RESUMEN
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.
Asunto(s)
Adipocitos/citología , Adipocitos/efectos de la radiación , Médula Ósea/efectos de la radiación , Osteoblastos/citología , Osteoblastos/efectos de la radiación , Irradiación Corporal Total , Adipocitos/enzimología , Fosfatasa Alcalina/metabolismo , Animales , Biomarcadores/metabolismo , Comunicación Celular/efectos de la radiación , Diferenciación Celular/efectos de la radiación , Movimiento Celular/efectos de la radiación , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Quimiocina CXCL12/metabolismo , Ensayo de Unidades Formadoras de Colonias , Regulación hacia Abajo/efectos de la radiación , Fémur/efectos de la radiación , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/efectos de la radiación , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/efectos de la radiación , Ratones , Neuropilina-1/metabolismo , Neutrófilos/citología , Neutrófilos/metabolismo , Neutrófilos/efectos de la radiación , Osteoblastos/enzimología , FenotipoRESUMEN
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.
Asunto(s)
Apoptosis/efectos de los fármacos , Leptina/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Ácido alfa-Linolénico/farmacología , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Médula Ósea/metabolismo , Médula Ósea/patología , Caspasas/metabolismo , Comunicación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Técnicas de Cocultivo , Medios de Cultivo/farmacología , Activación Enzimática/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Leptina/biosíntesis , Metabolismo de los Lípidos/efectos de los fármacos , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores de Leptina/metabolismoRESUMEN
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.
Asunto(s)
Ciclo Celular/fisiología , Células Madre Hematopoyéticas/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Receptores Acoplados a Proteínas G/fisiología , Receptores Histamínicos/fisiología , Animales , Proliferación Celular , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Silenciador del Gen , Células Madre Hematopoyéticas/citología , Humanos , Ratones , Receptores Histamínicos H4 , Transducción de SeñalRESUMEN
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.
Asunto(s)
Adipocitos/fisiología , Células de la Médula Ósea/citología , Factor Estimulante de Colonias de Granulocitos/antagonistas & inhibidores , Neuropilina-1/fisiología , Adipocitos/citología , Adipocitos/efectos de los fármacos , Animales , Antígenos CD/fisiología , Antígenos CD34/fisiología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/fisiología , Proteínas de Unión al Calcio , Diferenciación Celular , Cartilla de ADN , Regulación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Macrófagos/citología , Macrófagos/fisiología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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.
Asunto(s)
Células de la Médula Ósea/fisiología , Carcinoma/irrigación sanguínea , Neovascularización Coroidal/etiología , Neovascularización Patológica/etiología , Inhibidor 1 de Activador Plasminogénico/fisiología , Neoplasias Cutáneas/irrigación sanguínea , Animales , Trasplante de Médula Ósea , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Queratinocitos/patología , Queratinocitos/trasplante , Ratones , Ratones Transgénicos , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Inhibidor 1 de Activador Plasminogénico/genéticaRESUMEN
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.
Asunto(s)
Células de la Médula Ósea/citología , Diferenciación Celular , Fibroblastos/citología , Granulocitos/citología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células del Estroma/citología , Adipocitos/citología , Animales , Línea Celular , Supervivencia Celular , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/trasplante , Proteínas Fluorescentes Verdes/metabolismo , Células Madre Hematopoyéticas/metabolismo , Inmunohistoquímica , Ratones , Células del Estroma/trasplante , Factores de TiempoRESUMEN
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.
Asunto(s)
Adipocitos/metabolismo , Neuropilina-1/biosíntesis , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/genética , Médula Ósea , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , HumanosRESUMEN
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.
Asunto(s)
Trasplante de Médula Ósea/métodos , Trasplante Óseo/métodos , Hematopoyesis/inmunología , Células Madre Hematopoyéticas/inmunología , Huésped Inmunocomprometido/fisiología , Tolerancia al Trasplante/inmunología , Trasplante Heterólogo/métodos , Adolescente , Adulto , Animales , Trasplante de Médula Ósea/inmunología , Trasplante Óseo/inmunología , Comunicación Celular/inmunología , Supervivencia Celular/inmunología , Humanos , Antígenos Comunes de Leucocito/inmunología , Ratones , Ratones SCID , Persona de Mediana Edad , Modelos Animales , Bazo/citología , Bazo/inmunología , Células del Estroma/citología , Células del Estroma/inmunología , Trasplante Heterólogo/inmunología , Microglobulina beta-2/inmunologíaRESUMEN
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.
Asunto(s)
Apoptosis , Proteínas Proto-Oncogénicas/metabolismo , Timo/patología , Ubiquitina-Proteína Ligasas , Animales , Antiinflamatorios/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Western Blotting , Complejo CD3/inmunología , Epítopos/inmunología , Rayos gamma , Células HeLa/efectos de los fármacos , Células HeLa/metabolismo , Células HeLa/efectos de la radiación , Humanos , Hidrocortisona/farmacología , Células Jurkat/efectos de los fármacos , Células Jurkat/metabolismo , Células Jurkat/efectos de la radiación , Ratones , Ratones Noqueados , Pruebas de Precipitina , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-cbl , Timo/metabolismoRESUMEN
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.
Asunto(s)
Desoxiguanosina/análogos & derivados , Leucemia Inducida por Radiación/metabolismo , Linfoma/metabolismo , Neoplasias del Timo/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Trasplante de Médula Ósea , Respiración de la Célula , Transformación Celular Neoplásica , Desoxiguanosina/metabolismo , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Femenino , Citometría de Flujo , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Estrés Oxidativo/fisiología , Consumo de Oxígeno , Preleucemia/metabolismo , Timo/efectos de la radiación , Regulación hacia Arriba , Irradiación Corporal TotalRESUMEN
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.