RESUMO
Many studies have already examined the hematopoietic recovery after irradiation but paid with very little attention to the bone marrow microenvironment. Nonetheless previous studies in a murine model of reversible radio-induced bone marrow aplasia have shown a significant increase in alkaline phosphatase activity (ALP) prior to hematopoietic regeneration. This increase in ALP activity was not due to cell proliferation but could be attributed to modifications of the properties of mesenchymal stem cells (MSC). We thus undertook a study to assess the kinetics of the evolution of MSC correlated to their hematopoietic supportive capacities in mice treated with sub lethal total body irradiation. In our study, colony-forming units-fibroblasts (CFU-Fs) assay showed a significant MSC rate increase in irradiated bone marrows. CFU-Fs colonies still possessed differentiation capacities of MSC but colonies from mice sacrificed 3 days after irradiation displayed high rates of ALP activity and a transient increase in osteoblastic markers expression while pparγ and neuropilin-1 decreased. Hematopoietic supportive capacities of CFU-Fs were also modified: as compared to controls, irradiated CFU-Fs significantly increased the proliferation rate of hematopoietic precursors and accelerated the differentiation toward the granulocytic lineage. Our data provide the first evidence of the key role exerted by the balance between osteoblasts and adipocytes in spontaneous bone marrow regeneration. First, (pre)osteoblast differentiation from MSC stimulated hematopoietic precursor's proliferation and granulopoietic regeneration. Then, in a second time (pre)osteoblasts progressively disappeared in favour of adipocytic cells which down regulated the proliferation and granulocytic differentiation and then contributed to a return to pre-irradiation conditions.
Assuntos
Adipócitos/citologia , Adipócitos/efeitos da radiação , Medula Óssea/efeitos da radiação , Osteoblastos/citologia , Osteoblastos/efeitos da radiação , Irradiação Corporal Total , Adipócitos/enzimologia , Fosfatase Alcalina/metabolismo , Animais , Biomarcadores/metabolismo , Comunicação Celular/efeitos da radiação , Diferenciação Celular/efeitos da radiação , Movimento Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Quimiocina CXCL12/metabolismo , Ensaio de Unidades Formadoras de Colônias , Regulação para Baixo/efeitos da radiação , Fêmur/efeitos da radiação , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/efeitos da radiação , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos da radiação , Camundongos , Neuropilina-1/metabolismo , Neutrófilos/citologia , Neutrófilos/metabolismo , Neutrófilos/efeitos da radiação , Osteoblastos/enzimologia , FenótipoRESUMO
It is suspected that bone marrow (BM) microenvironmental factors may influence the evolution of chronic myeloid leukaemia (CML). In this study, we postulated that adipocytes and lipids could be involved in the progression of CML. To test this hypothesis, adipocytes were co-cultured with two BCR-ABL positive cell lines (PCMDS and K562). T cell (Jurkat) and stroma cell (HS-5) lines were used as controls. In the second set of experiments, leukemic cell lines were treated with stearic, oleic, linoleic or α-linolenic acids in presence or absence of leptin. Survival, proliferation, leptin production, OB-R isoforms (OB-Ra and OB-Rb), phosphoinositide 3-kinase (PI3k) and BCL-2 expression have been tested after 24h, 48h and 72h of treatment. Our results showed that adipocytes induced a decrease of CML proliferation and an increase in lipid accumulation in leukemic cells. In addition, CML cell lines induced adipocytes cell death. Chromatography analysis showed that BM microenvironment cells were full of saturated (SFA) and monounsaturated (MUFA) fatty acids, fatty acids that protect tumor cells against external agents. Stearic acid increased Bcl-2 expression in PCMDS, whereas oleic and linoleic acids had no effects. In contrast, α-linolenic acid decreased the proliferation and the survival of CML cell lines as well as BCL-2 and OB-R expression. The effect of α-linolenic acids seemed to be due to PI3K pathway and Bcl-2 inhibition. Leptin production was detected in the co-culture medium. In the presence of leptin, the effect of α-linolenic acid on proliferation, survival, OB-R and BCl-2 expression was reduced.
Assuntos
Apoptose/efeitos dos fármacos , Leptina/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ácido alfa-Linolênico/farmacologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Medula Óssea/metabolismo , Medula Óssea/patologia , Caspases/metabolismo , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Meios de Cultura/farmacologia , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Leptina/biossíntese , Metabolismo dos Lipídeos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores para Leptina/metabolismoRESUMO
The most recently characterized H4 histamine receptor (H4R) is expressed preferentially in the bone marrow, raising the question of its role during hematopoiesis. Here we show that both murine and human progenitor cell populations express this receptor subtype on transcriptional and protein levels and respond to its agonists by reduced growth factor-induced cell cycle progression that leads to decreased myeloid, erythroid and lymphoid colony formation. H4R activation prevents the induction of cell cycle genes through a cAMP/PKA-dependent pathway that is not associated with apoptosis. It is mediated specifically through H4R signaling since gene silencing or treatment with selective antagonists restores normal cell cycle progression. The arrest of growth factor-induced G1/S transition protects murine and human progenitor cells from the toxicity of the cell cycle-dependent anticancer drug Ara-C in vitro and reduces aplasia in a murine model of chemotherapy. This first evidence for functional H4R expression in hematopoietic progenitors opens new therapeutic perspectives for alleviating hematotoxic side effects of antineoplastic drugs.
Assuntos
Ciclo Celular/fisiologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Receptores Acoplados a Proteínas G/fisiologia , Receptores Histamínicos/fisiologia , Animais , Proliferação de Células , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inativação Gênica , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Receptores Histamínicos H4 , Transdução de SinaisRESUMO
Adipocytes are part of hematopoietic microenvironment, even though up to now in humans, their role in hematopoiesis is still questioned. We have previously shown that accumulation of fat cells in femoral bone marrow (BM) coincides with increased expression of neuropilin-1 (NP-1), while it is weakly expressed in hematopoietic iliac crest BM. Starting from this observation, we postulated that adipocytes might exert a negative effect on hematopoiesis mediated through NP-1. To test this hypothesis, we set up BM adipocytes differentiated into fibroblast-like fat cells (FLFC), which share the major characteristics of primitive unilocular fat cells, as an experimental model. As expected, FLFCs constitutively produced macrophage colony stimulating factor and induced CD34(+) differentiation into macrophages independently of cell-to-cell contact. By contrast, granulopoiesis was hampered by cell-to-cell contact but could be restored in transwell culture conditions, together with granulocyte colony stimulating factor production. Both functions were also recovered when FLFCs cultured in contact with CD34(+) cells were treated with an antibody neutralizing NP-1, which proved its critical implication in contact inhibition. An inflammatory cytokine such as interleukin-1 beta or dexamethasone modulates FLFC properties to restore granulopoiesis. Our data provide the first evidence that primary adipocytes exert regulatory functions during hematopoiesis that might be implicated in some pathological processes. Disclosure of potential conflicts of interest is found at the end of this article.
Assuntos
Adipócitos/fisiologia , Células da Medula Óssea/citologia , Fator Estimulador de Colônias de Granulócitos/antagonistas & inibidores , Neuropilina-1/fisiologia , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Animais , Antígenos CD/fisiologia , Antígenos CD34/fisiologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/fisiologia , Proteínas de Ligação ao Cálcio , Diferenciação Celular , Primers do DNA , Regulação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Macrófagos/citologia , Macrófagos/fisiologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
An adequate balance between serine proteases and their plasminogen activator inhibitor-1 (PAI-1) is critical for pathological angiogenesis. PAI-1 deficiency in mice is associated with impaired choroidal neovascularization (CNV) and tumoral angiogenesis. In the present work, we demonstrate unexpected differences in the contribution of bone marrow (BM)-derived cells in these two processes regulated by PAI-1. PAI-1(-/-) mice grafted with BM-derived from wild-type mice were able to support laser-induced CNV formation but not skin carcinoma vascularization. Engraftment of irradiated wild-type mice with PAI-1(-/-) BM prevented CNV formation, demonstrating the crucial role of PAI-1 delivered by BM-derived cells. In contrast, the transient infiltration of tumor transplants by local PAI-1-producing host cells rather than by BM cells was sufficient to rescue tumor growth and angiogenesis in PAI-1-deficient mice. These data identify PAI-1 as a molecular determinant of a local permissive soil for tumor angiogenesis. Altogether, the present study demonstrates that different cellular mechanisms contribute to PAI-1-regulated tumoral and CNV. PAI-1 contributes to BM-dependent choroidal vascularization and to BM-independent tumor growth and angiogenesis.
Assuntos
Células da Medula Óssea/fisiologia , Carcinoma/irrigação sanguínea , Neovascularização de Coroide/etiologia , Neovascularização Patológica/etiologia , Inibidor 1 de Ativador de Plasminogênio/fisiologia , Neoplasias Cutâneas/irrigação sanguínea , Animais , Transplante de Medula Óssea , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Queratinócitos/patologia , Queratinócitos/transplante , Camundongos , Camundongos Transgênicos , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Inibidor 1 de Ativador de Plasminogênio/genéticaRESUMO
The study of the human hematopoietic system would be facilitated by availability of a relevant animal model. Because the medullar microenvironment is made of different types of cells, interactions between hematopoietic cells and stromal cells are difficult to analyze in detail. As an approach for establishing an in vivo model to dissect these interactions, we grafted murine bone marrow fibroblastic cells (MS-5 cell line) with hematopoietic cells into the kidney capsule of syngenic mice. To identify the origin of cells present in the graft, we used green fluorescent protein-stable transfected MS-5 cells for the transplantation. To analyze the evolution of stromal cells and identify hematopoietic cells able to develop in these conditions, we performed morphology, histochemistry, and immunohistology on tissue sections at different times after transplantation. When injected alone, MS-5 cells differentiate into adipocytes. When injected with a bone marrow suspension or with isolated CD45+ cells (leukocytes), the stromal cells keep their fibroblastic morphology and their alkaline phosphatase expression and sustain granulopoiesis. When injected with hematopoietic stem cells called c-kit+ Sca-1+ Lin- suspension, clusters of hematopoietic cells are also observed: They do not present any granulopoietic activity and do not belong to B or T population nor to erythroid lineage. They are quiescent, induce bone marrow recovery and survival of lethally irradiated recipients, are able to form macroscopic colonies in the spleen, and are able to form very few colonies in vitro, suggesting that they are hematopoietic stem cells. In conclusion, our results show that reticular fibroblastic stromal cells MS-5 sustain the survival of stem cells and are not able to induce their differentiation. However, they can control differentiation, proliferation, and/or survival of hematopoietic cells engaged in myeloid lineage.
Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular , Fibroblastos/citologia , Granulócitos/citologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células Estromais/citologia , Adipócitos/citologia , Animais , Linhagem Celular , Sobrevivência Celular , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/transplante , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Imuno-Histoquímica , Camundongos , Células Estromais/transplante , Fatores de TempoRESUMO
Vascular endothelial growth factor (VEGF), its receptors (VEGFR-1, VEGFR-2) and neuropillin-1 (NRP-1) are expressed at variable levels in bone marrow. NRP-1expression is higher in fatty bone marrow than in hematopoietic marrow. Adipocytes are responsible for NRP-1 expression suggesting that they may play a role in hematopoiesis by producing NRP-1 or that NRP-1 may regulate adipocyte activity.
Assuntos
Adipócitos/metabolismo , Neuropilina-1/biossíntese , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/genética , Medula Óssea , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , HumanosRESUMO
Attempts were made to establish models to study interactions between marrow stromal cells and hematopoietic cells in vivo. The approach was to create a NOD-SCID-hu murine model of long-term human hematopoiesis by implantation of a human adult bone fragment. Nine to 12 weeks posttransplantation, human CD45+ cells were detected in the blood and the spleen of some mice. The histology of the human transplant showed that human bone fragment was viable at 9 weeks. Moreover, vessels of human origin, as assessed by immunohistochemical detection of human beta2-microglobulin, were observed in the mouse tissue surrounding the transplanted human fragment.
Assuntos
Transplante de Medula Óssea/métodos , Transplante Ósseo/métodos , Hematopoese/imunologia , Células-Tronco Hematopoéticas/imunologia , Hospedeiro Imunocomprometido/fisiologia , Tolerância ao Transplante/imunologia , Transplante Heterólogo/métodos , Adolescente , Adulto , Animais , Transplante de Medula Óssea/imunologia , Transplante Ósseo/imunologia , Comunicação Celular/imunologia , Sobrevivência Celular/imunologia , Humanos , Antígenos Comuns de Leucócito/imunologia , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Modelos Animais , Baço/citologia , Baço/imunologia , Células Estromais/citologia , Células Estromais/imunologia , Transplante Heterólogo/imunologia , Microglobulina beta-2/imunologiaRESUMO
This study investigates the involvement of the c-cbl proto-oncogene during the first stages of the apoptotic process. We have already shown that a c-Cbl aptotosis-related protein of 90 kDa (CARP 90) is detected very rapidly in the cytoplasm as well as in the nucleus of murine thymocytes after hydrocortisone (HC) treatment. We report here that this protein appeared as well after in vivo treatment of mice by gamma-irradiation or injection of anti-CD3 monoclonal antibody, two potent thymic apoptosis inductors, providing a close relationship between the occurrence of apoptosis and the appearance of CARP 90. We showed that CARP 90 and p120(cbl) share numerous epitopes strikingly suggesting that CARP 90 is coded by c-cbl. In addition, KO mice do not sustain CARP 90 appearance. We finally showed that CARP 90 contains N- and C-terminal end epitopes of p120(cbl), which suggests that CARP 90 is an alternative spliced form of c-cbl. This protein was also observed under gamma-irradiation in tissues of different origin, which enlarges the physiological significance of this phenomenon. The very rapid CARP 90 appearance under apoptotic conditions in the nucleus of cells originating in different tissues makes this protein if not a possible new actor of the apoptotic process, at least an interesting marker of this process.
Assuntos
Apoptose , Proteínas Proto-Oncogênicas/metabolismo , Timo/patologia , Ubiquitina-Proteína Ligases , Animais , Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Western Blotting , Complexo CD3/imunologia , Epitopos/imunologia , Raios gama , Células HeLa/efeitos dos fármacos , Células HeLa/metabolismo , Células HeLa/efeitos da radiação , Humanos , Hidrocortisona/farmacologia , Células Jurkat/efeitos dos fármacos , Células Jurkat/metabolismo , Células Jurkat/efeitos da radiação , Camundongos , Camundongos Knockout , Testes de Precipitina , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-cbl , Timo/metabolismoRESUMO
The evolution of mitochondrial oxidative phosphorylation was studied during cancer induction in a model of thymic radiolymphomagenesis in C57BL/Ka mice. During the preneoplastic period, thymuses displayed an increase of the cytochrome c oxidase activity and oxygen consumption together with oxidative DNA damage assessed by the presence of the 8-hydroxydeoxyguanine DNA base modification. These transient changes in mitochondrial functional activity were not observed in thymuses of mice rescued from lymphoma development by a bone marrow graft, suggesting an important role of mitochondria for neoplastic transformation in this model, which might therefore be of interest to test the utilization of antioxidants for the prevention of radiation-induced malignancies.
Assuntos
Desoxiguanosina/análogos & derivados , Leucemia Induzida por Radiação/metabolismo , Linfoma/metabolismo , Neoplasias do Timo/metabolismo , 8-Hidroxi-2'-Desoxiguanosina , Animais , Transplante de Medula Óssea , Respiração Celular , Transformação Celular Neoplásica , Desoxiguanosina/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Citometria de Fluxo , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Estresse Oxidativo/fisiologia , Consumo de Oxigênio , Pré-Leucemia/metabolismo , Timo/efeitos da radiação , Regulação para Cima , Irradiação Corporal TotalRESUMO
OBJECTIVES: To investigate the longterm effects (12 days) of nonsteroidal antiinflammatory drugs [NSAID: aceclofenac (ACECLO), sodium diclofenac (DICLO), indomethacin (INDO), nimesulide (NIM), rofecoxib (ROFE), celecoxib (CELE), piroxicam (PIROX), and ibuprofen (IBUP)] on the metabolism of human chondrocytes cultured in alginate beads. METHODS: Enzymatically isolated osteoarthritic (OA) chondrocytes were cultured in alginate beads in a well defined culture medium for 12 days. The DNA content was measured according to a fluorimetric method and cell proliferation was determined by the incorporation of 3H-thymidine in the newly synthesized DNA. Interleukin 6 (IL-6) and IL-8, stromelysin [matrix metalloproteinase-3 (MMP-3)], and aggrecan (AGG) production were assayed by specific enzyme amplified sensitivity immunoassays, and prostaglandin E2 (PGE2) production by specific radioimmunoassay. All NSAID were tested at the mean peak plasma concentration (Cmax) obtained after oral administration of a therapeutic dose. RESULTS: In alginate beads, chondrocytes synthesized high amounts of AGG, which were largely (98%) immobilized in the alginate matrix. A large amount (43%) of the IL-8 produced was stored in the alginate beads, whereas almost all IL-6 production (94%) was released in the culture supernatant. At the therapeutic concentration, all NSAID tested fully blocked PGE2 production. ACECLO, DICLO, INDO, NIM significantly inhibited basal and IL-1beta stimulated IL-6 production; CELE and IBUP only inhibited IL-1beta stimulated IL-6 production; and ROFE and PIROX had no significant effects. No NSAID showed significant effects on basal and IL-1beta stimulated IL-8 production, except CELE and IBUP, which slightly increased basal IL-8 production. ACECLO and INDO increased AGG content by 25% in the alginate beads, while the other NSAID were without significant effect. No NSAID were able to modify the inhibitory effect of IL-1beta on AGG production. NSAID did not modify MMP-3 production. CONCLUSION: The mechanism of action of NSAID seems to be multifactorial and not limited to the inhibition of cyclooxygenases. Further, in our culture conditions, at the Cmax and by comparison with other NSAID, ACECLO and INDO show an advantageous activity profile. They fully blocked PGE2 production, inhibited IL-6 synthesis, and increased aggrecan synthesis. These effects appear advantageous for the longterm treatment of chronic joint diseases such as osteoarthritis.