The two antigens, ALDOA and FH, were found following serological identification of antigens by means of recombinant cDNA expression cloning (SEREX). Serum IgG antibodies in TIA patients further recognized them. Additionally, we confirmed by Western blotting the presence of antibodies against ALDOA and FH in the patients’ serum (Fig. 2). Furthermore, by means of AlphaLISA, we evaluated the antibody levels. AlphaLISA also allowed us to compare the levels between patients and HDs. Our results show that, compared with HDs, the antibody levels of anti-ALDOA(ALDOA-Abs) and anti-FH(FH-Abs) were significantly elevated in both TIA patients and those with CI (Fig. 3). We gathered additional confirmation that these antibodies are independent predictors of TIA (Table 3). Of note, TIA has a tendency to develop into CI and is a clear CI risk factor [39]. As an independent early warning risk factor for TIA, these elevated antibody levels may also be predictive markers of CI. Therefore, confirmation was obtained by further statistical analysis of clinical data and prospective case-control studies nested in large community-based samples (Tables 3 and 4).
Earlier studies have shown that ALDOA, also known as fructose-bisphosphate aldolase A, represents one of the glycolytic enzymes that catalyze the reversible conversion of fructose-1, 6-bisphosphate to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate [40]. ALDOA is widely distributed in whole body tissues. As a catalytic enzyme, ALDOA represents one of the key enzymes in the glycolysis process. Of note, it plays a role in the hypoxia responses regulating both glucose and energy metabolism and can serve as hypoxia biomarkers [41]. Ischemic stroke represents a typical atherosclerosis-related disease. Its basic pathophysiological feature is represented by local tissue hypoxia. Studies have shown that ALDOA is a hypoxia-inducible gene expression product [42]. When brain tissue undergoes ischemia or hypoxia, brain cells react by stimulating both glucose uptake and metabolism. Their goal is to compensate for the reduction in energy production by inducing overexpression of ALDOA [43]. Hypoxia-inducible factor 1α (HIF-1α) is a transcription factor sensitive to hypoxia-inducible genes. HIF-1α up-regulates ALDOA’s expression in hypoxic cells [44], thereby enhancing its glycolysis metabolism. A previous study by Chang et al. proved that ALDOA and HIF-1α were able to co-act, both evoking and enhancing the expression of matrix metalloproteinases [45]. These results are in line with our previous study [27] where we found an increased specificity of anti-MMP1 antibodies in the serum of TIA patients. MMPs can degrade the main components of the vascular extracellular matrix, which is an important factor responsible for the induction of atherosclerosis. An intimate relationship between ALDOA and MMPs can be observed. We believe that such association proves that ALDOA's overexpression may not only be a sequential pathological process in TIA’s development, but it may also be related to TIA’s initiation and deterioration. Of note, we observed that ALDOA-Abs levels had a constant positive correlation with the following characteristics: age, smoking, HT, obesity (BMI), and CHD (Table 5). ALDOA is silent in the presence of adequate nutritional status (e.g., hyperglycemia, hyperlipidemia, high protein, etc.). As a consequence, ALDOA-Abs are not elevated in the serum of patients with the following conditions: hyperglycemia, DM, hyperlipidemia, and high protein status. These results suggest two explanations. First, that ALDOA-Abs may represent a biomarker of TIA. Second, that ALDOA-Abs represent a marker more specific for atherosclerosis-related diseases, but not associated with glucose tolerance or blood lipids.
FH is a key enzyme involved in the tricarboxylic acid (TCA) cycle. It can reversibly catalyze the conversion of fumaric acid to L-malate in the cells [46]. A primary function of the TCA cycle is the oxidation of pyruvate, supplied by the glycolytic pathway, with the goal of producing energy. In addition to its classical metabolic-related functions, FH has other non-metabolic-related functions under the stimulation of cells [47]. Reports have associated FH with tumorigenesis, specifically by altering the gene expression site and configuration of tumor cells [48]. In an earlier study, Xiao et al. found that FH could antagonize α-ketoglutarate-dependent demethylase through its metabolite fumarate, thereby affecting histone methylation [49]. In addition, Wang et al. also showed that FH exhibited adenosine monophosphate-activated protein kinase-mediated phosphorylation in the absence of glucose or hypoxia, which inhibited histone’s demethylation by lysine-specific demethylase 2A [50]. FH inhibits histone methylation by reducing the physiological activity of vascular endothelial growth factor (VEGF) [51]. This, in turn, affects the repair and remodeling of vascular endothelium following atherosclerosis. It is noteworthy that abnormal histone methylation is responsible for significant gene expression changes, including VEGF. It has been shown that FH plays an important regulatory role in atherosclerosis’ occurrence and development [52]. Based on this background, our findings can be explained. Specifically, FH-Abs levels in TIA and ischemic stroke patients are significantly higher than those in HDs. FH’s metabolic function is related to that of ALDOA, which can provide energy to the body through physiological reactions. However, as opposed to ALDOA, when hyperglycemia occurs, FH’s expression is induced by the excited TCA cycle. Therefore, FH is associated not only with common atherosclerotic risk factors (e.g., age, blood pressure, obesity, CHD) but also with DM. In the present study, we demonstrated that FH is a broader spectrum marker of atherosclerosis-associated diseases. Due to its well-defined vascular injury, FH can be a potential target for TIA warning and for the early treatment of ischemic stroke.
Here, we have analyzed the biological function of ALDOA and FH. Our goal was to explain the physiological mechanism behind the elevation of related antibody levels in TIA patients. Based on our results, we inferred that the elevated levels of ALDOA and FH antibody levels represent risk predictors of ischemic stroke. Next, we aimed at demonstrating that the elevated levels of ALDOA-Abs and FH-Abs were independent risk factors for TIA and ischemic stroke. To this end, 92 TIA patients and 285 HDs were pooled together to establish a logistic regression analysis model (n = 377). It is a well-known fact that there are numerous independent risk factors affecting the occurrence and development of atherosclerosis, other than age, smoking habits, DM, HT, obesity and CHDs [7, 26–29, 53]. In the present model, we evaluated the association between TIA risk factors and/or antibody markers with the occurrence of TIA events (Table 3). The results showed that age, HT, DM, hyperlipidemia, and CHD had a good correlation with this model. It is indicated that this model is satisfied to analyze whether the elevated levels of ALDOA-Abs and FH-Abs are independent risk factors for TIA. By univariate and multivariate logistic regression analysis, we demonstrated that ALDOA-Abs and FH-Abs were diagnostic markers of TIA (Table 3). Since TIA is one of prodromal stages of CI, ALDOA-Abs and FH-Abs may be used as risk predictors. Specifically, they may have a high predictive value for ischemic stroke. To test this hypothesis, we conducted a case-control study nested within the Japan Public Health Center-based Prospective Study (see methods for details). ALDOA and FH’s antibody levels were measured in 202 cases of incident cerebral infarction developed in patients of the cohort between the baseline and 2008, and in 202 controls with matching age, sex, and area. In order to estimate the levels of ALDOA and FH for CI, we used a conditional logistic regression model. Our results showed that ALDOA and FH's antibody levels were positively and strongly associated with the risk of cerebral infarction. As a consequence, we believe that such antibody markers can be applied to predictive diagnosis rather than simple risk evaluation.
ALDOA-Abs and FH-Abs represent promising biomarkers for TIA and CI. Positive rates of each marker may not be sufficiently high. This may be due to their association with different causes (e.g., hypoxemia, HT, and smoking habit). In our opinion, the diagnostic value will improve through a combination of the measurement of antibodies and clinical risk factors, including age, HT, DM, and hyperlipidemia, which were independent TIA predictive factors in the multivariate logistic regression analysis (Table 3). Take TIA as an example, we calculated in the cohort of 92 patients with TIA and 285 HDs the positive rates including the conventional risk factors, age, HT, and DM. We used the cutoff values of ALDOA-Abs and FH-Abs to detect TIA, 14,869 and 2,849, respectively, as mentioned above. Positive predictive values (PPVs) of age, HT, and DM alone were 48.0%, 51.3%, and 71.1%, respectively (Supplementary Table 1). On the contrary, PPVs of ALDOA-Abs combined with age, HT, and DM increased to 63.1%, 63.5%, and 91.3%, respectively. Similarly, PPVs of FH-Abs combined with age, HT, and DM were 61.9%, 56.9%, and 94.1%, respectively. Furthermore, PPVs with the combination of three factors, HT, DM, and ALDOA-Abs or age, DM, and FH-Abs, reached up to 100% (Supplementary Table 1). In fact, antibody levels combined with clinical risk factors improve the ability to predict TIA, and this same applies to early prediction CI. Additional studies are needed to establish the complete predictive diagnosis system of ischemic stroke.
Collectively, this study provided solid evidence that ALDOA-Abs and FH-Abs could be used to early predict TIA and CI. ALDOA-Abs and FH-Abs' expression levels were independent warning markers for TIA and CI, providing additional information to guide therapeutic strategies.