Kenshi Matsumoto

Receptors for proteins modified by advanced glycation endproducts (AGE)--their functional role in atherosclerosis.

Long-term incubation of proteins with glucose leads, through the formation of early stage products such as Schiff base and Amadori rearrangement products, to the formation of advanced glycation end products (AGE). Recent studies of AGE-structures as well as the receptor for AGE-proteins (AGE-receptors) have emphasized the involvement of protein modification by AGE in aging and age-enhanced disease processes. Immunohistochemical analyses of human atherosclerotic lesions using a monoclonal anti-AGE antibody have demonstrated diffuse extracellular AGE-deposition as well as dense intracellular AGE-deposition in macrophage- and vascular smooth muscle cell (SMC)-derived foam cells. In vitro experiments using both CHO cells overexpressing macrophage scavenger receptor-A (MSR-A) and peritoneal macrophages from MSR-A-knockout mice have shown that the MSR-A plays a major role in endocytic uptake of AGE-proteins by macrophages. Furthermore, in vitro experiments with rabbit arterial SMCs demonstrated a novel AGE-receptor mediating endocytosis of AGE-proteins. These in vivo and in vitro experiments suggest that AGE-proteins formed extracellularly in atherosclerotic lesions are endocytosed by macrophages through MSR-A in the early stage, and by SMCs through the novel AGE-receptor in the advanced stage, implicating functional contribution of the AGE-receptor-mediated interaction of AGE-proteins with these cells to atherosclerotic processes in arterial walls.

Endocytic uptake of advanced glycation end products by mouse liver sinusoidal endothelial cells is mediated by a scavenger receptor distinct from the macrophage scavenger receptor class A.

Previous studies with peritoneal macrophages obtained from macrophage scavenger receptor class A (MSR-A) knock-out mice showed that the endocytic uptake of advanced glycation end products (AGE) by macrophages was mediated mainly by MSR-A. However, it is controversial whether the endocytic uptake of intravenously injected AGE proteins by liver sinusoidal endothelial cells (LECs) is similarly explained by receptor-mediated endocytosis via MSR-A. The present study was conducted to compare the capacity to endocytose AGE proteins in LECs and peritoneal macrophages obtained from MSR-A knock-out and littermate wild-type mice. The endocytic degradation capacity of MSR-A knock-out LECs for AGE-BSA was indistinguishable from that of wild-type LECs, whereas that of MSR-A knock-out peritoneal macrophages for AGE-BSA was decreased to 30% of that in wild-type cells. Similarly, the endocytic degradation of MSR-A knock-out LECs for acetylated low-density lipoprotein (acetyl-LDL) did not differ from that of wild-type LECs, whereas the endocytic degradation of acetyl-LDL by MSR-A knock-out peritoneal macrophages was less than 20% of that in wild-type cells. Furthermore, formaldehyde-treated serum albumin (f-Alb), a ligand known to undergo scavenger-receptor-mediated endocytosis by LECs, was effectively taken up by MSR-A knock-out LECs at a capacity that did not differ from that of wild-type LECs. Moreover, the endocytic uptake of AGE-BSA by LECs was effectively competed for by unlabelled f-Alb or acetyl-LDL. These results indicate that the scavenger-receptor ligands AGE proteins, acetyl-LDL and f-Alb are endocytosed by LECs through a non-MSR-A pathway.

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.

Expression of two isozymes of acyl-coenzyme A: Cholesterol acyltransferase-1 and -2 in clear cell type renal cell carcinoma

Objectives:  The present study investigated the expression of acyl‐coenzyme A: cholesterol acyltransferase‐1 (ACAT‐1) and ‐2 in clear cell type renal cell carcinoma (RCC).

Immunohistochemical detection of advanced glycation end products in human bladder with specific monoclonal antibody

Objective:  To investigate the accumulation of advanced glycation end products (AGE) in human bladder.

Overactive Bladder in Female Patients with Chronic Diseases Visiting Primary Care Doctors: Effect of Age on Prevalence and Bothersomeness

Objectives: We evaluated the effects of age on the prevalence and bothersomeness of overactive bladder (OAB) in female patients with chronic diseases visiting primary care doctors.

Endocytic uptake of advanced glycation endproducts by mouse liver sinusoidal endothelial cells is mediated by a receptor distinct from the class A scavenger receptor

Abstract Previous studies using peritoneal macrophages obtained from macrophage scavenger receptor-A (MSR-A)-knockout mice showed that the endocytic uptake of advanced glycation endproducts (AGE) by macrophages was mainly mediated by MSR-A. However, it is controversial whether the endocytic uptake of intravenously injected AGE-proteins by liver endothelial cells (LECs) is similarly explained by receptor-mediated endocytosis via MSR-A. The present study was conducted to compare the capacity to endocytose AGE-proteins in LECs and peritoneal macrophages obtained from MSR-A-knockout and litter mate wild type mice. The endocytic degradation of MSR-A-knockout LECs for AGE-BSA was indistinguishable from that of wild type LECs, whereas that of MSR-A-knockout peritoneal macrophages for AGE-BSA was reduced to 30% of wild type cells. Furthermore, formaldehyde-treated serum albumin (f-Alb), a ligand known to undergo scavenger receptor-mediated endocytosis by LECs, was effectively taken up by MSR-A-knockout LECs at a capacity that did not differ from that of wild type LECs. Furthermore, the endocytic uptake of AGE-BSA by LECs was effectively competed for by unlabelled f-Alb. These data indicate that the scavenger receptor-ligands AGE-proteins are endocytosed by LECs through a non-MSR-A pathway.

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

We recently demonstrated immunologically the intraccllular accumulation of advanced glycation end products (AGEs) in foam cells derived from smooth muscle cells (SMCs) in advanced atherosclerotic lesions. To understand the mechanism of AGE-accumulation in these foam cells, the interaction of AGE-proteins with rabbit cultured arterial SMCs was studied in the present study. In experiments at 4°C, 125I-AGE-bovine serum albumin (AGE-BSA) showed dose-dependent saturable binding to SMCs with an apparent dissociation constant (Kd) of 4.0 μ/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, but not by acetylated low density lipoprotein (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)-1 H-imidazolc-hexanoic acid-BSA (FFI-BSA), a ligand for the other AGE receptors called p60 and p90, indicating that the endocytic uptake of AGE-proteins by SMCs is mediated by an AGE receptor distinct either from MSR, RAGE, p60 or p90. To examine the functional role of (his AGE receptor, the effects of AGE-BSA on the migration of SMCs were tested. Incubation with 1-50 μg/mL of AGE-BSA resulted in significant dose-dependent cell migration. The AGE-BSA-induced SMCs 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 SMCs by AGE-BSA was 7-fold higher than that of control, indicating that TGF-β is involved in the AGE-induced SMCs chemotaxis.

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.