Macrophages, Smooth Muscle Cells, and Tissue Factor in Unstable Angina
Implications for Cell-Mediated Thrombogenicity in Acute Coronary Syndromes
Background Macrophage expression of tissue factor may be responsible for coronary thrombogenicity in patients with plaque rupture. In patients without plaque rupture, smooth muscle cells may be the thrombogenic substrate. This study was designed to identify the cellular correlations of tissue factor in patients with unstable angina.
Methods and Results Tissue from 50 coronary specimens (1560 pieces) from patients with unstable angina and 15 specimens from patients with stable angina were analyzed. Total and segmental areas (in square millimeters) were identified with trichrome staining. Macrophages, smooth muscle cells, and tissue factor were identified by immunostaining. Tissue factor content was larger in unstable angina (42±3%) than in stable angina (18±4%) (P=.0001). Macrophage content was also larger in unstable angina (16±2%) than in stable angina (5±2%) (P=.002). The percentage of tissue factor located in cellular areas was larger in coronary samples from patients with unstable angina (67±8%) than in samples from patients with stable angina (40±5%) (P=.00007). Multiple linear stepwise regression analysis showed that coronary tissue factor content correlated significantly (r=.83, P<.0001) with macrophage and smooth muscle cell areas only in tissue from patients with unstable angina, with a strong relationship between tissue factor content and macrophages in the atheromatous gruel (r=.98, P<.0001).
Conclusions Tissue factor content is increased in unstable angina and correlates with areas of macrophages and smooth muscle cells, suggesting a cell-mediated thrombogenicity in patients with acute coronary syndromes.
Rupture of lipid-rich coronary plaques with subsequent thrombosis is probably the most important mechanism underlying the sudden onset of acute coronary syndromes.1 2 3 Macrophages may play a role in rupture of the fibrous cap, with subsequent exposure of the lipid-rich atheromatous gruel to circulating blood, resulting in coronary thrombosis.4 5 6 Tissue factor antigen is present in the atheromatous gruel,7 is upregulated in circulating and endothelium-adhered monocytes, and is also increased in coronary tissue from patients with unstable angina.8 9 10 Therefore, tissue factor is a candidate for the procoagulant activity of ruptured coronary plaques. Tissue factor requires insertion into membranes containing acidic phospholipids to function efficiently as a cofactor.11 Although atheromatous gruel contains many lipids and antigenically detected tissue factor, intact cells are detected only at the boundary of the necrotic core, so the mechanisms of increased thrombogenicity remain to be elucidated.
Smooth muscle cells (SMCs) have been implicated in the pathophysiology of acute ischemic syndromes,12 13 with an increased presence in thrombosed vessels from patients with unstable angina at rest.14 Furthermore, fatal coronary thrombosis may occur with only fissures or erosions of the fibrous cap without exposure of the atheromatous gruel.15 16 However, thrombogenicity of coronary SMCs is unclear and remains to be elucidated.
This study was designed to identify tissue factor location within the plaque and to determine cellular correlations with macrophages and SMCs by a systematic quantification approach in coronary plaque tissue from patients with stable and unstable angina.
From October 1991 to November 1994, 724 consecutive directional coronary atherectomy procedures were performed in the Cardiac Catheterization Laboratory of the Massachusetts General Hospital. Patients were required to meet the Braunwald definition of unstable angina.17 This study was performed simultaneously with a study that evaluated the role of macrophages in restenosis after coronary intervention in patients with unstable angina,18 so all patients required a follow-up angiography within 1 to 12 months after directional coronary atherectomy. Initially, we identified 106 patients who met both clinical and angiographic criteria. Fifty-six patients were excluded because of a previous procedure at the site of the culprit lesion (44 patients), follow-up angiography >12 months (4 patients), and atherectomy sample area <1.5 mm2 (8 patients). Fifty lesions from patients with unstable angina (38 men and 12 women; mean age, 62±12 years; range, 41 to 80 years) compose the study population. Fifteen lesions from patients with chronic stable angina (12 men and 3 women; mean age, 63±7 years; range, 49 to 71 years) constitute the control group. Patients were required to meet the Canadian Cardiovascular Society classification for stable angina.19
Multiple pieces of tissue were obtained from each lesion and were immediately immersed in 10% formalin. Tissue was then routinely processed for paraffin embedding according to conventional techniques. Sections were serially cut at 5 μm, mounted on lysine-coated slides, and stained with hematoxylin-eosin and by the modified elastic tissue–Masson's trichrome method.
Human Tissue Factor Antibody
Polyclonal anti–human tissue factor (TF) antibody was raised in rabbits to the extracellular domain of rTF (sTF), residues 1 to 218. An IgG fraction was prepared from the crude antiserum by adsorption to and elution from a protein-A Sepharose column (Pharmacia). Immunopurification was achieved by binding of sTF to an Affigel-10 column (BioRad) and then adsorption of the IgG fraction to the column. After exhaustive washing with buffer, specific anti-sTF antibody was eluted with 4 mol/L guanidinium hydrochloride. The antibody was dialyzed into Tris buffer before use. Specificity of the sTF antibody was shown by absorption with a 10-fold excess of TF antigen and staining.
Human macrophage antibody staining was done with 5-μm-thick sections deparaffinized and rehydrated with distilled water. Slides were placed in PBS, blocked with normal goat serum and 3% H2O2 in water, washed in PBS, and incubated with appropriate primary antibody from 0.5 to 2 hours at 37°C. Sections were stained with an anti–human panmacrophage antibody, 7.6 μg/mL anti–CD-68; KP-1 (M814 Dako); 0.1 μg/mL anti–smooth muscle α-actin antibody (Dako); and 0.5 μg/mL of the sTF antibody. Sections were washed in PBS, and primary antibodies were detected with a biotin-streptavidin amplified detection system (SuperSensitive Kit, Biogenex) for 20 minutes at room temperature. Slides were again washed in PBS and reacted with horseradish peroxidase–conjugated streptavidin for 20 minutes at room temperature and developed with diaminobenzidine. Sections were then rehydrated, coverslipped with Permount (Fisher Scientific), and examined. Positive control slides (placenta for sTF, spleen for KP-1, and intact arteries for α-actin), nonimmune negative controls, and processing controls were performed for each antigen stain.
Total specimen area and segmental area for each of the plaque components were quantified by planimetry for each specimen. Each sample was outlined manually from each stained section, and the area occupied by stained macrophages, SMCs, and tissue factor within each histologically defined component was measured. Total area in square millimeters and the percentage of the total area are reported. Specimens with total area <1.5 mm2 were excluded. The trichrome connective tissue stain was used to identify and quantify the following five components: (1) sclerotic tissue, composed of tissue with sparse cells and densely stained collagen (Fig 1A and 1B⇓⇓); (2) fibrocellular tissue, composed of tissue with abundant SMCs and densely stained collagen (Fig 1C⇓); (3) hypercellular tissue, composed of a loose connective tissue matrix containing numerous stellate cells (Fig 1D⇓); (4) atheromatous gruel, composed of necrotic cellular debris and cholesterol clefts without evidence of connective tissue matrix (Fig 1E⇓); and (5) thrombus, which stained red (Fig 1F⇓). Media was recognized by the presence of internal elastic lamina and more orderly SMCs and connective tissue matrix. To establish tissue factor location, colocalization planimetric analysis was performed with areas of macrophages and SMCs (intracellular) as well as with acellular areas (extracellular). Hypercellular and fibrocellular tissues were clearly defined as cellular areas. Cellularity of lipid-rich gruel and thrombus was evaluated by colocalization planimetric analysis as well as linear regression analysis between these areas and immunostained cells. Finally, sclerotic tissue with cells was defined as cellular, and sclerotic tissue deprived of cell nuclei was defined as acellular.
Results are expressed as mean±SEM of the individual specimen measurements. Values of P<.05 were considered statistically significant. For comparison of discrete variables (clinical and demographic data), Fisher's test was used. For comparison of two gaussian samples, a two-tailed Student's t test was used. For comparison of data not compatible with a normal frequency distribution (morphometric data), the two-tailed Student's t test was performed with logarithmic transformation of individual values. For multiple comparisons, a correction for the level of significance was performed according to the Bonferroni formula. Multiple stepwise linear regression analysis was performed with the five segmental areas described in the morphometry section and the areas of macrophages (KP-1) and SMCs (α-actin) included as independent variables. Tissue factor area was included as the outcome variable. Independent variables with significant association in the multiple analysis were examined by linear regression analysis for prediction, correlation, and summarization. Sample size, equation of the fitted line, fraction of variation, SD of regression, and residual variances are also reported.
The demographic characteristics are shown in Table 1⇓. There were no differences in age, sex, coronary risk factors, or distribution of the vessel containing the culprit lesion between tissue from patients with unstable and stable angina.
A total of 1560 pieces of tissue, 24±2 from each specimen (sum of four stainings), were quantified. Total, segmental, and percent area measurements are given in Table 2⇓. The percentage of total area occupied by sclerotic tissue was lower in coronary samples from patients with unstable angina (34±4%) than in samples from patients with stable angina (64±5%) (P=.0001). The percentage of total area occupied by hypercellular tissue was larger in coronary samples from patients with unstable angina (23±3%) than in samples from patients with stable angina (3±3%) (P=.0001). There was a tendency toward a larger percentage of atheromatous gruel in samples from patients with unstable angina (7±3%) than in samples from patients with stable angina (4±2%) (P=.06). Thrombus was present in 24 of 50 samples from patients with unstable angina and in 2 of 15 samples from patients with stable angina (P=.018). The percentage of total area occupied by tissue factor was larger in coronary samples from patients with unstable angina (42±3%) than in samples from patients with stable angina (18±4%) (P=.0001). The percentage of total area occupied by macrophages was larger in coronary samples from patients with unstable angina (16±2%) than in samples from patients with stable angina (5±2%) (P=.002). Finally, the percentage of total area occupied by α-actin–positive SMCs was not significantly different in coronary tissue from patients with unstable angina than in specimens from patients with stable angina (P=NS).
Tissue Factor Location (Intracellular Versus Extracellular)
Colocalization planimetric analyses of tissue factor content with cellular and acellular areas in both groups are shown in Table 3⇓. The percentage of tissue factor located in cellular areas was larger in coronary samples from patients with unstable angina (67±8%) than in samples from patients with stable angina (40±5%) (P=.00007). The percentage of tissue factor located in acellular areas was lower in coronary samples from patients with unstable angina (33±4%) than in samples from patients with stable angina (60±3%) (P=.0001). In cellular areas, tissue factor was present in macrophages, followed by SMCs and KP-1–negative/α-actin–negative cells. This latter group of cells was located in hypercellular and fibrocellular tissue only. Morphology suggested that these cells were mostly SMCs, but T cells and mast cells could not be excluded. In acellular areas, tissue factor antigen was present in sclerotic tissue as well as in the atheromatous gruel.
Correlation of Tissue Factor With Cellular and Acellular Areas
In tissue from patients with stable angina, multiple linear regression analysis showed no correlation between tissue factor and macrophages (r=.014, P=.67) (Fig 2A⇓) or between tissue factor and SMCs (r=.001, P=.8) (Fig 3A⇓).
In tissue from patients with unstable angina, univariate analysis correlated tissue factor with hypercellular tissue (P=.001), atheromatous gruel (P=.004), fibrocellular tissue (P=.003), SMCs (P<.0001), and macrophages (P<.0001). Multiple linear stepwise regression analysis identified macrophages (r=.67, P<.0001) (Figs 2B and 4⇑⇓) and SMCs (r=.68, P<.0001) (Figs 3B and 4⇑⇓) as independent predictors for tissue factor content on the basis of the following equation: y=0.588565×SMC area+0.696687×macrophages+0.142781 (r=.83, P<.0001).
Correlations of Tissue Factor Within Lipid-Rich Areas (Unstable Angina)
Lipid-rich atheromatous gruel was identified in 13 of 50 samples (26%). SMCs were identified in the lipid core of only one specimen. Macrophages were identified in all lipid-rich areas (100%). KP-1 identified intact macrophages (predominantly at the periphery) as well as antigen in acellular regions, most notably in the core. Tissue factor was identified in all lipid-rich areas (0.32±0.11 mm2). Colocalization planimetric analysis showed that tissue factor was located in intact macrophages (0.07±0.02 mm2) as well as in KP-1–positive acellular core (0.25±0.09 mm2). Linear regression analysis displayed a high correlation between KP-1–positive areas and tissue factor in the atheromatous gruel (r=.98, P<.0001) (Fig 5⇓).
Correlations of Tissue Factor Within Thrombotic Areas (Unstable Angina)
Thrombus was identified in 24 of 50 samples (49%). Macrophages were found in 19 of 24 samples (79%). Linear regression analysis displayed a high correlation between macrophages in thrombus and the total thrombotic area (r=.85, P<.0001) (Fig 6⇓). Tissue factor was identified in all thrombus areas (0.11±0.03 mm2). Linear regression analysis displayed a high correlation between macrophages and tissue factor in thrombus (r=.64, P<.0001).
Tissue factor, presumably the primary initiator of thrombogenesis and thrombin generation in vivo, is a specific transmembrane glycoprotein expressed by circulating monocytes, monocyte-derived macrophages, endothelial cells, and SMCs.8 9 20 21 Coronary thrombosis plays a major role in the pathophysiology of acute ischemic syndromes, and tissue factor may be responsible for this increased coronary thrombogenicity.1 2 3 7 10 This study was designed to identify tissue factor location and cellular correlations of tissue factor content by a systematic quantification approach for specific cellular components in human coronary plaque tissue from patients with stable and unstable angina.
The results reveal that coronary tissue factor content was larger in tissue from patients with unstable angina than in tissue from patients with stable angina (P=.0001). In addition, total macrophage content was also significantly larger in tissue from patients with unstable angina (P=.002), as previously demonstrated by our group.4 Tissue factor location within the plaque varied according to the clinical syndrome. In patients with stable angina, planimetric colocalization analysis showed that tissue factor was located predominantly in the acellular component of the plaque. However, multiple regression analysis did not identify any histological predictors for tissue factor content in tissue from patients with stable angina. In patients with unstable angina, planimetric colocalization analysis showed that tissue factor was located predominantly in the cellular component of the plaque. Multiple regression analysis revealed that coronary tissue factor content correlated significantly with macrophage and SMC areas (r=.83, P<.0001) only in tissue from patients with unstable angina. In this group of patients, tissue factor colocalized with intact macrophages (27±4%) and with macrophage-derived acellular debris (11±2%). Furthermore, macrophages correlated with tissue factor in both lipid-rich (r=.98, P<.0001) and thrombus areas (r=.64, P<.0001).
Disruption of coronary atherosclerotic plaques with exposure of the lipid-rich gruel to flowing blood is an important mechanism in the pathophysiology of acute coronary syndromes.1 2 3 Macrophages may be involved in the process of plaque rupture.22 23 Consequently, coronary plaques with a high content of macrophages may have both a higher risk for plaque rupture and a higher propensity for thrombosis related to an increased macrophage expression of tissue factor. Macrophage content of atherosclerotic plaques may be a link between the two phenomena, ie, plaque rupture and thrombosis.
The relative thrombogenicity of atherosclerotic plaque components was elegantly studied by Ferna´ndez-Ortiz et al,6 who found that the lipid core was the most potent substrate for platelet-rich thrombus formation in vitro. Recently, Toschi et al24 evaluated tissue factor thrombogenicity in the atheromatous gruel from human arterial segments exposed to flowing blood. 111In-platelet deposition (platelets/cm2×106) was highest in the gruel (712±213), followed by normal adventitia (203±58) and normal tunica media (134±41).
Human plaques from carotid endarterectomy specimens express tissue factor mRNA and protein in the peripheral macrophage-like cells adjacent to cholesterol clefts as well as in intimal SMCs.7 However, the correlation between tissue factor and cellular areas of macrophages and SMCs by quantified planimetry in patients with stable and unstable coronary syndromes has not been analyzed. We systematically studied and quantified the cellular correlations of tissue factor in coronary specimens of patients with stable and unstable angina. The high correlation of tissue factor with macrophage areas within the atheromatous core in patients with unstable angina suggests that lipid gruel thrombogenicity is related to macrophage expression of tissue factor. Tissue factor in the gruel was found in intact macrophages (20±1%) as well as in macrophage-derived acellular debris (80±1%), suggesting that macrophages contribute not only to the bulk of the atheromatous core25 but also to its thrombogenicity. Tissue factor was also identified in all thrombus areas in tissue from patients with unstable angina. Colocalization planimetric analysis displayed a high incidence of macrophages in thrombus (79%). Linear regression analysis demonstrated a high correlation between macrophages and tissue factor in thrombus (r=.64, P<.0001). Fresh mural thrombus is highly thrombogenic26 and may be responsible for coronary reocclusion after thrombolysis. Tissue factor inhibition prevents reocclusion after thrombolysis in a rabbit model of carotid artery thrombosis and might be suitable as adjunctive antithrombotic therapy in the treatment of acute coronary syndromes.27
In fact, platelet deposition is increased two to four times on residual thrombus compared with deeply injured arterial wall. Although thrombus formation triggered by residual thrombosis is inhibited by specific antithrombin treatment,26 the roles of tissue factor, macrophages, and SMCs in this phenomenon are unclear and remain to be elucidated.
Coronary tissue classification by the modified trichrome staining showed an increased content of hypercellular tissue in coronary samples from patients with unstable angina above that of plaque tissue from patients with stable angina (P=.0001). Planimetric analysis in tissue from patients with unstable angina showed that tissue factor colocalized with α-actin–positive SMCs (28±4%) followed by KP-1–negative/α-actin–negative cells. Multiple regression analysis identified SMCs as an independent predictor for tissue factor content in patients with unstable angina (r=.68, P<.0001), suggesting that these cells may also play a role as a thrombogenic substrate in patients with acute coronary syndromes.
Recently, SMCs have been implicated in the pathophysiology of unstable angina. Flugelman et al12 found abundant SMCs in coronary specimens from patients with unstable angina, suggestive of the importance of these cells in the mechanisms of transformation from stable to unstable coronary syndromes. Likewise, directional atherectomy studies have shown that SMCs are increasingly present in complex coronary lesions, reaching 100% in thrombosed plaques from patients with unstable angina at rest.13 14 Furthermore, autopsy studies have shown that 40% to 45% of fatal coronary thrombosis can occur with denuded or superficially eroded plaques with SMC-rich fibrous caps.15 16 In these circumstances, SMCs may be the active clotting substrate, but thrombogenicity of SMCs is not completely understood and remains to be elucidated. The expression of tissue factor by vascular SMCs is rapidly induced by growth factors and thrombin in vitro and by vascular injury in vivo.19 20 Moreover, the reproduced finding of SMCs in coronary tissue from patients with unstable angina12 13 14 and the positive correlation between tissue factor and SMCs in the same group of patients reported in this study suggest that the biological injury responsible for unstable syndromes may be associated with activation and proliferation of SMCs, but further studies are needed to completely resolve this issue.
Several limitations have to be addressed. First, the activity of tissue factor was not evaluated in this study. At the time this study was designed, the assay to evaluate tissue factor activity was in process, and we decided to quantify tissue factor content only. More recently, Marmur et al28 examined functional tissue factor activity in human coronary atherectomy specimens using a factor Xa generation assay. They found a high correlation between tissue factor activity and the tissue factor antibody staining. Second, T cell and mast cell antibodies were not used and precluded specific identification of these cells. Previous work has shown that activated T cells and mast cells may be involved in the process of plaque rupture.5 29 Tissue factor colocalized with KP-1–negative/α-actin–negative cells (14±3%). Loss of expression of α-actin by SMCs is associated with a phenotype change observed as a response to vascular injury.30 These cells were found in fibrocellular and hypercellular areas, and trichrome morphology suggests that they were mostly SMCs. However, other cell types cannot be excluded.
The results of this study support the hypothesis that macrophages and SMCs are responsible for coronary plaque tissue factor content in patients with unstable angina. Within the lipid gruel, tissue factor is associated with macrophages and macrophage-derived membranous debris.24 In other areas of the plaque, SMCs correlated with tissue factor content, suggesting a cell-mediated expression of thrombogenicity in patients with unstable angina. Despite potential sampling limitations,31 these results suggest that macrophages and SMCs play a crucial role in the thrombogenic response of atherosclerotic plaque tissue from patients with acute coronary syndromes.
Dr Bernardi was supported in part by the Anchorena Hospital in Buenos Aires, Argentina. We thank Veronica Gulle for her help in preparing tissue sections, John B. Newell for statistical analysis, Drs Vivian M. Abascal, Erling Falk, and Henry H. Ting for thoughtful comments, Dr Mark J. Semigran for technical support, and Robert E. Holt for editorial assistance.
Presented in part at the 45th Annual Scientific Sessions of the American College of Cardiology, Orlando, Fla, March 24-27, 1996, and published in abstract form (J Am Coll Cardiol. 1996;27:306A).
- Received January 25, 1996.
- Revision received July 24, 1996.
- Accepted July 30, 1996.
- Copyright © 1996 by American Heart Association
Fuster V. Lewis A. Conner Memorial Lecture. Mechanisms leading to myocardial infarction: insights from studies of vascular biology. Circulation. 1994;90:2126-2146.
Falk E, Shah PK, Fuster V. Coronary plaque disruption. Circulation. 1995;92:657-671.
Moreno PR, Shah PK, Falk E. Determinants of rupture of atherosclerotic coronary lesions. In: Willich SN, Muller JE, eds. Triggering of Acute Coronary Syndromes: Implications for Prevention. Dordrecht, Netherlands: Kluwer Academic Publishers; 1996:268-283.
Moreno PR, Falk E, Palacios IF, Newell JB, Fuster V, Fallon JT. Macrophage infiltration in acute coronary syndromes: implications for plaque rupture. Circulation. 1994;90:775-778.
Libby P. Molecular bases of the acute coronary syndromes. Circulation. 1995;91:2844-2850.
Wilcox JN, Smith KM, Schwartz SM, Gordon D. Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque. Proc Natl Acad Sci U S A. 1989;86:2839-2842.
Leathman EW, Bath PM, Tooze JA, Camm AJ. Increased monocyte tissue factor expression in coronary artery disease. Br Heart J. 1995;73:10-13.
Lo SK, Cheung A, Zheng Q, Silverstein RL. Induction of tissue factor on monocytes by adhesion to endothelial cells. J Immunol. 1995;154:4768-4777.
Annex BH, Denning SM, Keith MC, Sketch MH, Stack RS, Morrisey JH, Peters KG. Differential expression of tissue factor protein in directional atherectomy specimens from patients with stable and unstable coronary syndromes. Circulation. 1995;91:619-622.
Flugelman MY, Virmani R, Correa R, Yu ZX, Farb A, Leon MB, Elami A, Fu YM, Casscells W, Epstein SE. Smooth muscle cell abundance and fibroblast growth factors in coronary lesions of patients with nonfatal unstable angina: a clue to the mechanism of transformation from the stable to the unstable clinical state. Circulation. 1993;88:2493-2500.
Wilson JM, Capek P, McAllister HA, Vaughn WK, Ferguson JJ, Club FJ, Buja LM, Willerson JT. Coronary lesion histology in stable, unstable and evolving angina pectoris. J Am Coll Cardiol. 1995;(special issue):279A. Abstract.
Mann JM, Kaski JC, Arie S, Pereira WI, Pileggi F, Davies MJ. Plaque constituents in patients with stable and unstable angina: an atherectomy study. J Am Coll Cardiol. 1995;(special issue):34A. Abstract.
van der Wal AC, Becker AE, van der Loos CM, Das PK. Site of intimal rupture or erosion of thrombosed coronary atherosclerotic plaques is characterized by an inflammatory process irrespective of the dominant plaque morphology. Circulation. 1994;89:36-44.
Farb A, Burke AP, Tang AL, Liang Y, Mannan P, Smalek J, Virmani R. Coronary plaque erosion without rupture into a lipid core: a frequent cause of coronary thrombosis in sudden coronary death. Circulation. 1996;93:1354-1363.
Braunwald E. Unstable angina: a classification. Circulation. 1989;80:410-414.
Moreno PR, Bernardi V, Lo´pez-Cue´llar J, Newell JB, McMellon C, Gold HK, Palacios IF, Fuster V, Fallon JT. Macrophage infiltration predicts restenosis after coronary intervention in patients with unstable angina. Circulation. 1996;94:3098-3102.
Taubman MB, Marmur JD, Rosenfield C-L, Guha A, Nichtberger S, Nemerson Y. Agonist-mediated tissue factor expression in cultured vascular smooth muscle cells: role of Ca2+ mobilization and protein kinase C activation. J Clin Invest. 1993;91:547-552.
Marmur JD, Rossikhina M, Guha A, Fyfe B, Friedrich V, Mendlowitz M, Nemerson Y, Taubman MB. Tissue factor is rapidly induced in arterial smooth muscle after balloon injury. J Clin Invest. 1993;91:2253-2259.
Galis ZS, Sukhova G, Lark MW, Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest. 1994;86:2493-2503.
Shah PK, Falk E, Badimo´n JJ, Ferna´ndez-Ortiz A, Mailhac A, Villareal-Levy G, Fallon JT, Regnstrom J, Fuster V. Human monocyte–derived macrophages induce collagen breakdown in fibrous caps of atherosclerotic plaques: potential role of matrix-degrading metalloproteinases and implications for plaque rupture. Circulation. 1995;92:1565-1569.
Toschi V, Fallon JT, Gallo R, Lettino M, Ferna´ndez-Ortiz A, Badimo´n L, Chesebro JT, Nemerson Y, Fuster V, Badimo´n JJ. Tissue factor predicts thrombogenicity of human atherosclerotic plaque components. Circulation. 1995;92(suppl I):I-112. Abstract.
Meyer BJ, Badimo´n JJ, Mailhac A, Ferna´ndez-Ortiz A, Chesebro JH, Fuster V, Badimo´n L. Inhibition of growth of thrombus on fresh mural thrombus: targeting optimal therapy. Circulation. 1994;90:2432-2438.
Ragni M, Cirillo P, Pascucci I, Scognamiglio A, D'Andrea D, Eramo N, Ezekowitz MD, Pawashe AB, Chiariello M, Golino P. Monoclonal antibody against tissue factor shortens tissue plasminogen activator lysis time and prevents reocclusion in a rabbit model of carotid artery thrombosis. Circulation. 1996;93:1913-1918.
Marmur JD, Thiruvikraman SV, Fife BS, Guha A, Sharma SK, Ambrose JA, Fallon JT, Nemerson Y, Taubman MB. The identification of active tissue factor in human coronary atheroma. Circulation. 1996;94:1226-1232.
Kovanen PT, Kaartinen M, Paavonen T. Infiltrates of activated mast cells at the site of coronary atheromatous erosion or rupture in myocardial infarction. Circulation. 1995;92:1084-1088.
Schwartz SM, deBlois D, O'Brien ERM. The intima: soil for atherosclerosis and restenosis. Circ Res. 1995;77:445-465.