Adiponectin Receptor 2 Deficiency Results in Reduced Atherosclerosis in the Brachiocephalic Artery in Apolipoprotein E Deficient Mice

Adiponectin has been shown to have beneficial cardiovascular effects and to signal through the adiponectin receptors, AdipoR1 and AdipoR2. The original aim of this study was to investigate the effect of combined AdipoR1 and AdipoR2 deficiency (AdipoR1-/-AdipoR2-/-) on atherosclerosis. However, we made the interesting observation that AdipoR1 -/- AdipoR2 -/- leads to embryonic lethality demonstrating the critical importance of the adiponectin signalling system during development. We then investigated the effect of AdipoR2-ablation on the progression of atherosclerosis in apolipoprotein E deficient (ApoE -/-) mice. AdipoR2-/-ApoE-/- mice fed an atherogenic diet had decreased plaque area in the brachiocephalic artery compared with AdipoR2 +/+ApoE-/- littermate controls as visualized in vivo using an ultrasound biomicroscope and confirmed by histological analyses. The decreased plaque area in the brachiocephalic artery could not be explained by plasma cholesterol levels or inflammatory status. However, accumulation of neutral lipids was decreased in peritoneal macrophages from AdipoR2-/-ApoE-/- mice after incubation with oxidized LDL. This effect was associated with lower CD36 and higher ABCA1 mRNA levels in peritoneal macrophages from AdipoR2-/-ApoE-/- mice compared with AdipoR2+/+ApoE-/- controls after incubation with oxidized LDL. In summary, we show that adiponectin receptors are crucial during embryonic development and that AdipoR2-deficiency slows down the progression of atherosclerosis in the brachiocephalic artery of ApoE-deficient mice.

Several studies in experimental animals have demonstrated anti-diabetic [11][12][13] and anti-atherosclerotic [12][13][14][15] effects of adiponectin. In addition, adiponectin can protect the heart from ischemia-reperfusion injury [16] and can act as an endogenous anti-thrombotic factor [17]. The anti-atherosclerotic effects of adiponectin have been demonstrated at several stages in plaque development, ranging from endothelial dysfunction and plaque initiation to plaque progression and rupture (reviewed in 18). In vitro studies have shown that adiponectin suppresses monocyte adhesion and expression of endothelial cell adhesion molecules, such as intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin [7]. Furthermore, adiponectin inhibits foam cell formation by downregulating macrophage scavenger receptor A, resulting in reduced lipid accumulation in macrophages [19,20]. Studies have also shown that adiponectin can induce cholesterol efflux from macrophages via upregulation of the ATP-binding cassette transporter ABCA1 [20,21].
Adiponectin has been proposed to mediate its effects via at least two transmembrane receptors, adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2) [22]. We and others have demonstrated the physiological importance of these receptors using gene knock-out mice [23][24][25]. AdipoR1 -/mice showed decreased glucose tolerance [23,25]. In contrast, AdipoR2 -/-mice were resistant to high-fat diet induced obesity and exhibited improved glucose tolerance and decreased plasma cholesterol levels [23,24]. In addition, knock-down of AdipoR2 using antisense oligonucleotides reduced plasma glucose levels in insulin resistant leptin-deficient ob/ob mice [24]. Thus, AdipoR1 and AdipoR2 are clearly involved in glucose and energy metabolism but have opposing roles [23].
In this study, we could show that mice lacking both AdipoR1 and AdipoR2 were embryonically lethal, i.e. found dead in utero at day 16.5 with macroscopical swelling and microscopical autolysis and tissue disintegration. We then hypothesized that the favourable metabolic phenotype of AdipoR2 -/-mice we and others have observed previously [23,24] also could be associated with a protective effect against developing atherosclerosis. We generated mice deficient in both AdipoR2 and ApoE (AdipoR2 -/-ApoE -/-) and littermate control mice lacking only ApoE (AdipoR2 +/+ ApoE -/-) in order to study the impact of AdipoR2 deficiency on the atherosclerosis process. Interestingly, the progression of atherosclerosis was attenuated in the brachiocephalic artery of AdipoR2 deficient mice.

Ethics Statement
All experiments were approved by the Gothenburg Ethics Committee for Experimental Animals.

Ultrasound measurements
The mice were anaesthetized with isoflurane gas and maintained anaesthetized during the ultrasound measurement with an isoflurane dose of 1.5% in room air. Ultrasound measurements were performed at 8, 15 and 22 weeks of age. Ultrasound biomicroscopy equipment (Vevo 770, Visual Sonics, Toronto, Canada) with a transducer frequency of 40 MHz was used for vascular imaging. The brachiocephalic artery was visualized as described previously [26].

Blood samples, plasma and liver analyses
Blood samples were collected from Vena Saphena at 7 weeks of age (before switched to the Lard diet), at 11 and 18 weeks of age and at termination at 22 weeks of age. Lipids were extracted from plasma samples using the Folch method [27]. Triglycerides in total lipid extracts were separated using normal phase liquid chromatography, NPLC, and measured by evaporative light scattering detection, ELSD [28]. A calibration curve was obtained by injecting commercial lipid standard. Total plasma cholesterol levels were measured with an enzymatic colorimetric assay (Roche Diagnostics, Mannheim, Germany). Cholesterol distribution profiles were measured on 10 µl pooled plasma from 10 mice in each group, using a sizeexclusion high performance liquid chromatography system, SMART (GE Healthcare, Waukesha, WI). Plasma adiponectin levels were measured using a radioimmunoassay from Linco Research (St. Charles, MO) and plasma cytokines were determined using bead based multiplex suspension array kits with the Luminex technology on a BioPlex (Bio-Rad, CA). Liver triglyceride content was measured on an ABX Pentra 400 (HORIBA Medical, Kyoto, Japan) after homogenization in isopropanol (1 ml/50 mg), incubation at 4°C for 1 h, and centrifugation at 2,500 rpm for 5 min.

Histological analyses
Paraffin sections (4 µm) from the proximal brachiocephalic artery and the aortic arch were stained with Miller's Elastin and Picrosirius Red (Histolab Products AB, Gothenburg, Sweden) and used for measurement of plaque area and collagen content, respectively. A Mac-2 antibody (CL8942AP clone M3/38; Cedarlane, Hornby, Canada) at a dilution of 1:20,000 was used for quantification of macrophages and macrophagederived foam cells. For the en face analysis, the thoracic part of the descending aorta was cut open longitudinally and mounted in Tissue-Tek O.C.T compound (Sakura, Torrance, CA). All plaque analyses were done using BioPix iQ 2.1 Software (BioPix, Gothenburg, Sweden). Plaque area in the descending aorta is expressed as percentage of total vessel area.

Protein extraction and western blot
Liver protein was extracted by homogenization in T-PER Tissue Protein Extraction Reagent (Thermo Scientific, Waltham, MA) with added protease inhibitors according to the manufacturer's protocol. Protein concentrations were determined with the Pierce BCA Protein Assay kit (Thermo Scientific). 30 µg protein was separated on a 4-12% Bis-Tris gel and subsequently transferred onto a nitrocellulose membrane. The membrane was incubated with affinity purified primary antibodies against human AdipoR2 (1 μg/ml in phosphate-buffered saline with 0.05% Tween and 5% milk powder). The AdipoR2 antibodies were generated by immunizing rabbits with the peptide CSRTPEPDIRLRKGHQLDG (94% sequence homology with the corresponding mouse sequence). The secondary HRPconjugated goat anti-rabbit IgG antibody (Novus Biologics, Littleton, CO) was used at 1:10 000. The bands were visualized using the Novex ECL Chemiluminescent substrate reagent kit (Invitrogen, Carlsbad, CA).

Analyses of lipid accumulation, inflammatory response and gene expression in peritoneal macrophages
Primary peritoneal macrophages were isolated by flushing the peritoneum of 8 weeks old male AdipoR2 -/-ApoE -/-mice and AdipoR2 +/+ ApoE -/-littermate controls with PBS. Cells were collected and allowed to adhere to a cell culture dish for 2 h in serum-free RPMI 1640 media supplemented with sodiumpyruvate (2mM), non-essential amino acids, sodium bicarbonate (1.5 g/l), penicillin (100 U/ml) and streptomycin (100 mg/l) at 37°C. Cells not attached to the plastic after 2 h were removed by washing three times with PBS and adherent cells were cultured in supplemented RPMI 1640 media containing 10% FCS. For analysis of inflammatory response, peritoneal macrophages were incubated with or without oxidized LDL (oxLDL) (50 µg/ml) or LPS (0.1 mg/ml) for 24 h and cytokine levels in the culture medium were determined as described above. For analysis of lipid accumulation, macrophages were incubated with or without oxidized LDL (oxLDL) (50µg/ml) for 24 h, followed by staining with oil red O. Quantification of the total oil red O area was assessed using BioPix software. For gene expression analyses, total RNA was extracted from cultured macrophages using an RNeasy kit (Qiagen, Hilden, Germany) and cDNA was synthesized using a High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA). Real-time PCR analysis was performed with an ABI Prism 7900 HT Detection System (Applied Biosystems) using the following TaqMan Gene Expression assays: Mm00446214 for scavenger receptor class A (SR-A), Mm00450236 for SR-B1, Mm00432401 for CD36, Mm0043739 for ATP-binding cassette sub-family G member 1 (ABCG1) and Mm00442646 for ABC sub-family A member 1 (ABCA1). Primers and FAM/ TAMRA labelled fluorogenic probes were used for real-time PCR analysis using the following sequences: AdipoR1 Forward primer 5´-TGGCTGAAAGACAACGACTACCT-3´, Reverse primer 5´-TGAAGCA AGCCCGAAAGG-3´, Probe 5´-ACATGGCCACAG ACCACCTATGCCC-3´, interleukin-6 (IL-6) Forward primer 5´-ACACATGTTCTCTGGGAAATCGT-3´, Reverse primer 5´-AAGTGCATCATCGTTGTTCATACA-3´, Probe 5´-AAATGAGAAAAGAGTTGTGCAATGGCAATTCTG-3 , IL-10, Forward primer 5´-CCAGAGCCACATGCTCCTAGA-3 , Reverse primer 5´-GGTCCTTTGTTTGAAAGAAAGTCTTC -3 , Probe 5´-CTGCGGACTGCCTTCAGCCAGG-3´. All TaqMan Gene Expression assays, primers and probes were from Applied Biosystems. Expression data were normalized against ribosomal 18S RNA or mouse acidic ribosomal phosphoprotein P0 (m36B4). The relative expression levels were calculated according to the formula 2 -ΔCT , where ΔCT is the difference in cycle threshold (CT) values between the target and the ribosomal 18S RNA internal control.

Statistics
Values are expressed as means ± SEM. Comparisons between groups were made by Kruskal-Wallis test and Mann-Whitney U test. P<0.05 was considered statistically significant.

Generation of AdipoR1 AdipoR2 double deficient (AdipoR1 -/-AdipoR2 -/-) mice
The generation of AdipoR1 -/-and AdipoR2 -/-mice and their respective phenotypes in energy and glucose homeostasis have been described before [23]. In order to study the effects of combined AdipoR1 and AdipoR2 deficiency on atherosclerosis during ApoE deficiency, we first wanted to produce AdipoR1 -/-AdipoR2 -/-mice. Mice heterozygous for the two receptors (AdipoR1 +/-AdipoR2 +/-) were generated using AdipoR1 -/-males and AdipoR2 -/-females (backcrossed for eight generations towards C57Bl/6J). We used this strategy since AdipoR2 -/-males in contrast to AdipoR1 -/-males did not produce any offspring (Table 1). This is probably due to an atrophy of the seminiferous tubules and aspermia associated with reduced testes weight as we have described before [23].

Females
Males Again, none of these breedings produced any AdipoR1 -/-AdipoR2 -/-mice (Table 3). Next, we set up an identical breeding using the same parent mice analyzing the resulting embryos at embryonic day 16.5 (Table 4). At this time point, AdipoR1 -/-AdipoR2 -/-mouse embryos were found dead in utero with a swollen appearance macroscopically (Figure 1), and microscopical examination at this stage showed autolysis with accompanying early disintegration of organ and tissue structures. As a specific finding we noted an intravascular accumulation of immature nucleated blood cells (data not shown). We conclude that knocking out both AdipoR1 and AdipoR2 in mice results in embryonic lethality.

Generation of AdipoR2 ApoE double deficient AdipoR2 -/-ApoE -/-mice
Based on the previous finding by us and Liu et al [23,24] that AdipoR2 -/-mice are protected from high-fat diet-induced dyslipidemia and insulin resistance, we then hypothesized that AdipoR2 deficiency could potentially have a protective role also against atherosclerosis. Mice deficient in both AdipoR2 and ApoE (AdipoR2 -/-ApoE -/-) and littermate control mice lacking only ApoE (AdipoR2 +/+ ApoE -/-) were generated to study the effect of AdipoR2 deficiency on the progression of atherosclerosis. The absence of AdipoR2 protein in AdipoR2 -/-ApoE -/-mice was confirmed by western blot analysis of liver protein extracts (Figure 2).

Effects of AdipoR2-deficiency on body weight and plasma biochemistry
Body weights, body weight gain, WAT, liver weights and liver triglyceride content were similar between AdipoR2 -/-ApoE -/mice and AdipoR2 +/+ ApoE -/-controls (Table 5). Total cholesterol levels were followed during the study and measured at four time points (Table 6). Cholesterol levels were similar between the genotypes, except for the measurement at termination when AdipoR2 -/-ApoE -/-mice had higher plasma cholesterol levels compared with AdipoR2 +/+ ApoE -/-controls (Table 6). Since the plasma cholesterol levels were equal, or at termination even higher in AdipoR2 -/-ApoE -/-mice, the smaller plaque size in these mice must be explained by mechanisms other than reduction in total cholesterol levels. In addition, the cholesterol distribution profiles were almost identical between  the groups ( Figure 4A). Furthermore, the plasma triglyceride levels were higher at termination in AdipoR2 -/-ApoE -/-mice compared with AdipoR2 +/+ ApoE -/-controls ( Figure 4B). AdipoR2 deficiency had no effect on total plasma adiponectin levels (Table 6). Thus, neither plasma levels of lipids or adiponectin could help to explain the protective effect of AdipoR2 deficiency against atherosclerosis.

Effects of AdipoR2 deficiency on lipid accumulation and gene expression in peritoneal macrophages
To investigate if there was a difference in lipid accumulation, primary peritoneal macrophages were incubated with or without oxLDL for 24 h and the lipid accumulation was assessed with oil red O staining. AdipoR1 mRNA was expressed at similar levels in macrophages from both AdipoR2 -/-ApoE -/-and AdipoR2 +/+ ApoE -/-mice and there was no change in AdipoR1 mRNA levels after exposure to oxLDL (data not shown).
Interestingly, accumulation of neutral lipids was decreased by 27% in macrophages from AdipoR2 -/-ApoE -/-mice compared with macrophages from AdipoR2 +/+ ApoE -/-controls after incubation with oxLDL ( Figure 5A). In order to explain the decreased lipid accumulation in macrophages from AdipoR2 -/-ApoE -/-mice, gene expression levels for the scavenger receptors CD36, SR-B1 and SR-A1, and the cholesterol transporters ABCA1 and ABCG1 were measured in peritoneal macrophages incubated with or without oxLDL for 24 h. The expression levels of SR-A1, SR-B1 and ABCG1 mRNA were similar between the genotypes (data not shown). However, CD36 mRNA was downregulated by 26% in oxLDL treated macrophages from AdipoR2 -/-ApoE -/-mice compared with control macrophages (Figure 5B), which may indicate a decreased lipid uptake. Furthermore, ABCA1 mRNA was upregulated by 39% in oxLDL treated macrophages from AdipoR2 -/-ApoE -/-mice compared with control macrophages (Figure 5C), which could result in increased lipid efflux. These data may help to explain the reduced accumulation of lipids in macrophages from AdipoR2 -/-ApoE -/-mice compared with macrophages from AdipoR2 +/+ ApoE -/-littermate controls. Finally, mRNA expression levels of interleukin (IL)-6 ( Figure  5D) and IL-10 ( Figure 5E) were similar in macrophages obtained from AdipoR2 -/-ApoE -/-mice and AdipoR2 +/+ ApoE -/littermate controls indicating that AdipoR2-deficiency did not affect the activation pattern (classically activated versus alternatively activated) of the macrophages.

Discussion
In this study in male mice, we could for the first time show that knocking out both AdipoR1 and AdipoR2 results in embryonic lethality demonstrating the critical importance of these receptors and the adiponectin signalling system during embryonic development. We could also demonstrate the novel finding that AdipoR2 deficiency has a protective effect on the progression of atherosclerosis in ApoE -/-mice. AdipoR2 -/-ApoE -/mice had smaller plaque area in the brachiocephalic artery compared with AdipoR2 +/+ ApoE -/-littermate controls. The protective effect was unrelated to changes in plasma cholesterol levels, inflammatory status or differences in the distribution of cholesterol in lipoprotein fractions. However, the protective effect of AdipoR2 deficiency in the brachiocephalic artery could be associated with decreased lipid accumulation in macrophages as demonstrated in intraperitoneal macrophages upon incubation with oxLDL.
At embryonic day 16.5, an AdipoR1 -/-AdipoR2 -/-embryo was found dead in utero with macroscopical swelling and microscopical autolysis and tissue disintegration and intravascular accumulation of immature nucleated blood cells. Interestingly, adiponectin has been shown to stimulate angiogenesis both in vitro and in vivo [30,31], and it would be of interest for future studies to explore the contribution of the two adiponectin receptors in this process. Our finding that AdipoR1 -/-AdipoR2 -/-mice die during embryonic development is in contrast to a study by Yamauchi et al [25] in which viable AdipoR1 -/-AdipoR2 -/-mice were generated. The AdipoR2 -/-mice described by Yamauchi et al [25] was shown to express an aberrantly spliced AdipoR2 mRNA and the AdipoR2 -/-mice studied by us and Liu et al has been shown to express exon 3 and exon 7 [24] surrounding the deleted exon 5 (corresponding to several transmembrane parts of this seven membrane receptor). Importantly, using an antibody directed against a peptide sequence in exon 2 containing the ATG (outside the targeting construct), we could confirm lack of AdipoR2 protein in livers of AdipoR2 -/-ApoE -/-mice in this study and in AdipoR2 -/mice in our previous study [23], indicating that complete lack of adiponectin receptors results in embryonic lethality. However, the non viable AdipoR1 -/-AdipoR2 -/-embryos generated in this study were on a C57Bl/6J genetic background while the Table 7. Plasma levels of inflammatory mediators in AdipoR2 -/-ApoE -/-mice and AdipoR2 +/+ ApoE -/-littermate controls.

AdipoR2 +/+ ApoE -/-(n=25)
AdipoR2 -/-ApoE -/-(n=14) IL-1α (pg/ml) 19   AdipoR1 -/-AdipoR2 -/-mice generated by Yamauchi et al were on a more mixed 129/Sv x C57Bl/6 genetic background [25]. This may be the reason for the different results concerning generation of viable double receptor deficient mice between our laboratories. The difference in plaque area between AdipoR2 -/-ApoE -/mice and AdipoR2 +/+ ApoE -/-controls was seen in the brachiocephalic artery but not in the aortic arch or the descending aorta at the time of investigation. However, since the descending aorta was only analyzed using en face analysis and not cross-sectional analysis, we cannot rule out that also the descending aorta may have been affected. Site specific effects on atherosclerosis have been described previously in ApoE -/-mice in response to various compounds or genetic modifications [32][33][34]. This site specificity is thought to be related to differences in hemodynamic flow patterns, with sites of low shear stress, oscillatory flow or turbulent flow (e.g. at curvatures and branching points) being prone to develop atherosclerosis, whereas areas of laminar flow are more resistant [33]. The brachiocephalic artery (innominate artery) is the first branch in the aortic arch and constitutes a highsusceptibility site, where advanced lesions develop [33,[35][36][37]. Thus, the fact that there is a difference in plaque area between the genotypes in the brachiocephalic artery but not in the arotic arch or the descending aorta in the present study could be due to the length of the study but also indicate a mild overall protective effect of AdipoR2 deficiency against atherosclerosis. Adiponectin levels were found not to correlate with a suppression of the atherosclerosis process in mice using adiponectin deficient and overexpressing mice crossed with either low-density lipoprotein receptor or ApoE deficient mice [38]. AdipoR1 has been found to bind globular adiponectin with higher affinity than full-length adiponectin, while AdipoR2 seems to be an intermediate receptor [22]. Recently, AdipoR1 and AdipoR2 were found to form homo-and heteromeric complexes, with different interaction behaviours and signalling properties. Heteromers dissociated faster than homodimers in response to adiponectin binding and importantly, phosphorylation of AMP-activated protein kinase was delayed in response to adiponectin treatment in cells where heteromers were favoured [39]. It could therefore be speculated that lack of adiponectin receptor heteromers in the absence of AdipoR2 may have led to an improved AdipoR1 signalling. Thus, considering the results from the present study, AdipoR1 and AdipoR2 signalling may have opposing effects in atherosclerosis and it will be important for the future to investigate the effect of AdipoR1 deficiency on the atherosclerosis process.
Plaques in the brachiocephalic artery from AdipoR2 -/-ApoE -/mice were generally smaller and often rich in macrophages, whereas the majority of the plaques from AdipoR2 +/+ ApoE -/mice were larger and characterized by cholesterol clefts and a clear fibrous cap. Plaques from AdipoR2 -/-ApoE -/-mice contained less collagen and more macrophages compared with AdipoR2 +/+ ApoE -/-controls. Therefore, it cannot be ruled out that plaque stability was affected. However, macrophage infiltration and foam cell formation is an early process in plaque development, whereas extracellular matrix, including collagen, is produced in more mature lesions [40]. Thus, these compositional data indicate that plaques in AdipoR2 -/-ApoE -/mice represents earlier lesions with a higher content of macrophages, whereas more mature lesions are seen in the AdipoR2 +/+ ApoE -/-control mice.
We and Liu et al have previously shown that AdipoR2 -/-mice are resistant to high-fat diet induced obesity [23,24], lasting at least until 15 weeks of high-fat diet [23]. The fact that AdipoR2 -/-ApoE -/-mice and AdipoR2 +/+ ApoE -/-controls were comparable in terms of body weight gain or obesity in this study is however not surprising since ApoE-deficiency has been shown to attenuate diet-induced obesity in mice [41]. Further, we and Liu et al have shown that AdipoR2 deficiency leads to hypocholesterolemia [23,24]. AdipoR2 -/-male mice have a plasma cholesterol level of ~3 mmol/l [23], while AdipoR2 -/-ApoE -/-males have a plasma cholesterol level of ~40 mmol/l when fed a high-fat diet. Thus, the severe hypercholesterolemia in ApoE -/-mice attenuated the hypocholesterolemic phenotype seen in AdipoR2 -/-mice [23,24]. Therefore, the decreased atherosclerosis in AdipoR2 -/-ApoE -/-mice compared with AdipoR2 +/+ ApoE -/-controls must be explained by other mechanisms than reduced plasma cholesterol levels. Indeed, AdipoR2 -/-ApoE -/-mice had even higher plasma cholesterol levels compared with control mice at termination. Although high-fat diet fed ApoE -/-mice are regularly used in preclinical atherosclerosis studies, the cholesterol levels obtained are extremely high. Thus, it will be important for future studies to investigate the long-term consequences of AdipoR2-deficiency on atherosclerosis under more moderately elevated cholesterol levels. Since macrophages play an important role in atherogenesis as modulators of both lipid metabolism and immune responses, we isolated primary macrophages to study whether there was a difference in their capacity to accumulate lipids or secrete inflammatory mediators. The inflammatory response was similar in macrophages from AdipoR2 -/-ApoE -/-mice and AdipoR2 +/+ ApoE -/-controls. On the other hand, primary macrophages from AdipoR2 -/-ApoE -/-mice accumulated less neutral lipids compared with macrophages from AdipoR2 +/ + ApoE -/-mice upon incubation with oxLDL. Interestingly, macrophages from AdipoR2 -/-ApoE -/-mice had lower expression levels of CD36 mRNA and higher expression levels of ABCA1 mRNA compared with control macrophages. Studies have demonstrated that CD36-deficiency dramatically inhibits lesion development in ApoE -/-mice [42,43], and can also have a protective effect in late stages of atherosclerosis [44]. The protective effect of CD36-deficiency was shown to be macrophage-dependent, since mice lacking CD36 selectively in macrophages were protected against atherosclerosis, whilst reintroduction of CD36 expressing macrophages resulted in an increased lesion area [45]. Furthermore, macrophage-specific overexpression of ABCA1 inhibits the progression of atherosclerosis [46], whereas macrophage-specific inactivation of ABCA1 increased foam cell formation and atherosclerosis [47]. Thus, downregulation of CD36 and upregulation of ABCA1 in macrophages may have contributed to the slower progression of atherosclerosis in the brachiocephalic artery of AdipoR2 -/-ApoE -/-mice in the present study.
Interestingly, homozygotes and heterozygotes of the ADIPOR2 gene polymorphism rs767870 major allele were recently shown to have lower flow mediated dilation, increased intima-media thickness and increased ADIPOR2 protein levels in peripheral monocytes compared with homozygotes of the minor allele [48]. The same gene polymorphism has previously been associated with elevated liver fat content and fasting triglyceride levels [49] and type 2 diabetes [50]. Thus, high ADIPOR2 levels may be associated with an increased risk of developing cardiovascular disease in humans.
In summary, we could for the first time show that combined genetic deletion of both adiponectin receptors results in embryonic lethality demonstrating the crucial importance of the adiponectin signalling system during embryonic development. We could also show that genetic ablation of AdipoR2 slows down the progression of atherosclerosis in the brachiocephalic artery in atherosclerosis prone male mice lacking ApoE indicating that antagonizing AdipoR2 or activating AdipoR1 may be an attractive therapeutic approach for the treatment of atherosclerosis.