RESUMEN
OBJECTIVE: The signaling by thrombopoietin (TPO) via its receptor, c-MPL, plays a crucial role in the maintenance of hematopoietic stem cells (HSCs). Small-molecule c-MPL agonists have recently been shown to be beneficial in the treatment of thrombocytopenia. However, their effects on HSCs have not yet been explored. In this study, we evaluated the effects of NR-101, a novel small-molecule c-MPL agonist, on the ex vivo expansion of human cord blood (hCB) HSCs. MATERIALS AND METHODS: hCB CD34(+) or CD34(+)CD38(-) hematopoietic stem and progenitor cells were cultured for 7 days in the presence of thrombopoietin (TPO) or NR-101, and then subjected to flow cytometric analyses, colony-forming cell assays, and severe combined immunodeficiency-repopulating cell assays. RESULTS: During a 7-day culture of CD34(+) or CD34(+)CD38(-) hematopoietic stem and progenitor cells, NR-101 efficiently increased their numbers, with a greater than twofold increase compared to TPO, although its effect on megakaryocytopoiesis was comparable to that of TPO. Correspondingly, severe combined immunodeficiency-repopulating cells were increased 2.9-fold during a 7-day culture with NR-101 compared to freshly isolated CD34(+) cells, and 2.3-fold compared to that with TPO. Of note, NR-101 persistently activated signal transducer and activator of transcription (STAT) 5 but not signal transducer and activator of transcription 3. Furthermore, NR-101 induced a long-term accumulation of hypoxia-inducible factor-1alpha protein and enhanced activation of its downstream target genes. CONCLUSION: This is the first time that a small-molecule c-MPL agonist has been demonstrated to promote net expansion of HSCs. NR-101 is more efficient in ex vivo expansion of HSCs than TPO. NR-101 could be a useful tool for the therapeutic manipulation of human HSCs.
Asunto(s)
Células Madre Hematopoyéticas/efectos de los fármacos , Receptores de Trombopoyetina/agonistas , Trombopoyesis/efectos de los fármacos , Animales , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/genética , Línea Celular Tumoral/citología , Línea Celular Tumoral/efectos de los fármacos , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/trasplante , Trasplante de Células Madre de Sangre del Cordón Umbilical , Subunidad beta Común de los Receptores de Citocinas/genética , Subunidad beta Común de los Receptores de Citocinas/fisiología , ADN Complementario/genética , Evaluación Preclínica de Medicamentos , Sangre Fetal/citología , Regulación de la Expresión Génica/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Receptor de Interleucina-3/genética , Subunidad alfa del Receptor de Interleucina-3/fisiología , Leucemia Mieloide/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Quimera por Radiación , Receptores de Eritropoyetina/genética , Receptores de Eritropoyetina/fisiología , Receptores de Trombopoyetina/genética , Receptores de Trombopoyetina/fisiología , Proteínas Recombinantes de Fusión/fisiología , Transducción de Señal/efectos de los fármacos , Trombopoyetina/farmacologíaRESUMEN
Macrophage migration inhibitory factor acts via its intrinsic thiol-protein oxidoreductase activity to negatively regulate the neuronal chronotropic actions of angiotensin II in normotensive rat neurons. Because the chronotropic action of angiotensin II is potentiated in spontaneously hypertensive rat neurons, we investigated whether this negative regulatory mechanism is absent in these rats. Angiotensin II (100 nM) elicited an approximately 89% increase in neuronal firing in Wistar-Kyoto rat hypothalamus and brain stem cultured neurons and an increase in intracellular macrophage migration inhibitory factor levels in the same cells. The chronotropic action of angiotensin II was significantly greater (approximately 212% increase) in spontaneously hypertensive rat neurons, but angiotensin II failed to alter macrophage migration inhibitory factor expression in these cells. Intracellular application of recombinant macrophage migration inhibitory factor (0.8 nM) or its specific neuronal overexpression via Ad5-SYN-MIF (1x10(7) infectious units) significantly attenuated the chronotropic action of angiotensin II in spontaneously hypertensive rat neurons, similar to results from Wistar-Kyoto rat neurons. In contrast, C60S-macrophage migration inhibitory factor (0.8 nM), which lacks thiol-protein oxidoreductase activity, failed to alter the chronotropic action of angiotensin II in neurons from either rat strain. Thus, whereas macrophage migration inhibitory factor has the potential to depress the chronotropic action of angiotensin II in spontaneously hypertensive rat neurons, it is unlikely that this regulatory mechanism occurs, because angiotensin II does not increase the expression of this protein. The lack of this regulatory mechanism may contribute to the increased chronotropic action of angiotensin II in spontaneously hypertensive rat neurons.