Variation in the Human Matrix Metalloproteinase-9 Gene Is Associated With Arterial Stiffness in Healthy Individuals
Background— Arterial stiffness is an important determinant of cardiovascular risk. Elastin is the main elastic component of the arterial wall and can be degraded by a number of enzymes including serine proteases and matrix metalloproteinases (MMPs). Serum MMP-9 levels correlate with arterial stiffness and predict cardiovascular risk. Polymorphisms in the MMP-9 gene are also associated with large artery function in subjects with coronary artery disease. Therefore, we investigated the influence of known MMP-9 (−1562C>T, R279Q) polymorphisms on arterial stiffness in a large cohort of healthy individuals (n=865).
Methods and Results— Aortic pulse wave velocity (PWV) and augmentation index were assessed. Supine blood pressure, biochemical markers, MMP-9 levels, and serum elastase activity (SEA) were also determined. Genomic DNA was extracted and genotyping performed. Aortic PWV, serum MMP-9, and SEA were higher in carriers of the rare alleles for the −1562C>T and R279Q polymorphisms. These polymorphisms were also associated with aortic PWV after correction for other confounding factors. Stepwise regression models with known or likely determinants of arterial stiffness revealed that ≈60% of the variability in aortic PWV was attributable to age, mean arterial pressure, and genetic variants (P<0.001).
Conclusions— We have demonstrated for the first time that aortic stiffness and elastase activity are influenced by MMP-9 gene polymorphisms. This suggests that the genetic variation in this protein may be involved in the process of large artery stiffening.
- aortic pulse wave velocity
- augmentation index
- MMP-9 gene polymorphisms
- MMP-9 levels
- serum elastase activity
Stiffening of the large arteries has a number of adverse hemodynamic consequences, including widened pulse pressure (PP) and alteration in shear stress, which promote vascular and cardiac remodelling, and ultimately increase cardiovascular risk.1 Although the factors determining large artery stiffness are complex, it is now clear that arterial stiffness is, in part, a heritable and dynamic trait.2 However, the precise molecular pathways regulating stiffness are largely unknown.
The large arteries contain a number of different structural proteins, including elastin, which is the main elastic component, and collagen. These proteins are susceptible to chemical attack and degradation by proteolytic enzymes such as serine proteases and matrix metalloproteinases (MMPs). Increased total serum elastase activity (SEA) is associated with atherosclerosis and an increased risk of cardiovascular disease (CVD).3 MMP-9 (also known as 92-kDa type IV collagenase or gelatinase B) may be particularly important as it degrades elastin as well as other basement membrane proteins including fibronectin, laminin, and type IV collagen. Interestingly, MMP-9 levels are also associated with the risk of CVD.4 Recently, we demonstrated a relationship between serum MMP-9 levels and aortic stiffness in healthy individuals, and higher levels in subjects with isolated systolic hypertension—a condition of increased large artery stiffness.5 These data suggest that increased degradation of matrix components by proteolytic enzymes may influence aortic stiffening, thereby increasing the risk of cardiovascular events.
MMP-9 is an inducible protein, and tissue and plasma levels therefore reflect stimulatory and genetic factors. The MMP-9 gene is located on chromosome 20, and a number of polymorphisms in the promoter, coding, and untranslated regions that exhibit tight linkage disequilibrium have been reported.6 Of all the variants, the most extensively studied is the −1562C>T polymorphism, upstream from the transcription start in the promoter region. Previous data indicate that this variant is functional with increased transcriptional activity in macrophages.7 It has also been associated with presence and severity of CVD,4 increased MMP-9 levels,4 and systemic arterial stiffness8 in patients with coronary artery disease (CAD). A second nonconservative single nucleotide polymorphism (SNP) at codon 279, resulting in an amino acid substitution, has also been reported (R279Q). This is associated with increased MMP-9 levels and future cardiovascular events in patients with angina.4 We hypothesized that the two variants in the MMP-9 gene which are in linkage disequilibrium (LD) and associated with cardiovascular risk would be related to arterial stiffness and MMP-9 levels in healthy subjects. The aim of this study was to investigate the possible influence of the common variants in MMP-9 gene (−1562C>T and R279Q) on arterial stiffness, serum MMP-9 levels, and elastase activity in a large cohort of healthy individuals across a wide age range.
A total of 865 subjects, aged between 18 and 81 years, were studied as part of a community-based investigation into the factors influencing arterial stiffness.9–10 Individuals were selected at random from local General Practice lists by letter of invitation, and open-access cardiovascular risk assessment clinics across East Anglia and Wales (the overall response rate was 85%). Subjects with diabetes, hypercholesterolemia (total cholesterol ≥6.5 mmol/L), a history of cardiovascular disease (defined as a clinical history or evidence on examination) were excluded from the present analyses, as were subjects receiving cardiovascular medication. Approval for the study was obtained from the Local Research Ethics Committees, and written informed consent was obtained from each participant.
Protocol and Hemodynamic Measurements
All studies were conducted in a quiet temperature-controlled room. Height and weight were recorded, and body mass index was calculated. After 20 minutes supine rest, peripheral blood pressure was recorded in the dominant arm using a validated oscillometric device (HEM-705CP; Omron Corporation).11 Radial artery waveforms were recorded with a high fidelity micromanometer (SPC-301; Millar Instruments) from the wrist, and from this, a corresponding central waveform was generated using a validated transfer function (Sphygmocor; AtCor Medical),12–14 as previously described in detail.15 Augmentation index (AIx), a composite measure of systemic arterial stiffness and wave reflection,16 and heart rate were determined using the integral software. The aortic PWV was measured using the same device by sequentially recording ECG-gated carotid and femoral artery waveforms as described previously.15 All measurements were made in duplicate, and mean values used in the subsequent analysis.
Blood samples were drawn and the serum separated and stored at −80°C. Total cholesterol, triglycerides, and glucose were determined using standard methodology. Commercially available sandwich ELISA assays (Oncogene Research Products) were used to determine MMP-9 levels according to manufacturers instructions.5 In this study, MMP mass rather activity was assessed (n=564) as described previously.5 SEA was determined using a synthetic substrate, succinyl trialanine paranitroanilide, according to a modified colorimetric assay as previously described.5
Genomic DNA was extracted from the venous blood using standard methods.17 Subjects were genotyped for two variants in the MMP-9 gene, −1562C>T and R279Q, using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and a validated Taqman assay respectively.
Determination of −1562C>T Genotype by PCR-RFLP
The MMP-9 promoter polymorphism −1562C>T was amplified from 100 ng of genomic DNA using forward (5′-GCC TGG CAC ATA GTA GGC CC-3′) and reverse (5′-CTT CCT AGA CAG CCG GCA TC-3′) primers.8 The PCR conditions consisted of predenaturation at 94°C for 2 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 65°C for 1 minute 30 seconds, extension at 72°C for 1 minute 30 seconds, and a final extension at 72°C for 2 minutes. The amplified PCR product was then digested with 10 U of BbU I restriction enzyme overnight (Promega), and the product was run on 2% agarose gel. The two alleles were then visualized as 379-bp (C allele) and 320-bp (T allele) bands under ultraviolet light.
Determination of R279Q Genotypes by Taqman
For the exonic MMP-9/R279Q polymorphism in the coding region allelic discrimination was performed using the Assay-on-Demand (Applied Biosystems) on an ABI Prism (7700 Sequence Detecting System). For each sample, 100 ng of genomic DNA was mixed with 2× Taqman Universal Master Mix, No AmpErase UNG, labeled probe (FAM and VIC dye-labeled) and MQ H2O to a total volume of 15 μL. Probes are designed such that each probe is homologous to only one of the two possible alleles and has a single-base mismatch with the other allele. The two probes are fluorescently labeled with different reporter dyes. During PCR, the probes bind to their chosen allele and the reporter dye cleaved and released into solution by the 3′→5′ exonuclease activity of the Taq polymerase. Reporter dye intensity was then measured in real time using the ABI 7700. The thermal cycling conditions consisted of 50°C for 2 minutes, 95°C for 10 minutes×1, followed by 40 cycles each of 95°C for 15 seconds and then a final 60°C for 1 minute. The data were analyzed off-line with the sequence detection software (version 1.9).
The haplotype frequencies and linkage disequilibrium (LD) between the two MMP-9 SNPs were assessed using the SAS/Genetics package (SAS Institute Inc; version 9.0), which constructs the haplotypes using the expectation-maximization (EM) algorithm. The strength of LD between pairs of alleles at two markers is presented as Yule’s Q and testing for equilibrium between the two SNPs by the χ2 statistic.18–19 Individual haplotypes were also reconstructed from the population genotype data using a Bayesian approach as implemented in the program PHASE (version 2) that was downloaded from the author’s website (www.stat.washington.edu/stephens/software.html).
Other data were analyzed using SPSS (version 12.0) software. One-way analysis of variance (ANOVA) with post-hoc testing compared the differences for continuous parameters and χ2 testing compared differences for categorical variables between genotypes according to each gender. Two-way analysis of variance evaluated gender, genotype, and gender-genotype interactions. Logarithmic transformations were performed for distributions that were significantly skewed and these variables were used in the subsequent analysis. To test the effect of the allele in question, the variant allele carriers were compared with the common allele in regression models. Stepwise multiple regression analysis was performed to investigate the relationship between arterial stiffness and the genetic polymorphisms, and to identify the major determinants for arterial stiffness or circulating elastases. All values are expressed as means±SEM in tables and figures. SEM was used instead of SD for all the variables due to low frequency of the T allele and for consistency of results. A probability value of <0.01 was considered significant.
Because arterial stiffness depends on age, gender, and mean arterial pressure, and to adhere to the recommendations of Almasy et al,20 the baseline data were analyzed and presented separately for each gender according to the MMP-9 genotypes (Tables 2 and 3⇓). However, as the strength and magnitude of associations were similar in both men and women, and as no interactions were observed between gender and genotype for any of the variables for either SNP, the data were combined in subsequent analyses and results presented for the whole cohort (Figures 1 and 2⇓; Table 4).
As reported previously,6 the two SNPs studied were in linkage disequilibrium (Table 1). The degree of LD expressed as Yule’s Q was 0.7. These values are comparable to that reported by Zhang et al for the two polymorphisms (ρ=0.54).21 The frequencies of the −1562T and 279Q alleles were 15% and 39%, respectively, similar to those reported in other populations.4,8 The genotype distributions for the polymorphisms investigated were consistent with Hardy-Weinberg equilibrium. The estimated population frequencies for the four haplotypes are shown in Table 1. As expected, the −1562C/279R (CR) haplotype was the most frequent (56%) and −1562T/279R (TR) the rarest with an estimated frequency of just 5%.
The average values for all the parameters according to each gender and genotype are presented in Tables 2 and 3⇑. For the −1562C>T polymorphism (Table 2), there were no significant differences in smoking status, body mass index, blood pressure level (systolic, diastolic, pulse, and mean pressures), or serum markers (cholesterol, triglycerides and glucose) between genotypes for either men or women. But, as expected, men were significantly taller and heavier compared with women (P<0.001), whereas women had higher AIx values than men. A similar trend was reflected for the majority of parameters for the R279Q polymorphism between genotypes in both men and women, although systolic and diastolic pressures were significantly raised between genotypes in men only (P<0.01; Table 3).
Association Between Arterial Stiffness and MMP-9 Polymorphisms
Aortic PWV was significantly related to the two polymorphisms. In both men and women, mean aortic PWV values were significantly higher in the carriers of the −1562T (TT homozygotes and CT heterozygotes) and 279Q (QQ homozygotes and QR heterozygotes) alleles compared with respective homozygotes (Tables 2 and 3⇑). Combining the data for both genders revealed a dose-dependent effect of the polymorphisms on aortic stiffness (Figure 1A and 1B). The results of the two-way analysis of variance for the whole cohort further confirmed the influence of MMP-9 genotypes on aortic PWV (Tables 2 and 3⇑), but there was no influence of gender.
As the two SNPs showed linkage disequilibrium, we tested whether whole genotypes also affected aortic stiffness using univariate analysis of variance which incorporated the covariates. This analysis showed that whole genotypes significantly influenced aortic PWV (Figure 2A; R2=56%, P<0.01). To confirm that this effect of whole genotypes probably reflected the MMP-9 haplotype, the model was re-run with the whole genotypes replaced with the predicted individual haplotypes. This showed that the predicted haplotypes were also independently associated with aortic PWV and explained an almost identical amount of variation as the whole genotypes model (data not shown).
To assess the relationship between aortic PWV and the alleles in question, multivariate stepwise regression analysis was performed with known or likely factors that affect arterial stiffness in the whole cohort (Table 4). As expected, age and mean arterial pressure emerged as the major determinants of aortic PWV in these models. The −1562T and 279Q alleles were also independent determinants of aortic PWV and quantitatively as important as mean arterial pressure (Table 4). Further additional analysis which included the two SNPs in the regression model also demonstrated that the two SNPs are predictors of aortic velocity, and the observed associations are unlikely to be attributable to chance alone. Similar results were observed when the regression models were constructed for each gender separately, although the total variance explained by aortic PWV predictors was much greater in men (64 and 71%) than women (51 and 54%) for −1562C>T and R279Q polymorphisms, respectively (data not shown).
In contrast to aortic PWV, AIx was significantly different between the homozygotes (Tables 2 and 3⇑), but not after adjusting for known confounders such as age, gender, mean arterial pressure, heart rate, and height (data not shown).
Association Between Serum Elastase Activity, MMP-9 Levels, and MMP-9 Polymorphisms
Both total elastase activity and serum MMP-9 levels were related to the two polymorphisms (Tables 2 and 3⇑). Among men, total serum elastase activity was higher in individuals harboring the TT and QQ genotypes as compared with CC and RR genotypes (Tables 2 and 3⇑). As expected, the average serum MMP-9 levels were also higher in subjects carrying the −1562T (TT homozygotes and CT heterozygotes) and 279Q (QQ homozygotes and QR heterozygotes) alleles as compared with −1562CC and 279RR genotypes in men and women, respectively (Tables 2 and 3⇑). This pattern of genotype influence was more evident in the whole cohort, reflecting a dose-dependent effect of the polymorphisms on serum levels (data not shown). As expected, MMP-9 whole genotypes also significantly influenced serum MMP-9 levels in the whole cohort after correcting for covariates (Figure 2B; R2=13%, P<0.001).
Stiffening of the large arteries is associated with increased cardiovascular morbidity and is, in part, related to genetic variation. The current study examined, for the first time, the relationship between polymorphisms in the MMP-9 gene (−1562C>T, R279Q), arterial stiffness, and MMP-9 levels in healthy men and women. The main findings were that aortic PWV was increased in the promoter and coding polymorphisms in the MMP-9 gene. These associations remained significant even after correcting for potential confounding factors. In addition, MMP-9 levels and total SEA were also higher in the −1562C>T and R279Q polymorphisms in the whole population. Importantly, MMP-9 genotype and whole genotypes also influenced both aortic PWV and serum MMP-9 levels. To our knowledge, this is the first investigation that reports an independent association between MMP-9 gene polymorphisms, whole genotypes, predicted haplotypes, serum markers (MMP-9 levels, SEA), and aortic PWV.
Several studies have highlighted that aortic PWV, the gold standard measure of aortic stiffness, is an independent predictor of cardiovascular mortality.22–25 Recently, polymorphisms in the MMP-9 gene have been associated with severity of vascular disease4 and large artery stiffness in patients with CVD.8 Medley et al showed increased aortic stiffness and MMP-9 expression in carriers of the T allele compared with CC homozygotes. However, this study was carried out in a small group of individuals, which included mostly men with known CVD, a condition that itself is associated with increased stiffness.8 Therefore, the findings may not be representative of the general population. Medley et al also assessed input and characteristic impedance rather than aortic PWV, which is the current gold-standard measure of aortic stiffness. In the present study, we have extended these data to a normotensive population including both genders and also demonstrated that the polymorphism is associated with increased MMP-9 levels and higher aortic PWV, not only in the whole cohort but also in each gender separately.
A number of factors (age, gender, mean pressure) are known to influence aortic PWV. Therefore, we conducted stepwise multiple regression analysis to control for such factors. The results revealed that MMP-9 polymorphisms are important and independent predictors of aortic PWV. This suggests that large artery stiffness in individuals carrying the alleles in question may be attributable to enhanced degradation of the arterial wall matrix.
Although augmentation index, a measure of systemic stiffness, was significantly raised in the alleles in question at baseline, we did not observe any significant association with the polymorphisms after correcting for potential confounding variables. This suggests that aortic stiffness per se rather than enhanced wave reflection is a major phenotype influenced by variants in MMP-9 gene.
Increased MMP-9 levels predict cardiovascular risk, and the functional variant in the promoter region influences circulating MMP-9 levels.4 Although a number of cells secrete MMP-9, the predominant source of circulating MMP-9 is unclear. Neutrophils and monocytes are thought to release MMP-9 into the circulation as a consequence of a general proinflammatory state. Indeed, a variety of stimuli including cytokines, other inflammatory mediators, and mechanical stress influence MMP-9 expression.4,26–28 Moreover, genetic variants within the MMP-9 gene also appear to influence protein expression.4,8 In previous studies, we and others demonstrated a link between inflammation, serum MMP-9 levels, and arterial stiffness.5,9,29–32 In keeping with previous data concerning increased enzyme activity/expression,4 we found that individuals with −1562C>T polymorphism had elevated serum MMP-9 and total serum elastase activity. Similarly, we confirmed previous observation that the R279Q polymorphism in exon 6 is also associated with increased MMP-9 levels.4 These observations provide a potential explanation for the observed increased aortic PWV in carriers of the T and Q alleles, because elastin is a substrate for MMP-9 and loss of elastin has been associated with increased arterial stiffness. However, further studies are needed to examine this hypothesis.
Recently, haplotype association analysis has been suggested as a more powerful approach of identifying predisposing genes/alleles for complex conditions than any individual SNP,33–34 because haplotypes capture almost all the variation in the gene. In the present study, the two polymorphisms were in linkage disequilibrium and the whole genotypes influenced aortic PWV and serum MMP-9 levels. To our knowledge, this is the first study to observe a direct relationship between MMP-9 gene polymorphisms, whole genotypes, predicted haplotypes, MMP-9 levels, and arterial stiffness. These findings support the notion that genetic variation in this gene is involved with higher elastolytic activity, which in turn may influence large artery stiffening. Future studies are required to evaluate whether increased serum gelatinolytic/elastase activity in particular lead to elastin degradation in a genetically susceptible individual in the development of premature arterial stiffening and/or increased vascular risk.
Limitations and Strengths
The cross-sectional nature of the present study limits our ability to infer a causal relationship between genetic variants, circulating elastases, and arterial stiffness. We also assessed serum rather than tissue MMP-9 levels. However, serum levels are known to relate to outcome, and direct measurement of levels within the walls of large arteries during life is not feasible. Some strengths of our study include: firstly, the selection of a large sample of the general population rather than a case-control design; secondly, the exclusion of subjects with vascular disease or known cardiovascular risk factors; thirdly, the inclusion of both men and women and gender-specific analysis at baseline; lastly, whole genotype-phenotype analysis to confirm the role of MMP-9 gene regulation on serum enzymes (MMP-9, SEA) and aortic stiffness.
Large artery stiffness is an important determinant of cardiovascular risk. The present study indicates that aortic PWV, MMP-9 levels, and total SEA are modulated by the −1562C>T and R279Q polymorphisms in the MMP-9 gene. These polymorphisms also associated independently and significantly with aortic PWV in healthy individuals across a wide age-range. This strongly suggests that the genetic variation in the MMP-9 protein may be involved in the process of large artery stiffening presumably through effects on turnover of matrix proteins in the vessel wall. It also suggests that both MMP-9 and total SEA may be useful circulating biomarkers for cardiovascular disease. Large scale prospective studies will be needed to address these issues. But if confirmed, biomarkers such as MMP-9 and total SEA could offer a noninvasive index of clinically important pathological change in the arterial wall as well as identifying individuals at increased risk for cardiovascular disease.
We thank Sue Monteith for her help with the Taqman assays.
Source of Funding
This work was partly supported by a project grant from the British Heart Foundation.
Original received October 16, 2005; final version accepted May 8, 2006.
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