Takayuki Higashi

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.

Lipoprotein(a) induces cell growth in rat peritoneal macrophages through inhibition of transforming growth factor-β activation

To elucidate the atherogenicity of lipoprotein(a) (Lp(a)), we examined its growth-stimulating activity in rat resident peritoneal macrophages. When macrophages were incubated with Lp(a), cell numbers were increased 1.5-fold as compared with control macrophages. Furthermore, apolipoprotein(a) (apo(a)), a plasminogen-like glycoprotein which is covalently attached to a low density lipoprotein-like particle (Lp(a)), also induced macrophage growth, while the growth-stimulating effect of Lp(a-) was negligible. These results suggest that apo(a) plays an active role in the mitogenic activity of Lp(a). Lp(a)-induced macrophage growth was inhibited by exogenously added active transforming growth factor-beta (TGF-beta) dose-dependently, and also by the addition of plasmin, which converts latent TGF-beta to an active form. Moreover, the amounts of endogenous active TGF-beta in the medium were significantly reduced by the incubation with Lp(a). It is evident from these results that Lp(a) induces macrophage growth by inhibiting TGF-beta activation. The capacity of Lp(a) to stimulate macrophage growth shown here could be novel atherogenic function of Lp(a).

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.

β-Migrating very low density lipoproteins induce foam cell formation in mouse mesangial cells

To elucidate whether beta-migrating very low density lipoproteins (beta-VLDL) induce foam cell formation in mesangial cells or not, surface binding and foam cell formation with beta-VLDL were studied in mouse mesangial cells. Specific binding kinetics for beta-VLDL and low density lipoproteins (LDL) on the mesangial cells were observed with Kd = 3.8 and 13.7 micrograms/ml, and Bmax = 65.9 and 71.9 ng/ml cell protein at 4 degrees C, respectively. The binding of beta-VLDL was inhibited by excess amounts of LDL or beta-VLDL, but not by acetyl-low density lipoproteins. Ligand blotting using beta-VLDL or LDL and immunoblotting using anti-human LDL receptor monoclonal antibody detected the same apparent single protein (approx. 130 kDa). Incorporation of [14C]oleate into cholesteryl ester in mouse mesangial cells was enhanced by beta-VLDL to 3-fold higher than that by LDL, and it was inhibited by chloroquine or anti-human LDL receptor monoclonal antibody. The light microscopic findings also demonstrated that cholesteryl ester deposition increased in these cells incubated with beta-VLDL, but not with LDL. In conclusion, beta-VLDL was specifically taken up by receptor-mediated endocytosis in mouse mesangial cells through LDL receptors, resulting in foam cell formation.

Release of fructose and hexose phosphates from perivascular cells induced by low density lipoprotein and acceleration of protein glycation in vitro

We investigated whether low density lipoprotein (LDL) under oxidative stress might induce the release of fructose, glucose-6-phosphate and fructose-6-phosphate from perivascular cells, and also whether these substances might accelerate the formation of advanced glycation end products (AGE) from proteins in vitro. When vascular smooth muscle cells were incubated with LDL in Hams F10 at 37 degrees C for 48 h. release of all these substances was increased dose-dependently by oxidized LDL. Fructose release was increased in a dose-dependent manner by glucose. Indomethacin (20 microM) significantly (P < 0.01) suppressed the release of fructose (25.4 +/- 15.7% of control) and hexose phosphates (29.4 +/- 4.0) with the inhibition of release of lactate dehydrogenase (35.5 +/- 4.9) as well as probucol, whereas an aldose reductase inhibitor, epalrestat, significantly (P < 0.001) inhibited only the fructose release (0.9 +/- 0.8). Release of fructose and hexose phosphates from vascular endothelial cells was also induced by oxidized LDL. AGE immunoreactivities and AGE-related fluorescence formed from proteins and glucose were significantly increased (P < 0.001) in the presence of small amounts of the cellular glucose metabolites (6.6%) with glucose (93.4%). These data suggest that release of potent AGE initiators, fructose and hexose phosphates, from perivascular cells induced by oxidized LDL may be an important phenomenon for vascular complications.

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.

Macrophage Scavenger Receptor Mediates The Endocytic Uptake of Advanced Glycation End Products (AGEs)

Summary Cellular interactions of AGEs are mediated by AGE receptors. The AGE receptors so far reported are RAGE, galectin-3 and MSR (macrophage scavenger receptor). Macrophages or macrophage-derived cells are known to show the highest endocytic activity for AGE-proteins. Our recent study using CHO (Chinese Hamster Ovary) cells overexpressing MSR clearly showed that the endocytic uptake of AGE-proteins by macrophages is mediated by MSR. To strengthen this contention, the present study was undertaken to examine the interaction of AGE-proteins with peritoneal macrophages from MSR-deficient mice (MSR (-/-)). In experiments at 37°C, thioglycolate-induced peritoneal macrophages from MSR (-/-) showed a marked decrease (more than 80%) in the endocytic degradation capacity for 125 I-acetylated low-density lipoprotein (acetyl-LDL). Under parallel conditions, the degradation activity of 125 I-AGE-bovine serum albumin (BSA) by these MSR-deficient macrophages was less than 20%. The remaining endocytic capacity of 125 I-AGE-BSA by these MSR-deficient macrophages was not inhibited by acetyl-LDL, but was inhibited significantly by AGE-BSA, AGE-hemoglobin or polyanions such as dextran sulfate and polyinosinic acid. These results indicate that −80% of the endocytic uptake of AGE-proteins by macrophages is mediated by MSR, while the remaining part is mediated by other AGE receptors.

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.