右旋糖酐40,70对红细胞与内与细胞粘附的影响

采用流室显微观察系统，定量研究右旋糖酐４０（ＤＸ４０）和右旋糖酐７０对红细胞与内皮细胞动态粘附特性的影响。统计分析处理数据，到得反映应力与细胞间动态粘附数关系的经验权式及粘附特征常数ａ，ｂ值。     

右旋糖酐40、70对红细胞与内皮细胞粘附的影响

采用流室显微观察系统，定量研究右旋糖酐４０（ＤＸ４０）和右旋糖酐７０（ＤＸ７０）对红细胞与内皮细胞动态粘附特性的影响。统计分析处理数据，得到反映切应力与细胞间动态粘附数关系的经验公式及粘附特征常数ａ，ｂ值。ａ值反映红细胞的初始粘附数，ｂ值反映随切应力增大，粘附数减小的速率。ＤＸ７０实验组的ａ值大于ＤＸ４０组的，而ｂ值的情况相反。故结果显示切应力越大，粘附的红细胞越少；ＤＸ４０使红细胞的粘附明显减少；ＤＸ７０使红细胞粘附显著增加。由此提示不同长度的细胞桥接分子对红细胞的粘附影响不同，且临床上选用ＤＸ４０作血浆扩容剂较ＤＸ７０优越得多     

In vitro and in situ intercellular adhesion molecule-1 (ICAM-1) expression by endothelial cells lining a polyester fabric

In order to improve long-term patency of vascular grafts, the promising concept of endothelial cell seeding is actually under investigation. Our laboratory tested a polyester coated with albumin and chitosan which permits a rapid colonization by human umbilical vein endothelial cells (HUVEC) and it seems relevant to test in vitro the expression of adhesive molecules expressed by cells with regard to the inflammatory process. We studied intercellular adhesion molecule-1 (ICAM-1) expression and focused our work on the determination of ICAM-1 sites expressed per adherent cell lining the biomaterial, thus in situ, in comparison to control HUVEC on plastic wells: the results obtained by binding experiments were correlated to flow cytometry analyses and showed that the polyester does not induce a proinflammatory state and that HUVEC covering the structure are able to respond to a stimulus.     

Quantitation of polymorphonuclear leukocyte adherence to endothelial cells by electronic particle size discrimination

A method for quantitating the number of polymorphonuclear leukocytes (PMNs) adhering to endothelial cells in vitro is presented. Confluent human umbilical vein endothelial cells cultured in 24-well multiplates and treated with tumor necrosis factor (TNF ) were incubated subsequently wioth PMNs which adhere to the endothelial cell as a function of TNF concentration. Adherent PMNs and endothelial cells were proteolytically dissociated from the multiwell and, using an electronic particle counter, the number of endothelial cells and PMNs were determined simultaneously on the basis of size. The average number of PMNs adhering per endothelial cell was then calculated. The method is rapid and precise and offers an alternative to both tedious microscopic counting and the hazardous radiolabeling of PMNs.     

血管内皮细胞的活化有利于造血干细胞的归巢

目的:探讨影响造血干细胞归巢的因素,为提高造血干细胞移植的效率奠定基础。方法:传代培养的血管内皮细胞长成单层后,用血管内皮细胞生长因子(VEGF)、粒细胞集落刺激因子(G-CSF)和脂多糖(LPS)激活内皮细胞,再将经过去除红细胞、粒细胞、单核细胞和T淋巴细胞而富集的脐血造血干细胞与内皮细胞共同培养,使之发生粘附。用双抗体夹心ELISA法测定C-Kit⁺的造血干细胞与血管内皮细胞的粘附情况。并比较VCAM-1单克隆抗体封闭血管内皮细胞对造血干细胞粘附的影响。结果:静息状态的血管内皮细胞能够与C-Kit⁺的造血干细胞发生粘附,但粘附率较低。用VEGF、G-CSF和LPS激活血管内皮细胞后,造血干细胞的粘附显著增加。用粘附分子VCAM-1单克隆抗体预处理激活血管内皮细胞后,造血干细胞的粘附明显下降。结论:激活的血管内皮细胞明显增加对造血干细胞的粘附,这种粘附与粘附分子的作用有关。     

CD226分子参与内皮细胞黏附功能的形态学证据

目的：提供CD226分子参与内皮细胞黏附功能的形态学证据。方法：体外培养人脐静脉内皮细胞，加入融合蛋白CD226—Ig，采用倒置显微镜和扫描电子显微镜观测CD226分子在活化内皮细胞和活化PBMC黏附过程中的作用。结果：融合蛋白CD226／Ig可显著降低活化内皮细胞与活化PBMC间的黏附。结论：CD226分子是活化内皮细胞表面一种新的黏附分子，可能具有重要的生理和病理功能。     

VEGF-subtype specific protection of SCC and HUVECs from radiation induced cell death

The contribution of VEGF (vascular endothelial growth factor) to angiogenesis and tumor aggressiveness has been shown in several tumor types, but no comparative data regarding the impact of the VEGF-subtypes on endothelial and tumor cell protection are available. Therefore, we analysed the cytoprotective effects of the two major soluble VEGF-subtypes, VEGF-121 and -165, on HUVEC cell cultures (human umbilical vein endothelial cells) and squamous cell carcinoma (SCC) cell lines after ionizing radiation. We performed clonogenic analyses and proliferation assays for evaluation of cell survival and proliferative activity. Experiments were performed employing different intensities up to 8 Gy with or without added recombinant VEGF-121 or -165 and compared to non-irradiated cultures. To evaluate a possible contribution of the VEGF-receptors, we performed immunohistochemical stainings of VEGF-R1 (flt), -R2 (KDR,flk), and Neuropilin-1. In the SCC cell lines and HUVEC cultures, cell survival and proliferative activity were reduced in a dosage-dependent manner. The addition of VEGF-121 yielded a significant increase of resistance from 1.2 to 2.7 Gy (+125%) for killing 50% of subjected cells, while VEGF-165 was less effective in this regard (+83%). Conversely, in HUVEC cultures, 50%-survival was increased more strongly by VEGF-165 compared to VEGF-121 (+100% vs. +43%). Accordingly, proliferation was more intensely stimulated in HUVECs by VEGF-165 than by VEGF-121. VEGF-receptors were expressed in all cell cultures analysed at comparable levels. We conclude that the two VEGF-subtypes differentially increase the survival of tumor and endothelial cells after irradiation in vitro. We hypothesise, that the release of VEGF by the tumor protects tumor cells and endothelium resulting in increased radiation resistance.     

A preliminary investigation of cell growth after irradiation using a modulated x-ray intensity pattern

In this study we have investigated a spatial distribution of cell growth after their irradiation using a modulated x-ray intensity pattern. An A549 human non-small cell lung cancer cell line was grown in a 6-well culture. Two of the wells were the unirradiated control wells, whilst another two wells were irradiated with a modulated x-ray intensity pattern and the third two wells were uniformly irradiated. A number of plates were incubated for various times after irradiation and stained with crystal violet. The spatial distribution of the stained cells within each well was determined by measurement of the crystal violet optical density at multiple positions in the plate using a microplate photospectrometer. The crystal violet optical density for a range of cell densities was measured for the unirradiated well and this correlated with cell viability as determined by the MTT cell viability assay. An exponential dose response curve was measured for A549 cells from the average crystal violet optical density in the uniformly irradiated well up to a dose of 30 Gy. By measuring the crystal violet optical density distribution within a well the spatial distribution of cell growth after irradiation with a modulated x-ray intensity pattern can be plotted. This method can be used for in vitro investigation into the changes in radiation response associated with treatment using intensity modulated radiation therapy (IMRT).     

Effect of bacterial peptidoglycan on erythrocyte death and adhesion to endothelial cells

Peptidoglycans, bacterial wall components, have previously been shown to trigger eryptosis, the suicidal erythrocyte death, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Phosphatidylserine exposing erythrocytes adhere to the vascular wall at least partially by interaction of erythrocytic phosphatidylserine with endothelial CXC chemokine ligand 16 (CXCL16). The present study explored whether peptidoglycan exposure fosters the adhesion of erythrocytes to human umbilical vein endothelial cells (HUVEC). To this end, HUVEC were treated for 48 h with peptidoglycan (10 μg/ml) and CXCL16 abundance determined by confocal microscopy and FACS analysis. Moreover, human erythrocytes were exposed for 48 h to peptidoglycan (10 μg/ml) and phosphatidylserine exposure estimated from binding of fluorescent annexin-V, cell volume from forward scatter in FACS analysis and erythrocyte adhesion to human umbilical vein endothelial cells (HUVEC) from trapping of labeled erythrocytes in a flow chamber. As a result, bacterial peptidoglycan exposure was followed by increased CXCL16 expression in HUVEC as well as erythrocyte shrinkage, phosphatidylserine exposure and adhesion to HUVEC under flow conditions at arterial shear rates. The adhesion was significantly attenuated but not abrogated in the presence of either, erythrocyte phosphatidylserine-coating annexin-V (5 μl/ml) or CXCL16 neutralizing antibody directed against endothelial CXCL16 (4 μg/ml). In conclusion, exposure to peptidoglycan increases endothelial CXCL16 expression and leads to eryptosis followed by phosphatidylserine- and CXCL16-mediated adhesion of eryptotic erythrocytes to vascular endothelial cells.     

A novelin vitro assay system for transendothelial tumor cell invasion: significance of E-selectin and α3 integrin in the transendothelial invasion by HT1080 fibrosarcoma cells

The interaction of tumor cells with endothelial cells is a key event in tumor metastasis. We established anin vitro invasion assay system, in which the invasion of tumor cells after interaction with endothelial cells can be examined. Two chamber culture wells separated by porous membrane were used. Human umbilical vein endothelial cells (HUVEC) were placed on porous membranes coated with matrix components. The invasion by HT1080 fibrosarcoma cells was determined in this system by counting the number of cells that moved through the membranes from upper to lower chambers. HUVEC cells did not migrate through the membranes as judged by the staining with UEA-I. Observation by scanning electron microscopy revealed that HT1080 cells bound to HUVEC surfaces and migrated underneath the HUVEC monolayer. Effects of antibodies specific for cell surface adhesion molecules on the migration of HT1080 cells were examined. Invasion of uncoated membranes and membranes coated with HUVEC cells was compared. Antibody against E-selectin significantly suppressed an increase of HT1080 cell invasion of HUVEC monolayers stimulated by IL-1 or TNF. Antibody against integrin ₃ subunit remarkably inhibited the invasion of HUVEC cell-coated membranes, suggesting that integrins with the ₃ subunit may play an important role in the transendothelial invasion by HT1080 cells.     

Chemokine- and adhesion-dependent survival of neutrophils after transmigration through cytokine-stimulated endothelium

We examined the fate of neutrophils following transmigration through an endothelial monolayer cultured on "Transwell" membrane filters. Treatment of human umbilical vein endothelial cells (HUVEC) with increasing doses of tumor necrosis factor-alpha increased the efficiency of transmigration and markedly reduced apoptosis among the transmigrated neutrophils in a dose-dependent manner. Apoptosis was also inhibited after transmigration of neutrophils through HUVEC stimulated with interleukin (IL)-1beta but not so effectively after chemotaxis through unstimulated HUVEC driven by IL-8 added below the filter. Inhibition of beta2-integrin binding after transmigration or coating the lower chamber with a nonadhesive polymer (polyhydroxyl-ethyl-methacrylate) abrogated neutrophil survival. Although integrin engagement during migration itself was not essential to inhibit apoptosis, activation of neutrophils through CXC chemokine receptors was necessary. Quite brief exposure to the HUVEC (30-120 min) was effective in reducing subsequent apoptosis, although if coincubation with the HUVEC were prolonged, neutrophil apoptosis was reduced further. Neutralization of granulocyte macrophage-colony stimulating factor inhibited this additional effect. Thus, a complex interplay between migration- and activation-dependent signals and adhesive interaction in tissue may combine to effectively prolong the survival of neutrophils recruited during inflammation.     

轻度修饰低密度脂蛋白对血管内皮细胞粘附功能影响及其机理初探

目的:观察轻度修饰LDL(MM-LDL)对培养人脐静脉内皮细胞(HUVEC)与人类单核细胞系U937粘附功能及其表面粘附分子表达的影响。方法:利用计数法观察经MM-LDL作用的HUVEC与U937细胞的粘附率;用ELISA方法检测MM-LDL作用后HUVEC膜表面粘附分子血管细胞粘附分子-1(VCAM-1)、细胞间粘附分子-1(ICAM-1)及P选择素(P-selectin)的表达。结果:MM-LDL(75mg/L)作用HUVEC4h后,其对U937细胞粘附率明显增加(P<0.01),HUVEC膜表面未见VCAM-1,ICAM-1,P-selectin表达上调,作为阳性对照重组肿瘤坏死因子α(rTNF_α)5.0μg/L可显著诱导以上3种粘附分子表达。延长MM-LDL与HUVEC作用时间至18h可诱导产生P-selectin表达,对VCAM-1表达无影响。结论:MM-LDL诱导的HUVEC与U937粘附不是通过ICAM-1、VCAM-1介导的,P-selectin可能起一定的作用。     

Effect of cytokines on the expression of cell adhesion molecule and on the adhesion of melanoma cells to endothelial cells.

We examined the role of cell adhesion molecules (CAM) by which tumor cells bind to the endothelial cells using human umbilical vein endothelial cells (HUVEC) and cultured melanoma cells. Endothelial cells from human umbilical veins were isolated and examined for CAM expression and its modulation by tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), interleukin-6 (IL-6) or interferon-gamma (IFN-gamma). The expression of intercellular adhesion molecule 1 (ICAM-1) on HUVEC was increased by TNF-alpha, IL-1 and IFN-gamma when measured by ELISA or flow cytometric (FACS) analysis. IL-6 did not increase ICAM-1 expression on HUVEC. Two melanoma cell lines, Malme-3M and SK-Mel-28, showed increased expression of ICAM-1 after treatment with TNF-alpha, IL-1 and IFN-gamma in FACS analysis. IFN-gamma induced increased expression of HLA-DR only in SK-Mel-28 melanoma cells, not in Malme-3M melanoma cells. Neither HUVEC nor melanoma cells expressed lymphocyte function-associated antigen 1 (LFA-1) in either the basal (i.e., cytokine untreated) condition or the cytokine treated condition. Melanoma cells showed minimal increment in adhesion to TNF-alpha or IL-1 treated HUVEC than to cytokine untreated HUVEC. HUVEC and melanoma cells did not express LFA-1 and increased ICAM-1 expression by TNF-alpha, IL-1 and IFN-gamma treatment in FACS analysis did not coincide with minimal increase of melanoma cells adhesion to cytokine treated HUVEC. These results suggest that adhesion between melanoma cells and HUVEC is probably mediated by molecular interaction other than ICAM-1/LFA-1.     

Inhibitory effect of fluvastatin,an HMG-CoA reductase inhibitor,on the expression of adhesion molecules on human monocyte cell line

The effect of fluvastatin, an HMG-CoA reductase inhibitor, was investigated on the adhesive interaction between U937 cells, the human monocyte cell line, and human umbilical vein endothelial cells (HUVEC), focusing on the expression of adhesion molecules. U937 treated with fluvastatin lowered the capacity for binding to HUVEC. Fluvastatin at 0.1 μM or more inhibited the expression of lymphocyte function associated antigen-I (LFA-1) on U937 and intercellular adhesion molecule-1 (ICAM-I) on U937. The expression of ICAM-1 on HUVEC was not inhibited by fluvastatin. The inhibitory effects of fluvastatin on the expression of adhesion molecules on U937 were completely reversed by the addition of mevalonate. Because fluvastatin did not affect the expression of other cell surface markers, CD4 and CD71, the inhibitory effects of fluvastatin on adhesion molecule expression could not be attributed to the non-specific suppression of the cell. It is conceivable that cellular interaction between monocytes and endothelial cells is inhibited by fluvastatin, mediated via reducing the expression of adhesion molecules, particularly in the side of monocyte.     

X线电离辐射通过上调c-Myc表达促进肺癌A549细胞上皮间质转化

目的:探讨电离辐射对肺癌A549细胞上皮间质转化(EMT)的影响及其可能机制。方法:采用不同剂量(0 Gy、1 Gy、2 Gy、4 Gy和8 Gy)的X射线分别照射肺癌A549细胞不同时间,通过倒置显微镜观察X射线照射12 h、24 h和48 h时肺癌A549细胞形态的改变;Western blot检测X射线照射12 h与24 h时EMT相关蛋白vimentin、N-cadherin及E-cadherin和转录因子c-Myc的表达。结果:照射后肺癌A549细胞轮廓不清,突起增多,边缘不规则且呈煎蛋样塌陷,以8 Gy X射线照射48 h时肺癌A549细胞上皮间质化形态最明显。与0 Gy照射剂量对照组相比,vimentin虽然于4 Gy剂量照射12 h时下调,但是处理24 h时各剂量照射组vimentin均表现为上调,其中2 Gy剂量照射组其上调最明显(P0.01);与0 Gy照射剂量对照组相比,1 Gy、2 Gy及4 Gy照射组照射肺癌A549细胞24 h后其N-cadherin表达上调(P0.05);各照射剂量对肺癌A549细胞E-cadherin表达没有显著影响。照射24 h后肺癌A549细胞c-Myc表达上调,以4 Gy剂量照射组表达差异最明显(P0.01)。结论:X线电离辐射可能通过上调c-Myc表达促进肺癌A549细胞发生上皮间质转化。     

Vascular endothelial cells provide T cells with costimulatory signals via the OX40/gp34 system

We investigated whether gp34, the ligand of OX40, expressed on EC is involved in costimulation of T cells. Normal CD4+ T cells were stimulated with anti-CD3-coated beads, phytohemagglutinin (PHA), or concanavalin A (Con A) in the presence or absence of irradiated human umbilical vein endothelial cells (HUVEC). Stimulation of T cells with each of these mitogens results in significant T-cell proliferation only when HUVEC were present, and this proliferation was inhibited markedly by anti-OX40 or anti-gp34 monoclonal antibody (mAb). T cells cultured with HUVEC produced more interleukin (IL)-2 than those cultured without HUVEC. The addition of anti-IL-2R alpha chain and anti-IL-2R beta chain mAbs abolished the costimulatory effects of HUVEC. Thus, the augmentation of T-cell proliferation appears to be attributable to increased IL-2 production. These results suggest that gp34 expressed on HUVEC plays a role in potentiation of T-cell immune response by providing OX40+ T cells with costimulatory signals.     

Membrane related effects in endothelial cells induced by human cytomegalovirus

Summary Previously, we have reported on the increase in procoagulant activity of human umbilical vein endothelial cells (HUVEC) after infection with human cytomegalovirus (HCMV). When using microvascular endothelial cells from foreskin (MVEC), we also observe a significant increase in membrane perturbation and a concomittant increase in procoagulant activity. This effect is both observed with a laboratory HCMV strain (AD169) with low pathogenicity for endothelium and a HUVEC adapted strain (VHL-E) that readily infects endothelial cells. We compared the membrane perturbation of two types of endothelial cells, HUVEC and MVEC with human embryonal fibroblasts (HEF), being fully permissive for both strains. A membrane effect was only found in endothelial cells. Our results suggest that HCMV induces in MVEC more merocyanine-540 incorporation in the membrane as in HUVEC. The increase in the procoagulant activity induced by HCMV was more pronounced in MVEC than in HUVEC. Inactivated virus, as well as virus pre-incubated with heparin was unable to evoke membrane perturbation. It therefore appears that HCMV induces a rapid membrane response in vascular endothelium and that physical interaction of the virion and the endothelial cell is required to elicit this response.     

Rapid Adhesive Responses of Endothelial Cells and of Neutrophils Induced by Leukotriene B4 are Mediated by Leucocytic Adhesion Protein CD18

Controversy has existed as to the ability of leukotriene B4 (LTB4) to enhance adhesive properties of human neutrophils (PMN) and endothelial cells. We found that LTB4 induced a rapid but transient adhesion of PMN to an albumin-coated plastic surface and to cultured human umbilical vein endothelial cells (HUVEC). Although the adhesive response of PMN to the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) was longer lasting, peak hyperadherence was of similar magnitude as to LTB4 and was less susceptible to assay conditions. Adherence induced by either LTB4 or fMLP could be abrogated by the monoclonal antibody 60.3, indicating similar dependence on the leucocyte adhesion protein CD18. Lipoxin A did not induce PMN hyperadherence. Treating HUVEC with LTB4, but not with its omega-oxidized metabolites 20-OH- and 20-COOH-LTB4, lipoxin A, or with fMLP conferred a rapid, dose-related, enhanced adhesion of PMN. This effect was dependent on CD18 and on divalent cations. It disappeared with prolonged exposure to LTB4, required a metabolically active HUVEC, and was not due to passive binding of LTB4 to HUVEC. Thus, LTB4 induces a transient expression of hyperadhesiveness in HUVEC as well as in neutrophils, and both effects are dependent on expression of CD18.     

Leucocyte Endothelial Cell Adhesion: a Study comparing Human Umbilical Vein Endothelial Cells and the Endothelial Cell Line EA-hy-926

EA-hy-926 is a cell line produced by hybridizing human umbilical vein endothelial cells (HUVEC) and the epithelial cell line A549. To establish whether EA-hy-926 could be used as a model for endothelial cells (EC) in leucocyte-EC adhesion interactions, the effect of interleukin-4 (IL-4), tumour necrosis factor (TNF) or interferon-γ (IFN) stimulation on their adhesiveness and expression of E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) was compared with that of HUVEC and A549. Although HUVEC exhibited increased adhesiveness and adhesion molecule expression with IL-4, TNF or IFN, EA-hy-926 exhibited these responses only with TNF. CD11/CD 18-dependent binding accounted for a significant component of basal binding to HUVEC and EA-hy-926, but did not account for the increased binding of T cells, JY, J6, ICH-BJ or ICH-KM cell lines to TNF-stimulated monolayers. At least part of the CD1l/CD18-independent adhesion was attributable to VCAM-1 induction on HUVFC and FA-hy-926. TNF-stimulation also induced F-selectin expression on EA-hy-926 and HUVEC and an accompanying increase in neutrophil (PMN) binding. The EA-hy-926 cells used in this study, therefore, showed responses similar to HUVEC when stimulated with TNF but not when stimulated with IL-4 or IFN.     

Activation of Human Monocytes for Enhanced Production of Interleukin 8 During Transendothelial Migration in Vitro

Interleukin-8 (IL-8) is a chemokine for polymorphonuclear leukocytes (PMNs) and lymphocytes, which promotes the extravasation of these inflammatory cells. In this study, we investigated IL-8 synthesis induced by the adhesive interaction between monocytes and endothelial cells during transmigration and the capacity of transmigrated monocytes to produce IL-8. Cocultured human monocytes and human umbilical vein endothelial cell (HUVEC) monolayers induced the synefgistic production of IL-8, compared with cultures of either monocytes or HUVEC monolayers alone. Coculture-induced IL-8 production almost doubled after HUVECs were stimulated with IL-1. The induced IL-8 mRNA expression was consistent with the protein data, indicating the de novo synthesis of IL-8 by the coculture. Monoclonal antibodies (mAbs) against IL-8 inhibited the transendothelial chemotactic activity of the supernatants for PMNs by 55%. Immunohistochemistry revealed that both adherent and transmigrated monocytes and unstimulated HUVECs expressed IL-8 protein, whereas nonadherent monocytes did little. Transmigrated monocytes spontaneously secreted a 3.8-fold greater amount of IL-8 than the initial monocytes. Coculture-induced IL-8 production was inhibited about 30% by polyclonal Abs against IL-, IL-1, or tumor necrosis factor , while it was not affected by mAbs against intercellular adhesion molecule 1 or vascular cell adhesion molecule 1. The results suggested that adhesive interaction during the transmigration of monocytes through HUVEC monolayers activates both cell types to produce IL-8 and that transmigrated monocytes are capable of producing ample IL-8.