Hanying Sun

Sonic hedgehog protein promotes bone marrow-derived endothelial progenitor cell proliferation, migration and VEGF production via PI 3-kinase/ Akt signaling pathways

AbstractAim:To investigate the effects of Sonic hedgehog (shh) protein on bone marrow-derived endothelial progenitor cells (BM-EPC) proliferation, migration and vascular endothelial growth factor (VEGF) production, and the potential signaling pathways involved in these effects.Methods:Bone marrow-derived Flk-1+ cells were enriched using the MACS system from adult Kunming mice and then BM-EPC was cultured in gelatin-coated culture dishes. The effects of shh N-terminal peptide on BM-EPC proliferation were evaluated using the MTT colorimetric assay. Cell migration was assayed using a modified Boy den chamber technique. The production of VEGF was determined by ELISA and immunofluorescence analysis. The potential involvement of PKC and PI3K signaling pathways was explored using selective inhibitor or Western blot.Results:The proliferation, migration and VEGF production in BM-EPC could be promoted by endogenous shh N-terminal peptide at concentrations of 0.1 μg/mL to 10 μg/mL, and could be inhibited by anti-shh antibodies. Shh-mediated proliferation and migration in BM-EPC could be partly attenuated by anti-VEGF. Phospho-PI3-kinase expression in newly separated BM-EPC was low, and it increased significantly when exogenous shh N-terminal peptide was added, but could be attenuated by anti-human/mouse shh N-terminal peptide antibody. Moreover, the inhibitor of the PI3-kinase, but not the inhibitor of the PKC, significantly inhibited the shh-mediated proliferation, migration and VEGF production.Conclusion:Shh protein can stimulate bone marrow-derived BM-EPC proliferation, migration and VEGF production, which may promote neovascularization to ischemic tissues. This results also suggests that the PI3-kinase/Akt signaling pathways are involved in the angiogenic effects of shh.

Sonic hedgehog maintains survival and growth of chronic myeloid leukemia progenitor cells through β-catenin signaling

Sonic hedgehog (Shh) signaling plays an important role in many human cancers and cancer stem cells. Here we investigate the activity and functional role of Shh signaling in chronic myeloid leukemia (CML) and leukemia progenitor cells. Differential activation of Shh signaling was found in about 50% CML chronic phase samples, about 70% of CML accelerated phase samples, and >80% CML blast crisis phase samples. Deregulated activation of Shh signaling was observed in CD34(+) and c-kit(+) leukemia progenitor cells. Stimulation of Shh signaling with exogenous Shh peptide induced expansion of CD34(+) and c-kit(+) progenitor cells (p < 0.05), inversely, blocking the pathway with signal inhibitor induced cell apoptosis (p < 0.05). Low level of Shh protein was observed in CML bone marrow stromal cells, and CD34(+) progenitor cells are less sensitive to exogenous Shh peptide and more sensitive to cyclopamine than CD34(-) cells (p < 0.05), implying cell-autonomous activation of Shh signaling play a predominant role in progenitor cells. Coactivation of Shh and β-catenin signaling was found in CD34(+) and c-kit(+) progenitor cells. Administration of Shh-neutralizing antibody or Wnt3a-neutralizing antibody in c-kit(+) progenitor cells induced cell apoptosis; however, Wnt3a peptide could salvage cell apoptosis, while Shh peptide failed to revert anti-Wnt3a-induced cell apoptosis. C-MYC, GLI1, BCL-2, and P21 were also found to be downstream targets of Shh signaling, mediating apoptosis or G(2)/M cell cycle arrest of progenitor cells. Our results demonstrate that autoactivated Shh signaling provides survival and proliferative cues in CML progenitor cells through downstream β-catenin signaling, suggesting a novel therapeutic approach in CML.

Involvement of Wnt signaling in the injury of murine mesenchymal stem cells exposed to X-radiation.

Abstract Purpose: To investigate the injury of murine mesenchymal stem cells (mMSC) exposed to 4 Gy X-radiation and the role of canonical and non-canonical wingless-type (Wnt) signaling in the radiation injury. Materials and methods: C3H10T1/2 cells were submitted to 4 Gy X-radiation. At different time points after radiation, Hoechst33258 staining and Annexin V-fluorescein isothiocyanate (FITC) flow cytometry analysis were performed to assess cellular apoptosis. Senescence-associated β-galactosidase (SA-β-gal) staining was performed to analyze cellular senescence. Cell cycle was measured by flow cytometry. P53, p21, Wnt3a, Wnt5a, sonic hedgehog (Shh) mRNA was detected by Real time polymerase chain reaction (PCR) and Wnt5a protein was determined by Western blot. Results: A time-dependent cellular apoptosis was observed with a peak level 12 hours after radiation. Cellular senescence was detected 72 h after radiation. A remarkable up-regulation of Wnt5a mRNA expression (∼ 269-fold) and protein expression was seen 72 h after radiation. Conclusions: The effect of 4 Gy X-radiation to mMSC was time-dependent in the form of cellular apoptosis in the early period and cellular senescence in the late period. Non-canonical Wnt signaling may be involved in mMSC senescence induced by 4 Gy X-radiation.

Wnt and Notch signaling pathways selectively regulating hematopoiesis.

Hematopoietic stem and progenitor cells (HSPCs) are the source of all blood cells in the adult body. The pool of HSPCs is formed during embryogenesis process through a well-characterized succession of intra-embryonic regions and organs. The spatial and temporal restrictions in definitive hematopoietic development and the signaling molecules involved are of great interest as these may prove useful for generating and expanding these clinically important cell populations ex vivo. To elucidate the mechanism by which definitive HSPCs expand during this limited developmental time frame, we analyzed the spatial and temporal programmed gene expression patterns of Wnt and Notch signaling members during hematopoietic development. Genes related to the Wnt signaling pathway were up-regulated in E10.5 aorta-gonad-mesonephros (AGM) and E14.5 fetal liver corresponding to the inherent proliferation potential of hematopoietic progenitors, whereas genes related to the Notch signaling pathway were identified as up-regulated in E10.5 AGM, and bone marrow coincides with the maintenance of undifferentiation state of hematopoietic progenitors. Our findings suggest that Wnt and Notch signalings are integrated and are selectively regulating hematopoiesis. The spatial and temporal balance between Wnt and Notch signaling orchestrates the precise progression of hematopoietic progenitors.

Proliferation and apoptosis of bone marrow CD4+ T cells in patients with aplastic anemia and impacts of the secreted cytokines on hematopoietic stem cells from umbilical cord blood

Recent studies indicate that immune-associated aplastic anemia (AA) resembles such autoimmune diseases as insulin-dependent diabetes and chronic autoimmune thyroiditis that belong to organ-specific autoimmune diseases. Many independent investigation groups have successfully isolated the pathopoiesis-associated T cell clone causing hematopoiesis failure with a CD4 phenotype from peripheral blood and bone marrow (BM) in AA patients. In the current study, BM CD4+ T cells were isolated from AA patients and healthy controls with immunomagnetic beads sorting, and proliferation capability, apoptosis features and the impacts of their secreted cytokines on hematopoiesis stem/progenitor cells were compared between them. By 3H-TdR method, CD4+ T cells in AA group presented more enhanced proliferative activity. The stimulation index in control group and AA group was 1.47±0.24, and 2.51±0.34 respectively (P<0.01). After BM CD4+ T cells were induced by high concentration of CD3 monoclonal antibody for 18 h, evident apoptosis cells could be seen under the electron microscope in both control group and AA group. Flow cytometry revealed that apoptosis rates in the early and late stages of AA group were significantly higher than in control group (P<0.01). Early-stage apoptosis rate in control and AA groups was (6.85±1.48)% and (16.98±4.40)%, and late-stage apoptosis rate in control group and AA group was (2.65±1.57)% and (7.74±0.83)%, respectively (P<0.01). The CFU-GM count in AA group and control group was (74.50±9.50)/104 cells and (124.25±19.80)/104 cells respectively under an inverted microscope (P<0.01), and the expression levels of CyclinD3 mRNA and protein in cord blood CD34+ cells were both down-regulated induced by BM CD4+ T cell culture supernatant in AA patients. These results indicate that BM CD4+ T cells of AA patients are likely in an abnormally proliferative, and activated state which can correlate intimately with AA hematopoiesis damage. BM CD4+ T cells in AA patients can secret some soluble cytokines that can inhibit proliferation of hematopoietic stem cells by suppressing the expression of Cyclin D3, resulting in hematopoiesis failure.SummaryRecent studies indicate that immune-associated aplastic anemia (AA) resembles such autoimmune diseases as insulin-dependent diabetes and chronic autoimmune thyroiditis that belong to organ-specific autoimmune diseases. Many independent investigation groups have successfully isolated the pathopoiesis-associated T cell clone causing hematopoiesis failure with a CD4 phenotype from peripheral blood and bone marrow (BM) in AA patients. In the current study, BM CD4+ T cells were isolated from AA patients and healthy controls with immunomagnetic beads sorting, and proliferation capability, apoptosis features and the impacts of their secreted cytokines on hematopoiesis stem/progenitor cells were compared between them. By 3H-TdR method, CD4+ T cells in AA group presented more enhanced proliferative activity. The stimulation index in control group and AA group was 1.47±0.24, and 2.51±0.34 respectively (P<0.01). After BM CD4+ T cells were induced by high concentration of CD3 monoclonal antibody for 18 h, evident apoptosis cells could be seen under the electron microscope in both control group and AA group. Flow cytometry revealed that apoptosis rates in the early and late stages of AA group were significantly higher than in control group (P<0.01). Early-stage apoptosis rate in control and AA groups was (6.85±1.48)% and (16.98±4.40)%, and late-stage apoptosis rate in control group and AA group was (2.65±1.57)% and (7.74±0.83)%, respectively (P<0.01). The CFU-GM count in AA group and control group was (74.50±9.50)/104 cells and (124.25±19.80)/104 cells respectively under an inverted microscope (P<0.01), and the expression levels of CyclinD3 mRNA and protein in cord blood CD34+ cells were both down-regulated induced by BM CD4+ T cell culture supernatant in AA patients. These results indicate that BM CD4+ T cells of AA patients are likely in an abnormally proliferative, and activated state which can correlate intimately with AA hematopoiesis damage. BM CD4+ T cells in AA patients can secret some soluble cytokines that can inhibit proliferation of hematopoietic stem cells by suppressing the expression of Cyclin D3, resulting in hematopoiesis failure.

Fetal liver stromal cells promote hematopoietic cell expansion

Future application of hematopoietic stem and progenitor cells (HSPCs) in clinical therapies largely depends on their successful expansion in vitro. Fetal liver (FL) is a unique hematopoietic organ in which hematopoietic cells markedly expand in number, but the mechanisms involved remain unclear. Stromal cells (StroCs) have been suggested to provide a suitable cellular environment for in vitro expansion of HSPCs. In this study, murine StroCs derived from FL at E14.5, with a high level of Sonic hedgehog (Shh) and Wnt expression, were found to have an increased ability to support the proliferation of HSPCs. This effect was inhibited by blocking Shh signaling. Supplementation with soluble Shh-N promoted the proliferation of hematopoietic cells by activating Wnt signaling. Our findings suggest that FL-derived StroCs support proliferation of HSPCs via Shh inducing an autocrine Wnt signaling loop. The use of FL-derived StroCs and regulation of the Shh pathway might further enhance HPSC expansion.

Tcf-1 gene silence suppresses downstream gene expression in CD4+ T cells from bone marrow of aplastic anemia patients

CD4+ T cells play a crucial role in the pathogenesis of acquired aplastic anemia (AA). Tcf-1 gene regulates T cell development and function, and it is significantly upregulated in the bone marrow CD4+ T cells from patients with acquired AA. To explore the role of Tcf-1 in the pathogenesis of AA, we knocked down Tcf-1 gene in CD4+ T cells of AA patients and studied the effects of Tcf-1 silencing on its downstream gene expression. Upon transfection of psiRNA into marrow CD4+ T cells from bone marrow of aplastic anemia patients, the expression of Tcf-1 was significantly knocked down; consequently, expressions of c-Myc and CD44 were also significantly reduced. Our results suggest that Tcf-1 may contribute to pathogenesis of AA by regulating downstream gene expression such as c-myc and CD44.

Selection of HLA-A2 restricted CML28 peptide by artificial antigen-presenting cells.

Chronic myelogenous leukemia (CML) 28 is a broadly immunogenic antigen that can have an antileukemia effect. We adopted the SYFPEITHI database to predict human leukocyte antigen-A2 restricted CML28 peptide. We designed artificial antigen-presenting cells (aAPCs) by coating micro beads with human leukocyte antigen-A2–immunoglobulin dimer and CD28-specific antibody. We used the selected peptides-pulsed aAPCs to induce cytotoxic T lymphocytes (CTLs), to choose the CML28 peptide, which was best for inducing CTLs. The result showed that the peptides-pulsed aAPCs could induce CTLs and that the peptide VLTFALDSV was the best choice for significantly inducing specific CTLs.

Saline is a more appropriate solution for microvesicles for flow cytometric analyses

Microvesicles (MVs) are carriers of molecular and oncogenic signatures present in subsets of tumor cells and tumor-associated stroma, and a focus of cancer research. Although methods to detect MVs are mature, we were concerned that the buffer used could lead to false results when quantitating MVs by flow cytometry. In this work, we detected MVs by flow cytometry withthree different solutions: water, saline, and phosphate-buffered saline (PBS). The results demonstrated that PBS, when reacted with annexin V binding buffer, produced nano-sized vesicles even when there were no MVs in the sample. No similar events occurred in the saline and water groups (P < 0.01). Annexin V positive rate increased significantly when PBS was used as the buffer, compared to saline and water. These false negative results were also observed when we quantified some markers of MVs such as CD3 and CD19. A probable explanation for these findings is the production of insoluble Ca(H2PO4)2 or Ca3PO4 from calcium in the binding buffer and phosphate in PBS. Thus, considering the osmotic pressure of water, we suggest that saline is a more suitable buffer when counting MVs by flow cytometry.

In vitro effects of imatinib on CD34 + cells of patients with chronic myeloid leukemia in the megakaryocytic crisis phase

Imatinib is a tailored drug for the treatment of chronic myeloid leukemia (CML), and has substantial activity and a favorable safety profile when used as a single agent in patients with CML in myeloid blast crisis. The megakaryocytic blast crisis in CML occurs rarely and carries a poor prognosis. The aim of the present study was to investigate the effects of imatinib on cluster of differentiation (CD)34+ cells from patients with CML in the megakaryocytic crisis phase. Bone marrow mononuclear cells (BMNCs) were isolated from patients with CML in the megakaryocytic crisis phase. CD34+ cells were selected from BMNCs by positive immunomagnetic column separation. Imatinib significantly induced G1 arrest, reduced the phosphorylation of cyclin-dependent kinase 1 and retinoblastoma proteins and inhibited the proliferation of CD34+ cells from patients with CML in the megakaryocytic crisis phase. Annexin V/propidium iodide and caspase-3 activity showed that imatinib induced apoptosis. Western blot analysis and protein tyrosine kinase activity assays showed that imatinib inhibited BCR-ABL protein tyrosine kinase activity. The in vitro data thus markedly indicate a potential clinical application of imatinib for patients with CML in the megakaryocytic crisis phase.