Mo Yang

Thrombopoietin Protects Against In Vitro and In Vivo Cardiotoxicity Induced by Doxorubicin

Background— Doxorubicin (DOX) is an important antineoplastic agent. However, the associated cardiotoxicity, possibly mediated by the production of reactive oxygen species, has remained a significant and dose-limiting clinical problem. Our hypothesis is that the hematopoietic/megakaryocytopoietic growth factor thrombopoietin (TPO) protects against DOX-induced cardiotoxicity and might involve antiapoptotic mechanism exerted on cardiomyocytes. Methods and Results— In vitro investigations on H9C2 cell line and spontaneously beating cells of primary, neonatal rat ventricle, as well as an in vivo study in a mouse model of DOX-induced acute cardiomyopathy, were performed. Our results showed that pretreatment with TPO significantly increased viability of DOX-injured H9C2 cells and beating rates of neonatal myocytes, with effects similar to those of dexrazoxane, a clinically approved cardiac protective agent. TPO ameliorated DOX-induced apoptosis of H9C2 cells as demonstrated by assays of annexin V, active caspase-3, and mitochondrial membrane potential. In the mouse model, administration of TPO (12.5 &mgr;g/kg IP for 3 alternate days) significantly reduced DOX-induced (20 mg/kg) cardiotoxicity, including low blood cell count, cardiomyocyte lesions (apoptosis, vacuolization, and myofibrillar loss), and animal mortality. Using Doppler echocardiography, we observed increased heart rate, fractional shortening, and cardiac output in animals pretreated with TPO compared with those receiving DOX alone. Conclusions— These data have provided the first evidence that TPO is a protective agent against DOX-induced cardiac injury. We propose to further explore an integrated program, incorporating TPO with other protocols, for treatment of DOX-induced cardiotoxicity and other forms of cardiomyopathy.

Peroxisome Proliferator-Activated Receptor-γ Contributes to the Inhibitory Effects of Embelin on Colon Carcinogenesis

Down-regulation of XIAP (X-linked inhibitor of apoptosis protein) sensitizes colon cancer cells to the anticancer effect of peroxisome proliferator-activated receptor-gamma (PPARgamma) ligands in mice. The aims of this study were to evaluate the effect of embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone), an antagonist of XIAP, on colon cancer, with a particular focus on whether PPARgamma is required for embelin to exert its effect. A dominant-negative PPARgamma was used to antagonize endogenous PPARgamma in HCT116 cells. Cells were treated with or without embelin. Cell proliferation, apoptosis, and nuclear factor-kappaB (NF-kappaB) activity were measured. For in vivo studies, 1,2-dimethylhydrazine dihydrochloride (DMH) was s.c. injected to induce colon cancer in PPARgamma(+/+) and PPARgamma(+/-) mice. Mice were fed embelin daily for 10 days before DMH injection, and continued for 30 more weeks. Embelin inhibited proliferation and induced apoptosis in HCT116 cells with marked up-regulation of PPARgamma. In addition, embelin significantly inhibited the expressions of survivin, cyclin D1, and c-Myc. These effects were partially dependent on PPARgamma. PPARgamma(+/-) mice were more susceptible to DMH-induced colon carcinogenesis than PPARgamma(+/+) mice, and embelin significantly reduced the incidence of colon cancer in PPARgamma(+/+) mice but not in PPARgamma(+/-) mice. Embelin inhibited NF-kappaB activity in PPARgamma(+/+) mice but marginally so in PPARgamma(+/-) mice. Thus, reduced expression of PPARgamma significantly sensitizes colonic tissues to the carcinogenic effect of DMH. Embelin inhibits chemical carcinogen-induced colon carcinogenesis, but this effect is partially dependent on the presence of functional PPARgamma, indicating that PPARgamma is a necessary signaling pathway involved in the antitumor activity of normal organisms.

Promoting effects of serotonin on hematopoiesis : Ex vivo expansion of cord blood CD34+ stem/progenitor cells, proliferation of bone marrow stromal cells, and antiapoptosis

Serotonin is a monoamine neurotransmitter that has multiple extraneuronal functions. We previously reported that serotonin exerted mitogenic stimulation on megakaryocytopoiesis mediated by 5‐hydroxytryptamine (5‐HT)2 receptors. In this study, we investigated effects of serotonin on ex vivo expansion of human cord blood CD34+ cells, bone marrow (BM) stromal cell colony‐forming unit‐fibroblast (CFU‐F) formation, and antiapoptosis of megakaryoblastic M‐07e cells. Our results showed that serotonin at 200 nM significantly enhanced the expansion of CD34+ cells to early stem/progenitors (CD34+ cells, colony‐forming unit‐mixed [CFU‐GEMM]) and multilineage committed progenitors (burst‐forming unit/colony‐forming unit‐erythroid [BFU/CFU‐E], colony‐forming unit‐granulocyte macrophage, colony‐forming unit‐megakaryocyte, CD61+CD41+ cells). Serotonin also increased nonobese diabetic/severe combined immunodeficient repopulating cells in the expansion culture in terms of human CD45+, CD33+, CD14+ cells, BFU/CFU‐E, and CFU‐GEMM engraftment in BM of animals 6 weeks post‐transplantation. Serotonin alone or in addition to fibroblast growth factor, platelet‐derived growth factor, or vascular endothelial growth factor stimulated BM CFU‐F formation. In M‐07e cells, serotonin exerted antiapoptotic effects (annexin V, caspase‐3, and propidium iodide staining) and reduced mitochondria membrane potential damage. The addition of ketanserin, a competitive antagonist of 5‐HT2 receptor, nullified the antiapoptotic effects of serotonin. Our data suggest the involvement of serotonin in promoting hematopoietic stem cells and the BM microenvironment. Serotonin could be developed for clinical ex vivo expansion of hematopoietic stem cells for transplantation.

Partial neuroprotective effect of pretreatment with tanshinone IIA on neonatal hypoxia-ischemia brain damage.

Tanshinone IIA is a compound purified from the Chinese herb Danshen (Radix Salviae Miltiorrhiza Bge). The neuroprotective effect of tanshinone IIA was investigated in a neonatal rat model of hypoxia-ischemia brain damage. Hypoxia-ischemia encephalopathy was induced in rats at day 7 of postnatal age by ligation of the right common carotid artery, followed by 2 h of hypoxia. Tanshinone IIA (10 mg/kg, i.p.) was injected daily from day 2 before surgery for 9 or 16 d. Our results demonstrated significant and sustained brain damage in the hypoxia-ischemia– and vehicle-treated groups at 1 and 3 wk after surgery. Treatment with tanshinone IIA significantly reduced the severity of brain injury, as indicated by the increase in ipsilateral brain weight and neuron density, compared with those of sham-operated animals. The recovery of sensorimotor function and histology was observed in animals that received tanshinone IIA. The plasma of tanshinone IIA–treated rats exhibited higher antioxidant activities, as reflected by the oxygen radical absorbance capacity assay, compared with the vehicle-treated rats. In the neural progenitor cell line C17.2 that was subjected to 2,2′-azobis (2-amidino propane hydrochloride)–induced oxidative stress, tanshinone IIA increased cell viability and protected against mitochondrial damage (JC-1 assay). Our results suggest that tanshinone IIA has antioxidative activities and that treatment that is started before a hypoxic-ischemic insult is partially neuroprotective. Further studies are required to elucidate whether rescue treatment with tanshinone IIA is effective and to determine whether its protective effect is also associated with secondary cooling of the brain.

Platelet-derived growth factor enhances ex vivo expansion of megakaryocytic progenitors from human cord blood.

Infusion of ex vivo expanded megakaryocytic (MK) progenitor cells is a strategy for shortening the duration of thrombocytopenia after haematopoietic stem cell transplantation. The cell dose after expansion has emerged as a critical factor for achieving the desired clinical outcomes. This study aimed to establish efficient conditions for the expansion of the MK lineage from enriched CD34+ cells of umbilical cord blood and to investigate the effect of platelet-derived growth factor (PDGF) in this system. Our results demonstrated that thrombopoietin (TPO) alone produced a high proportion of CD61+CD41+ cells but a low total cell count and high cell death, resulting in an inferior expansion. The addition of interleukin-1β (IL-1β), Flt-3 ligand (Flt-3L) and to a lesser extent IL-3 improved the expansion outcome. The treatment groups with three to five cytokines produced efficient expansions of CFU-MK up to 400-fold with the highest yield observed in the presence of TPO, IL-1β, IL-3, IL-6 and Flt-3L. CD34+ cells were expanded by five to 22-fold. PDGF improved the expansion of all cell types with CD61+CD41+ cells, CFU-MK and CD34+ cells increased by 101%, 134% and 70%, respectively. On day 14, the CD61+ population consisted of diploid (86.5%), tetraploid (11.8%) and polyploid (8N–32N; 1.69%) cells. Their levels were not affected by PDGF. TPO, IL-1β, IL-3, IL-6, Flt-3L and PDGF represented an effective cytokine combination for expanding MK progenitors while maintaining a moderate increase of CD34+ cells. This study showed, for the first time, that PDGF enhanced the ex vivo expansion of the MK lineage, without promoting their in vitro maturation. PDGF might be a suitable growth factor to improve the ex vivo expansion of MK progenitors for clinical applications. Bone Marrow Transplantation (2001) 27, 1075–1080.

Platelet‐derived growth factor promotes ex vivo expansion of CD34+ cells from human cord blood and enhances long‐term culture‐initiating cells, non‐obese diabetic/severe combined immunodeficient repopulating cells and formation of adherent cells

Summary. Platelet‐derived growth factor (PDGF) is a major mitogen for connective tissue cells. In this study, we investigated the effects and mechanism of PDGF on the ex vivo expansion of cord blood CD34+ cells. Our data demonstrated that among various cytokine combinations of thrombopoietin (TPO), interleukin 1 beta (IL‐1β), IL‐3, IL‐6 and Flt‐3 ligand (Flt‐3L), TPO + IL‐6 + Flt‐3L was most efficient in promoting the expansion of CD34+ cells, CD34+CD38– cells, mixed‐lineage colony‐forming units (CFU‐GEMM) and long‐term culture‐initiating cells (LTC‐IC) by 21·7 ± 5·00‐, 103 ± 27·9‐, 10·7 ± 7·94‐ and 6·52 ± 1·51‐fold, respectively, after 12–14 d of culture. The addition of PDGF increased the yield of these early progenitors by 45·0%, 66·5%, 45·1% and 79·8% respectively. More significantly, PDGF enhanced the engraftment of human CD45+ cells and their myeloid subsets (CD33+, CD14+ cells) in non‐obese diabetic (NOD)/severe‐combined immunodeficient (SCID) mice. The expression of PDGF receptor (PDGFR)‐β was not detectable in fresh CD34+ cells but was upregulated after culture for 3 d. PDGF also enhanced the development of adherent cells/clusters that expressed the endothelial markers VE‐cadherin and CD31. These findings suggest that PDGF is an effective cytokine for the ex vivo expansion of early stem and progenitor cells. The mechanism could be mediated by PDGFR‐β on committed CD34+ progenitor cells and/or secondary to the stimulation of autologous, stromal feeder cells.

Ex vivo expansion of enriched CD34+ cells from neonatal blood in the presence of thrombopoietin, a comparison with cord blood and bone marrow

Neonatal blood (NB) contains substantial numbers of stem and progenitor cells which decline rapidly after birth. Using a combination of cord blood (CB) and NB, we performed a successful, sibling transplant for a thalassaemia patient, leading to the proposal that NB could be used as an adjunct to CB for transplantation. This study was aimed at addressing the feasibility of expanding NB and thus minimizing the volume needed from a NB collection. In the presence of early acting cytokines interleukin-1β (IL-1β), IL-3, IL-6, stem cell factor (SCF), flt-3 ligand with and without thrombopoietin (Tpo), we compared the expansion capacity of CD34+ enriched cells from CB, NB and bone marrow (BM). Flow cytometry and colony-forming unit (CFU) analyses show that Tpo significantly increased the expansion of CD34+ cells from CB and NB to early and committed progenitors. No significant difference was observed between the expansion of CB and NB at 7, 14 or 21 days of culture in terms of CFU, CD34+ and CD61+ cell subsets. The expansion capacity of BM was significantly lower than that of NB or CB, possibly related to the low proportion of CD34+CD38− cells observed at day 0. There was a relatively rapid expansion of NB which was evident at day 7 whilst the expansion of CB and BM remained low, suggesting a speedy maturation process in the postnatal infant. The expanded cells, being heterogeneous in their morphology and cell surface marker expression, were mostly of the myeloid lineage (CD45+, CD33+ and HLA-DR+). Our results showed that the expansion capacity of NB is comparable to that of CB and if transplanted, the expanded products of NB might contribute to the engraftment kinetics of the neutrophil and megakaryocyte lineage.

Recombinant PDGF enhances megakaryocytopoiesis in vitro

Summary. The effect of recombinant platelet‐derived growth factor (PDGF) on both murine and human megakaryocyte colony formation was studied in the plasma clot culture system. PDGF significantly stimulates megakaryocyte colony formation in a dose‐dependent manner. The minimum concentration which had a significant stimulating effect on colony forming unit megakaryocyte (CFU‐MK) was lOng/ml and maximum stimulation occurred at 50 ng/ml. The effect of PDGF was compared with that of interleukin (IL)‐3, IL‐6, granulocyte‐macrophage colony stimulating factor (GM‐CSF), erythropoietin (EPO) and acid fibroblast growth factor (aFGF) on megakaryocyte colony formation. The results showed that megakaryocyte colony stimulating activity of PDGF was slightly higher than those of GM‐CSF and aFGF, but lower than those of IL‐3, IL‐6 and EPO. The effect of PDGF in combination with IL‐3 or IL‐6 on megakaryocyte colony formation was also investigated. No synergistic action was found between PDGF and IL‐3 or IL‐6, but an additive effect was observed with IL‐3 plus IL‐6. We also studied the effects of PDGF in combination with anti‐IL6, anti‐IL‐3 or anti‐GM‐CSF antibody. The increase of megakaryocyte colony formation induced by PDGF was partially inhibited by anti‐IL‐6 or anti‐GM‐CSF antibody but not by anti‐IL‐3 antibody. These results indicate that PDGF is a positive regulator for megakaryocytopoiesis in vitro and IL‐6 and GM‐CSF may play a role in the mechanism whereby PDGF stimulates megakaryocytopoiesis.

Role of PCDH10 and its hypermethylation in human gastric cancer.

Epigenetic changes of genomic DNA are involved in the development and progression of many cancers. Aberrant methylation of CpG islands in the promoter regions of certain tumor-suppressor genes (TSG) is frequently observed in cancer cells. Protocadherin 10 (PCDH10), a member of the cadherin superfamily, is a recently identified putative TSG. PCDH10 is frequently silenced in many solid tumors. However, the role of PCDH10 in gastric cancer is largely unknown. In this study, we examined the expression and methylation status of PCDH10 in gastric cancer cells and tissues by real time PCR and methylation-specific PCR (MSP), and then investigated the biological function of PCDH10. We found that the expression of PCDH10 was markedly reduced in gastric cancer cells and tissues. The reduced expression correlated with hypermethylation of this gene in its promoter region, as demonstrated by MSP and bisulfite genomic sequencing (BGS) analysis. In addition, pharmacological demethylation using 5-Aza restored the expression of PCDH10 in gastric cancer cells. Over-expression of PCDH10 in gastric cancer cells suppressed cell proliferation and migration, but did not cause marked apoptosis. Over-expression of PCDH10 also suppressed growth of xenograft tumors in nude mice. Thus, PCDH10 functions as a TSG in gastric cancer, and might be a useful target for cancer therapy.

Dedifferentiation-Reprogrammed Mesenchymal Stem Cells with Improved Therapeutic Potential†‡§

Stem cell transplantation has been shown to improve functional outcome in degenerative and ischemic disorders. However, low in vivo survival and differentiation potential of the transplanted cells limits their overall effectiveness and thus clinical usage. Here we show that, after in vitro induction of neuronal differentiation and dedifferentiation, on withdrawal of extrinsic factors, mesenchymal stem cells (MSCs) derived from bone marrow, which have already committed to neuronal lineage, revert to a primitive cell population (dedifferentiated MSCs) retaining stem cell characteristics but exhibiting a reprogrammed phenotype distinct from their original counterparts. Of therapeutic interest, the dedifferentiated MSCs exhibited enhanced cell survival and higher efficacy in neuronal differentiation compared to unmanipulated MSCs both in vitro and in vivo, with significantly improved cognition function in a neonatal hypoxic–ischemic brain damage rat model. Increased expression of bcl‐2 family proteins and microRNA‐34a appears to be the important mechanism giving rise to this previously undefined stem cell population that may provide a novel treatment strategy with improved therapeutic efficacy. STEM CELLS 2011;29:2077–2089.