More Fibrosis and Thrombotic Complications but Similar Expression Patterns of Markers for Coagulation and Inflammation in Symptomatic Plaques from DM2 Patients
Laboratory of Experimental Internal Medicine (DWS,AB,BEvA,HtC) and Department of Cardiovascular Pathology (HP,CMvdL,ACvdW), Academic Medical Center, Amsterdam; The Netherlands; Department of Clinical Chemistry, Free University, Amsterdam, The Netherlands (CGS); and Department of Internal Medicine and Cardiovascular Research Institute Maastricht, Academic Hospital and University of Maastricht, Maastricht, The Netherlands (HtC)
Correspondence to: Dirkje W. Sommeijer, MD, Laboratory of Experimental Internal Medicine, G2-108, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail: d.w.sommeijer{at}amc.uva.nl
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Summary |
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(J Histochem Cytochem 52:11411149, 2004)
Key Words: diabetes mellitus type 2 atherothrombotic complications atherectomy tissue factor endothelial protein C receptor advanced glycation end products nuclear factor B
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Introduction |
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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 downregulation 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 B (NF-
B) pathway. NF-
B plays a central role in immune and inflammatory reactions. The usually transient NF-
B-dependent gene expression is exaggerated in pathological situations. NF-
B activation has been observed in atherosclerotic lesions (Brand et al. 1996
). There is evidence that NF-
B 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-B, 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.
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Patients and Methods |
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Immunohistochemistry
Sections were subjected to immunohistochemistry using cell-specific mouse monoclonal antibodies against vascular SMCs (anti--actin), macrophages (anti-CD68), endothelial cells (anti-vWF), EPCR, TF, CML, NF-
B (recognizing total p65 protein), the activated form of NF-
B (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-
B, anti-CD68, anti-CD3, and anti-
-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 streptavidinbiotin complex method was applied, and immunoreactivity was visualized with either diaminobenzidine or 3-amino 9-ethyl carbazole solution. Sections stained with antibodies against vWF,
-actin, CD3, and CD68 were counterstained with hematoxylin. Positive controls were according to literature kidney tissue (anti-TF), stomach epithelium (anti-NF-
B), 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.
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For evaluation of CD3, NF-B, and TF immunostaining, a semiquantitative score approach was chosen using the following criteria: 0, no staining; 1, <10% plaque tissue positive; 2, 1050% 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 2575 quartiles. For comparison between continuous variables of interest, Student's t-test was used. The association between categorical variables was assessed using the MannWhitney test or the Pearson Chi-square when appropriate. p values of <0.05 are considered statistically significant.
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Results |
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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).
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Discussion |
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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-B pathway is a possible mechanism that may contribute to the changed expression of coagulation factors by AGEs (Bierhaus et al. 2001
). But NF-
B activation may also be involved in other inflammatory atherogenic processes (Brand et al. 1996
). The detection of similar staining of activated NF-
B confirms a role for activation of the NF-
B 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.
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Acknowledgments |
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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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Footnotes |
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Received for publication November 18, 2003; accepted April 13, 2004
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