Abstract WMP44: Mir-145 Mediates Bone-marrow-stromal Cell Derived From Type-one Diabetes (t1dm) Rats Induced Neurorestorative Effects in T1dm Rats

Stroke ◽  
2016 ◽  
Vol 47 (suppl_1) ◽  
Author(s):  
Chengcheng Cui ◽  
Michael Chopp ◽  
Xinchun Ye ◽  
Alex Zacharek ◽  
Ruizhou Ning ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cortical Neurons ◽  
Target Genes ◽  
Capillary Tube ◽  
Border Zone ◽  
Therapeutic Effects ◽  
Ischemic Brain ◽  
Type One Diabetes

Objective: Treatment of stroke with bone-marrow-stromal cells (BMSCs) derived from type-one diabetes (T1DM) rats (DM-BMSCs) improves functional recovery compared to BMSCs derived from normal rats (Nor-BMSCs) and non treatment T1DM rats. In the study, we tested the mechanisms underlying the benefit of the treatment of T1DM stroke with DM-BMSCs. Methods: T1DM rats induced by streptozocin in male Wistar rats were subjected to 2h middle cerebral artery occlusion (MCAo) and were treated at 24h after MCAo via tail vein with: 1) vehicle control; 2) DM-BMSCs; 3) DM-BMSCs with miR-145 overexpression (miR-145+/+DM-BMSCs)(5x10^6) (n=8/group). A battery of functional tests, vascular,white matter (WM) measurements, and cell culture experiments were performed. Results: In vitro, DM-BMSCs exhibited reduced level of miR-145, and increased survival rate compared to Nor-BMSCs. miR-145+/+DM-BMSCs significantly decreased DM-BMSCs survival. DM-BMSCs media increased capillary tube formation and axonal outgrowth in cultured primary cortical neurons (PCNs) compared to Nor-BMSCs media. While miR-145+/+DM-BMSCs exhibited reverse effects compared to DM-BMSCs media. In vivo, DM-BMSCs improved functional outcome, vascular and WM remodeling in the ischemic border zone (IBZ) compared to T1DM-MCAo rats. However, miR-145+/+DM-BMSCs significantly attenuated DM-BMSCs induced beneficial effects. To further test the underlying mechanism of miR-145 mediated DM-BMSCs induced therapeutic effects in T1DM stroke rats, miR-145 target genes adenosine triphosphate-binding cassette transporter 1 (ABCA1) and insulin-like growth factor 1 receptor (IGFR-1) were measured in IBZ. ABCA1 and IGFR1 have neurorestorative effects. Reduction of IGF1 contributes ABCA1 deficiency induced damage in ischemic brain. We found that DM-BMSCs significantly decreased miR-145, increased ABCA1 and IGFR-1 expression in IBZ compared to Nor-BMSCs. While miR-145+/+DM-BMSCs significantly decreased ABCA1 and IGFR-1 expression in IBZ. Conclusion: DM-BMSCs exhibit decreased miR-145 expression and increase miR-145 target gene ABCA1 and IGFR-1 expression in ischemic brain. The miR-145/ABCA1/IGFR-1 pathway may contribute to DM-BMSCs induced neurorestorative effects in T1DM stroke.

scholarly journals Hyperoside Suppresses Renal Inflammation by Regulating Macrophage Polarization in Mice With Type 2 Diabetes Mellitus

2021 ◽  
Vol 12 ◽  
Author(s):  
Jialing Liu ◽  
Yanmei Zhang ◽  
Hongqin Sheng ◽  
Chunling Liang ◽  
Huazhen Liu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Macrophage Polarization ◽  
Fasting Blood Glucose ◽  
Therapeutic Effects ◽  
Protective Effects ◽  
Monocyte Chemoattractant Protein 1 ◽  
Anti Inflammatory

Accumulating evidence reveals that both inflammation and lymphocyte dysfunction play a vital role in the development of diabetic nephropathy (DN). Hyperoside (HPS) or quercetin-3-O-galactoside is an active flavonoid glycoside mainly found in the Chinese herbal medicine Tu-Si-Zi. Although HPS has a variety of pharmacological effects, including anti-oxidative and anti-apoptotic activities as well as podocyte-protective effects, its underlying anti-inflammatory mechanisms remain unclear. Herein, we investigated the therapeutic effects of HPS on murine DN and the potential mechanisms responsible for its efficacy. We used C57BLKS/6J Lepdb/db mice and a high glucose (HG)-induced bone marrow-derived macrophage (BMDM) polarization system to investigate the potentially protective effects of HPS on DN. Our results showed that HPS markedly reduced diabetes-induced albuminuria and glomerular mesangial matrix expansion, accompanied with a significant improvement of fasting blood glucose level, hyperlipidaemia and body weight. Mechanistically, pretreatment with HPS effectively regulated macrophage polarization by shifting proinflammatory M1 macrophages (F4/80+CD11b+CD86+) to anti-inflammatory M2 ones (F4/80+CD11b+CD206+) in vivo and in bone marrow-derived macrophages (BMDMs) in vitro, resulting in the inhibition of renal proinflammatory macrophage infiltration and the reduction in expression of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor (TNF-α) and inducible nitric oxide synthase (iNOS) while increasing expression of anti-inflammatory cytokine Arg-1 and CD163/CD206 surface molecules. Unexpectedly, pretreatment with HPS suppressed CD4+ T cell proliferation in a coculture model of IL-4-induced M2 macrophages and splenic CD4+ T cells while promoting their differentiation into CD4+IL-4+ Th2 and CD4+Foxp3+ Treg cells. Taken together, we demonstrate that HPS ameliorates murine DN via promoting macrophage polarization from an M1 to M2 phenotype and CD4+ T cell differentiation into Th2 and Treg populations. Our findings may be implicated for the treatment of DN in clinic.

scholarly journals Bone Marrow-Derived Mesenchymal Stem Cells Modified with Akt1 Ameliorates Acute Liver GVHD

2019 ◽  
Vol 21 (1) ◽  
Author(s):  
Lukun Zhou ◽  
Shuang Liu ◽  
Zhao Wang ◽  
Jianfeng Yao ◽  
Wenbin Cao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Liver Injury ◽  
Therapeutic Effects ◽  
Hematopoietic Stem ◽  
Ifn Γ ◽  
Immunomodulatory Function

Abstract Background Liver injury associated with acute graft-versus-host disease (aGVHD) is a frequent and severe complication of hematopoietic stem cell transplantation and remains a major cause of transplant-related mortality. Bone marrow-derived mesenchymal stem cells (BM-MSCs) has been proposed as a potential therapeutic approach for aGVHD. However, the therapeutic effects are not always achieved. In this study, we genetically engineered C57BL/6 mouse BM-MSCs with AKT1 gene and tested whether AKT1-MSCs was superior to control MSCs (Null-MSCs) for cell therapy of liver aGVHD. Results In vitro apoptosis analyses showed that, under both routine culture condition and high concentration interferon-γ (IFN-γ) (100ng/mL) stimulation condition, AKT1-MSCs had a survival (anti-apoptotic) advantage compared to Null-MSCs. In vivo imaging showed that AKT1-MSCs had better homing capacity and longer persistence in injured liver compared to Null-MSCs. Most importantly, AKT1-MSCs demonstrated an enhanced immunomodulatory function by releasing more immunosuppressive cytokines, such as IL-10. Adoptive transfer of AKT1-MSCs mitigated the histopathological abnormalities of concanavalin A(ConA)-induced liver injury along with significantly lowered serum levels of ALT and AST. The attenuation of liver injury correlated with the decrease of TNF-α and IFN-γ both in liver tissue and in the serum. Conclusions In summary, BM-MSCs genetically modified with AKT1 has a survival advantage and an enhanced immunomodulatory function both in vitro and in vivo and thus demonstrates the therapeutic potential for prevention and amelioration of liver GVHD and other immunity-associated liver injuries.

Abstract T P94: Differential Response to Treatment of Stroke in Rats With T1dm With Bone Marrow Stromal Cells Derived From Normal or T1DM Rats

Stroke ◽  
2015 ◽  
Vol 46 (suppl_1) ◽  
Author(s):  
Chengcheng Cui ◽  
Michael Chopp ◽  
Xinchun Ye ◽  
Alex Zacharek ◽  
Ruizhuo Ning ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cerebral Artery ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Border Zone ◽  
Response To Treatment ◽  
Vascular Density ◽  
Therapeutic Effects

Objective: Treatment of stroke with bone-marrow-stromal cells (BMSCs) derived from normal rats (Nor-BMSCs) at 24h after stroke onset improves functional recovery in non-DM rats, but not in type-one DM (T1DM) rats. In the present study, we tested the differential therapeutic effects and mechanisms of action of the treatment of T1DM stroke with BMSCs derived from DM rats (DM-BMSCs) or Nor-BMSCs. Methods: T1DM rats were subjected to 2h of middle cerebral artery occlusion (MCAo) and were treated with DM-BMSCs, Nor-BMSCs or vehicle control (n=8/group) initiated at 24h after MCAo. A battery of functional tests, and vascular and white matter (WM) changes and in vitro cell culture experiments were performed. Results: Compared with Nor-BMSCs, treatment of stroke in T1DM rats with DM-BMSCs significantly improved functional outcome. DM-BMSCs increased vascular remodeling identified by cerebral vascular density and cerebral artery density, increased Bielschowsky silver (axon marker) and Luxol fast blue (myelin marker) expression in the ischemic border zone (IBZ) compared to T1DM-MCAo rats (p<0.05). However, DM-BMSC treatment also increased artherosclerosis-like changes identified by an increased internal carotid wall thickness, artery intimae thickness, and decreased cerebral artery diameter (p<0.05) compared to T1DM-MCAo control. DM-BMSCs treatment significantly increased PDGF, but decreased RhoA and ROCK expression in the IBZ. In vitro, DM-BMSCs have higher miR133b (1.9 fold) expression compared to Nor-BMSCs. DM-BMSC conditioned medium increased brain endothelial cell, smooth muscle cell and oligodendrocyte progenitor cell survival, as well as increased subventricular zone explant cell migration and primary cortical neuron axon outgrowth compared to Nor-BMSC conditioned or control medium. Conclusion: Treatment of stroke in T1-DM rats with DM-BMSCs have dual effects on stroke, i.e. significantly increases neurovascular remodeling, improves neurological recovery, and induces artherosclerosis-like vascular changes. The miR133b/RhoA/ROCK pathway may play a role in the DM-BMSC induced neurorestorative effects.

scholarly journals Nanoparticles Delivering Antagomir-483-5p to Bone Marrow Mesenchymal Stem Cells Prevent Osteoporosis by Increasing Bone Formation

2021 ◽  
Author(s):  
Yue Zhou ◽  
Hao Jia ◽  
Aihua Hu ◽  
Rangru Liu ◽  
Xiangzhou Zeng ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Formation ◽  
Small Molecules ◽  
Target Genes ◽  
Osteoblastic Differentiation ◽  
Mitogen Activated Protein Kinase ◽  
Osteoporosis Prevention ◽  
Bone Formation Markers

Abstract Background: Promoting bone marrow mesenchymal stem cell (BMSC) osteoblastic differentiation is a promising therapeutic strategy for osteoporosis (OP). The present study demonstrates that miR-483-5p inhibits the osteogenic differentiation of BMSCs. Therefore, selectively delivering the nanoparticles carrying antagomir-483-5p (miR-483-5p inhibitor) to BMSCs is expected to become an effective treatment drug for OP.Methods: Real-time PCR assays were used to analyse miR-483-5p, ALP and Bglap levels in BMSCs of ovariectomized and aged osteoporotic mice. To selectively and efficiently deliver antagomir-483-5p to BMSCs in vivo, immunoglobulin G and poloxamer-188 were used to encapsulate the functional small molecules, and BMSC-targeting aptamer was employed to confirm the direction of the nanoparticles. Luciferase assays were used to determine the target genes of miR-483-5p. Western blot assays and immunohistochemistry staining were used to detect the targets in vitro and vivo.Results: miR-483-5p levels were increased in BMSCs of ovariectomized and aged osteoporotic mice. Inhibition of miR-483-5p levels in BMSCs by antagomir-483-5p in vitro promoted the expression of bone formation markers, such as ALP and Bglap. The FAM-BMSC-aptamer-nanoparticles carrying antagomir-483-5p were taken up by BMSCs, resulting in stimulation of BMSC osteoblastic differentiation in vitro and osteoporosis prevention in vivo. Furthermore, our research demonstrated that mitogen-activated protein kinase 1 (MAPK1) and SMAD family member 5 (Smad5) were direct targets of miR-483-5p in regulating BMSC osteoblastic differentiation and osteoporosis pathological processes. Conclusions: The important therapeutic role of FAM-BMSC-aptamer-nanoparticles carrying antagomir-483-5p in osteoporosis was established in our study. These nanoparticles are novel candidate for the clinical prevention and treatment of osteoporosis. The optimized targeted drug delivery platform for small molecules will provide new ideas for the treatment of clinical diseases.

scholarly journals Nestin+ Mesenchymal Precursors Generate Distinct Spleen Stromal Cell Subsets and Have Immunomodulatory Function

2021 ◽  
Author(s):  
Jing Huang ◽  
Minrong Li ◽  
Ronghai Deng ◽  
Weiqiang Li ◽  
Meihua Jiang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Therapeutic Potential ◽  
Inflammatory Diseases ◽  
Cell Phenotype ◽  
Therapeutic Effects ◽  
Inflammatory Bowel ◽  
Inflammatory Milieu

Abstract Background Mesenchymal stromal cells (MSCs) are known to be widespread in many tissues and possess a broad spectrum of immunoregulatory properties. They have been used in the treatment of a variety of inflammatory diseases; however, the therapeutic effects are still inconsistent owing to their heterogeneity. Spleen stromal cells have evolved to regulate the immune response at many levels as they are bathed in a complex inflammatory milieu during infection. Therefore, it is unknown whether they have stronger immunomodulatory effects than their counterparts derived from other tissues. Methods Here, using a transgenic mouse model expressing GFP driven by the Nestin (Nes) promoter, Nes-GFP+ cells from bone marrow and spleen were collected. Artificial lymphoid reconstruction in vivo was performed. Cell phenotype, inhibition of T cell inflammatory cytokines, and in vivo therapeutic effects were assessed. Results We observed Nes-GFP+ cells colocalized with splenic stromal cells and further demonstrated that these Nes-GFP+ cells had the ability to establish ectopic lymphoid-like structures in vivo. Moreover, we showed that the Nes-GFP+ cells possessed the characteristics of MSCs. Spleen-derived Nes-GFP+ cells exhibited greater immunomodulatory ability in vitro, and more remarkable therapeutic efficacy in inflammatory diseases, especially inflammatory bowel disease (IBD) than bone marrow-derived Nes-GFP+ cells. Conclusions Overall, our data showed that Nes-GFP+ cells contributed to subsets of spleen stromal populations and possessed the biological characteristics of MSCs with a stronger immunoregulatory function and therapeutic potential than bone marrow-derived Nes-GFP+ cells.

Abstract 14554: Angiogenic Agent Ono-1301 Enhanced Cardiac Contractile Proteins in Hipsc Derived Cardiac Tissue-like Construct With Functional Angiogenesis

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Xiang Qu ◽  
Junjun Li ◽  
Kota Suzuki ◽  
Akima Harada ◽  
Shigeru Miyagawa ◽  
...  
Keyword(s):  
Cardiac Tissue ◽  
Early Stage ◽  
Induced Pluripotent Stem Cell ◽  
Capillary Density ◽  
Border Zone ◽  
Nuclear Antigen ◽  
Therapeutic Effects ◽  
Nude Rat

Introduction: Transplantation of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) into myocardial infarcted areas has shown potential in healing myocardial infarction (MI). However, the survival rate of transplanted cells is yet considered limited, restricting the therapeutic effects of the transplantation. We assumed that adding angiogenic agent ONO-1301 could promote early stage angiogenesis between host and graft, and benefit the therapeutic effects of hiPSC-CM transplantation. Methods: This study is based on our former study of Cardiac Tissue-Like Construct (CTLC), where 5 x10e6 of hiPSC-CM was seeded on a 1x1cm poly(lactic-co-glycolic acid) (PLGA) nano fiber (Figure A). In addition, angiogenic agent ONO-1301 was mixed into the fibers. After 24h culture in vitro, 3 layers of CTLCs were transplanted into Nude rat MI models with or without ONO-1301. The rats were raised for another 4 or 8 weeks before sacrifice. Results: In vitro experiments have shown that supernatant of iPS-CM with ONO-1301 underwent a rise in VEGF during a 3 to 6 day culture (Figure B). The VEGF concentration in ONO-1301 positive group is much higer than ONO-1301 negative group (P=0.022) on day3. This difference narrows down over time. In vivo experiments on nude rat have shown that capillary density on MI border zone of ONO fiber+iPS-CM group is 29.95% higher than that of PLGA fiber+iPS-CM group (P=0.004) (figure C). HE and immunofluorescence staining shows remaining transplanted hiPSC-CMs in ONO fiber+iPS-CM group shows more significant TNT2 and human nuclear antigen(HNA) expression than that of PLGA fiber+iPS-CM group (Figure D and E). Also ONO fiber+iPS-CM group showed functional recovery compared to the sham.

An EVI1 Point Mutant Unable To Bind GATA1 Rescues EVI1−Induced Defects in Erythroid Differentiation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 775-775
Author(s):  
Leopoldo Laricchia-Robbio ◽  
Ciro R. Rinaldi ◽  
Raffaella Fazzina ◽  
Giuseppina Nucifora
Keyword(s):  
Bone Marrow ◽  
Target Genes ◽  
Chromosomal Abnormalities ◽  
Direct Interaction ◽  
Erythroid Differentiation ◽  
Erythroid Cells ◽  
Murine Bone Marrow ◽  
Differentiation Block

Abstract EVI1 is a nuclear oncoprotein deregulated by recurring chromosomal abnormalities in MDS. The expression of this gene in MDS patients represents a very poor prognostic marker and is associated with erythroid and megakaryocytic defects. We have previously shown that the forced expression of EVI1 in mice results in a fatal disease with features characteristic of MDS including dyserythropoiesis, dysmegakaryopoiesis, and anemia. More recently we have shown that EVI1 directly interacts with GATA1 and disrupts GATA1−binding to DNA leading to deregulation of GATA1−dependent genes. Here we describe the effects of an EVI1 mutant unable to bind GATA1, on the regulation of GATA1 target genes and on the erythroid differentiation of murine bone marrow progenitors. The structure of two zinc finger motifs of this mutant, EVI1(1+6Mut), were destroyed by His to Ala and Cys to Ala changes. Semi−quantitative RT−PCR showed that most of the analysed genes were down−regulated in 32Dcl3 cells expressing EVI1 but not EVI1(1+6Mut). Bone marrow lineage negative cells infected with EVI1, EVI1(1+6Mut), or the empty vector as control, selected in G418, and plated in presence of Epo were utilized to determine the progenitors’ potential to differentiate in response to this cytokine. The EVI1−expressing cells were virtually unable to generate erythroid colonies after Epo stimulation and only scarce small colonies were observed. In contrast, the EVI1(1+6Mut)−cells produced about 65% of the colonies formed by the control cells. The cells were recovered and their morphology was analysed after Wright−Giemsa staining. All EVI1−positive erythroid cells showed an impaired differentiation that was arrested at the basophilic−erythroblast stage. These cells were bi− and tetra−nucleated with chromatin bridges, budding nuclei and nuclear−cytoplasmatic maturative asynchronizations. All of these features are known as dysplastic erythroid aspects in MDS patients. These dysplastic characteristics were less prominent in EVI1(1+6Mut)−positive cells and were observed only in a minority of cells rather than in the entire population. As expected, the control cells had the appearance of normal erythroblasts and none of the aberrant features were observed. These results parallel the morphology observation of peripheral blood smears obtained from EVI1−mice and EVI1(1+6Mut)−mice in that erythropoiesis defects such as aniso−poikilocytosis and orthochromatic erythroblasts were less striking and observed only in a limited number of erythroid cells in EVI1(1+6Mut)−mice. Based on these results, we propose that EVI1 blocks erythroid differentiation by direct interaction with GATA1 leading to impairment of GATA1 regulation. This differentiation block is significantly reduced in vitro and in vivo with the EVI1(1+6Mut) mutant, which re−establishes to a high degree the normal functions of GATA1 and diminishes the erythroid dysplastic aspects observed in EVI1 cells.

Bone Marrow Interaction and Multiple Myeloma - Approximating Reality in Novel High-Throughput Multiple Myeloma Coculture Systems

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2243-2243
Author(s):  
Johannes Waldschmidt ◽  
Dagmar Wider ◽  
Anna Simon ◽  
Andreas R. Thomsen ◽  
Christine Aldrian ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
High Throughput ◽  
Cxcr4 Expression ◽  
Therapeutic Effects ◽  
Great Effort ◽  
Quiescent State ◽  
Made In

Abstract Introduction: In the past decade, substantial progress has been made in the understanding of multiple myeloma (MM) cell biology and its interaction with the bone marrow microenvironment (BMM). Binding of MM cells to BM stroma cells (BMSCs) alters the expression of SDF-1α and its receptor CXCR4, leading to the secretion of anti-apoptotic cytokines, promoting tumor growth, drug resistance and migration. MM cancer stem cells migrate to endosteal BM niches, where they escape therapies in a quiescent state causing relapse in the course of the disease. The development of novel agents that aim to target the MM and BMM interaction includes drugs as promising as 2nd and 3rdgeneration IMIDs or proteasome inhibitors. Despite these profound advances, the failure rate of preclinically proven cytotoxic single substances is sizeable, as preclinical models often lack the biological, genetic, etiological and immunological properties of the disease (Schüler, Expert. Opin. Biol. Ther. 2013; Kortüm. CLML 2014; Rongvaux. Annu Rev Immunol 2013). Methods & Results: We have previously demonstrated that BM interaction and homing to niches, mediated by the adhesion molecules CXCR4, CD49d and CD44, protect MM cell lines (MMCL) and primary plasma cells (PC) from the cytotoxic effect of anti-MM agents, such as bortezomib (Bor), vorinostat (Vor) and pomalidomide (Pom). Our in vitro and in vivo observed cytotoxic effects from Bor, Vor and Pom confirmed their potent cytotoxicity, whereas cocultivation with M2-10B4 substantially reduced apoptosis and induced tumor protective effects. Additional treatment with the CXCR4 inhibitor AMD3100 blocked CXCR4 in coculture, but left CD49d, CD44 and CD11a widely unchanged. Toxic or therapeutic effects from AMD3100 monotherapy were excluded for the doses used. Comparison of the CXCR4 antibody (ab)-clones 12G5, 44717 and 4G10 revealed that AMD3100 treatment of U266 cells reduced CXCR4 expression with use of 12G5 and 44717, whereas binding of both FITC- and PE-coupled 4G10 was not influenced, making the latter the most reliable for CXCR4 analysis. Use of image cytometry (IC) allowed accurate visualization of co-localisation of CXCR4 expression both on the cell surface and within the cytoplasm of MM cells. IC correlated with flow cytometry-determined CXCR4 expression and allowed the detailed assessment of treatment studies with and without anti-MM agents and AMD3100. Of note, AMD3100 resensitized MM cells to Bor, Vor and Pom (Waldschmidt. Blood 2012:2450), whereas carfilzomib (Cfz) reduced CXCR4 expression in MMCL and could not be antagonized by stroma coculture. Cfz sensitivity was not increased by adding AMD3100 (Simon. Blood 2013:3851). These preclinical studies need additional adaptation to the clinical setting in order to surpass prior drug failure rates, and there is a need to develop more broadly available and better predictive preclinical systems. Therefore, we are currently assessing a 3D co-culture MM model composed of agarose matrix interlayers, based on a novel liquid overlay technique. This model has been specifically adapted to MM cell and BM component interactions as described (Udi. BJH 2013; Zlei. Exp Hematol 2007; Schüler. EOBT 2013). MM cells are cultivated in conical microwells of a non-adherent agarose matrix after BMSCs were plated on the bottom of each plate, allowing the diffusion of soluble cytokines but no direct contact between BMSC and MMCL. Therein, we are presently testing novel anti-MM substances in comparison to our standard-coculture system. Conclusion: Targeting microenvironmental mediators, like SDF-1α and CXCR4, is a promising approach to expand the choice of antimyeloma agents and amplify the effects of established antimyeloma drugs, as previously shown by us and others for the combination of AMD3100 and Bor or Pom. However, as our knowledge on MM and its BMM has dramatically increased a great effort has been made in the preclinical testing of promising new anti-MM agents, and more complex high-throughput in vitro models are urgently needed to better predict the potency of these substances in order to reduce dropouts in clinical trials. We hereby provide a novel approach which better reflects the spatial growth of human MM samples in BMSC coculture, and more closely mimics the growth and proliferation of human MM clones in vivo. Disclosures No relevant conflicts of interest to declare.

Oncogenic Role for the Lck/ZAP70/PLCG2 Signaling Pathway in Pre-B-ALL Pathogenesis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 810-810 ◽  
Author(s):  
Jesus Duque-Afonso ◽  
Michael C. Wei ◽  
Chiou-Hong Lin ◽  
Jue Feng ◽  
Corina Buechele ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Bone Marrow Transplantation ◽  
Signaling Pathways ◽  
Signaling Pathway ◽  
Marrow Transplantation ◽  
Target Genes ◽  
Leukemia Cells

Abstract Although the treatment and prognosis of patients with pediatric acute lymphoblastic leukemia (ALL) have improved during the last decades, there is still a clinical need for more effective/selective and less toxic therapies. To address this, we have interrogated various signaling pathways in human ALL cells and mouse strains that express E2A-PBX1, which is present in 5-7% of pediatric ALL. Phospho-flow analysis revealed basal hyper-phosphorylation levels of PLCγ2 in mouse E2A-PBX1 leukemias, consistent with hyper-activation of upstream signaling pathways. Efficient shRNA-mediated depletion of PLCγ2 reduced colony formation of mouse E2A-PBX1+ leukemias in vitro and increased disease-free survival after secondary bone marrow transplantation in vivo. Furthermore, PLCγ2-depleted human ALL cell lines including E2A-PBX1+ cells, showed reduced proliferation. These data suggest a pathogenic role of hyperactivated PLCγ2 in pre-B-ALL. Bioinformatics analysis of E2A-PBX1 target genes in human ALLs revealed an enrichment of B- and T-cell activation pathways, which include the SRC-family kinase LCK and the cytoplasmic kinase ZAP70, upstream of PLCγ2. Comparative analyses of global transcriptional profiles in human primary and mouse leukemias and preleukemias induced by the E2A-PBX1 oncogene identified the signaling kinase ZAP70 as one of the earliest and most consistently up-regulated genes in E2A-PBX1 leukemias. Using a candidate gene approach, we identified LCK with increased expression levels in E2A-PBX1 leukemia cells compared to normal B-cell progenitors. Mouse and human E2A-PBX1 leukemia cells were dependent on the E2A-PBX1 target genes ZAP70 and LCK for proliferation and survival as confirmed by shRNA knock-down experiments. Hence, efficient depletion of these genes resulted in a decrease of phosphorylated PLCγ2, suggesting therapeutic targets in E2A-PBX1 leukemias. Combined suppression of ZAP70 and LCK using double-shRNA experiments showed an additive effect on inhibition of cell proliferation and decrease of phosphorylated PLCγ2. These results provide a rationale for combination therapy to block this hyper-activated signaling pathway at different levels. Several small molecule inhibitors were evaluated for their effects on PLCγ2 upstream pathways in E2A-PBX1 leukemia cells. SRC-family kinase inhibitors including dasatinib were most effective in reducing phosphorylation of PLCγ2 and inhibiting cell proliferation. Furthermore, dasatinib showed promising preclinical efficacy in vitro in colony forming assays and in vivo after secondary bone marrow transplantation of leukemias. In summary, our studies demonstrate that the proliferation and survival of E2A-PBX1 leukemias are dependent on PLCγ2 and upstream signaling pathways, which are suitable for pharmacological inhibition. Disclosures No relevant conflicts of interest to declare.

scholarly journals HIF1α Signaling in the Endogenous Protective Responses after Neonatal Brain Hypoxia-Ischemia

2018 ◽  
Vol 40 (5-6) ◽  
pp. 617-626 ◽  
Author(s):  
Xiao Liang ◽  
Xuemei Liu ◽  
Fuxin Lu ◽  
Yunling Zhang ◽  
Xiangning Jiang ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Target Genes ◽  
Apoptotic Cell ◽  
Glucose Deprivation ◽  
Brain Maturation ◽  
Maturation Stage ◽  
Hypoxia Ischemia

Hypoxia-inducible factor 1α (HIF1α) is a key regulator of oxygen homeostasis, and its target genes mediate adaptive, protective, and pathological processes. The role of HIF1α in neuronal survival is controversial and the brain maturation stage is important in determining its function in brain ischemia or hypoxia-ischemia (HI). In this study, we used neuron-specific HIF1α knockout mice at postnatal day 9 (P9), and immature cortical neurons (days 7–8 in vitro) treated with the HIF1α inhibitor 2-methoxyestradiol (2ME2) or stabilizer dimethyloxalylglycine (DMOG), to examine the function of neuronal HIF1α in neonatal HI in vivo (Vannucci model) and in vitro (oxygen glucose deprivation, OGD). Inhibition of HIF1α with 2ME2 in primary neurons or deletion of neuronal HIF1α in P9 mice increased both necrotic and apoptotic cell death following HI, as evaluated by the protein levels of 145/150-kDa and 120-kDa spectrin breakdown products 24 h after HI. DMOG attenuated neuronal death right after OGD. Acute pharmacological manipulation of HIF1α synchronously regulated the expression of its targets, vascular endothelial growth factor (VEGF) and erythropoietin (Epo), in the same manner. The in vivo findings agree with our previous data using the same HIF1α-deficient mice at an earlier age. This study confirms the role of neuronal HIF1α signaling in the endogenous protective responses following HI in the developing brain.

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