Abstract 454: Myeloid CD98hc Deficiency Reduces Atherosclerotic Plaque Development via Impaired Proliferation of Macrophages

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Sara McCurdy ◽  
William A Boisvert
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Atherosclerotic Plaque ◽  
Bone Marrow Cells ◽  
Transmembrane Protein ◽  
Mouse Bone Marrow ◽  
Plaque Development ◽  
Primary Mouse

Macrophage accumulation is a key process affecting all stages of atherosclerosis. Whether these cells accumulate in plaque solely by recruitment of monocytes from circulation or by proliferation within the plaque is an important question that has garnered much interest in recent years. Originally identified as a lymphocyte activation marker, CD98hc (SLC3A2) is a transmembrane protein involved in cell proliferation and survival via integrin signaling and MAP kinase activation. We hypothesized that CD98hc deficiency in myeloid cells would have a protective effect on atherosclerosis development and plaque composition by limiting macrophage proliferation. For the studies described, we utilized mice with myeloid-specific deletion of the CD98hc ( CD98hc fl/fl LysMCre + ) to determine the effects of CD98hc deficiency on macrophage function in the context of atherosclerosis . We performed in vitro assays to investigate the role of CD98hc in the proliferation and survival of primary mouse bone marrow derived macrophages. Although we found no differences in the number of bone marrow cells isolated from control or CD98hc -/- animals, after differentiation with MCS-F for 7 days, the number of macrophages obtained from CD98hc -/- mice was approximately 80% lower (7.2 ± 2.2 x 10 6 vs. 42.4 ± 4.6 x 10 6 per mouse) compared to control mice. Proliferation assays in vitro using EdU revealed approximately 50% (15.4 ± 2.5% vs. 7.5±1.8%) reduced cell proliferation in CD98hc -/- macrophages compared to control cells that could not be rescued with the addition M-CSF. In a 6-week atherosclerosis study using Ldlr -/- CD98hc fl/fl LysMCre + mice, Oil-Red O staining of whole aortae as well as aortic sinus sections showed that atherosclerotic plaque development was reduced compared to Ldlr -/- CD98hc fl/fl LysMCre - control mice. Additionally, immunohistochemical staining of atherosclerotic tissues revealed a reduction in macrophage abundance and proliferation within the plaque of Ldlr -/- CD98hc fl/fl LysMCre + mice compared to control mice. These findings support an important role of CD98hc in macrophage proliferation within the plaque environment, and provide a novel target for reducing atherosclerosis.

Overlapping but Distinct Role of p21WAF1/CDKN1 and Survivin in Hematopoietic Progenitor Cell Proliferation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1334-1334
Author(s):  
Seiji Fukuda ◽  
Mariko Abe ◽  
Seiji Yamaguchi ◽  
Louis M. Pelus
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Growth Factor ◽  
Bone Marrow Cells ◽  
Mouse Bone Marrow ◽  
Hematopoietic Progenitor ◽  
Primary Mouse ◽  
Marrow Cells

Abstract Survivin is a member of the inhibitor of apoptosis protein family that has been implicated in cell cycle control, anti-apoptosis and cell division. Our previous studies and others have shown that Survivin and the cyclin dependent kinase inhibitor p21WAF1/CDKN1 (p21) are functionally associated and are involved in cell cycle, anti-apoptosis and cytokinesis in cancer cells and in normal hematopoietic progenitor cells (HPC). P21 is highly expressed in quiescent hematopoietic stem cells (HSC) in steady state, but the proportion of quiescent HSCs in G0 phase is reduced in p21−/− mice. In contrast, p21 has been shown as positive regulator on cell cycle of normal HPC since p21 deficiency results in fewer total CFU in mouse bone marrow (BM) cells with fewer CFU in S-phase and retrovirus transduction of p21 in p21 deficient bone marrow cells restores total and cycling CFU. We have previously reported that Survivin increases the proliferation of mouse primary HPC and that this enhancing effect is on HPC proliferation is absent when p21 is functionally deleted, suggesting that p21 is required for Survivin to enhance HPC proliferation. In addition, ITD-Flt3 mutations that are normally expressed in patients with acute myeloid leukemia and associate poor prognosis increase expression of both Survivin and p21, implicating their involvement in aberrant proliferation of HPC expressing ITD-Flt3. Herein we have characterized the functional association between p21 and Survivin in normal and transformed cell proliferation. Antagonizing wild-type Survivin in mouse BaF3 cells by retrovirus transduction of a T34A dominant negative mutant Survivin or anti-sense increased p21 expression, even though Survivin requires p21 to enhance HPC proliferation. Ectopic p21 in Survivin+/+ primary mouse bone marrow cells increased the number of immunophenotypically defined c-kit+, lin− (KL) cells, which is consistent with a positive role of p21 in HPC proliferation, however; ectopic expression of p21 failed to increase HPC proliferation in Survivin deficient primary bone marrow cells, suggesting that p21 alone is not sufficient to substitute for Survivin’s enhancing function on normal HPC proliferation. Over-expression of ITD-Flt3 enhanced growth factor independent proliferation of primary mouse marrow c-kit+, Sca-1+, lin− (KSL) cell number; however, co-expression of p21 with ITD-Flt3 dramatically decreased the number of growth factor independent KSL cells (80±6% reduction: P<0.01). Furthermore, the inhibitory effect of p21 on KLS proliferation was further enhanced by Survivin knockout bone marrow cells (64±5% reduction compared with presence of Survivin: P<0.05). These findings indicate that Survivin and p21 have a overlapping but distinct roles in regulating normal HPC proliferation and that manipulating p21 and Survivin may represent a potential therapeutic target for acute leukemia cells expressing ITD-Flt3.

The effect of decitabine on megakaryocyte maturation and platelet release

2011 ◽  
Vol 106 (08) ◽  
pp. 337-343 ◽  
Author(s):  
Jianhui Wang ◽  
Zanhua Yi ◽  
Shiyang Wang ◽  
Zongdong Li
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Bone Marrow Cells ◽  
Mouse Bone Marrow ◽  
Human Cord Blood ◽  
Primary Mouse ◽  
Platelet Release ◽  
Mouse Bone Marrow Cells ◽  
Marrow Cells

SummaryThrombocytopenia is a common feature of myelodysplastic syndromes (MDS). 5-aza-2’-deoxycytidine (decitabine) has been used to treat MDS with an approximately 20% response rate in thrombocytopenia. However, the mechanism of how decitabine increases platelet count is not clear. In this study, we investigated the effect of decitabine on megakaryocyte maturation and platelet release in the mouse. The effect of decitabine on megakaryocyte maturation was studied in an in vitro megakaryocyte differentiation model utilising mouse bone marrow cells and mouse megakaryoblastic cell line L8057. Decitabine (2.5 μM) is able to induce L8057 cells to differentiate into a megakaryocyte-like polyploidy cells with positive markers of acetylcholinesterase and αIIb integrin (CD41). Higher expression of αIIb integrin was also found in primary mouse bone marrow cells and human cord blood CD34+ cells cultured with both thrombopoietin and decitabine as compared to thrombopoietin alone. In addition, we noted a 30% platelet count increase in Balb/c mice 12 hours after the injection of decitabine at a clinically relevant dose (15 mg/m2), suggesting a rapid platelet release from the spleen or bone marrow. Our data suggest that decitabine increases platelet counts by enhancing platelet release and megakaryocyte maturation.

CREB Plays a Critical Role in the Regulation of Normal and Malignant Hematopoiesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1224-1224
Author(s):  
Jerry C. Cheng ◽  
Dejah Judelson ◽  
Kentaro Kinjo ◽  
Jenny Chang ◽  
Elliot Landaw ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Leukemia Cells ◽  
Mouse Bone Marrow ◽  
T315i Mutation

Abstract The cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, memory, and glucose metabolism. We previously demonstrated that CREB overexpression is associated with an increased risk of relapse in a small cohort of adult acute myeloid leukemia (AML) patients. Transgenic mice that overexpress CREB in myeloid cells develop myeloproliferative/myelodysplastic syndrome after one year. Bone marrow cells from these mice have increased self-renewal and proliferation. To study the expression of CREB in normal hematopoiesis, we performed quantitative real-time PCR in both mouse and human hematopoietic stem cells (HSCs). CREB expression was highest in the lineage negative population and was expressed in mouse HSCs, common myeloid progenitors, granulocyte/monocyte progenitors, megakaryocyte/erythroid progenitors, and in human CD34+38- cells. To understand the requirement of CREB in normal HSCs and myeloid leukemia cells, we inhibited CREB expression using RNA interference in vitro and in vivo. Bone marrow progenitor cells infected with CREB shRNA lentivirus demonstrated a 5-fold decrease in CFU-GM but increased Gr-1/Mac-1+ cells compared to vector control infected cells (p<0.05). There were fewer terminally differentiated Mac-1+ cells in the CREB shRNA transduced cells (30%) compared to vector control (50%), suggesting that CREB is critical for both myeloid cell proliferation and differentiation. CREB downregulation also resulted in increased apoptosis of mouse bone marrow progenitor cells. Given our in vitro results, we transplanted sublethally irradiated mice with mouse bone marrow cells transduced with CREB or scrambled shRNA. At 5 weeks post-transplant, we observed increased Gr-1+/Mac-1+ cells in mice infused with CREB shRNA transduced bone marrow compared to controls. After 12 weeks post-transplant, there was no difference in hematopoietic reconstitution or in the percentage of cells expressing Gr-1+, Mac-1+, Gr-1/Mac-1+, B22-+, CD3+, Ter119+, or HSCs markers, suggesting that CREB is not required for HSC engraftment. To study the effects of CREB knockdown in myeloid leukemia cells, K562 and TF-1 cells were infected with CREB shRNA lentivirus, sorted for GFP expression, and analyzed for CREB expression and proliferation. Within 72 hours, cells transduced with CREB shRNA demonstrated decreased proliferation and survival with increased apoptosis. In cell cycle experiments, we observed increased numbers of cells in G1 and G2/M with CREB downregulation. Expression of cyclins A1 and D, which are known target genes of CREB, was statistically significantly decreased in TF-1 and K562 cells transduced with CREB shRNA lentivirus compared to controls. To study the in vivo effects of CREB knockdown on leukemic progression, we injected SCID mice with Ba/F3 cells expressing bcr-abl or bcr-abl with the T315I mutation and the luciferase reporter gene. Cells were transduced with either CREB or scrambled shRNA. Disease progression was monitored using bioluminescence imaging. The median survival of mice injected with CREB shRNA transduced Ba/F3 bcr-abl or bcr-abl with the T315I mutation was increased with CREB downregulation compared to controls (p<0.05). Our results demonstrate that CREB is a critical regulator of normal and neoplastic hematopoiesis both in vitro and in vivo.

Structural and Functional Characterization of the NHR2 and Runt Domains of AML1/ETO.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 482-482
Author(s):  
Matthew D. Cheney ◽  
Yizhou Liu ◽  
Yunpeng Zhou ◽  
Maksymilian Chruszcz ◽  
Thomas M. Laue ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Dominant Negative ◽  
Heptad Repeat ◽  
Mouse Bone Marrow ◽  
Runt Domain ◽  
Primary Mouse ◽  
Dominant Negative Activity ◽  
Marrow Cells

Abstract AML1/ETO is the chimeric fusion protein resulting from the t(8;21) found in AML of the M2 subtype. It contains the N-terminal 177 amino acids of RUNX1 and virtually all (575aa) of ETO. The RUNX1 component includes the Runt domain, which mediates both DNA binding and heterodimerization with CBFβ, but lacks the more C-terminal sequences required for transactivation. AML1/ETO occupies RUNX target genes in vivo and is associated with a repressive chromatin structure characterized by reduced levels of acetylated histone H3. AML1/ETO is thought to repress transcription by recruiting a SMRT (N-CoR)/Sin3A/HDAC complex to chromatin via sequences in ETO. ETO is the human homologue of the Drosophila Nervy protein and shares 4 regions of homology with Nervy called Nervy Homology Regions (NHR) 1–4. Deletion studies have shown that three of the AML1/ETO domains essential for its repressive function are the Runt domain, NHR2, and NHR4. The NHR2 domain is a hydrophobic heptad repeat that mediates oligomerization of AML1/ETO, interaction with ETO family members, and also with mSin3A and HDACs. We recently solved an x-ray structure of the NHR2 domain and found it to be an alpha-helical tetramer. Based on this structure we have introduced amino acid substitutions into the NHR2 domain that disrupt tetramer formation but not AML1/ETO stability. These mutations impair the ability of AML1/ETO to inhibit the differentiation of GR-1+/Mac-1+ cells following retroviral transduction into primary mouse bone marrow cells, and also inhibit the serial replating ability of AML1/ETO expressing bone marrow cells in vitro. We additionally show that mutations reported by Amann et al. (Mol Cell Biol. 21, 6470, 2001) to disrupt mSin3A binding to NHR2 do not affect the biological activity of AML1/ETO in vitro. We also introduced mutations in the Runt domain of AML1/ETO that disrupt CBFβ binding by defined amounts (40-fold, 200-fold, 500-fold), and demonstrated that CBFβ binding by AML1/ETO is essential for its dominant negative activity. The latter results suggest that small molecules designed to selectively impair heterodimerization of AML1/ETO with CBFβ could potentially block AML1/ETO’s dominant negative activity.

scholarly journals Important role of EP4, a subtype of prostaglandin (PG) E receptor, in osteoclast-like cell formation from mouse bone marrow cells induced by PGE2

1998 ◽  
Vol 158 (3) ◽  
pp. R1-R5 ◽  
Author(s):  
K Ono ◽  
T Akatsu ◽  
T Murakami ◽  
M Nishikawa ◽  
M Yamamoto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Cells ◽  
Bone Marrow Cells ◽  
Therapeutic Potential ◽  
Cell Formation ◽  
Mouse Bone Marrow ◽  
Dependent Manner ◽  
Clinical Conditions

Of various PGs, PGE1 and PGE2 are shown to be the most potent stimulators of osteoclastogenesis in vitro. PGE receptors have been classified into four subtypes, EP1-EP4. Little is known about PGE receptors functioning in bone cells. In this study, using mouse marrow culture, we investigated which PGE receptors are important in osteoclast-like cell (OCL) formation induced by PGE. 11-deoxy-PGE1 (EP2, EP3 and EP4 agonist) stimulated OCL formation potently. Butaprost (EP2 agonist) stimulated it slightly, while sulprostone (EP1 and EP3 agonist) and ONO-AP-324-01 (EP3 agonist) did not. AH23848B (EP4 antagonist) inhibited PGE2-induced OCL formation in a dose-dependent manner. The expression of EP4 mRNA in mouse bone marrow was confirmed by RT-PCR. The results indicate an important role of EP4 in PGE2-induced OCL formation in marrow cultures and suggest therapeutic potential of EP4 antagonists in some clinical conditions with accelerated bone resorption.

scholarly journals RUNX1 mutations promote leukemogenesis of myeloid malignancies in ASXL1-mutated leukemia

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Rabindranath Bera ◽  
Ming-Chun Chiu ◽  
Ying-Jung Huang ◽  
Tung-Huei Lin ◽  
Ming-Chung Kuo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Critical Role ◽  
Mouse Bone Marrow ◽  
Myeloid Malignancies ◽  
Inhibitor Of Dna Binding

Abstract Background Additional sex combs-like 1 (ASXL1) mutations have been described in all forms of myeloid neoplasms including chronic myelomonocytic leukemia (CMML) and associated with inferior outcomes, yet the molecular pathogenesis of ASXL1 mutations (ASXL1-MT) remains poorly understood. Transformation of CMML to secondary AML (sAML) is one of the leading causes of death in CMML patients. Previously, we observed that transcription factor RUNX1 mutations (RUNX1-MT) coexisted with ASXL1-MT in CMML and at myeloid blast phase of chronic myeloid leukemia. The contribution of RUNX1 mutations in the pathogenesis of myeloid transformation in ASXL1-mutated leukemia, however, remains unclear. Methods To evaluate the leukemogenic role of RUNX1-MT in ASXL1-mutated cells, we co-expressed RUNX1-MT (R135T) and ASXL1-MT (R693X) in different cell lines and performed immunoblot, co-immunoprecipitation, gene expression microarray, quantitative RT-PCR, cell proliferation, differentiation, and clonogenic assays for in vitro functional analyses. The in vivo effect was investigated using the C57BL/6 mouse bone marrow transplantation (BMT) model. Results Co-expression of two mutant genes increased myeloid stem cells in animal model, suggesting that cooperation of RUNX1 and ASXL1 mutations played a critical role in leukemia transformation. The expression of RUNX1 mutant in ASXL1-mutated myeloid cells augmented proliferation, blocked differentiation, and increased self-renewal activity. At 9 months post-BMT, mice harboring combined RUNX1 and ASXL1 mutations developed disease characterized by marked splenomegaly, hepatomegaly, and leukocytosis with a shorter latency. Mice transduced with both ASXL1 and RUNX1 mutations enhanced inhibitor of DNA binding 1 (ID1) expression in the spleen, liver, and bone marrow cells. Bone marrow samples from CMML showed that ID1 overexpressed in coexisted mutations of RUNX1 and ASXL1 compared to normal control and either RUNX1-MT or ASXL1-MT samples. Moreover, the RUNX1 mutant protein was more stable than WT and increased HIF1-α and its target ID1 gene expression in ASXL1 mutant cells. Conclusion The present study demonstrated the biological and functional evidence for the critical role of RUNX1-MT in ASXL1-mutated leukemia in the pathogenesis of myeloid malignancies.

WT1 Promotes Myeloid Development and Inhibits Lymphoid Development.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1539-1539
Author(s):  
Aschwin L. Menke ◽  
G.H. J. Knops ◽  
P.C. M. Linssen ◽  
G. Nikoloski ◽  
A. Pennings ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Biological Activities ◽  
Mouse Bone Marrow ◽  
Mutant Proteins ◽  
Empty Vector ◽  
Primary Mouse ◽  
Marrow Cells

Abstract The Wilms’ Tumor 1 (WT1) gene is highly expressed in bone marrow progenitor cells, and is downregulated during the differentiation towards mature blood cells. Several lines of evidence suggest that WT1 plays an important role in leukemia development. WT1 overexpression can be detected in more than 80% of acute leukemia’s and an inverse correlation has been found between the expression levels of WT1 and the overall survival of patients. In addition, in about 9% of AML cases and 3% of ALL cases, WT1 is mutated and the presence of these mutations may have an adverse effect on the survival of the patients. So far, little is known about the biological activities of the wildtype and the WT1-mutant proteins during hematopoiesis and the presence of different isoforms with different biological activities has hampered a clear interpretation of the results so far. In a comprehensive study, we have investigated the function of all four major WT1 isoforms in primary mouse bone marrow cells using in-vitro and in-vivo assays. In addition, we have studied for each isoform the effect of mutations on the biological activity. In-vitro studies: 4 wildtype WT1 isoforms, 6 mutants and an empty vector control were retrovirally transduced into primary murine bone marrow cells. Subsequently, the transduced cells were FACS-sorted and used for various assays. WT1 inhibited the in-vitro colony formation (CFU-GM) by 60–95%, depending on the expressed isoform. In contrast, expression of the corresponding WT1 mutant proteins had no effect on colony formation. To study the underlying mechanism, we cultured the WT1-transduced bone marrow cells and analyzed the cells each day for proliferation (Cell count & DNA histograms), differentiation (Mac1, Gr1) and apoptosis (Annexin V) using FACS analysis. In agreement with the colony assays, the expression of all 4 wildtype WT1 isoforms induced growth arrest and resulted in accelerated differentiation. Target genes: To investigate which genes may be involved in the observed phenotypes, we quantitatively analyzed the expression levels of 34 putative WT1-target genes in the transduced murine bone marrow cells. Briefly, primary mouse bone marrow cells were retrovirally transduced with 4 different wildtype WT1 isoforms, 4 different mutant isoforms or an empty vector control. Sixteen hours after transduction, the transduced cells were FACS-sorted and RNA was extracted for quantitative real-time RT-PCR analysis. We have identified a number of putative WT1-target genes that are differentially regulated by the 4 wildtype WT1 isoforms but not by the WT1 mutant proteins: E-cadherin, syndecan, NGF-receptor, Egr-1, TGF-b, c-Myc, Vitamin D-receptor, insulin-receptor thrombospondin and the taurine transporter. In-vivo studies: To study the effect of WT1 on more immature bone marrow stem cells, we have transplanted WT1-transduced primary mouse CD45.2 bone marrow cells together with empty-vector-transduced primary mouse CD45.1 bone marrow cells into ablatively irradiated syngenic CD45.1/CD45.2 heterozygous mice. Six weeks after transplantation, 5-colour FACS analysis of peripheral blood indicated that the expression of WT1 promotes myeloid differentiation (Mac1 & Gr1) and inhibits the formation of B- (IgM/B220) and T-cells (CD4 & CD8).

scholarly journals CONTRIBUTION OF A THYMIC HUMORAL FACTOR TO THE DEVELOPMENT OF AN IMMUNOLOGICALLY COMPETENT POPULATION FROM CELLS OF MOUSE BONE MARROW

1971 ◽  
Vol 134 (3) ◽  
pp. 786-800 ◽  
Author(s):  
Myra Small ◽  
Nathan Trainin
Keyword(s):  
Bone Marrow ◽  
Lymphoid Tissue ◽  
Host Response ◽  
Bone Marrow Cells ◽  
Immune Reactivity ◽  
Mouse Bone Marrow ◽  
Graft Versus Host ◽  
Peripheral Lymphoid Tissue ◽  
Marrow Cells

The hypothesis that cells located in mouse bone marrow can acquire immunological competence by a process that involves interaction with a noncellular component of the thymus was tested using an in vitro assay of graft-versus-host reactivity as a criterion of cell competence. When suspensions of C57BL bone marrow cells were incubated in thymus extract and injected into mice incapable of inducing a response in the graft-versus-host assay as a result of neonatal thymectomy, or adult thymectomy plus irradiation, or because of genetic similarity with the (C3H x C57BL)F1 tissue used for challenge in the assay, competent cells were recovered from the spleens of the injected mice. The reactive cells were shown to be of bone marrow origin since immune reactivity was related to the genetic makeup of the bone marrow cells rather than that of the intermediate recipients. A thymic factor was involved in the process leading to immune reactivity by these cells, as bone marrow cells incubated in xenogeneic or syngeneic thymic extracts induced a graft-versus-host response after passage through nonresponsive mice, whereas incubation of bone marrow cells in xenogeneic lymph node or spleen extracts or in culture medium only did not lead to subsequent reactivity. Participation of peripheral lymphoid tissue seemed essential in this process since bone marrow cells tested directly after exposure to thymic extract failed to induce a graft-versus-host response. C57BL bone marrow cells exposed to thymus extract and cultured together with fragments of (C3H x C57BL)F1 spleen tissue in vitro were competent to induce a graft-versus-host response; thus, these components would seem to be sufficient as well as necessary for the immunodifferentiation process leading to graft-versus-host activity. It is concluded that one step in the process by which bone marrow cells acquire competence vis-a-vis the graft-versus-host response depends upon a thymic agent that is noncellular and extractable, and that another stage in this process is under the influence of components found within the peripheral lymphoid tissue environment. It is suggested that differentiation of precursor cells to competence could occur by progressive development of the cells in separate compartments of the lymphoid system.

Sign in / Sign up

Export Citation Format

Share Document