Interactions between cytokines and growth hormone for resistance to experimental

Cytokine-activated human vein endothelial cells (HUVEC) may play an important role in resistance to Toxoplasma gondii infection. In this study, it was investigated the role of rTNF-alpha and GH in the induction of antitoxoplasmal activities in HUVEC. Co-treatment of HUVEC with rTNF-alpha plus GH induced both toxoplasmastatic activity and the intracellular killing of T. gondii (p <0.01 each vs untreated cells). Thus, these functions were inhibited by both neutralizing antibodies to IL-6 and GM-CSF (but not to IL-3) suggesting that these cytokines participate in the inhibitory process. Consistent with this hypothesis, the treatment of HUVEC with rIL-6 or rGM-CSF in the presence of rTNF-alpha, limited T. gondii multiplication in a dose-dependent manner (p <0.01 each vs untreated cells). In order to elucidate the inhibitory mechanism of HUVEC, it was assessed by L-arginine analogs (e.g., NG-monomethyl-arginine) whether NO2 molecules originating from HUVEC were directly or indirectly involved in the rTNF-alpha/GH-dependent induction of toxoplasmastatic activity. A good correlation was found between toxoplasmastatic activity and NO2 release during the activation phase, before infection of the HUVEC with T. gondii, but no correlation was found between the parasitostatic activity and NO2 release during the infection phase. These data indicate that NO2- itself does not directly affect toxoplasmastatic activity. Besides, the reduction of intracellular killing by monoclonal antibodies to ICAM-1 suggest that this adhesin plays a role in controlling T. gondii entry into cells.     

Human mononuclear phagocyte antiprotozoal mechanisms: oxygen-dependent vs oxygen-independent activity against intracellular Toxoplasma gondii

To determine if the oxygen-dependent and -independent antiprotozoal mechanisms with which the human mononuclear phagocyte is equipped to act against Leishmania donovani operate against other intracellular parasites, oxidatively intact and deficient cells were challenged with Toxoplasma gondii. Fresh monocytes and lymphokine- or gamma-interferon (IFN-gamma)-activated macrophages from normal individuals killed 35% and 50% of T. gondii within 6 hr, respectively, and each of these cell populations inhibited the replication of surviving parasites 20 hr after infection. This activity was associated with the capacity to release large amounts of H2O2 (572 to 971 nmol/mg) and to respond to toxoplasma ingestion with respiratory burst activity. Impairing the ability to generate oxygen intermediates by glucose deprivation or treatment with superoxide dismutase, catalase, or mannitol inhibited toxoplasmacidal activity by greater than 80% and permitted a 2.6- to 4.3-fold increase in the number of intracellular toxoplasmas. In contrast to normal cells, fresh monocytes from patients with chronic granulomatous disease (CGD) killed less than 8% of toxoplasmas and exerted 50% less toxoplasmastatic activity. However, although associated with the induction of only modest toxoplasmacidal effects (18 to 20% killing), lymphokine stimulation did induce CGD monocytes and macrophages as well as oxidatively inactive human endothelial cells to display near normal levels of toxoplasmastatic activity. Similar to oxygen-dependent mechanisms, the enhancement of oxygen-independent activity by crude lymphokines could be abolished by a monoclonal anti-IFN-gamma antibody and could be achieved by treatment with recombinant IFN-gamma alone. Unstimulated CGD monocytes, however, were found to lose all antitoxoplasma activity after two days in culture, whereas normal cells continued to effectively inhibit T. gondii replication, suggesting that oxygen-independent responses may not actually be required for the normal monocyte to act against T. gondii. Taken together with previous findings with L. donovani, these results indicate that the human mononuclear phagocyte possesses an oxygen-independent antiprotozoal mechanism and that its effects can be enhanced by lymphokines (IFN-gamma), but that nevertheless this cell's primary response to intracellular protozoa is largely oxygen dependent.     

Antimicrobial and immunoregulatory effects mediated by human lung cells: role of IFN-gamma-induced tryptophan degradation

Pneumonia caused by bacterial, viral and parasitic pathogens is one of the most common clinical problems facing primary and secondary care physicians. Staphylococcus aureus is a common cause of lung abscesses in humans and, in immunocompromised patients, herpes simplex virus type I and Toxoplasma gondii can cause severe life-threatening pneumonia. The authors focused their interest in the antimicrobial effects mediated by human lung cells against these pathogens. It was found that IFN-gamma-stimulated lung cells are capable of inhibiting T cell proliferation and restrict the replication of microorganisms such as T. gondii, S. aureus and herpes simplex virus. This immunoregulatory and antimicrobial effect was enhanced in the presence of IL-1 or tumor necrosis factor-alpha (TNF-alpha). Furthermore, the IFN-gamma-dependent antimicrobial effects of HBE4-E6/E7 (human lung bronchus epithelial cells) and A549 (human type II alveolar cells) correlated with the activation of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO). It was found that both the abrogation of IDO activity by the specific IDO-inhibitor 1-L-methyltryptophan and the supplementation of cultures with tryptophan result in an inhibition of IFN-gamma-induced antimicrobial effects mediated by lung cells. Therefore it is suggested that tryptophan depletion via IFN-gamma-mediated IDO induction is a major antibacterial, antiparasitic, antiviral and immunoregulatory mechanism in human lung cells.     

Induction of suppressor of cytokine signaling-1 by Toxoplasma gondii contributes to immune evasion in macrophages by blocking IFN-gamma signaling

Toxoplasma gondii is an intracellular parasite that survives and multiplies in professional phagocytes such as macrophages. Therefore, T. gondii has to cope with the panel of antimicrobial host immune mechanisms, among which IFN-gamma plays a crucial role. We report in this study that in vitro infection of murine macrophages with viable, but not with inactivated, parasites results in inhibition of IFN-gamma signaling within the infected cells. Thus, infection of RAW264.7 macrophages with tachyzoites inhibited IFN-gamma-induced STAT-1 tyrosine phosphorylation, mRNA expression of target genes, and secretion of NO. These effects were dependent on direct contact of the host cells with living parasites and were not due to secreted intermediates. In parallel, we report the induction of suppressor of cytokine signaling-1 (SOCS-1), which is a known feedback inhibitor of IFN-gamma receptor signaling. SOCS-1 was induced directly by viable parasites. SOCS overexpression in macrophages did not affect tachyzoite proliferation per se, yet abolished the inhibitory effects of IFN-gamma on parasite replication. The inhibitory effects of T. gondii on IFN-gamma were diminished in macrophages from SOCS-1-/- mice. The results suggest that induction of SOCS proteins within phagocytes due to infection with T. gondii contributes to the parasite's immune evasion strategies.     

Antimicrobial and immunoregulatory effects mediated by human lung cells: role of IFN-γ-induced tryptophan degradation

Pneumonia caused by bacterial, viral and parasitic pathogens is one of the most common clinical problems facing primary and secondary care physicians. Staphylococcus aureus is a common cause of lung abscesses in humans and, in immunocompromised patients, herpes simplex virus type I and Toxoplasma gondii can cause severe life-threatening pneumonia. The authors focused their interest in the antimicrobial effects mediated by human lung cells against these pathogens. It was found that IFN-γ-stimulated lung cells are capable of inhibiting T cell proliferation and restrict the replication of microorganisms such as T. gondii , S. aureus and herpes simplex virus. This immunoregulatory and antimicrobial effect was enhanced in the presence of IL-1 or tumor necrosis factor-α (TNF-α). Furthermore, the IFN-γ-dependent antimicrobial effects of HBE4-E6/E7 (human lung bronchus epithelial cells) and A549 (human type II alveolar cells) correlated with the activation of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO). It was found that both the abrogation of IDO activity by the specific IDO-inhibitor 1- l -methyltryptophan and the supplementation of cultures with tryptophan result in an inhibition of IFN-γ-induced antimicrobial effects mediated by lung cells. Therefore it is suggested that tryptophan depletion via IFN-γ-mediated IDO induction is a major antibacterial, antiparasitic, antiviral and immunoregulatory mechanism in human lung cells.     

NAD(P)H-oxidase presence in Toxoplasma gondii tachyzoite vacuole during interaction with IFN-gamma-activated human endothelial cells

Toxoplasma gondii invades and proliferates in human umbilical vein endothelial cells (HUVEC) where it resides in a parasitophorous vacuole (PV) preventing lysosomal fusion. To study the intracellular outcome of PV containing tachyzoites of T. gondii during interaction with IFN-gamma-activated HUVEC, a quantitative analysis of the T. gondii infection and multiplication was assayed. The quantification of PVs' fusion with lysosomes, ultrastructural examination of phagosome-lysosome fusion, and the localization of NAD(P)H-oxidase activity were also investigated. HUVEC activated with IFN-gamma inhibited T. gondii infection and multiplication by 67.5% and 91.0%, respectively. After 4 hr of infection, 10.2% of IFN-gamma-activated HUVEC exhibited phagosome-lysosome fusion assayed by fluorescence microscopy, which was also observed at the ultrastructural level. Furthermore, the enzyme NAD(P)H-oxidase present at the plasma membrane of activated HUVEC was internalized together with the parasite in 38.0% of the cells. In addition, colocalization of colloidal gold particles and reaction product of NAD(P)H-oxidase in the PV of some activated HUVEC was observed. These results suggest that NAD(P)H-oxidase may participate in a mechanism by which IFN-gamma-activated HUVEC inhibit T. gondii multiplication.     

Luteolin inhibits pyrogallol-induced apoptosis through the extracellular signal-regulated kinase signaling pathway

Luteolin is an antioxidative, antitumor and anti-inflammatory flavone. It has been shown to reduce endothelial dysfunction, but the mechanism is not clear. We set out to explore the effects of luteolin on apoptosis and its mechanism of action in endothelial cells. The effect of luteolin on pyrogallol-induced superoxide stress and the subsequent apoptosis was studied in the mouse heart capillary endothelial cell line H5V and human umbilical vein endothelial cells, by the use of flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, Hoechst staining, and western blot. Pyrogallol (0-400 μm) dose-dependently induced reactive oxygen species production, cytotoxicity, an annexin V-fluorescein isothiocyanate increase, mitochondrial transmembrane depolarization and DNA condensation in both H5V and human umbilical vein endothelial cells; these actions were reversed by luteolin (0.78-50 μm) in a concentration-dependent manner. Luteolin suppressed the poly (ADP-ribose) polymerase activation, caspase-8 cleavage and p38 mitogen-activated protein kinase activation triggered by pyrogallol, and stimulated the extracellular signal-regulated kinase signaling pathway to counteract the pyrogallol-induced apoptotic signals. Luteolin is an effective agent for the protection of endothelial cells from superoxide stress-induced apoptosis via the extracellular signal-regulated kinase signaling pathway.     

Anionic sites,fucose residues and class I human leukocyte antigen fate during interaction of Toxoplasma gondii with endothelial cells

Toxoplasma gondii invades and proliferates in human umbilical vein endothelial cells where it resides in a parasitophorous vacuole. In order to analyze which components of the endothelial cell plasma membrane are internalized and become part of the parasitophorous vacuole membrane, the culture of endothelial cells was labeled with cationized ferritin or UEA I lectin or anti Class I human leukocyte antigen (HLA) before or after infection with T. gondii. The results showed no cationized ferritin and UEA I lectin in any parasitophorous vacuole membrane, however, the Class I HLA molecule labeling was observed in some endocytic vacuoles containing parasite until 1 h of interaction with T. gondii. After 24 h parasite-host cell interaction, the labeling was absent on the vacuolar membrane, but presents only in small vesicles near parasitophorous vacuole. These results suggest the anionic site and fucose residues are excluded at the time of parasitophorous vacuole formation while Class I HLA molecules are present only on a minority of Toxoplasma-containing vacuoles.     

Safrole oxide induces human umbilical vein endothelial cell transdifferentiation to 5-hydroxytryptaminergic neuron-like cells through tropomyosin receptor kinase A/cyclooxygenase 2/nuclear factor-kappa B/interleukin 8 signaling

The phenomenon of endothelial-neural transdifferentiation has been observed for a long time, but the mechanism is not clear. We previously found that safrole oxide induced human umbilical vein endothelial cell transdifferentiation into neuron-like cells. In this study, we first validated that these cells induced by safrole oxide were functional 5-hydroxytryptaminergic neuron-like cells. Then, we performed microarray analysis of safrole oxide-treated and -untreated human umbilical vein endothelial cells. Safrole oxide elevated the levels of cyclooxygenase 2 (COX-2), interleukin-8 (IL-8) and reactive oxygen species (ROS), which was accompanied by nuclear factor-kappa B (NF-κB) nuclear translocation during the transdifferentiation. Blockade of tropomyosin receptor kinase A (TrkA) by an inhibitor or short hairpin RNA inhibited the levels of COX-2/IL-8 and the nuclear translocation of NF-κB but did not suppress the increased ROS level. As a result, cells underwent apoptosis. Therefore, via TrkA, safrole oxide may induce endothelial cell transdifferentiation into functional neuron-like cells. During this process, the increased levels of COX-2/IL-8 and the subsequent elevation of ROS production induced NF-κB nuclear translocation and IL-8 secretion. With the activity of TrkA inhibited, the inactive NF-κB regulated the ROS level in a negative feedback manner. Finally, the transdifferentiation pathway was blocked and cells became apoptotic. The TrkA/COX-2/IL-8 signal pathway may have an important role in endothelial-neural transdifferentiation, and safrole oxide may trigger this process by activating TrkA.     

Troglitazone-binding to LDL and its glycated modifications: its role in cell-catalysed and Cu-mediated LDL-oxidation

Troglitazone (T), an anti-diabetic drug improving insulin resistance, was studied as to its inhibition of copper ion-catalysed oxidation of native, glycated and glycoxidated low-density lipoprotein (LDL). A dose-dependent inhibition was noted in the concentration range 40-160 microg/ml. An almost complete inhibition of oxidation (2-8 h), as monitored by the formation of thiobarbituric acid-reactive substances, was observed for both native and glycated LDL at a concentration of 160 microg/ml T, while the maximal inhibition for glycoxidated LDL amounted only to 60% at this concentration of the drug. This is reflected by differences in the affinity of the drug for the different types of LDL modification: While the binding of T both to native or glycated LDL increased linearly with increasing T concentration and was not saturable in the concentration range tested (0-160 microg/ml), binding of the drug to glycoxidated LDL was already nearly saturated at 10 microg/ml. The nearly complete inhibitory action of T towards oxidation of native and glycated LDL was lost, however, upon increasing the total oxidation time to 24 h. In human umbilical vein endothelial cell-mediated oxidation of LDL, T at a concentration of 20 microg/ml significantly reduced formation of oxidation-dependent fluorescent chromophores and liberation of 8-epi-PGF2alpha. In contrast, generation of thiobarbituric acid-reactive substances was not significantly inhibited. As opposed to copper-mediated LDL-oxidation, different binding of T to LDL-modifications does not govern inhibition of human umbilical vein endothelial cell-mediated LDL-oxidation.     

Induction of oxidative stress by humic acid through increasing intracellular iron: a possible mechanism leading to atherothrombotic vascular disorder in blackfoot disease

Humic acid (HA), a potential toxin that has penetrated the drinking well water of blackfoot disease-endemic areas in Taiwan, has been implicated as an etiological factor of this disease. In this study, we investigated the effects of HA on the generation of reactive oxygen species (ROS) in cultured human umbilical vein endothelial cells (HUVECs). The generation of ROS was monitored by flow cytometry. Pretreatment of HUVECs with HA induced reactive oxygen species in a dose- and time-dependent manner. Xanthine oxidase inhibitor (Allopurinol), NADPH oxidase inhibitor (diphenylene iodomium) and calcium chelator (BAPTA) could not reduce the generation of ROS. Protein kinase C inhibitor (H7) could reduce the generation of ROS slightly, but the intracellular antioxidant glutathione monoethyl ester and the iron chelator desferrioxamine (DFO) could inhibit the generation of ROS completely. HA also enhanced the expression of ferritin and induced intracellular chelatable iron; however, HA reduced the expression of transferrin receptor. Pretreatment with DFO inhibited HA-mediated increases of ferritin synthesis and intracellular chelatable iron, but caused recovery of the inhibitory effect on transferrin receptor. Cotreatment with iron and HA induced more ROS and intracellular chelatable iron than iron or HA treatment alone. Furthermore, HA enhanced the accumulation of iron in endothelial cells. These data demonstrate that HA can increase the generation of ROS through enhancing the accumulation of intracellular iron. Taken together, our findings suggest that iron mediates HA-associated oxidative stress in endothelial cells, which may be a possible mechanism leading to atherothrombotic vascular injury observed for patients with blackfoot disease.     

Susceptibility of Eimeria bovis and Toxoplasma gondii to oxygen intermediates and a new mathematical model for parasite killing

Eimeria bovis and Toxoplasma gondii differ in their susceptibility to macrophages activated by lymphokines. Interferon-gamma can activate macrophages to totally inhibit E. bovis sporozoite development, whereas growth of T. gondii tachyzoites in macrophages is not totally affected. The susceptibility of these parasites to oxygen intermediates and their ability to evade the oxidative burst by macrophages were investigated in cell-free systems. Using a logistic model to assess growth inhibition, T. gondii growth was impaired by 50% at 10(-4.25) M (56 microM) H2O2, with 30 min as the optimum time for measuring inhibition. Preliminary results indicate that T. gondii follows mode-one and mode-two killing with relation to time after exposure to H2O2, implying a role for OH. and the induction of a DNA repair mechanism. The same model was used to assess inhibition of E. bovis growth that was more susceptible, being inhibited to 50% by 10(-5) M (10 microM) H2O2. Both parasites were susceptible to the effects of xanthine-xanthine oxidase that releases a full complement of oxygen intermediates (H2O2, OH., (1)O2, and O2-). Adding quenchers or scavengers to the system confirmed that T. gondii was susceptible to products of the interaction of O2- and H2O2 (OH. and (1)O2), and that E. bovis sporozoites were at least partially susceptible to H2O2 and O2-, but extremely susceptible to OH.. These data were supported by studies on scavenging enzymes present in the parasites. Toxoplasma gondii was rich in superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPO), and E. bovis had less catalase and SOD.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelial cell adhesiveness for human T lymphocytes is inhibited by transforming growth factor-beta 1.

Recombinant human transforming growth factor-beta (TGF-beta) was found to inhibit the adhesive phenotype of human umbilical vein endothelial cells for human PBL, purified T lymphocytes, and PHA-activated lymphoblasts. TGF-beta inhibited lymphocyte attachment to resting human umbilical vein endothelial cells and also to endothelial monolayers stimulated with the pro-inflammatory cytokines TNF-alpha and IL-1 beta. Our investigations also show that the ability of endothelial cells to respond to TGF-beta by altering their adhesiveness is lost with prolonged culture of the cells. However, this loss is selective as TGF-beta inhibits cell proliferation in both early and late passage endothelial cells. These results suggest that in vivo TGF-beta may inhibit the adhesive phenotype of endothelial cells and also may limit the immunologic response occurring at the endothelial cell barrier.     

Homocysteine induces oxidative stress by uncoupling of NO synthase activity through reduction of tetrahydrobiopterin

Hyperhomocysteinemia is a risk factor for cardiovascular diseases that induces endothelial dysfunction. Here, we examine the participation of endothelial NO synthase (eNOS) in the homocysteine-induced alterations of NO/O(2)(-) balance in endothelial cells from human umbilical cord vein. When cells were treated for 24 h, homocysteine dose-dependently inhibited thrombin-activated NO release without altering eNOS phosphorylation and independently of the endogenous NOS inhibitor, asymmetric dimethylarginine. The inhibitory effect of homocysteine on NO release was associated with increased production of reactive nitrogen and oxygen species (RNS/ROS) independent of extracellular superoxide anion (O(2)(-)) and was suppressed by the NOS inhibitor L-NAME. In unstimulated cells, L-NAME markedly decreased RNS/ROS formation and the ethidium red fluorescence induced by homocysteine. This eNOS-dependent O(2)(-) synthesis was associated with reduced intracellular levels of both total biopterins (-45%) and tetrahydrobiopterin (-80%) and increased release of 7,8-dihydrobiopterin and biopterin in the extracellular medium (+40%). In addition, homocysteine suppressed the activating effect of sepiapterin on NO release, but not that of ascorbate. The results show that the oxidative stress and inhibition of NO release induced by homocysteine depend on eNOS uncoupling due to reduction of intracellular tetrahydrobiopterin availability.     

Type V collagen selectively inhibits human endothelial cell proliferation

Type V collagen from human placenta remarkably inhibited human umbilical vein endothelial cell (HUVEC) proliferation in a dose-dependent manner when coated on the culture dishes. Other types of collagen (I, III, IV) and fibronectin enhanced HUVEC proliferation under the same conditions. The inhibitory activity of type V collagen was seen not only when it was coated on the dishes, but also when it was directly added into cell culture. The attachment effect of type V collagen did not differ from that of type I collagen. The inhibitory activity is a phenomenon selective for endothelial cells, since type V collagen did not affect the proliferation of human umbilical vein smooth muscle cells, aortic smooth muscle cells, or nasal mucosa fibroblasts.     

IFN-gamma-induced L-arginine-dependent toxoplasmastatic activity in murine peritoneal macrophages is mediated by endogenous tumor necrosis factor-alpha.

Activated murine peritoneal macrophages inhibit the intracellular proliferation of Toxoplasma gondii and produce a number of cytokines, such as TNF-alpha and IL-1. Both TNF-alpha and IL-1 have been reported to be involved in the immune response against various microorganisms, but the mechanisms responsible for these effects are not known. In the present study it was investigated whether endogenously produced TNF-alpha and IL-1 are involved in the activation of peritoneal macrophages by rIFN-gamma leading to toxoplasmastatic activity and the production of reactive nitrogen intermediates. The rIFN-gamma-induced toxoplasmastatic activity was inhibited by neutralizing antibodies against mouse TNF-alpha in a dose-dependent and time-dependent way, but neutralizing antibodies against mouse IL-1 alpha and IL-1 beta did not affect this activity. Involvement of TNF-alpha in the induction of toxoplasmastatic activity was confirmed by our finding that rTNF-alpha in combination with a nonactivating concentration of rIFN-gamma inhibited the intracellular proliferation of T. gondii. No synergistic activity of rIL-1 and rIFN-gamma on the inhibition of T. gondii proliferation was found. Both rTNF-alpha and rIL-1 alpha alone inhibited the intracellular proliferation of T. gondii only slightly. Because it has been reported recently that activated macrophages produce reactive nitrogen intermediates that are essential in the induction of toxoplasmastatic activity, we investigated whether these intermediates are involved in the TNF-dependent induction of toxoplasmastatic activity. Neutralizing antibodies against mouse TNF-alpha inhibited also the release of NO2- by rIFN-gamma-activated macrophages almost completely. Macrophages incubated with rTNF-alpha in combination with a nonactivating concentration of rIFN-gamma released substantial amounts of NO2-, but rTNF-alpha and rIL-1 alpha alone, and the combination of rIL-1 alpha and a nonactivating concentration of rIFN-gamma induced only little NO2(-)-release by macrophages. To assess whether reactive nitrogen intermediates act directly or indirectly on the intracellular proliferation of T. gondii, macrophages were incubated with the L-arginine analog NG-monomethyl-L-arginine or the NADPH-inhibitor diphenylene iodonium, both inhibitors of the generation of reactive nitrogen intermediates. Good correlation was found between toxoplasmastatic activity and the release of NO2- during the 24-h activation period before infection of the macrophages with T. gondii, but no correlation was found between toxoplasmastatic activity and the release of NO2- during infection of the macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)     

The antioxidant systems in Toxoplasma gondii and the role of cytosolic catalase in defence against oxidative injury

Superoxide dismutase, catalase, glutathione peroxidase and peroxiredoxins form an antioxidant network protecting cells against reactive oxygen species (ROS). Catalase is a potent H2O2-detoxifying enzyme, which is unexpectedly absent in some members of the Kinetoplastida and Apicomplexa, but present in Toxoplasma gondii. In T. gondii, catalase appears to be cytosolic. In addition, T. gondii also possesses genes coding for other types of peroxidases, including glutathione/thioredoxin-like peroxidases and peroxiredoxins. This study presents a detailed analysis of the role of catalase in the parasite and reports the existence of antioxidant enzymes localized in the cytosol and the mitochondrion of T. gondii. The catalase gene was disrupted and, in addition, T. gondii cell lines overexpressing either catalase or a cytosolic 1-cys peroxiredoxin, TgPrx2, under the control of a strong promoter were created. Analysis of these mutants confirmed that the catalase activity is cytosolic and is encoded by a unique gene in T. gondii. Furthermore, the catalase confers protection against H2O2 exposure and contributes to virulence in mice. The overexpression of Prx2 also increases protection against H2O2 treatment, suggesting that catalase and other peroxidases function as a defence mechanism against endogenously produced reactive oxygen intermediates and the oxidative stress imposed by the host.     

Critical effect of VEGF in the process of endothelial cell apoptosis induced by high glucose

The underlying molecular mechanism whereby hyperglycemia causes endothelial cell apoptosis is not well understood. This study aims to elucidate the role of survival factor VEGF involved in the apoptosis of endothelial cells induced by elevated glucose. The present study confirmed that high concentration of glucose (25 mmol/l) significantly increased the apoptotic cell number in cultured primary human umbilical vein endothelial cells (HUVEC). Up-regulation of Bax/Bcl-2 ratio and activation of caspase-3 induced by high glucose suggested that mitochondria apoptosis pathway was involved. High glucose significantly reduced VEGF expression in HUVEC both at mRNA and protein levels. p42/44 MAPK phosphorylation was transitory attenuated when exposed to high glucose and preceded VEGF reduction, thus suggesting down-regulation of VEGF through inhibition of p42/44 MAPK. Addition of VEGF prevented HUVEC apoptosis from high glucose exposure. Moreover, elevated reactive oxygen species (ROS) generation, calcium overload, Bax/Bcl-2 ratio, caspase-3 activation in HUVEC induced by high glucose were reversed by pre-challenge with VEGF. This may represent a mechanism for the anti-apoptotic effect of VEGF. These results suggest that down-regulation of VEGF plays a critical role in apoptosis of endothelial cells induced by high glucose and restoration of VEGF might have benefits in the early stage of diabetic endothelial dysfunction. Zhonghan Yang, Xuehua Mo, and Qing Gong have contributed equally to this study.     

促吞噬肽衍生物 T 肽对裸鼠术后残瘤 VEGF 表达的影响

目的：探讨促吞噬肽衍生物T肽抑制裸鼠术后残瘤生长的作用机理。方法：建立MCF-7乳腺癌裸鼠皮下移植瘤术后残瘤模型,观察8mg／kg剂量T肽对残余肿瘤组织的生长情况,并采用免疫组化和Western印迹检测给药后残瘤组织血管内皮生长因子（VEGF）的表达,采用RT-PCR检测不同T肽浓度对血管内皮细胞VEGF基因转录的影响。结果：T肽对MCF-7乳腺癌裸鼠皮下移植瘤术后残瘤生长表现出良好的抑制作用,按照瘤重得出的抑制率为68．2％,按照肿瘤体积得出的抑制率为67．6％,T肽给药组裸鼠残瘤组织中VEGF的表达量较空白对照组明显减少。血管内皮细胞中T肽呈剂量相关性抑制VEGF基因的转录。结论：T肽的抗癌作用与其抑制肿瘤微环境肿瘤组织和血管内皮细胞中VEGF的表达有密切联系,预示着T肽有潜力成为预防术后残瘤生长的抗癌新药。     

Wogonin suppresses tumor growth in vivo and VEGF-induced angiogenesis through inhibiting tyrosine phosphorylation of VEGFR2

Previous studies revealed that wogonin, a naturally occurring monoflavonoid extracted from Scutellariae radix, possessed anticancer activity both in vitro and in vivo. However, the molecular mechanism of its potent anticancer activity remains poorly understood and warrants further investigations. In this study, we found for the first time that wogonin inhibited the growth and tumor angiogenesis of human gastric carcinoma in nude mice. We explored the inhibitory effect of wogonin on angiogenesis stimulated by vascular endothelial growth factor (VEGF) in vitro. Wogonin suppressed the VEGF-stimulated migration and tube formation of human umbilical vein endothelial cells (HUVECs). It also restrained VEGF-induced tyrosine phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2). This inhibition of receptor phosphorylation was correlated with a significant decrease in VEGF-triggered phosphorylated forms of ERK, AKT and p38. Taken together, these findings strongly suggest that wogonin might be a promising antitumor drug.