (95% CI, 1.5 to 5.5) and 8% CX3CR1+ (95% CI, 5.7 to 11.7) (Figure 1). As previously reported,26,27 without lipid stimulation, CCR2+ monocytes decreased in a time-dependent manner to 20% (95% CI, 16 to 24) by 24 hours (Figure 1A). The frequency of CX3CR1+ monocytes also decreased to 20% (95% CI, 15.5 to 26) by 24 hours (Figure 1B). Compared with these benchmarks, after 24 hours in culture in the presence of 9-HODE or 13-HODE, the frequency of CCR2+ cells decreased by 65% to 4.7% (95% CI, 0.5 to 9) and 4.5% (95% CI, 2.7 to 6.2) respectively, consistent with previous reports,22 whereas the frequency of CX3CR1+ cells markedly increased under these same conditions (Figure 2A and 2
B). In contrast, monocyte treatment for 24 hours with 10 µg/mL PGPC or POV-PC, which are bioactive oxidized arachidonic acid derivatives of LDL found in plaque,28 had no effect on the expression of either receptor (Figure 2A and 2B). The same CCR2/CX3CR1 receptor switch also occurred when cells were incubated with oxidized LDL. In contrast, unmodified LDL slightly upregulated CCR2 surface expression, as reported previously,29 although in our study this increase did not reach statistical significance. Unmodified LDL had no effect on the frequency of CX3CR1+ cells (Figure I in the online-only Data Supplement).
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9-HODE and 13-HODE stimulated monocyte maturation because the oxidized LDL/scavenger receptor CD36 and the macrophage marker HAM56 were both specifically upregulated (Figure 2C through 2
F). The lipid-driven CCR2/CX3CR1 receptor switch occurred in the presence of endogenous tumor necrosis factor-, interferon-, and interleukin-1ß, which are present in human atherosclerotic lesions1 and are induced in monocytes by these lipids (Data Supplement Figure II).30,31 9-HODE and 13-HODE effects on CCR2, CX3CR1, and CD36 surface expression were consistent with effects on the corresponding steady-state mRNA levels (Figure 2G).
Oxidized Linoleic Acid Metabolites Enhance Adhesion of Macrophages to CASMCs
CX3CL1 and CX3CR1 were constitutively expressed on CASMCs cultured in vitro (Figure 3A). Unstimulated monocytes were poorly adherent to CASMCs under static conditions (Figure 3B). In contrast, 9-HODE or 13-HODE treatment of monocytes markedly increased their adhesion to CASMCs 5.2- and 5.1-fold, respectively. Preincubation of stimulated monocytes with saturating concentrations of soluble CX3CL1 or anti-CX3CR1 mAb specifically reduced adhesion by 75% to 80% (Figure 3B).
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Linoleic Acid Metabolites Induce CX3CR1 Expression in a PPAR-Dependent Manner: Pharmacological Analysis
9-HODE and 13-HODE are endogenous ligands and activators of PPAR.32 The synthetic PPAR agonist CAY 10410 oppositely regulated the frequency of CX3CR1+ and CCR2+ cells (Figure 4A), increasing the former by 2.5-fold to 44.8% (95% CI, 34 to 55) and decreasing the latter by 4.5-fold to 3.8% (95% CI, 1.2 to 6.4) at 100 µmol/L. CAY 10410 also increased the frequency of CD36+CX3CR1+ and HAM56+CX3CR1+ macrophages (Figure 4B and 4C) and decreased CCR2 mRNA while increasing accumulation of CX3CR1 and CD36 mRNA (Figure 4D). PPAR agonistic glitazones (pioglitazone, rosiglitazone, and troglitazone) at submicromolar concentrations also upregulated CX3CR1 and downregulated CCR2 in this system (Data Supplement Figure III).
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The synthetic PPAR antagonist GW 9662 had no effect on basal monocyte CX3CR1, CCR2, HAM56, and CD36 expression but blocked lipid-driven inhibition of CCR2 and induction of CX3CR1, CD36, and HAM56 in a dose-dependent manner both on the surface (Figure 5A through 5C) and at the RNA level (Figure 5D). These data suggest that oxidized LDL metabolites both downregulate CCR2 expression and upregulate CX3CR1 expression by a mechanism that requires PPAR activation.
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PPAR Interacts With the CX3CR1 Promoter in Intact Human Macrophages
To test directly whether PPAR interacts with endogenous CX3CR1, we first used the TRANSFAC version 4.0 transcription factor database to screen 1278 bp of CX3CR1 upstream of the major transcriptional start point, a region shown to contain promoter activity,33,34 for the presence of putative PPAR response elements (PPREs). Three PPRE consensus sites were identified at positions –904, –994, and –1054 relative to the transcriptional start point (Data Supplement Figure IV).
Stimulation of monocytes with either 9-HODE or 13-HODE resulted in binding of PPAR to portions of the CX3CR1 promoter containing the –904 and –994 PPREs but not the –1054 PPRE, as determined by chromatin immunoprecipitation (ChIP) analysis (Figure 6A). PPAR did not associate with these target genomic regions in unstimulated cells. After stimulation with 9-HODE and 13-HODE, PPAR was recruited to genomic DNA fragments containing both the –904 (Figure 6B) and –994 (Figure 6C) PPREs in a time-dependent fashion, with maximum complex formation 12 hours after stimulation. The absence of PPAR from the target DNA fragments in unstimulated monocytes (Figure 6A) is consistent with the lack of an effect of PPAR antagonist GW 9662 on constitutive CX3CR1 expression in unstimulated monocytes (Figure 5).
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Consistent with this, in monocytes cultured with 100 µmol/L CAY 10410 (PPAR agonist), PPAR bound selectively to genomic DNA fragments containing both the –904 and –994 PPREs (Figure 6D and 6E). Coimmunoprecipitation of PPAR with the 248- and 123-bp target sequences containing these PPREs occurred at equal levels in monocytes stimulated with 9-HODE and 13-HODE and in monocytes treated with CAY 10410 (Figure 6E). Consistent with the inhibitory effect of GW 9662 on CX3CR1 expression in monocytes stimulated with 9-HODE and 13-HODE, this PPAR antagonist inhibited PPAR binding to genomic fragments containing the –904 and –994 PPREs. The amounts of target DNA sequences in the PPAR coimmunoprecipitates from monocytes stimulated with 9-HODE or 13-HODE decreased on treatment with GW 9662 (Figures 6D and 6E). The 248- and 123-bp target CX3CR1 genomic fragments failed to coimmunoprecipitate with PPAR in unstimulated cells treated with GW 9662 (Figure 6E). It is important to note that although PPAR activity is necessary for oxidized LDL metabolite-induced upregulation of CX3CR1, it does not appear to support constitutive expression of CX3CR1 in monocytes. Thus, our data obtained by ChIP analysis are consistent with oxidized LDL metabolite-dependent regulation of CX3CR1 through effects on PPAR; however, further work is required to test whether the specific consensus PPRE sites on the 248- and 123-bp target CX3CR1 genomic fragments actually bind PPAR and function as PPREs.
To test directly whether PPAR promotes upregulation of CX3CR1 expression in monocytes exposed to oxidized LDL metabolites, we silenced its expression with specific sRNAi. We initially optimized experimental conditions for stealth RNAi-dependent knockdown of PPAR by nucleofection of primary monocytes. PPAR-specific sRNAi oligomers were transfected into 95% of cells (95% CI, 92.8 to 98.2) (Figure 7A) and specifically interfered with accumulation of the target mRNA in a dose-dependent manner (Data Supplement Figure V).
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Transfection of monocytes did not alter steady-state CCR2, CX3CR1 (Figure 7B and 7C), HAM56 (Figure 7D and 7E), or CD36 (Figure 7F and 7G) surface expression. In monocytes transfected with the negative control oligomer, stimulation with 9-HODE and 13-HODE as expected downregulated the frequency of CCR2+ cells by 63% to 4% (95% CI, 1.75 to 6.2) and 4.75% (95% CI, 2.7 to 6.7), respectively, whereas both lipids upregulated the frequency of CX3CR1+, HAM56+CX3CR1+, and CX3CR1+CD36+ cells (Figures 7B through 7G). In contrast, specific PPAR knockdown reversed the effects of linoleic acid metabolites on monocyte CX3CR1, CCR2, HAM56, and CD36 expression (Figure 7). These effects also occurred at the RNA level (Data Supplement Figure VI).
Oxidized Linoleic Acid Metabolite Induction of Macrophage-CASMC Adhesion Is PPAR Dependent
Transfection with either negative control or PPAR sRNAi had no effect on adhesion of unstimulated monocytes to CASMCs (Figure 8). Control sRNAi did not suppress 9-HODE or 13-HODE induction of PPAR mRNA (Data Supplement Figure VI) or macrophage-CASMC adhesion (Figure 8). In contrast, PPAR-specific sRNAi decreased induction of macrophage adhesion to CASMCs by 75.8% for both 9-HODE and 13-HODE (Figure 8).
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Discussion |
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The present study demonstrates in a model system of monocyte maturation to macrophages that oxidized linoleic acid metabolites, which are major components of oxidized LDL found at high concentrations in human atherosclerotic plaque,16–19 specifically induce a chemokine receptor switch—CCR2 off, CX3CR1 on—that is dependent on the transcription factor PPAR
. Switching CX3CR1 on plays a dominant and direct role in macrophage adhesion to CASMCs in this system. This is the first example in which CX3CL1/CX3CR1 interaction functions as a major adhesion system for primary cells. Together, the data support the inflammation theory of atherogenesis and provide a novel mechanism for macrophage accumulation in atherosclerotic plaques. Our CCR2 results confirm a previous pharmacological analysis by Han et al22 and extend it by direct genetic analysis. Han et al proposed a 2-step model for CCR2 regulation of atherogenesis in which the receptor first is used to recruit monocytes to the intima under the direction of CCL2 and then is downregulated by oxidized lipids in the intima, thus decreasing the physiological response of monocytes to CCL2 and promoting the pathological accumulation of monocytes in the intima.22 This model does not address adhesive mechanisms for macrophage retention in the vessel wall. Our results fill this gap and suggest a molecular and cellular mechanism of action for CX3CR1 in atherogenesis. CX3CR1 has previously been strongly implicated in atherogenesis by multiple lines of evidence, including direct detection of the receptor by immunohistochemistry in human atherosclerotic plaques,14 relative resistance of 2 independent lines of CX3CR1–/– mice to atherosclerosis after dietary challenge on an atherogenesis-prone apolipoprotein E–/– genetic background,4,6 and association of the defective human CX3CR1 allele CX3CR1-M280 with decreased risk of cardiovascular disease in multiple independent patient cohorts, including the Framingham Heart Study Offspring Cohort.7,35
CX3CR1 is an unusual dual-function chemokine receptor able to mediate leukocyte adhesion and migration in response to plasma membrane-tethered and -shed forms of CX3CL1, respectively.8,9 Prior work had focused on the role of CX3CR1 in adhesion of monocytes/macrophages to vascular endothelial cells,8,36 not to smooth muscle cells in which CX3CL1 function had remained undefined.13,14,37 In atherogenesis, this has become an important issue because direct immunohistochemical analysis of human atherosclerotic plaques has demonstrated CX3CL1 expression on smooth muscle cells and macrophages but not on endothelial cells.14 Our results are particularly relevant to atherogenesis because we tested primary human macrophages derived from blood monocytes and primary smooth muscle cells from human coronary arteries. It is noteworthy that CX3CL1 is not found in healthy coronary arteries but has been detected by immunohistochemistry in the intima, media, and adventitia of atherosclerotic vessels. Moreover, CX3CL1+ lesional cells colocalize with markers for macrophages and foam cells.14 Furthermore, electron microscopic and immunocytochemical analyses of human atherosclerotic lesions have shown that macrophages and foam cells are in direct contact with vascular smooth muscle cells.15 Because both CX3CL1 and CX3CR1 are expressed on macrophages,14 they also may mediate homotypic adhesion of foamy macrophages; however, additional work is needed to test this hypothesis.
The linoleic acid metabolites that we tested are the predominant oxidized LDL derivatives present in all stages of atherosclerotic lesions16–19 and are known to act as endogenous ligands and activators of PPAR, a member of the nuclear receptor superfamily.32 This information was the basis for the second major finding in our study, that oxidized linoleic acid metabolites activate PPAR20 to induce CX3CR1 mRNA accumulation, increased frequency of CX3CR1+ cells, and CX3CR1-dependent macrophage-CASMC adhesion. The mechanism appears to involve direct binding of PPAR to portions of the CX3CR1 promoter that contain consensus PPRE sites.
This result is the opposite of the inhibitory effect of PPAR on CCR2 expression.22 CCR2 and CX3CR1 are the only chemokine receptors found to be regulated by this factor so far. However, oxidized LDL also upregulates CXCR238 and stimulates release of the CXC chemokines CXCL1, CXCL5, and CXCL8,39 suggesting that PPAR may broadly regulate the chemokine system.
The precise mechanism by which PPAR regulates macrophage CCR2 expression has not been established, but its opposite effects relative to CX3CR1 suggest that other factors may exist that shape the specific effect of PPAR, most likely at the level of gene transcription, resulting in inhibition or stimulation. Additional work is needed to address this subject and to finely resolve the structural basis of PPAR induction of CX3CR1 expression and function.
PPAR is a multifunctional protein. In addition to the chemokine receptor switch that we have described, macrophage PPAR has been shown to regulate 2 other processes involved in lipid homeostasis: lipoprotein uptake and cholesterol efflux.20 In atherosclerosis, PPAR may promote oxidized LDL uptake by macrophages by increasing expression of the scavenger receptor CD36.40 This creates a positive feedback loop because internalized oxidized LDL delivers PPAR ligands to the cell.21,32
Consistent with our model, 2 mutations in human PPAR that reduce function, C161T and P12A, are both associated with reduced risk of atherosclerotic cardiovascular disease, implying that normal PPAR may facilitate atherogenesis.41,42 Moreover, for C161T, the association was independent of metabolic abnormalities, suggesting the possibility of a direct effect of PPAR on atherogenesis at the level of the arterial wall.42
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Conclusions |
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Our data are consistent with a proinflammatory model of atherogenesis in which oxidized linoleic acid–driven activation of macrophage PPAR
in the intima causes a proadhesive chemokine receptor switch–CCR2 off, CX3CR1 on–resulting in cessation of CCR2-dependent migration and activation of CX3CR1-dependent anchorage to CASMCs. Induction of CD36 expression by PPAR activation provides a positive feedback loop for internalization of PPAR ligands that may be exacerbated by conditions of chronic lipid overload associated with a Western diet and lifestyle. These results are consistent at the molecular and cellular levels with genetic evidence linking PPAR, CCR2, and CX3CR1 to cardiovascular disease and support the choice of these molecules as potential therapeutic targets. Moreover, the results identify macrophage binding to CASMCs as the first primary cell setting in which the CX3CL1/CX3CR1 ligand-receptor pair functions as the dominant adhesion system. |
Acknowledgments |
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Disclosures
None.
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References |
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The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.105.602359/DC1.
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