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Reactive oxygen (ROS) and nitrogen oxide (RNOS) species are produced as by-products of oxidative metabolism. A major function for ROS and RNOS is immunological host defense. Recent evidence indicate that ROS and RNOS may also function as signaling molecules. However,high levels of ROS and RNOS have been considered to potentially damage cellular macromolecules and have been implicated in the pathogenesis and progression of various chronic diseases. alpha-Lipoic acid and dihydrolipoic acid exhibit direct free radical scavenging properties and as a redox couple,with a low redox potential of -0.32 V,is a strong reductant. Several studies provided evidence that alpha-lipoic acid supplementation decreases oxidative stress and restores reduced levels of other antioxidants in vivo. However,there is also evidence indicating that alpha-lipoic acid and dihydrolipoic acid may exert prooxidant properties in vitro. alpha-Lipoic acid and dihydrolipoic acid were shown to promote the mitochondrial permeability transition in permeabilized hepatocytes and isolated rat liver mitochondria. Dihydrolipoic acid also stimulated superoxide anion production in rat liver mitochondria and submitochondrial particles. alpha-Lipoic acid was recently shown to stimulate glucose uptake into 3T3-L1 adipocytes by increasing intracellular oxidant levels and/or facilitating insulin receptor autophosphorylation presumably by oxidation of critical thiol groups present in the insulin receptor beta-subunit. Whether alpha-lipoic acid and/or dihydrolipoic acid-induced oxidative protein modifications contribute to their versatile effects observed in vivo warrants further investigation. View Publication

Small molecule inhibitors have proven extremely useful for investigating signal transduction pathways and have the potential for development into therapeutics for inhibiting signal transduction pathways whose activities contribute to human diseases. Transforming growth factor beta (TGF-beta) is a member of a large family of pleiotropic cytokines that are involved in many biological processes,including growth control,differentiation,migration,cell survival,adhesion,and specification of developmental fate,in both normal and diseased states. TGF-beta superfamily members signal through a receptor complex comprising a type II and type I receptor,both serine/threonine kinases. Here,we characterize a small molecule inhibitor (SB-431542) that was identified as an inhibitor of activin receptor-like kinase (ALK)5 (the TGF-beta type I receptor). We demonstrate that it inhibits ALK5 and also the activin type I receptor ALK4 and the nodal type I receptor ALK7,which are very highly related to ALK5 in their kinase domains. It has no effect on the other,more divergent ALK family members that recognize bone morphogenetic proteins (BMPs). Consistent with this,we demonstrate that SB-431542 is a selective inhibitor of endogenous activin and TGF-beta signaling but has no effect on BMP signaling. To demonstrate the specificity of SB-431542,we tested its effect on several other signal transduction pathways whose activities depend on the concerted activation of multiple kinases. SB-431542 has no effect on components of the ERK,JNK,or p38 MAP kinase pathways or on components of the signaling pathways activated in response to serum. View Publication

Transforming growth factor beta1 (TGF-beta1) is a potent fibrotic factor responsible for the synthesis of extracellular matrix. TGF-beta1 acts through the TGF-beta type I and type II receptors to activate intracellular mediators,such as Smad proteins,the p38 mitogen-activated protein kinase (MAPK),and the extracellular signal-regulated kinase pathway. We expressed the kinase domain of the TGF-beta type I receptor [activin receptor-like kinase (ALK)5] and the substrate,Smad3,and determined that SB-431542 is a selective inhibitor of Smad3 phosphorylation with an IC50 of 94 nM. It inhibited TGF-beta1-induced nuclear Smad3 localization. The p38 mitogen-activated protein kinase inhibitors SB-203580 and SB-202190 also inhibit phosphorylation of Smad3 by ALK5 with IC50 values of 6 and 3 microM,respectively. This suggests that these p38 MAPK inhibitors must be used at concentrations of less than 10 microM to selectively address p38 MAPK mechanisms. However,the p38 MAPK inhibitor SB-242235 did not inhibit ALK5. To evaluate the relative contribution of Smad signaling and p38 MAPK signaling in TGF-beta1-induced matrix production,the effect of SB-431542 was compared with that of SB-242235 in renal epithelial carcinoma A498 cells. All compounds inhibited TGF-beta1-induced fibronectin (FN) mRNA,indicating that FN synthesis is mediated in part via the p38 MAPK pathway. In contrast,SB-431542,but not the selective p38 MAPK inhibitor SB-242235,inhibited TGF-beta1-induced collagen Ialpha1 (col Ialpha1). These data indicate that some matrix markers that are stimulated by TGF-beta1 are mediated via the p38 MAPK pathway (i.e.,FN),whereas others seem to be activated via ALK5 signaling independent of the p38 MAPK pathway (i.e.,col Ialpha1). View Publication

The lysophospholipids,lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P),are now recognized as important extracellular signaling molecules. These lipid mediators are pleiotropic; among the most common cellular responses are mitogenesis,cell survival (anti-apoptosis),inhibition of adenylyl cyclase and calcium mobilization. Physiologic events associated with these mediators include platelet aggregation,vasopressor activity,wound healing,immune modulation,and angiogenesis. Many of the actions of LPA and S1P are mediated through a set of eight G protein-coupled receptors. Five of these are S1P-prefering while the remaining three are LPA receptors. These receptors are expressed widely and in aggregate signal through a variety of heterotrimeric G proteins. The lysophospholipid receptor family is referred to commonly as the Edg" group (e.g.� View Publication

BACKGROUND AND OBJECTIVES: Leptin receptors can be expressed by acute myelogenous leukemia (AML) cells,but the functional effects of leptin on native AML blasts have not been characterized in detail. We investigated systemic leptin levels in AML patients and in vitro effects of leptin on cultured AML blasts. DESIGN AND METHODS: Serum leptin levels were compared for patients with untreated AML and healthy controls. Native AML blasts were derived from a large group of consecutive patients,and effects of leptin on proliferation (suspension cultures and colony formation),constitutive cytokine secretion,differentiation and apoptosis regulation were assayed in vitro. RESULTS: Systemic leptin levels were decreased in patients with untreated AML,and leptin levels in acute leukemia patients were not altered during severe chemotherapy-induced cytopenia and complicating febrile neutropenia. In vitro studies demonstrated that leptin increased AML blast release of interleukin (IL) 1beta,IL6,tumor necrosis factor (TNF) alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF). This enhancing effect showed no correlation with CD34 expression and was not dependent on the presence of serum,induction of differentiation or alteration of caspase 3 activity with decreased in vitro apoptosis. Leptin also increased spontaneous AML blast proliferation,whereas divergent effects on blast proliferation were observed in the presence of exogenous cytokines. The in vitro effects were usually observed at concentrations exceeding the systemic levels. INTERPRETATION AND CONCLUSIONS: Our results suggest that systemic leptin levels alone do not have a major influence on native AML blasts,but the systemic levels in combination with local leptin release in the bone marrow may affect the functional characteristics of these cells. View Publication

PPAR gamma is activated by diverse ligands and regulates the differentiation of many cell types. Based on evidence that activation of PPAR gamma 2 by rosiglitazone stimulates adipogenesis and inhibits osteoblastogenesis in U-33/gamma 2 cells,a model mesenchymal progenitor of adipocytes and osteoblasts,we postulated that the increase in marrow fat and the decrease in osteoblast number that occur during aging are due to increased PPAR gamma 2 activation. Here,we show that the naturally occurring PPAR gamma ligands 9,10-dihydroxyoctadecenoic acid,and 15-deoxy-Delta(12,14)-PGJ(2),also stimulate adipocytes and inhibit osteoblast differentiation of U-33/gamma 2 cells. Strikingly,9,10-epoxyoctadecenoic acid and the thiazolidine acetamide ligand GW0072 [(+/-)-(2S,5S)-4-(4-(4-carboxyphenyl)butyl)-2-heptyl-4-oxo-5-thaizolidineN,N-dibenzyl-acetamide] prevent osteoblast differentiation,but do not stimulate adipogenesis,whereas 9-hydroxyoctadecadienoic acid stimulates adipogenesis but does not affect osteoblast differentiation. The divergent effects of PPAR gamma 2 ligands on osteoblast and adipocyte differentiation were confirmed in primary murine bone marrow cultures using rosiglitazone and GW0072. These findings indicate that the proadipogenic and antiosteoblastogenic effects of PPAR gamma 2 are mediated by distinct regulatory pathways that can be differentially modulated depending on the nature of the ligand,and they support the idea that increased fatty acid oxidation during aging may inhibit osteoblast differentiation. Moreover,there may be selective PPAR gamma 2 modulators that block the adverse effects of fatty acid oxidation products while retaining beneficial activities such as insulin sensitization. View Publication

In mice there are two families of MHC class I-specific receptors,namely the Ly49 and CD94/NKG2 receptors. The latter receptors recognize the nonclassical MHC class I Qa-1(b) and are thought to be responsible for the recognition of missing-self and the maintenance of self-tolerance of fetal and neonatal NK cells that do not express Ly49. Currently,how NK cells acquire individual CD94/NKG2 receptors during their development is not known. In this study,we have established a multistep culture method to induce differentiation of embryonic stem (ES) cells into the NK cell lineage and examined the acquisition of CD94/NKG2 by NK cells as they differentiate from ES cells in vitro. ES-derived NK (ES-NK) cells express NK cell-associated proteins and they kill certain tumor cell lines as well as MHC class I-deficient lymphoblasts. They express CD94/NKG2 heterodimers,but not Ly49 molecules,and their cytotoxicity is inhibited by Qa-1(b) on target cells. Using RT-PCR analysis,we also report that the acquisition of these individual receptor gene expressions during different stages of differentiation from ES cells to NK cells follows a predetermined order,with their order of acquisition being first CD94; subsequently NKG2D,NKG2A,and NKG2E; and finally,NKG2C. Single-cell RT-PCR showed coexpression of CD94 and NKG2 genes in most ES-NK cells,and flow cytometric analysis also detected CD94/NKG2 on most ES-NK cells,suggesting that the acquisition of these receptors by ES-NK cells in vitro is nonstochastic,orderly,and cumulative. View Publication

Types A and B Niemann-Pick disease (NPD) are lysosomal storage disorders resulting from loss of acid sphingomyelinase (ASM) activity. We have used a knockout mouse model of NPD (ASMKO mice) to evaluate the effects of direct intracerebral transplantation of bone marrow-derived mesenchymal stem cells (MSCs) on the progression of neurological disease in this disorder. MSCs were transduced with a retroviral vector to overexpress ASM and were injected into the hippocampus and cerebellum of 3-week-old ASMKO pups. Transplanted cells migrated away from the injection sites and survived at least 6 months after transplantation. Seven of 8 treated mice,but none of the untreated controls,survived for textgreater or = 7 months after transplant. Survival times were greater in sex-matched than in sex-mismatched transplants. Transplantation significantly delayed the Purkinje cell loss that is characteristic of NPD,although the protective effect declined with distance from the injection site. Overall ASM activity in brain homogenates was low,but surviving Purkinje cells contained the retrovirally expressed human enzyme,and transplanted animals showed a reduction in cerebral sphingomyelin. These results reveal the potential of treating neurodegenerative lysosomal storage disorders by intracerebral transplantation of bone marrow-derived MSCs. View Publication

Putative myogenic and endothelial (myo-endothelial) cell progenitors were identified in the interstitial spaces of murine skeletal muscle by immunohistochemistry and immunoelectron microscopy using CD34 antigen. Enzymatically isolated cells were characterized by fluorescence-activated cell sorting on the basis of cell surface antigen expression,and were sorted as a CD34+ and CD45- fraction. Cells in this fraction were approximately 94% positive for Sca-1,and mostly negative (textless3% positive) for CD14,31,49,144,c-kit,and FLK-1. The CD34+/45- cells formed colonies in clonal cell cultures and colony-forming units displayed the potential to differentiate into adipocytes,endothelial,and myogenic cells. The CD34+/45- cells fully differentiated into vascular endothelial cells and skeletal muscle fibers in vivo after transplantation. Immediately after sorting,CD34+/45- cells expressed only c-met mRNA,and did not express any other myogenic cell-related markers such as MyoD,myf-5,myf-6,myogenin,M-cadherin,Pax-3,and Pax-7. However,after 3 d of culture,these cells expressed mRNA for all myogenic markers. CD34+/45- cells were distinct from satellite cells,as they expressed Bcrp1/ABCG2 gene mRNA (Zhou et al.,2001). These findings suggest that myo-endothelial progenitors reside in the interstitial spaces of mammalian skeletal muscles,and that they can potentially contribute to postnatal skeletal muscle growth. View Publication

BACKGROUND: Serum-free cultures supplemented with stem cell factor (SCF) and IL-6 is reported to support the extensive growth of less functional human cord blood-derived mast cells. OBJECTIVE: To obtain more functional mast cells from cord blood,we developed a culture system combining a serum-free condition for 0-8 weeks of culture,and followed by a serum-supplemented culture condition and examined the function of the cells compared to the cells cultured continuously in serum-free condition. METHODS: Human cord blood progenitors were purified with anti-CD133 antibody. They were cultured in a serum-free medium StemSpan supplemented with SCF at 100 ng/ml and IL-6 at 50 ng/ml for 8 weeks. Then,an aliquot of the cultured cells were cultured in the above condition but further supplemented with 10% fetal calf serum (FCS). RESULTS: The addition of FCS after 8 weeks of culture significantly increased the amount of histamine per mast cell (3.8 pg/cell) when compared to the serum-free condition (0.7 pg/cell). The cells cultured with FCS after 8 weeks expressed more FcvarepsilonRI alpha and released textgreater30% of the histamine content upon anti-IgE stimulation than those cultured without serum. CONCLUSION: It is uncertain why FCS enhanced the functional maturation of mast cells when added after week 8 of culture but suppressed mast cell development when added at day 0 of culture. Yet,the present method combining a serum-free culture system with a serum-supplemented culture system seems to be beneficial for most of the laboratories to obtain functional human mast cells. View Publication

Retinoids are important mediators of cellular growth and differentiation. Retinoids modulate the growth of both normal and malignant cells through their binding to retinoid nuclear receptors and their subsequent activation. While retinoids have demonstrated therapeutic efficacy in the treatment of acute promyelocytic leukemia,their spectrum of activity remains limited. Other agents such as histone deacetylase inhibitors may significantly increase retinoid activity in a number of malignant cell types. The novel retinoids N-(4-hydroxyphenyl) retinamide (4-HPR) and 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437; AHPN) induce apoptosis in a wide variety of malignant cells. Their mechanism(s) of action remain unclear,although a number of potential targets have been identified. Whether the retinoid receptors are involved in 4-HPR and CD473/AHPN mediated apoptosis remains unclear. Both 4-HPR and CD437/AHPN display significant potential as therapeutic agents in the treatment of a number of premalignant and malignant conditions. View Publication

Excessive accumulation of smooth-muscle cells (SMCs) has a key role in the pathogenesis of vascular diseases. It has been assumed that SMCs derived from the outer medial layer migrate,proliferate and synthesize extracellular matrix components on the luminal side of the vessel. Although much effort has been devoted to targeting migration and proliferation of medial SMCs,there is no effective therapy that prevents occlusive vascular remodeling. We show here that in models of post-angioplasty restenosis,graft vasculopathy and hyperlipidemia-induced atherosclerosis,bone-marrow cells give rise to most of the SMCs that contribute to arterial remodeling. Notably,purified hematopoietic stem cells differentiate into SMCs in vitro and in vivo. Our findings indicate that somatic stem cells contribute to pathological remodeling of remote organs,and may provide the basis for the development of new therapeutic strategies for vascular diseases through targeting mobilization,homing,differentiation and proliferation of bone marrow-derived vascular progenitor cells. View Publication