Sublethal Irradiation Research Articles

Gene therapy improves dental manifestations in hypophosphatasia model mice.

Hypophosphatasia is a rare inherited skeletal disorder characterized by defective bone mineralization and deficiency of tissue non-specific alkaline phosphatase (TNSALP) activity. The disease is caused by mutations in the liver/bone/kidney alkaline phosphatase gene (ALPL) encoding TNSALP. Early exfoliation of primary teeth owing to disturbed cementum formation, periodontal ligament weakness and alveolar bone resorption are major complications encountered in oral findings, and discovery of early loss of primary teeth in a dental examination often leads to early diagnosis of hypophosphatasia. Although there are no known fundamental treatments or effective dental approaches to prevent early exfoliation of primary teeth in affected patients, several possible treatments have recently been described, including gene therapy. Gene therapy has also been applied to TNSALP knockout mice (Alpl-/- ), which phenocopy the infantile form of hypophosphatasia, and improved their systemic condition. In the present study, we investigated whether gene therapy improved the dental condition of Alpl-/- mice. Following sublethal irradiation (4 Gy) at the age of 2 d, Alpl-/- mice underwent gene therapy using bone marrow cells transduced with a lentiviral vector expressing a bone-targeted form of TNSALP injected into the jugular vein (n = 3). Wild-type (Alpl+/+ ), heterozygous mice (Alpl+/- ) and Alpl-/- mice were analyzed at 9 d of age (n = 3 of each), while Alpl+/+ mice and treated or untreated Alpl-/- mice were analyzed at 1 mo of age (n = 3 of each), and Alpl+/- mice and Alpl-/- mice with gene therapy were analyzed at 3 mo of age (n = 3 of each). A single mandibular hemi-section obtained at 1 mo of age was analyzed using a small animal computed tomography machine to assess alveolar bone formation. Other mandibular hemi-sections obtained at 9 d, 1 mo and 3 mo of age were subjected to hematoxylin and eosin staining and immunohistochemical analysis of osteopontin, a marker of cementum. Immunohistochemical analysis of osteopontin, a marker of acellular cementum, revealed that Alpl-/- mice displayed impaired formation of cementum and alveolar bone, similar to the human dental phenotype. Cementum formation was clearly present in Alpl-/- mice that underwent gene therapy, but did not recover to the same level as that in wild-type (Alpl+/+ ) mice. Micro-computed tomography examination showed that gene therapy improved alveolar bone mineral density in Alpl-/- mice to a similar level to that in Alpl+/+ mice. Our results suggest that gene therapy can improve the general condition of Alpl-/- mice, and induce significant alveolar bone formation and moderate improvement of cementum formation, which may contribute to inhibition of early spontaneous tooth exfoliation.

MEF2C protects bone marrow B-lymphoid progenitors during stress haematopoiesis.

DNA double strand break (DSB) repair is critical for generation of B-cell receptors, which are pre-requisite for B-cell progenitor survival. However, the transcription factors that promote DSB repair in B cells are not known. Here we show that MEF2C enhances the expression of DNA repair and recombination factors in B-cell progenitors, promoting DSB repair, V(D)J recombination and cell survival. Although Mef2c-deficient mice maintain relatively intact peripheral B-lymphoid cellularity during homeostasis, they exhibit poor B-lymphoid recovery after sub-lethal irradiation and 5-fluorouracil injection. MEF2C binds active regulatory regions with high-chromatin accessibility in DNA repair and V(D)J genes in both mouse B-cell progenitors and human B lymphoblasts. Loss of Mef2c in pre-B cells reduces chromatin accessibility in multiple regulatory regions of the MEF2C-activated genes. MEF2C therefore protects B lymphopoiesis during stress by ensuring proper expression of genes that encode DNA repair and B-cell factors.

Egf Signaling Directs Neoblast Repopulation by Regulating Asymmetric Cell Division in Planarians

A large population of proliferative stem cells (neoblasts) is required for physiological tissue homeostasis and post-injury regeneration in planarians. Recent studies indicate that survival of a few neoblasts after sublethal irradiation results in the clonalexpansion of the surviving stem cells and the eventual restoration of tissue homeostasis and regenerative capacity. However, the precise mechanisms regulating the population dynamics of neoblasts remain largely unknown. Here, we uncovered a central role for epidermal growth factor (EGF) signaling during invivo neoblast expansion mediated by Smed-egfr-3 (egfr-3) and its putative ligand Smed-neuregulin-7 (nrg-7). Furthermore, the EGF receptor-3 protein localizes asymmetrically on thecytoplasmic membrane of neoblasts, and the ratioof asymmetric to symmetric cell divisions decreases significantly in egfr-3(RNAi) worms. Our results not only provide the first molecular evidence of asymmetric stem cell divisions in planarians, but also demonstrate that EGF signaling likely functionsas an essential regulator of neoblast clonal expansion.

PreImplantation factor (PIF) therapy provides comprehensive protection against radiation induced pathologies.

Acute Radiation Syndrome (ARS) may lead to cancer and death and has few effective countermeasures. Efficacy of synthetic PIF treatment was demonstrated in preclinical autoimmune and transplantation models. PIF protected against inflammation and mortality following lethal irradiation in allogeneic bone marrow transplant (BMT) model. Herein, we demonstrate that PIF imparts comprehensive local and systemic protection against lethal and sub-lethal ARS in murine models. PIF treatment 2 h after lethal irradiation led to 100% survival and global hematopoietic recovery at 2 weeks after therapy. At 24 h after irradiation PIF restored hematopoiesis in a semi-allogeneic BMT model. PIF-preconditioning provided improved long-term engraftment. The direct effect of PIF on bone marrow cells was also demonstrated in vitro: PIF promoted pre-B cell differentiation and increased immunoregulatory properties of BM-derived mesenchymal stromal cells. PIF treatment also improved hematopoietic recovery and reduced systemic inflammatory cytokine production after sub-lethal radiation exposure. Here, PIF also prevented colonic crypt and basal membrane damage coupled with reduced nitric oxide synthetase (iNOS) and increased (B7h1) expression. Global upper GI gene pathway analysis revealed PIF's involvement in protein-RNA interactions, mitochondrial oxidative pathways, and responses to cellular stress. Some effects may be attributed to PIF's influence on macrophage differentiation and function. PIF demonstrated a regulatory effect on irradiated macrophages and on classically activated M1 macrophages, reducing inflammatory gene expression (iNOS, Cox2), promoting protective (Arg1) gene expression and inducing pro-tolerance cytokine secretion. Notably, synthetic PIF is stable for long-term field use. Overall, clinical investigation of PIF for comprehensive ARS protection is warranted.

Abstract 3499: Triptolide pro-drug decreases tumor burden and halts tumor progression in a murine model of acute myeloid leukemia

Abstract Introduction Standard treatment for acute myelogenous leukemia (AML) has not changed over the past few decades and relies primarily on the traditional “7 + 3″ regimen of daunorubicin, administered over 3 days and Cytarabine, administered over 7 days. This chemo-intensive regimen is poorly tolerated by patients and is characterized by a high rate of relapse and treatment failure. Triptolide is a diterpenoid tri-epoxide compound isolated from the Chinese herb Tripterygium wilfordii. The water-soluble pro-drug of triptolide, Minnelide has been shown by our group to be highly effective in a number of pre-clinical models of solid tumors and is currently undergoing Phase I Clinical trial for gastrointestinal tumors. Interim analysis of the Phase I data has shown that the equivalent mouse dose of 0.2 mg/kg/day is well tolerated by patients with no reported dose limiting toxicity. Methods Primary AML apheresis samples from patients with acute myeloid leukemia and multiple AML cell Lines (THP1, KG1, Kasumi1, HL-60) were treated with Triptolide at doses from of 2.5 nM to 50nM and cell viability was measured using a formazan based colorimetric assay. Apoptosis was measured using Annexin V via flowcytometry and colony forming ability of AML cell lines was measured using a methylcellulose based assay. To generate an engraftment model of AML, NOD.Rag1-/-;γcnull (NRG) animals expressing human interleukin-3 (IL-3) and human GM-CSF (NRGS) were injected luciferase expressing THP1 cells after sublethal irradiation of 250 cGy. These animals were then serially evaluated using In Vivo Imaging System (IVIS) and leukemic burden was calculated the total bioluminescent signal after intra-periotneal injection of luciferin. Treatment with vehicle or Minnelide at a dose of 0.1mg/kg/day and 0.15 mg/kg/day was started on the 10th day after confirming engraftment using IVIS. Results Triptolide at a dose range of 2.5 nM to 50 nM produced a dose and time dependent killing of leukemic cells in both cell lines and primary AML patient samples. The IC-50 for THP1, KG1, Kasumi1 and HL-60 cell lines with triptolide treatment at 48 hours were 5 ± 0.8 nM, 8 ± 0.7 nM, 7.2 ± 0.6 nM, 10 nM ± 2 nM respectively. Treatment with triptolide at a dose of 2.5 nM induced cell death and apoptosis as measured by Annexin V posiitvity in primary AML apheresis samples. Colony formation by THP1 cells and KG1 cells was completely abrogated by treatment with Triptolide at 2.5 nM and 25 nM respectively. Vehicle treated mice showed a rapid progression of disease burden when compared to Minnelide treated mice. At a dose of 0.1 mg/kg/day and 0.15 mg/kg/day, these mice had no appreciable increase in leukemic burden over normal mice when assessed by IVIS. Conclusion We show that Minnelide induces cell death at therapeutically relevant concentrations in vitro and decreased leukemic burden in a murine model of AML. Minnelide may emerge as a novel therapeutic strategy in treating patients with AML. Citation Format: Bhuwan Giri, Sulagna Banerjee, John George, Shrey Modi, Vineet Kumar Gupta, Mahendra K. Singh, Vikas Dudeja, Ashok K. Saluja. Triptolide pro-drug decreases tumor burden and halts tumor progression in a murine model of acute myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3499.

Generation of Pediatric Leukemia Xenograft Models in NSG-B2m Mice: Comparison with NOD/SCID Mice

Generation of orthotopic xenograft mouse models of leukemia is important to understand the mechanisms of leukemogenesis, cancer progression, its cross talk with the bone marrow microenvironment, and for preclinical evaluation of drugs. In these models, following intravenous injection, leukemic cells home to the bone marrow and proliferate there before infiltrating other organs, such as spleen, liver, and the central nervous system. Moreover, such models have been shown to accurately recapitulate the human disease and correlate with patient response to therapy and prognosis. Thus, various immune-deficient mice strains have been used with or without recipient preconditioning to increase engraftment efficiency. Mice homozygous for the severe combined immune deficiency (SCID) mutation and with non-obese diabetic background (NOD/SCID) have been used in the majority of leukemia xenograft studies. Later, NOD/SCID mice deficient for interleukin 2 receptor gamma chain (IL2Rγ) gene called NSG mice became the model of choice for leukemia xenografts. However, engraftment of leukemia cells without irradiation preconditioning still remained a challenge. In this study, we used NSG mice with null alleles for major histocompatibility complex class I beta2-microglobulin (β2m) called NSG-B2m. This is a first report describing the 100% engraftment efficiency of pediatric leukemia cell lines and primary samples in NSG-B2m mice in the absence of host preconditioning by sublethal irradiation. We also show direct comparison of the engraftment efficiency and growth rate of pediatric acute leukemia cells in NSG-B2m and NOD/SCID mice, which showed 80–90% engraftment efficiency. Secondary and tertiary xenografts in NSG-B2m mice generated by injection of cells isolated from the spleens of leukemia-bearing mice also behaved similar to the primary patient sample. We have successfully engrafted 25 acute lymphoblastic leukemia (ALL) and 5 acute myeloid leukemia (AML) patient samples with distinct cytogenetic characteristics in NSG-B2m mice, with the purpose of generating pediatric ALL and AML xenografts for preclinical evaluation of drugs. Thus, our data support the use of NSG-B2m mouse model for leukemia engraftment and in vivo preclinical drug efficacy studies.

Effects of γ-ray total body irradiation from a 60Co source on expression level of intrathymic forkhead box protein N1 in mice

Effects of γ-ray total body irradiation from a 60Co source on expression level of intrathymic forkhead box protein N1 in mice

Effects of Designer Hyper-Interleukin 11 (H11) on Hematopoiesis in Myelosuppressed Mice.

The incidence of cancer is constantly increasing. Chemo/radiotherapy is one of major methods of treating cancer. Although adverse chemo/radiotherapy events, such as anemia and neutropenia, can be successfully cured, thrombocytopenia is still problematic. We constructed the Hyper-IL11 (H11) cytokine by linking soluble interleukin 11 receptor alpha (sIL-11Ralpha) with IL-11. In vivo H11 activity was examined in myelosuppressed mice. Myelosuppression was induced by either i) sublethal irradiation and carboplatin administration or ii) sublethal irradiation. A dose of 100 μg/kg of H11 or IL-11 was administered subcutaneously for 7 days. IL-11 and H11 accelerated leukocyte, hematocrit and platelet recovery. The effect on the attenuation of thrombocytopenia was significant. Moreover, both cytokines increased the cellularity and numbers of megakaryocyte, erythroid, and granulocyte/macrophage progenitors in the bone morrow and spleen compared with the control. Although H11 was administered at a molar concentration that was three times lower, its effects were comparable with or better than those of IL-11; thus, the activity of H11 was superior to that of IL-11. Because no toxicity was observed after the intravenous administration of H11, this hyper-cytokine may be potentially useful for treatment of thrombocytopenia and other IL-11-dependent disorders.

Spleen hypoplasia leads to abnormal stress hematopoiesis in mice with loss of Pbx homeoproteins in splenic mesenchyme.

The spleen plays critical roles in immunity and also provides a permissive microenvironment for hematopoiesis. Previous studies have reported that the TALE-class homeodomain transcription factor Pbx1 is essential in hematopoietic stem and progenitor cells (HSPCs) for stem cell maintenance and progenitor expansion. However, the role of Pbx1 in the hematopoietic niche has not been investigated. Here we explored the effects that genetic perturbation of the splenic mesenchymal niche has on hematopoiesis upon loss of members of the Pbx family of homeoproteins. Splenic mesenchyme-specific inactivation of Pbx1 (SKO) on a Pbx2- or Pbx3-deficient genetic background (DKO) resulted in abnormal development of the spleen, which is dysmorphic and severely hypoplastic. This phenotype, in turn, affected the number of HSPCs in the fetal and adult spleen at steady state, as well as markedly impairing the kinetics of hematopoietic regeneration in adult mice after sub-lethal and lethal myelosuppressive irradiation. Spleens of mice with compound Pyx deficiency 8days following sublethal irradiation displayed significant downregulation of multiple cytokine-encoding genes, including KitL/SCF, Cxcl12/SDF-1, IL-3, IL-4, GM-CSF/Csf2 IL-10, and Igf-1, compared with controls. KitL/SCF and Cxcl12/SDF-1 were recently shown to play key roles in the splenic niche in response to various haematopoietic stresses such as myeloablation, blood loss, or pregnancy. Our results demonstrate that, in addition to their intrinsic roles in HSPCs, non-cell autonomous functions of Pbx factors within the splenic niche contribute to the regulation of hematopoiesis, at least in part via the control of KitL/SCF and Cxcl12/SDF-1. Furthermore, our study establishes that abnormal spleen development and hypoplasia have deleterious effects on the efficiency of hematopoietic recovery after bone marrow injury.

Results of Experimental Studies of the Immunotropic Action of Polyprenols Extracted from Alcea Nudiflora

Methods of extracting polyprenols from Alcea nudiflora and their immunotropic actions were studied. Administration of Alcea nudiflora polyprenols to mice simultaneously with immunization with sheep erythrocytes produced marked stimulation of primary antibody formation, apparent as an increase in the number of antibody-forming cells in the spleen. In addition, there were increases in the cellularity of the central and peripheral organs of immunity, along with increases in blood erythrocyte and leukocyte levels. The immunomodulatory action of Alcea nudiflora polyprenols was seen not only in normal animals, but also in animals with secondary immunodeficiency states developing in acute toxic hepatitis and sublethal irradiation. In terms of activity in these experiments, Alcea nudiflora polyprenols were as active as the known immunostimulatory substance Immunal.

Role of ADAM17 in invasion and migration of CD133-expressing liver cancer stem cells after irradiation.

We investigated the biological role of CD133-expressing liver cancer stem cells (CSCs) enriched after irradiation of Huh7 cells in cell invasion and migration. We also explored whether a disintegrin and metalloproteinase-17 (ADAM17) influences the metastatic potential of CSC-enriched hepatocellular carcinoma (HCC) cells after irradiation. A CD133-expressing Huh7 cell subpopulation showed greater resistance to sublethal irradiation and specifically enhanced cell invasion and migration capabilities. We also demonstrated that the radiation-induced MMP-2 and MMP-9 enzyme activities as well as the secretion of vascular endothelial growth factor were increased more predominantly in Huh7CD133+ cell subpopulations than Huh7CD133− cell subpopulations. Furthermore, we showed that silencing ADAM17 significantly inhibited the migration and invasiveness of enriched Huh7CD133+ cells after irradiation; moreover, Notch signaling was significantly reduced in irradiated CD133-expressing liver CSCs following stable knockdown of the ADAM17 gene. In conclusion, our findings indicate that CD133-expressing liver CSCs have considerable metastatic capabilities after irradiation of HCC cells, and their metastatic capabilities might be maintained by ADAM17. Therefore, suppression of ADAM17 shows promise for improving the efficiency of current radiotherapies and reducing the metastatic potential of liver CSCs during HCC treatment.

The immunogenicity of platelet-derived FVIII in hemophilia A mice with or without preexisting anti-FVIII immunity

AbstractEvidence shows that factor VIII (FVIII) ectopically expressed in platelets (2bF8) is therapeutic in FVIIInull mice even with anti-FVIII inhibitory antibodies (inhibitors). If current efforts to generate platelets in vitro succeed, genetically manipulated platelets containing FVIII may be used therapeutically in hemophilia A patients with inhibitors. One important concern is the immunogenicity of platelet-derived FVIII. To address this concern, we infused 2bF8 transgenic (2bF8Tg) platelets into naïve FVIIInull mice weekly for 8 weeks. No anti-FVIII antibodies were detected in the infused animals during the study course. We then explored whether platelet-derived FVIII is immunogenic in FVIIInull mice with inhibitors. The 2bF8Tg platelets were transfused into rhF8-primed FVIIInull mice, resulting in no augmentation of anti-FVIII antibodies. To investigate whether preconditioning affects the immune response, animals were sublethally irradiated and subsequently transfused with 2bF8Tg platelets. No anti-FVIII antibodies were detected in the recipients after platelet infusions. Following further challenge with rhF8, the inhibitor titer in this group was significantly lower than in naïve FVIIInull mice utilizing the same immunization protocol. Thus, our data demonstrate that infusion of platelets containing FVIII triggers neither primary nor memory anti-FVIII immune response in FVIIInull mice and that sublethal irradiation plus 2bF8Tg platelet infusion suppresses anti-FVIII immune response in FVIIInull mice.

Sox2 cooperates with Lkb1 loss to promote squamous cell lung cancer

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Production of BMP4 By Endothelial Cells Is Crucial for Endogenous Thymic Regeneration

Endogenous thymic regeneration is a crucial function that allows for renewal of immune competence following immunodepletion caused by cytoreductive chemotherapy or radiation; however, the mechanisms governing this regeneration remain poorly understood. Moreover, despite this capacity, prolonged T cell deficiency is a major clinical hurdle in recipients of hematopoietic stem cell transplantation (HSCT) and can precipitate high morbidity and mortality from opportunistic infections, and may even facilitate malignant relapse. We have recently described a central role for group 3 innate lymphoid cells (ILC3) in a complex cellular and molecular network that drives endogenous thymic regeneration (Dudakov 2012 Science 336:91). Although IL-22 contributes considerably towards thymic regeneration and mice deficient for IL-22 lag behind WT controls in recovery of thymic function, there is still some tissue regeneration in these mice, suggesting that other regeneration pathways also play a role. Unlike other lymphoid cells, ILC3 were extremely radio-resistant with little if any depletion of cells after even lethal doses of total body irradiation (TBI). However, comprehensive analysis of all thymus-resident cell subsets revealed that ILCs were not the only damage-resistant population in the thymus as endothelial cells (ECs) were also remarkably resistant to damage in multiple clinically relevant models of acute tissue injury including corticosteroids, chemotherapy and sublethal total body irradiation (SL-TBI, 550cGy) (Fig. 1a).Thymopoiesis is dependent on the close interaction between developing thymocytes and the non-hematopoietic stromal microenvironment, which includes highly specialized thymic epithelial cells (TECs) and ECs. While the role of TECs has been well studied, the contribution of ECs to thymopoiesis and thymic regeneration remains largely unclear. Here we demonstrate that rather than just being passive conduits that deliver oxygen and nutrients, ECs are active participants in organ function producing distinct paracrine factors that orchestrate thymic repair. Using a technique whereby ECs are transduced with the adenoviral gene E4ORF1 - ECs could be expanded ex vivo (exEC) and, when administered to mice after SL-TBI, significantly boost recovery of thymic function (Fig. 1b). Intriguingly, this trophic effect was only observed when exEC were derived from the thymus but not when they were derived from heart or kidney (Fig. 1b). Mechanistically, in vivo administration of exEC(Thy) induced the expression by TECs of Foxn1 (Fig. 1c), a key transcription factor required for thymus organogenesis, maintenance and regeneration. In vitro co-culture assays revealed that conditioned media (CM) from exEC derived from the thymus, but not the heart or the kidney, could induce Foxn1 expression by TECs (Fig. 1d), in addition to the FOXN1 target genes Kitl and Dll4; a Notch ligand itself critical for T cell development. These findings suggest that thymus-derived exEC produce a soluble factor that contributes toward thymic regeneration via activation of Foxn1. Transcriptome analysis of highly purified thymic ECs after SL-TBI identified that, among other things, expression of Bmp4 was significantly increased, offering a potential mechanism by which thymic ECs mediate their regeneration. Consistent with this hypothesis, not only could recombinant BMP4 promote the expression of Foxn1 by TECs in vitro, induction of Foxn1 by CM from exEC(Thy) was abrogated by Noggin, an inhibitor of BMP4 signaling (Fig. 1e). Moreover, exEC(Thy) produced significantly more BMP4 compared to exEC derived from the heart or kidney; and silencing Bmp4 expression by shRNA within exEC(Thy) limited their capacity to mediate exogenous thymic regeneration and failed to induce expression of Foxn1 and Dll4. Finally, strengthening its role in endogenous regeneration, administration of a pharmacologic BMP inhibitor inhibited thymic regeneration after SL-TBI; and inducible deletion of Bmp4 specifically in ECs reduced thymic regeneration after SL-TBI (Fig. 1f).These studies not only detail a novel pathway promoting endogenous thymic regeneration, but also offer an innovative clinical approach to enhance T cell immunity in recipients of allo-HSCT and for individuals with T cell deficiencies due to aging, infectious disease, and common cancer treatments such as chemo- and radiation-therapy. [Display omitted] DisclosuresRafii:Angiocrine Bioscience: Other: Founder of Angiocrine Biosceince, which is developing the technology behind endothelial cell propagation. van den Brink:Boehringer Ingelheim: Consultancy, Other: Advisory board attendee; Regeneron: Honoraria; Merck: Honoraria; Tobira Therapeutics: Other: Advisory board attendee; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Proteinase 3 Is Expressed in Stem Cells and Regulates Bone Marrow Hematopoiesis

Previously published data from our lab identified proteinase 3 (PR3) as a key protein in regulating spontaneous death of neutrophils. Our lab showed that PR3 could cleave caspase 3 in a caspase 9-independent manner in ageing neutrophils (Loison et al. JCI. 2014). Meanwhile, the maintenance of stem cell pool in the bone marrow and the development of mature blood cells are tightly regulated processes. In an effort to understand the hematopoietic consequences of absence of PR3, we analyzed stem and progenitor cell compartments in wild-type (WT) and PR3 knock-out (PR3-/-) mice.Of note, PR3 is thought to be a neutrophil-specific serine protease. Surprisingly, we observed PR3 expression in sorted Lin- Sca-1+ c-Kit+ (LSK) cells by gene expression analysis at mRNA level as well as Western blotting at protein level. Conventional flow cytometry methods also confirmed this expression.While bone marrow cellularity was similar between WT and PR3-/- mice, conventional flow cytometry studies showed that bone marrow from PR3-/- mice contained a higher frequency of LSK and Lin- Sca-1- c-Kit+ (LK) cells. Colony forming cell assays also suggested that both bone marrow cells and splenocytes from PR3-/- mice contained a higher number of progenitor cells compared to their WT counterparts. Histological analysis of bone marrow smears revealed that bone marrow from PR3-/- mice had a higher number of immature myeloid cells and a reduced number of mature leukoyctes and lymphocytes.Next, we asked if the enhanced stem and progenitor cell compartments in PR3-/- mice were functional in vivo. Toward this aim, we performed competitive reconstitution experiments using total bone marrow cells as well as sublethal irradiation experiments. PR3-/- donor-derived cells gave rise to a higher number of cells in the recipient mice compared to WT donor-derived cells in competitive reconstitution experiments. Also, after WT and PR3-/- mice were irradiated with 4 Gy, peripheral blood cell counts recovered faster in PR3-/- mice. Similarly, when mice were irradiated with 6 Gy, PR3-/- mice had a higher survival rate. These data suggest that enhanced stem and progenitor cell compartments in PR3-/- mice are functional. To understand if the enhanced hematopoietic activity in PR3-/- mice is an intrinsic feature of stem cells, another set of competitive reconstitution experiments were performed using equal numbers of sorted LSK cells. Again, PR3-/- donor-derived cells were more dominant than WT donor-derived cells in the recipient mice.Then, we wanted to understand the underlying mechanism. The rate of proliferation and senescence in LSK cells were analyzed by BrdU incorporation studies and C12FDG staining, respectively. No difference in proliferation or senescence was observed between WT and PR3-/- mice. We also looked at the apoptosis rate of LSK cells in vivo and in vitro. Untreated LSK cells from WT and PR3-/- mice exhibited similar levels of viability both in vivo and in vitro. However, when bone marrow macrophages responsible for clearance of apoptotic cells were depleted using clodronate liposomes, PR3-/- LSK cells exhibit enhanced viability compared to WT LSK cells as evidenced by annexin V and 7-AAD staining. Using a fluorogenic substrate of caspase 3, we also observed that the enhanced viability seen in PR3-/- LSK cells after depletion of macrophages is due to a reduction in caspase 3 activation.Additionally, we asked whether LSK cells from PR3-/- mice represent features associated with stem cells from aged mice such as an increase in DNA damage accumulation and reduced polarity. Immunofluorescent microscopy studies suggested that long-term hematopoietic stem cells from PR3-/- mice exhibit higher levels of p-gH2AX foci, a marker of DNA damage, and a reduced polarized distribution of a-tubulin. Finally, we saw a reduced lifespan in PR3-/- mice. These data indicate that PR3 regulates bone marrow hematopoiesis by contributing to the maintenance of a healthy stem cell pool in the bone marrow. DisclosuresNo relevant conflicts of interest to declare.

Prostaglandin E2 Promotes the Sequential Recovery of Bone Marrow Vasculature and the Megakaryocyte Lineage Following Radiation Injury

The sinusoidal vascular niche is the site of terminal megakaryopoiesis and platelet production in the bone marrow. The association of megakaryocytes (MKs) with the sinusoidal endothelium and microenvironmental cues from the vascular niche are critical for efficient thrombopoiesis. Thrombocytopenia is an important clinical problem that can cause significant morbidity for patients receiving genotoxic treatments such as chemotherapy and radiation therapy. Following myeloablative bone marrow injury, the recovery of both hematopoietic stem and progenitor cells (HSPCs) and the MK lineage is dependent on marrow vascular recovery (Hooper et al. Cell Stem Cell 2009, Avecilla et al. Nature 2004). Accordingly, we reasoned that improving recovery of the vasculature might serve as a rational therapeutic strategy for thrombocytopenia stemming from cytotoxic treatments. We focused our efforts on the arachidonic acid derivative prostaglandin E2 (PGE2), which regulates numerous physiologic processes including microvascular endothelial cell proliferation and angiogenesis. Within the context of the bone marrow, PGE2 also has diverse roles in HSPC expansion, survival, and trafficking. Importantly, systemic administration of the long-acting analogue 16,16-dimethyl PGE2 (dmPGE2) improves hematopoietic recovery and enhances overall survival following total body irradiation (TBI) in mice (Hoggatt et al. Blood Cells 2013). Intriguingly, following sublethal TBI, dmPGE2-induced recovery of peripheral platelets precedes restoration of red blood cells and neutrophils (Porter et al. Stem Cells 2013). We hypothesized that the preferential recovery of platelets following dmPGE2 administration is mediated by accelerated vascular recovery in the marrow. We tested this in a murine model of sublethal radiation injury in which we had previously characterized MK lineage kinetics following 4Gy TBI (Niswander et al. Blood 2014). First, the kinetics of MK progenitors (MKPs), maturing MKs, and platelets were determined at days 5, 7, and 10 in mice receiving 6mg/kg dmPGE2 or vehicle at 48 and 72 hours post-4Gy TBI. Mice receiving dmPGE2 had an early decrease in both MKPs and platelets, with no change in MKs at day 5 post-TBI. However, by day 10, dmPGE2-treated mice demonstrated significantly enhanced recovery of both MKs and circulating platelets compared to controls, but no difference in the number of immunophenotypic HSPCs. Having confirmed enhanced recovery of the MK lineage with dmPGE2 treatment, we next sought to characterize vascular recovery. Marked injury and dilation of the marrow sinusoidal vasculature occurs rapidly following TBI. Immunohistochemistry with morphometric analysis revealed no change in vascular dilation at day 5 post-TBI with dmPGE2 treatment. However, mice receiving dmPGE2 exhibited a more than 25% improvement in vascular dilation that was first evident at day 7 and was sustained at day 10 post-TBI. While vascular dilation post-TBI has been posited to correlate with loss of surrounding hematopoietic cells, improvement in vascular dilation with dmPGE2 treatment is contrastingly evident in the setting of slightly fewer (day 7) or equivalent (day 10) numbers of nucleated marrow cells compared to irradiated control mice. These data suggest that dmPGE2-mediated recovery of the vasculature coincides temporally with the accelerated onset of MK lineage recovery. In addition, immunohistochemical morphometric analyses indicate that dmPGE2-treated mice had an 80% increase in maturing MKs in physical contact with the vascular endothelium, the site of platelet production, at day 10 post-TBI. Taken together, these data support the concept that dmPGE2-mediated marrow vascular recovery following radiation injury enhances the recovery of the MK lineage and accelerates the production of circulating platelets. Modulation of vascular recovery with dmPGE2 may be a promising therapeutic strategy for thrombocytopenia secondary to myeloablative bone marrow injury. DisclosuresCalvi:Fate Therapeutics: Patents & Royalties.

Abstract B199: Novel anti-angiogenic cancer therapeutic strategy by targeting differentiated macrophage lineage cells through N-myc downstream regulated gene 1 (NDRG1)

Abstract [Background] Angiogenesis is essential for malignant progression of cancer. Over the last couple of decades, it was highlighted that the progenitor cells derived from bone marrow (BM) differentiated, became part of the tumor stroma, and involved in tumor angiogenesis. In particular, macrophages, which differentiated into tumor-associated macrophages in tumor microenvironment, are known to play crucial roles in tumor angiogenesis. However, the specific molecules and markers of these “angiogenesis-supporting macrophages” have not been fully defined (Qian and Pollard, Cell. 2010). N-myc downstream regulated gene 1 (NDRG1), a gene responsible for a hereditary motor and sensory neuropathy (Lon-Charcot-Marie disease), plays pleiotropic roles in cell proliferation, development, differentiation, and tumorigenesis. We have previously reported that NDRG1 expression levels in cancer cells were closely correlated with tumor angiogenesis (Maruyama et al., Cancer Res. 2006; Hosoi et al., Cancer Res. 2009; Ureshino et al., PLoS One. 2012; Murakami et al., J Biol Chem. 2013). However, how NDRG1 could affect tumor angiogenesis remains unclear. In this study, we asked how NDRG1 could specifically modulate tumor angiogenesis through its differentiation control of macrophage lineage cells by using NDRG1 knock out (KO) mice. [Material and methods] NDRG1 KO mice: The NDRG1 KO mice on C57BL6 background have been kindly donated by Dr. Toshiyuki Miyata (National Cerebral and Cardiovascular Center) (Okuda et al., Mol Cell Biol., 2004). Preparation of BM derived macrophages (BMDMs): BMDMs were obtained by flushing mouse femurs from mice. Cells were seeded in dishes and washed twice 3 hours later. Adherent cells were incubated in DMEM supplemented with 10% FBS and 20ng/ml M-CSF for 7 days. Matrigel plug assay in BM suppression mice: wild type (WT) recipient mice were exposed to 3-Gy sublethal whole-body irradiation to suppress BM and temporarily deplete leukocytes. Seven days after the irradiation, the mice were injected subcutaneously with growth factor-reduced Matrigel containing B16/BL6 cells, with or without BMDMs from WT or NDRG1 KO mice. [Results] In NDRG1 KO mice as compared to their WT counterparts, [1] serum levels of M-CSF and macrophage-related cytokines were all decreased, and BM cells showed much slower growth rates in response to M-CSF in vitro; [2] trabecular bone mass and number of osteoclasts were decreased in vivo; [3] tumor growth and angiogenesis were markedly suppressed, accompanying by decreased infiltration of tumor-associated macrophages; [4] the transfer of BMDMs from KO mice into BM-eradicated WT mice induced much less tumor angiogenesis than observed in WT mice; [5] VEGF-A expression in BMDMs from KO mice were much less than those in cells from WT mice when stimulated by LPS or IL-1β. [Conclusion] We discovered the central role of NDRG1 in differentiation of macrophage lineage cells, which affected bone remodeling and tumor angiogenesis. We will discuss which molecules are specifically regulated by NDRG1 during differentiation processes of monocytes/macrophages into angiogenesis-supporting macrophages, and whether these molecules in macrophages could have potentially important target for anti-angiogenesis cancer therapeutics. Citation Format: Kosuke Watari, Tomohiro Shibata, Hiroshi Nabeshima, Ai Shinoda, Yuichi Fukunaga, Akihiko Kawahara, Kazuyuki Karasuyama, Jun-ichi Fukushi, Yuichi Murakami, Michihiko Kuwano, Mayumi Ono. Novel anti-angiogenic cancer therapeutic strategy by targeting differentiated macrophage lineage cells through N-myc downstream regulated gene 1 (NDRG1). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B199.

Citrulline as a Biomarker in the Murine Total-Body Irradiation Model: Correlation of Circulating and Tissue Citrulline to Small Intestine Epithelial Histopathology.

The use of plasma citrulline as a biomarker for gastrointestinal acute radiation syndrome via exposure to total-body irradiation in a murine model was investigated. The radiation exposure covered lethal, mid-lethal, and sub-lethal gastrointestinal acute radiation syndrome. Plasma citrulline profiles were generated over the first 6 d following total-body irradiation exposure of 6-15 Gy. In addition, plasma citrulline was comprehensively evaluated in the context of matching small intestine citrulline and histopathology. Higher plasma citrulline was significantly associated with lower irradiation doses over the first 6 d following the irradiation insult. Furthermore, higher plasma citrulline was significantly associated with higher crypt survival. The correlation of the plasma citrulline to crypt survival was more robust for higher irradiation doses and for later time points. The data suggested plasma citrulline was most informative for reflecting gastrointestinal injury resulting from exposure to 9-15 Gy total-body irradiation covering time-points 2-5 d post the irradiation insult.

Autocrine protective mechanisms of human granulocyte colony-stimulating factor (G-CSF) on retinal ganglion cells after optic nerve crush

This study investigated the role of autocrine mechanisms in the anti-apoptotic effects of human granulocyte colony-stimulating factor (G-CSF) on retinal ganglion cells (RGCs) after optic nerve (ON) crush. We observed that both G-CSF and G-CSF receptor (G-CSFR) are expressed in normal rat retina. Further dual immunofluorescence staining showed G-CSFR immunoreactive cells were colocalized with RGCs, Müller cells, horizontal and amacrine cells. These results confirm that G-CSF is an endogenous ligand in the retina. The semi-quantitative RT-PCR finding demonstrated the transcription levels of G-CSF and G-CSFR were up-regulated after ON crush injury. G-CSF treatment further increased and prolonged the expression level of G-CSFR in the retina. G-CSF has been shown to enhance transdifferentiation of the mobilized hematopoietic stem cells into tissue to repair central nervous system injury. We test the hypothesis that the hematopoietic stem cells recruited by G-CSF treatment can transdifferentiate into RGCs after ON crush by performing sublethal irradiation of the rats 5 days before ON crush. The flow cytometric analysis showed the number of CD34 positive cells in the peripheral blood is significantly lower in the irradiated, crushed and G-CSF-treated group than the sham control group or crush and G-CSF treated group. Nevertheless, the G-CSF treatment enhances the RGC survival after sublethal irradiation and ON crush injury. These data indicate that G-CSF seems unlikely to induce hematopoietic stem cell transdifferentiation into RGCs after ON crush injury. In conclusion, G-CSF may serve an endogenous protective signaling in the retina through direct activation of intrinsic G-CSF receptors and downstream signaling pathways to rescue RGCs after ON crush injury, exogenous G-CSF administration can enhance the anti-apoptotic effects on RGCs.

Recombinant human thrombopoietin promotes hematopoietic reconstruction after severe whole body irradiation.

Recombinant human thrombopoietin (rHuTPO) is a drug that is used clinically to promote megakaryocyte and platelet generation. Here, we report the mitigative effect of rHuTPO (administered after exposure) against severe whole body irradiation in mice. Injection of rHuTPO for 14 consecutive days following exposure significantly improved the survival rate of lethally irradiated mice. RHuTPO treatment notably increased bone marrow cell density and LSK cell numbers in the mice after sub-lethal irradiation primarily by promoting residual HSC proliferation. In lethally irradiated mice with hematopoietic cell transplantation, rHuTPO treatment increased the survival rate and enhanced hematopoietic cell engraftment compared with the placebo treatment. Our observations indicate that recombinant human TPO might have a therapeutic role in promoting hematopoietic reconstitution and HSC engraftment.