Tumor necrosis factor-induced endothelial tissue factor is associated with subendothelial matrix vesicles but is not expressed on the apical surface.

Cultured endothelial cells can be induced by tumor necrosis factor/cachectin (TNF) and other cytokines to synthesize the procoagulant cofactor tissue factor (TF). Intact monolayers of TNF-treated endothelial cells showed only minimal TF activity. In contrast, after permeabilization of these monolayers with detergent (saponin, 0.02%), there was approximately 10- to 20-fold increase in TF-mediated, factor VIIa-dependent factor Xa formation. Extracellular matrix derived from TNF-treated endothelium, prepared after removing the cells by hypotonic lysis or ammonium hydroxide (0.1 N), also had similarly enhanced TF activity. Incubation with a blocking monoclonal antibody to TF inhibited the procoagulant activity of both TNF-stimulated endothelial cells, whether they were intact or permeabilized, and of their matrices. However, when the apical cell surface was pretreated with anti-TF antibody, washed, and then cells were lysed with water or permeabilized with saponin, similar augmentation of TF activity was still observed, suggesting the presence of a pool of TF to which the antibody did not initially gain access. Consistent with this concept, the presence of TF in the matrix of TNF-treated endothelial cells was shown by immunoblotting and morphologic studies; cultured endothelial monolayers and the native endothelium of aortic segments after exposure to TNF showed TF in extracellular matrix, associated with vesicles. In contrast, TF was virtually undetectable on the apical endothelial surface. Taken together, these findings suggest that endothelial TF can be present in a cryptic pool that only gains access to the blood after alteration in the integrity of the endothelial monolayer.     

Tumor necrosis factor and endothelial cell death

Tumor necrosis factor (TNF) is a multifunctional cytokine that elicits various responses in endothelial cells that can be summarized as being proinflammatory and procoagulant. Apoptosis refers to a genetically programmed form of cell death in which the cell participates in its own demise. Although TNF can activate the death pathway in normal human endothelial cells, apoptosis only results when protein synthesis is blocked. This finding indicates that TNF elicits both survival and death pathways. Endothelial cell apoptosis has been reported in various pathophysiologic situations from vascular development to immune disorders and atherosclerosis. The role of TNF-mediated endothelial death in these situations remains to be defined.     

The role of fatty acids and caveolin-1 in tumor necrosis factor alpha-induced endothelial cell activation

Hypertriglyceridemia and associated high circulating free fatty acids are important risk factors for atherosclerosis. In contrast to omega-3 fatty acids, linoleic acid, the major omega-6 unsaturated fatty acid in the American diet, may be atherogenic by amplifying an endothelial inflammatory response. We hypothesize that omega-6 and omega-3 fatty acids can differentially modulate tumor necrosis factor α (TNF-α)-induced endothelial cell activation and that functional plasma membrane microdomains called caveolae are required for endothelial cell activation. Caveolae are particularly abundant in endothelial cells and play a major role in endothelial trafficking and the regulation of signaling pathways associated with the pathology of vascular diseases. To test our hypothesis, endothelial cells were preenriched with either linoleic acid or α-linolenic acid before TNF-α-induced endothelial activation. Measurements included oxidative stress and nuclear factor κB-dependent induction of cyclooxygenase-2 (COX-2) and prostaglandin E₂ (PGE₂) under experimental conditions with intact caveolae and with cells in which caveolin-1 was silenced by small interfering RNA. Exposure to TNF-α induced oxidative stress and inflammatory mediators, such as p38 mitogen-activated protein kinase (MAPK), nuclear factor κB, COX-2, and PGE₂, which were all amplified by preenrichment with linoleic acid but blocked or reduced by α-linolenic acid. The p38 MAPK inhibitor SB203580 blocked TNF-α-mediated induction of COX-2 protein expression, suggesting a regulatory mechanism through p38 MAPK signaling. Image overlay demonstrated TNF-α-induced colocalization of TNF receptor type 1 with caveolin-1. Caveolin-1 was significantly induced by TNF-α, which was further amplified by linoleic acid and blocked by α-linolenic acid. Furthermore, silencing of the caveolin-1 gene completely blocked TNF-α-induced production of COX-2 and PGE₂ and significantly reduced the amplified response of linoleic acid plus TNF-α. These data suggest that omega-6 and omega-3 fatty acids can differentially modulate TNF-α-induced inflammatory stimuli and that caveolae and its fatty acid composition play a regulatory role during TNF-α-induced endothelial cell activation and inflammation.     

COPD患者肺泡巨噬细胞源性MMP-9的表达及其酶活性变化

目的探讨TNFΑ对慢性阻塞性肺疾病(COPD)患者肺泡巨噬细胞(AM)源性基质金属蛋白酶9(MMP-9)表达与酶活性的影响及其可能调节机制。方法从COPD患者支气管肺泡灌洗液中分离与培养AM,以0.1、1、5、10、20 NG/M L TNFΑ刺激24 H后,用半定量RT-PCR法检测MMP-9MRNA表达,明胶酶谱法检测MMP-9酶活性,凝胶阻滞分析实验检测NF-ΚB活性。结果当AM未被TNFΑ刺激时,MMP-9 MRNA表达吸光度比值为0.1826±0.0084,MMP-9酶活性的相对吸光度值为10 227±738。随着TNFΑ刺激剂量增加,MMP-9 MRNA表达相应增加,分别为0.4279±0.0204、0.5431±0.0058、0.6185±0.0208、0.6859±0.0080、0.7784±0.0208(P<0.05);MMP-9酶活性相应增强,分别为40 528±974、113 741±4547、190 696±4032、309 505±7639、427 305±9145(P<0.05)。以10 NG/M L TNFΑ刺激AM后,NF-ΚB的活性较刺激前显著增强(P<0.05)。结论TNFΑ能够诱导COPD患者AM表达MMP-9,TNFΑ诱导的AM源性MMP-9的表达可能与NF-ΚB介导的信号转导途径有关。     

Tumor necrosis factor alpha response elements in the HLA-DRA promoter: identification of a tumor necrosis factor alpha-induced DNA-protein complex in astrocytes.

The cytokine tumor necrosis factor alpha (TNF-alpha) alone does not induce class II major histocompatibility complex (MHC) expression in most primary cells but can regulate ongoing class II expression in either a positive or negative fashion. The mechanism(s) by which TNF-alpha enhances interferon gamma (IFN-gamma)-induced class II expression was examined in a primary cell type, the astrocyte, by transient transfection of the HLA-DRA promoter linked to a chloramphenicol acetyltransferase reporter gene (DRA-CAT). We show that TNF-alpha, while having no effect on its own, can synergize with IFN-gamma to increase the level of promoter activity of a DRA-CAT construct. Three known sequences--W, X, and Y--are required for TNF-alpha enhancement of IFN-gamma-induced promoter activity. The corollary effect of TNF-alpha on DNA-binding proteins specific for these elements was examined. A previous report described a DNA-binding protein, IFN-gamma-enhanced factor X (IFNEX), which is upregulated by IFN-gamma in astrocytes and is specific for the X box of the DRA promoter. In this study, we found that TNF-alpha alone did not induce any nuclear proteins; however, combined treatment of astrocytes with both IFN-gamma and TNF-alpha induced a DNA-protein complex of slower electrophoretic mobility than IFNEX. The TNF-alpha-induced complex (TIC-X) has specificity for the X element of the DRA promoter. These results suggest a mechanism by which TNF-alpha enhances IFN-gamma-induced class II MHC expression via the formation of TIC-X.     

Tumor necrosis factor type alpha, a potent inhibitor of endothelial cell growth in vitro, is angiogenic in vivo.

Tumor necrosis factor type alpha (TNF-alpha) inhibits endothelial cell proliferation in vitro. Basal cell growth (in the absence of exogenously added growth factor) and fibroblast growth factor (FGF)-stimulated cell proliferation are inhibited in a dose-dependent manner from 0.1 to 10 ng/ml with half-maximal inhibition occurring at 0.5-1.0 ng of TNF-alpha per ml. Bovine aortic and brain capillary endothelial and smooth muscle cells are similarly affected. TNF-alpha is a noncompetitive antagonist of FGF-stimulated cell proliferation. Its action on endothelial cells is reversible and noncytotoxic. Surprisingly, TNF-alpha does not seem to inhibit endothelial cell proliferation in vivo. In the rabbit cornea, even a high dose of TNF-alpha (10 micrograms) does not suppress angiogenesis induced by basic FGF. On the contrary, in this model system TNF-alpha stimulates neovascularization. The inflammatory response that is seen in the cornea after TNF-alpha implantation suggests that the angiogenic properties of this agent may be a consequence of leukocyte infiltration.     

Claudin 1 mediates tumor necrosis factor alpha-induced cell migration in human gastric cancer cells

AIM: To investigate the role of claudin 1 in the regulation of genes involved in cell migration and tumor necrosis factor alpha (TNF-α)-induced gene expression in human gastric adenocarcinoma cells.METHODS: Knockdown experiments were conducted with claudin 1 small interfering RNA (siRNA), and the effects on the cell cycle, apoptosis, migration and invasion were analyzed in human gastric adenocarcinoma MKN28 cells. The gene expression profiles of cells were analyzed by microarray and bioinformatics.RESULTS: The knockdown of claudin 1 significantly inhibited cell proliferation, migration and invasion, and increased apoptosis. Microarray analysis identified 245 genes whose expression levels were altered by the knockdown of claudin 1. Pathway analysis showed that the top-ranked molecular and cellular function was the cellular movement related pathway, which involved MMP7, TNF-SF10, TGFBR1, and CCL2. Furthermore, TNF- and nuclear frctor-κB were the top-ranked upstream regulators related to claudin 1. TNF-α treatment increased claudin 1 expression and cell migration in MKN28 cells. Microarray analysis indicated that the depletion of claudin 1 inhibited 80% of the TNF-α-induced mRNA expression changes. Further, TNF-α did not enhance cell migration in the claudin 1 siRNA transfected cells.CONCLUSION: These results suggest that claudin 1 is an important messenger that regulates TNF-α-induced gene expression and migration in gastric cancer cells. A deeper understanding of these cellular processes may be helpful in establishing new therapeutic strategies for gastric cancer.     

Chlamydia pneumoniae alters mildly oxidized low-density lipoprotein-induced cell death in human endothelial cells, leading to necrosis rather than apoptosis

BACKGROUND: Atherosclerosis is characterized by oxidative stress that induces lipid and protein oxidation in the vascular wall. Oxidized low-density lipoproteins (oxLDLs) are present in lesions, and one of their actions is to induce apoptosis or necrosis in vascular cells. A role for Chlamydia pneumoniae in atherosclerosis has been proposed, but the mechanisms involved remain largely unknown. METHODS: The in vitro effect of C. pneumoniae infection on apoptosis induced by mildly oxidized LDLs (moxLDLs) in human endothelial cells was studied. RESULTS: Infection inhibited apoptosis, as was demonstrated by a decrease in such apoptotic features as cytochrome c release, caspase activity, 89-kilodalton poly(ADP-ribose) polymerase (PARP) fragment formation, nuclear condensation and fragmentation, and DNA fragmentation. However, the inhibition of apoptosis did not favor cell survival, because infection promoted cell death with necrotic features, as was illustrated by an increase in lactate dehydrogenase release, an enhancement of necrotic cellular morphological characteristics, and generation of low-molecular-mass PARP fragments. The increase in occurrence of necrosis-like cell death was correlated with a strong increase in intracellular reactive oxygen species (ROS) concentration. Vitamin E inhibited ROS production and promoted cell survival, underscoring the involvement of ROS in cell death induced by the combination of C. pneumoniae and moxLDLs. CONCLUSION: C. pneumoniae infection enhances the inflammatory action of oxLDLs in the vascular wall, leading to cell necrosis rather than apoptosis.     

Septic acute kidney injury: the culprit is inflammatory apoptosis rather than ischemic necrosis

For a long time, acute kidney injury (AKI) was considered to be a primarily hemodynamic condition characterized by a reduction of renal blood flow, induced by either cardiogenic or distributive (septic) shock. Consequently, all efforts to treat AKI were essentially concentrated on increasing renal flow by enhancing cardiac flow output and perfusion pressure. At the beginning of this decade, Bellomo and co-workers produced new and intriguing data in an animal model of septic AKI that undermined existing concepts. They observed that medullar and cortical renal blood flow were both maintained and even increased in septic shock, underscoring that septic AKI was a totally different physiological phenomenon than nonseptic AKI. Also, apoptosis was found to play a more important role in sepsis and septic shock than pure necrosis. Despite these findings, the role of apoptosis as a main mechanism of organ dysfunction remains topic of debate.     

Activated factor X and thrombin formation triggered by tissue factor on endothelial cell matrix in a flow model: effect of the tissue factor pathway inhibitor

The procoagulant subcellular matrix of stimulated endothelial cells that contains tissue factor (TF) was used to investigate the mechanism by which TF pathway inhibitor (TFPI) inhibits thrombin formation initiated by TF/factor VIIa (FVIIa) under flow conditions. Purified coagulation factors VII, X, and V and prothrombin were perfused at a wall shear rate of 100 s-1 through a flow chamber containing a coverslip covered with matrix of cultured human umbilical vein endothelial cells. This resulted in a TF- and FVII-dependent FXa and thrombin generation as measured in the effluent at the outlet of the system. Inhibition of this TF/FVIIa-triggered thrombin formation by TFPI purified from plasma was dependent on the amount of TF present on the endothelial cell matrix. The rate of prothrombinase assembly and steady-state levels of thrombin formation were decreased by TFPI. Because persistent albeit decreased steady-state levels of thrombin formation occurred in the presence of TFPI, we conclude that plasma- TFPI does not inhibit FXa present in the prothrombinase complex. The addition of FIX and FVIII to perfusates containing FVII and FX increased the FXa generation on endothelial matrices, and counteracted the inhibition of thrombin formation on endothelial cell matrices by TFPI. Our data provide further evidence for the hypothesis that the rapid inactivation of TF/FVIIa by TFPI in combination with the absence of either FVIII or FIX causes the bleeding tendency of patients with hemophilia A or B.     

Bid is upstream of lysosome-mediated caspase 2 activation in tumor necrosis factor alpha-induced hepatocyte apoptosis

BACKGROUND & AIMS: During tumor necrosis factor alpha-mediated hepatocyte cytotoxicity, cathepsin B is released from lysosomes and contributes to apoptosis by indirectly promoting mitochondrial dysfunction. How this lysosomal pathway mediates mitochondrial dysfunction is unclear. Because Bcl-2 family proteins and caspase 2 have been implicated in proximal apoptosis-signaling pathways, we examined the role of these proteins in tumor necrosis factor alpha-induced lysosomal permeabilization and cathepsin B-mediated mitochondrial dysfunction. METHODS: Studies were performed in primary hepatocytes from wild-type cathepsin B knockout, Bid knockout, and caspase 2 knockout mice and in the rat hepatoma cell line McArdle7777 by using tumor necrosis factor alpha/actinomycin D. RESULTS: Studies in wild-type and Bid knockout hepatocytes showed that tumor necrosis factor alpha-mediated lysosomal permeabilization is Bid dependent. After tumor necrosis factor alpha/actinomycin D treatment, caspase 2 activity increased severalfold in wild-type hepatocytes, whereas minimal activity was observed in hepatocytes from cathepsin B knockout mice or in hepatoma cells treated with a cathepsin B inhibitor. In contrast, Bax was activated independently of cathepsin B. Pharmacological, genetic, or small interfering RNA-mediated inhibition of caspase 2 attenuated tumor necrosis factor alpha-mediated mitochondrial dysfunction, downstream caspase activation, and hepatocyte apoptosis. CONCLUSIONS: These data suggest that tumor necrosis factor alpha triggers Bid-dependent lysosomal permeabilization, followed by release of cathepsin B into the cytosol and activation of caspase 2. Caspase 2 then facilitates efficient mitochondrial cytochrome c release and apoptosis.     

肿瘤坏死因子α和基质金属蛋白酶家族与左室重构的关系

目的　探讨肿瘤坏死因子 (TNF)α与基质金属蛋白酶家族 (MMPs)MMP 2、3、9与心肌重构、心功能损害的关系。方法　采用双抗体夹心ELISA法测定心力衰竭 (心衰 )患者TNFα血浓度 ,Westernblot法对 31例风湿性心脏病 (RHD)心衰患者及 8例健康人心室肌组织中MMP 2、3、9蛋白表达含量进行半定量分析 ;RHD患者术前心功能指标采用超声心动图检查。结果　心衰患者TNFα血浓度随心衰加重而增高 (P <0 0 5或 <0 0 1) ,心肌组织MMP 2、3、9蛋白表达随心衰程度加重而增加 (P <0 0 5或程 <0 0 1) ,TNFα血浓度与心肌组织MMP 2、3、9蛋白表达存在明显的正相关关系 (P <0 0 1或 <0 0 0 1)。结论　TNFα可能通过刺激MMPs的表达 ,引起细胞外基质结构发生改变 ,加重心衰时的左室重构 ,引起心功能恶化     

Transport of insulin-like growth factor-I across endothelial cell monolayers and its binding to the subendothelial matrix.

Cultured human umbilical vein endothelial cells (HUVEC) were used as a model to study transendothelial IGF-I transport, and its deposition into the extracellular matrix (ECM). Specific binding of (125)I-IGF-I to HUVEC monolayers was demonstrated, which was inhibited by aIR-3, a specific antibody directed against the IGF-I receptor. ECM-associated (125)I-IGF-I was approximately 10% of cell-bound IGF-I at 22 degrees C, and increased 4.5-fold at 37 degrees C, indicating that endothelial metabolism is required for the transport. However, neither monensin and cytochalasin B, both of which block endocytosis, nor aIR-3 did inhibit transport of (125)I-IGF-I into the ECM. In order to characterize IGF-I binding to the subendothelial ECM, HUVEC were removed nonenzymatically by treatment with Triton X-100 and ammonia. Specific, saturable binding of (125)I-IGF-I to the isolated ECM was observed, which was protease-sensitive. Antibodies directed against vitronectin inhibited IGF-I binding to the matrix by 35%, while antibodies directed against other ECM proteins had no significant influence on IGF-I binding. Using radioimmunoassays the IGF binding protein-2 was detected in the ECM, while IGFBP-1 and IGFBP-3 were below the detection limits. In order to evaluate functional aspects of IGF-I binding to the matrix, HUVEC were incubated under serum-free conditions in the absence and presence of IGF-I. Under serum-free conditions 48% of cells rounded up and started to detach after 2 hours incubation, while only 23% of the cells started to detach in the presence of IGF-I. These data indicate that IGF-I is transported via a paracellular route across endothelial cells, and becomes bound to the subendothelial ECM. Vitronection seems to be involved in binding of IGF-I to the ECM. ECM-associated IGF-I might play a role in endothelial cell survival and stability.     

Tumor necrosis factor leads to the internalization and degradation of thrombomodulin from the surface of bovine aortic endothelial cells in culture

Tumor necrosis factor (TNF), a mediator of the inflammatory response, induces tissue factor and decreases the expression of thrombomodulin (TM) on endothelial cells, thus shifting the hemostatic properties of the endothelium. To determine the mechanism of TM downregulation, bovine aortic endothelial cells in culture were treated with TNF (2 nmol/L) and the fate of TM followed. Both surface expressed TM (antigen and activity), and the total TM pool (measured by radioimmunoassay and activity in detergent extracts) dropped to less than or equal to 20% of control values within 12 hours of TNF treatment. TM was not found in an immunologically recognizable form in the supernatants of treated cultures. Chloroquine (greater than or equal to 100 mumol/L) was able to abrogate the TNF effect on the total TM pool but not the effect on surface-expressed TM activity. We conclude that TNF induces the internalization and subsequent degradation of the TM molecule. None of the components of the protein C anticoagulant pathway, either alone or in combination, prevented the TNF-dependent downregulation of TM antigen.     

Tumor necrosis factor induces the production of urokinase-type plasminogen activator by human endothelial cells

Endothelial cells play an important role in the regulation of fibrinolysis by the production of several key regulatory proteins. The cytokines tumor necrosis factor (TNF), lymphotoxin, and interleukin-1 (IL-1), but not interleukin-6, increase the production of plasminogen activator inhibitor-1 (PAI-1) by endothelial cells, whereas they have no stimulatory effect on the production of tissue-type plasminogen activator (t-PA). Primary cultures of human endothelial cells release very little urokinase-type plasminogen activator (u-PA). We report here that TNF and lymphotoxin induce, in a concentration-dependent way, the production of both cellular and secreted u-PA antigen in primary and subcultured human endothelial cells. The TNF-induced increase was accompanied by a more than 10-fold increase in u-PA mRNA. Upon stimulation of early passage umbilical vein endothelial cells by TNF, u-PA was predominantly secreted at the basolateral side, whereas PAI activity and t-PA were found in more equal amounts at the apical and basolateral sides of the cell monolayers. TNF-stimulated u-PA secretion by subcultured human aorta endothelial cells showed only a marginal polarity. The u-PA antigen was present in a plasmin-activatable form (single chain u-PA) and in a nonactivatable form (probably u-PA: PAI-1 complex). During the induction of u-PA by TNF, the ratio between plasmin-activatable u-PA and total u-PA decreased markedly. This may indicate that TNF also increases the degree of u-PA activation. The parallel induction of the synthesis and secretion of both u-PA and PAI-1 by endothelial cells adds a new aspect to the alterations of the fibrinolytic system caused by inflammatory mediators. This aspect may be significant for the regulation of cell-associated and interstitial plasminogen activator activity.