Context- and Cell-Dependent Effects of Delta-Like 4 Targeting in the Bone Marrow Microenvironment

Delta-like 4 (Dll4) is a ligand of the Notch pathway family which has been widely studied in the context of tumor angiogenesis, its blockade shown to result in non-productive angiogenesis and halted tumor growth. As Dll4 inhibitors enter the clinic, there is an emerging need to understand their side effects, namely the systemic consequences of Dll4:Notch blockade in tissues other than tumors. The present study focused on the effects of systemic anti-Dll4 targeting in the bone marrow (BM) microenvironment. Here we show that Dll4 blockade with monoclonal antibodies perturbs the BM vascular niche of sub-lethally irradiated mice, resulting in increased CD31⁺, VE-Cadherin⁺ and c-kit⁺ vessel density, and also increased megakaryocytes, whereas CD105⁺, VEGFR3⁺, SMA⁺ and lectin⁺ vessel density remained unaltered. We investigated also the expression of angiocrine genes upon Dll4 treatment in vivo, and demonstrate that IGFbp2, IGFbp3, Angpt2, Dll4, DHH and VEGF-A are upregulated, while FGF1 and CSF2 are reduced. In vitro treatment of endothelial cells with anti-Dll4 reduced Akt phosphorylation while maintaining similar levels of Erk 1/2 phosphorylation. Besides its effects in the BM vascular niche, anti-Dll4 treatment perturbed hematopoiesis, as evidenced by increased myeloid (CD11b⁺), decreased B (B220⁺) and T (CD3⁺) lymphoid BM content of treated mice, with a corresponding increase in myeloid circulating cells. Moreover, anti-Dll4 treatment also increased the number of CFU-M and -G colonies in methylcellulose assays, independently of Notch1. Finally, anti-Dll4 treatment of donor BM improved the hematopoietic recovery of lethally irradiated recipients in a transplant setting. Together, our data reveals the hematopoietic (BM) effects of systemic anti-Dll4 treatment result from qualitative vascular changes and also direct hematopoietic cell modulation, which may be favorable in a transplant setting.     

Feedback Signals in Myelodysplastic Syndromes: Increased Self-Renewal of the Malignant Clone Suppresses Normal Hematopoiesis

Myelodysplastic syndromes (MDS) are triggered by an aberrant hematopoietic stem cell (HSC). It is, however, unclear how this clone interferes with physiologic blood formation. In this study, we followed the hypothesis that the MDS clone impinges on feedback signals for self-renewal and differentiation and thereby suppresses normal hematopoiesis. Based on the theory that the MDS clone affects feedback signals for self-renewal and differentiation and hence suppresses normal hematopoiesis, we have developed a mathematical model to simulate different modifications in MDS-initiating cells and systemic feedback signals during disease development. These simulations revealed that the disease initiating cells must have higher self-renewal rates than normal HSCs to outcompete normal hematopoiesis. We assumed that self-renewal is the default pathway of stem and progenitor cells which is down-regulated by an increasing number of primitive cells in the bone marrow niche – including the premature MDS cells. Furthermore, the proliferative signal is up-regulated by cytopenia. Overall, our model is compatible with clinically observed MDS development, even though a single mutation scenario is unlikely for real disease progression which is usually associated with complex clonal hierarchy. For experimental validation of systemic feedback signals, we analyzed the impact of MDS patient derived serum on hematopoietic progenitor cells in vitro: in fact, MDS serum slightly increased proliferation, whereas maintenance of primitive phenotype was reduced. However, MDS serum did not significantly affect colony forming unit (CFU) frequencies indicating that regulation of self-renewal may involve local signals from the niche. Taken together, we suggest that initial mutations in MDS particularly favor aberrant high self-renewal rates. Accumulation of primitive MDS cells in the bone marrow then interferes with feedback signals for normal hematopoiesis – which then results in cytopenia.     

Expression of the MDR1 and MRP Genes in Patients with Lymphoma with Primary Bone Marrow Involvement

Expression of MDR1 and MRP genes in patients with low‐grade and high‐grade non‐Hodgkin's lymphomas with primary bone marrow involvement before and after chemotherapy was investigated. The data demonstrate that overexpression of MDR1 and MRP genes in hematological malignancies elevates in patients after chemotherapy and correlates with poor clinic prognosis and more frequent recurrences of the malignancies.     

The Different Immunoregulatory Functions on Dendritic Cells between Mesenchymal Stem Cells Derived from Bone Marrow of Patients with Low-Risk or High-Risk Myelodysplastic Syndromes

Myelodysplastic syndrome (MDS) is a group of progressive,clonal, neoplastic bone marrow disorders characterized by hematopoietic stem cell dysregulation and abnormalities in the immune system. Mesenchymal stem cells (MSC) appear to modulate the immune system at the very first step of the immune response through the inhibition of dendritic cells (DCs) differentiation and maturation. However, it is still unclear whether the effects of MSC on the development of DCs will be altered with disease state. In addition, it is not clear whether there are differences in the effects between low-risk and high-risk MDS-MSC on DCs development. In this study, our data confirm that MDS-MSC mediate a potent inhibition of DCs differentiation. Additionaly, MDS-MSC greatly alter DCs functions, including endocytosis, IL-12 secretion, their ability to inhibit T cell proliferation. Moreover, our results show that there are major differences in DCs development and function between low-risk and high-risk MDS-MSC. Compared to high-risk MDS-MSC, low-risk MDS-MSC is characterized by a poor ability to inhibit DCs differentiation and maturation; and correspondingly, less dysfunctional DC endocytosis, mildly decreased IL-12 secretion, and a reduction in DC-mediated inhibition of T cell proliferation. Finally, our results demonstrate that MDS-MSC derived TGF-β1 is largely responsible for the inhitory effects. These results elucidate the different immunoregulatory role of MSC in low-risk and high-risk MDS on DCs development, which may be important for understanding the pathogenesis of MDS and the development of novel immune therapies for the treatment of MDS.     

Oncostatin M Maintains the Hematopoietic Microenvironment in the Bone Marrow by Modulating Adipogenesis and Osteogenesis

The bone marrow (BM) is an essential organ for hematopoiesis in adult, in which proliferation and differentiation of hematopoietic stem/progenitor cells (HSPC) is orchestrated by various stromal cells. Alterations of BM hematopoietic environment lead to various hematopoietic disorders as exemplified by the linking of fatty marrow with increased adipogenesis to anemia or pancytopenia. Therefore, the composition of mesenchymal stromal cell (MSC)-derived cells in the BM could be crucial for proper hematopoiesis, but the mechanisms underlying the MSC differentiation for hematopoiesis remain poorly understood. In this study, we show that Oncostatin M (OSM) knock out mice exhibited pancytopenia advancing fatty marrow with age. OSM strongly inhibited adipogenesis from BM MSC in vitro, whereas it enhanced their osteogenesis but suppressed the terminal differentiation. Intriguingly, OSM allowed the MSC-derived cells to support the ex vivo expansion of HSPC effectively as feeder cells. Furthermore, the administration of OSM in lethally irradiated wild-type mice blocked fatty marrow and enhanced the recovery of HSPC number in the BM and peripheral blood cells after engraftment of HSPC. Collectively, OSM plays multiple critical roles in the maintenance and development of the hematopoietic microenvironment in the BM at a steady state as well as after injury.     

Endocannabinoids Are Expressed in Bone Marrow Stromal Niches and Play a Role in Interactions of Hematopoietic Stem and Progenitor Cells with the Bone Marrow Microenvironment

Endocannabinoids are lipid signaling molecules that act via G-coupled receptors, CB₁ and CB₂. The endocannabinoid system is capable of activation of distinct signaling pathways on demand in response to pathogenic events or stimuli, hereby enhancing cell survival and promoting tissue repair. However, the role of endocannabinoids in hematopoietic stem and progenitor cells (HSPCs) and their interaction with hematopoietic stem cells (HSC) niches is not known. HSPCs are maintained in the quiescent state in bone marrow (BM) niches by intrinsic and extrinsic signaling. We report that HSPCs express the CB₁ receptors and that BM stromal cells secrete endocannabinoids, anandamide (AEA) (35 pg/10⁷ cells), and 2-AG (75.2 ng/10⁷ cells). In response to the endotoxin lipopolysaccharide (LPS), elevated levels of AEA (75.6 pg/10⁷ cells) and 2-AG (98.8 ng/10⁷ cells) were secreted from BM stromal cells, resulting in migration and trafficking of HSPCs from the BM niches to the peripheral blood. Furthermore, administration of exogenous cannabinoid CB₁ agonists in vivo induced chemotaxis, migration, and mobilization of human and murine HSPCs. Cannabinoid receptor knock-out mice Cnr1^−/− showed a decrease in side population (SP) cells, whereas fatty acid amide hydrolase (FAAH)^−/− mice, which have elevated levels of AEA, yielded increased colony formation as compared with WT mice. In addition, G-CSF-induced mobilization in vivo was modulated by endocannabinoids and was inhibited by specific cannabinoid antagonists as well as impaired in cannabinoid receptor knock-out mice Cnr1^−/−, as compared with WT mice. Thus, we propose a novel function of the endocannabinoid system, as a regulator of HSPC interactions with their BM niches, where endocannabinoids are expressed in HSC niches and under stress conditions, endocannabinoid expression levels are enhanced to induce HSPC migration for proper hematopoiesis.     

Purification of Bone Marrow Clonal Cells from Patients with Myelodysplastic Syndrome via IGF-IR

Malignant clonal cells purification can greatly benefit basic and clinical studies in myelodysplastic syndrome (MDS). In this study, we investigated the potential of using type 1 insulin-like growth factor receptor (IGF-IR) as a marker for purification of malignant bone marrow clonal cells from patients with MDS. The average percentage of IGF-IR expression in CD34⁺ bone marrow cells among 15 normal controls was 4.5%, 70% of which also express the erythroid lineage marker CD235a. This indicates that IGF-IR mainly express in erythropoiesis. The expression of IGF-IR in CD34⁺ cells of 55 MDS patients was significantly higher than that of cells from the normal controls (54.0 vs. 4.5%). Based on the pattern of IGF-IR expression in MDS patients and normal controls, sorting of IGF-IR-positive and removal of CD235a-positive erythroid lineage cells with combination of FISH detection were performed on MDS samples with chromosomal abnormalities. The percentage of malignant clonal cells significantly increased after sorting. The enrichment effect was more significant in clonal cells with a previous percentage lower than 50%. This enrichment effect was present in samples from patients with +8, 5q-/-5, 20q-/-20 or 7q-/-7 chromosomal abnormalities. These data suggest that IGF-IR can be used as a marker for MDS bone marrow clonal cells and using flow cytometry for positive IGF-IR sorting may effectively purify MDS clonal cells.     

Three-Dimensional Microfluidic Tri-Culture Model of the Bone Marrow Microenvironment for Study of Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) initiates and progresses in the bone marrow, and as such, the marrow microenvironment is a critical regulatory component in development of this cancer. However, ALL studies were conducted mainly on flat plastic substrates, which do not recapitulate the characteristics of marrow microenvironments. To study ALL in a model of in vivo relevance, we have engineered a 3-D microfluidic cell culture platform. Biologically relevant populations of primary human bone marrow stromal cells, osteoblasts and human leukemic cells representative of an aggressive phenotype were encapsulated in 3-D collagen matrix as the minimal constituents and cultured in a microfluidic platform. The matrix stiffness and fluidic shear stress were controlled in a physiological range. The 3-D microfluidic as well as 3-D static models demonstrated coordinated cell-cell interactions between these cell types compared to the compaction of the 2-D static model. Tumor cell viability in response to an antimetabolite chemotherapeutic agent, cytarabine in tumor cells alone and tri-culture models for 2-D static, 3-D static and 3-D microfluidic models were compared. The present study showed decreased chemotherapeutic drug sensitivity of leukemic cells in 3-D tri-culture models from the 2-D models. The results indicate that the bone marrow microenvironment plays a protective role in tumor cell survival during drug treatment. The engineered 3-D microfluidic tri-culture model enables systematic investigation of effects of cell-cell and cell-matrix interactions on cancer progression and therapeutic intervention in a controllable manner, thus improving our limited comprehension of the role of microenvironmental signals in cancer biology.     

Cell Signaling and Differential Protein Expression in Neuronal Differentiation of Bone Marrow Mesenchymal Stem Cells with Hypermethylated Salvador/Warts/Hippo (SWH) Pathway Genes

Human mesenchymal stem cells (MSCs) modified by targeting DNA hypermethylation of genes in the Salvador/Warts/Hippo pathway were induced to differentiate into neuronal cells in vitro. The differentiated cells secreted a significant level of brain-derived neurotrophy factor (BDNF) and the expression of BDNF receptor tyrosine receptor kinase B (TrkB) correlated well with the secretion of BDNF. In the differentiating cells, CREB was active after the binding of growth factors to induce phosphorylation of ERK in the MAPK/ERK pathway. Downstream of phosphorylated CREB led to the functional maturation of differentiated cells and secretion of BDNF, which contributed to the sustained expression of pERK and pCREB. In summary, both PI3K/Akt and MAPK/ERK signaling pathways play important roles in the neuronal differentiation of MSCs. The main function of the PI3K/Akt pathway is to maintain cell survival during neural differentiation; whereas the role of the MAPK/ERK pathway is probably to promote the maturation of differentiated MSCs. Further, cellular levels of protein kinase C epsilon type (PKC-ε) and kinesin heavy chain (KIF5B) increased with time of induction, whereas the level of NME/NM23 nucleoside diphosphate kinase 1 (Nm23-H1) decreased during the time course of differentiation. The correlation between PKC-ε and TrkB suggested that there is cross-talk between PKC-ε and the PI3K/Akt signaling pathway.     

稳态及衰老情况下骨髓微环境对造血干细胞的调控作用

稳态下,骨髓微环境(bone marrow microenvironment)被证实能通过多种信号通路和细胞因子调控造血干细胞(hematopoietic stem cells,HSCs)的自我更新、增殖、分化和迁移能力以维持造血系统的稳定.在衰老过程中,HSCs功能受损会导致造血系统功能的退化以及年龄相关的免疫应答的...     

Melatonin Signaling Modulates Clock Genes Expression in the Mouse Retina

Previous studies have shown that retinal melatonin plays an important role in the regulation of retinal daily and circadian rhythms. Melatonin exerts its influence by binding to G-protein coupled receptors named melatonin receptor type 1 and type 2 and both receptors are present in the mouse retina. Earlier studies have shown that clock genes are rhythmically expressed in the mouse retina and melatonin signaling may be implicated in the modulation of clock gene expression in this tissue. In this study we determined the daily and circadian expression patterns of Per1, Per2, Bmal1, Dbp, Nampt and c-fos in the retina and in the photoreceptor layer (using laser capture microdissection) in C3H-f+/+ and in melatonin receptors of knockout (MT₁ and MT₂) of the same genetic background using real-time quantitative RT-PCR. Our data indicated that clock and clock-controlled genes are rhythmically expressed in the retina and in the photoreceptor layer. Removal of melatonin signaling significantly affected the pattern of expression in the retina whereas in the photoreceptor layer only the Bmal1 circadian pattern of expression was affected by melatonin signaling removal. In conclusion, our data further support the notion that melatonin signaling may be important for the regulation of clock gene expression in the inner or ganglion cells layer, but not in photoreceptors.     

Evaluation of MicroRNA Expression in Patient Bone Marrow Aspirate Slides

Like formalin fixed paraffin embedded (FFPE) tissues, archived bone marrow aspirate slides are an abundant and untapped resource of biospecimens that could enable retrospective molecular studies of disease. Historically, RNA obtained from slides is limited in utility because of their low quality and highly fragmented nature. MicroRNAs are small (≈22 nt) non-coding RNA that regulate gene expression, and are speculated to preserve well in FFPE tissue. Here we investigate the use of archived bone marrow aspirate slides for miRNA expression analysis in paediatric leukaemia. After determining the optimal method of miRNA extraction, we used TaqMan qRT-PCR to identify reference miRNA for normalisation of other miRNA species. We found hsa-miR-16 and hsa-miR-26b to be the most stably expressed between lymphoblastoid cell lines, primary bone marrow aspirates and archived samples. We found the average fold change in expression of hsa-miR-26b and two miRNA reportedly dysregulated in leukaemia (hsa-miR-128a, hsa-miR-223) was <0.5 between matching archived slide and bone marrow aspirates. Differential expression of hsa-miR-128a and hsa-miR-223 was observed between leukaemic and non-leukaemic bone marrow from archived slides or flash frozen bone marrow. The demonstration that archived bone marrow aspirate slides can be utilized for miRNA expression studies offers tremendous potential for future investigations into the role miRNA play in the development and long term outcome of hematologic, as well as non-hematologic, diseases.     

STING在宿主天然免疫信号通路中的调节作用

STING(stimulator of interferon genes)是天然免疫信号通路中一种新发现的蛋白质,在防御病毒及胞内细菌感染、介导Ⅰ型IFN产生过程中发挥重要功能.来自病原体的B型DNA与5′-3p dsRNA暴露在宿主细胞中后被相应的模式识别受体识别,通过不同的通路传递信号给STING.STING随后通过相似的机制招募TBK1激活IRF3,诱导干扰素表达.对细菌中的环二核苷酸c-di-GMP和c-di-AMP,STING则可以直接作为模式识别受体引发Ⅰ型干扰素反应.此外STING还能激活STAT6诱导特异趋化因子产生,吸引各种免疫细胞抵抗病毒感染.本文通过对STING的发现、结构、定位、功能、机理以及调节机制进行综述,以期为揭示病毒逃逸天然免疫调节机制和抗病毒新型免疫调节剂提供新的思路.     

Integrated Genomic Analysis Implicates Haploinsufficiency of Multiple Chromosome 5q31.2 Genes in De Novo Myelodysplastic Syndromes Pathogenesis

Deletions spanning chromosome 5q31.2 are among the most common recurring cytogenetic abnormalities detectable in myelodysplastic syndromes (MDS). Prior genomic studies have suggested that haploinsufficiency of multiple 5q31.2 genes may contribute to MDS pathogenesis. However, this hypothesis has never been formally tested. Therefore, we designed this study to systematically and comprehensively evaluate all 28 chromosome 5q31.2 genes and directly test whether haploinsufficiency of a single 5q31.2 gene may result from a heterozygous nucleotide mutation or microdeletion. We selected paired tumor (bone marrow) and germline (skin) DNA samples from 46 de novo MDS patients (37 without a cytogenetic 5q31.2 deletion) and performed total exonic gene resequencing (479 amplicons) and array comparative genomic hybridization (CGH). We found no somatic nucleotide changes in the 46 MDS samples, and no cytogenetically silent 5q31.2 deletions in 20/20 samples analyzed by array CGH. Twelve novel single nucleotide polymorphisms were discovered. The mRNA levels of 7 genes in the commonly deleted interval were reduced by 50% in CD34+ cells from del(5q) MDS samples, and no gene showed complete loss of expression. Taken together, these data show that small deletions and/or point mutations in individual 5q31.2 genes are not common events in MDS, and implicate haploinsufficiency of multiple genes as the relevant genetic consequence of this common deletion.