RESUMEN
Although the molecular mechanisms underlying congenital heart disease (CHD) remain poorly understood, recent advances in genetic analysis have facilitated the exploration of causative genes for CHD. We reported that the pathogenic variant c.1617del of TMEM260, which encodes a transmembrane protein, is highly associated with CHD, specifically persistent truncus arteriosus (PTA), the most severe cardiac outflow tract (OFT) defect. Using whole-exome sequencing, the c.1617del variant was identified in two siblings with PTA in a Japanese family and in three of the 26 DNAs obtained from Japanese individuals with PTA. The c.1617del of TMEM260 has been found only in East Asians, especially Japanese and Korean populations, and the frequency of this variant in PTA is estimated to be next to that of the 22q11.2 deletion, the most well-known genetic cause of PTA. Phenotype of patients with c.1617del appears to be predominantly in the heart, although TMEM260 is responsible for structural heart defects and renal anomalies syndrome (SHDRA). The mouse TMEM260 variant (p.W535Cfs*56), synonymous with the human variant (p.W539Cfs*9), exhibited truncation and downregulation by western blotting, and aggregation by immunocytochemistry. In situ hybridization demonstrated that Tmem260 is expressed ubiquitously during embryogenesis, including in the development of cardiac OFT implicated in PTA. This expression may be regulated by a ~ 0.8 kb genomic region in intron 3 of Tmem260 that includes multiple highly conserved binding sites for essential cardiac transcription factors, thus revealing that the c.1617del variant of TMEM260 is the major single-gene variant responsible for PTA in the Japanese population.
Asunto(s)
Cardiopatías Congénitas , Proteínas de la Membrana , Animales , Femenino , Humanos , Masculino , Ratones , Pueblo Asiatico/genética , Pueblos del Este de Asia , Secuenciación del Exoma , Predisposición Genética a la Enfermedad , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/patología , Japón , Proteínas de la Membrana/genética , Linaje , FenotipoRESUMEN
BACKGROUND: Pulmonary arterial hypertension (PAH) is a fatal disease, with approximately 10% of cases associated with genetic variants. Recent genetic studies have reported pathogenic variants in the TBX4 gene in patients with PAH, especially in patients with childhood-onset of the disease, but the pathogenesis of PAH caused by TBX4 variant has not been fully uncovered. METHODS: We analysed the TBX4 gene in 75 Japanese patients with sporadic or familial PAH using a PCR-based bidirectional sequencing method. Detected variants were evaluated using in silico analyses as well as in vitro analyses including luciferase assay, immunocytochemistry and chromatin immunoprecipitation (ChIP) whether they have altered function. We also analysed the function of TBX4 using mouse embryonic lung explants with inhibition of Tbx4 expression. RESULTS: Putative pathogenic variants were detected in three cases (4.0%). Our in vitro functional analyses revealed that TBX4 directly regulates the transcriptional activity of fibroblast growth factor 10 (FGF10), whereas the identified TBX4 variant proteins failed to activate the FGF10 gene because of disruption of nuclear localisation signal or poor DNA-binding affinity. Furthermore, ex vivo inhibition of Tbx4 resulted in insufficiency of lung morphogenesis along with specific downregulation of Tie2 and Kruppel-like factor 4 expression. CONCLUSION: Our results implicate variants in TBX4 as a genetic cause of PAH in a subset of the Japanese population. Variants in TBX4 may lead to PAH through insufficient lung morphogenesis by disrupting the TBX4-mediated direct regulation of FGF10 signalling and pulmonary vascular endothelial dysfunction involving PAH-related molecules.
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Hipertensión Arterial Pulmonar , Proteínas de Dominio T Box , Animales , ADN , Hipertensión Pulmonar Primaria Familiar/genética , Factor 10 de Crecimiento de Fibroblastos , Ratones , Señales de Localización Nuclear , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factores de TranscripciónRESUMEN
Long-term complications after the Fontan procedure are important concerns for patients with pediatric and adult congenital heart disease. Although thrombocytopenia due to portal hypertension and hypersplenism is a well-known complication of the Fontan circulation, few studies have reported on its management. Herein we describe a young adult Fontan patient with thrombocytopenia and a splenic artery aneurysm caused by conduit stenosis. The patient required conduit replacement due to high venous pressure. We performed partial splenic artery embolization (PSE) and embolization of the aneurysm preoperatively to reduce the risk of bleeding, resulting in successful subsequent cardiac surgery. Preoperative evaluation of the splenic artery aneurysm was informative, and PSE was a safe and effective treatment option for thrombocytopenia to avoid bleeding during open-heart surgery in this patient.
Asunto(s)
Aneurisma , Embolización Terapéutica , Procedimiento de Fontan , Cardiopatías Congénitas , Trombocitopenia , Adulto , Niño , Embolización Terapéutica/efectos adversos , Procedimiento de Fontan/efectos adversos , Cardiopatías Congénitas/complicaciones , Humanos , Arteria Esplénica/cirugía , Trombocitopenia/etiología , Adulto JovenRESUMEN
Congenital heart diseases (CHDs) involving the outflow tract (OFT), such as persistent truncus arteriosus (PTA), lead to mortality and morbidity with implications not only in the heart, but also in the pulmonary vasculature. The mechanisms of pulmonary artery (PA) development and the etiologies underlying PA disorders associated with CHD remain poorly understood partly because of a specific marker for PA development is nonexistent. The three subtypes of inositol 1,4,5-trisphosphate receptors (IP3R1, 2, and 3) are intracellular Ca2+ channels that are essential for many tissues and organs. We discovered that IP3R2 was expressed in the vasculature and heart during development using transgenic mice, in which a LacZ marker gene was knocked into the IP3R2 locus. Whole-mount and section LacZ staining showed that IP3R2-LacZ-positive cells were detectable exclusively in the smooth muscle cells, or tunica media, of PA, merging into αSMA-positive cells during development. Furthermore, our analyses suggested that IP3R2-LacZ positive PA smooth muscle layers gradually elongate from the central PA to the peripheral PAs from E13.5 to E18.5, supporting the distal angiogenesis theory for the development of PA, whereas IP3R2-LacZ was rarely expressed in smooth muscle cells in the pulmonary trunk. Crossing IP3R-LacZ mice with mice hypomorphic for Tbx1 alleles revealed that PTA of Tbx1 mutants may result from agenesis or hypoplasia of the pulmonary trunk; thus, the left and right central to peripheral PAs connect directly to the dorsal side of the truncus arteriosus in these mutants. Additionally, we found hypercellular interstitial mesenchyme and delayed maturation of the lung endoderm in the Tbx1 mutant lungs. Our study identifies IP3R2 as a novel marker for clear visualization of PA during development and can be utilized for studying cardiopulmonary development and disease.
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Vasos Coronarios/metabolismo , Corazón/embriología , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Animales , Apoptosis , Arterias/metabolismo , Vasos Sanguíneos/metabolismo , Señalización del Calcio , Vasos Coronarios/embriología , Femenino , Corazón/fisiología , Inositol , Masculino , Ratones/embriología , Ratones Endogámicos C57BL , Miocardio/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas de Dominio T Box/metabolismo , Tronco Arterial Persistente/metabolismoRESUMEN
Skeletal overgrowth accompanied by de novo heterozygous activating mutations in PDGFRB (platelet-derived growth factor receptor beta), that is, p.Pro584Arg and p.Trp566Arg, defines Kosaki overgrowth syndrome (OMIM #616592). Emerging evidence suggests a role of PDGFRB in the genesis of cerebral aneurysms. The delineation of the range and progression of the vascular phenotype of Kosaki overgrowth syndrome is urgently needed. Herein, we conducted subsequent analyses of serial neurovascular imaging studies of two original patients with a de novo heterozygous mutation in PDGFRB, that is, p.Pro584Arg. The analysis showed the progressive dilation of basilar and vertebral arteries and coronary arteries commencing during the teenage years and early 20s. The radiographic appearance of the basilar vertebral aneurysms showed signs of arterial wall dilation, compatible with the known vascular pathology of vascular-type Ehlers-Danlos syndrome and Loeys-Dietz syndrome. The dolichoectasia in cerebrovascular arteries can lead to fatal complications, even with neurosurgical interventions. To prevent the progression of artery dilation, preventative and therapeutic medical measures using tyrosine kinase inhibitors may be necessary in addition to optimal control of the systemic blood pressure. Kosaki overgrowth syndrome is a clinically recognizable syndrome that can exhibit progressive dilatory and tortuous vascular changes in basilar/vertebral and coronary arteries as early as in the teenage years. We recommend careful counseling regarding the risk of future vascular complications, optimal blood pressure control, and regular systemic vascular screening during follow-up examinations.
Asunto(s)
Enfermedades del Desarrollo Óseo/genética , Aneurisma Coronario/genética , Mutación con Ganancia de Función , Aneurisma Intracraneal/genética , Mutación Missense , Mutación Puntual , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Insuficiencia Vertebrobasilar/genética , Adolescente , Edad de Inicio , Sustitución de Aminoácidos , Aneurisma/genética , Ceguera/etiología , Calcinosis/etiología , Enfermedades de las Arterias Carótidas/genética , Aneurisma Coronario/diagnóstico por imagen , Progresión de la Enfermedad , Femenino , Pérdida Auditiva Unilateral/etiología , Humanos , Aneurisma Intracraneal/diagnóstico por imagen , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/fisiología , Síndrome , Insuficiencia Vertebrobasilar/diagnóstico por imagenRESUMEN
BACKGROUND: The correlation between the Z-score of the left ventricular (LV) diameter and the LV volume-overload due to pulmonary over-circulation in children with ventricular septal defect (VSD) or patent ductus arteriosus (PDA) remains unclear.MethodsâandâResults:The present, retrospective study enrolled 70 children (aged 0.3-16.8 years; 33 males, 37 females) with a diagnosis of isolated VSD and/or PDA who underwent cardiac catheterization (CC) between 2015 and 2019. Patients with chromosomal/genetic anomalies, growth disorder, right-ventricular enlargement or other conditions causing LV enlargement were excluded. Echocardiographic parameters were retrospectively evaluated from the medical records, converted to a Z-score, then compared with CC data. The pulmonary-systemic flow ratio on CC (cQp/Qs) correlated significantly with the Z-score of both the LV end-diastolic diameter (Zd) (r=0.698, P<0.0001) and LV end-systolic diameter (r=0.593, P<0.0001). Regression analysis and curve-fitting were used to predict the cQp/Qs based on the Zd, and a significant regression equation was found on cubic regression (R2of 0.524, P<0.0001) showing a strong correlation with the cQp/Qs (r=0.724, P<0.0001). CONCLUSIONS: The Z-score of the LV diameter can be a useful, non-invasive marker for evaluating LV volume overload and determining the surgical indications in children with VSD or PDA because of its strong correlation with the cQp/Qs.
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Conducto Arterioso Permeable , Insuficiencia Cardíaca , Defectos del Tabique Interventricular , Niño , Conducto Arterioso Permeable/diagnóstico por imagen , Ecocardiografía/métodos , Femenino , Defectos del Tabique Interventricular/diagnóstico por imagen , Ventrículos Cardíacos , Humanos , Masculino , Estudios RetrospectivosRESUMEN
Erythropoietin (EPO) has been reported to exert a beneficial effect on glucose metabolism in obesity. However, the effect of EPO on lipid metabolism and non-alcoholic fatty liver disease (NAFLD) was unclear. Furthermore, the effect of long acting erythropoiesis stimulating agents (ESA) on metabolism has not been poorly understood. The objective of this study was to investigate the effect of EPO and long acting ESA on NAFLD and lipid metabolism. We administered EPO and darbepoetin alpha (DEPO), a long acting ESA, by intraperitoneally injection for 4 weeks to mice with high-fat-diet (HFD)-induced obesity. EPO and DEPO treatment reduced body weight, ameliorated glucose tolerance and insulin resistance, and prevented lipid accumulation in liver and white adipose tissue (WAT). Administration of EPO and DEPO suppressed lipid synthesis-related protein in liver, including sterol regulatory element-binding protein 1 (SREBP-1), acetyl-CoA carboxylase (ACC1) and fatty acid synthase (FAS). EPO and DEPO also increased lipolysis protein in visceral WAT, including hormone-sensitive lipase (HSL), atni-adipose triglyceride lipase (ATGL). EPO and DEPO increased phosphorylation signal transducer and activator of transcription 3 (STAT3) and STAT5, transcriptional factors with crucial roles of lipid metabolism. These data suggest that EPO and DEPO ameliorated NAFLD by improving lipid metabolism via EPO/EPOR-induced STAT3 and STAT5 activation. EPO and DEPO may be a therapeutic option for NAFLD.
Asunto(s)
Darbepoetina alfa/uso terapéutico , Eritropoyetina/uso terapéutico , Hematínicos/uso terapéutico , Lipogénesis/efectos de los fármacos , Lipólisis/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Animales , Masculino , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Receptores de Eritropoyetina/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Recent progress in surgical and intensive care has improved the prognosis of congenital heart disease (CHD) associated with heterotaxy syndrome. Less is known, however, about pulmonary vascular complications in these patients. MethodsâandâResults: We reviewed medical records of 236 patients who were diagnosed with polysplenia syndrome at 2 institutions for pediatric cardiology in Japan from 1978 to 2015. We selected and compared the clinical records of 16 patients with polysplenia who had incomplete atrioventricular septal defect (AVSD) as the polysplenia group, and 22 age-matched patients with incomplete AVSD without any syndromes including polysplenia as the control group. Although the severity of systemic to pulmonary shunt was not significantly different between the groups, mean pulmonary artery pressure (mPAP) and pulmonary vascular resistance index (PVRI) were significantly higher in the polysplenia group than the control (mPAP, 37.3 vs. 19.1 mmHg, P=0.001; PVRI, 5.7 vs. 1.4 WUâm2, P=0.014) before surgical intervention. On regression analysis, polysplenia influenced the development of pulmonary hypertension (PH) regardless of age at evaluation or degree of systemic to pulmonary shunt in the patients with incomplete AVSD. CONCLUSIONS: Polysplenia syndrome is an independent risk factor for CHD-associated PH. Earlier intervention may be required to adjust the pulmonary blood flow in polysplenia syndrome with CHD to avoid the progression of PH.
Asunto(s)
Progresión de la Enfermedad , Síndrome de Heterotaxia/complicaciones , Hipertensión Pulmonar/etiología , Adolescente , Adulto , Estudios de Casos y Controles , Niño , Preescolar , Cardiopatías Congénitas , Humanos , Lactante , Japón , Arteria Pulmonar/fisiopatología , Estudios Retrospectivos , Factores de Riesgo , Adulto JovenRESUMEN
Pulmonary arterial hypertension (PAH) is a progressive disease associated with vasoconstriction and remodeling. Intracellular Ca2+ signaling regulates the contraction of pulmonary arteries and the proliferation of pulmonary arterial smooth muscle cells (PASMCs); however, it is not clear which molecules related to Ca2+ signaling contribute to the progression of PAH. In this study, we found the specific expression of type 2 inositol 1,4,5-trisphosphate receptor (IP3R2), which is an intracellular Ca2+ release channel, on the sarco/endoplasmic reticulum in mouse PASMCs, and demonstrated its inhibitory role in the progression of PAH using a chronic hypoxia-induced PAH mouse model. After chronic hypoxia exposure, IP3R2-/- mice exhibited the significant aggravation of PAH, as determined by echocardiography and right ventricular hypertrophy, with significantly greater medial wall thickness by immunohistochemistry than that of wild-type mice. In IP3R2-/- murine PASMCs with chronic hypoxia, a TUNEL assay revealed the significant suppression of apoptosis, whereas there was no significant change in proliferation. Thapsigargin-induced store-operated Ca2+ entry (SOCE) was significantly enhanced in IP3R2-/- PASMCs in both normoxia and hypoxia based on in vitro fluorescent Ca2+ imaging. Furthermore, the enhancement of SOCE in IP3R2-/- PASMCs was remarkably suppressed by the addition of DPB162-AE, an inhibitor of the stromal-interacting molecule (STIM)-Orai complex which is about 100 times more potent than 2-APB. Our results indicate that IP3R2 may inhibit the progression of PAH by promoting apoptosis and inhibiting SOCE via the STIM-Orai pathway in PASMCs. These findings suggest a previously undetermined role of IP3R in the development of PAH and may contribute to the development of targeted therapies.
Asunto(s)
Apoptosis , Calcio/metabolismo , Hipertensión Pulmonar/fisiopatología , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Músculo Liso Vascular/metabolismo , Arteria Pulmonar/fisiopatología , Animales , Señalización del Calcio , Células Cultivadas , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/patología , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/patología , Arteria Pulmonar/patología , VasoconstricciónRESUMEN
The renin-angiotensin system is a key regulator of metabolism with beneficial effects of the angiotensin 1-7 (Ang 1-7) peptide. We hypothesized that the antiobesity effect of Ang 1-7 was related to the stimulation of brown adipose tissue (BAT). We administered Ang 1-7 (0.54 mg kg-1 day-1) for 28 days via implanted micro-osmotic pumps to mice with high-fat diet (HFD)-induced obesity. Ang 1-7 treatment reduced body weight, upregulated thermogenesis, and ameliorated impaired glucose homeostasis without affecting food consumption. Furthermore, Ang 1-7 treatment enlarged BAT and the increased expression of UCP1, PRDM16, and prohibitin. Alterations in PRDM16 expression correlated with increased AMPK and phosphorylation of mTOR. Ang 1-7 treatment elevated thermogenesis in subcutaneous white adipose tissue without altering UCP1 expression. These changes occurred in the context of decreased lipid accumulation in BAT from HFD-fed mice, preserved insulin signaling concomitant with phosphorylation of hormone-sensitive lipase and decreased expression of perilipin. These data suggest that Ang 1-7 induces brown adipocyte differentiation leading to upregulation of thermogenesis and improved metabolic profile in diet-induced obesity. Enhancing Ang 1-7 action represents a promising therapy to increase BAT and to reduce the metabolic complications associated with diet-induced obesity.
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Tejido Adiposo Pardo/efectos de los fármacos , Angiotensina I/farmacología , Dieta Alta en Grasa , Obesidad/prevención & control , Fragmentos de Péptidos/farmacología , Termogénesis/efectos de los fármacos , Adipocitos Marrones/efectos de los fármacos , Adipocitos Marrones/fisiología , Tejido Adiposo Pardo/metabolismo , Animales , Peso Corporal/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/etiologíaRESUMEN
Both human embryonic stem cell-derived cardiomyocytes (ESC-CMs) and human induced pluripotent stem cell-derived CMs (iPSC-CMs) can serve as unlimited cell sources for cardiac regenerative therapy. However, the functional equivalency between human ESC-CMs and iPSC-CMs for cardiac regenerative therapy has not been demonstrated. Here, we performed a head-to-head comparison of ESC-CMs and iPSC-CMs in their ability to restore cardiac function in a rat myocardial infarction (MI) model as well as their exosomal secretome. Human ESCs and iPSCs were differentiated into CMs using small molecule inhibitors. Fluorescence-activated cell sorting analysis confirmed â¼85% and â¼83% of CMs differentiated from ESCs and iPSCs, respectively, were positive for cardiac troponin T. At a single-cell level, both cell types displayed similar calcium handling and electrophysiological properties, with gene expression comparable with the human fetal heart marked by striated sarcomeres. Sub-acute transplantation of ESC-CMs and iPSC-CMs into nude rats post-MI improved cardiac function, which was associated with increased expression of angiogenic genes in vitro following hypoxia. Profiling of exosomal microRNAs (miRs) and long non-coding RNAs (lncRNAs) revealed that both groups contain an identical repertoire of miRs and lncRNAs, including some that are known to be cardioprotective. We demonstrate that both ESC-CMs and iPSC-CMs can facilitate comparable cardiac repair. This is advantageous because, unlike allogeneic ESC-CMs used in therapy, autologous iPSC-CMs could potentially avoid immune rejection when used for cardiac cell transplantation in the future. Stem Cells 2017;35:2138-2149.
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Células Madre Embrionarias Humanas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Diferenciación Celular , Células Cultivadas , Exosomas , HumanosAsunto(s)
Tirosinemias , Humanos , Tirosinemias/complicaciones , Tirosinemias/diagnóstico , HeptanoatosRESUMEN
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful platform for uncovering disease mechanisms and assessing drugs for efficacy/toxicity. However, the accuracy with which hiPSC-CMs recapitulate the contractile and remodeling signaling of adult cardiomyocytes is not fully known. We used ß-adrenergic receptor (ß-AR) signaling as a prototype to determine the evolution of signaling component expression and function during hiPSC-CM maturation. In "early" hiPSC-CMs (less than or equal to d 30), ß2-ARs are a primary source of cAMP/PKA signaling. With longer culture, ß1-AR signaling increases: from 0% of cAMP generation at d 30 to 56.8 ± 6.6% by d 60. PKA signaling shows a similar increase: 15.7 ± 5.2% (d 30), 49.8 ± 0.5% (d 60), and 71.0 ± 6.1% (d 90). cAMP generation increases 9-fold from d 30 to 60, with enhanced coupling to remodeling pathways (e.g., Akt and Ca(2+)/calmodulin-dependent protein kinase type II) and development of caveolin-mediated signaling compartmentalization. By contrast, cardiotoxicity induced by chronic ß-AR stimulation, a major component of heart failure, develops much later: 5% cell death at d 30vs 55% at d 90. Moreover, ß-AR maturation can be accelerated by biomechanical stimulation. The differential maturation of ß-AR functionalvs remodeling signaling in hiPSC-CMs has important implications for their use in disease modeling and drug testing. We propose that assessment of signaling be added to the indices of phenotypic maturation of hiPSC-CMs.-Jung, G., Fajardo, G., Ribeiro, A. J. S., Kooiker, K. B., Coronado, M., Zhao, M., Hu, D.-Q., Reddy, S., Kodo, K., Sriram, K., Insel, P. A., Wu, J. C., Pruitt, B. L., Bernstein, D. Time-dependent evolution of functionalvs remodeling signaling in induced pluripotent stem cell-derived cardiomyocytes and induced maturation with biomechanical stimulation.
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Diferenciación Celular/fisiología , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/citología , Transducción de Señal/fisiología , Fenómenos Biomecánicos , Calcio/metabolismo , Diferenciación Celular/genética , Células Cultivadas , AMP Cíclico/metabolismo , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Immunoblotting , Células Madre Pluripotentes Inducidas/metabolismo , Microscopía Confocal , Miocitos Cardíacos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Factores de TiempoRESUMEN
BACKGROUND: Human induced pluripotent stem cells (iPSCs) are attractive candidates for therapeutic use, with the potential to replace deficient cells and to improve functional recovery in injury or disease settings. Here, we test the hypothesis that human iPSC-derived cardiomyocytes (iPSC-CMs) can secrete cytokines as a molecular basis to attenuate adverse cardiac remodeling after myocardial infarction. METHODS AND RESULTS: Human iPSCs were generated from skin fibroblasts and differentiated in vitro with a small molecule-based protocol. Troponin(+) iPSC-CMs were confirmed by immunohistochemistry, quantitative polymerase chain reaction, fluorescence-activated cell sorting, and electrophysiological measurements. Afterward, 2×10(6) iPSC-CMs derived from a cell line transduced with a vector expressing firefly luciferase and green fluorescent protein were transplanted into adult NOD/SCID mice with acute left anterior descending artery ligation. Control animals received PBS injection. Bioluminescence imaging showed limited engraftment on transplantation into ischemic myocardium. However, magnetic resonance imaging of animals transplanted with iPSC-CMs showed significant functional improvement and attenuated cardiac remodeling compared with PBS-treated control animals. To understand the underlying molecular mechanism, microfluidic single-cell profiling of harvested iPSC-CMs, laser capture microdissection of host myocardium, and in vitro ischemia stimulation were used to demonstrate that the iPSC-CMs could release significant levels of proangiogenic and antiapoptotic factors in the ischemic microenvironment. CONCLUSIONS: Transplantation of human iPSC-CMs into an acute mouse myocardial infarction model can improve left ventricular function and attenuate cardiac remodeling. Because of limited engraftment, most of the effects are possibly explained by paracrine activity of these cells.
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Células Madre Pluripotentes Inducidas/fisiología , Microfluídica/métodos , Infarto del Miocardio/terapia , Miocitos Cardíacos/fisiología , Análisis de la Célula Individual/métodos , Trasplante de Células Madre , Animales , Línea Celular , Femenino , Humanos , Células Madre Pluripotentes Inducidas/trasplante , Ratones , Ratones Endogámicos NOD , Ratones SCID , Infarto del Miocardio/patología , Distribución AleatoriaRESUMEN
BACKGROUND: Despite the promise shown by stem cells for restoration of cardiac function after myocardial infarction, the poor survival of transplanted cells has been a major issue. Hypoxia-inducible factor-1 (HIF1) is a transcription factor that mediates adaptive responses to ischemia. Here, we hypothesize that codelivery of cardiac progenitor cells (CPCs) with a nonviral minicircle plasmid carrying HIF1 (MC-HIF1) into the ischemic myocardium can improve the survival of transplanted CPCs. METHODS AND RESULTS: After myocardial infarction, CPCs were codelivered intramyocardially into adult NOD/SCID mice with saline, MC-green fluorescent protein, or MC-HIF1 versus MC-HIF1 alone (n=10 per group). Bioluminescence imaging demonstrated better survival when CPCs were codelivered with MC-HIF1. Importantly, echocardiography showed mice injected with CPCs+MC-HIF1 had the highest ejection fraction 6 weeks after myocardial infarction (57.1±2.6%; P=0.002) followed by MC-HIF1 alone (48.5±2.6%; P=0.04), with no significant protection for CPCs+MC-green fluorescent protein (44.8±3.3%; P=NS) when compared with saline control (38.7±3.2%). In vitro mechanistic studies confirmed that cardiac endothelial cells produced exosomes that were actively internalized by recipient CPCs. Exosomes purified from endothelial cells overexpressing HIF1 had higher contents of miR-126 and miR-210. These microRNAs activated prosurvival kinases and induced a glycolytic switch in recipient CPCs, giving them increased tolerance when subjected to in vitro hypoxic stress. Inhibiting both of these miRs blocked the protective effects of the exosomes. CONCLUSIONS: In summary, HIF1 can be used to modulate the host microenvironment for improving survival of transplanted cells. The exosomal transfer of miRs from host cells to transplanted cells represents a unique mechanism that can be potentially targeted for improving survival of transplanted cells.
Asunto(s)
Células Madre Adultas/trasplante , Exosomas , Terapia Genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/uso terapéutico , MicroARNs/uso terapéutico , Células Madre Multipotentes/trasplante , Infarto del Miocardio/terapia , Isquemia Miocárdica/terapia , Animales , Comunicación Celular , Microambiente Celular , Terapia Combinada , Medios de Cultivo Condicionados , ADN Circular , Femenino , Vectores Genéticos/uso terapéutico , Supervivencia de Injerto , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , MicroARNs/genética , Infarto del Miocardio/genética , Infarto del Miocardio/cirugía , Isquemia Miocárdica/genética , Isquemia Miocárdica/cirugía , Neovascularización Fisiológica , Plásmidos , Distribución Aleatoria , TransfecciónRESUMEN
RATIONALE: Human embryonic stem cell (hESC) derivatives are attractive candidates for therapeutic use. The engraftment and survival of hESC derivatives as xenografts or allografts require effective immunosuppression to prevent immune cell infiltration and graft destruction. OBJECTIVE: To test the hypothesis that a short-course, dual-agent regimen of two costimulation-adhesion blockade agents can induce better engraftment of hESC derivatives compared to current immunosuppressive agents. METHODS AND RESULTS: We transduced hESCs with a double fusion reporter gene construct expressing firefly luciferase (Fluc) and enhanced green fluorescent protein, and differentiated these cells to endothelial cells (hESC-ECs). Reporter gene expression enabled longitudinal assessment of cell engraftment by bioluminescence imaging. Costimulation-adhesion therapy resulted in superior hESC-EC and mouse EC engraftment compared to cyclosporine therapy in a hind limb model. Costimulation-adhesion therapy also promoted robust hESC-EC and hESC-derived cardiomyocyte survival in an ischemic myocardial injury model. Improved hESC-EC engraftment had a cardioprotective effect after myocardial injury, as assessed by magnetic resonance imaging. Mechanistically, costimulation-adhesion therapy is associated with systemic and intragraft upregulation of T-cell immunoglobulin and mucin domain 3 (TIM3) and a reduced proinflammatory cytokine profile. CONCLUSIONS: Costimulation-adhesion therapy is a superior alternative to current clinical immunosuppressive strategies for preventing the post-transplant rejection of hESC derivatives. By extending the window for cellular engraftment, costimulation-adhesion therapy enhances functional preservation following ischemic injury. This regimen may function through a TIM3-dependent mechanism.
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Anticuerpos Monoclonales/farmacología , Ciclosporina/farmacología , Células Madre Embrionarias/trasplante , Rechazo de Injerto/prevención & control , Inmunoconjugados/farmacología , Inmunosupresores/farmacología , Prednisona/farmacología , Abatacept , Animales , Cardiotónicos/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Células Madre Embrionarias/citología , Células Madre Embrionarias/inmunología , Células Endoteliales/inmunología , Células Endoteliales/trasplante , Rechazo de Injerto/inmunología , Humanos , Tolerancia Inmunológica , Terapia de Inmunosupresión/métodos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Infarto del Miocardio/inmunología , Infarto del Miocardio/patología , Infarto del Miocardio/cirugía , Distribución AleatoriaRESUMEN
Advances in the understanding and treatment of cardiac disorders have been thwarted by the inability to study beating human cardiac cells in vitro. Induced pluripotent stem cells (iPSCs) bypass this hurdle by enabling the creation of patient-specific iPSC-derived cardiomyocytes (iPSC-CMs). These cells provide a unique platform to study cardiac diseases in vitro, especially hereditary cardiac conditions. To date, iPSC-CMs have been used to successfully model arrhythmic disorders, showing excellent recapitulation of cardiac channel function and electrophysiologic features of long QT syndrome types 1, 2, 3, and 8, and catecholaminergic polymorphic ventricular tachycardia (CPVT). Similarly, iPSC-CM models of dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) have shown robust correlation of predicted morphologic, contractile, and electrical phenotypes. In addition, iPSC-CMs have shown some features of the respective phenotypes for arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C), LEOPARD syndrome, Pompe's disease, and Friedriech's ataxia. In this review, we examine the progress of utilizing iPSC-CMs as a model for cardiac conditions and analyze the potential for the platform in furthering the biology and treatment of cardiac disorders.
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Cardiopatías Congénitas/patología , Cardiopatías Congénitas/fisiopatología , Modelos Cardiovasculares , Cardiopatías Congénitas/terapia , HumanosRESUMEN
A six-year-old boy presented with short stature and gingival fibromatosis (GF). Dysmorphic features included slant optic fissures, a high-arched palate, thick earlobes, and an edematous face. Laboratory tests showed low levels of serum insulin-like growth factor-1 and serum free thyroxine but normal serum thyrotropin levels. Provocative tests suggested growth hormone deficiency, central hypocortisolemia, and hypothalamic hypothyroidism. At 12 years old, hypogonadotropic hypogonadism was observed. Next-generation sequencing revealed a heterozygous missense variant, KCNQ1 p. (P369L), in the proband and mother. The coexistence of multiple pituitary hormone deficiencies and GF helps diagnose KCNQ1-variant dysmorphic syndrome through genetic testing.