Matrix metalloproteinases (MMPs) are involved in cardiac remodelling. We examined whether MMP-2 genetic polymorphisms are associated with hypertension and left ventricular (LV) remodelling in hypertensive patients. We studied 160 hypertensive patients and 123 healthy controls. Echocardiography was performed in all patients and the C−1306T (rs243865) and C−735T (rs 2285053) MMP-2 polymorphisms were analysed. Haplo.stats analysis was used to evaluate whether MMP-2 haplotypes are associated with hypertension and with extremes in LV mass index (LVMI). Multiple linear regression analysis was performed to assess whether MMP-2 genotypes or haplotypes affect LVMI and other echocardiography parameters. The C−1306T ‘CC’ genotype was associated with reduced LVMI and LV end-diastolic diameter (EDD) (P=0.0365 and P=0.0438, respectively). The haplotype ‘C, C’ was associated with reduced LVMI and EDD (P=0.0278 and P=0.0322, respectively). The comparison of upper and lower extremes of the LVMI phenotype showed that the ‘C, C’ haplotype was more common in the lower LVMI group (P=0.0060), whereas the ‘T, C’ haplotype was more common in the higher quartile of LVMI (P=0.0187), and this haplotype was associated with increased risk of higher LVMI values (odds ratio=3.5121, 95% confidence interval=1.3193–9.3494). The findings suggest that MMP-2 polymorphisms affect hypertension-induced LV remodelling.
Hypertension challenges health care systems because it affects millions of people and commonly leads to cardiovascular complications. One of the major complications associated with chronic pressure overload is left ventricular hypertrophy (LVH),1 which may progress to left ventricular (LV) dilatation and cardiac failure, a well-established risk factor for cardiovascular mortality.2
Among other mechanisms, mounting evidence indicates that altered activity of a group of structurally related, zinc-dependent enzymes, the matrix metalloproteinases (MMPs) is involved in LVH. Several MMPs are upregulated in cardiac hypertrophy and an imbalance between MMPs and their endogenous inhibitors (tissue inhibitor of metalloproteinases—TIMPs) may underlie the transition of compensated LVH to dilated LVH.3, 4, 5, 6, 7 In addition, experimental evidence suggests that nonspecific inhibition of MMPs, especially MMP-2, ameliorates hypertensive cardiovascular remodelling.8
MMPs activities are regulated at different levels including transcription, activation of latent forms of MMPs and inhibition by TIMPs5, 9 In this respect, genetic polymorphisms in MMP-2 gene may also affect MMP-2 expression or activity, as shown in several disease conditions.10, 11, 12 Although several polymorphisms in the MMP-2 gene have been described, there is limited information about their functionality. In the present report, we studied two functional polymorphisms in the MMP-2 gene: the C−1306T SNP (rs243865) and the C−735T (rs 2285053), both in the promoter region of MMP-2 gene. These polymorphisms affect MMP-2 expression12, 13 and have been associated with several diseases including cancer.14 and cardiovascular diseases.10, 15, 16, 17, 18, 19 However, no previous study has examined whether combinations of MMP-2 gene polymorphisms within haplotypes may affect LV modifications associated with hypertension. This issue is important because the study of MMP-2 genetic variations possibly affecting hypertensive LVH may help to identify subjects at increased cardiovascular risk.
In the present study, we compared MMP-2 allele and genotype distributions in healthy volunteers and hypertensive patients. We then examined MMP-2 haplotypes distributions in hypertensive subjects and assessed whether these haplotypes could influence LV remodelling.
Materials and methods
This study was approved by the Human Research Ethics Committee of the University of Campinas, and informed consent was obtained from each participant. This study included 160 hypertensive patients followed up at the Hypertension Unit of the University of Campinas and 123 healthy controls. Clinical data were on the basis of medical history, physical examination and routine analytical tests. Hypertension was defined as systolic blood pressure ⩾140 mm Hg, or diastolic blood pressure ⩾90 mm Hg or current antihypertensive medication use. Exclusion criteria were age under 18 years and evidence of moderate or severe cardiac valve disease, hypertrophic cardiomyopathy, previous myocardial infarction, neoplastic disease and suspected secondary hypertension.
Blood pressure was measured using a validated digital oscillometric device (Omron HEM-705CP, Omron Healthcare, Kyoto, Japan) with appropriate cuff sizes. Body mass index (BMI) was calculated as body weight divided by height squared (expressed in kg m–2).
Echocardiography studies were performed on each subject at rest in the left lateral decubitus position using a Vivid 3 Pro (General Electric, Milwaukee, WI, USA) apparatus equipped with a 2.5-MHz transducer as previously described.20 LV end-diastolic and end-systolic diameters, interventricular septum thickness, posterior wall thickness and LV mass were measured in accordance with the American Society of Echocardiography guidelines.21 Relative wall thickness was computed as twice the posterior wall thickness divided by LV end-diastolic diameter (EDD). LV mass index (LVMI) was considered as LV mass/height,2.7 and LVH was defined with the use of a cutoff point >51 g m–2.7.22 All the recordings were made by the same physician, who was unaware of other data regarding the subjects. The reproducibility of both acquiring and measuring LV mass was determined in recordings obtained from 10 subjects. Intraobserver LV mass variability was <8%.
Genotyping for the C−1306T and the C−735T polymorphisms in the 5′-flanking region of MMP-2 gene
Venous blood samples were collected and genomic DNA was extracted from the cellular component of 1 ml of whole blood and stored at −20 °C until analysed.
Genotypes for the C−1306T and the C−735T polymorphisms in the 5′-flanking region of MMP-2 gene were determined by Taqman Allele Discrimination assay (Applied Biosystems, Carlsbad, CA, USA). Probes and primers used for the C−1306T genotyping assay were customised as follows: forward 5′-IndexTermGCCATTGTCAATGTTCCCTAAAACA-3′, reverse 5′-IndexTermTGACTTCTGAGCTGAGACCTGAA-3′ and probes 5′-IndexTermCAGCACTC[T/C]ACCTCT-3′. TaqMan PCR was performed in a total volume of 12 μl (3 ng of DNA, 1 × TaqMan master mix, 1 × assay mix) placed in 96-well PCR plates. Fluorescence from PCR amplification was detected using Chromo 4 Detector (Bio-Rad Laboratories, Hercules, CA, USA) and analysed with manufacturer's software. Probes and primers used in MMP-2 C−735T assay were designed by Applied Biosystems (ID: C_26734093-20). TaqMan PCR and fluorescence reading were performed as described above for the C−1306T polymorphism.
Differences in genotypes and alleles distributions and deviations from the Hardy–Weinberg equilibrium were assessed using χ2-tests (Stat-View for Windows; SAS Institute, Cary, NC, USA). The Haplo.stats package (version 1.4.4; http://www.r-project.org) was used to estimate the haplotype frequencies. The function haplo.em computes maximum likelihood estimates of haplotype probabilities using the progressive insertion algorithm, which progressively inserts batches of loci into haplotypes of growing lengths. The function haplo.score was used to compute haplotype-specific score statistics to test for association,23 with the value of P<0.05 considered statistically significant. Only the haplotypes with frequencies >1% were taken into consideration for the haplotype-specific score analysis. The function haplo.cc was also performed to calculate odds ratio and 95% confidence intervals for each haplotype. The possible haplotypes including genetic variants of two polymorphisms in the MMP-2 gene studied, C−1306T and C−735T were: H1 (C, C); H2 (C, T); H3 (T, C); H4 (T, T).
Linear regression analysis and non-linear fitting routines were performed to assess univariate relations between variables (software JMP 5.0.1a; SAS Institute). In addition, a bivariate analysis was also used to assess for the potential confounding influence of each covariate on the relation between MMP-2 haplotypes and LVMI. The variables of clinical importance, as identified by the bivariate approach, were then included in the final multiple linear regression models. Septal thickness, posterior wall thickness, LVMI, relative wall thickness and EDD were considered as dependent variables. MMP-2 haplotypes, gender, BMI, age, pharmacological treatment and systolic blood pressure were considered as independent variables.
Clinical features of studied subjects are shown in Table 1. We found no differences in age, gender, race, cholesterol and triglycerides concentrations between the study groups. However, hypertensive patients had increased arterial pressure and BMI when compared with healthy controls (P<0.05; Table 1).
Alleles and genotypes distributions are presented in Table 2. The distribution of genotypes for each polymorphism showed no deviation from Hardy–Weinberg equilibrium. Genotypes and alleles distributions showed no significant differences when healthy and hypertensive groups were compared (P>0.05; Table 2). The haplotypes distribution showed no significant differences between healthy and hypertensive groups (global P=0.9748, Table 3).
To determine the influence of MMP-2 genotypes or haplotypes on echocardiography parameters, we performed a multiple linear regression analysis for genotypes (Table 4) and another for haplotypes (Table 5), both adjusted for gender, BMI, age, systolic blood pressure and pharmacological treatment.
The systolic blood pressure was significantly and positively associated with septal thickness and LVMI in both models and with posterior wall thickness in haplotypes model. BMI was significantly and positively associated with all parameters in both models. Female gender was associated with reduced posterior wall thickness, LV and septal thickness in both models, whereas age was associated with increased LVMI in haplotype model. Finally, the usage of diuretics was also positively associated with EDD. After adjustment for selected variables, we found that both LV EDD and LVMI were significantly influenced by MMP-2 genotypes and haplotypes (Tables 4 and Tables 5). Although the analysis of genotypes showed that the CC genotype for the C−1306T polymorphism was associated with reduced LV EDD (Estimate (B)=−2.1171, P=0.0438) and LVMI (B=−0.0499, P=0.0365), no effect was attributed to any of the C−735T genotypes. Moreover, the analysis of haplotypes showed that the ancestral H1 (C, C) haplotype is associated with reduced EDD (B=−0.9791, P=0.0322) and LVMI (B=−0.0228, P=0.0278). No other echocardiography parameters were affected by the studied polymorphisms and haplotypes.
On the basis of these results, we performed a new analysis using Haplostats (Table 6). We compared the upper and lower extreme quartiles of the LVMI phenotype. We found that while the H1 (C, C) haplotype was more common in the lower LVMI group (P=0.0060), the H3 (T, C) haplotype was more common in the highest quartile of LVMI (P=0.0187). In addition, the H3 haplotype showed a remarkable increased risk of higher LVMI values (odds ratio=3.5121, 95% confidence interval=1.3193–9.3494).
MMPs are major players in extracellular matrix turnover and myocardial remodelling.6, 7, 9 Given the relevance of MMP-2 in these processes, functional genetic polymorphisms of this gene may affect the prevalence of LVH in hypertensive patients. This is the first report to show a protective effect associated with the CC genotype of the C−1306T polymorphism against increases in EDD and LVMI in hypertensive subjects. This finding aligns with a protective effect previously reported after stroke in patients carrying the C allele of this polymorphism.10 Moreover, we found that the H1 (C, C) haplotype protects against the increases in EDD and LVMI found in hypertensive patients. A more sophisticated analysis comparing the extremes of LVMI phenotype (upper versus lower quartiles) confirmed the protective effect associated with this particular haplotype. As LV mass depends on both LV wall thickness and LV EDD, our results suggest that the influence of MMP-2 haplotypes on LVMI was related to modifications in chamber diameter rather than to alterations in wall thickness. To our knowledge, no previous study has examined whether MMP-2 polymorphisms or haplotypes are associated with hypertension, although experimental evidence has implicated MMP-2 in the pathogenesis of hypertension24, 25, 26, 27 and in hypertensive LVH.8
Previous studies suggest that genetic polymorphisms modify MMP-2 expression or activity.10, 11, 12 For instance, the C−1306T polymorphism disrupts a Sp1-type promoter site (CCACC box), thus causing a 1.6-fold increase in the promoter activity when the C allele is present as compared with the T allele.13 In addition, similar mechanisms explain a threefold increase in MMP-2 promoter activity when the C allele of the C−735T polymorphism is present.12 Consistent with these findings, the MMP-2 haplotype combining both C alleles of both polymorphisms causes remarkable increases in MMP-2 expression.12 In cardiac hypertrophy, it has been shown that the overall MMP gelatinolytic activity is increased and has crucial roles in the development of LVH and in the progression to decompensated LVH and heart failure.6, 7, 28 Although our results suggesting a protective role for a MMP-2 polymorphism and haplotype associated with higher MMP-2 expression may seem paradoxical, little is known about the specific role for each MMP in this process and the possible signalling pathways that depend on them.29 In this respect, transgenic models have contributed to the understanding of the roles for MMPs in LVH, and MMP-2 knockout mice present interesting reductions in LVH and its complications in models of heart hypertrophy.30 These mice have increased TIMP-4 levels in the heart, and TIMP-4 is a major MMPs inhibitor.31 Whether the reduced LVH seen in these animals is because of lack of MMP-2 activity or to the inhibitory effects of TIMP-4 on various MMPs is not known. In addition, other MMPs, including MMP-9, may be involved in LVH associated with hypertension.32 It is also relevant to mention that many other actions independent of matrix degradation have been attributed to MMPs, especially MMP-2, and these actions may be very relevant to cardiac remodelling.29, 33, 34 Although we do not have a mechanistic explanation for the associations being reported here, it is clear that complex interactions of factors regulate MMPs activities in the heart.
The present study has some limitations. First, we studied a relatively small number of patients. However, we found significant associations between MMP-2 gene variants or haplotypes and LVH in hypertensive patients. Second, the patients included in the present study were under pharmacological treatment. It is clearly unacceptable not to treat hypertensive patients. However, although our statistical analysis took into consideration this factor, it may have obscured the genetic influence of MMP-2 polymorphisms. Despite these limitations, it is important to consider that the H1 (C, C) and H3 (T, C) haplotypes are common haplotypes (>10% frequency), thus increasing their importance. Finally, we should state that few of our healthy controls may have subclinical hypertension or metabolic syndrome, as some of the clinical features of these subjects may be slightly above normal limits.
In conclusion, genetic polymorphisms in MMP-2 gene may be risk factors for the development of hypertension-induced LV remodelling. Further studies are necessary to validate our findings, and to examine whether these polymorphisms affect long-term outcomes in hypertensive patients and maybe targeted therapies such as MMPs inhibition before LVH is present.
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This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, Conselho Nacional de Desenvolvimento Científico e Tecnológico and Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior.
The authors declare no conflict of interest.
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Lacchini, R., Jacob-Ferreira, A., Luizon, M. et al. Common matrix metalloproteinase 2 gene haplotypes may modulate left ventricular remodelling in hypertensive patients. J Hum Hypertens 26, 171–177 (2012). https://doi.org/10.1038/jhh.2011.8
- left ventricular hypertrophy
- MMP-2 polymorphisms
- Heart remodelling
- MMP-2 haplotypes
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