Loss of G2A promotes macrophage accumulation in atherosclerotic lesions of low density lipoprotein receptor-deficient mice
2005; Elsevier BV; Volume: 46; Issue: 7 Linguagem: Inglês
10.1194/jlr.m500085-jlr200
ISSN1539-7262
AutoresBrian W. Parks, Ginger P. Gambill, Aldons J. Lusis, Janusz H. Kabarowski,
Tópico(s)Cholesterol and Lipid Metabolism
ResumoLysophosphatidylcholine (LPC) is considered a major proatherogenic component of oxidized low density lipoprotein based on its proinflammatory actions in vitro. LPC stimulates macrophage and T-cell chemotaxis via the G protein-coupled receptor G2A and may thus promote inflammatory cell infiltration during atherosclerotic lesion development. However, G2A also mediates proapoptotic effects of LPC and may therefore promote the death of inflammatory cells within developing lesions. To determine how these effects of LPC modify atherogenesis, we examined atherosclerotic lesion development in G2A-sufficient and G2A-deficient low density lipoprotein receptor knockout mice. Although LPC species capable of activating G2A-dependent responses were increased during lesion development, G2A-deficient mice developed lesions similar in size to those in their G2A-sufficient counterparts. Loss of G2A during atherosclerotic lesion development did not reduce macrophage and T-cell infiltration but instead resulted in increased lesional macrophage content associated with reduced numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeled cells and decreased collagen deposition.These data indicate that the ability of LPC to stimulate macrophage and T-cell chemotaxis via G2A is not manifested in vivo and that G2A-mediated proapoptotic rather than chemotactic action is most penetrant during atherogenesis and may modify the stability of atherosclerotic lesions by promoting macrophage death. Lysophosphatidylcholine (LPC) is considered a major proatherogenic component of oxidized low density lipoprotein based on its proinflammatory actions in vitro. LPC stimulates macrophage and T-cell chemotaxis via the G protein-coupled receptor G2A and may thus promote inflammatory cell infiltration during atherosclerotic lesion development. However, G2A also mediates proapoptotic effects of LPC and may therefore promote the death of inflammatory cells within developing lesions. To determine how these effects of LPC modify atherogenesis, we examined atherosclerotic lesion development in G2A-sufficient and G2A-deficient low density lipoprotein receptor knockout mice. Although LPC species capable of activating G2A-dependent responses were increased during lesion development, G2A-deficient mice developed lesions similar in size to those in their G2A-sufficient counterparts. Loss of G2A during atherosclerotic lesion development did not reduce macrophage and T-cell infiltration but instead resulted in increased lesional macrophage content associated with reduced numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeled cells and decreased collagen deposition. These data indicate that the ability of LPC to stimulate macrophage and T-cell chemotaxis via G2A is not manifested in vivo and that G2A-mediated proapoptotic rather than chemotactic action is most penetrant during atherogenesis and may modify the stability of atherosclerotic lesions by promoting macrophage death. Atherosclerosis is an inflammatory disease characterized by the subendothelial accumulation of oxidized low density lipoprotein (OxLDL) in the arterial wall (1Lusis A.J. Atherosclerosis.Nature. 2000; 407: 233-241Google Scholar). Proinflammatory actions of OxLDL are mediated to a large extent by its phospholipid components. For example, hydrolysis of oxidized phosphatidylcholine decomposition products by lipoprotein-associated platelet-activating factor-acetylhydrolase (PAF-A7H) generates the bioactive lysophospholipid lysophosphatidylcholine (LPC) during LDL oxidation (2Parthasarathy S. Barnett J. Phospholipase A2 activity of low density lipoprotein: evidence for an intrinsic phospholipase A2 activity of apoprotein B-100.Proc. Natl. Acad. Sci. USA. 1990; 87: 9741-9745Google Scholar). Proinflammatory actions of LPC on cultured macrophages and endothelial cells have led to the widely held view that it is a key proatherogenic component of OxLDL. Most notably, LPC is believed to contribute directly to the recruitment of inflammatory cells during atherosclerotic lesion development. For example, LPC induces intercellular adhesion molecule-1 and monocyte chemotactic protein-1 expression in endothelial cells (3Kume N. Ochi H. Nishi E. Gimbrone Jr., M.A. Kita T. Involvement of protein kinase C-independent mechanisms in endothelial ICAM-1 up-regulation by lysophosphatidylcholine.Ann. N. Y. Acad. Sci. 1995; 748: 541-542Google Scholar, 4Murugesan G. Sandhya Rani M.R. Gerber C.E. Mukhopadhyay C. Ransohoff R.M. Chisolm G.M. Kottke-Marchant K. Lysophosphatidylcholine regulates human microvascular endothelial cell expression of chemokines.J. Mol. Cell. Cardiol. 2003; 35: 1375-1384Google Scholar) and stimulates macrophage and T-cell chemotaxis in vitro (5Quinn M.T. Parthasarathy S. Steinberg D. Lysophosphatidylcholine: a chemotactic factor for human monocytes and its potential role in atherogenesis.Proc. Natl. Acad. Sci. USA. 1988; 85: 2805-2809Google Scholar). Although these and many other in vitro studies have provided insights into potential proatherogenic effects of LPC, most have remained poorly characterized mechanistically, as it has been difficult to distinguish nonspecific effects of LPC from those mediated by specific proximal effectors. Furthermore, the multiplicity of cellular and biochemical factors associated with atherosclerotic lesion development raises questions regarding the relevance of these potential atherogenic mechanisms in vivo. However, the recent identification of the G protein-coupled receptor (GPCR) G2A as an effector of LPC action (6Lin P. Ye R.D. The lysophospholipid receptor G2A activates a specific combination of G proteins and promotes apoptosis.J. Biol. Chem. 2003; 278: 14379-14386Google Scholar, 7Radu C.G. Yang L.V. Riedinger M. Au M. Witte O.N. T cell chemotaxis to lysophosphatidylcholine through the G2A receptor.Proc. Natl. Acad. Sci. USA. 2004; 101: 245-250Google Scholar, 8Han K.H. Hong K.H. Ko J. Rhee K.S. Hong M.K. Kim J.J. Kim Y.H. Park S.J. Lysophosphatidylcholine up-regulates CXCR4 chemokine receptor expression in human CD4 T cells.J. Leukoc. Biol. 2004; 76: 195-202Google Scholar, 9Yang L.V. Radu C.G. Wang L. Riedinger M. Witte O.N. Gi-independent macrophage chemotaxis to lysophosphatidylcholine via the immunoregulatory GPCR G2A.Blood. 2004; 105: 1127-1134Google Scholar, 10Chen G. Li J. Qiang X. Czura C.J. Ochani M. Ochani K. Ulloa L. Yang H. Tracey K.J. Wang P. et al.Suppression of HMGB1 release by stearoyl lysophosphatidylcholine: an additional mechanism for its therapeutic effects in experimental sepsis.J. Lipid Res. 2005; 46: 623-627Google Scholar, 11Wang L. Radu C.G. Yang L.V. Bentolila L.A. Riedinger M. Witte O.N. Lysophosphatidylcholine-induced surface redistribution regulates signaling of the murine G-protein-coupled receptor G2A.Mol. Biol. Cell. 2005; 16: 2234-2247Google Scholar) has provided a mechanistic framework with which to establish how LPC modifies atherosclerosis independently of its nonspecific actions. In vitro studies show that G2A induces diverse biological effects in response to, as well as independently of, exogenously added LPC. These effects include actin cytoskeleton reorganization and focal adhesion assembly (12Kabarowski J.H. Feramisco J.D. Le L.Q. Gu J.L. Luoh S.W. Simon M.I. Witte O.N. Direct genetic demonstration of G alpha 13 coupling to the orphan G protein-coupled receptor G2A leading to RhoA-dependent actin rearrangement.Proc. Natl. Acad. Sci. USA. 2000; 97: 12109-12114Google Scholar, 13Zohn I.E. Klinger M. Karp X. Kirk H. Symons M. Chrzanowska-Wodnicka M. Der C.J. Kay R.J. G2A is an oncogenic G protein-coupled receptor.Oncogene. 2000; 19: 3866-3877Google Scholar), LPC stimulation of macrophage and T-cell chemotaxis (7Radu C.G. Yang L.V. Riedinger M. Au M. Witte O.N. T cell chemotaxis to lysophosphatidylcholine through the G2A receptor.Proc. Natl. Acad. Sci. USA. 2004; 101: 245-250Google Scholar, 8Han K.H. Hong K.H. Ko J. Rhee K.S. Hong M.K. Kim J.J. Kim Y.H. Park S.J. Lysophosphatidylcholine up-regulates CXCR4 chemokine receptor expression in human CD4 T cells.J. Leukoc. Biol. 2004; 76: 195-202Google Scholar, 9Yang L.V. Radu C.G. Wang L. Riedinger M. Witte O.N. Gi-independent macrophage chemotaxis to lysophosphatidylcholine via the immunoregulatory GPCR G2A.Blood. 2004; 105: 1127-1134Google Scholar, 11Wang L. Radu C.G. Yang L.V. Bentolila L.A. Riedinger M. Witte O.N. Lysophosphatidylcholine-induced surface redistribution regulates signaling of the murine G-protein-coupled receptor G2A.Mol. Biol. Cell. 2005; 16: 2234-2247Google Scholar), LPC-dependent extracellular signal regulated kinase mitogen-activated protein kinase activation (11Wang L. Radu C.G. Yang L.V. Bentolila L.A. Riedinger M. Witte O.N. Lysophosphatidylcholine-induced surface redistribution regulates signaling of the murine G-protein-coupled receptor G2A.Mol. Biol. Cell. 2005; 16: 2234-2247Google Scholar, 14Kabarowski J.H. Zhu K. Le L.Q. Witte O.N. Xu Y. Lysophosphatidylcholine as a ligand for the immunoregulatory receptor G2A.Science. 2001; 293: 702-705Google Scholar), and LPC-mediated apoptosis (6Lin P. Ye R.D. The lysophospholipid receptor G2A activates a specific combination of G proteins and promotes apoptosis.J. Biol. Chem. 2003; 278: 14379-14386Google Scholar). Although G2A was originally described as a binding receptor for LPC (14Kabarowski J.H. Zhu K. Le L.Q. Witte O.N. Xu Y. Lysophosphatidylcholine as a ligand for the immunoregulatory receptor G2A.Science. 2001; 293: 702-705Google Scholar), we have been unable to reproduce the G2A/LPC binding originally reported in crude cell homogenates prepared from receptor-overexpressing cell lines as a result of the high nonspecific membrane binding of this lysophospholipid (14Kabarowski J.H. Zhu K. Le L.Q. Witte O.N. Xu Y. Lysophosphatidylcholine as a ligand for the immunoregulatory receptor G2A.Science. 2001; 293: 702-705Google Scholar, 15Witte O.N. Kabarowski J.H. Xu Y. Le L.Q. Zhu K. Retraction.Science. 2005; 307: 206Google Scholar; our unpublished data). Although we cannot rule out a direct interaction between G2A and LPC, a recent study has provided strong evidence for LPC-dependent mobilization of intracellular G2A pools to the plasma membrane as the molecular mechanism by which LPC activates cellular responses via G2A (11Wang L. Radu C.G. Yang L.V. Bentolila L.A. Riedinger M. Witte O.N. Lysophosphatidylcholine-induced surface redistribution regulates signaling of the murine G-protein-coupled receptor G2A.Mol. Biol. Cell. 2005; 16: 2234-2247Google Scholar). Furthermore, the human G2A receptor and three GPCRs related by sequence homology [G protein-coupled receptor 4 (GPR4), ovarian cancer G protein-coupled receptor 1 (OGR1), and T-cell death-associated gene 8 (TDAG8)] have recently been described as "proton-sensing" receptors (16Ludwig M.G. Vanek M. Guerini D. Gasser J.A. Jones C.E. Junker U. Hofstetter H. Wolf R.M. Seuwen K. Proton-sensing G-protein-coupled receptors.Nature. 2003; 425: 93-98Google Scholar, 17Wang J.Q. Kon J. Mogi C. Tobo M. Damirin A. Sato K. Komachi M. Malchinkhuu E. Murata N. Kimura T. et al.TDAG8 is a proton-sensing and psychosine-sensitive G-protein-coupled receptor.J. Biol. Chem. 2004; 279: 45626-45633Google Scholar, 18Murakami N. Yokomizo T. Okuno T. Shimizu T. G2A is a proton-sensing G-protein coupled receptor antagonized by lysophosphatidylcholine.J. Biol. Chem. 2004; 279: 42484-42491Google Scholar). However, a subsequent comparative study of each receptor confirmed GPR4, OGR1, and TDAG8 as bona fide proton-sensing GPCRs yet failed to detect pH-dependent activation of murine G2A and reported very weak responses of human G2A to extracellular acidification compared with those of GPR4, OGR1, and TDAG8 (19Radu C.G. Nijagal A. McLaughlin J. Wang L. Witte O.N. Differential proton sensitivity of related G protein-coupled receptors T cell death-associated gene 8 and G2A expressed in immune cells.Proc. Natl. Acad. Sci. USA. 2005; 102: 1632-1637Google Scholar). Thus, there is no evidence to support a proton-sensing function for murine G2A in addition to its role as an effector of LPC action, and the physiological significance of the weak pH sensitivity of the human receptor is questionable and requires further study. Although G2A-mediated effects of LPC have the potential to modify inflammatory events during atherosclerotic lesion development, their significance in vivo has not been tested. By breeding G2A-deficient mice onto the LDL receptor knockout (LDLR−/−) background, we directly assessed the role of G2A in atherosclerosis development. G2A deficiency resulted in increased lesional macrophage numbers associated with decreased apoptosis and reduced collagen content. Thus, G2A deficiency promotes macrophage accumulation, likely by suppressing the death-inducing effects of LPC, which in turn may promote lesion destabilization caused by increased levels of macrophage-derived collagen-degrading enzymes. Adherent murine peritoneal macrophages were isolated by culturing peritoneal exudates in DMEM containing 10% fetal calf serum (FCS) for 6 h. Thioglycolate-elicited peritoneal macrophages were obtained from peritoneal exudates of mice injected intraperitoneally 3 days earlier with 3 ml of 4% thioglycolate. All bone marrow-derived cells were cultured from total mononuclear bone marrow cells flushed from the femurs and tibias of mice. Bone marrow cells were cultured at 106/ml in DMEM, 10% FCS, 20% L cell-conditioned medium as a source of macrophage-colony stimulating factor, 50 μM 2-mercaptoethanol, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin for 5 days to obtain CD11b-positive macrophages. Bone marrow cells were cultured at 106/ml in DMEM, 10% FCS, 50 ng/ml recombinant granulocyte/macrophage-colony stimulating factor, 250 U/ml recombinant interleukin-4, 50 μM 2-mercaptoethanol, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin for 10 days with three medium changes to obtain CD11c-positive dendritic cells. Surface FcεR1 IgE receptor-positive mast cells were generated from bone marrow cells cultured at 106/ml in RPMI, 10% FCS, 20% WEHI-3B cell conditioned medium as a source of interleukin-3, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin for 5 weeks with regular medium changes. Platelet/edothelial cell adhesion molecule-1 (PECAM-1)-positive murine endothelial cells were obtained by incubating dissected aorta on Matrigel (BD Biosciences) with DMEM, 10% FCS, 90 μg/ml heparin, 60 μg/ml endothelial cell growth supplement (Collaborative Biomedical Products), 1% Fungizone, 100 U/ml penicillin, and 100 μg/ml streptomycin for 3 days with daily addition of fresh medium. Fifty percent confluent cultures were passaged by dispase treatment onto 100 mm tissue culture plates and incubated until confluent. α-Actin-positive murine aortic smooth muscle cells were cultured from aorta digested with 15 mM HEPES containing 2% BSA, 0.125 μg/ml elastase, 0.25 μg/ml soybean trypsin inhibitor, and 10 μg/ml collagenase D. Aortic digests were forced through a 70 μm filter and cultured in DMEM, 10% FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin until ∼80% confluent. B220+ B-cells and CD4+ and CD8+ T-cells were purified from spleens and lymph nodes of mice by immunomagnetic depletion of B-cells (MACS CD45R MicroBeads; Miltenyi Biotec GmbH) followed by incubation with either phycoerythrin-conjugated anti-CD4 or anti-CD8 antibodies (BD Pharmingen) and flow cytometric sorting on a FACS ARIA flow cytometer (Becton Dickinson). RNA was isolated using the Absolutely RNA RT-PCR Miniprep kit (Stratagene). One microgram of RNA was reverse-transcribed using an oligo-dT primer with the SuperScript First Strand Synthesis System (Invitrogen). Ten percent of the cDNA was subjected to PCR amplification (25 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min) for analysis of G2A, GPR4, or actin expression with the following primers: G2A, 5′-CTGCCTCAGGACTGGCTTGG and 3′-TCACACACGCAGAAATGGTGAC; GPR4, 5′-CTCTCTACATCTTCGTCATCGG and 3′-CGGTAGCACAGCAACATGAGTG; actin, 5′-CACAGGCATTGTGATGGACT and 3′-CTTCTGCATCCTGTCAGCCAA. G2A−/− mice backcrossed a total of nine generations (N9) onto the C57BL/6J background were bred with C57BL/6J LDLR−/− mice (Jackson Laboratory, Bar Harbor, ME), and the resulting compound heterozygotes (N10 G2A+/−LDLR+/−) were intercrossed to obtain G2A+/+LDLR−/− and G2A−/−LDLR−/− progeny. Mice were weaned at 4 weeks of age and maintained on a standard rodent chow diet containing 4% fat (5015; Harlan Teklad, Madison, WI). At 8 weeks of age, mice were fasted for 12 h, weighed, bled by retro-orbital puncture, and transferred onto a "Western" diet (42% fat, 0.15% cholesterol, 19.5% casein without sodium cholate) (88137; Harlan Teklad) for 6 or 12 weeks. Mice were subsequently fasted for 12 h, weighed, and bled by retro-orbital puncture for lipid profile analysis. Plasma samples were processed for measurement of total cholesterol, unesterified cholesterol, HDL cholesterol, triglycerides, and free fatty acids by enzymatic procedures described previously (20Hedrick C.C. Castellani L.W. Warden C.H. Puppione D.L. Lusis A.J. Influence of mouse apolipoprotein A-II on plasma lipoproteins in transgenic mice.J. Biol. Chem. 1993; 268: 20676-20682Google Scholar). Each sample was measured in triplicate, and Centers for Disease Control plasma samples with known lipid values were included as controls. LPC species were measured in snap-frozen aortas and plasma from five female LDLR−/− mice maintained on a regular chow diet or high-fat Western diet for 12 weeks by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) as described previously (21Xiao Y. Chen Y. Kennedy A.W. Belinson J. Xu Y. Evaluation of plasma lysophospholipids for diagnostic significance using electrospray ionization mass spectrometry (ESI-MS) analyses.Ann. N. Y. Acad. Sci. 2000; 905: 242-259Google Scholar, 22Sutphen R. Xu Y. Wilbanks G.D. Fiorica J. Grendys Jr., E.C. LaPolla J.P. Arango H. Hoffman M.S. Martino M. Wakeley K. et al.Lysophospholipids are potential biomarkers of ovarian cancer.Cancer Epidemiol. Biomarkers Prev. 2004; 13: 1185-1191Google Scholar). Briefly, lipids were extracted from plasma or aortic tissue as described by Sutphen et al. (22Sutphen R. Xu Y. Wilbanks G.D. Fiorica J. Grendys Jr., E.C. LaPolla J.P. Arango H. Hoffman M.S. Martino M. Wakeley K. et al.Lysophospholipids are potential biomarkers of ovarian cancer.Cancer Epidemiol. Biomarkers Prev. 2004; 13: 1185-1191Google Scholar) and resuspended in methanol-chloroform (2:1, v/v). Samples were spotted onto silica gel TLC plates and run in a solvent system composed of chloroform-methanol-ammonium hydroxide (65:35:5.5) with lipid standards (Avanti Polar Lipids). LPC bands were eluted from TLC plates, dried under nitrogen, and resuspended in 50 μl of methanol-water (1:1, v/v). To obtain standard curves, different amounts (5–300 pmol) of standard LPC solutions (6:0, 8:0, 10:0, 12:0, 14:0, 16:0, 18:0, 20:0, 22:0, and 24:0; Avanti Polar Lipids) were mixed with the same amount (50 pmol) of internal standard 17:0-LPC and ESI-MS/MS was performed using a Micromass Quattro II Triple Quadrupole Mass Spectrometer with a MassLynx data-acquisition system (Micromass, Inc., Beverly, MA). Peak intensity ratios (standard vs. internal standard) were plotted against molar ratios (standard vs. internal standard) to obtain standard curves. For quantitative analysis of LPC, 500 pmol of 17:0 LPC internal standard was added to each sample before lipid extraction. Lipid samples were delivered into the ESI source using a Waters 2690 autosampler (Waters, Milford, MA) in a mobile phase of methanol-water (1:1, v/v) and a flow rate of 100 μl/min. Parent scanning and MS/MS analyses were performed in the positive ion mode with multiple reaction monitoring and a dwell time of 100 ms using instrument settings identical to those described previously (22Sutphen R. Xu Y. Wilbanks G.D. Fiorica J. Grendys Jr., E.C. LaPolla J.P. Arango H. Hoffman M.S. Martino M. Wakeley K. et al.Lysophospholipids are potential biomarkers of ovarian cancer.Cancer Epidemiol. Biomarkers Prev. 2004; 13: 1185-1191Google Scholar). Monitoring ions were at m/z 483 (parent ion) and 184 (product ion) for 16:0 lyso-PAF, 496 and 184 for 16:0-LPC, 510 and 184 for internal standard 17:0-LPC, 524 and 184 for 18:0-LPC, 522 and 184 for 18:1-LPC, 520 and 184 for 18:2-LPC, 544 and 184 for 20:4-LPC, and 568 and 184 for 22:6 LPC. After euthanization, the heart was perfused with 20 ml of PBS and removed by cutting at the proximal aorta. The upper portion of the heart was placed into a tissue mold, covered with OCT (Tissue-Tek), and frozen. Ventricular tissue was sectioned in a Leica 1850 cryostat, and serial 8 μm sections were collected onto microscope slides at the first appearance of the aortic valve leaflets. One hundred alternate sections were collected for lesion quantification, and intervening sections were collected and stored at −20°C for immunohistochemistry. Sections for lesion quantification were stained with Oil Red O and counterstained with hematoxylin and fast green. Lesion areas were measured morphometrically by two blinded independent observers with a Zeiss Axiostar Plus microscope using a 1 mm square eyepiece grid (100 × 10,000 μm2) at 100× magnification. One hundred alternate frozen sections were collected from each animal onto 25 slides (four sections per slide). For six randomly chosen mice from each of the four experimental groups . G2A+/+LDLR−/−,. G2A−/−LDLR−/−,. G2A+/+LDLR−/−, and. G2A−/−LDLR−/−), each of the four sections on six consecutive slides representing similar parts of the aortic root were stained with one of the following antibodies: 1) rat anti-CD11b (BD Pharmingen); 2) rat anti-CD3 (BD Pharmingen); 3) rat anti-PECAM-1 (CD31) (BD Pharmingen); or 4) rabbit anti-smooth muscle α-actin (Spring Biosciences, Fremont, CA). Sections were fixed in acetone at room temperature, treated with 0.3% hydrogen peroxide in PBS, and blocked in PBS containing 4% BSA and 10% serum of the species from which the secondary antibody was derived before incubation with primary antibodies. After incubation with appropriate biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA) followed by HRP-conjugated streptavidin (Southern Biotechnology Associates), sections were developed with diaminobenzidine (Vector Laboratories) and counterstained with hematoxylin. The specificity of staining was confirmed using rat IgG2b isotype control for CD11b and CD3, IgG2a isotype control for PECAM-1, and rabbit IgG for smooth muscle α-actin. For T-cell quantification, CD3-positive cells were counted in lesions from each of the six randomly chosen. G2A+/+LDLR−/−,. G2A−/−LDLR−/−,. G2A+/+LDLR−/−, and. G2A−/−LDLR−/− mice. For macrophage quantification, the percentage of total lesion area in each section occupied by CD11b-positive cells was measured morphometrically. For visualization of lesional collagen deposition, eight alternate 8 μm frozen sections from five randomly chosen animals of each experimental group were fixed in Bouin's fixative and stained with Masson's Trichrome (NewcomerSupply, Middleton, WI). The percentage of lesion area occupied by collagen was measured morphometrically. For five randomly chosen female G2A+/+LDLR−/− and G2A−/− LDLR−/− mice, eight alternate 8 μm frozen sections from similar parts of the aortic root were fixed in 1% paraformaldehyde, permeabilized in cold ethanol-acetic acid (2:1, v/v) at −20°C, and subjected to terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining with or without terminal deoxynucleotidyl transferase using the ApopTag fluorescein in situ apoptosis detection kit (S7110; Chemicon International, Temecula, CA) according to the manufacturer's protocol. TUNEL-positive cells within lesions were quantified in color images captured on an Olympus BX60 fluorescence microscope. For colocalization of TUNEL staining with specific lesional cell types, aortic root sections were stained with anti-CD11b, anti-PECAM-1, or anti-CD3 antibodies followed by AlexaFluor-555-conjugated anti-rat antibody (BD Pharmingen). GPR4 has been described as a second effector of LPC action (23Zhu K. Baudhuin L.M. Hong G. Williams F.S. Cristina K.L. Kabarowski J.H. Witte O.N. Xu Y. Sphingosylphosphorylcholine and lysophosphatidylcholine are ligands for the G protein-coupled receptor GPR4.J. Biol. Chem. 2001; 276: 41325-41335Google Scholar). Therefore, we analyzed the expression of G2A and GPR4 in primary murine cell types involved in atherosclerosis (Fig. 1). By RT-PCR, we detected the expression of G2A in macrophages from various sources, T- and B-lymphocytes, bone marrow-derived mast cells and dendritic cells, and aortic endothelial cells. GPR4 expression was undetectable in these hematopoietic cell types and was found only in aortic endothelial cells. Finally, we detected no G2A or GPR4 expression in aortic smooth muscle cells. We also analyzed the same cell populations from G2A-deficient (G2A−/−) mice to confirm the specificity of G2A amplification and detected no GPR4 expression in any of the G2A−/− hematopoietic cell types examined. Furthermore, there was no significant difference in GPR4 expression in G2A−/− aortic endothelial cells compared with their wild-type (G2A+/+) counterparts (data not shown). Thus, there is no compensatory upregulation of GPR4 in the absence of G2A. A recent study also reported GPR4 expression in endothelial cells and further suggested that GPR4 expression is transcriptionally induced by inflammatory stress in human vascular endothelial cells (24Lum H. Qiao J. Walter R.J. Huang F. Subbaiah P.V. Kim K.S. Holian O. Inflammatory stress increases receptor for lysophosphatidylcholine in human microvascular endothelial cells.Am. J. Physiol. Heart Circ. Physiol. 2003; 285: H1786-H1789Google Scholar). However, this study did not detect G2A expression, which contradicts our observations. Although this may reflect the use of human versus murine cells, it is also possible that differences in their preparation, or immortalization in the case of cell lines, may influence endothelial cell receptor expression, as G2A is subject to transcriptional regulation by multiple stress stimuli (25Weng Z. Fluckiger A.C. Nisitani S. Wahl M.I. Le L.Q. Hunter C.A. Fernal A.A. Le Beau M.M. Witte O.N. A DNA damage and stress inducible G protein-coupled receptor blocks cells in G2/M.Proc. Natl. Acad. Sci. USA. 1998; 95: 12334-12339Google Scholar). Furthermore, it is possible that endothelial cells from different vascular sites exhibit varying patterns of receptor expression (24Lum H. Qiao J. Walter R.J. Huang F. Subbaiah P.V. Kim K.S. Holian O. Inflammatory stress increases receptor for lysophosphatidylcholine in human microvascular endothelial cells.Am. J. Physiol. Heart Circ. Physiol. 2003; 285: H1786-H1789Google Scholar). Nevertheless, our data show that G2A but not GPR4 is expressed in inflammatory cells, whereas GPR4 is the predominant receptor in aortic endothelial cells that also express lower levels of G2A. Of the major LPC species identified previously in human atherosclerotic tissue (26Thukkani A.K. McHowat J. Hsu F.F. Brennan M.L. Hazen S.L. Ford D.A. Identification of {alpha}-chloro fatty aldehydes and unsaturated lysophosphatidylcholine molecular species in human atherosclerotic lesions.Circulation. 2003; 108: 3128-3133Google Scholar), only 16:0 LPC, 18:0 LPC, and 18:1 LPC have been shown to elicit cellular and molecular responses via G2A (6Lin P. Ye R.D. The lysophospholipid receptor G2A activates a specific combination of G proteins and promotes apoptosis.J. Biol. Chem. 2003; 278: 14379-14386Google Scholar, 7Radu C.G. Yang L.V. Riedinger M. Au M. Witte O.N. T cell chemotaxis to lysophosphatidylcholine through the G2A receptor.Proc. Natl. Acad. Sci. USA. 2004; 101: 245-250Google Scholar, 9Yang L.V. Radu C.G. Wang L. Riedinger M. Witte O.N. Gi-independent macrophage chemotaxis to lysophosphatidylcholine via the immunoregulatory GPCR G2A.Blood. 2004; 105: 1127-1134Google Scholar, 10Chen G. Li J. Qiang X. Czura C.J. Ochani M. Ochani K. Ulloa L. Yang H. Tracey K.J. Wang P. et al.Suppression of HMGB1 release by stearoyl lysophosphatidylcholine: an additional mechanism for its therapeutic effects in experimental sepsis.J. Lipid Res. 2005; 46: 623-627Google Scholar). To determine whether these LPC species are significantly increased during atherogenesis in LDLR−/− mice, LPC levels were measured in plasma and the entire aorta of LDLR−/− mice before and after a 12 week period of high-fat Western diet intervention by ESI-MS/MS (21Xiao Y. Chen Y. Kennedy A.W. Belinson J. Xu Y. Evaluation of plasma lysophospholipids for diagnostic significance using electrospray ionization mass spectrometry (ESI-MS) analyses.Ann. N. Y. Acad. Sci. 2000; 905: 242-259Google Scholar, 22Sutphen R. Xu Y. Wilbanks G.D. Fiorica J. Grendys Jr., E.C. LaPolla J.P. Arango H. Hoffman M.S. Martino M. Wakeley K. et al.Lysophospholipids are potential biomarkers of ovarian cancer.Cancer Epidemiol. Biomarkers Prev. 2004; 13: 1185-1191Google Scholar). Levels of 16:0 lyso-PAF were measured and found to be increased significantly after Western diet intervention, confirming that a known product of PAF-AH hydrolysis was generated during atherogenesis (Fig. 2). Although levels of certain LPC species remained unchanged or were reduced by Western diet intervention in aortic tissue (18:2 LPC and 20:4 LPC) and plasma (18:0 LPC, 18:2 LPC, 20:4 LPC, and 22:6 LPC), several, including those previously established as major constituents of OxLDL (26Thukkani A.K. McHowat J. Hsu F.F. Brennan M.L. Hazen S.L. Ford D.A. Identification of {alpha}-chloro fatty aldehydes and unsaturated lysophosphatidylcholine molecular species in human atherosclerotic lesions.Circulation. 2003; 108: 3128-3133Google
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