| The acute and residual bone marrow injury induced by ionizing radiation is a major clinic concern for victims accidentally exposed to a moderate-to-high dose of irradiation in local war nowadays. HSCs may be challenged by diverse sources of stress, including oxidation, anemia, hypoxia, radiation, cytotoxic chemotherapy and inflammation, which can disrupt homeostasis and impair regeneration. Ionizing radiation and high-dose chemotherapy, which are commonly used to treat hematopoietic malignancies or solid malignancies, may induce damage to the bone marrow and microenvironment and limit the regeneration and differentiation potential of HSC by reducing their numbers and causing functional deficits among the remaining HSC.Various hematopoietic growth factors, such as G-CSF, IL-11, TPO and EPO, improve acute myelosuppression by stimulating hematopoietic stem/progenitor cell proliferation and differentiation in clinic. However the regenerative process that takes place after hematopoietic injury remains more elusive. Various signaling pathways including Notch signaling, apoptosis, TGF-β, cytokines and extracellular matrix proteins implicated in homeostasis have also been shown to be involved in regeneration and are mediated in part by the bone marrow vasculature.The bone marrow niche has mystified scientists for many years, leading to widespread investigation to shed light into its molecular and cellular composition. Recent advances in imaging and genetic manipulation of mouse models have allowed the identification of distinct vascular niches that have been shown to orchestrate the balance between quiescence, proliferation and regeneration of the bone marrow after injury. The cellular compositions of the HSC niche contain perivascular cells, sinusoid endothelial cells, osteoblasts, sympathetic nerves, nonmyelinating Schwann cells,and macrophages. Meanwhile, a complex milieu of components is responsible for HSC maintenance, including soluble mediators, intrinsic signaling pathways and microenvironmental signals, such as those mediated by adhesion molecules and local oxygen tension, as well as interactions with other cellular niche constituents. The molecular compositions of the HSC niche contain perivascular-derived SCF and CXCL12 axis, a complex and unresolved role for Wnt and Notch signaling, N-cadherin, TGF-β and other niche factors. Thus therapeutic implications targeted to cellular and environmental factors in stem cell pool highlights further investigations of new drug strategies to improve acute myelosuppression.Experimental evidence indicates that highly conserved Notch signaling pathway is required for the embryonic development of HSCs, and plays a key role in regulating multiple aspects within the hematopoietic system during vertebrate development, which requires intimate contact between signal-emitting and signal-receiving cells. Previous studies have shown that Notch receptors are expressed by HSCs and their progeny, while Notch ligands are expressed in surrounding niches in bone marrow, which give feasible implications to activate Notch pathway in bone marrow niche. Expression of Notch receptors early in hematopoiesis may be involved in cell differentiation decisions and may be used to identify specific progenitor cell types with a predetermined cell fate: Notch1 was found to promote T cell commitment and has been shown to specify megakaryocyte fate, whereas Notch2 marked primarily erythroid progenitor cells. Although the Notch signaling pathway plays an important role in regulating self-renewal and differentiation within HSC ex vivo, it remains under debate whether canonical Notch signaling contributes to HSC maintenance in vivo or whether this signaling pathway is dispensable. The difficulty in resolving opposing results regarding the role of Notch in hematopoiesis may lie in the innate complexity of the signaling pathway, the complex native bone marrow microenvironment and methodologies used to evaluate its function. Uncovering safe techniques to promote HSC expansion in vivo without inducing cancerous transformation, along with cellular and environmental factors that encourage HSC lodgment while maintaining stemness, could form the basis for new therapeutics and in turn result in expedited regeneration with improved clinical outcomes.This research proposed to clarify whether Notch signaling regulated hematopoietic recovery after stress in genetically modified mice. Furthermore, we generated a novel recombinant protein m D1 R targeting endothelial cells and activating Notch pathway specifically. After confirming the protein activity in cellular and molecular experiments, we confirmed its biological effects in ionizing radiation and chemicals caused acute myelosuppression models. Finally, the bioinformatics and molecular biology were conjunctively used to uncover the underlying mechanism during Notch signaling regulating bone marrow reconstruction. The main results were as follows: 1. Notch signaling knockout in bone marrow hampered the he matopoietic regeneration.RBP-J deficiency in bone marrow hampered the hematopoietic and myeloid regeneration, which indicated that the canonical Notch pathway was necessary to regulate bone marrow recovery and lineage differentiation after acute myelosuppression from irradiation. 2. A novel activator for Notch signaling, m D1 R, rescued myelosuppression and improved the reconstruction of hematopoietic stem cells and myeloid cells in vivo.We generated a recombinant protein, m D1 R, fused the Delta-Serrate-Lag-2 fragment of the mouse Notch ligand Delta like 1 with integrin αVβ3 specific ligand arginine-glycine-aspartate(RGD) motif, and proved m D1 R could triggered Notch signaling efficiently by targeting sinusoid endothelial cells in vascular niche. In wild type mice myelosuppression models induced by sublethal irradiation or chemical drugs, systemic intraperitoneal injection of m D1 R selectively pharmacologic activated Notch pathway, mitigated myelosuppression, promoted bone marrow hematopoietic mobilization, improved hematopoietic recovery both in intramedullary and extramedullary tissues, expanded hematopoietic stem/progenitor cells especially long-term HSC, and enhanced an inclinate myeloid lineage reconstitution. 3. m D1 R mitigated myelosuppression concentration-related.Additionally, we demonstrated an in vivo concentration-related effect of Notch ligand in stimulating hematopoietic stem cells regeneration and myeloid differentiation after acute DN A damage. Relative m D1 R densities quantitatively enhanced the generation of hematopoietic stem cells and myeloid cells, whereas extreme low concentration was invalid while high concentration restrained. This conclusion may have an important guiding significance in Notch clinical application in the future. 4. m D1 R and G-CSF had no synergistic effectCombining m D1 R with G-CSF acted no combinatorial pharmacologic efficacy in myeloid reconstitution after acute stress in mice. Considering the similar enlargement of myeloid differentiation and the robust supportment of hematopoietic stem/progenitor cells expansion to avoid exhausting stem cell pool, m D1 R may have a more broad application prospect for patients frequently suffered from infectious complications after myelosuppressive chemotherapy or radiotherapy or that undergoing stem cell transplantation. 5. m D1 R acted no toxicity and side effects in mice.In view of confirming no toxicity and side effects in mouse main organs by m D1 R in a previous experiment, we emphatically observed the changes in hematopoietic system in this subject. A continued observation for 3 to 6 months after stopping m D1 R application indicated that m D1 R treated mice achieved excellently intra- and extra- medullary hematopoietic reconstruction as well as normal unirradiated mice. No potential malignant transformation and toxicity were observed in hematopoietic system and solid organs.Otherwise, there is a rapid reconstruction(only for 14 days) in chemical damage model,which confirmed the safety of D1 R as a new drug in translational medicine. 6. Notch signaling increased CSF2RB2 expression and activated STAT3 survival signaling pathwaysBioinformatics analysis of gene chip hybridization and experimental confirmation indicated that Notch signaling activated by m D1 R significantly upregulated Csf2rb2 gene expression in vivo and in vitro. Reporter assay suggested that ICN1 enhanced Csf2rb2 expression through recognizing its RBP-J binding sites in promoter region. Chromatin immunoprecipitation assay demonstrated that Csf2rb2 was a direct transcriptional target of Notch signaling with ICN1 and RBP-J regulatory elements. The underlying cellular and molecular mechanisms were further uncovered that Notch pathway mediated hematopoietic recovery not related to increase proliferation but to inhibite apoptosis in hematopoietic stem/progenitor cells, which may associated with an activation of STAT3/Erk survival signaling pathway partially. 7. Notch signaling was positively correlated with the prognosis of hematopoietic stem cell transplantation patients.The positive relevance between nuclear localization of Notch signaling activation and hematopoietic reconstruction in blood cells was assessed by immunofluorescence staining. Meanwhile, expression of all examined Notch target genes including Csf2rb2 were upregulated in blood cells from hematopoietic stem cell transplantation patients by real-time quantitative PCR. It is possible that the feasibility of a potential prognostic value and a new translational medicine drug.In conclusion, our findings establish a previously unknown in vivo role for Notch signaling in promoting hematopoietic stem cells regeneration and in the commitment of progenitors toward myelopoiesis lineage response to chemo- and radiation- induced. This research provides the possibility to develop new adjuvant for myelosuppressive pateints in clinic. The main conclusions are as follows:1. Genetically modified myelosuppression mice model confirmes that Notch signal pathway positively regulates hematopoietic reconstruction directly after stress injury.2. We generates a novel Notch signaling activator targeted to sinusoid endothelial cells in vascular niche, and confirmes its improvement of hematopoietic stem/progenitor cells especially long-term HSC after myelosuppression thoroughly in muli- models and muli-aspects. The bone marrow injured mice achieve a rapid hematopoietic reconstitution, a decreased mortality rate, and a mitigable genetic toxicity from ionizing radiation and chemical drug.3. These results suggest that D1 R recombinant protein has an important role in regulating myeloid lineage regeneration after bone marrow injury. In the presence of homeostasis, Notch signaling does not modulate myloid differentiation, however Notch- mediated induction of hematopoietic stem/progenitor cells differentiating to myeloid progenitor cells contributes to the early recovery of neutrophils and monocytes, which enhances the ability to anti- infection. Translationally, we have elucidated a previously unknown function of Notch signaling promoting myeloid regeneration after myelosuppression.4. Here we show that D1 R treatment induces early survival and inhibites apoptosis after radiation exposure and that this effect is mediated through activation of the Notch/CSF2RB2/STAT3/Erk axis.5. Our observations that the activation of Notch signa ling may be informative about the prognosis in hematopoietic stem cell transplantation patients and expand the potential of new therapeutic applications. |
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