Regulatory role of peroxynitrite in advanced glycation end products mediated diabetic cardiovascular complications

The Advanced Glycation End Products(AGE)binding with its receptor can increase reactive oxygen species(ROS)generation through specific signaling mediators.The effect of superoxide(O2-)and O2-mediated ROS and reactive nitrogen species depends on their concentration and location of formation.Nitric oxide(NO)has anti-inflammatory and anticoagulant properties and a vasodilation effect,but NO can be deactivated by reacting with O_(2)^(-).This reaction between NO and O2-produces the potent oxidant ONOO−.Therefore,ONOO-'s regulatory role in AGEs in diabetic cardiovascular complications must considered as a regulator of cardiovascular complications in diabetes.

Advanced glycation end products:Key mediator and therapeutic target of cardiovascular complications in diabetes

The incidence of type 2 diabetes mellitus is growing in epidemic proportions and has become one of the most critical public health concerns.Cardiovascular complications associated with diabetes are the leading cause of morbidity and mortality.The cardiovascular diseases that accompany diabetes include angina,myocardial infarction,stroke,peripheral artery disease,and congestive heart failure.Among the various risk factors generated secondary to hyperglycemic situations,advanced glycation end products(AGEs)are one of the important targets for future diagnosis and prevention of diabetes.In the last decade,AGEs have drawn a lot of attention due to their involvement in diabetic pathophysiology.AGEs can be derived exogenously and endogenously through various pathways.These are a nonhomogeneous,chemically diverse group of compounds formed nonenzymatically by condensation between carbonyl groups of reducing sugars and free amino groups of protein,lipids,and nucleic acid.AGEs mediate their pathological effects at the cellular and extracellular levels by multiple pathways.At the cellular level,they activate signaling cascades via the receptor for AGEs and initiate a complex series of intracellular signaling resulting in reactive oxygen species generation,inflammation,cellular proliferation,and fibrosis that may possibly exacerbate the damaging effects on cardiac functions in diabetics.AGEs also cause covalent modifications and cross-linking of serum and extracellular matrix proteins;altering their structure,stability,and functions.Early diagnosis of diabetes may prevent its progression to complications and decrease its associated comorbidities.In the present review,we recapitulate the role of AGEs as a crucial mediator of hyperglycemia-mediated detrimental effects in diabetes-associated complications.Furthermore,this review presents an overview of future perspectives for new therapeutic interventions to ameliorate cardiovascular complications in diabetes.

The effect of protein oxidation on the formation of advanced glycation end products after chicken myofibrillar protein glycation

Investigation that protein oxidation to the formation of advanced glycation end products(AGEs)after chicken myofibrillar protein glycation is limited.Models of protein oxidation induced by different concentrations of hydroxyl radicals(·OH)were developed after the chicken myofibrillar protein mild glycation(MPG).Results exhibited that levels of AGEs and surface hydrophobicity(H_(0))steadily increased with the a ddition of h ydrogen peroxide(H_(2)O_(2))concentration.However,levels of s ulfhydryl group,free amino group,and particle size gradually decreased with the H_(2)O_(2)concentration.The protein carbonyl value increased in H_(2)O_(2)concentration until 10 mmol/L.Pearson's correlation indicated that MPG structure modification(unfolding and degradation)induced by protein oxidation were significantly positively correlated with AGEs concentration(P<0.05).Finally,a mechanism was proposed to hypothesize t he effect of protein oxidation on the formation of AGEs under MPG conditions.

Implications of receptor for advanced glycation end products for progression from obesity to diabetes and from diabetes to cancer

Obesity and type 2 diabetes mellitus(T2DM)are chronic pathologies with a high incidence worldwide.They share some pathological mechanisms,including hyperinsulinemia,the production and release of hormones,and hyperglycemia.The above,over time,affects other systems of the human body by causing tissue hypoxia,low-grade inflammation,and oxidative stress,which lay the pathophysiological groundwork for cancer.The leading causes of death globally are T2DM and cancer.Other main alterations of this pathological triad include the accumulation of advanced glycation end products and the release of endogenous alarmins due to cell death(i.e.,damage-associated molecular patterns)such as the intracellular proteins high-mobility group box protein 1 and protein S100 that bind to the receptor for advanced glycation products(RAGE)-a multiligand receptor involved in inflammatory and metabolic and neoplastic processes.This review analyzes the latest advanced reports on the role of RAGE in the development of obesity,T2DM,and cancer,with an aim to understand the intracellular signaling mechanisms linked with cancer initiation.This review also explores inflammation,oxidative stress,hypoxia,cellular senescence,RAGE ligands,tumor microenvironment changes,and the“cancer hallmarks”of the leading tumors associated with T2DM.The assimilation of this information could aid in the development of diagnostic and therapeutic approaches to lower the morbidity and mortality associated with these diseases.

Catalpol Prevents Glomerular Angiogenesis Induced by Advanced Glycation End Products via Inhibiting Galectin-3

Objective:The main characteristics of diabetic nephropathy(DN)at the early stage are abnormal angiogenesis of glomerular endothelial cells(GECs)and macrophage infiltration.Galectin-3 plays a pivotal role in the pathogenesis of DN via binding with its ligand,advanced glycation end products(AGEs).Catalpol,an iridoid glucoside extracted from Rehmannia glutinosa,has been found to ameliorate vascular inflammation,reduce endothelial permeability,and protect against endothelial damage in diabetic milieu.However,little is known about whether catalpol could exert an anti-angiogenesis and anti-inflammation effect induced by AGEs.Methods:Mouse GECs(mGECs)and RAW 264.7 macrophages were treated with different concentrations of AGEs(0,50,100,200 and 400μg/mL)for different time(0,6,12,24 and 48 h)to determine the optimal concentration of AGEs and treatment time.Cells were treated with catalpol(10μmol/L),GB1107(1μmol/L,galectin-3 inhibitor),PX-478(50μmol/L,HIF-1αinhibitor),adenovirus-green fluorescent protein(Ad-GFP)[3×10^(7)plaque-forming unit(PFU)/mL]or Ad-galectin-3-GFP(2×10^(8)PFU/mL),which was followed by incubation with 50μg/mL AGEs.The levels of galectin-3,vascular endothelial growth factor A(VEGFA)and pro-angiogenic factors angiopoietin-1(Ang-1),angiopoietin-2(Ang-2),tunica interna endothelial cell kinase-2(Tie-2)were detected by enzyme-linked immunosorbent assay(ELISA).Cell counting kit-8(CCK-8)assay was used to evaluate the proliferation of these cells.The expression levels of galectin-3,vascular endothelial growth factor receptor 1(VEGFR1),VEGFR2,and hypoxia-inducible factor-1α(HIF-1α)in mGECs and those of galectin-3 and HIF-1αin RAW 264.7 macrophages were detected by Western blotting and immunofluorescence(IF)staining.The rat DN model was established.Catalpol(100 mg/kg)or GB1107(10 mg/kg)was administered intragastrically once a day for 12 weeks.Ad-galectin-3-GFP(6×10^(7)PFU/mL,0.5 mL)or Ad-GFP(6×10^(6)PFU/mL,0.5 mL)was injected into the tail vein of rats 48 h before the sacrifice of the animals.The expression of galectin-3,VEGFR1,.VEGFR2,and HIF-1αin renal cortices was analyzed by Western blotting.The expression of galectin-3,F4/80(a macrophage biomarker),and CD34(an endothelium biomarker)in renal cortices was detected by IF staining,and collagen accumulation by Masson staining.Results:The expression levels of galectin-3 and VEGFA were significantly higher in mGECs and RAW 264.7 macrophages treated with 50μg/mL AGEs for 48 h than those in untreated cells.Catalpol and GB1107 could block the AGEs-induced proliferation of mGECs and RAW 264.7 macrophages.Over-expression of galectin-3 was found to reduce the inhibitory effect of catalpol on the proliferation of cells.Catalpol could significantly decrease the levels of Ang-1,Ang-2 and Tie-2 released by AGEs-treated mGECs,which could be reversed by over-expression of galectin-3.Catalpol could significantly inhibit AGEs-induced expression of galectin-3,HIF-1α,VEGFR1,and VEGFR2 in mGECs.The inhibitory effect of catalpol on galectin-3 in AGEs-treated mGECs was impaired by PX-478.Moreover,catalpol attenuated the AGEs-activated HIF-1α/galectin-3 pathway in RAW 264.7 macrophages,which was weakened by PX-478.Additionally,catalpol significantly inhibited the expression of galectin-3,macrophage infiltration,collagen accumulation,and angiogenesis in the kidney of diabetic rats.Over-expression of galectin-3 could antagonize these inhibitory effects of catalpol.Conclusion:Catalpol prevented the angiogenesis of mGECs and macrophage proliferation via inhibiting galectin-3.It could prevent the progression of diabetes-induced renal damage.

Nε-(carboxymethyl)lysine promotes lipid uptake of macrophage via cluster of differentiation 36 and receptor for advanced glycation end products

BACKGROUND Advanced glycation end products(AGEs)are diabetic metabolic toxic products that cannot be ignored.Nε-(carboxymethyl)lysine(CML),a component of AGEs,could increase macrophage lipid uptake,promote foam cell formation,and thereby accelerate atherosclerosis.The receptor for AGEs(RAGE)and cluster of differentiation 36(CD36)were the receptors of CML.However,it is still unknown whether RAGE and CD36 play key roles in CML-promoted lipid uptake.AIM Our study aimed to explore the role of RAGE and CD36 in CML-induced macrophage lipid uptake.METHODS In this study,we examined the effect of CML on lipid uptake by Raw264.7 macrophages.After adding 10 mmol/L CML,the lipid accumulation in macrophages was confirmed by oil red O staining.Expression changes of CD36 and RAGE were detected with immunoblotting and quantitative real-time polymerase chain reaction.The interaction between CML with CD36 and RAGE was verified by immunoprecipitation.We synthesized a novel N-succinimidyl-4-18Ffluorobenzoate-CML radioactive probe.Radioactive receptor-ligand binding assays were performed to test the binding affinity between CML with CD36 and RAGE.The effects of blocking CD36 or RAGE on CML-promoting lipid uptake were also detected.RESULTS The study revealed that CML significantly promoted lipid uptake by macrophages.Immunoprecipitation and radioactive receptor-ligand binding assays indicated that CML could specifically bind to both CD36 and RAGE.CML had a higher affinity for CD36 than RAGE.ARG82,ASN71,and THR70 were the potential interacting amino acids that CD36 binds to CML Anti-CD36 and anti-RAGE could block the uptake of CML by macrophages.The lipid uptake promotion effect of CML was significantly attenuated after blocking CD36 or RAGE.CONCLUSION Our results suggest that the binding of CML with CD36 and RAGE promotes macrophage lipid uptake.

Fluorescent advanced glycation end-products (ages) detected by spectro-photofluorimetry, as a screening tool to detect diabetic microvascular complications

BACKGROUND: Advanced glycation end-products (AGEs) are one of the mechanisms related to diabetic vascular complications. However, since AGEs are multiple and heterogeneous moieties, there is no universally accepted method to measure them for clinical purposes. The aim of this work was to study the utility of a simple fluorimetric assay as predictor of complications. METHODS: Blood samples from 102 type 2 diabetic patients were obtained to assess glucose, glycosylated haemoglobin, creatinine, lipoproteins and C Reactive Protein (CRP), fluorescent AGES by spectrophotofluorimetry and non-fluorescent AGEs by measurement of N(ε)-carboxymethyl-Lysine (CML) using an ELISA kit in a subsample of 82 patients. Urinary fluorescent AGEs, albumin and creatinine were also measured in a morning urine sample. Microvascular complications were studied by ophthalmologic examination, albuminuria and peripheral nerve conduction velocity. RESULTS: Patients without microvascular complications had significantly lower levels of both serum and urinary AGEs. CML was associated with retinopathy. Multiple regression analysis confirmed that AGEs, length of diabetes and glycosylated haemoglobin were all variables associated with diabetic complications, in this sample. CONCLUSIONS: A simple fluorimetric assay to measure low molecular weight fluorescent AGEs, and CML could be employed as screening tools to predict diabetic complications, at a primary care setting. AGEs should probably be considered as another therapeutic target in diabetes management.

Rosiglitazone inhibits expression of acyl-coenzyme A:cholesterol acyltransferase-1 in THP-1 macrophages induced by advanced glycation end-products

Objective:To investigate the effects of rosiglitazone,a synthetic ligand of peroxisome proliferators-activated receptor gamma(PPARγ),on the expression of acyl-coenzyme A:cholesterol acyltransferase-1(ACAT-1) in phorbol myristate acetate(PMA)-pretreated THP-1 cells after the inducement of advanced glycation end products(AGEs).Methods:After THP-1 cells were cultured in the presence of 0.1 μmol/L PMA for 72 h to induce phagocytic differentiation,the obtained THP-1 macrophages were treated with rosiglitazone for 4 h at different concentrations(1,5 or 10 μmol/L) and then exposed to AGEs-modified bovine serum albumin(AGEs-BSA) for 24 h at a concentration of 200 mg/L.Reverse transcription polymerase chain reaction(RT-PCR) and Western blot analysis were performed to detect the mRNA and protein expressions of ACAT-1 respectively.Results:Administration of AGEs-BSA(200 mg/L) into the THP-1 macrophages resulted in up-regulation of ACAT-1 at mRNA and protein levels when compared with the expressions in macrophages incubated with serum-free RPMI1640.Pretreatment of rosiglitazone inhibited significantly the increased expression of ACAT-1 induced by AGEs-BSA in a concentration-dependent manner.Conclusion:PPARγ activation by rosiglitazone down-regulates ACAT-1 expression induced by AGEs in THP-1 macrophages,which might provide a new way for treating atherogenesis in diabetic patients.

Individual and Combined Effects of Food Components in Attenuating the Formation of Advanced Glycation End Products (AGEs)

Advanced Glycation End Products (AGEs) have been associated as a possible cause in inflammation-mediated chronic diseases such as diabetes, Alzheimer’s and cardiovascular disorders. Thus, inhibition of AGE formation represents a prospective therapeutic target for the prevention and treatment of these complications. This study investigated the individual and combined effects of dietary ingredients, spices, on lowering AGEs formation in meat patties. In the study, Carboxymethyllysine (CML), a well-investigated AGE is used as a marker for AGEs and malondialdehyde (MDA) as an indicator for lipid peroxidation. Nine spices were selected based on their ability to inhibit the formation of AGEs at different stages of Maillard reactions. Individually, all the 9 selected spices significantly inhibited the formation of AGEs. Among the 33 combinations of spices, 26 combinations significantly inhibited the formation of AGEs. The highest reduction (84%) was found by the combination of Black Pepper-Rose-Cumin. The individual spices failed to significantly lower the MDA concentration;however, all 33 combinations were able to significantly reduce MDA concentration. The results of this study showed that spices when supplemented in combinations are more effective in inhibiting the formation of AGEs and in decreasing MDA concentration in meat patties.

Advanced glycation end product signaling and metabolic complications:Dietary approach

Advanced glycation end products(AGEs)are a heterogeneous collection of compounds formed during industrial processing and home cooking through a sequence of nonenzymatic glycation reactions.The modern western diet is full of heat-treated foods that contribute to AGE intake.Foods high in AGEs in the contemporary diet include processed cereal products.Due to industrialization and marketing strategies,restaurant meals are modified rather than being traditionally or conventionally cooked.Fried,grilled,baked,and boiled foods have the greatest AGE levels.Higher AGE-content foods include dry nuts,roasted walnuts,sunflower seeds,fried chicken,bacon,and beef.Animal proteins and processed plant foods contain furosine,acrylamide,heterocyclic amines,and 5-hydroxymethylfurfural.Furosine(2-furoil-methyl-lysine)is an amino acid found in cooked meat products and other processed foods.High concentrations of carboxymethyl-lysine,carboxyethyl-lysine,and methylglyoxal-O are found in heat-treated nonvegetarian foods,peanut butter,and cereal items.Increased plasma levels of AGEs,which are harmful chemicals that lead to age-related diseases and physiological aging,diabetes,and autoimmune/inflammatory rheumatic diseases such as systemic lupus erythematosus and rheumatoid arthritis.AGEs in the pathophysiology of metabolic diseases have been linked to individuals with diabetes mellitus who have peripheral nerves with high amounts of AGEs and diabetes has been linked to increased myelin glycation.Insulin resistance and hyperglycemia can impact numerous human tissues and organs,leading to long-term difficulties in a number of systems and organs,including the cardiovascular system.Plasma AGE levels are linked to all-cause mortality in individuals with diabetes who have fatal or nonfatal coronary artery disease,such as ventricular dysfunction.High levels of tissue AGEs are independently associated with cardiac systolic dysfunction in diabetic patients with heart failure compared with diabetic patients without heart failure.It is widely recognized that AGEs and oxidative stress play a key role in the cardiovascular complications of diabetes because they both influence and are impacted by oxidative stress.All chronic illnesses involve protein,lipid,or nucleic acid modifications including crosslinked and nondegradable aggregates known as AGEs.Endogenous AGE formation or dietary AGE uptake can result in additional protein modifications and stimulation of several inflammatory signaling pathways.Many of these systems,however,require additional explanation because they are not entirely obvious.This review summarizes the current evidence regarding dietary sources of AGEs and metabolism-related complications associated with AGEs.

Advanced glycation end products induce neural tube defects through elevating oxidative stress in mice

Our previous study showed an association between advanced glycation end products （AGEs） and neural tube defects （NTDs）. To understand the molecular mechanisms underlying the effect of AGEs on neural tube development, C57BL/6 female mice were fed for 4 weeks with com- mercial food containing 3% advanced glycation end product bovine serum albumin （AGE-BSA） or 3% bovine serum albumin （BSA） as a control. After mating mice, oxidative stress markers including malondialdehyde and H202 were measured at embryonic day 7.5 （E7.5） of ges- tation, and the level of intracellular reactive oxygen species （ROS） in embryonic cells was determined at E8.5. In addition to evaluating NTDs, an enzyme-linked immunosorbent assay was used to determine the effect of embryonic protein administration on the N-（carboxymethyl） lysine reactivity of acid and carboxyethyl lysine antibodies at E10.5. The results showed a remarkable increase in the incidence of NTDs at El0.5 in embryos of mice fed with AGE-BSA （no hyperglycemia） compared with control mice. Moreover, embryonic protein administration resulted in a noticeable increase in the reactivity of N-（carboxymethyl） lysine and N（ε）-（carboxyethyl） lysine antibodies. Malondialdehyde and H2O2 levels in embryonic cells were increased at E7.5, followed by increased intracellular ROS levels at E8.5. Vitamin E supplementation could partially recover these phenomena. Collectively, these results suggest that AGE-BSA could induce NTDs in the absence of hyperglycemia by an underlying mechanism that is at least partially associated with its capacity to increase embryonic oxidative stress levels.

Advanced Glycation End Products Promote Differentiation of CD4^+ T Helper Cells toward Pro-inflammatory Response

This study investigated the effect of advanced glycation end products（AGEs） on differentiation of na ve CD4＋T cells and the role of the receptor of AGEs（RAGE） and peroxisome proliferator-activated receptors（PPARs） activity in the process in order to gain insight into the mechanism of immunological disorders in diabetes. AGEs were prepared by the reaction of bovine serum albumin（BSA） with glucose. Human na ve CD4＋T cells, enriched from blood of healthy adult volunteers with negative selection assay, were cultured in vitro and treated with various agents including AGEs, BSA, high glucose, PGJ2 and PD68235 for indicated time. In short hairpin（sh） RNA knock-down experiment, na ve CD4＋T cells were transduced with media containing shRNA-lentivirus generated from lentiviral packaging cell line, Lent-XTM293 T cells. Surface and intracellular cytokine stainings were used for examination of CD4＋T cell phenotypes, and real-time PCR and Western blotting for detection of transcription factor mRNA and protein expression, respectively. The suppressive function of regulatory T（Treg） cells was determined by a [3H]-thymidine incorporation assay. The results showed that AGEs induced higher pro-inflammatory Th1/Th17 cells differentiated from na ve CD4＋T cells than the controls, whereas did not affect anti-inflammatory Treg cells. However, AGEs eliminated suppressive function of Treg cells. In addition, AGEs increased RAGE mRNA expression in na ve CD4＋T cells, and RAGE knock-down by shRNA eliminated the effect of AGEs on the differentiation of CD4＋T cells and the reduction of suppressive function of Treg cells. Furthermore, AGEs inhibited the mRNA expression of PPARγ, not PPARα; PPARγ agonist, PGJ2, inhibited the effect of AGEs on na ve CD4＋T cell differentiation and reversed the AGE-reduced suppressive function of Treg cells; on the other hand, PPARγ antagonist, PD68235, attenuated the blocking effect of RAGE shRNA on the role of AGEs. It was concluded that AGEs may promote CD4＋T cells development toward pro-inflammatory state, which is associated with increased RAGE mRNA expression and reduced PPARγ activity. ＋

Injury of cortical neurons is caused by the advanced glycation end products-mediated pathway

Advanced glycation end products lead to cell apoptosis, and cause cell death by increasing endoplasmic reticulum stress. Advanced glycation end products alone may also directly cause damage to tissues and cells, but the precise mechanism remains unknown. This study used primary cultures of rat cerebral cortex neurons, and treated cells with different concentrations of glycation end products （50, 100, 200, 400 mg/L）, and with an antibody for the receptor of advanced glycation end products before and after treatment with advanced glycation end products. The results showed that with increasing concentrations of glycation end products, free radical content increased in neurons, and the number of apoptotic cells increased in a dose-dependent manner. Before and after treatment of advanced glycation end products, the addition of the antibody against advanced glycation end-products markedly reduced hydroxyl free radicals, malondialdehyde levels, and inhibited cell apoptosis. This result indicated that the antibody for receptor of advanced glycation end-products in neurons from the rat cerebral cortex can reduce glycation end product-induced oxidative stress damage by suppressing glycation end product receptors. Overall, our study confirms that the advanced glycation end products-advanced glycation end products receptor pathway may be the main signaling pathway leading to neuronal damage.

Role of the advanced glycation end products receptor in Crohn's disease inflammation

AIM:To investigate the level of mucosal expression and the involvement of the receptor for the advanced glycation end products(RAGE)in delayed apoptosis and tumor necrosis factor(TNF)-αproduction in Crohn’s disease(CD).METHODS:Surgical and endoscopic specimens from both inflamed and non-inflamed areas of the ileum and/or colon were collected from 20 and 14 adult CD patients,respectively,and used for the assessment of RAGE expression by means of immunohistochemistry and western blotting analysis.Normal tissues from 21 control subjects were used for comparison.The same polyclonal anti-human RAGE antibody(R and D System)was used in all experimental conditions.RAGE staining was quantized by a score including both the amount of positive cells and intensity of immunoreactivity;cellular pattern was also described.The effects of RAGE blocking on apoptotic rate and TNF-αproduction were investigated on immune cells freshly isolated from CD mucosa and incubated both with and without the muramyl dipeptide used as antigenic stimulus.Statistical analysis was performed via the test for trend,with regression models to account for intra-patient correlations.A 2-sided P<0.05 was considered significant.RESULTS:In inflamed areas,RAGE expression in both the epithelial and lamina propria compartments was higher than control tissues(P=0.001 and 0.021,respectively),and a cluster of positive cells were usually found in proximity of ulcerative lesions.Similar results were obtained in the lamina propria compartment of non-inflamed areas(P=0.025).The pattern of staining was membranous and granular cytosolic at the epithelial level,while in the lamina propria it was diffuse cytosolic.When evaluating the amount of protein expression by immunoblotting,a significant increase of both surface area and band intensity(P<0.0001 for both)was observed in CD inflamed areas compared to control tissue,while in non-inflamed areas a significant increase was found only for band intensity(P<0.005).Moreover,a significantly lower expression in noninflamed areas in comparison with inflamed areas was found for both surface area and band intensity(P<0.0006 for both).Finally,RAGE blocking largely affects both the apoptotic rate of mucosal cells(towards an increase in both non-inflamed and inflamed areas of P<0.001 and<0.0001,respectively)and TNF-αsecretion(towards a decrease in both non-inflamed and inflamed areas of P<0.05 and<0.01,respectively),mainly in the presence of antigenic stimulation.CONCLUSION:RAGE is up-regulated in CD,especially in inflamed areas,and it appears to play a role in the mechanisms involved in chronic inflammation.

Increased plasma galectin-1 correlates with advanced glycation end products and interleukin-1β in patients with proliferative diabetic retinopathy

Dear Editor,Galectin-1, one of galactoside-binding lectin family proteins, has been shown to regulate cell growth, migration, and proliferation, where it binds many receptors depending on their glycosylation profile, rather than its specific receptors.

HEPATOCYTE GROWTH FACTOR PROTECTS AGAINST APOPTOSIS INDUCED BY ADVANCED GLYCATION END PRODUCTS IN ENDOTHELIAL CELLS

Objective To investigate the effects of hepatocyte growth factor(HGF)on vascular endothelial cells apoptosis induced by advanced glycation end products(AGEs)and its possible mechanism. Methods Human umbilical vein endothelial cells(HUVECs)were cultured in vitro and intervened by different concentrations of AGEs and HGF.The cell inhibitory rates of each group with different culture time(12, 24, 48, and 72 hours)were measured by methyl thiazolyl tetrazolium(MTT)assay. The early stage apoptosis was detected by flow cytometry with Annexin V-FITC/PI double staining, morphology of cell apoptosis was observed by hoechst 33258 fluorescence staining, and the expression of apoptosis-associated genes Bax and Bcl-2 were determined by Western blotting.The activity of caspase-3 was detected by enzyme-linked immunosorbent assay (ELISA).Results Morphological observation indicated that high concentration of AGEs induced characteristic apoptotic changes in HUVECs.Within a certain concentration range, HUVECs apoptosis inducing rates by AGEs were in both dose- and time-dependent manners.HGF significantly inhibited the apoptosis of HUVECs induced by AGEs (P< 0.05).AGEs significantly promoted expression of Bax protein, but not Bcl-2.Whereas HGF significantly promoted the expression of Bcl-2(P<0.01)and decreased the activity of caspase-3(P<0.05)without affecting Bax level.Conclusions AGEs can induce the apoptosis of endothelial cells in vitro.HGF may effectively attenuate AGEs-induced endothelial cells apoptosis through upregulating Bcl-2 gene expression and inhibiting caspase-3 activation.

Role of the receptor for advanced glycation end products in hepatic fibrosis

AIM:To study the role of advanced glycation end products(AGE)and their specifi c receptor(RAGE)in the pathogenesis of liver fi brogenesis.METHODS:In vitro RAGE expression and extracellular matrix-related gene expression in both rat and human hepatic stellate cells(HSC)were measured after stimulation with the two RAGE ligands,advanced glycation end product-bovine serum albumin(AGE-BSA)and Nε-(carboxymethyl)lysine(CML)-BSA,or with tumor necrosis factor-α(TNF-α).In vivo RAGE expression was examined in models of hepatic fi brosis induced by bile duct ligation or thioacetamide.The effects of AGE-BSA and CML-BSA on HSC proliferation,signal transduction and profi brogenic gene expression were studied in vitro.RESULTS:In hepatic fibrosis,RAGE expression was enhanced in activated HSC,and also in endothelial cells,inflammatory cells and activated bile duct epithelia.HSC expressed RAGE which was upregulated after stimulation with AGE-BSA,CML-BSA,and TNF-α.RAGE stimulation with AGE-BSA and CML-BSA did not alter HSC proliferation,apoptosis,fibrogenic signal transduction and fibrosis-or fibrolysis-related gene expression,except for marginal upregulation of procollagen α1(Ⅰ)mRNA by AGE-BSA.CONCLUSION:Despite upregulation of RAGE in activated HSC,RAGE stimulation by AGE does not alter their fibrogenic activation.Therefore,RAGE does not contribute directly to hepatic fibrogenesis.

Association of vascular endothelial growth factor-634G/C and receptor for advanced glycation end products G82S gene polymorphisms with diabetic retinopathy

AIMTo investigate the association of receptor for advanced glycation end products (RAGE) G82S and vascular endothelial growth factor (VEGF) -634 G/C gene polymorphisms with diabetic retinopathy (DR).METHODSOur cross-sectional study included 61 diabetic patients, 12 of them had proliferative diabetic retinopathy (PDR), 15 had non proliferative diabetic retinopathy (NPDR), 34 had no diabetic retinopathy (NDR) and 61 healthy controls. Participants were tested for RAGE G82S and VEGF -634 G/C polymorphisms by polymerase chain reaction-restriction fragment length polymorphism.RESULTSWe found a significant association between VEGF -634 G/C polymorphism and PDR as PDR patients had increased incidence of VEGF -634 CC genotype compared to NDR patients [odds ratio for CC vs (GC+GG)=6.5, 95% CI=1.5-27.8, P=0.021]. Also VEGF -634 CC genotype and C allele were significantly higher in the PDR than in NPDR patients, which is a novel finding in our study (P=0.024, 0.009 respectively). The mean triglycerides level was significantly higher in diabetic patients with CC genotype (P=0.01) as compared to patients with other genotypes. All cases and control subjects were of the same heterozygous RAGE 82G/S genotype.CONCLUSIONPatients carrying VEGF -634 C polymorphism have a higher risk of PDR development, so VEGF -634 G/C polymorphism could be used as a predictive marker for PDR in diabetic patients. We could not find a significant association between RAGE G82S polymorphism and DR.

Role of Protein Kinase C in Advanced Glycation End Products-induced Epithelial-Mesenchymal Transition in Renal Proximal Tubular Epithelial Cells

The role of protein kinase C （PKC） activation in advanced glycation end products （AGEs）-induced epithelial-mesenchymal transition in renal proximal tubular epithelial cells was investigated. HKC cells were divided into three groups： normal group, AGE-BSA group （100 mg/L AGE-BSA） and AGE-BSA＋PKC inhibitor （10 μmol/L chelerythrine chloride） group. PKC activity was measured by PKC assay kit. The expression of Vimentin, and phosphorylated β-catenin was detected by using Western blotting, and the content of TGF-β1 was examined by ELISA method. The intracellular disposition of Vimentin was observed by fluorescence microscopy. As compared with normal group, PKC activity was increased significantly in AGE-BSA group. The expression of Vimentin, phosphorylated β-catenin, and TGF-β1 was enhanced significantly in AGE-BSA group. The expression of Vimentin, phosphorylated β-catenin, and TGF-β1 was significantly blocked by chelerythrine chloride. High expression of Vimentin, phosphorylated β-catenin, and TGF-β1 induced by AGE-BSA may be mediated via the activation of PKC signal transduction pathway.

Effect of Italian Sour Cherry (<i>Prunus cerasus</i>L.) on the Formation of Advanced Glycation End Products and Lipid Peroxidation

Sweet and sour cherries contain several polyphenols that possess antioxidant and anti-inflammatory properties. Aim of this study was to investigate the effect of the maturity stage on phenol content and biological properties of extract of a local Morello-type of sour cherry (Prunus cerasus?L.), “visciola”. The study of total phenol content and total antioxidant potential was associated with the evaluation of the antioxidant property of extracts using a copper catalyzed human low density lipoproteins (LDL) oxidation as experimental model. Moreover, using albumin glycated by methylglyoxal, we evaluated the anti-glycation effect of fruit extract. The results demonstrated that fully ripened fruits exert higher antioxidant and anti-glycation properties when compared with partially ripened fruits. Information about the health-promoting components of “visciola” could lead to a better understanding and an increased consumption of these, including its use as functional food.