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author:("Wang, xiuning")
1.  Normal IgG Downregulates the Intracellular Superoxide Level and Attenuates Migration and Permeability in Human Aortic Endothelial Cells form a Hypertensive Patient 
Hypertension  2012;60(3):818-826.
The normal IgG, a circulating antibody, is maintained at a constant level in humans. However, little is known if normal IgG has effects on the function of vascular endothelial cells. The purpose of this study was to investigate if IgG affects superoxide (O2-) generation and cell permeability in human aortic endothelial cells (HAECs) isolated from a hypertensive patient. The effect of normal human IgG on endothelial cell function was investigated in cultured HAECs isolated from a hypertensive patient who died of stroke. The results demonstrated, for the first time, that normal IgG attenuated the intracellular O2- level and decreased cell migration, cell permeability, and stress fiber formation in HAECs. IgG significantly decreased Rac1 activity and NADPH oxidase activity but upregulated MnSOD expression in HAECs, which may contribute to the IgG-induced decrease in O2- level. It is noted that AMPK was activated by IgG as evidenced by increased phosphorylation of AMPK (p-AMPK). Interestingly, inhibition of AMPK by an AMPK inhibitor (AI) abolished IgG-induced decreases in Rac1 and NADPH oxidase activities and IgG-induced increases in MnSOD expression, suggesting that AMPK is an important mediator of the IgG-induced regulation of these enzymes. Importantly, inhibition of AMPK activity also prevented the IgG-induced decrease in O2- levels, cell migration, cell permeability, and stress fiber formation. Therefore, normal human IgG may protect HAECs via activation of AMPK and subsequent decreases in intracellular O2-. These findings reveal a previously unidentified role of normal IgG in regulating AMPK and endothelial cell function.
doi:10.1161/HYPERTENSIONAHA.112.199281
PMCID: PMC3421057  PMID: 22777940
IgG; superoxide; AMPK; NADPH oxidase; endothelial cell; permeability; migration
2.  AAV-Based RNAi Silencing of NADPH Oxidase gp91phox Attenuates Cold-Induced Cardiovascular Dysfunction 
Human Gene Therapy  2012;23(9):1016-1026.
Abstract
Clinical observations and epidemiological surveys indicated that the prevalence of hypertension and heart diseases is increased in cold regions or during winter. Cold exposure increased NADPH oxidase gp91phox protein expression in heart, kidneys, and aorta in rats. The aim of this study was to investigate if RNA interference (RNAi) silencing of gp91phox would attenuate cold-induced hypertension and cardiovascular and renal damage. The recombinant adeno-associated virus serotype 2 (AAV-2) vector carrying gp91phox-shRNA (gp91-shRNA) was constructed for inhibiting gp91phox protein expression in cold-exposed rats. Blood pressure (BP) was monitored using a telemetry system. BP was increased in the Control-shRNA and PBS groups within 1 week of exposure to moderate cold (5°C) and reached a plateau after 7 weeks. The cold-induced increase in BP was attenuated significantly by intravenous delivery of gp91-shRNA (1.25×1010 particles/rat, 0.5 mL). One single dose of gp91-shRNA controlled hypertension for up to 10 weeks. In addition, gp91-shRNA reversed cold-induced vascular dysfunction. gp91-shRNA abolished the cold-induced up-regulation of gp91phox protein expression in heart, kidneys, and aorta, confirming effective silencing of gp91phox. The cold-induced increases in NADPH oxidase activity and superoxide production were eliminated by silencing of gp91phox, suggesting that the cold-induced up-regulation of NADPH oxidase activity may be attributed to the increased gp91phox protein expression. RNAi silencing of gp91phox abolished cold-induced cardiac and renal hypertrophy and attenuated aortic, coronary, and renal remodeling. The up-regulation of gp91phox may play a critical role in cold-induced cardiovascular dysfunction and organ damage. AAV delivery of gp91-shRNA may be a new and effective therapeutic approach for cold-related cardiovascular disorders.
Wang and colleagues investigate silencing of gp91phox as a potential way to attenuate cold-induced hypertension and cardiovascular and renal damage. Recombinant adeno-associated virus serotype 2 (AAV2) carrying gp91phox–short hairpin RNA was constructed and delivered to cold-exposed rats. A single dose of vector controlled hypertension for up to 10 weeks and reversed cold-induced vascular dysfunction as well as renal hypertrophy.
doi:10.1089/hum.2012.078
PMCID: PMC3440021  PMID: 22888847
3.  Aging-related kidney damage is associated with a decrease in klotho expression and an increase in superoxide production 
Age  2010;33(3):261-274.
The purpose of this study was to determine changes in klotho, endothelin (ET) receptors, and superoxide production in kidneys of aged rats and whether these changes are exacerbated in aged rats with cognitive impairment. Twenty aged rats (male, 27 months) were divided into an Old Impaired group (n = 9) and an Old Intact group (n = 11) according to a cognitive function test. A group of 12-month-old rats (n = 10) was used as a Young Intact group. Serum creatinine was increased significantly in the Old Impaired group, suggesting impaired renal function. Aged rats showed glomerulosclerosis and tubulointerstitialfibrosis. These pathological changes were markedly aggravated in the old cognitively impaired than in the old cognitively intact animals. Notably, aged rats demonstrated a significant decrease in klotho protein expression in renal cortex and medulla. Protein expression of IL-6, Nox2, ETa receptors and superoxide production were increased whereas mitochondrial SOD (MnSOD) and ETb receptors expression were decreased in kidneys of the aged rats. Interestingly, these changes were more pronounced in the old impaired than in the old intact rats. In conclusion, the aging-related kidney damage was exacerbated in aged rats with cognitive impairment. Klotho, ETB, and MnSOD were downregulated but ETa, IL-6, Nox2, and superoxide production were upregulated in the aging-related kidney damage. These changes were more pronounced in rats with cognitive impairment.
doi:10.1007/s11357-010-9176-2
PMCID: PMC3168600  PMID: 20830528
Aging; Klotho; Glomerusclerosis; ET receptor; Superoxide; Interleukin-6
4.  Genetic IL-10 Deficiency Causes Vascular Remodeling via the Upregulation of Nox1 
Journal of Hypertension  2011;29(11):2116-2125.
Background & Hypothesis
IL-10 is an anti-inflammatory cytokine. Nox1 is a mitogenic oxidase (p65-mox). The objective of this study was to test a hypothesis that IL10 deficiency would cause vascular remodeling via the upregulation of Nox1.
Methods & Results
Recombinant AAV carrying short hairpin small interference RNA for Nox1 (AAV.Nox1shRNA) was constructed for in vivo specific inhibition of Nox1. Three groups of IL10 gene knockout (IL10KO) mice and 3 groups of wild-type (WT) mice were used. Three groups of each strain received intravenous delivery of AAV.Nox1shRNA, AAV.ScrambledshRNA, and PBS, respectively. Animals were euthanized at 3 weeks after gene delivery. IL10KO increased Nox1 protein expression, NADPH oxidase activity, and superoxide production in aortas. IL10KO also resulted in a significant decrease in aortic medial thickness, a loss of smooth muscle cells, and an increase in vascular collagen deposition, indicating vascular remodeling. The IL10KO-induced increases in NADPH oxidase activity and superoxide production and vascular remodeling were abolished by silencing of p65-mox, suggesting that these effects may be mediated by the upregulation of Nox1. In addition, IL10KO increased endothelin-1 (ET-1) levels in plasma and aortas, and this effect was partially blocked by silencing of Nox1. RNAi silencing of Nox1 obliterated the IL10KO-induced increases in IL-6 expression in aortas, superoxide production and MMP-9 activity in aortic smooth muscle cells (SMC), and SMC migration.
Conclusions
IL10 is essential to the maintenance of normal vasculature as IL10 deficiency resulted in vascular damage and remodeling. The IL10KO-induced vascular structure damage may be mediated by the up-regulation of Nox1.
doi:10.1097/HJH.0b013e32834b22a0
PMCID: PMC3373265  PMID: 21918473
vascular remodeling; vascular smooth muscle cell; AAV; IL-6; MMP-9; ET-1; NADPH oxidase; superoxide
5.  Post-Transcriptional Control of Type I Interferon Induction by Porcine Reproductive and Respiratory Syndrome Virus in Its Natural Host Cells 
Viruses  2012;4(5):725-733.
Porcine reproductive and respiratory syndrome virus (PRRSV) is not only a poor inducer of type I interferon but also inhibits the efficient induction of type I interferon by porcine transmissible gastroenteritis virus (TGEV) and synthetic dsRNA molecules, Poly I:C. However, the mechanistic basis by which PRRSV interferes with the induction of type I interferon in its natural host cells remains less well defined. The purposes of this review are to summarize the key findings in supporting the post-transcriptional control of type I interferon in its natural host cells and to propose the possible role of translational control in the regulation of type I interferon induction by PRRSV.
doi:10.3390/v4050725
PMCID: PMC3386621  PMID: 22754646
PRRSV; Type I interferon; post-transcriptional regulation; translational control
6.  Expression of cecropin B in Pichia pastoris and its bioactivity in vitro 
Natural cecropin B (CB), consisting of 35 amino acids, is a member of the cecropin family with the highest antibacterial activity. Here, a novel approach for the generation of recombinant CB in the methylotrophic yeast Pichia pastoris was explored. For this purpose, the CB gene was amplified by recursive PCR (rPCR) and cloned into the pPICZαA vector. The SacI-linearized plasmid pPICZαA-CB was transformed into P. pastoris SMD1168 by electroporation. The expression of recombinant CB was induced with 1.0% methanol at pH 5.0 for 60 h at 28°C. Recombinant CB was purified using cationic exchange chromatography; 5.0 mg of pure active CB was obtained from 100 ml of culture broth supernatant. Antimicrobial assays demonstrated that CB has a broad spectrum of antimicrobial activity against both Gram-positive and Gram-negative bacteria. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional CB for both research and industrial purposes.
doi:10.3892/etm.2011.262
PMCID: PMC3440743  PMID: 22977556
antibacterial activity; cecropin B; expression; Pichia pastoris; antimicrobial assays
7.  Thyroid hormone induces artery smooth muscle cell proliferation: discovery of a new TRα1-Nox1 pathway 
Background
Thyroid hormone (T3) can stimulate protein synthesis and cell growth. NOX1 is a mitogenic oxidase. The aim of this study was to test a novel hypothesis that T3 induces artery smooth muscle cell proliferation by up-regulating NOX1.
Methods and Results
Immunofluoresence confocal microscopy was used to visualize the sub-cellular localization of NOX1 and TRα1 in rat aorta smooth muscle (RASM) cells. Optical sectioning showed that TRα1 and NOX1 co-localized around the nucleus. T3 promoted RASM cell proliferation as determined by the fact that T3 significantly increased the number of cytokinesis cells, proliferating cellular nuclear antigen (PCNA), and smooth muscle α-actin (SM α-actin). T3 increased NOX1 expression at both the transcription (mRNA) and translation (protein) levels as evaluated by RT-PCR and western blot, respectively. T3 also significantly increased the intracellular ROS production based on the oxidation of 2’,7’-dichlorodihydrofluoresein (H2DCF) to a fluorescent 2’,7’-dichlorofluoresein (DCF). RNAi silence of TRα1 or NOX1 abolished T3-induced intracellular ROS generation and PCNA and SM α-actin expression, indicating that TRα1 and NOX1 mediated T3-induced RASM cell proliferation. Notably, RNAi silence of TRα1 blocked the T3-induced increase in NOX1 expression whereas silence of NOX1 did not affect TRα1 expression, disclosing a new pathway, i.e., T3-TRα1-NOX1-cell proliferation.
Conclusions
TRα1 and NOX1 co-localized around the nucleus. T3 induced RASM cell proliferation by up-regulating NOX1 in a TRα1-dependent manner.
PMCID: PMC2888973  PMID: 20414976
thyroid hormone; TRα1; NOX1; ROS; smooth muscle cell; proliferation
8.  Recombinant Murine Cytomegalovirus Vector Activates Human Monocyte-derived Dendritic Cells in a NF-κB Dependent Pathway 
Molecular immunology  2009;46(16):3462-3465.
To evaluate the potential use of recombinant murine cytomegalovirus (MCMV) as an antigen delivery vector, we examined the cytokine and CD80 and CD86 expression profiles of MCMV encoding either enhanced green fluorescent protein gene (MCMV-EGFP) or human immunodeficiency virus-1 glycoprotein gp120 gene (MCMV-gp120) infected monocyte-derived dendritic cells (Mo-DC) and investigated the role of nuclear factor kappa B (NF-κB) in Mo-DC activation. Results showed that MCMV triggered the induction of inflammatory cytokines and/or CD80 and CD86 up-regulation in Mo-DC. UV-inactivated MCMV exhibited a reduced production of inflammatory cytokines and a lowered expression of CD80 and CD86 compared with live MCMV infection. Treatment of cells with a NF-κB peptide inhibitor prior to MCMV infection reduced the induction of cytokines and CD80 and CD86 up-regulation. Overall, the results suggest that recombinant MCMV vectors activate human Mo-DC in a NF-κB dependent pathway. The abortive infection or de novo gene expression greatly enhances the activation of Mo-DC by MCMV vectors.
doi:10.1016/j.molimm.2009.08.001
PMCID: PMC2757491  PMID: 19716178
9.  Effects of glutathione reductase inhibition on cellular thiol redox state and related systems 
Although inhibition of glutathione reductase (GR) has been demonstrated to cause a decrease in reduced glutathione (GSH) and increase in glutathione disulfide (GSSG), a systematic study of the effects of GR inhibition on thiol redox state and related systems has not been noted. By employing a monkey kidney cell line as the cell model and 2-acetylamino-3-[4-(2-acetylamino-2-carboxy-ethylsulfanylthio-carbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) as a GR inhibitor, an investigation of the effects of GR inhibition on cellular thiol redox state and related systems was conducted. Our study demonstrated that, in addition to a decrease in GSH and increase in GSSG, 2-AAPA increased the ratios of NADH/NAD+ and NADPH/NADP+. Significant protein glutathionylation was observed. However, the inhibition did not affect the formation of reactive oxygen species or expression of antioxidant defense enzyme systems [GR, glutathione peroxidase, catalase, and superoxide dismutase)] and enzymes involved in GSH biosynthesis [γ-glutamylcysteine synthetase and glutathione synthetase].
doi:10.1016/j.abb.2009.03.001
PMCID: PMC2709784  PMID: 19272349
Glutathione reductase inhibitor; thiol redox state; protein thiols; GSH; GSSG
10.  Murine Cytomegalovirus Abortively Infects Human Dendritic Cells, Leading to Expression and Presentation of Virally Vectored Genes 
Journal of Virology  2003;77(13):7182-7192.
Dendritic cells (DC) are potent antigen-presenting cells that play a crucial role in antigen-specific immune responses. Thus, the targeting of exogenous antigens to DC has become a popular approach for cancer immunotherapy and vaccine development. In this report, we studied the interplay between murine cytomegalovirus (MCMV) and human monocyte-derived DC. The results showed that an enhanced green fluorescent protein (EGFP)-encoding, replication-competent MCMV vector underwent abortive infection in human DC; this was accompanied by the efficient expression of EGFP. Infection of human DC by this vector resulted in a modest increase in the expression of cell surface proteins associated with DC maturation and has no significant effect on the immunostimulatory function of the cells, as reflected by their ability to support T-cell proliferation in a mixed-lymphocyte reaction. Finally, an MCMV vector encoding the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein was constructed and used to infect cultured human DC. The infected DC were shown to be capable of stimulating the expansion of autologous, gp120-specific, class I-restricted T lymphocytes from an HIV-1-negative donor, as determined by tetramer staining and enzyme-linked immunospot analysis. Taken together, these results suggest that MCMV may have potential utility as a vector for human vaccine development.
doi:10.1128/JVI.77.13.7182-7192.2003
PMCID: PMC164805  PMID: 12805417
11.  Expression of Human Immunodeficiency Virus Type 1 gp120 from Herpes Simplex Virus Type 1-Derived Amplicons Results in Potent, Specific, and Durable Cellular and Humoral Immune Responses 
Journal of Virology  2002;76(11):5565-5580.
Herpes simplex virus type 1 (HSV-1) infects a wide range of cells, including dendritic cells. Consequently, HSV-1 vectors may be capable of eliciting strong immune responses to vectored antigens. To test this hypothesis, an HSV-1 amplicon plasmid encoding human immunodeficiency virus type 1 gp120 was constructed, and murine immune responses to helper virus-free amplicon preparations derived from this construct were evaluated. Initial studies revealed that a single intramuscular (i.m.) injection of 106 infectious units (i.u.) of HSV:gp120 amplicon particles (HSV:gp120) elicited Env-specific cellular and humoral immune responses. A potent, CD8+-T-cell-mediated response to an H-2Dd-restricted peptide from gp120 (RGPGRAFVTI) was measured by a gamma interferon ELISPOT and was confirmed by standard cytotoxic-T-lymphocyte assays. Immunoglobulin G enzyme-linked immunosorbent assay analysis showed the induction of a strong, Env-specific antibody response. An i.m. or an intradermal administration of HSV:gp120 at the tail base elicited a more potent cellular immune response than did an intraperitoneal (i.p.) inoculation, although an i.p. introduction generated a stronger humoral response. The immune response to HSV:gp120 was durable, with robust cellular and humoral responses persisting at 171 days after a single 106-i.u. inoculation. The immune response to HSV:gp120 was also found to be dose dependent: as few as 104 i.u. elicited a strong T-cell response. Finally, HSV:gp120 elicited significant Env-specific cellular immune responses even in animals that had been previously infected with wild-type HSV-1. Taken together, these data strongly support the use of helper-free HSV-1 amplicon particles as vaccine delivery vectors.
doi:10.1128/JVI.76.11.5565-5580.2002
PMCID: PMC137011  PMID: 11991985

Results 1-11 (11)