2-1 Study population
The study comprised 92 individuals (52 men and 40 women) with a mean age of 64.86±10.38 with aortic and mitral valve calcification, who were confirmed as the case group by a cardiologist (patient group). In addition, the Healthy group consisted of 92 sex- and age-matched people (43 men and 49 women), with a mean age of 63.77±7.46. There were no additional illnesses associated with matrix metalloproteinases 2 and 9 in the control group. Individuals with OVC were referred to Imam Ali Hospital based on a cardiologist's diagnosis; also, the control group had no history of cardiovascular illness. The study was done according to the guidelines of the 1975 Declaration of Helsinki.
2.2 Sample Collection and DNA Extraction
In each of the two groups, 10 mL of blood was taken and split into two portions of 5 mL to extract DNA and separate serum samples. The phenol-chloroform technique was used to extract DNA from peripheral blood cells (24-26), and samples were dissolved in ddH2O and stored at −70 °C.
On chromosomes 20 and 16, target areas (MMP-9 C/T -1562 and MMP-2 G/A -1575) were amplified using polymerase chain reaction (PCR) (27, 28). Primers were designed using the Primer3 online website (https:// prime r3. ut. ee/). The sequences of the primers were as the following:
(MMP-9, F: 5′- GCCT GGC ACATAG TAG GCCC -3′, R: 5′- CTTCCTAGCCAGCCGGCATC -3′) (MMP-2, F: 5′- AGG TTT GTC ACT GGG TCA GGC TG -3′, R: 5′- CCT AGG AAG GGG GCA GAT AGG AC -3′)
MMP-2 and MMP-9 were genotyped using restriction fragment length polymorphism (RFLP), with RCA1 and sph1 as restriction enzymes, respectively. PCR was performed with 25 μL volume (1 μL of DNA at the concentrations of 200–600 ng, 0.6 μL of each primer at a concentration of 10 pmol, 0.75 μL of MgCl2 at a concentration of 50 mM, 0.5 μL of dNTPs at a concentration of 200 μM, 0.5 μL Taq polymerase at a concentration of 5 U/μL, and 2.5 μL of 10 × PCR buffer. The PCR process for determining the MMP-2 genotypes was completed in 35 cycles under initial denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 68 °C for 60 s, extension at 72 °C for 30 s, and a final extension at 72 °C for 10 min. The PCR product was detected as a 301 bp on 1.5% agarose gel after electrophoresis. 10 μL of the PCR product was digested with RCA1 enzyme for 16 h at 37 °C. The RFLP product was electrophoresed in 2.0% agarose gel and stained with ethidium bromide. The PCR product in this position was not cleaved by the RCA1 enzyme since there was no mutation in the wild allele (G/G), resulting in a 301-bp fragment. In the heterozygote form (A/G), three fragments of 301, 189, and 112 bp were generated, and two fragments of 189 and 112 bp in the mutant homozygote form (A/A) (Fig. 1a). The PCR process for determining the MMP-9 genotypes was performed in 35 cycles under initial denaturation at 94 °C for 5 min, denaturation at 95 °C for thirty seconds, annealing at 64 °C for 60 s, extension at 72 °C for 30 s, and a final extension at 72 °C for 10 min. The PCR product was detected as a 435 bp on 1.5% agarose gel after electrophoresis. 10 μL of the PCR product was digested with sph1 enzyme for 16 h at 37 °C. The RFLP product was electrophoresed in 2.0% agarose gel and stained with ethidium bromide. Since there was no mutation in the form of wild allele (C/C), the PCR product in this site was not cleaved by the sph1 enzyme, and a fragment with a length of 435 bp was created. Three fragments of 435 bp, 244 bp, and 191 bp were developed in the heterozygote form (C/T), and two fragments of 244 bp and 191 bp in the mutant homozygote form (T/T). (Fig. 1b).
2.4 Measurement of Serum MMP‑2 and MMP‑9
Serum MMP-2 and MMP-9 levels were measured using a Quantikine ELISA kit (R&D Systems-USA), and the results were detected using the Awareness Elisa reader version Stat fax 2100 (ChroMate, Minneapolis-US).
2.5 Measurement of matrix metalloproteinase 2 and 9 enzyme activity
The gelatin zymography technique was used to evaluate the serum enzymatic activity of MMP-2 and MMP-9, as well as their proenzymes. In brief, the amount of total protein was measured by Bradford method and diluted to adjust the protein content to 2 μg/μL. In this experiment, we used sodium dodecyl sulfate (SDS) acrylamide gel (8%) containing 1 mg/mL gelatin and loaded 20 μL of sample in each well before running the test. Electrophoresis was performed and then, gels were washed with 100 mL renaturation buffer comprising 2.5% Triton X-100 to remove the SDS three times and incubated with developing buffer in the shaker incubator at 37 °C for 18 h. After this step, the gels were stained with Coomassie brilliant blue for 40 minutes before being rinsed with H2O, methanol, and acetic acid for 60 minutes. The gel image was analyzed using the software Image-J®program (NIH-USA)(Fig. 2a and 2b).
2.6 Biochemical and Clinical Parameters
In this study, the serum levels of urea, FBS, calcium, phosphorus, creatinine, total cholesterol, triacylglyceride (TG), high-density lipoprotein (HDL-C), and low-density lipoprotein (LDL-C) were measured by Pars Azmun kits-Iran using automated RA-1000 (AutoTechnicon, USA). The physician used a blood pressure monitor to measure SBP and DBP parameters (Omron, M2-HEM-7121-E-Vietnam). Angiography was used to diagnose AVC.
2.7 Statistical Analysis
The normal distribution of quantitative data was investigated by the One-Sample Kolmogorov–Smirnov test. Analysis of age, BMI, the serum levels and activity of matrix metalloproteinase 2 and 9, creatinine, TG, calcium, total cholesterol, urea, phosphorus, HDL-C, LDL-C, FBS, SBP, and DBP was performed by student’s t-test. Also, distribution of different qualitative data was compared between the groups using the Chi-square test (χ2). Simple and multiple logistic regression analyses were used for the calculation of the odds ratio. Moreover, we used the Pearson correlation test to assess the association between matrix metalloproteinase 2 and 9 with other biochemical and physiologic parameters. The statistical analysis was performed using SPSS (Ver 16, SPSS Inc., Chicago, USA) and P < 0.05 was considered to be statistically significant.