Noriyuki Sakata

In vivo and in vitro evidence for the glycoxidation of low density lipoprotein in human atherosclerotic plaques

Although there have been suggestions that the glycation and oxidation of low density lipoprotein (LDL) might increase its atherogenic potential, little is known about the presence of glycoxidative LDL in human atherosclerotic lesions. We developed specific antibodies against different immunological epitopes of AGE structures, including N(epsilon)-(carboxymethyl)lysine-protein adduct (CML), a glycoxidation product, and structure(s) other than CML (nonCML), and a monoclonal antibody against oxidized phosphatidylcholine (oxPC), as an epitope of oxidized LDL. Immunohistochemical analysis demonstrated that the CML- and oxPC-epitopes were accumulated mainly in macrophage-derived foam cells in atherosclerotic lesions, including fatty streaks and atherosclerotic plaques. On the other hand, the nonCML-epitope and apolipoprotein B were localized mainly in extracellular matrices of atherosclerotic lesions. The CML- and oxPC-epitopes were characterized by a model antigen-generating system using the copper ion-induced peroxidation and/or glucose-induced glycation of LDL. The glycoxidation of LDL caused the formation of CML-epitope with increasing concentrations of copper ion and glucose. It was also formed to some extent in LDL incubated with high concentrations (500 mM) of glucose. However, no CML-epitope was observed in oxidized LDL induced by copper ion alone. On the other hand, the formation of oxPC-epitope in LDL was dependent on copper ion-induced peroxidation, but independent of glucose-induced glycation. The addition of chelators, ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid, reduced the increase in electrophoretic mobility and TBARS caused by the peroxidation and glycoxidation of LDL, but had no effects on the formation of fructosamine caused by the glycation and glycoxidation of LDL. Chelators as well as aminoguanidine protected the formation of CML-epitope in glycated or glycoxidative LDL. Although the formation of oxPC-epitope was completely inhibited by the addition of chelators, it was partially protected by aminoguanidine. These in vitro results suggest that the glycoxidative modification of LDL may occur in the arterial intima, and may contribute to the development of human atherosclerotic lesions.

Glycoxidation and lipid peroxidation of low-density lipoprotein can synergistically enhance atherogenesis

OBJECTIVEnThe purpose of this study was to clarify the role of glycoxidation and lipid peroxidation of low-density lipoprotein (LDL) in atherogenesis.nnnMETHODS AND RESULTSnWe examined the formation of N(epsilon)-(carboxymethyl) lysine (CML), a glycoxidation product, and malondialdehyde (MDA), a lipid peroxidation product, in vitro and their co-localization in human atherosclerotic lesions. Immunochemical analysis revealed that CML was formed in a time-dependent manner by human LDL incubated with copper ions and glucose, i.e. an in vitro model of glycoxidation of LDL. When LDL was exposed to copper ions alone, a small amount of CML was formed, however this was significantly less in oxidized LDL than glycoxidative LDL. In contrast, MDA formation was observed in both oxidation and glycoxidation of LDL, but not in glycation of LDL. Hexitol-lysine (HL), an Amadori product, was formed by both glycation and glycoxidation of LDL, but not by oxidation of LDL. Immunohistochemical analysis showed that CML and MDA accumulated mainly in macrophage/foam cells, while pyrraline, a non-oxidative product of glycation, and apolipoprotein B were localized in the extracellular matrix in atherosclerotic lesions. Atheromas were positive for CML and MDA, but negative for pyrraline. Macrophage/foam cells in atherosclerotic lesions exhibited co-localization of macrophage scavenger receptor-A with CML and MDA, but not with pyrraline.nnnCONCLUSIONnOur results suggest that glycoxidation and lipid peroxidation of LDL synergistically promote the development of atherosclerotic lesions through interaction with macrophage scavenger receptor-A.

Carboxymethyllysine in Dermal Tissues of Diabetic and Nondiabetic Patients with Chronic Renal Failure: Relevance to Glycoxidation Damage

Carboxymethyllysine (CML) is currently recognized as a major advanced glycation end product and a marker for glycoxidation. Plasma CML levels are increased in patients with chronic renal failure (CRF). However, significance and mechanism of CML accumulation in these patients are poorly understood. The objective of the present study was to analyze CML in soluble and collagen-binding fractions of the dermis to investigate CML deposition and formation and collagen damage related to CML accumulation in patients with CRF. Skin samples (among them autopsy samples) were obtained from 33 subjects: 8 nondiabetic CRF patients, 7 diabetic predialysis patients with CRF (CRF-DM), 7 hemodialysis patients, and 11 control subjects without either CRF or DM. The dermal samples were extracted sequentially by phosphate-buffered normal saline, pepsin, and collagenase. The extracts were referred to as the soluble fraction and the proteinase-extracted fraction (including pepsin-extracted and collagenase-extracted fractions). Our ELISA assay for CML in dermal collagen from predialysis patients with CRF (CRF and CRF-DM groups) demonstrated that the levels of CML in both the soluble fraction (containing soluble CML which was mainly determined by serum clearance) and the structural collagen-binding proteinase-extracted fraction (in which high CML levels could be a strong indication of in situ formation) were increased and could not be completely reduced after hemodialysis in CRF-DM and CRF groups. These results suggest that accumulation of CML may be due to both a low serum clearance and/or increased in situ CML formation in CRF. CML contents in the proteinase extracted fraction inversely correlated with the susceptibility of collagen to extraction by proteinases (n = 33, r = –0.59, p < 0.001). Our results provide the first biochemical evidence that CML level is increased in both the soluble and collagen-binding fractions and that increased CML level resulted in increased fractions of proteinase-resistant collagen in dermal extracts of patients with CRF.

Possible Involvement of Altered RGD Sequence in Reduced Adhesive and Spreading Activities of Advanced Glycation End Product-Modified Fibronectin to Vascular Smooth Muscle Cells

Although fibronectin (FN) modified by advanced glycation end products (AGEs) has been shown to contribute to the development of diabetic vascular complications through its reduced adhesive activity to vascular cells, little is known about changes in the cell binding domain of AGE-modified FN. Here we examined the mechanism of reduced adhesive and spreading activities of AGE-modified FN to vascular smooth muscle cells (SMCs), particularly the contribution of modification of Arg-Gly-Asp (RGD) sequence. Incubation with glucose caused not only the formation of N-carboxymethyllysine and pentosidine, but also polymerization of FN in a dose- and time-dependent manner. AGE-modified FN had significantly low adhesive and spreading activities to cultured SMCs. On the other hand, multimeric FN formed by disulfide bonds did not show any effect on either cell adhesion or spreading. The adhesive activity of type I collagen, one of the RGD sequence-containing proteins, to SMCs also decreased by AGE-modification. The inhibitory effect of AGE-modification on cell adhesion was significantly greater in type I collagen than in FN. Although the extent of AGE-modification of type I collagen was indistinguishable from that of FN, AGE-modification decreased the arginine content of type I collagen by 69.5% and of FN by 30.6%, compared with their non-glycated forms. The addition of RGD peptides caused a decrease in adhesion of SMCs to non-glycated FN, but not to AGE-modified FN. Modification of RGD sequence with glyoxal eliminated its inhibitory effect on cell adhesion. Our results suggest that a marked decrease in adhesive and spreading activities of AGE-modified FN to SMCs might largely be due to a modification of its RGD sequence by AGE, thus suggesting a potential link between AGE modification of FN and the pathogenesis of diabetic angiopathy.

Increased glycoxidation and lipoperoxidation in the collagen of the myocardium in hemodialysis patients

OBJECTIVEnThe purpose of this study was to examine the glycoxidation and lipoperoxidation products in the collagen of the myocardium in hemodialysis (HD) patients and age-matched control subjects.nnnMETHODSnCardiac samples from 15 autopsied subjects (HD, n=6; control, n=9) were sequentially extracted with 0.9% NaCl and collagenase to obtain two fractions [soluble fraction (SF) and collagenase soluble fraction (CSF)]. The glycoxidation and lipoperoxidation products of these two fractions were measured by pentosidine-linked fluorescence (lambda(ex), 335; lambda(em), 385) and malondialdehyde (MDA)-linked fluorescence (lambda(ex), 390; lambda(em), 460), respectively.nnnRESULTSnBoth pentosidine- and MDA-linked fluorescence were found to have significantly increased more in the collagenase soluble fraction (CSF) extracted form the anterior and posterior wall in HD patients than in the controls (P<0. 05, control, n=9 vs. HD, n=6). Interestingly, the level of the lipid peroxides strongly correlated with that of the glycoxidation product in CSF (both P<0.0001 for the anterior and posterior wall). In contrast, in SF, which did not contain matrix collagen, neither significant difference nor correlation in the levels of pentosidine- and MDA-linked fluorescence was observed in these two groups.nnnCONCLUSIONnthe present study provides the first biochemical evidence for an increase in glycoxidation and a close link between glycoxidation and lipoperoxidation in the collagen of the myocardium in hemodialysis patients. These findings suggest that these two spontaneous chemical reactions in the collagen matrix of myocardium may synergistically contribute to cardiac damage in hemodialysis patients.

Causal relationship between conformational change and inhibition of domain functions of glycoxidative fibronectin.

Glycoxidative modification of various body proteins, including fibronectin (FN), has been shown to change their structural and functional properties, and be implicated in pathogenesis of diabetic complications. Little is known about the role of secondary structure of glycoxidative FN (gFN) in its domain functions. gFN was prepared by incubation with 25 and 200 mM glucose in 0.2 M sodium phosphate buffer at 37°C on a shaking plate under aerobic and sterile conditions for various time intervals up to 49 days, being defined as gFN25 and gFN200, respectively. Unmodified FN (uFN) was prepared by incubation in 0.2 M sodium phosphate buffer without any glucose at 4°C for 49 days. The extent of glycoxidative modification was examined using a noncompetitive enzyme-linked immunosorbent assay with an antibody against N-(carboxymethyl)lysine (CML), one of the major glycoxidation products. The binding activities of uFN and gFN to collagen, gelatin and heparin were determined by a solid phase enzyme immunoassay or heparin-affinity HPLC. Cell attachment was estimated by the extent of adhesion of FITC-labeled smooth muscle cells to uFN or gFN. Conformational change in gFN was detected by SDS-polyacrylamide gel electrophoresis and spectroscopy (circular dichroism). CML was detected in gFN25 and gFN200 after 49 and 21 days of incubation, respectively. Levels of CML were about six-fold higher in gFN200 than in gFN25 after 49 days. Both gFN25 and gFN200 showed a significant decrease in the ability of binding to collagen and gelatin after 7 days of incubation. The binding activity for heparin was significantly decreased in both gFN25 and gFN200 after one day. Cell attachment activity was reduced to 89% and 76% of the unmodified form in both gFN25 and gFN200 after 49 days, respectively. High molecular weight materials were found in gFN25 and gFN 200 after 21 and 7 days, respectively. CD spectrum showed that gFN25 had lost its native conformation after 3 days of incubation, depending upon the concentration and incubation interval of the applied glucose. These in vitro results suggest that the loss of native conformation may reduce the domain functions of gFN, including binding activity to macromolecular ligands and cell attachment, and may play a major role in the pathogenesis of diabetic complications.

Glycoxidation and lipoperoxidation in the collagen of the myocardium in hemodialysis patients

Abstract Objective: The purpose of this study is to examine the glycoxidation and lipoperoxidation products in the collagen of the myocardium in hemodialysis patients (HD group) and age-matched control subjects. Subjects and methods: Cardiac samples from 15 autopsied subjects (HD, n=6; Control, n=9) were sequentially extracted with 0.9% NaCl and collagenase to obtain two fractions (soluble fraction: SF and collagenase soluble fraction: CSF). The glycoxidation and lipoperoxidation products of these two fractions were measured by pentosidine-linked fluorescence (ex: 335/em: 385) and malondialdehyde (MDA)-linked fluorescence (ex: 390/em: 460), respectively. Results: Both pentosidine- and MDA-linked fluorescence were found to have significantly increased more in the collagenase soluble fraction (CSF) extracted form the anterior and posterior wall in HD patients than in the controls (p