Activation of CXCR7 Limits Atherosclerosis and Improves Hyperlipidemia by Increasing Cholesterol Uptake in Adipose Tissue
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Abstract
Background—
The aim of this study was to determine the role of the chemokine receptor CXCR7 in atherosclerosis and vascular remodeling. CXCR7 is the alternative receptor of CXCL12, which regulates stem cell–mediated vascular repair and limits atherosclerosis via its receptor, CXCR4.
Methods and Results—
Wire-induced injury of the carotid artery was performed in mice with a ubiquitous, conditional deletion of CXCR7 and in mice treated with the synthetic CXCR7 ligand CCX771. The effect of CCX771 treatment on atherosclerosis was studied in apolipoprotein E–deficient (Apoe−/−) mice fed a high-fat diet for 12 weeks. Lipoprotein fractions were quantified in the plasma of Apoe−/− mice by fast protein liquid chromatography. Uptake of DiI-labeled very low-density lipoprotein to adipose tissue was determined by 2-photon microscopy. We show that genetic deficiency of Cxcr7 increased neointima formation and lesional macrophage accumulation in hyperlipidemic mice after vascular injury. This was related to increased serum cholesterol levels and subsequent hyperlipidemia-induced monocytosis. Conversely, administration of the CXCR7 ligand CCX771 to Apoe−/− mice inhibited lesion formation and ameliorated hyperlipidemia after vascular injury and during atherosclerosis. Treatment with CCX771 reduced circulating very low-density lipoprotein levels but not low-density lipoprotein or high-density lipoprotein levels and increased uptake of very low-density lipoprotein into Cxcr7-expressing white adipose tissue. This effect of CCX771 was associated with an enhanced lipase activity and reduced expression of Angptl4 in adipose tissue.
Conclusions—
CXCR7 regulates blood cholesterol by promoting its uptake in adipose tissue. This unexpected cholesterol-lowering effect of CXCR7 is beneficial for atherosclerotic vascular diseases, presumably via amelioration of hyperlipidemia-induced monocytosis, and can be augmented with a synthetic CXCR7 ligand.
Introduction
Chemokines, acting through various mechanisms including the recruitment of immune cells and smooth muscle progenitor cells (SPCs) to the vessel wall,1,2 are crucial for vascular remodeling and atherosclerosis. In addition, chemokines are critical for monocyte and neutrophil homeostasis. Hyperlipidemia-induced monocytosis results from the combined action of various chemokine receptors, such as CCR2, which are expressed by immune cells (including monocytes), and facilitates atherosclerosis.3–5 By contrast, the CXCL12 receptor, CXCR4, protects against atherosclerosis by controlling the mobilization of neutrophils,6 whereas the mobilization and recruitment of SPCs by CXCL12 during vascular repair aggravates neointima formation, which indicates a context- specific role for this chemokine-receptor axis in arterial remodeling.7,8 Accordingly, increased levels of CXCL12 and SPCs are observed in patients with severe cardiac allograft vasculopathy.9 Systemic treatment with CXCL12 induces the release of SPCs, which accumulate in atherosclerotic lesions and thus lead to plaque stabilization.10 Whereas most of the effects of CXCL12 are linked to its interaction with CXCR4, the function of the alternative CXCL12 receptor, CXCR7, in atherogenesis and vascular remodeling is unclear.
Clinical Perspective on p 1253
CXCR7 binds to both CXCL12 and CXCL11 and promotes the growth and adhesion of tumor cells.11,12 Genetic deletion of Cxcr7 in mice results in a high level of perinatal lethality and abnormal cardiovascular development.13,14 In contrast to other chemokine receptors, CXCR7 is not expressed on leukocytes.15 Although structurally characterized as a G-protein–coupled receptor, CXCR7 does not induce classic G-protein–coupled receptor signaling events but can trigger the recruitment of β-arrestin-2, which leads to receptor internalization or activation of downstream signaling pathways, for example, mitogen-activated protein kinases.16,17 The interaction between CXCR7 and β-arrestin-2 controls the bioavailability of extracellular CXCL12.18,19 Furthermore, cross-regulation of CXCR4 by CXCR7 can affect CXCR4- dependent Gαi protein activation.13,20 Interestingly, the highly selective synthetic CXCR7 ligand CCX771 antagonizes the binding of CXCL12 to CXCR7 but induces the association of β-arrestin-2 with CXCR7 more effectively than its endogenous ligands.16 In light of this complex interplay between CXCL12, CXCR4, and CXCR7, we aimed to dissect the role played by CXCR7 during vascular remodeling and atherosclerosis and to assess the potential interactions between CXCR7 and the CXCL12/CXCR4 axis in vascular disease.
Here, we identify a key role for CXCR7 in the regulation of serum cholesterol levels by enhancing the uptake of very low-density lipoprotein (VLDL) and increasing the uptake of cholesterol in adipose tissue. The cholesterol-lowering effect of CXCR7 was associated with a reduction in hyperlipidemia- induced monocytosis and with decreased arterial lesion formation and macrophage accumulation.
Methods
Mice
Cxcr7LacZ/+ and Cxcr7flox mice (generated on a pure C57Bl/6 background) were provided by ChemoCentryx, Inc (Mountain View, CA).15CAG-CreERTM, 21 apolipoprotein E–deficient (Apoe−/−), and low-density lipoprotein receptor–deficient (Ldlr−/−) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice with a ubiquitous tamoxifen-inducible Cxcr7 knockout were generated by breeding CAG-CreERTMCxcr7WT/WT with Cxcr7flox/flox mice. CAG-CreERTMCxcr7flox/flox mice were further crossed with Apoe−/− mice to obtain CAG-CreERTMCxcr7flox/floxApoe−/− mice.
Animal Models
Wire injury of carotid arteries was performed in CAG-CreERTMCxcr7flox/flox, CAG-CreERTMCxcr7flox/floxApoe−/−, and Apoe−/− mice fed a high-cholesterol diet (HCD; 21% fat and 0.15% cholesterol; Altromin, Lage, Germany), as described previously.7 Diet-induced atherosclerosis was induced by feeding Apoe−/− mice an HCD for 12 weeks. Treatment with CCX771 (ChemoCentryx, Inc.) was given via a daily subcutaneous injection (10 mg·kg−1·d−1 dissolved in 10% Captisol [Ligand Pharmaceuticals, La Jolla, CA]; 100 μL per injection). Control mice were injected with Captisol alone (10%; 100 μL/mouse SC). Mice expressing tamoxifen-inducible Cre recombinase and the floxed Cxcr7 allele, as well as corresponding control mice, were treated with tamoxifen (1.5 mg per 20 g of body weight; Sigma- Aldrich Chemie GmbH, Munich, Germany) dissolved in neutral oil (Migyol, Sasol Germany GmbH, Hamburg, Germany) for 5 consecutive days (intraperitoneal injection). Wire injury of the carotid arteries was performed 15 days after the last injection of tamoxifen. One week before vascular injury, splenectomy was performed in some Apoe−/− mice after ligation of the splenic vessels. Bone marrow (BM) cells were harvested from femurs of CAG-Cre+Cxcr7floxApoe−/− or CAG-Cre−Cxcr7floxApoe−/− mice and injected (4×106 cells) into the tail vein of Apoe−/− recipient mice 24 hours after total body irradiation (2×6 Gy, 4-hour interval). Lipid metabolism was studied in Apoe−/− and Ldlr−/− mice fed an HCD after 4 weeks of treatment with CCX771 or Captisol.
The aorta and carotid arteries were harvested after in situ perfusion fixation with 4% paraformaldehyde (Carl Roth GmbH, Karlsruhe, Germany) or PAXgene (Qiagen GmbH, Hilden, Germany). All animal experiments were reviewed and approved by the local authorities (NRW LANUV [State Agency for Nature, Environment, and Consumer Protection of North Rhein-Westphalia]) according to German animal protection laws.
Histomorphometry
Serial (4 μm thick) cross sections of the left common carotid arteries (within a standardized distance [80–320 μm] from the bifurcation) and the aortic roots were obtained and subsequently stained with modified Movat pentachrome or elastic van Gieson stain (4–6 sections per mouse). Lesions and medial areas were determined by planimetry of digitized images with Diskus software (Hilgers, Königswinter, Germany). The thoracoabdominal aortas were prepared en face and stained with oil red O. The oil red O–stained area and total aortic surface area were quantified with ImageJ software.
Immunostaining
Expression of CXCR7 was studied in 4% paraformaldehyde-fixed cryosections after antigen retrieval with citrate buffer (20 minutes heating in a microwave oven) by incubation with an antibody against CXCR7 (clone 11G8, a gift from ChemoCentryx, Inc). A secondary fluorescein isothiocyanate-conjugated anti-mouse antibody (polyclonal goat; Jackson ImmunoResearch Europe Ltd, Suffolk, United Kingdom) was used to visualize the primary antibody. Sections from Cxcr7−/− mice were used for negative control staining.
The composition of the lesions was determined by immunostaining of α-smooth muscle actin (α-SMA; clone 1A4; Dako Deutschland GmbH, Hamburg, Germany) and macrophage-specific Mac2 (clone M3/38; Cedarlane, Burlington, Canada) in paraffin- embedded carotid arteries and aortic roots. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Vectashield, Vector Laboratories, Burlingame, CA). Nonspecific antibodies (Santa Cruz Biotechnology, Dallas, TX) were used for negative control staining. A secondary DyLight 594–, fluorescein isothiocyanate-, or Cy3-conjugated antibody (polyclonal goat; Jackson ImmunoResearch Europe Ltd) was used to visualize the primary antibody. Digital images from ≥4 sections per mouse were recorded with a fluorescence microscope (Leica DM 2500; Leica Microsystems GmbH, Wetzlar, Germany) equipped with a charge-coupled device camera (JVC Deutschland GmbH, Friedberg, Germany) with Diskus software. The numbers of Mac2-stained macrophages and α-SMA–stained smooth muscle cells (SMCs) within the lesions were determined by counting the number of nuclei surrounded by positive staining for Mac2 and α-SMA, respectively.
Statistical Analysis
Data represent the mean±SEM. Paired or unpaired Student t test, 1-way ANOVA (followed by the Newman-Keuls post test), or 2-way ANOVA (followed by an uncorrected Fisher least significant difference test) was used for statistical comparison between groups (Prism 5, GraphPad Software, La Jolla, CA). P values <0.05 were considered statistically significant.
Results
Cxcr7 Expression Patterns
Transcripts for both Cxcr7 and Cxcr4 were highly expressed in lung, spleen, and adipose tissue compared with liver and skeletal muscle (Figure IA and IB in the online-only Data Supplement). In contrast to Cxcr4, expression of Cxcr7 was also higher in arterial tissue, heart, and kidney than in blood and BM cells (Figure IA and IB in the online-only Data Supplement). CXCR7 was detected on cultured endothelial cells by flow cytometry but not on murine peripheral leukocytes, BM cells, or platelets (Figure IC in the online-only Data Supplement). X-gal staining of tissues from adult Cxcr7+/LacZ mice showed expression of Cxcr7 in the marginal or sinusoidal zones of the spleen, in epithelial or endothelial cells of the kidney and lung, in cells of the intestinal lamina propria, in adipocytes, and in cardiomyocytes but not in hepatocytes (Figure II in the online-only Data Supplement). Moreover, activation of the Cxcr7 promoter was observed in endothelial and adventitial cells of the aorta and in the endothelium covering the aortic valves (Figure II in the online-only Data Supplement). Expression of CXCR7 protein was detected in spleen, kidney, and endothelium of the aortic valves by immunostaining (Figure III in the online-only Data Supplement) but not in the carotid artery wall (Figure IV in the online-only Data Supplement).
Conditional Cxcr7 Deletion Exacerbates Neointimal Hyperplasia
Ubiquitous deletion of Cxcr7 was induced in Apoe−/− (CAG- Cre+Cxcr7floxApoe−/−) mice by tamoxifen treatment. Knockdown of Cxcr7 in CAG-Cre+Cxcr7flox mice was verified by CXCR7 immunostaining of the spleen (Figure 1A). Next, wire injury of the carotid artery was performed in CAG- Cre+Cxcr7floxApoe−/− mice fed an HCD. The neointimal but not the medial area was increased at 28 days after vascular injury in CAG- Cre+Cxcr7floxApoe−/− mice compared with that in tamoxifen-treated CAG- Cre−Cxcr7floxApoe−/− mice (Figure 1B). The lengths of the internal and external elastic laminae were similar in both groups (Figure 1B). The number of Mac2+ macrophages was higher in CAG- Cre+Cxcr7floxApoe−/− mice than in CAG- Cre−Cxcr7floxApoe−/− mice, as determined by Mac2 immunostaining (Figure 1C). Although the number of SMCs in the neointima tended to increase in CAG-Cre+Cxcr7floxApoe−/− mice, the difference was not statistically significant (Figure 1D). Moreover, the α-SMA+ neointimal area was comparable between CAG-Cre+Cxcr7floxApoe−/− and CAG-Cre−Cxcr7floxApoe−/− mice (Figure 1D).
Figure 1. Cxcr7 deficiency increases neointimal macrophage accumulation. A, CXCR7 immunostaining in spleen sections of apolipoprotein E–deficient mice (Apoe−/−) in which Cxcr7 was deleted by activation of a ubiquitously expressed Cre recombinase (CAG-Cre+Cxcr7floxApoe−/− mice) 2 weeks after tamoxifen treatment. Tamoxifen-treated Cxcr7floxApoe−/− mice not expressing Cre recombinase were used as controls (CAG-Cre−CxcrfloxApoe−/− mice). Representative images are shown. Arrows indicate positive staining for CXCR7. n=8 per group. B, Quantification of neointimal and medial areas and of internal (IEL) and external (EEL) elastic laminae length 28 days after wire injury to carotid arteries in CAG-Cre−Cxcr7floxApoe−/− and CAG-Cre+Cxcr7floxApoe−/− mice. C, Quantification of neointimal macrophage numbers and of Mac2-positive area was performed by Mac2 immunostaining (green; n=6–8 per group). D, Neointimal smooth muscle cell number and smooth muscle actin (SMA)–positive area were studied in the neointima with α-SMA immunostaining (green; n=6–8 per group). Nuclei were counterstained with DAPI (blue). Scale bars, 200 μm (A) and 100 μm (B–D). *P<0.05.
Mobilization of SPCs after vascular injury is mediated by CXCL121; however, the injury-induced increase in circulating Sca-1+Lin− cells was unaffected in CAG-Cre+Cxcr7floxApoe−/− mice compared with that in CAG-Cre−Cxcr7floxApoe−/− mice (Figure 2A). Moreover, CXCL12 levels in the plasma and BM of CAG-Cre+Cxcr7floxApoe−/− mice were higher than those in CAG-Cre−Cxcr7floxApoe−/− mice at 28 days after injury (Figure 2B). The expression of Cxcr4 mRNA in various tissues, such as blood cells, aorta, and adipose tissue, was not altered in CAG-Cre+Cxcr7floxApoe−/− mice (Figure 2C). Notably, neointima formation did not differ between CAG-Cre+Cxcr7floxApoe+/+ mice and CAG-Cre−Cxcr7floxApoe+/+ mice fed an HCD diet (Figure 2D), which indicates that the effect of CXCR7 is related to serum cholesterol levels. Apoe−/− mice showed markedly higher serum cholesterol levels than Apoe+/+ mice (Figure 2E). Although serum cholesterol and triglyceride concentrations were further increased in CAG-Cre+Cxcr7floxApoe−/− mice at 28 days after injury compared with those in CAG-Cre−Cxcr7floxApoe−/− mice, cholesterol and triglyceride levels in CAG-Cre+Cxcr7floxApoe+/+ mice and CAG-Cre−Cxcr7floxApoe+/+ mice did not differ (Figure 2E). Similarly, circulating monocyte counts were higher in Apoe−/− mice than in Apoe+/+ mice and were further increased in CAG-Cre+Cxcr7floxApoe−/− mice (but not in CAG-Cre+Cxcr7floxApoe+/+ mice) compared with the respective control groups (Figure 2F). In contrast, the neointimal area, serum cholesterol and triglyceride levels, and peripheral monocyte counts in tamoxifen-treated Apoe−/− mice harboring CAG-Cre+Cxcr7floxApoe−/− BM cells were not substantially different from the control group (Figure V in the online-only Data Supplement). These findings suggest that the effect of Cxcr7 deficiency on neointima formation was attributable to increased serum cholesterol levels, which indirectly led to monocytosis.
Figure 2. The effect of Cxcr7 deficiency on neointima formation is related to hyperlipidemia-induced monocytosis. A, The circulating Sca-1+/Lin− cell population was analyzed before and 24 hours after vascular injury (n=8–9 per group). B, CXCL12 levels were determined in plasma (n=6–7 per group) and bone marrow (BM; n=3 per group) 28 days after vascular injury. C, The expression level of Cxcr4 transcripts was quantified in various tissues of CAG-Cre−Cxcr7floxApoe−/− and CAG-Cre+Cxcr7floxApoe−/− mice (n=3–6 per group). SM indicates skeletal muscle; VAT, visceral adipose tissue. D, Neointima formation after carotid wire injury was quantified in tamoxifen (TMX)-treated CAG-Cre−Cxcr7floxApoe+/+ and CAG-Cre+Cxcr7floxApoe+/+ mice fed a high-cholesterol diet (n=7–8 per group). Serum cholesterol and triglyceride (TG) levels (E) and peripheral monocyte counts (F) were determined in TMX-treated CAG-Cre−Cxcr7flox and CAG-Cre+Cxcr7flox mice (either Apoe+/+ or Apoe−/−) after 28 days of high-cholesterol diet feeding (n=5–10 per group). G, Fat, lean, and total weights of CAG-Cre−Cxcr7floxApoe−/− and CAG-Cre+Cxcr7floxApoe−/− mice were determined by micro-computed tomography after 28 days of high-cholesterol diet feeding (n=4 per group). H, In addition, fasting blood glucose and plasma insulin levels were measured and the quantitative insulin sensitivity check index (QUICKI) was determined in these mice (n=4 per group). *P<0.05; **P<0.01.
To study the effects of deletion of the Cxcr7 gene on body composition, adipose tissue size was analyzed in CAG-Cre+Cxcr7floxApoe−/− and CAG-Cre−Cxcr7floxApoe−/− mice fed an HCD for 4 weeks by micro-computed tomography. Total and lean body weight and body fat mass were similar between CAG-Cre+Cxcr7floxApoe−/− and CAG-Cre−Cxcr7floxApoe−/− mice (Figure 2G). Moreover, visceral adipose tissue (VAT) and brown adipose tissue mass were not significantly different between CAG-Cre+Cxcr7floxApoe−/− and CAG-Cre−Cxcr7floxApoe−/− mice (Figure VI in the online-only Data Supplement). In addition, fasting glucose and insulin levels and the surrogate marker for insulin sensitivity, QUICKI, were similar between the 2 groups (Figure 2H).
CCX771 Ameliorates Neointimal Lesion Formation
To study the effect of a CXCR7 ligand in lesion formation, CCX771 (10 mg/kg) was injected into Apoe−/− mice, reaching mean CCX771 plasma levels of 336 ng/mL after 1 hour and 106 ng/mL after 12 hours (Figure VIIA in the online-only Data Supplement). Contrary to the effects of Cxcr7 deficiency, treatment with CCX771 reduced the neointimal area (Figure 3A and 3B) and macrophage accumulation as assessed by the number of lesional Mac2+ macrophages (Figure 3D) after wire injury in Apoe−/− mice fed an HCD compared with vehicle- injected controls. The medial area (Figure 3B), lengths of the internal and external elastic laminae (Figure 3C), and neointimal SMC content as determined by the number of SMA+ cells or the SMA+ area (Figure 3E) did not differ between CCX771- and vehicle-treated mice. Treatment with CCX771 did not alter the injury-induced mobilization of Sca-1+Lin− SPCs (Figure 4A) or CD34+c-kit+Lin− endothelial progenitor cells (Figure 4B). Similar to Cxcr7−/− mice, CXCL12 levels in plasma and BM were also higher in CCX771-treated mice than in vehicle-treated mice (Figure 4C); however, treatment with CCX771 for 4 weeks did not increase CXCL12 levels in the plasma and BM of Cre+Cxcr7floxApoe−/− mice compared with vehicle-injected controls (Figure VIII in the online-only Data Supplement). Furthermore, serum cholesterol and triglyceride levels (Figure 4D) and peripheral monocyte counts (Figure 4E) were reduced in CCX771-treated mice compared with those in vehicle-treated mice. The Gr-1high/Gr-1low monocyte ratio was unaffected by CCX771 treatment (Figure 4F). Aspartate transaminase and creatinine levels were not different between groups (Figure VIIB and VIIC in the online-only Data Supplement).
Figure 3. Treatment with the CXCR7 ligand, CCX771, reduces neointima formation in apolipoprotein E–deficient mice (Apoe−/−). The effects of CCX771 treatment (compared with vehicle treatment) on neointima formation, media size (A and B), and length of the internal (IEL) and external (EEL) elastic laminae (A and C) after vascular injury to the carotid arteries of Apoe−/− mice were determined by planimetry (n=7–8 per group). Macrophage numbers in the neointima were quantified by immunostaining of Mac2 (red; D and E). The relative number of smooth muscle cells and the neointimal smooth muscle actin (SMA)–positive area were measured by α-SMA staining (F; n=7–8 per group). Arrows delineate neointima. Nuclei were counterstained with DAPI (blue). *P<0.05. Scale bars, 100 μm.
Figure 4. CCX771 ameliorates hyperlipidemia independent of splenic Cxcr7 expression. The circulating Sca-1+/Lin− (A; n=8–9 per group) and the CD34+/c-kit+/Lin− (B; n=4–5 per group) cell population was determined before and 24 hours after vascular injury. C, CXCL12 plasma (n=6–7 per group) and bone marrow (BM; n=3 per group) levels were quantified 28 days after vascular injury. D, Serum cholesterol and triglyceride (TG) levels were determined in apolipoprotein E–deficient mice (Apoe−/−) fed a high-cholesterol diet and treated with CCX771 or vehicle 28 days after vascular injury (n=6 per group). Quantitative analysis of circulating monocytes (E; n=6–7 per group) and the ratio of Gr-1hi to Gr-1lo monocytes (F; n=4 per group) in mice treated with CCX771 or vehicle was performed 28 days after vascular injury. G and H, Splenectomized Apoe−/− mice fed a high-cholesterol diet were treated with vehicle or CCX771 for 28 days after vascular injury. Neointimal area in sections of the carotid artery (G; n=4 per group) and serum cholesterol levels (H; n=3–4 per group) were quantified. Representative images are shown. *P<0.05; **P<0.01. Scale bars, 200 μm.
To study whether Cxcr7 expression in the spleen plays a role in the effect of CCX771 on neointima formation and hypercholesterolemia, splenectomized Apoe−/− mice were treated with CCX771 after vascular injury. However, even in the absence of the spleen, treatment with CCX771 reduced both the neointimal area (Figure 4G) and serum cholesterol levels (Figure 4F).
CCX771 Lowers Cholesterol Levels and Promotes VLDL Uptake in Adipose Tissue
To determine the role of CXCR7 in lipoprotein metabolism, lipoprotein profiles were analyzed in uninjured Apoe−/− mice fed an HCD after treatment with CCX771 or vehicle for 4 weeks. Treatment with CCX771 reduced serum cholesterol and triglyceride concentrations in fasting (5 hours) and nonfasting mice but did not affect the postprandial increase in cholesterol and triglyceride levels (Figure 5A and 5B). The decrease in total serum cholesterol levels observed in CCX771-treated mice was caused by a reduced concentration of circulating VLDL cholesterol; however, LDL cholesterol and HDL cholesterol concentrations were unaltered by CCX771 (Figure 5C). In Ldlr−/− mice, treatment with CCX771 also reduced serum cholesterol and triglyceride levels after 4 weeks of an HCD compared with vehicle-treated controls (Figure IX in the online-only Data Supplement).
Figure 5. CCX771 lowers very low-density lipoprotein (VLDL) cholesterol levels. Lipid metabolism was studied in apolipoprotein E–deficient mice (Apoe−/−) after 28 days of feeding a high-cholesterol diet and treatment with CCX771 or vehicle. Fasting and nonfasting serum cholesterol (A) and triglyceride (TG; B) concentrations were studied (n=4–5 per group). C, Total serum cholesterol and VLDL, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) cholesterol were quantified by fast protein liquid chromatography (n=5–7 per group). D, TG production rate was calculated from serum TG concentrations before and 6 hours after treatment with poloxamer 407 (P-407; n=4 per group). E, Clearance of human apolipoprotein B100 (hApoB100) and DiI was studied at 3 minutes (control) and 60 minutes after intravenous injection of DiI-labeled VLDL (n=8–9 per group). *P<0.05.
To study the VLDL-lowering mechanism of CCX771, hepatic triglyceride secretion was analyzed by measuring the increase in serum triglyceride levels after injection of the lipase inhibitor P-407 (poloxamer 407).22 At 6 hours after P-407 administration, triglyceride levels increased to similar levels in CCX771- and vehicle-treated mice (Figure X in the online-only Data Supplement). Accordingly, the hepatic triglyceride production rate was not altered by treatment with CCX771 (Figure 5D). To investigate the effect of CCX771 on VLDL clearance, DiI-labeled human VLDL was injected into Apoe−/− mice treated with CCX771 or vehicle for 4 weeks. At 60 minutes after injection of DiI-labeled VLDL, human apolipoprotein B100 and DiI levels in plasma were significantly reduced in CCX771-treated mice (Figure 5E), which indicates that CCX771 enhances the clearance of VLDL.
Next, DiI-labeled VLDL uptake was determined in VAT, brown adipose tissue, skeletal muscle, and liver of Apoe−/− mice. The concentration of DiI was significantly increased in VAT but not in liver, brown adipose tissue, or skeletal muscle of CCX771-treated compared with vehicle-treated mice (Figure 6A and 6B; Figure XI in the online-only Data Supplement). Accordingly, the DiI fluorescent signal in adipocytes was enhanced in CCX771-treated mice, as assessed by in vivo multiphoton microscopy (Figure 6C). Moreover, cholesterol content was increased in VAT but not in the liver of CCX771-treated mice (Figure 6A and 6B). Uptake of DiI-labeled VLDL to the subendothelial space was not detectable in the carotid arteries of CCX771-treated mice and Cre+Cxcr7floxApoe−/− mice by multiphoton and epifluorescence microscopy, respectively (Figure XII in the online-only Data Supplement). Taken together, these findings indicate that CXCR7 promotes the uptake of VLDL-derived cholesterol to adipose tissue.
Figure 6. CCX771 enhances the uptake of very low-density lipoprotein (VLDL) into adipose tissue. DiI fluorescence intensity and cholesterol content in visceral adipose tissue (A; VAT) and liver (B) were determined 60 minutes after DiI-labeled VLDL administration in apolipoprotein E–deficient mice (Apoe−/−) fed a high-cholesterol diet and treated with CCX771 or vehicle for 28 days (n=4 per group). C, DiI-labeled VLDL (red) in epididymal adipose tissue from these mice was detected by in vivo multiphoton microscopy. D, DiI-labeled VLDL uptake to adipocytes was studied in untreated (Blank) and vehicle-, CCX704-, or CCX771-treated Simpson-Golabi-Behmel syndrome (SGBS) adipocytes. E, Expression of β-arrestin2 (ARRB2) mRNA in SGBS adipocytes treated with small interfering RNAs (siRNAs) against ARRB2 or nontargeting siRNAs (ctrl-siRNA). F, Effect of ARRB2 silencing on DiI-VLDL uptake to SGBS adipocytes treated with CCX771 or vehicle was determined. Data represent 3 independent experiments (D, E, and F). *P<0.05; ***P<0.001. Scale bars, 100 μm.
Effects of CCX771 on Adipose Tissue and Insulin Sensitivity
To study the mechanism of CXCR7-mediated uptake of VLDL, human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes, which expressed CXCR7 as detected by immunostaining (Figure XIII in the online-only Data Supplement), were treated with DiI-labeled VLDL. CCX771 significantly increased the basal uptake of DiI-VLDL compared with untreated, vehicle- treated, or CCX704 (an analog of CCX771 with low affinity for CXCR7)-treated adipocytes (Figure 6D). Silencing of β-arrestin-2 with small interfering RNAs (Figure 6E) prevented the uptake of DiI-labeled VLDL triggered by CCX771 (Figure 6F), which indicates that β-arrestin-2 promotes VLDL uptake to adipocytes mediated by CXCR7.
To assess the effect of CCX771 on lipoprotein lipase (Lpl), which plays an important role in the clearance of VLDL,23 lipase activity was determined in Apoe−/− mice after treatment with CCX771 or vehicle. Lipase activity was increased in VAT but not in plasma of CCX771-treated compared with vehicle- treated mice (Figure 7A). Angptl3 and Angptl4 are negative regulators of Lpl activity.24 Treatment with CCX771 reduced Angptl4 protein levels in VAT but not in plasma (Figure 7B), whereas Angptl3 protein levels were unaltered in the VAT and plasma in CCX771-treated compared with vehicle-treated mice (Figure 7C). Moreover, the Angptl4 mRNA expression level was diminished in the VAT of CCX771-treated mice, whereas expression of Lpl and Angptl3 transcripts remained unchanged (Figure 7D), which indicates that downregulation of Angptl4 may increase lipase activity in the VAT of CCX771-treated mice. In addition, the mRNA expression level of Pparg, a positive regulator of Angptl4 expression in adipocytes, was reduced in the VAT of CCX771-treated mice (Figure 7D). However, treatment with CCX771 did not substantially change the expression of Vldlr and Ldlr transcripts in the VAT (Figure XIVA in the online-only Data Supplement). Unlike Cxcr4, the expression of Cxcr7 in different VATs was comparable to that in the spleen (Figure XIVB in the online-only Data Supplement).
Figure 7. CCX771 increases lipase activity and inhibits Angptl4 expression in visceral adipose tissue (VAT). Apolipoprotein E–deficient mice (Apoe−/−) were fed a high-cholesterol diet and treated with CCX771 or vehicle for 28 days. Lipase activity (A) and protein concentration of Angptl4 (B) and Angptl3 (C) were measured in plasma and VAT (n=4–5 per group). D, mRNA expression levels of Lpl, Angptl4, Angptl3, and Pparg were quantified in VAT (n=5–6 per group). E, Fasting and nonfasting blood glucose concentrations (E; n=4–6 per group) and fasting plasma insulin levels were determined (F; n=3–4 per group). G, As a surrogate marker for insulin sensitivity, quantitative insulin sensitivity check index (QUICKI) was calculated (n=3–4 per group). *P<0.05.
To determine the impact of CCX771 on insulin sensitivity, Apoe−/− mice were fed an HCD and treated with CCX771 or vehicle for 4 weeks. There was no significant difference in fasting and nonfasting blood glucose levels detectable between CCX771-treated and vehicle-treated mice (Figure 7E). Fasting insulin levels in the plasma were also similar between the groups (Figure 7F). Accordingly, the quantitative insulin- sensitivity check index (QUICKI) was not different between CCX771- and vehicle-treated mice (Figure 7G), which indicates that the effects of CCX771 on adipose tissue do not affect insulin sensitivity.
Treatment With CCX771 Inhibits Atherosclerosis
To investigate the role of CCX771 in diet-induced atherosclerosis, Apoe−/− mice fed an HCD were treated with CCX771 or vehicle for 12 weeks. The size of the atherosclerotic lesions in the aortic root (Figure 8A) and thoracoabdominal aorta (Figure 8B) and the number of Mac2+ macrophages in the aortic root lesions (Figure 8C) were reduced by CCX771. However, the number of SM22+ SMCs and the SM22+ area in the lesions were unaltered by CCX771 (Figure 8D), and serum cholesterol and triglyceride levels (Figure 8E), as well as the peripheral monocyte count (Figure 8F), were reduced. Alanine transaminase and creatinine levels (Figure XV in the online-only Data Supplement) were comparable between CCX771- and vehicle-treated mice.
Figure 8. CCX771 treatment inhibits diet-induced atherosclerosis in mice by reducing hyperlipidemia and monocytosis. A, The size of the atherosclerotic lesions in the aortic roots of apolipoprotein E–deficient mice (Apoe−/−) treated with vehicle or CCX771 was determined after 12 weeks on a high-cholesterol diet. B, Lipid accumulation was quantified by oil red O staining of en face–prepared thoracoabdominal aortas. C, Macrophage number was studied in aortic root lesions by Mac2 immunostaining (red). D, The number of smooth muscle cells and the SM22+ area in the lesions was analyzed by SM22 staining. Serum cholesterol and triglyceride (TG) levels (E) and peripheral monocyte counts (F) were determined in Apoe−/− mice after 12 weeks on a high-cholesterol diet and treated with vehicle or CCX771. *P<0.05; **P<0.01; ***P<0.001; n=9 to 10 per group. Scale bars, 200 μm.
Discussion
The present results indicate a key role for CXCR7 in regulating lipoprotein metabolism and serum cholesterol levels in mice by enhancing the clearance of VLDL through adipose tissue under conditions of hyperlipidemia, thereby unveiling a new functional feature of chemokine receptors. Treatment with the CXCR7 ligand CCX771 reduced hyperlipidemia and subsequent monocytosis in Apoe−/− mice, thereby limiting atherosclerotic lesion formation. Thus, treatment with agents that activate CXCR7 appears to be a promising adjunct to lipid- lowering drugs that are used to prevent atherosclerosis, especially in the context of metabolic disorders characterized by increased VLDL levels.
The neointimal accumulation of SMCs after vascular injury is mediated by the CXCL12-dependent recruitment of SPCs from the BM.1,8 CXCR4 is expressed on SPCs, and inhibition of CXCR4 or CXCL12 prevents neointima formation.7,8,25 The present results did not indicate that CXCR7 regulates SPC mobilization or neointimal SMC accumulation. Although CXCR7 is not expressed on circulating leukocytes,15 we found that a genetic deficiency of Cxcr7 results in enhanced neointima formation because of increased accumulation of Mac2+ macrophages. Alternatively, Mac2 (also known as galectin-3) may accumulate in the extracellular space after being released from macrophages26; however, changes in lesional Mac2+ macrophage content correlate very well with those of F4/80+ macrophages, as determined by flow cytometry.10 The increased macrophage accumulation can be explained by findings showing that the monocytosis induced by severe hyperlipidemia3,5 is aggravated in conditional Cxcr7−/−Apoe−/− mice. In addition, hyperlipidemia may also promote the transformation of SMCs into foam cells and expression of the macrophage marker Mac2 in SMCs.27 By contrast, neointima formation and monocyte counts were not altered in mildly hyperlipidemic Cxcr7−/− mice, which indicates that the effect of CXCR7 on peripheral monocytes is indirect and dependent on the degree of hyperlipidemia. Concordantly, Cxcr7 deficiency further increased serum cholesterol levels only in HCD-fed Apoe−/− mice, which suggests a regulatory role for CXCR7 in lipid metabolism under severe hyperlipidemic conditions. Apoe itself controls hematopoietic stem cell proliferation (by autonomously promoting cholesterol efflux via ABC transporters), monocytosis, and lesional monocyte accumulation.28 However, it is unlikely that CXCR7 directly compensates for such effects, because it is not expressed on BM cells. Although CXCR7 expression has been reported previously in lesional macrophages,29 we did not detect CXCR7 in injured arteries, and genetic deletion of Cxcr7 in BM cells had no effect on neointima formation, which indicates a minor role of CXCR7 in macrophages during vascular repair.
Treatment of Apoe−/− mice with CCX771 led to the opposite phenotype to that expressed by Cxcr7−/− mice in terms of lesion formation, monocytosis, and hyperlipidemia; this indicates that CCX771 triggers the activation of CXCR7. Of note, CCX771 induces stronger β-arrestin-2 activation via CXCR7 than its canonical ligand, namely, CXCL12.16 In line with previous reports, we found that CXCR7 is expressed in cells within the marginal zone of the spleen, most likely in B cells,13 which are involved in protective immunity against atherosclerosis.30 However, removal of the spleen did not alter the effect of CCX771 on neointima formation and hyperlipidemia, which suggests that splenic CXCR7 expression does not play a role in atherosclerotic vascular disease. In contrast to neointima formation, both Cxcr7−/− and CCX771-treated mice showed increased CXCL12 levels in the plasma and BM, which indicates that the endocytotic removal of CXCL12 is impaired by Cxcr7 deficiency and by CCX771 treatment. This parallel increase in CXCL12 levels in the plasma and BM maintained the gradient of CXCL12 between these 2 compartments, which prevented the mobilization of SPCs.10 However, this effect on extracellular CXCL12 levels cannot explain the contrary change in neointima formation in Cxcr7−/− and CCX771-treated mice.
The type of dyslipidemia observed in Apoe−/− mice is characterized by an elevation of VLDL/intermediate-density lipoprotein–derived cholesterol levels, whereas LDL cholesterol levels are only mildly increased and HDL cholesterol levels are reduced.31 Treatment with CCX771 lowered only circulating VLDL levels in Apoe−/− mice by enhanced VLDL clearance and uptake to VAT, which is the primary storage site for unesterified cholesterol.32–34 Cholesterol uptake by adipocytes occurs mainly via triglyceride-rich lipoproteins, such as chylomicrons and VLDL.35 CCX771 treatment enhanced the uptake of cholesterol from VLDL to the VAT in a β-arrestin-2–dependent manner, which may explain the reduced serum cholesterol levels observed in CCX771-treated mice. Moreover, CCX771 treatment enhanced lipase activity in adipose tissue, presumably by downregulating Angptl4, which inhibits the lipolytic activity of Lpl by converting the active dimer into an inactive monomer.36 In addition to lipolysis, Lpl dimers enhance the cellular uptake of VLDL independent of its catalytic activity by bridging between lipoproteins and lipoprotein receptors or proteoglycans.37,38 Increased Lpl activity may cause the reduced VLDL cholesterol and triglyceride serum levels in Angptl4−/− mice,39–41 and Apoe−/− mice lacking the Angptl4 gene develop less atherosclerosis.39 Conversely, overexpression of Angptl4 impairs the clearance of VLDL and results in hypertriglyceridemia and hypercholesterolemia in mice.41,42 Moreover, chronic intermittent hypoxia increases VLDL levels, reduces adipose Lpl activity, and promotes atherosclerosis by upregulating Angptl4 expression in adipose tissue of Apoe−/− mice.43 However, Angptl4 can also limit lipid accumulation in macrophages, which may play a role in the protection against atherosclerosis in mice that overexpress Angptl4.44 In summary, these data indicate that activation of CXCR7 under hyperlipidemic conditions lowers Angptl4 expression in adipose tissue and thereby reduces serum cholesterol levels through enhanced Lpl-mediated uptake of VLDL to adipose tissue.
Taken together, these results suggest a novel role for the chemokine receptor CXCR7 in the uptake of VLDL into adipose tissue in Apoe−/− mice, which regulates serum cholesterol levels. Activation of CXCR7 by the synthetic ligand CCX771 protects against atherosclerosis, most likely by lowering serum lipid levels. Thus, treatment with CCX771 might be a promising therapeutic approach to treating atherogenic dyslipidemia.
Acknowledgments
We thank Stephanie Elbin, Melanie Garbe, Yuan Kong, Judit Corbalán Campos, and Roya Soltan for technical assistance. We thank Dr Lin Gan (University of Rochester, Rochester, MN) for generating the Cxcr7flox mice.
Sources of Funding
This work was supported by the
Disclosures
Drs Penfold and Schall are current employees of ChemoCentryx Inc and have equity ownership in that company. The other authors report no conflicts.
Footnotes
References
- 1.
Schober A . Chemokines in vascular dysfunction and remodeling.Arterioscler Thromb Vasc Biol. 2008; 28:1950–1959.LinkGoogle Scholar - 2.
Weber C, Noels H . Atherosclerosis: current pathogenesis and therapeutic options.Nat Med. 2011; 17:1410–1422.CrossrefMedlineGoogle Scholar - 3.
Combadière C, Potteaux S, Rodero M, Simon T, Pezard A, Esposito B, Merval R, Proudfoot A, Tedgui A, Mallat Z . Combined inhibition of CCL2, CX3CR1, and CCR5 abrogates Ly6C(hi) and Ly6C(lo) monocytosis and almost abolishes atherosclerosis in hypercholesterolemic mice.Circulation. 2008; 117:1649–1657.LinkGoogle Scholar - 4.
Tsou CL, Peters W, Si Y, Slaymaker S, Aslanian AM, Weisberg SP, Mack M, Charo IF . Critical roles for CCR2 and MCP-3 in monocyte mobilization from bone marrow and recruitment to inflammatory sites.J Clin Invest. 2007; 117:902–909.CrossrefMedlineGoogle Scholar - 5.
Swirski FK, Libby P, Aikawa E, Alcaide P, Luscinskas FW, Weissleder R, Pittet MJ . Ly-6Chi monocytes dominate hypercholesterolemia-associated monocytosis and give rise to macrophages in atheromata.J Clin Invest. 2007; 117:195–205.CrossrefMedlineGoogle Scholar - 6.
Zernecke A, Bot I, Djalali-Talab Y, Shagdarsuren E, Bidzhekov K, Meiler S, Krohn R, Schober A, Sperandio M, Soehnlein O, Bornemann J, Tacke F, Biessen EA, Weber C . Protective role of CXC receptor 4/CXC ligand 12 unveils the importance of neutrophils in atherosclerosis.Circ Res. 2008; 102:209–217.LinkGoogle Scholar - 7.
Schober A, Knarren S, Lietz M, Lin EA, Weber C . Crucial role of stromal cell-derived factor-1alpha in neointima formation after vascular injury in apolipoprotein E-deficient mice.Circulation. 2003; 108:2491–2497.LinkGoogle Scholar - 8.
Subramanian P, Karshovska E, Reinhard P, Megens RT, Zhou Z, Akhtar S, Schumann U, Li X, van Zandvoort M, Ludin C, Weber C, Schober A . Lysophosphatidic acid receptors LPA1 and LPA3 promote CXCL12-mediated smooth muscle progenitor cell recruitment in neointima formation.Circ Res. 2010; 107:96–105.LinkGoogle Scholar - 9.
Schober A, Hristov M, Kofler S, Forbrig R, Löhr B, Heussen N, Zhe Z, Akhtar S, Schumann U, Krötz F, Leibig M, König A, Kaczmarek I, Reichart B, Klauss V, Weber C, Sohn HY . CD34+CD140b+ cells and circulating CXCL12 correlate with the angiographically assessed severity of cardiac allograft vasculopathy.Eur Heart J. 2011; 32:476–484.CrossrefMedlineGoogle Scholar - 10.
Akhtar S, Gremse F, Kiessling F, Weber C, Schober A . CXCL12 promotes the stabilization of atherosclerotic lesions mediated by smooth muscle progenitor cells in Apoe-deficient mice.Arterioscler Thromb Vasc Biol. 2013; 33:679–686.LinkGoogle Scholar - 11.
Balabanian K, Lagane B, Infantino S, Chow KY, Harriague J, Moepps B, Arenzana-Seisdedos F, Thelen M, Bachelerie F . The chemokine SDF-1/CXCL12 binds to and signals through the orphan receptor RDC1 in T lymphocytes.J Biol Chem. 2005; 280:35760–35766.CrossrefMedlineGoogle Scholar - 12.
Burns JM, Summers BC, Wang Y, Melikian A, Berahovich R, Miao Z, Penfold ME, Sunshine MJ, Littman DR, Kuo CJ, Wei K, McMaster BE, Wright K, Howard MC, Schall TJ . A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development.J Exp Med. 2006; 203:2201–2213.CrossrefMedlineGoogle Scholar - 13.
Sierro F, Biben C, Martínez-Muñoz L, Mellado M, Ransohoff RM, Li M, Woehl B, Leung H, Groom J, Batten M, Harvey RP, Martínez-A C, Mackay CR, Mackay F . Disrupted cardiac development but normal hematopoiesis in mice deficient in the second CXCL12/SDF-1 receptor, CXCR7.Proc Natl Acad Sci U S A. 2007; 104:14759–14764.CrossrefMedlineGoogle Scholar - 14.
Gerrits H, van Ingen Schenau DS, Bakker NE, van Disseldorp AJ, Strik A, Hermens LS, Koenen TB, Krajnc-Franken MA, Gossen JA . Early postnatal lethality and cardiovascular defects in CXCR7-deficient mice.Genesis. 2008; 46:235–245.CrossrefMedlineGoogle Scholar - 15.
Berahovich RD, Zabel BA, Penfold ME, Lewén S, Wang Y, Miao Z, Gan L, Pereda J, Dias J, Slukvin II, McGrath KE, Jaen JC, Schall TJ . CXCR7 protein is not expressed on human or mouse leukocytes.J Immunol. 2010; 185:5130–5139.CrossrefMedlineGoogle Scholar - 16.
Zabel BA, Wang Y, Lewén S, Berahovich RD, Penfold ME, Zhang P, Powers J, Summers BC, Miao Z, Zhao B, Jalili A, Janowska-Wieczorek A, Jaen JC, Schall TJ . Elucidation of CXCR7-mediated signaling events and inhibition of CXCR4-mediated tumor cell transendothelial migration by CXCR7 ligands.J Immunol. 2009; 183:3204–3211.CrossrefMedlineGoogle Scholar - 17.
Rajagopal S, Kim J, Ahn S, Craig S, Lam CM, Gerard NP, Gerard C, Lefkowitz RJ . Beta-arrestin- but not G protein-mediated signaling by the “decoy” receptor CXCR7.Proc Natl Acad Sci U S A. 2010; 107:628–632.CrossrefMedlineGoogle Scholar - 18.
Luker KE, Steele JM, Mihalko LA, Ray P, Luker GD . Constitutive and chemokine-dependent internalization and recycling of CXCR7 in breast cancer cells to degrade chemokine ligands.Oncogene. 2010; 29:4599–4610.CrossrefMedlineGoogle Scholar - 19.
Naumann U, Cameroni E, Pruenster M, Mahabaleshwar H, Raz E, Zerwes HG, Rot A, Thelen M . CXCR7 functions as a scavenger for CXCL12 and CXCL11.PLoS One. 2010; 5:e9175.CrossrefMedlineGoogle Scholar - 20.
Levoye A, Balabanian K, Baleux F, Bachelerie F, Lagane B . CXCR7 heterodimerizes with CXCR4 and regulates CXCL12-mediated G protein signaling.Blood. 2009; 113:6085–6093.CrossrefMedlineGoogle Scholar - 21.
Hayashi S, McMahon AP . Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse.Dev Biol. 2002; 244:305–318.CrossrefMedlineGoogle Scholar - 22.
Millar JS, Cromley DA, McCoy MG, Rader DJ, Billheimer JT . Determining hepatic triglyceride production in mice: comparison of poloxamer 407 with Triton WR-1339.J Lipid Res. 2005; 46:2023–2028.CrossrefMedlineGoogle Scholar - 23.
Stein Y, Stein O . Lipoprotein lipase and atherosclerosis.Atherosclerosis. 2003; 170:1–9.CrossrefMedlineGoogle Scholar - 24.
Mattijssen F, Kersten S . Regulation of triglyceride metabolism by angiopoietin- like proteins.Biochim Biophys Acta. 2012; 1821:782–789.CrossrefMedlineGoogle Scholar - 25.
Hamesch K, Subramanian P, Li X, Dembowsky K, Chevalier E, Weber C, Schober A . The CXCR4 antagonist POL5551 is equally effective as sirolimus in reducing neointima formation without impairing re-endothelialisation.Thromb Haemost. 2012; 107:356–368.CrossrefMedlineGoogle Scholar - 26.
Cherayil BJ, Weiner SJ, Pillai S . The Mac-2 antigen is a galactose-specific lectin that binds IgE.J Exp Med. 1989; 170:1959–1972.CrossrefMedlineGoogle Scholar - 27.
Rong JX, Shapiro M, Trogan E, Fisher EA . Transdifferentiation of mouse aortic smooth muscle cells to a macrophage-like state after cholesterol loading.Proc Natl Acad Sci U S A. 2003; 100:13531–13536.CrossrefMedlineGoogle Scholar - 28.
Murphy AJ, Akhtari M, Tolani S, Pagler T, Bijl N, Kuo CL, Wang M, Sanson M, Abramowicz S, Welch C, Bochem AE, Kuivenhoven JA, Yvan-Charvet L, Tall AR . ApoE regulates hematopoietic stem cell proliferation, monocytosis, and monocyte accumulation in atherosclerotic lesions in mice.J Clin Invest. 2011; 121:4138–4149.CrossrefMedlineGoogle Scholar - 29.
Ma W, Liu Y, Ellison N, Shen J . Induction of C-X-C chemokine receptor type 7 (CXCR7) switches stromal cell-derived factor-1 (SDF-1) signaling and phagocytic activity in macrophages linked to atherosclerosis.J Biol Chem. 2013; 288:15481–15494.CrossrefMedlineGoogle Scholar - 30.
Caligiuri G . Protective immunity against atherosclerosis carried by B cells of hypercholesterolemic mice.J Clin Invest. 2002; 109:745–753.CrossrefMedlineGoogle Scholar - 31.
Zhang SH, Reddick RL, Piedrahita JA, Maeda N . Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E.Science. 1992; 258:468–471.CrossrefMedlineGoogle Scholar - 32.
Krause BR, Hartman AD . Adipose tissue and cholesterol metabolism.J Lipid Res. 1984; 25:97–110.CrossrefMedlineGoogle Scholar - 33.
Angel A, Farkas J . Regulation of cholesterol storage in adipose tissue.J Lipid Res. 1974; 15:491–499.CrossrefMedlineGoogle Scholar - 34.
Farkas J, Angel A, Avigan MI . Studies on the compartmentation of lipid in adipose cells, II: cholesterol accumulation and distribution in adipose tissue components.J Lipid Res. 1973; 14:344–356.CrossrefMedlineGoogle Scholar - 35.
Kovanen PT, Nikkilä EA . Cholesterol exchange between fat cells, chylomicrons and plasma lipoproteins.Biochim Biophys Acta. 1976; 441:357–369.CrossrefMedlineGoogle Scholar - 36.
Sukonina V, Lookene A, Olivecrona T, Olivecrona G . Angiopoietin-like protein 4 converts lipoprotein lipase to inactive monomers and modulates lipase activity in adipose tissue.Proc Natl Acad Sci U S A. 2006; 103:17450–17455.CrossrefMedlineGoogle Scholar - 37.
Merkel M, Kako Y, Radner H, Cho IS, Ramasamy R, Brunzell JD, Goldberg IJ, Breslow JL . Catalytically inactive lipoprotein lipase expression in muscle of transgenic mice increases very low density lipoprotein uptake: direct evidence that lipoprotein lipase bridging occurs in vivo.Proc Natl Acad Sci U S A. 1998; 95:13841–13846.CrossrefMedlineGoogle Scholar - 38.
Krapp A, Zhang H, Ginzinger D, Liu MS, Lindberg A, Olivecrona G, Hayden MR, Beisiegel U . Structural features in lipoprotein lipase necessary for the mediation of lipoprotein uptake into cells.J Lipid Res. 1995; 36:2362–2373.CrossrefMedlineGoogle Scholar - 39.
Adachi H, Fujiwara Y, Kondo T, Nishikawa T, Ogawa R, Matsumura T, Ishii N, Nagai R, Miyata K, Tabata M, Motoshima H, Furukawa N, Tsuruzoe K, Kawashima J, Takeya M, Yamashita S, Koh GY, Nagy A, Suda T, Oike Y, Araki E . Angptl 4 deficiency improves lipid metabolism, suppresses foam cell formation and protects against atherosclerosis.Biochem Biophys Res Commun. 2009; 379:806–811.CrossrefMedlineGoogle Scholar - 40.
Adachi H, Kondo T, Koh GY, Nagy A, Oike Y, Araki E . Angptl4 deficiency decreases serum triglyceride levels in low-density lipoprotein receptor knockout mice and streptozotocin-induced diabetic mice.Biochem Biophys Res Commun. 2011; 409:177–180.CrossrefMedlineGoogle Scholar - 41.
Köster A, Chao YB, Mosior M, Ford A, Gonzalez-DeWhitt PA, Hale JE, Li D, Qiu Y, Fraser CC, Yang DD, Heuer JG, Jaskunas SR, Eacho P . Transgenic angiopoietin-like (angptl)4 overexpression and targeted disruption of angptl4 and angptl3: regulation of triglyceride metabolism.Endocrinology. 2005; 146:4943–4950.CrossrefMedlineGoogle Scholar - 42.
Mandard S, Zandbergen F, van Straten E, Wahli W, Kuipers F, Müller M, Kersten S . The fasting-induced adipose factor/angiopoietin-like protein 4 is physically associated with lipoproteins and governs plasma lipid levels and adiposity.J Biol Chem. 2006; 281:934–944.CrossrefMedlineGoogle Scholar - 43.
Drager LF, Yao Q, Hernandez KL, Shin MK, Bevans-Fonti S, Gay J, Sussan TE, Jun JC, Myers AC, Olivecrona G, Schwartz AR, Halberg N, Scherer PE, Semenza GL, Powell DR, Polotsky VY . Chronic intermittent hypoxia induces atherosclerosis via activation of adipose angiopoietin-like 4.Am J Respir Crit Care Med. 2013; 188:240–248.CrossrefMedlineGoogle Scholar - 44.
Georgiadi A, Wang Y, Stienstra R, Tjeerdema N, Janssen A, Stalenhoef A, van der Vliet JA, de Roos A, Tamsma JT, Smit JW, Tan NS, Müller M, Rensen PC, Kersten S . Overexpression of angiopoietin-like protein 4 protects against atherosclerosis development.Arterioscler Thromb Vasc Biol. 2013; 33:1529–1537.LinkGoogle Scholar
CLINICAL PERSPECTIVE
Hypercholesterolemia caused by increased plasma levels of apolipoprotein B–containing lipoproteins, such as low-density lipoprotein or very low-density lipoprotein, drives the pathogenesis of atherosclerosis. Chemokines and their receptors mediate the hypercholesterolemia-induced mobilization and recruitment of monocytes that leads to accumulation of macrophages in atherosclerotic arteries. In contrast to other chemokine receptors, CXCR7 does not induce G-protein–coupled signaling and is not expressed on leukocytes but internalizes and degrades its ligands, such as CXCL12 and CXCL11. We studied the role of CXCR7 and the synthetic CXCR7 ligand CCX771 in mouse models of atherosclerosis. Our results reveal an unanticipated role of CXCR7 in the regulation of circulating very low-density lipoprotein cholesterol levels, which is unrelated to its effects on systemic CXCL12 levels. Treatment with CCX771 lowered very low-density lipoprotein levels in hyperlipidemic mice because of an enhanced uptake into adipose tissue and thus reduced hyperlipidemia-induced monocytosis and atherosclerotic lesion formation. This effect of CCX771 was probably attributable to increased lipoprotein lipase activity in adipose tissue in response to downregulation of Angptl4. Conversely, genetic deficiency of CXCR7 increased serum cholesterol and triglyceride levels and promoted lesion formation after vascular injury in hyperlipidemic mice. Thus, our findings indicate that adipose tissue regulates serum cholesterol levels under hyperlipidemic conditions through CXCR7-mediated uptake of very low-density lipoprotein. This cholesterol-buffering mechanism of adipose tissue may play an important role in the handling of an increased dietary lipid load. Pharmacological activation of CXCR7 can enhance its lipid-lowering effect and may thus provide a novel therapeutic approach to the treatment of atherogenic dyslipidemia.
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