Macrophage scavenger receptor-a-deficient mice are resistant against diabetic nephropathy through amelioration of microinflammation.

Microinflammation is a common major mechanism in the pathogenesis of diabetic vascular complications, including diabetic nephropathy. Macrophage scavenger receptor-A (SR-A) is a multifunctional receptor expressed on macrophages. This study aimed to determine the role of SR-A in diabetic nephropathy using SR-A-deficient (SR-A(-/-)) mice. Diabetes was induced in SR-A(-/-) and wild-type (SR-A(+/+)) mice by streptozotocin injection. Diabetic SR-A(+/+) mice presented characteristic features of diabetic nephropathy: albuminuria, glomerular hypertrophy, mesangial matrix expansion, and overexpression of transforming growth factor-beta at 6 months after induction of diabetes. These changes were markedly diminished in diabetic SR-A(-/-) mice, without differences in blood glucose and blood pressure levels. Interestingly, macrophage infiltration in the kidneys was dramatically decreased in diabetic SR-A(-/-) mice compared with diabetic SR-A(+/+) mice. DNA microarray revealed that proinflammatory genes were overexpressed in renal cortex of diabetic SR-A(+/+) mice and suppressed in diabetic SR-A(-/-) mice. Moreover, anti-SR-A antibody blocked the attachment of monocytes to type IV collagen substratum but not to endothelial cells. Our results suggest that SR-A promotes macrophage migration into diabetic kidneys by accelerating the attachment to renal extracellular matrices. SR-A may be a key molecule for the inflammatory process in pathogenesis of diabetic nephropathy and a novel therapeutic target for diabetic vascular complications.

Association of advanced glycation end products with A549 cells, a human pulmonary epithelial cell line, is mediated by a receptor distinct from the scavenger receptor family and RAGE.

Cellular interactions with advanced glycation end products (AGE)-modified proteins are known to induce several biological responses, not only endocytic uptake and degradation, but also the induction of cytokines and growth factors, combined responses that may be linked to the development of diabetic vascular complications. In this study we demonstrate that A549 cells, a human pulmonary epithelial cell line, possess a specific binding site for AGE-modified bovine serum albumin (AGE-BSA) (K(d) = 27.8 nM), and additionally for EN-RAGE (extracellular newly identified RAGE binding protein) (K(d) = 118 nM). Western blot and RT-PCR analysis showed that RAGE (receptor for AGE) is highly expressed on A549 cells, while the expression of other known AGE-receptors such as galectin-3 and SR-A (class A scavenger receptor), are below the level of detection. The binding of (125)I-AGE-BSA to these cells is inhibited by unlabeled AGE-BSA, but not by EN-RAGE. In contrast, the binding of (125)I-EN-RAGE is significantly inhibited by unlabeled EN-RAGE and soluble RAGE, but not by AGE-BSA. Our results indicate that A549 cells possess at least two binding sites, one specific for EN-RAGE and the other specific for AGE-BSA. The latter receptor on A549 cells is distinct from the scavenger receptor family and RAGE.

Peroxynitrite induces formation of N( epsilon )-(carboxymethyl) lysine by the cleavage of Amadori product and generation of glucosone and glyoxal from glucose: novel pathways for protein modification by peroxynitrite.

Accumulation of advanced glycation end products (AGEs) on tissue proteins increases with pathogenesis of diabetic complications and atherosclerosis. Here we examined the effect of peroxynitrite (ONOO(-)) on the formation of N( epsilon )-(carboxymethyl)lysine (CML), a major AGE-structure. When glycated human serum albumin (HSA; Amadori-modified protein) was incubated with ONOO(-), CML formation was detected by both enzyme-linked immunosorbent assay and high-performance liquid chromatography (HPLC) and increased with increasing ONOO(-) concentrations. CML was also formed when glucose, preincubated with ONOO(-), was incubated with HSA but was completely inhibited by aminoguanidine, a trapping reagent for alpha-oxoaldehydes. For identifying the aldehydes that contributed to ONOO(-)-induced CML formation, glucose was incubated with ONOO(-) in the presence of 2,3-diaminonaphthalene. This experiment led to identification of glucosone and glyoxal by HPLC. Our results provide the first evidence that ONOO(-) can induce protein modification by oxidative cleavage of the Amadori product and also by generation of reactive alpha-oxoaldehydes from glucose.

Specific interaction of oxidized low-density lipoprotein with thrombospondin-1 inhibits transforming growth factor-beta from its activation.

Oxidized LDL (Ox-LDL) plays atherogenic roles, whereas thrombospondin-1 (TSP-1) is thought to be anti-atherogenic through activation of TGF-beta that contributes to plaque stabilization. Ox-LDL was prepared by incubating of human LDL with CuSO4. Effect of Ox-LDL on TSP-1-induced TGF-beta activation was examined in the present study. Incubation of Ox-LDL with mouse peritoneal macrophages for 3 days resulted in reduction in amounts of active TGF-beta in the culture medium by 70-78% when compared with that of parallel incubation without Ox-LDL. TSP-1 could enhance conversion of latent TGF-beta1 into active TGF-beta1 in a cell-free system. This TSP-1-mediated latent TGF-beta1 activation was inhibited by 30% by Ox-LDL, suggesting the possible interaction of Ox-LDL with TSP-1. Incubation of TSP-1 with [125I]Ox-LDL or [125I]LDL, followed by immunoprecipitation with an anti-TSP-1 antibody demonstrated that a significant amount of [125I]Ox-LDL was co-precipitated with TSP-1 while precipitation of [125I]LDL was negligible. Furthermore, upon TSP-1-conjugated Sepharose 4B affinity chromatography, both [125I]Ox-LDL and [125I]latent TGF-beta1 bound to the affinity gel were eluted by unlabeled Ox-LDL. These findings indicate that Ox-LDL interacts with TSP-1 and suppresses subsequent TSP-1-dependent TGF-beta activation, revealing a novel atherogenic function of Ox-LDL.

CD36 is not involved in scavenger receptor-mediated endocytic uptake of glycolaldehyde- and methylglyoxal-modified proteins by liver endothelial cells.

Circulating proteins modified by advanced glycation end-products (AGE) are mainly taken up by liver endothelial cells (LECs) via scavenger receptor-mediated endocytosis. Endocytic uptake of chemically modified proteins by macrophages and macrophage-derived cells is mediated by class A scavenger receptor (SR-A) and CD36. In a previous study using SR-A knockout mice, we demonstrated that SR-A is not involved in endocytic uptake of AGE proteins by LECs [Matsumoto et al. (2000) Biochem. J. 352, 233-240]. The present study was conducted to determine the contribution of CD36 to this process. Glycolaldehyde-modified BSA (GA-BSA) and methylglyoxal-modified BSA (MG-BSA) were used as AGE proteins. 125I-GA-BSA and 125I-MG-BSA underwent endocytic degradation by these cells at 37 degrees C, and this process was inhibited by several ligands for the scavenger receptors. However, this endocytic uptake of 125I-GA-BSA by LECs was not inhibited by a neutralizing anti-CD36 antibody. Similarly, hepatic uptake of (111)In-GA-BSA after its intravenous injection was not significantly attenuated by co-administration of the anti-CD36 antibody. These results clarify that CD36 does not play a significant role in elimination of GA-BSA and MG-BSA from the circulation, suggesting that the receptor involved in endocytic uptake of circulating AGE proteins by LEC is not SR-A or CD36.

Glycolaldehyde-modified bovine serum albumin downregulates leptin expression in mouse adipocytes via a CD36-mediated pathway.

Previous observations by us have clarified that proteins modified by advanced glycation end products (AGEs) are recognized as effective ligands by CD36-overexpressed CHO cells and undergo receptor-mediated endocytosis. CD36, a member of the class B scavenger receptor family, also acts as a fatty acid transporter in adipocytes. Oxidized low-density lipoprotein (Ox-LDL), a ligand for CD36, is known to upregulate CD36 by activating peroxisome proliferator-activated receptor gamma (PPAR-gamma) in macrophages, whereas PPAR-gamma ligands such as troglitazone and 15-deoxy-delta12,14-prostaglandin J2 decrease leptin secretion from adipocytes. The purpose of this study was to examine effects of AGE ligands on leptin expression in adipocytes. Glycolaldehyde-modified bovine serum albumin (GA-BSA) decreased leptin expression at both the protein and mRNA levels in 3T3-L1 adipocytes and mouse epididymal adipocytes. The binding to and subsequent endocytic degradation of GA-BSA by 3T3-L1 adipocytes were effectively inhibited by a neutralizing anti-CD36 antibody. These results indicate that the ligand interaction of GA-BSA with CD36 leads to downregulation of leptin expression in 3T3-L1 adipocytes, suggesting that AGE-induced leptin downregulation is linked to reduction of the insulin sensitivity in metabolic syndrome.

Pathological roles of advanced glycation end product receptors SR-A and CD36.

The pathological significance of advanced glycation end product (AGE)-modified proteins deposited in several lesions is generally accounted for by their cellular interaction via the AGE receptors and subsequent acceleration of the inflammatory process. In this study, we focused on two AGE receptors-specifically, the role of SR-A in pathogenesis of diabetic nephropathy and the role of CD36 in AGE-induced downregulation of leptin by adipocytes. In terms of SR-A, diabetic wild-type mice exhibited increased urinary albumin excretion, glomerular hypertrophy, and mesangial matrix expansion, whereas SR-A-knockout mice showed reduced glomerular size and mesangial matrix area. In these diabetic SR-A-knockout mice, the number of macrophages that infiltrated into glomeruli was remarkably reduced (P < 0.05), suggesting that SR-A-dependent glomerular migration of macrophages plays an important role in the pathogenesis of diabetic nephropathy. In terms of CD36, incubation of glycolaldehyde-modified bovine serum albumin (GA-BSA) with 3T3-L1 adipocytes reduced leptin secretion by these cells. The binding of GA-BSA to these cells and subsequent endocytic degradation were effectively inhibited by a neutralizing anti-CD36 antibody. AGE-induced downregulation of leptin was protected by N-acetyl-cysteine, an antioxidant. These results indicate that the interaction of AGE ligands with 3T3-L1 adipocytes via CD36 induces oxidative stress and leads to inhibition of leptin expression by these cells, suggesting a potential link of this phenomenon to exacerbation of the insulin sensitivity in metabolic syndrome.

Acyl-coenzyme A:cholesterol acyltransferase-2 (ACAT-2) is responsible for elevated intestinal ACAT activity in diabetic rats.

OBJECTIVE: Diabetes-induced dyslipidemia is seen in streptozotocin-induced diabetic rats. This is caused, in part, by elevated intestinal acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity. Because two ACAT isozymes (ACAT-1 and ACAT-2) were identified, in the present study we determined which ACAT isozyme was involved in the elevated intestinal ACAT activity in diabetic rats. METHODS AND RESULTS: We cloned a full-length cDNA of rat ACAT-2. Its overexpression in ACAT-deficient AC29 cells demonstrated that the ACAT activity is derived from the cloned cDNA, and a 45-kDa protein of rat ACAT-2 cross-reacts with an anti-human ACAT-2 antibody. The tissue distribution of rat ACAT-2 mRNA revealed its restricted expression to liver and small intestine. Immunohistochemical analyses using an anti-human ACAT-2 antibody demonstrated that ACAT-2 is localized in villus-crypt axis of rat small intestine. The intestinal ACAT activity in diabetic rats was significantly immunodepleted by an anti-ACAT-2 antibody but not by an anti-ACAT-1 antibody. Finally, intestinal ACAT-2 in diabetic rats significantly increased at both protein and mRNA levels as compared with that in control rats. CONCLUSIONS: Our data demonstrate that ACAT-2 isozyme is responsible for the increased intestinal ACAT activity of diabetic rats, suggesting an important role of ACAT-2 for dyslipidemia in diabetic patients. Diabetic rats exhibit dyslipidemia caused, in part, by elevated intestinal acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity. We determined which ACAT isozyme (ACAT-1 or ACAT-2) was involved in the elevated intestinal ACAT activity in diabetic rats. We demonstrated an important role of ACAT-2, implicating its involvement in dyslipidemia in diabetic patients.

Scavenger receptors that recognize advanced glycation end products.

Scavenger receptors recognize modified low-density lipoproteins (LDLs) such as acetylated LDL and oxidized LDL. Advanced glycation end products (AGE), which are generated through long-term exposure of proteins to glucose, also behave as active ligands for some scavenger receptors, including class A scavenger receptor (SR-A) and class B scavenger receptors such as CD36 and scavenger receptor, class B, type I (SR-BI). SR-BI, the first identified high-density lipoprotein (HDL) receptor, plays key roles in reverse cholesterol transport by promoting selective uptake of cholesteryl esters (CE) in HDL by hepatocytes, and cholesterol efflux of unesterified cholesterol from peripheral cells to HDL. Using Chinese hamster ovary cells overexpressing SR-BI (CHO-SR-BI cells), it was demonstrated that AGE-bovine serum albumin binds to SR-BI and inhibits selective uptake of HDL-CE by CHO-SR-BI cells as well as cholesterol efflux from CHO-SR-BI cells to HDL, suggesting potential roles of AGE in diabetic dyslipidemia and accelerated atherosclerosis in diabetes.

Lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) serves as an endothelial receptor for advanced glycation end products (AGE).

Advanced glycation end products (AGE) are known to serve as ligands for the scavenger receptors such as SR-A, CD36 and SR-BI. In the current study, we examined whether AGE is recognized by lectin-like oxidized low density lipoprotein receptor-1 (LOX-1). Cellular binding experiments revealed that AGE-bovine serum albumin (AGE-BSA) showed the specific binding to CHO cells overexpressing bovine LOX-1 (BLOX-1), which was effectively suppressed by an anti-BLOX-1 antibody. Cultured bovine aortic endothelial cells also showed the specific binding for AGE-BSA, which was suppressed by 67% by the anti-BLOX-1 antibody. Thus, LOX-1 is identified as a novel endothelial receptor for AGE.

CD36-mediated endocytic uptake of advanced glycation end products (AGE) in mouse 3T3-L1 and human subcutaneous adipocytes.

Interaction of advanced glycation end products (AGE) with AGE receptors induces several cellular phenomena potentially relating to diabetic complications. We here show that AGE-modified bovine serum albumin (BSA) is endocytosed by adipocytes via CD36. Upon differentiation, 3T3-L1 and human subcutaneous adipose cells showed marked increases in endocytic uptake and subsequent degradation of [(125)I]AGE-BSA, which were inhibited effectively by the anti-CD36 antibody. Ligand specificity of CD36 for modified BSAs was compared with that of LOX-1 and scavenger receptor class A. Effect of fucoidan on [(125)I]AGE-BSA binding showed a sharp contrast to that on [(125)I]-oxidized low density lipoprotein. These results implicate that CD36-mediated interaction of AGE-modified proteins with adipocytes might play a pathological role in obesity or insulin-resistance.

Reactive oxygen species accelerate production of vascular endothelial growth factor by advanced glycation end products in RAW264.7 mouse macrophages.

Advanced glycation end products (AGEs) are believed to play an important role in the development of angiopathy in diabetes mellitus. Previous reports suggested a correlation between accumulation of AGEs and production of vascular endothelial growth factor (VEGF) in human diabetic retina. However, the mechanisms involved were not revealed. In this study, we investigated the transcriptional regulation of the expression of vascular endothelial growth factor (VEGF) by AGEs, and possible involvement of reactive oxygen species (ROS) in the induction. We employed an AGE of bovine serum albumin (BSA) prepared by an incubation of BSA with D-glucose for 40 weeks and N(epsilon)-(carboxymethyl)lysine (CML), a major AGE. The expression of VEGF was induced by CML-BSA in RAW264.7 mouse macrophage-like cells. CML-BSA stimulated the DNA-binding activity of activator protein-1 (AP-1). Promoter assay showed that the induction of VEGF was dependent on AP-1. The activity of Ras/Raf-1/MEK/ERK1/2 was involved in the CML-BSA-stimulated signaling pathways to activate the AP-1 transcription with a peak at 1 h. AGE-BSA also induced VEGF mediated by AP-1, however, there was a difference of effect between AGE-BSA and CML-BSA in the activation of AP-1. AGE-BSA-stimulated AP-1 activity showed a peak at 5 h, which paralleled the formation of ROS. Reduction of AGE-BSA with NaBH(4) or addition of vitamin E attenuated the AGE-BSA-stimulated signaling pathways leading to the same pattern as for CML-BSA-stimulated signals. These results suggest an important role for AGEs in stimulation of the development of angiogenesis observed in diabetic complications, and that ROS accelerates the AGE-stimulated VEGF expression.

Activated protein C inhibits lipopolysaccharide-induced tumor necrosis factor-alpha production by inhibiting activation of both nuclear factor-kappa B and activator protein-1 in human monocytes.

Activated protein C (APC), an important natural anticoagulant, inhibits tumor necrosis factor-alpha (TNF-alpha) production and attenuates various deleterious events induced by lipopolysaccharide (LPS), contributing thereby to a significant reduction of mortality in patients with severe sepsis. In this study, we investigated the mechanism(s) by which APC inhibits TNF-alpha production by LPS-stimulated human monocytes in vitro. Although APC inhibited LPS-induced TNF-alpha production in a concentration-dependent fashion, diisopropyl fluorophosphate-treated APC, an active-site-blocked APC, had no effect. APC inhibited both the binding of nuclear factor-kappa B (NF-kappa B) to target sites and the degradation of I kappa B alpha. APC also inhibited both the binding of activator protein-1 (AP-1) to target sites and the activation of mitogen-activated protein kinase pathways. These observations strongly suggest that APC inhibited LPS-induced TNF-alpha production by inhibiting the activation of both NF-kappa B and AP-1 and that the inhibitory activity of APC might depend on its serine protease activity. These results would at least partly explain the mechanism(s) by which APC reduces the tissue injury seen in animal models of sepsis and in patients with sepsis.

Possible mechanism for medial smooth muscle cell injury in diabetic nephropathy: glycoxidation-mediated local complement activation.

BACKGROUND: Although recent studies have emphasized the pathogenic role of intrarenal muscular arteries in patients with diabetic nephropathy, notice has not been taken of their pathological characteristics. We focused on medial smooth muscle cell (SMC) injury and the involvement of glycoxidation and complement activation. METHODS: Renal samples were obtained at autopsy from patients with diabetes mellitus (DM), patients with hypertension without renal involvement (n = 9), patients with benign nephrosclerosis (n = 7), and age-matched control subjects (n = 12). Patients with DM had glomerulosclerosis classified as severe (n = 9; DM-sev), moderate (n = 11; DM-mod), and minimal (n = 7). Renal samples were immunohistochemically determined. Activation of plasma complement from healthy subjects using advanced glycation end products (AGEs) also was performed. RESULTS: A marked SMC loss was noted in the media of patients with DM-sev and DM-mod. A membrane attack complex (MAC) observed in the area with SMC loss correlated well with the loss. Considerable carboxymethyllysine (CML), an oxidatively modified AGE, was deposited in the area with SMC loss in patients with DM-mod and DM-sev. Degrees of MAC deposition, SMC loss, and CML deposition were greater in the DM-sev group than the non-DM groups. Another oxidative product, acrolein, colocalized with CML. Plasma complements were not activated by AGE-modified bovine serum albumin in our in vitro assays, which included a complement hemolytic activity assay and determination of complement fragments, including C4d, C3bB, iC3b, and MAC. CONCLUSION: It is strongly suggested that medial SMC injury in intrarenal arteries is caused by interaction between glycoxidation and complement activation and contributes to the progression of diabetic nephropathy.

Acyl-coenzyme A:cholesterol acyltransferase inhibitors for controlling hypercholesterolemia and atherosclerosis.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT; Sterol O-acyltransferase/SOAT) is an intracellular enzyme that catalyzes the formation of cholesteryl esters from cholesterol and fatty acyl-coenzyme A. ACAT inhibitors reduce plasma cholesterol levels by suppressing absorption of dietary cholesterol and by suppressing the assembly and secretion of apolipoprotein B-containing lipoproteins such as very low density lipoprotein in liver and chylomicron in intestine. Moreover, ACAT inhibitors prevent the conversion of macrophages into foam cells in the arterial walls. Thus, ACAT inhibitors are under investigation for controlling hypercholesterolemia and the development of atherosclerosis. Some potent ACAT inhibitors have been tested for their efficacy and safety in humans.

Creatine plays a direct role as a protein modifier in the formation of a novel advanced glycation end product.

Pentosidine, a cross-link structure between lysine and arginine residues, is one of the major advanced glycation end products (AGE). It is formed by the reaction of ribose with lysine and arginine. The pentosidine concentration produced by in vitro incubation of plasma obtained from uremic patients was reported to be higher than in normal plasma, indicating that uremic plasma contains an enhancer(s) for pentosidine formation [Miyata, T., Ueda, Y., Yamada, Y., Izuhara, Y., Wada, T., Jadoul, M., Saito, A., Kurokawa, K., and Strihou, C.Y. (1998) J. Am. Soc. Nephrol. 9, 2349-2356]. Since our preliminary study using a monoclonal anti-pentosidine antibody identified creatine as the most effective enhancer, the purpose of the present study was to clarify the mechanism by which creatine contributes to pentosidine formation. Lysine was incubated with ribose in the presence of creatine and analyzed by reverse phase high performance liquid chromatography. A novel fluorescent peak (lambda(ex/em) = 335/385 nm) was detected at 8 min, under conditions at which the authentic pentosidine (lysine was incubated with ribose in the presence of arginine under identical conditions) eluted at 12 min. Structural analyses of this compound revealed a pentosidine-like structure in which the arginine residue was replaced by creatine. This novel AGE-structure, named here creatine-derived pentosidine (C-pentosidine), was detected in the plasma of patients on hemodialysis. These results indicate that creatine increases the formation of C-pentosidine but not authentic pentosidine. This study indicates that creatine plays a direct role as a protein modifier in C-pentosidine formation, although the clinical significance of C-pentosidine is still unknown.

Conventional antibody against Nepsilon-(carboxymethyl)lysine (CML) shows cross-reaction to Nepsilon-(carboxyethyl)lysine (CEL): immunochemical quantification of CML with a specific antibody.

Immunological strategies for the detection of N(epsilon)-(carboxymethyl)lysine (CML), one of the major antigenic structures of advanced glycation end products (AGE), are widely applied to demonstrate the contribution of CML to the pathogeneses of diabetic complications and atherosclerosis. Recent studies have indicated that methylglyoxal (MG), which is generated intracellularly through the Embden-Meyerhof and polyol pathways, reacts with proteins to form MG-derived AGE structures such as N(epsilon)-(carboxyethyl)lysine (CEL). In order to accurately measure the CML contents of the proteins by means of an immunochemical method, we prepared CML-specific antibodies since conventionally prepared polyclonal anti-CML antibody and monoclonal anti-CML antibody (6D12) cross-reacted with CEL. To prepare polyclonal CML-specific antibody, CML-keyhole limpet hemocyanin (CML-KLH) were immunized with rabbit and CEL-reactive antibody was removed by CEL-conjugated affinity chromatography. Monoclonal antibody specific for CML (CMS-10) was obtained by immunization with CML-KLH, followed by successive screening according to CML-bovine serum albumin (CML-BSA)-positive but CEL-BSA-negative criteria. Both polyclonal CML-specific antibody and CMS-10 significantly reacted with CML-proteins but not with CEL-proteins. It is likely therefore that these antibodies can recognize the difference of one methyl group between CML and CEL. Moreover, CMS-10 significantly reacted with BSA modified with several aldehydes and its reactivity was highly correlated with the CML content, which was determined by high performance liquid chromatography, whereas 6D12 showed a low correlation. These results indicate that CMS-10 can be used to determine the CML contents of modified proteins in a more specific way.

Nepsilon-(Carboxymethyl)lysine and 3-DG-imidazolone are major AGE structures in protein modification by 3-deoxyglucosone.

The levels of plasma 3-deoxyglucosone (3-DG) increase under hyperglycemic conditions and are associated with the pathogenesis of diabetic complications because of the high reactivity of 3-DG with proteins to form advanced glycation end products (AGE). To investigate potential markers for 3-DG-mediated protein modification in vitro and in vivo, we compared the yield of several 3-DG-derived AGE structures by immunochemical analysis and HPLC and measured their localization in human atherosclerotic lesions. When BSA was incubated with 3-DG at 37 degrees C for up to 4 wk, the amounts of N(epsilon)-(carboxymethyl)lysine (CML) and 3-DG-imidazolone steeply increased with incubation time, whereas the levels of pyrraline and pentosidine increased slightly by day 28. In contrast, significant amounts of pyrraline and pentosidine were also observed when BSA was incubated with 3-DG at 60 degrees C to enhance AGE-formation. In atherosclerotic lesions, CML and 3-DG-imidazolone were found intracellularly in the cytoplasm of most foam cells and extracellularly in the atheromatous core. A weak-positive immunoreaction with pyrraline was found in the extracellular matrix and a few foam cells in aortic intima with atherosclerotic lesions. Our results provide the first evidence that CML and 3-DG-imidazolone are major AGE structures in 3-DG-modified proteins, and that 3-DG-imidazolone provides a better marker for protein modification by 3-DG than pyrraline.

Advanced glycation end products (AGE) inhibit scavenger receptor class B type I-mediated reverse cholesterol transport: a new crossroad of AGE to cholesterol metabolism.

Advanced glycation end products (AGE) -modified proteins behave as active ligands for several receptors belonging to the scavenger receptor family. Scavenger receptor class B type I (SR-BI) was identified as the first high density lipoprotein (HDL) receptor that mediates selective uptake of HDL-cholesteryl esters (HDL-CE). This study investigated whether AGE proteins serve as ligands for SR-BI and affect SR-BI-mediated cholesterol transport using Chinese hamster ovary (CHO) cells overexpressing hamster SR-BI (CHO-SR-BI cells). [125I] AGE-bovine serum albumin (AGE-BSA) underwent active endocytosis and subsequent lysosomal degradation by CHO-SR-BI cells, indicating that SR-BI serves as an AGE receptor. SR-BI-mediated selective uptake of HDL-CE by CHO-SR-BI cells was efficiently inhibited by AGE-BSA although AGE-BSA had no effect on HDL binding to CHO-SR-BI cells. In addition, AGE-BSA significantly inhibited the efflux of [3H] cholesterol from CHO-SR-BI cells to HDL. These findings suggest the possibility that AGE proteins in the circulation interfere with the functions of SR-BI in reverse cholesterol transport by inhibiting the selective uptake of HDL-CE, as well as cholesterol efflux from peripheral cells to HDL, thereby accelerating diabetes-induced atherosclerosis.

Vascular change of hippocampal capillary is associated with vascular change of retinal capillary in aging.

Vascular deficiency, such as deleterious change of endothelial cells, becomes the prominent feature of hippocampal microvessels during the processes of aging in rodents and it seems to be associated with deficiency of intellectual behavior in aged subjects. The hippocampal microvessels and hippocampal pyramidal neurons form and accumulate intermediates of advanced Maillard reaction (glycation) end products, specifically N()-carboxymethyl lysine (CML) and CML in rodents during the processes of aging. CML facilitates proliferation of endothelial cells in culture. However, further conjugation of CML with the substance(s) seems to occur in the microvessels and pyramidal neurons of hippocampus and it brings about deleterious change of endothelial cells and pyramidal neuron death. This would cause deficiency of recognition and reference memory in rodents during the processes of aging. In man in Alzheimer's disease (AD), one might speculate that formation and accumulation of CML in the hippocampal microvessels initiate the accumulation of amyloid to produce cerebral amyloid angiopathy and it brings about hypoglycemia and hypoxia in the hippocampal pyramidal neurons. Furthermore, formation and accumulation of CML in the hippocampal pyramidal neurons initiate the deposition of neurofibrillary tangles and senile plaques which cause neuronal death. In this way, vascular deficiency of hippocampal microvessels seems to be associated with the demented disease, the atrophic process of the brain and accumulation of amyloid in the brain in man. In terms of vascular deficiency concerns, the vascular change of the retinal capillaries becomes also a prominent feature during the processes of aging and it has a positive correlation with the vascular change of hippocampal capillary. In man during senescence, one might also speculate that vascular change of eye capillaries would become the early market for diagnosis of dementia in AD.