The effect of thrombin on fibronectin in cultured human endothelial cells

Cultures of human endothelial cells (EC) incubated for periods up to 24 h with highly purified thrombin (2 NIH u/ml) contained considerably less cell-associated fibronectin fibrils than corresponding controls. The loss of fibronectin fibrils was evident after 4 h and was accompanied by a 2-3 fold increase in the concentration of fibronectin in the incubation medium. Hirudin inhibited the effects of thrombin. Thrombin also induced characteristic shape changes of EC. These shape changes were reversible within a 4-6 h period and could not be reinvoked by new additions of thrombin. Thus, structural refractoriness to thrombin coincided temporally with a period when EC-associated fibronectin fibrils were markedly reduced.     

Hemorheological effect of ticlopidine in the rat

Hemorheological effect of ticlopidine was studied with rats in various tests including whole blood viscosity, blood filtrability through a micropore membrane, mechanical hemolysis and shear stress-induced red cell deformability which was studied by a new technique developed by us.Ticlopidine treatment (30–300 mg/kg, p.o.) resulted in decrease in viscosity of heparinized whole blood, increase in blood filtrability and red cell stability against mechanical hemolysis. Ticlopidine was also found to increase reversible deformability of the red cells in response to shear stress which was evoked by passing whole blood through a thin tube (0.15 mm Ø) in a high speed (0.1 ml/0.2 sec). However, ticlopidine did not affect hematocrit values, plasma fibrinogen levels and plasma viscosity. Red cell morphology was not affected under the conditions without shear stress.These findings indicate that ticlopidine decreases whole blood viscosity by increasing red cell deformability, and that the effect may be favorable for improvement of microcirculation disorders.     

Inhibition by ticlopidine of Paf-acether-induced in vitro aggregation of rabbit and human platelets

Ticlopidine was incubated $\text{in vitro}$ with rabbit or human washed platelets and aggregations were triggered by submaximal concentrations of adenosine-5′-diphosphate (ADP), arachidonic acid (AA) and Paf-acether (platelet-activating factor), the mediators of the three known pathways of platelet activation. Inhibition of Paf-acether-induced rabbit platelet aggregation was proportionnal to the concentrations of Ticlopidine used. The same range of inhibition by Ticlopidine was observed when aggregations were triggered by the two other agonists. Human platelet aggregation induced by Paf-acether was also inhibited by Ticlopidine. Inhibition was increased when platelets were rendered insensitive to ADP and AA. Our results show that Ticlopidine inhibits human and rabbit platelet aggregation triggered by Paf-acether through a mechanism not related to the inhibition of the ADP and prostaglandin pathways.     

Effect of ticlopidine on human platelet responsiveness ex vivo: Comparison with aspirin

Platelet responsiveness in a variety of tests was measured in seven volunteers twice prior to and once after treatment with ticlopidine (250mg bds for seven days). The drug caused moderate but significant inhibition of both collagen-induced aggregation and the release of [¹⁴C]5-HT associated with aggregation induced by collagen, ADP and adrenaline. Platelet responses to ADP after ticlopidine treatment were unusual in that a marked tendency to disaggregate was observed. First-phase aggregation in response to ADP and adrenaline was not affected by ticlopidine, nor was platelet retention in glass bead columns or adhesion to collagen. A single low dose of aspirin (300mg) had a significantly greater effect than seven days treatment with ticlopidine on aggregation and [¹⁴C]5-HT release, and also reduced adhesion to collagen. However, aspirin did not induce disaggregation. Although ticlopidine seems relatively weak as an anti-aggregant drug when tested in humans $\text{ex vivo}$, its capacity to induce disaggregation distinguishes it from aspirin and could be important in a potential antithrombotic drug.     

Comparison between the antiaggregating effect of ticlopidine in whole blood,platelet-rich plasma and washed platelets of the rat

Ticlopidine, an inhibitor of platelet activation enhances the anti-aggregating effect of prostaglandins (PG) El and I2 when applied to rat whole blood or platelet-rich plasma. Aggregation by ADP was inhibited to a similar extent when evaluated on whole blood and on PRP of ticlopidine-treated rats showing that circulating PGs are probably not directly responsible for the antiaggregating effects of the drug. When platelets collected from ticlopidine-treated rats were separated from their plasma, they became largely refractory to ADP. PGE₂ was equally effective in potentiating platelet aggregation of washed platelets of treated and untreated animals, but failed to fully restore the impaired responsiveness to ADP of washed platelets from ticlopidine-treated rats. Our results indicate that ticlopidine does not prevent platelet aggregation by enhancing the antiaggregating effect of circulating PGs, and that ticlopidine or a metabolite, interact directly with the rat platelet, reducing its responsiveness to ADP.     

Antithrombotic effect of ticlopidine in a platelet-independent model of venous thrombosis

The antithrombotic activity of ticlopidine demonstrated in a variety of experimental models of thrombosis has been explained by its antiaggregating properties. This study describes the antithrombotic effect of ticlopidine in a platelet independent model of venous thrombosis. In the rat, ligature of the inferior vena cava induces thrombosis. Antiaggregating drugs (acetylsalicylic acid, dipyridamole, sulfinpyrazone) are inactive while anticoagulants (heparin, acenocoumarol) are highly antithrombotic. Ticlopidine reduces thrombus weight significantly and dose-dependently (ED 50 = 150 mg/kg/day X 3 days). Thrombocytopenia induced by injection of anti-platelet anti-serum was found not to modify thrombus formation. Yet, even in these conditions, ticlopidine remains active. Acetylsalicylic acid treatment does not prevent the antithrombotic effect of ticlopidine, indicating that its action is independent of PGI2 synthesis. These results demonstrate that ticlopidine acts as an antithrombotic agent in a venous thrombosis model in which platelets play a minor role.     

The effect of ticlopidine on platelet function in normal volunteers and in patients with platelet hyperaggregability

The effect of 500 mg Ticlopidine has been studied in 8 normal volunteers and in 18 patients with platelets, which in vitro show hyperaggregability as defined by low threshold concentrations of ADP and epinephrine. Ticlopidine significantly inhibits ADP, epinephrine and collagen induced platelet aggregation and release function as illustrated by reduction of serotonin release, and increase of the platelet disaggregation. Maximal effect is seen after around 24 hours in normal controls, but not until after 72 hours in patients with hyperaggregability, which is significantly later than the peak concentration in serum. We found no correlation between Ticlopidine concentrations and inhibitory effect.     

高浓度葡萄糖诱导血管内皮细胞凋亡及对VEGF表达的影响

目的 探讨高血糖对血管内皮细胞（VEC-304）的生长抑制作用及其分子机制。方法 以不同浓度（7.5～60mmol/L）的葡萄糖作用于体外培养脐静脉内皮细胞（VEG-304）24～72h，MTT法检测细胞生长抑制率，流式细胞仪检测细胞凋亡率，单细胞凝胶电泳法检测细胞的DNA损伤，免疫组化法检测内皮细胞生长因子（VEGF）蛋白的表达。结果 不同浓度的葡萄糖作用VEC-30424h后可显著抑制VEC-304的生长，诱导细胞发生凋亡，凋亡率8.46～74.16%，明显高于对照组（P〈0.01）；单细胞凝胶电泳结果显示葡萄糖作用6h后的EVC-304细胞表现为彗星样拖尾，尾长24.36～72.18μm，亦高于对照组（P〈0.01）；免疫组化结果提示VEGF蛋白表达下调，并呈浓度依赖性。结论 葡萄糖能显著抑制VEC-304的生长，诱导其凋亡，并下调VEGF的表达，这可能是高浓度葡萄糖损伤血管内皮细胞的机制之一。     

人胚胎干细胞在无血清mTeSR®1培养基中维持培养和向内皮细胞的诱导分化

背景：传统的人胚胎干细胞培养扩增方法中应用含动物血清培养基,并依赖饲养层细胞培养,这种培养方法显著制约了干细胞的体外培养规模;另外异源动物血清成分介入,使病原污染及免疫排斥的概率显著增加。目的：明确应用无血清培养基mTeSR®1对人胚胎干细胞进行长期体外培养的可行性,并建立诱导人胚胎干细胞分化为血管内皮细胞的相关技术平台。方法：采用无血清培养基mTeSR®1以非饲养层细胞依赖的方式体外培养、扩增人胚胎干细胞株H9。经过40余次体外传代后,于倒置显微镜下观察其生长形态,并利用免疫荧光染色方法评估其细胞表型。此外,应用条件培养基诱导H9细胞株向内皮细胞方向分化。利用免疫荧光染色技术,定量RT-PCR以及低密度脂蛋白摄取实验对该胚胎干细胞源内皮细胞的表型及功能进行评价、分析。结果与结论：mTeSR®1培养基能够支持H9细胞株在体外以非饲养层依赖的方式进行长期扩增,同时维持其未分化的干细胞潜能。添加血管内皮细胞的条件培养基能够定向诱导H9细胞向内皮细胞方向分化。该胚胎干细胞源内皮细胞不但表达内皮细胞的标志基因(kdr,pecam)和标记蛋白CD31,而且还能够摄取低密度脂蛋白,形成类似微血管结构。提示实验中所提供的培养及诱导分化体系能够支持胚胎干细胞的增殖与分化行为。     

纤维蛋白对大鼠脑血管内皮细胞血管内皮生长因子表达的影响

目的观察纤维蛋白对大鼠脑血管内皮细胞血管内皮生长因子(VEGF)转录及蛋白水平表达的影响。方法大鼠脑血管内皮细胞分离后培养,加入不同浓度的纤维蛋白,通过Real-time PCR检测VEGF转录水平,应用酶联免疫方法(ELISA)定量检测培养基和细胞裂解液中的VEGF水平。结果纤维蛋白可以特异性诱导大鼠脑血管内皮细胞表达VEGF;加入不同浓度的纤维蛋白(0.03mg/ml、0.1mg/ml、0.3mg/ml和1.0mg/ml),24h后,1.0mg/ml纤维蛋白组的培养基VEGF水平显著增高(P<0.001);1.0mg/ml纤维蛋白与大鼠脑血管内皮细胞分别培养0、2、4、8、24、48h,VEGF浓度在共培养2h已经升高,8h时显著升高,在24h时仍然保持在显著升高表达水平(P<0.005),48h有所下降;Real-time PCR结果提示,大鼠脑血管内皮细胞中VEGF mRNA的上调呈现出剂量和时间依赖性增加。结论纤维蛋白可以上调大鼠血管内皮细胞中的VEGF。     

人脐带血内皮祖细胞体外培养和鉴定

传统免疫磁珠法分选内皮祖细胞的方法操作复杂、费用大,细胞获得率较低,且内皮祖细胞的增生及分化受限。 目的：尝试改良人脐带血内皮祖细胞体外诱导分化及鉴定的方法,为实现内皮祖细胞移植、改善血管功能提供足量细胞来源。 方法：采用密度梯度离心方法获得人带血单个核细胞,体外进行诱导、分化和扩增,通过免疫组织化学、免疫荧光和流式细胞仪等技术对脐血来源的内皮祖细胞进行鉴定。 结果与结论：脐带血单个核细胞在培养过程中出现梭形贴壁和铺路石样等形态;在细胞培养至第7天,免疫组织化学显示：CD31、vWF在体外培养过程中的表达阳性;免疫荧光染色表明：(83.0±4.3)%的贴壁细胞双染呈阳性,并且DiI-acLDL标记的内皮祖细胞红色荧光在体外可以持续6周以上;第7天贴壁细胞流式细胞仪分析显示：CD34、CD133和KDR分别为(17.8±3.7)%、(22.1±4.4)%、(81.5±5.0)%。结果提示,实验成功从脐带血单个核细胞中分离培养出内皮祖细胞,在其体外可扩增并向为内皮细胞方向分化。     

噻氯匹啶与阿司匹林预防缺血性脑卒中前瞻性随机对照研究

目的 观察噻氯匹啶（ＴＩＣ）预防缺血性卒中（ＩＳ）的临床疗效及安全性,并与小剂量阿司匹林（ＡＳＡ）预防ＩＳ的疗效作对照。方法 选择３２９例ＴＩＡ与轻型ＩＳ病人,随机分为ＴＩＣ组（１６５例）与ＡＳＡ组（１６４例）。ＴＩＣ组口服ＴＩＣ ２５０ｍｇ／ｄ,ＡＳＡ组口服ＡＳＡ ５０ｍｇ／ｄ,禁服其他抗血小板药物。每例病人１～２个月随访１次,共随访６～１８个月。结果 ＴＩＣ组卒中死亡率（８．３％）低于ＡＳＡ组     

体外诱导羊膜来源间充质干细胞分化为血管内皮细胞

背景：羊膜来源间充质干细胞植入机体不同类型组织后是否可以分化为相应组织靶细胞呢？ 目的：检测血管内皮细胞生长因子在体外诱导人羊膜间充质干细胞分化为血管内皮细胞的可行性。 方法：分离培养羊膜间充质干细胞,鉴定其表面抗原表达,用含体积分数2%胎牛血清以及50 μg/L血管内皮生长因子的条件培养基诱导,诱导后细胞通过内皮细胞标志物血管内皮生长因子受体2以及v-WF染色鉴定。 结果与结论：羊膜间充质干细胞表面抗原CD29、CD44、CD105阳性,CD34、CD45、CD106以及HLA-DR阴性。诱导后细胞形态明显改变,内皮细胞标志物血管内皮细胞生长因子受体2以及v-WF染色结果阳性。提示羊膜间充质干细胞在体外具有分化为血管内皮细胞的能力。     

Factor V in human vascular endothelium and in endothelial cells in culture

Human blood vessels and human umbilical vein endothelial cells in culture were examined for the presence and synthesis of factor V. Factor V antigen was detected on the luminal surface of blood vessels washed with buffers containing calcium but was absent from segments of the same vessels perfused with buffers containing EDTA. Very low levels of endogenous factor V antigen were found in endothelial cells in culture but these cells did not synthesise factor V in sufficient quantities to be detected by the present methods. Factor V activity was not detected in any of the present cell preparations.     

Meningeal lymphatic endothelial cells fulfill scavenger endothelial cell function and cooperate with microglia in waste removal from the brain

Brain lymphatic endothelial cells (BLECs) constitute a group of loosely connected endothelial cells that reside within the meningeal layer of the zebrafish brain without forming a vascular tubular system. BLECs have been shown to readily endocytose extracellular cargo molecules from the brain parenchyma, however, their functional relevance in relation to microglia remains enigmatic. We here compare their functional uptake efficiency for several macromolecules and bacterial components with microglia in a qualitative and quantitative manner in 5-day-old zebrafish embryos. We find BLECs to be significantly more effective in the uptake of proteins, polysaccharides and virus particles as compared to microglia, while larger particles like bacteria are only ingested by microglia but not by BLECs, implying a clear distribution of tasks between the two cell types in the brain area. In addition, we compare BLECs to the recently discovered scavenger endothelial cells (SECs) of the cardinal vein and find them to accept an identical set of substrate molecules. Our data identifies BLECs as the first brain-associated SEC population in vertebrates, and demonstrates that BLECs cooperate with microglia to remove particle waste from the brain.     

Long-term effects of ticlopidine on fibrinogen and haemorheology in patients with peripheral arterial disease

The effects of ticlopidine treatment (250 mg b.i.d. for 21 months) on fibrinogen and other rheological variables, as compared to placebo, were studied in 44 patients with intermittent claudication due to peripheral arterial occlusive disease. Blood samples were collected every 3 months during this double-blind, randomised placebo-controlled trial which lasted 21 months. Consistently lower values of fibrinogen, haematocrit and whole blood viscosity at high and low shear rate levels were found in the ticlopidine group; the intergroup differences were statistically significant at most but not all follow-up examinations. A significant time-related variance was observed in the ticlopidine group for the measured variables, also after correction for the variability found in the placebo group. Thus, the observed changes in the ticlopidine group are mainly treatment related. These effects on fibrinogen and haemorheology may contribute, besides the known antiplatelet activity of the drug, to the clinical improvement reported in a larger group of claudicants to which the present subset of patients belong.     

人脐静脉内皮细胞单层通透性改变的效应因子

背景：外源性刺激引起血管屏障功能损伤的分子机制是血管病理生理学尚未阐明的热点问题之一。 目的：探讨炎症递质脂多糖诱导的人脐静脉内皮细胞单层通透性改变的效应分子,寻找有效治疗靶点。 方法：应用脂多糖刺激并观察人脐静脉内皮细胞骨架蛋白F-actin和细胞单层通透性的改变。应用荧光免疫组化和Western blot方法检测脂多糖刺激前后细胞中RhoA和SRF等信号分子的改变。并通过阻断实验证实RhoA-SRF信号通路的作用。 结果与结论：100 µg/L脂多糖刺激6 h可引起人脐静脉内皮细胞中F-actin快速重构并形成大量应力纤维,细胞单层通透性明显增强。细胞中活化RhoA的表达明显增加,SRF发生明显的入核转位现象。应用特异性分子抑制剂Y27632抑制RhoA的活化后,细胞中F-actin重构现象消失,细胞单层通透性增加也受到明显抑制,SRF蛋白发生明显的出现转位。而应用Latrunculin B抑制脂多糖刺激的人脐静脉内皮细胞中F-actin应力纤维形成,对抗通透性增加,但RhoA活化未受到干扰,SRF入核现象则受到抑制。提示RhoA-SRF通路的活化介导了脂多糖诱导的人脐静脉内皮细胞中F-actin重构和内皮单层通透性增加,特异性抑制F-actin也可以阻断脂多糖引起的血管内皮单层通透性增加,同时反馈抑制SRF的入核活化现象。     

RNAi对细胞因子信号转导抑制因子1在内皮细胞中表达的沉默作用

背景：利用RNAi技术引发细胞因子信号转导抑制因子1沉默可促进神经内分泌肿瘤细胞的凋亡,然而对于内皮细胞是否有类似的作用,目前尚不清楚。 目的：实验拟验证内皮细胞中是否有细胞因子信号转导抑制因子1表达,并在所设计的siRNA中,筛选出1对对细胞因子信号转导抑制因子1沉默效率最高的siRNA。 设计、时间及地点：随机对照,体外细胞学实验,于2007-12/2008-06在南昌大学医学院第二附属医院所属的江西省分子医学重点实验室完成。 材料：人脐静脉内皮细胞由南京凯基生物公司提供。 方法：设计并化学合成4对靶向细胞因子信号转导抑制因子1的siRNA：siRNA-1、siRNA-2、siRNA-3、siRNA-4。培养人脐静脉内皮细胞,利用RT-PCR检测人脐静脉内皮细胞是否有细胞信号转导抑制因子1表达。将带有荧光标记的阴性对照siRNA配制成25,50,75,100 nmol/L的4组,利用脂质体转染细胞,并在荧光倒置显微镜下观察转染效率,对比得出有最佳转染效率的浓度。然后,实验根据不同的处理因素分成8组：siRNA-1、siRNA-2、siRNA-3、siRNA-4和阳性对照组、阴性对照组、转染试剂组、空白对照组4个对照组。将均为有最佳转染效率浓度的各组siRNA转染细胞,48 h后提取RNA。 主要观察指标：利用RT-PCR测定细胞因子信号转导抑制因子1 mRNA的相对表达水平。 结果：人脐静脉内皮细胞高表达细胞因子信号转导抑制因子1。siRNA在50 nmol/L时有较高的转染效率。4对siRNA干扰后,细胞因子信号转导抑制因子1 mRNA的表达水平与阴性对照、转染试剂组和空白对照组相比明显下调(P < 0.05),与阳性对照组相比显著下调(P < 0.01),其中siRNA-3的沉默效率最高(P < 0.05),阴性对照、转染试剂组和空白对照组与阳性对照组相比明显下调(P < 0.05),阴性对照、转染试剂组和空白对照组之间差异无显著性意义(P > 0.05)。 结论：内皮细胞中存在细胞因子信号转导抑制因子1表达。RNAi抑制细胞因子信号转导抑制因子1在内皮细胞中的表达,实验筛选出1对沉默效率最高的siRNA。