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Mitochondria are indispensable in maintaining hematopoietic stem cells (HSCs), and mitochondrial complex II (MCII) has been recognized as a key component of HSCs. However, the physiological role of MCII on long-term hematopoiesis and hematopoietic reconstitution capacity remains unknown. Hence, this study evaluated the impact of MCII dysfunctions on long-term HSC maintenance and hematopoietic homeostasis among conditional transgenic mice with a missense mutation in the succinate dehydrogenase complex subunit C gene (SdhcV69E). HSCs collected from SdhcV69E mice had a higher reactive oxygen species (ROS) accumulation and DNA damage in response to mitochondrial activation. Via the aging stress response, MCII dysfunctions caused decreased white blood cell count with myeloid-skewing property, macrocytic anemia, and thrombocytosis. Moreover, the HSCs of aged SdhcV69E mice exhibited greater ROS accumulation and lower membrane potential. Transplantation-induced replicative stress also caused premature senescent hematopoiesis. Furthermore, accelerated ROS accumulation and profound DNA damage in HSCs were observed in the SdhcV69E-derived cell recipients. The long-term hematopoietic reconstitution capacity was remarkably impaired in HSCs from the SdhcV69E-derived cell recipients. Taken together, MCII plays an essential role in long-term hematopoiesis, and MCII dysfunctions with aging or replicative stresses caused excessive ROS accumulation and DNA damage in HSCs, leading to premature senescence.
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Senilidade Prematura , Camundongos , Animais , Espécies Reativas de Oxigênio/metabolismo , Senilidade Prematura/genética , Senilidade Prematura/metabolismo , Transporte de Elétrons , Células-Tronco Hematopoéticas/metabolismo , Envelhecimento/genética , Camundongos Transgênicos , Hematopoese/genética , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Anti-programmed cell death 1 antibodies (anti-PD-1 Abs) are widely used for advanced melanoma, but the efficacy of an anti-PD-1 Abs is limited in the Asian population. There remains an unmet need to improve the therapeutic effects of anti-PD-1 Abs treatment, particularly in melanoma patients who are refractory to anti-PD-1 Abs. The aim was to evaluate anti-PD-1 Abs treatment in combination with TM5614 (plasminogen activator inhibitor-1: PAI-1 inhibitor) in patients with unresectable melanoma. METHODS: The TM5614-MM study was a multicentre, open-label, single-arm, phase 2 clinical trial to evaluate the efficacy and safety of nivolumab in combination with TM5614 in patients with advanced, unresectable malignant melanoma recruited at 7 Japanese institutes between 13 September 2021 and 31 March 2023. Patients with metastatic or unresectable melanoma previously treated with anti-PD-1 Abs were enrolled. Nivolumab 480 mg was administered intravenously every 4 weeks for 8 weeks, while TM5614 was administered orally at a dose of 120 mg (0-4 weeks) and 180 mg once daily (5-8 weeks). The primary endpoint was the overall response rate after 8 weeks of concomitant use of TM5614. RESULTS: Thirty nine patients were enrolled, and 34 patients in the anti-PD-1 Abs-refractory cohort. The overall response rate at 8 weeks was 25.9% (95% CI: 12.9-44.9%; P = .027) in 27 anti-PD-1-Abs refractory patients by investigator assessment in the protocol per set cohort. Seven patients discontinued treatment due to progressive disease or adverse events. Treatment-related grade 3 or higher adverse events occurred in 3 of 39 patients (7.7%) in the intention-to-treat cohort. CONCLUSIONS: TM5614 in combination with nivolumab is well-tolerated and effective in anti-PD-1 Abs-refractory, unresectable melanoma. TRIAL REGISTRATION: This trial was registered with Clinical Trial gov, jRCT2021210029.
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Bone marrow adipocytes (BMAs) have recently been recognized as a niche component with a suppressive function. Obese individuals with abundant BMAs exhibit impaired hematopoietic regeneration after hematopoietic stem cell transplantation (HSCT). We hypothesized that plasminogen activator inhibitor type-1 (PAI-1), an adipokine that regulates the fibrinolytic system, contributes to impaired hematopoiesis in bone marrow (BM) microenvironment with abundant BMAs. We demonstrated that BMAs differentiated in vitro could secrete PAI-1 and were positive for PAI-1 in vivo. In addition, the abundance of BMAs was associated with high levels of PAI-1 expression. The BMA-rich microenvironment exhibited impaired hematopoietic regeneration after HSCT when compared with a BMA-less microenvironment. The impaired hematopoietic regeneration in BMA-rich microenvironment was significantly alleviated by PAI-1 knockout or PAI-1 inhibitor treatment. Obese mice with abundant BMAs, compared with normal-weight mice, exhibited higher bone marrow PAI-1 concentrations, increased fibrinolytic system suppression, and lower stem cell factor (SCF) concentrations after HSCT. PAI-1 inhibitor administration significantly activated the fibrinolytic system in obese mice, contributing to the higher SCF concentration. Moreover, PAI-1 inhibitor treatment significantly alleviated the impaired hematopoietic regeneration in obese mice both after 5-fluorouracil injection and HSCT. These results indicate that PAI-1 hinders hematopoietic regeneration in BMA-rich microenvironments. The blockade of PAI-1 activity could be a novel therapeutic means of facilitating hematopoietic reconstitution in BMA-rich patients.
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Adipócitos/metabolismo , Medula Óssea/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Obesidade/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Animais , Antimetabólitos/farmacologia , Medula Óssea/metabolismo , Fluoruracila/farmacologia , Técnicas de Inativação de Genes , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/genética , Obesidade/terapia , Inibidor 1 de Ativador de Plasminogênio/genética , Regeneração/efeitos dos fármacos , Fator de Células-Tronco/metabolismo , Nicho de Células-Tronco/efeitos dos fármacosRESUMO
Therapeutic strategies that target leukemic stem cells (LSCs) provide potential advantages in the treatment of chronic myeloid leukemia (CML). Here, we show that selective blockade of plasminogen activator inhibitor-1 (PAI-1) enhances the susceptibility of CML-LSCs to tyrosine kinase inhibitor (TKI), which facilitates the eradication of CML-LSCs and leads to sustained remission of the disease. We demonstrated for the first time that TGF-ß-PAI-1 axis was selectively augmented in CML-LSCs in the bone marrow (BM), whereby protecting CML-LSCs from TKI treatment. Furthermore, the combined administration of TKI plus a PAI-1 inhibitor, in a mouse model of CML, significantly enhanced the eradication of CML cells in the BM and prolonged the survival of CML mice. The combined therapy of imatinib and a PAI-1 inhibitor prevented the recurrence of CML-like disease in serially transplanted recipients, indicating the elimination of CML-LSCs. Interestingly, PAI-1 inhibitor treatment augmented membrane-type matrix metalloprotease-1 (MT1-MMP)-dependent motility of CML-LSCs, and the anti-CML effect of PAI-1 inhibitor was extinguished by the neutralizing antibody for MT1-MMP, underlining the mechanistic importance of MT1-MMP. Our findings provide evidence of, and a rationale for, a novel therapeutic tactic, based on the blockade of PAI-1 activity, for CML patients.
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Proteínas de Fusão bcr-abl , Leucemia Mielogênica Crônica BCR-ABL Positiva , Animais , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Camundongos , Células-Tronco Neoplásicas , Inibidor 1 de Ativador de Plasminogênio , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Serpina E2RESUMO
Epstein-Barr virus (EBV) causes various diseases in the elderly, including B-cell lymphoma such as Hodgkin's lymphoma and diffuse large B-cell lymphoma. Here, we show that EBV acts in trans on noninfected macrophages in the tumor through exosome secretion and augments the development of lymphomas. In a humanized mouse model, the different formation of lymphoproliferative disease (LPD) between 2 EBV strains (Akata and B95-8) was evident. Furthermore, injection of Akata-derived exosomes affected LPD severity, possibly through the regulation of macrophage phenotype in vivo. Exosomes collected from Akata-lymphoblastoid cell lines reportedly contain EBV-derived noncoding RNAs such as BamHI fragment A rightward transcript (BART) micro-RNAs (miRNAs) and EBV-encoded RNA. We focused on the exosome-mediated delivery of BART miRNAs. In vitro, BART miRNAs could induce the immune regulatory phenotype in macrophages characterized by the gene expressions of interleukin 10, tumor necrosis factor-α, and arginase 1, suggesting the immune regulatory role of BART miRNAs. The expression level of an EBV-encoded miRNA was strongly linked to the clinical outcomes in elderly patients with diffuse large B-cell lymphoma. These results implicate BART miRNAs as 1 of the factors regulating the severity of lymphoproliferative disease and as a diagnostic marker for EBV+ B-cell lymphoma.
Assuntos
Infecções por Vírus Epstein-Barr/complicações , Exossomos/virologia , Herpesvirus Humano 4/genética , Inflamação/virologia , Linfoma/virologia , RNA Viral/genética , Animais , Carcinogênese/genética , Carcinogênese/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/virologia , Exossomos/genética , Exossomos/imunologia , Herpesvirus Humano 4/imunologia , Herpesvirus Humano 4/isolamento & purificação , Humanos , Inflamação/etiologia , Inflamação/genética , Inflamação/imunologia , Linfoma/etiologia , Linfoma/genética , Linfoma/imunologia , Camundongos , MicroRNAs/análise , MicroRNAs/genética , RNA Viral/análise , Análise de Sequência de RNA , Microambiente TumoralRESUMO
BACKGROUND: The transmembrane glycoprotein podoplanin (PDPN) is upregulated in some tumors and has gained attention as a malignant tumor biomarker. PDPN molecules have platelet aggregation-stimulating domains and, are therefore, suggested to play a role in tumor-induced platelet activation, which in turn triggers epithelial-to-mesenchymal transition (EMT) and enhances the invasive and metastatic activities of tumor cells. In addition, as forced PDPN expression itself can alter the propensity of certain tumor cells in favor of EMT and enhance their invasive ability, it is also considered to be involved in the cell signaling system. Nevertheless, underlying mechanisms of PDPN in tumor cell invasive ability as well as EMT induction, especially by platelets, are still not fully understood. METHODS: Subclonal TE11A cells were isolated from the human esophageal squamous carcinoma cell line TE11 and the effects of anti-PDPN neutralizing antibody as well as PDPN gene knockout on platelet-induced EMT-related gene expression were measured. Also, the effects of PDPN deficiency on cellular invasive ability and motility were assessed. RESULTS: PDPN-null cells were able to provoke platelet aggregation, suggesting that PDPN contribution to platelet activation in these cells is marginal. Nevertheless, expression of platelet-induced EMT-related genes, including vimentin, was impaired by PDPN-neutralizing antibody as well as PDPN deficiency, while their effects on TGF-ß-induced gene expression were marginal. Unexpectedly, PDPN gene ablation, at least in either allele, engendered spontaneous N-cadherin upregulation and claudin-1 downregulation. Despite these seemingly EMT-like alterations, PDPN deficiency impaired cellular motility and invasive ability even after TGF-ß-induced EMT induction. CONCLUSIONS: These results suggested that, while PDPN seems to function in favor of maintaining the epithelial state of this cell line, it is indispensable for platelet-mediated induction of particular mesenchymal marker genes as well as the potentiation of motility and invasion capacity.
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Upon hematopoietic stem cell transplantation (HSCT), the availability of recipients' niches in the bone marrow (BM) is one of the factors that influence donor HSC engraftment and hematopoietic reconstitution. Therefore, myeloablative conditioning, such as irradiation and/or chemotherapy, which creates empty niches in the recipients' BM, is required for the success of HSCT. However, the conventional myeloablation causes extensive damages to the patients' BM, which results in the treatment-induced severe complications and even mortality. Thus, alternative and mild conditioning could fulfill the need for safer HSCT-based therapies for hematological and nonhematological disorders. Recently, we have demonstrated that pharmacological inhibition of plasminogen activator inhibitor-1 (PAI-1) activity increases cellular motility and cause detachment of HSCs from the niches. In this study, we performed HSCT using a PAI-1 inhibitor without any myeloablative conditioning. Donor HSCs were transplanted to recipient mice that were pretreated with saline or a PAI-1 inhibitor. Saline pretreated nonmyeloablative recipients showed no engraftment. In contrast, donor cell engraftment was detected in the PAI-1 inhibitor pretreated recipients. Multilineage differentiation, including lymphoid and myeloid cells, was observed in the PAI-1 inhibitor pretreated recipients. Donor-derived cells that exhibited multilineage reconstitution as well as the existence of stem/progenitor cells were detected in the secondary recipients, confirming the maintenance of donor HSCs in the BM of PAI-1 inhibitor pretreated primary recipients. The results indicate that the PAI-1 blockade vacates functional niches in the recipients' BM, which allows the engraftment of long-term multilineage HSCs without myeloablative conditioning.
Assuntos
Medula Óssea/metabolismo , Transplante de Células-Tronco Hematopoéticas , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Nicho de Células-Tronco , Condicionamento Pré-Transplante , Animais , Linhagem da Célula , Células-Tronco Hematopoéticas/metabolismo , CamundongosRESUMO
Hematopoietic stem and progenitor cells (HSPCs) reside in the supportive stromal niche in bone marrow (BM); when needed, however, they are rapidly mobilized into the circulation, suggesting that HSPCs are intrinsically highly motile but usually stay in the niche. We questioned what determines the motility of HSPCs. Here, we show that transforming growth factor (TGF)-ß-induced intracellular plasminogen activator inhibitor (PAI)-1 activation is responsible for keeping HSPCs in the BM niche. We found that the expression of PAI-1, a downstream target of TGF-ß signaling, was selectively augmented in niche-residing HSPCs. Functional inhibition of the TGF-ß-PAI-1 signal increased MT1-MMP-dependent cellular motility, causing a detachment of HSPCs from the TGF-ß-expressing niche cells, such as megakaryocytes. Furthermore, consistently high motility in PAI-1-deficient HSPCs was demonstrated by both a transwell migration assay and reciprocal transplantation experiments, indicating that intracellular, not extracellular, PAI-1 suppresses the motility of HSPCs, thereby causing them to stay in the niche. Mechanistically, intracellular PAI-1 inhibited the proteolytic activity of proprotein convertase Furin, diminishing MT1-MMP activity. This reduced expression of MT1-MMP in turn affected the expression levels of several adhesion/deadhesion molecules for determination of HSPC localization, such as CD44, VLA-4, and CXCR4, which then promoted the retention of HSPCs in the niche. Our findings open up a new field for the study of intracellular proteolysis as a regulatory mechanism of stem cell fate, which has the potential to improve clinical HSPC mobilization and transplantation protocols.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Espaço Intracelular/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Nicho de Células-Tronco , Fator de Crescimento Transformador beta/metabolismo , Animais , Medula Óssea/metabolismo , Movimento Celular , Espaço Extracelular/metabolismo , Furina/metabolismo , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Humanos , Metaloproteinase 14 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Multipotentes/metabolismo , Transdução de SinaisRESUMO
Adult bone mass is maintained through a balance of the activities of osteoblasts and osteoclasts. Although Notch signaling has been shown to maintain bone homeostasis by controlling the commitment, differentiation, and function of cells in both the osteoblast and osteoclast lineages, the precise mechanisms by which Notch performs such diverse and complex roles in bone physiology remain unclear. By using a transgenic approach that modified the expression of delta-like 1 (DLL1) or Jagged1 (JAG1) in an osteoblast-specific manner, we investigated the ligand-specific effects of Notch signaling in bone homeostasis. This study demonstrated for the first time that the proper regulation of DLL1 expression, but not JAG1 expression, in osteoblasts is essential for the maintenance of bone remodeling. DLL1-induced Notch signaling was responsible for the expansion of the bone-forming cell pool by promoting the proliferation of committed but immature osteoblasts. However, DLL1-Notch signaling inhibited further differentiation of the expanded osteoblasts to become fully matured functional osteoblasts, thereby substantially decreasing bone formation. Osteoblast-specific expression of DLL1 did not alter the intrinsic differentiation ability of cells of the osteoclast lineage. However, maturational arrest of osteoblasts caused by the DLL1 transgene impaired the maturation and function of osteoclasts due to a failed osteoblast-osteoclast coupling, resulting in severe suppression of bone metabolic turnover. Taken together, DLL1-mediated Notch signaling is critical for proper bone remodeling as it regulates the differentiation and function of both osteoblasts and osteoclasts. Our study elucidates the importance of ligand-specific activation of Notch signaling in the maintenance of bone homeostasis. J. Cell. Physiol. 232: 2569-2580, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc.
Assuntos
Remodelação Óssea , Fêmur/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Osteoblastos/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Tíbia/metabolismo , Animais , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Proteínas de Ligação ao Cálcio , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Células Cultivadas , Feminino , Fêmur/embriologia , Fêmur/crescimento & desenvolvimento , Genótipo , Idade Gestacional , Homeostase , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Osteoclastos/metabolismo , Osteogênese , Fenótipo , Tíbia/embriologia , Tíbia/crescimento & desenvolvimentoRESUMO
Analysis of the hematopoiesis of non-human primates is important to clarify the evolution of primate-specific hematopoiesis and immune regulation. However, the engraftment and development of the primate hematopoietic system are well-documented only in humans and are not clear in non-human primates. Callithrix jacchus (common marmoset, CM) is a New World monkey with a high rate of pregnancy and small size that lives in closed colonies. As stem cell factor (SCF) is an essential molecule for hematopoietic stem cell development in mice and humans, we focused on CD117, the SCF receptor, and examined whether CD117-expressing cells possess the hematopoietic stem/progenitor cell characteristics of newborn marmoset-derived hematopoietic cells that can develop into T cells and B cells. When CD117(+) cell fractions of the bone marrow were transplanted into immunodeficient NOD (non-obese diabetic)/Shi-scid, common γc-null (NOG) mice, these cells engrafted efficiently in the bone marrow and spleens of the NOG mice. The CD117(+) cells developed into myeloid lineage cells, CD20(+) B cells and CD3(+) T cells, which could express CM cytokines in vivo. The development of B cells did not precede that of T cells. The development of CD8(+) T cells was dominant in NOG mice. The engraftment was comparable for both CD117(+)CD34(+) cells and CD117(+)CD34(-) cells. These results suggest that the CD117(+) cell fraction can differentiate into all three cell lineages, and the development of marmoset immunity in the xenogeneic environment follows diverse developmental pathways compared with human immunity.
Assuntos
Diferenciação Celular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Animais , Animais Recém-Nascidos , Antígenos de Superfície/metabolismo , Callithrix , Autorrenovação Celular , Sangue Fetal/citologia , Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunofenotipagem , Linfócitos/citologia , Linfócitos/metabolismo , Camundongos , Células Mieloides/citologia , Células Mieloides/metabolismo , FenótipoRESUMO
RUNX1/AML1 mutations have been identified in myelodysplastic syndromes (MDSs). In a mouse bone marrow transplantation model, a RUNX1 mutant, D171N, was shown to collaborate with Evi1 in the development of MDSs; however, this is rare in humans. Using enforced expression in human CD34(+) cells, we showed that the D171N mutant, the most frequent target of mutation in the RUNX1 gene, had an increased self-renewal capacity, blocked differentiation, dysplasia in all 3 lineages, and tendency for immaturity, but no proliferation ability. BMI1 overexpression was observed in CD34(+) cells from the majority of MDS patients with RUNX1 mutations, but not in D171N-transduced human CD34(+) cells. Cotransduction of D171N and BMI1 demonstrated that BMI1 overexpression conferred proliferation ability to D171N-transduced cells in both human CD34(+) cells and a mouse bone marrow transplantation model. Stepwise transduction of D171N followed by BMI1 in human CD34(+) cells resulted in long-term proliferation with a retained CD34(+) cell fraction, which is quite similar to the phenotype in patients with higher-risk MDSs. Our results indicate that BMI1 overexpression is one of the second hit partner genes of RUNX1 mutations that contribute to the development of MDSs.
Assuntos
Transformação Celular Neoplásica/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Mutação/genética , Síndromes Mielodisplásicas/patologia , Complexo Repressor Polycomb 1/metabolismo , Idoso , Animais , Antígenos CD34/metabolismo , Western Blotting , Transplante de Medula Óssea , Diferenciação Celular , Proliferação de Células , Citometria de Fluxo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Fenótipo , Complexo Repressor Polycomb 1/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The prognosis of patients undergoing hematopoietic stem cell transplantation (HSCT) depends on the rapid recovery and sustained life-long hematopoiesis. The activation of the fibrinolytic pathway promotes hematopoietic regeneration; however, the role of plasminogen activator inhibitor-1 (PAI-1), a negative regulator of the fibrinolytic pathway, has not yet been elucidated. We herein demonstrate that bone marrow (BM) stromal cells, especially osteoblasts, produce PAI-1 in response to myeloablation, which negatively regulates the hematopoietic regeneration in the BM microenvironment. Total body irradiation in mice dramatically increased the local expression levels of fibrinolytic factors, including tissue-type plasminogen activator (tPA), plasmin, and PAI-1. Genetic disruption of the PAI-1 gene, or pharmacological inhibition of PAI-1 activity, significantly improved the myeloablation-related mortality and promoted rapid hematopoietic recovery after HSCT through the induction of hematopoiesis-promoting factors. The ability of a PAI-1 inhibitor to enhance hematopoietic regeneration was abolished when tPA-deficient mice were used as recipients, thus indicating that PAI-1 represses tPA-dependent hematopoietic regeneration. The PAI-1 inhibitor not only accelerated the expansion of the donor HSCs during the early-stage of regeneration, but also supported long-term hematopoiesis. Our results indicate that the inhibition of PAI-1 activity could be a therapeutic approach to facilitate the rapid recovery and sustained hematopoiesis after HSCT.
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Medula Óssea/metabolismo , Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Regeneração , Serpina E2/antagonistas & inibidores , Nicho de Células-Tronco , Aloenxertos , Animais , Humanos , Camundongos , Serpina E2/metabolismo , Células Estromais/metabolismoRESUMO
Background: Increased levels of plasminogen activator inhibitor-1 (PAI-1) in tumors have been found to correlate with poor clinical outcomes in patients with cancer. Although abundant data support the involvement of PAI-1 in cancer progression, whether PAI-1 contributes to tumor immune surveillance remains unclear. The purposes of this study are to determine whether PAI-1 regulates the expression of immune checkpoint molecules to suppresses the immune response to cancer and demonstrate the potential of PAI-1 inhibition for cancer therapy. Methods: The effects of PAI-1 on the expression of the immune checkpoint molecule programmed cell death ligand 1 (PD-L1) were investigated in several human and murine tumor cell lines. In addition, we generated tumor-bearing mice and evaluated the effects of a PAI-1 inhibitor on tumor progression or on the tumor infiltration of cells involved in tumor immunity either alone or in combination with immune checkpoint inhibitors. Results: PAI-1 induces PD-L1 expression through the JAK/STAT signaling pathway in several types of tumor cells and surrounding cells. Blockade of PAI-1 impedes PD-L1 induction in tumor cells, significantly reducing the abundance of immunosuppressive cells at the tumor site and increasing cytotoxic T-cell infiltration, ultimately leading to tumor regression. The anti-tumor effect elicited by the PAI-1 inhibitor is abolished in immunodeficient mice, suggesting that PAI-1 blockade induces tumor regression by stimulating the immune system. Moreover, combining a PAI-1 inhibitor with an immune checkpoint inhibitor significantly increases tumor regression. Conclusions: PAI-1 protects tumors from immune surveillance by increasing PD-L1 expression; hence, therapeutic PAI-1 blockade may prove valuable in treating malignant tumors.
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Antígeno B7-H1 , Inibidor 1 de Ativador de Plasminogênio , Evasão Tumoral , Animais , Feminino , Humanos , Camundongos , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Evasão da Resposta Imune , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Neoplasias/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Transdução de Sinais , Microambiente Tumoral/imunologiaRESUMO
Stem cells of highly regenerative organs including blood are susceptible to endogenous DNA damage caused by both intrinsic and extrinsic stress. Response mechanisms to such stress equipped in hematopoietic stem cells (HSCs) are crucial in sustaining hematopoietic homeostasis but remain largely unknown. In this study, we demonstrate that serial transplantation of human HSCs into immunodeficient mice triggers replication stress that induces incremental elevation of intracellular reactive oxygen species (ROS) levels and the accumulation of persistent DNA damage within the human HSCs. This accumulation of DNA damage is also detected in HSCs of clinical HSC transplant patients and elderly individuals. A forced increase of intracellular levels of ROS by treatment with a glutathione synthetase inhibitor aggravates the extent of DNA damage, resulting in the functional impairment of HSCs in vivo. The oxidative DNA damage activates the expression of cell-cycle inhibitors in a HSC specific manner, leading to the premature senescence among HSCs, and ultimately to the loss of stem cell function. Importantly, treatment with an antioxidant can antagonize the oxidative DNA damage and eventual HSC dysfunction. The study reveals that ROS play a causative role for DNA damage and the regulation of ROS have a major influence on human HSC aging.
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Proliferação de Células , Dano ao DNA/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Estresse Oxidativo/fisiologia , Animais , Sobrevivência Celular , Células Cultivadas , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Subunidade gama Comum de Receptores de Interleucina/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Espécies Reativas de Oxigênio/metabolismoRESUMO
mRNAs produced in a cell are almost always translated within the same cell. Some mRNAs are transported to other cells of the organism through processes involving membrane nanotubes or extracellular vesicles. A recent report describes a surprising new phenomenon of encapsulating mRNAs inside virus-like particles (VLPs) to deliver them to other cells in a process that was named SEND (Selective Endogenous eNcapsidation for cellular Delivery). Although the seminal work demonstrates the SEND process in cultured cells, it is unknown whether this phenomenon occurs in vivo . Here, we demonstrate the SEND process in living organisms using specially designed genetically engineered mouse models. Our proof of principle study lays a foundation for the SEND-VLP system to potentially be used as a gene therapy tool to deliver therapeutically important mRNAs to tissues.
RESUMO
In this study, we examined the antileukemic effects of pterostilbene, a natural methylated polyphenol analog of resveratrol that is predominantly found in berries and nuts, using various human and murine leukemic cells, as well as bone marrow samples obtained from patients with leukemia. Pterostilbene administration significantly induced apoptosis of leukemic cells, but not of non-malignant hematopoietic stem/progenitor cells. Interestingly, pterostilbene was highly effective in inducing apoptosis of leukemic cells harboring the BCR/ABL fusion gene, including ABL tyrosine kinase inhibitor (TKI)-resistant cells with the T315I mutation. In BCR/ABL+ leukemic cells, pterostilbene decreased the BCR/ABL fusion protein levels and suppressed AKT and NF-κB activation. We further demonstrated that pterostilbene along with U0126, an inhibitor of the MEK/ERK signaling pathway, synergistically induced apoptosis of BCR/ABL+ cells. Our results further suggest that pterostilbene-promoted downregulation of BCR/ABL involves caspase activation triggered by proteasome inhibition-induced endoplasmic reticulum stress. Moreover, oral administration of pterostilbene significantly suppressed tumor growth in mice transplanted with BCR/ABL+ leukemic cells. Taken together, these results suggest that pterostilbene may hold potential for the treatment of BCR/ABL+ leukemia, in particular for those showing ABL-dependent TKI resistance.
Assuntos
Apoptose/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Leucemia/tratamento farmacológico , Leucemia/genética , Mutação/genética , Estilbenos/farmacologia , Administração Oral , Animais , Caspases/metabolismo , Estresse do Retículo Endoplasmático , Células-Tronco Hematopoéticas/patologia , Humanos , Leucemia/patologia , Camundongos , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Estilbenos/administração & dosagem , Células Tumorais CultivadasRESUMO
T-cell development in the thymus is dependent on Notch signaling induced by the interaction of Notch1, present on immigrant cells, with a Notch ligand, delta-like (Dll) 4, on the thymic epithelial cells. Phylogenetic analysis characterizing the properties of the Dll4 molecule suggests that Dll4 emerged from the common ancestor of lobe- and ray-finned fishes and diverged into bony fishes and terrestrial organisms, including mammals. The thymus evolved in cartilaginous fishes before Dll4, suggesting that T-cell development in cartilaginous fishes is dependent on Dll1 instead of Dll4. In this study, we compared the function of both Dll molecules in the thymic epithelium using Foxn1-cre and Dll4-floxed mice with conditional transgenic alleles in which the Dll1 or Dll4 gene is transcribed after the cre-mediated excision of the stop codon. The expression of Dll1 in the thymic epithelium completely restored the defect in the Dll4-deficient condition, suggesting that Dll1 can trigger Notch signaling that is indispensable for T-cell development in the thymus. Moreover, using bone marrow chimeras with Notch1- or Notch2-deficient hematopoietic cells, we showed that Dll1 is able to activate Notch signaling, which is sufficient to induce T-cell development, with both the receptors, in contrast to Dll4, which works only with Notch1, in the thymic environment. These results strongly support the hypothesis that Dll1 regulates T-cell development via Notch1 and/or Notch2 in the thymus of cartilaginous fishes and that Dll4 has replaced Dll1 in inducing thymic Notch signaling via Notch1 during evolution.
Assuntos
Proteínas de Ligação ao Cálcio , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Epitélio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligantes , Mamíferos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , FilogeniaRESUMO
Mesenchymal stem cells (MSCs) have emerged as a new therapeutic modality for reconstituting the hematopoietic microenvironment by improving engraftment in stem cell transplantation. However, the availability of conventional bone marrow (BM)-derived MSCs (BMSCs) is limited. Recent studies showed that a large number of MSCs can be easily isolated from fat tissue (adipose tissue-derived MSCs [ADSCs]). In this study, we extensively evaluated the hematopoiesis-supporting properties of ADSCs, which are largely unknown. In vitro coculture and progenitor assays showed that ADSCs generated significantly more granulocytes and progenitor cells from human hematopoietic stem cells (HSCs) than BMSCs. We found that ADSCs express the chemokine CXCL12, a critical regulator of hematopoiesis, at levels that are three fold higher than those with BMSCs. The addition of a CXCL12 receptor antagonist resulted in a lower yield of granulocytes from ADSC layers, whereas the addition of recombinant CXCL12 to BMSC cocultures promoted the growth of granulocytes. In vivo cell homing assays showed that ADSCs facilitated the homing of mouse HSCs to the BM better than BMSCs. ADSCs injected into the BM cavity of fatally irradiated mice reconstituted hematopoiesis more promptly than BMSCs and subsequently rescued mice that had received a low number of HSCs. Secondary transplantation experiments showed that ADSCs exerted favorable effects on long-term HSCs. These results suggest that ADSCs can be a promising therapeutic alternative to BMSCs.
Assuntos
Tecido Adiposo/citologia , Células da Medula Óssea/fisiologia , Hematopoese/fisiologia , Células-Tronco Mesenquimais/fisiologia , Animais , Células da Medula Óssea/citologia , Transplante de Medula Óssea , Linhagem Celular , Quimiocina CXCL12/metabolismo , Humanos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: To understand how myelodysplastic syndrome cells evolve from normal stem cells and gain competitive advantages over normal hematopoiesis, we established a murine xenograft model harboring bone marrow cells from patients with myelodysplastic syndromes or acute myeloid leukemia with myelodysplasia-related changes. DESIGN AND METHODS: Bone marrow CD34(+) cells obtained from patients were injected, with or without human mesenchymal stem cells, into the bone marrow of non-obese diabetic/severe combined immunodeficient/IL2Rγ(null) hosts. Engraftment and differentiation of cells derived from the patients were investigated by flow cytometry and immunohistochemical analysis. RESULTS: Co-injection of patients' cells and human mesenchymal stem cells led to successful engraftment of patient-derived cells that maintained the immunophenotypes and genomic abnormalities of the original patients. Myelodysplastic syndrome-originated clones differentiated into mature neutrophils, megakaryocytes, and erythroblasts. Two of the samples derived from patients with acute myeloid leukemia with myelodysplasia-related changes were able to sustain neoplastic growth into the next generation while these cells had limited differentiation ability in the murine host. The hematopoiesis of mice engrafted with patients' cells was significantly suppressed even when human cells accounted for less than 1% of total marrow mononuclear cells. Histological studies revealed invasion of the endosteal surface by patient-derived CD34(+) cells and disruption of extracellular matrix architecture, which probably caused inhibition of murine hematopoiesis. CONCLUSIONS: We established murine models of human myelodysplastic syndromes using cells obtained from patients: the presence of neoplastic cells was associated with the suppression of normal host hematopoiesis. The efficiency of engraftment was related to the presence of an abnormality in chromosome 7.
Assuntos
Modelos Animais de Doenças , Síndromes Mielodisplásicas/patologia , Transplante Heterólogo , Animais , Antígenos CD34/metabolismo , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Camundongos , Camundongos Knockout , Camundongos SCID , Síndromes Mielodisplásicas/metabolismoRESUMO
Notch signaling primarily determines T-cell fate. However, the molecular mechanisms underlying the maintenance of T-lineage potential in pre-thymic progenitors remain unclear. Here, we established two murine Ebf1-deficient pro-B cell lines, with and without T-lineage potential. The latter expressed lower levels of Lmo2; their potential was restored via ectopic expression of Lmo2. Conversely, the CRISPR/Cas9-mediated deletion of Lmo2 resulted in the loss of the T-lineage potential. Introduction of Bcl2 rescued massive cell death of Notch-stimulated pro-B cells without efficient LMO2-driven Bcl11a expression but was not sufficient to retain their T-lineage potential. Pro-B cells without T-lineage potential failed to activate Tcf7 due to DNA methylation; Tcf7 transduction restored this capacity. Moreover, direct binding of LMO2 to the Bcl11a and Tcf7 loci was observed. Altogether, our results highlight LMO2 as a crucial player in the survival and maintenance of T-lineage potential in T-cell progenitors via the regulation of the expression of Bcl11a and Tcf7.