Endothelial urocortin has potent antioxidative properties and is upregulated by inflammatory cytokines and pitavastatin

BACKGROUND: Urocortin, a neuropeptide discovered in the midbrain, is a member of the corticotropin-releasing factor family and is expressed in heart tissues. Urocortin exerts potent cardioprotective effects under various pathological conditions including ischemia/reperfusion. However, the regulation and function of vascular urocortin are unknown. METHODS AND RESULTS: Immunohistochemistry showed definitive expression of urocortin in endothelial cells of coronary large arteries and microvessels from autopsied hearts. RT-PCR confirmed the expression of urocortin in human umbilical vein endothelial cells (HUVECs). Urocortin (10(-8) M) potently suppressed the generation of angiotensin II-induced reactive oxygen species (ROS) in HUVECs. Tumor necrosis factor-alpha and interferon-gamma increased the urocortin mRNA levels and its release from HUVECs. Incubation with pitavastatin (0.1-3.0 microM) significantly increased the urocortin mRNA levels and its release from HUVECs. Furthermore, treatment with pitavastatin (2 mg/day) for 4 weeks increased the serum urocortin level from 11.0 +/- 6.5 to 16.4 +/- 7.3 ng/ml in healthy volunteers. CONCLUSION: Endothelial urocortin was upregulated by inflammatory cytokines and pitavastatin and suppressed ROS production in endothelial cells. Treatment with pitavastatin increased the serum urocortin level in human subjects. Thus, endothelial urocortin might protect cardiomyocytes in inflammatory lesions. Urocortin might partly explain the mechanisms of various pleiotropic effects of statins.     

The effect of selective phosphodiesterase isoenzyme inhibition on neutrophil function in vitro

Neutrophil-derived proteases such as neutrophil elastase (NE) and matrix metalloproteinase (MMP) are implicated in the pathogenesis of Chronic Obstructive Pulmonary Disease (COPD). In this study, the effects of selective phosphodiesterase (PDE) inhibition on NE and MMP-9 release, as well as Myeloperoxidase (MPO) activity and integrin-mediated neutrophil adhesion to human umbilical vein endothelial cells (HUVECs), were investigated. Human neutrophils were treated with PDE inhibitors (10(-11)-10(-4)M) in the absence and presence of TNF-alpha (tumour necrosis factor) (100 U ml(-1)) for 30 min, prior to fMLP activation. After 45 min, the cells were removed and NE, MPO and MMP-9 release assessed. In the adhesion studies, the neutrophils were radio-labelled with 51Cr, stimulated and immediately transferred to cultured HUVEC monolayers for 30 min, prior to assessment of adhesion. TNF-alpha (100 U ml(-1)) acted synergistically with fMLP in stimulating azurophil degranulation with respect to both MPO activity (P<0.01) and NE release (P<0.01). In contrast, an additive effect was observed with TNF-alpha and fMLP with regard to MMP-9 release and neutrophil adhesion to HUVECs. The PDE4 inhibitors, roflumilast, roflumilast N-oxide, cilomilast and rolipram significantly suppressed MPO, NE and MMP-9 release in both the presence and absence of TNF-alpha (P<0.05; n=6-10) and also reduced neutrophil adhesion to HUVECs. In contrast, milrinone, a PDE3 inhibitor and the non-selective PDE inhibitor, theophylline did not inhibit azurophil degranulation under any of the experimental conditions. These data provide further evidence that selective PDE4 isoenzyme inhibitors can inhibit neutrophil degranulation, effects not shared by PDE3 inhibitors or theophylline.     

Matrix metalloproteinase-1 expression by interaction between monocytes and vascular endothelial cells

There is accumulating evidence of complicated interactions among vascular cells, i.e. endothelial cells, smooth muscle cells and monocytes/macrophages, in the regulation of vascular function and remodeling. We have investigated the mechanisms responsible for matrix metalloproteinase (MMP)-1 expression by interactions between monocytes and vascular endothelial cells. THP-1 cells (human monocytic cell line) and human umbilical vein endothelial cells (HUVECs) were cocultured. MMP-1 levels in the culture medium were measured by enzyme-linked immunosorbent assays. Collagenolytic activity in the culture medium was measured by fluorescence labeled-collagen digestion. Immunohistochemistry using an anti-MMP antibody was carried out to determine which types of cell produce MMP-1. The addition of THP-1 cells to HUVECs for 48 h induced increases in MMP-1 levels and collagenolytic activity, which were 5- and 2-fold relative to those of HUVECs alone, respectively. A separate coculture experiment revealed that direct contact of THP-1 cells and HUVECs contributed to enhanced MMP-1 production in the cocolture. Immunohistochemical analysis revealed that both types of cell produce MMP-1 in the coculture. Neutralizing anti-interleukin-1 beta and tumor necrosis factor- alpha antibodies inhibited MMP-1 production by the coculture. The Src kinase and MEK inhibitors significantly inhibited MMP-1 production by the coculture. Coculture of THP-1 cells and HUVECs induced significant increases in Src and mitogen activated protein (MAP) kinase activities. Enhanced MMP-1 expression induced by monocyte-endothelial cell interactions may play an important role in the pathogenesis of atherosclerosis and plaque rupture.     

洛伐他汀抑制人脐静脉内皮细胞基质金属蛋白酶9表达和活性

目的观察洛伐他汀对人脐静脉内皮细胞基质金属蛋白酶9表达的影响。方法体外培养人脐静脉内皮细胞,加入20μg/L肿瘤坏死因子α诱导后,再用0.05～1.35μmol/L洛伐他汀处理0～24 h,蛋白印迹以及逆转录聚合酶链反应分别检测基质金属蛋白酶9的蛋白和mRNA表达情况,明胶酶谱法测定基质金属蛋白酶9活性。结果肿瘤坏死因子α能诱导人脐静脉内皮细胞的基质金属蛋白酶9表达和活性增强,洛伐他汀处理后,基质金属蛋白酶9的mRNA水平、蛋白量及活性均降低,且呈明显的时间和剂量依赖性,其中0.05μmol/L洛伐他汀能显著降低基质金属蛋白酶9的mRNA水平,但在蛋白水平和基质金属蛋白酶9活性上,0.05μmol/L洛伐他汀并没有表现出显著的降低效果,其余洛伐他汀各浓度均能对基质金属蛋白酶9的mRNA水平、蛋白量及活性产生显著的抑制作用,以1.35μmol/L洛伐他汀的抑制效果最显著。结论洛伐他汀能抑制肿瘤坏死因子α诱导的人脐静脉内皮细胞基质金属蛋白酶9的表达。     

High-density lipoprotein cholesterol regulates endothelial progenitor cells by increasing eNOS and preventing apoptosis

OBJECTIVE: Endothelial progenitor cells (EPCs) are implicated as an important marker of endothelial function and cardiovascular risk. In the present study, we examined whether high-density lipoprotein (HDL) cholesterol plays a role in the peripheral EPC levels and its underlying mechanisms in the HDL cholesterol-induced elevation of EPCs. METHODS: For the clinical study, vascular risk factors and blood markers were measured and EPC colony forming units were counted after 7 days of culture. For the in vitro study, after 7 days of culture, EPCs were incubated in the presence or absence of HDL for 24h followed by measurements of eNOS and pro-MMP-9 expression and caspase-3 activity. RESULTS: EPC colony levels significantly correlated with HDL levels (P=0.017). HDL treatment significantly increased eNOS protein expression in EPCs (P<0.001) while it significantly decreased pro-MMP-9 levels at the concentration of 50 microg/mL (P=0.002). Homocysteine treatment significantly increased caspase-3 activity whereas HDL significantly decreased it as compared to the homocysteine-only treated group. INTERPRETATION: The data demonstrate that EPC colony levels are significantly lower in individuals with low HDL and that HDL increases eNOS and decreases pro-MMP-9 in EPCs. HDL also prevents EPC apoptosis through inhibition of caspase-3 activity suggesting a possible mechanism for its positive effects on circulating EPC numbers.     

Enhancement of MMP-9 activity in THP-1 cells by 7-ketocholesterol and its suppression by the HMG-CoA reductase inhibitor fluvastatin

We investigated the effect of 7-ketocholesterol (7-KCHO) on the activity of matrix metalloproteinase (MMP-9) in human monocytic THP-1 cells, and the inhibition of this effect by fluvastatin. In cells incubated with 7-KCHO or cholesterol, the activity of MMP-9 was enhanced, accompanying an increase in the secretion of MMP-9 proenzyme (pro-MMP-9). However, the activity of MMP-9 and the amount of pro-MMP-9 were significantly greater following incubation with 7-KCHO than cholesterol. Neither 7-KCHO nor cholesterol influenced the amount of tissue inhibitor of metalloproteinase (TIMP)-1 secreted by THP-1 cells. When fluvastatin was added to the cells, the MMP-9 activity stimulated by 7-KCHO or cholesterol decreased significantly, accompanying a decrease in the secretion of pro-MMP-9 and TIMP-1. The inhibition of pro-MMP-9 secretion by fluvastatin was stronger in the cells incubated with 7-KCHO than with cholesterol. These results suggest that 7-KCHO activates macrophage and enhances MMP-9 activity, and its effects may be inhibited by fluvastatin.     

Impairment of endothelial function after a high-fat meal in patients with coronary artery disease.

OBJECTIVE: To determine the effect of postprandial lipid changes on endothelial function in patients with coronary artery disease (CAD) after a high-fat meal. METHODS: We studied 50 CAD patients and 25 control participants, who were all normocholesterolemic. Flow-mediated vasodilatation of the brachial artery was evaluated by the high-resolution ultrasound technique before and after a single high-fat meal (800 calories; 50 g fat). RESULTS: Postprandial serum triglyceride level increased significantly at 2-7 h and mean flow-mediated vasodilatation was impaired significantly (from 4.22 +/- 0.44 to 2.75 +/- 0.33%, P < 0.01) for 75 subjects. The increment in 2 h serum triglyceride level correlated positively with the decrement in postprandial flow-mediated vasodilatation (r = 0.459, P < 0.01). Postprandial triglyceride level was significantly higher in CAD patients than in control participants. Flow-mediated vasodilatation was significantly impaired in CAD patients (from 3.04 +/- 0.39 to 1.69 +/- 0.23%, P < 0.01) and control participants (from 6.58 +/- 0.52 to 4.87 +/- 0.19%, P < 0.05) after a high-fat meal. The impairment of flow-mediated dilatation was more severe in CAD patients (44.41%) than in control participants (25.99%, P < 0.01). CONCLUSION: Postprandial endothelium-dependent vasodilatation after a single high-fat meal was severely impaired in normocholesterolemic CAD patients and control participants. The disordered postprandial metabolism of triglyceride-rich lipoproteins may play an atherogenic role by inducing endothelial dysfunction.     

Insulin-mediated stimulation of protein kinase Akt: A potent survival signaling cascade for endothelial cells

Insulin exerts potent antiapoptotic effects in neuronal cells and has been suggested to promote angiogenesis. Therefore, we investigated whether insulin inhibits tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in human umbilical vein endothelial cells (HUVECs). Because insulin has been shown to stimulate the protein kinase Akt, we investigated whether activation of Akt contributes to the apoptosis-suppressive effect of insulin and characterized the downstream signaling pathway. Incubation with insulin dose-dependently prevented apoptosis induced by TNF-alpha (50 ng/mL). The extent of apoptosis suppression by insulin was similar to the effect of vascular endothelial growth factor. Pharmacological inhibition of Akt activation or overexpression of a dominant-negative Akt mutant prevented the antiapoptotic effect of insulin. Furthermore, we investigated the effect of TNF-alpha on Akt phosphorylation by Western blot analysis with the use of a phosphospecific Akt antibody. Incubation of HUVECs with TNF-alpha induced a marked dephosphorylation of Akt. Insulin counteracted this TNF-alpha-induced dephosphorylation of Akt. Furthermore, we investigated the downstream signaling events. Akt has been shown to mediate its apoptosis-suppressive effects via phosphorylation of Bad or caspase-9. However, incubation with insulin did not lead to enhanced phosphorylation of Bad at Ser 136 or Ser 112. In contrast, insulin inhibited caspase-9 activity and prevented caspase-9-induced apoptosis. Mutation of the Akt site within caspase-9 significantly reduced the apoptosis-suppressive effect of insulin. The present study demonstrates an important role for insulin-mediated Akt activation in the prevention of endothelial cell apoptosis, which may importantly contribute to cell homeostasis and the integrity of the endothelium. In endothelial cells, Akt seems to mediate its antiapoptotic effect, at least in part, via phosphorylation of caspase-9 rather than Bad.     

冠心病患者摄入不同量脂肪餐后血脂水平的变化 及其对血管内皮功能的影响

目的探讨冠心病患者餐后血脂代谢情况及其对血管内皮功能的影响。 方法将40例血清总胆固醇(TC)水平不高的冠心病患者随机分为两组,禁食12小时后,分别接受高脂肪餐饮食(高脂餐组,n=20)和低脂肪餐饮食(低脂餐组,n=20)。于空腹、餐后2、4、5、7小时测定血清TC、血清甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C),并同时于空腹状态及餐后4小时测定内皮依赖性及非依赖性血管舒张功能。 结果高脂餐组,餐后血清TG水平显著升高,TC、LDL-C、HDL-C无明显改变,内皮依赖性血管舒张功能由空腹状态的(3.38±2.34)%降至餐后4小时的(1.27±2.31)%,差异有非常显著意义(P＜0.01),内皮非依赖性血管舒张功能无明显改变,餐后4小时内皮依赖性血管舒张功能的受损程度与餐后2小时血清TG升高值呈显著性正相关(r=0.599,P＜0.01);低脂餐组餐后血脂水平、内皮依赖性及非依赖性血管舒张功能无明显改变。 结论冠心病患者进食高脂餐后内皮依赖性血管舒张功能明显受损,且与餐后血清TG的升高程度密切相关。     

Effects of a high-fat diet on postabsorptive and postprandial testosterone responses to a fat-rich meal.

Postprandial testosterone concentrations have been shown to significantly decrease after a fat-rich meal, which may be due to inhibition of testosterone production by chylomicrons. We examined the effects of a high-fat diet known to reduce postprandial chylomicrons on the testosterone response to a fat-rich meal. Total testosterone (TT), free testosterone (FT), cortisol, and insulin responses to a high-fat test meal containing 5.44 MJ (1,300 kcal, 11% carbohydrate, 3% protein, 86% fat) were determined before (week 0) and after (week 8) an 8-week high-fat diet (64% fat) in 11 healthy men. The high-fat diet resulted in significant reductions in postabsorptive and postprandial serum triacylglycerols (55% and 50%, respectively). There were no significant changes in postabsorptive serum TT, FT, and cortisol, but insulin concentrations were significantly (P < or = .05) lower at week 8 (-28%). There was a significant reduction 1 hour after the fat-rich meal for TT (-22%) and FT (-23%), which remained significantly below baseline for 8 hours. Postprandial TT and FT responses were not significantly different after the 8-week high-fat diet. Postprandial serum cortisol concentrations were significantly reduced 1 hour after the meal. There were no significant differences before and after the high-fat diet. Insulin was significantly increased at the 0-, 1-, and 2-hour postprandial time points before and after the high-fat diet. Compared with week 0, insulin concentrations were significantly lower prior to and immediately after the fat-rich meal at week 8. These data indicate a fat-rich meal results in a prolonged reduction in TT and FT concentrations that is not altered by lowering postprandial chylomicrons. Alternative mechanisms (eg, higher uptake at the receptor level of cells) other than chylomicron-induced or insulin-induced inhibition of steroidogenesis are likely responsible for the reduction in TT and FT after a fat-rich meal.     

Regulation of vascular endothelial growth factor by melatonin in human breast cancer cells

Melatonin exerts oncostatic effects on breast cancer by interfering with the estrogen‐signaling pathways. Melatonin reduces estrogen biosynthesis in human breast cancer cells, surrounding fibroblasts and peritumoral endothelial cells by regulating cytokines that influence tumor microenvironment. This hormone also exerts antiangiogenic activity in tumoral tissue. In this work, our objective was to study the role of melatonin on the regulation of the vascular endothelial growth factor (VEGF) in breast cancer cells. To accomplish this, we cocultured human breast cancer cells (MCF‐7) with human umbilical vein endothelial cells (HUVECs). VEGF added to the cultures stimulated the proliferation of HUVECs and melatonin (1 mm ) counteracted this effect. Melatonin reduced VEGF production and VEGF mRNA expression in MCF‐7 cells. MCF‐7 cells cocultured with HUVECs stimulated the endothelial cells proliferation and increased VEGF levels in the culture media. Melatonin counteracted both stimulatory effects on HUVECs proliferation and on VEGF protein levels in the coculture media. Conditioned media from MCF‐7 cells increased HUVECs proliferation, and this effect was significantly counteracted by anti‐VEGF and 1 mm melatonin. All these findings suggest that melatonin may play a role in the paracrine interactions between malignant epithelial cells and proximal endothelial cells through a downregulatory action on VEGF expression in human breast cancer cells, which decrease the levels of VEGF around endothelial cells. Lower levels of VEGF could be important in reducing the number of estrogen‐producing cells proximal to malignant cells as well as decreasing tumoral angiogenesis.     

Effects of a single administration of acarbose on postprandial glucose excursion and endothelial dysfunction in type 2 diabetic patients: a randomized crossover study

CONTEXT: Postprandial hyperglycemia has been reported to elicit endothelial dysfunction and provoke future cardiovascular complications. A reduction of postprandial blood glucose levels by the alpha-glucosidase inhibitor acarbose was associated with a risk reduction of cardiovascular complications, but effects of acarbose on endothelial function have never been elucidated. DESIGN: This study was aimed to assess the efficacy of acarbose on postprandial metabolic parameters and endothelial function in type 2 diabetic patients. Postprandial peakglucose (14.47 +/- 1.27 vs. 8.50 +/- 0.53 mmol/liter), plasma glucose excursion (PPGE), and change in the area under the curve (DeltaAUC)glucose after a single loading of test meal (total 450 kcal; protein 15.3%; fat 33.3%; carbohydrate 51.4%) were significantly higher in diet-treated type 2 diabetic patients (n = 14) than age- and sex-matched controls (n = 12). RESULTS: The peak forearm blood flow response and total reactive hyperemic flow (flow debt repayment) during reactive hyperemia, indices of resistance artery endothelial function on strain-gauge plethysmography, were unchanged before and after meal loading in controls. But those of diabetics were significantly decreased 120 and 240 min after the test meal. A prior administration of acarbose decreased postprandial peakglucose, PPGE, and DeltaAUCglucose. The peak forearm blood flow and flow debt repayment were inversely well correlated with peakglucose, PPGE, and DeltaAUCglucose but not with DeltaAUCinsulin or the other lipid parameters. CONCLUSIONS: Even a single loading of test meal was shown to impair endothelial function in type 2 diabetic patients, and the postprandial endothelial dysfunction was improved by a prior use of acarbose. Acarbose might reduce macrovascular complication by avoiding endothelial injury in postprandial hyperglycemic status.     

阿托伐他汀抑制同型半胱氨酸诱导的内皮细胞凋亡涉及NADPH氧化酶及p38MAPK途径

目的观察阿托伐他汀对同型半胱氨酸（Hcy）诱导的人脐静脉内皮细胞凋亡及活性氧的影响,并探讨其作用机制。方法Hcy单独或联合阿托伐他汀、N-乙酰半胱氨酸（NAC）、还原型辅酶Ⅱ（NADPH）氧化酶抑制剂（DPI）或p38促细胞分裂剂激活性蛋白激酶（p38MAPK）阻断剂（SB203580）处理人脐静脉内皮细胞后,检测细胞凋亡率,活性氧水平,NADPH氧化酶活性,caspase-3 mRNA的表达和p-p38MAPK的表达。结果阿托伐他汀明显抑制Hcy诱导的人脐静脉内皮细胞凋亡及活性氧的产生,并能拮抗Hcy诱导的NADPH氧化酶的激活,p38MAPK蛋白的磷酸化及caspase-3 mRNA表达的增加。NAC、DPI、SB203580可产生相同的作用。结论阿托伐他汀可能通过抑制NADPH氧化酶的激活,p38MAPK磷酸化途径抑制Hcy诱导的人脐静脉内皮细胞活性氧的产生和细胞凋亡。     

细胞外调节蛋白激酶在1型糖尿病颈动脉内皮及炎症表达中的作用

目的探讨细胞外调节蛋白激酶(ERK)在1型糖尿病小鼠颈动脉内皮功能紊乱及炎症表达中的作用。方法选择C57BL/6野生型雄性小鼠8只为对照组,同窝INS2~(AKITA)雄性小鼠16只分为1型糖尿病组和干预组,每组8只,分别腹腔注射0.1%DMSO 50μl/d;PD98059 10 mg/(kg·d),连续8周。检测血糖及NO、内皮素1水平,凝胶电泳迁移率实验检测颈动脉NO、一氧化氮合酶(NOS)、髓过氧化物酶(MPO)水平及信号转导与转录活化因子3(STAT-3)活性,Western blot检测ERK和基质金属蛋白酶9(MMP-9)蛋白表达,免疫组织化学检测颈动脉内皮MMP-9阳性表达。结果与对照组比较,1型糖尿病组血清内皮素1和颈动脉MPO明显增高,血清和颈动脉NO、NOS明显减低(P<0.05);ERK和MMP-9蛋白表达、颈动脉STAT-3活性及颈动脉内皮MMP-9阳性表达明显增高(P<0.05)。与1型糖尿病组比较,干预组上述各项均明显变化,差异有统计学意义(P<0.05)。结论 ERK信号途径可通过STAT-3参与1型糖尿病小鼠颈动脉内皮功能紊乱及MMP-9等炎性因子表达。     

Assessment of the clinical significance of gelatinase activity in patients with juvenile idiopathic arthritis using quantitative protein substrate zymography

OBJECTIVE: To measure gelatinase activities in paired synovial fluid (SF) and serum of patients with juvenile idiopathic arthritis (JIA), and to assess how these activities relate to clinical and laboratory measures of disease activity. METHODS: A quantitative protein substrate zymography method was adapted and validated for use with serum and SF. Bands of activity were measured by densitometry and correlated with standard laboratory indicators of inflammation: erythrocyte sedimentation rate and platelet count. RESULTS: Gelatinase activity was found consistently in patients with JIA, with reproducible, quantified bands of activity corresponding to pro-matrix metalloproteinase-9 (pro-MMP-9), including the neutrophil associated lipocalin complex, and pro- and active forms of MMP-2. Both active MMP-2 and pro-MMP-9 were higher in JIA serum than in controls, though no differences were seen between patients grouped according to age, disease duration, or JIA subtype. However, SF MMP-9 correlated significantly with the laboratory indicators of inflammation, as did the relative level of active MMP-2. CONCLUSIONS: Both MMP-2 and MMP-9 gelatinolytic activities are raised during active JIA and associated with inflammatory activity regardless of age and disease duration, supporting a role for MMPs in the breakdown of joint components from early in disease. These MMPs may be specific markers of active joint destruction linked to inflammatory JIA, MMP-9 as a product of infiltrating cells, and the activation of MMP-2 produced within the joint.     

那格列奈与阿卡波糖对2型糖尿病患者餐后血清游离脂肪酸水平的影响

目的比较那格列奈和阿卡波糖对新诊断2刑糖尿病患者餐后血清游离脂肪酸（FFA）和甘油三酯（TG）的影响。方法16例新诊断2型糖尿病患者随机分为两组,进行为期9周的自身交叉对照研究。前4周两组分别给于阿卡波糖（50mg,日三次）和那格列奈（120mg,日三次）,而后停药清洗药物1周。后4周两组交换药物。在第1周和第6周的第1天,所有患者进行标准餐试验,同时服用阿卡波糖50mg或那格列奈120mg,测定0、30、60、120min的血糖、胰岛素、FFA及TG。在第4周和第9周末,测定空腹血糖、胰岛素、FFA、TG。结果单剂景那格列奈和阿卡波糖降低标准餐后血糖的效果相似。与阿卡波糖相比,那格列奈可显著增加2型糖尿病患者标准餐后30min胰岛素分泌水平,降低餐后120min血清FFA水平,但对餐后血清TG水平兀明显影响。那格列奈或阿卡波糖治疗4周前后,空腹FFA和TG水平无显著改变。结论那格列奈在降低餐后血清FFA方而优于阿号波糖,这可能与其部分恢复早时相胰岛素分泌有关。     

Aging enhances the sensitivity of endothelial cells toward apoptotic stimuli: important role of nitric oxide

Advanced aging leads to impaired endothelial NO synthesis and enhanced endothelial cell apoptosis; therefore, we investigated the sensitivity of aged endothelial cells toward apoptotic stimuli and determined the role of NO. Human umbilical vein endothelial cells (HUVECs) were cultured until 14th passage. In aged cells, oxLDL and tumor necrosis factor-alpha-induced apoptosis and caspase-3-like activity were significantly enhanced more than 3-fold compared with young cells (passage 3). Because NO contributes to protection against endothelial cell death via S-nitrosylation of caspases, we determined endothelial NO synthase (eNOS) protein expression and the content of S-nitrosylated proteins. Aged HUVECs showed significantly reduced eNOS expression (35+/-10%) and a decrease in the overall S-NO content (33+/-3%), suggesting that eNOS downregulation may be involved in age-dependent increase of apoptosis sensitivity. Indeed, eNOS knockout endothelial cells showed a significantly enhanced apoptosis induction. Exogenous NO donors abolished increased apoptosis and caspase-3-like activity. In contrast, the application of shear stress, which exerts a profound apoptosis inhibitory effect via upregulation of NO synthesis in young cells, failed to inhibit apoptosis in aged cells. Moreover, no upregulation of eNOS protein expression and S-NO content in response to shear stress was detected in aged cells. Overexpression of wild-type eNOS completely restored the antiapoptotic effect of shear stress, whereas only a partial inhibitory effect was detected under steady conditions. Strikingly, transfection of constitutively active phosphomimetic eNOS (S1177D) further abrogated apoptosis in aged HUVECs. Thus, aging of endothelial cells is associated with decreased NO synthesis and concomitantly increased sensitivity of apoptosis, which may contribute to functional impairment of the endothelial monolayer.     

抵抗素对内皮细胞活性氧生成及AMPK磷酸化水平的影响

目的探讨抵抗素对内皮细胞功能影响的作用机制。方法体外培养人脐静脉内皮细胞,50、100ng/ml抵抗素干预24h,分别检测活性氧（ROS）生成和腺苷酸活化蛋白激酶（AMPK）磷酸化水平,并应用AICAR激活AMPK观察其对内皮ROS生成的影响。结果50、100ng/ml抵抗素作用组与对照组相比,内皮细胞AMPK磷酸化水平明显下降,而ROS生成无明显改变;50ng/ml抵抗素作用组加用AICAR干预后,AMPK磷酸化水平显著增高,同时伴ROS生成显著降低。结论抵抗素可影响内皮细胞功能,其机制可能通过抑制内皮细胞AMPK的磷酸化水平,参与内皮细胞炎症过程的调节,激活内皮AMPK,减少ROS生成,对内皮损伤起保护性作用。     

Comparison of two consecutive fat-rich and carbohydrate-rich meals on postprandial myeloperoxidase response in women with and without type 2 diabetes mellitus

Patients with type 2 diabetes mellitus (DM2) have an increased risk of cardiovascular disease (CVD). Myeloperoxidase (MPO), expressed in leukocytes and released upon activation, is associated with CVD and endothelial dysfunction. Postprandial leukocyte recruitment and activation with subsequent MPO release may contribute to atherosclerosis and CVD. We hypothesized that MPO may increase in the postprandial state because of postprandial leukocyte recruitment and/or activation, especially in subjects with DM2. One hundred postmenopausal women, aged 50 to 65 years (66 with normal glucose metabolism [NGM] and 34 with DM2), received 2 consecutive fat-rich meals and 2 consecutive carbohydrate-rich meals on separate occasions. Blood samples were taken before (t = 0) and at 2, 4, and 8 hours after breakfast; lunch was given at t = 4. Plasma MPO concentration was measured by sandwich enzyme-linked immunosorbent assay. The number of leukocytes in fasting blood samples was higher in DM2 compared with NGM (6.1 +/- 1.4 and 5.4 +/- 1.2 x 10(9)/L, respectively; P < .05). Baseline MPO concentration did not significantly differ between NGM and DM2 (51.4 +/- 12.9 and 54.5 +/- 18.4 mug/L, respectively; P = .39). Baseline MPO was positively associated with leukocytes (r = 0.20, P < .05) and inversely associated with high-density lipoprotein cholesterol (r = -0.22, P < .05). Leukocytes increased from 5.0 +/- 1.5 to 6.1 +/- 1.5 x 10(9)/L and from 5.8 +/- 1.4 to 6.6 +/- 1.4 x 10(9)/L in NGM and DM2, respectively (both P < .01), after the fat-rich meals. In contrast to our hypothesized increase in MPO, we found a significant decrease in MPO in NGM (both meal types) and DM2 (fat-rich meals only). Our findings provide no support to our initial hypothesis that meal-induced release of MPO might be a mechanism that contributes to CVD risk.