RESUMEN
Anemia is a common complication of systemic inflammation. Proinflammatory cytokines both decrease erythroblast sensitivity to erythropoietin (EPO) and increase the levels of the hepatic hormone hepcidin, sequestering iron in stores and causing functional iron deficiency. Anemia of chronic kidney disease (CKD) is a peculiar form of anemia of inflammation, characterized by impaired EPO production paralleling progressive kidney damage. Traditional therapy based on increased EPO (often in combination with iron) may have off-target effects due to EPO interaction with its non-erythroid receptors. Transferrin Receptor 2 (Tfr2) is a mediator of the iron-erythropoiesis crosstalk. Its deletion in the liver hampers hepcidin production, increasing iron absorption, whereas its deletion in the hematopoietic compartment increases erythroid EPO sensitivity and red blood cell production. Here, we show that selective hematopoietic Tfr2 deletion ameliorates anemia in mice with sterile inflammation in the presence of normal kidney function, promoting EPO responsiveness and erythropoiesis without increasing serum EPO levels. In mice with CKD, characterized by absolute rather than functional iron deficiency, Tfr2 hematopoietic deletion had a similar effect on erythropoiesis but anemia improvement was transient because of limited iron availability. Also, increasing iron levels by downregulating only hepatic Tfr2 had a minor effect on anemia. However, simultaneous deletion of hematopoietic and hepatic Tfr2, stimulating erythropoiesis and increased iron supply, was sufficient to ameliorate anemia for the entire protocol. Thus, our results suggest that combined targeting of hematopoietic and hepatic Tfr2 may be a therapeutic option to balance erythropoiesis stimulation and iron increase, without affecting EPO levels.
Asunto(s)
Anemia , Eritropoyetina , Deficiencias de Hierro , Insuficiencia Renal Crónica , Ratones , Animales , Hierro/metabolismo , Eritropoyesis/genética , Hepcidinas/genética , Hepcidinas/metabolismo , Modelos Animales de Enfermedad , Anemia/etiología , Anemia/genética , Eritropoyetina/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/complicaciones , Receptores de Transferrina/genética , Insuficiencia Renal Crónica/complicaciones , Insuficiencia Renal Crónica/tratamiento farmacológico , Insuficiencia Renal Crónica/genéticaRESUMEN
ß-thalassemia is a genetic disorder caused by mutations in the ß-globin gene, and characterized by anemia, ineffective erythropoiesis and iron overload. Patients affected by the most severe transfusion-dependent form of the disease (TDT) require lifelong blood transfusions and iron chelation therapy, a symptomatic treatment associated with several complications. Other therapeutic opportunities are available, but none is fully effective and/or applicable to all patients, calling for the identification of novel strategies. Transferrin receptor 2 (TFR2) balances red blood cells production according to iron availability, being an activator of the iron-regulatory hormone hepcidin in the liver and a modulator of erythropoietin signaling in erythroid cells. Selective Tfr2 deletion in the BM improves anemia and iron-overload in non-TDT mice, both as a monotherapy and, even more strikingly, in combination with iron-restricting approaches. However, whether Tfr2 targeting might represent a therapeutic option for TDT has never been investigated so far. Here, we prove that BM Tfr2 deletion improves anemia, erythrocytes morphology and ineffective erythropoiesis in the Hbbth1/th2 murine model of TDT. This effect is associated with a decrease in the expression of α-globin, which partially corrects the unbalance with ß-globin chains and limits the precipitation of misfolded hemoglobin, and with a decrease in the activation of unfolded protein response. Remarkably, BM Tfr2 deletion is also sufficient to avoid long-term blood transfusions required for survival of Hbbth1/th2 animals, preventing mortality due to chronic anemia and reducing transfusion-associated complications, such as progressive iron-loading. Altogether, TFR2 targeting might represent a promising therapeutic option also for TDT.
Asunto(s)
Sobrecarga de Hierro , Receptores de Transferrina , Talasemia beta , Animales , Transfusión Sanguínea , Modelos Animales de Enfermedad , Hierro/metabolismo , Sobrecarga de Hierro/genética , Sobrecarga de Hierro/metabolismo , Ratones , Receptores de Transferrina/genética , Globinas beta , Talasemia beta/genética , Talasemia beta/terapiaRESUMEN
Inflammatory cells can either promote or inhibit tumor growth. Here we studied whether CD40, a key molecule for adaptive immune response, has any role in mammary carcinogenesis of BALB/NeuT transgenic tumor-prone mice. We transferred the HER2/neu oncogene into CD40-null background to obtain the CD40-KO/NeuT strain. CD40-KO/NeuT mice showed delayed tumor onset and reduced tumor multiplicity. BM (BM) transplantation experiments excluded a role of BM-derived cells in the reduced tumorigenicity associated with CD40 deficiency. Rather, CD40 expressed by endothelial cells (ECs) takes part to the angiogenic process. Accordingly, large vessels, well organized around the tumor lobular structures, characterize BALB/NeuT tumors, whereas tiny numerous vessels with scarce extracellular matrix are dispersed in the parenchyma of poorly organized CD40-KO/NeuT tumors. Activated platelets, which may interact with and activate ECs, are a possible source of CD40L. Their localization within tumor vessels prompted the idea of treating BALB/NeuT and CD40-KO/NeuT mice chronically with the anti-platelet drug clopidogrel, known to inhibit platelet CD40L expression. Treatment of BALB/NeuT mice reduced tumor growth to a level similar to CD40-deficient mice, whereas CD40-KO/NeuT mice treated or not showed the same attenuated tumor outgrowth, indicating that activated platelets are the likely source of CD40L in this model. Collectively, these data point to a participation of CD40/CD40L in the angiogenic processes associated with mammary carcinogenesis of BALB/NeuT mice.
Asunto(s)
Antígenos CD40/fisiología , Proliferación Celular , Endotelio Vascular/inmunología , Endotelio Vascular/metabolismo , Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/patología , Animales , Antígenos CD40/genética , Endotelio Vascular/patología , Femenino , Neoplasias Mamarias Experimentales/irrigación sanguínea , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , RatasRESUMEN
Adherent fibroblast-like cells have been reported to appear in cultures of human endocrine or exocrine pancreatic tissue during attempts to differentiate human beta cells from pancreatic precursors. A thorough characterization of these mesenchymal cells has not yet been completed, and there are no conclusive data about their origin.We demonstrated that the human mesenchymal cells outgrowing from cultured human pancreatic endocrine or exocrine tissue are pancreatic mesenchymal stem cells (pMSC) that propagate from contaminating pMSC. The origin of pMSC is partly extrapancreatic both in humans and mice, and by using green fluorescent protein (GFP(+)) bone marrow transplantation in the mouse model, we were able to demonstrate that these cells derive from the CD45(+) component of bone marrow. The pMSC express negligible levels of islet-specific genes both in basal conditions and after serum deprivation or exogenous growth factor exposure, and might not represent optimal candidates for generation of physiologically competent beta-cells. On the other hand, when cotransplanted with a minimal pancreatic islet mass, pMSC facilitate the restoration of normoglycemia and the neovascularization of the graft. These results suggest that pMSCs could exert an indirect role of "helper" cells in tissue repair processes.
Asunto(s)
Células de la Médula Ósea/metabolismo , Diferenciación Celular , Movimiento Celular , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos/cirugía , Células Madre Mesenquimatosas/metabolismo , 5'-Nucleotidasa/análisis , Antígeno AC133 , Proteínas Angiogénicas , Animales , Antígenos CD/análisis , Glucemia/metabolismo , Células de la Médula Ósea/inmunología , Trasplante de Médula Ósea , Diferenciación Celular/genética , Proliferación Celular , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/cirugía , Células Endoteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Glicoproteínas/análisis , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Humanos , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/irrigación sanguínea , Islotes Pancreáticos/citología , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/metabolismo , Antígenos Comunes de Leucocito/análisis , Masculino , Células Madre Mesenquimatosas/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , Neovascularización Fisiológica , Péptidos/análisis , Factores de TiempoRESUMEN
Gene therapy clinical trials require rigorous non-clinical studies in the most relevant models to assess the benefit-to-risk ratio. To support the clinical development of gene therapy for ß-thalassemia, we performed in vitro and in vivo studies for prediction of safety. First we developed newly GLOBE-derived vectors that were tested for their transcriptional activity and potential interference with the expression of surrounding genes. Because these vectors did not show significant advantages, GLOBE lentiviral vector (LV) was elected for further safety characterization. To support the use of hematopoietic stem cells (HSCs) transduced by GLOBE LV for the treatment of ß-thalassemia, we conducted toxicology, tumorigenicity, and biodistribution studies in compliance with the OECD Principles of Good Laboratory Practice. We demonstrated a lack of toxicity and tumorigenic potential associated with GLOBE LV-transduced cells. Vector integration site (IS) studies demonstrated that both murine and human transduced HSCs retain self-renewal capacity and generate new blood cell progeny in the absence of clonal dominance. Moreover, IS analysis showed an absence of enrichment in cancer-related genes, and the genes targeted by GLOBE LV in human HSCs are well known sites of integration, as seen in other lentiviral gene therapy trials, and have not been associated with clonal expansion. Taken together, these integrated studies provide safety data supporting the clinical application of GLOBE-mediated gene therapy for ß-thalassemia.
RESUMEN
Gene therapy of genetic diseases requires persistent and position-independent expression of a therapeutic transgene. Transcriptional enhancers binding chromatin-remodeling and modifying complexes may play a role in shielding transgenes from repressive chromatin effects. We tested the activity of the HS2 enhancer of the GATA1 gene in protecting the expression of a ß-globin minigene delivered by a lentiviral vector in hematopoietic stem/progenitor cells. Gene expression from proviruses carrying GATA1-HS2 in both LTRs was persistent and resistant to silencing at most integration sites in the in vivo progeny of human hematopoietic progenitors and murine long-term repopulating stem cells. The GATA1-HS2-modified vector allowed correction of murine ß-thalassemia at low copy number without inducing clonal selection of erythroblastic progenitors. Chromatin immunoprecipitation studies showed that GATA1 and the CBP acetyltransferase bind to GATA1-HS2, significantly increasing CBP-specific histone acetylations at the LTRs and ß-globin promoter. Recruitment of CBP by the LTRs thus establishes an open chromatin domain encompassing the entire provirus, and increases the therapeutic efficacy of ß-globin gene transfer by reducing expression variegation and epigenetic silencing.