Effect of advanced glycosylation end products on the expression of receptor for advanced glycosylation end products in human monocyte-derived dendritic cells

AIM: To investigate the effect of advanced glycosylation end products on the expression of receptor for advanced glycosylation end products in human monocyte-derived dendritic cells. METHODS: Monocytes were purified (over 98%) using anti-CD14＋ microbeads. After 8 d culture in RPMI-1640 medium containing rhGM-CSF (100 μg/L) and rhIL-4 (50 μg/L), immature MDCs were derived, then exposed to AGE-BSA (0 or 200 mg/L) for 24 h. Expression of RAGE was semi-quantified by RT-PCR and Western blotting. At the same time, supernatants were collected. IFN-γ and IL-12 were analyzed by ELISA. RESULTS: mRNA and protein of RAGE incubated by 200 mg/L AGE-BSA was higher than that in control at 24 h. Treatment of DCs with AGE-BSA resulted in about two-fold increase in the expression of RAGE (P<0.05). The concentrations of IFN-γ and IL-12 were both significantly higher than that in control (P<0.05). CONCLUSION: AGEs up-regulates the expression of RAGE and induces the secretion of IFN-γ and IL-12 by DCs. These findings may provide insight into the effect of DCs on the processes of atherosclerosis.     

Advanced glycation end products induce apoptosis of human ovarian granulosa COV434 cells and up-regulate pro-inflammatory cytokine HMGB1 autocrine

AIM:To study whether advanced glycation end products (AGEs) induce the apoptosis of human ovarian granulosa COV434 cells, and to explore the possible mechanism. METHODS:Human ovarian granulosa COV434 cells were treated with AGEs at different concentrations. Flow cytometry was used to observe the apoptotic rate. The protein levels of caspase-3 and cleaved caspase-3 were determined by Western blot. The release of high mobility group box 1 protein (HMGB1) in the culture supernatant was measured by ELISA. RESULTS:Compared with control group, early apoptotic rate and late apoptotic rate in 100 mg/L AGEs group and 200 mg/L AGEs group were significantly increased (P<0.05). No obvious difference of caspase-3 protein level in each group was observed, while the protein levels of cleaved caspase-3 in 100 mg/L AGEs group and 200 mg/L AGEs group were significantly increased compared with control group (P<0.05). In addition, compared with control group, pro-inflammatory factor HMGB1 in the culture medium in 100 mg/L AGEs group and 200 mg/L AGEs group was significantly increased. CONCLUSION:The apoptosis of human ovarian granulosa COV434 cells induced by AGEs may be related to pro-inflammatory reaction.     

Effect of advanced glycosylation end products on function of human adipose tissue-derived stem cells in vitro

AIM：To explore the effect of advanced glycosylation end products (AGEs) on the function of human adipose-derived stem cells (hADSCs) in promoting wound healing. METHODS：hADSCs were isolated by conventional method in vitro and divided into control bovine serum albumin (BSA) group, low-dose AGE-BSA group and high-dose AGE-BSA group. The proliferation and migration of hADSCs with different treatments were determined by WST-8 assay and Transwell assay, respectively. In addition, the expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1(IGF-1) at mRNA and protein levels was determined by real-time PCR and ELISA analysis. RESULTS：Compared with control group, the proliferation and migration abilities were significantly inhibited in the hADSCs of AGE-BSA group. The mRNA expression of VEGF, HGF and IGF-1 in AGE-BSA group was obviously lower than that in control group. The contents of VEGF, HGF and IGF-1 in hADSCs-conditioned me-dium in AGE-BSA group were also obviously lower than those in control group. CONCLUSION：AGEs alter the intrinsic properties of hADSCs and impair their functions in promoting wound healing, thus affecting the therapeutic potential of hADSCs in the treatment of diabetic ulcers.     

The mechanism of intercellular adhesion molecule-1 expression in endothelial cells stimulated by advanced glycosylation end products

AIM: To explore the relationship between intercellular adhesion molecule-1(ICAM-1)expression in endothelial cells(EC) and advanced glycosylation end products(AGEs) stimulation. METHODS: Murine bone marrow derived ECs was stimulated by AGEs after pretreated with anti-AGEs, anti-IL-1β and N-acetylcysteine(NAC),then SOD activity and ICAM-1 concentration and adhesion rate(AR) were evaluated. RESULTS: ECs which expressed ICAM-1 induced by AGEs showed lower SOD activity . The ICAM-1 expression as well as the increase of AR caused by AGEs stimulation could be suppressed by anti-AGEs(0.12±0.01) and NAC(0.11±0.05). Anti-IL-1β had no influence on these changes. CONCLUSION: AGEs could induce endothelial cells to express ICAM-1 in vitro, most probably due to the formation of free radicals. Besides, AGEs may stimulate other cells to secrete cytokines resulting in ICAM-1 expression in endothelial cells.     

Progress in advanced glycosylation end products and diabetic vascular complications

The formation of advanced glycosylation end products (AGEs) is enhanced in diabetes mellitus, closely associated with diabetic vascular complication.In this review, biochemical properties and structures of AGEs, AGEs receptors and binding proteins, pathogenic properties of AGEs, deposition and turnover of AGEs, inhibitors of AGEs were summarized.     

Inhibitory effect of catalpol on inflammation and expression of RAGE in EA.hy926 cells induced by advanced glycation end products

AIM: To investigate the inhibitory effect of catalpol on inflammation in EA.hy926 cells induced by advanced glycation end products(AGEs) and to explore its antioxidant mechanisms.METHODS: Human endothelial cell line EA.hy926 was cultured and randomly divided into control group, catalpol(0.5 mmol/L) group, AGEs group, high-dose(0.5 mmol/L) catalpol+AGEs group, middle-dose(0.25 mmol/L) catalpol+AGEs group and low-dose(0.05 mmol/L) catalpol+AGEs group. Intracellular reative oxygen species(ROS) production was detected by laser scanning confocal microscopy. The levels of monocyte chemotactic protein-1(MCP-1), tumor necrosis factor-α(TNF-α) and vascular cell adhesion molecule-1(VCAM-1) in culture supernatant were detected by commercial ELISA kits. The expression of MCP-1, TNF-α, VCAM-1 and receptor for advanced glycation end products(RAGE) in the EA.hy926 cells were detected by Western blot.RESULTS: In high-dose catalpol+AGEs and middle-dose catalpol+AGEs groups, the generation of ROS was decreased significantly. The levels of MCP-1, TNF-α and VCAM-1, and protein expression of MCP-1, TNF-α and VCAM-1 were significantly lower. The expression of RAGE protein in EA.hy926 cells were significantly inhibited(P<0.05).CONCLUSION: Catalpol effectively inhibits the AGEs-induced oxidative stress and inflammation in EA.hy926 cells, which may be associated with a decrease in the expression of RAGE.     

Effect of fluvastatin on expression of SGK1 and CTGF induced by aldosterone in rat mesangial cells

AIM: To investigate the effect of fluvastatin on the expression of serum and glucocorticoid inducible kinase 1 (SGK1) and connective tissue growth factor (CTGF) induced by aldosterone (Ald) in rat mesangial cells (GMCs). METHODS: GMCs were divided into (1) control group; (2) aldosterone group with different concentrations and times; (3) Ald (10-7 mol/L)+spironolactone (10-9mol/L) group; (4) Ald (10-7mol/L)+LY294002 (20 μmol/L) group; (5) Ald (10-7mol/L) +SB203580 (20 mmol/L) group; (6) the group of Ald (10-7mol/L)+ fluvastatin at different concentrations (10-7, 10-6, 10-5 mol/L); (7) Ald (10-7mol/L) +fluvastatin (10-5mol/L)+mevalonate (10-4 mol/L) group; (8) Ald (10-7mol/L) +fluvastatin (10-5mol/L)+FPP (farnesyl pyrophosphate, 10-4 mol/L) group; (9) Ald (10-7mol/L) +fluvastatin (10-5 mol/L) +GGPP (geranylgerany pyrophosphate, 10-4 mol/L) group. The protein levels of SGK1 and CTGF were determined by Western blotting. The levels of fibronection (FN), monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1) in the supernatants were determined by enzyme-linked immunosorbant assay (ELISA). RESULTS: Aldosterone stimulated the protein expression of SGK1 and CTGF in cultured mesangial cells in a dose-dependent manner (P<0.01). SGK1 expression was increased at as early as 6 h (P<0.05), peaked at 12 h after aldosterone treatment (P<0.01). This stimulatory effect of aldosterone on SGK1 and CTGF was blocked by mineralocorticoid receptor (MR) inhibitor and LY294002 (a specific inhibitor of PI3K). Incubation of cells with fluvastatin significantly inhibited the aldosterone-induced the activations of SGK1 and CTGF, and the expression of MCP-1 and ICAM was in a dose-dependent manner (P<0.05). Exogenous mevalonate prevented the effect of fluvastatin on SGK1 expression in GMCs. CONCLUSION: Fluvastatin reduces aldosterone-induced SGK1 expression via mineralocorticoid receptor and PI3K pathway in rat mesangial cells. Such effect of flurastatin is partly blocked by mevalonate.     

Advanced glycation end products-modified protein up-regulates expression of adhesion molecules in human umbilical vein endothelial cells

AIM: Advanced glycation end products (AGEs)-modified proteins are found in plasma and atherosclerosis lesion of diabetes mellitus patients. The study was conducted to elucidate the effect of AGE on expression of adhesion molecules in human endothelial cells. METHODS: Human endothelial cells derived from umbilical veins (HUVECs) were coincubated in vitro with native human serum albumin (HSA) or HSA modified with advanced glycation end products (AGE-HSA). The expression of adhesion molecule intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were determined by immunofluorescent staining and flow cytometry analysis. RESULTS: ICAM-1 and VCAM-1 were constitutively expressed on HUVECs. AGE-HAS enhanced the expression of ICAM-1 and VCAM-1 on HUVECs in a time- and dose-dependent manner. HSA had no effect on the expression of adhesion molecules. CONCLUSIONS: AGE-HSA up-regulates the expression of adhesion molecules in human endothelial cells. AGEs may therefore promote the infiltration of monocytes in the vascular endothelium and have an important effect in the generation and progress of atherosclerosis.     

Transferrin receptor and Fcα/μ receptor are not the major IgA1 receptor on human mesangial cells

AIM: To investigate whether transferrin receptor (TfR) and Fc α/μ R are the major IgA1 receptor on human mesangial cells (HMC). METHODS: Serum IgA1 was isolated by jacalin affinity chromatography and heated to aggregated form (aIgA1). RT-PCR was performed to investigate the expression of TfR mRNA and Fcα/μR mRNA. Binding capacity of aIgA1 to human mesangial cell line (HMCL) was evaluated by radio-ligand binding assay. Binding specificity was determined by competitive inhibition assay and phosphorylation of extracellular signal-regulated kinase (ERK) was determined by Western blot. RESULTS: TfR cDNA and Fcα/μR cDNA products were amplified from HMC but not from HMCL. aIgA1 was found to bind to HMCL in a dose-dependent, saturable manner and the binding was inhibited by BSA. Scatchard analysis revealed a Kd of (6.4±1.7)×10-7 mol/L and the binding sites were (3.0±1.2)×106/cells. Both aIgA1 from patients with IgAN and healthy controls were able to induce the phosphorylation of ERK in a similar time-dependent manner, but the effect of aIgA1 from patients with IgAN was much stronger (P＜0.01) and the duration was much longer (P＜0.05) than those from healthy controls. CONCLUSION: There might be a novel IgA1 receptor on HMCL, but TfR and Fc α/μ R are not the major candidates.     

HCMV-infected human THP-1 cells induce expression of HLA-G and its receptors

AIM: To investigate the differential expression of human leukocyte antigen-G (HLA-G) isoforms and its receptors in human monocyte line THP-1 after human cytomegalovirus (HCMV) infection for exploring the role of HLA-G in HCMV escaping the immune response of the organism.METHODS: THP-1 cells were infected with HCMV Towne strain. The expression of HLA-G isoforms at mRNA and protein levels was determined by RT-PCR and Western blot, respectively. The surface expression of HLA-G and its receptors ILT2/ILT4 and the cell viability were analyzed by flow cytometry. The levels of soluble HLA-G (sHLA-G) and IL-10 were measured by ELISA.RESULTS: After infection of the THP-1 cells with HCMV, no obvious apoptosis in the cells was observed, and the viability of the cells was high. A significant up-regulation of HLA-G1, -G3, -G4 and -G5 at mRNA expression level 1 d after infection was found, while the protein expression of HLA-G1 and HLA-G5 isoforms was mainly detected. The expression of HLA-G/ILT2/ILT4 was evidently up-regulated 1 d after infection. The level of sHLA-G was significantly increased 1 d after infection as compared with control group (P<0.01). The expression of IL-10 was obviously up-regulated 1 d post-infection as compared with control group.CONCLUSION: The differential expression of HLA-G isoforms and secretion of the receptors ILT2/ILT4 and IL-10 in the THP-1 cells are induced after HCMV infection. This study provides experimental evidence for evaluating the immune mechanism of HCMV infection.     

Effect of probucol and simvastatin on reactive oxygen species production and HO-1 expression induced by advanced glycation end products in rat renal microvascular endothelial cells

AIM: To investigate the effect of probucol and simvastatin on the production of reactive oxygen species (ROS) and heme oxygenase-1 (HO-1) expression induced by advanced glycation end products (AGEs) in rat renal microvascular endothelial cells (RMECs). METHODS: RMECs isolated and cultured from rat kidney were divided into 4 groups: normal control group, AGEs group, probucol group and simvastatin group. The levels of ROS were determined by the molecular probes of DCFH-DA. The expression of HO-1 at mRNA and protein levels was detected by RT-PCR and Western blotting, respectively. RESULTS: (1) AGEs up-regulated ROS production and HO-1 expression in RMECs. (2) Probucol up-regulated HO-1 expression in RMECs, and inhibited the increasing level of ROS and expression of HO-1 in RMECs induced by AGEs. (3) Simvastatin also inhibited the increasing level of ROS in RMECs induced by AGEs, but it had no effect on HO-1 expression in RMECs with or without AGEs.CONCLUSION: Protective effect of probucol on the dysfunction of RMECs induced by AGEs may be related with its effect on the expression of HO-1 at mRNA and protein levels. Simvastatin also plays roles in antioxidation and renal protection, but is ineffective in the modulation of HO-1 expression.     

Inflammation disrupts cholesterol efflux in human kidney mesangial cells via PPARγ-LXRα-ABCA1 pathway

AIM: To investigate the perturbative effects of inflammatory stress on cholesterol efflux in human kidney mesangial cells (HMCs) and the relation to peroxisome proliferators activated receptor-γ (PPARγ)-1iver X activated receptor-α (LXRα)-and ATP-binding cassette transporter A1 (ABCA1) pathway. METHODS: HMCs were cultured and divided into control group (incubated with serum free medium), high lipid group , inflammatory stress group or combination treatment group . The mRNA and protein levels of PPARγ, LXRα,ABCA1 were examined by real-time polymerase chain reaction (PCR) and Western blotting. cholesterol assay was performed to evaluate the efflux of cholesterol by liquid scintillation counter. Oil red O staining was used to evaluate lipid droplet accumulation in the cells. Intracellular cholesterol level was measured by enzymic assay. RESULTS: : LDL increased the expression of PPARγ, LXRα and ABCA1 at mRNA and protein levels in HMCs, while TNF-α reduced the expression of these genes at mRNA and protein levels. The cholesterol efflux was increased after LDL loading. However, inflammatory stress inhibited cholesterol efflux in the absence or presence of LDL loading. Oil red O staining and quantitative analysis showed that LDL loading increased the intracellular cholesterol level in HMCs and inflammatory stress further exacerbated the lipid accumulation. CONCLUSION: Inflammatory cytokine reduces cholesterol efflux by inhibiting the expression of PPARγ, LXRα and ABCA1, thereby causing lipid accumulation in HMCs.     

Role of MAPK signaling pathways in advanced glycosylation end products-induced morphological changes of actin cytoskeleton in human umbilical vein endothelial cells

AIM：To investigate the effect of advanced glycosylation end products (AGEs) modified protein on morphological changes of actin cytoskeleton in primary endothelial cells and the role of MAPK signaling pathways in this pathological procedure.METHODS：Human umbilical vein endothelial cells (HUVECs) were incubated with AGEs modified human serum albumin (AGE-HSA) at concentrations of 12.5,25,50,and 100 mg/L,respectively,for 2,4,8,12 and 24 hours.The cells were treated with different chemical compounds of inhibitors,or adenoviral deliver approach (either dominant positive or negative adenoviral constructs) of MAP kinases to specifically block or activate certain signal transduction pathways under above situations.As control,HSA of the same concentration was administered to cells at the same time.The treated cells were incubated with FITC-phalloidin to stain F-actin.RESULTS：F-actin in HUVECs was rearranged greatly by AGEs in a concentration and time-dependent manners.The unmodified HSA did not influence F-actin distributions.The AGEs-induced changes were blocked by pretreatment with SB203580,PD98059 for 30 min,or pre-infection with recombinant virus of dominant negative form of MKK 6b ［MKK6b (A)］,MEK1 ［MEK1 (A)］ for 24 h,while SP600125 and dominant negative form of MKK7 ［MKK7 (A)］ failed to inhibit the effects of AGEs.Furthermore,the infection of recombinant virus of constitutive active form of MKK6b ［MKK6b (E)］ or MEK1 ［MEK1 (E)］ could also induced re-arrangement of F-actin,respectively,while the effect elicited by ［MKK6b (E)］ was abolished by co-infection with recombinant adeno-virus of dominant negative p38α.CONCLUSION：AGEs-induced morphological changes of F-actin in endothelial cells are mediated by p38-and ERK MAPK signal pathways.     

Effects of advanced glycation end products on protein and mRNA expression of macrophage inflammatory protein-1α in cultured human umbilical vein endothelial cells

AIM: To investigate the effects of advanced glycation end products (AGEs) on protein and mRNA expression of macrophage inflammatory protein-1α (MIP-1α) in cultured human umbilical vein endothelial cells(HUVECs). METHODS: HUVECs were cultured with AGEs at different concentrations for 24 h and at a concentration of 400 mg/L for different time.The levels of mRNA and protein expression of MIP-1α in cultured HUVEC were detected by in situ hybridization and Western blot, respectively. RESULTS: In situ hybridization showed that after exposure of HUVECs to AGEs at different concentrations (100 mg/L, 200 mg/L, 400 mg/L) for 24 h, the average integrated optical density values (18.76±3.17, 26.58±1.61, 34.23±2.25) of MIP-1α mRNA expression in HUVECs were higher than that in control group (13.83±1.24, P0.05). After exposure of HUVECs to AGEs at a concentration of 400 mg/L for 12 h, 24 h and 36 h, the average integrated optical density values of MIP-1α mRNA expression in HUVECs were 22.67±1.46, 34.23±2.25 and 42.28±3.14, higher than that in 0 h group (12.56±1.24, P0.05). Western Blot showed that exposure of HUVECs to AGEs at different concentrations(100 mg/L, 200 mg/L, 400 mg/L) for 24 h resulted in a 1.34-fold, 1.87-fold and 2.46-fold increase in the expression of MIP-1α protein in HUVECs compared with BSA control group (P<0.05). Meanwhile, exposure of HUVECs to AGEs at a concentration of 400 mg/L for 12 h, 24 h and 36 h resulted in a 1.82-fold, 2.71-fold and 3.34-fold increase in MIP-1α protein expression in HUVECs compared with 0 h group (P<0.05). CONCLUSION:These data suggest that AGEs could induce a high expression of MIP-1αmRNA and protein in cultured HUVECs in a dose-dependent and time-dependent manner.     

Effect of insulin on the SGK1 expression and extracellular matrix synthesis in human mesangial cells cultured in high glucose

AIM:To study the effect of insulin on the serum and glucocorticoid-inducible kinase 1 (SGK1) expression and extracellular matrix synthesis in human glomerular mesangial cells (HMC) cultured in high glucose. METHODS:The HMCs were cultured in the presence of 5.5 or 25 mmol/L glucose with or without 100 nmol/L insulin (i.e. NG, HG, NI and HI groups). 4 h latter, expressions of SGK1, insulin receptor substrate-1 (IRS1) and IRS2 in corresponding groups were detected by immunofluorescence or examined by Western blotting. The phosphorylation of IRS1 and IRS2 was measured by immunoprecipitation. 24 h latter, connective tissue growth factor(CTGF) and fibronectin (FN) were also examined by RT-PCR and ELISA, respectively. RESULTS:Compared with NG, the SGK1 protein expression in HG, NI and HI groups was significantly higher (P<0.01). High glucose mainly caused IRS2 protein and its phosphorylation level increase (P<0.01). When treated with 100 nmol/L insulin, IRS1 protein and its phosphorylation in HI group apparently elevated while slight inhibition of IRS2 protein expression and its phosphorylation were observed (HI vs HG, P<0.05). High glucose enhanced the expression of CTGF and FN, and insulin strengthened this effect.CONCLUSION:Insulin and high glucose up-regulate the expression of SGK1 in mesangial cells through different target molecular pathways and ultimately enhance ECM synthesis. The effect of insulin is highly associated with IRS1 signaling cascades.