The Association of Serum Levels of stromolysin-1 and connexin-37 with the severity of coronary artery disease

Background Coronary Artery Disease (CAD) is a common form of heart disease that is considered a serious health problem in society. Atherosclerosis is widely recognized as a chronic inammatory disease of the vessels and can lead to CAD and myocardial infarction. The aim of the present study was to investigate serum levels of connexin-37 and stromelysin-1 as signicant biomarkers of CAD and their correlation with the extent of CAD. Methods and results Sixty CAD patients with one-vessel (1VD), two-vessel (2VD), and three-vessel (3VD) disease were enrolled in this study. For comparison with the results, 20 healthy control subjects were also included in this study. Serum concentrations of connexin-37 and stromelysin-1 were determined using commercial ELISA kits. Serum connexin-37 concentrations were not signicantly different between the patient and control groups (p < 0.05). The analysis showed a statistically signicant difference between subjects with one-vessel disease, subjects with two-vessel disease, and subjects with three-vessel disease. Serum Stromelysin-1 concentration was signicantly higher in the patients than in the control group (p < 0.05). Conclusions The results of our study indicate that serum levels of stromelysin-1, but not connexin-37, may contribute to the prediction of the occurrence and progression of CAD.


Introduction
Coronary artery disease (CAD) is frequently diagnosed in patients with cardiovascular complications [1]. According to recent ndings, excessive in ammatory response in the sub arterial space increases the possibility of thrombus formation [2]. Factors such as hypertension, dyslipidemia, atherosclerosis, smoking, and inappropriate lifestyle may increase the risk of CAD [3,4]. Atherosclerosis is associated with endothelial dysfunction and correlates with the formation of coronary necrotic plaques that may detach in the early stages. A link between atherosclerosis and the possibility of myocardial infarction has already been demonstrated [5].
Connexins are transmembrane proteins and are encoded by 20 genes in various tissues [6,7]. These proteins connect adjacent cells using gap junctions and channels and facilitate the passive mutual diffusion of small molecules [8,9]. One connexin protein, namely connexin-37, plays a key role in regulating the in ammatory response. Connexin-37 can control the attachment of monocyte endothelial cells and reduce pathological remodeling [10]. There is evidence that a mutation in the gene allele C1019T predisposes men to cardiovascular disease [11]. Patients who are prone to atherosclerotic plaques may have a single nucleotide polymorphism (SNP) detected in their genome [12]. Sei et al. have shown that the presence of a polymorphism in the connexin 37 genes can alter the complications of acute myocardial infarction (AMI) [13]. It has been elucidated that suppression of the connexin 37 gene in apolipoprotein (Apo) E de cient mice can lead to the formation of atherosclerotic plaques [14].
Stromelysin-1, also known as matrix metalloproteinase-3 (MMP-3), is involved in the pathological changes during AMI and CAD [15]. Stromelysin-1 digests the extracellular matrix (ECM) and is frequently found in atherosclerotic plaques [16]. The hyperactivity of MMPs such as stromelysin-1 may promote coronary atheroma due to connective tissue remodeling. Along with these changes, atherosclerotic plaques are detached, leading to coronary artery thrombosis [15].
This study aimed to determine the serum levels of connexin-37 and stromolysin-1 in patients with CAD and to nd a possible association between these factors and the severity of CAD. For this purpose, we selected CAD patients with different numbers of affected vessels (1, 2, and 3 vessels).

Ethical considerations
The protocol used in this study was approved by the local ethics committee of Tabriz University of Medical Sciences (IR.TBZMED.REC.1398.1181). All patients and healthy volunteers were asked to complete an informed consent form before participating in this study.

Sample preparation
This study was a cross-sectional analysis of 80 cases (aged 40-70 years), 60 CAD patients and 20 controls. In this study, patients with symptoms of heart disease were studied. The possibility of heart disease was con rmed by angiography. The exclusion criteria were hyperlipidemia, diabetes mellitus, respiratory disease, liver dysfunction, renal disease, and cancer. Healthy volunteers with no history of heart disease or other conditions were asked to participate in the current study to compare results. Blood samples were collected from CAD patients referred to the Cardiology Unit of Shahid Madani Hospital, a liated with Tabriz University of Medical Science (Tabriz, Iran). Based on the results of angiography, CAD patients were classi ed into three categories as follows: patients with one-vessel disease (1VD), patients with two-vessel disease (2VD), and patients with three-vessel disease (3VD). All information, such as blood pressure, height, weight, family history, hyperlipidemia, age, and sex, was collected by appropriate questionnaires.

Blood Sampling
For this purpose, 10 ml of fasting venous blood was drawn from each patient and collected in tubes containing EDTA. The samples were centrifuged at 3000 g for 20 minutes. After completion of the centrifugation procedure, the samples were immediately placed in the − 80˚C freezer and stored until use.

Biochemical assays
Serum levels of triglycerides (TG), total cholesterol, and high-density lipoprotein (HDL) were measured with spectrophotometry and commercially available kits (Pars Azmoun, Iran). To calculate low-density lipoprotein cholesterol (LDL-C), we used the formula of Fried Wald (17). LDL-C was measured directly in the samples with a higher value of TG (400 mg/dl). An enzyme-linked immunosorbent assay (ELISA) was used for the determination of connexin-37 (Cat. No.: E67777Hu; Bioassay Technology) and stromelysin-1 (Cat. No.: E1722Hu; Bioassay Technology).

Statistical analysis
Statistical analysis was performed using SPSS version 16. Differences between the patient and control groups were analyzed by t-test and ANOVA. All variables were expressed as mean ± SD. P < 0.05 was considered statistically signi cant.

Laboratory ndings
The data showed that the mean concentrations of cholesterol (TC), triglycerides (TG), and LDL-C were signi cantly higher in the patients with CAD compared with the control group. In contrast, serum levels of HDL-C were signi cantly lower in the patient group (Table 2). Statistical analysis revealed no signi cant difference in connexin-37 levels between the two groups (p > 0.05; Table 3). We found that the mean connexin-37 level in the patient group CAD reached 7.3 ± 1.63 ng/ml, while this value in the control group was 7.28 ± 2.47 ng/ml. Of note, these changes were statistically signi cant between the patient groups with CAD (patients with one-vessel disease, patients with two-vessel disease, and patients with threevessel disease) (Tables 3 and 4). According to our data, the serum concentration of connexin-37 was higher in the 2VD group than in the 3VD and 1VD groups. The 1VD group had the lowest levels of connexin-37.   The case group with one blocked artery) 1VD); two blocked arteries (2VD); three blocked arteries (3VD) The ELISA results showed that the mean serum level of stromelysin-1 in the CAD patients was 15.2 ± 8.65 ng/ml, and this value reached about 9.3 ± 5.93 ng/ml in the control group. Independent-sample ttest analysis showed a statistically signi cant difference in the stromelysin-1 levels of CAD patients compared with healthy controls (Table 5). In the one-way analysis ANOVA, the mean difference between stromelysin-1 was statistically signi cant at 1VD, 2VD, and 3VD. Accordingly, the serum concentration of stromelysin-1 was higher in the 2VD group than in the 3VD and 1VD subgroups. According to our data, these levels were higher in the 1VD subgroup than in the 3VD CAD patients (Table 6).  The case group with one blocked artery) 1VD); two blocked arteries (2VD); three blocked arteries (3VD)

Discussion
Atherosclerosis is a pathological condition characterized by the accumulation of lipids on the luminal surface of vessels, which promotes the recruitment of macrophages and T lymphocytes. It has been suggested that dyslipidemia may sensitize coronary arterioles to atherosclerotic plaques. On this basis, the increase in TC, TG, LDL-C and the decrease in HDL-C are predisposing factors in myocardial infarction patients [18]. Here, we showed that TG, TC, and LDL-C were signi cantly increased in CAD patients compared with control subjects. These conditions coincided with a signi cant decrease in HDL-C levels in CAD patients compared with control subjects. Remarkably, we also found that the intensity of CAD and vascular complications (3VD patients) was closely associated with the change in TG, TC, LDL-C, and HDL-C. We found no signi cant differences in serum levels of connexin-37 between the control group and the CAD group. Connexin-37 is thought to regulate the bioactivity of endothelial cells by participating in various signal transductions and juxtacrine connections between the endothelium and smooth muscle cells [16]. According to previous experiments, connexin-37 is involved in the formation of hemichannels that prevent atherosclerotic plaques by suppressing the interaction between endothelial cells and leukocytes [19]. According to our results, the occurrence of CAD may not affect the serum levels of connexin-37, but the intensity of vascular complications in CAD patients leads to signi cant differences between 3VD and other subgroups. Stromelysin-1 (MMP3) has been reported to be elevated in CAD patients compared with healthy controls. It has been suggested that elevation of MMPs may lead to the formation of atherosclerotic plaques in the carotid artery [20]. In agreement with our data, Tengiz and coworkers con rmed the elevation of stromelysin-1 in patients with coronary aneurysms compared with the control group [21]. Stromelysin-1 is a speci c proteinase released by connective tissue cells. This enzyme can degrade ECM when activated [22]. In support of our data, Wu and colleagues investigated the prognosis of different plasma levels of MMPs in patients with CAD [23]. They found that the intensity of vasculitis and the levels of high-sensitivity C-reactive protein and stromelysin-1 were closely correlated with the progression of cardiovascular events. However, it appears that plasma stromelysin-1 levels are an independent prognostic factor for cardiovascular disease, suggesting that this factor should be used to classify CAD.

Conclusion
This study shows that stromelysin-1, but not connexin-37, was increased in CAD patients, indicating a close relationship between serum levels of stromelysin-1 and CAD. In patients at risk for atherosclerosis, monitoring serum levels of stromelysin-1 along with other biochemical biomarkers may help predict the possibility of the occurrence of CAD and the intensity of vascular involvement.

Declarations
Compliance with ethical standards

Con ict of interest
The authors declare that there are no con icts of interest.

Ethical approval
The Ethics Committee of Tabriz University of Medical Sciences approved the study protocols according to the Declaration of Helsinki.

Satisfaction with publication
All authors agree to publish the data of this study.