Generation and characterization of a human induced pluripotent stem cell (iPSC) line from a patient with congenital heart disease (CHD).

Epstein-Barr virus (EBV) immortalized lymphoblastoid cell lines (LCLs) are widely used for banking. This bioresource could be leveraged for creating human iPSC lines to model diseases including CHD. We generated an LCL-derived iPSC line (NCHi001-A) from a patient with congenital aortic valve stenosis. NCHi001-A was EBV and transgenes free, exhibited stem cell-like morphology, expressed pluripotency markers, has a normal karyotype, and could be differentiated into cells of three germ layers in vitro. Relationship inference via a microarray-based analysis showed NCHi001-A is identical to the parental cell line. NCHi001-A can be used for disease modeling, drug discovery, and cell therapy development.

Silencing neuroglobin enhances neuronal vulnerability to oxidative injury by down-regulating 14-3-3gamma.

AIM: To explore the protective role and mechanism of endogenous neuroglobin (Ngb) in neuronal cells under oxidative stress. METHODS: A stable N2a neuroblastoma cell line expressing the Ngb-siRNA plasmid (N2a/Ngb-siRNA) was established by neomycin screening. Reverse transcription (RT)-PCR and Western blot analysis were used to detect Ngb gene and protein levels. Hydrogen peroxide was used to induce oxidative stress in N2a cells. Cytotoxicity and cell viability were measured by lactate dehydrogenase (LDH) and WST-8 assays. Apoptotic cells were detected by Hoechst staining. RESULTS: Cotransfection of Ngb-siRNA with Ngb-GFP plasmids suppressed the expression of Ngb-GFP in N2a cells. RT-PCR and Western blot analysis showed that the expression of endogenous Ngb was successfully knocked down to about 20% in N2a/Ngb-siRNA cells compared with control cells. A WST-8 assay demonstrated that viability was significantly decreased in N2a/Ngb-siRNA cells and N2a cells transiently transfected with Ngb-siRNA plasmids compared with controls following hydrogen peroxide treatment. An LDH assay demonstrated a time-dependent increase in the death of Ngb-siRNA-transfected N2a cells following hydrogen peroxide treatment. Hoechst staining demonstrated that the quantity of apoptotic cells among N2a/Ngb-siRNA cells following hydrogen peroxide treatment significantly increased compared with controls. In N2a/Ngb-siRNA cells, the expression level of activated caspase-3 significantly increased, whereas the expression of 14-3-3gamma decreased compared with that of N2a/vec cells. Transfection of 14-3-3gamma plasmids significantly enhanced the viability of N2a/Ngb-siRNA cells following hydrogen peroxide treatment compared with vector controls. CONCLUSION: Ngb contributes to neuronal defensive machinery against oxidative injuries by regulating 14-3-3gamma expression.Acta Pharmacologica Sinica (2009) 30: 913-918; doi: 10.1038/aps.2009.70.

Role of histamine in airway remodeling of asthmatic guinea pig.

To investigate the role of histamine in airway remodeling, 50 healthy guinea pigs were divided into 5 groups: control group: nebulized inhalation of distilled water for 8 weeks; asthma model group: nebulized inhalation of ovalbumin (OVA) for 8 weeks after sensitization; continued asthma model group: nebulized inhalation of OVA for 14 weeks after sensitization; histamine group: nebulized inhalation of OVA for 14 weeks after sensitization and histamine was added in the last 6 weeks; antagonist group: nebulized inhalation of OVA for 14 weeks after sensitization and histamine receptor antagonists were added in the last 6 weeks. For each group, the concentration of histamine, sodium ion (Na(+)), chlorine ion (Cl(-)), arterial partial pressure of oxygen (PaO2), arterial partial pressure of carbon dioxide (PaCO2), pH, actual bicarbonate (AB), standard bicarbonate (SB) in serum, and thickness of airway mucosa, base membrane and smooth muscle were measured and compared with each other. The results showed that: (1) the concentration of histamine in serum and the thickness of airway increased, the following order was, the control group, the asthma model group, the continued asthma model group and histamine group (P<0.01); and the concentration of histamine in serum and the thickness of airway of antagonist group was lower than that of the continued asthma model group (P<0.05, 0.01). (2) PaO2 of the asthma model group was lower than that of the normal control group (P<0.01); PaO2, pH, AB, SB decreased, the following order was, the asthma model group, the continued asthma model group and the histamine group (P<0.01); and PaO2, pH, AB, SB of the antagonist group was higher than that of the continued asthma model group (P<0.01); but for PaCO2, the order was converse (P<0.01); For the concentration of Na(+) and Cl(-) in serum, there was no difference among these groups. It is concluded that: (1) Histamine is one of the mediators in the airway remodeling of asthma. (2) Histamine receptor antagonists may play a role in preventing and treating airway remodeling. (3) There is a negative correlation between the PaO2, pH and the wall thickness of the airway (P<0.01), while a positive correlation between the PaCO2, anion gap (AG) and the wall thickness of the airway (P<0.01).

[Effects of histamine on endothelial nitric oxide synthase expression in pulmonary artery endothelial cells].

All three nitric oxide synthase (NOS) isoforms are found in the lungs. It has been demonstrated that eNOS-derived NO plays an important role in modulating pulmonary vascular tone and inhibiting pulmonary vascular remodeling. Histamine induces pulmonary vasoconstriction by activating H(1)-receptor on the smooth muscle cells and vasodilation by stimulating H(2)-receptor. It remains unclear whether histamine also modulates the pulmonary vascular tone by regulating eNOS gene expression and NO production in pulmonary artery endothelial cells. Therefore, the present study was performed on cultured primary porcine pulmonary artery endothelial cells (PAECs) to investigate the effects of histamine on eNOS gene expression, and to explore the role of CaMK II in eNOS gene expression. After treatment with different concentrations histamine for different times, the levels of eNOS mRNA and protein were measured by RT-PCR and Western blot, respectively. The results showed that histamine upregulated eNOS mRNA and protein levels in a concentration- and time-dependent manner. Incubation with 10 micromol/L histamine for 24 h could increase eNOS mRNA and protein level to 160.8+/-12.2% (P<0.05) and 136.2+/-11.2% (P<0.05), respectively, of the control values. These up-regulation effects were prevented by selective CaMK II inhibitor, KN-93 (10 micromol/L). To investigate whether or not histamine increases eNOS expression by upregulating eNOS gene transcription, PAECs were transiently transfected with 1.6-kb fragment of the human eNOS promoter driving a luciferase reporter gene. The results suggested that eNOS gene promoter activity was enhanced to 148.2+/-33.7% (P<0.05) of the control after PAECs were incubated with 10 micromol/L histamine for 24 h. The nitrite and nitrate content in culture media measured by colorimetric method after incubation with 10 micromol/L histamine for 24 h indicated that the NO production in PAECs was increased. These results suggest that histamine up-regulates eNOS gene transcription and enhances NO production in PAECs by a signaling pathway involving CaMK II, which might be one of the mechanisms of histamine modulating pulmonary vascular tone.

[Effects of hypoxia on endothelial nitric oxide synthase gene expression in pulmonary artery endothelial cells and the role of protein kinase C isoforms].

OBJECTIVE: To explore the regulation of eNOS gene expression in pulmonary arterial endothelial cells (PAECs) by protein kinase C (PKC) and its isoforms during hypoxia. METHODS: Primary cultured porcine PAECs were exposed to 5%O(2) for 2, 6, 12, 24, 48 hours. The eNOS mRNA level was measured by RT-PCR. Western blot technology was used to detect the contents of eNOS protein and the 8 PKC isoforms. After addition of selective PKC inhibitors, bisindolylmaleimideI (BIMI, 1 micro mol/L) or Gö6983 (1 micro mol/L), PAECs were exposed to 5%O(2) for 24 hours, then the expression of eNOS mRNA was detected by RT-PCR. Promoter activity of eNOS gene was determined by luciferase reporter gene assay. PAECs were transfected transiently with 1.6 kb fragment of the human eNOS promoter driving a luciferaes reporter gene, then exposed to 5%O(2). 24 h later, the activity of luciferase and beta-galactosidase was examined and the relative luciferase activity, representing the eNOS promotor activity, was calculated. After addition of actinomycine D (5 micro g/ml) and exposure to 5%O(2) or normoxia for 6, 12, 24 hours, and eNOS mRNA in PAECs was measured by RT-PCR. RESULTS: After exposed to hypoxia for 24 hours, the expression of eNOS mRNA and protein level increased by 171% +/- 18% (P < 0.05) and 166% +/- 21% (P < 0.01) respectively. These up-regulation effects were prevented by BIM I and Gö6983. Further experiments showed that among 8 isoforms of PKC detected in this study, only nPKCepsilon protein expression was changed in PAECs after exposure to hypoxia for 24 h. After exposure to hypoxia nPKCepsilon was translocated from cytosol to cell membrane, showing the activation of nPKCepsilon during hypoxia. Reporter gene assay showed that hypoxia enhanced eNOS promoter activity up to 2.3 +/- 0.7 fold. In addition, hypoxia did not change the stability of eNOS mRNA. CONCLUSION: Hypoxia may up-regulate eNOS expression in PAECs by transcriptional mechanism through nPKCepsilon signaling pathway. Higher levels of mRNA observed during hypoxia are due to increased transcription, not to increased stability of mRNA.