The 5A/6A polymorphism in the promoter of the stromelysin-1 (MMP-3) gene predicts progression of angiographically determined coronary artery disease in men in the LOCAT gemfibrozil study
Introduction
Members of the matrix metalloproteinase (MMP) family have been implicated in connective tissue remodelling during atherogenesis 1, 2, 3. Since the level of expression of these enzymes in the plaque is essentially impossible to measure, this is an area of CAD where a specific genotype, if it predicted high or low enzyme levels, may add information to measurable plasma risk factors for estimating CAD risk. The presence of stromelysin-1 (MMP-3) in coronary atherosclerotic plaques was originally demonstrated by in situ mRNA hybridization [4]. Stromelysin-1 is an MMP with a broad substrate specificity, degrading collagen types II, IV and IX, proteoglycans, laminin, fibronectin, gelatins and elastin [5]. Expression of stromelysin-1 is primarily regulated at the level of transcription, where the promoter of the gene responds to various stimuli, including growth factors and cytokines 6, 7, 8mediated through a number of cis-elements located in the stromelysin-1 promoter.
Recently [9], we have identified a common polymorphism in the stromelysin-1 gene promoter, located roughly 600 bp upstream from the start of transcription, defined by either a run of six or five adenosines (designated 6A or 5A allele). In vitro studies of promoter strength showed that the 5A allele expressed higher activity than the 6A allele in both cultured fibroblasts and vascular smooth muscle cells [10], and band shift assays showed that a nuclear protein bound more strongly to the 6A than the 5A sequence, suggesting that this protein may be a repressor of transcriptional activity. This suggests that, compared with other genotypes, individuals homozygous for the 6A allele would have lower stromelysin-1 levels in their arterial walls because of reduced gene transcription, and this lower level of proteolytic activity might therefore favour deposition of extracellular matrix in the atherosclerotic lesions. This would lead to the development of an atherosclerotic plaque with a thick cap, and result in a more rapid progression of angiographically defined stenosis.
Support for this hypothesis has come from genetic analyses of two studies showing the effect of lipid-lowering treatment on progression of angiographically determined coronary atherosclerosis, the St. Thomas' Atherosclerosis Regression Study (STARS) [11], and the REGRESS study [12]. In both studies, in patients where lipid levels were lowered by treatment (and who had less progression of angiographically determined disease in their coronary arteries), stromelysin-1 genotype was not associated with differences in progression of disease, but in those whose lipid levels remained high the 5A allele was associated with less progression of angiographic stenosis, and the 6A allele with greater progression 9, 13. To investigate this further we have determined the relationship between stromelysin-1 genotype and progression of coronary artery disease in the LOCAT trial [14]. In this trial 395 post-coronary-bypass men with low (<1.1 mmol/l) high-density lipoprotein (HDL) cholesterol as their main lipid abnormality were randomly allocated to receive gemfibrozil or placebo for an average of 32 months. The trial has reported that, compared with placebo, gemfibrozil significantly retarded the reduction of average coronary diameters (P=0.009) and minimum diameters of stenosis (P=0.002), as well as the appearance of new lesions in bypass grafts (P<0.001) [15]. In this study we have examined the relationship between stromelysin-1 genotype and the progression and regression of coronary artery stenosis in the LOCAT subjects.
Section snippets
Patients and drug treatment
Details of the entry criteria and the screening process were described previously [14]. Three hundred and ninety-five men, 70 years or younger, were randomly assigned in a double-blind fashion to receive either slow-release gemfibrozil (Lopid SR), 1200 mg once daily, or a matching placebo. All patients had previously undergone coronary bypass surgery. They fulfilled the following lipid criteria at two consecutive screening visits: HDL cholesterol 1.1 mmol/l or less, low-density lipoprotein
Results
For determining the stromelysin-1 5A/6A genotype, a rapid and precise fluorescent-based PCR method was developed. Fig. 1 shows the print-out of the fluorogram from subjects with the three different genotypes, which had previously been determined by direct sequencing. As reported earlier [15], 372 of the 395 randomised patients completed the study, all of whom had had coronary artery bypass surgery. Genotype was missing in one subject, therefore, this report is based on 371 patients (187
Discussion
Two previous lipid-lowering studies of coronary artery disease progression, STARS and REGRESS have reported that the 5A allele of the stromelysin-1 promoter polymorphism was associated with reduced progression, and the 6A allele was associated with greater progression of angiographically determined disease 9, 11. The observation here of a similar pattern of effects in a third independent study puts beyond doubt the validity of the association. These results thus confirm the link between genetic
Acknowledgements
This work was supported by the British Heart Foundation (PG007 and 86-77) and the Commission of the European Communities (HIFMECH study, contract BMH4-CT96-0272).
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For the LOCAT Study Group: members of the LOCAT Study Group are listed in Circulation 1997;96:2137–2143.