RESUMEN
BACKGROUND: Epicardial adipose tissue volume and coronary artery disease are strongly associated, even after accounting for overall body mass. Despite its pathophysiological significance, the origin and paracrine signaling pathways that regulate epicardial adipose tissue's formation and expansion are unclear. METHODS: We used a novel modified mRNA-based screening approach to probe the effect of individual paracrine factors on epicardial progenitors in the adult heart. RESULTS: Using 2 independent lineage-tracing strategies in murine models, we show that cells originating from the Wt1+ mesothelial lineage, which includes epicardial cells, differentiate into epicardial adipose tissue after myocardial infarction. This differentiation process required Wt1 expression in this lineage and was stimulated by insulin-like growth factor 1 receptor (IGF1R) activation. IGF1R inhibition within this lineage significantly reduced its adipogenic differentiation in the context of exogenous, IGF1-modified mRNA stimulation. Moreover, IGF1R inhibition significantly reduced Wt1 lineage cell differentiation into adipocytes after myocardial infarction. CONCLUSIONS: Our results establish IGF1R signaling as a key pathway that governs epicardial adipose tissue formation in the context of myocardial injury by redirecting the fate of Wt1+ lineage cells. Our study also demonstrates the power of modified mRNA -based paracrine factor library screening to dissect signaling pathways that govern progenitor cell activity in homeostasis and disease.
Asunto(s)
Adipocitos/metabolismo , Células Madre Mesenquimatosas/citología , Infarto del Miocardio/patología , Pericardio/citología , Receptor IGF Tipo 1/metabolismo , Adipocitos/citología , Animales , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Células Madre Mesenquimatosas/metabolismo , Ratones , Infarto del Miocardio/metabolismo , Comunicación Paracrina , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor IGF Tipo 1/genética , Proteínas Represoras/metabolismo , Transducción de Señal , Proteínas WT1RESUMEN
The Heart of Glass-Cerebral Cavernous Malformation (Heg-CCM) pathway is essential for normal cardiovascular development in zebrafish and mouse. In zebrafish, the Heg-CCM pathway mutants santa(ccm1/san), valentine (ccm2/vtn), and heart of glass (heg) exhibit severely dilated hearts and inflow tracts and a complete absence of blood circulation. We identified a novel gene based on its sequence identity with ccm2, which we have named ccm2-like (ccm2l), and characterized its role in cardiovascular development. Disruption of ccm2l by morpholino injection causes dilation of the atrium and inflow tract and compromised blood circulation. Morpholino co-injection experiments identify ccm2l as an enhancer of the characteristic Heg-CCM dilated heart phenotype, and we find that ccm2 overexpression can partially rescue ccm2l morphant defects. Finally, we show that Ccm2l binds Ccm1 and perform deletion and mutational analyses to define the regions of Ccm1 that mediate its binding to Ccm2l and its previously established interactors Ccm2 and Heg. These genetic and biochemical data argue that ccm2l is a necessary component of the Heg-CCM pathway.
Asunto(s)
Sistema Cardiovascular/embriología , Glicoproteínas de Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Fenotipo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/fisiología , Proteínas de Pez Cebra/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sistema Cardiovascular/metabolismo , Cartilla de ADN/genética , Immunoblotting , Hibridación in Situ , Proteína KRIT1 , Ratones , Proteínas de Microfilamentos/genética , Microscopía , Proteínas Asociadas a Microtúbulos/genética , Datos de Secuencia Molecular , Morfolinos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Transducción de Señal/genéticaRESUMEN
Genome editing of human induced pluripotent stem cells (hiPSCs) offers unprecedented opportunities for in vitro disease modeling and personalized cell replacement therapy. The introduction of Cas9-directed genome editing has expanded adoption of this approach. However, marker-free genome editing using standard protocols remains inefficient, yielding desired targeted alleles at a rate of â¼1-5%. We developed a protocol based on a doxycycline-inducible Cas9 transgene carried on a piggyBac transposon to enable robust and highly efficient Cas9-directed genome editing, so that a parental line can be expeditiously engineered to harbor many separate mutations. Treatment with doxycycline and transfection with guide RNA (gRNA), donor DNA and piggyBac transposase resulted in efficient, targeted genome editing and concurrent scarless transgene excision. Using this approach, in 7 weeks it is possible to efficiently obtain genome-edited clones with minimal off-target mutagenesis and with indel mutation frequencies of 40-50% and homology-directed repair (HDR) frequencies of 10-20%.