Juan J. Badimon

Regression of atherosclerotic lesions by high density lipoprotein plasma fraction in the cholesterol-fed rabbit

The effects of homologous plasma HDL and VHDL fractions on established atherosclerotic lesions were studied in cholesterol-fed rabbits. Atherosclerosis was induced by feeding the animals a 0.5% cholesterol-rich diet for 60 d (group 1). Another group of animals were maintained on the same diet for 90 d (group 2). A third group was also fed the same diet for 90 d but received 50 mg HDL-VHDL protein per wk (isolated from normolipemic rabbit plasma) during the last 30 d (group 3). Aortic atherosclerotic involvement at the completion of the study was 34 +/- 4% in group 1, 38.8 +/- 5% in group 2, and 17.8 +/- 4% in group 3 (P less than 0.005). Aortic lipid deposition was also significantly reduced in group 3 compared with group 1 (studied at only 60 d) and group 2. This is the first in vivo, prospective evidence of the antiatherogenic effect of HDL-VHDL against preexisting atherosclerosis. Our results showed that HDL plasma fractions were able to induce regression of established aortic fatty streaks and lipid deposits. Our results suggest that it may be possible not only to inhibit progression but even to reduce established atherosclerotic lesions by HDL administration.

Tissue Factor Modulates the Thrombogenicity of Human Atherosclerotic Plaques

BACKGROUND The thrombogenicity of a disrupted atherosclerotic lesion is dependent on the nature and extent of the plaque components exposed to flowing blood together with local rheology and a variety of systemic factors. We previously reported on the different thrombogenicity of the various types of human atherosclerotic lesions when exposed to flowing blood in a well-characterized perfusion system. This study examines the role of tissue factor in the thrombogenicity of different types of atherosclerotic plaques and their components. METHODS AND RESULTS Fifty human arterial segments (5 foam cell-rich, 9 collagen-rich, and 10 lipid-rich atherosclerotic lesions and 26 normal, nonatherosclerotic segments) were exposed to heparinized blood at high shear rate conditions in the Badimon perfusion chamber. The thrombogenicity of the arterial specimens was assessed by 111In-labeled platelets. After perfusion, specimens were stained for tissue factor by use of an in situ binding assay for factor VIIa. Tissue factor in specimens was semiquantitatively assessed on a scale of 0 to 3. Platelet deposition on the lipid-rich atheromatous core was significantly higher than on all other substrates (P = .0002). The lipid-rich core also exhibited the most intense tissue factor staining (3 +/- 0.1 arbitrary units) compared with other arterial components. Comparison of all specimens showed a positive correlation between quantitative platelet deposition and tissue factor staining score (r = .35, P < .01). CONCLUSIONS Our results show that tissue factor is present in lipid-rich human atherosclerotic plaques and suggest that it is an important determinant of the thrombogenicity of human atherosclerotic lesions after spontaneous or mechanical plaque disruption.

Noninvasive In Vivo Human Coronary Artery Lumen and Wall Imaging Using Black-Blood Magnetic Resonance Imaging

BACKGROUND High-resolution MRI has the potential to noninvasively image the human coronary artery wall and define the degree and nature of coronary artery disease. Coronary artery imaging by MR has been limited by artifacts related to blood flow and motion and by low spatial resolution. METHODS AND RESULTS We used a noninvasive black-blood (BB) MRI (BB-MR) method, free of motion and blood-flow artifacts, for high-resolution (down to 0.46 mm in-plane resolution and 3-mm slice thickness) imaging of the coronary artery lumen and wall. In vivo BB-MR of both normal and atherosclerotic human coronary arteries was performed in 13 subjects: 8 normal subjects and 5 patients with coronary artery disease. The average coronary wall thickness for each cross-sectional image was 0.75+/-0.17 mm (range, 0.55 to 1.0 mm) in the normal subjects. MR images of coronary arteries in patients with >/=40% stenosis as assessed by x-ray angiography showed localized wall thickness of 4.38+/-0.71 mm (range, 3.30 to 5.73 mm). The difference in maximum wall thickness between the normal subjects and patients was statistically significant (P<0.0001). CONCLUSIONS In vivo high-spatial-resolution BB-MR provides a unique new method to noninvasively image and assess the morphological features of human coronary arteries. This may allow the identification of atherosclerotic disease before it is symptomatic. Further studies are necessary to identify the different plaque components and to assess lesions in asymptomatic patients and their outcomes.

Characterization of the relative thrombogenicity of atherosclerotic plaque components: Implications for consequences of plaque rupture☆

OBJECTIVES The purpose of this study was to determine whether different components of human atherosclerotic plaques exposed to flowing blood resulted in different degrees of thrombus formation. BACKGROUND It is likely that the nature of the substrate exposed after spontaneous or angioplasty-induced plaque rupture is one factor determining whether an unstable plaque proceeds rapidly to an occlusive thrombus or persists as a nonocclusive mural thrombus. Although observational data show that plaque rupture is a potent stimulus for thrombosis, and exposed collagen is suggested to have a predominant role in thrombosis, the relative thrombogenicity of different components of human atherosclerotic plaques is not well established. METHODS We investigated thrombus formation on foam cell-rich matrix (obtained from fatty streaks), collagen-rich matrix (from sclerotic plaques), collagen-poor matrix without cholesterol crystals (from fibrolipid plaques), atheromatous core with abundant cholesterol crystals (from atheromatous plaques) and segments of normal intima derived from human aortas at necropsy. Specimens were mounted in a tubular chamber placed within an ex vivo extracorporeal perfusion system and exposed to heparinized porcine blood (mean [+/- SEM] activated partial thromboplastin time ratio 1.5 +/- 0.04) for 5 min under high shear rate conditions (1,690 s-1). Thrombus was quantitated by measurement of indium-labeled platelets and morphometric analysis. Under similar conditions, substrates were perfused with heparinized human blood (2 IU/ml) in an in vitro system, and thrombus formation was similarly evaluated. RESULTS Thrombus formation on atheromatous core was up to sixfold greater than that on other substrates, including collagen-rich matrix (p = 0.0001) in both heterologous and homologous systems. Although the atheromatous core had a more irregular exposed surface and thrombus formation tended to increase with increasing roughness, the atheromatous core remained the most thrombogenic substrate when the substrates were normalized by the degree of irregularity as defined by the roughness index (p = 0.002). CONCLUSIONS The atheromatous core is the most thrombogenic component of human atherosclerotic plaques. Therefore, plaques with a large atheromatous core content are at high risk of leading to acute coronary syndromes after spontaneous or mechanically induced rupture because of the increased thrombogenicity of their content.

Plaque Neovascularization Is Increased in Ruptured Atherosclerotic Lesions of Human Aorta Implications for Plaque Vulnerability

Background—Growth of atherosclerotic plaques is accompanied by neovascularization from vasa vasorum microvessels extending through the tunica media into the base of the plaque and by lumen-derived microvessels through the fibrous cap. Microvessels are associated with plaque hemorrhage and may play a role in plaque rupture. Accordingly, we tested this hypothesis by investigating whether microvessels in the tunica media, the base of the plaque, and the fibrous cap are increased in ruptured atherosclerotic plaques in human aorta. Methods and Results—Microvessels, defined as CD34-positive tubuloluminal capillaries recognized in cross-sectional and longitudinal profiles, were quantified in 269 advanced human plaques by bicolor immunohistochemistry. Macrophages/T lymphocytes and smooth muscle cells were defined as CD68/CD3-positive and &agr;-actin–positive cells. Total microvessel density was increased in ruptured plaques when compared with nonruptured plaques (P=0.0001). Furthermore, microvessel density was increased in lesions with severe macrophage infiltration at the fibrous cap (P=0.0001) and at the shoulders of the plaque (P=0.0001). In addition, microvessel density was also increased in lesions with intraplaque hemorrhage (P=0.04) and in thin-cap fibroatheromas (P=0.038). Logistic regression analysis identified plaque base microvessel density (P=0.003) as an independent correlate to plaque rupture. Conclusions—Thus, neovascularization as manifested by the localized appearance of microvessels is increased in ruptured plaques in the human aorta. Furthermore, microvessel density is increased in lesions with inflammation, with intraplaque hemorrhage, and in thin-cap fibroatheromas. Microvessels at the base of the plaque are independently correlated with plaque rupture, suggesting a contributory role for neovascularization in the process of plaque rupture.

Rapamycin inhibits vascular smooth muscle cell migration.

Abnormal vascular smooth muscle cell (SMC) proliferation and migration contribute to the development of restenosis after percutaneous transluminal coronary angioplasty and accelerated arteriopathy after cardiac transplantation. Previously, we reported that the macrolide antibiotic rapamycin, but not the related compound FK506, inhibits both human and rat aortic SMC proliferation in vitro by inhibiting cell cycle-dependent kinases and delaying phosphorylation of retinoblastoma protein (Marx, S.O., T. Jayaraman, L.O. Go, and A.R. Marks. 1995. Circ. Res. 362:801). In the present study the effects of rapamycin on SMC migration were assayed in vitro using a modified Boyden chamber and in vivo using a porcine aortic SMC explant model. Pretreatment with rapamycin (2 ng/ml) for 48 h inhibited PDGF-induced migration (PDGF BB homodimer; 20 ng/ml) in cultured rat and human SMC (n = 10; P < 0.0001), whereas FK506 had no significant effect on migration. Rapamycin administered orally (1 mg/kg per d for 7 d) significantly inhibited porcine aortic SMC migration compared with control (n = 15; P < 0.0001). Thus, in addition to being a potent immunosuppressant and antiproliferative, rapamycin also inhibits SMC migration.

Effects of Lipid-Lowering by Simvastatin on Human Atherosclerotic Lesions A Longitudinal Study by High-Resolution, Noninvasive Magnetic Resonance Imaging

Background—This study was designed to investigate the effects of lipid-lowering by simvastatin on human atherosclerotic lesions. Methods and Results—Eighteen asymptomatic hypercholesterolemic patients with documented aortic and/or carotid atherosclerotic plaques were selected for the study. A total of 35 aortic and 25 carotid artery plaques were detected. Serial black-blood MRI of the aorta and carotid artery of the patients was performed at baseline and 6 and 12 months after lipid-lowering therapy with simvastatin. The effects of the treatment on atherosclerotic lesions were measured as changes in lumen area, vessel wall thickness, and vessel wall area, a surrogate of atherosclerotic burden. Simvastatin induced a significant (P <0.01) reduction in total and LDL cholesterol levels at 6 weeks that was maintained thereafter. At 6 months, no changes in lumen area, vessel wall thickness, or vessel wall area were observed. However, at 12 months, significant reductions in vessel wall thickness and vessel wall area, without changes in lumen area, were observed in both aortic and carotid arteries (P <0.001). Conclusions—This in vivo human study demonstrates that effective and maintained lipid-lowering therapy by simvastatin is associated with a significant regression of atherosclerotic lesions. Our observation suggests that statins induce vascular remodeling, as manifested by reduced atherosclerotic burden without changes in the lumen.

Inhibition of Intimal Thickening After Balloon Angioplasty in Porcine Coronary Arteries by Targeting Regulators of the Cell Cycle

BACKGROUND Although percutaneous transluminal coronary angioplasty (PTCA) is a highly effective procedure to reduce the severity of stenotic coronary atherosclerotic disease, its long-term success is significantly limited by the high rate of restenosis. Several cellular and molecular mechanisms have been implicated in the development of restenosis post-PTCA, including vascular smooth muscle cell (VSMC) activation, migration, and proliferation. Recently, our group demonstrated that rapamycin, an immunosuppressant agent with antiproliferative properties, inhibits both rat and human VSMC proliferation and migration in vitro. In the present study, we investigated (1) whether rapamycin administration could reduce neointimal thickening in a porcine model of restenosis post-PTCA and (2) the mechanism by which rapamycin inhibits VSMCs in vivo. METHODS AND RESULTS PTCA was performed on a porcine model at a balloon/vessel ratio of 1.7+/-0.2. Coronary arteries were analyzed for neointimal formation 4 weeks after PTCA. Intramuscular administration of rapamycin started 3 days before PTCA at a dose of 0.5 mg/kg and continued for 14 days at a dose of 0.25 mg/kg. Cyclin-dependent kinase inhibitor (CDKI) p27(kip1) protein levels and pRb phosphorylation within the vessel wall were determined by immunoblot analysis. PTCA in the control group was associated with the development of significant luminal stenosis 4 weeks after the coronary intervention. Luminal narrowing was a consequence of significant neointimal formation in the injured areas. Rapamycin administration was associated with a significant inhibition in coronary stenosis (63+/-3.4% versus 36+/-4.5%; P<0.001), resulting in a concomitant increase in luminal area (1.74+/-0.1 mm2 versus 3. 3+/-0.4 mm2; P<0.001) after PTCA. Inhibition of proliferation was associated with markedly increased concentrations of the p27(kip1) levels and inhibition of pRb phosphorylation within the vessel wall. CONCLUSIONS Rapamycin administration significantly reduced the arterial proliferative response after PTCA in the pig by increasing the level of the CDKI p27(kip1) and inhibition of the pRb phosphorylation within the vessel wall. Therefore, pharmacological interventions that elevate CDKI in the vessel wall and target cyclin-dependent kinase activity may have a therapeutic role in the treatment of restenosis after angioplasty in humans.

In Vivo Magnetic Resonance Evaluation of Atherosclerotic Plaques in the Human Thoracic Aorta A Comparison With Transesophageal Echocardiography

BACKGROUND The structure and composition of aortic atherosclerotic plaques are associated with the risk of future cardiovascular events. Magnetic resonance (MR) imaging may allow accurate visualization and characterization of aortic plaques. METHODS AND RESULTS We developed a noninvasive MR method, free of motion and blood flow artifacts, for submillimeter imaging of the thoracic aortic wall. MR imaging was performed on a clinical MR system in 10 patients with aortic plaques identified by transesophageal echocardiography (TEE). Plaque composition, extent, and size were assessed from T1-, proton density-, and T2- weighted images. Comparison of 25 matched MR and TEE cross-sectional aortic plaque images showed a strong correlation for plaque composition (chi(2) = 43.5, P<0.0001; 80% overall agreement; n = 25) and mean maximum plaque thickness (r = 0.88, n = 25; 4.56+/-0.21 mm by MR and 4.62+/-0.31 mm by TEE). Overall aortic plaque extent as assessed by TEE and MR was also statistically significant (chi(2) = 61.77, P<0.0001; 80% overall agreement; n = 30 regions). CONCLUSIONS This study demonstrates that noninvasive MR evaluation of the aorta compares well with TEE imaging for the assessment of atherosclerotic plaque thickness, extent, and composition. This MR method may prove useful for the in vivo study of aortic atherosclerosis.

Thrombus formation on atherosclerotic plaques: pathogenesis and clinical consequences.

Ischemic heart disease is the most common cause of death worldwide (1). Thrombi that form on atherosclerotic lesions in coronaries are responsible for myocardial ischemia and progression of atherosclerosis (2-5). Recent pathologic, experimental, and clinical findings have led to a better understanding of the cellular and molecular mechanisms underlying thrombus formation on atherosclerotic plaques. In this review, we describe pathophysiologic mechanisms that lead to arterial thrombosis, the clinical impact of thrombosis on arterial lesions, and new therapeutic developments. With regard to the impact of arterial thrombosis on human mortality, we focus on the thrombotic process in atherosclerotic disease. Recent reviews have summarized the pathophysiologic and clinical aspects of the vulnerable plaque in the development of atherosclerotic lesions (6-9). Methods We searched MEDLINE for English-language reports on thrombosis and atherosclerosis published from 1966 to the present. Experimental, clinical, and epidemiologic studies related to the pathogenesis and pathophysiology of thrombosis on atherosclerotic lesions were reviewed, and references from identified articles were also selected. Abstracts on new aspects of therapeutic options that were presented at recent international meetings of the International Society of Thrombosis and Haemostasis and the American Heart Association were selected. Therapeutic approaches were derived from experimental studies and large clinical investigations. Pathophysiology and Clinical Impact of Thrombus Formation on Atherosclerotic Lesions The Virchow Triad As described by Virchow more than 100 years ago, occurrence of arterial thrombosis depends on the arterial vessel wall substrates, the local rheologic characteristics of blood flow, and systemic factors in the circulating blood (Table 1) (2, 3, 8, 10, 11). Table 1. The Virchow Triad Local Substrates for Thrombosis: Atherosclerotic Plaques Vulnerable atherosclerotic plaques may cause most acute coronary syndromes (12, 13). Atherosclerotic lesions are found in most major arteries, including the aorta, carotid, iliofemoral, and medium-sized arteries (such as the coronaries) (13). Focal intimal lesions may first develop in the human fetus before birth (14). Autopsy studies in persons without clinical cardiovascular disease showed that intimal alterations occur in the different vascular beds within the first 15 to 20 years of life (15-17). Using histologic characteristics, the American Heart Association has developed a standardized classification of distinct plaque types (Figure 1). Advanced atheromatous lesions are the substrates for arterial thrombosis. In advanced atheromatous lesions, the lipid core of the plaque contains pultaceous debris, apoptotic cells (such as dead macrophages and smooth-muscle cells), mesenchymal cells, and abundant free cholesterol crystals (fatty gruel) (18). The lipid core of these type IV and V lesions is rich in tissue factor, which, upon plaque rupture and exposure to the circulating blood, initiates the coagulation cascade and thrombin generation (Figure 2) (19). Smoking increases tissue factor expression in atherosclerotic plaques. Tissue factor is associated with and probably generated by activated macrophages within the plaque. The degree of plaque disruption (erosion, fissure, or ulceration) and the amount of stenosis caused by the disrupted plaque and the overlying mural thrombus are key factors for determining thrombogenicity at the local arterial site. When deep ulceration occurs, tissue factor from the atherosclerotic lipid core is exposed to flowing blood and released into the lumen (20, 21). Tissue factor interacts with factor VII and subsequently activates factor X, which leads to conversion of prothrombin to thrombin in the prothrombinase complex (Figure 2) (22, 23). The high tissue factor activity contributes to the procoagulant activity of disrupted atherosclerotic lesions and the superimposed mural thrombi (20). Disruption of advanced plaques with exposure of the highly thrombotic lipid core to the flowing blood triggers the formation of thrombi up to 6 times larger than thrombi generated by exposure of other components of the arterial wall (18). Mural thrombus formation may contribute to arterial stenosis, release vasoconstrictors from platelets, and cause ischemic symptoms (6, 8, 24, 25). The vulnerability of a plaque includes the pathoanatomic features that are related to size of the lipid pool, thickness of the fibrous cap, content and metabolic activity of lipids (26, 27), activity and density of macrophages (28, 29), and matrix metalloproteinases (30, 31). The external physical forces that expose the vessel wall to blood flow at different shear rates also influence the occurrence and progression of plaque disruption, thrombosis, and arteriosclerosis (32-35). Figure 1. Relation of lesion morphologic characteristics and phases of progression of coronary atherosclerosis to clinical findings. Figure 2. The tissue factor pathway activation of coagulation. Blood Rheology and Thrombus Formation Acute changes in rheologic characteristics induced by vasoconstriction, plaque disruption, and thrombus formation induce changes in the shear rate of flowing blood. Thrombus formation increases with increasing shear force (32). Shear force is directly related to flow velocity and inversely related to the third power of the lumen diameter. Thus, acute platelet deposition after plaque disruption depends on arterial size and the geometric changes and degree of narrowing after disruption. Changes in geometry may increase platelet deposition, whereas a sudden protrusion of plaque contents or growth of thrombus at the injury site may create severe stenosis and thrombotic occlusion. Most platelets are deposited at the apex of a stenosis, which is the site of maximal shear force (32, 36). Mechanical forces associated with blood flow influence the vascular tone, arterial structure, and location of arterial lesions (32, 36). Thrombin causes vasoconstriction when endothelium is absent or dysfunctional. Arterial vasoconstriction increases the shear force. Systemic risk factors for atherosclerosis, such as smoking, hyperlipidemia, or diabetes mellitus, may cause endothelial dysfunction, promote vasoconstriction, and increase shear force and platelet deposition. Contribution of Systemic Risk Factors to Thrombosis Systemic factors, including changes in lipid and hormonal metabolism, hyperglycemia, hemostasis, fibrinolysis, and platelet and leukocyte function, are associated with increased blood thrombogenicity or a systemic hypercoagulable state (37). Approximately one third of patients with acute myocardial infarction and coronary thrombosis have plaque erosions that occurred on nonruptured, moderately stenotic plaques. The patients who developed coronary thrombosis on erosions had systemic risk factors associated with a hypercoagulable state (38). Lipoprotein(a), a known risk factor for coronary heart disease, has a structure similar to that of plasminogen and may reduce plasmin formation and impair thrombolysis (39). Elevated low-density lipoprotein cholesterol levels increase blood thrombogenicity (40) and growth of thrombus under defined rheologic conditions (41). Reducing low-density lipoprotein cholesterol levels using statins decreased thrombus growth by approximately 20% (41). The reduction of total vascular events, including death, coronary events, and stroke, by lipid-lowering therapy with statins was documented in several large prospective clinical trials (42-48). Reduced low-density lipoprotein cholesterol levels may also decrease vasoconstriction and the size of the lipid core (41). High plasma levels of cathecholamines potentiate platelet activation, enhance vasospasm, and increase the incidence of sudden death after emotional and physical stress in patients with acute cardiovascular events. Smoking increases catecholamine release, causes endothelial dysfunction, and is associated with increased levels of fibrinogen (49). Increased plasma fibrinogen level is an independent risk factor for complications in patients with atherosclerotic disease (50-52). Fibrinogen, factor VIIIc, and von Willebrand factor were shown to be positively related to C-reactive protein (53). C-reactive protein, like fibrinogen, is a protein of the acute-phase response and a sensitive marker of low-grade inflammation. Increased levels of C-reactive protein have been reported to predict acute coronary events (54-56). C-reactive protein seems to be a useful marker for predicting risk for thrombotic events. Whether C-reactive protein reflects only the extent of the acute-phase reaction in response to nonspecific events, such as myocardial ischemia or atherosclerosis, or whether it may directly participate in the process of thrombus formation at the site of the atherosclerotic vessel is not known (56). Diabetic patients, especially those whose diabetes is poorly controlled, have increased blood thrombogenicity, due in part to glycosylation of collagen and proteins and increased levels of plasma fibrinogen and plasminogen activator inhibitor-1 (57-62). Platelets from patients with diabetes have increased reactivity and hyperaggregability and expose a variety of activation-dependent adhesion proteins (63, 64). Abnormal platelet function is reflected by increased platelet consumption and prolonged accumulation of thrombocytes on the altered vessel wall (64-66). In addition, more leukocyteplatelet aggregates circulate in the blood of patients with diabetes and diabetic vasculopathy (64). The prothrombotic state in diabetes is also associated with increased expression of monocyte procoagulant activity in the presence of diabetic microalbuminuria (67). Increased procoagulant activity in diabetes is attributed to leukocytes (64, 65, 67), which may in part activate the tissue factor pathway (68) and contribute to the high blood thro