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[This retracts the article DOI: 10.1016/j.isci.2019.06.017.].
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[This retracts the article DOI: 10.1016/j.isci.2019.04.009.].
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The inability to effectively stimulate cardiomyocyte proliferation remains a principle barrier to regeneration in the adult human heart. A tightly regulated, acute inflammatory response mediated by a range of cell types is required to initiate regenerative processes. Prostaglandin E2 (PGE2), a potent lipid signaling molecule induced by inflammation, has been shown to promote regeneration and cell proliferation; however, the dynamics of PGE2 signaling in the context of heart regeneration remain underexplored. Here, we employ the regeneration-competent zebrafish to characterize components of the PGE2 signaling circuit following cardiac injury. In the regenerating adult heart, we documented an increase in PGE2 levels, concurrent with upregulation of cox2a and ptges, two genes critical for PGE2 synthesis. Furthermore, we identified the epicardium as the most prominent site for cox2a expression, thereby suggesting a role for this tissue as an inflammatory mediator. Injury also drove the opposing expression of PGE2 receptors, upregulating pro-restorative ptger2a and downregulating the opposing receptor ptger3. Importantly, treatment with pharmacological inhibitors of Cox2 activity suppressed both production of PGE2, and the proliferation of cardiomyocytes. These results suggest that injury-induced PGE2 signaling is key to stimulating cardiomyocyte proliferation during regeneration.
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Dinoprostona/metabolismo , Lesiones Cardíacas/metabolismo , Corazón/fisiología , Regeneración , Animales , Animales Modificados Genéticamente , Proliferación Celular , Regulación hacia Abajo , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Hibridación in Situ , Inflamación , Lípidos/química , Miocitos Cardíacos/metabolismo , Transducción de Señal , Pez CebraRESUMEN
The adult zebrafish is capable of regenerating heart muscle, resolving collagen tissue, and fully restoring heart function throughout its life. In this study, we show that the highly upregulated, epicardium-enriched microRNA let-7i functions in wound closure and cardiomyocyte proliferation. RNA sequencing experiments identified upregulated expression of members of the tumor necrosis factor (TNF) signaling pathway in the absence of let-7. Importantly, co-suppression of TNF and let-7 activity rescued epicardium migration and cardiomyocyte proliferation defects induced by depletion of let-7 alone. Sensitizing animals to low levels of TNF activity before injury culminated in repressed cardiomyocyte proliferation and wound closure defects, suggesting that levels of inflammation at the onset of injury are critical for heart regeneration. Our studies indicate that injury-induced reduction in TNF signaling by let-7 in the epicardium creates a pro-regenerative environment for cardiomyocyte proliferation during adult heart regeneration.
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Stimulating cardiomyocyte regeneration after an acute injury remains the central goal in cardiovascular regenerative biology. While adult mammals respond to cardiac damage with deposition of rigid scar tissue, adult zebrafish and salamander unleash a regenerative program that culminates in new cardiomyocyte formation, resolution of scar tissue, and recovery of heart function. Recent studies have shown that immune cells are key to regulating pro-inflammatory and pro-regenerative signals that shift the injury microenvironment toward regeneration. Defining the genetic regulators that control the dynamic interplay between immune cells and injured cardiac tissue is crucial to decoding the endogenous mechanism of heart regeneration. In this review, we discuss our current understanding of the extent that macrophage and regulatory T cells influence cardiomyocyte proliferation and how microRNAs (miRNAs) regulate their activity in the injured heart.
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Regeneration is an endogenous process of tissue repair that culminates in complete restoration of tissue and organ function. While regenerative capacity in mammals is limited to select tissues, lower vertebrates like zebrafish and salamanders are endowed with the capacity to regenerate entire limbs and most adult tissues, including heart muscle. Numerous profiling studies have been conducted using these research models in an effort to identify the genetic circuits that accompany tissue regeneration. Most of these studies, however, are confined to an individual injury model and/or research organism and focused primarily on protein encoding transcripts. Here we describe RegenDbase, a new database with the functionality to compare and contrast gene regulatory pathways within and across tissues and research models. RegenDbase combines pipelines that integrate analysis of noncoding RNAs in combination with protein encoding transcripts. We created RegenDbase with a newly generated comprehensive dataset for adult zebrafish heart regeneration combined with existing microarray and RNA-sequencing studies on multiple injured tissues. In this current release, we detail microRNA-mRNA regulatory circuits and the biological processes these interactions control during the early stages of heart regeneration. Moreover, we identify known and putative novel lncRNAs and identify their potential target genes based on proximity searches. We postulate that these candidate factors underscore robust regenerative capacity in lower vertebrates. RegenDbase provides a systems-level analysis of tissue regeneration genetic circuits across injury and animal models and addresses the growing need to understand how noncoding RNAs influence these changes in gene expression.
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Elucidating the spatial resolution of gene transcripts provides important insight into potential gene function. MicroRNAs are short, singled-stranded noncoding RNAs that control gene expression through base-pair complementarity with target mRNAs in the 3' untranslated region (UTR) and inhibiting protein expression. However, given their small size of ~22- to 24-nt and low expression levels, standard in situ hybridization detection methods are not amendable for microRNA spatial resolution. Here, I describe a technique that employs RNAscope probe design and propriety amplification technology that provides simultaneous single molecule detection of individual microRNA and its target gene. This method allows for rapid and sensitive detection of noncoding RNA transcripts in frozen tissue sections.
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Regulación de la Expresión Génica , Hibridación in Situ/métodos , MicroARNs/genética , Sondas ARN/metabolismo , Animales , Antígenos/metabolismo , Corazón , Procesamiento de Señales Asistido por Computador , Pez Cebra/genéticaRESUMEN
Regenerative medicine holds substantial promise for repairing or replacing tissues and organs damaged by disease, injury, and degeneration. Much of the field has focused on development of cell-based therapeutics, gene-based therapeutics, and tissue engineering-based therapeutics. In contrast, development of small molecule regenerative medicine therapies is an emerging area. Using the adult zebrafish as a novel screening platform, we identified MSI-1436 as a first-in-class regenerative medicine drug candidate. MSI-1436 is a naturally occurring aminosterol that inhibits protein tyrosine phosphatase 1B. Treatment of adult zebrafish by intraperitoneal injection of MSI-1436 increased the rate of regeneration of the amputated caudal fin, which is comprised of bone, connective, skin, vascular and nervous tissues and also increased the rate of adult zebrafish heart regeneration. Intraperitoneal administration of MSI-1436 to adult mice for 4 weeks after induction of myocardial infarction increased survival, improved heart function, reduced infarct size, reduced ventricular wall thinning and increased cardiomyocyte proliferation. Satellite cell activation in injured mouse skeletal muscle was stimulated by MSI-1436. MSI-1436 was well tolerated by patients in Phase 1 and 1b obesity and type 2 diabetes clinical trials. Doses effective at stimulating regeneration are 5-50-times lower than the maximum well tolerated human dose. The demonstrated safety and well established pharmacological properties of MSI-1436 underscore the potential of this molecule as a novel treatment for heart attack and multiple other degenerative diseases.
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A volume-regulated anion channel (VRAC) has been electrophysiologically characterized in innumerable mammalian cell types. VRAC is activated by cell swelling and mediates the volume regulatory efflux of Cl(-) and small organic solutes from cells. Two groups recently identified the mammalian leucine-rich repeat containing protein LRRC8A as an essential VRAC component. LRRC8A must be coexpressed with at least one of the other four members of this gene family, LRRC8B-E, to reconstitute VRAC activity in LRRC8(-/-) cells. LRRC8 genes likely arose with the origin of chordates. We identified LRRC8A and LRRC8C-E orthologs in the zebrafish genome and demonstrate that zebrafish embryo cells and differentiated adult cell types express a swelling-activated Cl(-) current indistinguishable from mammalian VRAC currents. Embryo cell VRAC currents are virtually eliminated by morpholino knockdown of the zebrafish LRRC8A ortholog lrrc8aa VRAC activity is fully reconstituted in LRRC8(-/-) human cells by coexpression of zebrafish lrrc8aa and human LRRC8C cDNAs. lrrc8aa expression varies during zebrafish embryogenesis and lrrc8aa knockdown causes pericardial edema and defects in trunk elongation and somatogenesis. Our studies provide confirmation of the importance of LRRC8A in VRAC activity and establish the zebrafish as a model system for characterizing the molecular regulation and physiological roles of VRAC and LRRC8 proteins.
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Tamaño de la Célula , Canales de Cloruro/fisiología , Desarrollo Embrionario/fisiología , Transporte Iónico/fisiología , Leucina/metabolismo , Proteínas/metabolismo , Pez Cebra/genética , Animales , Proteínas de Ciclo Celular/genética , Cloruros/metabolismo , Técnicas de Inactivación de Genes/métodos , Humanos , Canales Iónicos/metabolismo , Proteínas Repetidas Ricas en Leucina , Potenciales de la Membrana/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos BiológicosRESUMEN
BACKGROUND: Although regenerative capacity is evident throughout the animal kingdom, it is not equally distributed throughout evolution. For instance, complex limb/appendage regeneration is muted in mammals but enhanced in amphibians and teleosts. The defining characteristic of limb/appendage regenerative systems is the formation of a dedifferentiated tissue, termed blastema, which serves as the progenitor reservoir for regenerating tissues. In order to identify a genetic signature that accompanies blastema formation, we employ next-generation sequencing to identify shared, differentially regulated mRNAs and noncoding RNAs in three different, highly regenerative animal systems: zebrafish caudal fins, bichir pectoral fins and axolotl forelimbs. RESULTS: These studies identified a core group of 5 microRNAs (miRNAs) that were commonly upregulated and 5 miRNAs that were commonly downregulated, as well as 4 novel tRNAs fragments with sequences conserved with humans. To understand the potential function of these miRNAs, we built a network of 1,550 commonly differentially expressed mRNAs that had functional relationships to 11 orthologous blastema-associated genes. As miR-21 was the most highly upregulated and most highly expressed miRNA in all three models, we validated the expression of known target genes, including the tumor suppressor, pdcd4, and TGFß receptor subunit, tgfbr2 and novel putative target genes such as the anti-apoptotic factor, bcl2l13, Choline kinase alpha, chka and the regulator of G-protein signaling, rgs5. CONCLUSIONS: Our extensive analysis of RNA-seq transcriptome profiling studies in three regenerative animal models, that diverged in evolution ~420 million years ago, reveals a common miRNA-regulated genetic network of blastema genes. These comparative studies extend our current understanding of limb/appendage regeneration by identifying previously unassociated blastema genes and the extensive regulation by miRNAs, which could serve as a foundation for future functional studies to examine the process of natural cellular reprogramming in an injury context.
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Ambystoma mexicanum/fisiología , Extremidades/fisiología , Peces/fisiología , Redes Reguladoras de Genes , MicroARNs/genética , Regeneración/fisiología , Pez Cebra/fisiología , Aletas de Animales/fisiología , Animales , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , MicroARNs/metabolismo , ARN no Traducido/genética , Análisis de Secuencia de ARN , Transducción de SeñalRESUMEN
Cardiovascular disease is the leading cause of morbidity and mortality in the Western world owing to the limited regenerative capacity of the mammalian cardiovascular system. In lieu of new muscle synthesis, the human heart replaces necrotic tissue with deposition of a noncontractile scar. By contrast, the adult zebrafish is endowed with a remarkable regenerative capacity, capable of de novo cardiomyocyte (CM) creation and scar tissue removal when challenged with an acute injury. In these studies, we examined the contributions of the dynamically regulated microRNA miR-101a during adult zebrafish heart regeneration. We demonstrate that miR-101a expression is rapidly depleted within 3 days post-amputation (dpa) but is highly upregulated by 7-14 dpa, before returning to uninjured levels at the completion of the regenerative process. Employing heat-inducible transgenic strains and antisense oligonucleotides, we demonstrate that decreases in miR-101a levels at the onset of cardiac injury enhanced CM proliferation. Interestingly, prolonged suppression of miR-101a activity stimulates new muscle synthesis but with defects in scar tissue clearance. Upregulation of miR-101a expression between 7 and 14 dpa is essential to stimulate removal of the scar. Through a series of studies, we identified the proto-oncogene fosab (cfos) as a potent miR-101a target gene, stimulator of CM proliferation, and inhibitor of scar tissue removal. Importantly, combinatorial depletion of fosab and miR-101a activity rescued defects in scar tissue clearance mediated by miR-101a inhibition alone. In summation, our studies indicate that the precise temporal modulation of the miR-101a/fosab genetic axis is crucial for coordinating CM proliferation and scar tissue removal during zebrafish heart regeneration.
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Regulación del Desarrollo de la Expresión Génica , Corazón/fisiología , MicroARNs/genética , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas de Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo , Animales , Animales Modificados Genéticamente , Sistema Cardiovascular , Proliferación Celular , Cicatriz/patología , Perfilación de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Miocitos Cardíacos/citología , Necrosis , Oligonucleótidos Antisentido/genética , Proto-Oncogenes Mas , Regeneración/fisiología , Factores de TiempoRESUMEN
The first critical stage in salamander or teleost appendage regeneration is creation of a specialized epidermis that instructs growth from underlying stump tissue. Here, we performed a forward genetic screen for mutations that impair this process in amputated zebrafish fins. Positional cloning and complementation assays identified a temperature-sensitive allele of the ECM component laminin beta 1a (lamb1a) that blocks fin regeneration. lamb1a, but not its paralog lamb1b, is sharply induced in a subset of epithelial cells after fin amputation, where it is required to establish and maintain a polarized basal epithelial cell layer. These events facilitate expression of the morphogenetic factors shha and lef1, basolateral positioning of phosphorylated Igf1r, patterning of new osteoblasts, and regeneration of bone. By contrast, lamb1a function is dispensable for juvenile body growth, homeostatic adult tissue maintenance, repair of split fins, or renewal of genetically ablated osteoblasts. fgf20a mutations or transgenic Fgf receptor inhibition disrupt lamb1a expression, linking a central growth factor to epithelial maturation during regeneration. Our findings reveal transient induction of lamb1a in epithelial cells as a key, growth factor-guided step in formation of a signaling-competent regeneration epidermis.
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Aletas de Animales/fisiología , Laminina/genética , Regeneración , Proteínas de Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Epidermis/fisiología , Femenino , Factores de Crecimiento de Fibroblastos/fisiología , Laminina/metabolismo , Masculino , Datos de Secuencia Molecular , Transducción de Señal , Activación Transcripcional , Cicatrización de Heridas , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
PURPOSE: The blood-brain barrier (BBB) essentially restricts therapeutic drugs from entering into the brain. This study tests the hypothesis that brain endothelial cell derived exosomes can deliver anticancer drug across the BBB for the treatment of brain cancer in a zebrafish (Danio rerio) model. MATERIALS AND METHODS: Four types of exosomes were isolated from brain cell culture media and characterized by particle size, morphology, total protein, and transmembrane protein markers. Transport mechanism, cell uptake, and cytotoxicity of optimized exosome delivery system were tested. Brain distribution of exosome delivered anticancer drugs was evaluated using transgenic zebrafish TG (fli1: GFP) embryos and efficacies of optimized formations were examined in a xenotransplanted zebrafish model of brain cancer model. RESULTS: Four exosomes in 30-100 diameters showed different morphologies and exosomes derived from brain endothelial cells expressed more CD63 tetraspanins transmembrane proteins. Optimized exosomes increased the uptake of fluorescent marker via receptor mediated endocytosis and cytotoxicity of anticancer drugs in cancer cells. Images of the zebrafish showed exosome delivered anticancer drugs crossed the BBB and entered into the brain. In the brain cancer model, exosome delivered anticancer drugs significantly decreased fluorescent intensity of xenotransplanted cancer cells and tumor growth marker. CONCLUSIONS: Brain endothelial cell derived exosomes could be potentially used as a carrier for brain delivery of anticancer drug for the treatment of brain cancer.
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Antineoplásicos/farmacología , Barrera Hematoencefálica/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Doxorrubicina/farmacología , Sistemas de Liberación de Medicamentos/métodos , Células Endoteliales/metabolismo , Exosomas/metabolismo , Paclitaxel/farmacología , Animales , Animales Modificados Genéticamente , Antineoplásicos/química , Antineoplásicos/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Permeabilidad Capilar , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Química Farmacéutica , Modelos Animales de Enfermedad , Doxorrubicina/química , Doxorrubicina/metabolismo , Endocitosis , Genes Reporteros , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Xenoinjertos , Humanos , Trasplante de Neoplasias , Paclitaxel/química , Paclitaxel/metabolismo , Tamaño de la Partícula , Tecnología Farmacéutica/métodos , Factores de Tiempo , Carga Tumoral/efectos de los fármacos , Pez CebraRESUMEN
Basement membrane, a specialized ECM that underlies polarized epithelium of eumetazoans, provides signaling cues that regulate cell behavior and function in tissue genesis and homeostasis. A collagen IV scaffold, a major component, is essential for tissues and dysfunctional in several diseases. Studies of bovine and Drosophila tissues reveal that the scaffold is stabilized by sulfilimine chemical bonds (S = N) that covalently cross-link methionine and hydroxylysine residues at the interface of adjoining triple helical protomers. Peroxidasin, a heme peroxidase embedded in the basement membrane, produces hypohalous acid intermediates that oxidize methionine, forming the sulfilimine cross-link. We explored whether the sulfilimine cross-link is a fundamental requirement in the genesis and evolution of epithelial tissues by determining its occurrence and evolutionary origin in Eumetazoa and its essentiality in zebrafish development; 31 species, spanning 11 major phyla, were investigated for the occurrence of the sulfilimine cross-link by electrophoresis, MS, and multiple sequence alignment of de novo transcriptome and available genomic data for collagen IV and peroxidasin. The results show that the cross-link is conserved throughout Eumetazoa and arose at the divergence of Porifera and Cnidaria over 500 Mya. Also, peroxidasin, the enzyme that forms the bond, is evolutionarily conserved throughout Metazoa. Morpholino knockdown of peroxidasin in zebrafish revealed that the cross-link is essential for organogenesis. Collectively, our findings establish that the triad-a collagen IV scaffold with sulfilimine cross-links, peroxidasin, and hypohalous acids-is a primordial innovation of the ECM essential for organogenesis and tissue evolution.
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Membrana Basal/metabolismo , Evolución Biológica , Iminas/química , Compuestos de Azufre/química , Secuencia de Aminoácidos , Animales , Colágeno Tipo IV/química , Reactivos de Enlaces Cruzados/química , Drosophila melanogaster , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/química , Hemo/química , Espectrometría de Masas , Datos de Secuencia Molecular , Péptidos/química , Peroxidasa/química , Peroxidasas/química , Estructura Terciaria de Proteína , Análisis de Secuencia de ARN , Homología de Secuencia de Aminoácido , Pez Cebra , PeroxidasinaRESUMEN
Pacemaker cells are specialized cell types that drive biological rhythms like the heartbeat and intestinal peristalsis. What determines whether a cell functions as a pacemaker? Studies in Caenorhabditis elegans suggest that pacemaking activity may be controlled in part by microRNAs.
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Relojes Biológicos , Caenorhabditis elegans/metabolismo , Señalización del Calcio , MicroARNs/metabolismo , Animales , Proteínas de Caenorhabditis elegans/metabolismo , Mucosa Intestinal/metabolismoRESUMEN
Zebrafish regenerate cardiac muscle after severe injuries through the activation and proliferation of spared cardiomyocytes. Little is known about factors that control these events. Here we investigated the extent to which miRNAs regulate zebrafish heart regeneration. Microarray analysis identified many miRNAs with increased or reduced levels during regeneration. miR-133, a miRNA with known roles in cardiac development and disease, showed diminished expression during regeneration. Induced transgenic elevation of miR-133 levels after injury inhibited myocardial regeneration, while transgenic miR-133 depletion enhanced cardiomyocyte proliferation. Expression analyses indicated that cell cycle factors mps1, cdc37, and PA2G4, and cell junction components cx43 and cldn5, are miR-133 targets during regeneration. Using pharmacological inhibition and EGFP sensor interaction studies, we found that cx43 is a new miR-133 target and regeneration gene. Our results reveal dynamic regulation of miRNAs during heart regeneration, and indicate that miR-133 restricts injury-induced cardiomyocyte proliferation.
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Corazón/fisiología , MicroARNs/metabolismo , Miocitos Cardíacos/fisiología , Regeneración , Pez Cebra/fisiología , Animales , Proliferación Celular , Lesiones Cardíacas/fisiopatología , MicroARNs/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , TransgenesRESUMEN
Appendage regeneration is a complex and fascinating biological process exhibited in vertebrates by urodele amphibians and teleost fish. A current focus in the field is to identify new molecules that control formation and function of the regeneration blastema, a mass of proliferative mesenchyme that emerges after limb or fin amputation and serves as progenitor tissue for lost structures. Two studies published recently have illuminated new molecular regulators of blastemal proliferation. After amputation of a newt limb, the nerve sheath releases nAG, a blastemal mitogen that facilitates regeneration. In amputated zebrafish fins, regeneration is optimized through depletion of the microRNA miR-133, a mechanism that requires Fgf signaling. These discoveries establish research avenues that may impact the regenerative capacity of mammalian tissues.
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Regulación de la Expresión Génica/efectos de los fármacos , Mitógenos/farmacología , Regeneración/efectos de los fármacos , Regeneración/genética , Vertebrados/fisiología , Proteínas Anfibias/farmacología , Animales , Extremidades/fisiología , Regulación de la Expresión Génica/fisiología , MicroARNs/fisiología , Modelos Biológicos , Tejido Nervioso/efectos de los fármacos , Tejido Nervioso/fisiología , Regeneración/fisiología , Salamandridae/fisiología , Vertebrados/genética , Pez Cebra/genética , Pez Cebra/fisiologíaRESUMEN
Appendage regeneration is defined by rapid changes in gene expression that achieve dramatic developmental effects, suggesting involvement of microRNAs (miRNAs). Here, we find dynamic regulation of many miRNAs during zebrafish fin regeneration. In particular, miR-133 levels are high in uninjured fins but low during regeneration. When regeneration was blocked by Fibroblast growth factor (Fgf) receptor inhibition, high miR-133 levels were quickly restored. Experimentally increasing amounts of miR-133 attenuated fin regeneration. Conversely, miR-133 antagonism during Fgf receptor inhibition accelerated regeneration through increased proliferation within the regeneration blastema. The Mps1 kinase, an established positive regulator of blastemal proliferation, is an in vivo target of miR-133. Our findings identify miRNA depletion as a new regulatory mechanism for complex tissue regeneration.