Publications by authors named "Aizhen Yang"

29 Publications

  • Page 1 of 1

Plasma kallikrein contributes to ambient particulate matter-induced lung injury.

Biochem Biophys Res Commun 2019 10 23;518(3):409-415. Epub 2019 Aug 23.

Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Protection, Soochow University, Suzhou, China; The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, 19140, USA. Electronic address:

Particulate matter (PM) is a key component of air pollutants and is associated with mortality of cardiovascular and respiratory diseases. PM-induced tissue injury involves inflammation and coagulation. Plasma prekallikrein (pKal), along with coagulation factor XII (FXII) and high-molecular-weight kininogen (HK), form the plasma kallikrein-kinin system (KKS), a component of the innate immune response that generates proinflammatory products in response to injury. When the KKS proteins contact with activation surface such as negatively charged molecules, this system becomes activated. Activated kallikrein (Kal) activates FXII to initiate the intrinsic coagulation pathway, and cleaves HK to release bradykinin to enhance vascular permeability and systemic inflammation. In his study we determined the role of plasma pKal in the PM-induced lung injury. Using TALEN technology, we generated a new mouse strain lacking the gene for pKal. In PM-induced lung injury model, Klkb1 mice exhibited a decrease in total protein, cells numbers in bronchoalveolar lavage fluid (BALF) and histologic lung injury score. The TNF-α and IL-6 levels in BALF were significantly decreased in PM-treated Klkb1 mice. Plasma thrombin-antithrombin (TAT) complex levels were significantly decreased in PM-treated Klkb1 mice. PM induces pKal activation, HK cleavage and bradykinin production. PM-induced HK cleavage in plasma was completely blocked by a Kal inhibitor, as well as in pKal-deficient plasma. PM markedly induced thrombin generation in human plasma and wild-type mouse plasma, which was inhibited by both blockade and deficiency of pKal. Taken together, plasma pKal is activated by PM and the activated Kal plays an important role in PM-induced lung injury.
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http://dx.doi.org/10.1016/j.bbrc.2019.07.060DOI Listing
October 2019

Ginsenoside Rg3 Combined with Oxaliplatin Inhibits the Proliferation and Promotes Apoptosis of Hepatocellular Carcinoma Cells via Downregulating PCNA and Cyclin D1.

Biol Pharm Bull 2019 Jun 29;42(6):900-905. Epub 2019 Mar 29.

Department of Oncology, Jiangsu Province Hospital of TCM, the Affiliated Hospital of Nanjing University of Chinese Medicine.

The present study aims to investigate the effects of ginsenoside Rg3 combined with oxaliplatin on the proliferation and apoptosis of hepatocellular carcinoma cells and the related mechanism. In this study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was applied to examine the proliferation rate of hepatocellular carcinoma cell SMMC-7721 with different treatment. Flow cytometry was performed to examine apoptosis rate of hepatocellular carcinoma cells with different treatment. Immunofluorescence and Western blot methods were used to evaluate the expressions of proliferating cell nuclear antigen (PCNA) and cyclin D1 in different groups. We found that ginsenoside Rg3, oxaliplatin or ginsenoside Rg3 + oxaliplatin significantly suppressed the proliferation and promoted the apoptosis of SMMC-7721. Meanwhile, ginsenoside Rg3, oxaliplatin or ginsenoside Rg3 + oxaliplatin also significantly inhibited the expressions of PCNA and cyclin D1. Moreover, compared with ginsenoside Rg3 group and oxaliplatin group, the effect of ginsenoside Rg3 + oxaliplatin was more remarkable. Taken together, cells treated with oxaliplatin+ ginsenoside enhanced the anti-tumor effect and may inhibit the proliferation and promoted apoptosis of hepatocellular carcinoma via regulating the expression of PCNA and cyclin D1.
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http://dx.doi.org/10.1248/bpb.b18-00852DOI Listing
June 2019

Tyro3, Axl, and Mertk receptors differentially participate in platelet activation and thrombus formation.

Cell Commun Signal 2018 12 12;16(1):98. Epub 2018 Dec 12.

Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Protection, Soochow University, Suzhou, 215123, China.

Background: Previously, several studies have shown that Tyro3, Axl, and Mertk (TAM) receptors participate in platelet activation and thrombosis. However, the role of individual receptors is not fully understood.

Methods: Using single receptor-deficient platelets from TAM knockout mice in the C57BL/6 J strain, we performed a knockout study using single TAM-deficient mice. We treated platelets isolated from TAM knockout mice with the Glycoprotein VI (GPVI) agonists convulxin, poly(PHG), and collagen-related triple-helical peptide (CRP), as well as thrombin for in-vitro experiments. We used a laser-induced cremaster arterial injury model for thrombosis experiments in vivo.

Results: Deficiency of the tyrosine kinase receptors, Axl or Tyro3, but not Mertk, inhibited aggregation, spreading, JON/A binding, and P-selectin expression of platelets in vitro. In vivo, platelet thrombus formation was significantly decreased in Axl and Tyro3 mice, but not in Mertk mice. Upon stimulation with glycoprotein VI (GPVI) agonists, tyrosine phosphorylation of signaling molecules, including spleen tyrosine kinase (Syk) and phospholipase C-γ2 (PLCγ2), was decreased in Axl and Tyro3 platelets, but not in Mertk platelets. While platelet aggregation induced by agonists did not differ in the presence or absence of the Gas6 neutralizing antibody, the platelet aggregation was inhibited by anti-Axl or anti-Tyro3 neutralizing antibodies antibody, but not the anti-Mertk antibody. Additionally, the recombinant extracellular domain of Axl or Tyro3, but not that of Mertk, also inhibited platelet aggregation.

Conclusions: These data suggest that Axl and Tyro3, but not Mertk, have an important role in platelet activation and thrombus formation, and mechanistically may do so by a pathway that regulates inside to outside signaling and heterotypic interactions via the extracellular domains of TAMs.
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http://dx.doi.org/10.1186/s12964-018-0308-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6291976PMC
December 2018

The transmembrane protein disulfide isomerase TMX1 negatively regulates platelet responses.

Blood 2019 01 13;133(3):246-251. Epub 2018 Nov 13.

Sol Sherry Thrombosis Research Center, Division of Hematology, Department of Medicine, Temple University School of Medicine, Philadelphia, PA.

Secreted platelet protein disulfide isomerases, PDI, ERp57, ERp5, and ERp72, have important roles as positive regulators of platelet function and thrombosis. Thioredoxin-related transmembrane protein 1 (TMX1) was the first described transmembrane member of the protein disulfide isomerase family of enzymes. Using a specific antibody, the recombinant extracellular domain of TMX1 (rTMX1) protein, a knockout mouse model, and a thiol-labeling approach, we examined the role of TMX1 in platelet function and thrombosis. Expression of TMX1 on the platelet surface increased with thrombin stimulation. The anti-TMX1 antibody increased platelet aggregation induced by convulxin and thrombin, as well as potentiated platelet ATP release. In contrast, rTMX1 inhibited platelet aggregation and ATP release. TMX1-deficient platelets had increased aggregation, ATP release, αIIbβ3 activation, and P-selectin expression, which were reversed by addition of rTMX1. TMX1-knockout mice had increased incorporation of platelets into a growing thrombus in an FeCl-induced mesenteric arterial injury model, as well as shortened tail-bleeding times. rTMX1 oxidized thiols in the αIIbβ3 integrin and TMX1-deficient platelets had increased thiols in the β3 subunit of αIIbβ3, consistent with oxidase activity of rTMX1 against αIIbβ3. Thus, TMX1 is the first identified extracellular inhibitor of platelet function and the first disulfide isomerase that negatively regulates platelet function.
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http://dx.doi.org/10.1182/blood-2018-04-844480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6337875PMC
January 2019

The Plasma Kallikrein-Kininogen Pathway Is Critical in the Pathogenesis of Colitis in Mice.

Front Immunol 2018 6;9:21. Epub 2018 Feb 6.

Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, Soochow University, Suzhou, China.

The kallikrein-kinin system (KKS) consists of two serine proteases, prekallikrein (pKal) and factor XII (FXII), and a cofactor, high-molecular-weight kininogen (HK). Upon activation of the KKS, HK is cleaved to release bradykinin. Although the KKS is activated in humans and animals with inflammatory bowel disease (IBD), its role in the pathogenesis of IBD has not been characterized. In the present study, we determined the role of the KKS in the pathogenesis of IBD using mice that lack proteins involved in the KKS. In two colitis models, induced by dextran sulfate sodium (DSS) or 2,4,6-trinitrobenzene sulfonic acid (TNBS), mice deficient in HK, pKal, or bradykinin receptors displayed attenuated phenotypes, including body weight loss, disease activity index, colon length shortening, histological scoring, and colonic production of cytokines. Infiltration of neutrophils and inflammatory monocytes in the colonic lamina propria was reduced in HK-deficient mice. Reconstitution of HK-deficient mice through intravenous injection of HK recovered their susceptibility to DSS-induced colitis, increased IL-1β levels in the colon tissue and bradykinin concentrations in plasma. In contrast to the phenotypes of other mice lacking other proteins involved in the KKS, mice lacking FXII had comparable colonic inflammation to that observed in wild-type mice. The concentration of bradykinin was significantly increased in the plasma of wild-type mice after DSS-induced colitis. analysis revealed that DSS-induced pKal activation, HK cleavage, and bradykinin plasma release were prevented by the absence of pKal or the inhibition of Kal. Unlike DSS, TNBS-induced colitis did not trigger HK cleavage. Collectively, our data strongly suggest that Kal, acting independently of FXII, contributes to experimental colitis by promoting bradykinin release from HK.
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http://dx.doi.org/10.3389/fimmu.2018.00021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5808240PMC
February 2019

The Procoagulant Activity of Apoptotic Cells Is Mediated by Interaction with Factor XII.

Front Immunol 2017 25;8:1188. Epub 2017 Sep 25.

Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, Soochow University, Suzhou, China.

Apoptotic cells, by externalizing phosphatidylserine (PS) as a hallmark feature, are procoagulant. However, the mechanism by which apoptotic cells activate coagulation system remains unknown. Intrinsic coagulation pathway is initiated by coagulation factor XII (FXII) of contact activation system. The purpose of this study was to determine whether FXII is involved in procoagulant activity of apoptotic cells. Using western blotting and chromogenic substrate assay, we found that incubation with apoptotic cells, but not with viable cells, resulted in rapid cleavage and activation of FXII in the presence of prekallikrein and high molecular weight kininogen (HK), other two components of contact activation system. As detected by flow cytometry, FXII bound to apoptotic cells in a concentration-dependent manner, which was inhibited by annexin V and PS liposome. Direct association of FXII with PS was confirmed in a surface plasmon resonance assay. Clotting time of FXII-deficient plasma induced by apoptotic cells was significantly prolonged, which was fully reversed by replenishment with FXII. Corn trypsin inhibitor, a FXII inhibitor, completely prevented apoptotic cells-induced intrinsic tenase complex formation. Consistently, apoptotic cells significantly increased thrombin production in normal plasma, which was not affected by an inhibitory anti-tissue factor antibody. However, blocking of PS by annexin V, inhibition of FXII, or the deficiency of FXII suppressed apoptotic cells-induced thrombin generation. Addition of purified FXII to FXII-deficient plasma recovered thrombin generation to the normal plasma level. In conclusion, FXII binds to apoptotic cells PS and becomes activated, thereby constituting a novel mechanism mediating the procoagulant activity of apoptotic cells.
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http://dx.doi.org/10.3389/fimmu.2017.01188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5622377PMC
September 2017

A critical role for plasma kallikrein in the pathogenesis of autoantibody-induced arthritis.

FASEB J 2017 12 14;31(12):5419-5431. Epub 2017 Aug 14.

Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, Soochow University, Suzhou, China;

The plasma kallikrein-kinin system (KKS) consists of serine proteases, prekallikrein (pKal) and factor XII (FXII), and a cofactor, high-MW kininogen (HK). Upon activation, activated pKal and FXII cleave HK to release bradykinin. Activation of this system has been noted in patients with rheumatoid arthritis, and its pathogenic role has been characterized in animal arthritic models. In this study, we generated 2 knockout mouse strains that lacked pKal and HK and determined the role of KKS in autoantibody-induced arthritis. In a K/BxN serum transfer-induced arthritis (STIA) model, mice that lacked HK, pKal, or bradykinin receptors displayed protective phenotypes in joint swelling, histologic changes in inflammation, and cytokine production; however, FXII-deficient mice developed normal arthritis. Inhibition of Kal ameliorated arthritis severity and incidence at early stage STIA and reduced the levels of major cytokines in joints. In addition to releasing bradykinin from HK, Kal directly activated monocytes to produce proinflammatory cytokines, up-regulated their C5aR and FcRIII expression, and released C5a. Immune complex increased pKal activity, which led to HK cleavage. The absence of HK is associated with a decrease in joint vasopermeability. Thus, we identify a critical role for Kal in autoantibody-induced arthritis with pleiotropic effects, which suggests that it is a new target for the inhibition of arthritis.-Yang, A., Zhou, J., Wang, B., Dai, J., Colman, R. W., Song, W., Wu, Y. A critical role for plasma kallikrein in the pathogenesis of autoantibody-induced arthritis.
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http://dx.doi.org/10.1096/fj.201700018RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5690395PMC
December 2017

An essential role of high-molecular-weight kininogen in endotoxemia.

J Exp Med 2017 09 9;214(9):2649-2670. Epub 2017 Aug 9.

Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, Soochow University, Suzhou, China

In this study, we show that mice lacking high-molecular-weight kininogen (HK) were resistant to lipopolysaccharide (LPS)-induced mortality and had significantly reduced circulating LPS levels. Replenishment of HK-deficient mice with human HK recovered the LPS levels and rendered the mice susceptible to LPS-induced mortality. Binding of HK to LPS occurred through the O-polysaccharide/core oligosaccharide, consistent with the ability to bind LPS from , , , and different strains. Binding of LPS induced plasma HK cleavage to the two-chain form (HKa, containing a heavy chain [HC] and a light chain [LC]) and bradykinin. Both HKa and the LC, but not the HC, could disaggregate LPS. The light chain bound LPS with high affinity ( = 1.52 × 10 M) through a binding site in domain 5 (DHG15). A monoclonal antibody against D5 significantly reduced LPS-induced mortality and circulating LPS levels in wild-type mice. Thus, HK, as a major LPS carrier in circulation, plays an essential role in endotoxemia.
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http://dx.doi.org/10.1084/jem.20161900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5584120PMC
September 2017

Differential responses of peach (Prunus persica) seedlings to elevated ozone are related with leaf mass per area, antioxidant enzymes activity rather than stomatal conductance.

Environ Pollut 2017 Aug 5;227:380-388. Epub 2017 May 5.

State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Shuangqing Road 18, Haidian District, Beijing, 100085, China; College of Resources and Environment, University of Chinese Academy of Sciences, Shijingshan District, Beijing 100049, China; Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture PR China, Beijing University of Agriculture, Beijing 102206, China. Electronic address:

To evaluate the ozone (O) sensitivity among peach tree (Prunus persica) cultivars widely planted in Beijing region and explore the possible eco-physiological response mechanisms, thirteen cultivars of peach seedlings were exposed to either charcoal-filtered air or elevated O (E-O, non-filtered ambient air plus 60 ppb) for one growing season in open-top chambers. Leaf structure, stomatal structure, gas exchange and chlorophyll a fluorescence, photosynthetic pigments, antioxidant defense system and lipid peroxidation were measured in three replicated chambers. Results showed that E-O significantly reduced abaxial epidemis thickness, but no effects on the thicknesses of adaxial epidemis, palisade parenchyma and spongy parenchyma. Stomatal area, density and conductance were not significantly affected by E-O. E-O significantly accelerated leaf senescence, as indicated by increased lipid peroxidation and more declines in light-saturated photosynthetic rate and pigments contents. The reduced ascorbate content (ASC) was decreased but antioxidant enzyme activity (CAT, APX and SOD) and total antioxidant capacity (TAC) were significantly increased by E-O among cultivars. The cultivars with visible symptoms also had more reductions in net photosynthetic rate than those without visible symptoms. Ozone sensitivity among cultivars was strongly linked to leaf mass per area (LMA), antioxidant enzymes activity e.g. SOD, APX rather than stomatal parameters (stomatal area, density and conductance) and ASC. Results could provide a theoretical basis for selecting and breeding the ozone-resistant cultivars of peach trees grown in high O-polluted regions.
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http://dx.doi.org/10.1016/j.envpol.2017.04.068DOI Listing
August 2017

Quantitative Proteomics Analysis of Developmental Reprogramming in Protoplasts of the Moss Physcomitrella patens.

Plant Cell Physiol 2017 05;58(5):946-961

College of Life Sciences, Capital Normal University, Beijing, China.

The moss Physcomitrella patens is a model system for studying Plant developmental processes. To better understand the biochemical and physiological changes involved in developmental reprogramming, we conducted a quantitative proteomics analysis for protonemata, protoplasts made therefrom and protoplasts regenerated for 2 d. Using an iTRAQ peptide labeling strategy and liquid chromatography-tandem mass spectrometry (LC-MS/MS), >3,000 peptides and 1,000 proteins were quantified. Of these, 162 proteins were identified as having differential abundances during developmental reprogramming. These proteins were involved in various biological functions, such as defense, energy production, translation, metabolism, protein destination and storage, transcription, transport, cell growth/division, cell structure and signal transduction. Of these, the proteins involved in energy production and translation increased in abundance, while many of the metabolism and defense proteins decreased in abundance. In addition, most of the cell growth/division, protein stability and cell structure proteins were also down-regulated. This is the first report on the metabolic changes involved in developmental reprogramming in protoplasts. The significance of metabolic networks in developmental programming is beginning to emerge. Our study suggested that stress signals, energy metabolism and ribosomal proteins are pivotal components during developmental programming.
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http://dx.doi.org/10.1093/pcp/pcx039DOI Listing
May 2017

Moderate drought did not affect the effectiveness of ethylenediurea (EDU) in protecting Populus cathayana from ambient ozone.

Sci Total Environ 2016 Nov 14;569-570:1536-1544. Epub 2016 Jul 14.

State key laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Shuangqing Road 18, Haidian District, Beijing, 100085, China; University of Chinese Academy of Sciences, Shijingshan District, Beijing 100049, China; Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture P. R. China, Beijing University of Agriculture, Beijing 102206, China. Electronic address:

A field study was conducted to evaluate the effects of ambient ozone (O3) on an O3-sensitive poplar (Populus cathayana) by using ethylenediurea (EDU) as a chemical protectant under two soil water treatments (well-watered (WW) and moderate drought (MD, 50-60% of WW in volumetric soil water content). EDU was applied as foliar spray at 0, 300, 450, and 600ppm. Photosynthetic parameters, pigment contents, leaf nitrogen, antioxidant capacity, growth, and biomass were measured. The 8h (9:00-17:00) average ambient O3 concentration was 71.7ppb, and AOT40 was 29.2ppmh during the experimental period (9 June to 21 September), which was high enough to cause plant injury. MD had significantly negative effects on P. cathayana, as indicated by reduced photosynthesis, growth, and biomass, and higher MDA contents. On the other hand, EDU significantly increased photosynthesis rate, chlorophyll a fluorescence, Vcmax and Jmax, photosynthetic pigments, total antioxidant capacity, tree growth and biomass accumulation, and reduced lipid peroxidation, but there was no significant interaction between EDU and drought for most parameters, indicating that EDU can efficiently protect Populus cathayana against ambient O3 and the protection was not affected by soil water contents when soil water reached moderate drought level. Among all doses, EDU at 450ppm provided maximum protection. Comparison of EDU-treated and non-treated P. cathayana could be used as a biomarker system in risk assessment of the effects of ambient O3 on forest health.
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http://dx.doi.org/10.1016/j.scitotenv.2016.06.247DOI Listing
November 2016

Focal adhesion kinase overexpression and its impact on human osteosarcoma.

Oncotarget 2015 Oct;6(31):31085-103

Department of Orthopedics, The Third Affiliated Hospital of Soochow University, The First People's Hospital of Changzhou, Changzhou 213003, Jiangsu Province, P.R.China.

Focal adhesion kinase (FAK) has been implicated in tumorigenesis in various malignancies. We sought to examine the expression patterns of FAK and the activated form, phosphorylated FAK (pFAK), in human osteosarcoma and to investigate the correlation of FAK expression with clinicopathologic parameters and prognosis. In addition, the functional consequence of manipulating the FAK protein level was investigated in human osteosarcoma cell lines. Immunohistochemical staining was used to detect FAK and pFAK in pathologic archived materials from 113 patients with primary osteosarcoma. Kaplan-Meier survival and Cox regression analyses were performed to evaluate the prognoses. The role of FAK in the cytological behavior of MG63 and 143B human osteosarcoma cell lines was studied via FAK protein knock down with siRNA. Cell proliferation, migration, invasiveness and apoptosis were assessed using the CCK8, Transwell and Annexin V/PI staining methods. Both FAK and pFAK were overexpressed in osteosarcoma. There were significant differences in overall survival between the FAK-/pFAK- and FAK+/pFAK- groups (P = 0.016), the FAK+/pFAK- and FAK+/pFAK+ groups (P = 0.012) and the FAK-/pFAK- and FAK+/pFAK+ groups (P < 0.001). There were similar differences in metastasis-free survival between groups. The Cox proportional hazards analysis showed that the FAK expression profile was an independent indicator of both overall and metastasis-free survival. SiRNA-based knockdown of FAK not only dramatically reduced the migration and invasion of MG63 and 143B cells, but also had a distinct effect on osteosarcoma cell proliferation and apoptosis. These results collectively suggest that FAK overexpression and phosphorylation might predict more aggressive biologic behavior in osteosarcoma and may be an independent predictor of poor prognosis.
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http://dx.doi.org/10.18632/oncotarget.5044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4741590PMC
October 2015

Combretastatin A4 phosphate treatment induces vasculogenic mimicry formation of W256 breast carcinoma tumor in vitro and in vivo.

Tumour Biol 2015 Nov 31;36(11):8499-510. Epub 2015 May 31.

Laboratory of Translational Medicine, Jiangsu Province Academy of Traditional Chinese Medicine, No. 100, Shizi Street, Hongshan Road, Nanjing, 210028, Jiangsu Province, People's Republic of China.

The purpose of this study was to investigate the effect of combretastatin A4 phosphate (CA4P) on vasculogenic mimicry (VM) channel formation in vitro and in vivo after a single-dose treatment and the underlying mechanism involved in supporting VM. In vitro model of three-dimensional cultures was used to test the effect of CA4P on the tube formation of Walker 256 cells. Western blot analysis was conducted to assess the expression of hypoxia-inducible factor (HIF)-1α and VM-associated markers. W256 tumor-bearing rat model was established to demonstrate the effect of CA4P on VM formation and tumor hypoxia by double staining and a hypoxic marker pimonidazole. Anti-tumor efficacy of CA4P treatment was evaluated by tumor growth curve. Under hypoxic conditions for 48 h in vitro, W256 cells formed VM network associated with increased expression of VM markers. Pretreatment with CA4P did not influence the amount of VM in 3-D culture as well as the expression of these key molecules. In vivo, W256 tumors showed marked intratumoral hypoxia after CA4P treatment, accompanied by increased VM formation. CA4P exhibited only a delay in tumor growth within 2 days but rapid tumor regrowth afterward. VM density was positively related to tumor volume and tumor weight at day 8. CA4P causes hypoxia which induces VM formation in W256 tumors through HIF-1α/EphA2/PI3K/matrix metalloproteinase (MMP) signaling pathway, resulting in the consequent regrowth of the damaged tumor.
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http://dx.doi.org/10.1007/s13277-015-3508-xDOI Listing
November 2015

High molecular weight kininogen binds phosphatidylserine and opsonizes urokinase plasminogen activator receptor-mediated efferocytosis.

J Immunol 2014 May 31;192(9):4398-408. Epub 2014 Mar 31.

Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, First Affiliated Hospital, Soochow University, Suzhou 215006, China;

Phagocytosis of apoptotic cells (efferocytosis) is essential for regulation of immune responses and tissue homeostasis and is mediated by phagocytic receptors. In this study, we found that urokinase plasminogen activator receptor (uPAR) plays an important role in internalization of apoptotic cells and also characterized the underlying mechanisms. In a flow cytometry-based phagocytic assay, uPAR-deficient macrophages displayed significant defect in internalization but not tethering of apoptotic cells. When uPAR-deficient mice were challenged with apoptotic cells, they exhibited pronounced splenomegaly resulting from accumulation of abundant apoptotic cells in spleen. Overexpression of uPAR in HEK-293 cells enhanced efferocytosis, which was inhibited by Annexin V and phosphatidylserine (PS) liposome, suggesting that uPAR-mediated efferocytosis is dependent on PS. In serum lacking high m.w. kininogen (HK), a uPAR ligand, uPAR-mediated efferocytosis was significantly attenuated, which was rescued by replenishment of HK. As detected by flow cytometry, HK selectively bound to apoptotic cells, but not viable cells. In purified systems, HK was specifically associated with PS liposome. HK binding to apoptotic cells induced its rapid cleavage to the two-chain form of HK (HKa) and bradykinin. Both the H chain and L chain of HKa were associated with PS liposome and apoptotic cells. HKa has higher binding affinity than HK to uPAR. Overexpression of Rac1/N17 cDNA inhibited uPAR-mediated efferocytosis. HK plus PS liposome stimulated a complex formation of CrkII with p130Cas and Dock-180 and Rac1 activation in uPAR-293 cells, but not in control HEK-293 cells. Thus, uPAR mediates efferocytosis through HK interaction with PS on apoptotic cells and activation of the Rac1 pathway.
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http://dx.doi.org/10.4049/jimmunol.1302590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4134950PMC
May 2014

A role for bradykinin in the development of anti-collagen antibody-induced arthritis.

Rheumatology (Oxford) 2014 Jul 5;53(7):1301-6. Epub 2014 Mar 5.

Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, First Affiliated Hospital, Soochow University, Suzhou, China and Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, USA.Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, First Affiliated Hospital, Soochow University, Suzhou, China and Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, USA.

Objectives: Clinical and experimental observations have suggested that bradykinin, a major activation product of the plasma kallikrein-kinin system, is involved in the pathogenesis of arthritis, but the pathogenic role of bradykinin receptors remains inconclusive. In this study we examined whether bradykinin receptors are important in the pathogenesis of anti-collagen antibody-induced arthritis (CAIA) using double receptor-deficient (B1RB2R(-/-)) mice.

Methods: CAIA was induced in B1RB2R(+/+) and B1RB2R(-/-) mice by injection of an anti-collagen antibody cocktail on day 0 and lipopolysaccharide on day 3. Severity of disease was evaluated by measurement of joint diameter and histological analysis. The expression of proinflammatory cytokines in joint tissue and peripheral mononuclear cells was determined by ELISA and real-time RT-PCR.

Results: The absent expression of B1R and B2R mRNA in B1RB2R(-/-) mice was confirmed by RT-PCR. Although B1RB2R(+/+) mice developed severe CAIA, the severity of the disease was significantly attenuated in B1RB2R(-/-) mice. In B1RB2R(+/+) mice bearing CAIA, both B1R and B2R mRNA levels were increased in joint tissue and peripheral mononuclear cells. Compared with B1RB2R(+/+) mice, the production of IL-1β and IL-6 in joint tissue and their mRNA expression in peripheral mononuclear cells were remarkably reduced in B1RB2R(-/-) mice.

Conclusion: These observations provide genetic evidence that bradykinin plays an important role in the pathogenesis of CAIA. B1R, whose expression is induced in inflamed joint tissue and peripheral inflammatory cells, is important in the development of CAIA.
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http://dx.doi.org/10.1093/rheumatology/keu015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4065007PMC
July 2014

Basic fibroblast growth factor is a key factor that induces bone marrow mesenchymal stem cells towards cells with Schwann cell phenotype.

Neurosci Lett 2014 Jan 3;559:82-7. Epub 2013 Dec 3.

Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Nantong, Jiangsu Province 226001, PR China. Electronic address:

Bone marrow mesenchymal stem cells (MSCs) can be differentiate towards a Schwann cells (SCs) lineage when exposed to pre-inducing reagents β-mercaptoethanol (BME) and retinoic acid (RA), followed by inducing factors: forskolin (FSK), basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), and heregulin (HRG). However, the underlying mechanisms remain unclear. Here, we investigated the individual effects of these inducing factors on the differentiation of MSCs towards SC phenotype in rats. We show that the omission of either HRG or PDGF from the induction medium is not sufficient to change the SC-like phenotype or the expression level of the SC marker, S100β. However, the omission of bFGF from the induction medium effectively blocked neural induction of the MSCs. Moreover, only bFGF was found to inhibit MSC proliferation during differentiation. To clarify the mechanism responsible for the effect of bFGF, we also investigated the activation of the extracellular signal-regulated kinase (ERK) pathway in the induced cells. Our results suggest that morphological changes in MSCs induced by bFGF depend on the activation of ERK, and bFGF may be an indispensable factor that induces MSCs to differentiate into cells with SCs phenotype.
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http://dx.doi.org/10.1016/j.neulet.2013.11.044DOI Listing
January 2014

Prophylactic and therapeutic functions of drug combinations against noise-induced hearing loss.

Hear Res 2013 Oct 18;304:33-40. Epub 2013 Jun 18.

Department of Otolaryngology, Center for Aging, Washington University School of Medicine, 4560 Clayton Avenue, St. Louis, MO 63110, USA. Electronic address:

Noise is the most common occupational and environmental hazard. Noise-induced hearing loss (NIHL) is the second most common form of sensorineural hearing deficit, after age-related hearing loss (presbycusis). Although promising approaches have been identified for reducing NIHL, currently there are no effective medications to prevent NIHL. Development of an efficacious treatment has been hampered by the complex array of cellular and molecular pathways involved in NIHL. We turned this difficulty into an advantage by asking whether NIHL could be effectively prevented by targeting multiple signaling pathways with a combination of drugs already approved by U.S. Food and Drug Administration (FDA). We previously found that antiepileptic drugs blocking T-type calcium channels had both prophylactic and therapeutic effects for NIHL. NIHL can also be reduced by an up-regulation of glucocorticoid (GC) signaling pathways. Based on these findings, we tested a combination therapy for NIHL that included ethosuximide and zonisamide (anticonvulsants) and dexamethasone and methylprednisolone (synthetic GCs) in mice under exposure conditions typically associated with dramatic permanent threshold shifts (PTS). We first examined possible prophylactic effects for each drug when administered alone 2 h before noise, and calculated the median effective dose (ED50). We then tested for synergistic effects of two-drug combinations (anticonvulsant + GC), and identified combinations with the strongest synergy against NIHL, based on a previously established combination index (CI) metric. We repeated similar tests to determine their therapeutic effects when administered the same drugs 24 h after the noise exposure. Our study shows the feasibility of developing pharmacological intervention in multiple pathways, and discovering drug combinations with optimal synergistic effects in preventing permanent NIHL.
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http://dx.doi.org/10.1016/j.heares.2013.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3769468PMC
October 2013

Role of plasma kallikrein-kinin system activation in synovial recruitment of endothelial progenitor cells in experimental arthritis.

Arthritis Rheum 2012 Nov;64(11):3574-82

Temple University School of Medicine, Philadelphia, Pennsylvania, USA.

Objective: To examine whether activation of the plasma kallikrein-kinin system (KKS) mediates synovial recruitment of endothelial progenitor cells (EPCs) in arthritis.

Methods: EPCs were isolated from Lewis rat bone marrow, and expression of progenitor cell-lineage markers and functional properties were characterized. EPCs were injected intravenously into Lewis rats with arthritis, and their recruitment and formation of de novo blood vessels in inflamed synovium were evaluated. The role of plasma KKS was examined using a plasma kallikrein inhibitor (EPI-KAL2) and an antikallikrein antibody (13G11). A transendothelial migration assay was used to determine the role of bradykinin and its receptor in EPC mobilization.

Results: EPCs from Lewis rats exhibited a strong capacity to form tubes and vacuoles and expressed increased levels of bradykinin type 2 receptor (B2R) and progenitor cell markers CD34 and Sca-1. In Lewis rats with arthritis, EPCs were recruited into inflamed synovium at the acute phase of disease and formed de novo blood vessels. Inhibition of plasma kallikrein by EPI-KAL2 and 13G11 significantly suppressed synovial recruitment of EPCs and hyperproliferation of synovial cells. Bradykinin stimulated transendothelial migration of EPCs in a concentration-dependent manner. This was mediated by B2R, as demonstrated by the finding that knockdown of B2R with silencing RNA completely blocked bradykinin-stimulated transendothelial migration. Moreover, bradykinin selectively up-regulated expression of the homing receptor CXCR4 in EPCs.

Conclusion: These observations demonstrate a novel role of plasma KKS activation in the synovial recruitment of EPCs in arthritis, acting via kallikrein activation and B2R-dependent mechanisms. B2R might be involved in the mobilization of EPCs via up-regulation of CXCR4.
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http://dx.doi.org/10.1002/art.34607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3477304PMC
November 2012

mRNA profiles of cytokine receptors in unstimulated peripheral blood mononuclear cells from patients with chronic idiopathic urticaria.

J Biomed Res 2011 Mar;25(2):141-7

Department of Dermatology, Haidian Hospital, Beijing 10080, China ; Section of Clinical Immunology, Institute of Dermatology, Chinese Academy of Medical Sciences & Peking Union Medical College, Nanjing 210042, Jiangsu, China.

This present study was aimed to investigate the roles of the receptors of Th1/Th2 cytokines and chemokines in the pathogenesis of chronic idiopathic urticaria (CIU). Thirty patients with CIU, 30 patients with dermographism and 30 healthy controls were randomly enrolled. Reverse transcription-PCR (RT-PCR) was used to analyze the mRNA of cytokine receptors in peripheral blood mononuclear cells (PBMCs). The mRNA levels of tumor necrosis factor receptor (TNFR), interferon-γ receptor (IFN-γR), and interleukin-10 receptor (IL-10R) were statistically increased in the CIU group (P < 0.05), while IL-2R, IL-4R, IL-6R, and IL-13R showed no significant differences between the CIU and other groups. The mRNA levels of CCR3 and CCR6 were statistically increased in the CIU group (P < 0.05). The toll-like receptor 2 (TLR2) mRNA level was significantly lower in the CIU group than the healthy control group (P < 0.05). These findings indicate that the regulation of mRNA of TNFR, IFN-γR, IL-10R, CCR3, CCR6 and TLR2 may be involved in the pathogenesis of CIU.
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http://dx.doi.org/10.1016/S1674-8301(11)60018-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3596706PMC
March 2011

Relationship of semicircular canal size to vestibular-nerve afferent sensitivity in mammals.

J Neurophysiol 2007 Dec 3;98(6):3197-205. Epub 2007 Oct 3.

Department of Otolaryngology, Head and Neck Surgery, Washington University in St Louis School of Medicine, St Louis, MO 63110, USA.

The relationship between semicircular canal radius of curvature and afferent sensitivity has not been experimentally determined. We characterized mouse semicircular canal afferent responses to sinusoidal head rotations to facilitate interspecies and intraspecies comparisons of canal size to sensitivity. The interspecies experiment compared the horizontal canal afferent responses among animals ranging in size from mouse to rhesus monkey. The intraspecies experiment compared afferent responses from the larger anterior canal to those from the smaller horizontal canal of mice. The responses of mouse vestibular-nerve afferents showed a low- and high-frequency phase lead and high-frequency gain enhancement. Regular horizontal-canal afferents showed a sensitivity to 0.5-Hz sinusoidal rotations of 0.10 +/- 0.03 (SD) spike . s(-1)/deg . s(-1) and high-gain irregular afferents showed a sensitivity of 0.25 +/- 0.11 spike . s(-1)/deg . s(-1). The interspecies comparison showed that the sensitivity of regular afferents was related to the radius of curvature R according to the formula G(r) = 0.23R - 0.09 (r(2) = 0.86) and the sensitivity of irregular afferents was related to radius according to the formula G(i) = 0.32R + 0.01 (r(2) = 0.67). The intraspecies comparison showed that regularly firing anterior canal afferents were significantly more sensitive than those from the relatively smaller horizontal canal, with G(r) = 0.25R. This suggests that canal radius of curvature is closely related to afferent sensitivity both among and within species. If the relationship in humans is similar to that demonstrated here, the sensitivity of their regular vestibular-nerve afferents to 0.5-Hz rotations is likely to be about 0.67 spike . s(-1)/deg . s(-1) and of their high-gain irregular afferents about 1.06 spikes . s(-1)/deg . s(-1).
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http://dx.doi.org/10.1152/jn.00798.2007DOI Listing
December 2007

Inhibitory effects of clofilium on membrane currents associated with Ca channels, NMDA receptor channels and Na+, K+-ATPase in cortical neurons.

Pharmacology 2005 Mar 3;73(3):162-8. Epub 2005 Jan 3.

Department of Pharmaceutical Sciences, College of Pharmacy, Medical University of South Carolina, Charleston, SC 29425, USA.

The class III antiarrhythmic agent 4-chloro-N,N-diethyl-N-heptyl-benzene butanaminium (clofilium) is known as a K+ channel open-channel blocker and has either anti- or proapoptotic property due to undefined mechanisms. Based on the evidence that neuronal viability is largely, sometimes critically, affected by voltage- and ligand-gated Ca2+ channels and the Na+, K+-ATPase, we tested the hypothesis that clofilium might additionally act on Ca2+ permeable ion channels and the Na+, K+-ATPase. Membrane currents associated with activities of voltage-gated Ca2+ channels, N-methyl-D-aspartate (NMDA) receptor channels and Na+, K+-ATPase were recorded using whole-cell recordings in cultured murine cortical neurons. Clofilium (0.1-100 micromol/l) inhibited high voltage-activated Ca2+ currents in concentration- and use-dependent manners. Clofilium acted as a potent antagonist of NMDA receptor channels, preferably blocked the NMDA steady-state current at a low concentration (0.1 micromol/l). At concentrations of >100 micromol/l, clofilium blocked both peak and steady-state NMDA currents in a voltage-independent manner. Clofilium also inhibited the Na+, K+-ATPase current with an IC50 of 7.5 micromol/l. Our data suggest that the pharmacological action of clofilium is far more complex than recognized before; the multiple actions of clofilium on membrane conductance may explain its diverse effects on cellular events and cell viability.
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http://dx.doi.org/10.1159/000083072DOI Listing
March 2005

Effects of chloride and potassium channel blockers on apoptotic cell shrinkage and apoptosis in cortical neurons.

Pflugers Arch 2004 Jun 1;448(3):325-34. Epub 2004 Apr 1.

Department of Pathology, Medical University of South Carolina, Charleston, SC 29425, USA.

K+ and Cl- homeostasis have been implicated in cell volume regulation and apoptosis. We addressed the hypothesis that K+ and Cl- efflux may contribute to apoptotic cell shrinkage and apoptotic death in cultured cortical neurons. CLC-2 and CLC-3 chloride channels were detected in cultured cortical neurons. The Cl- channel blockers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) inhibited the outwardly rectifying Cl- current, prevented apoptotic cell shrinkage, and mildly attenuated cell death induced by staurosporine, C2-ceramide, or serum deprivation. Cl- channel blockers, however, at concentrations that prevented cell shrinkage had no significant effects on caspase activation and/or DNA fragmentation. Cell death in the presence of a Cl- channel blocker was still sensitive to blockade by the caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethyl ketone (z-VAD-fmk). Electron microscopy revealed that, although DIDS prevented apoptotic cell shrinkage, certain apoptotic ultrastructural alterations still took place in injured neurons. On the other hand, the K+ channel blocker tetraethylammonium (TEA), clofilium, or the caspase inhibitor z-VAD-fmk prevented cell shrinkage as well as caspase activation and/or DNA damage, and showed stronger neuroprotection against apoptotic alterations and cell death. The results indicate that neurons may undergo apoptotic process without cell shrinkage and imply distinct roles for Cl- and K+ homeostasis in regulating different apoptotic events.
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http://dx.doi.org/10.1007/s00424-004-1277-2DOI Listing
June 2004

Potassium attenuates zinc-induced death of cultured cortical astrocytes.

Glia 2004 Apr;46(1):18-27

Department of Neurology and Center for the Study of Nervous System Injury, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

Transient global ischemia induces CA1 hippocampal neuronal death without astrocyte death, perhaps mediated in part by the toxic translocation of zinc from presynaptic terminals to postsynaptic neurons. We tested the hypothesis that cellular depolarization, which occurs in the ischemic brain due to increased extracellular potassium and energy failure, might contribute to astrocyte resistance to zinc-induced death. We previously reported that neurons in mixed cortical neuronal-astrocyte cultures were more vulnerable to a 5-15-min exposure to Zn(2+) than astrocytes in the same cultures. In the present report, we show that (1) neurons in isolation or in conjunction with astrocytes were 2-3-fold more sensitive to a 15-min nondepolarizing Zn(2+) exposure than are glia; (2) KCl-induced depolarization attenuated glial vulnerability to zinc toxicity but potentiated neuronal vulnerability to zinc toxicity; (3) Zn(2+)-induced glial death was attenuated by T-type Ca(2+) channel blockade, as well as compounds that increase NAD(+) levels; and (4) both astrocytic (65)Zn(2+) accumulation and the increase in astrocytic [Zn(2+)](i) induced by Zn(2+) exposure were also attenuated by depolarization or T-type Ca(2+) channel blockers. Zn(2+)-induced cell death in astrocytes was at least in part apoptotic, as caspase-3 was activated, and the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone partially attenuated Zn(2+)-induced death. The levels of peak [Zn(2+)](i) achieved in astrocytes during this toxic nondepolarizing Zn(2+) exposure (250 nM) were substantially greater than those achieved in neurons (40 nM). In glia, exposure to 400 microM Zn(2+) induced a 13-mV depolarization, which can activate T-type Ca(2+) channels. This Zn(2+)-induced astrocyte death, like neuronal death, was attenuated by the addition of pyruvate or niacinamide to the exposure medium.
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http://dx.doi.org/10.1002/glia.10313DOI Listing
April 2004

Apoptotic insults impair Na+, K+-ATPase activity as a mechanism of neuronal death mediated by concurrent ATP deficiency and oxidant stress.

J Cell Sci 2003 May 1;116(Pt 10):2099-110. Epub 2003 Apr 1.

Center for the Study of Nervous System Injury and Department of Neurology, Washington University School of Medicine, St Louis, MO 63110, USA.

The Na+, K+-ATPase (Na+, K+-pump) plays critical roles in maintaining ion homeostasis. Blocking the Na+, K+-pump may lead to apoptosis. By contrast, whether an apoptotic insult may affect the Na+, K+-pump activity is largely undefined. In cultured cortical neurons, the Na+, K+-pump activity measured as a membrane current Ipump was time-dependently suppressed by apoptotic insults including serum deprivation, staurosporine, and C2-ceramide, concomitant with depletion of intracellular ATP and production of reactive oxygen species. Signifying a putative relationship among these events, Ipump was highly sensitive to changes in ATP and reactive oxygen species levels. Moreover, the apoptosis-associated Na+, K+-pump failure and serum deprivation-induced neuronal death were antagonized by pyruvate and succinate in ATP- and reactive-oxygen-species-dependent manners. We suggest that failure of the Na+, K+-pump as a result of a combination of energy deficiency and production of reactive oxygen species is a common event in the apoptotic cascade; preserving the pump activity provides a neuroprotective strategy in certain pathological conditions.
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http://dx.doi.org/10.1242/jcs.00420DOI Listing
May 2003

Block of Na+,K+-ATPase and induction of hybrid death by 4-aminopyridine in cultured cortical neurons.

J Pharmacol Exp Ther 2003 May 21;305(2):502-6. Epub 2003 Jan 21.

Center for the Study of Nervous System Injury and Department of Neurology, Washington University School of Medicine, St. Louis, Missouri, USA.

K(+) channel blockers such as 4-aminopyridine (4-AP) can be toxic to neurons; the cellular mechanism underlying the toxicity, however, is obscure. In cultured mouse cortical neurons, we tested the hypothesis that the toxic effect of 4-AP might result from inhibiting the Na(+),K(+)-ATPase (Na(+),K(+)-pump) and thereafter induction of a hybrid death of concomitant apoptosis and necrosis. The Na(+),K(+)-pump activity, monitored as whole-cell membrane currents, was markedly blocked by 4-AP in concentration- and voltage-dependent manners in low millimolar ranges. At similar concentrations, 4-AP induced a neuronal death sensitive to attenuation by the caspase inhibitor Z-VAD-FMK (Z-Val-Ala-Asp(OMe)-fluoromethyl ketone) or Ca(2+) chelator BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester). Electron microscopy confirmed hybrid ultrastructural features of coexisting apoptotic and necrotic components in same cells. We suggest that 4-AP is a potent antagonist of the Na(+),K(+)-ATPase and an inducer of the hybrid death of central neurons.
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http://dx.doi.org/10.1124/jpet.102.045013DOI Listing
May 2003

Double lox targeting for neural cell transgenesis.

Brain Res Mol Brain Res 2003 Feb;110(2):220-33

Department of Anatomy and Neurobiology, Box 8108, Washington University School of Medicine, 660 S. Euclid Avenue, St Louis, MO 63110, USA.

ES cells differentiated along the neural lineage in vitro are an attractive model system. Here we have developed ES cell lines that are suitable for inserting transgenes at a single chromosomal site. ES cell line CE1 (for Cassette Exchange) contains one "acceptor" module (CE1) that allows for efficient double lox targeting. The site is also permissive for gene expression in neural progenitor cells, as well as differentiated neurons and glia. Line CE2 was derived by swapping a puromycin resistance cassette into CE1. Neural progenitors derived from this line are puromycin-resistant. A beta-actin/GFP expression cassette was inserted into the CE1 site to create CE3. The CE3 cell line was differentiated into neural cells and displayed strong EGFP expression in neural progenitors, differentiated neurons and glia. Differentiated CE3 ES cells (4-/4+ RA) were transplanted into the injured rat somatosensory cortex where many of the transplanted cells survived and differentiated into neurons expressing GFP. This strategy for creating sets of transgenic lines with multiple cassettes inserted into a single chromosomal site provides a powerful tool for studying development and function of ES cell-derived neural cells. Many of these will be useful in transplantation research.
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http://dx.doi.org/10.1016/s0169-328x(02)00651-4DOI Listing
February 2003

Slight impairment of Na+,K+-ATPase synergistically aggravates ceramide- and beta-amyloid-induced apoptosis in cortical neurons.

Brain Res 2002 Nov;955(1-2):253-9

Department of Neurology, Box 811, School of Medicine, Washington University, St Louis, MO 63110, USA.

Dysfunction of the Na(+),K(+)-ATPase (Na(+),K(+)-pump), due to reduced energy supply or increased endogenous ouabain-like inhibitors, likely occurs under pathological conditions in the central nervous system. In cultured mouse cortical neurons, we examined the hypothesis that a mild non-toxic inhibition of the Na(+),K(+)-ATPase could synergistically sensitize the vulnerability of neurons to normally non-lethal apoptotic signals. Ouabain at a low concentration of 0.1 microM slightly lessened the Na(+),K(+)-pump activity measured as an ouabain-sensitive current, yet did not affect K(+) homeostasis and viability of cortical neurons. Co-exposure to 0.1 microM ouabain plus non-lethal C(2)-ceramide (5 microM) or beta-amyloid 1-42 (5 microM), however, induced marked intracellular K(+) loss, caspase-3 cleavage, DNA laddering, and synergistically triggered neuronal death. The caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-FMK) predominantly blocked the caspase activation and neuronal death. These results suggest that slight impairment of Na(+),K(+)-pump activity may amplify the disruption of K(+) homeostasis in the presence of a non-lethal apoptotic insult, leading to activation of apoptotic cascade and substantial neuronal injury.
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http://dx.doi.org/10.1016/s0006-8993(02)03472-8DOI Listing
November 2002

Multiple channel interactions explain the protection of sympathetic neurons from apoptosis induced by nerve growth factor deprivation.

J Neurosci 2002 Jan;22(1):114-22

Department of Neurology and Center for the Study of Nervous System Injury, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

We investigated the neuroprotective properties of two M-type K+ channel blockers, linopirdine and its analog XE991, in rat sympathetic neurons deprived of nerve growth factor (NGF). Linopirdine and XE991 promoted sympathetic neuronal survival 48-72 hr after NGF withdrawal in a concentration-dependent manner. Both drugs prevented neuronal apoptosis by blocking the pathway leading to the release of cytochrome c and development of "competence-to-die" after NGF deprivation. Fura-2 Ca2+ imaging showed no significant difference in intracellular free Ca2+ ([Ca2+]i) in the presence or absence of NGF; linopirdine and XE991, on the other hand, caused membrane depolarization and increases in [Ca2+]i. Whole-cell recordings showed that linopirdine and XE991 selectively blocked the M current at neuroprotective concentrations, although they additionally inhibited other K+ currents at high concentrations. Membrane depolarization and [Ca2+]i increases induced by linopirdine and XE991 were blocked by the Na+ channel blocker tetrodotoxin (TTX) or by the L-type Ca2+ channel antagonist nifedipine. TTX and nifedipine also prevented the neuroprotection elicited by linopirdine or XE991. We propose that Na+ channel activation amplifies the membrane depolarization produced by M channel blockade and is essential for subsequent Ca2+ entry via the L-type Ca2+ channel. The interaction of these three classes of ion channels highlights an integrated anti-apoptosis mechanism in sympathetic neurons.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6757625PMC
January 2002