Identification of 92-kD Gelatinase in Human Coronary Atherosclerotic Lesions
Association of Active Enzyme Synthesis With Unstable Angina
Background Acute coronary ischemia is usually initiated by rupture of atherosclerotic plaque, leading to intracoronary thrombosis and clinical sequelae. The proximate cause of plaque rupture is unknown. Accordingly, we investigated the potential role of the 92-kD gelatinase member of the matrix metalloproteinase family in acute coronary ischemia.
Methods and Results Coronary atherectomy specimens from patients with atherosclerosis and an acute ischemic syndrome consistent with recent plaque rupture (unstable angina) (n=12) were immunostained for the presence of 92-kD gelatinase; the results were compared with those obtained by identical study of atherectomy specimens from patients with atherosclerosis and angina but without acute ischemia (stable angina) (n=12). Positive immunostaining for 92-kD gelatinase was present in 83% of specimens from both unstable and stable angina patients. However, intracellular localization of enzyme (indicating active synthesis) was documented in 10 of 10 positively stained specimens from patients with unstable angina compared with 3 of 10 positively stained specimens from patients with stable angina. Macrophages and smooth muscle cells were the major sources of 92-kD gelatinase in all specimens examined by immunostaining of adjacent sections.
Conclusions 92-kD gelatinase is commonly expressed in coronary arterial atherosclerotic lesions. Active synthesis of 92-kD gelatinase by macrophages and smooth muscle cells in atherosclerotic lesions may play a pathogenic role in the development of acute coronary ischemia.
The acute coronary ischemic syndromes, ie, unstable angina, acute myocardial infarction, and sudden cardiac death, represent a clinical continuum linked by a common pathophysiological event, intracoronary thrombosis.1 2 3 4 Thrombosis is usually initiated by rupture of atherosclerotic plaque and exposure of highly thrombogenic plaque constituents to coronary blood flow. The extent of the resulting thrombus and its location in the coronary circulation contribute to the different resultant clinical syndromes. Although the proximate cause of plaque rupture is not known, pathological studies performed on patients who died suddenly of acute coronary thrombosis have documented a consistent relation between sites of plaque rupture and the presence of intense macrophage infiltration.3 4
Macrophages synthesize and secrete a diverse array of proteolytic enzymes capable of degradation of plaque constituents. One such family of enzymes, the matrix metalloproteinases, is capable of degrading all macromolecular constituents of the extracellular matrix. Macrophages release the metalloproteinases interstitial collagenase, 92-kD gelatinase, and stromelysin as major products along with 72-kD gelatinase as a minor product.5 The mRNA for stromelysin has been identified in human coronary atherosclerotic plaques.6 Stromelysin, interstitial collagenase, and 92-kD gelatinase are present and enzymatically active in atherosclerotic plaque.7 These enzymes appear to function as a proteolytic cascade of which the 92-kD gelatinase is a downstream member, whose activation is initiated after that of stromelysin.8 In addition to collagen types IV, V, and XI and denatured collagens (gelatin), 92-kD gelatinase degrades proteoglycans and elastin, which are found in atherosclerotic lesions and are resistant to degradation by the matrix metalloproteinases stromelysin and interstitial collagenase.9 10
Because of its unique and broad substrate specificity and its distal position in the matrix proteolytic cascade, we hypothesized that excessive expression of 92-kD gelatinase may play an important role in matrix degradation and rupture of the coronary arterial atherosclerotic plaque. To test this hypothesis, we performed immunohistochemical examination of atherectomy specimens obtained from patients with unstable angina for expression of the 92-kD gelatinase and compared the results with its expression in normal internal mammary arteries and in atherectomy specimens from patients with coronary atherosclerosis but with stable angina.
Directional atherectomy was performed on patients with symptomatic ischemic heart disease enrolled in the Coronary Angioplasty Versus Excisional Atherectomy Trial (CAVEAT).11 This study was approved by the Institutional Review Board of each participating institution and was conducted according to the principles of the Declaration of Helsinki. For this study, patients were considered to have unstable angina if they presented with a crescendo pattern of ischemic symptoms, including chest pain at rest and ECG evidence of ischemia during symptoms. Atherectomy specimens from all 12 patients in the CAVEAT trial who met this strict definition of unstable angina were used for this study. Culprit lesions were identified by the individual operator on the basis of clinical, ECG, and angiographic data. Patients were considered to have stable angina if their symptoms were predictably exertional without any of the components of unstable angina. Ninety-two patients enrolled in CAVEAT underwent atherectomy for stable angina. Of this group, 12 specimens were randomly chosen for inclusion in this study. Specimens of the left internal mammary artery were obtained surgically from 2 patients undergoing coronary artery bypass surgery and prepared and fixed in the same manner as the atherectomy specimens.
Antibodies were prepared as previously described,12 either by immunizing rabbits with the 92-kD form of neutrophil gelatinase obtained by preparative gel electrophoresis (IS3-70) or by immunizing rabbits with the purified native form of the proteinase (MH-1). Both antibodies have been shown to be monospecific by immunoblotting techniques13 and gave identical results when the tissues used for this study were immunostained.
All tissue specimens were prefixed in 4% paraformaldehyde for 2 hours, followed by incubation in a 30% sucrose solution overnight. A portion of the specimen was then postfixed in 10% formalin for light-microscopic analysis. These portions were ultimately embedded in paraffin and stained with hematoxylin and eosin or elastic tissue trichrome.
The remaining portion of each specimen was embedded in OCT (Miles Diagnostics) and stored at −70°C before staining. In preparation for staining, specimens 6 μm thick were cut, applied to slides, and incubated in PBS for 5 minutes. Endogenous peroxidase activity was blocked by washing with 0.3% H2O2 for 5 minutes followed by two rinses in PBS. Slides were incubated with normal goat serum for 20 minutes. Primary antibody or negative control (normal rabbit serum), diluted 1:1000 in PBS/1% BSA, was applied for 45 minutes and followed by two 5-minute washes with PBS. Biotin-conjugated anti-rabbit secondary antibody (Vector Laboratories) was added for 45 minutes, after which two 5-minute washes with PBS were performed. Slides were incubated in avidin-biotin complex (Vector Laboratories) for 30 minutes and then washed twice in PBS. Diaminobenzidine was applied to slides for 5 minutes, followed by rinsing with distilled water. A 10-second counterstain with hematoxylin was performed. Staining of peripheral blood smears was used as a positive control. Negative staining with nonspecific rabbit IgG was documented for each experiment (results not shown).
Adjacent Section Immunostaining
Four frozen sections adjacent to those that stained positively for 92-kD gelatinase were cut, applied to slides, and incubated in PBS for 5 minutes. Slides were then incubated in 0.3% H2O2 for 5 minutes. Nonspecific protein binding was blocked by incubating slides in normal horse serum for 20 minutes. A different primary mouse antibody, HAM56 (IgM) for macrophages14 (Enzo Diagnostics), HHF35 (IgG1) for smooth muscle cells15 (Enzo Diagnostics), or DAKO-LCA (IgG1) (DAKO) for lymphocytes16 or negative control (nonspecific mouse IgG, Sigma Chemical Co) was added to each of the four slides for 20 minutes. Slides were then rinsed in PBS. Secondary anti-mouse antibody (Signet Laboratories) was added for 20 minutes and followed by washing in PBS. Slides were then incubated with avidin-biotin complex (Signet Laboratories) for 20 minutes, followed by a PBS wash. 3-Amino-9-ethylcarbazole substrate was added to the slides for 20 minutes. The slides were then washed with distilled water for 5 minutes and counterstained with hematoxylin for 10 seconds. The identity of cells that stained positively for 92-kD gelatinase was determined by identifying, in the three adjacent sections stained with cell type–specific antibodies, the cell type responsible for a positive immunoperoxidase reaction.
The stained specimens were analyzed by a cardiovascular pathologist (J.M.I.) blinded to the patient’s clinical diagnosis. The specimens were analyzed for thrombus, calcium, cholesterol clefts, foam cells, and cellularity.
Histological findings and immunohistochemical data were compared by χ2 analysis. Statistical significance was assigned if P<.05.
Expression of 92-kD Gelatinase in Normal Internal Mammary Artery
Specimens of the internal mammary artery from 2 different patients undergoing bypass surgery were evaluated for expression of the 92-kD gelatinase. Both specimens were histologically normal, without atherosclerosis. In neither artery was there any immunohistochemical evidence of 92-kD gelatinase expression (Fig 1a⇓ and 1b⇓).
Expression of 92-kD Gelatinase in Coronary Atherectomy Specimens From Patients With Unstable and Stable Angina
Of the coronary atherectomy specimens from the 12 patients with unstable angina, 10 (83%) stained positively for the 92-kD gelatinase. Fig 2a⇓ and 2b⇓ illustrates representative positive and negative specimens from patients with unstable angina. Nine of 12 specimens (75%) from patients with stable angina stained positively for the 92-kD gelatinase. Fig 3a⇓ and 3b⇓ demonstrates representative positive and negative staining patterns.
Extracellular Versus Intracellular Staining Pattern
Localization of positive staining differed significantly in specimens retrieved from patients with unstable versus stable angina. All 10 of the positively stained specimens retrieved from patients with unstable angina demonstrated intracellular localization of the 92-kD gelatinase (Fig 4⇓). In 3 of these specimens, positive staining for gelatinase was also observed in the extracellular space. In contrast, of the 9 positively stained specimens retrieved from patients with stable angina, only 3 displayed a pattern of intracellular staining (P<.01). In the remaining 6 positively stained specimens, weak immunostaining was limited to the extracellular space (Fig 5⇓).
For 7 of the patients with unstable angina in whom positive intracellular staining was observed, adjacent sections were successfully examined with antibodies specific for smooth muscle cells, macrophages, and lymphocytes. (In the remaining 3 patients with positive intracellular staining, the quantity of retrieved specimen was insufficient to perform adjacent section staining.) Positive immunostaining for the 92-kD gelatinase was localized to macrophages in 7 of 7 specimens (Fig 6a⇓ and 6b⇓). Staining was also demonstrated in smooth muscle cells in 3 of 7 positive specimens (Fig 7a⇓ and 7b⇓). In 7 of 7 of these positively stained specimens, 92-kD gelatinase was identified in a small number of lymphocytes (results not shown).
Similarly, macrophages and smooth muscle cells were the major sources of 92-kD gelatinase in each of the 3 specimens obtained from patients with stable angina that demonstrated intracellular staining (data not shown).
Specimens retrieved from patients with stable versus unstable angina did not differ with regard to histological demonstration of calcific deposits (50% of specimens versus 33% of specimens), foam cells (8% versus 0%), cholesterol clefts (8% versus 8%), and hypocellularity (100% versus 100%). Furthermore, the percentage of specimens in which components of the coronary artery media (50% versus 33%) and adventitia (0% versus 8%) were observed was not significantly different between the stable and unstable angina groups. However, thrombus was identified in 67% of specimens from patients with unstable angina versus only 33% of specimens obtained from patients with stable angina (P=.04).
Acute coronary ischemia has been shown to be precipitated often by atherosclerotic plaque rupture and subsequent intracoronary thrombosis.1 2 3 4 The biochemical factors predisposing to plaque rupture are poorly understood. The mRNA of stromelysin, one member of a family of proteases specific for constituents of the extracellular matrix, has been identified in atherosclerotic coronary arteries.6 In the present report, we provide evidence for the involvement of a second member of the metalloproteinase family, 92-kD gelatinase, in atherosclerotic coronary arterial lesions. In particular, the present findings are the first, to the best of our knowledge, to (1) identify evidence for a metalloproteinase in coronary arterial atherosclerotic lesions at the protein level and (2) relate the pattern of metalloproteinase expression to an acute ischemic coronary syndrome often precipitated by rupture of atherosclerotic plaque, namely, unstable angina. This gelatinase was found in 83% of specimens obtained from the “culprit” coronary lesions of patients who met a stringent definition of unstable angina. In this small study, macrophages and smooth muscle cells appeared to represent the major source of this metalloproteinase. The intracellular localization of the 92-kD gelatinase in macrophages from these specimens can be interpreted as evidence of active synthesis of this enzyme because macrophages do not store this metalloproteinase.17 In 3 of the 10 specimens that demonstrated intracellular staining, 92-kD gelatinase was also documented extracellularly.
The pattern of immunostaining of specimens from patients with stable angina was markedly different from that observed among patients with unstable angina. Only 3 of the 10 positive specimens in the stable angina group exhibited intracellular staining. The remaining 7 specimens demonstrated only weak extracellular staining. Thus, if active synthesis of enzyme, as suggested by intracellular staining, is considered to be pathophysiologically relevant in unstable angina, 83% of unstable angina specimens in this study were positive, compared with 25% of patients with stable angina. Despite the difference in 92-kD gelatinase staining pattern, the histological milieu of the lesions appears very similar with regard to cellularity, calcification, presence of foam cells, and cholesterol clefts, suggesting that stable and unstable atherosclerotic lesions are closely related histologically and that differences in plaque behavior stem from episodic differences in the presence of as yet undetermined stimuli for specific expression of one or more proteins capable of disrupting plaque stability. The statistically greater association of 92-kD gelatinase expression with histological demonstration of thrombus in specimens from patients with unstable angina suggests that 92-kD gelatinase may be involved in matrix degradation leading to plaque rupture.
There are several possible explanations for why not all specimens from patients with unstable angina were positive for 92-kD gelatinase expression. One is the sampling error inherent in the atherectomy procedure itself. The atherectomy catheter samples only an incomplete fraction of the atherosclerotic plaque. Thus, it is possible that during the course of an atherectomy for unstable angina, the actual site of plaque rupture was not excised. Furthermore, unstable angina, by definition, is an episodic phenomenon. It is therefore possible that those patients with unstable angina in whom specimens were negatively stained were in a quiescent phase of the syndrome, which either occurred spontaneously or was induced by treatment with heparin, nitrates, and/or aspirin.
Evidence of plaque rupture has been found at autopsy in sections of coronary arteries of asymptomatic patients who died of noncardiac diseases.18 Thus, silent plaque rupture resulting in nonocclusive thrombosis can occur in patients who do not develop the clinical hallmarks of any of the syndromes associated with plaque rupture. The 25% of patients in this study with clinically stable angina who nevertheless demonstrated intracellular staining for the 92-kD gelatinase may have been in this category. Alternatively, these patients, had they not undergone atherectomy, might have soon progressed to frank plaque rupture and one of the acute coronary ischemic syndromes.
This study has documented only the presence of the 92-kD gelatinase protein in coronary lesions. Gelatinase activity at a specific site in the coronary artery requires both production of the protein in excess of its natural inhibitors and activation of the inactive precursor form of the protease. Factors regulating the expression of matrix metalloproteinases in atherosclerotic lesions are incompletely understood. Certain cytokines, such as interleukin 1 and tumor necrosis factor, whose presence has been documented in atherosclerotic lesions, appear to increase metalloproteinase expression without increasing expression of metalloproteinase inhibitors.19 20 The expression of endogenous metalloproteinase inhibitors in coronary lesions of patients with acute ischemic syndromes was not investigated in this study but is worthy of future investigation. The antibodies to 92-kD gelatinase used in this study recognize both the active and inactive forms of the protease. Thus, the presence of protein in these lesions does not necessarily prove the presence of gelatinase activity. However, recent evidence suggests that in noncoronary atheromata, 92-kD gelatinase is present in an active form.7 Rupture of atherosclerotic plaque was not observed in any of the atherectomy samples examined, presumably as a result of the tissue disruption and distortion involved in the atherectomy procedure. Nevertheless, given the stringent definition of unstable angina used for this study and the strong correlation of unstable angina with plaque rupture in autopsy series,4 we think that plaque rupture is still very likely to have been the inciting event in the unstable angina patients.
In conclusion, the findings reported in this study demonstrate the high prevalence of 92-kD gelatinase in coronary atherosclerotic lesions and provide evidence that active synthesis of 92-kD gelatinase by macrophages and smooth muscle cells is strongly associated with the clinical syndrome of unstable angina, possibly by metalloproteinase-induced matrix degradation promoting plaque rupture. These findings are potentially important from a fundamental standpoint because they suggest a pathogenetic role for at least one of the metalloproteinases in the development of unstable angina. From a practical standpoint, these findings raise the possibility that inhibitors of metalloproteinase activity, and specifically of the 92-kD gelatinase, might provide a novel form of therapy for stabilization of the atherosclerotic lesions of patients with coronary artery disease and prevention of acute ischemic syndromes.
Presented in part at the 43rd Scientific Session of the American College of Cardiology, Atlanta, Ga, March 13-17, 1994.
- Received January 24, 1995.
- Accepted January 27, 1995.
- Copyright © 1995 by American Heart Association
Constantinides P. Plaque fissuring in human coronary thrombosis. J Atheroscler Res. 1966;6:1-17.
Falk E. Plaque rupture with severe pre-existing stenosis precipitating coronary thrombosis: characteristics of coronary atherosclerotic plaques underlying fatal occlusive thrombi. Br Heart J. 1983;50:127-134.
Davies MJ, Thomas AC. Plaque fissuring: the cause of acute myocardial infarction, sudden ischemic death, and crescendo angina. Br Heart J. 1985;53:363-373.
Welgus HG, Campbell EJ, Curry JD, Eisen AZ, Senior RM, Wilhelm SM, Goldberg GI. Neutral metalloproteinases produced by human mononuclear phagocytes: enzyme profile, regulation and cellular differentiation. J Clin Invest. 1990;86:1496-1502.
Henney AM, Wakeley PR, Davies MJ, Foster K, Hembry T, Murphy G, Humphries S. Localization of stromelysin gene expression in atherosclerotic plaques by in situ hybridization. Proc Natl Acad Sci U S A. 1991;88:8154-8158.
Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest. 1994;94:2493-2503.
Goldberg GI, Strongin A, Collier IE, Genrich T, Marmer BL. Interaction of 92-kD type IV collagenase with the tissue inhibitor of metalloproteinases prevents dimerization, complex formation with interstitial collagenase, and activation of the proenzyme with stromelysin. J Biol Chem. 1992;267:4583-4591.
Hibbs MS, Hoidal JR, Kang AH. Expression of a metalloproteinase that degrades native type V collagen and denatured collagens by cultured human alveolar macrophages. J Clin Invest. 1987;80:1644-1650.
Wilhelm SM, Collier IE, Marmer BL, Eisen AZ, Grant GA, Goldberg GI. SV-40-transformed human lung fibroblasts secrete a 92kDa type IV collagenase which is identical to that secreted by normal human macrophages. J Biol Chem. 1989;264:17213-17221.
Topol EJ, Leya F, Pinkerton CA, Whitlow PL, Hofling B, Simonton CA, Masden RR, Serruys PW, Leon MB, Williams DO, King SB III, Mark DB, Isner JM, Holmes DR, Ellis SG, Lee KL, Keeler GP, Berdan LG, Hinohara T, Califf RM, for the CAVEAT Study Group. A comparison of directional atherectomy with coronary angioplasty in patients with coronary artery disease. N Engl J Med. 1993;329:221-227.
Hibbs MS, Hasty KA, Seyer JS, Kang AH, Mainardi CL. Biochemical and immunological characterization of the secreted forms of neutrophil gelatinase. J Biol Chem. 1985;260:2493-2500.
Hibbs MS, Bainton DF. Human neutrophil gelatinase is a component of specific granules. J Clin Invest. 1989;84:1395-1402.
Gown AM, Vogel AM, Gordon D, Lu PL. A smooth muscle-specific monoclonal antibody recognizes smooth muscle actin isozymes. J Cell Biol. 1985;100:807-813.
Hibbs MS. Expression of 92 kDa phagocyte gelatinase by inflammatory and connective tissue cells. Matrix. 1992;suppl 1:51-57.
Davies M, Bland J, Hangartner J, Angelini A, Thomas AC. Factors influencing the presence or absence of acute coronary thrombi in sudden ischaemic death. Eur Heart J. 1989;10:203-208.
Galis ZS, Muszynski M, Sukhova GK, Simon-Morrissey E, Unemori EN, Lark MW, Amento E, Libby P. Cytokine-stimulated human vascular smooth muscle cells synthesize a complement of enzymes required for extracellular matrix digestion. Circ Res. 1994;75:181-189.