Matrix metalloproteinase-3 gene polymorphism and dilatative pathology of ascending thoracic aorta

Vaiva Lesauskaitė, Giedrė Šinkūnaitė, Rimantas Benetis, Vilius Grabauskas1, Jolanta Vaškelytė, Alina Smalinskienė, Sandrita Šimonytė, Giedrė Jarienė, Vacis Tatarūnas, Jūratė Klumbienė1, Janina Petkevičienė1, Šarūnas Kinduris2, Saulius Giedraitis2, Juozas Sakalauskas2, Ramūnas Bolys2, Edmundas Širvinskas2, Tadas Lenkutis2, Dalia Pangonytė Institute of Cardiology, 1Department of Preventive Medicine, 2Department of Cardiac, Thoracic and Vascular Surgery, Kaunas University of Medicine, Lithuania


Introduction
The etiology of thoracic aortic aneurysm formation mechanism remains uncertain. Only small part of them is caused by inflammation, as in giant cell arteritis and syphilitic aortitis (1); by hereditary connective tissue disorders, such as mutations in the fibrillin genes in Marfan syndrome and Ehlers-Danlos syndrome (2); or by atherosclerosis (3). However, it is recognized that disarrangement of smooth muscle cells (SMCs) and the extracellular matrix (ECM) appeared to be crucial in pathogenesis of aortic aneurysm formation (4). Recent studies indicate that SMCs participate in remodeling of aortic wall by localized production of MMPs and their tissue inhibitors in aortic media (1,4).
MMPs may play a key role in physiologic and pathologic remodeling of tissues, including embryogenesis and tissue morphogenesis, wound repair, inflammatory diseases, cancerogenesis, etc. (5,6). These multiple actions of MMPs are due to their ability to degrade extracellular matrices, basement membranes, and other proteins. Functional regulation of MMPs is controlled primarily by gene transcription and synthesis, whereas the activity is regulated by posttranslational activation of zymogens and by interactions of secreted MMPs with tissue inhibitors of metalloproteinases (TIMPs) (7)(8)(9). A common adenine insertion/ MMP-3 gene polymorphism and dilatative pathology of ascending thoracic aorta deletion polymorphism (5A/6A) at a position -1171 of the MMP3 (stromelysin-1) gene promoter (National Center for Biotechnology Information SNP Identification No. rs3025039) influences MMP-3 promoter activity and transcription factor binding (10). It was shown that the increased number of 5A alleles leads to increased expression of MMP-3 (11)(12)(13)(14)(15)(16) and higher plasma MMP-3 activity (15). The latter phenomenon suggests relation between MMP-3 genotype and the development of pathological states caused by connective tissue degradation, for example rupture of atherosclerotic plaque leading to the acute myocardial infarction or unstable angina pectoris (13,14), spreading of carcinoma (6).
Therefore, in the present study we investigated whether the 5A/6A polymorphism in the promoter region of MMP-3 gene is associated with the development of DP ATA.

Materials and methods
The study enrolled 76 patients (55 males, 21 females) who underwent aortic reconstruction due to DP ATA at the Clinic of Cardiac, Thoracic, and Vascular Surgery, Kaunas University of Medicine Hospital, Lithuania. The age of patients ranged from 31 to 81 years (median, 64 years). The patients were operated on due to dissection of the thoracic aorta (n=24, dissection subgroup), poststenotic dilatation of the ascending aorta due to aortic stenosis (n=21, valvular subgroup), and chronic aneurysm of the thoracic aorta (n=31, aneurysm subgroup). All patients underwent preoperative transthoracic echocardiography. Specimens of aortic tissue taken during aortic reconstruction surgery were fixed in 10% neutral buffered formalin for 24 hours and then processed for routine paraffin embedding.
The persons (244 males and 366 females) screened within the framework of the international CINDI (Countrywide Integrated Noncommunicable Diseases Intervention) study (17) were used for comparative analysis. A random sample of the population aged 25-64 years was taken from the inhabitants' lists of primary health care centers in three administrative regions of Lithuania (Kaišiadorys, Kretinga, and Varėna). The health examination of selected sample was performed in 2007. During the examination, venous blood for genetic investigation was taken in 3-mL K3EDTA vacutainers. They were stored at -80°C for subsequent DNA extraction.
Ethical approval was obtained from the Ethics Committee of Kaunas University of Medicine and Lithuanian Bioethical Committee ( March 23, 2006, No. 11).

DNA extraction
Standard procedure described by Sepp et al. (18) and Shi et al. (19) was used to extract genomic DNA from aortic wall sections taken during aortic reconstruction surgery. Six-mm-thick 150 sections from paraffin-embedded aortic wall were collected into an autoclaved plastic microtube and lysed with digestion buffer (100 mM NaCl; 10 mM Tris-Cl, pH8; 25 mM sodium dodecyl sulfate) and 20 mL of proteinase K (20 mg/mL, Fermentas, Lithuania) by incubation overnight at 55°C. The samples were incubated at 98°C to inactivate proteinase K and centrifuged at 5000×g for 15 min (18). The lysis solution was transferred into a new test tube, mixed with equal amount of phenol/chloroform/isoamyl alcohol (Sigma) in a 25:24:1 ratio and centrifuged at 13 000×g for 15 min at 4°C. The procedure was repeated twice with equal amount of chloroform (Sigma) to wash out phenol remains. The aqueous supernatant was transferred into a micro test tube, mixed with equal volume of isopropanol (Sigma), incubated for 1 hour at -20°C, and centrifuged at 13 000×g for 15 min. The precipitated total DNA was rinsed with 70% ethanol and then resuspended in RNase-free water (19).
Genomic DNA from blood was isolated by using Sorpoclean Genomic DNA Extraction Module kit (Sorpo Diagnostics, Vilnius, Lithuania) according to manufacturer's instructions.
DNA quality and concentration were estimated by spectrophotometrical analysis and by ethidium bromide-stained agarose gel under ultraviolet light. The latter method was used to evaluate DNA degradation level in the samples from paraffin-embedded aortic tissue. Photographs were documented through a video documentation system, the BioDocAnalyse 2.0 (Biometra, Göttingen, Germany).

Primers and probes for real-time polymerase chain reaction (RT PCR)
Primer-probe set was made using the TibMolBiol design service (Berlin, Germany) for MMP-3 genotyping. Two fluorogenic minor groove binder probes, 5¢-6FAM AAG ACA TGG TTT TTC CCC CCA TCAA BBQ-3¢ and 5¢-YAK AAG ACA TGG TTT TTT CCC CCC ATC AA BBQ-3¢, were used to detect the adenine insertion/deletion mutation. The dyes 6-carboxyfluorescein (FAM; excitation, 494 nm) and Yakima Yellow (YAK; excitation, 526 nm) are easily differentiated in the Applied Biosystems Prism 7900HT PCR system. The primers were the following: forward primer, 5¢-GTG GCC AAA TAT TTT CCC TGT ATTT-3¢; reverse primer, 5¢-GGC ACC TGG CCT AAA GAC ATT-3¢. RT PCR was performed using 12.5 mL of TaqMan 2X Universal PCR master mix (Applied Biosystems, Darmstadt, Germany), 0.4 mL of primers (20 mM) (TibMolBiol, Berlin, Germany), 0.125 mL of probes (20 mM) (TibMolBiol, Berlin, Germany), 10.45 mL of RNase-and DNasefree water (Ambion), and 1 mL of sample DNA, in a total volume of 25 mL per single tube reaction. DNasefree water used as nontemplate control and DNA of known MMP-3 genotypes used as a positive control were included in each assay run. Assay conditions were 2 min at 50°C, 10 min at 95°C, and 40 cycles of 95°C for 15 s and 60°C for 1 min. The SNP assay was set up using SDS, version 2.3, software (Applied Biosystems, USA).

Statistics
Statistical analysis was done using the SPSS statistical package (version 12.0; Chicago, IL). The nonparametric Kruskal-Wallis test was used for comparison between groups. A P value of <0.05 was accepted as statistically significant.

Results
The frequency of MMP-3 5A/6A genotypes and alleles did not differ between males and females in the group of DP ATA and random sample of population. Therefore, the data presented in Table are combined for both genders. The distributions of genotypes in the DP ATA and random sample of population were compatible with the Hardy-Weinberg equilibrium. The prevalence of MMP-3 genotypes and 5A/6A alleles was similar between group of DP ATA and random sample of population (Table). When we divided group of DP ATA into dissection, valvular, and aneurysm subgroups, the distribution of genotypes in each subgroup did not differ significantly. However, it was tendency of higher prevalence of 5A/5A genotype in dissection subgroup (33.3%) as compared with valvular and aneurysm subgroups (19.05% and 25.81%, respectively).
Figure presents data on the minimal, maximal, and median age according to MMP-3 5A/6A genotypes in males and females with DP ATA and in random sample of population. Males carrying 5A/5A genotype from the group of DP ATA were significantly younger than those with 6A/6A genotype (P<0.05). There was no significant difference in age between MMP-3 genotypes in both genders from random sample of population and in females with DP ATA.

Discussion
Previous understanding of the pathogenesis of aortic aneurysms based on the biomechanical factors, such as arterial hemodynamics and wall mechanics, has been increased by the role of biological factors (20). Destruction of the elastic media appears to depend on proteinases, which are members of the MMP family (4). The latter have been implicated in the etiopathogenesis of thoracic aneurysm formation (21,22). It was shown that alterations at the genetic level contribute to the overexpression of MMPs (10, 21, 22).
MMP-3 or stromelysin-1 is a member of stromelysin family. It is capable of degrading proteoglycan, fibronectin, laminin, and type IV collagen (9). In vitro studies of MMP-3 promoter strength showed that the 5A allele expressed higher activity than the 6A allele in both cultured fibroblasts and vascular smooth muscle cells (23). It is suggested that because of

MMP-3
Patients with DP ATA, n (%) RSP, n (%) P value (n=76)  (11,24), while others do not find such asso-ciation (25). The frequency of the 5A allele in our random sample of Lithuanian population is 0.514. It is consistent with published MMP-3 control population data for United Kingdom (24,25), Belgium, Canada (26), Sweden (27), and Italy (12). The frequency of the 5A allele in French (28) and Austrian (29) control populations (0.46 and 0.469, respectively) was smaller than in random sample of Lithuanian population. It is of interest that the 5A allele frequency in the Japanese (14,30), the South Korean (13), and in Taiwan control populations (15) is only 0.18, 0.15, and 0.18, respectively.
Kamijima et al. (4) showed that phenotypic modulation of SMCs in the aortic wall is a critical event in the enlargement of aortic abdominal aneurysm and MMP enhancement. Investigation of DP ATA demonstrated that transition of medial SMCs from the contractile to the synthetic type led to the increased production of MMPs by SMCs, as well (1). A trigger inducing the transition of medial SMC phenotype remains obscure. Our results show that the frequency of MMP-3 promoter 5A/6A genotypes did not differ between the group of patients with DP ATA and the random sample of population, but the male patients carrying 5A/5A genotype were significantly younger compared with those with the 6A/6A genotype. In addition, it was tendency of higher prevalence (33.3%) of 5A/5A genotype in dissection subgroup as compared to the random sample of population (25.99%). Our data lead to the hypothesis that MMP-3 promoter 5A/6A genotypes are not associated with the transition of medial SMCs from the contractile to the synthetic phenotype in the thoracic aorta. On the other hand, when phenotypic modulation of SMCs leading to DP ATA occurs, males with 5A/5A genotype require aortic reconstruction surgery at the younger age than males carrying 6A/6A genotype at the MMP-3 pro-moter region. Our study is limited by the small number of patients in the subgroups of DP ATA. For example, dissection subgroup included only 24 patients. Therefore, we were not able to obtain significant results on the impact of 5A/5A genotype on aortic dissection. Second, we had no possibility to carry out transthoracic echocardiography for the randomly selected population. Because of this, we did not design a control group composed only of the persons free from any aortic dilatative pathology.