RESUMEN
RATIONALE: The impact of diabetes mellitus on bone marrow (BM) structure is incompletely understood. OBJECTIVE: Investigate the effect of type-2 diabetes mellitus (T2DM) on BM microvascular and hematopoietic cell composition in patients without vascular complications. METHODS AND RESULTS: Bone samples were obtained from T2DM patients and nondiabetic controls (C) during hip replacement surgery and from T2DM patients undergoing amputation for critical limb ischemia. BM composition was assessed by histomorphometry, immunostaining, and flow cytometry. Expressional studies were performed on CD34(pos) immunosorted BM progenitor cells (PCs). Diabetes mellitus causes a reduction of hematopoietic tissue, fat deposition, and microvascular rarefaction, especially when associated with critical limb ischemia. Immunohistochemistry documented increased apoptosis and reduced abundance of CD34(pos)-PCs in diabetic groups. Likewise, flow cytometry showed scarcity of BM PCs in T2DM and T2DM+critical limb ischemia compared with C, but similar levels of mature hematopoietic cells. Activation of apoptosis in CD34(pos)-PCs was associated with upregulation and nuclear localization of the proapoptotic factor FOXO3a and induction of FOXO3a targets, p21 and p27(kip1). Moreover, microRNA-155, which regulates cell survival through inhibition of FOXO3a, was downregulated in diabetic CD34(pos)-PCs and inversely correlated with FOXO3a levels. The effect of diabetes mellitus on anatomic and molecular end points was confirmed when considering background covariates. Furthermore, exposure of healthy CD34(pos)-PCs to high glucose reproduced the transcriptional changes induced by diabetes mellitus, with this effect being reversed by forced expression of microRNA-155. CONCLUSIONS: We provide new anatomic and molecular evidence for the damaging effect of diabetes mellitus on human BM, comprising microvascular rarefaction and shortage of PCs attributable to activation of proapoptotic pathway.
Asunto(s)
Células de la Médula Ósea/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Angiopatías Diabéticas/metabolismo , Factores de Transcripción Forkhead/metabolismo , Células Madre Hematopoyéticas/metabolismo , MicroARNs/metabolismo , Microvasos/metabolismo , Transducción de Señal , Nicho de Células Madre , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD34/metabolismo , Apoptosis , Biomarcadores/metabolismo , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/patología , Examen de la Médula Ósea , Estudios de Casos y Controles , Linaje de la Célula , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Angiopatías Diabéticas/genética , Angiopatías Diabéticas/patología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Femenino , Citometría de Flujo , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/patología , Humanos , Inmunohistoquímica , Isquemia/genética , Isquemia/metabolismo , Isquemia/patología , Masculino , MicroARNs/genética , Microvasos/inmunología , Microvasos/patología , Persona de Mediana Edad , Enfermedad Arterial Periférica/genética , Enfermedad Arterial Periférica/metabolismo , Enfermedad Arterial Periférica/patología , TransfecciónRESUMEN
RNA editing, a common and potentially highly functional form of RNA modification, encompasses two different RNA modifications, namely adenosine to inosine (A-to-I) and cytidine to uridine (C-to-U) editing. As inosines are interpreted as guanosines by the cellular machinery, both A-to-I and C-to-U editing change the nucleotide sequence of the RNA. Editing events in coding sequences have the potential to change the amino acid sequence of proteins, whereas editing events in noncoding RNAs can, for example, affect microRNA target binding. With advancing RNA sequencing technology, more RNA editing events are being discovered, studied, and reported. However, RNA editing events are still often overlooked or discarded as sequence read quality defects. With this position paper, we aim to provide guidelines and recommendations for the detection, validation, and follow-up experiments to study RNA editing, taking examples from the fields of cardiovascular and brain disease. We discuss all steps, from sample collection, storage, and preparation, to different strategies for RNA sequencing and editing-sensitive data analysis strategies, to validation and follow-up experiments, as well as potential pitfalls and gaps in the available technologies. This paper may be used as an experimental guideline for RNA editing studies in any disease context.