ROS-induced NLRP3 inflammasome priming and activation mediate PCB 118- induced pyroptosis in endothelial cells.

Growing epidemiological evidence has shown that exposure to polychlorinated biphenyls (PCBs) is harmful to the cardiovascular system. However, how PCB 118-induced oxidative stress mediates endothelial dysfunction is not fully understood. Here, we explored whether and how PCB 118 exposure-induced oxidative stress leads to NLRP3 inflammasome-dependent pyroptosis in endothelial cells. As expected, PCB 118 was cytotoxic to HUVECs and induced caspase-1 activation and cell membrane disruption, which are characteristics of pyroptosis. Moreover, PCB 118-induced pyroptosis may have been due to the activation of the NLRP3 infammasomes. PCB 118 also induced excessive reactive oxygen species (ROS) in HUVECs. The ROS scavenger (±)-α-tocopherol and the NFκB inhibitor BAY11-7082 reversed the upregulation of NLRP3 expression and the increase in NLRP3 inflammasome activation induced by PCB 118 exposure in HUVECs. Additionally, PCB 118-induced oxidative stress and pyroptosis were dependent on Aryl hydrocarbon receptor (AhR) activation and subsequent cytochrome P450 1A1 upregulation, which we confirmed by using the AhR selective antagonist CH 223191. These data suggest that PCB 118 exposure induces NLRP3 inflammasome activation and subsequently leads to pyroptosis in endothelial cells in vitro and in vivo. AhR-mediated ROS production play a central role in PCB 118-induced pyroptosis by priming NFκB-dependent NLRP3 expression and promoting inflammasome activation.

2,3',4,4',5-Pentachlorobiphenyl impairs insulin-induced NO production partly through excessive ROS production in endothelial cells.

Polychlorinated biphenyls (PCBs) have been reported to be associated with increased risk to hypertension, atherosclerosis, cardiovascular disease, etc. 2,3',4,4',5-Pentachlorobiphenyl, known as PCB-118, is a member of coplanar PCBs which renders their structure similar to polychlorinated dibenzo-p-dioxins (PCDDs) and has dioxin-like activity. In our current study, we investigated the effect of PCB-118 exposure on nitric oxide (NO) production and the underlying mechanisms in vitro. Exposure of PCB-118 impaired insulin-induced NO production and endothelial nitric oxide synthase (eNOS) activity in human umbilical vein endothelial cells (HUVECs) with no significant effect on cell viability. Furthermore, PCB-118 treatment induced oxidative stress. In addition, scavenging of reactive oxygen species (ROS) by 10 µM N-acetyl-l-cysteine (NAC) partly rescued impaired insulin-induced eNOS activities and NO productions induced by PCB-118 in HUVECs. Taken together, these results indicate that PCB-118 mediates lower eNOS activity and impairs insulin-induced NO production partly through excessive ROS production in endothelial cells.

PCB 118-induced endothelial cell apoptosis is partially mediated by excessive ROS production.

Endothelial cell apoptosis, which may alter the integrity of the endothelium and lead to plaque instability, plays a critical role in the development and pathogenesis of atherosclerosis. Exposure of polychlorinated biphenyls (PCBs) is associated with increased risk of atherosclerosis and cardiovascular disease. In our present study, we explored whether exposure to PCB 118 influences endothelial cell apoptosis in vitro and the underlying mechanisms involved. As expected, exposure to PCB 118 increased the intracellular reactive oxygen species (ROS) levels in HUVECs. Increases in apoptosis and Bax/Bcl-2 ratios were observed in PCB 118-treated HUVECs. N-acetyl-l-cysteine (NAC), a ROS scavenger, partially reduced PCB 118-induced apoptosis and Bax/Bcl-2 ratios in HUVECs. Taken together, PCB 118-induced endothelial cell apoptosis was partially initiated by excessive ROS production.

Roles of Rho/Rock signaling pathway in silica-induced epithelial-mesenchymal transition in human bronchial epithelial cells.

OBJECTIVE: To investigate the roles of Rho/Rock signaling pathway in silica-induced Epithelial-mesenchymal transition (EMT) in human bronchial epithelial cells (BEC) in vitro. METHODS: Human BEC were incubated with silica with various concentrations for indicated times. Cell viability was assayed by MTT test. Morphologic Changes were observed by microscope. Mesenchymal marker α-smooth muscle actin (α-SMA), vimentin (Vim), and epithelial marker E-cadherin (E-cad) were analyzed by Western Blot. The pull-down assay was used to measure Rho activity. In the prevention experiments, the specific inhibitor for Rho effector ROCK (Y27632) was used to inhibit the activity of Rho. RESULTS: Human BEC stimulated with silica were converted from a "cobblestone" epithelial structure into an elongated fibroblast-like shape structure. Incubation of human BEC with silica induced de novo expression of α-SMA and Vim, and loss of E-cad. Also, silica treatment resulted in Rho activation in human BEC. Y27632 up-regulated the E-cad expression but attenuated α-SMA and Vim expression in silica-stimulated cells. CONCLUSION: The activation of Rho/ROCK signaling pathways is most likely involved in Silica-induced EMT in human bronchial epithelial cells.

[The influence of SiO2 on epithelial-mesenchymal transition (EMT) in human bronchial epithelial cells].

OBJECTIVE: To investigate SiO2-induced EMT in human bronchial epithelial cells HBE in vitro. METHODS: HBE cells were cultured and then stimulated with indicated doses of SiO2 (0, 50, 100, 200, 300 µg/ml). The morphological changes were observed by microscope. In addition, Western blot was per-formed to detect the expression of E-cad, α-SMA and Vim. The changes of migration ability were examined by wound-healing assay in vitro. RESULTS: (1) After exposure to SiO2, HBE cells lost contact with their neighbor and displayed a spindle-shape, fibroblast-like morphology. (2) Compared with the control, the E-cad (300 µg/ml group) expression downregulated 2.98 fold (P < 0.05), and the Vim (300 µg/ml group) and α-SMA (200 µg/ml group) expression upregulated 4.46 fold and 3.55 fold (P < 0.05). There were significant differences between 100, 200, 300 µg/ml groups and the control group (P < 0.05). (3) In the test group, the percentage of wound-healing areas/wound areas were larger than those in control group (P < 0.05). CONCLUSIONS: SiO2 could induce EMT in human bronchial epithelial cells.

The effect of retroviral vector on uptake of human lactoferrin DNA by Yak (Bos Grunniens) spermatozoa and their fertilizability in vitro.

The objectives of this study were to evaluate the transfection effectiveness of retroviral vector PLNCX2 in yak sperm-mediated gene transfer (SMGT) and the effect of SMGT on sperm motility and fertilizability. Human lactoferrin (hLF) DNA was ligated into PLNCX2 to construct recombinant plasmid PLNCX2-hLF, then, using PLNCX2-hLF+FuGene 6 to generate SMGT yak spermatozoa for fertilizing bovine oocytes. The result showed that DNA-binding rate increased with the extension of incubation period and DNA treatment did not decrease sperm motility. Oocytes inseminated with SMGT-spermatozoa had a lower (P < 0.05) cleavage rate (57.7%) than the control (73.4%), but development up to blastocyst stage was not different (26.8 vs. 31.7%). It appears that PLNCX2 is useful for generating transgenic yaks by SMGT.

ATP2B1 Gene Silencing Increases NO Production Under Basal Conditions Through the Ca2+/calmodulin/eNOS Signaling Pathway in Endothelial Cells.

Emerging epidemiological and experimental evidence has shown that the ATP2B1 gene is associated with blood pressure control. Impaired eNOS activity and NO production may be among the mechanisms involved. However, little is known about how PMCA1, which is encoded by the ATP2B1 gene, regulates the activity of eNOS and NO production. In the present study, we investigated the role of the ATP2B1 gene in regulating eNOS activity and NO production under basal conditions in HUVECs and explored the mechanisms involved. Silencing ATP2B1 gene expression resulted in higher NO production and eNOS activity under basal conditions in HUVECs. Additionally, ATP2B1 gene silencing resulted in enhanced intracellular calcium concentrations compared to that in the negative siRNA-transfected HUVECs. The enhanced eNOS activity mediated by ATP2B1 gene silencing was Ca2+/calmodulin dependent, as verified by the administration of the calcium chelator BAPTA-AM or the calmodulin-specific antagonist W7. Taken together, silencing ATP2B1 gene expression results in higher NO production and eNOS activity under basal conditions in HUVECs. Furthermore, the enhanced eNOS activity induced by ATP2B1 gene silencing may be mediated via higher levels of intracellular Ca2+, and the effect was confirmed to be dependent on the eNOS-calmodulin interaction.

ATP2B1 gene Silencing Increases Insulin Sensitivity through Facilitating Akt Activation via the Ca2+/calmodulin Signaling Pathway and Ca2+-associated eNOS Activation in Endothelial Cells.

Endothelial cell insulin resistance may be partially responsible for the higher risk of atherosclerosis and cardiovascular disease in populations with insulin resistance and type 2 diabetes mellitus (T2DM). A genome-wide association study revealed a significant association between the ATPase plasma membrane Ca2+ transporting 1 (ATP2B1) gene and T2DM in two community-based cohorts from the Korea Association Resource Project. However, little is known about the implication of the ATP2B1 gene on T2DM. In the present study, we investigated the role of the ATP2B1 gene in endothelial cell insulin sensitivity. ATP2B1 gene silencing resulted in enhanced intracellular calcium concentrations and increased insulin-induced Akt activation compared to that in the negative siRNA-transfected HUVECs (Human Umbilical Vein Endothelial Cells). The elevated insulin sensitivity mediated by ATP2B1 gene silencing was Ca2+/calmodulin-dependent, as verified by administration of the calcium chelator BAPTA-AM or the calmodulin-specific antagonist W7. Moreover, higher levels of phosphorylation of eNOS (Ser1177) were observed in ATP2B1-silenced HUVECs. In addition to BAPTA-AM and W7, L-NAME, an eNOS antagonist, abolished insulin-induced Akt phosphorylation at Ser473 in both si-Neg and si-ATP2B1-transfected endothelial cells. These results indicate that the enhanced insulin sensitivity in ATP2B1-silenced endothelial cells is alternatively dependent on an increase in intracellular Ca2+ and the subsequent activation of the Ca2+/calmodulin/eNOS/Akt signaling pathway. In summary, ATP2B1 gene silencing increased insulin sensitivity in endothelial cells by directly modulating the Ca2+/calmodulin signaling pathway and via the Ca2+/calmodulin/eNOS/Akt signaling pathway alternatively.

Developmental competence of embryos derived from reciprocal in vitro fertilization between Yak (Bos grunniens) and cattle (Bos taurus).

The purpose of this study was to investigate fertilization ability and embryo development to the blastocyst stage after reciprocal in vitro fertilization (IVF) between yak and cattle in an attempt to clarify the problem of low conception rate after mating yak females with cattle bulls. In vitro-matured (IVM) cattle and yak oocytes were inseminated with either Holstein or yak spermatozoa, and after an 18-h of coincubation period, a proportion of the oocytes was fixed and examined for sperm penetration, polyspermy and male pronuclear formation. The remaining oocytes were cultured in vitro and evaluated for cleavage and blastocyst formation rates. The percentage of IVM oocytes penetrated by spermatozoa ranged from 78.5 to 90.5%, and the formation of one or two pronuclei and the incidence of polyspermy did not differ among the different combinations. The cleavage and blastocyst rates were not affected by the species of the sperm, but they were affected by the species of the oocytes (P<0.05), with cattle oocytes having a higher (P<0.05) cleavage and blastocyst rates (69.9 and 31.3%) than yak oocytes (62.7 and 11.5%). The blastocyst formation rate was calculated from the cleaved zygotes. The interaction between sire and oocytes species (P<0.05) influenced blastocyst formation rate, with the highest blastocyst rate occurring in cattle oocytes fertilized with yak spermatozoa (36.5%) and the lowest rate occurring in yak oocytes fertilized with yak spermatozoa (9.4%). The effect of heterosis was apparent at the blastocyst stage, but there was a large reciprocal difference in blastocyst production between crosses. It was concluded that the low conception rate that results from crossing yaks with cattle is not due to either a species-specific block of fertilization or the developmental competence of the early stage embryo.