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Although osteoporosis is a relatively common complication after allogeneic stem cell transplantation,the role of bisphosphonates in its management has not yet been completely established. Thirty-two patients who underwent allogeneic stem cell transplantation were prospectively evaluated for bone mineral density (BMD) at the lumbar spine (LS) and femoral neck (FN) after a median period of 12.2 months. Then,15 of the patients with osteoporosis or rapidly progressing osteopenia (bone loss textgreater 5%/yr) received three monthly doses of 4 mg zoledronic acid iv. Fifteen patients were followed up without treatment,and all 30 patients were reevaluated after 12 months for BMD and bone turnover markers. By using enriched mesenchymal stem cells in the colony-forming units fibroblast (CFU-F) assay,we evaluated the osteogenic stromal lineage. This procedure was performed in both groups of patients at study entry and after 12 months. The average BMD loss was 3.42% at LS and 3.8% at FN during a 1-yr longitudinal evaluation in 32 patients. Subsequently,BMD increased at both LS and FN (9.8 and 6.4%,respectively) in the zoledronic acid-treated cohort. Hydroxyproline excretion decreased,and serum bone-specific alkaline phosphatase increased significantly,whereas serum osteocalcin increase did not reach the limit of significance. A significant increase in CFU-F growth in vitro was induced by in vivo zoledronic acid administration. In the untreated group,no significant change was observed in bone turnover markers,LS BMD (-2.1%),FN BMD (-2.3%),and CFU-F colony number. In conclusion,short-term zoledronic acid treatment consistently improved both LS and FN BMD in transplanted patients who were at high risk for fast and/or persistent bone loss,partly by increasing the osteogenic progenitors in the stromal cell compartment. View Publication

Autosomal recessive osteopetrosis (OP) is a rare,lethal disorder in which osteoclasts are absent or nonfunctional,resulting in a bone marrow cavity insufficient to support hematopoiesis. Because osteoclasts are derived from hematopoietic precursors,allogeneic hematopoietic cell transplantation can cure the bony manifestations of the disorder. However,high rates of graft failure have been observed in this population. It is not possible to harvest bone marrow from these patients for reinfusion should graft failure be observed. We report that 8 of 10 patients with OP had high numbers of circulating CD34(+) cells (3% +/- 0.9%). This increased proportion of peripheral CD34(+) cells made it possible to harvest 2 x 10(6) CD34(+) cells per kilogram with a total volume of blood ranging from 8.3 to 83.7 mL (1.3-11.6 mL/kg). In addition,colony-forming assays documented significantly more colony-forming unit-granulocyte-macrophage and burst-forming unit-erythroid in the blood of osteopetrotic patients compared with controls; the numbers of colony-forming units approximated those found in control marrow. We conclude that OP patients with high levels of circulating CD34(+) are candidates for peripheral blood autologous harvest by limited exchange transfusion. These cells are then available for reinfusion should graft failure be observed in patients for whom retransplantation is impractical. View Publication

BACKGROUND Transplantation of endothelial progenitor cells (EPCs) improves neovascularization after ischemia,but patients with coronary artery disease (CAD) or diabetes mellitus show a reduced number of EPCs and impaired functional activity. Therefore,we investigated the effects of risk factors,such as glucose and TNF-alpha,on the number of EPCs in vitro to elucidate the underlying mechanisms. METHODS AND RESULTS EPCs of patients or healthy subjects were isolated from peripheral blood. Incubation with glucose or TNF-alpha dose-dependently reduced the number of EPCs (79.9+/-1.3% and 74.3+/-8.1% of control; Ptextless0.05,respectively). This reduction was not caused by apoptosis. TNF-alpha and glucose induced a dose- and time-dependent activation of the p38 MAP kinase,the downstream kinase mitogen- and stress-activated kinase 1,and the transcription factor cAMP-responsive element-binding protein (CREB),in EPCs. Moreover,EPCs from CAD patients had significantly higher basal p38-phosphorylation levels (1.83+/-0.2-fold increase; Ptextless0.05) compared with healthy subjects. The inhibition of the p38-kinase by SB203580 or infection with a dominant negative p38 kinase adenovirus significantly increased basal number of EPCs (136.7+/-6.3% and 142.9+/-18% versus control,respectively). Likewise,ex vivo cultivation of EPCs from patients with CAD with SB203580 significantly increased the number of EPCs and partially reversed the impaired capacity for neovascularization of EPCs in vivo (relative blood flow: 0.40+/-0.03 versus 0.64+/-0.08,Ptextless0.05). The increased numbers of EPCs by SB203580 were associated with an augmentation of EPC proliferation and a reduction of cells expressing the monocytic marker proteins CD14 and CD64,suggesting that p38 regulates proliferation and differentiation events. CONCLUSIONS These results demonstrate that p38 MAP kinase plays a pivotal role in the signal transduction pathways regulating the number of EPCs ex vivo. SB203580 can prevent the negative effects of TNF-alpha and glucose on the number of EPCs and may be useful to improve the number of EPCs for potential cell therapy. View Publication

NK cells from individuals with X-linked lymphoproliferative (XLP) disease exhibit functional defects when stimulated through the NK receptor,2B4 (CD244). These defects are likely a consequence of aberrant intracellular signaling initiated by mutations of the adaptor molecule SLAM-associated protein. In this report,we show that NK cells from individuals with XLP but not healthy individuals fail to phosphorylate and thereby inactivate glycogen synthase kinase-3 (GSK-3) following 2B4 stimulation. Lack of GSK-3 phosphorylation prevented the accumulation of the transcriptional coactivator beta-catenin in the cytoplasm and its subsequent translocation to the nucleus. Potential signaling pathways leading from 2B4 stimulation to GSK-3 phosphorylation were also investigated. Ligation of 2B4 resulted in the phosphorylation of the guanine nucleotide exchange factor,Vav-1,and subsequent activation of the GTP-binding protein Rac-1 (but not Ras) and the serine-threonine kinase Raf-1 in healthy but not XLP-derived NK cells. In addition,the activity of MEK-2 (but not MEK-1) was up-regulated,and Erk1/2 was phosphorylated in normal NK cells but not those from an individual with XLP suggesting that these proteins relay SLAM-associated protein-dependent signals from 2B4. Finally,inactivation of GSK-3 using a specific inhibitor of GSK-3beta increased the cytotoxicity and cytokine secretion of both healthy and XLP NK cells. These data indicate that the signaling of 2B4 in NK cells is mediated by GSK-3 and beta-catenin,possibly through a signal transduction pathway that involves Vav-1,Rac-1,Raf-1,MEK-2,and Erk1/2 and that this pathway is aberrant in individuals with XLP. View Publication

Zebularine (2(1H)-pyrimidinone riboside,Zeb),a synthetic analogue of cytidine that is a potent inhibitor of cytidine deaminase,has been recently identified as a general inhibitor of DNA methylation. This inhibition of DNA methyltransferase (DNMT) is hypothesized to be mechanism-based and result from formation of a covalent complex between the enzyme and zebularine-substituted DNA. Metabolic activation of Zeb thus requires that it be phosphorylated and incorporated into DNA. We have quantitatively assessed the phosphorylation and DNA incorporation of Zeb in T24 cells using 2-[(14)C]-Zeb in conjunction with gradient anion-exchange HPLC and selected enzymatic and spectroscopic analyses. The corresponding 5'-mono-,di- and triphosphates of Zeb were readily formed in a dose- and time-dependent manner. Two additional Zeb-containing metabolites were tentatively identified as diphosphocholine (Zeb-DP-Chol) and diphosphoethanolamine adducts. Intracellular concentrations of Zeb-TP and Zeb-DP-Chol were similar and greatly exceeded those of other metabolites. DNA incorporation occurred but was surpassed by that of RNA by at least seven-fold. Equivalent levels and similar intracellular metabolic patterns were also observed in the Molt-4 (human T-lymphoblasts) and MC38 (murine colon carcinoma) cell lines. For male BALB/c nu/nu mice implanted s.c. with the EJ6 variant of T24 bladder carcinoma and treated i.p. with 500mg/kg 2-[(14)C]-Zeb,the in vivo phosphorylation pattern of Zeb in tumor tissue examined 24h after drug administration was similar to that observed in vitro. The complex metabolism of Zeb and its limited DNA incorporation suggest that these are the reasons why it is less potent than either 5-azacytidine or 5-aza-2'-deoxycytidine and requires higher doses for equivalent inhibition of DNMT. View Publication

Although prolonged transgene expression in progenitor cells might be desirable for modified cell therapy,the viral promoter-based expression vector tends to promote transgene expression only for a limited period. Here,we examined the ability of cellular promoters from elongation factor-1alpha (EF-1alpha) and ubiquitin C to drive gene expression in hematopoietic TF-1 and mesenchymal progenitor cells. We compared the expression levels and duration of a model gene,interleukin-2,generated by the cellular promoters to those by the cytomegalovirus (CMV) promoter. The EF-1alpha and ubiquitin C promoters drove prolonged gene expression in hematopoietic TF-1 and mesenchymal progenitor cells,whereas the CMV promoter did not. At day 7 after transfection in TF-1 cells,the mRNA expression levels of interleukin-2 driven by the EF-1alpha and ubiquitin C promoters were 118- and 56-fold higher,respectively,than those driven by the CMV promoter. Similarly,in mesenchymal progenitor cells,the expression levels of interleukin-2 driven by the EF-1alpha and ubiquitin C promoters were 98- and 20-fold higher,respectively,than that driven by the CMV promoter-encoding plasmid. Moreover,the ubiquitin C promoter directed higher levels of green fluorescence protein expression in mesenchymal progenitor cells than did the CMV promoter. These results indicate that the use of cellular promoters such as those for EF-1alpha and ubiquitin C might direct prolonged gene expression in hematopoietic and mesenchymal progenitor cells. View Publication

Hereditary hemochromatosis (HH),an iron overload disease associated with mutations in the HFE gene,is characterized by increased intestinal iron absorption and consequent deposition of excess iron,primarily in the liver. Patients with HH and Hfe-deficient (Hfe-/-) mice manifest inappropriate expression of the iron absorption regulator hepcidin,a peptide hormone produced by the liver in response to iron loading. In this study,we investigated the contribution of Hfe expression in macrophages to the regulation of liver hepcidin levels and iron loading. We used bone marrow transplantation to generate wild-type (wt) and Hfe-/- mice chimeric for macrophage Hfe gene expression. Reconstitution of Hfe-deficient mice with wt bone marrow resulted in augmented capacity of the spleen to store iron and in significantly decreased liver iron loading,accompanied by a significant increase of hepatic hepcidin mRNA levels. Conversely,wt mice reconstituted with Hfe-deficient bone marrow had a diminished capacity to store iron in the spleen but no significant alterations of liver iron stores or hepcidin mRNA levels. Our results suggest that macrophage Hfe participates in the regulation of splenic and liver iron concentrations and liver hepcidin expression. View Publication

Murine embryonic stem cells can differentiate in vitro to form cystic embryoid bodies (CEB) that contain different structures and cell types. The blood islands are one such structure that consist of immature hematopoietic cells surrounded by endothelial cells,the first identifiable vascular cells. CEBs differentiated in vitro developed blood islands initially,and subsequently these blood islands matured to form vascular channels containing hematopoietic cells. Phase contrast microscopy demonstrated the presence of channels in mature CEBs grown in suspension culture,and high resolution light and electron microscopy showed that the cells lining these channels were endothelial cells. The channels appeared less organized than the vasculature of the mature yolk sac. The hematopoietic cells were occasionally seen 'flowing' through the CEB channels,although their numbers were reduced relative to the yolk sac. Analysis of primary CEB cultures showed the presence of cells with two characteristics of endothelial cells: approximately 30% of the cells labelled with fluorescent acetylated low density lipoprotein and a small number of cells were positive for von Willebrand's factor by immunostaining. Thus we conclude that a primitive vasculature forms in CEBs differentiated in vitro,and that not only primary differentiation of endothelial cells but also some aspects of vascular maturation are intrinsic to this cell culture system. CEBs are therefore a useful model for the study of developmental blood vessel formation. View Publication

Purmorphamine is a novel small molecule with osteogenesis-inducing activity in multipotent mesenchymal progenitor cells,but there has been no evaluation of its effect on human cells to date. The aim of this study was to investigate the induction of osteogenic activity by purmorphamine in human osteoblasts differentiated from bone marrow mesenchymal cells. Cells were cultured in 24-well plates at a density of 2x10(4)/well in medium containing 1,2 or 3 microM purmorphamine,or vehicle. At 7,14 and 21 days,cell proliferation,viability,and alkaline phosphatase (ALP) activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Purmorphamine did not affect cell proliferation or viability,but increased ALP activity and bone-like nodule formation. These results indicate that events related to osteoblast differentiation,including increased ALP activity and bone-like nodule formation,are enhanced by purmorphamine. View Publication

The monocyte population in blood is considered a possible source of endothelial precursors. Because endothelial-specific receptor tyrosine kinases act as regulators of endothelial cell function,we investigated whether expression of the vascular endothelial growth factor receptor-2 (VEGFR-2) on monocytes is important for their endothelial-like functional capacity. Peripheral-blood monocytes expressing vascular endothelial growth factor receptor-2 (VEGFR-2),or CD14+/VEGFR-2+,were isolated,and their phenotypic,morphologic,and functional capacities were compared with those of monocytes negative for this marker (CD14+/VEGFR-2-). CD14+/VEGFR-2+ cells constituted approximately 2% +/- 0.5% of the total population of monocytes and 0.08% +/- 0.04% of mononuclear cells in blood. CD14+/VEGFR-2+ cells exhibited the potential to differentiate in vitro into cells with endothelial characteristics. The cells were efficiently transduced by a lentiviral vector driving expression of the green fluorescence protein (GFP). Transplantation of GFP-transduced cells into balloon-injured femoral arteries of nude mice significantly contributed to efficient reendothelialization. CD14+/VEGFR-2- did not exhibit any of these characteristics. These data demonstrate that the expression of VEGFR-2 on peripheral blood monocytes is essential for their endothelial-like functional capacity and support the notion of a common precursor for monocytic and endothelial cell lineage. Our results help clarify which subpopulations may restore damaged endothelium and may participate in the maintenance of vascular homeostasis. View Publication

BACKGROUND/AIMS The Rho-ROCK signaling pathways play an important role in the activation of hepatic stellate cells (HSCs). We investigated the effects of fasudil hydrochloride hydrate (fasudil),a Rho-kinase (ROCK) inhibitor,on cell growth,collagen production,and collagenase activity in HSCs. METHODS Rat HSCs and human HSC-derived TWNT-4 cells were cultured for studies on stress fiber formation and alpha-smooth muscle actin (alpha-SMA) expression. Proliferation was measured by BrdU incorporation,and apoptosis by TUNEL assay. The phosphorylation states of the MAP kinases (MAPKs),extra cellular signal -regulated kinase 1/2 (ERK1/2),c-jun kinase (JNK),and p38 were evaluated by western blot analysis. Type I collagen,matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) production and gene expression were evaluated by ELISA and real-time PCR,respectively. Collagenase activity (active MMP-1) was also evaluated. RESULTS Fasudil (100 microM) inhibited cell spreading,the formation of stress fibers,and expression of alpha-SMA with concomitant suppression of cell growth,although it did not induce apoptosis. Fasudil inhibited phosphorylation of ERK1/2,JNK,and p38. Treatment with fasudil suppressed the production and transcription of collagen and TIMP,stimulated the production and transcription of MMP-1,and enhanced collagenase activity. CONCLUSION These findings demonstrated that fasudil not only suppresses proliferation and collagen production but also increases collagenase activity. View Publication

The homeostatic adult bone marrow (BM) is a complex tissue wherein physical and biochemical interactions serve to maintain a balance between the hematopoietic and nonhematopoietic compartments. To focus on soluble factor interactions occurring between mesenchymal and hematopoietic cells,a serum-free adhesion-independent culture system was developed that allows manipulation of the growth of both mesenchymal and hematopoietic human BM-derived progenitors and the balance between these compartments. Factorial experiments demonstrated a role for stem cell factor (SCF) and interleukin 3 (IL-3) in the concomitant growth of hematopoietic (CD45+) and nonhematopoietic (CD45-) cells,as well as their derivatives. Kinetic tracking of IL-3alpha receptor (CD123) and SCF receptor (CD117) expression on a sorted CD45- cell population revealed the emergence of CD45-CD123+ cells capable of osteogenesis. Of the total fibroblast colony-forming units (CFU-Fs) and osteoblast colony-forming units (CFU-O),approximately 24% of CFU-Fs and about 22% of CFU-Os were recovered from this population. Cell-sorting experiments demonstrated that the CD45+ cell population secreted soluble factors that positively affect the survival and proliferation of CFU-Fs and CFU-Os generated from the CD45- cells. Together,our results provide insight into the intercellular cytokine network between hematopoietic and mesenchymal cells and provide a strategy to mutually culture both mesenchymal and hematopoietic cells in a defined scalable bioprocess. View Publication