Inhibitory effect of GSPE on RAGE expression induced by advanced glycation end products in endothelial cells

Advanced glycation end products' (AGEs) engagement of a cell-surface receptor for AGEs (RAGE) has been causally implicated in the pathogenesis of diabetic vascular complications via induction of reactive oxygen species (ROS) and subsequent alteration of many gene expressions, including RAGE itself. Grapeseed proanthocyanidin extract (GSPE), which is a naturally occurring polyphenolic compound, has been reported to possess potent radical-scavenging and antioxidant properties and to display significant cardiovascular protective action. In this study, we investigated whether GSPE could inhibit AGE-induced RAGE expression through interference with ROS generation in human umbilical-vein endothelial cells (HUVECs). AGE-modified bovine serum albumin (AGE-BSA) was prepared by incubating BSA with high-concentration glucose. Stimulation of cultured HUVECs with 200 microg/mL of AGE-BSA significantly enhanced intracellular ROS formation and subsequently upregulated the protein and mRNA expression of RAGE; unmodified BSA and GSPE alone had no effect. However, GSPE preincubation markedly downregulated AGE-induced surface expression of RAGE in a time- and concentration-dependent manner. In AGE-stimulated HUVECs, GSPE also dose-dependently decreased RAGE mRNA levels and inhibited AGE-induced ROS generation at defined time periods. These results demonstrate that GSPE can inhibit enhanced RAGE expression in AGE-exposed endothelial cells by suppressing ROS generation, thereby limiting the AGE-RAGE interaction. Hence, GSPE may have therapeutic potential in the prevention and treatment of vascular complications in diabetic patients.     

Nifedipine inhibits gene expression of receptor for advanced glycation end products (RAGE) in endothelial cells by suppressing reactive oxygen species generation

Advanced glycation end products (AGEs), the senescent macroprotein derivatives that form in increased amounts in diabetes, have been implicated in the pathogenesis of diabetic vascular complications. Indeed, AGEs elicit oxidative stress generation in vascular wall cells through an interaction with their receptor (RAGE), thus playing an important role in vascular inflammation and altered gene expression of growth factors and cytokines. We have previously shown that nifedipine, one of the most popular dihydropyridine-based calcium antagonists, blocked tumor necrosis factor-alpha-induced monocyte chemoattractant protein-1 expression in endothelial cells (ECs) through its antioxidative properties. However, the effects of nifedipine on AGE-exposed ECs remain to be elucidated. In this study we investigated whether nifedipine could inhibit the AGE-induced reactive oxygen species (ROS) generation and subsequent RAGE gene expression in human umbilical vein endothelial cells (HUVEC). Nifedipine completely inhibited AGE-induced ROS generation in HUVEC. Furthermore, nifedipine was found to prevent up-regulation of RAGE mRNA levels in AGE-exposed HUVEC. These results demonstrate that nifedipine can inhibit RAGE overexpression in AGE-exposed ECs by suppressing ROS generation. Our present study suggests that nifedipine may have therapeutic potential in the treatment of patients with AGE-related disorders such as diabetic vascular complications.     

Selective inhibition by grape seed proanthocyanidin extracts of cell adhesion molecule expression induced by advanced glycation end products in endothelial cells

The interaction of advanced glycation end products (AGE) with their cell surface receptors for AGEs (RAGE) has been causally implicated in the pathogenesis of diabetic vascular complications and has been shown to stimulate cell adhesion molecule expression in endothelial cells via induction of reactive oxygen species (ROS). Alternatively, grape seed proanthocyanidin extracts (GSPE), which are naturally occurring polyphenolic compounds, have been reported to possess potent radical scavenging and antioxidant properties and to display significant cardiovascular protective action. In this study, we investigated whether GSPE could inhibit AGE-induced cell adhesion molecule expression through interference with ROS generations in human umbilical vein endothelial cells. AGE-modified bovine serum albumin (AGE-BSA) was prepared by incubating BSA with a high concentration of glucose. Stimulation of cultured human umbilical vein endothelial cells with 200 microg/mL of AGE-BSA significantly enhanced intracellular ROS formation and subsequently upregulated the expression of vascular cell adhesion molecule-1 (VCAM) and intercellular adhesion molecule-1 (ICAM-1), whereas both unmodified BSA and GSPE alone were without effect. However, preincubation of different concentrations of GSPE markedly downregulated AGE-BSA-induced VCAM-1 expression at the surface protein and mRNA level in a concentration-dependent manner, but the increased ICAM-1 expression was not affected by GSPE treatment. Meanwhile, the inhibition by GSPE of intracellular ROS generation was also observed at defined time periods. These results demonstrate that GSPE can inhibit the enhanced VCAM-1 expression but not ICAM-1 in AGE-exposed endothelial cells by suppressing ROS generation. Hence, GSPE may have therapeutic potential in the prevention and treatment of vascular complications in patients with diabetes.     

西红花酸对晚期糖基化终产物诱导牛血管内皮细胞E-选择素表达的抑制作用

目的：研究西红花酸（crocetin）对晚期糖基化终产物（advanced glycation end products,AGEs）诱导牛主动脉血管内皮细胞（bovine Endothelial cells,BECs）E-选择素（E-selectin）表达的抑制作用。方法：分离纯化新生牛全血的中性粒细胞与单核细胞,用虎红染色法测定西红花酸对牛单核细胞/中性粒细胞与BECs黏附的影响,Cell-based ELISA法和sABC免疫细胞化学法测定E-选择素蛋白表达变化。结果：AGEs（100μg/mL）能促进中性粒细胞和单核细胞对BECs的黏附（P〈0.01 vs control）,用西红花酸（1,0.1,0.01μmol/L）预孵内皮细胞12h后,再用AGEs作用24h,中性粒细胞和单核细胞对BECs的黏附较AGEs模型组降低,且呈剂量依赖性关系（P〈0.05或0.01）。Cell-based ELISA测定结果显示,AGEs作用4h内,BECs中的E-selectin表达水平上升,之后E-selectin表达下降,8h时,恢复接近正常水平。而西红花酸（1,0.1μmol/L）能使E-selectin表达下降。sABC免疫化学结果也证实西红花酸对AGEs诱导BECs中E-selectin表达有抑制作用。结论：西红花酸可通过抑制黏附分子E-selcetin蛋白表达,从而抑制中性粒细胞和单核细胞对内皮细胞的黏附作用,这可能是西红花酸减轻糖尿病血管病变的作用机制之一。     

Role of advanced glycation end products in diabetic neuropathy

Diabetic neuropathy is the commonest form of peripheral neuropathy in the developed countries of the world. In diabetic patients, the presence of peripheral neuropathy increases their risks for developing foot ulceration and subsequent necrosis that necessitates lower limb amputation. Although the precise mechanisms underlying diabetic neuropathy remain unclear, there is evidence that hyperglycemia-induced formation of advanced glycation end products (AGEs) is related to diabetic neuropathy; AGE-modified peripheral nerve myelin is susceptible to phagocytosis by macrophages and contributes to segmental demyelination; modification of major axonal cytoskeletal proteins such as tubulin, neurofilament, and actin by AGEs results in axonal atrophy/degeneration and impaired axonal transport; and glycation of extracellular matrix protein laminin leads to impaired regenerative activity in diabetic neuropathy. Recently, the receptor for AGEs (RAGE) has been found to colocalize with AGEs in diabetic peripheral nerves. This suggests that, in diabetic neuropathy, AGEs and AGE/RAGE interactions induce oxidative stress, result in upregulation of nuclear factor (NF)-kappaB and various NF-kappaB-mediated proinflammatory genes, and exaggerate neurological dysfunction, including altered pain sensation. Additionally, AGE/RAGE-induced oxidative stress further accelerates formation of glycoxidation products such as Nepsilon-(carboxymethyl)lysine and pentosidine. Although new drugs that inhibit the formation of AGEs and block the AGE-RAGE interaction are being studied, no effective treatment modalities against AGE-induced nerve injury are currently available clinically. A therapeutic strategy to prevent and ameliorate diabetic neuropathy using anti-AGE agents needs to be established. In this review, the current issues involved in the role of the glycation process and the potential treatment options for diabetic neuropathy are explored.     

外源性糖基化终末产物对体外培养的内皮细胞损伤机制的研究

目的:探讨外源性糖基化终末产物(AGEs)对体外培养的人脐静脉内皮细胞(HUVEC)损伤的作用机制.方法:外源性AGEs与HUVEC共孵12 h,以组胺(Hi)和过氧化氢酶(CAT)为对照,检测内皮细胞的活性、单层通透性、凋亡率及细胞培养液生化指标,并且观察内皮细胞形态学改变.结果:外源性AGEs呈剂量依赖性(40 mg/L、120 mg/L、160 mg/L)地降低了内皮细胞的活性、增加了内皮细胞的单层通透性及凋亡率、增加了细胞培养液中丙二醛(MDA)含量,并降低了过氧化物歧化酶(SOD)的含量(均P < 0.05),与阳性对照组Hi的作用相当(均P ＞ 0.05).CAT明显抑制了外源性AGEs对内皮细胞的损伤作用.结论:外源性AGEs诱导血管内皮细胞损伤,其机制可能与氧化应激有关.     

Inhibition of reactive oxygen species/extracellular signal-regulated kinases pathway by pioglitazone attenuates advanced glycation end products-induced proliferation of vascular smooth muscle cells in rats

Advanced glycation end products (AGEs) have been shown to induce the proliferation of vascular smooth muscle cells (VSMCs) and contribute to atherogenesis and diabetes. In the present study, we investigated the effects of pioglitazone, a peroxisome proliferator activated receptor gamma (PPARγ) agonist, on AGE-induced rat VSMC growth and the underlying mechanism. In cultured rat VSMCs, AGE treatment induced VSMC proliferation in time- and dose-dependent manner, while down-regulated the expression of PPARγ. Pretreatment of pioglitazone not only prevented the down-regulation of PPARγ, but inhibited VSMC proliferation and prevented S-phase entry of cell via a G0-G1 block in the presence of AGEs. Western blotting analysis showed that AGE treatment potentiated to activate extracelluar signal-regulated kinases (ERK1/2) by the induction of intracellular reactive oxygen species (ROS) production, since ROS scavenger N-acetyl-L-cysteine pretreatment significantly inhibited AGE-induced ERK1/2 activation. Further, pretreatment with either N-acetyl-L-cysteine or the inhibitor of ERK1/2 activation suppressed AGE-induced proliferation of VSMCs, suggesting a role of ROS/ERK1/2 signaling. Notably, we demonstrated that pretreatment of pioglitazone significantly attenuated AGE-induced ROS and ERK1/2 activation. Collectively, these results suggest that pioglitazone inhibits AGE-induced VSMC proliferation via increasing PPARγ expression and inhibiting ROS/ERK1/2 signaling pathway.     

Grape seed proanthocyanidin extracts inhibit vascular cell adhesion molecule expression induced by advanced glycation end products through activation of peroxisome proliferators-activated receptor gamma

Although evidence has shown that grape seed proanthocyanidin extracts (GSPE) can selectively inhibit cell adhesion molecule expression induced by advanced glycation end products (AGEs), the underlying molecular mechanism has not been extensively characterized. To study the antiinflammation mechanism of GSPE, we investigated the effect of GSPE on Von Willebrand factor (vWF) content and the expression of vascular cell adhesion molecule-1 (VCAM-1) induced by AGEs and the effect of GSPE on peroxisome proliferators-activated receptor gamma (PPAR gamma) and receptor for AGEs (RAGE) expression in human umbilical vein endothelial cells (HUVEC). HUVEC were preincubated with or without GSPE of different concentrations (10 mg/L, 50 mg/L, and 100 mg/L) for 4 hours before being treated with 200 mg/L AGEs or unmodified bovine serum albumin (BSA) for 24 hours. The expression of RAGE and PPAR gamma was investigated by Western blot. VCAM-1 expression was measured by flow cytometry and vWF content by enzyme-linked immunosorbent assay (ELISA). Results showed that GSPE significantly inhibited the expression of VCAM-1 in HUVEC and reduced the content of vWF in culture fluid induced by AGEs in a dose-dependent manner. AGEs activated the expression of RAGE and inhibited PPAR gamma expression in HUVEC, whereas GSPE inhibited the expression of RAGE through activation of PPAR gamma in HUVEC simultaneously. These findings indicated that GSPE inhibited the cell inflammatory factor expression and protected the function of endothelial cell through activation of PPAR gamma expression and inhibition of RAGE expression.     

Involvement of hypoxia-inducible factor-1α in the oxidative stress induced by advanced glycation end products in murine Leydig cells

Hyperglycemia increases the formation of advanced glycation end products (AGEs), triggers oxidative impairments and influences inducible factor (HIF)-1α protein levels and transactivation function. Compromised HIF-1α in testis leads to male infertility. The aim of the study was to investigate the role of HIF-1α in oxidative stress induced by AGEs in murine Leydig TM3 cells. TM3 cells were treated with 50 μg/ml of AGEs, or HIF-1α siRNA or 500 μM of DMOG (dimethyloxalylglycine) respectively. The cells were also pretreated with HIF-1α siRNA or 500 μM of DMOG and then were treated with 50 μg/ml of AGEs. The formation of reactive oxygen species (ROS) and cell apoptosis was evaluated. The expression of caspase-3, Heme oxygenase (HO)-1, steroidogenic acute regulatory protein (StAR) and cytochrome P450 17α polypeptide 1 (CYP17A1) was examined by Western blotting. AGEs increased ROS production, induced apoptosis and activated HIF-1α and HO-1 in TM3 cells. HIF-1α attenuated the AGE-induced ROS formation and promoted apoptosis via the upregulation of caspase-3. Knockdown of HIF-1α inhibited the expression of CYP17A1 and StAR, and enhanced the inhibition of StAR and CYP17A1 by AGEs. These findings indicate that attenuated HIF-1α exacerbates the oxidative stress injury by AGEs in murine Leydig cells, and contributes to diabetic male infertility.     

Antioxidant effects of aqueous extract of Terminalia chebula in vivo and in vitro

The ripe fruit of Terminalia chebula RETZIUS (T. chebula RETZ) (Combretsceae), which is a native plant in India and Southeast Asia, has traditionally been used as a popular folk medicine for homeostatic, antitussive, laxative, diuretic, and cardiotonic treatments. The objective of this study was to evaluate the protective effects of an aqueous extract of fruit of T. chebula on the tert-butyl hydroperoxide (t-BHP)-induced oxidative injury observed in cultured rat primary hepatocytes and rat liver. Both treatment and pretreatment of the hepatocytes with the T. chebula extract (TCE) significantly reversed the t-BHP-induced cell cytotoxicity and lactate dehydrogenase leakage. In addition, TCE exhibited in vitro ferric-reducing antioxidant activity and 2,2-diphenyl-1-picryhydrazyl free radical-scavenging activities. The in vivo study showed that pretreatment with TCE (500 or 1000 mg/kg) by gavage for 5 d before a single dose of t-BHP (0.1 mmol/kg i.p.) significantly lowered the serum levels of the hepatic enzyme markers aspartate aminotransferase and alanine aminotransferase and reduced the indicators of oxidative stress in the liver, such as the glutathine disulfide content and lipid peroxidation, in a dose-dependent manner. Histopathologic examination of the rat livers showed that TCE reduced the incidence of liver lesions, including hepatocyte swelling and neutrophilic infiltration, and repaired necrosis induced by t-BHP. Based on the results described above, we speculate that TCE has the potential to play a role in the hepatic prevention of oxidative damage in living systems.     

Protective effects of sciadopitysin against methylglyoxal-induced degeneration in neuronal SK-N-MC cells

The accumulation of advanced glycation end products (AGEs) causes metabolic dysfunction and neuronal cell damage. Methylglyoxal (MG) is a major glycating agent that reacts with basic residues present in proteins and promotes the formation of AGEs. Sciadopitysin, a type of biflavonoid, exerts protective effects against neuronal cell damage; however, the underlying mechanisms have not been studied. This study aimed to investigate the mechanisms underlying the protective effects of sciadopitysin against MG-mediated cytotoxicity in SK-N-MC neuroblastoma cells. Our results demonstrated that pretreatment of SK-N-MC cells with sciadopitysin improved the cell viability that was inhibited by MG and inhibited the apoptosis induced by MG. Sciadopitysin attenuated intracellular Ca²⁺, NOX4 levels, oxidative stress, and MG-protein adduct levels, and increased nuclear Nrf2 and glyoxalase 1 levels in the presence of MG. These results suggest that sciadopitysin exerts neuroprotective effects against MG-induced death of human SK-N-MC cells via its antioxidative action. This study highlights sciadopitysin as a promising candidate for antioxidant therapy and designing natural drugs against AGE-induced neurodegenerative disorders.     

Inhibitory effects of Luobuma tea and its components against glucose-mediated protein damage.

Luobuma tea, prepared from the leaves of Apocynum venetum L., is a popular beverage in China. In this study, the activity of Luobuma leaf extract and its components against the formation of advanced glycation endproducts (AGEs), which are largely involved in the pathogenesis of diabetic vascular complications, was examined using the in vitro glycation reaction. Strong inhibitory activity against the formation of AGEs was shown by Luobuma aqueous extract. Following further fractionation of this extract, seven polyphenolic compounds, i.e. (+/-)-gallocatechin, (-)-epigallocatechin, (+/-)-catechin, (-)-epicatechin, epicatechin-(4beta-8)-gallocatechin, epigallocatechin-(4beta-8)-epicatechin and procyanidin B-2, were isolated by Sephadex LH-20 column chromatography. These purified compounds also exerted inhibitory activities that were more potent than the positive control, aminoguanidine. Our findings may help to explain the beneficial effects of this plant against atherosclerosis.     

二甲双胍对糖基化终末产物诱导的成骨细胞氧化应激和凋亡的影响

目的:观察二甲双胍对糖基化终末产物(AGEs)诱导的大鼠颅骨成骨细胞内氧化应激产物活性氧簇(ROS)、细胞早期凋亡的影响.方法:分离培养大鼠颅骨成骨细胞,2 '7'-二乙酰二氯荧光素(DCFH-DA)作为荧光探针,流式细胞检测技术检测细胞内ROS水平,膜联蛋白V-异硫氰酸荧光素/碘化丙啶(Annexin V-FITC/ PI)双染色分析细胞早期凋亡率.结果:与未经葡萄糖处理的牛血清白蛋白(BSA)组对比,500 μg/mLAGEs显著促进成骨细胞内ROS形成和细胞凋亡(均P＜ 0.01);给予二甲双胍(100～500μmol/L)呈浓度依赖性抑制BSA组及AGEs组成骨细胞内ROS形成和细胞凋亡,浓度分别为500、400 μmol/L时,对细胞内ROS形成和细胞凋亡的抑制作用达到最强.结论:AGEs显著诱导原代成骨细胞内ROS的形成和细胞凋亡,而二甲双胍能够呈浓度依赖性抑制AGEs诱导的成骨细胞内ROS的形成和细胞凋亡,减轻AGEs对成骨细胞的损害.     

Nifedipine inhibits tumor necrosis factor-alpha-induced monocyte chemoattractant protein-1 overexpression by blocking NADPH oxidase-mediated reactive oxygen species generation

There is a growing body of evidence that dihydropyridine-based calcium antagonists (DHPs) improve endothelial function, thus slowing the development and progression of atherosclerosis. However the molecular mechanisms by which DHPs normalize endothelial dysfunction, an initial step in atherosclerosis, are not fully understood. Monocyte recruitment and firm adhesion to endothelial cells play a central role in the pathogenesis of atherosclerosis. In this study, we investigated whether nifedipine, one of the most popular DHPs, could inhibit tumor necrosis factor-alpha (TNF-alpha)-induced reactive oxygen species (ROS) generation and subsequent monocyte chemoattractant protein-1 (MCP-1) expression in human umbilical vein endothelial cells (HUVEC). TNF-alpha significantly increased intracellular ROS generation in HUVEC, which was completely blocked by nifedipine. Nifedipine completely inhibited TNF-alpha-induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in HUVEC. Furthermore, nifedipine was found to significantly inhibit upregulation of MCP-1 messenger RNA levels in TNF-alpha-exposed HUVEC. The results demonstrate that nifedipine could inhibit TNF-alpha-induced MCP-1 overexpression in HUVEC by suppressing NADPH oxidase-mediated ROS generation. Our present study suggests that nifedipine may play a protective role in the development and progression of atherosclerosis through its antioxidative properties.     

Rosiglitazone prevents advanced glycation end products-induced renal toxicity likely through suppression of plasminogen activator inhibitor-1.

In the development of diabetic nephropathy, advanced glycation end products (AGEs) play a causative role via induction of extracellular matrix (ECM) accumulation. Plasminogen activator inhibitor-1 (PAI-1), as a major inhibitor of plasminogen activator that plays an important role in degrading ECM, was found to significantly increase in renal fibrotic diseases. Activation of peroxisome proliferator-activated receptor (PPAR)-gamma prevented diabetic nephropathy. The present study, therefore, was to define whether or not AGE-induced renal ECM accumulation and renal dysfunction are mediated by upregulation of PAI-1 expression and whether or not PPAR-gamma agonist can attenuate these AGE effects via suppressing PAI-1 expression. Rats were given AGEs alone by iv injection at 100 mg/kg daily with or without oral supplementation of PPAR-gamma agonist rosiglitazone (RGZ) at 2 mg/kg daily for 6 weeks. Results showed that AGEs induced a renal ECM accumulation, as shown by increases in periodic acid-Schiff-positive materials, fibronectin, and type IV collagen (Col IV) contents in glomeruli, and a mild renal dysfunction, as shown by an increase in urinary proteins. AGEs also caused an increase in PAI-1 expression and a decrease in plasminogen activator bioactivity in the kidney. Treatment with RGZ significantly ameliorated AGE-induced renal ECM accumulation, proteinuria, and PAI-1 upregulation. Direct exposure of rat mesangial cells to AGEs in vitro induced increases in fibronectin and Col IV syntheses along with an increase in PAI-1 expression, effects significantly attenuated by RGZ. Preincubation of PAI-1 antibody to AGE-treated mesangial cells completely prevented AGE-induced fibronectin and Col IV production. These results suggest that upregulation of PAI-1 expression plays a critical role in AGE-induced renal ECM accumulation. Renal protection of RGZ from AGEs may be associated with the suppression of PAI-1 expression through PPAR-dependent and independent mechanisms.     

A PKC-beta inhibitor prompts the HUVECs apoptosis-induced by advanced glycation end products

The accumulation of advanced glycation end products (AGEs) on micro-vasculature has been demonstrated to be a key factor in diabetes mellitus development. Evidence suggests that AGEs triggered apoptotic changes in human umbilical vein endothelial cells (HUVECs) and protein kinase C (PKC)-beta plays a pivotal role in AGEs-induced micro-vascular dysfunction. Thus the effect of the selective PKC-beta inhibitor (LY333531) on AGEs-induced HUVEC apoptosis and proliferation was investigated. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to determine the cells viability after being incubated with AGEs and LY333531. Acridine orange/ethidium bromide (AO/EB) fluorescence detection was applied to observe the pro-apoptosis effects of AGEs and LY333531. Bcl-2, Bax and Bad proteins' expression were determined by StreptAvidin-Biotin-enzyme Complex (SABC) immunocytochenistry. The results showed that pretreatment with LY333531 strikingly decreased the chance of HUVEC survival and the effect of LY333531 on apoptotic cell death in HUVEC significantly increased compared with the AGEs group. Blockade of PKC-beta up-regulated the expression of Bax and Bad proteins and down- regulated the expression of Bcl-2 protein. Moreover, LY333531 reduced the ratio of Bcl-2/Bax. The results indicate that the selective PKC-beta inhibitor, LY333531, can further prompt AGEs-induced endothelial cells apoptosis. The increased expression of Bax, Bad and decreased expression of Bcl-2 and Bcl-2/Bax ratio are associated with the apoptotic process.     

Advanced glycation end products (AGEs) activate mast cells

BACKGROUND AND PURPOSE

Advanced glycation endproducts (AGEs) represent one of the many types of chemical modifications that occur with age in long-lived proteins. AGEs also accumulate in pathologies such as diabetes, cardiovascular diseases, neurodegeneration and cancer. Mast cells are major effectors of acute inflammatory responses that also contribute to the progression of chronic diseases. Here we investigated interactions between AGEs and mast cells.

EXPERIMENTAL APPROACHES

Histamine secretion from AGEs-stimulated mast cells was measured. Involvement of a receptor for AGEs, RAGE, was assessed by PCR, immunostaining and use of inhibitors of RAGE. Production of reactive oxygen species (ROS) and cytokines was measured.

KEY RESULTS

Advanced glycation endproducts dose-dependently induced mast cell exocytosis with maximal effects being obtained within 20 s. RAGE mRNA was detected and intact cells were immunostained by a specific anti-RAGE monoclonal antibody. AGEs-induced exocytosis was inhibited by an anti-RAGE antibody and by low molecular weight heparin, a known RAGE antagonist. RAGE expression levels were unaltered after 3 h treatment with AGEs. AGE-RAGE signalling in mast cells involves Pertussis toxin-sensitive G_i-proteins and intracellular Ca²⁺ increases as pretreatment with Pertussis toxin, caffeine, 2-APB and BAPTA-AM inhibited AGE-induced exocytosis. AGEs also rapidly stimulated ROS production. After 6 h treatment with AGEs, the pattern of cytokine secretion was unaltered compared with controls.

CONCLUSIONS AND IMPLICATIONS

Advanced glycation endproducts activated mast cells and may contribute to a vicious cycle involving generation of ROS, increased formation of AGEs, activation of RAGE and to the increased low-grade inflammation typical of chronic diseases.     

Nafamostat Mesilate Inhibits TNF-α-Induced Vascular Endothelial Cell Dysfunction by Inhibiting Reactive Oxygen Species Production

Nafamostat mesilate (NM) is a serine protease inhibitor with anticoagulant and anti-inflammatory effects. NM has been used in Asia for anticoagulation during extracorporeal circulation in patients undergoing continuous renal replacement therapy and extra corporeal membrane oxygenation. Oxidative stress is an independent risk factor for atherosclerotic vascular disease and is associated with vascular endothelial function. We investigated whether NM could inhibit endothelial dysfunction induced by tumor necrosis factor-α (TNF-α). Human umbilical vein endothelial cells (HUVECs) were treated with TNF-α for 24 h. The effects of NM on monocyte adhesion, vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) protein expression, p38 mitogen-activated protein kinase (MAPK) activation, and intracellular superoxide production were then examined. NM (0.01~100 µg/mL) did not affect HUVEC viability; however, it inhibited the increases in reactive oxygen species (ROS) production and p66shc expression elicited by TNF-α (3 ng/mL), and it dose dependently prevented the TNF-α-induced upregulation of endothelial VCAM-1 and ICAM-1. In addition, it mitigated TNF-α-induced p38 MAPK phosphorylation and the adhesion of U937 monocytes. These data suggest that NM mitigates TNF-α-induced monocyte adhesion and the expression of endothelial cell adhesion molecules, and that the anti-adhesive effect of NM is mediated through the inhibition of p66shc, ROS production, and p38 MAPK activation.