Modification of elastin by pentosidine is associated with the calcification of aortic media in patients with end-stage renal disease
Noriyuki Sakata1,
Akiko Noma2,
Yuji Yamamoto2,
Kouji Okamoto2,
Jing Meng1,
Shigeo Takebayashi1,
Ryoji Nagai3 and
Seikoh Horiuchi3
1 Department of Pathology, School of Medicine, Fukuoka University, Fukuoka, 2 Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Iizuka, Fukuoka and 3 Department of Biochemistry, Kumamoto University School of Medicine, Kumamoto, Japan
Correspondence and offprint requests to: Noriyuki Sakata, MD, Department of Pathology, School of Medicine, Fukuoka University, 45-1, 7-chome Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan. Email: nysakata{at}fukuoka-u.ac.jp
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Abstract
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Background. Calcification of the media of arteries is common in patients with end-stage renal disease (ESRD) undergoing haemodialysis and is a major cause of arteriosclerosis. The aim of this study was to clarify the role of glycoxidative modification of elastin in the calcification of aortic media in this group of patients.
Methods. Samples of tunica media were obtained from non-atherosclerotic areas of the aortas of cadavers of seven non-diabetic patients with ESRD (age 65.5 ± 10.6 years) and 10 age-matched controls (age 61.1 ± 10.3 years). The localization of pentosidine, a major glycoxidation product, and calcium deposits in the media were examined using immunohistochemical and von Kossa staining, followed by orcein staining for elastin fibres. Tissue levels of pentosidine and calcium were measured in elastase-digested media using reversed high-performance liquid chromatography and atomic absorption spectrophotometry, respectively.
Results. In aortic media, but not intima, immunostained pentosidine was observed along elastin fibres or in the extracellular spaces between them. Early calcification was manifest as small punctate calcified deposits along elastin fibres in the media. Advanced calcification was found as large, confluent calcified deposits in extracellular spaces between elastin fibres. Double staining showed co-localization of pentosidine and calcified deposits in the media. Both the staining density of pentosidine and calcification were more prominent in ESRD patients than in controls. The mean medial contents of both elastin-associated pentosidine and calcium were significantly higher in ESRD patients than in controls. In ESRD patients, the level of calcium in elastase-digested media correlated significantly with pentosidine levels, which increased in parallel with the duration of haemodialysis.
Conclusions. Our results indicate that glycoxidative modification of elastin in aortic media may be involved in the enhancement of medial calcification in ESRD patients on haemodialysis.
Keywords: arteriosclerosis; elastin; end-stage renal disease; medial wall calcification; pentosidine
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Introduction
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Most cardiovascular complications in patients with end-stage renal disease (ESRD) on haemodialysis are caused by two distinct arterial disorders: atherosclerosis and arteriosclerosis [1]. Atherosclerosis is defined as an intimal disease of arteries, characterized by the formation of intimal fibrous plaques that often have a central lipid-rich core. Arteriosclerosis, on the other hand, results in diffuse fibroelastic intimal thickening, an increase in medial ground substance and collagen and the fragmentation of elastic lamellae with secondary fibrosis and medial calcification [1]. Calcification can take place in the vascular intima in association with atherosclerosis and in the media due largely to alterations in mineral metabolism [2]. Abnormalities in serum phosphorus, the calciumphosphorus product and parathyroid hormone levels are reported to result in vascular and visceral calcification in patients in chronic renal failure [3]. However, precisely how medial calcification arises in ESRD patients remains unclear.
Glycoxidation products, including pentosidine and N '-(carboxymethyl)lysine, are increased in plasma proteins and the collagen of skin and myocardium in uraemic patients [4,5]. Glycoxidative modification alters structural and functional properties of these proteins [68]. Increased glycation of elastin is related to aortic calcium deposition in diabetic rats [9]. Furthermore, glycated elastin and collagen have been shown to have an increased affinity for metal ions, including iron and copper [7]. We therefore hypothesized that glycoxidative modification of elastin might be related to aortic medial calcification in ESRD patients.
In this study, we measured the quantity of elastin-associated pentosidine and calcium in aortic media without atherosclerotic lesions. We also examined the localization of pentosidine and calcified deposits in media in relation to elastin fibres by immunohistochemical and von Kossa staining techniques and studied the association between glycoxidative modification of elastin and medial calcification in ESRD patients.
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Subjects and methods
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Subjects and samples
Aortic samples were obtained from 17 autopsy cases that included seven non-diabetic ESRD patients and 10 age-matched control subjects. All ESRD patients had undergone haemodialysis with bicarbonate dialysate under systemic heparinization two or three times weekly. Autopsy was performed within 26 h of death. The aorta was slit open longitudinally and the intima was washed with 0.01 M phosphate-buffered saline (PBS) and then examined macroscopically. The materials were taken from areas of thoracic aorta free of atherosclerotic changes, such as fatty streaks, plaques and complicated lesions. These samples were then cut into three pieces for light microscopy, immunohistochemistry and biochemical analysis. The samples for biochemical analysis were rapidly frozen and stored at -40°C until used. Our study conforms to the principles outlined in the Declaration of Helsinki.
Reagents
The Envision immunostaining kit, pronase, anti-smooth muscle actin antibody and horseradish peroxidase-conjugated rabbit IgG antibody were purchased from DAKO (Carpinteria, CA, USA). Albumin-conjugated desmosine and anti-desmosinekeyhole limpet haemocyanin antibody were obtained from Elastin Products Co. (Pacific, MO, USA). Keyhole limpet haemocyanin, Freunds complete and incomplete adjuvants, o-phenylenediamine dihydrochloride, acetonitrile, triethylamine and bacterial collagenase (type VII) were purchased from Sigma Chemical Co. (St Louis, MO, USA). Calcium standard solution was purchased from Wako Co. (Osaka, Japan). Elastase (from porcine pancreas; 326 units/mg) was obtained from Eisai Co. (Tokyo, Japan). No trypsin-like activity was detected in this enzyme preparation. All other chemicals were of the best grade available from commercial sources.
Light microscopy
The samples for light microscopy were fixed with 10% buffered formalin. After dehydration with ethanol, the tissues were embedded in paraffin. Paraffin sections were prepared and subjected to haematoxylin and eosin, Weigerts resorcinol fuchsin, orcein and Masson trichrome stains. To examine the localization of aortic calcium deposits, sections were stained with the von Kossa stain, followed by eosin and orcein stains.
Immunohistochemistry
Polyclonal and monoclonal antibodies against pentosidine were prepared according to a method described previously [10]. In our preliminary study, the staining pattern of pentosidine in the tissue samples was similar whether stained with monoclonal or polyclonal antibodies. Monoclonal antibody yielded a slightly greater degree of positive staining for pentosidine than did polyclonal antibody. Therefore, we used monoclonal anti-pentosidine antibody in this study. Tissue samples for immunohistochemistry were fixed with Carnoys fixative and embedded in paraffin. Immunohistochemical localization of pentosidine was performed using the Envision technique. Briefly, serial tissue sections were de-paraffinized and then subjected to protease pre-treatment (0.05% Pronase) at 37°C for 15 min. After treatment with 2% skim milk for 40 min, slides were incubated overnight with the primary antibody against pentosidine (1.0 µg/ml; monoclonal) at 4°C. After three additional washes in PBS, the slides were subjected to the Envision system using the manufacturers protocol. Negative control tests for specificity of immunostaining included the substitution of non-immune sera or PBS for the primary antibody and did not show any red alkaline phosphatase reaction products in tissue sections. The co-localization of calcium deposits with pentosidine and elastin in aorta was assessed by a double-staining technique. Briefly, in the first step, de-paraffinized sections were incubated with monoclonal anti-pentosidine antibody and anti-smooth muscle actin antibody, as described. After visualization with the alkaline phosphatase colouring reaction, the sections were stained with the von Kossa and orcein stains.
Extraction of collagen and elastin
Aortic media was dissected from the intima and adventitia and cut on ice into small pieces with a razor blade. After the samples of media were rinsed two times (with 4 mmol/l EDTA, 0.1 mol/l phenylmethylsulphonyl fluoride, 1 mmol/l N-ethylmaleimide and 0.1 µg/ml pepstatin A), the collagen and elastin fractions were prepared according to a method described previously [11].
Assay of collagen and elastin contents
Collagen content was determined according to the method of Stegemann and Stalder [12]. The elastin content was determined by measuring desmosine in samples with a competitive enzyme-linked immunosorbent assay [13]. The calculation of elastin content was based on the assumption that desmosine constitutes 0.9% of elastin.
HPLC assay for pentosidine
Pentosidine formation was assessed using high-performance liquid chromatography (HPLC) [4]. Elastin samples were acid-hydrolysed in 2 ml 6 N HCl for 24 h. The acid was evaporated and pentosidine was assayed by HPLC after reconstituting samples with water. HPLC materials consisted of a Shimadzu HPLC (Tokyo) with a 150 x 4.6 mm PRODIGY-ODS C18 (5 µm) analytical column (Showa Chemical Industries, Kyoto, Japan), SCL-10 Avp system controller, LC-10 AT pumps and RF-10A spectrofluorometer. The effluent was monitored at 335/385 nm (excitation/emission). The mobile phase A consisted of 140 mM sodium acetate with triethylamine, titrated to pH 5.05 with phosphoric acid. The mobile phase B consisted of 60% acetonitrile in water. Separations were made by applications of a gradient system of 065% of mobile phase B at a flow rate of 1 ml/min. Synthesized pentosidine was used as a standard.
Calcium content of elastin
The elastin samples were hydrolysed in 1 ml 6 N HCl at 100°C for 24 h. The solution was evaporated completely at 70°C with a continuous flow of nitrogen in the tube. The residue in the test tube was dissolved in 1 ml distilled water. Calcium content was quantified by atomic absorption spectrophotometry (Atomic Absorption Spectrophotometer Model Z-8000; Hitachi, Tokyo), using calcium standards in buffer [14].
Statistical analysis
Numerical data were expressed as means ± SD. The MannWhitney U-test was used to evaluate differences between the two groups. Pearsons correlation was used to identify correlations between different variables, including pentosidine and calcium levels and duration of haemodialysis. A P-value of <0.05 was considered significant.
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Results
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Subjects
The clinical and laboratory characteristics are shown in Table 1. The mean ages were 65.5 ± 10.6 and 61.1 ± 10.3 years for ESRD patients and controls, respectively. The mean duration of haemodialysis was 8.42 ± 6.00 years in ESRD patients. Significant differences were observed between the two groups in the levels of blood urea nitrogen (BUN), serum creatinine and systolic blood pressure. ESRD patients trended towards decreased levels of red blood cells, haemoglobin and haematocrit and increased levels of triglycerides and phosphorus, but the differences were not significant. The levels of other parameters, including calcium, fasting blood sugar, body mass index, total cholesterol and C-reactive protein, were not different between the two groups. Table 2 shows pathological diagnoses and the severity of aortic atherosclerosis. No pathological evidence of diabetes mellitus was detected in any subject. The degree of plaque formation, calcification and ulceration in the aorta was significantly greater in ESRD patients than in control subjects.
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Table 2. Renal diseases, pathological diagnoses and degree of aortic atherosclerosis in ESRD and non-ESRD patients enrolled in the study
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Immunohistochemistry of pentosidine
Figure 1 shows the immunohistochemical localization of pentosidine in an aorta. Positive staining for pentosidine was observed in the media in both groups, whereas immunoreactivity for pentosidine in the intima was positive in most ESRD patients but not in controls (Figure 1A and B). As shown in Figure 1D, controls exhibited faint and scattered immunoreactivity for pentosidine in the media. In contrast, ESRD patients showed more extensive immunoreactivity for pentosidine in the media (Figure 1C). Pentosidine was mainly localized along elastin fibres or in the areas between elastin fibres in both groups (Figure 1C and D).

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Fig. 1. Localization of pentosidine (AD) and calcium deposits (EH) in the aorta of an ESRD patient and a control. Specimens were obtained from a 62-year-old ESRD patient (A, C, E and G) and a 59-year-old non-ESRD control (B, D, F and H). Pentosidine was stained immunohistochemically using monoclonal anti-pentosidine antibody and the Envision technique. Calcium deposits were identified with von Kossa staining, followed by eosin. Intima was positive for pentosidine and calcium deposits in the ESRD patient (A and E), but not in the control (B and F). Both the ESRD patient and the control exhibited accumulation of pentosidine and calcified deposits along elastin fibres or in the areas between them. Both pentosidine staining and calcified deposits in the media were greater in the ESRD patient (C and G) than in the control (D and H). I, intima; M, media. Bar: 100 µm.
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Calcification
As shown in Figure 1, early calcified deposits were small and punctate and are here referred to as stippling calcification (Figure 1H). With advanced calcification there is an increase in the number and size of calcified deposits, which are large and confluent, here described as morula calcification (Figure 1G). In the control group, some cases showed stippling calcification in the media, but not in the intima (Figure 1F and H). Conversely, calcification was increased in both the intima and media of most ESRD patients, compared with controls (Figure 1E and G). ESRD patients showed both stippling and morula calcifications diffusely in extracellular spaces between elastin fibres (Figure 1G).
Co-localization of calcified deposits, pentosidine and elastin fibres
Figure 2 illustrates co-localization of calcium deposits with pentosidine and elastin fibres. As shown in Figure 2A and B, pentosidine coexisted with calcium deposits in the media. Double staining with von Kossa and orcein stains showed that the small, punctate calcified deposits were invariably associated with elastin fibres (Figure 2D, arrows). Intermediate or late calcifications appeared in extracellular spaces between elastin fibres as morula formations (Figure 2D, asterisks). Smooth muscle cells were rarely observed in areas of calcification (Figure 2E).

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Fig. 2. Co-localization of pentosidine and calcium deposits in association with elastin fibres in the aortic media of an ESRD patient. The specimen was obtained from a 79-year-old ESRD patient. Double staining of calcified deposits with pentosidine (B), elastin fibres (D) and smooth muscle cells (E) was performed as described in the Subjects and methods. Pentosidine (A) and calcified deposits (B) were identified by immunostaining with anti-pentosidine monoclonal antibody and von Kossa staining, respectively. Note co-localization of pentosidine and calcified deposits in the media (A and B). Early calcification was found mainly along elastin fibres as small punctate calcified deposits (stippling form, arrows). Large, confluent calcified deposits (morula form, asterisks) were found in extracellular spaces between elastin fibres. Smooth muscle cells (E) were rarely associated with calcifications. Bar: 100 µm.
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Biochemical analysis
Table 3 shows the collagen and elastin contents of aortas of ESRD patients and controls. Collagen and elastin contents extracted from aortic media did not differ between the two groups. The mean proportion of collagen and elastin in aortic tissues was 64.9% and 65.9%, in patients and controls, respectively. Figure 3 shows representative chromatograms of synthesized pentosidine. The retention time of the peak of synthesized pentosidine was 11.3 min. Its peak area increased in a dose-dependent manner (Figure 3, inset). Figures 4 and 5, respectively, show the mean contents of elastin-associated pentosidine and calcium in aortic media. The amount of elastin-associated pentosidine varied widely among the patients but the mean value (88.0 ± 97.5 pmol/mg elastin) was significantly higher than in the controls (15.6 ± 12.4 pmol/mg elastin; P < 0.01). Furthermore, calcium content in the elastin fraction was significantly higher in ESRD patients (9.87 ± 4.60 µg/mg elastin) than in the controls (3.57 ± 4.16 µg/mg elastin; P < 0.05). There was a significant correlation between pentosidine and calcium levels in the elastin fraction of aortic media in ESRD patients (r = 0.79, P < 0.05; Figure 6A), but not in controls (Figure 6B). Furthermore, the duration of haemodialysis correlated significantly with pentosidine levels (r = 0.82, P < 0.05; Figure 7A), but not with calcium levels (Figure 7B) in elastin of the aortic media.

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Fig. 3. Representative profile of HPLC chromatography of synthesized pentosidine. Retention time of the peak of synthesized pentosidine is 11.3 min. Its peak area increases in a dose-dependent manner (inset). (a) 0.1 nmol/l, (b) 1.0 nmol/l and (c) 10 nmol/l.
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Fig. 4. Pentosidine content of the elastin fraction in ESRD patients (ESRD) and controls (Cont). The pentosidine content was determined by HPLC. The pentosidine content of the elastin fraction was significantly greater in ESRD patients than in the controls. Values are expressed as means ± SD (pmol/mg elastin). *P < 0.05 vs control.
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Fig. 5. Calcium content of the elastin fraction in ESRD patients (ESRD) and controls (Cont). The calcium content was determined by atomic absorption spectrophotometry. The calcium content of the elastin fraction was significantly greater in ESRD patients than in controls. Values are expressed as means ± SD (µg/mg elastin). *P < 0.05 vs control.
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Fig. 6. Correlation between levels of pentosidine and calcium in the elastin fraction of media in ESRD patients (A) and controls (B). There was a significant correlation between levels of pentosidine and calcium in the elastin fraction of media of ESRD patients (r = 0.79, P < 0.05), but not of the control.
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Fig. 7. Correlation of the duration of haemodialysis with levels of pentosidine (A) and calcium (B) in the elastin fraction of media in ESRD patients. The level of pentosidine (r = 0.82, P < 0.05), but not calcium, correlated significantly with duration of haemodialysis.
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Discussion
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The main findings of this study were the increased elastin-associated pentosidine and calcium in the media of atherosclerosis-free aortas in ESRD patients on haemodialysis. This observation raises important questions concerning the role of impaired pentosidine metabolism in the calcification of the media of the aortic wall in those patients, irrespective of atherosclerosis.
Vascular calcification is enhanced in ESRD patients [1] and can arise from calcification of the intimal layer of arteries as a result of atherosclerosis or of the medial layer due largely to alterations in mineral metabolism [2,3]. However, it is uncertain whether elastin contributes to the medial calcification of aorta in ESRD patients. In this study, ESRD patients showed an enhancement of aortic atherosclerosis, including calcification, ulceration and plaque formation. At the same time, compared with controls these patients had increased calcified deposits in atherosclerosis-free media. The early calcium deposits (stippling calcification) were often observed along elastin fibres of aortic media. The calcium content of elastin extracted from atherosclerosis-free media was significantly greater in ESRD patients than in controls. These results indicate that elastin-associated calcification of the aortic media might be enhanced in ESRD patients, irrespective of atherosclerosis. Collagen is another abundant structural protein in the arterial wall and is prone to calcification [15]. In particular, morula deposits were found in extracellular spaces between elastin fibres in aortic media, indicating that calcification can also involve collagen fibres. However, this study could not determine the calcium content of the collagen fraction, as this fraction contained calcium ions added in vitro to enhance collagenase activity. Further studies are needed to elucidate the involvement of other components in medial calcification in ESRD patients.
Proteins in long-lived tissue components, such as collagen, lens crystalline and elastin, are known to be modified by glycation, resulting in the formation of advanced glycation end-products (AGEs) associated with aging, diabetes and renal dysfunction [16,17]. AGEs, generated by glycation and oxidation, are recognized as glycoxidation products, such as N'-(carboxymethyl)lysine and pentosidine. Based on several reports [5,18], these products increase in the plasma and tissue collagen of uraemic patients, suggesting their involvement in the development of long-term complications associated with chronic renal failure and dialysis. However, little is known about the modification of aortic elastin by glycoxidation in ESRD. In this study, immunohistochemical analysis revealed pentosidine staining along elastin fibres and in the extracellular spaces between these fibres. The staining intensity and pentosidine-positive area in aortic media were increased in ESRD patients. HPLC analysis showed the significantly higher pentosidine content of the elastin fraction in ESRD patients compared with controls. These results are indicative of enhanced modification of elastin fibres in aortic media by pentosidine in ESRD patients.
Glycoxidation can alter structural and functional properties of various tissue components, including collagen, fibronectin and elastin [68]. Elastin is implicated in various arterial diseases (including arteriosclerosis, atherosclerosis and aneurysm) either through the loss of its mechanical properties or because it acts as a substrate for calcium and lipid deposition. Calcium deposits have been shown to increase in association with an accumulation of glycated elastin in aortas of diabetic rats [9]. A significant correlation between aortic stiffness and increasing glycation of connective tissues and their components (including collagen and elastin) has been shown in diabetes, hypertension and chronic renal failure [19,20]. In vitro glycation of elastin increases its stiffness and binding activity to transition metals [6,7]. In our study, the samples were taken from non-atherosclerotic areas of the aorta to avoid the possibly confounding influence of atherosclerosis. ESRD patients had more pentosidine and calcium in the elastin of their aortic media than controls. Double staining revealed co-localization of pentosidine and calcium deposits in media, which were detected along and between elastin fibres. Finally, the level of elastin-derived calcium significantly correlated with that of elastin-associated pentosidine in aortic media of ESRD patients. The latter increased with the duration of haemodialysis in these patients. In our preliminary study, electron microscopy revealed an increased accumulation of dense materials around elastin fibres in aortic media in ESRD patients. Thus, we speculate that the marked increase in elastin-associated pentosidine might be related to the enhancement of medial calcification of the aortas of ESRD patients.
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Acknowledgments
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This work was supported in part by a Grant-in-Aid for Scientific Research (C) (no. 14570171) from the Ministry of Education, Science and Culture of Japan and in part by funds from the Central Research Institute of Fukuoka University.
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References
|
---|
- London GM, Drüeke TB. Atherosclerosis and arteriosclerosis in chronic renal failure. Kidney Int 1997; 51: 16781695[ISI][Medline]
- Goodman WG, Salusky IB. Non-invasive assessments of cardiovascular disease in patients with renal failure. Curr Opin Nephrol Hypertens 2001; 10: 365369[CrossRef][ISI][Medline]
- Block GA, Port FK. Re-evaluation of risks associated with hyperphosphatemia and hyperparathyroidism in dialysis patients: recommendations for a change in management. Am J Kidney Dis 2000; 35: 12261237[ISI][Medline]
- Miyata T, Taneda S, Kawai R et al. Identification of pentosidine as a native structure for advanced glycation end products in beta2-microglobulin-containing amyloid fibrils in patients with dialysis-related amyloidosis. Proc Natl Acad Sci USA 1996; 93: 23532358[Abstract/Free Full Text]
- Meng J, Sakata N, Imanaga Y, Takebayashi S, Nagai R, Horiuchi S. Carboxymethyllysine in dermal tissues of diabetic and nondiabetic patients with chronic renal failure: relevance to glycoxidation damage. Nephron 2001; 88: 3035[CrossRef][ISI][Medline]
- Winlove CP, Parker KH, Avery NC, Bailey AJ. Interactions of elastin and aorta with sugars in vitro and their effects on biochemical and physical properties. Diabetologia 1996; 39: 11311139[ISI][Medline]
- Qian M, Liu M, Eaton JW. Transition metals bind to glycated proteins forming redox active glycochelates: implications for the pathogenesis of certain diabetic complications. Biochem Biophys Res Comm 1998; 250: 385389[CrossRef][ISI][Medline]
- Sakata N, Sasatomi Y, Ando S et al. Causal relationship between conformational change and inhibition of domain functions of glycoxidative fibronectin. Conn Tissue Res 2000; 41: 117129[ISI][Medline]
- Tomizawa H, Yamazaki M, Kunika K, Itakura M, Yamashita K. Association of elastin glycation and calcium deposit in diabetic rat aorta. Diabetes Res Clin Practice 1993; 19: 18[CrossRef][ISI][Medline]
- Kato S, Horiuchi S, Liu J et al. Advanced glycation endproduct-modified superoxide dismutase-1 (SOD1)-positive inclusions are common to familial amyotrophic sclerosis patients with SOD1 gene mutations and transgenic mice expressing human SOD1 with a G85R mutation. Acta Neuropathol 2000; 100: 490505[CrossRef][ISI][Medline]
- Yamamoto Y, Meng J, Sakata N et al. Possible involvement of increased glycoxidation and lipid peroxidation of elastin in atherogenesis in haemodialysis patients. Nephrol Dial Transplant 2002; 17: 630636[Abstract/Free Full Text]
- Stegemann H, Stalder K. Determination of hydroxyproline. Clin Chim Acta 1967; 18: 267273[CrossRef][ISI][Medline]
- Osakabe T, Seyama Y, Yamashita S. Comparison of ELISA and HPLC for determination of desmosine or isodesmosine in aortic tissue elastin. J Clin Lab Anal 1995; 9: 293296[ISI][Medline]
- Vyavahare N, Ogle M, Schoen FJ, Levy RJ. Elastin calcification and its prevention with aluminum chloride pretreatment. Am J Pathol 1999; 155: 973982[Abstract/Free Full Text]
- Schoen FJ, Levy RJ, Nelson AC, Bernhard WF, Nashef A, Hawley M. Onset and progression of experimental bioprosthetic heart valve calcification. Lab Invest 1985; 52: 523532[ISI][Medline]
- Beisswenger PJ, Moore LL, Brink-Johnsen T. Increased collagen-linked pentosidine levels and advanced glycosylation end products in early diabetic nephropathy. J Clin Invest 1993; 92: 212217[ISI][Medline]
- Chen JR, Takahashi M, Kushida K et al. Direct detection of crosslinks of collagen and elastin in the hydrolysates of human yellow ligament using single-column high performance liquid chromatography. Anal Biochem 2000; 278: 99105[CrossRef][ISI][Medline]
- Miyata T, Ueda Y, Saito A, Kurokawa K. Carbonyl stress and dialysis-related amyloidosis. Nephrol Dial Transplant 2000; 15 [Suppl]: 125128[Free Full Text]
- Sims TJ, Rasmussen LM, Oxlund H, Bailey AJ. The role of glycation cross-links in diabetic vascular stiffening. Diabetologia 1996; 39: 946951[CrossRef][ISI][Medline]
- Mizutani K, Ikeda K, Kawai Y, Yamori Y. Biomechanical properties and chemical composition of the aorta in genetic hypertensive rats. J Hypertens 1999; 17: 481487[CrossRef][ISI][Medline]
Received for publication: 15. 3.02
Accepted in revised form: 31. 1.03