技术资料

BACKGROUND: Endothelial progenitor cells (EPCs) are present in peripheral blood and can promote postnatal angiogenesis. The number and function of circulating EPCs are altered in diabetics. This study sought to investigate whether the number and functional properties of EPCs from patients with type II diabetes could be improved by pioglitazone. METHODS: For this randomized controlled study,we recruited 36 type II diabetic patients on metformin monotherapy with a glycohemoglobin A1c of textless7%. Patients were separated into pioglitazone (n = 24) and control (n = 12) groups. The number and functional activity of EPCs,and the brachial artery flow-mediated dilation were determined before and after pioglitazone treatment (8 weeks) as an add-on therapy to metformin. In addition,direct effects of pioglitazone on EPCs were also investigated. RESULTS: After pioglitazone treatment,the numbers of circulating EPCs significantly increased (from 0.44% +/- 0.14% to 0.89% +/- 0.29%,P = .01). The migratory response and the adhesive capacity to fibronectin and collagen were improved by 158%,34%,and 83%,respectively (all P textless .05). Treatment with pioglitazone significantly lowered triglyceride,very low density lipoprotein cholesterol,and high-sensitivity C-reactive protein (hsCRP) levels,and increased high-density lipoprotein levels and insulin sensitivity (all P textless .05). The increase in the number of circulating EPCs and the improvement in the migratory response after pioglitazone treatment were independently correlated to the decrease in hsCRP levels (P textless or = .01). The increase in the adhesive capacity was independently correlated to the decreases in very low density lipoprotein cholesterol (P = .01) and hsCRP levels (P = .03). In addition,pioglitazone was also demonstrated to have direct effects on increasing EPC proliferation and colony formation,and attenuating EPC apoptosis (all P textless .05,versus the controls). There were no significant changes in flow-mediated dilation in either group. CONCLUSIONS: Pioglitazone significantly increased the number and improved the functional properties of EPCs in type II diabetic patients through direct effects and/or anti-inflammation and lipid modification effects. View Publication

Human embryonic stem cells (hESCs) provide great opportunities for regenerative medicine,pharmacological and toxicological investigation,and the study of human embryonic development. These applications require proper derivation,maintenance,and extensive characterization of undifferentiated cells before being used for differentiation into cells of interest. Undifferentiated hESCs possess several unique features,including their extensive proliferation capacity in the undifferentiated state,ability to maintain a normal karyotype after long-term culture,expression of markers characteristic of stem cells,high constitutive telomerase activity,and capacity to differentiate into essentially all somatic cell types. This chapter will summarize the current development in culture conditions and provide technical details for the evaluation and characterization of hESCs. View Publication

The essentially infinite expansion potential and pluripotency of human embryonic stem cells (hESCs) makes them attractive for cell-based therapeutics. In contrast to mouse embryonic stem cells (mESCs),hESCs normally undergo high rates of spontaneous apoptosis and differentiation,making them difficult to maintain in culture. Here we demonstrate that p53 protein accumulates in apoptotic hESCs induced by agents that damage DNA. However,despite the accumulation of p53,it nevertheless fails to activate the transcription of its target genes. This inability of p53 to activate its target genes has not been observed in other cell types,including mESCs. We further demonstrate that p53 induces apoptosis of hESCs through a mitochondrial pathway. Reducing p53 expression in hESCs in turn reduces both DNA damage-induced apoptosis as well as spontaneous apoptosis. Reducing p53 expression also reduces spontaneous differentiation and slows the differentiation rate of hESCs. Our studies reveal the important roles of p53 as a critical mediator of human embryonic stem cells survival and differentiation. View Publication

The mTOR complex 2 (mTORC2) containing mTOR and rictor is thought to be rapamycin insensitive and was recently shown to regulate the prosurvival kinase AKT by phosphorylation on Ser473. We investigated the molecular effects of mTOR inhibition by the rapamycin derivatives (RDs) temsirolimus (CCI-779) and everolimus (RAD001) in acute myeloid leukemia (AML) cells. Unexpectedly,RDs not only inhibited the mTOR complex 1 (mTORC1) containing mTOR and raptor with decreased p70S6K,4EPB1 phosphorylation,and GLUT1 mRNA,but also blocked AKT activation via inhibition of mTORC2 formation. This resulted in suppression of phosphorylation of the direct AKT substrate FKHR and decreased transcription of D-cyclins in AML cells. Similar observations were made in samples from patients with hematologic malignancies who received RDs in clinical studies. Our study provides the first evidence that rapamycin derivatives inhibit AKT signaling in primary AML cells both in vitro and in vivo,and supports the therapeutic potential of mTOR inhibition strategies in leukemias. View Publication

BACKGROUND: The purpose of this study was to determine whether murine mesenchymal stem cells (MSC) are able to home to the viable myocardium when injected intravenously and attenuate cardiac dysfunction and ventricular remodeling associated with myocardial infarction. METHODS AND RESULTS: Murine bone marrow cells were negatively selected for lineage markers and adherent MSC differentiated into adipocytes and osteocytes following treatment in culture. Two weeks after coronary occlusion that resulted in a permanent transmural infarct we observed a significant drop in LV systolic pressure,dP/dt(max),dP/dt(min),ESPVR and E(max) and a significant increase in end-diastolic volume in vivo. Femoral vein injection of MSC 1 h after occlusion attenuated the cardiac dysfunction without altering infarct size,or end-diastolic volume. Injected MSC pre-labeled with fluorescent paramagnetic microspheres were observed scattered in noninfarcted regions of the myocardium. Flow cytometry of whole heart digests after intravenous injection of MSC labeled with either fluorescent microspheres or fluorescent PKH26 dye demonstrated that infarcted hearts from mice that received MSC injections contained significantly more cells that integrated into the heart (20x) than those from uninfarcted controls. CONCLUSION: We conclude that intravenously injected MSC were able to home to viable myocardium and preserve systolic function by 2 weeks following ligation. The preserved contractility is likely an MSC-mediated paracrine response since infarct morphology was unchanged and labeled cells observed at two weeks exhibited the same characteristics as the injected MSC. These data underscore the importance of using MSC as a potential therapeutic intervention in preserving cardiac function following infarction. View Publication

OBJECTIVE Pancreatic cancer continues to be an urgent clinical problem. We used the novel DNA methyltransferase inhibitor Zebularine and the histone deacetylase inhibitor SAHA to investigate the epigenetic influence on viability and differentiation of the pancreatic cancer cell lines YAP C,DAN G and Panc-89 in vitro and in vivo. MATERIAL AND METHODS Cell vitality,proliferation and expression of PDX-1,cytokeratin 7 and 20,chromogranin A,vimentin,bax and bcl-2 were determined on the protein and mRNA level in vitro and in a subcutaneous xenograft model. RESULTS A time- and dose-dependent increase of apoptosis,paralleled by decreased proliferation,was observed after incubation with single agents or a combination therapy with lower concentrations. This was associated with up-regulation of pro-apoptotic bax and a phenotypic stabilization by the enhanced expression of cytokeratin 7. In vivo,growth of xenografts was delayed with the most pronounced effect in Panc-89 after 1 week of daily intraperitoneal injections of Zebularine paralleled with CK7 up-regulation and down-regulation of dedifferentiation markers. CONCLUSIONS Epigenetic modulation via inhibition of DNA methyltransferase and histone deacetylase induces apoptosis in human pancreatic cancer cells in vitro and delays xenograft growth in vivo,which is associated with a morphological/molecular phenotypic stabilization. These compounds may therefore be suitable as adjunctive therapeutic agents in the treatment of pancreatic cancer. View Publication

Macroautophagy (hereafter referred to as autophagy) is a well-conserved intracellular degradation process. Recent studies examining cells lacking the autophagy genes Atg5 and Atg7 have demonstrated that autophagy plays essential roles in cell survival during starvation,in innate cell clearance of microbial pathogens,and in neural cell maintenance. However,the role of autophagy in T lymphocyte development and survival is not known. Here,we demonstrate that autophagosomes form in primary mouse T lymphocytes. By generating Atg5-/- chimeric mice,we found that Atg5-deficient T lymphocytes underwent full maturation. However,the numbers of total thymocytes and peripheral T and B lymphocytes were reduced in Atg5 chimeras. In the periphery,Atg5-/- CD8+ T lymphocytes displayed dramatically increased cell death. Furthermore,Atg5-/- CD4+ and CD8+ T cells failed to undergo efficient proliferation after TCR stimulation. These results demonstrate a critical role for Atg5 in multiple aspects of lymphocyte development and function and suggest that autophagy may be essential for both T lymphocyte survival and proliferation. View Publication

Defining how cancer-associated mutations perturb signaling networks in stem/progenitor populations that are integral to tumor formation and maintenance is a fundamental problem with biologic and clinical implications. Point mutations in RAS genes contribute to many cancers,including myeloid malignancies. We investigated the effects of an oncogenic Kras(G12D) allele on phosphorylated signaling molecules in primary c-kit(+) lin(-/low) hematopoietic stem/progenitor cells. Comparison of wild-type and Kras(G12D) c-kit(+) lin(-/low) cells shows that K-Ras(G12D) expression causes hyperproliferation in vivo and results in abnormal levels of phosphorylated STAT5,ERK,and S6 under basal and stimulated conditions. Whereas Kras(G12D) cells demonstrate hyperactive signaling after exposure to granulocyte-macrophage colony-stimulating factor,we unexpectedly observe a paradoxical attenuation of ERK and S6 phosphorylation in response to stem cell factor. These studies provide direct biochemical evidence that cancer stem/progenitor cells remodel signaling networks in response to oncogenic stress and demonstrate that multi-parameter flow cytometry can be used to monitor the effects of targeted therapeutics in vivo. This strategy has broad implications for defining the architecture of signaling networks in primary cancer cells and for implementing stem cell-targeted interventions. View Publication

An urgent need exists to devise strategies to augment antiviral immune responses in patients with HIV who are virologically well controlled and immunologically stable on highly active antiretroviral therapy (HAART). The objective of this study was to compare the immunomodulatory effects of the cytokines interleukin (IL)-21 with IL-15 on CD8 T cells in patients with HIV RNA of less than 50 copies/mL and CD4 counts greater than 200 cells/mm.(3) Patient CD8 T cells displayed skewed maturation and decreased perforin expression compared with healthy controls. Culture of freshly isolated patient peripheral-blood mononuclear cells (PBMCs) for 5 hours to 5 days with IL-21 resulted in up-regulation of perforin in CD8 T cells,including memory and effector subsets and virus-specific T cells. IL-21 did not induce T-cell activation or proliferation,nor did it augment T-cell receptor (TCR)-induced degranulation. Treatment of patient PBMCs with IL-15 resulted in induction of perforin in association with lymphocyte proliferation and augmentation of TCR-induced degranulation. Patient CD8 T cells were more responsive to cytokine effects than the cells of healthy volunteers. We conclude that CD8 T cells of patients with HIV can be modulated by IL-21 to increase perforin expression without undergoing overt cellular activation. IL-21 could potentially be useful for its perforin-enhancing properties in anti-HIV immunotherapy. View Publication

To identify previously unknown platelet receptors we compared the transcriptomes of in vitro differentiated megakaryocytes (MKs) and erythroblasts (EBs). RNA was obtained from purified,biologically paired MK and EB cultures and compared using cDNA microarrays. Bioinformatical analysis of MK-up-regulated genes identified 151 transcripts encoding transmembrane domain-containing proteins. Although many of these were known platelet genes,a number of previously unidentified or poorly characterized transcripts were also detected. Many of these transcripts,including G6b,G6f,LRRC32,LAT2,and the G protein-coupled receptor SUCNR1,encode proteins with structural features or functions that suggest they may be involved in the modulation of platelet function. Immunoblotting on platelets confirmed the presence of the encoded proteins,and flow cytometric analysis confirmed the expression of G6b,G6f,and LRRC32 on the surface of platelets. Through comparative analysis of expression in platelets and other blood cells we demonstrated that G6b,G6f,and LRRC32 are restricted to the platelet lineage,whereas LAT2 and SUCNR1 were also detected in other blood cells. The identification of the succinate receptor SUCNR1 in platelets is of particular interest,because physiologically relevant concentrations of succinate were shown to potentiate the effect of low doses of a variety of platelet agonists. View Publication