Morphology of coronary atherosclerotic lesions in patients with end-stage renal failure

Ute Schwarz1,3, Moriz Buzello3, Eberhard Ritz1, Günter Stein2, Gerd Raabe5, Gabriele Wiest4, Gerhard Mall4 and Kerstin Amann3

1 Departments of Internal Medicine, Heidelberg, 2 Department of Internal Medicine, Jena, 3 Department of Pathology, Heidelberg, 4 Department of Pathology, Darmstadt and 5 Department of Pathology, Jena, Germany

Correspondence and offprint requests to: Prof. Dr Kerstin Amann, Department of Pathology, University of Erlangen-Nürnberg, Krankenhausstr. 8–10, D-91054 Erlangen, Germany. e-mail: kerstin.amann{at}patho.med.uni-erlangen.de.



   Abstract
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Background. An excessive rate of cardiac death is a well-known feature of renal failure. Coronary heart disease is frequent and the possibility has been raised that the natural history of the coronary plaque is different in uraemic patients. We assessed the morphology of coronary arteries in patients with end-stage renal failure and compared them with coronary arteries of matched non-uraemic control patients.

Methods. Fifty-four cases were identified at autopsy who met the inclusion criteria: cases, end-stage renal disease (n=27); controls, non-renal patients with coronary artery disease (n=27). At autopsy all three coronary arteries were prepared at corresponding sites for investigations: (i) qualitative analysis (after Stary), (ii) quantitative measurements of intima and media thickness (by planimetry), (iii) immunohistochemical analysis of the coronary plaques and (iv) X-ray diffraction of selected calcified plaques.

Results. Qualitative analysis of the coronary arteries showed significantly more calcified plaques of coronary arteries in patients with end-stage renal failure. Plaques of non-uraemic patients were mostly fibroatheromatous. Media thickness of coronary arteries was significantly higher in uraemic patients (187±53 µm vs 135±29 µm in controls) and intima thickness tended to be higher (158±38 µm vs 142±31 µm) but this difference was not statistically significant. Plaque area (4.09±1.50 mm2 vs 4.39±0.88 mm2) was comparable in both groups. Lumen area, however, was significantly lower in end-stage renal patients. Immunohistochemical analysis of the cellular infiltrate in coronary arteries showed no major differences in these advanced plaques of uraemic and non-uraemic subjects.

Conclusion. Coronary plaques in patients with end-stage renal failure are characterized by increased media thickness and marked calcification. In contrast to the previous opinion the most marked difference compared to non-uraemic controls does not concern the size, but the composition of the plaque. Deposition of calcium within the plaques may contribute to the high complication rate in uraemic patients.

Keywords: atherosclerosis; uraemia; coronary artery disease; plaque structure; calcification



   Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Cardiovascular events are the most frequent cause of death in patients with chronic renal failure [1,2]. The risk is 20-fold higher than in the background population. It is thus even much higher than in diabetic patients, a group with widely quoted excessive cardiac risk. Lindner et al. [3] advanced the hypothesis that the high rate of myocardial infarction resulted from accelerated atherogenesis and inferred that the probability of dying from cardiovascular diseases approached 90% by the 13th year of haemodialysis. The concept of accelerated atherogenesis would predict that the rate of ischaemic heart disease should progress with increasing duration on haemodialysis, but this is not seen in observational studies. Autopsy [4,5] and clinical investigations [6,7] documented that the prevalence of coronary plaques was higher in dialysed patients compared to matched non-uraemic patients.

Many risk factors may contribute to the high prevalence of atherosclerotic lesions in uraemia, i.e. dyslipidemia, particularly increased Lp(a) concentrations, hyperhomocysteinaemia, hypertension, increased PTH concentrations and others. The question arises whether the evolution of coronary plaque is different in uraemia. Different composition of the plaque might also explain the excessively high reocclusion rate after PTCA, i.e. 70% within 1 year in uraemic patients compared to 40% in diabetic patients and 20% in non-diabetic patients [8]. These considerations prompted us to perform a controlled postmortem study, comparing coronary plaque size and composition in uraemic patients and age and sex matched non-uraemic patients. Although the study design does not answer the question whether atherosclerosis is accelerated in renal failure it should provide additional data on plaque morphology.



   Material and methods
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Patient selection and evaluation (Table 1Go)
Between April 1994 and December 1995, 54 consecutive cases with coronary atherosclerosis were identified at postmortem in the Departments of Pathology Darmstadt (Germany) and Jena (Germany). Patients with end-stage renal disease (ESRD), i.e. predialysis or on dialysis, and coronary atherosclerosis were included and information concerning risk factors, e.g. hypertension, smoking or diabetes mellitus was obtained from hospital records. In addition, causes of death were recorded and analysed. The patients were compared with non-renal patients with coronary atherosclerosis who were matched for age and gender (Table 1Go).


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Table 1. Patient and clinical data
 
Tissue preparation
During autopsy the hearts of the patients were examined by a pathologist and specimens of all three coronary arteries were obtained at comparable sites: ramus circumflexus of the left coronary artery (LCX), right coronary artery (RCA), ramus interventricularis anterior of the left coronary artery (RIA). The segment of the vessel with the most severe stenosis was selected for histological examination. The tissues were dissected into a few slices of 2–3 mm width. For morphological investigation tissue was fixed in formaldehyde 8%, embedded in paraffin, sectioned (4 µm slices) and stained (haematoxylin and eosin; Kossa technique for demonstration of phosphates; Elastica-van Gieson for detection of fibrous tissue).

Immunohistological investigations (Table 2Go)
A specimen of the coronary artery was also shock frozen in liquid nitrogen, sectioned (5 µm) and examined immunohistochemically as described elsewhere [9].


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Table 2. List of antibodies used for immunohistochemical staining of coronary arteries
 
Antibodies with a potential role in the pathogenesis of atherosclerosis were used in order to characterize the cellular infiltrate and the expression of surface antigens (see Table 2Go). In a pilot study we evaluated for each antibody in a dilution series the concentration which yielded optimal staining. The sections were analysed using a semiquantitative score system in a blinded fashion (light microscopy; magnification x200). The degree of staining was ranked on an arbitrary scale: grade 0, no staining; grade 1, faintly positive staining; grade 2, positive staining involving up to 50% of the field of view; grade 3, positive staining involving more than 50%; grade 4, positive staining of all structures within the field of view [9].

Semiqualitative evaluation of the coronary arteries (Table 3Go)
On the basis of composition and structure of plaques and degree of intimal injury atherosclerotic plaques can be categorized into eight classes [10]. The histological sections of the coronary arteries of each patient were investigated using haematoxylin and eosin stained paraffin sections. Using light microscopy (x25 magnification) the lesions were scored according to Stary (Table 3Go). For evaluation of the presence of calcified areas and elastic tissue in coronary arteries, Kossa and Elasticalvan Giesson stained paraffin sections were investigated as well.


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Table 3. Categorization of atherosclerotic lesions according to Stary (10)
 
Quantitative evaluation of the coronary arteries
Intima and media thickness, lumen and plaque area were determined on paraffin sections (magnification x25) using planimetry and a semiautomatic image analysing system (IBAS II, Kontron Co., Eching, Germany).

X-ray diffraction analysis
Of selected patients (n=5) calcified areas were investigated using X-ray diffraction analysis. Calcified plaques were freed of soft tissue and kept frozen. For analysis the specimens were dried at room temperature or heated at 130°, 200°, 500° and 900°C in a muffle furnace, or both. The dried tissues were ground to fine powder and mounted on a glass slide for diffraction studies. Powder X-ray diffraction studies were performed with an automized Philips Norelco diffractometer (CuK{alpha}1+2, Ni-filter, scintillation counter; courtesy Department of Mineralogy, University Heidelberg, Germany). The diffraction range covered was 12° to 60° at a scanning speed of 1°/min. Qualitative phase analysis was carried out using the PDF 1 database (powder diffraction file) containing XRD reference patterns for more that 60000 inorganic compounds.

Statistics
Data are given as mean±SD. Mann–Whitney test or chi square test for pair differences were chosen to test whether the differences between the two groups were significant. The working hypothesis was rejected at a P<0.05.



   Results
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Patients (Table 1Go)
Age, body weight and gender distribution were comparable in the two groups as were the frequencies of hypertension and diabetes. Patients without renal disease had a slightly higher number of non-fatal and fatal myocardial infarctions. However, the number of deaths from cardiac failure of unknown causes (presumably due to sudden cardiac death from arrhythmias) was significantly higher in patients with end stage renal failure.

Categorization of the coronary plaques (Table 2Go, Figure 1Go)
Scoring of the coronary plaques showed more advanced, i.e. calcified lesions in patients with ESRD (Table 1Go). In non-renal control patients fibroatheroma (seven patients) and atheroma (two patients) were frequent. In the majority of patients with ESRD (n=17) plaques were heavily calcified (Figure 1Go).



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Fig. 1. Coronary artery plaque of uraemic patient with moderate calcification. Kossa stain; magnification: 1:100.

 
Quantitative analysis (Table 3Go, Figure 2Go)
Media thickness was significantly higher in patients with ESRD compared to controls. In contrast, intima thickness was not significantly different between the two groups nor was extension of plaques, i.e. plaque area. Lumen area, however, was significantly lower in patients with end-stage renal failure.



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Fig. 2. Representative coronary arteries of a non-renal control patient (A) and a patient with end stage renal disease (B). Please note thickening of the media and intima in renal disease. Elastica-van-Gieson stain; magnification: 1:200.

 
X-ray diffraction (Figure 3Go)
The X-ray analysis of the calcified plaques of patients with ESRD showed hydroxylapatite and calciumphosphate, but no calciumoxalate crystals.



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Fig. 3. Demonstration of hydroxylapatite by X-ray diffraction analysis in an atheromatous coronary plaque of a hyperphosphatemic dialysis patient. (Courtesy of Dr Torvar, Department of Mineralogy, University of Heidelberg, Germany.).

 
Immunohistological analysis (Figure 4Go)
Except for the expression of {alpha}-smooth muscle actin (Figure 4Go) which was significantly higher in vessels of end-stage renal patients no significant differences in the antibody pattern were noted.



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Fig. 4. Markedly enhanced expression of {alpha}-smooth muscle actin in the media and intima of the coronary artery of a patient with end-stage renal failure. Immunohistochemistry; magnification: 1:150.

 


   Discussion
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
The present study was designed to compare morphology and immunohistology of coronary arteries and coronary plaques of patients with ESRD and matched non-renal patients who had died from cardiac causes in the presence of documented coronary disease.

There is very little detailed information available in the literature concerning plaque morphology in uraemic patients. By coronarography 30–40% of patients [6,7,11], particularly diabetic patients [12] are found to have coronary lesions. This frequency corresponds approximately to the frequency that has been noted in autopsy studies [4]. Ibels [13] provided an analysis of the morphology of large elastic arteries; he noted marked intimal hyperplasia and striking medial calcification. Experimental data are not available because the usual animal models of uraemia do not develop spontaneous coronary atherosclerosis.

Although the present study does not provide an answer to the question of accelerated atherosclerosis in renal failure it clearly documents important differences between patients with ESRD and matched non-renal control patients concerning plaque morphology. The plaques of uraemic patients were significantly more severe according to the classification of Stary [10]. While plaques of non-renal patients used to be mostly fibroatheromatous, those of uraemic patients were usually heavily calcified. This information is of particular interest in view of the recent development of non-invasive techniques to monitor coronary calcification and its progression, e.g. using electron beam CT [14] to detect and grade calcified coronary atheroma. Using this technique, a high prevalence and rate of progression of coronary plaques has been noted in chronic dialysis patients [15]. In addition, plaque morphology is important for therapeutic strategy options in renal patients, i.e. treatment of coronary artery stenosis by PTCA or by bypass grafting.

In view of elevated plasma oxalate concentrations in renal failure [16], the question arose whether calcium was deposited as phosphate or as oxalate. Our data are clearcut and point to phosphate as the culprit. This information is important because in a large analysis based on 7096 dialysis patients hyperphosphataemia has recently been recognized as an independent predictor of overall mortality and particularly of death from coronary causes [17]. Heavy calcification of the plaques may also explain the notoriously high reocclussion rate following percutaneous transluminal coronary artery dilatation [8]. Unfortunately, we do not have data on serum levels of calcium, phosphate and PTH in all of our patients.

In the past, examining small resistance arteries and large elastic arteries of uraemic animals we noted thickening of the media layer [18,19]. We were able to show that this reflects both hypertrophy and hyperplasia of vascular smooth muscle cells [20,21]. The present analysis shows significantly greater media thickness in the coronary conduit arteries as well. This finding confirms findings of Demuth [22] and Barenbrock [23] in the aorta and the A. carotis and may have functional implications since vascular smooth muscle cells have been identified as important players in the evolution of the plaque [24]. A number of studies using different methodological approaches have documented endothelial cell dysfunction in uraemic animals or patients, respectively [25]. Against this background it is of interest that intima thickness, in contrast to previous observations of Ibels in elastic arteries [13] was not significantly altered in the coronaries of the patients under study.

It has recently been recognized that coronary plaques are dynamic structures reflecting a state of microinflammation, with a number of intermediary steps, the final outcome of which depends, among others, on the balance between proteolytic activity, mainly via activated macrophages, and protein synthesis, mainly generating a reparative fibrous cap. We probed a number of indicators, using immunohistochemical techniques, but failed to note major differences in these relatively advanced lesions between uraemic and non-renal controls.

Due to the study design (case-control study) our analysis does not directly address the hypothesis that atherogenesis is accelerated in end-stage renal failure [3]. It deserves comment, however, that the observations of more advanced transformation, but not greater extension of the plaque, does not directly support this hypothesis. Particularly, we failed to note a greater number of early lesions. We acknowledge, however, that the present sample is small and the biostatistical power of the analysis is limited. In addition, a potential sample bias has to be taken into consideration since only the most advanced lesion per vessel was investigated. However, cohort studies using the now available echo colour Doppler technique may be useful to compare the progression of atherosclerosis in uraemic patients with non-uraemic subjects sharing the same cardiovascular risk profile at baseline.

It is of further note that in our population we had a very low number of smokers. This is of particular interest since smoking is a well known confounder in clinical studies.

Taken together our morphological study of coronary plaques documents increased media thickness and the common presence of advanced stages of coronary plaques with heavy calcification in patients with end-stage renal failure.


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Table 4. Quantitative morphological analysis of intima- and media changes and of plaque size of coronary arteries
 


   Acknowledgments
 
Dedicated to Prof. A. Davison on the occasion of his 60th birthday. This work was supported by the `Else- Kröner Freseniusstiftung' and in part by the Deutsche Forschungsgemeinschaft (Am 93/2-2/3). Dr Ute Schwarz and Dr Moriz Buzello were supported by a scholarship from the `Graduiertenkolleg Nieren- und Kreislaufregulation' of Deutsche Forschungsgemeinschaft. Part of the results were presented at the `28. Kongress der Deutschen Gesellschaft für Nephrologie 1997' (Nieren Hochdruckkrankheiten 12: 435A, 1997). The contribution of Dr M. Torvar (Department of Mineralogy, University of Heidelberg) in performing X-ray diffraction analysis and the skillful technical assistance of G. Gorsberg, D. Lutz and H. Ziebart is gratefully acknowledged.



   References
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 

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