Morphologic Findings of Coronary Atherosclerotic Plaques in Diabetics
http://www.100md.com
《动脉硬化血栓血管生物学》
Allen P. Burke; Frank D. Kolodgie; Arthur Zieske; David R. Fowler; Deena K. Weber; P. Jacob Varghese; Andrew Farb; Renu Virmani
From the Department of Cardiovascular Pathology (A.P.B., F.D.K., D.K.W., A.F., R.V.), Armed Forces Institute of Pathology, Washington, DC; Louisiana State University Health Science Center (A.Z.), New Orleans, La; the University of Maryland (D.R.F.), Baltimore, Md; and the Department of Cardiology (J.P.V.), George Washington University Medical Center, Washington, DC.
ABSTRACT
Objective— Coronary atherosclerotic plaque composition of diabetic subjects and localization of receptor for advanced glycation end products (RAGE) and its ligands have not been extensively studied.
Methods and Results— Hearts from diabetic subjects and age, race, and sex-matched nondiabetic subjects dying suddenly were examined. Coronary arteries were dissected and lesions were evaluated for plaque burden, necrotic core size, and inflammatory infiltrate. The expression of RAGE, the RAGE-binding protein (S100-A12, EN-RAGE), and cell death (apoptosis) were also determined. Lesions from type II diabetic subjects had larger mean necrotic cores (P=0.01) and greater total and distal plaque load (P<0.001) than nondiabetic subjects. Necrotic core size correlated positively with diabetic status, independent of other risk factors. Intimal staining for macrophages, T-cells, and HLA-DR was also significantly greater in diabetic subjects (P=0.03, P=0.003, and P<0.0001), respectively. The association of increased macrophage infiltrate was independent of cholesterol levels and patient age. Expression of RAGE and EN-RAGE was significantly greater in diabetic subjects (P=0.004) and was associated with apoptotic smooth muscle cells and macrophages.
Conclusions— In sudden coronary death, inflammation and necrotic core size play a greater role in the progression of atherosclerosis in diabetic subjects. The expression of RAGE and EN-RAGE may further compromise cell survival and promote plaque destabilization.
In sudden coronary death, there is a prominent role of plaque-infiltrating macrophages and T cells in the progression of atherosclerosis in diabetic subjects. The expression of RAGE (receptor for advanced glycation end products) and the binding protein (S100 A12, EN-RAGE) may further compromise cell survival and promote plaque destabilization.
Key Words: diabetes mellitus ? sudden death ? atherosclerosis ? receptor for advanced glycation end products ? S100A12
Introduction
Diabetic patients experience increased morbidity and cardiovascular complications as compared with nondiabetic subjects.1,2 An angioscopic study demonstrated a higher rate of thrombosis in unstable angina patients with diabetes,3 and intravascular ultrasound has suggested decreased adaptive remodeling.4 Few detailed pathologic studies comparing the characteristics of atherosclerotic plaques in diabetic and nondiabetic patients are available. As demonstrated in early postmortem studies, diabetes and glucose intolerance are associated with premature atherosclerosis.5 Whether morphological characteristics of advanced symptomatic coronary plaques differ between diabetic and nondiabetic subjects is not clear.
Advanced glycation end products (AGEs) have been implicated in the structural and functional alterations of proteins that occur during aging and long-term hyperglycemia.6 Serum AGEs have been correlated with renal damage in diabetic subjects.7 The various AGEs bind RAGE (a receptor of advanced glycation end products),8 which is upregulated in diabetic glomeruli9 and associated with microangiopathic disease.10 RAGE expression can be demonstrated by immunohistochemical techniques11 and is present in atherosclerotic carotid plaques of diabetic and nondiabetic subjects.12 Further, RAGE also binds the non-AGEs (nonadvanced glycation end products), particularly S100A12, termed extracellular newly identified RAGE-binding protein (EN-RAGE), which may further promote inflammation within the plaque.13
The characteristics of coronary artery plaques in autopsy samples of diabetic subjects dying with severe coronary disease were compared with lesions from nondiabetic subjects emphasizing the cellular components. The expression of RAGE, EN-RAGE, and incidence of apoptosis was also correlated with plaque morphology to investigate potential mechanisms of inflammation and cell death.
Methods
Sudden coronary deaths with acute coronary thrombi or severe atherosclerosis in the absence of noncoronary causes of death or previous surgery were studied.14–18 From a series of 270 hearts, 66 cases were selected based on a history of type I diabetes mellitus treated with insulin or the presence of type II diabetes. A previous history of oral hypoglycemics or a postmortem glycohemoglobin value of >10% was used to document type II diabetes. Age, race, and gender-matched controls (n=66) were then selected from the remaining 204 cases based on normal postmortem glycohemoglobin (<8%) and known medical history, which excluded type I or treated type II diabetes. Epicardial arteries were serially sectioned at 3- to 4-mm intervals, and all areas of 50% cross-sectional luminal narrowing were studied histologically. Culprit plaques with acute thrombi from ruptures and erosions were evaluated (only 7 erosions from diabetic patients were identified and were therefore excluded from the analysis of inflammation).
Morphometric assessment of lipid core size, calcified matrix area, plaque area, and IEL area were assessed in each segment as previously described16 using the area of greatest maximal luminal narrowing of the 4 major coronary branches. To assess remodeling, internal elastic lamina (IEL) area was adjusted for distance from the coronary ostium by a tapering formula used previously.19 Fibrous cap atheroma, thin cap atheroma (traditional vulnerable plaque), and healed plaque ruptures were identified.20
Heart weight was corrected for body weight.15 A healed infarct was defined as an area of scarring 1 cm in greatest diameter. Plaque burden was calculated by adding the maximal percent cross-sectional area luminal narrowing in 4 arterial beds (range 0 to 400) as reported;15 a similar number was obtained for distal arteries. Risk factors (smoking, glycohemoglobin, total cholesterol and high-density lipoprotein cholesterol) were determined based on analysis of postmortem blood and sera, as well as history and renal histologic findings.5,14,15,18 Hypercholesterolemia was determined as the presence of either TC >200 mg/dL, or TC/HDL cholesterol ratio of >5.
Immunohistochemical stains included the macrophage marker CD68 (KP-1 clone; Dako, Carpinteria, Calif), anti-RAGE (Research Diagnostics Inc, Flanders, NJ), T cells (CD45RO), and anti-HLA-DR. DNA fragmentation staining for the identification of apoptosis was performed using terminal deoxyribonucleotide transferase (TdT)-mediated nick end-labeling (TACS; Trevigen, Gaithersburg, Md).21
Quantification of apoptotic nuclei involved a 500-μm zone surrounding necrotic cores. Double labeling against RAGE and apoptosis was performed on selected sections. Macrophage density was determined by computerized planimetry. The extent of HLA-DR reactivity was semi-quantified by light microscopy and is expressed as a percentage of the total amount of plaque area. The extent of T cells was scored as 0 to 3+: 0=absent; 1=up to 20 cells; 2=21 to 50 cells; and 3=>50 cells per section. RAGE staining was assessed semi-quantitatively on a 1 to 4+ score in various plaque components. All lesions were evaluated with the observer (A.P.B. or R.V.) blinded to diabetic status.
Rabbit polyclonal antiserum against S100A12 (EN-RAGE) generously provided by Philippe A. Tessier (Laboratoire d’infectiologie, Université Laval, Québec, Canada) was also used to stain selected cryosections of coronary plaques. Double labeling studies with either macrophage or smooth muscle cell markers and S100A12 were performed by immunofluorescence staining with the corresponding secondary antibodies labeled with AlexaFluor488 or AlexaFluor594 (Molecular Probes, Eugene, Ore). Nuclei were counterstained with DAPI and antibody fluorescence was observed using a Leica TCS SP2 spectral confocal imaging system (Leica, Heidelberg, Germany).
Statistical Analysis
Means were compared among the 3 groups by means table ANOVA with Fisher’s post-hoc test, with the exception of ISEL counts, which were not normally distributed. These were compared using the Mann-Whitney U test for nonpaired nonparametric data. Log-normalized data were used for nonparametric distributions of morphometric data (percent calcified area, macrophage area, and necrotic core area). The multivariate relationship between mean percent necrotic core and risk factors (glycohemoglobin, TC, HDL cholesterol, smoking, and age) was determined by multiple regression as was the independent effects of plaque area and presence of diabetes on IEL area. Multivariate analysis for the effect of plaque size and diabetes group (type I and type II versus nondiabetic) on IEL was performed using MANCOVA. Tests were performed using Statview software (SAS Institute).
Results
Demographic Data and Risk Factors
The numbers of hearts in each group are given in Table 1. The mean age and proportion of blacks and women were similar in all groups; races other than white and black were not represented. The mean body mass index was significantly greater in the type II diabetic group than in other groups.
TABLE 1. Risk Factors and Cardiac Findings
The ratio of TC to HDL lipoprotein cholesterol was significantly higher in type II diabetic subjects compared with nondiabetic subjects. Acute thrombi were relatively uncommon in type I diabetic subjects (Table 1); in type II diabetic subjects, the proportion of thrombi was similar to that of nondiabetic subjects. Mean heart weight was significantly greater in type II diabetes, along with the incidence of healed infarcts.
Plaque Characteristics
The mean percent plaque area composed of necrotic core was greater in type I (P=0.05) and type II (P=0.004) diabetic subjects compared with nondiabetic subjects. Macrophage plaque area and T-cell infiltration were also significantly greater in diabetic than nondiabetic patients (P=0.03), along with HLA-DR expression (Figure 1, and please see Figure I, available online at http://atvb.ahajournals.org). The mean percent calcified area was greatest in type II diabetic subjects and smallest in type I diabetic subjects, but the differences were not significant (P0.1). The mean number of fibrous cap atheromas was greater in type II diabetic subjects as compared with nondiabetic subjects (Table 2, P=0.02), but there was no significant difference in the numbers of thin-cap atheromas. The numbers of healed plaque ruptures were greatest in type I and II diabetic subjects. Plaque burden was increased in diabetic subjects, and distal burden increased in type II diabetic subjects versus controls.
Figure 1. Inflammation in diabetic coronary arteries. A, Coronary fibroatheromas illustrating the extent of macrophage (M) and T cells, (CD45RO) and HLA-DR expression in patients with type I and II diabetes mellitus (DM) and nondiabetic subjects.
TABLE 2. Plaque Characteristics in Type I and II Diabetic Subjects vs Nondiabetic Subjects
The multivariate effect of risk factors on 3 dependent morphological variables was studied separately (Table 3). There was a positive correlation between necrotic core size and glycohemoglobin, independent of HDL lipoprotein cholesterol, TC/HDL cholesterol ratio, age, smoking, and gender (P=0.005, t=2.8). There was a similar correlation with body mass index. A strong correlation between macrophage area and glycohemoglobin (P=0.0004, T=2.9, Table 3) was observed. There was also a stronger relationship between numbers of fibrous cap atheromas and TC/HDL cholesterol than with other risk factors, including glycohemoglobin.
TABLE 3. Relationship of Risk Factors, Including Diabetes, to Plaque Characteristics in a Multivariate Analysis
Combined Effect of Hypercholesterolemia and Diabetes on Necrotic Core Size and Macrophage Content
Patients were divided as follows: normoglycemic patients or diabetic patients (I or II) with or without hyperlipidemia. By univariate analysis, the degree of macrophage infiltrate and necrotic core size as assessed by morphometry was significantly higher in diabetic patients with normal cholesterol or hyperlipidemia compared with nondiabetic patients (Figure 2).
Figure 2. Combined effect of hyperlipidemia (hyperlipidemia defined as total cholesterol >200 mg/dL or TC/HDL cholesterol ratio >5) and diabetes on mean macrophage percent (log-normalized, left) and mean necrotic core size (log-normalized, right). There is a significant difference between diabetic patients (DM) and nondiabetic patients (non-DM) when cases were separated by the presence or absence of hyperlipidemia.
Evidence of Remodeling
The mean IEL (adjusted for distance from coronary ostium) was greater in type I and II diabetic subjects compared with nondiabetic subjects (18.2±6.6 mm2, 16.5±4.4 mm2, 16.0±4.5 mm2, respectively). The mean IEL was also significantly greater in type I (P=0.001) and II diabetic subjects (P=0.01). By multivariable analysis, there was a correlation between type I diabetes and IEL area independent of heart weight, percent necrotic core, plaque area, and percent plaque calcification (P=0.0004). This analysis (percent necrotic core, P=0.05; plaque area, P<0.0001; heart weight, P=0.05) was positively correlated with IEL area.
Immunolocalization of RAGE and EN-RAGE
Although RAGE was found localized to macrophages, smooth muscle cells, erythrocytes, and endothelial cells in both diabetic and nondiabetic subjects, the overall expression of RAGE, as graded semi-quantitatively, was significantly greater in diabetic subjects (16.8±6.2 for diabetic subjects, 10.0±6.1 for nondiabetic subjects, P=0.004) (please see Figure II, available online at http://atvb.ahajournals.org). Extensive RAGE staining was present in macrophages around and within necrotic cores of both diabetic and nondiabetic subjects, with its overall expression dependent on extent of cellular infiltrate. Fewer smooth muscle cells stained with RAGE, but the intensity was greater in diabetic subjects. RAGE expression was often associated with apoptotic macrophages and smooth muscle cells (please see Figure III, available online at http://atvb.ahajournals.org). EN-RAGE expression in diabetic patients was most prominent in macrophages and, to a lesser degree, in smooth muscle cells in the regions surrounding the necrotic core (Figure 3).
Figure 3. Expression of S100A12/EN-RAGE (a RAGE-binding protein) in human coronary lesions from sudden coronary death victims with diabetes (A–E) and without diabetes (F–J). A, Lesion from the proximal left circumflex coronary artery stained by Movat pentachrome. B to E, High magnification micrographs of the black box represented in (A). B, HHF-35 staining for smooth muscle cells (SMCs) in the shoulder region of the plaque. C, Adjacent section stained for CD68, demonstrating numerous inflammatory macrophages. D, The same region demonstrated intense staining for EN-RAGE. B to D, The counterstain is Gill’s hematoxylin. E, Double-labeled immunofluorescent staining of CD68-positive macrophages (green) and EN-RAGE (red); nuclei (blue) were counterstained with DAPI; areas of overlap appear as yellow–green. EN-RAGE was primarily found in macrophage-rich areas. F, A lesion from the proximal left anterior descending coronary artery from a nondiabetic patient stained by Movat pentachrome. G to I, Immunoreactivity to SMC, macrophage, and EN-RAGE, respectively. Overall, macrophage infiltrate was significantly less in nondiabetic patients, corresponding to less immunoreactivity to EN-RAGE. J, Double staining for EN-RAGE and macrophages.
ISEL Quantification
The density of apoptotic-staining cells surrounding necrotic cores was greater in diabetic (26.9 nuclei/mm2) versus nondiabetic subjects (6.5 nuclei/mm2, P=0.002; please see online Table I, www.ahajounals.org). The highest density was present in type I diabetic subjects (P=0.02 versus nondiabetic subjects), followed by type II diabetic subjects (P=0.04 versus controls).
Discussion
Sudden cardiac death in type II diabetic subjects in the absence of known previous symptoms is associated with extensive atherosclerosis compared with nondiabetic subjects, including distal coronary involvement. The increase in plaque burden is partly related to the observation of a greater number of healed ruptures in type II diabetic subjects. Further, healed myocardial infarction and cardiomegaly are also more frequent in type II diabetic subjects, without differences in the incidence of acute thrombosis. An additive effect of hypercholesterolemia on necrotic core size and macrophage infiltrate was apparent for individuals with type I and II diabetes together with T-cell infiltration and HLA-DR expression. The degree of RAGE expression and greater frequency of apoptosis in smooth muscle cells and macrophages together with increased expression of EN-RAGE (a known chemokine) may explain the severity of macrophage infiltration and larger necrotic cores in diabetic patients.
Total and distal plaque burden was significantly greater in type II diabetic compared with nondiabetic subjects, whereas only increased total plaque was evident in type I diabetic subjects. Although the precise cause of diffuse coronary disease in type II diabetic subjects is unclear, the extent of thrombus formation may be involved. Recent studies suggest that relative amount of thrombus may be dependent of the expression of plasminogen activator inhibitor-1 (PAI-1). Notably, individuals with type II diabetes show elevated levels of PAI- I22; in contrast, PAI-1 levels in type I diabetes are normal.23
An increased frequency of healed myocardial infarction and cardiomegaly suggests that the mechanism of sudden death may differ between diabetic and nondiabetic patients. Specifically, the high rate of healed infarcts suggests a propensity for arrhythmia in type II diabetic subjects. Of importance are the relatively low rates of acute thrombosis and healed infarcts in patients with type I diabetes. These data support a potential role for nonanatomic factors, such as autonomic neuropathy, long QT syndrome, or hypoglycemia, in the precipitation of sudden death in type I diabetic patients.24
Clinical studies have shown that diabetes is associated with positive25 or negative/absence of remodeling.26 The current study supports the notion that diabetic subjects are more likely to show positive remodeling. These data are consistent with previous findings from our laboratory that necrotic core and macrophage infiltrates are associated with expansion of the internal elastic lamina independent of plaque size.16
The inflammatory response was greater in diabetic than in nondiabetic plaques. However, T cells were only significantly greater for type I diabetic subjects. The fact that type I diabetes is an autoimmune disease with a common genetic susceptibility to other disorders like autoimmune thyroiditis may also be of pathophysiologic significance in coronary plaques.27
The result of increased numbers of RAGE-expressing smooth muscle cells and macrophages in plaques of diabetic subjects rather than in nondiabetic subjects provides a link between RAGE and apoptosis in coronary atherosclerosis. An association between RAGE-expressing macrophages and upregulation of inflammatory NF-kappa-?, COX-2/mPGES-1, and matrix metalloproteinases has been shown in carotid arteries of diabetic patients.28,29 The current study demonstrates an increase in apoptotic macrophages and smooth muscle cells in coronary arteries of diabetic patients, which may be related to necrotic core expansion, thinning of the fibrous cap, and plaque instability. Experimental studies of murine models of diabetic aortic atherosclerosis have demonstrated that RAGE blockade by soluble RAGE decreases atheroma formation and the development of less complex lesions.30
Although the interactions between EN-RAGE–modulated proteins and RAGE receptors in the fine regulation of leukocyte trafficking and proliferation have been recently reviewed,31 this mechanism in atherosclerosis-related inflammation has not been extensively studied. Several reports suggest that EN-RAGE is chemotactic and, in the nanomolar range, stimulates the migration of neutrophils, monocytes, and macrophages.32,33 Moreover, the increase in EN-RAGE protein is sensitive to pioglitazone and thiazolidinedione, suggesting the involvement of the peroxisome proliferator-activated receptor-. Thus, the role of RAGE/EN-RAGE upregulation in the atherosclerotic plaque is likely complex and may be linked with recruitment, apoptosis, and macrophage cell death.
Lesion calcification in the present study was more prevalent in patients with type II diabetes in comparison to nondiabetic subjects. Although the mechanisms of calcification are undoubtedly complex, a greater incidence of intimal cell death in coronary lesions from diabetic subjects may be responsible for this increase. In contrast, type I diabetes is an immune-mediated disease and in some respects is similar to transplant vasculopathy, in which arterial lesions show little or no calcification. Although coronary calcification measured with electron beam computed tomography in nondiabetic patients is greater in males than in females, this relationship is lost in patients with type I diabetes, of whom woman have a higher prevalence of calcification than men.34,35
Limitations
As an autopsy study, there is an inherent bias restricting cases to those of sudden unexpected death. Given the high rate of sudden death as a presenting symptom of coronary artery disease, however, the findings are applicable to a relatively large proportion of acute coronary syndromes. Another limitation is the classification of diabetic subjects based on postmortem data instead of clinical history. The use of glycohemoglobin as a tool for classification of diabetes has been accepted, however, in several studies.5,18
Conclusion
Diabetic atherosclerotic plaques have a higher content of inflammatory cell infiltrate and HLA-DR expression than do nondiabetic atherosclerotic plaques. This may be associated with larger necrotic core size, and a greater extent of smooth muscle cell and macrophage apoptosis. These finding may be related to the greater expression of RAGE and EN-RAGE in diabetic plaques. Through better understanding of plaque morphology in diabetic subjects, it may be possible to design more effective treatment that would prevent the atherosclerotic complication of frequent plaque ruptures, diffuse disease, and healed infarction associated with premature death in diabetic subjects.
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From the Department of Cardiovascular Pathology (A.P.B., F.D.K., D.K.W., A.F., R.V.), Armed Forces Institute of Pathology, Washington, DC; Louisiana State University Health Science Center (A.Z.), New Orleans, La; the University of Maryland (D.R.F.), Baltimore, Md; and the Department of Cardiology (J.P.V.), George Washington University Medical Center, Washington, DC.
ABSTRACT
Objective— Coronary atherosclerotic plaque composition of diabetic subjects and localization of receptor for advanced glycation end products (RAGE) and its ligands have not been extensively studied.
Methods and Results— Hearts from diabetic subjects and age, race, and sex-matched nondiabetic subjects dying suddenly were examined. Coronary arteries were dissected and lesions were evaluated for plaque burden, necrotic core size, and inflammatory infiltrate. The expression of RAGE, the RAGE-binding protein (S100-A12, EN-RAGE), and cell death (apoptosis) were also determined. Lesions from type II diabetic subjects had larger mean necrotic cores (P=0.01) and greater total and distal plaque load (P<0.001) than nondiabetic subjects. Necrotic core size correlated positively with diabetic status, independent of other risk factors. Intimal staining for macrophages, T-cells, and HLA-DR was also significantly greater in diabetic subjects (P=0.03, P=0.003, and P<0.0001), respectively. The association of increased macrophage infiltrate was independent of cholesterol levels and patient age. Expression of RAGE and EN-RAGE was significantly greater in diabetic subjects (P=0.004) and was associated with apoptotic smooth muscle cells and macrophages.
Conclusions— In sudden coronary death, inflammation and necrotic core size play a greater role in the progression of atherosclerosis in diabetic subjects. The expression of RAGE and EN-RAGE may further compromise cell survival and promote plaque destabilization.
In sudden coronary death, there is a prominent role of plaque-infiltrating macrophages and T cells in the progression of atherosclerosis in diabetic subjects. The expression of RAGE (receptor for advanced glycation end products) and the binding protein (S100 A12, EN-RAGE) may further compromise cell survival and promote plaque destabilization.
Key Words: diabetes mellitus ? sudden death ? atherosclerosis ? receptor for advanced glycation end products ? S100A12
Introduction
Diabetic patients experience increased morbidity and cardiovascular complications as compared with nondiabetic subjects.1,2 An angioscopic study demonstrated a higher rate of thrombosis in unstable angina patients with diabetes,3 and intravascular ultrasound has suggested decreased adaptive remodeling.4 Few detailed pathologic studies comparing the characteristics of atherosclerotic plaques in diabetic and nondiabetic patients are available. As demonstrated in early postmortem studies, diabetes and glucose intolerance are associated with premature atherosclerosis.5 Whether morphological characteristics of advanced symptomatic coronary plaques differ between diabetic and nondiabetic subjects is not clear.
Advanced glycation end products (AGEs) have been implicated in the structural and functional alterations of proteins that occur during aging and long-term hyperglycemia.6 Serum AGEs have been correlated with renal damage in diabetic subjects.7 The various AGEs bind RAGE (a receptor of advanced glycation end products),8 which is upregulated in diabetic glomeruli9 and associated with microangiopathic disease.10 RAGE expression can be demonstrated by immunohistochemical techniques11 and is present in atherosclerotic carotid plaques of diabetic and nondiabetic subjects.12 Further, RAGE also binds the non-AGEs (nonadvanced glycation end products), particularly S100A12, termed extracellular newly identified RAGE-binding protein (EN-RAGE), which may further promote inflammation within the plaque.13
The characteristics of coronary artery plaques in autopsy samples of diabetic subjects dying with severe coronary disease were compared with lesions from nondiabetic subjects emphasizing the cellular components. The expression of RAGE, EN-RAGE, and incidence of apoptosis was also correlated with plaque morphology to investigate potential mechanisms of inflammation and cell death.
Methods
Sudden coronary deaths with acute coronary thrombi or severe atherosclerosis in the absence of noncoronary causes of death or previous surgery were studied.14–18 From a series of 270 hearts, 66 cases were selected based on a history of type I diabetes mellitus treated with insulin or the presence of type II diabetes. A previous history of oral hypoglycemics or a postmortem glycohemoglobin value of >10% was used to document type II diabetes. Age, race, and gender-matched controls (n=66) were then selected from the remaining 204 cases based on normal postmortem glycohemoglobin (<8%) and known medical history, which excluded type I or treated type II diabetes. Epicardial arteries were serially sectioned at 3- to 4-mm intervals, and all areas of 50% cross-sectional luminal narrowing were studied histologically. Culprit plaques with acute thrombi from ruptures and erosions were evaluated (only 7 erosions from diabetic patients were identified and were therefore excluded from the analysis of inflammation).
Morphometric assessment of lipid core size, calcified matrix area, plaque area, and IEL area were assessed in each segment as previously described16 using the area of greatest maximal luminal narrowing of the 4 major coronary branches. To assess remodeling, internal elastic lamina (IEL) area was adjusted for distance from the coronary ostium by a tapering formula used previously.19 Fibrous cap atheroma, thin cap atheroma (traditional vulnerable plaque), and healed plaque ruptures were identified.20
Heart weight was corrected for body weight.15 A healed infarct was defined as an area of scarring 1 cm in greatest diameter. Plaque burden was calculated by adding the maximal percent cross-sectional area luminal narrowing in 4 arterial beds (range 0 to 400) as reported;15 a similar number was obtained for distal arteries. Risk factors (smoking, glycohemoglobin, total cholesterol and high-density lipoprotein cholesterol) were determined based on analysis of postmortem blood and sera, as well as history and renal histologic findings.5,14,15,18 Hypercholesterolemia was determined as the presence of either TC >200 mg/dL, or TC/HDL cholesterol ratio of >5.
Immunohistochemical stains included the macrophage marker CD68 (KP-1 clone; Dako, Carpinteria, Calif), anti-RAGE (Research Diagnostics Inc, Flanders, NJ), T cells (CD45RO), and anti-HLA-DR. DNA fragmentation staining for the identification of apoptosis was performed using terminal deoxyribonucleotide transferase (TdT)-mediated nick end-labeling (TACS; Trevigen, Gaithersburg, Md).21
Quantification of apoptotic nuclei involved a 500-μm zone surrounding necrotic cores. Double labeling against RAGE and apoptosis was performed on selected sections. Macrophage density was determined by computerized planimetry. The extent of HLA-DR reactivity was semi-quantified by light microscopy and is expressed as a percentage of the total amount of plaque area. The extent of T cells was scored as 0 to 3+: 0=absent; 1=up to 20 cells; 2=21 to 50 cells; and 3=>50 cells per section. RAGE staining was assessed semi-quantitatively on a 1 to 4+ score in various plaque components. All lesions were evaluated with the observer (A.P.B. or R.V.) blinded to diabetic status.
Rabbit polyclonal antiserum against S100A12 (EN-RAGE) generously provided by Philippe A. Tessier (Laboratoire d’infectiologie, Université Laval, Québec, Canada) was also used to stain selected cryosections of coronary plaques. Double labeling studies with either macrophage or smooth muscle cell markers and S100A12 were performed by immunofluorescence staining with the corresponding secondary antibodies labeled with AlexaFluor488 or AlexaFluor594 (Molecular Probes, Eugene, Ore). Nuclei were counterstained with DAPI and antibody fluorescence was observed using a Leica TCS SP2 spectral confocal imaging system (Leica, Heidelberg, Germany).
Statistical Analysis
Means were compared among the 3 groups by means table ANOVA with Fisher’s post-hoc test, with the exception of ISEL counts, which were not normally distributed. These were compared using the Mann-Whitney U test for nonpaired nonparametric data. Log-normalized data were used for nonparametric distributions of morphometric data (percent calcified area, macrophage area, and necrotic core area). The multivariate relationship between mean percent necrotic core and risk factors (glycohemoglobin, TC, HDL cholesterol, smoking, and age) was determined by multiple regression as was the independent effects of plaque area and presence of diabetes on IEL area. Multivariate analysis for the effect of plaque size and diabetes group (type I and type II versus nondiabetic) on IEL was performed using MANCOVA. Tests were performed using Statview software (SAS Institute).
Results
Demographic Data and Risk Factors
The numbers of hearts in each group are given in Table 1. The mean age and proportion of blacks and women were similar in all groups; races other than white and black were not represented. The mean body mass index was significantly greater in the type II diabetic group than in other groups.
TABLE 1. Risk Factors and Cardiac Findings
The ratio of TC to HDL lipoprotein cholesterol was significantly higher in type II diabetic subjects compared with nondiabetic subjects. Acute thrombi were relatively uncommon in type I diabetic subjects (Table 1); in type II diabetic subjects, the proportion of thrombi was similar to that of nondiabetic subjects. Mean heart weight was significantly greater in type II diabetes, along with the incidence of healed infarcts.
Plaque Characteristics
The mean percent plaque area composed of necrotic core was greater in type I (P=0.05) and type II (P=0.004) diabetic subjects compared with nondiabetic subjects. Macrophage plaque area and T-cell infiltration were also significantly greater in diabetic than nondiabetic patients (P=0.03), along with HLA-DR expression (Figure 1, and please see Figure I, available online at http://atvb.ahajournals.org). The mean percent calcified area was greatest in type II diabetic subjects and smallest in type I diabetic subjects, but the differences were not significant (P0.1). The mean number of fibrous cap atheromas was greater in type II diabetic subjects as compared with nondiabetic subjects (Table 2, P=0.02), but there was no significant difference in the numbers of thin-cap atheromas. The numbers of healed plaque ruptures were greatest in type I and II diabetic subjects. Plaque burden was increased in diabetic subjects, and distal burden increased in type II diabetic subjects versus controls.
Figure 1. Inflammation in diabetic coronary arteries. A, Coronary fibroatheromas illustrating the extent of macrophage (M) and T cells, (CD45RO) and HLA-DR expression in patients with type I and II diabetes mellitus (DM) and nondiabetic subjects.
TABLE 2. Plaque Characteristics in Type I and II Diabetic Subjects vs Nondiabetic Subjects
The multivariate effect of risk factors on 3 dependent morphological variables was studied separately (Table 3). There was a positive correlation between necrotic core size and glycohemoglobin, independent of HDL lipoprotein cholesterol, TC/HDL cholesterol ratio, age, smoking, and gender (P=0.005, t=2.8). There was a similar correlation with body mass index. A strong correlation between macrophage area and glycohemoglobin (P=0.0004, T=2.9, Table 3) was observed. There was also a stronger relationship between numbers of fibrous cap atheromas and TC/HDL cholesterol than with other risk factors, including glycohemoglobin.
TABLE 3. Relationship of Risk Factors, Including Diabetes, to Plaque Characteristics in a Multivariate Analysis
Combined Effect of Hypercholesterolemia and Diabetes on Necrotic Core Size and Macrophage Content
Patients were divided as follows: normoglycemic patients or diabetic patients (I or II) with or without hyperlipidemia. By univariate analysis, the degree of macrophage infiltrate and necrotic core size as assessed by morphometry was significantly higher in diabetic patients with normal cholesterol or hyperlipidemia compared with nondiabetic patients (Figure 2).
Figure 2. Combined effect of hyperlipidemia (hyperlipidemia defined as total cholesterol >200 mg/dL or TC/HDL cholesterol ratio >5) and diabetes on mean macrophage percent (log-normalized, left) and mean necrotic core size (log-normalized, right). There is a significant difference between diabetic patients (DM) and nondiabetic patients (non-DM) when cases were separated by the presence or absence of hyperlipidemia.
Evidence of Remodeling
The mean IEL (adjusted for distance from coronary ostium) was greater in type I and II diabetic subjects compared with nondiabetic subjects (18.2±6.6 mm2, 16.5±4.4 mm2, 16.0±4.5 mm2, respectively). The mean IEL was also significantly greater in type I (P=0.001) and II diabetic subjects (P=0.01). By multivariable analysis, there was a correlation between type I diabetes and IEL area independent of heart weight, percent necrotic core, plaque area, and percent plaque calcification (P=0.0004). This analysis (percent necrotic core, P=0.05; plaque area, P<0.0001; heart weight, P=0.05) was positively correlated with IEL area.
Immunolocalization of RAGE and EN-RAGE
Although RAGE was found localized to macrophages, smooth muscle cells, erythrocytes, and endothelial cells in both diabetic and nondiabetic subjects, the overall expression of RAGE, as graded semi-quantitatively, was significantly greater in diabetic subjects (16.8±6.2 for diabetic subjects, 10.0±6.1 for nondiabetic subjects, P=0.004) (please see Figure II, available online at http://atvb.ahajournals.org). Extensive RAGE staining was present in macrophages around and within necrotic cores of both diabetic and nondiabetic subjects, with its overall expression dependent on extent of cellular infiltrate. Fewer smooth muscle cells stained with RAGE, but the intensity was greater in diabetic subjects. RAGE expression was often associated with apoptotic macrophages and smooth muscle cells (please see Figure III, available online at http://atvb.ahajournals.org). EN-RAGE expression in diabetic patients was most prominent in macrophages and, to a lesser degree, in smooth muscle cells in the regions surrounding the necrotic core (Figure 3).
Figure 3. Expression of S100A12/EN-RAGE (a RAGE-binding protein) in human coronary lesions from sudden coronary death victims with diabetes (A–E) and without diabetes (F–J). A, Lesion from the proximal left circumflex coronary artery stained by Movat pentachrome. B to E, High magnification micrographs of the black box represented in (A). B, HHF-35 staining for smooth muscle cells (SMCs) in the shoulder region of the plaque. C, Adjacent section stained for CD68, demonstrating numerous inflammatory macrophages. D, The same region demonstrated intense staining for EN-RAGE. B to D, The counterstain is Gill’s hematoxylin. E, Double-labeled immunofluorescent staining of CD68-positive macrophages (green) and EN-RAGE (red); nuclei (blue) were counterstained with DAPI; areas of overlap appear as yellow–green. EN-RAGE was primarily found in macrophage-rich areas. F, A lesion from the proximal left anterior descending coronary artery from a nondiabetic patient stained by Movat pentachrome. G to I, Immunoreactivity to SMC, macrophage, and EN-RAGE, respectively. Overall, macrophage infiltrate was significantly less in nondiabetic patients, corresponding to less immunoreactivity to EN-RAGE. J, Double staining for EN-RAGE and macrophages.
ISEL Quantification
The density of apoptotic-staining cells surrounding necrotic cores was greater in diabetic (26.9 nuclei/mm2) versus nondiabetic subjects (6.5 nuclei/mm2, P=0.002; please see online Table I, www.ahajounals.org). The highest density was present in type I diabetic subjects (P=0.02 versus nondiabetic subjects), followed by type II diabetic subjects (P=0.04 versus controls).
Discussion
Sudden cardiac death in type II diabetic subjects in the absence of known previous symptoms is associated with extensive atherosclerosis compared with nondiabetic subjects, including distal coronary involvement. The increase in plaque burden is partly related to the observation of a greater number of healed ruptures in type II diabetic subjects. Further, healed myocardial infarction and cardiomegaly are also more frequent in type II diabetic subjects, without differences in the incidence of acute thrombosis. An additive effect of hypercholesterolemia on necrotic core size and macrophage infiltrate was apparent for individuals with type I and II diabetes together with T-cell infiltration and HLA-DR expression. The degree of RAGE expression and greater frequency of apoptosis in smooth muscle cells and macrophages together with increased expression of EN-RAGE (a known chemokine) may explain the severity of macrophage infiltration and larger necrotic cores in diabetic patients.
Total and distal plaque burden was significantly greater in type II diabetic compared with nondiabetic subjects, whereas only increased total plaque was evident in type I diabetic subjects. Although the precise cause of diffuse coronary disease in type II diabetic subjects is unclear, the extent of thrombus formation may be involved. Recent studies suggest that relative amount of thrombus may be dependent of the expression of plasminogen activator inhibitor-1 (PAI-1). Notably, individuals with type II diabetes show elevated levels of PAI- I22; in contrast, PAI-1 levels in type I diabetes are normal.23
An increased frequency of healed myocardial infarction and cardiomegaly suggests that the mechanism of sudden death may differ between diabetic and nondiabetic patients. Specifically, the high rate of healed infarcts suggests a propensity for arrhythmia in type II diabetic subjects. Of importance are the relatively low rates of acute thrombosis and healed infarcts in patients with type I diabetes. These data support a potential role for nonanatomic factors, such as autonomic neuropathy, long QT syndrome, or hypoglycemia, in the precipitation of sudden death in type I diabetic patients.24
Clinical studies have shown that diabetes is associated with positive25 or negative/absence of remodeling.26 The current study supports the notion that diabetic subjects are more likely to show positive remodeling. These data are consistent with previous findings from our laboratory that necrotic core and macrophage infiltrates are associated with expansion of the internal elastic lamina independent of plaque size.16
The inflammatory response was greater in diabetic than in nondiabetic plaques. However, T cells were only significantly greater for type I diabetic subjects. The fact that type I diabetes is an autoimmune disease with a common genetic susceptibility to other disorders like autoimmune thyroiditis may also be of pathophysiologic significance in coronary plaques.27
The result of increased numbers of RAGE-expressing smooth muscle cells and macrophages in plaques of diabetic subjects rather than in nondiabetic subjects provides a link between RAGE and apoptosis in coronary atherosclerosis. An association between RAGE-expressing macrophages and upregulation of inflammatory NF-kappa-?, COX-2/mPGES-1, and matrix metalloproteinases has been shown in carotid arteries of diabetic patients.28,29 The current study demonstrates an increase in apoptotic macrophages and smooth muscle cells in coronary arteries of diabetic patients, which may be related to necrotic core expansion, thinning of the fibrous cap, and plaque instability. Experimental studies of murine models of diabetic aortic atherosclerosis have demonstrated that RAGE blockade by soluble RAGE decreases atheroma formation and the development of less complex lesions.30
Although the interactions between EN-RAGE–modulated proteins and RAGE receptors in the fine regulation of leukocyte trafficking and proliferation have been recently reviewed,31 this mechanism in atherosclerosis-related inflammation has not been extensively studied. Several reports suggest that EN-RAGE is chemotactic and, in the nanomolar range, stimulates the migration of neutrophils, monocytes, and macrophages.32,33 Moreover, the increase in EN-RAGE protein is sensitive to pioglitazone and thiazolidinedione, suggesting the involvement of the peroxisome proliferator-activated receptor-. Thus, the role of RAGE/EN-RAGE upregulation in the atherosclerotic plaque is likely complex and may be linked with recruitment, apoptosis, and macrophage cell death.
Lesion calcification in the present study was more prevalent in patients with type II diabetes in comparison to nondiabetic subjects. Although the mechanisms of calcification are undoubtedly complex, a greater incidence of intimal cell death in coronary lesions from diabetic subjects may be responsible for this increase. In contrast, type I diabetes is an immune-mediated disease and in some respects is similar to transplant vasculopathy, in which arterial lesions show little or no calcification. Although coronary calcification measured with electron beam computed tomography in nondiabetic patients is greater in males than in females, this relationship is lost in patients with type I diabetes, of whom woman have a higher prevalence of calcification than men.34,35
Limitations
As an autopsy study, there is an inherent bias restricting cases to those of sudden unexpected death. Given the high rate of sudden death as a presenting symptom of coronary artery disease, however, the findings are applicable to a relatively large proportion of acute coronary syndromes. Another limitation is the classification of diabetic subjects based on postmortem data instead of clinical history. The use of glycohemoglobin as a tool for classification of diabetes has been accepted, however, in several studies.5,18
Conclusion
Diabetic atherosclerotic plaques have a higher content of inflammatory cell infiltrate and HLA-DR expression than do nondiabetic atherosclerotic plaques. This may be associated with larger necrotic core size, and a greater extent of smooth muscle cell and macrophage apoptosis. These finding may be related to the greater expression of RAGE and EN-RAGE in diabetic plaques. Through better understanding of plaque morphology in diabetic subjects, it may be possible to design more effective treatment that would prevent the atherosclerotic complication of frequent plaque ruptures, diffuse disease, and healed infarction associated with premature death in diabetic subjects.
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