Pigment epithelium-derived factor inhibits advanced glycation end-products-induced cytotoxicity in retinal pericytes

Aim

This study investigated the effects of pigment epithelium-derived factor (PEDF) on advanced glycation end-product (AGE)-induced cytotoxicity in porcine retinal pericytes and the signalling mechanism involved.

Methods

Retinal pericytes were isolated from porcine eyes and characterized by immunocytochemistry. The effect of AGEs and PEDF on cell proliferation was determined by bromodeoxyuridine (BrdU) assay. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was analyzed by luminescence assay. Reactive oxygen species (ROS), nitric oxide (NO), superoxide dismutase (SOD) and glutathione peroxidase (GSH) were determined by biochemical assays. Induction of apoptosis was determined by caspase-3 colorimetric assay and DNA fragmentation analysis. Src activity was assessed by transient transfection analysis, and the status of Src phosphorylation at Y419 was analyzed by a competitive ELISA method.

Results

AGEs significantly increased intracellular ROS generation in pericytes via NADPH oxidase and induced cell death via caspase-3 enzyme activation, whereas PEDF increased cell proliferation in a dose-dependent manner. In addition, PEDF inhibited AGE-induced ROS generation by increasing levels of SOD and GSH, and also blocked the activation of caspase-3. Furthermore, PEDF induced cell survival via the Src pathway by Src phosphorylation at Y419, as evidenced by a pharmacological inhibitor and Src mutants.

Conclusion

These results suggest that PEDF abrogates AGE-induced oxidative stress and apoptosis in retinal pericytes via the Src pathway, thereby suggesting that PEDF is an effective therapeutic agent for the treatment of loss of pericytes in early diabetic retinopathy.     

PEDF对人肾小球系膜细胞VEGF表达及活性氧簇产生的影响

观察不同浓度高糖培养下及人重组色素上皮衍生因子(PEDF)干预后人肾小球系膜细胞活性氧簇水平、血管内皮生长因子(VEGF)mRNA和蛋白的改变.结果 显示活性氧簇水平、VEGF mRNA和蛋白的表达随糖浓度的升高而增加;PEDF干预后,高糖环境下活性氧簇水平及VEGF的表达明显下降,且呈浓度依赖性.提示PEDF可能通过改善血管通透性和抑制氧化应激延缓糖尿病肾病的进展.     

Pigment epithelium-derived factor inhibits leptin-induced angiogenesis by suppressing vascular endothelial growth factor gene expression through anti-oxidative properties

Leptin, a circulating hormone secreted mainly from adipose tissues, is involved in the control of body weight. Recently, leptin was found to be an angiogenic factor, and its vitreous levels are associated with angiogenic eye diseases such as proliferative diabetic retinopathy. However, the molecular mechanism for leptin-elicited angiogenesis remains to be elucidated. Pigment epithelium-derived factor (PEDF) has been shown to be the most potent natural inhibitor of angiogenesis in the mammalian eye, and its levels in the vitreous were decreased in angiogenic eye diseases. In this study, we investigated whether and how PEDF could inhibit the leptin-induced DNA synthesis in microvascular endothelial cells (EC), a key step of angiogenesis. Leptin significantly increased intracellular reactive oxygen species (ROS) generation in microvascular EC. PEDF was found to inhibit the leptin-induced ROS generation in EC. An anti-oxidant, N-acetylcysteine, or PEDF completely prevented the leptin-induced upregulation of vascular endothelial growth factor (VEGF) mRNA levels as well as any increase in DNA synthesis in microvascular EC. Polyclonal antibodies against human VEGF were also found to completely inhibit DNA synthesis in leptin-exposed EC. The present study suggests that leptin could elicit angiogenesis through autocrine VEGF production via intracellular ROS generation. PEDF may block the angiogenic effects of leptin through its anti-oxidative properties.     

色素上皮衍生因子抑制糖基化终产物介导人肾小球系膜细胞糖基化终产物受体表达的研究

目的 观察色素上皮衍生因子（PEDF）、晚期糖基化终产物受体（RAGE）的表达及重组PEDF对RAGE表达的抑制作用,探讨PEDF与DN的关系及对DN的保护作用. 方法 采用糖化小牛血清白蛋白（AGE-BSA）体外诱导人肾小球系膜细胞（HRMCs）,Western blot及RT-PCR法分别检测RAGE、PEDF蛋白和mRNA表达. 结果 （1） AGE-BSA（100～400 mg/L）呈浓度梯度减少HRMCsPEDF表达（P＜0.01）,升高RAGE表达（P＜0.01）;（2）重组PEDF蛋白（5～40 nmol/L）呈浓度依赖性抑制AGE-BSA介导RAGE蛋白在HRMCs的表达（P＜0.05）. 结论 AGEs通过降低PEDF表达,增加RAGE表达参与DN的发生,PEDF可能通过抑制AGE-RAGE轴对DN发挥保护作用.     

糖化终末产物对MIN6细胞活力及活性氧(ROS)水平的影响

探讨糖化终末产物(AGE)对小鼠胰岛细胞株MIN6细胞活力及活性氧(ROS)水平的影响.制备BSA-AGE,用不同浓度AGE(100、200、400 mg/L)干预MIN6细胞不同时间后,MTF比色法检测细胞活力变化.以活性氧捕获剂双氢-乙酰乙酸二氯荧光黄(DCFH-DA)孵育细胞,通过流式细胞仪检测细胞内二氯荧光黄(DCF)的荧光强度而测得细胞内活性氧水平,并测定胰岛素分泌的变化.随着AGE浓度的升高和作用时间的延长,细胞活力明显下降(P＜0.05).经DCFH-DA孵育后流式细胞仪检测显示,AGE处理组细胞内DCF平均荧光强度较对照组明显升高(P＜0.05).胰岛素分泌量随着AGE浓度的增高和时间的延长,呈下降趋势(P＞0.05).提示BSA-AGE抑制MIN6活力,使细胞内活性氧生成增加,诱导MIN6细胞氧化应激.

Abstract：

To explore the effect of advanced glycation end-products(AGEs)on cell viability and level of reactive oxygen species(ROS)in MIN6 cells. After intervention of various concentrations(100,200, and 400 mg/L)of AGEs for some time, cell viability was detected by MTT assay. 2', 7'-dichlorofluorescein diacetate(DCFH-DA)was used as a reactive oxygen species capture agent. The fluorescent intensity of 2', 7'-dichlorofluorescein(DCF), which was the product of cellular oxidation of DCFH-DA, was detected by flow cytometry. The level of ROS and insulin secretion was thus measured. Viability of MIN6 cells was inhibited by AGEs in a dose and time dependent manner(P＜0.05).Intracellular fluorescent intensity of DCF was markedly elevated in the AGEs groups as compared with that in the control group(P＜0.05).Insulin secretion was decreased in the AGEs groups than that in the control group(P＞0.05). The results suggest that AGEs inhibit the viability and induce oxidative stress in MIN6 cells by overproduction of ROS.     

蛹虫草（北冬虫夏草）提取物对高糖诱导的内皮细胞衰老和细胞内活性氧的影响

目的：探讨蛹虫草乙酸乙酯提取物（GSCME）对高糖诱导人脐静脉内皮细胞（HUVECs）的衰老和细胞内活性氧的影响。方法：体外培养HUVECs,分3组：对照组（D-葡萄糖5.5mmol/L）、高糖组（D-葡萄糖40mmol/L）、GSCME组（GSCME 50μg/ml＋D-葡萄糖40mmol/L）,检测各组细胞增殖情况,SA-β-gal阳性衰老细胞,周期分布和细胞内活性氧（ROS）含量。结果：与对照组相比,高糖干预24、48、72h后细胞增殖活性显著下降[OD：48h：（0.599±0.062）比（0.375±0.013）],P均〈0.01;高糖孵育48h后SA-β-gal阳性细胞率[（50.70±1.49）%比（89.63±0.69）%]、G0/G1期细胞百分率[（49.15±2.56）%比（76.59±3.58）%]、细胞内活性氧（ROS）水平[（1.03±0.15）比（3.60±0.36）]均明显增加,P均〈0.01。与高糖组相比,GSCME孵育48h、72hHUVECs增殖率显著上升[48h：（0.375±0.013）比（0.424±0.023）],P均〈0.05,孵育48h显著降低SA-β-gal阳性细胞率[（89.63±0.69）%比（65.81±1.50）%]、G0/G1期细胞百分率[（76.59±3.58）%比（52.70±5.64）%]及细胞内ROS水平[（3.60±0.36）比（1.67±0.21）],P〈0.05～0.01。结论：蛹虫草提取物可延缓高糖诱导的人脐静脉内皮细胞衰老进程,降低细胞内活性氧从而减轻细胞氧化损伤可能是其作用的机制之一。     

Vitamin C blocks vascular dysfunction and release of interleukin-6 induced by endothelin-1 in humans in vivo

OBJECTIVE: Inflammation of the vessel wall is of importance in atherosclerosis. Endothelin-1 (ET-1) exerts pro-inflammatory effects and contributes to endothelial dysfunction. The objective was to test whether ET-1 impairs vascular function by increasing oxidative stress and release of pro-inflammatory cytokines in humans. METHODS: Forearm blood flow (FBF) was determined in 12 young healthy males with venous occlusion plethysmography. RESULTS: Intra-brachial infusion of ET-1 (20 pmol/min) decreased both endothelium-dependent and -independent vasodilatation (P<0.001). ET-1 also increased venous IL-6 levels (0.96+/-0.14-1.40+/-0.15 ng/ml; P<0.001). Administration of Vitamin C (24 mg/min) following the ET-1 infusion did not restore vascular function. However, pre-treatment with Vitamin C before ET-1 prevented the decrease in endothelium-dependent and -independent vasodilatation as well as the increase in IL-6 levels (1.20+/-0.28 versus 1.29+/-0.27 ng/ml; P=0.57). Infusion of a control vasoconstrictor substance, noradrenaline (80 ng/min) for 30 min did not affect IL-6 levels. CONCLUSIONS: ET-1 impairs endothelium-dependent and -independent vasodilatation and stimulates release of IL-6 in humans in vivo. These effects are inhibited by pre-treatment with the antioxidant Vitamin C. This suggests that the mechanism by which ET-1 impairs vascular function and stimulates release of IL-6 involves increased oxidative stress.     

Cigarette smoke exposure impairs VEGF-induced endothelial cell migration: role of NO and reactive oxygen species

Endothelial dysfunction is one of the earliest pathological effects of cigarette smoking. Vascular endothelial growth factor (VEGF) has been shown to be an important regulator of endothelial healing and growth. Accordingly, we tested the hypothesis that cigarette smoke exposure impairs VEGF actions in endothelial cells. In human umbilical vein endothelial cells (HUVECs), cigarette smoke extracts (CSE) inhibited VEGF-induced tube formation in the matrigel assay. CSE did not affect HUVECs proliferation, but significantly reduced cellular migration in response to VEGF. This impaired migratory activity was associated with a reduced expression of alpha(v)beta(3), alpha(v)beta(5), alpha(5)beta(1) and alpha(2)beta(1) integrins. The Akt/eNOS/NO pathway has been shown to be important for VEGF-induced endothelial cell migration. We found that CSE inhibited Akt/eNOS phosphorylation and NO release in VEGF-stimulated HUVECs. This was associated with an increased generation of reactive oxygen species (ROS). Importantly, in HUVECs exposed to CSE, treatment with antioxidants (NAC, vitamin C) reduced ROS formation and rescued VEGF-induced NO release, cellular migration and tube formation. Moreover, treatment with NO donors (SNAP, SNP) or a cGMP analog (8-Br-cGMP) rescued integrin expression, cellular migration and tube formation in endothelial cells exposed to CSE. (1) Cigarette smoke exposure impairs VEGF-induced endothelial cell migration and tube formation. (2) The mechanism involves increased generation of ROS, decreased expression of surface integrins together with a blockade of the Akt/eNOS/NO pathway. (3) These findings could contribute to explain the negative effect of cigarette smoking on endothelial function and vessel growth.     

Effects of dietary sodium on reactive oxygen species formation and endothelial dysfunction in low-density lipoprotein receptor-deficient mice on high-fat diet

Hypertension and high serum cholesterol level are important risk factors for atherosclerosis and coronary heart disease. In the present study we tested the hypothesis whether high sodium intake, when given in combination with Western type high-fat diet, induces endothelial dysfunction and promotes atherosclerosis. Furthermore, the role and enzyme sources of increased oxidative stress were examined. Low-density lipoprotein receptor-deficient mice (LDLR^−/−) and control C57Bl/6 mice received either high-fat, normal-sodium diet (fat 18% and cholesterol 0.5%; NaCl 0.7%; w/w) or high-fat, high-sodium diet (7% NaCl w/w) for 12 weeks. Superoxide formation was assessed by lucigenin enhanced chemiluminescence, endothelial functions were examined ex vivo, and atherosclerotic lesions from the aorta were assessed by light microscopy. High-fat, high-sodium diet increased systolic blood pressure in LDLR^−/− mice but not in C57Bl/6 mice, whereas it induced cardiac hypertrophy in both mouse strains. Dietary combination of fat and sodium induced endothelial dysfunction in LDLR^−/− mice. Preincubation with a superoxide scavenger Tiron normalized endothelial dysfunction, whereas the hydrogen peroxide scavenger catalase did not alter endothelial function. High sodium intake induced superoxide formation in LDLR^−/− mice on high-fat diet. Stimulation of muscarinic receptors in the endothelial cells by acetylcholine increased superoxide generation, whereas preincubation with the nitric oxide synthase (NOS) inhibitor L-arginine methyl ester or endothelium removal reduced superoxide production. Inhibition of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase by apocynin decreased vascular superoxide formation whereas the xanthine oxidase inhibitor oxypurinol did not significantly affect oxidative stress in LDLR^−/− mice. In conclusion, the detrimental effects of dietary sodium on endothelial function and progression of atherosclerosis in LDLR^−/− mice on high-fat diet are mediated by increased ROS formation mainly through uncoupled NOS and NADPH oxidase. The present study also underscores the importance of superoxide and endothelial NOS uncoupling in the pathogenesis of endothelial dysfunction.     

Effects of ethanol on the properties of platelets and endothelial cells in model experiments

AIM: To investigate effects of ethanol on activity markers of atherosclerosis in an in vitro endothelial cell model. METHODS: After 24 h incubation with ethanol (0.0095%), human umbilical vein endothelial cells were stimulated for 1 h with lipopolysaccharide, and were then incubated in direct contact with activated platelets. Following this incubation, the expression of CD40L and CD62P on platelets, and the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), urokinase plasminogen activator receptor (uPAR), and membrane-type 1 matrix metalloproteinase (MT1-MMP) on endothelial cells were measured by flow cytometry. RESULTS: The increased expression of VCAM-1 and uPAR on endothelial cells by proinflammatory stimulation with activated platelets was significantly reduced through pre-incubation with ethanol (P<0.05). Furthermore, platelets in direct contact with ethanol and with endothelial cells pre-incubated in ethanol showed a significant reduction in their CD40L expression (P<0.05). Ethanol had no significant effect on ICAM-1 and MT1-MMP expression on endothelial cells. CONCLUSION: Ethanol directly attenuates platelet activation and has significant endothelial cell-mediated effects on selected markers of atherosclerosis in vitro . These findings underline possible protective effects of ethanol on atherosclerosis.     

Reduction in generation of reactive oxygen species and endothelial dysfunction during postprandial state

Background and aims

To characterise changes in generation of cellular reactive oxygen species (ROS) in healthy males during the postprandial state, and to analyse the influence of the postprandial state on endothelial ROS generation and endothelial dysfunction.

Methods and results

Seventeen healthy subjects were recruited. Blood samples were collected in the fasting state and 2, 4, 6 and 8 h after liquid-meal intake (composition: 25% fat, 55% dextromaltose and 14% protein), providing 40 g fat m⁻² body surface. Plasma lipids, apolipoproteins, glucose and insulin were measured during this period. Peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation. The influence of postprandial state on intracellular ROS generation was measured by two different methods in PBMCs and in a human immortalised endothelial cell line (ECV 304). Artery flow-mediated vasodilation (FMD) was used to evaluate the endothelial function, and oxygen consumption by PBMCs was measured. Reduced ROS generation was observed in all methods and cells during the postprandial period. FMD was impaired 8 h after meal intake (23 ± 6 vs. 13 ± 2, P < 0.05 vs. baseline). The consumption of oxygen was reduced in PBMCs (−14% into 2 h, P < 0.05 vs. baseline and −27% after 4 h, P < 0.01 vs. baseline). ROS generation was correlated with plasma lipids, insulin, apolipoproteins and oxygen consumption.

Conclusions

In contrast to the previously reported elevation of postprandial oxidative stress, this study shows reduced ROS generation in PBMCs and in ECV 304. Data obtained in both cellular models suggest the existence of a protective response against plasma postprandial oxidative stress.     

MicroRNA-223 inhibits tissue factor expression in vascular endothelial cells

Objective: Atherosclerosis is a chronic inflammatory process, in which vascular endothelial cells (ECs) become dysfunctional owing to the effects of chemical substances, such as inflammatory factor and growth factors. Tissue factor (TF) expression is induced by the above chemical substances in activated ECs. TF initiates thrombosis on disrupted atherosclerotic plaques which plays an essential role during the onset of acute coronary syndromes (ACS). Increasing evidences suggest the important role of microRNAs as epigenetic regulators of atherosclerotic disease. The aim of our study is to identify if microRNA-223 (miR-223) targets TF in ECs. Methods and results: Bioinformatic analysis showed that TF is a target candidate of miR-223. Western blotting analysis revealed that tumor necrosis factor α (TNF-α) increased TF expression in aorta of C57BL/6J mice and cultured ECs (EA.hy926 cells and HUVEC) after 4 h treatment. In TNF-α treated ECs, TF mRNA was also increased measured by real-time PCR. Real-time PCR results showed that miR-223 levels were downregulated in TNF-α-treated aorta of C57BL/6J mice and cultured ECs. Transfection of ECs with miR-223 mimic or miR-223 inhibitor modified TF expression both in mRNA and protein levels. Luciferase assays confirmed that miR-223 suppressed TF expression by binding to the sequence of TF 3′-untranslated regions (3′UTR). TF procoagulant activity was inhibited by overexpressing miR-223 with or without TNF-α stimulation. Conclusions: MiR-223-mediated suppression of TF expression provides a novel molecular mechanism for the regulation of coagulation cascade, and suggests a clue against thrombogenesis during the process of atherosclerotic plaque rupture.     

川芎嗪对氧化低密度脂蛋白诱导内皮细胞炎症反应的影响

目的探讨川芎嗪对氧化低密度脂蛋白(ox-LDL)诱导内皮细胞炎症反应的影响及其分子机制。方法体外培养的人冠状动脉内皮细胞,随机分为正常对照组(无干预)、ox-LDL组(50mg/Lox-LDL)、川芎嗪组(1、10、100μmol/L川芎嗪+50mg/Lox-LDL),观察川芎嗪对细胞间黏附分子1(ICAM-1)和环氧酶2基因和蛋白表达的影响;进一步检测与其耦联的p38丝裂原活化蛋白激酶(p38MAPK)和核转录因子κB(NF-κB)信号通路活化的影响。结果与ox-LDL组比较,川芎嗪(10、100μmol/L)降低ICAM-1的基因表达(1.49±0.26、1.81±0.05比2.36±0.34,P<0.01),抑制环氧酶2的蛋白表达(0.21±0.03、0.13±0.04比0.48±0.01,P<0.05);川芎嗪(1、10μmol/L)降低ICAM-1的蛋白表达(0.16±0.03、0.14±0.02比0.87±0.05),抑制上述黏附分子和炎症因子耦联的信号分子p38MAPK的磷酸化激活(0.30±0.10、0.12±0.06比0.66±0.15),抑制信号分子NF-κB蛋白(0.32±0.08、0.20±0.11比0.62±0.10)表达水平的升高(均P<0.01)。结论川芎嗪具有对抗ox-LDL诱导的内皮细胞炎症和黏附反应,并抑制MAPK和NF-κB信号通路的激活。     

同型半胱氨酸对血管内皮细胞E-选择素、IL-6分泌的影响

目的探讨同型半胱氨酸（homocysteine,Hcy）对人脐静脉内皮细胞E-选择素、白细胞介素6（IL6）分泌的影响。方法在人脐静脉内皮细胞ECV304培养基中加入不同浓度Hcy,孵育不同时间,用ELISA法分别测定培养上清液中E-选择素及IL6的含量。结果终浓度为250μmol/L的Hcy孵育内皮细胞,E选择素的分泌在4h显著上调,6h达峰值;IL6的分泌在6h开始升高,24h达峰值,之后随着时间的延长逐渐下降。Hcy呈浓度依赖性（50μmol/L～500μmol/L）促进内皮细胞E选择素及IL6的分泌。结论Hcy能刺激血管内皮细胞分泌E选择素及IL6。     

Urotensin Ⅱ-induced insulin resistance is mediated by NADPH oxidase-derived reactive oxygen species in Hep G2 cells

AIM: To investigated the effects of urotensin Ⅱ(UII) on hepatic insulin resistance in Hep G2 cells and the potential mechanisms involved.METHODS: Human hepatoma Hep G2 cells were cultured with or without exogenous UII for 24 h, in the presence or absence of 100 nmol/L insulin for the last 30 min. Glucose levels were detected by the glucoseoxidase method and glycogen synthesis was analyzed by glycogen colorimetric/fluorometric assay. Reactive oxygen species(ROS) levels were detected with a multimode reader using a 2′,7′-dichlorofluorescein diacetate probe. The protein expression and phosphorylation levels of c-Jun N-terminal kinase(JNK), insulin signal essential molecules such as insulin receptor substrate-1(IRS-1), protein kinase B(Akt), glycogen synthase kinase-3β(GSK-3β), and glucose transporter-2(Glut 2), and NADPH oxidase subunits such as gp91 phox, p67 phox, p47 phox, p40 phox, and p22 phox were evaluated by Western blot.RESULTS: Exposure to 100 nmol/L UII reduced the insulin-induced glucose consumption(P 0.05)and glycogen content(P 0.01) in Hep G2 cells compared with cells without UII. UII also abolished insulin-stimulated protein expression(P 0.01) and phosphorylation of IRS-1(P 0.05), associated with down-regulation of Akt(P 0.05) and GSK-3β(P 0.05) phosphorylation levels, and the expression of Glut 2(P 0.001), indicating an insulin-resistance state in Hep G2 cells. Furthermore, UII enhanced the phosphorylation of JNK(P 0.05), while the activity of JNK, insulin signaling, such as total protein of IRS-1(P 0.001), phosphorylation of IRS-1(P 0.001) and GSK-3β(P 0.05), and glycogen synthesis(P 0.001) could be reversed by pretreatment with the JNK inhibitor SP600125. Besides, UII markedly improved ROS generation(P 0.05) and NADPH oxidase subunit expression(P 0.05). However, the antioxidant/NADPH oxidase inhibitor apocynin could decrease UII-induced ROS production(P 0.05), JNK phosphorylation(P 0.05), and insulin resistance(P 0.05) in HepG 2 cells. CONCLUSION: UII induces insulin resistance, and this can be reversed by JNK inhibitor SP600125 and antioxidant/NADPH oxidase inhibitor apocynin targeting the insulin signaling pathway in HepG 2 cells.