Human neutrophils transform prostaglandins by a myeloperoxidase-dependent mechanism

Intact human neutrophils, incubated with the soluble stimulant phorbol myristate acetate, discharge lysosomal components, generate oxygen metabolites, and transform exogenous 6-keto-prostaglandin F1 alpha, prostaglandin E2, and prostaglandin F2 alpha as assessed by thin layer radiochromatography. Neutrophils alone were incapable of transforming the prostaglandins. The addition of catalase or the myeloperoxidase inhibitor, azide, protected all three prostaglandins from the phorbol-stimulated neutrophils. Neither superoxide dismutase, heat-inactivated catalase, nor albumin had any inhibitory effect in this system. A model system consisting of glucose-glucose oxidase, as a source of H2O2, purified myeloperoxidase, and chloride was also able to transform the prostaglandins in an identical fashion. Neither glucose-glucose oxidase alone nor glucose-glucose oxidase and myeloperoxidase under chloride-free conditions were able to mediate this transformation. Thus, it appears that intact human neutrophils can transform prostaglandins by a mechanism dependent on H2O2, the lysosomal enzyme myeloperoxidase, and chloride. Given the importance of prostaglandins in regulating immune function, neutrophil-dependent prostaglandin transformation could play a novel role in modulating the inflammatory response.     

Mast cell-mediated apoptosis of endothelial cells in vitro: a paracrine mechanism involving TNF-alpha-mediated down-regulation of bcl-2 expression

Degranulated mast cells are present in the subendothelial space of eroded (de-endothelialized) coronary atheromas. Upon degranulation, mast cells secrete into the surrounding tissue an array of preformed and newly synthesized mediators, including proapoptotic molecules, such as chymase and TNF-alpha. In a co-culture system involving rat serosal mast cells and rat cardiac (microvascular) endothelial cells, we could show, by means of competitive RT-PCR, immunoblotting, immunocytochemistry, annexin staining, flow cytometry, and DNA-laddering, that stimulation of mast cells with ensuing degranulation rapidly (within 30 min) down-regulated the expression of both bcl-2 mRNA and protein, with subsequent induction of apoptosis in the endothelial cells. The major effect of bcl-2 down-regulation resided in the exocytosed granule remnants, a minor effect also being present in the granule remnant-free supernatant. No significant changes were observed in the expression levels of the pro-apoptotic protein, bax. The mast cell-mediated apoptotic effect was partially (70%) dependent on the presence of TNF-alpha and involved the translocation of cytochrome C from mitochondria into cytoplasm. These results are the first to show that one of the cell types present in the atherosclerotic plaques, namely the mast cell, by releasing both granule-remnant-bound and soluble TNF-alpha, may contribute to the erosion of atherosclerotic plaques by inducing apoptosis in adjacent endothelial cells. Published 2003 Wiley-Liss, Inc.     

Human endothelial gelatinases and angiogenesis

Endothelial cell invasion is an essential event during angiogenesis (formation of new blood vessels). The process involves the degradation of the basement membrane and the underlying interstitium. The matrix metalloproteinase (MMP) family is considered to be primarily responsible for matrix degradation. Two members of the family, gelatinase A and B play an important role in angiogenesis. This review outlines recent findings on their regulation in human endothelial cells. Latent gelatinase B is secreted from endothelial cells. This enzyme can also accumulate in the cytosol as an active enzyme, free of TIMP-1. In contrast, latent gelatinase A is constitutively secreted from the cells. Unlike other MMPs, gelatinase A activation occurs on the cell membrane and is mediated by MT1-MMP. A number of physiological activators have recently been described. These include thrombin and activated protein C, both of which activate gelatinase A independent of the MT1-MMP pathway. These new findings may lead to therapeutic interventions for the treatment of angiogenic-dependent diseases such as cancer and arthritis.     

Synthesis of platelet-activating factor by polymorphonuclear neutrophils stimulated with interleukin-8.

Human interleukin-8 (IL-8) was evaluated for its capability to induce the synthesis and release of platelet-activating factor (PAF) from human polymorphonuclear neutrophils (PMN). IL-8 promotes in a dose-dependent fashion (1-100 ng/ml) a rapid synthesis of PAF, which is only partially released. The synthesis of PAF is preceded by the activation of acetyl-CoA: 1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyl-transferase, suggesting that IL-8 activates the "remodeling pathway" of PAF synthesis. By thin layer chromatography and reverse-phase high pressure liquid chromatography, we demonstrated that PAF synthesized by human PMN stimulated with IL-8 is heterogeneous: the 2-acetylated phospholipids having the biological and physicochemical characteristics of PAF include the 1-O-alkyl form, which is produced in large extent (51%), and the 1-acyl form (20%). The analysis of the individual molecular species of radyl chain indicated nine peaks, 16:0 and 18:0 being the predominant forms. These results identify PAF as a direct product of IL-8 stimulation in PMN.     

Differential synthesis of two interleukin-1 receptor antagonist variants and interleukin-8 by peripheral blood neutrophils

With a short lifespan and containing only few ribosomes and endoplasmic reticulum structures, neutrophils are thought to have a limited capacity for protein synthesis. We here show that peripheral blood polymorphonuclear neutrophils (PMN) are able react to stimulants with differential production of two interleukin (IL)-1 receptor antagonist (IL-1ra) isoforms, secreted IL-1ra (sIL-1ra) and the 16kDa intracellular form of IL-1ra (icIL-1ra3), as well as IL-8. Neutrophils of a high purity and with a low degree of preactivation upregulate mRNA and de novo synthesize protein of both IL-1ra variants and IL-8 in response to granulocyte-macrophage colony-stimulating factor and lipopolysaccharide. The cytokines are differentially regulated and distributed in two intracellular compartments. In comparison with peripheral blood mononuclear cells (PBMC), PMN produce distinctly more sIL-1ra but significantly less IL-8. This may indicate an anti-inflammatory role, enabling PMN to antagonize proinflammatory signals. It is therefore possible that PMN play an important role in immune regulation by counteracting a dysregulation of the inflammatory process.     

Lyssavirus matrix protein induces apoptosis by a TRAIL-dependent mechanism involving caspase-8 activation

Lyssaviruses, which are members of the Rhabdoviridae family, induce apoptosis, which plays an important role in the neuropathogenesis of rabies. However, the mechanisms by which these viruses mediate neuronal apoptosis have not been elucidated. Here we demonstrate that the early induction of apoptosis in a model of lyssavirus-infected neuroblastoma cells involves a TRAIL-dependent pathway requiring the activation of caspase-8 but not of caspase-9 or caspase-10. The activation of caspase-8 results in the activation of caspase-3 and caspase-6, as shown by an increase in the cleavage of the specific caspase substrate in lyssavirus-infected cells. However, neither caspase-1 nor caspase-2 activity was detected during the early phase of infection. Lyssavirus-mediated cell death involves an interaction between TRAIL receptors and TRAIL, as demonstrated by experiments using neutralizing antibodies and soluble decoy TRAIL-R1/R2 receptors. We also demonstrated that the decapsidation and replication of lyssavirus are essential for inducing apoptosis, as supported by UV inactivation, cycloheximide treatment, and the use of bafilomycin A1 to inhibit endosomal acidification. Transfection of cells with the matrix protein induced apoptosis using pathways similar to those described in the context of viral infection. Furthermore, our data suggest that the matrix protein of lyssaviruses plays a major role in the early induction of TRAIL-mediated apoptosis by the release of a soluble, active form of TRAIL. In our model, Fas ligand (CD95L) appears to play a limited role in lyssavirus-mediated neuroblastoma cell death. Similarly, tumor necrosis factor alpha does not appear to play an important role.     

Increased elastase release by CF neutrophils is mediated by tumor necrosis factor-alpha and interleukin-8

Cystic fibrosis (CF) is a lethal, hereditary disorder characterized by a neutrophil-dominated inflammation of the lung. We sought to determine whether neutrophils from individuals with CF release more neutrophil elastase (NE) than neutrophils from normal subjects. Our results showed that peripheral blood neutrophils (PBNs) from normal subjects and individuals with CF contained similar amounts of NE, but after preincubation with CF bronchoalveolar lavage (BAL) fluid, significantly more NE was released by CF PBNs, a release that was amplified further by incubation with opsonized Escherichia coli. To determine which components of CF BAL fluid stimulated this excessive NE release from CF PBNs, we repeated the experiments after neutralization or immunoprecipitation of tumor necrosis factor (TNF)-alpha and interleukin (IL)-8 in CF BAL fluid. We found that subsequent NE release from CF PBNs was reduced significantly when TNF-alpha and IL-8 were removed from CF BAL fluid. When TNF-alpha and IL-8 were used as activating stimuli, CF PBNs released significantly greater amounts of NE compared with PBNs from control subjects and individuals with bronchiectasis. These results indicate that CF PBNs respond abnormally to TNF-alpha and IL-8 in CF BAL fluid and react to opsonized bacteria by releasing more NE. This may help explain the increased NE burden seen in this condition.     

间充质细胞外泌体促进小鼠胰岛内皮细胞血管生成的研究

目的探讨间充质细胞（MSC）外泌体对低氧条件下胰岛内皮细胞（MS-1）血管生成的影响。 方法MSC无血清低氧条件培养48?h,超滤离心法富集条件培养基中的外泌体,采用电镜和Western Blot的方法进行鉴定;通过血管形成试验比较分析不同条件下：常氧培养组（NOR组,21﹪O₂、5﹪CO₂）、低浓度氧培养组（HYP组,2﹪O₂、5﹪CO₂）、外泌体+低浓度氧共培养组（HYP+EXO组,2﹪O₂、5﹪CO₂）,MS-1细胞的血管形成能力;image J软件分析血管形成长度;PCR、Q-PCR检测血管内皮生长因子（VEGF） RNA水平的表达,Western Blot检测VEGF、HIF1α蛋白水平表达以及mTOR信号通路激活情况。采用单因素方差分析和SNK-q检验统计学分析。 结果超滤离心法富集的MSC条件培养基中的外泌体,大小为30 ~ 100 nm,表达CD9,CD63,CD81等外泌体表面标志物;血管形成试验结果显示,低氧促进MS-1血管生成,HYP+EXO组形成明显的血管网状结构;HYP+EXO组血管形成相对长度（2386.0±137.7）像素与NOR组（393.3±174.2）像素和HYP组（1467.0±230.0）像素相比增强,差异有统计学意义（t = 12.30,P?= 0.0065;t = 15.74,P = 0.0040）;PCR结果显示,HYP+EXO组VEGF相对表达量（20.26±9.972）较常氧对照组（1.000）和低氧组（6.521±3.501）均增强,差异有统计学意义（t = 5.462,P = 0.0009;t = 4.238,P = 0.0038）;同时,Western Blot结果显示VEGF蛋白水平表达升高,HIF1-α表达上调,mTOR发生磷酸化。 结论MSC外泌体可促进低氧条件下的小鼠胰岛内皮细胞血管生成。MSC外泌体可能通过上调HIF1-α,调节VEGF表达,激活mTOR信号通路,促进胰岛内皮细胞血管生成。     

Human adipose tissue endothelial cells promote preadipocyte proliferation

Adipogenesis is preceded by development of a microvascular network, and optimal functioning of adipose tissue as an energy store and endocrine organ is dependent on extensive vascularization. We have examined the role of endothelial cell-derived factors that influence the proliferation of human preadipocytes. Microvascular endothelial cells and preadipocytes were isolated from human omental and subcutaneous adipose tissue biopsies by use of a developed procedure of collagenase digest, immunoselection, and differential trypsinization. Conditioned medium from microvascular endothelial cell cultures promoted the proliferation of preadipocytes (P = <0.001) and (to a lesser extent) other cell types. No depot-specific differences in mitogenic capacity of microvascular endothelial cell medium or of preadipocyte response were observed. These results indicate that adipose tissue endothelial cells secrete soluble adipogenic factor(s).     

Membrane type 1-matrix metalloproteinase is regulated by chemokines monocyte-chemoattractant protein-1/ccl2 and interleukin-8/CXCL8 in endothelial cells during angiogenesis

We have investigated the putative role and regulation of membrane type 1-matrix metalloproteinase (MT1-MMP) in angiogenesis induced by inflammatory factors of the chemokine family. The absence of MT1-MMP from null mice or derived mouse lung endothelial cells or the blockade of its activity with inhibitory antibodies resulted in the specific decrease of in vivo and in vitro angiogenesis induced by CCL2 but not CXCL12. Similarly, CCL2- and CXCL8-induced tube formation by human endothelial cells (ECs) was highly dependent on MT1-MMP activity. CCL2 and CXCL8 significantly increased MT1-MMP surface expression, clustering, activity, and function in human ECs. Investigation of the signaling pathways involved in chemokine-induced MT1-MMP activity in ECs revealed that CCL2 and CXCL8 induced cortical actin polymerization and sustained activation of phosphatidylinositol 3-kinase (PI3K) and the small GTPase Rac. Inhibition of PI3K or actin polymerization impaired CCL2-induced MT1-MMP activity. Finally, dimerization of MT1-MMP was found to be enhanced by CCL2 in ECs in a PI3K- and actin polymerization-dependent manner. In summary, we identify MT1-MMP as a molecular target preferentially involved in angiogenesis mediated by CCL2 and CXCL8, but not CXCL12, and suggest that MT1-MMP dimerization might be an important mechanism of its regulation during angiogenesis.     

alpha-Tocopheryl succinate inhibits angiogenesis by disrupting paracrine FGF2 signalling

Malignant mesothelioma (MM) cells enhanced proliferation of endothelial cells (ECs) as well as their angiogenesis in vitro by secretion of fibroblast growth factor-2 (FGF2). This effect was suppressed by pre-treating MM cells with alpha-tocopheryl succinate (alpha-TOS), which inhibited FGF2 secretion by inducing mitochondria-dependent generation of reactive oxygen species. The role of FGF2 was confirmed by its down-regulation by treating MM cells with siRNA, abolishing EC proliferation and wound healing enhancement afforded by MM cells. We conclude that alpha-TOS disrupts angiogenesis mediated by MM cells by inhibiting FGF2 paracrine signalling.     

Colorectal cancer cell-derived microvesicles containing microRNA-1246 promote angiogenesis by activating Smad 1/5/8 signaling elicited by PML down-regulation in endothelial cells

Emerging studies on circulating microRNAs (miRNAs) or microvesicles (MVs) have shown the potential of them to be novel biomarkers and therapeutic targets for cancer. However, the biological roles of these miRNAs and MVs have not been validated yet. To determine the biological significance of MVs, we used human colorectal cancer cells as the MV donor and endothelial cells (HUVECs) as the MV recipient and demonstrated the transfer of colorectal cancer cell-derived MVs (CRC-MVs) to HUVECs and evaluated the roles of these MVs and their cargo in tumor angiogenesis. Consequently, the incubation of HUVECs with CRC-MVs promoted the proliferation, migration, and tube formation activities of these cells. Among the cargoes shuttled by the MVs, miR-1246 and TGF-β were considered to be responsible for the pro-angiogenic function of MVs by activating Smad 1/5/8 signaling in the HUVECs. These results suggest that colorectal cancer cells secreted MVs to contribute to tumor angiogenesis.     

Human herpesvirus 8 encodes a homolog of interleukin-6.

Kaposi's sarcoma is a multifocal lesion that is reported to be greatly influenced by cytokines such as interleukin-6 (IL-6) and oncostatin M. DNA sequences of a novel human gammaherpesvirus, termed human herpesvirus 8 (HHV-8) or Kaposi sarcoma-associated herpesvirus, have been identified in all epidemiological forms of Kaposi's sarcoma with high frequency. The presence of HHV-8 DNA is also clearly associated with certain B-cell lymphomas (body cavity-based lymphomas) and multicentric Castleman's disease. Sequence analysis of a 17-kb fragment revealed that adjacent to a block of conserved herpesvirus genes (major DNA-binding protein, glycoprotein B, and DNA polymerase), the genome of HHV-8 encodes structural homolog of IL-6. This cytokine is involved not only in the pathogenesis of Kaposi's sarcoma but also in certain B-cell lymphomas and multicentric Castleman's disease. The viral counterpart of IL-6 (vIL-6) has conserved important features such as cysteine residues involved in disulfide bridging or an amino-terminal signal peptide. Most notably, the region known to be involved in receptor binding is highly conserved in vIL-6. This conservation of essential features and the remarkable overlap between diseases associated with HHV-8 and diseases associated with IL-6 disregulation clearly suggest that vIL-6 is involved in HHV-8 pathogenesis.     

Ensembles of endothelial and mural cells promote angiogenesis in prenatal human brain

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Human immunodeficiency virus type 1 enters primary human brain microvascular endothelial cells by a mechanism involving cell surface proteoglycans independent of lipid rafts

Several studies have reported a crucial role for cholesterol-enriched membrane lipid rafts and cell-associated heparan sulfate proteoglycans (HSPGs), a class of molecules that can localize in lipid rafts, in the entry of human immunodeficiency virus type 1 (HIV-1) into permissive cells. For the present study, we examined the role of these cell surface moieties in HIV-1 entry into primary human brain microvascular endothelial cells (BMVECs), which represent an important HIV-1 central nervous system-based cell reservoir and a portal for neuroinvasion. Cellular cholesterol was depleted by exposure to beta-cyclodextrins and 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase inhibitors (statins), the loss of cholesterol was quantitated, and disruption of membrane rafts was verified by immunofluorescence. Nevertheless, these treatments did not affect binding of several strains of HIV-1 virions to BMVECs at 4 degrees C or their infectivities at 37 degrees C. In contrast, we confirmed that cholesterol depletion and raft disruption strongly inhibited HIV-1 binding and infection of Jurkat T cells. Enzymatic digestion of cell-associated HSPGs on human BMVECs dramatically inhibited HIV-1 infection, and our data from quantitative HIV-1 DNA PCR analysis strongly suggest that cell-associated chondroitin sulfate proteoglycans greatly facilitate infective entry of HIV-1 into human BMVECs. These findings, in combination with our earlier work showing that human BMVECs lack CD4, indicate that the molecular mechanisms for HIV-1 entry into BMVECs are fundamentally different from that of viral entry into T cells, in which lipid rafts, CD4, and probably HSPGs play important roles.     

Human neutrophils oxidize low-density lipoprotein by a hypochlorous acid-dependent mechanism: the role of vitamin C

Oxidatively modified low-density lipoprotein (LDL) has been strongly implicated in the pathogenesis of atherosclerosis. Peripheral blood leukocytes, such as neutrophils, can oxidize LDL by processes requiring superoxide and redox-active transition metal ions; however, it is uncertain whether such catalytic metal ions are available in the artery wall. Stimulated leukocytes also produce the reactive oxidant hypochlorous acid (HOCl) via the heme enzyme myeloperoxidase. Since myeloperoxidase-derived HOCl may be a physiologically relevant oxidant in atherogenesis, we investigated the mechanisms of neutrophil-mediated LDL modification and its possible prevention by the antioxidant ascorbate (vitamin C). As a sensitive marker of LDL oxidation, we measured LDL thiol groups. Stimulated human neutrophils (5x10(6) cells/ml) incubated with human LDL (0.25 mg protein/ml) time-dependently oxidized LDL thiols (33% and 79% oxidized after 10 and 30 min, respectively). Supernatants from stimulated neutrophils also oxidized LDL thiols (33% oxidized after 30 min), implicating long-lived oxidants such as N-chloramines. Experiments using specific enzyme inhibitors and oxidant scavengers showed that HOCl, but not hydrogen peroxide nor superoxide, plays a critical role in LDL thiol oxidation by neutrophils. Ascorbate (200 microM) protected against neutrophil-mediated LDL thiol oxidation for up to 15 min of incubation, after which LDL thiols became rapidly oxidized. Although stimulated neutrophils accumulated ascorbate during oxidation of LDL, pre-loading of neutrophils with ascorbate did not attenuate oxidant production by the cells. Thus, activated neutrophils oxidize LDL thiols by HOCl- and N-chloramine-dependent mechanisms and physiological concentrations of vitamin C delay this process, most likely due to scavenging of extracellular oxidants, rather than by attenuating neutrophil oxidant production.