Circulating Anti-Coatomer Protein Complex Subunit Epsilon (COPE) Autoantibodies as a Potential Biomarker for Cardiovascular and Cerebrovascular Events in Patients with Obstructive Sleep Apnea
Although moderate to severe obstructive sleep apnea (OSA) is an independent risk factor for severe arteriosclerotic diseases such as cardiovascular disease (CVD) and stroke, the development of atherosclerosis-related diseases cannot yet be predicted in patients with OSA. In a pilot study, we identified autoantibodies against the coatomer protein complex, subunit epsilon [circulating anti-coatomer protein complex subunit epsilon autoantibody (COPE-Ab)], a cytosolic complex that mediates protein transport in the Golgi compartment, as a potential novel biomarker of atherosclerosis. This study aimed to evaluate whether COPE-Ab levels had an association with cardiovascular and cerebrovascular events in patients with OSA.
Eighty-two adult patients with a diagnosis of OSA via polysomnography and 64 healthy donors were studied. Serum COPE-Ab levels were measured using an amplified luminescence proximity homogeneous assay. Then, clinical factors related to atherosclerosis were evaluated with respect to COPE-Ab levels.
Significant differences in COPE-Ab levels were observed in terms of OSA severity. COPE-Ab levels were significantly higher in patients with OSA and also CVD and/or stroke, hypertension, and a high body mass index. Univariate and multivariate logistic regression analyses of patients with OSA identified elevated COPE-Ab level as a significant predictor of CVD and/or stroke.
An elevated COPE-Ab level may be a potential predictor of the risks of cardiovascular and cerebrovascular events in patients with OSA. Therefore, patients with higher COPE-Ab levels may require more careful and intensive treatment.
A commentary on this article appears in this issue on page 361.
Matsumura T, Terada J, Kinoshita T, Sakurai Y, Yahaba M, Ema R, Amata A, Sakao S, Nagashima K, Tatsumi K, Hiwasa T. Circulating anti-coatomer protein complex subunit epsilon (COPE) autoantibodies as a potential biomarker for cardiovascular and cerebrovascular events in patients with obstructive sleep apnea. J Clin Sleep Med. 2017;13(3):393–400.
Obstructive sleep apnea (OSA) is characterized by repetitive obstruction of the upper airway during sleep, which causes recurrent intermittent hypoxia. Patients with OSA often exhibit excessive daytime sleepiness related to sleep fragmentation because of frequent arousal with apnea. Although OSA is considered a local and upper airway disorder, moderate to severe OSA has recently been recognized as a systemic disease that is associated with the potential risks of brain stroke, fatal or non-fatal cardiovascular events, and death from causes mainly related to the atherosclerotic process.1–4
Current Knowledge/Study Rationale: Moderate to severe OSA is an independent risk factor for arteriosclerosis-related disease; however, the development of severe atherosclerosis-related diseases such as cardiovascular and cerebrovascular events cannot yet be predicted in patients with OSA. The objective of this study was to evaluate whether COPE-Ab levels had an association with cardiovascular and cerebrovascular events in patients with OSA.
Study Impact: This study demonstrates that significant differences in COPE-Ab levels are observed when patients are stratified by OSA severity. An elevated COPE-Ab level is significantly predictive of CVD and/or stroke in patients with OSA; therefore, patients with higher COPE-Ab levels may require more careful and intensive treatment.
OSA-related intermittent hypoxia promotes oxidative stress by promoting the production of reactive oxygen species and angiogenesis, inducing sympathetic activation with blood pressure elevation. It causes systemic, adipose tissue, and vascular inflammation with endothelial dysfunction; taken together, these factors lead to the development of atherosclerosis and eventually cardiovascular and cerebrovascular diseases.5–7 Although various modalities for the evaluation of arterial atherosclerosis, such as brachial-ankle pulse wave velocity (baPWV), the ankle-brachial pressure index (ABI), carotid artery ultrasonography, fundus examination, and microalbuminuria status, have been introduced, their use cannot sufficiently predict the development of devastating conditions such as cardiovascular disease (CVD) and stroke. Additionally, considerable time and effort may be required to obtain reproducible and reliable results using these modalities. In the meantime, several studies have demonstrated the clinical benefit of continuous positive airway pressure (CPAP) therapy for patients with OSA in relation to atherosclerosis-related parameters such as inflammatory markers, lipid profile, and blood pressure.8–10 However, reported CPAP therapy usage frequency ranges from 29% to 85%, suggesting that positive adherence has not yet been achieved.11 Therefore, the development of a simple predictive modality for patients with OSA who require CPAP therapy is needed.
Circulating (i.e., serum) autoantibodies against atherosclerosis-specific antigens have been considered candidate markers of cardiovascular risk. Previously, we have identified autoantibodies recognized by immunoglobulin G (IgG) antibodies in the sera of patients with atherosclerosis-related diseases such as myocardial infarction, stroke, and diabetes.12,13 In our previous studies, we were the first to introduce the serological identification of antigens by recombinant complementary DNA expression cloning (SEREX) method to screen for clones with immunoreactivity against IgG antibodies in patient sera. Subsequently, we used an amplified luminescence proximity homogeneous assay (AlphaLISA) to evaluate the levels of selected antibodies in these patients. In another pilot study, we used SEREX screening to identify the coatomer protein complex subunit epsilon (COPE; accession number CR456886) (clone S33b7) as an antigen recognized by serum antibodies in patients with atherosclerosis.
In this report, we describe our investigation of circulating circulating anti-coatomer protein complex subunit epsilon autoantibody (COPE-Ab) levels in the sera of patients with general OSA in an attempt to evaluate whether COPE-Ab levels had an association with cardiovascular and cerebrovascular events in patients with OSA.
All study procedures involving human participants were conducted in accordance with the ethical standards of institutional (The Local Ethical Review Board of Chiba University, Graduate School of Medicine) and/or national research committees and conformed to the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.
Patients and Healthy Donors
Eighty-two Japanese adult patients in whom OSA was diagnosed by polysomnography (PSG) in our Hospital from June 2012 to January 2014 were studied (56 men and 26 women; median age: 59.0 y). Sixty-four healthy volunteer donors (HD) who underwent medical checkups and had no history of OSA were used as control subjects (38 men and 26 women; median age: 42.5 y). All HD were enrolled at Chiba University. Patients with autoimmune diseases were excluded from this study, and all individual study participants provided informed consent.
Blood Sampling and Purification
Blood samples were collected from each patient upon study admission. Each serum sample was centrifuged at 3,000 × g for 10 min at room temperature, and the supernatant was stored at −80°C until use. Repeated thawing and freezing of samples was avoided.
Classic risk factors for atherosclerosis, including age, sex, body mass index (BMI), smoking status, hypertension, diabetes, hyperlipidemia, CVD, and stroke, were determined from clinical records. Patients were divided into three groups according to smoking status (i.e., never smoked, ex-smoker, and current smoker). Hypertension was defined as a history of systolic blood pressure greater than 140 mmHg, diastolic blood pressure greater than 90 mmHg, or antihypertensive agent use, and diabetes was defined as the use of antidiabetic therapy or a history of diabetes. Hyperlipidemia was defined as a history of a total cholesterol level greater than 220 mg/dL, triglyceride level greater than 150 mg/dL, or lipid lowering agent use. CVD was defined as a history of myocardial infarction or angina pectoris. Stroke was defined as a history of cerebral infarction or cerebral hemorrhage. Cardiovascular and cerebrovascular events were defined as having a history of CVD and/or stroke.
The 2007 American Academy of Sleep Medicine alternative criteria was used to score the PSG.14 Apnea was defined as a reduction in the nasal airflow to less than 10% of the baseline for 10 sec or more, whereas hypopnea was defined as a reduction in the nasal airflow signal amplitude of 50% or higher for 10 sec or more in association with either an oxygen desaturation of 3% or higher or electroencephalographic arousal. OSA was defined as an apnea-hypopnea index (AHI) five events or more per hour combined with predominantly obstructive respiratory events. OSA severity was classified according to AHI values as follows: mild, 5–15; moderate, 15–30; and severe, 30 or higher.
Expression and Purification of Antigenic Glutathione-S-Transferase (GST)-Fusion Proteins
Recombinant GST-tagged proteins were constructed by recombining pBluescript insertion sequences into the pGEX-4T3 plasmid (GE Healthcare Life Sciences, Pittsburgh, PA, USA). A region comprising bases 35–1,131 of COPE messenger RNA was cloned into pBluescript II. As the actual coding sequence was located between bases 43 and 969 of the total 1,134 bases, the full-length coding sequence of COPE was recombined into pGEX-4T3. The gene product was purified as described in our previous reports.12,15,16
Amplified Luminescence Proximity Homogeneous Assay (AlphaLISA)
AlphaLISA was performed in 384-well microtiter plates (white opaque ProxiPlate, PerkinElmer, Waltham, MA, USA) containing 2.5 μL of 1/100-diluted sera and 2.5 μL of GST or a GST-fusion protein (10 μg/mL) in AlphaLISA buffer (25 mM 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid [HEPES], pH 7.4, 0.1% casein, 0.5% Triton X-100, 1 mg/mL dextran-500, and 0.05% Proclin-300). The resulting reaction mixture was incubated at room temperature for 6 to 8 h. Next, anti-human IgG-conjugated acceptor beads (2.5 μL of 40 μg/mL) and glutathione-conjugated donor beads (2.5 μL of 40 μg/mL) were added, and the samples were subjected to an additional 14-day incubation at room temperature in the dark. The chemical emissions of samples were read on an EnSpire Alpha micro-plate reader (PerkinElmer) as previously described.13,15,16 Specific reactions were calculated by subtracting the alpha values of GST control samples from those of samples containing GST-fusion proteins.
All statistical analyses were performed using the JMP Pro 12.2.0 software program (SAS Institute Inc., Cary, NC, USA), and all data are expressed as medians (interquartile ranges). The Mann–Whitney U or Kruskal-Wallis test was used to determine the significance of differences in baseline characteristics between groups. The association between COPE-Ab levels and OSA severity was compared among the HD group, the mild OSA group, moderate OSA group, and severe OSA group using the Kruskal-Wallis test. Each OSA group was compared with the HD group as a post hoc analysis using the Steel test. The pooled all-OSA group was compared with the HD group using the Mann–Whitney U test. Correlations were evaluated using a Spearman correlation analysis, and Fisher exact test was used to determine differences in the proportions of groups. The cutoff value of COPE-Ab levels for predicting CVD and/or stroke among all patients with OSA was determined using receiver operating characteristic (ROC) curve analysis at the value that maximized the sums of sensitivity and specificity. Univariate and multivariate logistic regression analyses were used to identify the set of variables that would classify patients according to CVD and/or stroke status. Eight covariates were included in the models: age (year), sex, obesity (BMI ≥ 25 kg/m2), smoking (current or ex-smoker), hypertension, diabetes, hyperlipidemia, and elevated COPE-Ab levels. All tests were two-tailed, and statistical significance was defined as p < 0.05.
Characteristics of Patients with OSA and the HD Group
Patients with OSA were divided into three groups corresponding to mild, moderate, and severe OSA; clinical characteristics of patients with OSA and the HD group are shown in Table 1. Sixty-four individuals categorized into the HD group and 82 patients with OSA (11 mild, 17 moderate, and 54 severe) were enrolled in the current study. Patients with OSA were significantly older and had higher BMI than those in the HD group. The histories of each atherosclerosis-related disease were more frequently observed as OSA severity increased. An AlphaLISA analysis of the serum COPE-Ab levels revealed significantly higher levels in moderate OSA, severe OSA, and the pooled all-patients with OSA relative to the HD group (p = 0.030, p < 0.001, and p < 0.001, respectively) (Figure 1). Regarding some outliers of COPE-Ab levels in the HD group and patients with OSA in Figure 1, a sensitivity analysis with log transformation of the COPE-Ab data was performed. After the log transformation, there were less serious outliers, and the data were close to a normal distribution. The results did not change much: significant differences were observed among the four groups using analysis of variance (p < 0.001), and post hoc analysis using the Dunnett test revealed significant differences between the severe OSA and HD groups (p < 0.001) and moderate OSA and HD groups (p = 0.031). Student t-test revealed significant differences between the pooled all-OSA and HD groups (p < 0.001).
Association of COPE-Ab Levels and Clinical Parameters in Patients with OSA
The relationships of COPE-Ab levels in patients with OSA with clinical parameters other than disease severity are shown in Figure 2. A moderate association was observed between COPE-Ab level and BMI (ρ = 0.33, p = 0.003, Figure 2c), mean peripheral saturation of oxygen, or SpO2 (ρ = −0.36, p < 0.001, Figure 2d), lowest SpO2 (ρ = −0.31, p = 0.004, Figure 2e), and arousal index (ρ = 0.29, p = 0.008, Figure 2f), whereas significantly higher COPE-Ab levels were observed in patients with hypertension (p = 0.013, Figure 2h), CVD (p = 0.039, Figure 2k), and CVD and/or stroke (p = 0.046, Figure 2l). In contrast, no significant differences in COPE-Ab levels were observed with respect to other parameters, including age, sex, smoking status, diabetes, or hyperlipidemia.
Associations between CVD and/or Stroke and Clinical Parameters in Patients with OSA
The cutoff value of COPE-Ab levels for predicting CVD and/or stroke was determined to be 1,050 using ROC curve analysis. The area under the curve (AUC) was 0.676 (95% confidence interval [CI]: 0.468–0.832).
The results of the univariate and multivariate logistic regression analyses are shown in Table 2. Using the COPE-Ab cutoff value derived from the ROC curve analysis, this univariate logistic regression analysis revealed associations between elevated COPE-Ab levels and male sex with the risk of CVD and/ or stroke (odds ratio [OR]: 4.50, 95% CI: 1.32–18.1, p = 0.016 and OR: 6.82, 95% CI: 1.23–127.8, p = 0.025, respectively). The multivariate logistic regression analysis, which included variables that differed significantly in the univariate analysis, revealed an independent association between elevated COPE-Ab levels and CVD and/or stroke (OR: 3.90, 95% CI: 1.11–16.0, p = 0.034).
In this study, we discovered two important features of COPE-Ab in patients with untreated OSA. First, COPE-Ab levels were significantly higher in patients with OSA, particularly those with moderate to severe OSA, than in the HD group. Furthermore, patients with OSA and also CVD and/or stroke, hypertension, and a high BMI had elevated levels of COPE-Ab. Second, an elevated COPE-Ab level was the significant predictor of a history of CVD and/or stroke in a multivariate analysis of patients with OSA. According to our results, COPE-Ab might therefore be a biomarker of the risk of cardiovascular and cerebrovascular events in patients with OSA.
According to recent studies, circulating autoantibodies exist in the sera of patients with atherosclerosis, including autoantibodies against phospholipids in patients with acute coronary syndrome,17,18 apolipoprotein A-1 in patients exhibiting atherosclerotic plaque vulnerability,19 and oxidized low-density lipoprotein, which is associated with plaque formation and coronary risk in some patients with systemic lupus erythematosus.20 Previously, we have also reported the identification of atherosclerosis antigens via expression cloning from a phage complementary DNA library, as well as the association of the levels of antibodies against several autoantigens (e.g., RPA2, ATP2B4, BMP-1) with atherosclerosis-related risk factors, including stroke, hypertension, and/or smoking habits.12,16 Antibody markers appear to be highly sensitive and therefore are expected to be predictive markers for other atherosclerotic diseases, such as the onset of stroke and CVD, in patients with OSA. In the preliminary stage of the current study, COPE-Ab levels were the most closely associated with OSA severity among our candidate markers. Therefore, we believed that this antibody marker might be useful for evaluating lethal atherosclerotic disease onset in patients with OSA, and thus, we initiated the current study and analysis.
COPE (36 kDa) is one of seven subunits of the coatomer protein complex known as coat protein complex I (COPI). COPI, which is among the best-characterized coat complexes, is a cytosolic complex that coats Golgi-derived vesicles and is involved in protein transport from the Golgi apparatus to the endoplasmic reticulum via recruitment by Arf1.21–23 Although the precise cellular mechanisms of COPI, and circulating COPE-Ab, in patients with OSA were not revealed by the current study, lipid inflammation might be a key element associated with elevated COPE-Ab levels in this patient population. Previous studies have reported that the Arf1–COPI vesicular transport machinery regulates droplet morphology and lipid storage/utilization within the vesicle-trafficking pathway.24–26 This implies a link between COPI and lipid storage (e.g., atherosclerosis-related) diseases. In the mechanism underlying atherosclerosis, macrophages and smooth muscle cells infiltrate into vascular endothelial cells to uptake lipids, resulting in differentiation into foam cells. Foam cells aggregate to form an atheroma, leading to the occlusion of blood vessels.27 OSA is often accompanied by obesity; therefore, serum levels of COPI and its subunit, COPE, may be elevated to store excessive lipids in the cells in patients with OSA. This speculation is consistent with the association of elevated COPE-Ab levels and BMI in the present study (Figure 2c). Furthermore, preceding a CVD and/or stroke, atheromatous plaques are partially ruptured, which can lead to the leakage of excessively expressed COPE into extracellular or intravascular spaces. Then, repetition of this COPE leakage may result in an elevated expression of COPE-Ab in serum.
Additionally, chronic intermittent hypoxia, which is experienced by most patients with OSA, has been known to modulate lipid metabolism via lipid peroxidation at a cellular level28; additionally, this condition increases sympathetic activity, which affects the blood pressure and heart rate, promotes free radical production and adhesion molecule expression, and induces insulin and leptin resistance, by altering peripheral (carotid bodies) and central (nucleus tractus solitarius, hypothalamus, and ventral medulla) chemoreflex pathways.29,30 Another study found that intermittent hypoxia caused adipose tissue inflammation in patients with OSA by increasing macrophage recruitment and the secretion of interleukin-6 and tumor necrosis factor-alpha, leading to the development of atherosclerosis.31 These potential COPE-mediated effects of chronic intermittent hypoxia on lipid metabolism might be reflected in the observed higher levels of COPE-Ab in untreated patients with OSA, although hyperlipidemia per se did not significantly correlate with the COPE-Ab level in the current study (p = 0.193, Figure 2j). In Figure 1, the COPE-Ab levels in patients with mild OSA did not differ from those in the HD group. This results can be explained by the level of (intermittent and/or sustained) hypoxia, because elevated COPE-Ab levels were associated with lower oxygen levels during sleep, as shown in Figure 2d and Figure 2e, which are relatively milder in patients with mild OSA than those in patients with moderate to severe OSA. Additionally, this result of mild OSA is consistent with a previous study; mild OSA has no clear association with the risk of atherosclerosis-related diseases.2
As the prevention of future cardiovascular and cerebrovascular events is the main purpose of OSA treatment, COPE-Ab might serve as a simple and easy biomarker for the evaluation of patients with OSA who clearly require continuous CPAP therapy. Generally, atherosclerosis in patients with OSA is evaluated via the clinical analyses of coronary artery calcium levels, carotid intima media thickness, baPWV, and flow-mediated dilation.32 Of these, increases in baPWV, intima media thickness, and carotid diameter were reported to be potential early signs of atherosclerosis in patients with OSA.33 However, when these parameters are evaluated, the autoantibody approach described in this study is clearly more simple and convenient, because it only requires a patient serum sample. Several markers of inflammation and endothelial dysfunction related to cardiovascular or cerebrovascular disease have also been described as atherogenic proinflammatory markers in patients with OSA; these include soluble tumor necrosis factor receptor, tumor necrosis factor-beta, interleukin-6, and soluble intercellular cell adhesion molecule-1. However, the usefulness of these biomarkers as simple predictors of future cardiovascular and cerebrovascular events remains to be determined. Accordingly, analysis of circulating COPE-Ab levels could prove useful for predicting the risk of lethal events in patients with OSA, although prospective cohort studies are needed to confirm the efficacy of COPE-Ab as a marker of atherosclerosis in this population.
We must note some limitations associated with our study. First, the number of patients with mild or moderate OSA was relatively small, as was the number of patients with stroke (just 5 of 82 patients with OSA). Accordingly, the cutoff value of COPE-Ab levels for predicting CVD and/or stroke among all patients with OSA determined via ROC curve analysis could be inaccurate because of the small number of CVD and/or stroke events. In addition, as the 64 controls were healthy volunteer donors, potential confounding factors between patients with OSA and controls (e.g., age, BMI, hypertension, diabetes, and hyperlipidemia) were not adjusted in the analysis in the current study. Second, some classic risk factors (e.g., hyperlipidemia and diabetes mellitus) of atherosclerosis were not associated with elevated COPE-Ab levels in this study. As antihypertensive agents, statins, and antiplatelet agents are generally known to affect the pathogenesis of atherosclerosis,34–37 we must consider the potential modulatory effects of these drugs on COPE-Ab levels. Third, we did not conduct physiological testing, such as ABI, baPWV, or coronary artery calcification, to evaluate atherosclerosis in subjects subjected to the COPE-Ab analysis. Nevertheless, these tests might be expected to confirm the results of the current study. Finally, the study population included only Japanese patients. Further studies are required in patients who are not taking drugs that can affect atherosclerosis, and in other ethnic groups.
In conclusion, patients with OSA had higher COPE-Ab levels than did those in the HD group. Notably, the COPE-Ab levels were significantly higher in patients with OSA and also CVD and/or stroke, hypertension, and a high BMI. The results suggest that elevated COPE-Ab levels could serve as a potential biomarker of the risk of cardiovascular and cerebrovascular events in this patient population. Therefore, patients with higher COPE-Ab levels may require more careful and intensive treatment.
Institution where the study was performed: Chiba University Hospital. This work was supported, in part, by a research grant from the Japan Agency for Medical Research and Development (AMED), JSPS KAKENHI (Grant number 16K09528) and Grants-in-Aid of the Japan Science and Technology Agency (JST) in Japan (Exploratory Research No. 14657335). The authors have indicated no financial conflicts of interest.
ankle-brachial pressure index
amplified luminescence proximity homogeneous assay
area under the curve
brachial-ankle pulse wave velocity
body mass index
circulating anti-coatomer protein complex subunit epsilon
circulating anti-coatomer protein complex subunit epsilon autoantibody
coat protein complex I
continuous positive airway pressure
healthy volunteer donor
obstructive sleep apnea
receiver operating characteristic
peripheral saturation of oxygen
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9 Impact of obstructive sleep apnea treatment by continuous positive airway pressure on cardiometabolic biomarkers: a systematic review from sham CPAP randomized controlled trials. Sleep Med Rev; 2015;21:23-38, 25220580.
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13 Identification of cerebral infarctionspecific antibody markers from autoantibodies detected in patients with systemic lupus erythematosus. J Mol Biomark Diagn; 2015;6:219.
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16 Elevated levels of autoantibodies against ATP2B4 and BMP-1 in sera of patients with atherosclerosis-related diseases. Immunome Res; 2015;11097.
20 Effects of autoimmune antibodies anti-lipoprotein lipase, anti-low density lipoprotein, and anti-oxidized low density lipoprotein on lipid metabolism and atherosclerosis in systemic lupus erythematosus. Rev Bras Reumatol; 2010;505:539-551, 21125190.
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29 Chronic intermittent hypoxia and hypercapnia inhibit the hypothalamic paraventricular nucleus neurotransmission to parasympathetic cardiac neurons in the brain stem. Hypertension; 2014;643:597-603, 24958501.