技术资料

CONTEXT AND OBJECTIVE: Impaired nitric oxide (NO) bioavailability and low levels of circulating endothelial progenitor cells (EPC) are correlated to an increased risk for development of cardiovascular diseases. We investigated whether improved systemic NO bioavailability and increased levels of EPC after GH treatment are related and mediated by the IGF-I. DESIGN,PATIENTS,AND RESULTS: Healthy middle-aged volunteers (n = 16) were treated for 10 d with recombinant human GH. Before and after GH treatment,we analyzed markers of NO bioavailability and EPC levels. GH treatment was responded by significant increases in plasma IGF-I levels. Urinary cGMP levels were increased and diastolic blood pressure reduced after GH treatment (P textless 0.05). Likewise,plasma nitrate and nitrite levels were increased,whereas the NO synthase inhibitor asymmetric dimethylarginine was reduced. Correspondingly,IGF-I treatment increased expression of the asymmetric dimethylarginine-metabolizing enzyme dimethylarginie dimethylaminohydrolase-1 and dimethylarginie dimethylaminohydrolase-2 in cultured human endothelial cells. IGF-I levels correlated with cGMP concentrations (r = 0.51; P textless 0.05). EPC numbers were increased after GH treatment and correlated with markers for NO bioavailability. These findings were also observed in mice treated with GH for 7 d. GH treatment additionally increased aortic endothelial NO synthase expression of mice. Importantly,blocking of the IGF-I receptor in vivo abolished the GH-mediated effects on markers of increased NO bioavailability. CONCLUSIONS: GH treatment induced markers of increased NO bioavailability and enhanced circulating EPC numbers in healthy volunteers. Animal data demonstrate increased NO availability to be mediated via an increase in IGF-I plasma levels. Thus,GH treatment enhances systemic NO bioavailability via IGF-I and may be beneficial in certain cardiovascular diseases. View Publication

AMP-activated protein kinase (AMPK) plays a key role in maintaining energy homeostasis. Activation of AMPK in peripheral tissues has been shown to alleviate the symptoms of metabolic diseases,such as type 2 diabetes,and consequently AMPK is a target for treatment of these diseases. Recently,a small molecule activator (A-769662) of AMPK was identified that had beneficial effects on metabolism in ob/ob mice. Here we show that A-769662 activates AMPK both allosterically and by inhibiting dephosphorylation of AMPK on Thr-172,similar to the effects of AMP. A-769662 activates AMPK harboring a mutation in the gamma subunit that abolishes activation by AMP. An AMPK complex lacking the glycogen binding domain of the beta subunit abolishes the allosteric effect of A-769662 but not the allosteric activation by AMP. Moreover,mutation of serine 108 to alanine,an autophosphorylation site within the glycogen binding domain of the beta1 subunit,almost completely abolishes activation of AMPK by A-769662 in cells and in vitro,while only partially reducing activation by AMP. Based on our results we propose a model for activation of AMPK by A-769662. Importantly,this model may provide clues for understanding the mechanism by which AMP leads to activation of AMPK,which in turn may help in the identification of other AMPK activators. View Publication

A novel population of tissue-resident endothelial precursors (TEPs) was isolated from small blood vessels in dermal,adipose,and skeletal muscle of mouse based on their ability to be grown as spheres. Cellular and molecular analyses of these cells revealed that they were highly related regardless of the tissue of origin and distinct from embryonic neural stem cells. Notably,TEPs did not express hematopoietic markers,but they expressed numerous characteristics of angiogenic precursors and their differentiated progeny,such as CD34,Flk-1,Tie-1,CD31,and vascular endothelial cadherin (VE-cadherin). TEPs readily differentiated into endothelial cells in newly formed vascular networks following transplantation into regenerating skeletal muscle. Taken together,these experiments suggest that TEPs represent a novel class of endothelial precursors that are closely associated with small blood vessels in muscle,adipose,and dermal tissue. This finding is of particular interest since it could bring new insight in cancer angiogenesis and collateral blood vessels developed following ischemia. Disclosure of potential conflicts of interest is found at the end of this article. View Publication

The transcription factor Runx1/AML1 is an important regulator of hematopoiesis and is critically required for the generation of the first definitive hematopoietic stem cells (HSCs) in the major vasculature of the mouse embryo. As a pivotal factor in HSC ontogeny,its transcriptional regulation is of high interest but is largely undefined. In this study,we used a combination of comparative genomics and chromatin analysis to identify a highly conserved 531-bp enhancer located at position + 23.5 in the first intron of the 224-kb mouse Runx1 gene. We show that this enhancer contributes to the early hematopoietic expression of Runx1. Transcription factor binding in vivo and analysis of the mutated enhancer in transient transgenic mouse embryos implicate Gata2 and Ets proteins as critical factors for its function. We also show that the SCL/Lmo2/Ldb-1 complex is recruited to the enhancer in vivo. Importantly,transplantation experiments demonstrate that the intronic Runx1 enhancer targets all definitive HSCs in the mouse embryo,suggesting that it functions as a crucial cis-regulatory element that integrates the Gata,Ets,and SCL transcriptional networks to initiate HSC generation. View Publication

Normal prion protein (PrP(c)),an essential substrate for development of prion disease,is widely distributed in hematopoietic cells. Recent evidence that variant Creutzfeldt-Jakob disease can be transmitted by transfusion of red cell preparations has highlighted the need for a greater understanding of the biology of PrP(c) in blood and blood-forming tissues. Here,we show that in contrast to another glycosylphosphoinositol-anchored protein CD59,PrP(c) at the cell surface of cultured human erythroblasts is rapidly internalized through the endosomal pathway,where it colocalizes with the tetraspanin CD63. In the plasma membrane,PrP(c) colocalizes with the tetraspanin CD81. Cross-linking with anti-PrP(c) or anti-CD81 causes clustering of PrP(c) and CD81,suggesting they can share the same microdomain. These data are consistent with a role for tetraspanin-enriched microdomains in trafficking of PrP(c). These results,when taken together with recent evidence that exosomes released from cells as a result of endosomal-mediated recycling to the plasma membrane contain prion infectivity,provide a pathway for the propagation of prion diseases. View Publication

Sirt1,a NAD(+)-dependent histone deacetylase,may regulate senescence,metabolism,and apoptosis. In this study,primary pig preadipocytes were cultured in DMEM/F12 medium containing 10% fetal bovine serum (FBS) with or without reagents affecting Sirt1 activity. The adipocyte differentiation process was visualized by light microscopy after Oil red O staining. Proliferation and differentiation of preadipocytes was measured using methylthiazolyldiphenyl-tetrazolium bromide (MTT) and Oil red O extraction. Expression of Sirt1,FoxO1,and adipocyte specific genes was detected with semi-quantitive RT-PCR. The results showed that Sirt1 mRNA was widely expressed in various pig tissues from different developmental stages. Sirt1 mRNA was expressed throughout the entire differentiation process of pig preadipocytes. Resveratrol significantly increased Sirt1 mRNA expression,but decreased the expression of FoxO1 and adipocyte marker gene PPARgamma2. Resveratrol significantly inhibited pig preadipocyte proliferation and differentiation. Nicotinamide decreased the expression of Sirt1 mRNA,but increased the expression of FoxO1 and adipocyte specific genes. Nicotinamide greatly stimulated the proliferation and differentiation of pig preadipocytes. In conclusion,these results indicate that Sirt1 may modulate the proliferation and differentiation of pig preadipocytes. Sirt1 may down-regulate pig preadipocytes proliferation and differentiation through repression of adipocyte genes or FoxO1. View Publication

The human HDAC (histone deacetylase) family,a well-validated anticancer target,plays a key role in the control of gene expression through regulation of transcription. While HDACs can be subdivided into three main classes,the class I,class II and class III HDACs (sirtuins),it is presently unclear whether inhibiting multiple HDACs using pan-HDAC inhibitors,or targeting specific isoforms that show aberrant levels in tumours,will prove more effective as an anticancer strategy in the clinic. To address the above issues,we have tested a number of clinically relevant HDACis (HDAC inhibitors) against a panel of rhHDAC (recombinant human HDAC) isoforms. Eight rhHDACs were expressed using a baculoviral system,and a Fluor de Lystrade mark (Biomol International) HDAC assay was optimized for each purified isoform. The potency and selectivity of ten HDACs on class I isoforms (rhHDAC1,rhHDAC2,rhHDAC3 and rhHDAC8) and class II HDAC isoforms (rhHDAC4,rhHDAC6,rhHDAC7 and rhHDAC9) was determined. MS-275 was HDAC1-selective,MGCD0103 was HDAC1- and HDAC2-selective,apicidin was HDAC2- and HDAC3-selective and valproic acid was a specific inhibitor of class I HDACs. The hydroxamic acid-derived compounds (trichostatin A,NVP-LAQ824,panobinostat,ITF2357,vorinostat and belinostat) were potent pan-HDAC inhibitors. The growth-inhibitory effect of the HDACis on HeLa cells showed that both pan-HDAC and class-I-specific inhibitors inhibited cell growth. The results also showed that both pan-HDAC and class-I-specific inhibitor treatment resulted in increased acetylation of histones,but only pan-HDAC inhibitor treatment resulted in increased tubulin acetylation,which is in agreement with their activity towards the HDAC6 isoform. View Publication

BACKGROUND AND PURPOSE: Glycogen synthase kinase-3 (GSK-3) affects neuropathological events associated with Alzheimeŕs disease (AD) such as hyperphosphorylation of the protein,tau. GSK-3beta expression,enzyme activity and tau phosphorylated at AD-relevant epitopes are elevated in juvenile rodent brains. Here,we assess five GSK-3beta inhibitors and lithium in lowering phosphorylated tau (p-tau) and GSK-3beta enzyme activity levels in 12-day old postnatal rats. EXPERIMENTAL APPROACH: Brain levels of inhibitors following treatment in vivo were optimized based on pharmacokinetic data. At optimal doses,p-tau (Ser(396)) levels in brain tissue was measured by immunoblotting and correlated with GSK-3beta enzyme activities in the same tissues. Effects of GSK inhibitors on p-tau,GSK-3beta activities and cell death were measured in a human neuronal cell line (LUHMES). KEY RESULTS: Lithium and CHIR98014 reduced tau phosphorylation (Ser(396)) in the cortex and hippocampus of postnatal rats,while Alsterpaullone and SB216763 were effective only in hippocampus. AR-A014418 and Indirubin-3'-monoxime were ineffective in either brain region. Inhibition of p-tau in brain required several-fold higher levels of GSK inhibitors than the IC(50) values obtained in recombinant or cell-based GSK-3beta enzyme activity assays. The inhibitory effect on GSK-3beta activity ex vivo correlated with protection against cell death and decrease of p-tau- in LUHMES cells,using low microM inhibitor concentrations. CONCLUSIONS AND IMPLICATIONS: Selective small-molecule inhibitors of GSK-3 reduce tau phosphorylation in vivo. These findings corroborate earlier suggestions that GSK-3beta may be an attractive target for disease-modification in AD and related conditions where tau phosphorylation is believed to contribute to disease pathogenesis. View Publication

There are a number of leukemogenic protein-tyrosine kinases (PTKs) associated with leukemic transformation. Although each is linked with a specific disease their functional activity poses the question whether they have a degree of commonality in their effects upon target cells. Exon array analysis of the effects of six leukemogenic PTKs (BCR/ABL,TEL/PDGFRbeta,FIP1/PDGFRalpha,D816V KIT,NPM/ALK,and FLT3ITD) revealed few common effects on the transcriptome. It is apparent,however,that proteome changes are not directly governed by transcriptome changes. Therefore,we assessed and used a new generation of iTRAQ tagging,enabling eight-channel relative quantification discovery proteomics,to analyze the effects of these six leukemogenic PTKs. Again these were found to have disparate effects on the proteome with few common targets. BCR/ABL had the greatest effect on the proteome and had more effects in common with FIP1/PDGFRalpha. The proteomic effects of the four type III receptor kinases were relatively remotely related. The only protein commonly affected was eosinophil-associated ribonuclease 7. Five of six PTKs affected the motility-related proteins CAPG and vimentin,although this did not correspond to changes in motility. However,correlation of the proteomics data with that from the exon microarray not only showed poor levels of correlation between transcript and protein levels but also revealed alternative patterns of regulation of the CAPG protein by different oncogenes,illustrating the utility of such a combined approach. View Publication

Thiazolidinediones (TZD) are widely prescribed for the treatment of Type 2 diabetes. Increased loss of bone mass and a higher incidence of fractures have been associated with the use of this class of drugs in post-menopausal women. In vitro studies performed in rodent cell models indicated that rosiglitazone (RGZ),one of the TZD,inhibited osteoblastogenesis and induced adipogenesis in bone marrow progenitor cells. The objective of the present study was to determine for the first time the RGZ-dependent shift from osteoblastogenesis toward adipogenesis using a human cell model. To this purpose,bone marrow-derived mesenchymal stem cells were characterized and induced to differentiate along osteogenic and adipogenic lineages. We found that the exposure to RGZ potentiated adipogenic differentiation and shifted the differentiation toward an osteogenic phenotype into an adipogenic phenotype,as assessed by the appearance of lipid droplets. Accordingly,RGZ markedly increased the expression of the typical marker of adipogenesis fatty-acid binding protein 4,whereas it reduced the expression of Runx2,a marker of osteoblastogenesis. This is the first demonstration that RGZ counteracts osteoblastogenesis and induces a preferential differentiation into adipocytes in human mesenchymal stem cells. View Publication

Human bone marrow multipotent mesenchymal stromal cells are progenitor cells that can be expanded in vitro and differentiate into various cells of mesodermal origin. They contribute to the bone marrow reticular niche,where mature B cells and long-lived plasma cells are maintained. Multipotent mesenchymal stromal cells were recently shown to modulate T- and B-cell proliferation and differentiation,dendritic cell maturation,and natural killer activity. These immunoregulatory properties encouraged a possible use of these cells to modulate autoimmune responses in humans. We studied the influence of bone marrow mesenchymal stem cells on highly purified B-cell subsets isolated from healthy donors and total B cells from pediatric systemic lupus erythematosus patients. Bone marrow mesenchymal stem cells promoted proliferation and differentiation into immunoglobulin-secreting cells of transitional and naive B cells stimulated with an agonist of Toll-like receptor 9,in the absence of B cell receptor triggering. They strongly enhanced proliferation and differentiation into plasma cells of memory B-cell populations. A similar effect was observed in response to polyclonal stimulation of B cells isolated from pediatric patients with systemic lupus erythematosus. This study casts important questions on bone marrow mesenchymal stem cells as a therapeutic tool in autoimmune diseases in which B-cell activation is crucially implicated in the pathogenesis of the disease. View Publication

We have previously developed an antibody fusion protein composed of a mouse/human chimeric IgG3 specific for the human transferrin receptor genetically fused to avidin (anti-hTfR IgG3-Av) as a universal delivery system for cancer therapy. This fusion protein efficiently delivers biotinylated FITC into cancer cells via TfR-mediated endocytosis. In addition,anti-hTfR IgG3-Av alone exhibits intrinsic cytotoxic activity and interferes with hTfR recycling,leading to the rapid degradation of the TfR and lethal iron deprivation in certain malignant B-cell lines. We now report on the cytotoxic effects of a conjugate composed of anti-hTfR IgG3-Av and biotinylated saporin 6 (b-SO6),a toxin derived from the plant Saponaria officinalis that inhibits protein synthesis. Conjugation of anti-hTfR IgG3-Av with b-SO6 enhances the cytotoxic effect of the fusion protein in sensitive cells and also overcomes the resistance of malignant cells that show low sensitivity to the fusion protein alone. Our results show for the first time that loading anti-hTfR IgG3-Av with a biotinylated toxin enhances the cytotoxicity of the fusion protein alone. These results suggest that anti-hTfR IgG3-Av has great potential as a therapeutic agent for a wide range of applications due to its intrinsic cytotoxic activity plus its ability to deliver biotinylated molecules into cancer cells. View Publication