Induction of aldose reductase in cultured human microvascular endothelial cells by advanced glycation end products

Accelerated formation and accumulation of advanced glycation end products, as well as increased flux of glucose through polyol pathway, have been implicated in the pathogenesis of diabetic vascular complications. We investigated effects of advanced glycation end products on the levels of aldose reductase mRNA, protein, and activity in human microvascular endothelial cells. When endothelial cells were cultured with highly glycated bovine serum albumin, aldose reductase mRNA in endothelial cells demonstrated concentration-dependent elevation. The increase in aldose reductase mRNA was accompanied by elevated protein expression and enzyme activity. Significant increase in the enzyme expression was also observed when endothelial cells were cultured with serum obtained from diabetic patients with end-stage renal disease. Pretreatment of the endothelial cells with probucol or vitamin E prevented the advanced glycation end products-induced increases in aldose reductase mRNA and protein. Electrophoretic mobility shift assays using the nuclear extracts of the endothelial cells treated with advanced glycation end products showed enhancement of specific DNA binding activity for AP-1 consensus sequence. These results indicate that accelerated formation of advanced glycation end products in vivo may elicit activation of the polyol pathway, possibly via augmented oxidative stress, and amplify endothelial cell damage leading to diabetic microvascular dysfunction.     

缬沙坦对人肾小球系膜细胞糖基化终产物受体表达的影响

目的：本实验探讨缬沙坦对糖基化终产物诱导的人肾小球系膜细胞氧化应激水平及糖基化终产物受体（RAGE）表达的影响。方法：体外常规培养人肾小球系膜细胞,运用糖基化修饰的牛血清白蛋白（AGE-BSA）和缬沙坦进行干预,流式细胞术检测细胞内活性氧（ROS）,RT-PCR法检测NADPH氧化酶的亚基p47^phox的mRNA表达,RT-PCR和细胞免疫化学法检测RAGE的表达量。结果：缬沙坦干预组人肾小球系膜细胞的ROS产生量、NADPH氧化酶的亚基p47^phox mRNA表达量、RAGE表达量均低于AGE-BSA组（P〈0．05）,且缬沙坦的抑制作用呈浓度和时间依赖性。结论：缬沙坦可能通过降低氧化应激水平来抑制RAGE的表达。     

Sitagliptin augments protective effects of GLP-1 against advanced glycation end product receptor axis in endothelial cells

Sitagliptin is a stable inhibitor of dipeptidyl peptidase-IV, a responsible enzyme that mainly inactivates glucagon-like peptide-1 (GLP-1), and now one of the widely used agents for the treatment of diabetes. However, effects of sitagliptin on vascular injury are largely unknown. Since advanced glycation end products (AGEs) and their receptor (RAGE) axis contribute to vascular damage in diabetes, we investigated here whether sitagliptin inhibits the AGE-RAGE-induced endothelial cell damage in vitro. Although effects of 10?pM GLP-1 or 0.5?μM sitagliptin monotherapy on RAGE gene and protein expression were modest, combination therapy completely blocked the AGE-induced increase in RAGE mRNA and protein levels in human umbilical vein endothelial cells (HUVEC). AGEs induced reactive oxygen species (ROS) generation and reduced endothelial nitric oxide synthase (eNOS) mRNA level in HUVEC, both of which were also completely blocked by the treatment with 10?pM GLP-1 and 0.5?μM sitagliptin, but not with GLP-1 or sitagliptin monotherapy. Further, anti-RAGE antibody restored the decrease in eNOS mRNA level in AGE-exposed HUVEC. The present study suggests that sitagliptin augments the effects of GLP-1 on eNOS mRNA level in AGE-exposed HUVEC by suppressing RAGE expression and subsequent ROS generation. Sitagliptin may work as a vasoprotecitve agent in diabetes by blocking the AGE-RAGE axis.     

Bridging advanced glycation end product, receptor for advanced glycation end product and nitric oxide with hormonal replacement/estrogen therapy in healthy versus diabetic postmenopausal women: a perspective

Cardiovascular diseases (CVD) are the most significant cause of death in postmenopausal women. The loss of estrogen biosynthesis with advanced age is suggested as one of the major causes of higher CVD in postmenopausal women. While some studies show beneficial effects of estrogen therapy (ET)/hormonal replacement therapy (HRT) in the cardiovascular system of healthy postmenopausal women, similar studies in diabetic counterparts contradict these findings. In particular, ET/HRT in diabetic postmenopausal women results in a seemingly detrimental effect on the cardiovascular system. In this review, the comparative role of estrogens is discussed in the context of CVD in both healthy and diabetic postmenopausal women in regard to the synthesis or expression of proinflammatory molecules like advanced glycation end products (AGEs), receptor for advanced glycation end products (RAGEs), inducible nitric oxide synthases (iNOS) and the anti-inflammatory endothelial nitric oxide synthases (eNOS). The interaction of AGE-RAGE signaling with molecular nitric oxide (NO) may determine the level of reactive oxygen species (ROS) and influence the overall redox status of the vascular microenvironment that may further determine the ultimate outcome of the effects of estrogens on the CVD in healthy versus diabetic women.     

Advanced glycation end product ligands for the receptor for advanced glycation end products: biochemical characterization and formation kinetics

Advanced glycation end products (AGEs) accumulate with age and at an accelerated rate in diabetes. AGEs bind cell-surface receptors including the receptor for advanced glycation end products (RAGE). The dependence of RAGE binding on specific biochemical characteristics of AGEs is currently unknown. Using standardized procedures and a variety of AGE measures, the present study aimed to characterize the AGEs that bind to RAGE and their formation kinetics in vitro. To produce AGEs with varying RAGE binding affinity, bovine serum albumin (BSA) AGEs were prepared with 0.5M glucose, fructose, or ribose at times of incubation from 0 to 12 weeks or for up to 3 days with glycolaldehyde or glyoxylic acid. The AGE-BSAs were characterized for RAGE binding affinity, fluorescence, absorbance, carbonyl content, reactive free amine content, molecular weight, pentosidine content, and N-epsilon-carboxymethyl lysine content. Ribose-AGEs bound RAGE with high affinity within 1 week of incubation in contrast to glucose- and fructose-AGE, which required 12 and 6 weeks, respectively, to generate equivalent RAGE ligands (IC50=0.66, 0.93, and 1.7 microM, respectively). Over time, all of the measured AGE characteristics increased. However, only free amine content robustly correlated with RAGE binding affinity. In addition, detailed protocols for the generation of AGEs that reproducibly bind RAGE with high affinity were developed, which will allow for further study of the RAGE-AGE interaction.     

晚期糖化终产物诱导内皮细胞通透性增高

本文探讨了晚期糖化终产物(advanrced glycation end products,AGEs)修饰蛋白对内皮细胞通透性及细胞骨架肌动蛋白的形态学影响,以及特异的AGEs受体(receptors for AGEs,RAGE)、氧化应激和p38 MAPK通路在此病理过程中的作用。用不同浓度的AGEs修饰人血清白蛋白(AGE-HSA)与人脐静脉内皮细胞株ECV304在体外共同培养不同时间,并设立对照组进行比较,采用TRITC荧光标记白蛋白漏出法测定单层内皮细胞的通透系数Pa值,荧光染色法示细胞骨架的形态学改变。与对照组相比,AGE-HSA以时间和剂量依赖的方式引起单层内皮细胞通透性的升高及细胞骨架肌动蛋白F-actin形态的改变;可溶性RAGE的抗体(anti-RAGE IgG)、NADPH氧化酶抑制剂(apocynin)及p38抑制剂SB203580均可减轻AGEs对内皮细胞屏障功能和形态的影响。结果提示,AGEs修饰蛋白对单层内皮细胞通透性及骨架重排的作用可能通过与内皮细胞上的RAGE结合,引起细胞内的氧化应激,并激活p38 MAPK通路所介导。     

Characterization of the advanced glycation end-product receptor complex in human vascular endothelial cells

Advanced glycation end products (AGEs) have been implicated as causal factors in the vascular complications of diabetes and it is known that these products interact with cells through specific receptors. The AGE-receptor complex, originally described as p60 and p90, has been characterised in hemopoietic cells and the component proteins identified and designated AGE-R1, -R2 and -R3. In the current study we have characterised this receptor in human umbilical vein endothelial cells (HUVECs) and elucidated several important biological properties which may impact on AGE mediated vascular disease. 125I-AGE-BSA binding to HUVEC monolayers was determined with and without various cold competitors. The synthetic AGE, 2-(2-furoyl)-4(5)-furanyl-1H-imidazole (FFI)-BSA, failed to compete with AGE-BSA binding unlike observations already reported in hemopoietic cells. The ability of 125I-AGE-BSA to bind to separated HUVEC plasma membrane (PM) proteins was also examined and the binding at specific bands inhibited by antibodies to each component of the AGE-receptor complex. Western blotting of whole cell and PM fractions, before and after exposure to AGE-BSA, revealed that AGE-R1, -R2 and -R3 are subject to upregulation upon exposure to their ligand, a phenomenon which was also demonstrated by immunofluorescence of non-permeabilised cells. mRNA expression of each AGE-receptor component was apparent in HUVECs, with the AGE-R2 and -R3 gene expression being upregulated upon exposure to AGEs in a time-dependent manner. A phosporylation assay in combination with AGE-R2 immunoprecipitation demonstrated that this component of the receptor complex is phosphorylated by acute exposure to AGE-BSA. These results indicate the presence of a conserved AGE-receptor complex in vascular endothelium which demonstrates subtle differences to other cell-types. In response to AGE-modified molecules, this complex is subject to upregulation, while the AGE-R2 component also displays increased phosphorylation possibly leading to enhanced signal transduction.     

Advanced glycation alters expression of the 67kDa laminin receptor in retinal microvascular endothelial cells

The 67kDa laminin receptor (67LR) plays an important role in vascular cell function and dysfunction. The present study has examined 67LR expression in retinal microvascular endothelial cells after exposure to AGEs. Retinal microvascular endothelial cells were exposed to either AGE-BSA, or were grown on methylglyoxal-modified laminin or Matrigel and expression of 67LR analysed by Western Blotting and RT-PCR/Southern blotting. Western blotting of plasma membrane and RT-PCR/Southern blotting revealed a significant upregulation of 67LR protein/mRNA expression after exposure to AGEs (p<0.05-0.01). The results show that 67LR is upregulated in cells exposed to AGEs and suggests a previously unrecognised role for this receptor in retinal microvascular endothelial cell interaction with glycated substrates.     

Overexpression of receptor of advanced glycation end products hypersensitizes cells for amyloid beta peptide-induced cell death

Receptor of advanced glycation end products (RAGE) was identified as one of the receptors for amyloid beta peptide (Abeta). There is evidence for controversial functions of RAGE such as a mediator of cell death or differentiation. In this report, we demonstrate that RAGE mediates Abeta toxicity. Transient transfection of RAGE already induced cell death. For further analysis, stable clones of hemagglutinin (HA)-tagged RAGE were selected. Analysis of cellular localization of HA-tagged RAGE protein revealed, in addition to the expected cell surface expression, a novel intracellular localization. Stable RAGE-expressing cells were hypersensitive to nanomolar amounts of Abeta. Only cells expressing RAGE at the cell surface showed hypersensitivity to Abeta.     

Hepatocyte growth factor protects human endothelial cells against advanced glycation end products-induced apoptosis

Advanced glycation end products (AGEs) form by a non-enzymatic reaction between reducing sugars and biological proteins, which play an important role in the pathogenesis of atherosclerosis. In this study, we assessed AGEs effects on human umbilical vein endothelial cells (HUVECs) growth, proliferation and apoptosis. Additionally, we investigated whether hepatocyte growth factor (HGF), an anti-apoptotic factor for endothelial cells, prevents AGEs-induced apoptosis of HUVECs. HUVECs were treated with AGEs in the presence or absence of HGF. Treatment of HUVECs with AGEs changed cell morphology, decreased cell viability, and induced DNA fragmentation, leading to apoptosis. Apoptosis was induced by AGEs in a dose- and time-dependent fashion. AGEs markedly elevated Bax and decreased NF-kappaB, but not Bcl-2 expression. Additionally, AGEs significantly inhibited cell growth through a pro-apoptotic action involving caspase-3 and -9 activations in HUVECs. Most importantly, pretreatment with HGF protected against AGEs-induced cytotoxicity in the endothelial cells. HGF significantly promoted the expression of Bcl-2 and NF-kappaB, while decreasing the activities of caspase-3 and -9 without affecting Bax level. Our data suggest that AGEs induce apoptosis in endothelial cells. HGF effectively attenuate AGEs-induced endothelial cell apoptosis. These findings provide new perspectives in the role of HGF in cardiovascular disease.     

Impact of estrogen receptor alpha and beta agonists on delayed alternation in middle-aged rats

Estrogens act in the adult brain to modulate cognition, enhancing performance on some learning tests and impairing performance on others. Our previous research has revealed an impairing effect of chronic 17β-estradiol treatment in young and aged rats on a prefrontally-mediated working memory task, delayed spatial alternation (DSA). Little is known about the mechanisms of these impairing effects. The current study examined the effects of selective estrogen receptor (ER) α or ERβ activation on DSA performance in middle-aged female rats. Ovariectomized 12 month old Long–Evans (LE) rats were treated by subcutaneous injection with the ERα agonist propyl pyrazole triol (PPT) or the ERβ agonist diarylpropionitrile (DPN) at 0.02, 0.08, or 0.20 mg/kg/day, or with oil vehicle and tested on an operant variable delay DSA task. A 17β-estradiol group (10% in cholesterol) was included as a positive control group. We replicated our previous finding of a 17β-estradiol induced deficit on DSA performance and this effect was paralleled by low dose (0.02 mg/kg/day) DPN treatment. Higher doses of DPN failed to produce a significant change in performance. The highest dose of PPT (0.20 mg/kg/day) also impaired performance, but this effect was subtle and limited to the longest delay during the final block of testing. These data confirm our earlier findings that chronic 17β-estradiol treatment has an impairing effect on the DSA task, and suggest that ERβ activation may underlie the deficit.     

Differential distribution and modulation of expression of alpha 1/beta 1 integrin on human endothelial cells

In this paper we report that the integrin complex alpha 1/beta 1, a laminin/collagen receptor, is expressed on cultured foreskin microvascular endothelium, but is absent on endothelial cells from large vessels such as the aorta and umbilical and femoral veins. The restricted expression of integrin alpha 1/beta 1 to microvascular endothelium was also demonstrated in vivo, by immunohistochemical staining of human tissue sections. Alpha 1 specific antibodies reacted strongly with endothelial cells of small blood vessels and capillaries in several tissues, but not with endothelium of vein and arteries of umbilical cord. Expression of integrin alpha 1 can be induced in cultured umbilical vein endothelial cells by treatment with 5 ng/ml tumor necrosis factor alpha (TNF alpha). Induction of alpha 1 subunit expression also occurred after treatment of umbilical vein endothelium with 10(-5) M retinoic acid or with 10 nM PMA; Maximal induction of alpha 1 integrin was reached after 48 h of treatment and costimulation with TNF alpha and PMA resulted in a synergistic effect. The induction of alpha 1 integrin changed the adhesive properties of umbilical vein endothelial cells, by increasing the adhesiveness to collagen, laminin, and laminin fragment P1, while adhesion to fibronectin and laminin fragment E8 remained constant. The alpha 1 integrin is thus a marker of a specific population of endothelial cells and its expression confers distinctive properties of interaction with the underlying basal membrane.     

Impact of estrogen receptor alpha and beta agonists on delayed alternation in middle-aged rats

Estrogens act in the adult brain to modulate cognition, enhancing performance on some learning tests and impairing performance on others. Our previous research has revealed an impairing effect of chronic 17β-estradiol treatment in young and aged rats on a prefrontally-mediated working memory task, delayed spatial alternation (DSA). Little is known about the mechanisms of these impairing effects. The current study examined the effects of selective estrogen receptor (ER) α or ERβ activation on DSA performance in middle-aged female rats. Ovariectomized 12 month old Long–Evans (LE) rats were treated by subcutaneous injection with the ERα agonist propyl pyrazole triol (PPT) or the ERβ agonist diarylpropionitrile (DPN) at 0.02, 0.08, or 0.20 mg/kg/day, or with oil vehicle and tested on an operant variable delay DSA task. A 17β-estradiol group (10% in cholesterol) was included as a positive control group. We replicated our previous finding of a 17β-estradiol induced deficit on DSA performance and this effect was paralleled by low dose (0.02 mg/kg/day) DPN treatment. Higher doses of DPN failed to produce a significant change in performance. The highest dose of PPT (0.20 mg/kg/day) also impaired performance, but this effect was subtle and limited to the longest delay during the final block of testing. These data confirm our earlier findings that chronic 17β-estradiol treatment has an impairing effect on the DSA task, and suggest that ERβ activation may underlie the deficit.     

Detection and determination of glyceraldehyde-derived advanced glycation end product

Protein is modified by carbonyl compound in the Maillard reaction, and the irreversible structure is formed as the advanced glycation end product (AGE). We identified GLAP (glyceraldehyde-derived pyridinium compound) as an AGE formed from glyceraldehyde and lysine residue of protein. In the present study, we investigated detection and determination of GLAP from glycated protein using fluorescence HPLC method. Albumin (BSA) and carbonyls (glyceraldehyde, glycolaldehyde, methylglyoxal, glyoxal, three pentoses or three hexoses) were dissolved in phosphate buffed solution (pH 7.4), and incubated at 37 degrees C for a week. GLAP was formed only in the glyceraldehyde-modified BSA. It is suggested that GLAP was specific AGE derived from glyceraldehyde. In addition, GLAP depressed the intracellular glutathione level and induced the reactive oxygen species (ROS) in HL-60 cells. GLAP caused the oxidative stress. Therefore, GLAP will be a biomarker in the AGE related disease such as diabetic complications or chronic renal failure.     

Differential human interferon alpha receptor expression on proliferating and non-proliferating cells

The expression of interferon-alpha (IFN-alpha) receptors was studied on a variety of human cells, using monoiodinated IFN-alpha 2 probes. Steady-state binding at 4 degrees C revealed a single class of non-interacting IFN receptor on peripheral blood lymphocytes, and tonsillar B lymphocytes, which are both known to be G0/G1 resting cell populations. The binding affinity of this class of receptor was found to be on the order of 5 X 10(-10) M, expressed as an apparent dissociation constant (Kd). However, cells proliferating either in culture or in vivo were found to express a heterogeneity in IFN-alpha 2 binding. Such binding could be objectively resolved (by a version of the LIGAND program of P. Munson) into a two-site receptor model. Hill plots of binding to proliferating cells indicated a negative cooperativity in the interaction of IFN and receptor. The high-affinity component, expressed on proliferating cells, typically exhibits a Kd of (1-10) X 10(-11) M, while the lower-affinity component indicates a Kd of (1-10) X 10(-9) M. Furthermore, the low-affinity component is apparently expressed on the order of 10-200 times the copy number, per cell, of the high-affinity site. Affinity-labeling experiments revealed that, in addition to the 140-160-kDa IFN-binding complex reported by others, both the proliferating and non-proliferating cell populations possess a novel IFN-binding component of 60 kDa.     

Advanced glycation end product recognition by the receptor for AGEs

Nonenzymatic protein glycation results in the formation of advanced glycation end products (AGEs) that are implicated in the pathology of diabetes, chronic inflammation, Alzheimer's disease, and cancer. AGEs mediate their effects primarily through a receptor-dependent pathway in which AGEs bind to a specific cell surface associated receptor, the Receptor for AGEs (RAGE). N(?)-carboxy-methyl-lysine (CML) and N(?)-carboxy-ethyl-lysine (CEL), constitute two of the major AGE structures found in tissue and blood plasma, and are physiological ligands of RAGE. The solution structure of a CEL-containing peptide-RAGE V domain complex reveals that the carboxyethyl moiety fits inside a positively charged cavity of the V domain. Peptide backbone atoms make specific contacts with the V domain. The geometry of the bound CEL peptide is compatible with many CML (CEL)-modified sites found in plasma proteins. The structure explains how such patterned ligands as CML (CEL)-proteins bind to RAGE and contribute to RAGE signaling.     

Minimum stable structure of the receptor for advanced glycation end product possesses multi ligand binding ability

The receptor for advanced glycation end products (RAGE) is a multi-ligand receptor involved in the development of diabetic complications. Although the soluble form of the extracellular domain maintains the ability to bind multi-ligands, it is unstable and degrades into several peptide species during storage. Proteolysis with thrombin or factor Xa revealed several protease sensitive sites. Most sensitive site is located between Arg228 and Val229, and peptide bond next to Arg216, Arg116, Arg114 and Trp271 are also cleaved. Seven truncated extracellular domains of RAGE were engineered in order to obtain a stable soluble fragment. RAGE 143 (Ala23-Thr143) is not only protease resistant but also shows the same ligand-binding ability as that of the full-length extracellular domain. The resultant minimum RAGE 143 works as a stable recognition devise to detect advanced glycation end products (AGEs).     

Participation of the receptor for advanced glycation end products in efferocytosis

Clearance of apoptotic cells by macrophages and other phagocytic cells, called efferocytosis, is a central process in the resolution of inflammation. Although the receptor for advanced glycation end products (RAGE) has been shown to participate in a variety of acute and chronic inflammatory processes in the lungs and other organs, a role for RAGE in efferocytosis has not been reported. In the present studies, we examined the potential involvement of RAGE in efferocytosis. Macrophages from transgenic RAGE(-/-) mice showed a decreased ability to engulf apoptotic neutrophils and thymocytes. Pretreatment of RAGE(+/+) macrophages with advanced glycation end products, which competitively bind to RAGE, or Abs against RAGE diminished phagocytosis of apoptotic cells. Overexpression of RAGE in human embryonic kidney 293 cells resulted in an increased ability to engulf apoptotic cells. Furthermore, we found that incubation with soluble RAGE enhances phagocytosis of apoptotic cells by both RAGE(+/+) and RAGE(-/-) macrophages. Direct binding of RAGE to phosphatidylserine (PS), an "eat me" signal highly expressed on apoptotic cells, was shown by using solid-phase ELISA. The ability of RAGE to bind to PS on apoptotic cells was confirmed in an adhesion assay. Decreased uptake of apoptotic neutrophils by macrophages was found under in vivo conditions in the lungs and peritoneal cavity of RAGE(-/-) mice. These results demonstrate a novel role for RAGE in which it is able to enhance efferocytosis through binding to PS on apoptotic cells.