Regulation of FasL/Fas in human trophoblasts: possible implications for chorioamnionitis

Chorioamnionitis is a common cause of premature birth and is associated with significant morbidity and mortality in the mother and infant. Preterm birth shares similarities with rejection of the fetal allograft, which is characterized by increased apoptosis of placental trophoblasts. We hypothesized that there is increased trophoblast apoptosis in chorioamnionitis and that this increased apoptosis is mediated by the Fas ligand (FasL)/Fas pathway. To test our hypothesis, we examined placental villous tissues from patients with chorioamnionitis and used the TUNEL assay to demonstrate enhanced trophoblast apoptosis in patients with chorioamnionitis. When the same samples were stained for Fas, there was increased trophoblast Fas expression in patients with chorioamnionitis. To define the mechanisms responsible for this increase in trophoblast apoptosis, we cultured villous explants from uncomplicated term placentas with proinflammatory cytokines and demonstrated a marked increase in trophoblast apoptosis. By blocking FasL, we reduced tumor necrosis factor alpha-induced and interferon gamma-induced apoptosis. These data suggest that chorioamnionitis is associated with increased trophoblast apoptosis and enhanced trophoblast Fas expression. As a complement to our in vivo study, we demonstrated that cytokine-induced trophoblast apoptosis is mediated in part by the FasL/Fas pathway, suggesting that cytokines promote sensitivity to Fas-mediated apoptosis. These mechanisms may be important in perpetuating inflammation in the placental microenvironment and may contribute to the pathogenesis of chorioamnionitis.     

Apoptosis in human cultured trophoblasts is enhanced by hypoxia and diminished by epidermal growth factor

Preeclampsia and fetal growthrestriction are associated with placental hypoperfusion and villoushypoxia. The villous response to this environment includes diminishedtrophoblast differentiation and enhanced apoptosis. We tested thehypothesis that hypoxia induces apoptosis in cultured trophoblasts, andthat epidermal growth factor (EGF), an enhancer of trophoblastdifferentiation, diminishes hypoxia-induced apoptosis. Trophoblastsisolated from placentas of term-uncomplicated human pregnancies werecultured up to 72 h in standard (PO₂ = 120 mmHg) or hypoxic (PO₂ < 15 mmHg) conditions. Exposure to hypoxia for 24 h markedly enhanced trophoblast apoptosis as determined by DNA laddering, internucleosomal in situ DNA fragmentation, and histomorphology, as well as by thereversibility of the apoptotic process with a caspase inhibitor. Apoptosis was accompanied by increased expression of p53 and Bax anddecreased expression of Bcl-2. Addition of EGF to cultured trophoblastsor exposure of more differentiated trophoblasts to hypoxiasignificantly lowered the level of apoptosis. We conclude that hypoxiaenhances apoptosis in cultured trophoblasts by a mechanism thatinvolves an increase in p53 and Bax expression. EGF and enhancement ofcell differentiation protect against hypoxic-induced apoptosis.

     

Growth factors protect in vitro cultured embryos from the consequences of oxidative stress

The aim of the study was to evaluate the effect of insulin-like growth factors (IGF1 and IGF2), stem cell factor (SCF) and epidermal growth factor (EGF) on the development of embryos exposed to oxidative stress. C3B6F1 female mice were stimulated with 5 IU of pregnant mare serum gonadotropin and 5 IU of equine chorionic gonadotropin (eCG). Two-cell embryos were flushed out from the fallopian tubes 40 h after eCG administration and mating with DBA males. In each experiment embryos were divided into three groups and cultured in (1) control medium, (2) control medium with 0.1 mM hydrogen peroxide and (3) control medium with hydrogen peroxide and separately with IGF1, IGF2, SCF or EGF in concentrations of 1 ng/ml, 10 ng/ml and 100 ng/ml. Under phase-contrast microscopy, 8-cell and compacted embryos, and early, expanded, hatched and outgrown blastocysts were counted at 24 h. The total blastocyst (TB) and inner cell mass (ICM) cell numbers were established by differential staining. Blastocyst cell viability was examined under fluorescence microscopy. To detect apoptosis, TUNEL was performed and visualized under a laser scanning confocal microscope. Hydrogen peroxide decreased embryo growth, blastocyst rates, blastocyst cell viability as well as TB and ICM counts. The TUNEL reaction revealed significantly more apoptotic cells in oxidative stress conditions. Tested factors revealed a varying extent of protective activity against oxidative stress caused by hydrogen peroxide. In media containing hydrogen peroxide and one of the four tested factors (IGF1, IGF2, SCF or EGF) the embryos developed faster than in media with hydrogen peroxide alone. IGF1, IGF2 and EGF increased both TB and (or) ICM counts in embryos exposed to hydrogen peroxide. All tested factors reduced the number of apoptotic cells (TUNEL) in embryos exposed to hydrogen peroxide.     

Cadmium-induced apoptosis of primary epithelial lung cells: Involvement of Bax and p53, but not of oxidative stress

The lung is a target organ for cadmium (Cd) toxicity. Apoptosis induced by cadmium acetate (CdAc) was studied in alveolar type 2 cells and Clara cells isolated from rat lung. Relatively low concentrations of CdAc (1–10 μmol/L) induced apoptosis after exposure for 20 h. Type 2 cells were more sensitive than Clara cells to Cd-induced apoptosis and loss of cell viability. On exposure to 10 μmol/L CdAc, the levels of the apoptosis-modulating proteins p53 and Bax were increased at 2 h and 5–12 h, respectively. The expression of p53 preceded the expression of Bax and the apoptotic process. The exposure to 10 μmol/L CdAc did not significantly increase the formation of cellular reactive oxygen species (ROS). However, after exposure to a high concentration of CdAc (100 μmol/L), a 30% increase of the ROS level was observed. No significant nitric oxide production was measured following CdAc exposure. Catalase, superoxide dismutase, dimethyl sulfoxide, or tetramethylthiourea did not protect against Cd-induced apoptosis. In conclusion, the results show that Clara cells and type 2 cells are sensitive to Cd-induced apoptosis. Increased levels of p53 and Bax are suggested to be involved in the apoptosis. The apoptosis did not appear to be mediated by oxidative pathways. This revised version was published online in August 2006 with corrections to the Cover Date.     

Rapid suppression of multidrug resistance of leukemic cells by oxidative srtess

The effect of a short-time (1 h) oxidative stress on multidrug resistance (MDR) of murine leukemic P388VR cells has been investigated. We studied the production of reactive oxygen species (ROS) in cells depending on the composition of medium and the concentration of cells and hydrogen peroxide, as well as the effect of hydrogen peroxide on MDR of cells. MDR was determined from the transport of calcein acetoxymethyl ester out of the cells and from a change in cell sensitivity to vincristine. The amount of ROS arising in cells was determined using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH₂-DA). It was shown that the rate of ROS formation in cells decreases after the addition of serum to the medium and with an increase of the cell number. By the action of hydrogen peroxide, the amount of ROS increases directly with its concentration. Oxidative stress generated by 30–300 μM hydrogen peroxide decreases the MDR of the cells. The effect of hydrogen peroxide increases with the treatment duration and concentration of hydrogen peroxide. MDR determined by the criterion of the efflux of calcein ester from cells is completely suppressed after 1-h exposure to 300 μM hydrogen peroxide. At a concentration of hydrogen peroxide of 60 μM and treatment duration of 1 h, the sensitivity of P388VR cells to vincristine increases to reach the sensitivity of the wild-type P388 cells. Rapid (about 1 h) suppression of MDR is caused by inhibition of the activity of transport proteins. MDR decrease induced by oxidative stress can be used in therapy of tumors resistant to anticancer drugs.     

Inhibition of trophoblast cell invasion by TGFB1, 2, and 3 is associated with a decrease in active proteases

Invasion of extravillous trophoblast cells into the uterus in human pregnancy is tightly regulated. The transforming growth factor-beta (TGFB) family has been suggested to play a role in controlling this process. We hypothesized that TGFB1, 2, and 3 would inhibit the invasive capacity of extravillous trophoblast cells. We also studied trophoblast apoptosis and proliferation and secreted protease levels as potential mechanisms by which these cytokines may act. Inhibition of endogenous TGFB1, 2, and 3 with neutralizing antibodies increased the invasive capacity of extravillous trophoblast cells derived from placental explants. Similarly, addition of exogenous TGFB1, 2, and 3 inhibited the invasive capacity of these cells in a dose-dependent manner. Proliferation of trophoblast in the placental explants did not alter in response to any of the cytokines tested. Apoptosis of villous and extravillous trophoblast did not alter in response to TGFB1, 2, and 3. There was a reduction in secreted levels of matrix metalloproteinase (MMP) 9 and urokinase plasminogen activator in response to all three cytokines. MMP2 and tissue inhibitor of metalloproteinase 1 and 3 levels were not altered. These results suggest that TGFB1, 2, and 3 inhibit trophoblast invasion by a mechanism dependent on reduced protease activity.     

Preeclampsia Is Associated with Alterations in the p53-Pathway in Villous Trophoblast

Background

Preeclampsia (PE) is characterized by exaggerated apoptosis of the villous trophoblast of placental villi. Since p53 is a critical regulator of apoptosis we hypothesized that excessive apoptosis in PE is mediated by abnormal expression of proteins participating in the p53 pathway and that modulation of the p53 pathway alters trophoblast apoptosis in vitro.

Methods

Fresh placental villous tissue was collected from normal pregnancies and pregnancies complicated by PE; Western blotting and real-time PCR were performed on tissue lysate for protein and mRNA expression of p53 and downstream effector proteins, p21, Bax and caspases 3 and 8. To further assess the ability of p53 to modulate apoptosis within trophoblast, BeWo cells and placental villous tissue were exposed to the p53-activator, Nutlin-3, alone or in combination with the p53-inhibitor, Pifithrin-α (PFT- α). Equally, Mdm2 was knocked-down with siRNA.

Results

Protein expression of p53, p21 and Bax was significantly increased in pregnancies complicated by PE. Conversely, Mdm2 protein levels were significantly depleted in PE; immunohistochemistry showed these changes to be confined to trophoblast. Reduction in the negative feedback of p53 by Mdm2, using siRNA and Nutlin-3, caused an imbalance between p53 and Mdm2 that triggered apoptosis in term villous explants. In the case of Nutlin, this was attenuated by Pifithrin-α.

Conclusions

These data illustrate the potential for an imbalance in p53 and Mdm2 expression to promote excessive apoptosis in villous trophoblast. The upstream regulation of p53 and Mdm2, with regard to exaggerated apoptosis and autophagy in PE, merits further investigation.     

Effect of human parathyroid hormone on the cAMP production and the endocrine functions of trophoblast cells from first trimester placenta.

Our previous study on teratocarcinoma cells suggested the role of human parathyroid hormone (hPTH) in early development of the placenta. The purpose of this study was to evaluate the possible role of hPTH on the functions of first trimester trophoblast cells. Adenylate cyclase activity in crude membranes from first trimester human placental villous tissue is stimulated 2-fold by hPTH (1-34) (10(-6) mol.l-1) from 265 +/- 32 to 532 +/- 80 pmol of cAMP/mg protein/15 min. A similar stimulation of adenylate cyclase is observed in human term placental villous tissue but not in 3 different choriocarcinoma cell lines. In order to evaluate the possible role of hPTH on the functions of first trimester human trophoblast cells, these cells were isolated by dispase and cultured (2 x 10(5) cells per plate) in DMEM supplemented with 20% fetal calf serum with or without 100 ng/ml of epidermal growth factor (EGF), for 4 d. On d 2 of culture, hPTH (10(-7) mol.l-1) stimulates cAMP production of these cells from 0.52 +/- 0.2 to 2.58 +/- 0.57 pmol.h-1 per 10(6) cells (mean +/- SEM). As compared to control (30 ng/ml), the output of hCG is increased by 1.5- (NS), 2- (P less than 0.01) and 3- (P less than 0.01) fold by EGF, hPTH, and hPTH added with EGF, respectively. Dibutyryl cAMP (10(-3) mol.l-1) increased hCG secretion by 3-fold (P less than 0.05). EGF and hPTH added separately or together significantly stimulated (P less than 0.01) the secretion of free alpha subunit 2-fold from 35 ng/ml to 70 ng/ml. In contrast, hPTH and EGF added separately did not change the secretion of free beta hCG. However, added together, they significantly increased (P less than 0.01) the secretion of free beta hCG after 48 h of culture, maximal stimulation (2.5 fold) being observed at d 4 of culture. In conclusion, human trophoblast cells are target cells for hPTH. hPTH acts in association with EGF in promoting expression of endocrine activity of these cells, such as hCG secretion. Trophoblast cells provide a model for the study of the cooperative effect between a peptide hormone and a growth factor in the regulation of endocrine function.     

Epidermal growth factor inhibits ceramide-induced apoptosis and lowers ceramide levels in primary placental trophoblasts

The activation of sphingomyelinase and the subsequent generation of ceramide are emerging as important components of signaling pathways leading to apoptosis. The combination of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) induces apoptosis of primary placental trophoblasts in vitro. This apoptosis is inhibited completely by cotreatment with epidermal growth factor (EGF). We therefore examined the role of sphingomyelinase and ceramide in trophoblast apoptosis and how this may be influenced by EGF. Exogenous C₁₆-ceramide (20 μM) and acid sphingomyelinase induced trophoblast apoptosis, an effect abrogated completely by cotreatment with 10 ng/ml EGF. Neutral sphingomyelinase also increased ceramide levels but did not induce apoptosis. Treatment with EGF alone decreased cellular ceramide levels. This decrease could be blocked by cotreatment with the acid ceramidase inhibitor N-oleoylethanolamine (OE). OE alone increased ceramide levels and induced apoptosis that could not be blocked by cotreatment with EGF. In contrast, the alkaline ceramidase inhibitor D-MAPP, although it also increased ceramide levels, did not induce apoptosis nor did it affect TNF-α/IFN-α-induced cell death. These results implicate sphingolipids as important mediators in trophoblast apoptosis and suggest that the antiapoptotic properties of EGF can in part be explained by its control of ceramide concentrations in trophoblasts. J. Cell. Physiol. 180:263–270, 1999. © 1999 Wiley-Liss, Inc.     

Fungicidal effect of hydrogen peroxide on fungal infection of rainbow trout eggs

Exposure to 1,500 μg/ml of hydrogen peroxide (H₂O₂) for 60 min at 13°C was found to be injurious to rainbow trout eggs. On the other hand, the concentration which effectively inhibited pathogenic fungi in vitro was substantially less than this toxic dosage; specifically, 500 μg/ml for 60 min at 20°C to inhibit the zoosporic stage and 1,000 μg/ml for 60 min at 20°C to inhibit the vegetative stage. From in vivo tests, treatment with 1,000 μg/ml of H₂O₂ for 60 min at 13°C was found to be the most effective procedure to control fungal infection and increase the hatching rate of rainbow trout eggs.     

Vitamin B6 suppresses apoptosis of NM-1 bovine endothelial cells induced by homocysteine and copper

Hyperhomocysteinemia is an important risk factor for atherosclerosis. We previously reported that formation of early atherosclerosis in the rat aorta was associated with hyperhomocysteinemia and reduction of antioxidant activity caused by low concentration of vitamin B₆in vivo. In the present study, we examined effects of vitamin B₆ on apoptosis of bovine endothelial cells (NM-1 cells) treated with homocysteine and copper. Homocysteine and copper induced extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. Cell viability was reduced to 30% compared to that of control cells. On the other hand, pyridoxal treatment as well as EDTA treatment increased viability of NM-1 cells treated with homocysteine and copper to about 60%, and significantly decreased extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. The treatment of catalase recovered cell viability and reduced the level of extracellular hydrogen peroxide and intracellular ROS. Cell death by homocysteine and copper was confirmed to be due to apoptosis by evaluation of DNA fragmentation and by TUNEL assay. However, apoptosis of NM-1 cells induced by homocysteine and copper was due to a caspase-independent pathway as it was not inhibited by the caspase inhibitor, Z-VAD-fmk. Apoptosis of NM-1 cells induced by homocysteine and copper accompanied with mitochondrial permeability but not cytochrome c release. These results suggest that pyridoxal treatment suppresses apoptosis of NM-1 cells induced by homocysteine and copper, most likely through antioxidant effects.     

Syncytiotrophoblast is a barrier to maternal-fetal transmission of herpes simplex virus

Herpes simplex virus (HSV)-1 has been discovered in placental tissue from spontaneous miscarriages, but reports of transplacental transmission and fetal infection are extremely rare. Previously, we demonstrated that the villous syncytiotrophoblast, which forms a continuous layer between the maternal and fetal circulation, is resistant to HSV entry. Here, we tested our hypothesis that the villous syncytiotrophoblast prevents transplacental transmission of HSV secondary to decreased expression of HSV entry mediators (HveA, HveB, and HveC). In addition, we investigated the ability of HSV to infect extravillous trophoblast cells, which mediate placental attachment to the uterine wall, and the expression of HSV receptors in these cells. We performed fluorescence-activated cell sorting (FACS) analyses and immunostaining to demonstrate that HveA, HveB, and HveC were not expressed in third-trimester villous trophoblast cells. Consequently, villous explants obtained from third-trimester placentas were resistant to infection by a recombinant HSV-1 vector, HSV-1 KOS, but approximately 20% of mesenchymal cells within the villous core were infected when villous explants were pretreated with trypsin to disrupt the villous trophoblast layer. Conversely, FACS analysis and immunostaining demonstrated that extravillous trophoblast cells expressed HveA, HveB, and HveC, and these cells were efficiently infected by HSV vectors. Infection of extravillous trophoblast cells by HSV-1 was not reduced when the cells were pretreated with an antibody against HveA but was partially reduced when the cells were pretreated with antibodies directed against HveB and HveC. Thus, the decreased expression of herpesvirus entry mediators in villous syncytiotrophoblast prevents placental villous infection, thereby limiting maternal-fetal transmission of HSV.     

Testing the vicious cycle theory of mitochondrial ROS production: effects of H2O2 and cumene hydroperoxide treatment on heart mitochondria

Vicious cycle theories of aging and oxidative stress propose that ROS produced by the mitochondrial electron transport chain damage the mitochondria leading exponentially to more ROS production and mitochondrial damage. Although this theory is widely discussed in the field of research on aging and oxidative stress, there is little supporting data. Therefore, in order to help clarify to what extent the vicious cycle theory of aging is correct, we have exposed mitochondria in vitro to different concentrations of hydrogen peroxide or cumene-hydroperoxide (0, 30, 100 and 500 μM). We have found that 30 μM hydrogen peroxide (or higher concentrations) inhibit oxygen consumption in state 3 and increase ROS production with pyruvate/malate but not with succinate as substrate, indicating that these effects occur specifically at complex I. Similar levels of cumene-OOH inhibit state 3 respiration with both kinds of substrates, and increase ROS production in both state 4 and state 3 with pyruvate/malate and with succinate. The effects of cumene-OOH on ROS generation are due to action of the peroxide in the complex III or in the complex III plus complex I ROS generators. In all cases, the increase in ROS production occurred at a threshold level of peroxide exposure without further exponential increase in ROS generation. These results are consistent with the idea that ROS production can contribute to increase oxidative stress in old animals, but the results do not fit with a vicious cycle theory in which peroxide generation leads exponentially to more and more ROS production with age.     

Beryllium-stimulated reactive oxygen species and macrophage apoptosis

Beryllium (Be), the etiologic agent of chronic beryllium disease, is a toxic metal that induces apoptosis in human alveolar macrophages. We tested the hypothesis that Be stimulates the formation of reactive oxygen species (ROS) which plays a role in Be-induced macrophage apoptosis. Mouse macrophages were exposed to 100 microM BeSO4 in the absence and presence of the catalytic antioxidant MnTBAP (100 microM). Apoptosis was measured as the percentage of TUNEL+ and caspase-8+ cells. ROS production was measured by flow cytometry using the fluorescence probes, dihydroethidine (DHE) and dichlorofluorescein diacetate (DCFH-DA). Be-exposed macrophages had increased TUNEL+ cells (15+/-1% versus controls 1+/-0.2%, P<0.05) and increased caspase-8+ cells (18.7+/-2% versus controls 1.8+/-0.4%, P<0.05). Be-induced caspase-8 activation, and a 4-fold increase in ROS formation, was ameliorated by exposure to MnTBAP. Hydrogen peroxide (30 microM) exposure potentiated Be-induced caspase-8 activation, and was also attenuated by MnTBAP. Our data are the first to demonstrate that Be stimulates macrophage ROS formation which plays an important role in Be-induced macrophage apoptosis.     

IGF2 actions on trophoblast in human placenta are regulated by the insulin-like growth factor 2 receptor, which can function as both a signaling and clearance receptor

Insulin-like growth factor 2 (IGF2) enhances proliferation and survival of human first-trimester cytotrophoblasts (CTB) by signaling through the insulin-like growth factor 1 receptor (IGF1R). However, the role of the IGF2 receptor (IGF2R) in regulating trophoblast kinetics is unclear: It could act as a clearance receptor for trafficking excess ligand to lysosomes for degradation and/or directly mediate IGF2 signaling. We used an IGF2R knockdown strategy in BeWo cells and placental villous explants to investigate trophoblast proliferation and survival in response to stimulation by IGF. Both IGF1 and IGF2 significantly (P < 0.001) increased mitosis and reduced apoptosis in serum-starved BeWo cells. Small interfering RNA (siRNA)-mediated knockdown of IGF2R further enhanced IGF2-stimulated mitosis (P < 0.01), and IGF2-mediated rescue of apoptosis (P < 0.001) in these cells. Leu(27)IGF2, an IGF2 analogue that binds to IGF2R but not IGF1R, also protected IGF2R-expressing BeWo cells from apoptosis but did not increase mitosis. IGF treatment of term placental villous explants with reduced syncytial expression of IGF2R increased CTB proliferation (P < 0.001) and decreased apoptosis (P < 0.01) compared to untreated controls. Moreover, IGF2-mediated rescue of CTB apoptosis was significantly greater than that in tissue with normal IGF2R expression. Leu(27)IGF2 promoted mitogenesis and survival only in explants with intact IGF2R expression. Given that altered CTB turnover is observed in pregnancies complicated by fetal growth restriction, the development of strategies to manipulate the IGF2R signaling axis in the syncytiotrophoblast may provide a therapeutic avenue for treating this condition.     

Intra-uterine growth restriction is associated with increased apoptosis and altered expression of proteins in the p53 pathway in villous trophoblast

Intrauterine growth restriction (IUGR) affects 3–8% of pregnancies and is associated with altered cell turnover in the villous trophoblast, an essential functional cell type of the human placenta. The intrinsic pathway of apoptosis, particularly p53, is important in regulating placental cell turnover in response to damage. We hypothesised that expression of proteins in the p53 pathway in placental tissue would be altered in IUGR. Expression of constituents of the p53 pathway was assessed using real-time PCR, Western blotting and immunohistochemistry. p53 mRNA and protein expression was increased in IUGR, which localised to the syncytiotrophoblast. Similar changes were noted in p21 and Bax expression. There was no change in the expression of Mdm2, Bak and Bcl-2. The association between altered trophoblast cell turnover in IUGR and increased p53 expression is reminiscent of that following exposure to hypoxia. These observations provide further insight into the potential pathogenesis of IUGR. Further research is required to elicit the role and interactions of p53 and its place in the pathogenesis of IUGR.     

Dose-related influence of sodium selenite on apoptosis in human thyroid follicles in vitro induced by iodine, EGF, TGF-β, and H2O2

Apoptosis of thyroid follicular cells is induced by high doses of iodide, epidermal growth factor (EGF), transforming growth factor-β (TGF-β), as well as H₂O₂ and might be attenuated by antioxidants. Therefore, we examined the apoptotic index induced by these substances in selenium-treated vs untreated human thyroid follicular cells. Reconstituted human thyroid follicles were incubated with sodium selenite (10 or 100 nM) for 72 h; controls received none. The follicles were then distributed to 24-well plates and incubated with potassium iodide (5, 10, or 20 nM), EGF (5 ng/mL), TGF-β (5 ng/mL), or H₂O₂ (100 μM). Apoptosis was determined by a mitochondrial potential assay and the number of apoptotic cells counted by two independent, experienced technicians and the glutathione peroxidase (GPx) activity was determined. A significant increase of apoptic cells was obtained in control thyroid follicles treated with iodine (5, 10, or 20 μM), thyroid-stimulating hormone (TSH) 1, or 10 mU/mL in combination with 5 and 10 μM iodine, EGF (5 ng/mL) and TGF-β (5 ng/mL), or H₂O₂ (100 μM) (p<0.001). In contrast, in thyroid follicles preincubated with 10 or 100 nM sodium selenite, the apoptototic index was identical to the basal rate. In H₂O₂-treated follicles, the apoptotic index was still significantly elevated but 50% lower compared to control cells. The GPx activity increased from 1.4±0.2 to 2.25±0.4 mU/μg DNA with 10 nM selenite and 2.6+0.4 mU/μg DNA with 100 nM selenite. Sodium selenite might increase the antioxidative potential in human thyroid follicles in vitro and therefore diminish the apoptosis induced by TGF-β, EGF, iodide, and even H₂O₂     

Fasudil Mesylate Protects PC12 Cells from Oxidative Stress Injury via the Bax-Mediated Pathway

We previously reported that fasudil mesylate (FM) improves neurological deficit and neuronal damage in rats with ischemia following middle cerebral artery occlusion and reperfusion in vivo. In this study, the properties of FM on hydrogen peroxide (H₂O₂)-induced oxidative stress insult in cultured PC12 cells as well as the underlying mechanisms were investigated in vitro. Pretreatment with FM (5, 10 μM) prior to H₂O₂ exposure significantly elevated cell viability, reduced cell apoptosis by MTT assay, LDH assay, Hoechst 33258 dye staining, and FM also decreased the accumulation of reactive oxygen species (ROS) by DCFH-DA staining and NBT test. Furthermore, FM also reversed the upregulation of Bax/Bcl-2 ratio, the downstream cascade following ROS. FM protected PC12 cells from oxidative stress insult via down-regulating the Bax/Bcl-2 ratio. These findings indicate that a direct effect of fasudil mesylate on PC12 cells may be partly responsible for its protective effect against oxidative stress injury.     

Vitamin B6 suppresses apoptosis of NM-1 bovine endothelial cells induced by homocysteine and copper

Hyperhomocysteinemia is an important risk factor for atherosclerosis. We previously reported that formation of early atherosclerosis in the rat aorta was associated with hyperhomocysteinemia and reduction of antioxidant activity caused by low concentration of vitamin B(6)in vivo. In the present study, we examined effects of vitamin B(6) on apoptosis of bovine endothelial cells (NM-1 cells) treated with homocysteine and copper. Homocysteine and copper induced extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. Cell viability was reduced to 30% compared to that of control cells. On the other hand, pyridoxal treatment as well as EDTA treatment increased viability of NM-1 cells treated with homocysteine and copper to about 60%, and significantly decreased extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. The treatment of catalase recovered cell viability and reduced the level of extracellular hydrogen peroxide and intracellular ROS. Cell death by homocysteine and copper was confirmed to be due to apoptosis by evaluation of DNA fragmentation and by TUNEL assay. However, apoptosis of NM-1 cells induced by homocysteine and copper was due to a caspase-independent pathway as it was not inhibited by the caspase inhibitor, Z-VAD-fmk. Apoptosis of NM-1 cells induced by homocysteine and copper accompanied with mitochondrial permeability but not cytochrome c release. These results suggest that pyridoxal treatment suppresses apoptosis of NM-1 cells induced by homocysteine and copper, most likely through antioxidant effects.     

Evaluation of mouse preimplantation embryos exposed to oxidative stress cultured with insulin-like growth factor I and II, epidermal growth factor, insulin, transferrin and selenium

Blastocyst culture requires strictly defined culture media to sustain its viability and quality. Although blastocyst media are commercially available, they do not meet all the needs and research focused on blastocyst-promoting agents is on the way. The aims of the study were to evaluate the significance of insulin-like growth factors I (IGF-I) and II (IGF-II); epidermal growth factor (EGF) and a mixture of insulin, transferrin and selenium (ITS) on the development of embryos exposed to oxidative stress. C3B6F1 mice were stimulated with 5 IU of pregnant mare serum gonadotropin following by administration of 5 IU of equine chorionic gonadotropin and mating with DBA males. The mice were killed 40 h after eCG injection by cervical dislocation and then the 2 cell embryos were flushed out from the fallopian tubes. To evaluate whether the growth factors may compensate the unfavorable--oxidative milieu created by hydrogen peroxide (H2O2), the embryos were transferred to 1/ control medium, 2/ control medium+0.1 mM (H2O2) or 3/ control medium+H2O2 enriched with 10(-7) g/ml of IGF-I, IGF-II, EGF or a mixture of insulin (5x10(-6) g/ml), transferrin (5 x10(-6) g/ml) and selenium (5x10(-9) g/ml; ITS). Embryos were evaluated 96-144 hours following eCG injection. In the study the dynamics of embryo development and blastocyst cell numbers (including inner cell mass) were assessed. The morphological evaluation comprised viability and apoptosis (TUNEL). In oxidative stress setting, IGF-I, IGF-II, EGF and ITS minimized the negative influence of H2O2, and embryos developed faster than in control conditions. Blastocysts cultured with hydrogen peroxide and growth factors or ITS displayed normal morphology and had more cells--also within the inner cell mass--than those treated only with H2O2. The positive TUNEL reactions were sporadically observed in embryos cultured with hydrogen peroxide supplemented with growth factors. IGF-I, IGF-II, EGF and ITS have a positive effect on pre-implantation embryo development in detrimental culture conditions of oxidative stress.