Initial screening of AP3D1 antigens using SEREX.
As an initial SEREX screening, sera of patients with atherosclerosis were used to search for antigens that could be recognized by serum IgG antibodies, one of which was AP3D1 (accession no. NM_003938.8). The region of 2490-4347 of the AP3D1 gene was then isolated and recombined into the EcoRI/NotI site of pGEX-4T-1, followed by confirmation by DNA sequencing. The cDNA was then expressed in E. coli, purified by affinity chromatography, and employed as an antigen in order to examine the serum antibody levels.
The levels of anti-AP3D1 antibodies were elevated inpatients with AIS and TIA.
The serum anti-AP3D1-antibody (s-AP3D1-Ab) levels in patients with AIS and TIA were examined using AlphaLISA. AIS and TIA sera were provided by Chiba Prefectural Sawara Hospital, Chiba Rosai Hospital, and Chiba Aoba Municipal Hospital, whereas samples of HDs were obtained from Chiba University, Port Square Kashiwado Clinic, and Chiba Prefectural Sawara Hospital. The average ages [± standard deviations (SDs)] of the HDs and patients with AIS and TIA were 52.33 ± 8.92, 57.99 ± 7.97, and 69.45 ± 11.64 years, respectively (Table 1a). The levels of s-AP3D1-Abs were determined to be significantly higher in patients with AIS and TIA than those in HDs (Fig. 1a). At a cutoff value equivalent to the average plus two SDs of the HD values, the s-AP3D1-Ab-positive rates in HDs and patients with AIS and TIA were 2.4%, 10.1%, and 10.4%, respectively (Table 1b). ROC curve analysis revealed that the area under the curve (AUC) values for s-AP3D1-Abs vs. AIS and vs. TIA were 0.616 and 0.662, respectively (Figs. 1b, c). No significant difference was found in the positive rates and the AUC values between AIS and TIA.
Elevation of s-AP3D1-Abs levelsin patients with DM.
The levels of s-AP3D1-Abs were also examined for DM. Sera of HD were obtained from Chiba University, whereas the sera of patients with DM were provided by the Chiba University Hospital. The average ages (± SDs) of the HDs and patients with DM were 45.20 ± 10.95 and 63.12 ± 12.04 years, respectively. The AlphaLISA results revealed that s-AP3D1-Ab levels were significantly higher in patients with DM than in the HDs (Fig. 2a). When the positive samples for which the AlphaLISA counts exceeded the cutoff value were scored, the positive rates of s-AP3D1-Abs in the HDs and the patients with DM were 3.7% and 41.8%, respectively (Table 2b). The AUC value of s-AP3D1-Abs vs. DM was as high as 0.791 (Fig. 2b). Therefore, it can be concluded that the s-AP3D1-Ab levels were closely associated with DM.
The s-AP3D1-Abs levels were associated with CVD.
For the next step, the antibody levels in samples from CVD patients were examined. The samples of CVD patients were obtained from Chiba University Hospital, and those in HDs were from Chiba University, Port Square Kashiwado Clinic, and Chiba Prefectural Sawara Hospital. The average ages (± SDs) of the HDs and CVD patients were 45.27 ± 11.20 and 66.07 ± 11.32 years, respectively. Compared with HDs, s-AP3D1-Abs levels were significantly higher in patients with CVD (Fig. 3a), and the s-AP3D1-Ab positivity rates in HDs and patients with CVD were 5.1% and 24.0%, respectively (Table 3). ROC curve analysis revealed that AUC of s-AP3D1-Abs for CVD was 0.758 (Fig. 3b).
The s-AP3D1-Ab levels were closely related to CKD.
The antibody levels in the sera of CKD patients were examined, which were assumed to be closely related to atherosclerosis. The sera of the CKD patients were obtained from the Kumamoto cohort [22, 23], including 145 from patients with diabetic kidney disease (type 1 CKD), 32 from patients with nephrosclerosis (type 2 CKD), and 123 from patients with glomerulonephritis (type 3 CKD). The sera of HDs (82 specimens) were obtained from Chiba University, Chiba Prefectural Sawara Hospital, and the National Hospital Organization of Shimoshizu Hospital (Table 4a). Patients from all three groups of CKD were found to have significantly higher levels of s-AP3D1-Abs compared to that in HDs (Fig. 4a). The s-AP3D1-Ab-positive rates in HDs and patients with type 1, type 2, and type 3 CKD were 4.9%, 27.6%, 37.5%, and 22.8%, respectively (Table 4). ROC curve analysis revealed AUC of s-AP3D1-Abs of type 1, type 2, and type 3 CKD to be 0.791, 0.874, and 0.735, respectively (Figs. 4b, c, d). Type 2 CKD showed the highest AUC value among all diseases examined.
Association of s-AP3D1-Ab levels with ESCC or CRC.
The s-AP3D1-Abs levels were also measured in serum samples from the HDs and patients with ESCC or CRC. AlphaLISA results revealed that s-AP3D1Ab levels were significantly higher in patients with ESCC and CRC than in HDs (Fig. 5a). The positivity rates of s-AP3D1-Abs in HDs and patients with ESCC and CRC were 3.1%, 42.2%, and 15.6%, respectively (Table 5). The AUC values were 0.872 and 0.743 for ESCC and CRC, respectively (Figs. 5b, c).
Correlation analysis.
Correlation analysis of s-AP3D1-Ab levels and subject data was performed using 635 specimens from Chiba Prefectural Sawara Hospital, including 139 samples from HDs, 121 from patients with deep and subcortical white matter hyperintensity, 17 from patients with asymptomatic cerebral infarction, 43 from patients with TIA, 228 from patients with AIS, 57 from patients with chronic-phase cerebral infarction, and 30 from other diseases. Using the Mann–Whitney U test, the s-AP3D1-Ab levels were compared between participants with body mass index (BMI) < 25 and BMI ≥ 25; participants with or without diseases of DM, hypertension (HT), CVD, and dyslipidemia; and between those patients who were smokers or nonsmokers and those who consumed alcohol or not. The analysis showed that the s-AP3D1-Ab levels were significantly higher in the subjects with HT than in those without HT and those with DM than without DM (Table 6). Conversely, no significant differences in s-AP3D1-Ab levels were observed in the other categories.
Performing a logistic regression analysis of the predictors for AIS using the results of the Sawara Hospital was also considered, which included 139 samples from HDs and 228 from patients with AIS. An elevated s-AP3D1-Ab level was associated with an increased risk of AIS as shown by the univariate logistic regression analysis (P < 0.0001). A multivariate logistic regression analysis has identified age, HT, and DM, but not s-AP3D1-Ab, as independent predictors of AIS (Table 7).
Next, correlation analysis was performed using a Spearman application in order to determine the correlation between s-AP3D1-Ab levels and subject parameters, including general information such as age, body height, weight, BMI, and the degree of artery stenosis [the maximum intima-media thickness (max IMT)]. The serum s-AP3D1-Ab levels were determined to be closely correlated with age (P < 0.001), max IMT (P < 0.001), blood pressure (BP) (P < 0.001), and smoking period (P < 0.001) (Table 8). Conversely, inverse correlation was observed between s-AP3D1-Ab levels and height, weight, Ca, and low-density lipoprotein cholesterol. Blood glucose and glycated hemoglobin, which are identified as DM markers, were not significantly correlated with the s-AP3D1-Abs levels. These results suggest that s-AP3D1-Ab reflected atherosclerosis and its causal HT and smoking.
JPHC cohort analysis.
To determine whether s-AP3D1-Ab marker can be applied to predict the onset of AIS, JPHC cohort samples were examined. The antibody level of AP3D1 protein was positively and strongly associated with the risk of AIS: the ORs (95% CI) were 1.40 (0.75–2.63), 1.97 (1.07–3.65), and 2.28 (1.26–4.13) for the samples with the second, third, and highest quartiles of antibody level, respectively, compared with the lowest quartile (Table 9). These results indicate that the antibody markers against AP3D1 are useful in predicting the onset of AIS.