scholarly journals Fibrin, Bone Marrow Cells and macrophages interactively modulate cardiomyoblast fate.

2022 ◽  
Author(s):  
Ines Borrego ◽  
Aurelien FROBERT ◽  
Guillaume AJALBERT ◽  
Jeremy VALENTIN ◽  
Cyrielle KALTENRIEDER ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Cardiac Function ◽  
Conditioned Medium ◽  
Bone Marrow Cells ◽  
Cardiac Repair ◽  
Cardiac Cell ◽  
Anti Inflammatory ◽  
Marrow Cells

Interactions between macrophages, cardiac cells and the extracellular matrix are crucial for cardiac repair following myocardial infarction (MI). The paracrine effects of cell-based treatments of MI might modulate these interactions and impact cardiac repair. The immunomodulatory capacity of the therapeutic cells is therefore of interest and could be modulated by the use of biomaterials. We first showed that bone marrow cells (BMC) associated with fibrin could treat MI. Then, we interrogated the influence of fibrin, as a biologically active scaffold, on the secretome of BMC and the impact of their association on macrophage fate and cardiomyoblast proliferation. Methods: In vivo, two weeks post-MI, rats were treated with epicardial implantation of BMC and fibrin or sham-operated. High-resolution echocardiography was performed to evaluate the heart function and structure changes after 4 weeeks. Histology and immunostaining were performed on harvested hearts. In vitro, BMC were first primed with fibrin. Second, non-polarized macrophages were differentiated toward either pro-inflammatory or anti-inflammatory phenotypes and stimulated with the conditioned medium of fibrin-primed BMC (F-BMC). Proteomic, cytokine levels quantification, and RT-PCR were performed. EdU incorporation and real-time cell analysis assessed cell proliferation. Results: The epicardial implantation of fibrin and BMC reduced the loss of cardiac function induced by MI, increased wall thickness and prevented the fibrotic scar expansion. After 4 and 12 weeks, the infarct content of CD68+ and CD206+ was similar in control and treated animals. In vitro, we showed that fibrin profoundly influenced the gene expression and the secretome of BMC, simultaneously upregulating both pro- and anti-inflammatory mediators. Furthermore, the conditioned medium from F-BMC significantly increased the proliferation of macrophages in a subsets dependent manner and modulated their gene expression and cytokines secretion. For instance, F-BMC significantly downregulated the expression of Nos2, Il6 and Ccl2/Mcp1 while Arg1, Tgfb and IL10 were upregulated. Interestingly, macrophages educated by F-BMC increased cardiomyoblast proliferation. In conclusion, our study provides evidence that BMC/fibrin-based treatment lowered the infarct extent and improved cardiac function. The macrophage content was unmodified when measured at a chronic stage. Nevertheless, acutely and in vitro, the F-BMC secretome promotes an anti-inflammatory response that stimulates cardiac cell growth. Finally, our study emphases the acute impact of F-BMC educated macrophages on cardiac cell fate.

scholarly journals Manipulations of cholinesterase gene expression modulate murine megakaryocytopoiesis in vitro.

1990 ◽  
Vol 10 (11) ◽  
pp. 6046-6050 ◽  
Author(s):  
D Patinkin ◽  
S Seidman ◽  
F Eckstein ◽  
F Benseler ◽  
H Zakut ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Conditioned Medium ◽  
Xenopus Oocytes ◽  
Bone Marrow Cells ◽  
Murine Bone Marrow ◽  
Marrow Cells

Megakaryocytopoiesis was selectively inhibited in cultured murine bone marrow cells by a 15-mer oligodeoxynucleotide complementary to the initiator AUG region in butyrylcholinesterase mRNA. Furthermore, conditioned medium from Xenopus oocytes producing recombinant butyrylcholinesterase stimulated megakaryocytopoiesis. These observations implicate butyrylcholinesterase in megakaryocytopoiesis and suggest application of oligodeoxynucleotides for modulating bone marrow development.

Manipulations of cholinesterase gene expression modulate murine megakaryocytopoiesis in vitro

1990 ◽  
Vol 10 (11) ◽  
pp. 6046-6050
Author(s):  
D Patinkin ◽  
S Seidman ◽  
F Eckstein ◽  
F Benseler ◽  
H Zakut ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Conditioned Medium ◽  
Xenopus Oocytes ◽  
Bone Marrow Cells ◽  
Murine Bone Marrow ◽  
Marrow Cells

Megakaryocytopoiesis was selectively inhibited in cultured murine bone marrow cells by a 15-mer oligodeoxynucleotide complementary to the initiator AUG region in butyrylcholinesterase mRNA. Furthermore, conditioned medium from Xenopus oocytes producing recombinant butyrylcholinesterase stimulated megakaryocytopoiesis. These observations implicate butyrylcholinesterase in megakaryocytopoiesis and suggest application of oligodeoxynucleotides for modulating bone marrow development.

Frequent in Vivo redundancy of C/EBPβ and C/EBPε during Myeloid Development

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2440-2440
Author(s):  
Nils Heinrich Thoennissen ◽  
Tadayuki Akagi ◽  
Sam Abbassi ◽  
Daniel Nowak ◽  
Ann George ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Transcription Factors ◽  
Bone Marrow Cells ◽  
Double Knockout ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Marrow Cells

Abstract CCAAT/enhancer binding protein (C/EBP) transcription factors are involved in a variety of cellular responses including proliferation and differentiation. Although C/EBPβ and C/EBPε are believed to be most important for macrophage and granulocyte activity, respectively, experiments by others and ourselves suggest a possible overlap in their function in myelopoiesis. In order to explore further this potential redundancy, we assessed the in vivo and in vitro function of both transcription factors by generating a double knockout (KO) germline murine model (C/EBPβ/ε−/−/−/−) and compared their hematopoiesis to those of single deficient (C/EBPβ−/−, C/EBPε−/−) and wild-type (WT) mice. Gene expression analysis of bone marrow cells showed expression of C/EBPβ in C/EBPε−/− and WT mice, and vice versa. The weight of the double-KO mice was significantly less as measured at 4 weeks of age (11.5 ± 0.9 g) compared to WT (13.4 ± 0.6 g), C/EBPβ−/− (14.5 ± 1.4 g), and C/EBPε−/− mice (15.4 ± 2.3 g) (p < 0.05). The double-KO mice were prone to infections of the eyes, lungs, liver, and peritoneum. In contrast, C/EBPβ−/−, C/EBPε−/− and WT mice demonstrated no signs of infection. Microscopic imaging of peripheral blood showed metamyelocytes and myelocytes in the double-KO mice. FACS analysis found that the fraction of bone marrow cells which were Lin(−) (no expression of B220, CD3, Gr1, Ter119, and Mac1) were modestly elevated in double-KO and C/EBPβ−/− mice (8.42 % and 8.1 %, respectively) compared to C/EBPε−/− (4.24 %) and WT (3.93 %) mice. A subanalysis highlighted an elevated level of B220(−)/Gr1(−) bone marrow cells in the double-KO mice (54 %) compared to the levels in the C/EBPβ−/− (31 %), C/EBPε−/− (33 %) and WT (21.5 %) mice. Moreover, the proportion of hematopoietic stem cells in the bone marrow were significantly increased in the hematopoietic stem cell compartment [Sca1(+)/c-Kit(+)] in the double-KO mice (20.8 %) compared to the C/EBPβ−/− (6.9 %), C/EBPε−/− (5.9 %) and WT (6.9 %) mice. When given a cytotoxic stress (5-FU) to kill cycling hematopoietic progenitor cells, the mean neutrophil count at their nadir (day 4) was 0.14 × 109 cells/L in the double-KO mice compared to 0.71 × 109 cells/L in the WT mice (p < 0.001); both reached normal values again on day 10. Taken together, these results indicated a relatively higher percentage of immature hematopoietic cells in the double-KO mice compared to the WT mice. Nevertheless, clonogenic assays in methylcellulose using bone marrow cells of the double-KO showed a significant decreased number of myeloid colonies. For example, in the presence of G-CSF, GM-CSF, and SCF, a mean of 83 ± 10 hematopoietic colonies formed in the double-KO mice compared to 135 ± 6 in C/EBPβ−/−, 159 ± 12 in C/EBPε−/− and 165 ± 2 in WT mice (p < 0.001, double-KO vs. WT). Similar clonogenic results occurred when bone marrow cells were stimulated with either G-CSF, GM-CSF or SCF/G-CSF alone. Although our in vitro experiments suggested that double-KO mice had a decreased clonogenic response to G-CSF, their bone marrow cells had normal levels of phosphorylated STAT3 protein when stimulated with G-CSF. Hence, the G-CSFR and its secondary signaling pathway seemed to be intact. In further experiments, downstream targets of the C/EBP transcription factors were examined. Bone marrow macrophages activated with LPS and IFNγ from both double-KO and C/EBPβ−/− mice had decreased gene expression of IL6, IL12p35, TNFα, and G-CSF compared to the levels detected in macrophages of C/EBPε−/− and WT. Interestingly, expression levels of cathelicidin antimicrobial peptide (CAMP) were similarly robust in the macrophages from C/EBPβ−/−, C/EBPε−/−, and WT mice. In sharp contrast, CAMP expression was undetectable in the activated macrophages of the double-KO mice. In conclusion, the phenotype of the double-KO mice was often distinct from the C/EBPβ−/− and C/EBPε−/− mice suggesting a redundancy of activity of both transcription factors in myeloid hematopoiesis.

MN1 Inhibits Myeloid Differentiation by Transcriptional Repression of EGR2

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 229-229
Author(s):  
Michael Heuser ◽  
Eric Yung ◽  
Courteney Lai ◽  
Bob Argiropoulos ◽  
Florian Kuchenbauer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Function Analysis ◽  
Myeloid Differentiation ◽  
Marrow Cells

Abstract Abstract 229 Overexpression of MN1 (meningioma 1) is a negative prognostic factor in acute myeloid leukemia (AML) patients with normal cytogenetics, and induces a rapidly lethal AML in mice. We have shown previously that MN1, a transcription cofactor of retinoic acid receptor alpha (RARA), increases resistance to all-trans retinoic acid (ATRA) by greater than 3000-fold in an in-vitro differentiation model. We investigated the molecular mechanisms involved in the MN1-induced myeloid differentiation block by fusing potent transcriptional activation or repression domains to MN1, conducting a structure-function analysis of MN1, gene expression profiling, ChIP-on chip experiments, and functional validation of MN1 target genes. We found that (1) MN1 inhibits myeloid differentiation through transcriptional repression; (2) the C-terminal domain of MN1 is critical for induction of resistance to ATRA; (3) EGR2 is a putative direct target of MN1 and RARA that is repressed in MN1 leukemias; and (4) that constitutive upregulation of EGR2 in MN1 leukemias permits differentiation and prevents engraftment of transplanted cells. To investigate whether MN1 impacts on myeloid differentiation through transcriptional activation or repression we fused a strong transcriptional activation domain (VP16) or repression domain (M33) to MN1. MN1VP16 immortalized murine bone marrow cells, however, these cells could differentiate to mature granulocytes, and succumbed to cell cycle arrest upon treatment with ATRA. Mice receiving transplants of MN1VP16 cells had a median survival of 143 days (n=16) compared to 35 days in mice receiving MN1-transduced cells (n=18; p<.001). Morphologic analysis of bone marrow mostly showed mature granulocytes with less than 20 percent immature forms consistent with a diagnosis of myeloproliferative-like disease. Conversely, mice receiving transplants with cells transduced with the fusion of MN1 to the transcriptional repression domain of M33 (n=7) developed leukemia with a similar latency and phenotype as mice receiving transplants from MN1-transduced cells (survival, P=.6). These data suggest that MN1 inhibits myeloid differentiation by transcriptional repression rather than activation of its target genes. A structure-function analysis was performed to identify the domain(s) of MN1 required to inhibit myeloid differentiation. Consecutive stretches of 200 amino acids of MN1 were interrogated The deletion constructs were subsequently transduced into bone marrow cells immortalized by NUP98-HOXD13 (ND13). ND13 cells are very sensitive to ATRA-induced differentiation and cell cycle arrest with an IC50 of 0.1 μ M, whereas overexpression of MN1 increases resistance greater than 3000-fold. Interestingly, deletion of the 200 C-terminal amino acids of MN1 restored ATRA sensitivity of ND13 cells compared to full-length MN1, suggesting that the C-terminus of MN1 is required for inhibition of myeloid differentiation. To identify MN1-regulated genes important for the myeloid differentiation block we performed gene expression profiling of MN1- and MN1VP16-transduced bone marrow cells. To further identify genes that might be directly regulated by MN1 we performed ChIP-on-chip using anti-MN1 and anti-RARA antibodies. EGR2, CCL5, CMAH, among others, were identified as targets of both MN1 and RARA whose gene expression was low in MN1 but high in MN1VP16 cells. Overexpression of these genes in MN1-transduced leukemic cells was used to validate their function. Blast percentage of in vitro cultured bone marrow cells was 93, 58, 83, and 41 percent in MN1+CTL cells, MN1+EGR2, MN1+CCL5, and MN1+CMAH cells, respectively. MN1+EGR2 cell engraftment in peripheral blood of mice declined from 2.2 percent at 4 weeks to undetectable levels at 8 weeks (n=4), whereas MN1+CCL5 and MN1+CMAH cell engraftment was 23 (n=4) and 26 (n=4) percent at 4 weeks, and 14 and 30 percent at 8 weeks, respectively. At time of death, EGR2 was not detectable in mice whereas leukemias of mice receiving MN1+CCL5 or MN1+CMAH- transduced cells were positive for CCL5 or CMAH, respectively. In conclusion, our data suggest that MN1 inhibits myeloid differentiation by transcriptional repression of a subset of its target genes, and that re-expression of EGR2, a zinc-finger transcription factor, may prevent outgrowth of MN1 leukemias in mice. Pharmacologic activation of EGR2 may become a novel antileukemic strategy. Disclosures: No relevant conflicts of interest to declare.

Cellular and Molecular Targets of MLL-AF9 in a Novel Conditional Mouse Model

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1280-1280
Author(s):  
Vaia Stavropoulou ◽  
Susanne Kaspar ◽  
Laurent Brault ◽  
Sabine Juge ◽  
Stefano Morettini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Mouse Model ◽  
Prognostic Markers ◽  
Bone Marrow Cells ◽  
Median Latency ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract Abstract 1280 Previous studies have shown that the expression of several leukemia-associated mixed lineage leukemia (MLL) fusion genes transformed human and mouse bone marrow cells in vitro and in vivo. In order to dissect the molecular and cellular targets of the MLL-AF9 fusion, we generated a novel inducible doxycycline (DOX)-regulated transgenic mouse model. Conditional ex vivo activation of MLL-AF9 induced aberrant self-renewal and impaired differentiation of long-term or short-term hematopoietic stem (LT-HSC and ST-HSC), common myeloid progenitor (CMP) and granulocyte-macrophage progenitor (GMP) cells in a fully reversible manner. Direct activation of the fusion in vivo or after transplantation of transgenic bone marrow cells into irradiated hosts induced an aggressive and transplantable disease after a median latency of 80days characterized as acute myelo-monocytic leukemia closely mimicking the human disease. Fusion gene expression and leukemia induction was DOX dosage dependent and reversible upon DOX removal. Activation of MLL-AF9 in isolated LT-HSC or GMP cells in vitro or in vivo resulted in the accumulation of immature blast-like cells with similar immunophenotypes. However, MLL-AF9-expressing stem and progenitor cells displayed distinct properties such as colony formation, differentiation and resistance to chemotherapeutic drugs. Turning-off the fusion resulted in multi-lineage differentiation of LT-HSC-derived cells, whereas GMP-derived cells were limited to mature macrophages and granulocytes suggesting partial maintenance of their original identity. In line with these in vitro observations, lower cell numbers of transplanted LT-HSCs induced a more aggressive leukemia with a significantly shorter latency as compared to ST-HSC, CMP or GMPs. Immunophenotypically 15% of the LT-HSC derived leukemias displayed a CMP–like phenotype and had a median latency of 37d (“early”) whereas the rest of the cases displayed a GMP-like phenotype with a median latency of 73d (“late”). In contrast, only GMP-like phenotypes and longer latencies were observed upon transplanting ST-HSCs (75d), CMPs (72d) or GMPs (100d). Transplantation of blasts from “early” LT-HSC- and GMP-derived leukemias into secondary recipients induced the disease after similar latency, however, cytarabine (Ara-C) treatment significantly delayed only the disease induced by GMP- but not by LT-HSC-derived blasts. Gene expression profiling in immortalized pre-leukemic cells revealed down-regulation of over 300 genes, including several well-known MLL targets such as Meis1, HoxA5, HoxA9 and HoxA10 upon reducing the levels of MLL-AF9 expression. Likewise, we observed a global decrease in histone H3 lysine 79 dimethylation consistent with a Dot1l function in MLL-AF9 driven leukemia. LT-HSC-derived (“early”) blasts displayed distinct genetic signatures with > 400 genes highly and > 1300 genes lowly expressed (p001 fc1.5), clearly separating them from the GMP-derived blasts. Evi-1 and Erg, two prognostic markers in patient-derived gene signatures, stood out among these genes. The aggressive “early” LT-derived murine leukemias showed high Evi-1 and Erg expression levels (Evi-1 high, Erg high) as compared to the “late” LT-derived (Evi-1 low, Erg high) or the GMP-derived leukemias (Evi-1 low, Erg low). These observations suggest that the previously reported poor prognosis associated with elevated EVI-1 and/or ERG expression might directly reflect the cell of origin of the disease. We are currently exploiting our highly informative MLL-AF9 disease model to evaluate the functional relevance of novel origin-dependent MLL-AF9 target genes and to identify novel prognostic markers and therapeutic targets. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Gene Expression in Response to Low-Intensity Pulsed Ultrasound Treatment of Bone Marrow Cells under Osteogenic Conditions In Vitro

2016 ◽  
Vol 25 (2) ◽  
pp. 137-148 ◽  
Author(s):  
Daisuke Yamaguchi ◽  
Kazuo Takeuchi ◽  
Hiroki Furuta ◽  
Shin Miyamae ◽  
Hiroshi Murakami ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Ultrasound Treatment ◽  
Pulsed Ultrasound ◽  
Low Intensity Pulsed Ultrasound ◽  
Low Intensity ◽  
Marrow Cells

scholarly journals Evidence that Contamination by Lipopolysaccharide Confounds in Vitro Studies of Adiponectin Activity in Bone

Endocrinology ◽  
2012 ◽  
Vol 153 (5) ◽  
pp. 2076-2081 ◽  
Author(s):  
Dorit Naot ◽  
Garry A Williams ◽  
Jian-ming Lin ◽  
Jillian Cornish ◽  
Andrew Grey
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Molecular Mechanisms ◽  
Bone Marrow Cells ◽  
Global Changes ◽  
Mouse Bone Marrow ◽  
Type I ◽  
Mineral Density ◽  
Marrow Cells

Adiponectin, a hormone produced and secreted from adipose tissue, circulates at levels that are inversely related to visceral fat mass and bone mineral density. Adiponectin receptors are expressed in bone cells, and several studies have shown that adiponectin affects bone phenotype and might play a role in the cross talk between fat and bone tissues. In the current study, we determined global changes in gene expression induced by adiponectin in mouse bone marrow cells, in order to identify the molecular mechanisms that mediate adiponectin's effect to inhibit osteoclast differentiation in these cultures. The gene signature that was produced by microarray analysis was very similar to a signature produced by activation of type I interferons (IFN), and we therefore tested the hypothesis that the adiponectin preparation, although marketed as “lipopolysaccharide (LPS) free”, was contaminated with LPS that induced an IFN response in the bone marrow cells. Heat inactivation of the adiponectin preparation and the use of small interfering RNA to knockdown the AdipoR1 receptor had not diminished the activity of the adiponectin preparation to induce the IFN target genes Ccl5 and Irf7. Thus, the changes in gene expression determined in the bone marrow cultures are likely to be the result of a combination of adiponectin and LPS effects. Our study suggests that the purity of commercially available proteins needs to be verified and that experimental results of adiponectin activity in vitro should be interpreted cautiously.

scholarly journals Normal Granulocyte Colony-forming Cells in the Bone Marrow of Yemenite Jews With Genetic Neutropenia

Blood ◽  
1973 ◽  
Vol 41 (6) ◽  
pp. 745-751 ◽  
Author(s):  
Uri Mintz ◽  
Leo Sachs
Keyword(s):  
Bone Marrow ◽  
Conditioned Medium ◽  
Human Embryo ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Release Mechanism ◽  
Neutropenic Patients ◽  
Yemenite Jews ◽  
Marrow Cells

Abstract The frequency of normal granulocyte colony-forming cells in the bone marrow of Yemenite Jews with genetic, absolute neutropenia and no special tendency to infection has been studied with conditioned medium from human embryo fibroblasts. Cells from these neutropenic patients gave an average of about twice the number of granulocyte colonies as bone marrow cells from nonneutropenic patients. No morphologic abnormalities were observed in the granulocytes in bone marrow smears or in colonies formed in vitro. The results indicate that the neutropenia in these patients was not due to a deficiency of granulocyte colony-forming cells. It is suggested that the neutropenia is due to a defect in the release mechanism of mature granulocytes from the bone marrow to the peripheral blood.

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