elastin and pentosidine

The most serious late complication of ageing and diabetes mellitus follow similar patterns in the dysfunction of retinal capillaries, renal tissue, and the cardiovascular system. The changes are accelerated in diabetic patients owing to hyerglycaemia and are the major cause of premature morbidity and mortality. These tissues and their optimal functioning are dependent on the integrity of their supporting framework of collagen. It is the modification of the properties by glycation that results in many of the damaging late complications. Initially glycation affects the interactions of collagen with cells and other matrix components, but the most damaging effects are caused by the formation of glucose-mediated intermolecular cross-links. These cross-links decrease the critical flexibility and permeability of the tissues and reduce turnover. In contrast to the renal and retinal tissue, the cardiovascular system also contains a significant proportion of other fibrous connective tissue protein elastin, and its properties are similarly modified by glycation. The nature of these glycation cross-links is now being unravelled and this knowledge is crucial in any attempt to inhibit these deleterious glycation reactions.

Topics: Aging; Animals; Arginine; Collagen; Cross-Linking Reagents; Diabetes Complications; Diabetes Mellitus; Elastin; Glycation End Products, Advanced; Glycosylation; Humans; Imidazoles; Lysine; Maillard Reaction; Malondialdehyde; Norleucine; Pyrroles

Among tissue grafts used for reconstruction of the anterior cruciate ligament (ACL), the pateller tendon (PT) and semitendinosus tendon (ST) are commonly used. It was thought that there were differences in the biochemical composition and process of healing between PT and ST. The aim of this study was to investigate the biochemical difference between ACL and the graft tissues used for reconstruction of the ACL. Hydroxyproline and crosslinks of collagen and elastin were measured from samples of 29 knees from cadavers preserved in formalin solutions. The results of measurements were hydroxyproline: ACL 0.522, PT 0.577, ST 0.463 (micromol/mg dry weight); pyridinoline/collagen: ACL 0.381, PT 0.272, ST 0.244 (mol/mol); and pentosidine/collagen: ACL 0.0434, PT 0.0558, ST 0.0799 (mol/mol). The biochemical properties of PT was not so different from ST. Pentosidine also was measured in the present study to aid in the comparison of the ligament and tendons of the knee joint.

Topics: Age Factors; Amino Acids; Anterior Cruciate Ligament; Arginine; Chromatography, High Pressure Liquid; Collagen; Elastin; Humans; Hydroxyproline; Lysine; Patellar Ligament

The aim of this study was to detect crosslinks of collagen and elastin in formalin-fixed tissue, to perform quantification of these crosslinks, and to investigate the effects of formalin fixation on crosslink contents in human yellow ligament and cartilage. Pyridinoline (Pyr) is a stable and nonreducible crosslink of collagen. Pentosidine (Pen) is a senescent crosslink formed between arginine and lysine in matrix proteins, including collagen. Desmosine (Des) and its isomer isodesmosine (Isodes) are crosslinks specifically found in elastin. It is useful to measure crosslink contents of collagen and elastin as a way of investigating the properties of various tissues or their pathological changes. If it is possible to evaluate crosslinks of collagen and elastin in formalin-fixed tissues, we can investigate crosslinks in a wide variety of tissues. We used HPLC to compare the concentrations of Pyr, Pen, Des, and Isodes in the formalin-fixed tissues with their concentrations in the frozen tissues. Pyr and Pen were detected in both the formalin-fixed yellow ligament and the cartilage, and their concentrations were not significantly affected by or related to the duration of formalin fixation. Des and Isodes were detected in the formalin-fixed yellow ligament but in significantly lower amounts compared to the frozen samples. We concluded that crosslinks of collagen were preserved in formalin, but crosslinks of elastin were not preserved in it. The reason for this might be that formalin did not fix elastin tissues sufficiently or it destroyed, masked, or altered elastin crosslinks.

Topics: Aged; Aged, 80 and over; Amino Acids; Arginine; Cartilage; Chromatography, High Pressure Liquid; Collagen; Cross-Linking Reagents; Desmosine; Elastin; Formaldehyde; Freezing; Hip Joint; Humans; Isodesmosine; Ligaments; Lysine; Tissue Fixation

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.. 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.. 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.. 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.

Topics: Aged; Aorta; Arginine; Calcinosis; Calcium; Elastin; Female; Humans; Immunohistochemistry; Kidney Failure, Chronic; Lysine; Male; Middle Aged; Tunica Media

Previous studies have shown that biomechanical analysis of aorta from diabetic subjects reveals a marked increase in stiffness compared to aorta from age-matched control subjects. In the present paper we have proposed that this increased stiffness can be attributed to glycation-induced inter-molecular cross-links based on a direct analysis of the two known glycation cross-links, the fluorescent pentosidine and the non-fluorescent NFC-1. There was a significant difference in the increase in concentration of both cross-links with increasing age for both the intima (p < 0.0025) and the media (p < 0.0005) from the diabetic compared to the control subjects, but no correlation with the mature enzymic cross-link hy droxylysyl-pyridinoline. Finally, we have obtained a significant correlation of stiffness with both glycation cross-links (NFC-1, r = 0.86; p < 0.005 and pentosidine r = 0.75, p < 0.05), but the concentration of NFC-1 is about 50 times greater than that of pentosidine, indicating that it is the major glycation cross-link responsible for the stiffening of the aorta.

Topics: Adult; Aged; Aging; Amino Acids; Aorta, Thoracic; Arginine; Collagen; Cross-Linking Reagents; Diabetes Mellitus, Type 1; Diabetic Angiopathies; Elastin; Glycation End Products, Advanced; Humans; Lysine; Middle Aged; Time Factors

Stiffening of blood vessel walls occurs in the early stages of atherosclerosis, and this process is known to occur earlier in diabetic subjects. The effect could be due, in part, to glycation. Although collagen is responsible for ensuring the ultimate tensile strength of the tissue, elastin largely determines the compliance of the vessel wall in the normal physiological range of pressures and this appears to be closely matched to haemodynamic requirements. Changes in elastin are therefore likely to affect optimal function of the tissue. We have investigated the susceptibility of elastin to glycation and effects of glycation on its mechanical and physicochemical properties. We found that purified elastin and a collagen-elastin preparation from the porcine thoracic aorta rapidly incorporated glucose and ribose, the extent increasing linearly with increasing concentration and reaching a maximum after 7 days at 37 degrees C. Biochemical analysis showed that about one of the five lysines available per elastin monomer was glycated after 12 days incubation at a sugar concentration of 250 mmol/l. In long-term incubations glycation was associated with the appearance of the advanced glycation end products, the fluorescent cross-link pentosidine and the non-fluorescent putative cross-link NFC-1. In both purified elastin and the whole elastin-collagen matrix the slope of the force-extension curve increased significantly with glycation. The greatest increase in stiffness was observed in the elastin-collagen preparation after ribose incubation (250 mmol/l for 1 month), where the slope, at large strain, increased by 56 +/- 19% (mean +/- SD, n = 12). The diameter of the tissue at 1 N force also changed: for elastin there was an increase in length of approximately 5%, but for the elastin-collagen there was a decrease of similar magnitude indicating that glycation introduces differential strains within the fibrous protein matrix. Potentiometric titration demonstrated that glycation was associated both with loss of basic groups and shifts in pK of the acidic groups, which indicated changes in the environment of the charge groups due to conformational rearrangements. Changes in ion binding were dependent on pH, and were consistent with a reduction in effective anionic charge. Calcium binding to elastin was increased at acid pH, but decreased at higher pH. We suggest that these effects are not only due to changes in the charge profile, but also in the conformation of the mole

Topics: Amino Acid Sequence; Animals; Aorta, Thoracic; Arginine; Carbon Radioisotopes; Cross-Linking Reagents; Desmosine; Elastin; Glucose; Glycosylation; In Vitro Techniques; Lysine; Molecular Sequence Data; Muscle, Smooth, Vascular; Platelet Activating Factor; Potentiometry; Protein Structure, Secondary; Radioisotope Dilution Technique; Ribose; Swine