More Fibrosis and Thrombotic Complications but Similar Expression Patterns of Markers for Coagulation and Inflammation in Symptomatic Plaques from DM2 Patients

Abstract

Objective: One of the possible pathological mechanisms behind the increased vascular injury in diabetes mellitus type 2 (DM2) is the formation of advanced glycation end products (AGEs). The aim of this study was to investigate whether the presence of AGEs and specific markers for coagulation and inflammation in symptomatic atherosclerotic plaques from DM2 patients differs from plaques from nondiabetics. Methods and results: Carotid atherectomies were obtained from DM2 patients (n = 11) and controls without DM2 matched for age and other cardiovascular risk factors (n = 12) who were treated for symptomatic carotid artery stenosis. Plaques were graded according to the American Heart Association classification of lesions. More fibrosis and more thrombotic complications (p = 0.007) were observed in carotid atherectomies from DM2 patients. Percentages of immunostained smooth muscle cells and macrophages in the lesions, quantified planimetrically, did not differ between the two groups. No differences were found in the immunostaining for T cells, tissue factor (TF), endothelial protein C receptor (EPCR), nuclear factor κB, and the AGE carboxymethyllysine. Conclusions: These findings demonstrate that DM2 is associated with increased plaque complications; however, a local changed presence of AGEs, TF, and EPCR seems not to be involved in this end stage of atherosclerosis.

Keywords

diabetes mellitus type 2 atherothrombotic complications atherectomy tissue factor endothelial protein C receptor advanced glycation end products nuclear factor κB

Diabetes mellitus type 2 (DM2) is a risk factor for cardiovascular disease attributable to an accelerated process of atherosclerosis. An increasing body of evidence indicates that the presence of hyperglycemia is the primary causal factor for vascular complications in diabetic patients (Turner 1998; Khaw et al. 2001; Aronson and Rayfield 2002). An important mediator of hyperglycemia-induced vascular injury may be the formation of advanced glycation end products (AGEs) (Vlassara and Bucala 1996; Wautier and Guillausseau 1998). AGEs are the result of nonenzymatic glycation and glycoxidation during normal aging. Their production is increased during hyperglycemia. The presence of AGEs and its binding to specific receptors can lead to cellular dysfunction and changed expression of coagulation factors, which could play a role in diabetic atherosclerosis (Esposito et al. 1989; Khechai et al. 1997; Ichikawa et al. 1998; Sano et al. 1999).

Tissue factor (TF) is a key player in initiating the activation of the coagulation cascade and is thought to be involved in the development of atherosclerosis and its thrombotic complications (Taubman et al. 1997; Moons et al. 2002). TF is found on the cell membrane of various cell types. Increased expression of TF has been reported in different animal models of diabetes mellitus (Samad et al. 1998; Kislinger et al. 2001). Studies in patients with type 2 diabetes reported increased expression of TF on microparticles and leukocytes (Ichikawa et al. 1998; Diamant et al. 2002). One of the possible mechanisms of this diabetes-related increase in TF expression could be the binding of AGEs to specific receptors (Khechai et al. 1997; Ichikawa et al. 1998; Kislinger et al. 2001). A role for increased TF expression has been suggested in the greater risk for cardiovascular complications in diabetic patients (Tschoepe 1997).

The endothelial protein C receptor (EPCR) plays a major role in the activation of the protein C anticoagulant pathway, which is an important mechanism in downregulating thrombus formation. It has been suggested that decreased expression of EPCR plays a role in the atherosclerotic process (Laszik et al. 2001). Several studies of patients with DM2 have reported a possible relation between hyperglycemia and the down-regulation of the protein C system (Esposito et al. 1989; Gabazza et al. 1996; Pannacciulli et al. 2001; Hafer-Macko et al. 2002). However it is not known whether the expression of EPCR is changed in diabetic vessels.

One of the signal transduction pathways that could play a role in the changed expression of coagulation factors by AGEs is the nuclear factor κB (NF-κB) pathway. NF-κB plays a central role in immune and inflammatory reactions. The usually transient NF-κB-dependent gene expression is exaggerated in pathological situations. NF-κB activation has been observed in atherosclerotic lesions (Brand et al. 1996). There is evidence that NF-κB activation is increased during hyperglycemia (Pieper and Riaz-ul-Haq 1997; Bierhaus et al. 2001), which may be involved in the changed expression of proteins in DM2.

Our hypothesis is that expression of TF and EPCR might be changed in diabetic atherosclerosis by an increased presence of AGEs. To test this hypothesis, we have immunohistochemically analyzed the presence and tissue localization of TF, EPCR, NF-κB, and N∊- (carboxymethyl)lysine (CML), which is reported to be a major AGE (Reddy et al. 1995), in carotid endarterectomies from DM2 patients and matched control patients with symptomatic carotid stenosis.

Patients and Methods

Patient Group

For this study, a database was used that contained paraffin-embedded specimens of various arteries and veins from 244 patients who consecutively underwent peripheral vascular surgery at the Academic Medical Center (AMC), Amsterdam, The Netherlands, between 1994 and 1998. To use this database to compare specimens from patients with DM2 and controls, a nested case-control study was performed. First, a selection was made of all the vascular specimens of patients who had undergone carotid atherectomy because of symptomatic arterial stenosis. Second, carotid atherectomy specimens were selected from a subpopulation of patients with known DM2 at the time of intervention, treated with insulin and/or oral anti-diabetics (n = 11). In addition, a matched control group (n = 12) was selected, matched for coronary risk factors such as age, known hypertension [treated or known with hypertension (blood pressure >160/90 mmHg)], hypercholesterolemia [treated or known with hypercholesterolemia (total cholesterol >6 mmol/liter)], and smoking. For group characteristics, see Table 1. Informed consent was obtained before surgery, and the study was approved by the local ethical committee of the AMC.

Table 1

Patient characteristics

Characteristic	Patients without DM2 (n = 12)	Patients with DM2 (n = 11)	p
Age (years)	69.5 ± 2.2	69.8 ± 2.7	0.471
Men	9 (75%)	7 (64%)	0.554
Glucose (mg/dl)	109.4 ± 4.0	173.6 ± 19.3	0.004
Hypercholesterolemia, statin treatment	1 (8%)	2 (18%)	0.732
Hypercholesterolemia, no treatment	4 (33%)	4 (36%)	0.732
Hypertension, treatment	8 (67%)	8 (73%)	0.868
Hypertension, no treatment	2 (17%)	1 (9%)	0.865
Smoking	3 (25%)	3 (27%)	0.951
Stopped smoking	4 (33%)	3 (38%)	0.951

Tissue Processing and Histomorphology

Carotid atherectomies were obtained and immediately fixed in 4% buffered formalin after surgical removal. Tissues were routinely processed for paraffin embedding, and sections were serially cut at 5 μm from each specimen. Per atherectomy sample, one to three sites were available for testing (total control sites, n = 17; total diabetes sites, n = 13). To evaluate the morphology of atherosclerotic plaques, one section was stained with hematoxylin and eosin and one with elastic van Gieson. The severity of plaque formation was graded according to the American Heart Association (AHA) classification: type I lesion, intimal thickening with an increase in macrophages and formation of scattered macrophage foam cells; type II, fatty streak consisting of layers of macrophage foam cells and lipid-laden smooth muscle cells (SMCs); type III, preatheroma, potentially symptom producing; type IV, atheroma with a more disruptive core of extracellular lipid; type Va, fibroatheroma, lipid core containing thick layers of fibrous connective tissue; type Vb, largely calcified plaque; type Vc, plaque consists mainly of fibrous connective tissue with little or no accumulated lipid or calcium; type VI, complicated plaque with fissure, hematoma, and thrombus (Stary et al. 1995). The presence of thrombus was identified on the basis of the presence of platelet aggregates and erythrocytes, with or without areas or layers of granulocytes, and in continuity with plaque material. Thrombus may also show ingrowth of SMCs, indicating thrombus organization. Adjacent serial sections were mounted for immunostaining.

Table 2

Antibody characteristics

Antibody	Monoclonal	Specificity	Concentration (μg/ml)	Source
Anti-α-actin smooth muscle	Mouse	Vascular SMCs	0.35	Dako, Glostrup, Denmark (1D4)
Anti-CD3	Rabbit (polyclonal)	T-lymphocytes	2.5	Dako
Anti-CD68	Mouse	Macrophages	3.6	Dako (PG-M1)
Anti-vWF	Mouse	Von Willebrand factor on endothelial cells	4.4	Dako (F8/86)
Anti-CML	Mouse	CML (AGE product)	1.3	Dr C.G. Schalkwijk, Free University Academic Hospital, Amsterdam, The Netherlands (Schalkwijk et al. 2004)
Anti-TF	Mouse	TF	10	American Diagnostica, Stamford, CT (4509)
Anti-EPCR	Mouse	EPCR	1.5	Dr C.T. Esmon, University of Oklahoma Health Sciences Center, Oklahoma City (HEPCR 1489) (Laszik et al. 1997)
Anti-NF-κB p65	Mouse	Total p65, activated and nonactivated form of NF-κB	20	Santa Cruz Biotechnology, Santa Cruz, CA (sc-8008)
Anti-NF-κB p65	Mouse	Nuclear location signal of p65, activated form of NF-κB	10	Chemicon International, Temecula, CA (MAB3026)

Immunohistochemistry

Sections were subjected to immunohistochemistry using cell-specific mouse monoclonal antibodies against vascular SMCs (anti-α-actin), macrophages (anti-CD68), endothelial cells (anti-vWF), EPCR, TF, CML, NF-κB (recognizing total p65 protein), the activated form of NF-κB (selectively recognizing the anti-p65 subunit overlapping the nuclear location signal), and rabbit monoclonal antibodies against T-lymphocytes (anti-CD3) (Table 2). Before immunostaining with anti-EPCR, anti-activated NF-κB, anti-CD68, anti-CD3, and anti-α-actin, the sections were pretreated with 10 mmol/liter citric buffer (pH 6.0). Before immunostaining with anti-vWF and anti-CML, the sections were pretreated with pepsin. A streptavidin-biotin complex method was applied, and immunoreactivity was visualized with either diaminobenzidine or 3-amino 9-ethyl carbazole solution. Sections stained with antibodies against vWF, α-actin, CD3, and CD68 were counterstained with hematoxylin. Positive controls were according to literature kidney tissue (anti-TF), stomach epithelium (anti-NF-κB), larger vessel endothelium (anti-EPCR), and atherosclerotic plaque (anti-CML). Staining with murine monoclonal IgG1 antibodies (Dako A/S, Glostrup, Denmark) or mouse serum was used as a negative control. Rabbit IgG was used as a negative control for anti-CD3.

Morphometric Analysis

Results of anti-α-actin and anti-CD68 antibodies were planimetrically quantified using image analysis software (Image Pro Plus; Media Cybernetics, Inc., Silver Spring, MD) on a personal computer connected with a video-mounted microscope. The total tissue area of each immunostained tissue section was outlined manually on the video screen and measured. Tissue areas of the immunopositive stained areas in the section were measured automatically using gray scale detection with a fixed threshold. Subsequently, SMCs and macrophage areas were calculated as percentages of the total tissue area.

For evaluation of CD3, NF-κB, and TF immunostaining, a semiquantitative score approach was chosen using the following criteria: 0, no staining; 1, <10% plaque tissue positive; 2, 10–50% plaque tissue positive; 3, >50% plaque tissue positive. Anti-CML staining was evaluated for immunoreactivity in SMCs, macrophages, and endothelial cells separated, in combination with the cell-specific antibodies for actin, CD68, and vWF, using serial sections. Anti-EPCR staining was evaluated in combination with the anti-vWF-stained section only. For each cell type, we used the following semiquantitative score criteria: 0, no staining; 1, only scarce cells positive; 2, ≃50% of cells positive; 3, most cells in section positive.

Observers were blinded to the clinical status of the patients.

Statistical Analysis

Results are expressed as means ± SEM for continuous variables. Categorical data are expressed as medians with 25–75 quartiles. For comparison between continuous variables of interest, Student's t-test was used. The association between categorical variables was assessed using the Mann–Whitney test or the Pearson Chi-square when appropriate. p values of <0.05 are considered statistically significant.

Results

Patient Group

For patient characteristics, see Table 1. There were no significant differences between diabetic patients and control patients with regard to age, gender, hypercholesterolemia, hypertension, and smoking. The average serum glucose levels of the diabetic patients (173.6 ± 19.3 mg/dl) were significantly higher than those of the control patients (109.4 ± 4.0 mg/dl) (p = 0.004).

Figure 1.

Example of an atherosclerotic plaque with thrombus (type VI lesion) from a patient with DM2. Tissue is stained with hematoxylin and eosin (magnification, ×40).

Morphological Features of Atherectomies

The occurrence of different lesion types was compared between control and diabetic atherectomies. To make comparison possible between the relatively low frequencies per lesion type, lesion types were grouped as either type III, IV, and Va lesions or type Vc and VI lesions. Carotid atherectomies from DM2 patients showed significantly more fibrotic lesions (type Vc lesions) and more thrombotic complications (type VI lesions) (for a typical example, see Figure 1) compared with control lesions (85% versus 31%) (p = 0.007) (Figure 2). No differences were observed in the amount of SMCs, macrophages, and T-cells between atherectomies from DM2 patients and controls (Tables 3 and 4).

Figure 2.

Histological grading of plaque morphology according to the AHA classification in diabetic and control patients. Significantly more type Vc and VI lesions were observed in diabetic patients compared with controls (p = 0.007, Pearson Chi-square test).

Table 3

Morphometric analysis of SMC and macrophage immunostaining in diabetic patients and control patientsa

Immunostaining	Controls	Diabetic patients	p
SMCs	0.08 ± 0.019	0.05 ± 0.007	0.21
Macrophages	0.03 ± 0.003	0.02 ± 0.005	0.22

Data are presented as means ± SEM.

Table 4

Semiquantitative analysis of TF, NF-κB, CML, EPCR, and T-cells in diabetic and control patientsa

Immunostaining	Controls	DM2 patients	p
TF	2.0 (2.0, 3.0)	2.0 (1.0, 2.0)	0.37
NF-κB	1.0 (0.5, 2.0)	1.0 (0.0, 2.0)	0.71
Activated NF-κB	1.0 (1.0, 1.0)	1.0 (1.0, 1.0)	0.76
CML SMCs in intima	1.0 (1.0, 1.8)	1.0 (1.0, 1.8)	0.95
CML SMCs in media	1.0 (1.0, 1.0)	1.0 (1.0, 1.5)	0.14
CML extracellular matrix	0.5 (0.0, 1.0)	1.0 (0.0, 1.0)	0.23
CML endothelium	1.0 (0.0, 1.0)	1.0 (0.0, 1.0)	1.00
EPCR endothelium plaque	1.0 (1.0, 2.0)	1.0 (1.0, 2.0)	0.91
EPCR microvascular endothelium	0.0 (0.0, 0.5)	0.0 (0.0, 1.0)	0.78
T-cells	2.0 (1.0, 2.0)	2.0 (1.0, 2.5)	0.83

Data are presented as medians with 25–75 quartiles.

Immunohistochemistry of TF, EPCR, NF-κB, and CML

No differences were observed in the presence of TF, EPCR, NF-κB, and CML staining in lesions from DM2 patients and controls (Table 4). Staining for CML varied widely from nearly absent to strong throughout the different plaques. However, no differences were found between DM2 patients and controls. CML was located in SMCs, macrophages, and the extracellular matrix (Figures 3C and 4C). Staining for TF was located in SMCs and macrophages (Figure 4B). In 87% of the plaques, colocalization of CML and TF in macrophages was observed, using two adjacent sections. There was no difference in the number of patients showing colocalization of CML and TF between the diabetic and nondiabetic groups. EPCR staining of the atherosclerotic plaque endothelium was weakly positive in endothelium of all atherosclerotic plaques (Figure 5B). EPCR staining in microvessel endothelium ranged from absent to weakly positive (Figure 5A). There were no differences in EPCR staining of the plaque endothelium or the microvessel endothelium in the DM2 group compared with the control group (Table 4). In nearly all atherectomies, only weak NF-κB-p65 staining was observed, mainly located in SMCs, macrophages, and endothelial cells (Figures 3D and 4D). To identify the activation of NF-κB, we used an antibody specifically against activated NF-κB (MAB3026). A weak staining pattern of activated NF-κB was observed, which was located in nuclei of SMCs, macrophages, and endothelial cells (Figure 6). No difference in staining was observed between diabetic and control plaques (Table 4).

Figure 3.

(A) Carotid artery atherosclerotic plaque classified as a type Va lesion according to the AHA classification (magnification, ×20). Tissue section is stained with elastic van Gieson (collagen = red, elastin = black, cells = yellow/brown). The plaque shows extensive fibrosis (F), a fibrocellular area with neovascularization (FC), and a deeply located atheroma (asterisk). L, lumen. (B–D) show details of the boxed area in (A), immunostained in adjacent serial sections. (B) Immunostaining with anti-vWF, showing the immunoreactivity of endothelial cells lining microvessels inside the plaque (magnification, ×80). (C) Immunostaining with anti-CML antibody, showing immunostaining of endothelia of all microvessels (magnification, ×80). (D) Immunostaining with anti-NF-κB antibody, showing immunostaining of microvascular endothelia and scattered inflammatory cells (magnification, ×64).

Discussion

Hyperglycemia is considered to play a major role in diabetic cardiovascular disease. In particular, the formation of AGEs appears to be an important mediator of hyperglycemia-induced vascular injury (Brownlee et al. 1988; Wautier and Guillausseau 1998; Aso et al. 2000). Several experimental studies showed that AGEs influence the production of proteins involved in the coagulation process in different cell types, which might play a role in the increased risk of thrombotic complications in diabetic patients (Esposito et al. 1989; Khechai et al. 1997; Ichikawa et al. 1998; Yamagishi et al. 1998; Min et al. 1999). In the present study, the simultaneous tissue localization of AGEs and coagulation proteins was studied in symptomatic atherosclerotic plaques from diabetic patients and their matched controls. Our data showed that plaques from DM2 patients had a significantly greater number of fibrotic lesions and lesions with thrombotic complications compared with those of matched control patients, confirming the well-known observation that DM2 is associated with an increased incidence of cardiovascular complications. Despite these differences, a consistent lack of differences in immunostaining of AGEs, TF, EPCR, and NF-κB was observed.

Figure 4.

(A) Carotid artery plaque type Va lesion stained with elastic van Gieson. F, fibrous tissue; L, lumen; M, media (magnification, ×20). The boxed area is at the border zone of a partly fibrosed lipid core and fibrous cap tissue (B–D) (magnification, ×80). (B) Immunoreactivity of TF in foam cell macrophages bordering the atheroma. (C) Cellular immunolocalization of AGEs at the same place shown in (B). (D) Anti-NF-κB-stained section showing immunostaining of foam cell macrophages and SMC in the same area.

Figure 5.

Fibrous plaque (type Va), stained with anti-EPCR antibody. (A) Immunoreactivity of scarce endothelial cells lining microvessels inside the plaque (magnification, ×80). (B) Prominent continuous immunoreactivity of arterial endothelium covering the plaque surface (magnification, ×55). L, lumen; P, plaque.

Figure 6.

Nuclear localization of activated NF-κB in endothelial cells and SMCs (arrows) stained with antibody specifically against activated NF-κB (magnification, ×160).

The lack of difference in AGE staining in the present study might be explained by the symptomatic, end-stage atherosclerosis that was studied and is in agreement with earlier studies that reported that AGEs are associated with atherosclerotic lesions regardless of the presence of diabetes (Niwa et al. 1997; Sakata et al. 1998). In particular, several studies showed that the degree of AGE staining correlated with the extent of the atherosclerotic changes (Kume et al. 1995; Nerlich and Schleicher 1999) in nondiabetic and diabetic patients. These findings suggest that the quantity of AGEs in atherosclerotic lesions is more related to the severity of the atherosclerosis than to the presence of hyperglycemia. The age of the studied patients (mean of 70 years) is a second possible explanation for the lack of increased AGE staining found in the DM2 group. It has been observed that AGE accumulation in tissue is related to aging (Schleicher et al. 1997; Sakata et al. 1998; Nerlich and Schleicher 1999). It may be possible that the role of AGEs is more important in an earlier stage of the accelerated atherosclerosis in diabetes mellitus and that differences in AGE localization are more distinct in plaques of younger diabetic patients or in less complicated plaques. This could explain why in other studies differences were observed between atherosclerotic lesions from diabetic patients and controls.

TF, the main initiator of the coagulation cascade, is widely expressed in atherosclerotic plaques and is thought to play a role in the development of acute arterial thrombosis. In agreement with earlier observations (Taubman et al. 1997), we clearly observed TF in macrophages, SMCs, and foam cells. However, this staining pattern was similar in control and diabetic plaques. This is in contrast to animal studies that showed that TF is increased in tissues and plaques from diabetic mice (Samad et al. 1998; Kislinger et al. 2001), which is probably related to accelerated atherosclerosis and enhanced AGE formation. The latter could explain the lack of difference in TF expression in the present study. AGEs, however, still might have been involved in TF expression in both the diabetic and nondiabetic plaques, because the TF pattern colocalized with the CML staining in almost 90% of both control and diabetic lesions.

Activation of the NF-κB pathway is a possible mechanism that may contribute to the changed expression of coagulation factors by AGEs (Bierhaus et al. 2001). But NF-κB activation may also be involved in other inflammatory atherogenic processes (Brand et al. 1996). The detection of similar staining of activated NF-κB confirms a role for activation of the NF-κB pathway in both diabetic and nondiabetic atherosclerosis.

EPCR expression was found to be decreased in endothelial cells covering atherosclerotic lesions compared with the expression in endothelial cells in control arteries (Laszik et al. 2001). In agreement with these findings, a weakly positive staining of EPCR on endothelium covering the atherosclerotic plaque was observed in our study. Although it has been suggested that the protein C system may be downregulated in DM2 (Gabazza et al. 1996; Pannacciulli et al. 2001; Hafer-Macko et al. 2002), we could not find a difference between the expression of EPCR in atherectomies of patients with DM2 and controls in this study. Neither did we find a relationship between AGEs and EPCR staining. This might be attributable to other regulatory mechanisms that have an effect on EPCR expression in atherosclerosis.

In summary, we found a significantly higher number of fibrotic lesions and lesions with thrombotic complications in plaques from DM2 patients. However, we did not find evidence that this could be related to the increased presence of AGEs and the changed expression of coagulation proteins in diabetic atherosclerotic lesions. It may be that in this end stage of atherosclerosis, no more local differences in the presence of these proteins can be distinguished, although they might have played a role in an earlier stage of the process. Preexisting differences in the expression of coagulation and inflammation markers may have been obscured by the advanced stage of atherosclerosis. Thus, to elucidate the question of whether DM2 leads to a changed expression of inflammation and coagulation markers and whether this plays a role in the accelerated atherosclerotic process, studies of less advanced presymptomatic atherosclerotic lesions may be necessary.

Footnotes

Acknowledgements

HtC is a Clinical Established Investigator of the Netherlands Heart Foundation. CGS is financially supported by a grant from the Diabetes Fonds Nederland.

We thank Angelique P.A. Groot from the Laboratory for Experimental Internal Medicine (AMC) and Wilfried P. Meun from the Department of Pathology (AMC) for their excellent technical assistance.

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