氯通道对血小板活化标志物PAC-1和P-选择素的影响

目的：探讨氯通道对血小板活化标志物PAC-1和P-选择素的影响。方法：采用Western blot和免疫荧光技术检测ClC-3在人外周血来源的血小板上的表达;ADP(20μM)不同时间点(15、30、60、120、180 min)处理血小板后,Western blot检测ClC-3蛋白的表达变化;利用流式细胞术检测空白对照组, ADP组,DIDS(100μM)+ADP组,NFA(100μM)+ADP组,NPPB(100μM)+ADP和低氯对照组,低氯+ADP组的血小板活化分子标志物PAC-1和P-选择素的表达变化。结果：在健康成人外周血分离的血小板上表达ClC-3蛋白;ADP时间依赖性地处理血小板,ClC-3蛋白表达呈增加趋势,15 min相较于空白对照组已有统计学意义(P<0.05);ADP组PAC-1(32.13%±2.0%)及P-选择素(52.32%±5.31%)表达均高于空白对照组(1.76%±0.41%,1.89%±0.32%,P<0.01);低氯对照组PAC-1(8.01%±1.36%)和P-选择素(12.19%±0.84%)的表达与空白对照组相比,均上调(P<0.05);与ADP组PAC-1相比, DIDS+ADP(5.62%±1.22%),NFA+ADP(1.96%±0.54%)和NPPB+ADP(3.56%±0.79%)组表达降低,低氯+ADP组(45.26%±4.43%)表达增加(P<0.01);与ADP组P-选择素相比,DIDS+ADP(16.64%±1.52%), NFA+ADP(4.97%±0.64%)和NPPB+ADP(8.56%±2.04%)组表达均降低,低氯+ADP组(73.33%±3.80%)表达增加(P<0.01)。结论：人血小板上ClC-3氯通道参与血小板的活化,氯通道阻断剂抑制ADP诱导的血小板活化,降低血小板胞外氯浓度可促进ADP诱导的血小板的活化。     

Thrombin inhibition by dabigatran attenuates atherosclerosis in ApoE deficient mice

Introduction

Atherosclerosis is a chronic inflammatory disease characterized by endothelial cell damage, infiltration, proliferation and accumulation of macrophages, lymphocytes and transformed vascular smooth muscle cells within the vascular wall and procoagulation processes involving activation of plasmatic coagulation events and platelets. Numerous studies suggested a close interaction between thrombin action and atherogenesis, but possibly underlying mechanisms are multiple and specific treatment options were missing until now.

Material and methods

Atherosclerosis prone 12 weeks old ApoE^–/– mice were fed a cholesterol rich diet for 4 weeks and were concomitantly treated orally with placebo or the thrombin inhibitor dabigatran (1.2 g/kg/day).

Results

The thrombin time (HEMOCLOT^®) was significant extended in dabigatran treated animals. Vascular oxidative stress was significantly reduced during thrombin inhibition, as assessed by L012 chemiluminescence in aortic segments (212 ±84 vs. 69 ±21 RLU/s/mg dry weight, p = 0.048). Organ chamber experiments of isolated aortic rings showed that dabigatran treatment significantly improved endothelium-derived vasorelaxation (p < 0.001). Dabigatran treated mice developed less atherosclerotic lesions (6.2 ±0.2% vs. 9 ±1.1%, p = 0.037) and showed less infiltration of atherosclerotic lesions with macrophages (2.59 ±0.3% vs. 5.14 ±0.7%, p = 0.0046), as determined by systematic histological and immunohistological analyses of the aortic root. Blood pressure, body weight and food intake were not altered by the treatment.

Conclusions

The thrombin inhibitor dabigatran reduces vascular oxidative stress and inflammation, improves endothelial function and decreases atherosclerosis in mice.     

Hypoxia attenuates effector-target cell interaction in the airway and pulmonary vascular compartment

Leucocyte infiltration is known to play an important role in hypoxia-induced tissue damage. However, little information is available about hypoxia and interaction of effector (neutrophils) with target cells (alveolar epithelial cells, AEC; rat pulmonary artery endothelial cells, RPAEC). The goal of this study was to elucidate hypoxia-induced changes of effector-target cell interaction. AEC and RPAEC were exposed to 5% oxygen for 2-6 h. Intercellular adhesion molecule-1 (ICAM-1) expression was determined and cell adherence as well as cytotoxicity assays were performed. Nitric oxide and heat shock protein 70 (HSP70) production was assessed in target cells. Under hypoxic conditions enhanced ICAM-1 production was found in both cell types. This resulted in an increase of adherent neutrophils to AEC and RPAEC. The death rate of hypoxia-exposed target cells decreased significantly in comparison to control cells. Nitric oxide (NO) concentration was enhanced, as was production of HSP70 in AEC. Blocking NO production in target cells resulted in increased cytotoxicity in AEC and RPAEC. This study shows for the first time that target cells are more resistant to effector cells under hypoxia, suggesting hypoxia-induced cell protection. An underlying mechanism for this phenomenon might be the protective effect of increased levels of NO in target cells.     

Melatonin attenuates hypoxia-induced ultrastructural changes and increased vascular permeability in the developing hippocampus

Abstract Hypoxic injury in the perinatal period may be involved in damaging the developing hippocampus. The damage may be mediated by excess production of vascular endothelial growth factor (VEGF) and nitric oxide (NO). We examined the hippocampus of neonatal Wistar rats subjected to hypoxia for VEGF and NO production. The mRNA and protein expression of hypoxia inducible factor-1alpha, endothelial, neuronal, inducible nitric oxide synthase and VEGF was found to be up-regulated significantly after the hypoxic exposure. Tissue VEGF concentration and NO production were also increased. By electron microscopy, swollen dendrites, vacuolated axons and hypertrophic astrocyte end feet associated with blood vessels were observed in hypoxic animals. In hypoxic rats, the passage of rhodamine isothiocyanate (RhIC) and horseradish peroxidase, administered intraperitoneally or intravenously, was observed through vascular walls. Furthermore, immunoglobulin G was localized in the neuropil and neurons. We suggest that increased VEGF and NO production in hypoxia had resulted in increased vascular permeability, leading to structural alteration of the dendrites and axons. Melatonin administration reduced VEGF and NO levels as well as leakage of RhIC, suggesting that it has a therapeutic potential in reducing hypoxia-associated damage in the developing hippocampus.     

Platelet Function Analyzer: Shear Activation of Platelets in Microchannels

Platelet function is suggestive of pathological conditions in cardiovascular diseases. With current insufficient prognostic devices, the need exists for a device to assess complete platelet function. This work presents a preliminary microfluidic device for such analysis based on platelet adhesion under shear flow conditions. In this novel device, polydimethylsiloxane (PDMS) microchannels were coated using a layer-by-layer self-assembly technique to provide controlled nanometer-thick layers of fibrinogen. Anticoagulated platelet rich plasma labeled with a fluorescein isothiocynate-tagged anti-glycoprotein IIb/IIIa-antibody and acridine orange was passed through these micro-channels at various time-averaged shear rate values. Fluorescence assays confirmed shear-dependent adhesion of platelets in the microchannels. Control experiments showed that the extent of adhesion on bare PDMS surfaces was less than on the surfaces coated with fibrinogen at similar shear rates. Fluorescent microscopy demonstrated that the extent of platelet adhesion to the fibrinogen substrate depended on shear rate. The extent of adhesion was modeled as a third order polynomial in shear rate.     

Extract from Aronia melanocarpa fruits potentiates the inhibition of platelet aggregation in the presence of endothelial cells

Introduction

Some polyphenolic compounds extracted from Aronia melanocarpa fruits (AM) have been reported to be cardioprotective agents. In this study we evaluated the ability of AM extract to increase the efficacy of human umbilical vein endothelial cells (HUVECs) to inhibit platelet functions in vitro.

Material and methods

This study encompasses two models of monitoring platelet reactivity: optical aggregation and platelet degranulation (monitored as the surface CD62P expression) in PRP upon the stimulation with ADP.

Results

We observed that only at low concentrations (5 µg/ml) did AM extract significantly improve antiplatelet action of HUVECs towards ADP-activated platelets in the aggregation test.

Conclusions

It is concluded that the potentiating effect of AM extract on the endothelial cell-mediated inhibition of platelet aggregation clearly depends on the used concentrations of Aronia-derived active compounds. Therefore, despite these encouraging preliminary outcomes on the beneficial effects of AM extract polyphenols, more profound dose-effect studies should certainly be considered before the implementation of Aronia-originating compounds in antiplatelet therapy and the prevention of cardiovascular diseases.     

Nitric oxide attenuates vascular endothelial cadherin‐mediated vascular integrity in human chronic inflammation

In this study, we examined the role of nitric oxide (NO) in controlling vascular integrity mediated by vascular endothelial (VE)‐cadherin in chronic inflammation. Periapical granulomas were analysed for the expression of inducible NO synthase (iNOS) and VE‐cadherin, and more iNOS expression than VE‐cadherin was shown. Human umbilical vein endothelial cells (HUVECs) were stimulated with proinflammatory cytokines and lipopolysaccharide extracted from Porphyromonas gingivalis and it induced iNOS expression, whereas it reduced VE‐cadherin expression, compared with negative controls. On the other hand, pre‐incubation with 1400W, an iNOS‐specific inhibitor, markedly reduced iNOS expression in stimulated HUVECs and restored VE‐cadherin expression to its control level, suggesting that vascular integrity was modulated in conjunction with the reduction of NO. Immunocytochemistry confirmed the functional role of NO in cultured HUVEC monolayers with or without 1400W. These data are consistent with a hypothesis suggesting that NO could attenuate VE‐cadherin‐mediated vascular integrity in human chronic inflammation.     

Effects of adenosine on ex vivo filtragometry platelet aggregation in relation to plasma levels

The aim of the present study was to investigate the concentration effect of adenosine on unstimulated platelet aggregation in humans. Adenosine infusion was given intravenously to 12 volunteers in the antecubital vein with infusion rates increasing from 20 to 100 μg kg^?1 min^?1. Filtragometry measurements were obtained from the contralateral antecubital vein before and during 100 μg kg^?1 min^?1 or during maximal tolerable infusion rate. In another set of experiments with 10 volunteers, basal filtragometry measurements were obtained before and after infusion of various concentrations of adenosine into the filtragometer test unit. With intravenous infusion aggregation time tended to increase from 333±42 to 418±8 s (mean±SEM) and increased the venous plasma adenosine concentration from 0.42±0.09 μM to 1.52±0.38 μM . Adenosine infusion into the filtragometer tubing system dose-dependently inhibited aggregation (P<0.05). Adenosine was rapidly eliminated with a half-life of adenosine in the filtragometry tubing system calculated to be about 6 s. These data extend our knowledge from an in vitroto an ex vivo situation that adenosine dose-dependently has a platelet antiaggregatory effect.     

Edaravone attenuates white matter lesions through endothelial protection in a rat chronic hypoperfusion model

A multicenter randomized clinical trial demonstrated that acute ischemic stroke patients treated with edaravone, a scavenger of hydroxyl radicals, had significant functional improvement. We tested the hypothesis that edaravone has protective effects against white matter lesions (WML) and endothelial injury, using a rat chronic hypoperfusion model. Adult Wistar rats underwent ligation of bilateral common carotid artery (LBCCA) and were divided into the edaravone group (injected once only immediately after LBCCA [n=39, ED₁]; and injected on three consecutive days [n=39, ED₃]), the vehicle group (n=39), and the sham group (n=15). Cerebral blood flow, Morris water maze performance, footprint test for locomotor function, immunohistochemical analyses and Western blot analysis were performed before and after LBCCA. The ED₃ group upregulated endothelial nitric oxide synthase and attenuated Evans Blue extravasation at day 3 after LBCCA (P<0.05). Edaravone markedly suppressed accumulation of 4-hydroxy-2-nonenal-modified protein and 8-hydroxy-deoxyguanosine (P<0.01), and loss of oligodendrocytes (P<0.05) in the cerebral white matter at days 3, 7, 14, 21 and 28 after LBCCA. These results were more evident in the ED₃ group. Moreover, at day 21 after LBCCA, spatial memory but not motor function, and axonal damage were significantly improved by three-time treatment of edaravone (P<0.05). Our results indicated that 3-day treatment with edaravone provides protection against WML through endothelial protection and free radical scavenging and suggested that edaravone is potentially useful for the treatment of cognitive impairment.     

束缚应激对大鼠血小板-氧化氮释放的影响

目的 观察急性应激对大鼠血小板一氧化氮(NO)释放的影响及其机制.方法 大鼠浸水-束缚应激(WRS)2、4和8 h,以胃溃疡指数(UI)作为应激损伤的标识,采用Greiss法测定血小板孵育液中亚硝酸盐(NO2-)含量;同位素法测其一氧化氮合酶(NOS)活性和L精氨酸(L-Arg)转运量.结果 WRS 2 h血小板L-Arg转运量、NOS活性和孵育液NO2-含量较对照组显著增加,但随应激时间延长,其呈下降趋势,应激8 h时均显著低于对照组,胃溃疡逐渐加重.结论 WRS应激早期阶段可上调血小板L-Arg/NO通路,促进血小板NO生成;长时间应激下调L-Arg/NO通路,减少NO释放.     

Effects of endothelin receptor type A antagonism and nitric oxide synthase inhibition on cerebral blood flow in hypertensive rats

Effects of the endothelin receptor type A antagonist BQ 123 and the NO synthase inhibitor L -NMMA on cerebral blood flow were studied in vivo in anaesthetized hypertensive (SHR) and normotensive (WKY) rats. The effects of acetylcholine following pre-treatment with these drugs were also studied with the microsphere method for blood flow determination in the cortex, thalamus, caudatus, pons, medulla, cerebellum and hypophysis. BQ 123 (1 mg kg^?1) induced only minor effects on cerebral blood flow in both strains (n = 8), whereas L -NMMA (N = 8; 20 mg kg^?1) reduced regional cerebral blood flow significantly in most regions (21–54%) in the hypertensive, but not in the normotensive rat. In normotensive rats pre-treated with BQ 123 intravenous administration of acetylcholine (2 μg kg^?1 min^?1) induced a widespread significant increase (20–50%) in cerebral blood flow despite a reduction of the mean arterial blood pressure, while no significant effects were seen in hypertensive animals. Intravenous infusion of acetylcholine in animals pre-treated with L -NMMA did not affect cerebral blood flow in most regions in either of the two rat strains. In conclusion, a vasodilatory response to acetylcholine was found following endothelin receptor A antagonism in the WKY rat only, suggesting a role for endothelin in the control of cerebral blood flow in this strain. Furthermore, a higher basal vasodilating nitric oxide-tone seems to be present in the hypertensive rat compared with the normotensive rat.     

维药伊木萨克片对大鼠阴茎勃起功能的影响

目的:探讨维药伊木萨克片对雄性大鼠阴茎勃起功能的影响及其机制.方法:性功能正常SD雄性大鼠用伊木萨克连续干预6周后,进行阿普吗啡(APO)阴茎勃起实验和交配实验,并检测阴茎海绵体窦压(ICP);用放射免疫法检测外周血清中睾酮(T)、促黄体生成素(LH)和促卵泡刺激素(FSH)含量,镜检睾丸的组织形态学改变;用免疫组织化学技术检测两组大鼠阴茎组织中eNOS、nNOS表达.结果:(1)与正常对照组相比较,伊木萨克组APO勃起所需时间显著缩短,插入次数显著增加,且基础ICP值与电刺激诱导ICP值均显著升高;(2)T水平在伊术萨克组显著高于正常对照组,而LH与FSH在两组问无显著性差异;(3)eNOS表达在伊木萨克组显著高于正常对照组,nNOS在两组间无昆著性差异.结论:维药伊木萨克片能够显著提高正常大鼠的阴茎勃起功能,其机制可能和提高雄激素水平与eNOS表达有关.     

人内皮型一氧化氮合酶cDNA在COS-7细胞中的表达

本研究从人脐静脉血管内皮细胞中提取总RNA，采用逆转录PCR(RT-PCR)方法，扩增出人内皮型一氧化氮合酶(heNOS)cDNA，总长度为3731bp。将其克隆入pUCm—T载体质粒，序列分析表明，克隆所得片段含有完整开放阅读框架，与GenBank中heNOScDNA序列同源性达99．93％，在此基础上发现有若干核酸多态性。将该基因片段亚克隆到真核表达载体pcDNA3．0上，用脂质体转染法将pcDNA3．0／heNOS转染到COS-7细胞株，RT—PCR、Western blot分别检测到外源性eNOS基因在mRNA水平和蛋白质水平的表达，分析表明所表达的heNOS蛋白质分子量为145ku；L-^14C-精氨酸掺入同位素法检测证实所表达的eNOS蛋白具有生物学活性，能将精氨酸氧化为瓜氨酸。     

尿酸抑制人脐静脉内皮细胞合成一氧化氮

目的 研究尿酸(UA)对人脐静脉内皮细胞(HUVEC)表达内皮型一氧化氮合酶(eNOS)及分泌一氧化氮(NO)的影响.方法 不同浓度UA(0、0.5、1、1.5及2 mg/L)及50 mg/L ox-LDL(阳性对照)分别作用HUVEC 24、48及72 h,用real-time PCR法测定HUVEC eNOS mRNA;Western blot法检测细胞eNOS蛋白;酶法检测上清液NO的含量.结果 UA 0.5 mg/L组eNOS mRNA表达水平明显高于对照组(P＜0.05);随着UA浓度升高(1、1.5及2 mg/L组),及其作用时间延长,HUVEC eNOS mRNA及蛋白表达水平及上清液NO分泌最相比对照均明显下降(72 h N02-/N03-2 mg/L组与对照组分别为0.52±0.18与1.00±0.10,P＜0.05),且趋势与ox-LDL组相同.结论 0.5 mg/L以上浓度的UA呈浓度及时间依赖性抑制HUVEC eNOS表达及NO合成,提示高浓度的UA可能损伤血管内皮功能.     

Nitric oxide synthase inhibition attenuates l-DOPA-induced dyskinesias in a rodent model of Parkinson's disease

Chronic l-DOPA pharmacotherapy in Parkinson's disease is often accompanied by the development of abnormal and excessive movements known as l-DOPA-induced dyskinesia. Rats with 6-hydroxydopamine lesion of dopaminergic neurons chronically treated with l-DOPA develop a rodent analog of this dyskinesia characterized by severe axial, limb, locomotor and orofacial abnormal involuntary movements. While the mechanisms by which these effects occur are not clear, they may involve the nitric oxide system. In the present study we investigate if nitric oxide synthase inhibitors can prevent dyskinesias induced by repeated administration of l-DOPA in rats with unilateral 6-hydroxydopamine lesion. Chronic l-DOPA (high fixed dose, 100 mg/kg; low escalating dose, 10–30 mg/kg) treatment induced progressive dyskinesia changes. Two nitric oxide synthase inhibitors, 7-nitroindazole (1–30 mg/kg) and NG-nitro-l-arginine (50 mg/kg), given 30 min before l-DOPA, attenuate dyskinesia. 7-Nitroindazolee also improved motor performance of these animals in the rota-rod test. These results suggest the possibility that nitric oxide synthase inhibitors may be useful to treat l-DOPA-induced dyskinesia.