NOX2 and NOX4 mediate proliferative response in endothelial cells

Increased levels of reactive oxygen species (ROS) contribute to many cardiovascular diseases. In neutrophils, ROS are generated by a NADPH oxidase containing p22phox and NOX2. NADPH oxidases are also major sources of vascular ROS. Whereas an active NOX2-containing enzyme has been described in endothelial cells, the contribution of recently identified NOX homologues to endothelial ROS production and proliferation has been controversial. The authors, therefore, compared the role of NOX2 with NOX4 and NOX1 in endothelial EaHy926 and human microvascular endothelial cells. NOX2 and NOX4 were abundantly expressed, whereas NOX1 expression was less prominent. NOX2, NOX4, and NOX1 were simultaneously present in a single cell in a perinuclear compartment. NOX2 and NOX4 co-localized with the endoplasmic reticulum (ER) marker calreticulin. Additionally, NOX2 co-localized with F-actin at the plasma membrane. NOX2 and NOX4, which interacted with p22phox, as was shown by bimolecular fluorescent complementation, contributed equally to endothelial ROS production and proliferation, whereas NOX1 depletion did not alter ROS levels under basal conditions. These data show that endothelial cells simultaneously express NOX2, NOX4, and NOX1. NOX2 and NOX4, but not NOX1, equally contributed to ROS generation and proliferation under basal conditions, indicating that a complex relation between NOX homologues controls endothelial function.     

Intracellular localization of natural and modified oligonucleotides in primary human endothelial cells

Intracellular localization of natural and fluorescent-labeled oligonucleotides in primary human endothelial cells was studied by means of fluorescence microscopy and radioisotope analysis. Transport and distribution of oligonucleotides in endotheliocytes depended on their structure and resistance to hydrolysis under the effect of cell nucleases. Modification of 5′-terminal phosphate and 3′-terminal oligonucleotide increased the stability and ensures nuclear localization of oligonucleotides in cells. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 2, pp. 163–165, February, 2007     

NK4基因在人胰腺癌细胞中的表达及其对血管内皮生长的影响

目的:构建NK4基因真核表达载体并进行转染研究。方法:对重组质粒pcDNA3/hNK4进行酶切,将目的基因NK4克隆到较强的真核表达载体pRC/CMV2中,应用脂质体将NK4基因转染到胰腺癌细胞株SW1990中,G418筛选获得稳定表达克隆,采用RT-PCR及Western blot鉴定及筛选出高转录和高表达的细胞克隆。以MTT法检测转染重组质粒pRC/CMV2-hNK4的细胞克隆表达产物对血管内皮细胞生长的影响。结果:在转染NK4基因的SW1990细胞克隆中,RT-PCR能扩增出NK4 mRNA预期的453bp片段,但强弱不同,Western blot显示均有约50 kD的NK4蛋白的表达。其上清可显著抑制血管内皮细胞的生长。结论:重组质粒pRC／CMV2-hNK4是一种高效真核表达载体,并且能够在SW1990细胞中高表达;NK4能够抑制血管内皮细胞的生长,提示其在肿瘤基因治疗中可能具有重要意义。     

Intracellular localization of Borrelia burgdorferi within human endothelial cells.

The later stages of infection by the Lyme disease pathogen, Borrelia burgdorferi, are characterized by the persistence of the organism in individuals possessing a strong anti-Borrelia immune response. This suggests that the organism is sequestered in a tissue protected from the immune system of the host or there is a reservoir of the organism residing within the cells of the host. In this report, the ability of B. burgdorferi to gain entrance into human umbilical vein endothelial cells was explored as a model for invasion. Incubation of B. burgdorferi with human umbilical vein endothelial cells at ratios ranging from 200:1 to 5,000:1 resulted in the intracellular localization of 10 to 25% of B. burgdorferi in 24 h. The intracellular location of the spirochetes was demonstrated by the incorporation of radiolabeled B. burgdorferi into a trypsin-resistant compartment and was confirmed by double-immunofluorescence staining which differentiated intracellular from extracellular organisms. Actin-containing microfilaments were required for the intracellular localization, indicating that the host cell participates in the internalization process. Activation of endothelial cells by agents known to increase the expression of several adhesion molecules had no effect on the interaction of B. burgdorferi with the endothelial monolayer. This indicates that the endothelial receptor for B. burgdorferi is constitutively expressed and that internalization is not dependent upon adhesion molecules whose expression is induced by inflammatory mediators. The demonstration of B. burgdorferi within endothelial cells suggest that intracellular localization may be a potential mechanism by which the organism escapes from the immune response of the host and may contribute to persistence of the organism during the later stages of Lyme disease.     

CD 4-positive lymphoid cells rescue HIV-1 replication from abortively infected human primary endothelial cells

Summary Human primary endothelial cell cultures, derived from umbilical vein (HUVEC), can be infected by different strains of HIV-1, but mature virus production remains undetectable both in supernatants and in cellular extracts. Yet viral DNA is transiently detectable during the first days of infection, but progressively declines during the subsequent days. This finding is characteristic of abortive infections. Co-culture of HUVEC carrying HIV DNA with activated peripheral blood mononuclear cells or with CD 4-positive lymphoid cells elicited a massive cpe (syncytia formation and cell degeneration) in the latter cells, caused by the establishment of productive HIV-1 infection. HUVEC infected in the presence of AZT were significantly impaired in the ability to transmit the infection of CD 4-positive cells, indicating that active DNA synthesis is required in HUVEC before rescue by CD 4-positive cells.These results are of interest in view of the possibility that endothelial cells can play a role in the transmission of HIV-1 infection from infected pregnant women to the foetuses, and, more generally, suggest a potential role of endothelial cells as a transient reservoir of HIV-1.     

登革病毒域型引起原代HDMECs 通透性变化机制的初步研究

目的:了解登革病毒域型(Dengue virus Type 2,DENV-2)感染原代人真皮微血管内皮细胞(Primary Human dermal micro-vascular endothelial cells,pHDMECs)引起细胞通透性改变的机制。方法:用103 TCID50 的DENV-2 感染pHDMECs;于4、8、12、24、48 h Real time-PCR、免疫荧光法及流式细胞术检测DENV-2 NS1 部分序列及蛋白;Transwell 法检测细胞通透性;Real time -PCR 和双抗体夹心ELISA 法检测IL-6 和IL-8 的变化;流式细胞术检测24、48、72 h 细胞凋亡。结果:DENV-2 感染的pHDMECs 病毒NS1 基因相对表达上调,但未检测到病毒NS1 蛋白;DENV-2 感染的pHDMECs 通透性在24、48 h 显著升高;pHDMECs 被感染72 h 后凋亡也无明显变化; IL-6 和IL-8 mRNA 分别在8、24 h 相对表达上调[IL-6:(2.490.5)倍,P<0.05;IL-8:(6.82±1.69)倍,P<0.05];对照组和感染组分泌的IL-6 于8 h 分别为(869.6±50.7)、(1 248.8±86.9)pg/ ml,P<0.05;IL-8 于48 h 分别为(967.6±156.6)、(1 331.0±86.3)pg/ ml,P<0.05。结论:DENV-2 能感pHDMECs;pHDMECs 被DENV-2 感染后,细胞的通透性增加与凋亡无关,与促炎性细胞因子IL-6 和IL-8 明显上调关系密切。     

骨肉瘤细胞中ArgBP2的表达和定位

目的:构建真核表达载体GFP-hArgBP2并检测在骨肉瘤细胞内的表达及定位。方法:以pcDNA3.1-hArgBP2为模板,利用PCR扩增hArgBP2基因cDNA全长,并将其克隆至真核表达载体pEGFP-C1中。进一步将构建的重组质粒进行酶切和测序鉴定,并转染到骨肉瘤细胞MG-63中,提取细胞蛋白进行Western blot检测。同时利用共聚焦激光扫描显微镜观察GFP-hArgBP2在MG-63细胞中的定位,免疫沉淀的方法纯化hArgBP2蛋白。结果:hArgBP2基因cDNA全长成功构建到真核表达载体pEGFP-C1中,Western blot检测到了GFP-hArgBP2融合蛋白表达,相对分子质量(Mr)约为97 000。GFP-hArgBP2在骨肉瘤细胞MG-63中主要定位于细胞质和核周,并成功纯化hArgBP2蛋白。结论:成功地构建了GFP-hArgBP2真核表达质粒,同时鉴定了GFP-hArgBP2融合蛋白的表达,并纯化hArgBP2蛋白。GFP-hArgBP2蛋白主要定位在细胞质和核周。     

Interferon production by a primary culture of human endothelial cells

N. F. Gamaleya Research Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR. All-Union Cardiologic Scienctific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academican of the Academy of Medical Sciences of the USSR P. A. Vershilova.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 108, No. 11, pp. 580–583, November 1989.     

骨肉瘤细胞中Plk1的表达和定位

目的构建真核表达载体GFP-hPlk1并检测在骨肉瘤细胞内的表达及定位。方法以pcDNA3.1-hPlk1为模板,利用聚合酶链反应扩增hPlk1基因的cDNA全长,并将其克隆至pEGFP-C1真核表达载体中。进一步将构建的重组质粒进行酶切和测序鉴定,并转染到U2OS骨肉瘤细胞中,提取细胞总蛋白进行Western blot检测。同时利用激光共聚焦扫描显微镜观察GFP-hPlk1在U2OS细胞中的定位,免疫沉淀的方法纯化hPlk1蛋白。结果 hPlk1基因cDNA全长成功构建到真核表达载体pEGFP-C1中,Western blot检测到了GFP-hPlk1融合蛋白表达,分子质量Mr约为93 000Da。GFP-hPlk1在骨肉瘤细胞U2OS中主要定位于细胞质和核周,并成功纯化hPlk1蛋白。结论成功地构建了GFP-hPlk1真核表达质粒,并在骨肉瘤细胞U2OS中表达。     

Atherosclerosis- and age-related multinucleated variant endothelial cells in primary culture from human aorta

Endothelial cells were cultured from human aortas and inferior venae cavae of autopsied subjects ranging in age from infancy to 85 years. Endothelial cells in 32 of more than 100 attempted cultures were pure enough for evaluation. Emerged endothelial cells in primary culture were classified into two types: typical endothelium and variant endothelium. Typical endothelial cells were small, round to polygonal shaped, and were arranged uniformly. Their diameter ranged from 50 to 70 microns. Variant endothelial cells were larger, ranging from 100 to 200 microns in diameter, and giant endothelial cells measuring more than 250 microns in diameter were scattered among them. Variant endothelial cells were usually multinucleated and possessed endothelium-specific markers of vWF and Weibel-Palade bodies. No incorporation of [3H]thymidine was found in the nuclei of cultured variant endothelial cells. Although most cultured endothelial cells were of the typical type, variant endothelial cells were interspersed throughout the culture. The ratio of variant endothelial cells to typical cells correlated well with the severity of atherosclerosis, but less so with aging. The number of variant endothelial cells in cultures from inferior venae cavae was slight and constant throughout all age groups. The presence of multinucleated endothelial cells in in vivo aortas was confirmed by both scanning and transmission electron microscopy. They sometimes existed in colonies in the aortas from elderly subjects with intimal-thickened or advanced atherosclerotic lesions. These results indicate that variant endothelial cells were present in vivo and their ratio in primary culture reflected the in vivo population. It is likely that these cells were formed by adhesion of adjacent typical endothelial cells and that this process was affected more by atherosclerosis than by aging. Although it is not clear if the multinucleated variant cells were formed before the formation of atherosclerotic plaque or after the plaque formation, they will contribute to further development of atherosclerotic lesions, which in turn cause malfunction of the cell membrane. We suggest that there is a cyclic effect of these processes for multiplication of the variant endothelial cells and advancement of atherosclerotic lesions.     

TLR2和TLR4在人慢性根尖周病组织肥大细胞上的表达

 目的: 观察人不同类型慢性根尖周病组织中肥大细胞(mast cells,MCs)上Toll样受体2(Toll-like receptor 2,TLR2)和TLR4的表达情况,探讨TLR2-类胰蛋白酶(tryptase)和TLR4-tryptase双阳性MCs在慢性根尖周疾病发病机制中的作用。方法: 将60例自愿参与本研究的受试者按根尖周病分类标准分为3组:(1)正常对照组;(2)根尖周肉芽肿组;(3)根尖周囊肿组。将根尖周组织标本置于4%甲醛固定液中浸泡48 h以上,制作连续组织切片。HE染色,光学显微镜下观察各组根尖周标本的组织学变化;免疫荧光双染色后于荧光显微镜下观察TLR2-tryptase和TLR4-tryptase双阳性MCs在根尖周组织中的分布情况。结果: 根尖周病变组TLR2-tryptase和TLR4-tryptase双阳性MCs比正常牙周膜组明显增多(P<0.01);与根尖肉芽肿组相比,根尖囊肿组TLR2-tryptase及TLR4-tryptase双阳性MCs数目明显增高(P<0.01)。结论: TLR2及TLR4在慢性根尖周疾病组织中MCs上表达增加,提示TLR2-tryptase及TLR4-tryptase双阳性MCs可能参与慢性根尖周病发病过程的免疫调节。     

Lovastatin stimulates p75 TNF receptor (TNFR2) expression in primary human endothelial cells

HMG-CoA reductase inhibitors (statins) exert pleiotropic physiological effects. Among others they attenuate cellular responses to genotoxic and inflammatory stress. We investigated the effect of lovastatin on the expression level of TNF receptors (TNFR) in primary human endothelial cells (HUVEC). ELISA, FACS and immunocytochemical analyses show that lovastatin selectively increases the cell surface expression of TNFR2 without affecting the expression level of TNFR1. This effect of lovastatin is independent from inhibition of cell-cycle progression since cells both in G1- and G2-phase showed elevated levels of TNFR2 after lovastatin treatment. To analyze the physiological relevance of lovastatin-mediated upregulation of TNFR2, we investigated the expression of the cell adhesion molecule E-selectin, which is inducible by TNFalpha. While lovastatin on its own did not change the number of HUVEC expressing E-selectin protein, it promoted the TNFalpha-stimulated increase in the percentage of E-selectin expressing endothelial cells in a dose-dependent manner. This indicates that lovastatin sensitizes HUVEC towards TNFalpha-induced signaling by upregulation of TNFR2 expression. Based on the data, we suggest that statins have impact on endothelial responses to inflammatory stress by modulation of the expression of cytokine receptors.     

4-羟基壬烯酸诱导人主动脉内皮细胞和心肌细胞凋亡

我们应用4-羟基壬烯酸(HNE)诱导培养的人主动脉内皮细胞和心肌细胞的氧化应激,观察不同浓度的HNE促使培养的主动脉内皮细胞和心肌细胞凋亡的程度。1材料与方法1·1人主动脉内皮细胞(HAOEC)和心肌细胞的培养:人主动脉内皮细胞(购自美国细胞应用公司CA 92121)和人心肌细胞(购自ATCC公司CRL-1446)培养于改良Eagle’s培养基中(DMEM培养基),37℃条件下进行原代和传代培养。所有的实验在2周内完成。1·2在培养的细胞中加入HNE:在加入HNE(Calb iochem公司)之前,先用含有钙、镁离子和葡萄糖的磷酸盐缓冲液洗去细胞培养液,然后分别加入1…     

Ebola virus glycoprotein-mediated anoikis of primary human cardiac microvascular endothelial cells

Ebola virus glycoprotein (EGP) has been implicated for the induction of cytotoxicity and injury in vascular cells. On the other hand, EGP has also been suggested to induce massive cell rounding and detachment from the plastic surface by downregulating cell adhesion molecules without causing cytotoxicity. In this study, we have examined the cytotoxic role of EGP in primary endothelial cells by transduction with a replication-deficient recombinant adenovirus expressing EGP (Ad-EGP). Primary human cardiac microvascular endothelial cells (HCMECs) transduced with Ad-EGP displayed loss of cell adhesion from the plastic surface followed by cell death. Transfer of conditioned medium from EGP-transduced HCMEC into naive cells did not induce loss of adhesion or cell death, suggesting that EGP needs to be expressed intracellularly to exert its cytotoxic effect. Subsequent studies suggested that HCMEC death occurred through apoptosis. Results from this study shed light on the EGP-induced anoikis in primary human cardiac endothelial cells, which may have significant pathological consequences.     

Cytocompatibility study of NHLBI primary reference materials using human endothelial cells

Four different materials, low density polyethylene (LDPE), polydimethylsiloxane (PDMS), polyvinylcholoride (PVC) and cellulose, were selected by the Devices and Technology Branch of the National Heart Lung and Blood Institute (NHLBI) as primary reference materials for blood contacting. Among the wide variety of tests proposed to assess hemocompatibility of short-term blood contacting catheters, it was desirable to rule out whether these materials could release toxics for vascular cells of the physiological environment. Thus, the cytocompatibility of these materials have been checked towards human umbilical vein endothelial cells: the method used avoids direct contact between cells and materials but evaluates the effect of possible toxic substances leached from materials. These substances were obtained under defined conditions according to a standard. The results show that the extracts of cellulose and LDPE provoke an important cytotoxic effect on the endothelial cell cultures, while the extracts of PDMS and PVC allow the obtention of endothelial cell lining of the reference surface, with a correct global metabolic activity and the intracellular presence of von Willebrand factor.