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Platelet homeostasis is essential for vascular integrity and immune defence1,2. Although the process of platelet formation by fragmenting megakaryocytes (MKs; thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of MKs by their progenitor cells (megakaryopoiesis) remains unclear3,4. Here we use intravital imaging to track the cellular dynamics of megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as homeostatic sensors that monitor the bone marrow for apoptotic MKs and deliver IFNα to the MK niche triggering local on-demand proliferation and maturation of MK progenitors. This pDC-dependent feedback loop is crucial for MK and platelet homeostasis at steady state and under stress. pDCs are best known for their ability to function as vigilant detectors of viral infection5. We show that virus-induced activation of pDCs interferes with their function as homeostatic sensors of megakaryopoiesis. Consequently, activation of pDCs by SARS-CoV-2 leads to excessive megakaryopoiesis. Together, we identify a pDC-dependent homeostatic circuit that involves innate immune sensing and demand-adapted release of inflammatory mediators to maintain homeostasis of the megakaryocytic lineage.
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Células Dendríticas , Homeostase , Megacariócitos , Trombopoese , Animais , Feminino , Humanos , Masculino , Camundongos , Apoptose , Plaquetas/citologia , Medula Óssea , Linhagem da Célula , Proliferação de Células , Células Dendríticas/imunologia , Células Dendríticas/citologia , Retroalimentação Fisiológica , Imunidade Inata , Microscopia Intravital , Megacariócitos/citologia , Megacariócitos/imunologia , Camundongos Endogâmicos C57BL , SARS-CoV-2/imunologia , COVID-19/imunologia , COVID-19/fisiopatologia , COVID-19/virologiaRESUMO
Cardiac macrophages are heterogenous in phenotype and functions, which has been associated with differences in their ontogeny. Despite extensive research, our understanding of the precise role of different subsets of macrophages in ischemia/reperfusion (I/R) injury remains incomplete. We here investigated macrophage lineages and ablated tissue macrophages in homeostasis and after I/R injury in a CSF1R-dependent manner. Genomic deletion of a fms-intronic regulatory element (FIRE) in the Csf1r locus resulted in specific absence of resident homeostatic and antigen-presenting macrophages, without affecting the recruitment of monocyte-derived macrophages to the infarcted heart. Specific absence of homeostatic, monocyte-independent macrophages altered the immune cell crosstalk in response to injury and induced proinflammatory neutrophil polarization, resulting in impaired cardiac remodeling without influencing infarct size. In contrast, continuous CSF1R inhibition led to depletion of both resident and recruited macrophage populations. This augmented adverse remodeling after I/R and led to an increased infarct size and deterioration of cardiac function. In summary, resident macrophages orchestrate inflammatory responses improving cardiac remodeling, while recruited macrophages determine infarct size after I/R injury. These findings attribute distinct beneficial effects to different macrophage populations in the context of myocardial infarction.
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Macrófagos , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos , Animais , Macrófagos/imunologia , Camundongos , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Isquemia Miocárdica/imunologia , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/imunologia , Masculino , Traumatismo por Reperfusão Miocárdica/imunologia , Traumatismo por Reperfusão Miocárdica/patologia , Camundongos Endogâmicos C57BL , Miocárdio/patologia , Miocárdio/imunologia , Modelos Animais de DoençasRESUMO
BACKGROUND: Preclinical studies have shown the therapeutic potential of VEGF-B (vascular endothelial growth factor B) in revascularization of the ischemic myocardium, but the associated cardiac hypertrophy and adverse side effects remain a concern. To understand the importance of endothelial proliferation and migration for the beneficial versus adverse effects of VEGF-B in the heart, we explored the cardiac effects of autocrine versus paracrine VEGF-B expression in transgenic and gene-transduced mice. METHODS: We used single-cell RNA sequencing to compare cardiac endothelial gene expression in VEGF-B transgenic mouse models. Lineage tracing was used to identify the origin of a VEGF-B-induced novel endothelial cell population and adeno-associated virus-mediated gene delivery to compare the effects of VEGF-B isoforms. Cardiac function was investigated using echocardiography, magnetic resonance imaging, and micro-computed tomography. RESULTS: Unlike in physiological cardiac hypertrophy driven by a cardiomyocyte-specific VEGF-B transgene (myosin heavy chain alpha-VEGF-B), autocrine VEGF-B expression in cardiac endothelium (aP2 [adipocyte protein 2]-VEGF-B) was associated with septal defects and failure to increase perfused subendocardial capillaries postnatally. Paracrine VEGF-B led to robust proliferation and myocardial migration of a novel cardiac endothelial cell lineage (VEGF-B-induced endothelial cells) of endocardial origin, whereas autocrine VEGF-B increased proliferation of VEGF-B-induced endothelial cells but failed to promote their migration and efficient contribution to myocardial capillaries. The surviving aP2-VEGF-B offspring showed an altered ratio of secreted VEGF-B isoforms and developed massive pathological cardiac hypertrophy with a distinct cardiac vessel pattern. In the normal heart, we found a small VEGF-B-induced endothelial cell population that was only minimally expanded during myocardial infarction but not during physiological cardiac hypertrophy associated with mouse pregnancy. CONCLUSIONS: Paracrine and autocrine secretions of VEGF-B induce expansion of a specific endocardium-derived endothelial cell population with distinct angiogenic markers. However, autocrine VEGF-B signaling fails to promote VEGF-B-induced endothelial cell migration and contribution to myocardial capillaries, predisposing to septal defects and inducing a mismatch between angiogenesis and myocardial growth, which results in pathological cardiac hypertrophy.
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Cardiomegalia , Linhagem da Célula , Endocárdio , Células Endoteliais , Camundongos Transgênicos , Fator B de Crescimento do Endotélio Vascular , Animais , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Cardiomegalia/genética , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Fator B de Crescimento do Endotélio Vascular/metabolismo , Fator B de Crescimento do Endotélio Vascular/genética , Camundongos , Endocárdio/metabolismo , Endocárdio/patologia , Comunicação Parácrina , Proliferação de Células , Comunicação Autócrina , Camundongos Endogâmicos C57BL , Feminino , Masculino , Movimento CelularRESUMO
Fibrogenesis is part of a normal protective response to tissue injury that can become irreversible and progressive, leading to fatal diseases. Senescent cells are a main driver of fibrotic diseases through their secretome, known as senescence-associated secretory phenotype (SASP). Here, we report that cellular senescence, and multiple types of fibrotic diseases in mice and humans are characterized by the accumulation of iron. We show that vascular and hemolytic injuries are efficient in triggering iron accumulation, which in turn can cause senescence and promote fibrosis. Notably, we find that senescent cells persistently accumulate iron, even when the surge of extracellular iron has subdued. Indeed, under normal conditions of extracellular iron, cells exposed to different types of senescence-inducing insults accumulate abundant ferritin-bound iron, mostly within lysosomes, and present high levels of labile iron, which fuels the generation of reactive oxygen species and the SASP. Finally, we demonstrate that detection of iron by magnetic resonance imaging might allow non-invasive assessment of fibrotic burden in the kidneys of mice and in patients with renal fibrosis. Our findings suggest that iron accumulation plays a central role in senescence and fibrosis, even when the initiating events may be independent of iron, and identify iron metabolism as a potential therapeutic target for senescence-associated diseases.
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Senescência Celular , Fenótipo Secretor Associado à Senescência , Humanos , Ferro , Rim , FibroseRESUMO
Disruption of the physiologic sleep-wake cycle and low melatonin levels frequently accompany cardiac disease, yet the underlying mechanism has remained enigmatic. Immunostaining of sympathetic axons in optically cleared pineal glands from humans and mice with cardiac disease revealed their substantial denervation compared with controls. Spatial, single-cell, nuclear, and bulk RNA sequencing traced this defect back to the superior cervical ganglia (SCG), which responded to cardiac disease with accumulation of inflammatory macrophages, fibrosis, and the selective loss of pineal gland-innervating neurons. Depletion of macrophages in the SCG prevented disease-associated denervation of the pineal gland and restored physiological melatonin secretion. Our data identify the mechanism by which diurnal rhythmicity in cardiac disease is disturbed and suggest a target for therapeutic intervention.
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Ritmo Circadiano , Cardiopatias , Macrófagos , Melatonina , Glândula Pineal , Transtornos do Sono do Ritmo Circadiano , Gânglio Cervical Superior , Animais , Humanos , Camundongos , Cardiopatias/fisiopatologia , Melatonina/metabolismo , Glândula Pineal/patologia , Glândula Pineal/fisiopatologia , Sono , Transtornos do Sono do Ritmo Circadiano/fisiopatologia , Gânglio Cervical Superior/patologia , Gânglio Cervical Superior/fisiopatologia , Macrófagos/imunologia , FibroseRESUMO
Recent studies of severe acute inflammatory lung disease including COVID-19 identify macrophages to drive pulmonary hyperinflammation and long-term damage such as fibrosis. Here, we report on the development of a first-in-class, carbohydrate-coupled inhibitor of microRNA-21 (RCS-21), as a therapeutic means against pulmonary hyperinflammation and fibrosis. MicroRNA-21 is among the strongest upregulated microRNAs in human COVID-19 and in mice with acute inflammatory lung damage, and it is the strongest expressed microRNA in pulmonary macrophages. Chemical linkage of a microRNA-21 inhibitor to trimannose achieves rapid and specific delivery to macrophages upon inhalation in mice. RCS-21 reverses pathological activation of macrophages and prevents pulmonary dysfunction and fibrosis after acute lung damage in mice. In human lung tissue infected with SARS-CoV-2 ex vivo, RCS-21 effectively prevents the exaggerated inflammatory response. Our data imply trimannose-coupling for effective and selective delivery of inhaled oligonucleotides to pulmonary macrophages and report on a first mannose-coupled candidate therapeutic for COVID-19.
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COVID-19 , MicroRNAs , Pneumonia , Camundongos , Humanos , Animais , COVID-19/patologia , SARS-CoV-2 , Pulmão/patologia , Macrófagos , Pneumonia/patologia , MicroRNAs/genética , MicroRNAs/farmacologia , FibroseRESUMO
In barley (Hordeum vulgare), signalling rat sarcoma homolog (RHO) of plants guanosine triphosphate hydrolases (ROP GTPases) support the penetration success of Blumeria graminis f. sp. hordei but little is known about ROP activation. Guanine nucleotide exchange factors (GEFs) facilitate the exchange of ROP-bound GDP for GTP and thereby turn ROPs into a signalling-activated ROP-GTP state. Plants possess a unique class of GEFs harbouring a plant-specific ROP nucleotide exchanger domain (PRONE). Here, we performed phylogenetic analyses and annotated barley PRONE-GEFs. The leaf epidermal-expressed PRONE-GEF HvGEF14 undergoes a transcriptional down-regulation on inoculation with B. graminis f. sp. hordei and directly interacts with the ROP GTPase and susceptibility factor HvRACB in yeast and in planta. Overexpression of activated HvRACB or of HvGEF14 led to the recruitment of ROP downstream interactor HvRIC171 to the cell periphery. HvGEF14 further supported direct interaction of HvRACB with a HvRACB-GTP-binding CRIB (Cdc42/Rac Interactive Binding motif) domain-containing HvRIC171 truncation. Finally, the overexpression of HvGEF14 caused enhanced susceptibility to fungal entry, while HvGEF14 RNAi provoked a trend to more penetration resistance. HvGEF14 might therefore play a role in the activation of HvRACB in barley epidermal cells during fungal penetration.
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Hordeum , Ascomicetos , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Trifosfato/metabolismo , Hordeum/microbiologia , Filogenia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Saccharomyces cerevisiae/metabolismo , Internalização do VírusRESUMO
Background & Aims: Increased sensitivity towards tumor necrosis factor (TNF)-induced cell death in virus-infected hepatocytes has revealed a so far unrecognized hepatocyte-intrinsic antiviral immune surveillance mechanism, for which no in vitro or ex vivo model is available. We aimed to establish precision-cut liver slices (PCLS) as a model system to study hepatocyte-intrinsic regulation of apoptosis. Methods: Preparation of PCLS from mouse and human liver tissue was optimized for minimal procedure-associated apoptosis. Functionality of liver cells in PCLS was characterized using extracellular flux analysis to determine mitochondrial respiration, and viral infection with recombinant adenovirus and lymphocytic choriomeningitis virus (LCMV) was used to probe for hepatocyte-intrinsic sensitivity towards apoptosis in PCLS. Apoptosis was detected by immunohistochemical staining for cleaved-caspase 3 and quantified by detection of effector caspase activity in PCLS. Results: We established an optimized protocol for preparation of PCLS from human and mouse models using agarose-embedding of liver tissue to improve precision cutting and using organ-protective buffer solutions to minimize procedure-associated cell death. PCLS prepared from virus-infected livers showed preserved functional metabolic properties. Importantly, in PCLS from adenovirus- and LCMV-infected livers we detected increased induction of apoptosis after TNF challenge ex vivo. Conclusion: We conclude that PCLS can be used as model system to ex vivo characterize hepatocyte-intrinsic sensitivity to cell death. This may also enable researchers to characterize human hepatocyte sensitivity to apoptosis in PCLS prepared from patients with acute or chronic liver diseases. Lay summary: Virus-infected hepatocytes in vivo show an increased sensitivity towards induction of cell death signaling through the TNF receptor. Studying this hepatocyte-intrinsic antiviral immune surveillance mechanism has been hampered by the absence of model systems that reciprocate the in vivo finding of increased apoptosis of virus-infected hepatocytes challenged with TNF. Herein, we report that an optimized protocol for generation of precision-cut liver slices can be used to study this hepatocyte-intrinsic surveillance mechanism ex vivo.
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Cardiosphere-derived cells (CDCs) generated from human cardiac biopsies have been shown to have disease-modifying bioactivity in clinical trials. Paradoxically, CDCs' cellular origin in the heart remains elusive. We studied the molecular identity of CDCs using single-cell RNA sequencing (sc-RNAseq) in comparison to cardiac non-myocyte and non-hematopoietic cells (cardiac fibroblasts/CFs, smooth muscle cells/SMCs and endothelial cells/ECs). We identified CDCs as a distinct and mitochondria-rich cell type that shared biological similarities with non-myocyte cells but not with cardiac progenitor cells derived from human-induced pluripotent stem cells. CXCL6 emerged as a new specific marker for CDCs. By analysis of sc-RNAseq data from human right atrial biopsies in comparison with CDCs we uncovered transcriptomic similarities between CDCs and CFs. By direct comparison of infant and adult CDC sc-RNAseq data, infant CDCs revealed GO-terms associated with cardiac development. To analyze the beneficial effects of CDCs (pro-angiogenic, anti-fibrotic, anti-apoptotic), we performed functional in vitro assays with CDC-derived extracellular vesicles (EVs). CDC EVs augmented in vitro angiogenesis and did not stimulate scarring. They also reduced the expression of pro-apoptotic Bax in NRCMs. In conclusion, CDCs were disclosed as mitochondria-rich cells with unique properties but also with similarities to right atrial CFs. CDCs displayed highly proliferative, secretory and immunomodulatory properties, characteristics that can also be found in activated or inflammatory cell types. By special culture conditions, CDCs earn some bioactivities, including angiogenic potential, which might modify disease in certain disorders.
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Células Endoteliais , Adulto , Humanos , Miócitos Cardíacos , Análise de Sequência de RNA , Células-TroncoRESUMO
Genetically engineered mouse models (GEMMs) transformed the study of organismal disease phenotypes but are limited by their lengthy generation in embryonic stem cells. Here, we describe methods for rapid and scalable genome engineering in somatic cells of the liver and pancreas through delivery of CRISPR components into living mice. We introduce the spectrum of genetic tools, delineate viral and nonviral CRISPR delivery strategies and describe a series of applications, ranging from gene editing and cancer modeling to chromosome engineering or CRISPR multiplexing and its spatio-temporal control. Beyond experimental design and execution, the protocol describes quantification of genetic and functional editing outcomes, including sequencing approaches, data analysis and interpretation. Compared to traditional knockout mice, somatic GEMMs face an increased risk for mouse-to-mouse variability because of the higher experimental demands of the procedures. The robust protocols described here will help unleash the full potential of somatic genome manipulation. Depending on the delivery method and envisaged application, the protocol takes 3-5 weeks.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Neoplasias , Animais , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes/métodos , Fígado , Camundongos , Camundongos Knockout , Neoplasias/genética , PâncreasRESUMO
Abnormalities of ventricular action potential cause malignant cardiac arrhythmias and sudden cardiac death. Here, we aim to identify microRNAs that regulate the human cardiac action potential and ask whether their manipulation allows for therapeutic modulation of action potential abnormalities. Quantitative analysis of the microRNA targetomes in human cardiac myocytes identifies miR-365 as a primary microRNA to regulate repolarizing ion channels. Action potential recordings in patient-specific induced pluripotent stem cell-derived cardiac myocytes show that elevation of miR-365 significantly prolongs action potential duration in myocytes derived from a Short-QT syndrome patient, whereas specific inhibition of miR-365 normalizes pathologically prolonged action potential in Long-QT syndrome myocytes. Transcriptome analyses in these cells at bulk and single-cell level corroborate the key cardiac repolarizing channels as direct targets of miR-365, together with functionally synergistic regulation of additional action potential-regulating genes by this microRNA. Whole-cell patch-clamp experiments confirm miR-365-dependent regulation of repolarizing ionic current Iks. Finally, refractory period measurements in human myocardial slices substantiate the regulatory effect of miR-365 on action potential in adult human myocardial tissue. Our results delineate miR-365 to regulate human cardiac action potential duration by targeting key factors of cardiac repolarization.
Assuntos
Potenciais de Ação/fisiologia , Arritmias Cardíacas/metabolismo , MicroRNAs/metabolismo , Arritmias Cardíacas/genética , Perfilação da Expressão Gênica , Células HEK293 , Ventrículos do Coração/fisiopatologia , Humanos , Síndrome do QT Longo/genética , MicroRNAs/genética , Miocárdio , Miócitos CardíacosRESUMO
BACKGROUND: Cardiac macrophages (cMPs) are increasingly recognized as important regulators of myocardial homeostasis and disease, yet the role of noncoding RNA in these cells is largely unknown. Small RNA sequencing of the entire miRNomes of the major cardiac cell fractions revealed microRNA-21 (miR-21) as the single highest expressed microRNA in cMPs, both in health and disease (25% and 43% of all microRNA reads, respectively). MiR-21 has been previously reported as a key microRNA driving tissue fibrosis. Here, we aimed to determine the function of macrophage miR-21 on myocardial homeostasis and disease-associated remodeling. METHODS: Macrophage-specific ablation of miR-21 in mice driven by Cx3cr1-Cre was used to determine the function of miR-21 in this cell type. As a disease model, mice were subjected to pressure overload for 6 and 28 days. Cardiac function was assessed in vivo by echocardiography, followed by histological analyses and single-cell sequencing. Cocultures of macrophages and cardiac fibroblasts were used to study macrophage-to-fibroblast signaling. RESULTS: Mice with macrophage-specific genetic deletion of miR-21 were protected from interstitial fibrosis and cardiac dysfunction when subjected to pressure overload of the left ventricle. Single-cell sequencing of pressure-overloaded hearts from these mice revealed that miR-21 in macrophages is essential for their polarization toward a M1-like phenotype. Systematic quantification of intercellular communication mediated by ligand-receptor interactions across all cell types revealed that miR-21 primarily determined macrophage-fibroblast communication, promoting the transition from quiescent fibroblasts to myofibroblasts. Polarization of isolated macrophages in vitro toward a proinflammatory (M1-like) phenotype activated myofibroblast transdifferentiation of cardiac fibroblasts in a paracrine manner and was dependent on miR-21 in cMPs. CONCLUSIONS: Our data indicate a critical role of cMPs in pressure overload-induced cardiac fibrosis and dysfunction and reveal macrophage miR-21 as a key molecule for the profibrotic role of cMPs.
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Insuficiência Cardíaca/patologia , MicroRNAs/metabolismo , Miocárdio/patologia , Miofibroblastos/metabolismo , Animais , Comunicação Celular , Fibroblastos/metabolismo , Fibrose , Insuficiência Cardíaca/metabolismo , Macrófagos/metabolismo , Camundongos , MicroRNAs/genética , Miocárdio/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Renal ischemia-reperfusion (I/R) injury is a major cause of AKI. Noncoding RNAs are intricately involved in the pathophysiology of this form of AKI. Transcription of hypoxia-induced, long noncoding RNA H19, which shows high embryonic expression and is silenced in adults, is upregulated in renal I/R injury. METHODS: Lentivirus-mediated overexpression, as well as antisense oligonucleotide-based silencing, modulated H19 in vitro. In vivo analyses used constitutive H19 knockout mice. In addition, renal vein injection of adeno-associated virus 2 (AAV2) carrying H19 caused overexpression in the kidney. Expression of H19 in kidney transplant patients with I/R injury was investigated. RESULTS: H19 is upregulated in kidney biopsies of patients with AKI, in murine ischemic kidney tissue, and in cultured and ex vivo sorted hypoxic endothelial cells (ECs) and tubular epithelial cells (TECs). Transcription factors hypoxia-inducible factor 1-α, LHX8, and SPI1 activate H19 in ECs and TECs. H19 overexpression promotes angiogenesis in vitro and in vivo. In vivo, transient AAV2-mediated H19 overexpression significantly improved kidney function, reduced apoptosis, and reduced inflammation, as well as preserving capillary density and tubular epithelial integrity. Sponging of miR-30a-5p mediated the effects, which, in turn, led to target regulation of Dll4, ATG5, and Snai1. CONCLUSIONS: H19 overexpression confers protection against renal injury by stimulating proangiogenic signaling. H19 overexpression may be a promising future therapeutic option in the treatment of patients with ischemic AKI.
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Injúria Renal Aguda/etiologia , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Adulto , Animais , Técnicas de Cultura de Células , Dependovirus , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Humanos , Isquemia/complicações , Isquemia/metabolismo , Isquemia/patologia , Masculino , Camundongos , Pessoa de Meia-IdadeRESUMO
Fibrosis is a pathognomonic feature of structural heart disease and counteracted by distinct cardioprotective mechanisms, e.g. activation of the phosphoinositide 3-kinase (PI3K) / AKT pro-survival pathway. The Cullin-RING E3 ubiquitin ligase 7 (CRL7) was identified as negative regulator of PI3K/AKT signalling in skeletal muscle, but its role in the heart remains to be elucidated. Here, we sought to determine whether CRL7 modulates to cardiac fibrosis following pressure overload and dissect its underlying mechanisms. For inactivation of CRL7, the Cullin 7 (Cul7) gene was deleted in cardiac myocytes (CM) by injection of adeno-associated virus subtype 9 (AAV9) vectors encoding codon improved Cre-recombinase (AAV9-CMV-iCre) in Cul7flox/flox mice. In addition, Myosin Heavy Chain 6 (Myh6; alpha-MHC)-MerCreMer transgenic mice with tamoxifen-induced CM-specific expression of iCre were used as alternate model. After transverse aortic constriction (TAC), causing chronic pressure overload and fibrosis, AAV9-CMV-iCre induced Cul7-/- mice displayed a ~50% reduction of interstitial cardiac fibrosis when compared to Cul7+/+ animals (6.7% vs. 3.4%, p<0.01). Similar results were obtained with Cul7flox/flox Myh6-Mer-Cre-MerTg(1/0) mice which displayed a ~30% reduction of cardiac fibrosis after TAC when compared to Cul7+/+ Myh6-Mer-Cre-MerTg(1/0) controls after TAC surgery (12.4% vs. 8.7%, p<0.05). No hemodynamic alterations were observed. AKTSer473 phosphorylation was increased 3-fold (p<0.01) in Cul7-/- vs. control mice, together with a ~78% (p<0.001) reduction of TUNEL-positive apoptotic cells three weeks after TAC. In addition, CM-specific expression of a dominant-negative CUL71152stop mutant resulted in a 16.3-fold decrease (p<0.001) of in situ end-labelling (ISEL) positive apoptotic cells. Collectively, our data demonstrate that CM-specific ablation of Cul7 restrains myocardial fibrosis and apoptosis upon pressure overload, and introduce CRL7 as a potential target for anti-fibrotic therapeutic strategies of the heart.
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Apoptose , Cardiomiopatias/enzimologia , Miócitos Cardíacos/enzimologia , Transdução de Sinais , Animais , Cardiomiopatias/genética , Cardiomiopatias/patologia , Cardiomiopatias/fisiopatologia , Proteínas Culina , Dependovirus , Fibrose , Camundongos , Camundongos Knockout , Miócitos Cardíacos/patologia , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução GenéticaRESUMO
Arterial macrophages have different developmental origins, but the association of macrophage ontogeny with their phenotypes and functions in adulthood is still unclear. Here, we combine macrophage fate-mapping analysis with single-cell RNA sequencing to establish their cellular identity during homeostasis, and in response to angiotensin-II (AngII)-induced arterial inflammation. Yolk sac erythro-myeloid progenitors (EMP) contribute substantially to adventitial macrophages and give rise to a defined cluster of resident immune cells with homeostatic functions that is stable in adult mice, but declines in numbers during ageing and is not replenished by bone marrow (BM)-derived macrophages. In response to AngII inflammation, increase in adventitial macrophages is driven by recruitment of BM monocytes, while EMP-derived macrophages proliferate locally and provide a distinct transcriptional response that is linked to tissue regeneration. Our findings thus contribute to the understanding of macrophage heterogeneity, and associate macrophage ontogeny with distinct functions in health and disease.
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Artérias/citologia , Arterite/imunologia , Diferenciação Celular/fisiologia , Homeostase/fisiologia , Macrófagos/fisiologia , Envelhecimento/fisiologia , Angiotensina II/administração & dosagem , Angiotensina II/imunologia , Animais , Artérias/fisiologia , Medula Óssea/fisiologia , Transplante de Medula Óssea , Linhagem da Célula , Modelos Animais de Doenças , Feminino , Células-Tronco Hematopoéticas/fisiologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , RNA-Seq , Regeneração/fisiologia , Análise de Célula Única , Quimeras de TransplanteRESUMO
Vast parts of mammalian genomes are actively transcribed, predominantly giving rise to non-coding RNA (ncRNA) transcripts including microRNAs, long ncRNAs, and circular RNAs among others. Contrary to previous opinions that most of these RNAs are non-functional molecules, they are now recognized as critical regulators of many physiological and pathological processes including those of the cardiovascular system. The discovery of functional ncRNAs has opened up new research avenues aiming at understanding ncRNA-related disease mechanisms as well as exploiting them as novel therapeutics in cardiovascular therapy. In this review, we give an update on the current progress in ncRNA research, particularly focusing on cardiovascular physiological and disease processes, which are under current investigation at the ESC Working Groups of Myocardial Function and Cellular Biology of the Heart. This includes a range of topics such as extracellular vesicle-mediated communication, neurohormonal regulation, inflammation, cardiac remodelling, cardio-oncology as well as cardiac development and regeneration, collectively highlighting the wide-spread involvement and importance of ncRNAs in the cardiovascular system.
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Cardiopatias/metabolismo , Miocárdio/metabolismo , RNA não Traduzido/metabolismo , Animais , Regulação da Expressão Gênica , Terapia Genética , Cardiopatias/genética , Cardiopatias/fisiopatologia , Cardiopatias/terapia , Humanos , Miocárdio/patologia , RNA não Traduzido/genética , Recuperação de Função Fisiológica , Regeneração , Transdução de Sinais , Função Ventricular Esquerda , Remodelação VentricularRESUMO
BACKGROUND: miR-21 is a central regulator of cardiac fibrosis, and its inhibition in small-animal models has been shown to be an effective antifibrotic strategy in various organs, including the heart. Effective delivery of therapeutic antisense micro-ribonucleic acid (antimiR) molecules to the myocardium in larger organisms is challenging, though, and remains to be established for models of chronic heart failure. OBJECTIVES: The aims of this study were to test the applicability and therapeutic efficacy of local, catheter-based delivery of antimiR-21 in a pig model of heart failure and determine its effect on the cardiac transcriptomic signature and cellular composition. METHODS: Pigs underwent transient percutaneous occlusion of the left coronary artery and were followed up for 33 days. AntimiR-21 (10 mg) was applied by intracoronary infusion at days 5 and 19 after the injury. Cardiac function was assessed in vivo, followed by histological analyses and deep ribonucleic acid sequencing (RNA-seq) of the myocardium and genetic deconvolution analysis. RESULTS: AntimiR-21 effectively suppressed the remodeling-associated increase of miR-21. At 33 days after ischemia/reperfusion injury, LNA-21-treated hearts exhibited reduced cardiac fibrosis and hypertrophy and improved cardiac function. Deep RNA-seq revealed a significant derepression of the miR-21 targetome in antimiR-21-treated myocardium and a suppression of the inflammatory response and mitogen-activated protein kinase signaling. A genetic deconvolution approach built on deep RNA-seq and single-cell RNA-seq data identified reductions in macrophage and fibroblast numbers as the key cell types affected by antimiR-21 treatment. CONCLUSIONS: This study provides the first evidence for the feasibility and therapeutic efficacy of miR-21 inhibition in a large animal model of heart failure.
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Cardiomegalia/terapia , Fibrose/terapia , MicroRNAs/antagonistas & inibidores , Miocárdio/patologia , Traumatismo por Reperfusão/terapia , Remodelação Ventricular , Animais , Cardiomegalia/genética , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibrose/genética , Macrófagos/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Oligonucleotídeos/química , Remodelação Ventricular/genéticaRESUMO
Improved therapy of cancer has significantly increased the lifespan of patients. However, cancer survivors face an increased risk of cardiovascular complications due to adverse effects of cancer therapies. The chemotherapy drug doxorubicin is well known to induce myofibril damage and cardiac atrophy. Our aim was to test potential counteracting effects of the pro-hypertrophic miR-212/132 family in doxorubicin-induced cardiotoxicity. In vitro, overexpression of the pro-hypertrophic miR-212/132 cluster in primary rodent and human iPSC-derived cardiomyocytes inhibited doxorubicin-induced toxicity. Next, a disease model of doxorubicin-induced cardiotoxicity was established in male C57BL/6N mice. Mice were administered either adeno-associated virus (AAV)9-control or AAV9-miR-212/132 to achieve myocardial overexpression of the miR-212/132 cluster. AAV9-mediated overexpression limited cardiac atrophy by increasing left ventricular mass and wall thickness, decreased doxorubicin-mediated apoptosis, and prevented myofibril damage. Based on a transcriptomic profiling we identified fat storage-inducing transmembrane protein 2 (Fitm2) as a novel target and downstream effector molecule responsible, at least in part, for the observed miR-212/132 anti-cardiotoxic effects. Overexpression of Fitm2 partially reversed the effects of miR-212/132. Overexpression of the miR-212/132 family reduces development of doxorubicin-induced cardiotoxicity and thus could be a therapeutic entry point to limit doxorubicin-mediated adverse cardiac effects.
Assuntos
Doxorrubicina/efeitos adversos , MicroRNAs/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Cardiotoxicidade , Caspase 3/metabolismo , Caspase 7/metabolismo , Dependovirus/genética , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Miocárdio/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , RatosRESUMO
Oomycetes such as the potato blight pathogen Phytophthora infestans deliver RXLR effectors into plant cells to manipulate host processes and promote disease. Knowledge of where they localize inside host cells is important in understanding their function. Fifty-two P. infestans RXLR effectors (PiRXLRs) up-regulated during early stages of infection were expressed as fluorescent protein (FP) fusions inside cells of the model host Nicotiana benthamiana. FP-PiRXLR fusions were predominantly nucleo-cytoplasmic, nuclear, or plasma membrane-associated. Some also localized to the endoplasmic reticulum, mitochondria, peroxisomes, or microtubules, suggesting diverse sites of subcellular activity. Seven of the 25 PiRXLRs examined during infection accumulated at sites of haustorium penetration, probably due to co-localization with host target processes; Pi16663 (Avr1), for example, localized to Sec5-associated mobile bodies which showed perihaustorial accumulation. Forty-five FP-RXLR fusions enhanced pathogen leaf colonization when expressed in Nicotiana benthamiana, revealing that their presence was beneficial to infection. Co-expression of PiRXLRs that target and suppress different immune pathways resulted in an additive enhancement of colonization, indicating the potential to study effector combinations using transient expression assays. We provide a broad platform of high confidence P. infestans effector candidates from which to investigate the mechanisms, singly and in combination, by which this pathogen causes disease.