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Exosomes are double-layered lipid membranous nanovesicles that are endosomal in origin and secreted by almost all cells. They are 30-130 nm in size and contain various molecular signatures such as miRNAs, mRNAs, DNA, lipids, and proteins. Due to their highly heterogeneous content, exosomes have a major role in influencing cellular physiology and pathology. Although exosome research has been in progress for a long time, its biomedical applications have recently been expanding due to its bio-friendly nature. However, the most challenging part is its isolation to obtain quality exosomes with good yield. Therefore, in this chapter, we have described appropriate protocols for exosome isolation and characterization along with alternative purification methods.
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Exosomas , Exosomas/química , Exosomas/metabolismo , Humanos , Fraccionamiento Celular/métodos , Ultracentrifugación/métodosRESUMEN
Cardiac fibrosis is the hallmark of cardiovascular disease (CVD), which is leading cause of death worldwide. Previously, we have shown that interleukin-10 (IL10) reduces pressure overload (PO)-induced cardiac fibrosis by inhibiting the recruitment of bone marrow fibroblast progenitor cells (FPCs) to the heart. However, the precise mechanism of FPC involvement in cardiac fibrosis remains unclear. Recently, exosomes and small extracellular vesicles (sEVs) have been linked to CVD progression. Thus, we hypothesized that pro-fibrotic miRNAs enriched in sEV-derived from IL10 KO FPCs promote cardiac fibrosis in pressure-overloaded myocardium. Small EVs were isolated from FPCs cultured media and characterized as per MISEV-2018 guidelines. Small EV's miRNA profiling was performed using Qiagen fibrosis-associated miRNA profiler kit. For functional analysis, sEVs were injected in the heart following TAC surgery. Interestingly, TGFß-treated IL10-KO-FPCs sEV increased profibrotic genes expression in cardiac fibroblasts. The exosomal miRNA profiling identified miR-21a-5p as the key player, and its inhibition with antagomir prevented profibrotic signalling and fibrosis. At mechanistic level, miR-21a-5p binds and stabilizes ITGAV (integrin av) mRNA. Finally, miR-21a-5p-silenced in sEV reduced PO-induced cardiac fibrosis and improved cardiac function. Our study elucidates the mechanism by which inflammatory FPC-derived sEV exacerbate cardiac fibrosis through the miR-21a-5p/ITGAV/Col1α signalling pathway, suggesting miR-21a-5p as a potential therapeutic target for treating hypertrophic cardiac remodelling and heart failure.
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Energetic demand and nutrient supply fluctuate as a function of time-of-day, in alignment with sleep-wake and fasting-feeding cycles. These daily rhythms are mirrored by 24-hour oscillations in numerous cardiovascular functional parameters, including blood pressure, heart rate, and myocardial contractility. It is, therefore, not surprising that metabolic processes also fluctuate over the course of the day, to ensure temporal needs for ATP, building blocks, and metabolism-based signaling molecules are met. What has become increasingly clear is that in addition to classic signal-response coupling (termed reactionary mechanisms), cardiovascular-relevant cells use autonomous circadian clocks to temporally orchestrate metabolic pathways in preparation for predicted stimuli/stresses (termed anticipatory mechanisms). Here, we review current knowledge regarding circadian regulation of metabolism, how metabolic rhythms are synchronized with cardiovascular function, and whether circadian misalignment/disruption of metabolic processes contribute toward the pathogenesis of cardiovascular disease.
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Relojes Circadianos , Ritmo Circadiano , Corazón , Relojes Circadianos/fisiología , Sueño/fisiología , Miocardio/metabolismoRESUMEN
Exosomes are nanosized vesicles that carry biologically diverse molecules for intercellular communication. Researchers have been trying to engineer exosomes for therapeutic purposes by using different approaches to deliver biologically active molecules to the various target cells efficiently. Recent technological advances may allow the biodistribution and pharmacokinetics of exosomes to be modified to meet scientific needs with respect to specific diseases. However, it is essential to determine an exosome's optimal dosage and potential side effects before its clinical use. Significant breakthroughs have been made in recent decades concerning exosome labelling and imaging techniques. These tools provide in situ monitoring of exosome biodistribution and pharmacokinetics and pinpoint targetability. However, because exosomes are nanometres in size and vary significantly in contents, a deeper understanding is required to ensure accurate monitoring before they can be applied in clinical settings. Different research groups have established different approaches to elucidate the roles of exosomes and visualize their spatial properties. This review covers current and emerging strategies for in vivo and in vitro exosome imaging and tracking for potential studies.
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Enfermedades Cardiovasculares , Exosomas , Humanos , Exosomas/metabolismo , Enfermedades Cardiovasculares/terapia , Enfermedades Cardiovasculares/metabolismo , Distribución Tisular , Comunicación CelularRESUMEN
Among several known RNA modifications, N6-methyladenosine (m6A) is the most studied RNA epitranscriptomic modification and controls multiple cellular functions during development, differentiation, and disease. Current research advancements have made it possible to examine the regulatory mechanisms associated with RNA methylation and reveal its functional consequences in the pathobiology of many diseases, including heart failure. m6A methylation has been described both on coding (mRNA) and non-coding RNA species including rRNA, tRNA, small nuclear RNA and circular RNAs. The protein components which catalyze the m6A methylation are termed methyltransferase or 'm6A writers'. The family of proteins that recognize this methylation are termed 'm6A readers' and finally the enzymes involved in the removal of a methyl group from RNA are known as demethylases or 'm6A erasers'. At the cellular level, different components of methylation machinery are tightly regulated by many factors to maintain the m6A methylation dynamics. The m6A methylation process impacts different stages of mRNA metabolism and the biogenesis of long non-coding RNA and miRNA. Although, mRNA methylation was initially described in the 1970s, its regulatory roles in various diseases, including cardiovascular diseases are broadly unexplored. Recent investigations suggest the important role of m6A mRNA methylation in both hypertrophic and ischaemic heart diseases. In the present review, we evaluate the significance of m6A methylation in the cardiovascular system, in cardiac homeostasis and disease, all of which may help to improve therapeutic intervention for the treatment of heart failure. RNA methylation in cardiovascular diseases: altered m6A RNA (coding and non-coding RNA) methylation is identified during different cardiovascular diseases. Increased cardiac hypertrophy is observed following METTL3 overexpression. In contrast, reduced FTO level was seen in mice following myocardial infarction. Increased cardiac fibroblasts activation or increased atherosclerotic plaques were also co-related with m6A RNA methylation.
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Enfermedades Cardiovasculares , Sistema Cardiovascular , Insuficiencia Cardíaca , ARN Largo no Codificante , Animales , Biología , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/terapia , Sistema Cardiovascular/metabolismo , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Endotoxemia triggers life-threatening immune and cardiovascular response that leads to tissue damage, multi-organ failure, and death. The understanding of underlying molecular mechanisms is still evolving. N6-methyladenosine (m6A)-RNA modification plays key regulatory role in numerous biological processes. However, it remains unclear whether endotoxemia alters RNA methylation in the myocardium. In the current study, we investigated the effect of lipopolysaccharide (LPS)-induced endotoxemia on m6A-RNA methylation and its implications on myocardial inflammation and left ventricular (LV) function. Following LPS administration, mice showed increases in m6A-RNA methylation in the myocardium with a corresponding decrease in the expression of fat mass and obesity-associated protein (FTO, an m6A eraser/demethylase). The changes were associated with a significant increase in expression of myocardial inflammatory cytokine genes, such as IL-6, TNF-α, IL-1ß, and reduced LV function. Moreover, rat cardiomyoblasts (H9c2) exposed to LPS showed similar changes (with increase in m6A-RNA methylation and inflammatory cytokine genes, whereas downregulation of FTO). Furthermore, methylated RNA immunoprecipitation assay showed hypermethylation and increase in the expression of IL-6 and TNF-α genes in LPS-treated H9c2 cells as compared to untreated cells. Interestingly, FTO knockdown in cardiomyocytes mimicked the above effects. Taken together, these data suggest that endotoxemia-induced m6A methylation might play a critical role in expression of cardiac proinflammatory cytokines, and modulation of m6A methylation might limit myocardial inflammation and dysfunction during endotoxemia.
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Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/biosíntesis , Endotoxemia/metabolismo , Miocarditis/metabolismo , Miocardio/metabolismo , Procesamiento Postranscripcional del ARN , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Animales , Línea Celular , Endotoxemia/inducido químicamente , Endotoxemia/genética , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/metabolismo , Lipopolisacáridos/toxicidad , Ratones , Miocarditis/inducido químicamente , Miocarditis/genéticaRESUMEN
Cardiovascular disease is the leading cause of morbidity and mortality world-wide. Recently, the role of inflammation in the progression of diseases has significantly attracted considerable attention. In addition, various comorbidities, including diabetes, obesity, etc. exacerbate inflammation in the cardiovascular system, which ultimately leads to heart failure. Furthermore, cytokines released from specialized immune cells are key mediators of cardiac inflammation. Here, in this review article, we focused on the role of selected immune cells and cytokines (both pro-inflammatory and anti-inflammatory) in the regulation of cardiac inflammation and ultimately in cardiovascular diseases. While IL-1ß, IL-6, TNFα, and IFNγ are associated with cardiac inflammation; IL-10, TGFß, etc. are associated with resolution of inflammation and cardiac repair. IL-10 reduces cardiovascular inflammation and protects the cardiovascular system via interaction with SMAD2, p53, HuR, miR-375 and miR-21 pathway. In addition, we also highlighted recent advancements in the management of cardiac inflammation, including clinical trials of anti-inflammatory molecules to alleviate cardiovascular diseases.
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Antiinflamatorios/uso terapéutico , Enfermedades Cardiovasculares/tratamiento farmacológico , Animales , Corazón , Humanos , Inflamación/tratamiento farmacológico , Enfermedades Metabólicas/tratamiento farmacológicoRESUMEN
Background: Endothelial cells (ECs) play a critical role in the maintenance of vascular homeostasis and in heart function. It was shown that activated fibroblast-derived exosomes impair cardiomyocyte function in hypertrophic heart, but their effect on ECs is not yet clear. Thus, we hypothesized that activated cardiac fibroblast-derived exosomes (FB-Exo) mediate EC dysfunction, and therefore modulation of FB-exosomal contents may improve endothelial function. Methods and Results: Exosomes were isolated from cardiac fibroblast (FB)-conditioned media and characterized by nanoparticle tracking analysis and electron microscopy. ECs were isolated from mouse heart. ECs were treated with exosomes isolated from FB-conditioned media, following FB culture with TGF-ß1 (TGF-ß1-FB-Exo) or PBS (control) treatment. TGF-ß1 significantly activated fibroblasts as shown by increase in collagen type1 α1 (COL1α1), periostin (POSTN), and fibronectin (FN1) gene expression and increase in Smad2/3 and p38 phosphorylation. Impaired endothelial cell function (as characterized by a decrease in tube formation and cell migration along with reduced VEGF-A, Hif1α, CD31, and angiopoietin1 gene expression) was observed in TGF-ß1-FB-Exo treated cells. Furthermore, TGF-ß1-FB-Exo treated ECs showed reduced cell proliferation and increased apoptosis as compared to control cells. TGF-ß1-FB-Exo cargo analysis revealed an alteration in fibrosis-associated miRNAs, including a significant increase in miR-200a-3p level. Interestingly, miR-200a-3p inhibition in activated FBs, alleviated TGF-ß1-FB-Exo-mediated endothelial dysfunction. Conclusions: Taken together, this study demonstrates an important role of miR-200a-3p enriched within activated fibroblast-derived exosomes on endothelial cell biology and function.
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BACKGROUND: Endothelial cells dysfunction has been reported in many heart diseases including acute myocardial infarction, and atherosclerosis. The molecular mechanism for endothelial dysfunction in the heart is still not clearly understood. We aimed to study the role of m6A RNA demethylase alkB homolog 5 (ALKBH5) in ECs angiogenesis during ischemic injury. METHODS AND RESULTS: ECs were treated with ischemic insults (lipopolysaccharide and 1% hypoxia) to determine the role of ALKBH5 in ECs angiogenesis. siRNA mediated ALKBH5 gene silencing was used for examining the loss of function. In this study, we report that ALKBH5 levels are upregulated following ischemia and are associated with maintaining ischemia-induced ECs angiogenesis. To decipher the mechanism of action, we found that ALKBH5 is required to maintain eNOS phosphorylation and SPHK1 protein levels. ALKBH5 silencing alone or with ischemic stress significantly increased SPHK1 m6A mRNA methylation. In contrast, METTL3 (RNA methyltransferase) overexpression resulted in the reduced expression of SPHK1. CONCLUSION: We reported that ALKBH5 helps in the maintenance of angiogenesis in endothelial cells following acute ischemic stress via reduced SPHK1 m6A methylation and downstream eNOS-AKT signaling.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Exosomes are a group of extracellular microvesicles that deliver biologically active RNAs, proteins, lipids and other signaling molecules to recipient cells. Classically, exosomes act as a vehicle by which cells or organs communicate with each other to maintain cellular/tissue homeostasis and to respond to pathological stress. Most multicellular systems, including the cardiovascular system, use exosomes for intercellular communication. In heart, endogenous exosomes from cardiac cells or stem cells aid in regulation of cell survival, cell proliferation and cell death; and thus tightly regulate cardiac biology and repair processes. Pathological stimulus in heart alters secretion and molecular composition of exosomes, thus influencing the above processes. The past decade has yielded increasing interest in the role of exosomes in the cardiovascular system and significant contribution of cardiac fibroblast (CF) and mediated cardiac fibrosis in heart failure, in this review we had overviewed the relevant literatures about fibroblast exosomes, its effect in the cardiovascular biology and its impact on cardiovascular disease (CVD). This review briefly describes the communication between fibroblasts and other cardiac cells via exosomes, the influence of such on myocardial fibrosis and remodeling, and the possibilities to use exosomes as biomarkers for acute and chronic heart diseases.
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Circular RNAs are generated from many protein-coding genes, but their role in cardiovascular health and disease states remains unknown. Here we report identification of circRNA transcripts that are differentially expressed in post myocardial infarction (MI) mouse hearts including circFndc3b which is significantly down-regulated in the post-MI hearts. Notably, the human circFndc3b ortholog is also significantly down-regulated in cardiac tissues of ischemic cardiomyopathy patients. Overexpression of circFndc3b in cardiac endothelial cells increases vascular endothelial growth factor-A expression and enhances their angiogenic activity and reduces cardiomyocytes and endothelial cell apoptosis. Adeno-associated virus 9 -mediated cardiac overexpression of circFndc3b in post-MI hearts reduces cardiomyocyte apoptosis, enhances neovascularization and improves left ventricular functions. Mechanistically, circFndc3b interacts with the RNA binding protein Fused in Sarcoma to regulate VEGF expression and signaling. These findings highlight a physiological role for circRNAs in cardiac repair and indicate that modulation of circFndc3b expression may represent a potential strategy to promote cardiac function and remodeling after MI.
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Fibronectinas/genética , Infarto del Miocardio/metabolismo , Isquemia Miocárdica/metabolismo , ARN Circular/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Apoptosis/fisiología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Isquemia Miocárdica/genética , Isquemia Miocárdica/patología , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , ARN Circular/biosíntesis , ARN Circular/genética , Proteína FUS de Unión a ARN/genéticaAsunto(s)
Glucógeno Sintasa Quinasa 3 beta , Glucógeno Sintasa Quinasa 3 , Corazón , Humanos , Obesidad , FosforilaciónRESUMEN
The physiological cardiac hypertrophy is an adaptive condition without myocyte cell death, while pathological hypertrophy is a maladaptive condition associated with myocyte cell death. This study explores the miRNome of α-2M-induced physiologically hypertrophied cardiomyocytes and the role of miRNA-99 family during cardiac hypertrophy. Physiological and pathological cardiac hypertrophy was induced in H9c2 cardiomyoblast cell lines using α-2M and isoproterenol respectively. Total RNA isolation and small RNA sequencing were executed for physiological hypertrophy model. The differentially expressed miRNAs and its target mRNAs were validated in animal models. Transcription factor binding sites were predicted in the promoter of specific miRNAs and validated by ChIP-PCR. Subsequently, the selected miRNA was functionally characterized by overexpression and silencing. The effects of silencing of upstream regulator and downstream target gene were studied. Analysis of small RNA reads revealed the differential expression of a large set of miRNAs during hypertrophy, of which miR-99 family was highly downregulated upon α-2M treatment. However, this miR-99 family expression was upregulated during pathological hypertrophy and confirmed in animal models. ChIP-PCR confirms the binding of Egr-1 transcription factor to the miR-99 promoter. Further, silencing of Egr-1 decreased the expression of miR-99. The overexpression or silencing of miR-99 diverges the physiological hypertrophy to pathological hypertrophy and vice versa by regulating Akt-1 pathway. Silencing of Akt-1 replicates the effect of overexpression of miR-99. CONCLUSION: The results proved Egr-1 mediated regulation of miR-99 family that plays a key role in determining the fate of cardiac hypertrophy by regulating Akt-1 signaling.
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Cardiomegalia/genética , Cardiomegalia/patología , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , MicroARNs/genética , Miocitos Cardíacos/patología , Animales , Línea Celular , Regulación hacia Abajo/genética , Regiones Promotoras Genéticas/genética , Ratas , Ratas Wistar , Transducción de Señal/genética , Regulación hacia Arriba/genéticaRESUMEN
Endothelial progenitor cell (EPC)-based therapy has immense potential to promote cardiac neovascularization and attenuate ischemic injury. Functional benefits of EPCs and other adult stem cell therapies largely involve paracrine mechanisms and exosomes secreted by stem cells are emerging as pivotal paracrine entity of stem/progenitor cells. However, modest outcomes after EPC-/stem cell-based clinical trials suggest that stem cell/exosome function might be modulated by stimuli they encounter in ischemic tissues, including systemic inflammation. We hypothesized that EPCs under inflammatory stress might produce exosomes of altered and dysfunctional content, which may compromise EPC repair in ischemic heart disease. We have previously shown that EPCs obtained from interleukin-10 knockout (IL-10KO) mice (model mimicking systemic inflammation) display impaired angiogenic functions. Whether IL-10KO-EPC-derived exosomes inherit their parental dysfunctional phenotype and whether inflammatory environment alters the cargo of their secreted exosomes are not known. After cell expansion from IL-10KO and wild-type (WT) mice, we isolated exosomes and compared their functions in terms of effect on cell survival, proliferation, migration, and angiogenic capacity in vitro. WT-EPC-Exo treatment enhanced endothelial cell proliferation and tube formation, and inhibited apoptosis, whereas IL-10KO-Exo exhibited impaired or even detrimental effects, suggesting that the reparative capacity of WT-EPC-Exo is lost in exosomes derived from IL-10-KO-EPCs. Deep RNA sequencing and proteomic analyses to compare WT and IL-10KO-Exo revealed drastically altered exosome cargo. Importantly, IL-10KO-EPC-Exo were highly enriched in microRNAs and proteins that promote inflammation and apoptosis and inhibit angiogenesis. Through modulation of a specific enriched miRNA (miR-375), we partially rescued IL-10KO-EPC-Exo dysfunction. Thus, our study revealed that EPC exosomes display impaired function under inflammatory stimulus through changed exosome contents, and the dysfunction can be rescued by modulation of a specific target packed in exosomes.
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Células de la Médula Ósea/metabolismo , Células Progenitoras Endoteliales/metabolismo , Exosomas/metabolismo , Interleucina-10/deficiencia , Animales , Perfilación de la Expresión Génica , Interleucina-10/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/metabolismo , FenotipoRESUMEN
Mitochondrial dysfunction and associated oxidative stress are strongly linked to cardiovascular, neurodegenerative, and age associated disorders. More specifically cardiovascular diseases are common in patients with diabetes and significant contributor to the high mortality rates associated with diabetes. Studies have shown that the heart failure risk is increased in diabetic patients even after adjusting for coronary artery disease and hypertension. Although the actual basis of the increased heart failure risk is multifactorial, increasing evidences suggest that imbalances in mitochondrial function and associated oxidative stress play an important role in this process. This review summarizes these abnormalities in mitochondrial function and discusses potential underlying mechanisms. This article is part of a Special Issue entitled: Oxidative Stress and Mitochondrial Quality in Diabetes/Obesity and Critical Illness Spectrum of Diseases - edited by P. Hemachandra Reddy.
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Enfermedad de la Arteria Coronaria/metabolismo , Cardiomiopatías Diabéticas/metabolismo , Hipertensión/metabolismo , Mitocondrias Cardíacas/metabolismo , Miocardio/metabolismo , Estrés Oxidativo , Animales , Enfermedad de la Arteria Coronaria/patología , Cardiomiopatías Diabéticas/patología , Humanos , Hipertensión/patología , Mitocondrias Cardíacas/patología , Miocardio/patologíaRESUMEN
BACKGROUND: Although autophagy is an essential cellular salvage process to maintain cellular homeostasis, pathological autophagy can lead to cardiac abnormalities and ultimately heart failure. Therefore, a tight regulation of autophagic process would be important to treat chronic heart failure. Previously, we have shown that IL-10 strongly inhibited pressure overload-induced hypertrophy and heart failure, but role of IL-10 in regulation of pathological autophagy is unknown. Here we tested the hypothesis that IL-10 inhibits angiotensin II-induced pathological autophagy and this process, in part, leads to improve cardiac function. METHODS AND RESULTS: Chronic Ang II strongly induced mortality, cardiac dysfunction in IL-10 Knockout mice. IL-10 deletion exaggerated pathological autophagy in response to Ang II treatment. In isolated cardiac myocytes, IL-10 attenuated Ang II-induced pathological autophagy and activated Akt/mTORC1 signaling. Pharmacological or molecular inhibition of Akt and mTORC1 signaling attenuated IL-10 effects on Ang II-induced pathological autophagy. Furthermore, lysosomal inhibition in autophagic flux experiments further confirmed that IL-10 inhibits pathological autophagy via mTORC1 signaling. CONCLUSION: Our data demonstrate a novel role of IL-10 in regulation of pathological autophagy; thus can act as a potential therapeutic molecule for treatment of chronic heart disease.
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Autofagia , Cardiomegalia/patología , Interleucina-10/metabolismo , Angiotensina II/administración & dosificación , Animales , Animales Recién Nacidos , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia/efectos de los fármacos , Beclina-1 , Cardiomegalia/complicaciones , Regulación hacia Abajo/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Eliminación de Gen , Insuficiencia Cardíaca/complicaciones , Insuficiencia Cardíaca/patología , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/ultraestructura , Interleucina-10/deficiencia , Interleucina-10/farmacología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Poor survival and function of transplanted cells in ischemic and inflamed myocardium likely compromises the functional benefit of stem cell-based therapies. We have earlier reported that co-administration of interleukin (IL)-10 and BMPAC enhances cell survival and improves left ventricular (LV) functions after acute myocardial infarction (MI) in mice. We hypothesized that IL-10 regulates microRNA-375 (miR-375) signaling in BMPACs to enhance their survival and function in ischemic myocardium after MI and attenuates left ventricular dysfunction after MI. miR-375 expression is significantly upregulated in BMPACs upon exposure to inflammatory/hypoxic stimulus and also after MI. IL-10 knockout mice display significantly elevated miR-375 levels. We report that ex vivo miR-375 knockdown in BMPAC before transplantation in the ischemic myocardium after MI significantly improve the survival and retention of transplanted BMPACs and also BMPAC-mediated post-infarct repair, neovascularization, and LV functions. Our in vitro studies revealed that knockdown of miR-375-enhanced BMPAC proliferation and tube formation and inhibited apoptosis; over expression of miR-375 in BMPAC had opposite effects. Mechanistically, miR-375 negatively regulated 3-phosphoinositide-dependent protein kinase-1 (PDK-1) expression and PDK-1-mediated activation of PI3kinase/AKT signaling. Interestingly, BMPAC isolated from IL-10-deficient mice showed elevated basal levels of miR-375 and exhibited functional deficiencies, which were partly rescued by miR-375 knockdown, enhancing BMPAC function in vitro and in vivo. Taken together, our studies suggest that miR-375 is negatively associated with BMPAC function and survival and IL-10-mediated repression of miR-375 enhances BMPAC survival and function.
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Células de la Médula Ósea/metabolismo , Interleucina-10/metabolismo , MicroARNs/metabolismo , Infarto del Miocardio/metabolismo , Miocardio/metabolismo , Trasplante de Células Madre , Células Madre/metabolismo , Animales , Células de la Médula Ósea/patología , Técnicas de Silenciamiento del Gen , Interleucina-10/genética , Ratones , Ratones Noqueados , MicroARNs/genética , Infarto del Miocardio/genética , Infarto del Miocardio/terapia , Miocardio/patología , Células Madre/patologíaRESUMEN
RATIONALE: Embryonic stem cells (ESCs) hold great promise for cardiac regeneration but are susceptible to various concerns. Recently, salutary effects of stem cells have been connected to exosome secretion. ESCs have the ability to produce exosomes, however, their effect in the context of the heart is unknown. OBJECTIVE: Determine the effect of ESC-derived exosome for the repair of ischemic myocardium and whether c-kit(+) cardiac progenitor cells (CPCs) function can be enhanced with ESC exosomes. METHODS AND RESULTS: This study demonstrates that mouse ESC-derived exosomes (mES Ex) possess ability to augment function in infarcted hearts. mES Ex enhanced neovascularization, cardiomyocyte survival, and reduced fibrosis post infarction consistent with resurgence of cardiac proliferative response. Importantly, mES Ex augmented CPC survival, proliferation, and cardiac commitment concurrent with increased c-kit(+) CPCs in vivo 8 weeks after in vivo transfer along with formation of bonafide new cardiomyocytes in the ischemic heart. miRNA array revealed significant enrichment of miR290-295 cluster and particularly miR-294 in ESC exosomes. The underlying basis for the beneficial effect of mES Ex was tied to delivery of ESC specific miR-294 to CPCs promoting increased survival, cell cycle progression, and proliferation. CONCLUSIONS: mES Ex provide a novel cell-free system that uses the immense regenerative power of ES cells while avoiding the risks associated with direct ES or ES-derived cell transplantation and risk of teratomas. ESC exosomes possess cardiac regeneration ability and modulate both cardiomyocyte and CPC-based repair programs in the heart.