Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation. 2006;114:583-590
Published online before print July 31, 2006, doi: 10.1161/CIRCULATIONAHA.105.540583
CLINICAL PERSPECTIVE
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
114/6/583    most recent
CIRCULATIONAHA.105.540583v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Aslanian, A. M.
Right arrow Articles by Charo, I. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aslanian, A. M.
Right arrow Articles by Charo, I. F.
Related Collections
Right arrow Pathophysiology
Right arrow Genetically altered mice

(Circulation. 2006;114:583-590.)
© 2006 American Heart Association, Inc.

Molecular Cardiology

Targeted Disruption of the Scavenger Receptor and Chemokine CXCL16 Accelerates Atherosclerosis

Ara M. Aslanian, PhD; Israel F. Charo, MD, PhD

From the Gladstone Institute of Cardiovascular Disease (A.M.A., I.F.C.), San Francisco, Calif, and Cardiovascular Research Institute (I.F.C.), Department of Medicine, University of California, San Francisco, Calif.

Correspondence to Israel F. Charo, MD, PhD, Gladstone Institute of Cardiovascular Disease, 1650 Owens St, San Francisco, CA 94158. E-mail icharo{at}gladstone.ucsf.edu

Received February 15, 2006; revision received May 5, 2006; accepted May 30, 2006.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Background— The uptake of oxidized low-density lipoprotein (OxLDL) by macrophage scavenger receptors is thought to be a key process in the formation of foam cells, the hallmark of early atherosclerotic lesions. CXCL16/scavenger receptor for phosphatidylserine and OxLDL is a multifunctional chemokine that exhibits scavenger receptor activity toward oxidized lipids in a membrane-bound configuration and may be shed to serve as a chemoattractant for T helper 1–polarized T lymphocytes. These properties, as well as the expression of CXCL16 in human and mouse atheroma, suggest that CXCL16 plays a role in atherosclerosis.

Methods and Results— To examine the role of CXCL16 in plaque formation, we created CXCL16-deficient mice (CXCL16–/–) and bred them with mice deficient in the LDL receptor (LDLR–/–). In vitro, macrophages from CXCL16–/– mice have a significant reduction in the capacity to bind and internalize OxLDL. We found that CXCL16–/–/LDLR–/– mice have accelerated atherosclerosis, enhanced macrophage recruitment to the aortic arch, and more abundant mRNA for monocyte chemotactic protein-1 and tumor necrosis factor-{alpha}.

Conclusions— These data suggest that scavenger receptor activity mediated by CXCL16 in vivo is atheroprotective, and they contrast with studies that document protection from atherosclerosis in scavenger receptor class A- and CD36-deficient mice.


Key Words: atherosclerosis • leukocytes • lipids • chemokines


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Early atherosclerosis is characterized by the accumulation of lipid-laden macrophages in the vessel wall. Several in vivo studies support the hypothesis that scavenger receptors play a key role in this process by mediating the unrestricted uptake of modified lipoproteins. Oxidized lipoprotein (OxLDL) is the most potent atherogenic lipoprotein species1,2 and is recognized by several receptors, including scavenger receptor (SR) class A (SR-A),3 CD36,4,5 the lectinlike OxLDL receptor (LOX-1),6 and CD68.7,8 However, the relative contribution of scavenger receptors to plaque progression in vivo is controversial. Recently, scavenger receptor for phosphatidylserine and OxLDL (SR-PSOX) was identified by expression cloning from a human phorbol myristate acetate-stimulated THP-1 cDNA library.9 SR-PSOX exhibited scavenger receptor activity in vitro and was subsequently found to be identical to the chemokine CXCL16.10,11

Clinical Perspective p 590

CXCL16/SR-PSOX (henceforth referred to as CXCL16) is expressed on endothelial and smooth muscle cells,12 as well as on macrophages9 and dendritic cells.11 It has been identified in human carotid endarterectomy samples13 and in lesions of ApoE-null mice fed a Western diet,14 which suggests a role in atherogenesis. CXCL16 is expressed in 2 distinct forms. Membrane-bound CXCL16 consists of a C-terminal chemokine domain atop a glycosylated mucinlike stalk with a single helical transmembrane domain.10,11 In this configuration, CXCL16 promotes firm adhesion of cells expressing its cognate receptor, known as CXCR6.15 Human CXCL16 also mediates the binding and internalization of bacteria16 and OxLDL.9

Proteolytic cleavage of membrane-bound CXCL16 results in the release of soluble CXCL16,17,18 which acts as a chemoattractant for CXCR6+ cells. In humans and mice, CXCR6+ cells represent a highly polarized T helper 1 T-cell compartment,19,20 and lymphocytes expressing CXCR6 are enriched in chronically inflamed tissues, such as rheumatoid synovium and inflamed liver.20 As a soluble protein, CXCL16 may promote the directed migration of effector T cells to atherosclerotic lesions, a key process in the development of the proatherogenic inflammatory response.

To investigate the role of CXCL16 in atherosclerosis, we generated CXCL16–/– mice, crossed them with mice deficient in the LDL receptor (LDLR–/– mice), and quantified lesion development after placing the offspring on a diet. Unexpectedly, we found that CXCL16–/–/LDLR–/– mice had accelerated atherosclerosis and increased macrophage recruitment to the aortic arch compared with CXCL16+/+/LDLR–/– controls, despite the fact that macrophages from CXCL16–/– mice had markedly reduced scavenger receptor activity for OxLDL. We found no differences in the ability of macrophages from CXCL16–/– mice to bind high-density lipoprotein (HDL) or phagocytose apoptotic cells. Thus, the present data indicate that CXCL16 is atheroprotective and, in the context of a recent study of SR-A- and CD36-deficient mice that shows similar results,21 suggest that scavenger receptor activity does not contribute to atherosclerosis and may in fact be atheroprotective.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Generation of Targeted Embryonic Stem Cells
A mouse 129/Sv strain bacterial artificial chromosome library (RPCI-22.C, ResGen [Invitrogen], Carlsbad, Calif) was screened with a probe generated by polymerase chain reaction (PCR) specific for the coding region of CXCL16. Flanking regions of the CXCL16 gene were amplified by PCR with an Expand High Fidelity Kit (Roche, Basel, Switzerland). An upstream region of 4643 bp was generated with primers containing EcoRI and SalI restriction sites (5'-GGAATTCCGGATCTTCAGGGATTGCTTGTTC-3' and 5'-ACGCGTCGACAATGACTCGGTTGGGTTCGG-3'). A downstream region of 3019 bp was generated with primers containing PacI and NotI restriction sites (5'-CCTTAATTAATGCCTCTA-CCTCCTAGATTCTGCG-3' and 5'-ATAAGAATGCGGCCGCT-GTCCAAGGAAAAGACTAACGAGC-3'). The amplicons were ligated into the pSV-Neo-TK vector,22 which resulted in the complete removal of the coding region of CXCL16.

The CXCL16 targeting vector was linearized with NotI, electroporated into RF8 embryonic stem (ES) cells, and selected with G418 and FIAU as described previously.23 Colonies were screened by PCR with primers located within the neo cassette and downstream of the 3' targeting arm (5'-TCGCCTTCTATCGCCTTCTTG-3' and 5'-CAATAACCGCCTCTTTCCACC-3'). The resulting amplicon was 4032 bp in correctly targeted clones and recombination was confirmed by Southern blotting of XmaI-digested genomic DNA.

Mice
CXCL16-null mice (CXCL16–/–) were backcrossed 5 generations (N5) onto the C57BL/6 genetic background, achieving 96.87% C57BL/6 as determined by MAX-BAX marker-assisted backcrossing analysis (Charles River Laboratories, Wilmington, Mass). Subsequent mating with fully backcrossed LDLR-null mice (LDLR–/–) (The Jackson Laboratory, Bar Harbor, Me) generated CXCL16–/–/LDLR–/– double knockouts. CXCL16+/–/LDLR–/– mice were crossed to generate littermate controls for all experiments.

Mice were weaned at 21 days of age and fed a chow diet for 14 additional days. They were then fed a high-fat, cholate-free diet containing 1.25% (wt/wt) cholesterol (D12108, Research Diets, New Brunswick, NJ). Mice were matched for gender in each control and experimental group for all atherosclerosis and mRNA analyses.

Atherosclerosis Analysis
The extent of atherosclerosis was quantified in the aortic arch and the aortic root by digital morphometry without knowledge of the genotype, as described previously.24 The aortic arch was taken as the region 3 mm inferior to the branch point of the left subclavian artery to the heart.

Lipid Analysis
Plasma samples were collected by cardiac puncture at the time the animals were euthanized. Total cholesterol analysis and size fractionation of lipoprotein particles was performed as described previously.25

Real-Time PCR
Quantitation of mRNA was determined by TaqMan (Applied Biosystems, Foster City, Calif) real-time PCR with the cyclophilin gene as an internal control as described previously.25 The sequences for CXCR6 were 5'-CCCTTTTGGGCCTATGCAG-3' (forward), 5'-ATGCCTCGAAGAGTTTTGCAC-3' (reverse), 5'-6-FAM-CACCTATGAGTGGGTCTTTGGCACAGTCA-BHQ-1-3' (probe), CXCL16, 5'-TGACCTCGTCCCAACAAGCT-3' (forward), 5'-CGCAAGAGACAAGGGTCCAA-3' (reverse), and 5'-6-FAM-AGAAGCCGCAGATGAGGCGGG-BHQ-1-3' (probe). The monocyte chemotactic protein (MCP)-1 probe set was purchased as predeveloped assay reagents from Applied Biosystems. The CD68 and tumor necrosis factor-{alpha} primers and probe were identical to published sequences.26,27 cDNA (2 µL) was amplified for all control and experimental reactions.

Scavenger Receptor Assay
Thioglycolate-elicited macrophages were harvested from wild-type or CXCL16–/– mice by peritoneal lavage with ice-cold Ca+-free phosphate-buffered saline. Cells were resuspended in minimum essential medium supplemented with 10% fetal bovine serum and seeded at a density of 1x106 cells/well in a 48-well plate. After 2 hours, the medium was replaced to remove nonadherent cells, and the attached macrophages were cultured for an additional 48 hours. Scavenger receptor activity was determined by the method of Teusper et al.28 Nonspecific uptake was found to be &7% of the total cell-associated 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-LDL.

Immunohistochemistry
Visualization of CXCL16 and macrophages in the aortic root was performed with a tyramide amplification plus kit (Perkin Elmer, Boston, Mass) as described previously.24 Primary biotinylated CXCL16 antibody (R&D Systems, Minneapolis, Minn) was incubated with a 1:1000 dilution of streptavidin-horseradish peroxidase secondary antibody and visualized with rhodamine tyramide reagent.

Apoptosis Analysis
Apoptotic cells were identified in the aortic sinus by TUNEL staining with an in situ death detection kit (Roche). Hearts were removed and maintained in formalin buffer for 4 to 5 hours before equilibration in 20% sucrose to completely fix the samples. To quantify apoptotic cells, 9- to 10-µm sections of each heart centered on the orifice of the left coronary artery were incubated in 0.1% Triton X-100/0.1 mol/L citric acid for 2 minutes on ice before staining.

Statistical Analysis
Statistical differences between experimental groups were analyzed with the Mann-Whitney nonparametric U test. Probability values less than 0.05 were considered significant.

The authors had full access to the data and take full responsibility for its integrity. All authors have read and agree to the manuscript as written.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Generation of CXCL16–/– Mice
To selectively delete the CXCL16 gene, we constructed a targeting vector that replaced the entire 5-exon coding region of CXCL16 with a neo cassette (Figure 1A).Heterozygous offspring were intercrossed to obtain homozygous CXCL16–/– mice at the F2 generation (Figure 1B). CXCL16–/– mice had no overt developmental or morphological abnormalities. To confirm the absence of CXCL16 expression in the mutant animals, CXCL16 mRNA was measured in thioglycolate-elicited peritoneal macrophages from wild-type, CXCL16+/–, and CXCL16–/– mice by real-time PCR (Figure 1C).


Figure 1176948
View larger version (24K):
[in this window]
[in a new window]
  		Figure 1. Targeted disruption of CXCL16. A, Map of the CXCL16 genomic locus with the targeting sequence shown above. The entire 5-exon coding region of CXCL16 was replaced by a neomycin cassette. The predicted configuration of the genomic locus after correct recombination and the location of the hybridization probe used for screening of genomic DNA are shown. XmaI digestion produces fragments of 7.5 and 11 kilobase (kb) in the wild-type and targeted loci, respectively. B, Southern blot of XmaI-digested genomic DNA from F2 generation mice showing correct wild-type (+/+), heterozygous (+/–), and knockout (–/–) fragments. C, Absence of CXCL16 expression in CXCL16–/– mice. CXCL16 mRNA was measured in thioglycolate-elicited macrophages from wild-type, CXCL16+/–, and CXCL16–/– mice by real-time PCR. Data are presented as a fraction of mRNA abundance in wild-type mice.

CXCL16 Is Highly Expressed in the Aortic Arch
To determine whether CXCL16 was expressed in the aortic arch under atherogenic conditions, LDLR–/– mice were fed either a chow or Western diet for 4 or 8 weeks, and the aortas were analyzed for CXCL16 mRNA by real-time PCR. At 4 weeks, CXCL16 mRNA was 52% more abundant in mice fed the Western diet than in those fed chow (P=0.0002; Figure 2). At 8 weeks, mice fed the Western diet had 150% more CXCL16 mRNA than mice fed the same diet for 4 weeks (P<0.0001); however, mice fed the chow diet showed no increase in the amount of CXCL16 mRNA during this time period. Given these data, we determined that CXCL16 mRNA is highly induced in atherosclerosis-prone areas of the aorta under hypercholesterolemic conditions and that time points of 6 and 10 weeks were optimal for subsequent analysis in the LDLR–/– mouse model. Serial sections of the aortic root stained with anti-CXCL16 and MOMA-2 antibodies revealed that the primary cells expressing CXCL16 in the lesion were macrophage foam cells (Figure 3).


Figure 2176948
View larger version (21K):
[in this window]
[in a new window]
  		Figure 2. CXCL16 expression in the aortic arch. CXCL16 mRNA was measured by real-time PCR in mice fed a chow or Western diet for 4 or 8 weeks. CXCL16 mRNA was significantly more abundant in mice fed the Western diet at both time points than in those fed chow (*P=0.0002; **P<0.0001).


Figure 3176948
View larger version (38K):
[in this window]
[in a new window]
  		Figure 3. Localization of CXCL16 in atherosclerotic lesions. Serial sections of aortic roots from CXCL16+/+/LDLR–/– and CXCL16–/–/LDLR–/– mice fed a Western diet for 10 weeks were incubated with antibodies and analyzed by immunofluorescence. Staining of CXCL16 (red; A, B) and the macrophage marker MOMA-2 (pseudocolored green; C, D) in sections from wild-type (A, C) and CXCL16–/– (B, D) mice. Nuclei were stained with DAPI (blue). Original magnification x40.

Scavenger Receptor Activity of CXCL16
To determine whether murine CXCL16 had detectable scavenger receptor activity in vivo, we measured DiI-OxLDL uptake in macrophages from CXCL16–/– mice. Overall, macrophages from CXCL16–/– mice had significantly less OxLDL uptake than wild-type controls (Figure 4). At a concentration of 40 µg/mL DiI-OxLDL, macrophages from CXCL16–/– mice had &34% less cell-associated DiI-OxLDL than wild-type mice (Figure 4), which indicates that CXCL16 mediates a significant portion of OxLDL uptake in vivo.


Figure 4176948
View larger version (20K):
[in this window]
[in a new window]
  		Figure 4. Scavenger receptor activity of CXCL16 in vivo. Thioglycolate-elicited peritoneal macrophages from CXCL16+/+ and CXCL16–/– mice were incubated at 37°C for 5 hours with the indicated concentrations of DiI-OxLDL. After incubation, total cell-associated fluorescence was measured and expressed as micrograms of DiI-OxLDL per milligram of total cellular protein. Values shown are the average (±SD) of n=7 mice for each condition (*P<0.05). The experiment was performed twice with very similar results.

CXCL16–/–/LDLR–/– Mice Have Accelerated Atherosclerosis Progression
To assess the role of CXCL16 in atherosclerosis, we crossed CXCL16–/– mice with LDLR–/– mice and placed CXCL16–/–/ LDLR–/– and CXCL16+/+/LDLR–/– littermates on a Western diet for 6 or 10 weeks. There were no significant differences in total cholesterol or weight between CXCL16–/–/LDLR–/– and CXCL16+/+/LDLR–/– mice (Tables 1 and 2Down). Analysis of lipoprotein profiles for CXCL16–/–/LDLR–/– and CXCL16+/+/LDLR–/– mice showed indistinguishable particle size distributions after 6 or 10 weeks on the Western diet (data not shown).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Total Cholesterol Level of Mice Fed the Western Diet


View this table:
[in this window]
[in a new window]
  		TABLE 2. Weight of Mice Fed the Western Diet

To quantify lesion progression, en face analysis was performed. After 6 weeks, average lesion sizes were 46% larger in CXCL16–/–/LDLR–/– mice than in CXCL16+/+/LDLR–/– mice (P=0.025; Figure 5A). After 10 weeks, CXCL16–/–/LDLR–/– mice had an average lesion size 27% larger than CXCL16+/+/LDLR–/– mice (P=0.025). Very similar results were obtained when serial sections of the aortic root were stained with oil red O. At the 10-week time point, lesion size was 56% larger in the CXCL16–/–/LDLR–/– mice than in controls (Figure 5C).


Figure 5176948
View larger version (31K):
[in this window]
[in a new window]
  		Figure 5. Lesion area in CXCL16+/+/LDLR–/– and CXCL16–/–/LDLR–/– mice fed a Western diet for 6 or 10 weeks. A, Each data point represents the area of Sudan IV staining in the aortic arch of a single animal. At 6 weeks, mean lesion area was 46% larger in CXCL16–/–/LDLR–/– mice than in CXCL16+/+/LDLR–/– mice. At 10 weeks, mean lesion area was 27% larger in CXCL16–/–/LDLR–/– mice than in CXCL16+/+/LDLR–/– mice. Equal numbers of male and female mice were used at each condition. *P=0.025 vs controls. B, Representative photographs of average staining from 10-week CXCL16+/+/LDLR–/– and CXCL16–/–/LDLR–/– mice. C, Quantitation of lesion area in the aortic root in CXCL16+/+/LDLR–/– and CXCL16–/–/LDLR–/– mice fed a Western diet for 10 weeks. Mean lesion area was 57% larger in CXCL16–/–/LDLR–/– mice than in CXCL16+/+/LDLR–/– controls (*P=0.0087).

To determine whether macrophage recruitment was enhanced in CXCL16–/–/LDLR–/– mice, CD68 mRNA was quantified in the aortic arch. At 10 weeks, the level of CD68 mRNA was 44% greater in CXCL16–/–/LDLR–/– mice than in littermate controls (P=0.002; Figure 6A). Taken together, these data show that the absence of CXCL16 results in acceleration of atherosclerosis and enhanced macrophage recruitment to the aortic arch.


Figure 6176948
View larger version (16K):
[in this window]
[in a new window]
  		Figure 6. Macrophage and T-cell recruitment to the aortic arch. Levels of CD68 (A) and CXCR6 (B) mRNA were measured by real-time PCR in the aortic arch of CXCL16+/+/LDLR–/– and CXCL16–/–/LDLR–/– mice fed a Western diet for 6 or 10 weeks (*P=0.002 vs controls).

CXCR6+ Cell Recruitment to Lesions in CXCL16–/–/LDLR–/– Mice
To determine whether CXCR6+ cells are recruited to atherosclerosis-prone areas of the aorta under hypercholesterolemic conditions and whether the absence of CXCL16 affects the migration of these cells, we quantified CXCR6 mRNA in the aortic arch. CXCR6 mRNA was 123% more abundant in CXCL16+/+/LDLR–/– mice from 6 to 10 weeks on the Western diet (P=0.032; Figure 6B). There was a trend toward a decrease in the abundance of CXCR6 mRNA in CXCL16–/–/LDLR–/– versus CXCL16+/+/LDLR–/– mice at 10 weeks, although this difference did not reach statistical significance (P=0.3). The total number of T cells, as determined by mRNA for CD3, did not differ between CXCL16+/+/LDLR–/– and CXCL16–/–/LDLR–/– mice (data not shown).

Apoptotic Bodies in Lesions of CXCL16–/– Mice
SR-PSOX was initially identified by expression cloning based on binding to phosphatidylserine-coated plates.9 Because apoptotic cells express high levels of phosphatidylserine on their surface, we hypothesized that CXCL16–/– mice might be impaired in their ability to phagocytose apoptotic cells. In agreement with Harada et al,29 who observed an average of 1 TUNEL-positive cell per section in the aortic roots of LDLR-deficient mice fed a Western diet for 3.5 months, lesions from CXCL16+/+/LDLR–/– and CXCL16–/–/LDLR–/– mice exhibited few cells with evidence of DNA fragmentation (Figure 7A). However, CXCL16–/–/LDLR–/– mice contained 80% more apoptotic cells (P=0.026) throughout the plaque than CXCL16+/+/LDLR–/– controls (Figure 7B).


Figure 7176948
View larger version (20K):
[in this window]
[in a new window]
  		Figure 7. TUNEL analysis of CXCL16+/+/LDLR–/– and CXCL16–/–/LDLR–/– mice. A, Example shows 1 TUNEL-positive cell (white arrow) with clear nuclear fragmentation of DNA. B, Each point represents the average number of TUNEL-positive cells in 9 sections of the aortic root centered on the left coronary artery from each mouse. Apoptotic cells were 80% more numerous in aortic root sections from CXCL16–/–/LDLR–/– mice than CXCL16+/+/LDLR–/– mice (*P=0.026 vs controls).

Increased Inflammation in CXCL16–/– Mice
We used real-time PCR to quantify mRNA for MCP-1 and tumor necrosis factor-{alpha}. The expression of these cytokines after 10 weeks was increased by 264% (P=<0.0001; Figure 8A) and 76% (P=0.006; Figure 8B), respectively, in CXCL16–/–/–/–/LDLR–/– mice compared with controls. No significant differences between CXCL16–/–/LDLR–/– and CXCL16+/+/LDLR–/– mice were seen for interferon-{gamma}, interleukin-10, or transforming growth factor-ß1 (data not shown).


Figure 8176948
View larger version (17K):
[in this window]
[in a new window]
  		Figure 8. Inflammatory cytokines in the aortic arch. Levels of MCP-1 (A) and tumor necrosis factor-{alpha} (B) mRNA were measured by real-time PCR in the aortic arch of CXCL16+/+/LDLR–/– and CXCL16–/–/LDLR–/– mice fed a Western diet for 6 or 10 weeks. *P<0.0001; **P=0.006 vs controls.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
To study the role of CXCL16 in atherosclerosis, we generated mice in which the entire coding region of the gene was deleted by homologous recombination in ES cells. CXCL16–/– mice were viable and developed normally. Despite the reduced capacity of macrophages from CXCL16–/– mice to bind and internalize OxLDL, we found that CXCL16–/–/LDLR–/– mice fed a Western-type died had larger lesions, enhanced macrophage recruitment, and increased expression of inflammatory cytokine mRNAs in the aortic arch than littermate controls. These data suggest that the accumulation of oxidized lipids by CXCL16 may be an atheroprotective process and stand in contrast to studies performed in SR-A- and CD36-deficient mice that documented protection from disease.

Although human SR-PSOX/CXCL16 had been shown to bind OxLDL,9 it was not known whether this function was shared by the murine homolog. Here, we report a decrease in Cu2+-OxLDL binding and internalization of &34% in macrophages from CXCL16–/–mice compared with wild-type controls. This result closely approximates the values obtained in SR-A-deficient macrophages,3 which suggests that a significant portion of SR-A-independent accumulation of OxLDL is mediated by CXCL16.

In addition to its role as a scavenger receptor, CXCL16 functions as a chemokine and adhesion receptor for CXCR6+ cells.10,11 We observed a trend towards reduced abundance of CXCR6+ mRNA in the aortic arch of CXCL16–/–/LDLR–/– mice. These results suggest that CXCL16 contributes, along with other chemokines such as CXCL9 (Mig) and CXCL10 (IP-10),30 to the recruitment of activated T cells to lesions. However, despite the relative reduction in the presence of potentially proatherogenic CXCR6+ T cells, atherosclerotic lesions in CXCL16–/–/LDLR–/– mice were larger than in littermate controls.

The strong cell-surface expression of CXCL16 on macrophages in the plaque, combined with its high avidity for phosphatidylserine, prompted us to consider whether the atheroprotective role of CXCL16 was due to the binding and clearance of apoptotic cells, which are proatherogenic, from the lesion.31,32 Our finding of increased numbers of apoptotic cells in the atheroma of CXCL16–/– mice was consistent with such a mechanism, but macrophages from wild-type and CXCL16–/– mice bound and internalized apoptotic thymocytes equally well in both in vitro and in vivo assays (data not shown). These data suggest that clearance of apoptotic cells is not a major function of CXCL16.

The role of scavenger receptors in foam cell formation and atherogenesis is controversial. Mice deficient in SR class B type I have severely accelerated occlusive atherosclerotic disease,33 and it has been proposed that the hepatic uptake of HDL cholesterol mediated by this receptor is atheroprotective.34 We considered the possibility that CXCL16 might be playing a similar role but found no difference in HDL binding in macrophages from CXCL16–/– mice compared with wild-type controls (data not shown). In vitro OxLDL uptake assays do not address the possibility that in vivo, soluble CXCL16 might bind to OxLDL and block its uptake by other scavenger receptors; however, saturating levels of recombinant CXCL16 had no effect on OxLDL binding and accumulation (data not shown). Given our failure to detect differences in phagocytosis of apoptotic cells or HDL binding in CXCL16–/– macrophages, we hypothesize that the atheroprotection conferred by CXCL16 is due to its scavenger receptor activity.

In vitro experiments have shown that SR-A and CD36, the 2 most thoroughly characterized scavenger receptors, are responsible for the majority of Cu2+-oxidized LDL uptake. Macrophages from SR-A–/–/CD36–/– double-knockout mice have a 75% to 90% reduction in uptake of oxidized lipids and an almost complete absence of cholesterol ester formation.35 Early in vivo experiments with mice deficient in either SR-A or CD36 appeared to validate the hypothesis that the uptake of modified lipids by scavenger receptors is proatherogenic, with reductions in lesion sizes of 60% and 23% reported in SR-A–/– mice on the ApoE–/–36 and LDLR–/–37 backgrounds, respectively. Similar to results obtained for SR-A, dramatic decreases in lesion size at the aortic sinus (70% to 80%) were observed in CD36–/–/ApoE–/– mice5 and irradiated ApoE–/– mice reconstituted with CD36–/–/ApoE–/– bone marrow.38 Recently, Moore et al21 have reexamined this question and found either no protection against or in some cases frank exacerbation of atherosclerosis in SR-A–/–/ApoE–/– and CD36–/–/ApoE–/– mice, despite profound reductions in the in vitro uptake of oxidized lipids. The authors postulated that mixed 129/C57BL6 genetic backgrounds present in the earlier studies confounded the results and led to misinterpretation of the role of scavenger receptors in atherosclerotic lesion formation. In the present study, using LDLR–/– mice extensively backcrossed onto C57BL6, we find that the absence of CXCL16 significantly increase increases lesion size and the abundance of inflammatory cytokines.

These data support the notion that scavenger receptor activity is potentially atheroprotective; however, details concerning the mechanism are unclear. It was postulated by Moore et al21 and in a related commentary39 that nonreceptor-mediated accumulation of native LDL by aggregation, Fc-mediated binding, and/or macropinocytosis might account for the formation of foam cells and lesion development in the absence of SR-A or CD36. The uptake of aggregated native LDL is mediated by the LDL receptor pathway,40 and thus, the present findings in LDLR–/– mice suggest that increased lesion formation occurs in the absence of both CXCL16 scavenger receptor activity and internalization of aggregated LDL. We also observed no differences in the plasma levels of immunoglobulin G or immunoglobulin M antibodies to Cu2+-OxLDL or malondialdehyde-modified LDL (data not shown). Although it is unclear whether circulating titers accurately reflect the potential of antibodies to coat OxLDL within the vessel wall, it appears that there is no overt compensation in CXCL16–/–/LDLR–/– mice to produce excess anti-OxLDL antibodies. The uptake of LDL by fluid-phase macropinocytosis, as described by Kruth et al,41 remains a possible route of scavenger receptor-independent foam cell formation in CXCL16–/– mice.

We considered the possibility that genetic deletion of CXCL16 may have altered the expression and function of other scavenger receptors, but the strong induction of cytokines, particularly MCP-1, which is upregulated by vessel wall cells by minimally oxidized LDL,42 suggests that alternate scavenger receptors are not clearing the blood of modified lipids. Moreover, direct measurement of SR-A and CD36 mRNA levels revealed no differences between CXCL16–/– and wild-type macrophages (data not shown). It remains possible that CXCL16 specifically mediates the uptake and sequestration of a particularly atherogenic species of lipoprotein that is not present in Cu+2-modified LDL.

Recently, 2 human studies have addressed the correlation of CXCL16 expression in coronary artery disease patients. In the first study, the effect of an alanine-to-valine polymorphism of CXCL16 that is postulated to influence the release of CXCL16 from the cell surface was examined in post-myocardial infarction subjects.43 The results showed that carriers of the polymorphism had more severe coronary artery stenosis. In another analysis, a cohort of stable angina pectoris patients were found to have significantly lower circulating levels of CXCL16 protein than healthy control subjects.44 These patient studies further underscore the link between CXCL16 and coronary artery disease and indicate that CXCL16 may be atheroprotective in the context of human disease.

In summary, we have shown that genetic deletion of the chemokine and scavenger receptor CXCL16 exacerbates atherosclerosis in LDLR–/– mice, even though CXCL16–/– macrophages exhibit marked reductions in the uptake of oxidized lipids in vitro. Atherosclerotic lesions in CXCL16–/– mice have increased numbers of macrophages and apoptotic cells and upregulation of inflammatory cytokine mRNA. These data support the notion that scavenger receptor-mediated clearance of modified lipids is an atheroprotective function and suggest that therapeutic blockade of such receptors might be detrimental to the treatment of the disease.


*    Acknowledgments
 
We thank Dr Joseph Witzum for analysis of oxidized lipoproteins, Sarah Slaymaker for assistance with real-time PCR assays, Dr Robert Pitas for a critical reading of the manuscript, John Carroll for preparation of the figures, Gary Howard and Stephen Ordway for editorial assistance, and Mijoung Chang for manuscript preparation.

Sources of Funding

This work was funded in part by NIH grants R01 HL52773-11 and R01 HL63894-05 (to Dr Charo) and F32 HL74514-02 (to Dr Aslanian).

Disclosures

None.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Parthasarathy S, Steinberg D, Witztum JL. The role of oxidized low-density lipoproteins in the pathogenesis of atherosclerosis. Annu Rev Med. 1992; 43: 219–225.[CrossRef][Medline] [Order article via Infotrieve]
  2. Steinberg D. Low density lipoprotein oxidation and its pathobiological significance. J Biol Chem. 1997; 272: 20963–20966.[Free Full Text]
  3. Lougheed M, Lum CM, Ling W, Suzuki H, Kodama T, Steinbrecher U. High affinity saturable uptake of oxidized low density lipoprotein by macrophages from mice lacking the scavenger receptor class A type I/II. J Biol Chem. 1997; 272: 12938–12944.[Abstract/Free Full Text]
  4. Podrez EA, Febbraio M, Sheibani N, Schmitt D, Silverstein RL, Hajjar DP, Cohen PA, Frazier WA, Hoff HF, Hazen SL. Macrophage scavenger receptor CD36 is the major receptor for LDL modified by monocyte-generated reactive nitrogen species. J Clin Invest. 2000; 105: 1095–1108.[Medline] [Order article via Infotrieve]
  5. Febbraio M, Podrez EA, Smith JD, Hajjar DP, Hazen SL, Hoff HF, Sharma K, Silverstein RL. Targeted disruption of the class B scavenger receptor CD36 protects against atherosclerotic lesion development in mice. J Clin Invest. 2000; 105: 1049–1056.[Medline] [Order article via Infotrieve]
  6. Moriwaki H, Kume N, Sawamura T, Aoyama T, Hoshikawa H, Ochi H, Nishi E, Masaki T, Kita T. Ligand specificity of LOX-1, a novel endothelial receptor for oxidized low density lipoprotein. Arterioscler Thromb Vasc Biol. 1998; 18: 1541–1547.[Abstract/Free Full Text]
  7. Ramprasad MP, Terpstra V, Kondratenko N, Quehenberger O, Steinberg D. Cell surface expression of mouse macrosialin and human CD68 and their role as macrophage receptors for oxidized low density lipoprotein. Proc Natl Acad Sci U S A. 1996; 93: 14833–14838.[Abstract/Free Full Text]
  8. Ramprasad MP, Fischer W, Witztum JL, Sambrano GR, Quehenberger O, Steinberg D. The 94- to 97-kDa mouse macrophage membrane protein that recognizes oxidized low density lipoprotein and phosphatidylserine-rich liposomes is identical to macrosialin, the mouse homologue of human CD68. Proc Natl Acad Sci U S A. 1995; 92: 9580–9584.[Abstract/Free Full Text]
  9. Shimaoka T, Kume N, Minami M, Hayashida K, Kataoka H, Kita T, Yonehara S. Molecular cloning of a novel scavenger receptor for oxidized low density lipoprotein, SR-PSOX, on macrophages. J Biol Chem. 2000; 275: 40663–40666.[Abstract/Free Full Text]
  10. Wilbanks A, Zondlo SC, Murphy K, Mak S, Soler D, Langdon P, Andrew DP, Wu L, Briskin M. Expression cloning of the STRL33/BONZO/TYMSTR ligand reveals elements of CC, CXC, and CX3C chemokines. J Immunol. 2001; 166: 5145–5154.[Abstract/Free Full Text]
  11. Matloubian M, David A, Engel S, Ryan JE, Cyster JG. A transmembrane CXC chemokine is a ligand for HIV-coreceptor Bonzo. Nat Immunol. 2000; 1: 298–304.[CrossRef][Medline] [Order article via Infotrieve]
  12. Hofnagel O, Luechtenborg B, Plenz G, Robenek H. Expression of the novel scavenger receptor SR-PSOX in cultured aortic smooth muscle cells and umbilical endothelial cells. Arterioscler Thromb Vasc Biol. 2002; 22: 710–711.[Free Full Text]
  13. Minami M, Kume N, Shimaoka T, Kataoka H, Hayashida K, Akiyama Y, Nagata I, Ando K, Nobuyoshi M, Hanyuu M, Komeda M, Yonehara S, Kita T. Expression of SR-PSOX, a novel cell-surface scavenger receptor for phosphatidylserine and oxidized LDL in human atherosclerotic lesions. Arterioscler Thromb Vasc Biol. 2001; 21: 1796–1800.[Abstract/Free Full Text]
  14. Wuttge DM, Zhou X, Sheikine Y, Wågsäter D, Stemme V, Hedin U, Stemme S, Hansson GK, Sirsjö A. CXCL16/SR-PSOX is an interferon-{gamma}–regulated chemokine and scavenger receptor expressed in atherosclerotic lesions. Arterioscler Thromb Vasc Biol. 2004; 24: 750–755.[Abstract/Free Full Text]
  15. Shimaoka T, Nakayama T, Fukumoto N, Kume N, Takahashi S, Yamaguchi J, Minami M, Hayashida K, Kita T, Ohsumi J, Yoshie O, Yonehara S. Cell surface-anchored SR-PSOX/CXC chemokine ligand 16 mediates firm adhesion of CXC chemokine receptor 6-expressing cells. J Leukoc Biol. 2004; 75: 267–274.[Abstract/Free Full Text]
  16. Shimaoka T, Nakayama T, Kume N, Takahashi S, Yamaguchi J, Minami M, Hayashida K, Kita T, Ohsumi J, Yoshie O, Yonehara S. Cutting edge: SR-PSOX/CXC chemokine ligand 16 mediates bacterial phagocytosis by APCs through its chemokine domain. J Immunol. 2003; 171: 1647–1651.[Abstract/Free Full Text]
  17. Gough PJ, Garton KJ, Wille PT, Rychlewski M, Dempsey PJ, Raines EW. A disintegrin and metalloproteinase 10-mediated cleavage and shedding regulates the cell surface expression of CXC chemokine ligand 16. J Immunol. 2004; 172: 3678–3685.[Abstract/Free Full Text]
  18. Abel S, Hundhausen C, Mentlein R, Schulte A, Berkhout TA, Broadway N, Hartmann D, Sedlacek R, Dietrich S, Muetze B, Schuster B, Kallen K-J, Saftig P, Rose-John S, Ludwig A. The transmembrane CXC-chemokine ligand 16 is induced by IFN-{gamma} and TNF-{alpha} and shed by the activity of the disintegrin-like metalloproteinase ADAM10. J Immunol. 2004; 172: 6362–6372.[Abstract/Free Full Text]
  19. Unutmaz D, Xiang W, Sunshine MJ, Campbell J, Butcher E, Littman DR. The primate lentiviral receptor Bonzo/STRL33 is coordinately regulated with CCR5 and its expression pattern is conserved between human and mouse. J Immunol. 2000; 165: 3284–3292.[Abstract/Free Full Text]
  20. Kim CH, Kunkel EJ, Boisvert J, Johnston B, Campbell JJ, Genovese MC, Greenberg HB, Butcher EC. Bonzo/CXCR6 expression defines type 1–polarized T-cell subsets with extralymphoid tissue homing potential. J Clin Invest. 2001; 107: 595–601.[Medline] [Order article via Infotrieve]
  21. Moore KJ, Kunjathoor VV, Koehn SL, Manning JJ, Tseng AA, Silver JM, McKee M, Freeman MW. Loss of receptor-mediated lipid uptake via scavenger receptor A or CD36 pathways does not ameliorate atherosclerosis in hyperlipidemic mice. J Clin Invest. 2005; 115: 2192–2201.[CrossRef][Medline] [Order article via Infotrieve]
  22. Haskell CA, Hancock WW, Salant DJ, Gao W, Csizmadia V, Peters W, Faia K, Fituri O, Rottman JB, Charo IF. Targeted deletion of CX3CR1 reveals a role for fractalkine in cardiac allograft rejection. J Clin Invest. 2001; 108: 679–688.[CrossRef][Medline] [Order article via Infotrieve]
  23. Meiner VL, Cases S, Myers HM, Sande ER, Bellosta S, Schambelan M, Pitas RE, McGuire J, Herz J, Farese RV Jr. Disruption of the acyl-CoA:cholesterol acyltransferase gene in mice: evidence suggesting multiple cholesterol esterification enzymes in mammals. Proc Natl Acad Sci U S A. 1996; 93: 14041–14046.[Abstract/Free Full Text]
  24. Lesnik P, Haskell CA, Charo IF. Decreased atherosclerosis in CX3CR1–/– mice reveals a role for fractalkine in atherogenesis. J Clin Invest. 2003; 111: 333–340.[CrossRef][Medline] [Order article via Infotrieve]
  25. Aslanian AM, Chapman HA, Charo IF. Transient role for CD1d-restricted natural killer T cells in the formation of atherosclerotic lesions. Arterioscler Thromb Vasc Biol. 2005; 25: 628–632.[Abstract/Free Full Text]
  26. Trogan E, Choudhury RP, Dansky HM, Rong JX, Breslow JL, Fisher EA. Laser capture microdissection analysis of gene expression in macrophages from atherosclerotic lesions of apolipoprotein E-deficient mice. Proc Natl Acad Sci U S A. 2002; 99: 2234–2239.[Abstract/Free Full Text]
  27. Overbergh L, Giulietti A, Valckx D, Decallonne R, Bouillon R, Mathieu C. The use of real-time reverse transcriptase PCR for the quantification of cytokine gene expression. J Biomol Tech. 2003; 14: 33–43.[Medline] [Order article via Infotrieve]
  28. Teupser D, Thiery J, Walli AK, Seidel D. Determination of LDL- and scavenger-receptor activity in adherent and non-adherent cultured cells with a new single-step fluorometric assay. Biochim Biophys Acta. 1996; 1303: 193–198.[Medline] [Order article via Infotrieve]
  29. Harada K, Chen Z, Ishibashi S, Osuga J-I, Yagyu H, Ohashi K, Yahagi N, Shionoiri F, Sun L, Yazaki Y, Yamada N. Apoptotic cell death in atherosclerotic plaques of hyperlipidemic knockout mice. Atherosclerosis. 1997; 135: 235–239.[CrossRef][Medline] [Order article via Infotrieve]
  30. Mach F, Sauty A, Iarossi AS, Sukhova GK, Neote K, Libby P, Luster AD. Differential expression of three T lymphocyte-activating CXC chemokines by human atheroma-associated cells. J Clin Invest. 1999; 104: 1041–1050.[Medline] [Order article via Infotrieve]
  31. Takahashi T, Tanaka M, Brannan CI, Jenkins NA, Copeland NG, Suda T, Nagata S. Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand. Cell. 1994; 76: 969–976.[CrossRef][Medline] [Order article via Infotrieve]
  32. Aprahamian T, Rifkin I, Bonegio R, Hugel B, Freyssinet J-M, Sato K, Castellot JJ Jr, Walsh K. Impaired clearance of apoptotic cells promotes synergy between atherogenesis and autoimmune disease. J Exp Med. 2004; 199: 1121–1131.[Abstract/Free Full Text]
  33. Braun A, Trigatti BL, Post MJ, Sato K, Simons M, Edelberg JM, Rosenberg RD, Schrenzel M, Krieger M. Loss of SR-B1 expression leads to the early onset of occlusive atherosclerotic coronary artery disease, spontaneous myocardial infarctions, severe cardiac dysfunction, and premature death in apolipoprotein E-deficient mice. Circ Res. 2002; 90: 270–276.[Abstract/Free Full Text]
  34. Trigatti BL, Krieger M, Rigotti A. Influence of the HDL receptor SR-BI on lipoprotein metabolism and atherosclerosis. Arterioscler Thromb Vasc Biol. 2003; 23: 1732–1738.[Abstract/Free Full Text]
  35. Kunjathoor VV, Febbraio M, Podrez EA, Moore KJ, Andersson L, Koehn S, Rhee JS, Silverstein R, Hoff HF, Freeman MW. Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages. J Biol Chem. 2002; 277: 49982–49988.[Abstract/Free Full Text]
  36. Suzuki H, Kurihara Y, Takeya M, Kamada N, Kataoka M, Jishage K, Ueda O, Sakaguchi H, Higashi T, Suzuki T, Takashima Y, Kawabe Y, Cynshi O, Wada Y, Honda M, Kurihara H, Aburatani H, Doi T, Matsumoto A, Azuma S, Noda T, Toyoda Y, Itakura H, Yazaki Y, Horiuchi S, Takahashi K, Kruijt JK, van Berkel TJC, Steinbrecher UP, Ishibashi S, Maeda N, Gordon S, Kodama T. A role for macrophage scavenger receptors in atherosclerosis and susceptibility to infection. Nature. 1997; 386: 292–296.[CrossRef][Medline] [Order article via Infotrieve]
  37. Sakaguchi H, Takeya M, Suzuki H, Hakamata H, Kodama T, Horiuchi S, Gordon S, van der Laan LJW, Kraal G, Ishibashi S, Kitamura N, Takahashi K. Role of macrophage scavenger receptors in diet-induced atherosclerosis in mice. Lab Invest. 1998; 78: 423–434.[Medline] [Order article via Infotrieve]
  38. Febbraio M, Guy E, Silverstein RL. Stem cell transplantation reveals that absence of macrophage CD36 is protective against atherosclerosis. Arterioscler Thromb Vasc Biol. 2004; 24: 2333–2338.[Abstract/Free Full Text]
  39. Witztum JL. You are right too! J Clin Invest. 2005; 115: 2072–2075.[CrossRef][Medline] [Order article via Infotrieve]
  40. Khoo JC, Miller E, McLoughlin P, Steinberg D. Enhanced macrophage uptake of low density lipoprotein after self-aggregation. Arteriosclerosis. 1988; 8: 348–358.[Abstract/Free Full Text]
  41. Kruth HS, Jones NL, Huang W, Zhao B, Ishii I, Chang J, Combs CA, Malide D, Zhang WY. Macropinocytosis is the endocytic pathway that mediates macrophage foam cell formation with native low density lipoprotein. J Biol Chem. 2005; 280: 2352–2360.[Abstract/Free Full Text]
  42. Reddy S, Hama S, Grijalva V, Hassan K, Mottahedeh R, Hough G, Wadleigh DJ, Navab M, Fogelman AM. Mitogen-activated protein kinase phosphatase 1 activity is necessary for oxidized phospholipids to induce monocyte chemotactic activity in human aortic endothelial cells. J Biol Chem. 2001; 276: 17030–17035.[Abstract/Free Full Text]
  43. Lundberg GA, Kellin A, Samnegård A, Lundman P, Tornvall P, Dimmeler S, Zeiher AM, Hamsten A, Hansson GK, Eriksson P. Severity of coronary artery stenosis is associated with a polymorphism in the CXCL16/SR-PSOX gene. J Intern Med. 2005; 257: 415–422.[CrossRef][Medline] [Order article via Infotrieve]
  44. Sheikine Y, Bang CS, Nilsson L, Samnegard A, Hamsten A, Jonasson L, Eriksson P, Sirsjo A. Decreased plasma CXCL16/SR-PSOX concentration is associated with coronary artery disease. Atherosclerosis. Published online before print, December 2005.
 

CLINICAL PERSPECTIVE

Accumulation of lipid-laden macrophages in the blood vessel wall leads to fatty streak formation and is an early harbinger of the development of atherosclerosis. Although the mechanism of lipid accumulation by the macrophages is not well understood, a group of receptors that take up oxidized lipids in an unregulated manner, the "scavenger receptors," have been implicated in promoting fatty streak formation and atherosclerosis. Results in this report indicate that loss of one such receptor, CXCL16, unexpectedly exacerbates atherosclerosis. These data, and similar results obtained with mice lacking other key scavenger receptors, suggest that pharmacological blockade of scavenger receptors may not be beneficial.




This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
C. Smith, B. Halvorsen, K. Otterdal, T. Waehre, A. Yndestad, B. Fevang, W. J. Sandberg, U. M. Breland, S. S. Froland, E. Oie, et al.
High levels and inflammatory effects of soluble CXC ligand 16 (CXCL16) in coronary artery disease: down-regulatory effects of statins
Cardiovasc Res, July 1, 2008; 79(1): 195 - 203.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. Meijer, J. Ogink, B. Kreike, D. Nuyten, K. E. de Visser, and E. Roos
The Chemokine Receptor CXCR6 and Its Ligand CXCL16 Are Expressed in Carcinomas and Inhibit Proliferation
Cancer Res., June 15, 2008; 68(12): 4701 - 4708.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
D. M. Okamura, J. M. Lopez-Guisa, K. Koelsch, S. Collins, and A. A. Eddy
Atherogenic scavenger receptor modulation in the tubulointerstitium in response to chronic renal injury
Am J Physiol Renal Physiol, August 1, 2007; 293(2): F575 - F585.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
S. Collot-Teixeira, J. Martin, C. McDermott-Roe, R. Poston, and J. L. McGregor
CD36 and macrophages in atherosclerosis
Cardiovasc Res, August 1, 2007; 75(3): 468 - 477.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
114/6/583    most recent
CIRCULATIONAHA.105.540583v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Aslanian, A. M.
Right arrow Articles by Charo, I. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aslanian, A. M.
Right arrow