Protective effects of timosaponin B-II on high glucose-induced apoptosis in human umbilical vein endothelial cells

This study was designed to investigate the action of timosaponin B-II, a main bioactive compound in Anemarrhena asphodeloides Bunge, on the prevention from high glucose-induced cytotoxicity and apoptosis in human umbilical vein endothelial cells (HUVECs) and the potential mechanisms involved. The results showed that compared with the normal control group, exposure of HUVECs to high glucose media for 72 h resulted in a significant increase in lactates dehydrogenise release, reactive oxygen species production, Caspase-3 activity and the percentage of apoptotic cells (p < 0.01). However, pretreatment with timosaponin B-II significantly increased the viability of HUVECs and decreased lactates dehydrogenise release, Caspase-3 activity and the apoptosis rate in a concentration-dependent manner (p < 0.05). In addition, timosaponin B-II notably decreased the amount of reactive oxygen species and malondialdehyde, as well as promoted glutathione peroxidase activity, endothelial nitric oxide synthase activity and nitric oxide release (p < 0.05). These results suggest that timosaponin B-II has the antiapoptotic effect in endothelial cells through inhibition of high glucose-induced oxidative stress and has the potential for preventing diabetic cardiovascular complications.     

等鞭金藻多肽IEC对脂多糖诱导血管内皮炎症的保护作用研究

目的 探究氨基酸序列为Ile-Ile-Ala-Val-Glu-Ala-Gly-Cys的等鞭金藻多肽IEC对LPS诱导人脐静脉内皮细胞（HUVEC）炎症反应的保护作用。方法 用噻唑蓝（MTT）法确定细胞活力,DCFH-DA探针法和DAF-FM DA探针法分别测定活性氧（ROS）和NO的表达情况,蛋白免疫印迹法检测细胞间Toll样受体（TLR4）、一氧化氮合酶（NOS-2）和环氧合酶（COX-2）蛋白的表达水平,酶联免疫吸附法测定白细胞介素-6 （IL-6）和肿瘤坏死因子（TNF-α）的释放量以及分子对接分析IEC与COX-2、TLR-4蛋白的相互作用。结果 MTT法证明IEC对HUVEC细胞无明显毒性作用(P>0.05);与对照组相比,随着实验组多肽浓度增加,可以明显降低ROS和NO的释放(P<0.05),炎症相关细胞因子TLR-4、COX-2、NOS-2和IL-6、TNF-α的表达也明显逐渐减少(P<0.000 1);分子对接结果表明,IEC与COX-2、TLR4能形成稳定的化学键,说明存在相互作用的可能性。结论 IEC具有良好的抑制ROS和NO生成效果,并可明显抑制TLR-4/NOS-2/COX-2信号通路的激活以及抑制炎症因子的表达,是潜在的炎症抑制剂。     

Arsenic trioxide induces procoagulant activity through phosphatidylserine exposure and microparticle generation in endothelial cells

Background

Coagulopathy is a major cause of early death when arsenic trioxide (As₂O₃) therapy fails. In addition to the procoagulant properties of blast cells, the cytotoxic therapy may contribute to the coagulation disorders. The aim of the present study was to evaluate the possible impact of As₂O₃ on membrane alterations, including phosphatidylserine (PS) exposure and microparticle generation, and the consequent procoagulant properties of endothelial cells.

Methods

Procoagulant activity (PCA) of human umbilical vein endothelial cells (HUVECs) was assessed by measuring clotting time and through purified coagulation complex assays. PS exposure on HUVEC membrane was observed by confocal microscopy and quantified with flow cytometry. In addition, counts and PCA of endothelial microparticles were determined by flow cytometry and plasma coagulation assay.

Results

As₂O₃ increased the ability of HUVECs to accelerate coagulation process and promote formation of coagulation complexes. Procoagulant activity corresponded to PS exposed on HUVECs. In coincidence with the PS externalization, As₂O₃ increased the production of PS-bearing microparticles, which then accelerated fibrin strand formation significantly. By blocking PS, lactadherin was able to inhibit over 90% of the intrinsic tenase/prothrombinase activity of As₂O₃-treated HUVECs, and restored coagulation times of As₂O₃-treated cells and microparticles to control levels.

Conclusions

As₂O₃ increases PCA of HUVECs through PS exposure and PS-bearing microparticle generation, which might cause thrombosis and act as a contributing factor in As₂O₃ therapy-related coagulopathy.     

pSilencer1.0-U6-Ang2/Tie2-siRNA重组质粒的构建及其在体外人脐静脉内皮细胞中抑制Ang2.Tie2基因的表达

目的 探讨应用RNA干扰(RNAi)技术抑制Ang2、Tie2基因的表达情况,为进一步研究其在体外抑制血管生成以及抑制肿瘤血管生成的动物实验研究奠定基础.方法 构建pSilencer 1.0-U6-Ang2/Tie2-siRNA重组质粒各2条并予以鉴定.用pSilencer 1.0-U6-Ang2/Tie2-siRNA重组质粒转染人脐静脉内皮细胞(HUVECs),采用免疫细胞化学染色和RT-PCR方法检测各组HUVECs中Ang2及Tie2的蛋白及mRNA的表达状况.结果 成功构建pSilencer 1.0-U6-Ang2/Tie2-siRNA重组质粒各2条,用其转染HUVECs后,免疫细胞化学染色和RT-PCR检测结果显示,Ang2、Tie2在蛋白和mRNA的表达水平均受到明显抑制(P＜0.05),并且siRNA的2条重组质粒之间均差异无统计学意义(P＞0.05).结论 pSilencer 1.0-u6-Ang2/Tie2-siRNA在体外能够抑制HUVECs的Ang2和Tie2的蛋白及mRNA的表达.     

Innate response of human endothelial cells infected with mycobacteria

Endothelial cells are susceptible to infection by several pathogens, but little is known about mycobacterial infection. We analyzed some features of mycobacteria-endothelial cell interactions and the innate response to the infection. Intracellular growth in human umbilical vein endothelial cells (HUVECs) of three Mycobacterium species: M. tuberculosis (MTB), M. abscessus (MAB) and M. smegmatis (MSM) was analyzed. M. smegmatis was eliminated; M. abscessus had an accelerate intracellular replication and M. tuberculosis did not replicate or was eliminated. M. abscessus infection induced profound cytoskeleton rearrangements, with M. tuberculosis infection changes were less marked, and with MSM were slight. Nitric oxide (NO) production was induced differentially: M. abscessus induced the highest levels followed by M. tuberculosis and M. smegmatis; the contrary was true for reactive oxygen species (ROS) production. Only M. tuberculosis infection caused beta-1 defensin over-expression. As a whole, our results describe some aspects of the innate response of HUVEC infected by mycobacteria with different virulence and suggest that a strong cytoskeleton mobilization triggers a high NO production in these cells.     

洛铂和顺铂对血管内皮细胞及肝、肾细胞损伤的体外实验研究

目的分析洛铂(LBP)和顺铂(DDP)对传代培养的人脐静脉内皮细胞株(HUVECs)、人肝细胞株(QSG-7701)和人近端肾小管上皮细胞(HK-2细胞)的抑制作用,并初步探讨其可能机制。方法采用MTT法检测LBP和DDP对HUVECs、QSG-7701和HK-2细胞抑制作用的差异,并测定HK-2细胞培养液中丙二醛(MDA)和超氧化物歧化酶(SOD)含量,观察LBP和DDP对HK-2细胞脂质过氧化过程的影响。结果在相同浓度、相同作用时间下,DDP组对HK-2细胞抑制率明显高于LBP组(P<0.05),而LBP组对HUVECs细胞的抑制率明显高于DDP组(P<0.05);LBP组和DDP组对QSG-7701抑制率的差异无统计学意义(P>0.05);此外,与空白对照组比较,LBP组和DDP组均使HK-2细胞培养液中MDA含量明显升高,SOD活性明显降低(P均<0.05),但LBP组和DDP组间差异无统计学意义(P>0.05)。结论 LBP和DDP均对肝、肾细胞有较强的抑制作用,LBP的肾细胞毒性较DDP低,LBP组对正常人脐静脉内皮细胞抑制作用明显强于DDP组,提示LBP可能具有更强的抑制血管形成的能力;氧化性损伤可能是两者造成肾毒性的机制。     

Estradiol pretreatment attenuated nicotine-induced endothelial cell apoptosis via estradiol functional membrane receptor

Cigarette smoking is highly associated with increased cardiovascular disease complications. The female population, however, manifests reduced cardiovascular morbidity. We define nicotine's effect upon human umbilical vein endothelial cells (HUVECs), determine whether estradiol might ameliorate endothelial dysfunction via its membrane estrogen receptor (mER), and attempt to elucidate the underlying mechanisms. Endothelial cells were pretreated with estradiol-BSA and measured resultant ion flux across the cells via the patch clamp technique to assess mER is functionality. Estradiol-BSA administration was associated with 30% decreased nicotine-induced apoptosis and also attenuated nicotine-activated phosphorylation of p38 and ERK. Pretreatment of estradiol-BSA triggered a low calcium influx, suggesting ahead low influx calcium played a critical role in the underlying protective mechanisms of estradiol. Furthermore, this estradiol-BSA protection against apoptosis remained effective in the presence of tamoxifen, an intracellular estrogen receptor (iER) inhibitor. Additionally, tamoxifen did not abolish estradiol-BSA's inhibitory effect upon p38 and ERK's activation, giving evidence to the obligatory role of p38 and ERK signaling in the estradiol-BSA's anti-apoptotic action via mER. Our study provides evidence that nicotine enhances endothelial cell apoptosis, but estrogen exerts anti-apoptotic effect through its functional membrane estrogen receptor. Clinically, the nicotine in cigarettes might contribute to endothelial dysfunction, whereas ambient estradiol may provide cellular protection against nicotine-induced injury through its functional membrane receptor via MAPK pathway downregulation.     

10-Hydroxy-2-decenoic acid, a major fatty acid from royal jelly, inhibits VEGF-induced angiogenesis in human umbilical vein endothelial cells

Vascular endothelial growth factor (VEGF) is reported to be a potent pro-angiogenic factor that plays a pivotal role in both physiological and pathological angiogenesis. Royal jelly (RJ) is a honeybee product containing various proteins, sugars, lipids, vitamins and free amino acids. 10-Hydroxy-2-decenoic acid (10HDA), a major fatty acid component of RJ, is known to have various pharmacological effects; its antitumor activity being especially noteworthy. However, the mechanism underlying this effect is unclear. We examined the effect of 10HDA on VEGF-induced proliferation, migration and tube formation in human umbilical vein endothelial cells (HUVECs). Our findings showed that, 10HDA at 20 microM or more significantly inhibited such proliferation, migration and tube formation. Similarly, 10 microM GM6001, a matrix metalloprotease inhibitor, prevented VEGF-induced migration and tube formation. These findings indicate that 10HDA exerts an inhibitory effect on VEGF-induced angiogenesis, partly by inhibiting both cell proliferation and migration. Further experiments will be needed to clarify the detailed mechanism.     

Altered detection of molecules associated with leukocyte traffic in HUVECs derived from newborns with a strong family history of myocardial infarction

Atherosclerosis is a chronic inflammatory disease. As such, recruitment of immune cells is a significant event. Tightly controlled signaling molecules regulate leukocyte adhesion and migration to the tissues. The aim of this study was to determine if human umbilical vein endothelial cells (HUVECs) derived from healthy newborns with a strong family history of myocardial infarction (FHMI) showed variations in the presence of molecules related with leukocyte traffic and migration, in comparison to control healthy newborns. For this purpose, we evaluated the labeling of sialic acid containing glycoproteins, tight junction claudins and the cytoskeleton, using lectin- and immunocytochemistry in HUVECs from individuals with and without a strong FHMI. Our results show important differences in the labeling of alpha-2,3 or alpha-2,6 sialic acid-containing glycoconjugates, a disarrangement of actin filaments secondary to the absence of cytoplasmic claudin-5 immunopositivity and an increase in the binding of FHMI HUVECs to CD3+ Jurkat cells. It is possible that these differences relate to a predisposition for early appearance of atherosclerotic lesions.