克拉霉素抑制肺癌细胞诱导血管内皮细胞迁移的实验研究

目的 观察克拉霉素（CAM）对人小细胞癌细胞表达VEGF其诱导血管内皮细胞迁移的影响，探讨CAM抗血管生成的机制。方法 采用免疫组化和图象分析技术，观察不同浓度的CAM作用下人小细胞肺癌细胞（NCI－H446）中VEGF蛋白表达的变化。采用共培养法（Marigel invasion chamber)，观察CAM为NCI－H446细胞诱导人血管内皮细胞（ECV－304）迁移的抑制作用。结果 CAM达到30mmol/L对其诱导的ECV－304细胞迁移表现出明显的抑制作用，达到40mmol/L可以抑制CI－H446细胞表达VEGH，CAM浓芳在30，40和50mmol/L时，抑制率分别为19．7％，24．3％和25．0％，呈现出明显剂量－反应关系（r=-0.764,P=0.001)。结论 CAM能够抑制肺癌细胞诱导的血管内皮细胞迁移，对其表达VEGF也具有抑制作用，以上作用可能是CAM抗血管生成的机制之一。     

Compound exocytosis of granules in human neutrophils

Human neutrophils are of prime importance for the immune defense. Recent data from eosinophils and pancreatic beta cells have indicated that granules, upon exocytosis, occasionally fuse with each other in the cytosol prior to their subsequent fusion with the plasma membrane. This is termed compound exocytosis. We therefore studied exocytosis of single granules from human neutrophils by the high-resolution cell-attached patch-clamp capacitance technique. We found that 1.5% of the capacitance steps was greater than 5 fF, i.e., significantly larger than steps expected for exocytosis of single granules. The mean step size of these events was 20.5 fF, corresponding to compounds formed by at least five granules. The capacitance input from compound steps contributed more than 20% of the total capacitance increase. Electron microscopy captured morphological manifestations of transient exocytic events, confirming the functional results obtained by capacitance measurements. Compound exocytosis may be a mechanism for efficient targeting of release during exocytosis.     

Relationship between antibody-dependent tumour cell lysis and primary granule exocytosis by human neutrophils.

Neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) against Raji target cells and neutrophil degranulation during the ADCC process were evaluated in the presence and in the absence of different agents able to interfere with the neutrophil release of granule components (anion channel blockers, colchicine, isoproterenol, dimethylxanthine, cAMP). When used at concentrations incapable of preventing the target cell recognition by neutrophils, the majority of these agents inhibited both the ADCC and the release of myeloperoxidase (MPO, primary granule marker) and lysozyme (LZM, primary and secondary granule marker). The inhibition of the ADCC correlated strictly with the inhibition of the MPO release. Thus, the results are consistent with the hypothesis that neutrophil primary granules play a major role in the cytolytic process.     

The inhibitory effect of quercetin on IL-6 production by LPS-stimulated neutrophils

Quercetin is a herbal flavonoid derived from various foods of plant origin and plays a role in anti-inflammation. Although a number of researches in the field have been done, the mechanism of anti-inflammatory effect of quercetin should be further darified. In the present study, we investigated the effects of quercetin on IL-6 production by LPS-stimulated neutrophils in human. Neutrophils were were pre-treated with quercetin at the final concentrations of ranging from 0-80 ~M for 30 rain, or not treated, and then incubated in the presence or absence of lipopolysaccharide （LPS） at a final concentration of 100 ng/ml for indicated time. The secretion level of IL-6 in the culture supernatants was assayed by ELISA, the intracellular level of IL-6 was detected by flow cytometry and the expression of IL-6 mRNA was analyzed by RT-PCR. The experiment results showed that neutrophils cultured with medium or quercetin alone did not express IL-6, but LPS （100 ng/ml） induced IL-6 expression of neutrophils. However, after pre-treatment of neutrophils with quercetin （40 ~Vl） for 30 rain, the inducible effects of LPS on the increase of IL-6 secretion, intracellular IL-6 level and IL-6 mRNA expression by neutrophils were abrogated. IL-6 is one of the important pro-inflammatory factors, especially in early phage of inflammation. Thus, our data suggested that quercetin might exert its anti-inflammatory effect through negatively modulating pro-inflammatory factors, such as IL-6. The inhibitory effects of quercetin on IL-6 production by neutrophils may provide a theoretical basis on future therapy of inflammation. Cellular ＆ Molecular Immunology. 2005;2（6）：455-460.     

Degranulation of primary and secondary granules in adherent human neutrophils

The aim of this study was to investigate whether neutrophil adhesion to extracellular matrix proteins like fibronectin, fibrinogen, and albumin influence the release proteins from primary and secondary granules of neutrophils stimulated by phorbol-myristate-acetate (PMA) and formyl-methionyl-leucyl-phenylalanine (f-MLP). Isolated granulocytes plated on wells coated with fibronectin, fibrinogen, and albumin were stimulated with f-MLP (10-7 mol/l), PMA (10-9 mol/l), Mn2+ (5 mmol/l), or combinations of these stimuli, and the degree of adhesion to protein-coated surfaces and the amount of granule proteins released was quantified during 90 min of incubation. PMA, in combination with Mn2+, induced a maximum release of approximately 80% of the intracellular content of lactoferrin and human neutrophil lipocalin (HNL) and 15-20% of the myeloperoxidase (MPO) content regardless of the protein used. PMA or f-MLP alone induced 30-40% release of lactoferrin and HNL depending on the protein that the cells were plated on. Adhesion and release of lactoferrin and HNL were quantitatively related when induced by PMA and PMA plus Mn2+, but not by f-MLP. The mean release of lactoferrin and HNL showed a significant negative relationship to the viability of the cells. In conclusion, adhesion modulates neutrophil degranulation, but it is not always quantitatively related or related in time.     

Properties of concanavalin A-elicited granule exocytosis from human polymorphonuclear neutrophils

Exposure of human neutrophils to concanavalin A (Con A) resulted in a time- and concentration-dependent extracellular release of granule-associated lysozyme but not-glucuronidase or cytosolic lactate dehydrogenase. Maximum extrusion of lysozyme occurred 30 min after cell contact with Con A. The percent of total granule enzyme activity discharged is insignificant when cells are not preincubated with cytochalasin B prior to being exposed to Con A (5–80g/ml). Granule enzyme release from Con A-treated cells is markedly inhibited by-methyl-d-mannoside. Con A-elicited extrusion of lysozyme is reduced significantly, but not abolished, in the absence of extracellular calcium. However, contact between neutrophils and EGTA in calcium-free medium had no effect on Con A-stimulated release of granule enzymes. 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), an antagonist of intracellular calcium, caused a dose-dependent inhibition of lysozyme discharge from Con A-treated neutrophils. The activity of TMB-8 could be abrogated with the addition of calcium, but not magnesium, to the extracellular medium. Therefore, Con A and TMB-8 should serve as useful tools for elucidating the mechanism of granule enzyme release from neutrophils.     

Protective role of bovine neutrophils in Pasteurella haemolytica-mediated endothelial cell damage.

The purpose of this study was to determine if Pasteurella haemolytica can directly injure bovine pulmonary endothelial cells (EC) and if neutrophils have a beneficial or detrimental role in bacterium-EC interaction. Various combinations of live P. haemolytica, heat-killed P. haemolytica, anti-P. haemolytica immune serum, polymyxin B, and bovine neutrophils were added to confluent monolayers of bovine EC. Monitoring included determination of 51Cr release from EC, phase microscopy, and transmission electron microscopy. Although toxic changes were not evident at 5 h postinoculation, both live and heat-killed P. haemolytica produced extensive EC damage by 22 h postinoculation. Damage by live P. haemolytica was prevented only when both neutrophils and immune serum were used. Polymyxin B effectively prevented the toxic effect of heat-killed P. haemolytica, suggesting that lipopolysaccharide was the major toxic factor. Morphological studies showed close apposition of P. haemolytica to EC membranes, neutrophil activation, and adherence to EC but no evidence of neutrophil-associated EC membrane damage. These studies demonstrate that neutrophils and immune serum in combination are effective in preventing EC damage mediated by live P. haemolytica.     

Live Bartonella henselae enhances endothelial cell proliferation without direct contact

The proliferation of human umbilical vein endothelial cells (HUVECs) cocultivated with live B. henselae was enhanced in a bacterial dose-dependent manner, and the stimulatory effect was specific to vascular endothelial cells. The inactivation of B. henselae by UV or heat treatment abolished its stimulatory activity, suggesting that live bacteria is necessary for the growth stimulation effect. To investigate the role of direct contact, live B. henselae were separated from HUVECs by a filter membrane (Millicell-CM insert). Even under this condition, an enhanced proliferation of HUVECs was observed. However, no morphological changes in the HUVECs were apparent compared to the B. henselae -infected cells. Furthermore, we isolated a nonpiliated strain of B. henselae that is unable to attach to and enter into endothelial cells. The nonpiliated strain possessed the ability to stimulate the proliferation of cocultivated HUVECs the same as the piliated strain. Moreover, the culture supernatants of B. henselae were also able to induce HUVEC proliferation. Our results indicate that the stimulation of HUVEC proliferation by B. henselae is mediated by soluble factor(s) secreted from the bacteria.     

Activation of a 66-kilodalton human endothelial cell matrix metalloprotease by Streptococcus pyogenes extracellular cysteine protease.

Human umbilical vein endothelial cells (HUVECs) were used to gain insight into the molecular mechanism whereby the major extracellular protease from group A streptococci damages host tissue. HUVECs exposed to streptococcal cysteine protease (SCP) for various times exhibited cytopathic effect and cell detachment from the culture vessel. Gelatin substrate zymography showed that a time- and concentration-dependent increase in the level of activity of an approximately 66-kDa gelatinase occurred in culture medium taken from cells exposed to enzymatically active SCP. This gelatinase comigrated in gelatin zymograms with the activated form of purified recombinant matrix metalloprotease 2 (MMP-2) and had type IV collagenase activity. In contrast, medium taken from cells exposed to inactivated (boiled) SCP and cells exposed to SCP inhibited by treatment with N-benzyloxycarbonyl-leucyl-valyl-glycine diazomethyl ketone lacked the 66-kDa gelatinase. Appearance of the 66-kDa gelatinase activity was also prevented by 1,10-phenanthroline, a zinc chelator and MMP inhibitor. Inasmuch as proteolytically active SCP is required for the emergence of this gelatinase and MMP activation occurs by proteolytic processing, the 66-kDa gelatinase may be a proteolytic cleavage product of a latent MMP expressed extracellularly by HUVECs. Direct SCP treatment of culture supernatant taken from HUVECs not exposed to SCP also produced the 66-kDa gelatinase. The data show that SCP activates an MMP produced by human endothelial cells, a process that may contribute to endothelial cell damage, tissue destruction, and hemodynamic derangement observed in some patients with severe, invasive group A streptococcal infection.     

Pulmonary endothelial cell killing by human neutrophils. Possible involvement of hydroxyl radical

Human blood neutrophils stimulated by a variety of agents were shown to have cytotoxic effects on bovine pulmonary artery endothelial cells. Effective agonists included immune complexes, opsonized zymosan and 12-O-tetradecanoyl phorbol acetate. Unstimulated human neutrophils and neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine or with platelet-activating factor failed to induce significant killing even though secretory release of lysosomal enzymes occurred. In comparing the effects of the different agonists, endothelial cell killing showed a better correlation with the production of H2O2 than with the generation of O2-. Endothelial cell killing by stimulated human neutrophils was inhibited by catalase but not by soybean trypsin inhibitor or superoxide dismutase. Killing was also inhibited by two scavengers (N, N-dimethylthiourea and D-mannitol) of hydroxyl radical and by deferoxamine mesylate, an iron-chelator. Iron-saturated deferoxamine mesylate was significantly less effective in protecting the endothelial cells against killing. Agents that were protective against endothelial cell killing did not interfere with the generation of O2- in stimulated neutrophils. These results suggest that leukocyte-induced endothelial cell killing in vitro can be induced by some but not all agonists for neutrophils and that the killing is oxygen-dependent and may be directly due to hydroxyl radical production.     

Stable lymphocyte contact induces remodeling of endothelial cell matrix receptor complexes

Endothelial cells (EC) actively participate in lymphocyte transendothelial migration by remodeling their actin cytoskeleton. We studied the endothelial cell abluminal matrix receptor (focal adhesion, FA) complexes to determine if these structures were remodeled following lymphocyte adhesion. Lymphocytes (PBL) were isolated from whole blood and added to cultured EC. Lectin-stimulated PBL adhered to EC spontaneously, whereas adhesion of freshly isolated lymphocytes to EC was induced by pre-treatment with MCP-1 or activating anti-CD11a mAb. Sustained adhesion between lymphocytes and EC resulted in a significant, contact-dependent decrease in paxillin incorporation into the FA following 15, but not 5, min of contact. EC FA remodeling was associated with increased phosphorylation of pp125 FA kinase. Pretreatment of the EC with an activating beta1 integrin monoclonal antibody, TS2/16, prevented lymphocyte-stimulated FA remodeling. Further, TS2/16 pretreatment inhibited transendothelial migration of lymphocytes and beta1 integrin-deficient JY lymphoblasts. These data demonstrate that sustained lymphocyte adhesion induces remodeling of EC FA structures and that this remodeling event is required for efficient lymphocyte transendothelial migration in vitro.     

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.     

肿瘤细胞向血管外游走过程中内皮细胞调控机制的研究

目的：阐明肿瘤细胞向血管外游走过程中内皮细胞肌球蛋白轻链激酶(MLCK)及Rho／Rho激酶的调控作用。方法：利用肿瘤细胞向血管外游走的体外模型，观察肿瘤细胞的血管外游走状况，并测定肿瘤细胞游走过程中血管内皮单细胞层的电阻变化；通过Western blot评价肿瘤游走过程中内皮细胞肌球蛋白轻链(MLC)的磷酸化水平。结果：肿瘤细胞可以穿过血管内皮细胞游走至血管外；并且肿瘤细胞向血管外游走过程中跨血管内皮细胞层的电阻下降、内皮细胞MLC的磷酸化水平升高；而内皮细胞肌球蛋白轻链激酶抑制剂(ML-7)及Rho抑制剂(C3转移酶)、Rho激酶抑制剂(Y-27632)能够抑制上述变化。结论：内皮细胞MLCK及Rho／Rho激酶可以通过控制MLC的磷酸化水平来引起内皮细胞骨架蛋白的改变，从而调控肿瘤细胞穿过内皮细胞间缝隙向血管外游走。     

Aortic endothelial cell migration. I. Matrix requirements and composition.

Endothelial cell migration was studied following a mechanical injury produced in cultured confluent monolayers of calf aortic endothelium with the use of a quantitative migration assay. In this method the cells were grown on glass coverslips coated with scarlet red-containing Formvar. At confluency, the cultures were cut in half with a blade; one half was removed with the pigmented Formvar, and the other was returned to culture. Migration was linear for a least 96 hours, and was due to cell motility, not proliferation. Since it was blocked in the presence of L-azetidine carboxylic acid or cis-hydroxyproline, inhibitors of collagen secretion, endothelial cell migration appeared to be dependent on the continual secretion of collagen. Furthermore, the types, apparent relative amounts, and localizations of the collagens as well as laminin changed during the migratory process. These studies support the notion that the aortic endothelial cell migratory response to injury is a dynamic one requiring the continual secretion and modulation of matrix molecules.     

Endometrial endothelial cell proliferation during the menstrual cycle

Adult endometrium is a tissue in which physiological angiogenesisoccurs regularly and provides an accessible source of materialfor study. Endometrial biopsies and immunohistochemistry wereused to test two hypotheses: firstly, that there are peaks ofendothelial cell proliferation in human endometrium during themenstrual cycle, and secondly that in-vitro endothelial cellmigratory signal production by human endometrium (measured ina previous study) accompanies endothelial cell proliferativeactivity in vivo. Proliferating cells were identified usinganti-proliferating cell nuclear antigen, and endothelial cellswere identified using anti-CD34. The method was validated bya smaller, separate study using bromodeoxyuridine incorporationand detection with formalin-fixed biopsies, and comparison withproliferating cell nuclear antigen staining. The main study(n=50) showed that significant peaks of endometrial endothelialcell proliferation could not be identified, due to the largevariability in endothelial cell proliferative activity betweenindividuals at the same stage of the menstrual cycle. Endometrialendothelial cell migratory signal production did not correlatewith endothelial cell proliferation (n = 27). Results suggestthat blood vessel growth in the endometrium may occur by a processthat differs from the traditional concept of angiogenesis, whichhas been derived mainly from in-vitro or in-vivo experimentalstudies     

Angiomotin regulates endothelial cell migration during embryonic angiogenesis

The development of the embryonic vascular system into a highly ordered network requires precise control over the migration and branching of endothelial cells (ECs). We have previously identified angiomotin (Amot) as a receptor for the angiogenesis inhibitor angiostatin. Furthermore, DNA vaccination targeting Amot inhibits angiogenesis and tumor growth. However, little is known regarding the role of Amot in physiological angiogenesis. We therefore investigated the role of Amot in embryonic neovascularization during zebrafish and mouse embryogenesis. Here we report that knockdown of Amot in zebrafish reduced the number of filopodia of endothelial tip cells and severely impaired the migration of intersegmental vessels. We further show that 75% of Amot knockout mice die between embryonic day 11 (E11) and E11.5 and exhibit severe vascular insufficiency in the intersomitic region as well as dilated vessels in the brain. Furthermore, using ECs differentiated from embryonic stem (ES) cells, we demonstrate that Amot-deficient cells have intact response to vascular endothelial growth factor (VEGF) in regard to differentiation and proliferation. However, the chemotactic response to VEGF was abolished in Amot-deficient cells. We provide evidence that Amot is important for endothelial polarization during migration and that Amot controls Rac1 activity in endothelial and epithelial cells. Our data demonstrate a critical role for Amot during vascular patterning and endothelial polarization.     

Endocytosis is required for exocytosis and priming of respiratory burst activity in human neutrophils

Objective and design

Neutrophil generation of reactive oxygen species (ROS) is enhanced by exposure to pro-inflammatory agents in a process termed priming. Priming is depending on exocytosis of neutrophil granules and p47^phox phosphorylation-dependent translocation of cytosolic NADPH oxidase components. Clathrin-mediated endocytosis was recently reported to be necessary for priming, but the mechanism linking endocytosis to priming was not identified. The present study examined the hypothesis that endocytosis regulates neutrophil priming by controlling granule exocytosis.

Materials and methods

Clathrin-mediated endocytosis by isolated human neutrophils was inhibited by chlorpromazine, monodansylcadaverine, and sucrose. Exocytosis of granule subsets was measured as release of granule components by ELISA or chemiluminescence. ROS generation was measured as extracellular release of superoxide as reduction of ferrocytochrome c. p38 MAPK activation and p47^phox phosphorylation were measured by immunoblot analysis. Statistical analysis was performed using a one-way ANOVA with the Tukey–Kramer multiple-comparison test.

Results

Inhibition of endocytosis prevented priming of superoxide release by TNFα and inhibited TNFα stimulation and priming of exocytosis of all four granule subsets. Inhibition of endocytosis did not reduce TNFα-stimulated p38 MAPK activation or p47^phox phosphorylation. Inhibition of NADPH oxidase activity blocked TNFα stimulation of secretory vesicle and gelatinase granule exocytosis.

Conclusions

Endocytosis is linked to priming of respiratory burst activity through ROS-mediated control of granule exocytosis.