Pathology of Diabetic Atherosclerosis: Composition, Characteristics, and Distribution

Diabetes mellitus is associated with the development of accelerated atherosclerotic coronary artery disease, which results in increased morbidity and mortality from cardiovascular complications including acute myocardial infarction and stroke. ^, Atherothrombosis is the leading cause of death worldwide despite major progress in the understanding of the role of traditional risk factors in its etiopathogenesis.

In this chapter we address morphologic characteristics of coronary and carotid plaques in individuals with type 1 and 2 diabetes as compared with those without diabetes, and we discuss the involvement of endothelial cells, macrophages, and smooth muscle cells as well as the signaling transduction receptors for advanced glycation endproducts (RAGEs) that accumulate in diabetes and are associated with acceleration of atherosclerosis.

Plaque Morphology and Inflammation in Diabetic Atherosclerosis

Diabetes is associated with increased prevalence of hyperlipidemia, hypertension, obesity, and a hypercoagulable state, all of which contribute to higher incidence of coronary, carotid, and peripheral artery diseases that are associated with high mortality and morbidity, as reviewed in Chapters 7 , 27 , and 28 .

Coronary and Carotid Artery Disease

Moreno and colleagues have evaluated coronary atherectomy specimens obtained from 47 patients with type 2 diabetes and compared them with specimens from 48 nondiabetic individuals, and demonstrated that patients with diabetes exhibited a larger content of lipid-rich atheroma (7% ± 2%) than those without diabetes (2 ± 1%). In addition, macrophage infiltration was significantly greater in patients with diabetes (22 ± 3%) than in those without diabetes (12 ± 1%), and the incidence of thrombus was higher in individuals with diabetes (62%) than in those without diabetes (40%).

Cipollone and colleagues examined carotid endarterectomy specimens from patients with type 2 diabetes (n = 30) and compared them with specimens from patients without diabetes (n = 30) and showed that the plaques from patients with diabetes were richer in macrophages and T lymphocytes and also had a more frequent expression of human leukocyte antigen–DR (HLA-DR). Immunohistochemistry revealed greater reactivity of the RAGEs in patients with diabetes versus those without diabetes, especially in areas rich in macrophages and angiogenesis. The activity of nuclear factor kappa B (NF-κB) was greater in patients with diabetes than in those without diabetes and showed a concordance with RAGE expression. Also, cyclooxygenase 2 (COX-2) membrane-associated protein eicosanoid and glutathione metabolism synthase 1 (mPGES-1), matrix metalloproteinases (MMPs), and gelatinolytic activity were increased in patients with diabetes compared with those without diabetes. Patients with diabetes had reduced collagen content and increased lipid and oxidized low-density lipoprotein content as compared with those without diabetes. In this study, RAGE, COX-2/mPGES-1, and MMP expression was linearly correlated with plasma concentration of hemoglobin A1c (HbA1c). Therefore, in individuals with diabetes, RAGE overexpression along with enhanced inflammatory reaction and COX-2/mPGES-1 expression in macrophages may contribute to plaque destabilization.

The linear correlation between RAGE and HbA1c in the aforementioned study indicates that RAGE may be downregulated by improving glycemic control ; the same group also reported the possibility of a pharmacologic modulation of glucose-independent RAGE generation. Patients with type 2 diabetes and asymptomatic carotid artery stenosis were randomized to diet plus simvastatin (40 mg/day) or diet alone for 4 months before endarterectomy. Plaques from the simvastatin group showed significantly less immunoreactivity for myeloperoxidase (MPO), AGEs, RAGE, p65, COX-2, mPGES-1, MMP-2, MMP-9, lipids, and oxidized low-density lipoprotein, along with reduced gelatinolytic activity, increased procollagen 1 and collagen content, and fewer macrophages, T lymphocytes, and HLA-DR–positive cells. RAGE inhibition by simvastatin was also identified in plaque-derived macrophages and was reverted by addition of AGEs in vitro. These results suggest that simvastatin inhibits RAGE expression by decreasing MPO-dependent AGE generation, which may contribute to plaque stabilization.

Hyperglycemia is known to increase lipolysis, which leads to the release of nonesterified fatty acids (NEFAs) into the bloodstream. ^, Elevated serum levels of NEFAs are associated with vascular damage in type 2 diabetes. Mas and colleagues showed a significant increase in the quantity of NEFAs in carotid endarterectomy specimens retrieved from patients with type 2 diabetes as compared with those without diabetes by using time-of-flight secondary ion mass spectrometry. Although plasma levels of NEFAs were greater in patients with diabetes than in those without diabetes, tissue NEFA levels did not correlate with plasma NEFA levels. Laser-capture microdissection with quantitative reverse transcription polymerase chain reaction RT-PCR revealed that mRNA expressions of lipoprotein lipase (LPL) and monocyte chemoattractant protein 1 (MCP-1) were greater in NEFA-rich areas than in NEFA-poor areas. Conventional immunohistochemistry and in situ Southwestern hybridization also demonstrated that those with diabetes had greater protein expression of LPL and MCP-1, greater infiltration of macrophages and T lymphocytes, and greater activated NF-κB–positive nuclei than those without diabetes, where the patterns of distribution were similar to those of NEFA. These findings indicate that NEFA may be produced locally and contribute to local inflammation within the atherosclerotic plaques in patients with type 2 diabetes.

A recent study using carotid endarterectomy specimens showed that neovascularization, only in the shoulder regions of the plaques, was more frequent in patients with type 2 diabetes than in those without diabetes (52% versus 26%) with no differences in macrophage content in the entire section of the plaque. In addition, patients with diabetes had greater expression of vascular endothelial growth factor receptor 2 (VEGFR-2) as compared with those without diabetes. The increased vascularization in the shoulder region suggests a higher risk of atherosclerotic vascular complications, such as plaque rupture ( Fig. 8-1 ) followed by healing in patients with diabetes. These findings parallel those shown in aortic and coronary arteries of diabetic patients.

Figure 8-1, Acute plaque rupture with occlusive thrombus.

In summary, atherosclerotic plaques retrieved from patients with hyperglycemia (diabetes) show a higher expression of AGE and its receptor RAGE on endothelial and smooth muscle cells, which are involved in the induction of plaques that are highly inflamed with greater infiltration by macrophages, T cells, and HLA-DR-positive cells. Patients with diabetes show vascular dysfunction that likely occurs from increased production of reactive oxygen species as well as activation of platelet. Furthermore, carotid plaques from individuals with diabetes have demonstrated higher expression of protein kinase C (PKC) and NF-κB, with large necrotic cores, hemorrhage, and an increase in angiogenesis, especially in the shoulder regions. In addition, patients with diabetes had greater expression of VEGFR-2 than those without diabetes. These carotid plaques have highly expressed MPO, p65, COX-2, mPGES-1, MMP-2, MMP-9, lipids, and oxidized low-density lipoprotein, along with an increase in gelatinolytic activity and greater collagen content ( Fig. 8-2 ).

Figure 8-2, Mechanisms of hyperglycemia-induced vascular damage.

Coronary Atherosclerosis in Sudden Death

Sudden death victims with type 2 diabetes show greater prevalence of coronary artery disease as a cause of death than those without diabetes ( Fig. 8-3A ). We have reported morphologic findings in patients with type 1 and those with type 2 diabetes and compared them with age- and gender-matched individuals without diabetes who died suddenly from coronary artery atherosclerotic disease. The underlying inclusion criterion for sudden coronary death was presence of an acute coronary thrombus or severe epicardial coronary atherosclerosis (> 75% cross-sectional area luminal narrowing) of one or more major arteries and the absence of noncoronary causes of death at autopsy.

Figure 8-3, Extent of coronary artery disease (CAD) in sudden death victims with and without diabetes mellitus (DM).

Sixty-six individuals with diabetes were selected on the basis of history of type 1 diabetes mellitus treated with insulin or the presence of type 2 diabetes. Type 2 diabetes was ascertained by history of oral hypoglycemics or postmortem glycohemoglobin 10% or higher in the absence of type 1 diabetes. A total of 16 patients with type 1 diabetes and 50 with type 2 diabetes were included. The findings in these patients were compared with 66 age- and gender-matched individuals without diabetes who died from severe coronary artery disease ( Table 8-1 ). The prevalence of smoking and hypertension in patients with type 1 and type 2 diabetes was comparable to the prevalence in those without diabetes. The body mass index (BMI) in individuals with type 2 diabetes (30.5 ± 7.4 kg/m ² ) was significantly greater than in those without diabetes (26.6 ± 5.4 kg/m ² , P = 0.001), whereas in individuals with type 1 diabetes BMI (25.6 ± 6.4 kg/m ² ) was similar to that in patients without diabetes ( P = 0.7). Individuals with type 2 diabetes showed a trend toward higher levels of total cholesterol (TC) and lower levels of high-density lipoprotein cholesterol (HDL-C) than those without diabetes (TC 227 ± 83 versus 211 ± 79 mg/dL, P = 0.3; HDL-C 33 ± 16 versus 38 ± 18 mg/dL, P = 0.1). The ratio of TC to HDL-C was significantly higher in individuals with type 2 diabetes than in those without diabetes (7.9 ± 3.9 versus 6.3 ± 3.4, P = 0.02). On the contrary, individuals with type 1 diabetes had a trend toward lower levels of TC (183 ± 52 mg/dL) and comparable levels of HDL-C (37 ± 14 mg/dL) and TC-to–HDL-C ratio (5.8 ± 2.9) relative to those without diabetes.

Table 8-1

Patient Demographics, Risk Factors, and Cardiac Findings in Patients with Type 1 Diabetes and Those with Type 2 Diabetes Versus Nondiabetic Patients from Sudden Coronary Death Registry

Data from Burke AP, Kolodgie FD, Zieske A, et al: Morphologic findings of coronary atherosclerotic plaques in diabetics: a postmortem study, Arterioscler Thromb Vasc Biol 24:1266-1271, 2004.

	Type 1 DM (n = 16)	Type 2 DM (n = 50)	Non-DM (n = 66)	P value (Type 1 DM Versus non-DM)	P value (Type 2 DM Versus non-DM)
Age (year)	50.3 ± 13.2	50.2 ± 11.0	50.6 ± 12.3	0.9	0.9
Women	25%	30%	29%	0.8	0.9
Blacks	20%	30%	29%	0.7	0.9
HbA1c (%)	12.2 ± 2.5	10.7 ± 2.6	6.2 ± 0.6	0.0001	0.0001
Smokers	42%	58%	55%	0.4	0.8
Hypertension	29%	35%	30%	0.9	0.6
Body mass index (kg/m ² )	25.6 ± 6.4	30.5 ± 7.4	26.6 ± 5.4	0.7	0.001
TC (mg/dL)	183 ± 52	227 ± 83	211 ± 79	0.3	0.3
HDL cholesterol (mg/dL)	37 ± 14	33 ± 16	38 ± 18	0.8	0.1
TC/HDL cholesterol	5.8 ± 2.9	7.9 ± 3.9	6.3 ± 3.4	0.7	0.02
Heart weight (g)	425 ± 119	524 ± 140	434 ± 121	0.7	0.004
Corrected heart weight (g) *	428 ± 94	508 ± 134	460 ± 106	0.3	0.03
Healed infarcts	33%	73%	37%	0.7	0.0001

Values are expressed as mean ± standard deviation or percentage.

DM = diabetes mellitus; HDL = high-density lipoprotein; TC = total cholesterol.

* Corrected for body weight.

The percent necrotic core area (necrotic core area divided by plaque area) was greater in individuals with type 1 (12.0% ± 5.7%) and type 2 (11.6% ± 8.4%) diabetes than in those without diabetes (9.4% ± 9.3%; P = 0.05 versus type 1, P = 0.004 versus type 2 diabetics) ( Table 8-2 ). The percent calcified area was greater in individuals with type 2 diabetes (12.1% ± 11.2%) than in those without diabetes (11.4% ± 13.5%, P = 0.05), and individuals with type 1 diabetes had a comparable percent calcified area (7.8% ± 9.1%) compared with those without diabetes. The number of fibroatheromas was greater in individuals with type 2 diabetes (8.8 ± 4.3) than in those without diabetes (6.9 ± 4.7, P = 0.02), whereas those with type 1 diabetes had a similar number of fibroatheromas (7.1 ± 5.0) compared with those without diabetes. The number of thin-cap fibroatheromas was comparable among the groups. The number of healed plaque ruptures in individuals with type 2 diabetes was greater than in those without diabetes (2.6 ± 1.8 versus 1.9 ± 1.8, P = 0.04), while those with type 1 diabetes only showed a trend toward a greater number of healed ruptures (2.6 ± 2.1) as compared with those without diabetes.

Table 8-2

Plaque Characteristics in Patients with Type 1 Diabetes and Those with Type 2 Diabetes Versus Nondiabetic Patients from Sudden Coronary Death Registry

Data from Burke AP, Kolodgie FD, Zieske A, et al: Morphologic findings of coronary atherosclerotic plaques in diabetics: a postmortem study, Arterioscler Thromb Vasc Biol 24:1266-1271, 2004.

	Type 1 DM (n = 16)	Type 2 DM (n = 50)	Non-DM (n = 66)	P value (Type 1 DM versus non-DM)	P value (Type 2 DM versus non-DM)
Acute coronary thrombi	21%	42%	51%	0.03	0.2
Acute plaque rupture	6%	32%	27%	0.09	0.6
Plaque erosion	6%	12%	29%	0.02	0.04
Necrotic core area (%) *	12.0 ± 5.7	11.6 ± 8.4	9.4 ± 9.3	0.05 ^†	0.004 ^†
Calcified matrix area (%) *	7.8 ± 9.1	12.1 ± 11.2	11.4 ± 13.5	0.9 ^†	0.05 ^†
Fibroatheroma (n)	7.1 ± 5.0	8.8 ± 4.3	6.9 ± 4.7	0.9	0.02
Thin-cap fibroatheroma (n)	1.0 ± 1.3	0.8 ± 0.8	0.7 ± 0.8	0.5	0.8
Healed plaque rupture (n)	2.6 ± 2.1	2.6 ± 1.8	1.9 ± 1.8	0.2	0.04
Total plaque burden (%)	275 ± 129	358 ± 114	232 ± 128	0.04	0.0001
Distal plaque burden (%)	310 ± 114	630 ± 263	331 ± 199	0.8	0.0001
Macrophage area (mm ² )	0.15 ± 0.02	0.13 ± 0.03	0.10 ± 0.02 ^‡	0.03 ^†	0.03 ^†

Values are expressed as mean ± standard deviation or percentage.

DM = diabetes mellitus.

* Divided by plaque area.

† P values calculated using log-normalized data.

‡ P = 0.006 versus type 1 and 2 diabetes combined.

By multivariable analysis ( Table 8-3 ) there was a positive correlation between mean percent necrotic core area and glycohemoglobin, independent of HDL-C, ratio of TC to HDL-C, age, smoking, and gender (T = 2.8, P = 0.005). Similarly, the ratio of TC to HDL-C (T = 2.5, P = 0.01) and BMI (T = 3.5, P = 0.006) correlated positively with percent necrotic core area. There was a significant relationship between numbers of fibroatheroma and ratio of TC to HDL-C (T = 3.0, P = 0.0003). Glycohemoglobin correlated positively with number of fibroatheromas, although the relationship was not statistically significant (T = 1.7, P = 0.09).

Table 8-3

Relationship of Risk Factors, Including Diabetes, to Plaque Characteristics: A Multivariate Analysis

Data from Burke AP, Kolodgie FD, Zieske A, et al: Morphologic findings of coronary atherosclerotic plaques in diabetics: a postmortem study, Arterioscler Thromb Vasc Biol 24:1266-1271, 2004.

Independent Variables	% Necrotic Core Area *		Number of Fibroatheromas ^†		% Macrophage Area ^‡
(Risk Factors)	T	P Value	T	P Value	T	P Value
Glycohemoglobin (%)	2.8	0.005	1.7	0.09	2.9	0.004
TC/HDL cholesterol	2.5	0.01	3.0	0.0003	1.3	0.19
Body mass index	3.5	0.006	0.57	0.57	1.5	0.14
Smoking	− 0.4	0.7	− 1.1	0.24	− 0.6	0.5
Age	− 1.2	0.2	− 1.2	0.2	− 5.4	0.0001

The population for this table is the 132 patients with three separate one-way analysis of variance (ANOVA) analyses correlating three dependent variables (mean % necrotic core area, mean % macrophage area).

* Mean percent necrotic core area (of the four arteries studies per patient).

† Mean number of fibroatheromas per heart.

‡ Mean macrophage area (of the four arteries per patient).

Macrophage plaque area and T-cell infiltration were significantly greater in individuals with diabetes than in those without diabetes ( P = 0.03), along with HLA-DR expression (see Table 8-2 ; Figs. 8-4 and 8-5 ). The fact that T-cell infiltration was greater in individuals with type 1 diabetes is consistent with the fact that type 1 diabetes is an autoimmune disease with a common genetic susceptibility to other disorders, like autoimmune thyroiditis, which may also be of pathophysiological significance in coronary plaque pathology. There was a strong positive correlation between macrophage area and glycohemoglobin, independent of HDL-C, ratio of TC to HDL-C, age, smoking, and gender (T = 2.9, P = 0.004) (see Table 8-3 ). The combined effect of hypercholesterolemia and diabetes on macrophage infiltration and necrotic core size were further evaluated. The degree of macrophage infiltrate and necrotic core size as assessed by morphometry were significantly greater in diabetic patients with normal cholesterol or hyperlipidemia as compared to nondiabetic patients ( Fig. 8-6 ).