低浓度泰素抑制血管内皮细胞管道形成的实验研究

目的 体外实验研究低浓度泰素（人工半合成紫杉醇Pacitaxel）抑制血管内皮细胞组装形成管道的能力。方法 体外培养的人脐静脉血管内皮细胞（Human Umbilical Vein Endothelial Cell，HUVEC）在含生长因子的胶原（Matrigcl）上能自行组装成管道结构，在细胞培养液中加入低浓度泰素，观察其抑制HUVEC组装管道的能力．计算管道总长度，与对照组相比较。结果发现泰素浓度在0．5—50μg/ml时，几乎完全抑制了HUVEC的管道形成。当泰素浓度降低到1、2、5和10ng／ml时，HUVEC形成的管道总长度分别为对照组的57％、51％、34％和27％。结论 ng／ml级的低浓度泰素在体外可抑制HUVEC约50％-70％的血管形成能力，而μg／ml级浓度泰素几乎可完全抑制HUVEC的血管形成能力。     

静脉表型分子COUP-TFⅡ表达降低对血管内皮细胞衰老的影响

目的:观察静脉表型分子鸡卵清蛋白上游启动子转录因子Ⅱ(COUP-TFⅡ)表达对血管内皮细胞衰老的影响和分子机制。方法:通过RT-qPCR检测比较人冠状动脉内皮细胞(HCAEC)和人脐静脉内皮细胞(HUVEC)中COUP-TFⅡ的表达情况。使用10~(-5)mol/L血管紧张素Ⅱ(AngⅡ)诱导HUVEC衰老,通过COUP-TFⅡ特异性小干扰RNA(siCOUP-TFⅡ)转染HUVEC使其COUP-TFⅡ的表达降低,利用β-半乳糖苷酶染色、Western blot、CCK-8、细胞计数等方法分别观察siCOUP-TFⅡ对AngⅡ诱导的血管内皮细胞衰老及增殖的影响,通过Western blot检测Akt信号分子的表达变化。结果:与HCAEC相比,HUVEC中COUP-TFⅡ呈显著高表达;用siCOUP-TFⅡ抑制HUVEC的COUP-TFⅡ表达时,能显著促进AngⅡ诱导的内皮细胞衰老并抑制内皮细胞增殖和p-Akt蛋白水平;加入Akt激动剂SC79(4 mg/L)能够部分逆转siCOUP-TFⅡ对AngⅡ诱导的HUVEC衰老和增殖的影响。结论:COUP-TFⅡ表达降低可促进血管内皮细胞衰老并抑制内皮细胞增殖,其机制可能与调节Akt信号有关。     

骨形态发生蛋白4通过ERK/MAPK信号通路影响人脐静脉内皮细胞的成血管能力

目的 研究不同浓度的BMP-4对于脐静脉内皮细胞体外成血管能力的影响并对其作用机制做初步探讨.方法 BrdU参入法检测细胞增殖情况,划痕实验检测细胞迁移情况,基质胶法检测细胞体外成血管能力,Western blot 检测phospho ERK1/2表达情况.结果 BMP-4在低浓度时具有抑制HUVEC的增殖,迁移和成血管,然而随着BMP-4浓度的升高,这种抑制作用逐渐减弱,并有恢复至正常水平的趋势.结论 不同浓度的BMP4对于HUVEC的增殖,迁移及成血管能力具有不同的影响,BMP-4可能是通过ERK/MAPK信号通路影响HUVEC成血管能力的.     

血管紧张素Ⅱ促进内皮细胞凋亡和NOX4表达

目的分析高浓度血管紧张素Ⅱ(AngⅡ)刺激人脐静脉内皮细胞(HUVECs)时细胞内活性氧(ROS)、NOX4 mRNA水平和细胞凋亡的变化。方法倒置显微镜下观察人脐静脉内皮细胞形态;免疫组化法检测人脐静脉内皮细胞Ⅷ因子相关抗原的表达;RT-PCR检测HUVECs中NOX4的表达;流式细胞仪检测各组细胞内ROS生成量和细胞凋亡率,Hoechst染色分析细胞凋亡。结果高AngⅡ刺激HUVECs时,NOX4 mRNA表达上调,细胞内ROS生成增加,细胞凋亡增加。结论高AngⅡ上调HUVCEs内NOX4mR-NA表达并促进细胞内ROS生成和细胞凋亡。     

骨髓间充质细胞来源的血管内皮样细胞与脐静脉血管内皮细胞抗凝基因表达的比较

目的观察不同来源的血管内皮细胞抗凝能力的差异。方法采用密度梯度离心及贴壁培养法对骨髓间充质干细胞(BMMSCs)进行体外培养、纯化和扩增,用含有VEGF(10μg/L)的诱导培养液对其进行体外诱导分化,1周后免疫组化染色鉴定Ⅷ因子相关抗原(VWF),用RT-PCR法检测BMMSCs来源的血管内皮样细胞及脐静脉血管内皮细胞(HUVEC)的主要抗凝血基因的表达。结果BMMSCs能够在体外成功诱导分化为血管内皮样细胞,但不表达主要抗凝基因的mRNA,而HUVEC能够表达这些基因的mRNA。结论虽然BMSCs能够成功诱导分化为血管内皮样细胞,但它的抗凝能力较HUVEC弱。     

小檗碱对人脐静脉内皮细胞增殖与凋亡的作用

目的：观察小檗碱对人脐静脉内皮细胞增殖与凋亡的作用，以探讨小檗碱抑制肿瘤生长与转移的机制。 方法： 用小檗碱与体外培养的人脐静脉内皮细胞（HUVEC）共同孵育，以MTT法检测细胞的增殖活性，免疫细胞化学法检测增殖细胞核抗原（PCNA）的表达，荧光染色法观察凋亡细胞核形态，用Rhodamine123染色，激光共聚焦显微镜检测线粒体膜电位。 结果： 不同浓度小檗碱与HUVEC共同孵育可明显抑制HUVEC的增殖(P<0.05，P<0.01)，呈现一定的浓度依赖性和时效性；20 mg/L小檗碱与HUVEC共同孵育48 h，可显著降低细胞核PCNA的表达（P<0.01），并可见凋亡细胞数增多、线粒体膜电位明显降低（P<0.01）。 结论： 小檗碱抑制血管内皮细胞的增殖，并促进血管内皮细胞凋亡，从而抑制肿瘤血管形成，可能是小檗碱抑制肿瘤生长与转移的机制之一。     

稳定层流剪应力对内皮细胞骨架调节蛋白VASP表达的影响

为探讨生理强度的稳定层流剪应力对内皮细胞骨架actin相关蛋白VASP特征影响规律，我们采用胰蛋白酶消化法分离人脐静脉内皮细胞(HUVECs)，模拟体内流动环境，建立平行板流动腔模型。利用细胞图像分析系统和ALEXA488—若丹明一次毒蕈环肽双标记法，观察内皮细胞在稳态层流下形态、actin排列变化与VASP分布变化之间的规律。采用Western blot定量动态检测细胞内VASP表达及磷酸化的水平。结果表明，内皮细胞在10dyn／cm^2剪切作用后，随时间细胞逐渐延长，长轴趋于剪应力作用方向排列，细胞与静息态的细胞相比，细胞内骨架沿剪应力方向重组，与此同时VASP表达增强，沿着actin纤维呈点状分布，尤其集中在细胞膜下actin末端区域；Western blot检测显示在剪切后，细胞内VASP出现快速磷酸化，VASP总体表达量增加，2h达高峰后逐渐恢复，8h后再次逐渐升高。以上结果提示血流动力学特性中剪应力引起了细胞胞质内骨架蛋白分子重组，血管内皮细胞形态改变，在此过程中，VASP发挥骨架调节蛋白的作用。     

基因突变和转基因技术评价TLR-4信号受体在流体切应力诱导血管内皮细胞IL-8基因转录激活中的作用

用基因突变和转基因技术，评价TLR-4信号受体在层流低切应力刺激诱导血管内皮细胞IL-8基因转录激活中的作用，RT-PCR，Northern杂交和免疫荧光细胞化学染色均显示脐静脉血管内皮细胞表达TLR-4，同时RT-PCR和Northern杂交显示，层流切应力刺激1h后血管内皮细胞TLR-4表达增强，用RT-PCR技术从血管内皮细胞扩增出胞内区段缺失突变TLR-4cDNA，用PCR技术从其基因组DNA中扩增出IL-8上游调控序列，分别克隆于真核表达质粒pcDNA3和绿色荧光增强蛋白报告基因pEGFP1质粒，构建出重组TLR-4缺失突变基因真核表达质粒pcDNA3-mTLR4和IL-8报告基因表达质粒pEGFP1-IL8USCS。用pEGFP1-IL8USCS转染或pcD-NA3-mTLR4和pEGFP1-IL8USCS共转染ECV304细胞，4.2dyne/cm^2层流切应力刺激3h后，流式细胞仪观察荧光蛋白表达强度变化，用pEGFP1-IL8USCS转染细胞，经层流切应力刺激3h后荧光蛋白表达增强（1.06:2.71)，同样用pcDNA3-mTLR4和pEGFP1-IL8USCS共转染细胞，层流切应力刺激3h后荧光蛋白表达未明显增强，提示TLR4/NF-кB信号传导通路可能介导层流切应力诱导血管内皮细胞IL-8基因的表达。     

小檗碱抗肿瘤新生血管形成作用机制的研究

目的：研究小檗碱对bFGF活化人脐静脉内皮细胞（HUVEC）增殖、细胞周期及细胞凋亡的影响，探讨其对肿瘤新生血管形成作用的机制。方法：MTT法检测小檗碱对bFGF活化HUVEC的增殖作用；流式细胞仪检测用药后细胞周期的变化；激光共聚焦扫描显微镜下观察药物对细胞形态、细胞内钙的影响；流式细胞仪检测小檗碱对细胞凋亡的作用。结果：小檗碱能明显抑制bFGF活化的HUVEC增殖，且存在剂量依赖关系；使细胞在G0-G1期的比例明显增多；使细胞核浓缩、甚至裂解成碎块，同时使细胞内钙增多；并诱导活化HUVEC发生细胞凋亡。结论：小檗碱可能通过将bFGF活化的HUVEC细胞周期阻滞在G0-G1期，抑制活化HUVEC的增殖：诱导活化HUVEC细胞发生凋亡等机制，阻止新生血管形成，发挥其抗肿瘤作用。     

Up regulation of DLL4 expression in lung cancer A549 cells in hypoxia induced by CoCl2 and secreted promote HUVEC migration material

目的 探讨氯化钴( CoCl2)化学模拟低氧对肺癌细胞(A549) DLL4表达的影响及其与内皮细胞迁移的关系,为寻找抗肿瘤血管新生的潜在靶点提供理论依据.方法 A549分为对照组和CoCl2干预低氧组,EdU掺入法测定细胞增殖;用免疫荧光细胞染色和Western blot测定DLL4和低氧诱导因子HIF-1α在A549细胞中的表达;细胞划痕实验检测脐静脉内皮细胞(HUVEC)的迁移变化.结果 低浓度氯化钴(200 μmol/L)干预12 h后,A549细胞增殖没有明显抑制,DLL4和HIF-1α的表达均明显上调,低氧组DLL4蛋白表达为0.194 ±0.028显著高于对照组的0.098 ±0.015(P ＜0.05).培养上清液促进HUVEC迁移.结论 低氧明显上调A549细胞DLL4的表达,其对内皮细胞的迁移趋化作用提示其为抗肿瘤血管新生治疗的潜在靶点.     

Effects of Cytotoxic Necrotizing Factor 1 and Lethal Toxin on Actin Cytoskeleton and VE-Cadherin Localization in Human Endothelial Cell Monolayers

Integrity of the vascular endothelium is largely dependent on endothelial cell shape and establishment of intercellular junctions. Certain pathogenic bacterial toxins alter the cytoskeletal architecture of intoxicated cells by modulating the GTPase activity of p21 Rho family proteins. In the present study we have analyzed the effect of Rho-directed toxins on the actin cytoskeleton and monolayer integrity of endothelial cells. We report here that Escherichia coli cytotoxic necrotizing factor 1 (CNF1) activates Rho in human umbilical vein endothelial cells (HUVEC). In confluent monolayers, CNF1 treatment induces prominent stress fiber formation without significantly modifying peripheral localization of VE-cadherin, a specific marker of vascular endothelial cell adherens junctions. Further, Rho activation with CNF1 blocks thrombin-induced redistribution of VE-cadherin staining and gap formation in HUVEC monolayers. Inhibition of Rho by prolonged treatment of cells with C3 exoenzyme (Clostridium botulinum) eliminates actin stress fibers without disrupting the continuity of VE-cadherin staining, indicating that Rho-dependent stress fibers are not required for maintaining this adhesion receptor at sites of intercellular contact. Lethal toxin (Clostridium sordellii), an inhibitor of Rac as well as Ras and Rap, potently disrupts the actin microfilament system and monolayer integrity in HUVEC cultures.     

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.     

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.     

Synergistic effect of high-affinity binding and flow preconditioning on endothelial cell adhesion

The current study examined whether the combined introduction of high-affinity avidin-biotin bonds and fibronectin-integrin bonds (i.e., dual ligand treatment) would further augment the adhesion of flow-preconditioned endothelial cells to model substrates via contributions to the actin cytoskeleton and the formation of focal contacts. Human umbilical vein endothelial cells (HUVEC) were grown under static conditions or exposed to a flow-preconditioning regimen for 24 h. Cell retention was determined by exposure to 75 dynes/cm(2). The combination of flow preconditioning and the dual ligand treatment yielded higher cell retention under flow compared to the cells adherent via fibronectin-integrin bonds only. This increase in adhesion strength correlated with a greater focal contact area. Elongation of the HUVEC occurred after exposure to flow preconditioning; however, orientation of dual ligand adherent cells was restricted due to the presence of the high-affinity ligand. Flow-preconditioned cells showed increased stress fiber formation compared to nonconditioned cells although the stress fibers per cell for flow-preconditioned cells were the same on both the ligand systems employed. The results indicate that enhanced adhesion strength is due to a combination of increased focal contact area, stress fiber formation, and cell alignment.     

In-vitro inhibitory effect of EGFL7-RNAi on endothelial angiogenesis in glioma

Objective: To investigate the role and mechanism of epidermal growth factor like domain 7 (EGFL7) in glioma angiogenesis by cell co-culture and RNA interference. Methods: NSCs-HUVECs co-culture system was established using Transwell culturing techniques. The interactions between glioma and endothelial cells were simulated in-vitro. Cellular expression of EGFL7 in NSCs and HUVEC was targeted and suppressed by lentiviral vector carrying siRNA. The effect of EGFL7 on angiogenesis in glioma in-vitro micro-environment was detected by endothelial cell proliferation, adhesion and tube formation assay. Results: Following EGFL7 gene silencing, expression of EGFL7 in HUVECs was reduced and cell adhesion capability was inhibited significantly. Endothelial cells failed to form a lumen-like structure after EGFL7 gene silencing, shown by the tube formation assay. Conclusion: By regulating endothelial cell adhesion, EGFL7 plays a key role in the regulation of glioma angiogenesis.     

HB-EGF promotes angiogenesis in endothelial cells via PI3-kinase and MAPK signaling pathways

OBJECTIVE: Heparin-binding EGF-like growth factor (HB-EGF) belongs to the epidermal growth factor (EGF) superfamily of ligands. It has been implicated as a regulator of angiogenesis. However, the mechanisms by which HB-EGF promotes angiogenesis are unknown. The goal of the present study was to define the pathways by which HB-EGF stimulates angiogenesis in endothelial cells. METHODS: To characterize the angiogenic activity of HB-EGF, we treated human umbilical vein endothelial cells (HUVEC) with HB-EGF and analyzed the effects on cell proliferation, migration and tube formation. Side-by-side assays with EGF were used for comparison. RESULTS: Both HB-EGF and EGF stimulated HUVEC migration in scratch assays and promoted vascular tube formation in 2D-angiogenesis assays, without inducing cell proliferation. HB-EGF- and EGF-induced HUVEC migration and capillary tube formation were dependent upon activation of PI3K, MAPK and eNOS. Importantly, HB-EGF-and EGF-induced tube formation was comparable to, but were independent of tube formation induced by VEGF. CONCLUSIONS: We have demonstrated that HB-EGF and EGF induce angiogenesis via activation of PI3K, MAPK and eNOS in a VEGF-independent fashion. Thus, the role played by HB-EGF in stimulating physiologic processes such as wound healing in vivo may be dependent, in part, on its ability to promote angiogenesis.     

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.     

Legumain对肿瘤细胞侵润力和血管内皮细胞管腔形成的作用

目的探讨肿瘤组织中过度表达的一种天冬酰胺肽链内切酶Legumain在肿瘤细胞侵润性生长和血管内皮导管形成中的作用及相关机制。方法采用体外细胞培养的方法,观察在缺氧条件下人乳腺癌细胞MDA MB231和人静脉血管内皮细胞HUVEC中Legumain的蛋白表达量：观察Legumain对MDA MB231细胞的侵润能力的影响以及人工合成的Legumain抑制物（asparaginly endopepidase inhibitor．AEPI）对人静脉血管内皮细胞HUVEC小管腔形成能力的影响;用免疫组织化学的方法观察Legumain与整合素（Integrin）β1的结合情况。结果Western—blot的结果表明。在缺氧条件下．体外培养的人乳腺癌细胞MDA MB231和人静脉血管内皮细胞HUVEC的Legumain蛋白表达量增加。应用免疫荧光双染色法对缺氧条件下培养的MDA MB231细胞进行染色．与正常条件下培养的细胞相比,缺氧条件下培养的MDA MB231细胞体积增大,胞浆内含有大量绿色的Legumain,成点状分布,并有部分Legumain分布在迁移中的细胞表面前缘,与红色的整合素β1重叠,呈现为黄色。在血管内皮细胞小管腔形成试验中,加入AEPI的实验组,HUVEC细胞的小管形成数量明显减少,分别为（13．2±0．85）,（5．33±0．35）,（0．57±0．23）,与对照组（21．3±1．73）相比差异均具有统计学意义（P〈0．001）。细胞的侵润能力试验结果表明,加入激活的Legumain后,实验组中的MDA MB231细胞的侵袭能力增高．分别为（170．90±59．27和（321．22±22．39）,与对照组（67．56±7．58）相比差异具有统计学意义（P〈0．05）。结论缺氧条件下．MDA MB231细胞株和HUVEC细胞株的Legumain表达增加,能促进肿瘤细胞的迁移和血管内皮细胞的小管形成。其分子机理之一可能是通过与整合素β1结合。     

β2-glycoprotein I,lipopolysaccharide and endothelial TLR4: Three players in the two hit theory for anti-phospholipid-mediated thrombosis

The thrombogenic effect of β2-glycoprotein I (β2GPI)-dependent anti-phospholipid antibodies (aPL) in animal models was found to be LPS dependent. Since β2GPI behaves as LPS scavenger, LPS/β2GPI complex was suggested to account for in vitro cell activation through LPS/TLR4 involvement being LPS the actual bridge ligand between β2GPI and TLR4 at least in monocytes/macrophages. However, no definite information is available on the interaction among β2GPI, LPS and endothelial TLR4 in spite of the main role of endothelial cells (EC) in clotting. To analyse at the endothelial level the need of LPS, we investigated the in vitro interaction of β2GPI with endothelial TLR4 and we assessed the role of LPS in such an interaction.To do this, we evaluated the direct binding and internalization of β2GPI by confocal microscopy in living TLR4-MD2 transfected CHO cells (CHO/TLR4-MD2) and β2GPI binding to CHO/TLR4-MD2 cells and human umbilical cord vein EC (HUVEC) by flow cytometry and cell-ELISA using anti-β2GPI monoclonal antibodies in the absence or presence of various concentrations of exogenous LPS. To further investigate the role of TLR4, we performed anti-β2GPI antibody binding and adhesion molecule up-regulation in TLR4-silenced HUVEC.Confocal microscopy studies show that β2GPI does interact with TLR4 at the cell membrane and is internalized in cytoplasmic granules in CHO/TLR4-MD2 cells. β2GPI binding to CHO/TLR4-MD2 cells and HUVEC is also confirmed by flow cytometry and cell-ELISA, respectively. The interaction between β2GPI and TLR4 is confirmed by the reduction of anti-β2GPI antibody binding and by the up-regulation of E-selectin or ICAM-1 by TLR4 silencing in HUVEC. β2GPI binding is not affected by LPS at concentrations comparable to those found in both β2GPI and antibody preparations. Only higher amount of LPS that can activate EC and up-regulate TLR4 expression are found to increase the binding.Our findings demonstrate that β2GPI interacts directly with TLR4 expressed on EC, and that such interaction may contribute to β2GPI-dependent aPL-mediated EC activation. At variance of monocytic cells, we also showed a threshold effect for the action of LPS, that is able to enhance anti-β2GPI antibody EC binding only at cell activating concentrations, shown to increase TLR4 expression. This in vitro model may explain why LPS behaves as a second hit increasing the expression of β2GPI in vascular tissues and triggering aPL-mediated thrombosis in experimental animals.     

A quantitative ELISA-based co-culture angiogenesis and cell proliferation assay

Since solid tumours and metastases depend on adequate blood supply, much research is focused on inhibition of angiogenesis. Unfortunately, most known angiogenesis inhibitors have serious side effects when used as therapeutic agents in man. It is therefore important to develop methods to identify well-tolerated and efficient angiogenesis inhibitors. As a method for identification of new angiogenesis inhibitors we have further developed the procedure described by Bishop et al. (Angiogenesis 1999;3:335-44) to a quantitative ELISA-based fibroblast and endothelial cell co-culture angiogenesis assay. In each well of a 96-microwell plate, human umbilical vein endothelial cells (HUVEC) are seeded onto normal human dermal fibroblasts (NHDF) and propagated in co-culture for 72 h with or without a potential angiogenesis inhibitor. The effect on total cell proliferation is evaluated by quantitative immunochemical measurement of DNA, and on endothelial tube formation by quantification of CD 31, von Willebrand factor, and collagen IV. After ELISA reading, the morphology of the tubular structures formed by HUVEC is visualised with BCIP/NBT, permitting a quantitative result and a qualitative evaluation of cell morphology from the same well. We have used the assay to demonstrate the effect of well-known angiogenesis inhibitors on HUVEC tube formation.