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1.
Artigo em Inglês | MEDLINE | ID: mdl-31756538

RESUMO

Polyphenols are a group of chemical substances found in plants, with immunomodulatory, antiproliferative, and anti-inflammatory properties that might be useful in the prophylaxis and treatment of graft-versus-host disease (GVHD). Polyphenolic extract (PE) obtained from extra virgin olive oil (EVOO) decreased the activation and proliferation of activated T cells. In addition, a decreased production of proinflammatory cytokines was observed upon exposure to PE. Western blot assays showed a marked inhibition of Akt phosphorylation and nuclear translocation of NF-κB in activated T cells. In a murine model of acute GVHD, we observed that mice that received a diet supplemented in PE (600 ppm) presented a higher survival rate and lower risk of developing GVHD when compared with the group that received a control diet. Histopathologic examination showed a significantly lower gut involvement in mice receiving PE, with a decrease in proinflammatory cytokines (IL-2, IL-17, and TNF-α) in serum and the reestablishment of butyrate concentration in the gut. In conclusion, PE obtained from EVOO exerted a potent immunomodulatory effect, reducing the activation and proliferation of activated T cells and the production of proinflammatory cytokines. In a murine model of acute GVHD, a PE-supplemented diet reduced the incidence and severity of the disease and increased survival after transplantation.

2.
Stem Cells ; 37(10): 1357-1368, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31184411

RESUMO

Mesenchymal stromal cells (MSC) may exert their functions by the release of extracellular vesicles (EV). Our aim was to analyze changes induced in CD34+ cells after the incorporation of MSC-EV. MSC-EV were characterized by flow cytometry (FC), Western blot, electron microscopy, and nanoparticle tracking analysis. EV incorporation into CD34+ cells was confirmed by FC and confocal microscopy, and then reverse transcription polymerase chain reaction and arrays were performed in modified CD34+ cells. Apoptosis and cell cycle were also evaluated by FC, phosphorylation of signal activator of transcription 5 (STAT5) by WES Simple, and clonal growth by clonogenic assays. Human engraftment was analyzed 4 weeks after CD34+ cell transplantation in nonobese diabetic/severe combined immunodeficient mice. Our results showed that MSC-EV incorporation induced a downregulation of proapoptotic genes, an overexpression of genes involved in colony formation, and an activation of the Janus kinase (JAK)-STAT pathway in CD34+ cells. A significant decrease in apoptosis and an increased CD44 expression were confirmed by FC, and increased levels of phospho-STAT5 were confirmed by WES Simple in CD34+ cells with MSC-EV. In addition, these cells displayed a higher colony-forming unit granulocyte/macrophage clonogenic potential. Finally, the in vivo bone marrow lodging ability of human CD34+ cells with MSC-EV was significantly increased in the injected femurs. In summary, the incorporation of MSC-EV induces genomic and functional changes in CD34+ cells, increasing their clonogenic capacity and their bone marrow lodging ability. Stem Cells 2019;37:1357-1368.

3.
Nutrients ; 11(5)2019 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-31109078

RESUMO

Stroke is one of the leading causes of death worldwide and while there is increasing evidence that a Mediterranean diet might decrease the risk of a stroke, the effects of dietary fat composition on stroke outcomes have not been fully explored. We hypothesize that the brain damage provoked by a stroke would be different depending on the source of dietary fat. To test this, male C57BL/6J mice were fed for 4 weeks with a standard low-fat diet (LFD), a high-fat diet (HFD) rich in saturated fatty acids (HFD-SFA), an HFD containing monounsaturated fatty acids (MUFAs) from olive oil (HFD-OO), or an HFD containing MUFAs from olive oil plus polyunsaturated fatty acids (PUFAs) docosahexaenoic acid/eicosapentaenoic acid (DHA/EPA) (HFD-OO-ω3). These mice were then subjected to transient middle cerebral artery occlusion (tMCAo). Behavioural tests and histological analyses were performed 24 and/or 48 h after tMCAo in order to elucidate the impact of these diets with different fatty acid profiles on the ischemic lesion and on neurological functions. Mice fed with HFD-OO-ω3 displayed better histological outcomes after cerebral ischemia than mice that received an HFD-SFA or LFD. Furthermore, PUFA- and MUFA-enriched diets improved the motor function and neurological performance of ischemic mice relative to those fed with an LFD or HFD-SFA. These findings support the use of DHA/EPA-omega-3-fatty acid supplementation and olive oil as dietary source of MUFAs in order to reduce the damage and protect the brain when a stroke occurs.


Assuntos
Isquemia Encefálica/tratamento farmacológico , Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Azeite de Oliva/farmacologia , Animais , Antioxidantes/metabolismo , Comportamento Animal , Dieta Hiperlipídica/efeitos adversos , Ácidos Docosa-Hexaenoicos/administração & dosagem , Ingestão de Alimentos , Ácido Eicosapentaenoico/administração & dosagem , Marcha , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Artéria Cerebral Média , Azeite de Oliva/administração & dosagem , Perda de Peso/efeitos dos fármacos
4.
Exp Hematol ; 72: 60-71.e11, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30769020

RESUMO

BCR-ABL-negative myeloproliferative neoplasms (MPNs) are driven by JAK-STAT pathway activation, but epigenetic alterations also play an important pathophysiological role. These can be pharmacologically manipulated with histone deacetylase inhibitors (HDACIs), which have proven to be clinically effective in the treatment of MPNs but exhibit dose-limiting toxicity. The treatment of primary MPN cells with vorinostat modulates the expression of genes associated with apoptosis, cell cycle, inflammation, and signaling. The induction of this transcriptional program results in decreased cellular viability, paralleled by a decrease in levels of reactive oxygen species (ROS). In vitro manipulation of ROS levels revealed that the reduction of ROS levels promoted apoptosis. When vorinostat was combined with antioxidant agents, the apoptosis of MPN cells increased in a synergistic manner. The results described here suggest a novel and promising therapeutic strategy combining HDACIs with ROS-reducing agents to treat MPNs.


Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Hematológicas/tratamento farmacológico , Transtornos Mieloproliferativos/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismo , Vorinostat/farmacologia , Adulto , Idoso , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos Mieloproliferativos/metabolismo , Transtornos Mieloproliferativos/patologia , Transdução de Sinais/efeitos dos fármacos , Vorinostat/agonistas
5.
Front Immunol ; 9: 1971, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30233577

RESUMO

The relevance of the immune system in cancer has long been studied. Autologous adoptive T cell therapies, based on the use of tumor infiltrating lymphocytes (TILs), have made great progress in recent years for the treatment of solid tumors, especially melanoma. However, further work is needed to isolate tumor-reactive T cells among patients diagnosed with hematologic malignancies. The dynamics of the interaction between T cells and antigen presenting cells (APC) dictate the quality of the immune responses. While stable joints between target cells and T lymphocytes lead to the induction of T cell activation and immune response, brief contacts contribute to the induction of immune-tolerance. Taking advantage of the strong interaction between target cell and activated T-cells, we show the feasibility to identify and isolate tumor-specific cytotoxic T lymphocytes (CTLs) from acute myeloid leukemia (AML) patients by flow cytometry. Using this technology, CTLs bound through T cell receptor (TCR) to tumor cells can be identified in peripheral blood and bone marrow and subsequently selected and isolated by FACS-based cell sorting. These CTLs display higher percentage of effector cells and marked cytotoxic activity against AML blasts. In conclusion, we have developed a new procedure to identify and select specific cytotoxic T cells in patients diagnosed with acute myeloid leukemia.


Assuntos
Vacinas Anticâncer/imunologia , Citometria de Fluxo/métodos , Imunoterapia Adotiva/métodos , Leucemia Mieloide Aguda/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos T Citotóxicos/imunologia , Apresentação do Antígeno , Antígenos de Neoplasias/imunologia , Separação Celular , Células Cultivadas , Citotoxicidade Imunológica , Humanos , Tolerância Imunológica , Vigilância Imunológica , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/transplante , Evasão Tumoral , Microambiente Tumoral
6.
J Tissue Eng Regen Med ; 12(3): e1450-e1460, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28865091

RESUMO

Biocompatibility studies, especially innate immunity induction, in vitro and in vivo cytotoxicity, and fibrosis, are often lacking for many novel biomaterials including recombinant protein-based ones, such as elastin-like recombinamers (ELRs), and has not been extensively explored in the scientific literature, in contrast to traditional biomaterials. Herein, we present the results from a set of experiments designed to elucidate the preliminary biocompatibility of 2 types of ELRs that are able to form extracellular matrix-like hydrogels through either physical or chemical cross-linking both of which are intended for different applications in tissue engineering and regenerative medicine. Initially, we present in vitro cytocompatibility results obtained upon culturing human umbilical vein endothelial cells on ELR substrates, showing optimal proliferation up to 9 days. Regarding in vivo cytocompatibility, luciferase-expressing hMSCs were viable for at least 4 weeks in terms of bioluminescence emission when embedded in ELR hydrogels and injected subcutaneously into immunosuppressed mice. Furthermore, both types of ELR-based hydrogels were injected subcutaneously in immunocompetent mice and serum TNFα, IL-1ß, IL-4, IL-6, and IL-10 concentrations were measured by enzyme-linked immunosorbent assay, confirming the lack of inflammatory response, as also observed upon macroscopic and histological evaluation. All these findings suggest that both types of ELRs possess broad biocompatibility, thus making them very promising for tissue engineering and regenerative medicine-related applications.


Assuntos
Materiais Biocompatíveis/farmacologia , Reagentes para Ligações Cruzadas/farmacologia , Elastina/farmacologia , Hidrogéis/farmacologia , Proteínas Recombinantes/farmacologia , Medicina Regenerativa/métodos , Engenharia Tecidual/métodos , Animais , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Rastreamento de Células , Citocinas/sangue , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamação/patologia , Injeções Subcutâneas , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos
7.
Biol Blood Marrow Transplant ; 24(3): 443-451, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29155314

RESUMO

Bone marrow mesenchymal stromal cells (MSCs) are precursors of adipocytes and osteoblasts and key regulators of hematopoiesis. Irradiation is widely used in conditioning regimens. Although MSCs are radio-resistant, the effects of low-dose irradiation on their behavior have not been extensively explored. Our aim was to evaluate the effect of 2.5 Gy on MSCs. Cells from 25 healthy donors were either irradiated or not (the latter were used as controls). Cells were characterized following International Society for Cellular Therapy criteria, including in vitro differentiation assays. Apoptosis was evaluated by annexin V/7-amino-actinomycin staining. Gene expression profiling and reverse transcriptase (RT)-PCR of relevant genes was also performed. Finally, long-term bone marrow cultures were performed to test the hematopoietic-supporting ability. Our results showed that immunophenotypic characterization and viability of irradiated cells was comparable with that of control cells. Gene expression profiling showed 50 genes differentially expressed. By RT-PCR, SDF-1 and ANGPT were overexpressed, whereas COL1A1 was downregulated in irradiated cells (P = .015, P = .007, and P = .031, respectively). Interestingly, differentiation of irradiated cells was skewed toward osteogenesis, whereas adipogenesis was impaired. Higher expression of genes involved in osteogenesis as SPP1 (P = .039) and lower of genes involved in adipogenesis, CEBPA and PPARG (P = .003 and P = .019), together with an increase in the mineralization capacity (Alizarin Red) was observed in irradiated cells. After differentiation, adipocyte counts were decreased in irradiated cells at days 7, 14, and 21 (P = .018 P = .046, and P = .018, respectively). Also, colony-forming unit granulocyte macrophage number in long-term bone marrow cultures was significantly higher in irradiated cells after 4 and 5 weeks (P = .046 and P = .007). In summary, the irradiation of MSCs with 2.5 Gy improves their hematopoietic-supporting ability by increasing osteogenic differentiation and decreasing adipogenesis.


Assuntos
Adipogenia/efeitos da radiação , Diferenciação Celular/efeitos da radiação , Raios gama , Hematopoese/efeitos da radiação , Células-Tronco Mesenquimais/metabolismo , Osteogênese/efeitos da radiação , Adulto , Idoso , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/patologia , Pessoa de Meia-Idade
8.
PLoS One ; 12(8): e0182470, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28796790

RESUMO

There is evidence of continuous bidirectional cross-talk between malignant cells and bone marrow-derived mesenchymal stromal cells (BM-MSC), which favors the emergence and progression of myeloproliferative neoplastic (MPN) diseases. In the current work we have compared the function and gene expression profile of BM-MSC from healthy donors (HD-MSC) and patients with MPN (JAK2V617F), showing no differences in the morphology, proliferation and differentiation capacity between both groups. However, BM-MSC from MPN expressed higher mean fluorescence intensity (MIF) of CD73, CD44 and CD90, whereas CD105 was lower when compared to controls. Gene expression profile of BM-MSC showed a total of 169 genes that were differentially expressed in BM-MSC from MPN patients compared to HD-MSC. In addition, we studied the ability of BM-MSC to support the growth and survival of hematopoietic stem/progenitor cells (HSPC), showing a significant increase in the number of CFU-GM colonies when MPN-HSPC were co-cultured with MPN-MSC. Furthermore, MPN-MSC showed alteration in the expression of genes associated to the maintenance of hematopoiesis, with an overexpression of SPP1 and NF-kB, and a downregulation of ANGPT1 and THPO. Our results suggest that BM-MSC from JAK2+ patients differ from their normal counterparts and favor the maintenance of malignant clonal hematopoietic cells.


Assuntos
Neoplasias Hematológicas/patologia , Janus Quinase 2/metabolismo , Células-Tronco Mesenquimais/fisiologia , Adulto , Idoso , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Técnicas de Cocultura , Feminino , Expressão Gênica , Neoplasias Hematológicas/sangue , Neoplasias Hematológicas/enzimologia , Hematopoese , Humanos , Masculino , Pessoa de Meia-Idade
9.
Oncotarget ; 8(17): 28187-28202, 2017 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-28390197

RESUMO

Histone deacetylases (HDACs) are involved in epigenetic modulation and their aberrant expression has been demonstrated in myeloproliferative neoplasms (MPN). HDAC8 inhibition has been shown to inhibit JAK2/STAT5 signaling in hematopoietic cells from MPN. Nevertheless, the role of HDAC8 expression in bone marrow-mesenchymal stromal cells (BM-MSC) has not been assessed. In the current work we describe that HDAC8 is significantly over-expressed in MSC from in JAK-2 positive MPN compared to those from healthy-donors (HD-MSC). Using a selective HDAC8 inhibitor (PCI34051), we verified that the subsequent decrease in the protein and mRNA expression of HDAC8 is linked with an increased apoptosis of malignant MSC whereas it has no effects on normal MSC. In addition, HDAC8 inhibition in MPN-MSC also decreased their capacity to maintain neoplastic hematopoiesis, by increasing the apoptosis, cell-cycle arrest and colony formation of JAK2+-hematopoietic cells. Mechanistic studies using different MPN cell lines revealed that PCI34051 induced their apoptosis, which is enhanced when were co-cultured with JAK2V617F-MSC, decreased their colony formation and the phosphorylation of STAT3 and STAT5. In summary, we show for the first time that the inhibition of HDAC8 in MSC from JAK2+ MPN patients selectively decreases their hematopoietic-supporting ability, suggesting that HDAC8 may be a potential therapeutic target in this setting by acting not only on hematopoietic cells but also on the malignant microenvironment.


Assuntos
Histona Desacetilases/genética , Janus Quinase 2/metabolismo , Células-Tronco Mesenquimais/metabolismo , Transtornos Mieloproliferativos/genética , Proteínas Repressoras/genética , Apoptose/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Expressão Gênica , Hematopoese/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Humanos , Janus Quinase 2/genética , Células-Tronco Mesenquimais/efeitos dos fármacos , Terapia de Alvo Molecular , Mutação , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo
10.
Exp Eye Res ; 154: 64-69, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27818317

RESUMO

Dry eye disease is one of the most frequent pathological events that take place in the course of the graft versus host disease (GVHD), and is the main cause of deterioration in quality of life for patients. Thus, demonstration of dry eye signs in murine models of oGVHD is crucial for the validation of these models for the study of the disease. Given the increasing evidence that tear osmolarity is an important player of dry eye disease, our purpose in this study was to validate the use of a reliable method to assess tear osmolarity in mice: the electrical impedance method. Then, we wanted to test its utility with an oGVHD model. Tear volume assessment was also performed, using the phenol red thread test. We found differences in tear osmolarity in mice that received a transplant with cells from bone marrow and spleen (the GVHD group) when compared with mice that only received bone marrow cells (the BM group) at day 7 (362 ± 8 mOsm/l and 345 ± 9 mOsm/l respectively; P < 0.01) and day 21 (348 ± 19 mOsm/l vs. 326 ± 15 mOsm/l; P < 0.05). We found also differences in tear volume at day 14 (2.30 ± 0.61 mm in oGVHD group and 2.89 ± 0.62 mm in BM group; P = 0.06) and at day 21 (2.10 ± 0.30 mm in oGVHD group and 2.89 ± 0.32 mm in BM group; P < 0.01). Besides this, we observed reduction in epithelial thickness between the GVHD and BM groups (37.0 ± 6.2 µm and 43.6 ± 3.3 µm respectively; P < 0.05). These data show the usefulness of the electrical impedance method to measure tear osmolarity in mice. We can also conclude that this oGVHD model mimics the tear film alterations found in human dry eye disease, what contributes to give relevance to this model for the study of GVHD.


Assuntos
Síndromes do Olho Seco/diagnóstico , Epitélio Anterior/metabolismo , Doença Enxerto-Hospedeiro/diagnóstico , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Lágrimas/metabolismo , Animais , Modelos Animais de Doenças , Síndromes do Olho Seco/etiologia , Síndromes do Olho Seco/metabolismo , Epitélio Anterior/patologia , Doença Enxerto-Hospedeiro/complicações , Doença Enxerto-Hospedeiro/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Concentração Osmolar
11.
PLoS One ; 11(2): e0146722, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26836120

RESUMO

Exosomes/microvesicles (MVs) provide a mechanism of intercellular communication. Our hypothesis was that mesenchymal stromal cells (MSC) from myelodysplastic syndrome (MDS) patients could modify CD34+ cells properties by MVs. They were isolated from MSC from MDS patients and healthy donors (HD). MVs from 30 low-risk MDS patients and 27 HD were purified by ExoQuick-TC™ or ultracentrifugation and identified by transmission electron microscopy, flow cytometry (FC) and western blot for CD63. Incorporation of MVs into CD34+ cells was analyzed by FC, and confocal and fluorescence microscopy. Changes in hematopoietic progenitor cell (HPC) properties were assessed from modifications in microRNAs and gene expression in CD34+ cells as well as viability and clonogenic assays of CD34+ cells after MVs incorporation. Some microRNAs were overexpressed in MVs from patients MSC and two of them, miR-10a and miR-15a, were confirmed by RT-PCR. These microRNAs were transferred to CD34+ cells, modifying the expression of MDM2 and P53 genes, which was evaluated by RT-PCR and western blot. Finally, examining CD34+ cells properties after incorporation, higher cell viability (p = 0.025) and clonogenic capacity (p = 0.037) were observed when MVs from MDS patients were incorporated. In summary, we show that BM-MSC release MVs with a different cargo in MDS patients compared with HD. These structures are incorporated into HPC and modify their properties.


Assuntos
Comunicação Celular , Exossomos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Síndromes Mielodisplásicas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD34 , Sobrevivência Celular , Microambiente Celular , Feminino , Expressão Gênica , Células-Tronco Hematopoéticas/imunologia , Humanos , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/genética
12.
Stem Cell Reports ; 5(4): 516-31, 2015 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-26344907

RESUMO

The regulation of hematopoietic stem cells (HSCs) depends on the integration of the multiple signals received from the bone marrow niche. We show the relevance of the protein tyrosine phosphatase PTPN13 and ß-catenin as intracellular signaling molecules to control HSCs adhesiveness, cell cycling, and quiescence. Lethally irradiated mice transplanted with Lin(-) bone marrow cells in which PTPN13 or ß-catenin had been silenced showed a significant increase of long-term (LT) and short-term (ST) HSCs. A decrease in cycling cells was also found, together with an increase in quiescence. The decreased expression of PTPN13 or ß-catenin was linked to the upregulation of several genes coding for integrins and several cadherins, explaining the higher cell adhesiveness. Our data are consistent with the notion that the levels of PTPN13 and ß-catenin must be strictly regulated by extracellular signaling to regulate HSC attachment to the niche and the balance between proliferation and quiescence.


Assuntos
Células da Medula Óssea/citologia , Células-Tronco Hematopoéticas/citologia , Linfopoese , Proteína Tirosina Fosfatase não Receptora Tipo 13/metabolismo , Trombopoese , beta Catenina/metabolismo , Animais , Células da Medula Óssea/metabolismo , Adesão Celular , Comunicação Celular , Linhagem Celular , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteína Tirosina Fosfatase não Receptora Tipo 13/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Nicho de Células-Tronco , beta Catenina/genética
13.
Leuk Res ; 39(8): 921-4, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26105049

RESUMO

The expression of BCR-ABL in hematopoietic stem cells is a well-defined primary event in chronic myeloid leukemia (CML). Some reports have described the presence of BCR-ABL on endothelial cells from CML patients, suggesting the origin of the disease in a primitive hemangioblastic cell. On the other hand, extracellular vesicles (EVs) released by CML leukemic cells are involved in the angiogenesis modulation process. In the current work we hypothesized that EVs released from BCR-ABL(+) cells may carry inside the oncogene that can be transferred to endothelial cells leading to the expression of both BCR-ABL transcript and the oncoprotein. EVs from K562 cells and plasma of newly diagnosed CML patients were isolated by ultracentrifugation. RT-PCR analysis detected the presence of BCR-ABL RNA in the EVs isolated from both K562 cells and plasma of CML patients. The incorporation of these EVs into endothelial cells was demonstrated by flow cytometry and fluorescence microscopy showed that after 24h of incubation most EVs were incorporated. BCR-ABL transcripts were detected in all experiments on endothelial cells incubated with EVs from both sources. The presence of BCR-ABL on endothelial cells incubated with Philadelphia(+) EVs was also confirmed by Western blot assays. In summary, endothelial cells acquire BCR-ABL RNA and the oncoprotein after incubation with EVs released from Ph(+) positive cells (either from K562 cells or from plasma of newly diagnosed CML patients). This results challenge the hypothesis that endothelial cells may be part of the Philadelphia(+) clone in CML.


Assuntos
Células-Tronco Hematopoéticas/fisiologia , Células Endoteliais da Veia Umbilical Humana/fisiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Células-Tronco Neoplásicas/patologia , Vesículas Secretórias/fisiologia , Células Cultivadas , Células Clonais/metabolismo , Células Clonais/patologia , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Células-Tronco Hematopoéticas/patologia , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo
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