Seikoh Horiuchi

beta 2-Microglobulin modified with advanced glycation end products is a major component of hemodialysis-associated amyloidosis.

beta 2-Microglobulin (beta 2M) is a major constituent of amyloid fibrils in hemodialysis-associated amyloidosis, a complication of long-term hemodialysis patients. Amyloid fibril proteins were isolated from connective tissues forming carpal tunnels in hemodialysis patients with carpal tunnel syndrome. Two-dimensional polyacrylamide gel electrophoresis and Western blotting demonstrated that most of the beta 2M forming amyloid fibrils exhibited a more acidic pI value than normal beta 2M. This acidic beta 2M was also found in a small fraction of beta 2M in sera and urine from these patients, whereas heterogeneity was not observed in healthy individuals. We purified acidic and normal beta 2M from the urine of long-term hemodialysis patients and compared their physicochemical and immunochemical properties. Acidic beta 2M, but not normal beta 2M, was brown in color and fluoresced, both of which are characteristics of advanced glycation end products (AGEs) of the Maillard reaction. Immunochemical studies showed that acidic beta 2M reacted with anti-AGE antibody and also with an antibody against an Amadori product, an early product of the Maillard reaction, but normal beta 2M did not react with either antibody. Incubating normal beta 2M with glucose in vitro resulted in a shift to a more acidic pI, generation of fluorescence, and immunoreactivity to the anti-AGE antibody. The beta 2M forming amyloid fibrils also reacted with anti-AGE antibody. These data provided evidence that AGE-modified beta 2M is a dominant constituent of the amyloid deposits in hemodialysis-associated amyloidosis.

The relationship between accumulation of advanced glycation end products and expression of vascular endothelial growth factor in human diabetic retinas

Summary Both advanced glycation end products and vascular endothelial growth factor are believed to play a role in the pathogenesis of diabetic retinopathy. It is known that vascular endothelial growth factor causes retinal neovascularization and a breakdown of the blood-retinal barrier; how advanced glycation end products affect the retina, however, remains largely unclear. The substance Ne-(carboxymethyl)lysine is a major immunologic epitope, i. e. a dominant advanced glycation end products antigen. We generated an anti-Ne-(carboxymethyl)lysine antibody to investigate the relationship between the localization of advanced glycation end products and that of vascular endothelial growth factor in 27 human diabetic retinas by immunohistochemistry. Nine control retinas were also examined. In all 27 diabetic retinas, Ne-(carboxymethyl)lysine was located in the thickened vascular wall. In 19 of the 27 retinas, strand-shaped Ne-(carboxymethyl)lysine immunoreactivity was also observed around the vessels. In all 27 diabetic retinas, vascular endothelial growth factor revealed a distribution pattern similar to that of Ne-(carboxymethyl)lysine. Vascular endothelial growth factor was also located in the vascular wall and in the perivascular area. Neither Ne-(carboxymethyl)lysine nor vascular endothelial growth factor immunoreactivity was detected in the 9 control retinas. Vessels with positive immunoreactivity for Ne-(carboxymethyl)lysine and/or vascular endothelial growth factor were counted. A general association was noted between accumulation of Ne-(carboxymethyl)lysine and expression of vascular endothelial growth factor in the eyes with non-proliferative diabetic retinopathy (p < 0.01) and proliferative diabetic retinopathy (p < 0.05). [Diabetologia (1997) 40: 764–769]

Morphological evidence for lipid peroxidation and protein glycoxidation in spinal cords from sporadic amyotrophic lateral sclerosis patients.

For determining whether both the spinal cord motor neurons and glial cells are exposed to increased oxidative stress in amyotrophic lateral sclerosis (ALS), we performed an immunohistochemical investigation of end products of lipid peroxidation and protein glycoxidation in spinal cords from seven sporadic ALS patients and seven age-matched control individuals. In the ALS spinal cords, immunoreactivities for adducts of 4-hydroxy-2-nonenal-histidine and crotonaldehyde-lysine as markers of lipid peroxidation, N(epsilon)-(carboxymethyl)lysine as a marker of lipid peroxidation or protein glycoxidation, and pentosidine as a marker of protein glycoxidation were localized in the gray matter neuropil and almost all of the motor neurons, reactive astrocytes and microglia/macrophages, whereas none of the immunoreactivities for N(epsilon)-(carboxyethyl)lysine or argpyrimidine as markers of protein glycoxidation or enzymatic glycolysis, or pyrraline or imidazolone as markers of nonoxidative protein glycation were detectable. The control spinal cords displayed no significant immunoreactivities for any of these examined products. Our results indicate that in sporadic ALS, both lipid peroxidation and protein glycoxidation are enhanced in the spinal cord motor neurons and glial cells, and suggest that the formation of certain products in these abnormal reactions is implicated in motor neuron degeneration.

Advanced glycation end products-cytokine-nitric oxide sequence pathway in the development of diabetic nephropathy: Aminoguanidine ameliorates the overexpression of tumour necrosis factor-α and inducible nitric oxide synthase in diabetic rat glomeruli

Aims/hypothesis. Advanced glycation end products are believed to contribute to diabetic microvascular complications by inducing glomerular damage but their role has not been fully clarified. In this study, we explain their central role in the induction of inducible nitric oxide synthase and production of nitric oxide (NO) in streptozotocin-induced diabetic rat glomeruli. Methods. Localization of carboxymethyllysine, which is one of the chemical components of advanced glycation end products, glomerular expression of inducible nitric oxide synthase and urinary excretion and glomerular production of NO2–/NO3– were examined at 0, 26, 51, and 52 weeks after the induction of diabetes. Therapeutic effects of aminoguanidine were also examined. Results. Carboxymethyllysine was detected in the mesangial area in glomeruli and it progressively accumulated during 52 weeks of observation. Immunohistochemistry and hybridization studies in situ showed that the number of inducible nitric oxide synthase-positive cells was notably increased in diabetic rat glomeruli at 52 weeks. Further, this augmented expression paralleled intraglomerular expression of TNF-α and NO2–/NO3– in diabetic rat glomeruli. Treatment with aminoguanidine reduced the expression of TNF-α, inducible nitric oxide synthase and intraglomerular NO2–/NO3– production. It also ameliorated proteinuria in diabetic rats. Conclusion/interpretation. This study showed that carboxymethyllysine possibly enhances the expression of inducible nitric oxide synthase by stimulating the expression of TNF-α in diabetic rat glomeruli. The carboxymethyllysine-cytokine-NO sequence pathway could be one of the major mechanisms in the development of diabetic nephropathy. [Diabetologia (1999) 42: 878–886]

Scavenger receptors for oxidized and glycated proteins

Summary. Our present knowledge on chemically modified proteins and their receptor systems is originated from a proposal by Goldstein and Brown in 1979 for the receptor for acetylated LDL which is involved in foam cell formation, one of critical steps in atherogenesis. Subsequent extensive studies using oxidized LDL (OxLDL) as a representative ligand disclosed at least 11 different scavenger receptors which are collectively categorized as “scavenger receptor family”. Advanced glycation endproducts (AGE) and their receptor systems have been studied independently until recent findings that AGE-proteins are also recognized as active ligands by scavenger receptors including class A scavenger receptor (SR-A), class B scavenger receptors such as CD36 and SR-BI, type D scavenger receptor (LOX-1) and FEEL-1/FEEL-2. Three messages can be summarized from these experiments; (i) endocytic uptake of OxLDL and AGE-proteins by macrophages or macrophage-derived cells is mainly mediated by SR-A and CD36, which is an important step for foam cell formation in the early stage of atherosclerosis, (ii) selective uptake of cholesteryl esters of high density lipoprotein (HDL) mediated by SR-BI is inhibited by AGE-proteins, suggesting a potential pathological role of AGE in a HDL-mediated reverse cholesterol transport system, (iii) a novel scavenger receptor is involved in hepatic clearance of plasma OxLDL and AGE-proteins.

Expression of ACAT-1 Protein in Human Atherosclerotic Lesions and Cultured Human Monocytes-Macrophages

The acyl coenzyme A:cholesterol acyltransferase (ACAT) gene was first cloned in 1993 (Chang et al, J Biol Chem. 1993;268:20747-20755; designated ACAT-1). Using affinity-purified antibodies raised against the N-terminal portion of human ACAT-1 protein, we performed immunohistochemical localization studies and showed that the ACAT-1 protein was highly expressed in atherosclerotic lesions of the human aorta. We also performed cell-specific localization studies using double immunostaining and showed that ACAT-1 was predominantly expressed in macrophages but not in smooth muscle cells. We then used a cell culture system in vitro to monitor the ACAT-1 expression in differentiating monocytes-macrophages. The ACAT-1 protein content increased by up to 10-fold when monocytes spontaneously differentiated into macrophages. This increase occurred within the first 2 days of culturing the monocytes and reached a plateau level within 4 days of culturing, indicating that the increase in ACAT-1 protein content is an early event during the monocyte differentiation process. The ACAT-1 protein expressed in the differentiating monocytes-macrophages was shown to be active by enzyme assay in vitro. The high levels of ACAT-1 present in macrophages maintained in culture can explain the high ACAT-1 contents found in atherosclerotic lesions. Our results thus support the idea that ACAT-1 plays an important role in differentiating monocytes and in forming macrophage foam cells during the development of human atherosclerosis.

Protective effect of lipoproteins containing apoprotein A-I on Cu2+ - catalyzed oxidation of human low density lipoprotein

Two apoprotein A‐I (apoA‐I)‐containing lipoproteins, one containing apoA‐I and apoA‐II (LpA‐I/A‐II) and the other containing only apoA‐I (LpA‐I), were examined for their effect on Cu2+‐mediated oxidation of low density lipoprotein (LDL). The presence of LpA‐I or LpA‐I/A‐II prevented LDL oxidation when assessed by the electrophoretic mobility, apoprotein B fragmentation and amounts of thiobarbituric acid‐reactive substances. The protection of LDL oxidation by these lipoproteins was effective for up to 6 h, with LpA‐I being more active than LpA‐I/A‐II. Results from these in vitro model experiments raise a possibility that LpA‐I mayplay a role in protecting LDL from Cu2+ ‐mediated oxidation.

Localization in human diabetic peripheral nerve of Nɛ-carboxymethyllysine-protein adducts, an advanced glycation endproduct

Summary The present study was designed to elucidate in situ distribution of advanced glycation end-products (AGE) in human peripheral nerve and whether the reaction products were excessive in the diabetic condition. For the detection of AGE, immunoperoxidase staining was undertaken on peripheral nerve samples obtained from 5 non-insulin-dependent diabetic patients and 5 non-diabetic control subjects. The anti-AGE antibody used in this study contained an epitope against Nɛ-carboxymethyllysine. Light microscopically, AGE localized in the perineurium, endothelial cells and pericytes of endoneurial microvessels as well as myelinated and unmyelinated fibers. At the submicroscopic level, AGE deposition appeared focally as irregular aggregates in the cytoplasm of endothelial cells, pericytes, axoplasm and Schwann cells of both myelinated and unmyelinated fibers. Interstitial collagens, basement membranes of the perineurium also reacted with this antibody. The AGE depositions were detected in both control and diabetic nerves, but were more intense in the latter. The excessive AGE deposition correlated with a reduction in myelinated fiber density. However, the localization of AGE was not directly associated with degeneration of nerve fibers and the link between AGE deposition and nerve fiber degeneration is yet to be determined. The present study thus demonstrated the excessive deposition of intra- and extracellular AGE in human diabetic peripheral nerve and strengthened the contention that the enhanced glycation may play a role in the development of diabetic neuropathy. [Diabetologia (1997) 40: 1380–1387]

Identification in Human Atherosclerotic Lesions of GA-pyridine, a Novel Structure Derived from Glycolaldehyde-modified Proteins

Glycolaldehyde (GA) is formed from serine by action of myeloperoxidase and reacts with proteins to form several products. Prominent among them isN ε-(carboxymethyl)lysine (CML), which is also known as one of the advanced glycation end products. Because CML is formed from a wide range of precursors, we have attempted to identify unique structures characteristic of the reaction of GA with protein. To this end, monoclonal (GA5 and 1A12) and polyclonal (non-CML-GA) antibodies specific for GA-modified proteins were prepared. These antibodies specifically reacted with GA-modified and with hypochlorous acid-modified BSA, but not with BSA modified by other aldehydes, indicating that the epitope of these antibodies could be a specific marker for myeloperoxidase-induced protein modification. By HPLC purification from GA-modifiedN α-(carbobenzyloxy)-l-lysine, GA5-reactive compound was isolated, and its chemical structure was characterized as 3-hydroxy-4-hydroxymethyl-1-(5-amino-5-carboxypentyl) pyridinium cation. This compound named as GA-pyridine was recognized both by 1A12 and non-CML-GA, indicating that GA-pyridine is an important antigenic structure in GA-modified proteins. Immunohistochemical studies with GA5 demonstrated the accumulation of GA-pyridine in the cytoplasm of foam cells and extracellularly in the central region of atheroma in human atherosclerotic lesions. These results suggest that myeloperoxidase-mediated protein modification via GA may contribute to atherogenesis.

Increased advanced glycation end products in atherosclerotic lesions of patients with end-stage renal disease

Although advanced glycation end products (AGEs) are increased in the serum and tissues of patients with end-stage renal disease, little is known about the role of AGEs in atherogenesis. We therefore carried out an immunohistochemical study on the accumulation of AGEs and apolipoprotein B in the human aortas of diabetic and nondiabetic subjects with end-stage renal disease. The atherosclerotic lesions included diffuse intimal thickening, fatty streaks and atherosclerotic plaque. We used antibodies against two different epitopes of AGE structures, i.e. an Nepsilon-(carboxymethyl)lysine-protein adduct (CML) and a structure(s) other than CML (nonCML). The area that was positive for an antigen as a percentage of the total area (%Ar) was determined morphometrically, using an NIH-image program. In diffuse intimal thickening, atherosclerotic plaque and tunica media, the %Ar of CML and nonCML was significantly greater in diabetic or nondiabetic subjects with end-stage renal disease than in control subjects without end-stage renal disease. In fatty streaks, the %Ar of nonCML was significantly greater in nondiabetic subjects with end-stage renal disease than in control subjects, while no difference in the %Ar of CML was found between the subjects with or without end-stage renal disease. Nondiabetic subjects with end-stage renal disease showed a significantly increased %Ar of apolipoprotein B in fatty streaks and atherosclerotic plaque than the control subjects. The %Ar of CML and nonCML significantly correlated with the duration of hemodialysis in diffuse intimal thickening and atherosclerotic plaque of subjects with end-stage renal disease, but not in fatty streaks. On the other hand, the %Ar was not related to the duration of diabetes in any of the lesions in the diabetic subjects with end-stage renal disease. In diffuse intimal thickening and atherosclerotic plaque, subjects with end-stage renal disease showed a significant correlation between the %Ar of apolipoprotein B and AGEs (CML and nonCML), as well as their immunohistochemical colocalization. These results suggest that impaired AGE clearance may cause the increased accumulation of AGEs in the aortic wall of subjects with end-stage renal disease, thus resulting in the rapid progression of atherosclerosis. The accumulation of AGEs may be related to an enhanced LDL deposition in atherosclerotic lesions of subjects with end-stage renal disease.