Evaluation of MMP-9, IL-6, TNF-α levels and peripheral blood mononuclear cells genes expression of MMP-9 and TIMP-1 in Iranian patients with coronary artery disease

Introduction: Coronary artery disease (CAD) is the main cause of death and is characterized by atherosclerosis in coronary arteries. Inflammation plays a crucial role in the progression and development of atherosclerosis. Methods: The present study consisted of 132 Iranian individuals who underwent coronary angiography, 65 patients with CAD, and 67 controls. The matrix metalloproteinase-9 (MMP-9), TNF-α, IL-6, and vitamin D serum levels were measured by the ELISA technique. The gene expression of MMP-9 and tissue inhibitors of metalloproteinase (TIMP-1) was estimated by real-time PCR assay. Results: A considerable increase in levels and PBMC gene expression of MMP-9 and serum levels of IL-6 and TNF-α were found in CAD patients compared with controls. A significant decrease was detected in vitamin D levels of CAD patients in comparison with controls. A considerable direct correlation was found between MMP-9 levels and MMP-9 and TIMP1 gene expression in CAD patients. MMP-9 levels positively correlated with LDL-C in CAD patients. The correlation between TIMP1 gene expression and IL-6 levels was also negatively significant. There were positive correlations between MMP-9 levels with IL-6 and TNF-α serum levels in CAD patients. Conclusion: This study showed that the interaction between MMPs, TIMP1, and cytokines could play a role in the pathogenesis of atherosclerosis. The present study suggested that high levels of TNF-α and IL-6 and vitamin D deficiency in our studied patients could disturb the MMP-9/TIMP-1 balance and lipid metabolism, leading to plaque formation/ rupture in predisposed CAD patients.

4][15][16] To our knowledge, both TNF-α and IL-6 possess the potential to activate transcription factors, most notably PPAR-γ and NF-κB pathways.This activation can trigger the transcytosis of LDL across the endothelium, thereby starting the progression of atherosclerosis.Furthermore, it can result in the activation of reactive oxygen species (ROS) and inflammasomes. 15,16merging evidence has suggested that Vitamin-D (Vit-D) plays a crucial role in CAD aetiology, and Vit-D insufficiency is associated with CAD severity.Vitamin D plays a protective role in preventing atherosclerosis through its anti-inflammatory and anti-oxidant effects.It also regulates the growth, migration, differentiation, and immune system modulation of vascular cells, while inhibiting fibrotic signaling. 17,18][9][10][11][12][13][14][15][16][17][18] Vit-D deficiency in heart diseases leads to inflammation and ECM remodeling by activating of inflammatory transcription factors such as NF-κB and AP-1. 19,20ccording to recent findings, MMPs and inflammatory factors contribute to the development and progression of cardiovascular diseases.Hence, the present study aimed to explore the interrelationship between the lipid profile, Vit-D, Zn 2 + , Ca 2 + , TNF-α and IL-6 with PBMC gene expression of MMP-9, TIMP-1 and MMP-9 levels in patients with CAD and controls.

Population
A cohort of 132 Iranian subjects underwent a study at Hazrat-e-Rasoul Hospital during coronary angiography, at the Iran University of Medical Sciences.This study was conducted between April 2020 and February 2021 and using the confidence interval method, sixty-five patients with CAD and sixty-seven healthy controls were recruited.Inclusion criteria was CAD diagnosis by coronary angiography.
The diagnosis of CAD is typically made by a cardiologist when an individual has at least one major coronary artery with stenosis greater than 50%.Conversely, healthy controls are identified from participants whose coronary arteries exhibit less than 25% stenosis. 19ll participants were subject to exclusion criteria, which included having a history of chronic diseases, chemotherapy, steroid hormone therapy, cancer, liver diseases, diabetes, renal failure and anti-inflammatory medication in the last six months.Additionally, the study did not include individuals with unstable angina, carotid plaque, a history of cardio-cerebrovascular disease, acute coronary syndrome, or peripheral artery disease as controls.The studied subjects' medical history, demographic information, and medication consumption were collected by questionnaire.Anthropometric information was measured, including systolic blood pressure (SBP) and diastolic blood pressure (DBP), weight, height and body mass index (BMI).The study was approved by the Ethics Committee of Iran University of Medical Sciences, Tehran, Iran.(IR.IUMS.FMD.REC.1398.059).According to the Declaration of Helsinki, informed written consent was obtained from all participants (IR.IUMS.FMD.REC.1398.059).

Blood
Collection and laboratory parameter measurements Fifteen mL Venous blood was collected following night fasting (8 to 12 hours) and stored at -80°C.The lipid profile, which is shown in Table 1 and the levels of fasting blood sugar (FBS) were measured (Pars Azmon kit, Iran).

PBMCs isolation and RNA-Extraction
As previously described, 10 mL whole blood was collected in test tubes containing EDTA for PBMC isolation by Ficoll- Hypaque density-gradient centrifugation. 21Extracting total RNA from cell lysates was done by miRNeas mini kit, following the instructions provided by the manufacturer (QIAGEN, USA, Cat.No. / ID: 74004).For evaluating the purity and concentration of extracted RNA, agarose gel electrophoresis (1.5%) (Figure 1) (Bio-Rad, USA, Cat.No: 161-0722) and the Nano-drop spectrophotometer were used (thermo-scientific, US).

Synthesis of cDNA and mRNA Quantitation
To evaluate gene expression, the real-time PCR (RT-PCR) method was utilized.The process involved converting total RNA to cDNA and then running RT-PCR on an ABI-Step One system (Takara, Japan: Cat.#6110A and Applied Biosystems, USA).Real Plus 2x Master Mix Green in our RT-PCR was used, with one µg of RNA (Amplicon, Denmark: Cat.No.: A323402).To ensure accuracy, GAPDH was used as an internal control and designed specific primers (Table 2) for amplification of the studied genes.

Statistical Analysis
The statistical analysis was conducted using the SPSS software (V.
According to Table 3, in controls, a significant positive association was observed between MMP-9 Levels with gene expression of MMP-9, and BMI with TIMP1 gene  expression (all P < 0.05).moreover, a positive correlation between MMP-9 and IL-6 levels was detected (P = 0.01).
As it shown in Figure 3, TNF-α and IL-6 levels of CAD patients were more than controls significantly (all P < 0.01) As shown in Figure 4 and Table 2, TIMP-1 gene expression no differ between CAD patients and controls.
Figure 3 shows that the gene expression of MMP-9 and the MMP-9 levels in CAD patients were considerably higher than those in the control group (all P < 0.01).

Discussion
3][24][25] In this study, serum levels of cytokines of IL-6 and TNF-α were greatly higher than controls (P < 0.05).In line with our results, Wen et al. reported in patients with acute aortic dissection (AD) levels of IL-6, TNF-α and CRP significantly increased than control, although serum MMP-9 significantly increased in Chronic aortic dissection than in AD patients and controls. 25Their results indicated a gradual increase in TNF-α levels leads to MMP-9 upregulation in neutrophils and macrophages.Thus, a noteworthy link between TNF-α levels and the AD progression confirmed that inflammatory response has a critical role in MMP-9 activation and other inflammatory responses. 24][28] Our study revealed that in CAD patients' MMP-9

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serum levels and gene expression were significantly higher than in the control group.Furthermore, MMP-9 levels are significantly positively associated with PBMC MMP-9 gene expression in CAD patients.Our results agree with previous studies that have also revealed that MMP-1 and MMP-9 levels increased in individuals with CAD. 9,22However, contrary to our finding, some studies showed no significant difference between MMP-9 in CAD patients and controls. 29Our findings indicate no significant variation in TIMP-1 gene expression between both groups.However, we observed a significant positive correlation between TIMP-1 gene expression and the gene and serum concentration of MMP-9 in the CAD group (P < 0.05).Therefore, this association between both genes revealed a molecular mechanism for modulation of MMP-9 gene expression and activity (P < 0.05). 9-14Jordakieva et al. suggested that patients who did not survive in the ICU unit had significantly higher levels of MMP-9 and TIMP-1 compared to those who did survive.They suggested that levels of MMP-9 and TIMP-1 could be recognized as prognostic biomarkers and independent predictors of survival in systemic inflammation and acute organ failure. 24There is significant debate on the role of TIMP-1 in heart disorders, which has led to conflicting reports.Though our study failed to find any differences in TIMP-1 gene expression in patients with CAD, patients with atrial fibrillation, cardiomyopathy or ischemic revealed that HDL-C significantly reduced in CAD patients than in controls, while lipid profile didn't differ between groups.Furthermore, a significant correlation was observed between MMP-9 and LDL-C levels in our CAD patients, whereas an inverse correlation was observed between TC and MMP-9 gene expression in controls.Hernandez-Anzaldo et al. suggested that MMP-9 alters cholesterol metabolism through phospholipase A2 secretion and transcriptional responses in the liver. 33n diabetic patients with coronary heart disease (CHD) levels of MMP-9, Apo-Protein-E (APO-E), high sensitive CRP (hs-CRP) and lipid profile were increased, and a significant correlation was observed between MMP-6 with APO-E, hs-CRP, TC, TG, LDL and HDL levels. 34] In CAD patients, hypertension induces vascular remodeling through modifications in ECM structure and composition, leading to vascular endothelium damage and muscle contractility, consequently affects blood pressure.However, in experimental for hypertension and atherosclerosis, MMP inhibition could attenuate arterial remodeling and human arterial remodeling. 34In the present study, there was a significant correlation between DBP and MMP-9 levels in CAD patients, whereas MMP-9 levels had no significant correlation with SBP.][37][38] Additionally, MMP-2, MMP-9, and TIMP-1 levels tend to be higher in obese subjects with BMI over 24, although there was a controversy among researchers. 37,38The present study revealed that in CAD patients, there was an inverse significant association between BMI with PBMC TIMP-1 gene expression and MMP-9 levels (all P < 0.05).In line with our results, other studies reported that in subjects with obesity and overweight, levels of MMP-9 and TIMP-1 were increased and positively related to BMI. [37][38][39] According to NHANES III study, Vit-D deficiency was associated with CVD. 38Furthermore, in recent decades, a growing literature has revealed Vit-D suppresses the inflammatory response and NF-κB pathway, thereby attenuating the progression of CAD. 39,40In the present study, Vti-D levels of CAD patients were significantly lower than controls (P < 0.05).0][41][42] Recent findings have suggested that Vit-D supplementation inhibits inflammation process, VCAM activation and NF-κB pathways, although these findings are still inconclusive and more research is needed to fully understand their effects. 41,42ur ROC analysis results in CAD patients showed that the MMP-9 gene, MMP-9 and TNF-α levels can be valid biomarkers for evaluation and follow up of CAD.Therefore, increased MMP-9 gene expression, serum MMP-9 and TNF-α concentration in CAD patients could have key roles in pathophysiological state of vascular injury, susceptibility to plaque formation and atherosclerosis.It is suggested that MMP-9 and TNF-α levels have regulatory effects on ongoing inflammation, lipid metabolism and atherosclerosis.
Taken together, it is worthy to note that the positive correlation between of MMP-9 and IL-6 levels and negative association between MMP-9 gene expression and total cholesterol levels in CAD patients could be considered as the novelty of present study.According to our ROC curve analysis result, serum MMP-9 concertation is suggested as a hallmark in CAD prognosis and monitoring.
A small sample size, instability of total-RNA, and the variation in demographic and socioeconomic characteristics of subjects were major study limitations that might influence our findings.

Conclusion
The results indicated that the relationship between MMPs, TIMP1, and some cytokines could have a key role in the pathogenesis of atherosclerosis.Based on the present study high levels of TNF-α and IL-6 as well as vitamin D deficiency in studied participants could disturb the MMP-9/TIMP-1 balance and lipid metabolism, leading to plaque formation/ rupture in predisposed CAD patients.

Table 1 . Anthropometric and laboratory profiles of study population Control (n = 67) CAD (n = 65) Mean ± SD Mean ± SD P value
P<0.05 is statistically significant.
We utilized Pearson's correlation coefficient or Spearmen's correlation test to assess any connections between variables.The Receiver Operating Characteristic (ROC) was employed to evaluate the diagnostic ability of the variables of interest.A P value < 0.05 is considered significant.
20, IBM, Chicago, IL, USA).Results were presented as mean ± standard deviation and median values.Both parametric and non-parametric variables were analyzed using the student t-test and Mann-Whitney U test, respectively.To analyze categorical data, we conducted the χ 2 test and presented the results in frequency and percentage.