Decreased Adiponectin-Mediated Signaling Through the AdipoR2 Pathway Is Associated With Carotid Plaque Instability
Background and Purpose—Adiponectin, the most abundantly secreted anti-inflammatory adipokine, protects against all stages of atherosclerotic plaque formation by acting on its receptors, AdipoR1 (adiponectin receptor 1) and AdipoR2 (adiponectin receptor 2). Through binding of AdipoR1, adiponectin leads to the activation of the AMPK (adenosine monophosphate–activated protein kinase) pathway, whereas stimulation of PPAR-α (peroxisome proliferator–activated receptor-α) is attributed to the binding of AdipoR2. However, the role of adiponectin and its receptors in plaque instability remains to be characterized. Thus, we aimed to investigate whether the adiponectin–AdipoR pathway is associated with carotid atherosclerotic plaque instability.
Methods—The instability of plaque specimens obtained from patients who underwent a carotid endarterectomy (n=143) was assessed using gold standard histological classifications.
Results—Using immunohistochemistry, we showed that adiponectin and AdipoR1/AdipoR2 are expressed in human carotid plaques and that their expression was localized most abundantly in areas of macrophage and foam cell accumulation. Unstable plaques expressed more adiponectin protein (Western blot, P<0.05) and less AdipoR2 mRNA (2.11-fold decrease, P<0.05) than stable plaques, whereas AdipoR1 expression remained similar between stable and unstable plaques. Beyond AdipoR1/AdipoR2 expression, a graded decrease in PPAR-α protein levels was observed in relation to carotid plaque instability (P<0.001), whereas AMPK phosphorylation was increased (P<0.05). Our in vitro model of plaque instability, involving the induction of foam cells from human THP-1 (Tamm–Horsfall protein 1) macrophages treated with acetylated low-density lipoprotein, supported our in vivo conclusions.
Conclusions—An overall abundance of adiponectin with a decrease in AdipoR2 expression and activity was observed in unstable plaques, suggesting that reduced signaling through the AdipoR2 pathway, and not through AdipoR1, may contribute to plaque instability.
Carotid atherosclerotic plaque rupture is a major cause of acute cerebrovascular ischemic events, such as stroke.1 Plaque morphology and composition, in addition to progressive stenosis of the vessel lumen, are major determinants for plaque instability and rupture.1
Adipokines, immunomodulatory proteins secreted by adipose tissue, can regulate vascular homeostasis.2 The majority of adipokines are proinflammatory and exert adverse effects on the vasculature. However, the most abundantly secreted adipokine is adiponectin, which is widely recognized for its anti-inflammatory, antiatherogenic, and vascular protective properties.2,3 Hypoadiponectinemia is associated with obesity, type 2 diabetes mellitus, coronary artery disease (CAD), and progression of carotid atherosclerosis.4–6 The atheroprotective effects of adiponectin have been demonstrated at all stages in atherosclerotic plaque formation, from endothelial dysfunction, plaque initiation and progression, to plaque rupture and thrombosis; these effects are attributed to adiponectin’s actions on all major cell types present in the vasculature, including macrophages, endothelial, and smooth muscle cells.3 Adiponectin exerts its effects via 2 transmembrane receptors, AdipoR1 (adiponectin receptor 1) and AdipoR2 (adiponectin receptor 2),7 which play a key role in glucose and energy metabolism through potentially distinct pathways.8,9 Adiponectin mainly leads to the activation of the AMPK (adenosine monophosphate–activated protein kinase) pathway through binding of AdipoR1, whereas AdipoR2 stimulates the PPAR-α (peroxisome proliferator–activated receptor-α) pathway.8 However, the precise physiological role of these receptors in the vasculature and in plaque infiltrating cells remains to be clarified.
Because of its antiatherosclerotic activities, it is reasonable to hypothesize that a decrease in plaque adiponectin-mediated signaling through the AdipoR1/AdipoR2 pathways may reflect plaque instability in humans. Therefore, the aim of this study was to investigate whether the adiponectin–AdipoR pathway is associated with plaque instability in humans.
Consecutive patients with high-grade carotid stenosis, scheduled for a carotid endarterectomy (CEA), were recruited from the Vascular Surgery preoperative clinics at the McGill University Health Centre (MUHC) and the Jewish General Hospital (JGH) in Montreal, Canada, as described previously.10,11 Patients were excluded from participation in the study if they had previous interventions on the same carotid artery (CEA, carotid artery stenting). Further details concerning patient recruitment are provided in the online-only Data Supplement.
The McGill University’s Institutional Ethics Review Board and Transplant Quebec granted ethics approval (A12-M145-09B) for this study. All subjects provided written informed consent before study participation.
Fasting blood samples were collected from each subject the day of the CEA before surgical intervention. Blood processing and measurements were performed as described in the online-only Data Supplement.
Human carotid atherosclerotic plaques were obtained immediately after surgical resection and processed as explained in the online-only Data Supplement. Furthermore, plaque instability was assessed as previously described10,11 and outlined in detail in the online-only Data Supplement.
To serve as healthy control tissue, carotid artery specimens free of atherosclerosis (termed herein as healthy carotid arteries) were obtained from recently deceased organ transplant donors, who consented for research through Transplant Quebec.
Techniques for Histology/IHC and RNA/Protein Analyses
Other techniques used in this study can be found in full detail in the online-only Data Supplement.
In Vitro Model of Plaque Instability
We developed an in vitro model of plaque instability, using THP-1 (Tamm–Horsfall protein 1) cells, a human monocytic cell line (American Type Tissue Culture Collection, Camden, NJ), to confirm our in vivo findings. These cells were first differentiated into macrophages and then transformed into foam cells using human acetylated low-density lipoprotein. Details about cell culture and acetylated low-density lipoprotein preparation and validation of the transformation of THP-1 macrophages into foam cells by quantification of intracellular cholesterol ester content and Oil Red O staining are described in Figures II and III in the online-only Data Supplement.
The statistical methods are presented in the online-only Data Supplement.
Demographic, clinical, and biological parameters of the total CEA population (n=143), as well as of the population split according to the plaque instability classification (definitely stable, intermediate stable, and definitely unstable), are presented in the Table. The total population had a mean age (±SD) of 69.1±9.0 years, was predominantly male (70.6%), and had a median circulating adiponectin level of 8.54 (4.28–13.92) µg/mL. The proportion of patients with CAD differed significantly across all plaque groups (P=0.044), with the definitely stable group having the greatest proportion of patients with CAD than the intermediate stable and definitely unstable group. Differences in sex and ever smoker status were approaching significance (P=0.058 and P=0.056, respectively), with a greater proportion of women (52.9%) within the definitely stable group and a greater proportion of ever smokers (96.7%) within the definitely unstable group.
Circulating adiponectin levels did not differ significantly among the plaque groups (Table). However, adiponectin was positively correlated with high-density lipoprotein (r=0.413; P<0.001) and negatively with body mass index (r=−0.366; P<0.001) and triglycerides (r=−0.286; P=0.001).
Symptomatology and Plaque Instability
The overall relationship between cerebrovascular symptomatology and plaque stability was not significant (Table; P=0.246), suggesting that symptomatology is not a good indicator of the stability of a plaque. In addition, the nature of the cerebrovascular event (amaurosis fugax, transient ischemic attack, or stroke), as well as the average time between the cerebrovascular event and the CEA, did not differ significantly in relation to plaque stability (Table).
Among the CEA patients who experienced a cerebrovascular ischemic event (amaurosis fugax, transient ischemic attack, or stroke), 23% had their CEA within the acute phase (≤7 days). The time since event ranged from 1 to 169 days, with an average time of 5.0 (3.0–6.0) days for subjects who underwent a CEA within the acute phase (≤7 days) and 30.0 (20.0–60.5) days for subjects who underwent a CEA after the acute phase (>7 days). However, the nature of the event (amaurosis fugax versus transient ischemic attack versus stroke) remained similar between the 2 groups (acute: 13%/52.5%/34.8%; nonacute: 20.8%/41.6%/37.7%; P=0.591). In addition, the stability of the plaque (definitely stable versus intermediate stable versus definitely unstable) in relation to acute and nonacute cerebrovascular ischemic events did not differ (acute: 8.7%/69.6%/21.7%; nonacute: 9.1%/66.2%/24.7%; P=0.953), even when the relationship was assessed according to the nature of the event.
Graded Expression of Adiponectin in Carotid Atherosclerotic Plaques in Relation to Plaque Instability
Using quantitative real-time polymerase chain reaction, adiponectin mRNA was not detected in carotid plaques or in healthy carotid arteries, suggesting that adiponectin mRNA transcripts are not produced by cells present in the plaque or in the intimal and medial layer of the healthy vasculature (Figure IV in the online-only Data Supplement). In contrast, using Western blot, adiponectin protein was detected in both atherosclerotic plaques and in healthy carotid arteries and a graded increase in adiponectin expression was noted from healthy carotid arteries, to definitely stable plaques, to definitely unstable plaques (P=0.023; Figure 1).
The associations between plaque adiponectin protein expression and plaque instability, according to specific clinical covariates (sex, smoking, diabetes mellitus, CAD, hypertension, symptomatology, and statin use), are presented in Table I in the online-only Data Supplement. Furthermore, Table II in the online-only Data Supplement presents differences in plaque adiponectin protein levels in relation to these clinical variables.
Differential Expression of AdipoR1/AdipoR2 in Carotid Atherosclerotic Plaques in Relation to Plaque Instability
AdipoR1 and AdipoR2 mRNA transcripts were present in both human atherosclerotic lesions and healthy carotid arteries. AdipoR1 expression did not differ between atherosclerotic plaques and healthy arteries (1.092±0.270 arbitrary units [AU] versus 1.000±0.158 AU, respectively; P=0.870; Figure 2A), whereas there was a trend for a 1.73-fold reduction of AdipoR2 expression in atherosclerotic plaques compared with healthy carotid arteries (0.578±0.349 AU versus 1.000±0.305 AU, respectively; P=0.293; Figure 2B). AdipoR1 expression remained similar across all grades of instability (P=0.659; Figure 2C), whereas AdipoR2 differed significantly (P=0.019; Figure 2D). Specifically, there was a graded decrease in AdipoR2 expression from definitely stable to intermediate stable to definitely unstable plaques. When compared with definitely stable plaques, there was a 1.68-fold and a 2.11-fold decrease in AdipoR2 expression in intermediate stable (0.596±0.345 AU versus 1.000±0.382 AU; P=0.068) and definitely unstable plaques (0.474±0.342 AU versus 1.000±0.382 AU; P=0.014), respectively. Furthermore, in plaques that had a high presence of foam cells (≥50 cells), AdipoR2 mRNA expression decreased significantly by 1.37-fold compared with plaques with a lower presence of foam cells (<50 cells) (0.729±0.332 AU versus 1.000±0.364 AU, respectively; P=0.033; Figure V in the online-only Data Supplement). Table III in the online-only Data Supplement presents AdipoR1 and AdipoR2 mRNA expression in relation to other plaques features, including infiltration of inflammatory cells in the cap, which was found to be trending in association with lower AdipoR2 expression.
Circulating adiponectin levels were negatively correlated with AdipoR1 mRNA expression in the plaque (r=−0.243; P=0.004) but not with AdipoR2 mRNA (r=−0.056; P=0.508). Neither AdipoR1 nor AdipoR2 mRNA were correlated with clinical variables (Table II in the online-only Data Supplement). Table I in the online-only Data Supplement presents the association between plaque AdipoR2 mRNA expression and plaque instability, according to specific clinical covariates (sex, smoking, diabetes mellitus, CAD, hypertension, symptomatology, and statin use).
Adiponectin-Mediated Signaling in Relation to Plaque Instability
A graded decrease in PPAR-α protein levels was observed in relation to carotid plaque instability (P<0.001; Figure 3B). A 1.92- and 1.84-fold reduction in PPAR-α was noted in definitely unstable plaques when compared with healthy carotid arteries and definitely stable plaques, respectively (P<0.001; Figure 3B), whereas AMPK phosphorylation (activity) was significantly increased in definitely unstable plaques (P<0.05; Figure 3C).
Cellular Localization of Adiponectin, AdipoR1, and AdipoR2
Healthy Carotid Arteries
Using immunohistochemical (IHC), adiponectin was highly detected in the endothelial layer and to a lower extent in the smooth muscle cells. AdipoR1 and AdipoR2 were also found expressed in endothelial cells and smooth muscle cells (Figure VI in the online-only Data Supplement).
Carotid Atherosclerotic Plaques
Adiponectin, AdipoR1, and AdipoR2 were expressed in endothelial cells (Figure VII in the online-only Data Supplement) and vascular smooth muscle cells (Figure VIII in the online-only Data Supplement). Importantly, expression of adiponectin, AdipoR1, and AdipoR2 was most abundant in macrophage and foam cell-rich areas in the fibrous cap and surrounding the lipid core (Figure 4). Immunofluorescence staining demonstrated colocalization of AdipoR1 and AdipoR2 with CD68 (Figures IX and X in the online-only Data Supplement), a marker for macrophage/foam cells, indicating that both receptors are expressed on macrophages/foam cells in human atherosclerotic plaques.
In Vitro Model of Plaque Instability
THP-1 macrophages on treatment with adiponectin resulted in a significant upregulation in AdipoR2 and PPAR-α protein expression (P<0.01; Figure 5B) compared with nontreated macrophages, whereas no change in expression was observed among foam cells treated with adiponectin (Figure 5C). However, adiponectin treatment of both macrophages and foam cells did not result in a significant change in AdipoR1 expression or activity (AMPK phosphorylation; Figure 5B and 5C). Figure XIA in the online-only Data Supplement presents the absolute change in expression of AdipoR1, AdipoR2, AMPK-phosphorylated, and PPAR-α between treated and nontreated macrophages and foam cells. Furthermore, in the presence of adiponectin, foam cells had significantly lower AdipoR2 and PPAR-α expression than macrophages (P<0.001; Figure 5D), whereas in the absence of adiponectin, foam cells and macrophages demonstrated comparable AdipoR2 and PPAR-α protein levels than macrophages (Figure XIB in the online-only Data Supplement). However, AMPK phosphorylation was observed to be significantly higher in foam cells treated with adiponectin when compared with macrophages treated with adiponectin (P<0.05; Figure 5D), whereas no significant change in AdipoR1 was observed. We noted a similar significant increase in AMPK phosphorylation in foam cells not treated with adiponectin when compared with macrophages not treated with adiponectin (P<0.05; Figure XIB in the online-only Data Supplement), with no observed changes in AdipoR1 expression. A summary diagram of these results is present in Figure XII in the online-only Data Supplement.
This study represents an essential step in identifying a novel association between decreased adiponectin-mediated signaling through the AdipoR2 pathway and plaque instability. We identified that definitely unstable plaques express more adiponectin and less overall AdipoR2 than definitely stable plaques, whereas AdipoR1 expression remained similar among all groups of plaque instability. In parallel, we noted a decrease in AdipoR2 activity, specifically through PPAR-α, in definitely unstable plaques, whereas an upregulation was observed in AMPK phosphorylation, which is a downstream target of AdipoR1. Furthermore, we created an in vitro model of plaque instability, using human THP-1 macrophages, which supported our in vivo hypotheses.
Adiponectin exerts atheroprotective and anti-inflammatory effects on the vasculature.3 It can attenuate monocyte attachment and migration into the intima, suppress macrophage transformation into foam cells, and induce the expression of tissue inhibitor of matrix metalloproteinase-1, ultimately reducing the risk of a rupture in the fibrous cap.12–14 Its deficiency, both clinically and experimentally, plays a significant role in the development of various vascular complications. Hypoadiponectinemia is associated with endothelial dysfunction, CAD, and progression of carotid atherosclerosis. In animal models, adiponectin deficiency enhances neointimal formation in response to acute vascular damage and impaired endothelium-dependent vasodilation, whereas treatment with adiponectin decreases atherosclerotic lesion formation in apolipoprotein-E knockout mice and in a rabbit model of abdominal aortic atherosclerosis.5,6,15–18
Adiponectin protein has been detected in the vasculature of normal and atherosclerotic mice.19 We demonstrated that adiponectin protein is also present in healthy human carotid arteries, as well as human carotid atherosclerotic plaques. Interestingly, we noted a lack of adiponectin mRNA in healthy arteries (composed of only the intimal and medial layer) and carotid plaques. This indicates that adiponectin protein in the plaque area or in the intimal and medial layer of healthy vasculature is not because of de novo cellular expression. Instead, adiponectin must enter these layers from outside sources.19–21 Indeed, one murine study assessing the ultrastructural localization of adiponectin within endothelial cells of the aortic wall observed the presence of adiponectin in endocytic vesicles, suggesting that adiponectin undergoes endocytosis from the circulation into the endothelial cells.19 Others have suggested that adiponectin in the vascular wall is derived from the adventitia and surrounding perivascular adipose tissue, an abundant producer and secretor of adiponectin.20,21 Herein, we have confirmed the abundant expression of adiponectin in perivascular adipose tissue (surrounding healthy carotid arteries), using polymerase chain reaction and IHC analyses (Figures IV and XIII in the online-only Data Supplement).
Furthermore, in our study, IHC staining of healthy carotid arteries demonstrated that adiponectin protein is highly localized to the endothelial layer and to a lower extent on smooth muscle cells, whereas in carotid plaques, adiponectin was mainly detected in areas of high macrophage and foam cell abundance. Differences in the localization pattern of adiponectin have also been detected within the aortic wall of normal versus atherosclerotic mice.19 We are the first to observe an increase in adiponectin expression within atherosclerotic plaques that exhibit unstable features when compared with definitely stable plaques or healthy arteries. Lending support to the hypothesis that the origin of adiponectin in the plaque may be derived from the surrounding perivascular adipose tissue, a recent study demonstrated higher adiponectin expression in the perivascular adipose tissue of neurologically symptomatic patients versus asymptomatic patients who underwent a CEA.21 Our findings, along with previous experimental evidence,13,22 suggest that adiponectin may accumulate in unstable lesions or in the damaged vascular wall as a protective mechanism in response to injury or because of increased endothelial permeability that is associated with vascular damage and atherosclerotic lesion progression.23
The beneficial effects of adiponectin are mediated through its receptors, AdipoR1 and AdipoR2,7,8 which we observed to be expressed most abundantly on macrophages/foam cells, and to a lower extent on endothelial and smooth muscle cells in atherosclerotic plaques. An overall decrease in AdipoR2 expression was noted in definitely unstable plaques. Furthermore, AdipoR2 signaling through PPAR-α was significantly impaired in definitely unstable plaques, suggesting not only a decrease in AdipoR2 expression but also in its activity. In contrast, AdipoR1 expression remained unchanged between definitely stable and definitely unstable plaques. However, a significant upregulation in AMPK-phosphorylated activity was observed in association with greater plaque instability. Although PPAR-α and AMPK are main downstream signaling components of AdipoR1 and AdipoR2, respectively, they are not limited to these receptors and can also be triggered by other factors present in carotid lesions.24,25 Thus, we used the in vitro model of foam cell formation (a critical process of plaque instability), to confirm our in vivo observations.
On treatment with recombinant adiponectin, a significant upregulation in AdipoR2 and PPAR-α protein expression was observed in macrophages but resulted in no change in AdipoR1 expression and AMPK activity. The recombinant adiponectin used was primarily composed of the higher molecular weight isoforms of adiponectin (Figure I in the online-only Data Supplement); thus, it was not surprising to observe significant changes in the AdipoR2 pathway but not in AdipoR1 since AdipoR1 possesses lower affinity than AdipoR2 for the high-molecular-weight isoforms of adiponectin.7 AdipoR2 or PPAR-α expression was not upregulated in adiponectin-treated foam cells, suggesting impairment in the response of AdipoR2 to its ligand and a resulting loss in its downstream signaling activity. The baseline values (ie, in the absence of adiponectin) of AdipoR2 and PPAR-α remained unchanged after the transformation of macrophages into foam cells. However, the response of foam cells to adiponectin was impaired on transformation from macrophages, resulting in decreased AdipoR2 expression and activity (through PPAR-α) compared with adiponectin-treated macrophages (summary diagram, Figure XII in the online-only Data Supplement), which mirrors the decrease in expression noted between definitely unstable plaques (ie, contains greater presence of foam cells), definitely stable plaques, and healthy carotid arteries. We hypothesize that decreased signaling downstream of AdipoR2 may impair the atheroprotective actions of adiponectin and cause adiponectin resistance, thereby contributing to plaque instability and an accumulation of adiponectin in vulnerable lesions. In contrast, AMPK activity was upregulated in adiponectin-treated foam cells compared with macrophages, whereas no changes in AdipoR1 were noted, as similarly observed in definitely unstable versus definitely stable plaques. However, this increase in AMPK activity was also noted between foam cells and macrophages not treated with adiponectin. Thus, we think that the increase in AMPK activity associated with foam cell induction, as well as plaque instability, is independent of the adiponectin–AdipoR1 pathway.
Interestingly, previous evidence supports a differential role of the 2 receptors. In mice, the 2 receptors demonstrated opposite effects on glucose tolerance and energy expenditure, where AdipoR2 deficiency promoted diabetes mellitus.9,26 However, sufficient evidence remains lacking to elucidate their independent role in atherosclerotic disease.
Our study has several strengths, including the use of human carotid arteries, pointing to its translational aspect, the combination of in vivo and in vitro experiments to unravel the observed associations, and the novelty of the topic in the area of atherosclerotic disease. We acknowledge that our study also contains limitations. No causative associations can be determined; because the study analyzed carotid specimens, a cross-sectional design was inevitable. Furthermore, as we used human data, adiponectin and its receptors can be influenced by several factors. However, because the stable and unstable patient groups were similar in terms of patient comorbidities and clinical characteristics, and analyses performed according to specific clinical covariates did not affect the significance of the results, we are confident that the observed differences are not because of interindividual variability. Furthermore, our in vitro experiments led support to our in vivo findings.
In addition, although beyond the scope of this study, another limitation is that the origin of adiponectin in the plaque could not be determined, as it is not feasible to collect perivascular adipose tissue from our CEA subjects. A recently published article demonstrated higher adiponectin expression in the perivascular adipose tissue (surrounding the carotid plaques) of neurologically symptomatic patients versus asymptomatic patients who underwent a CEA,21 thus mirroring the elevation in adiponectin protein levels we observed in unstable versus stable carotid plaques versus healthy carotid arteries. To provide some supporting evidence, we performed polymerase chain reaction analysis on the removed perivascular adipose tissue that was surrounding the healthy carotid arteries and demonstrated that the adiponectin mRNA expression was a 1100-fold higher than in healthy carotid arteries and in carotid plaques (Figure IV in the online-only Data Supplement), confirming that adiponectin is highly expressed by perivascular adipose tissue and not by plaque tissue or the healthy vasculature that has been stripped of its surrounding adventitial and adipose tissue layers. We also performed IHC analyses of the removed perivascular adipose tissue, which demonstrated adiponectin protein to be expressed by the adipocytes (Figure XIII in the online-only Data Supplement).
Our findings suggest that a decrease in AdipoR2 signaling in unstable plaques may define a state of adiponectin resistance, which partly explains the compensatory elevation in adiponectin levels observed in unstable plaques. This study including a large number of carotid plaque specimens provided human evidence that AdipoR2 may be a key player in the context of plaque instability, whereas the adiponectin–AdipoR1 pathway plays no significant role (summary model in Figure XIV in the online-only Data Supplement). However, as this is a relatively untapped area of research, investigations are ongoing to further elucidate the distinct effect of AdipoR2 on plaque instability.
We are grateful to the vascular surgeons (Drs Steinmetz, MacKenzie, Corriveau, and Obrand) at the McGill University Health Centre (MUHC) and Jewish General Hospital (JGH), in Montreal, Canada, as well as Ms Gorgui and Ms Gomez for their help with subject recruitment.
Sources of Funding
The work was supported by the Fonds de recherche du Québec-Santé (FRQ-S)  and the Research Institute of the MUHC, as well as the Canadian Institutes of Health Research (CIHR) Catalyst Grant  and the CIHR Project Scheme Grant . Dr Daskalopoulou is a Chercheur-Boursier Clinicien supported by the FRQ-S. K. Gasbarrino is supported by a CIHR Doctoral Studentship. The funding sources had no involvement in the study.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.116.015145/-/DC1.
- Received August 23, 2016.
- Revision received January 5, 2017.
- Accepted January 17, 2017.
- © 2017 American Heart Association, Inc.
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