Adriano M. Henney

Matrix Metalloproteinases and Cardiovascular Disease

The vessel wall is an integrated functional component of the circulatory system that is continually remodeling in response to hemodynamic conditions and disease states. The endothelium releases locally active mediators, such as nitric oxide and endothelin, which have immediate vasoactive properties and longer-term trophic effects on the medial SMCs. Vascular tone and compliance are determined by these SMCs, which not only actively control wall tension but also synthesize the major structural components of the vessel wall: collagens types I, III, IV, and V, elastin, proteoglycans, and glycoproteins. These components interact to form a complex network that gives blood vessels their elastic physical characteristics. Continual mechanical stresses will cause weakening of the vessel wall if the structural integrity and physical properties of the matrix are not maintained. The matrix composition determines not only the physical elastic properties of the vessel wall but also its cellular components via stored growth factors in the matrix and growth factor activation by MMPs. The matrix, therefore, rather than being merely a system of scaffolding for the surrounding cells, is a dynamic structure that is central to the control of vascular remodeling. Connective tissue repair and remodeling to maintain matrix integrity involves the synthesis and removal of these proteins, a process that depends on the action of a range of proteases and their inhibitors. Evidence suggests that there are two systems that predominate and interact to achieve homeostasis within the vessel wall: the plasminogen activator-plasmin system and the MMPs. This review focuses on discussing the increasing evidence that supports a role for enzymes of the MMP family in the pathogenesis of atherosclerosis and postangioplasty restenosis. MMPs are a family of Zn2+- and Ca2+-dependent enzymes, which are important in the resorption of extracellular matrices in both normal physiological processes and pathological states. Nine …

Functional Polymorphism in the Regulatory Region of Gelatinase B Gene in Relation to Severity of Coronary Atherosclerosis

BACKGROUND Gelatinase B, a matrix metalloproteinase that has proteolytic activity against connective tissue proteins, has been suggested to be important in the connective tissue remodeling processes associated with atherogenesis and plaque rupture. This study tested the hypothesis that sequence variation in the promoter region of the gelatinase B gene influences its expression, predisposing individuals carrying certain genetic variants to more severe atherosclerosis. METHODS AND RESULTS Single-strand conformation polymorphism analysis was carried out to search the promoter region of the gene encoding gelatinase B for naturally occurring genetic variation. As a result, an unreported common polymorphism was detected, which arose from a cytosine (C) to thymidine (T) transition at position -1562 relative to the start of transcription. Transient transfection experiments and DNA-protein interaction assays indicated that the T allele had a higher promoter activity than the C allele, which appeared to be due to preferential binding of a putative transcription repressor protein to the C allelic promoter. A sample of 584 male patients with myocardial infarction and 645 age-matched male healthy control subjects were genotyped. The allele frequencies were not significantly different between the cases and control subjects. However, in 374 patients with available angiographic data, 26% of those carrying 1 or 2 copies of the T allele had >50% stenosis in 3 coronary arteries, whereas only 15% of C/C homozygotes had triple-vessel disease. CONCLUSIONS These data suggest that this functional genetic variation influences gelatinase B gene promoter activity in an allele-specific manner and has an effect on atherosclerotic phenotype.

Genetic variation at the plasminogen activator inhibitor-1 locus is associated with altered levels of plasma plasminogen activator inhibitor-1 activity.

Plasminogen activator inhibitor-1 (PAI-1), a rapid inhibitor of tissue-type plasminogen activator, has been shown to be an independent risk factor for recurrent myocardial infarction (MI) at a young age. To investigate whether genetic variation in the PAI-1 gene is affecting plasma PAI-1 levels, a sample of 145 patients with an MI before the age of 45 years was genotyped for two polymorphisms at the PAI-1 locus, together with a sample of 95 healthy individuals of a similar age. All individuals were measured for plasma PAI-1 levels as well as for other fibrinolytic and metabolic risk indicators. A HindIII restriction fragment length polymorphism (RFLP) was used in this study in conjunction with a previously unreported eight-allele dinucleotide repeat polymorphism at the PAI-1 locus. The dinucleotide repeat polymorphism and HindIII RFLP were in strong linkage disequilibrium. There was no difference in the frequency of alleles of either polymorphism between patient and control groups. However, the smaller dinucleotide repeat alleles were significantly associated (p = 0.03) with higher plasma PAI-1 levels in the patient sample. This association was also apparent in the control sample but not at significant levels. Differences in regression coefficients for the effect of triglycerides on plasma PAI-1 levels suggest that triglyceride regulation of PAI-1 is genotype specific. Our data suggest that genetic variation at this locus contributes to between-individual differences in the level of plasma PAI-1, which is important in fibrinolysis and the pathogenesis of MI.

The status of PAI-1 as a risk factor for arterial and thrombotic disease: A review

Plasminogen activator inhibitor-1 (PAI-1) is a rapid inhibitor of tissue plasminogen (tPA) in vivo. Evidence suggests that the level of plasma PAI-1 activity is responsible for the regulation of the whole fibrinolytic process through this tPA/PAI-1 interaction. Levels of PAI-1 have therefore emerged as a candidate for a thrombotic risk factor. Recent epidemiological data supports the view that high plasma levels of PAI-1 may be important in the pathogenesis of arterial and thrombotic disease. These data are reviewed and their significance discussed. PAI-1 expression has been shown to be regulated by many different factors in vitro and the relevance of these data to in vivo physiology is addressed. The current knowledge of the biochemistry, expression and genetics of PAI-1 is also presented and the significance of this to disease pathogenesis is discussed.

Allele-Specific Regulation of Matrix Metalloproteinase-12 Gene Activity Is Associated With Coronary Artery Luminal Dimensions in Diabetic Patients With Manifest Coronary Artery Disease

Both the processes of atherosclerosis and plaque rupture are indicated to be influenced by matrix metalloproteinase (MMP) activity. We therefore searched for common functional variation in the matrix metalloelastase (MMP-12) gene locus that may be implicated in coronary artery disease. Single-strand conformation polymorphism analysis of DNA from healthy individuals detected a common polymorphism within the MMP-12 gene promoter (an A-to-G substitution at position -82). The frequency of the G allele was 0. 19. The polymorphism influences the binding of the transcription factor activator protein-1 (AP-1) in electromobility shift assay. A higher binding affinity of AP-1 to the A allele was associated with higher MMP-12 promoter activity in vitro in transient transfection studies in U937 and murine lung macrophage (MALU) cells. Phorbol 12-myristate 13-acetate (PMA) and insulin, 2 known activators of AP-1, increased the binding of AP-1 to the MMP-12 promoter, with higher affinity for the A allele. In transfection experiments, both the A and the G alleles responded to insulin and PMA, the A allele showing higher promoter activity than the G allele. Furthermore, Western blot analysis demonstrated that insulin increased MMP-12 protein production. To analyze whether the -82 A/G polymorphism is associated with coronary artery disease, 367 consecutive patients who underwent percutaneous transluminal coronary angiography with stent implantation were genotyped. In patients (n=71) with diabetes, the A allele was associated with a smaller luminal diameter. In conclusion, a common functional polymorphism within the MMP-12 promoter influences coronary artery luminal dimensions in diabetic patients with manifest coronary artery disease.

Polymorphisms of the Human Matrix Gla Protein ( MGP ) Gene, Vascular Calcification, and Myocardial Infarction

The matrix Gla protein (MGP) is an important inhibitor of vessel and cartilage calcification that is strongly expressed in human calcified, atherosclerotic plaques and could modulate plaque calcification and coronary heart disease risk. Using a genetic approach, we explored this possibility by identifying polymorphisms of the MGP gene and testing their possible association with myocardial infarction (MI) and plaque calcification. Eight polymorphisms were identified in the coding and 5′-flanking sequences of the MGP gene. All polymorphisms were investigated in 607 patients with MI and 667 control subjects recruited into the ECTIM Study (Etude Cas-Témoins de l’Infarctus du Myocarde) and in 717 healthy individuals with echographically assessed arterial calcification and atherosclerosis who were participating in the AXA Study. In the ECTIM Study, alleles and genotypes were distributed similarly in patients and controls in the whole study group; in only 1 subgroup of subjects defined as being at low risk for MI were the concordant A −7 and Ala 83 alleles more frequent in patients with MI than in controls (P <0.003). In the AXA Study among subjects with femoral atherosclerosis, the same alleles were more common in the presence than the absence of plaque calcification (P <0.025). The other MGP polymorphisms were not associated with any investigated clinical phenotype. Transient transfection experiments with allelic promoter-reporter gene constructs and DNA-protein interaction assays were carried out to assess possible in vitro functionality of the promoter variants detected at positions −814, −138, and −7 relative to the start of transcription. When compared with the −138 T allele, the minor −138 C allele consistently conferred a reduced promoter activity of −20% (P <0.0001) in rat vascular smooth muscle cells and of −50% (P <0.004) in a human fibroblast cell line, whereas the other polymorphisms, including −7, displayed no evidence of in vitro functionality. We conclude that the A −7 or Ala 83 alleles of the MGP gene may confer an increased risk of plaque calcification and MI; however, the observed relationships are weak or limited to subgroups of patients and therefore need confirmation.

Expression of Matrix Metalloproteinases and Their Inhibitor TIMP-1 in the Rat Carotid Artery After Balloon Injury

The temporal relationship of matrix metalloproteinases (MMPs) and a specific tissue inhibitor (TIMP-1) has been examined by reverse transcription-polymerase chain reaction and substrate zymography, after balloon catheter angioplasty of the rat carotid artery. The contralateral uninjured carotid artery was used as a comparative control. Of the MMPs examined, only MMP-2 (72-kDa gelatinase) was produced constitutively by normal uninjured arteries. After injury, MMP-2 mRNA levels fell compared with the uninjured arteries; by 24 hours, levels had increased 2-fold. Zymography showed that the inactive form of MMP-2 predominated in uninjured vessels, but after injury, the level of the active form was increased. MMP-9 (92-kDa gelatinase) mRNA levels and activity peaked at 6 hours after injury and were still detectable at 7 days. MMP-3 (stromelysin) expression was detectable at low levels as early as 2 hours after injury and showed an approximate 2-fold increase of expression at 7 days. The presence of the active protein paralleled the mRNA expression. The inhibitor TIMP-1 mRNA was first detected 6 hours after injury and showed a marked peak of expression at 24 hours; however, no expression was detected by 7 days. The presence of a constitutively expressed, low molecular weight caseinolytic enzyme (27 kDa) was observed, and the induction of a caseinolytic enzyme (30 kDa) was noted that was induced as early as 2 hours after injury, peaked at 6 hours, and was barely detectable by 7 days. These results demonstrate that the process of extracellular matrix breakdown by MMPs after balloon catheter-induced injury is controlled by a tightly regulated temporal response by the genes responsible for the production of these enzymes and their inhibitor and by post-translational activation of the proenzymes.

Unravelling the familial tendency to aneurysmal disease: Popliteal aneurysm, hypertension and fibrillin genotype

PURPOSE To screen patients with abdominal aortic aneurysm for popliteal aneurysm and investigate cardiovascular and genetic risk factors associated with aneurysmal disease at more than one site (generalised aneurysmal disease). SUBJECTS, DESIGN AND SETTING: All patients referred to the Regional Vascular Surgical Service at Charing Cross Hospital with unruptured abdominal aortic aneurysm between 1989 and 1993 were screened for popliteal aneurysms, using ultrasonography. MAIN OUTCOME MEASURES Palpation of a popliteal aneurysm or ultrasonographic detection of popliteal dilatation, where the ratio maximum popliteal fossa diameter/suprageniculate popliteal diameter was > or = 1.5, in relation to cardiovascular and genetic risk factors. RESULTS Clinical examination detected popliteal aneurysms in only 11/232 patients (5%), but ultrasonography demonstrated the presence of popliteal aneurysm in a further 13 patients, 24/232 in total (10%). Multivariate regression identified four independent factors associated with popliteal dilatation disease: age (p = 0.046), height (p = 0.006), systolic hypertension (p = 0.037) and triglyceride concentration (p = 0.009). Generalised aneurysmal disease and systolic blood pressure were associated with polymorphic variation in the fibrillin-1 gene, but not with variations in the apolipoprotein B and type III collagen genes. CONCLUSIONS Few patients with abdominal aortic aneurysm (10%) also have popliteal aneurysms: the risk of popliteal dilatation increases with age, height, systolic blood pressure, triglyceride concentration and fibrillin genotype. The strong interaction between fibrillin genotype and blood pressure may contribute to the familial tendency to aortic aneurysm.

Porcine heart valves produce a protein that induces cell-mediated connective tissue degradation: I. Biological properties.

Cultured porcine heart valves secrete a factor that induces cells to degrade their extracellular matrix. This activity was routinely monitored by measuring the release of glycosaminoglycans from proteoglycan in cultured bovine nasal cartilage. It was observed that factor-mediated matrix degradation was dose dependent and required live target cells, while factor production by valves was inhibited by cycloheximide and declined with reduced cell viability. The effect of various steroidal and nonsteroidal anti-inflammatory drugs on the production and activity of the factor is discussed with reference to similar cytokines. It is concluded that there is a close similarity between the cardiac catabolic factor described here and catabolin, a protein isolated from porcine synovia and leukocytes, as shown by the neutralization of biological activity with antisera raised to porcine catabolin (interleukin-1).

Porcine heart valves produce a protein that induces cell-mediated connective tissue degradation: II. Biochemical properties of the partially purified protein.

A cardiac catabolic factor (CCF) has been partially purified from serum-free medium conditioned by minced porcine heart valves. CCF was prepared by a series of chromatographic techniques and compared directly with porcine synovial catabolin purified by the same protocol. CCF displayed a somewhat higher molecular weight (Mr 21,000) and isoelectric point (pi 5.2) than did synovial catabolin (Mr 18,000 and pi 4.8), but the two factors clearly resemble one another closely. CCF stimulated the release of glycosaminoglycans from cultured cartilage and mitral valve and provoked porcine valves to degrade their own collagen extracellular matrix. The release of hydroxyproline was inhibited by corticosteroids, whereas proteoglycan breakdown was not. Partially pure preparations of CCF and synovial catabolin stimulated murine thymocyte proliferation; moreover, that activity was almost totally abolished by an antibody raised against pure porcine interleukin-1. These observations suggest that CCF may represent a catabolic factor that belongs to the interleukin-1 family and that it could potentially regulate the composition of valvular connective tissue.