Adiponectin receptor 1 expression is associated with carotid plaque stability
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.115063
Source of Support: None, Conflict of Interest: None
Background: Adiponectin is a hormone secreted exclusively by adipose tissue, and is important in the regulation of tissue inflammation and insulin sensitivity. Adiponectin exerts its effects through two cell-surface receptors: Adiponectin receptor 1 (ADR1) and ADR2. However, the relationship between ADR1/2 expression and progression of atherosclerosis or plaque vulnerability remains unclear. Aims: To investigate the relationship between ADR1/2 expression and plaque characteristics in patients with carotid artery atherosclerosis. Materials and Methods: Forty-three patients who underwent carotid endarterectomy for treatment of carotid artery stenosis were reviewed. Immunohistochemical staining for ADR1 and ADR2 was performed in the specimens of carotid plaque. The relationships between ADR1/2 expression and clinical characteristics were analyzed statistically. Results: Plaque was stable in 7 patients and vulnerable in 36 patients. ADR1 expression was considered weak in 29 patients and strong in 14 patients. The formation of vulnerable plaques was significantly correlated with weak ADR1 expression (P < 0.003). ADR2 expression was considered weak in 14 patients and strong in 29 patients. Rates of formation of vulnerable plaque did not differ between patients with weak and strong ADR2 expression. Conclusions: Based on previous and the present results, ADR1 may be strongly related to the stabilization of established atherosclerotic plaques via inactivating macrophages. Enhancement of ADR1 expression could serve as a therapeutic target for the prevention of the formation of vulnerable plaque.
Keywords: Adiponectin receptor, atherosclerosis, carotid artery stenosis, carotid plaque, stability
Atherosclerosis is a chronic disease characterized by cholesterol plaque formation within the blood vessel wall, and occurrence in the carotid artery is the cause of a substantial proportion of ischemic strokes.  In particular, vulnerable/unstable plaques are likely to result in stroke.  The conversion of a stable plaque to a vulnerable plaque involves many processes, including inflammation, cellular breakdown, expansion of the acellular component, thinning of the fibrous cap, formation of a large lipid core, and intraplaque hemorrhage. ,, Adiponectin is a hormone secreted exclusively by adipose tissue, and is important in the regulation of tissue inflammation and insulin sensitivity.  Adiponectin acts through two cell-surface receptors: Adiponectin receptor 1 (ADR1) and adiponectin receptor 2 (ADR2), ,, which activate the adenosine monophosphate-activated protein kinase (AMPK) pathways and/or peroxisome proliferator-activated receptor (PPAR)-α pathways.,, ADR1/2 are expressed in monocytes, macrophages,  and atherosclerotic lesions.  It has been reported that obesity is associated with not only low plasma adiponectin level but also decreased ADR1/2 expression. ,,
Numerous experimental studies have suggested that adiponectin is involved in the maintenance of vascular homeostasis through many pathways, such as increasing endothelial nitric oxide production, inhibiting endothelial cell activation and endothelium-leukocyte interaction, and suppressing macrophage activation and macrophage-to-foam cell transformation. ,,, Therefore, adiponectin may have a protective effect in atherosclerosis and cardiovascular disease. ,, Low plasma adiponectin concentration is associated with increased risk of coronary artery disease, , and increased plaque vulnerability in patients with coronary artery disease. ,, More recently, low plasma adiponectin concentration was suggested to be related to the progression of carotid artery atherosclerosis. , These findings indicate that adiponectin may contribute to the stabilization of plaque. However, the relationship between ADR1/2 expression and the progression of atherosclerosis or plaque vulnerability remains unclear. The present study investigated the relationship between ADR1/2 expression and plaque characteristics in patients with carotid artery atherosclerosis.
This study reviewed the records of 45 consecutive patients who underwent carotid endarterectomy (CEA) for treatment of carotid artery stenosis between March 2004 and February 2012. Specimens of carotid plaque were unavailable in 2 patients, hence 43 patients were included in this study. Indications for CEA included symptomatic carotid artery stenosis ≥50% or asymptomatic carotid artery stenosis ≥70%.  Preoperative imaging evaluation included digital subtraction angiography in 14 patients, computed tomography angiography in 27 patients, or carotid duplex ultrasonography in 2 patients. The medical records of the 43 patients were examined, and age, sex, past history, statin medication, symptom, stenosis degree, and laboratory data were recorded. The stenosis degree was evaluated using the North American Symptomatic Carotid Endarterectomy Trial criteria.  Patients were defined as symptomatic if focal symptoms of cerebral ischemia were present in the hemisphere ipsilateral to the carotid lesion, such as transient ischemic attack or stroke within the past 6 months.
Histological procedures and immunostaining
The specimens of the carotid plaque were collected intraoperatively immediately after CEA and fixed in 10% buffered formalin for 24 hours. After decalcification, if necessary, specimens were embedded in paraffin wax using conventional techniques, and these paraffin tissue blocks were stored at room temperature until analysis. For analysis, these blocks were cut transversely into 5-μm thick slices, and stained with hematoxylin and eosin. The sections with the most stenotic segment of the plaques were chosen for analysis. The number of sections per specimen was 6.9 ± 2.5. Immunohistochemical analyses were performed using the Universal Immuno-peroxidase Polymer method. After deparaffinization and hydration, the endogenous peroxidase activity was blocked with 3% hydrogen peroxidase. The sections were incubated overnight at 4°C with the following primary antibodies: goat antihuman ADR1 antibody (1:250; Novus Biologicals, Littleton, CO, USA) and goat anti-human ADR2 (1:250; Novus Biologicals). The sections were washed with phosphate buffered saline (PBS) and incubated with Histofine Simple Stain MAX-PO (G) (Nichirei Co, Tokyo, Japan) for 30 minutes at room temperature. The sections were rinsed with PBS. Peroxidase activity was detected with diaminobenzidine.
Criteria for defining plaque characteristics
Plaque sections were classified as either vulnerable or stable. The slides were independently evaluated by two reviewers who were unaware of the imaging results. Criteria for defining vulnerable plaque were presence of thin fibrous cap with a large lipid core, superficial platelet aggregation and lumen thrombosis, plaque surface ulceration and fissuration, intraplaque hemorrhage, and superficial calcified nodule. ,, Other plaques were classified as stable.
Analysis of ADR1/2 expression
All sections were analyzed for ADR1/2 expression. The amounts of immunoreactivity for ADR1/2 were based on estimated percentages of immunopositive cells (0-10%, negative; 10-50%, moderate staining; 50-100%, intense staining) in a high-power field (×400), and were graded by two reviewers (HO and NO) who were unaware of the clinical data or findings of plaque characteristics. Faint diffuse staining was not scored as positive. Negative and moderate staining was considered to be weak expression, and intense staining was considered to be strong expression for the statistical analysis.
All statistical analyses were performed with the Statistical Package for the Social Sciences version 11.0 (IBM Corporation, Armonk, NY, USA). The comparisons between clinical characteristics and the degree of ADR1/2 expression were determined by the Mann-Whitney U test, Chi-square, or Fisher's exact tests. P < 0.05 was considered to be statistically significant.
The clinical characteristics of the patients are summarized in [Table 1]. Forty patients were male and three were female, aged 60-81 years (mean, 70 years). Plaque was stable in 7 patients and vulnerable in 36 patients. All 36 vulnerable plaques had a thin fibrous cap with a large lipid core.
ADR1 expression and clinical characteristics
ADR1 staining was negative in 29 patients, moderate in 7 patients, and intense in 7 patients. Thus, ADR1 expression was considered weak in 29 patients and strong in 14 patients [Table 2]. In most cases, the immunopositive cells were macrophages and foam cells. Carotid plaques were vulnerable in 28 of the 29 patients with weak ADR1 expression and in 8 of the 14 patients with strong ADR1 expression [Figure 1] and [Figure 2]. The formation of vulnerable plaque was significantly correlated with weak ADR1 expression (P < 0.003) [Table 2]. Other characteristics were not associated with ADR1 expression.
ADR2 expression and clinical characteristics
ADR2 staining was negative in 14 patients, moderate in 20 patients, and intense in 9 patients. Thus, ADR2 expression was considered weak in 14 patients and strong in 29 patients [Table 3]. In most cases, the immunopositive cells were macrophages and foam cells. Carotid plaques were vulnerable in 10 of the 14 patients with weak ADR2 expression and in 26 of the 29 patients with strong ADR2 expression. Rates of formation of vulnerable plaques did not differ between the patients with weak and strong ADR2 expression. No other characteristics were associated with ADR2 expression.
Ischemic strokes including transient ischemic attacks are frequently caused by cerebral embolism from athero-thrombotic plaque or thrombosis at the site of plaque rupture.  Vulnerable carotid plaque carries a high risk of stroke, so intensive efforts are warranted to identify therapeutic targets that prevent the formation of vulnerable plaques.
Adiponectin and its signal pathways are reported to participate in the development of plaque. ,,, Low plasma adiponectin levels might be associated with the presence of a thin fibrous cap and a large lipid core, , indicating that high plasma adiponectin levels are a potential therapeutic target to prevent the formation of vulnerable plaques. The mechanism by which adiponectin inhibits proatherogenic processes includes enhancement of endothelial nitric oxide synthase activity, inhibition of inflammatory changes that lead to increased expression of endothelial adhesion molecules, and suppression of macrophage activation required for development of foam cells. , In particular, the differentiation of macrophages into lipid-laden foam cells is a crucial process during the initiation and development of atherosclerosis, because foam cells produce various bioactive molecules, such as cytokines and growth factors, , indicating that suppression of macrophage activity can prevent the formation of vulnerable plaques as well as the initiation of atherosclerosis. ADR1 and ADR2 were first cloned from a skeletal muscle complementary deoxyribonucleic acid library, 7 and expression has been detected in various tissues, including macrophages.  ADR1 and ADR2 expressions are the important factors for adiponectin to exert its antiatherogenic effects, ,, so these receptors are new therapeutic targets for atherosclerosis.
The present study showed that weak ADR1 expression, but not ADR2, was related to the formation of vulnerable plaque. Both ADR1 and ADR2 can activate AMPK pathways and/or PPAR-α pathways,,, but ADR1 and ADR2 exhibited a functional differences. , An in vivo mouse study showed that ADR1 was more tightly linked to the activation of the AMPK pathways, whereas ADR2 was more involved with activation of the PPAR-α pathways. AMPK activation can protect against the acceleration of atherosclerosis by inactivating macrophages. , In addition, ADR1 was more potent in the suppression of inflammatory cytokines.  Taken together with our results, these findings suggest that ADR1 may be more strongly related to the stabilization of established atherosclerotic plaques via inactivating macrophages.
Therefore, we can speculate that not only increasing adiponectin level but also enhancing ADR1 expression are potential therapeutic targets for the prevention of the formation of vulnerable plaques. Exercise combined with a hypocaloric diet increases both ADR1 and ADR2 expression in skeletal muscle in obese adults. 
There are several limitations in the current study. Retrospective analyses are subject to observational and assessment biases. We could not perform the analysis about the correlation between ADR1/2 expression and plasma adiponectin level. Furthermore, the number of patients in this series was small, which reduced the statistical power and increased the chance of type-2 errors. Further studies are required to investigate whether upregulation of ADR1 can suppress the development of plaques.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]