Yoshiteru Jinnouchi

The Receptor for Advanced Glycation End Products Mediates the Chemotaxis of Rabbit Smooth Muscle Cells

Long-term incubation of proteins with glucose leads to advanced glycation end products (AGEs) with fluorescence and a brown color. We recently demonstrated immunologically the intracellular AGE accumulation in smooth muscle cell (SMC)–derived foam cells in advanced atherosclerotic lesions. To understand the mechanism of AGE accumulation in these foam cells, we have now characterized the interaction of AGE proteins with rabbit-cultured arterial SMCs. In experiments at 4°C, 125I-labeled AGE-bovine serum albumin (AGE-BSA) showed a dose-dependent saturable binding to SMCs with an apparent dissociation constant (Kd) of 4.0 μg/ml. In experiments at 37°C, AGE-BSA underwent receptor-mediated endocytosis and subsequent lysosomal degradation. The endocytic uptake of 125I-AGE-BSA was effectively inhibited by unlabeled AGE proteins such as AGE-BSA and AGE-hemoglobin, but not by acetylated LDL and oxidized LDL, well-known ligands for the macrophage scavenger receptor (MSR). Moreover, the binding of 125I-AGE-BSA to SMCs was affected neither by amphoterin, a ligand for one type of the AGE receptor, named RAGE, nor by 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole–hexanoic acid–BSA, a ligand for the other AGE receptors, p60 and p90. This indicates that the endocytic uptake of AGE proteins by SMCs is mediated by an AGE receptor distinct from MSR, RAGE, p60, and p90. To examine the functional role of this AGE receptor, the migratory effects of AGE-BSA on these SMCs were tested. Incubation with 1–50 μg/ml of AGE-BSA for 14 h resulted in significant dose-dependent cell migration. The AGE-BSA–induced SMC migration was chemotactic in nature and was significantly inhibited (∼80%) by an antibody against transforming growth factor-β (TGF-β), and the amount of TGF-β secreted into the culture medium from SMC by AGE-BSA was sevenfold higher than that of control, indicating that TGF-β is involved in the AGE-induced SMC chemotaxis. These data suggest that AGE may play a role in SMC migration in advanced atherosclerotic lesions.

Advanced Glycation End Products and Their Recognition by Macrophage and Macrophage-Derived Cells

Modification of proteins by long-term incubation with glucose leads to the formation of advanced glycation end products (AGEs). AGE proteins are taken up by macrophages via the AGE receptor, which is similar to the macrophage scavenger receptor (MSR). In the present study, we compared the ligand specificity of the AGE receptor with that of MSR by three different experiments. The endocytic uptake of 125I-acetyl-LDL by RAW cells was effectively inhibited by unlabeled AGE-bovine serum albumin (BSA), whereas the inhibitory effect of acetyl-LDL on 125I-AGE-BSA was partial. Polyanions showing an effective inhibition for endocytic uptake of AGE-BSA were not always inhibitory for endocytic degradation of acetyl-LDL. These data, together with those obtained by three-dimensional fluorescence-activated cell sorter analysis, indicate that AGE proteins are recognized by more than two receptors, of which MSR is at least one. Finally, we examined whether MSR could mediate the endocytic uptake of AGE proteins by Chinese hamster ovary cells overexpressing bovine type II MSR (CHO-SRII cells). 125I-AGE-BSA underwent endocytic degradation by CHO-SRII cells, and this was effectively inhibited by unlabeled acetyl-LDL. These results clearly show that MSR mediates the endocytic uptake of AGE proteins, suggesting a new role of MSR in biological recognition of AGE in vivo.

INSULIN ENHANCES MACROPHAGE SCAVENGER RECEPTOR-MEDIATED ENDOCYTIC UPTAKE OF ADVANCED GLYCATION END PRODUCTS

Hyperglycemia accelerates the formation and accumulation of advanced glycation end products (AGE) in plasma and tissue, which may cause diabetic vascular complications. We recently reported that scavenger receptors expressed by liver endothelial cells (LECs) dominantly mediate the endocytic uptake of AGE proteins from plasma, suggesting its potential role as an eliminating system for AGE proteins in vivo (Smedsrød, B., Melkko, J., Araki, N., Sano, H., and Horiuchi, S. (1997) Biochem. J. 322, 567–573). In the present study we examined the effects of insulin on macrophage scavenger receptor (MSR)-mediated endocytic uptake of AGE proteins. LECs expressing MSR showed an insulin-sensitive increase of endocytic uptake of AGE-bovine serum albumin (AGE-BSA). Next, RAW 264.7 cells expressing a high amount of MSR were overexpressed with human insulin receptor (HIR). Insulin caused a 3.7-fold increase in endocytic uptake of 125I-AGE-BSA by these cells. The effect of insulin was inhibited by wortmannin, a phosphatidylinositol-3-OH kinase (PI3 kinase) inhibitor. To examine at a molecular level the relationship between insulin signal and MSR function, Chinese hamster ovary (CHO) cells expressing a negligible level of MSR were cotransfected with both MSR and HIR. Insulin caused a 1.7-fold increase in the endocytic degradation of 125I-AGE-BSA by these cells, the effect of which was also inhibited by wortmannin and LY294002, another PI3 kinase inhibitor. Transfection of CHO cells overexpressing MSR with two HIR mutants, a kinase-deficient mutant, and another lacking the binding site for insulin receptor substrates (IRS) resulted in disappearance of the stimulatory effect of insulin on endocytic uptake of AGE proteins. The present results indicate that insulin may accelerate MSR-mediated endocytic uptake of AGE proteins through an IRS/PI3 kinase pathway.

Immunochemical Approaches to AGE-Structures—Characterization of Anti-AGE Antibodies

Summary Recent immunological approaches have greatly helped broaden our understanding of the biomedical significance of AGEs (advanced glycation end-products) in aging and age-enhanced disease processes. We previously prepared a monoclonal anti-AGE antibody (6D12) that recognized a common AGE-structure(s) as a major immunochemical epitope. Subsequently, Nɛ-(carboxymethyl)lysine (CML), one of the glycoxidation products of AGEs, was demonstrated to be a major immunological epitope among AGEs, and 6D12 turned out to recognize CML as an epitope. In the present study, 13 different polyclonal anti-AGE antibodies were characterized in order to obtain the other epitope structure(s), other than CML (non-CML). We used CML-bovine serum albumin as an authentic CML-protein and AGE-lysozyme as an authentic non-CML-protein. The results indicated that these antibodies were classified into 3 groups (Group I, II & III). Group I was specific for CML, but both Group II and Group III were unreactive to CML. Group II, but not Group III, recognized AGE-lysozyme, suggesting Group II and III were specific for non-CML but different epitopes. The epitope of Group II was formed much earlier than that of Group III during incubation of BSA with glucose in vitro. Furthermore, we made two monoclonal anti-AGE antibodies (M-1 and M-2) whose epitope structures appeared to be identical or closely similar to Group III and Group II, respectively. These results indicate that AGE-proteins express two major non-CML epitopes in addition to CML.

Lower urinary tract dysfunction in type 1 familial amyloidotic polyneuropathy in Kumamoto, Japan.

Objective: To evaluate lower urinary tract dysfunction of type 1 familial amyloidotic polyneuropathy (FAP) patients in Kumamoto, Japan.

Neurologic toxicity associated with interferon α therapy for renal cell carcinoma

Abstract  A 67‐year‐old man received interferon α (IFN α) therapy for lung metastases of renal cell carcinoma (RCC). Multiple pulmonary metastases disappeared completely. However, neurological toxicity was detected by magnetic resonance imaging (MRI) as abnormal brain lesions. After discontinuation of IFN α therapy, his neurological symptoms and abnormal lesions on MRI disappeared completely. Complete remission of RCC has continued, and results of neurological study have remained normal for 5 years after discontinuation of IFN α therapy.

Insulin Accelerates the Endocytic Uptake and Degradation of Advanced Glycation End-Products Mediated by The Macrophage Scavenger Receptor

Summary The macrophage scavenger receptor (MSR), one of the receptors for advanced glycation end-products (AGEs), mediates endocytic uptake and degradation of AGE-proteins in several cell types. In the present study, we examined whether MSR function was regulated by insulin signaling. Co-expression of human insulin receptor (IR) with MSR in Chinese hamster ovary (CHO) cells showed that insulin accelerated the degradation of AGE proteins to 160% of the control. The insulin-enhanced endocytic uptake of AGE-proteins was significantly inhibited by phosphatidylinositol-3-OH kinase (PI(3)K) inhibitors, wortmannin and LY294002. Thus, insulin signaling through the PI(3)K pathway may regulate MSR-mediated endocytic uptake of AGE-proteins.

Endocytic uptake of AGE-modified low-density lipoprotein by macrophages leads to cholesteryl ester accumulation in vitro

Recent studies disclosed that proteins modified by advanced glycation endproducts (AGE) are taken up by macrophages or macrophage-derived cells by the macrophage scavenger receptor (MSR) in vitro and that AGE-proteins are accumulated in foam cells in the human atherosclerotic lesions in vivo , suggesting a possibility that AGE-modified low density lipoprotein (AGE-LDL) in situ might be involved in atherogenic processes in vivo . As a first step, AGE-LDL was prepared by incubating LDL with glycolaldehyde (GA), a highly reactive intermediate of the Maillard reaction. GA-modified LDL (GA-LDL) is characterized by an increase in negative charge, fluorescent intensity and reactivities to anti-AGE antibodies, suggesting that these physicochemical and immunochemical properties of GA-LDL were highly similar to those of AGE-proteins. Furthermore, studies of cellular interaction of GA-LDL with mouse peritoneal macrophages showed that GA-LDL is recognized and endocytosed, followed by intralysosomal degradation by these cells. Endocytic uptake of GA-LDL by these cells was competitively inhibited by acetylated LDL (acetyl-LDL), a representative ligand for MSR. Endocytic degradation of 125 I-acetyl-LDL was competed for by GA-LDL. Furthermore, incubation of GA-LDL effectively converted them into foam cells. Similar results were obtained from CHO cells overexpressing MSR. These results suggest that LDL modified by AGE in situ is taken up by macrophages mainly via MSR and contributed to foam cell formation in the early atherosclerotic lesions.