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Senescence plays a key role in various physiological and pathological processes. We reported that injury-induced transient senescence correlates with heart regeneration, yet the multi-omics profile and molecular underpinnings of regenerative senescence remain obscure. Using proteomics and single-cell RNA sequencing, here we report the regenerative senescence multi-omic signature in the adult mouse heart and establish its role in neonatal heart regeneration and agrin-mediated cardiac repair in adult mice. We identified early growth response protein 1 (Egr1) as a regulator of regenerative senescence in both models. In the neonatal heart, Egr1 facilitates angiogenesis and cardiomyocyte proliferation. In adult hearts, agrin-induced senescence and repair require Egr1, activated by the integrin-FAK-ERK-Akt1 axis in cardiac fibroblasts. We also identified cathepsins as injury-induced senescence-associated secretory phenotype components that promote extracellular matrix degradation and potentially assist in reducing fibrosis. Altogether, we uncovered the molecular signature and functional benefits of regenerative senescence during heart regeneration, with Egr1 orchestrating the process.
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Proliferación Celular , Senescencia Celular , Proteína 1 de la Respuesta de Crecimiento Precoz , Miocitos Cardíacos , Regeneración , Animales , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Regeneración/fisiología , Senescencia Celular/fisiología , Miocitos Cardíacos/metabolismo , Ratones Endogámicos C57BL , Neovascularización Fisiológica/fisiología , Transducción de Señal , Fibroblastos/metabolismo , Ratones , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células Cultivadas , Animales Recién Nacidos , Modelos Animales de Enfermedad , Fenotipo Secretor Asociado a la Senescencia , Proteómica , Análisis de la Célula Individual , Masculino , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ratones Noqueados , Quinasa 1 de Adhesión FocalRESUMEN
Biocompatible fluorescent agents are key contributors to the theranostic paradigm by enabling real-time in vivo imaging. This study explores the optical properties of phenylenediamine carbon dots (CDs) and demonstrates their potential for fluorescence imaging in cells and brain blood vessels. The nonlinear absorption cross-section of the CDs was measured and achieved values near 50 Goeppert-Mayer (GM) units with efficient excitation in the 775-895 nm spectral range. Mesoporous vaterite nanoparticles were loaded with CDs to examine the possibility of a biocompatible imaging platform. Efficient one- and two-photon imaging of the CD-vaterite composites uptaken by diverse cells was demonstrated. For an in vivo scenario, CD-vaterite composites were injected into the bloodstream of a mouse, and their flow was monitored within the blood vessels of the brain through a cranial window. These results show the potential of the platform for high-brightness biocompatible imaging with the potential for both sensing and simultaneous drug delivery.
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Encéfalo , Carbono , Puntos Cuánticos , Animales , Carbono/química , Ratones , Encéfalo/diagnóstico por imagen , Puntos Cuánticos/química , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Carbonato de Calcio/química , Humanos , Nanopartículas/química , Colorantes Fluorescentes/químicaRESUMEN
Cardiomyocyte proliferation and dedifferentiation have fueled the field of regenerative cardiology in recent years, whereas the reverse process of redifferentiation remains largely unexplored. Redifferentiation is characterized by the restoration of function lost during dedifferentiation. Previously, we showed that ERBB2-mediated heart regeneration has these two distinct phases: transient dedifferentiation and redifferentiation. Here we survey the temporal transcriptomic and proteomic landscape of dedifferentiation-redifferentiation in adult mouse hearts and reveal that well-characterized dedifferentiation features largely return to normal, although elements of residual dedifferentiation remain, even after the contractile function is restored. These hearts appear rejuvenated and show robust resistance to ischemic injury, even 5 months after redifferentiation initiation. Cardiomyocyte redifferentiation is driven by negative feedback signaling and requires LATS1/2 Hippo pathway activity. Our data reveal the importance of cardiomyocyte redifferentiation in functional restoration during regeneration but also protection against future insult, in what could lead to a potential prophylactic treatment against ischemic heart disease for at-risk patients.
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Significance: rPySight brings a flexible and highly customizable open-software platform built around a powerful multichannel digitizer; combined, it enables performing complex photon counting-based experiments. We exploited advanced programming technology to share the photon counting stream with the graphical processing unit (GPU), making possible real-time display of two-dimensional (2D) and three-dimensional (3D) experiments and paving the road for other real-time applications. Aim: Photon counting improves multiphoton imaging by providing better signal-to-noise ratio in photon-deprived applications and is becoming more widely implemented, as indicated by its increasing presence in many microscopy vendor portfolios. Despite the relatively easy access to this technology offered in commercial systems, these remain limited to one or two channels of data and might not enable highly tailored experiments, forcing most researchers to develop their own electronics and code. We set to develop a flexible and open-source interface to a cutting-edge multichannel fast digitizer that can be easily integrated into existing imaging systems. Approach: We selected an advanced multichannel digitizer capable of generating 70M tags/s and wrote an open software application, based on Rust and Python languages, to share the stream of detected events with the GPU, enabling real-time data processing. Results: rPySight functionality was showcased in real-time monitoring of 2D imaging, improved calcium imaging, multiplexing, and 3D imaging through a varifocal lens. We provide a detailed protocol for implementing out-of-the-box rPySight and its related hardware. Conclusions: Applying photon-counting approaches is becoming a fundamental component in recent technical developments that push well beyond existing acquisition speed limitations of classical multiphoton approaches. Given the performance of rPySight, we foresee its use to capture, among others, the joint dynamics of hundreds (if not thousands) of neuronal and vascular elements across volumes, as is likely required to uncover in a much broader sense the hemodynamic transform function.
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Nature-inspired nanosized formulations based on an imageable, small-sized inorganic core scaffold, on which biomolecules are assembled to form nanobiomimetics, hold great promise for both early diagnostics and developed therapeutics. Nevertheless, the fabrication of nanobiomimetics that allow noninvasive background-free mapping of pathological events with improved sensitivity, enhanced specificity, and multiplexed capabilities remains a major challenge. Here, we introduce paramagnetic glyconanofluorides as small-sized (<10 nm) glycomimetics for immunotargeting and sensitive noninvasive in vivo19F magnetic resonance imaging (MRI) mapping of inflammation. A very short T1 relaxation time (70 ms) of the fluorides was achieved by doping the nanofluorides' solid crystal core with paramagnetic Sm3+, resulting in a significant 8-fold enhancement in their 19F MRI sensitivity, allowing faster acquisition and improved detectability levels. The fabricated nanosized glycomimetics exhibit significantly enhanced uptake within activated immune cells, providing background-free in vivo mapping of inflammatory activity, demonstrated in both locally induced inflammation and clinically related neuropathology animal models. Fabricating two types of nanofluorides, each with a distinct chemical shift, allowed us to exploit the color-like features of 19F MRI to map, in real time, immune specificity and preferred targetability of the paramagnetic glyconanofluorides, demonstrating the approach's potential extension to noninvasive multitarget imaging scenarios that are not yet applicable for nanobiomimetics based on other nanocrystal cores.
Asunto(s)
Imagen por Resonancia Magnética , Nanopartículas , Animales , FluorurosRESUMEN
Cardiomyocyte loss after injury results in adverse remodelling and fibrosis, inevitably leading to heart failure. The ERBB2-Neuregulin and Hippo-YAP signalling pathways are key mediators of heart regeneration, yet the crosstalk between them is unclear. We demonstrate that transient overexpression of activated ERBB2 in cardiomyocytes (OE CMs) promotes cardiac regeneration in a heart failure model. OE CMs present an epithelial-mesenchymal transition (EMT)-like regenerative response manifested by cytoskeletal remodelling, junction dissolution, migration and extracellular matrix turnover. We identified YAP as a critical mediator of ERBB2 signalling. In OE CMs, YAP interacts with nuclear-envelope and cytoskeletal components, reflecting an altered mechanical state elicited by ERBB2. We identified two YAP-activating phosphorylations on S352 and S274 in OE CMs, which peak during metaphase, that are ERK dependent and Hippo independent. Viral overexpression of YAP phospho-mutants dampened the proliferative competence of OE CMs. Together, we reveal a potent ERBB2-mediated YAP mechanotransduction signalling, involving EMT-like characteristics, resulting in robust heart regeneration.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Transición Epitelial-Mesenquimal , Insuficiencia Cardíaca/metabolismo , Infarto del Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Receptor ErbB-2/metabolismo , Regeneración , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas de Ciclo Celular/genética , Células Cultivadas , Citoesqueleto/metabolismo , Citoesqueleto/patología , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibrosis , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Mecanotransducción Celular , Ratones Transgénicos , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/patología , Fosforilación , Receptor ErbB-2/genética , Proteínas Señalizadoras YAPRESUMEN
BACKGROUND: Ischemic heart diseases are leading causes of death and reduced life quality worldwide. Although revascularization strategies significantly reduce mortality after acute myocardial infarction (MI), a large number of patients with MI develop chronic heart failure over time. We previously reported that a fragment of the extracellular matrix protein agrin promotes cardiac regeneration after MI in adult mice. METHODS: To test the therapeutic potential of agrin in a preclinical porcine model, we performed ischemia-reperfusion injuries using balloon occlusion for 60 minutes followed by a 3-, 7-, or 28-day reperfusion period. RESULTS: We demonstrated that local (antegrade) delivery of recombinant human agrin to the infarcted pig heart can target the affected regions in an efficient and clinically relevant manner. A single dose of recombinant human agrin improved heart function, infarct size, fibrosis, and adverse remodeling parameters 28 days after MI. Short-term MI experiments along with complementary murine studies revealed myocardial protection, improved angiogenesis, inflammatory suppression, and cell cycle reentry as agrin's mechanisms of action. CONCLUSIONS: A single dose of agrin is capable of reducing ischemia-reperfusion injury and improving heart function, demonstrating that agrin could serve as a therapy for patients with acute MI and potentially heart failure.
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Agrina/farmacología , Infarto del Miocardio/tratamiento farmacológico , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Recuperación de la Función/efectos de los fármacos , Animales , Humanos , Ratones , Infarto del Miocardio/metabolismo , Infarto del Miocardio/fisiopatología , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/fisiopatología , Proteínas Recombinantes/farmacología , PorcinosRESUMEN
The adult mammalian heart regenerates poorly after injury and, as a result, ischemic heart diseases are among the leading causes of death worldwide. The recovery of the injured heart is dependent on orchestrated repair processes including inflammation, fibrosis, cardiomyocyte survival, proliferation, and contraction properties that could be modulated in patients. In this work we designed an automated high-throughput screening system for small molecules that induce cardiomyocyte proliferation in vitro and identified the small molecule Chicago Sky Blue 6B (CSB). Following induced myocardial infarction, CSB treatment reduced scar size and improved heart function of adult mice. Mechanistically, we show that although initially identified using in vitro screening for cardiomyocyte proliferation, in the adult mouse CSB promotes heart repair through (i) inhibition of CaMKII signaling, which improves cardiomyocyte contractility; and (ii) inhibition of neutrophil and macrophage activation, which attenuates the acute inflammatory response, thereby contributing to reduced scarring. In summary, we identified CSB as a potential therapeutic agent that enhances cardiac repair and function by suppressing postinjury detrimental processes, with no evidence for cardiomyocyte renewal.
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Corazón/efectos de los fármacos , Infarto del Miocardio/metabolismo , Miocitos Cardíacos , Azul de Tripano/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cicatriz/metabolismo , Femenino , Ratones , Ratones Endogámicos ICR , Miocardio/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismoRESUMEN
Brain metastases are prevalent in various types of cancer and are often terminal, given the low efficacy of available therapies. Therefore, preventing them is of utmost clinical relevance, and prophylactic treatments are perhaps the most efficient strategy. Here, we show that systemic prophylactic administration of a toll-like receptor (TLR) 9 agonist, CpG-C, is effective against brain metastases. Acute and chronic systemic administration of CpG-C reduced tumor cell seeding and growth in the brain in three tumor models in mice, including metastasis of human and mouse lung cancer, and spontaneous melanoma-derived brain metastasis. Studying mechanisms underlying the therapeutic effects of CpG-C, we found that in the brain, unlike in the periphery, natural killer (NK) cells and monocytes are not involved in controlling metastasis. Next, we demonstrated that the systemically administered CpG-C is taken up by endothelial cells, astrocytes, and microglia, without affecting blood-brain barrier (BBB) integrity and tumor brain extravasation. In vitro assays pointed to microglia, but not astrocytes, as mediators of CpG- C effects through increased tumor killing and phagocytosis, mediated by direct microglia-tumor contact. In vivo, CpG-C-activated microglia displayed elevated mRNA expression levels of apoptosis-inducing and phagocytosis-related genes. Intravital imaging showed that CpG-C-activated microglia cells contact, kill, and phagocytize tumor cells in the early stages of tumor brain invasion more than nonactivated microglia. Blocking in vivo activation of microglia with minocycline, and depletion of microglia with a colony-stimulating factor 1 inhibitor, indicated that microglia mediate the antitumor effects of CpG-C. Overall, the results suggest prophylactic CpG-C treatment as a new intervention against brain metastasis, through an essential activation of microglia.
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Neoplasias Encefálicas/complicaciones , Neoplasias Encefálicas/metabolismo , Microglía/metabolismo , Microglía/patología , Oligodesoxirribonucleótidos/uso terapéutico , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Factores Estimulantes de Colonias/antagonistas & inhibidores , Factores Estimulantes de Colonias/metabolismo , Femenino , Humanos , Neoplasias Pulmonares/complicaciones , Neoplasias Pulmonares/metabolismo , Masculino , Melanoma/complicaciones , Melanoma/metabolismo , Ratones , Minociclina/metabolismo , Fagocitosis/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Acute MI induces leptin expression in the heart, however the role of myocardial leptin in cardiac ischemia and reperfusion (IR) remains unknown. To shed light on the effects of elevated levels of leptin in the myocardium, we overexpressed cardiac leptin and assessed local remodeling and myocardial function in this context. METHODS AND RESULTS: Cardiac leptin overexpression was stimulated in mice undergoing IR by a single intraperitoneal injection of leptin antagonist (LepA). All mice exhibited a normal pattern of body weight gain. A rapid, long-term upregulation of leptin mRNA was demonstrated in the heart, adipose, and liver tissues in IR/LepA-treated mice. Overexpressed cardiac leptin mRNA extended beyond postoperative day (POD) 30. Plasma leptin peaked 7.5 hours postoperatively, especially in IR/LepA-treated mice, subsiding to normal levels by 24 hours. On POD-30 IR/LepA-treated mice demonstrated cardiomyocyte hypertrophy and perivascular fibrosis compared to IR/saline controls. Echocardiography on POD-30 demonstrated eccentric hypertrophy and systolic dysfunction in IR/LepA. We recorded reductions in Ejection Fraction (p<0.001), Fraction Shortening (p<0.01), and Endocardial Fraction Area Change (p<0.01), and an increase in Endocardial Area Change (p<0.01). Myocardial remodeling in the context of IR and cardiac leptin overexpression was associated with increased cardiac TGFß ligand expression, activated Smad2, and downregulation of STAT3 activity. CONCLUSIONS: Cardiac IR coinciding with increased myocardial leptin synthesis promotes cardiomyocyte hypertrophy and fibrosis and potentiates myocardial dysfunction. Plasma leptin levels do not reflect cardiac leptin synthesis, and may not predict leptin-related cardiovascular morbidity. Targeting cardiac leptin is a potential treatment for cardiac IR damage.
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Leptina/genética , Infarto del Miocardio/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Miocardio/metabolismo , Disfunción Ventricular Izquierda/etiología , Animales , Modelos Animales de Enfermedad , Ecocardiografía , Leptina/antagonistas & inhibidores , Leptina/metabolismo , Masculino , Ratones , Infarto del Miocardio/complicaciones , Infarto del Miocardio/genética , Daño por Reperfusión Miocárdica/complicaciones , Daño por Reperfusión Miocárdica/genética , Factor de Transcripción STAT3/metabolismo , Proteína Smad2/metabolismo , Disfunción Ventricular Izquierda/sangre , Remodelación VentricularRESUMEN
Endothelial activation with up-regulation of E-selectin adhesion molecules mediates leukocyte rolling along the vascular wall and controls inflammation in many diseases including atherosclerosis and heart failure. Therefore, we aimed to test the hypothesis that inhibition of E-selectin-mediated interactions by a new E-selectin-targeted copolymer could inhibit the progression of atherosclerosis. To target E-selectin on activated endothelium, we developed a new N-(2-hydroxypropyl)methacrylamide (HPMA)-based E-selectin binding copolymer with or without dexamethasone (Dex) (designated P-(Esbp)-Dex and P-Esbp, respectively). To determine the effect of P-(Esbp)-Dex and P-Esbp on atherosclerosis, we allocated ApoE (-/-) mice on a high fat diet, to weekly intra-peritoneal injections of either 1) P-Esbp; 2) P-(Esbp)-Dex; 3) free Dex (1â¯mg/kg) or 4) saline, for four weeks. Aortic atherosclerosis and left ventricular (LV) remodeling and function were assessed by serial ultrasound studies and histology. Monocytes and macrophages were characterized by flow cytometry. After four weeks of treatment, P-Esbp effectively targeted aortic atherosclerotic lesions. Both P-Esbp and P-(Esbp)-Dex reduced wall thickening of the ascending aortas. However, only the drug-free copolymer (P-Esbp) significantly decreased the areas of necrotic core in the plaques and switched spleen macrophages toward an anti-inflammatory (M2) phenotype. Furthermore, P-Esbp attenuated adverse LV remodeling and dysfunction in ApoE (-/-) mice. In summary, P-Esbp copolymer targets activated endothelial cells, regresses and stabilizes atherosclerotic plaques, and prevents adverse LV remodeling and dysfunction in ApoE (-/-) mice. Our results suggest a new, drug-free macromolecular therapy to treat vascular inflammation.
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Antiinflamatorios/administración & dosificación , Aterosclerosis/tratamiento farmacológico , Selectina E/antagonistas & inhibidores , Metacrilatos/administración & dosificación , Disfunción Ventricular Izquierda/tratamiento farmacológico , Remodelación Ventricular/efectos de los fármacos , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Aterosclerosis/metabolismo , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Dexametasona/administración & dosificación , Selectina E/metabolismo , Macrófagos/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Monocitos/efectos de los fármacos , Disfunción Ventricular Izquierda/metabolismo , Disfunción Ventricular Izquierda/patología , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
Developing efficient brain imaging technologies by combining a high spatiotemporal resolution and a large penetration depth is a key step for better understanding the neurovascular interface that emerges as a main pathway to neurodegeneration in many pathologies such as dementia. This review focuses on the advances in two complementary techniques: multi-photon laser scanning microscopy (MPLSM) and functional ultrasound imaging (fUSi). MPLSM has become the gold standard for in vivo imaging of cellular dynamics and morphology, together with cerebral blood flow. fUSi is an innovative imaging modality based on Doppler ultrasound, capable of recording vascular brain activity over large scales (i.e., tens of cubic millimeters) at unprecedented spatial and temporal resolution for such volumes (up to 10µm pixel size at 10kHz). By merging these two technologies, researchers may have access to a more detailed view of the various processes taking place at the neurovascular interface. MPLSM and fUSi are also good candidates for addressing the major challenge of real-time delivery, monitoring, and in vivo evaluation of drugs in neuronal tissue.
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Encéfalo/fisiología , Circulación Cerebrovascular/fisiología , Neuronas/fisiología , Animales , Humanos , Microscopía Confocal/métodos , Ultrasonografía/métodosRESUMEN
The adult mammalian heart is non-regenerative owing to the post-mitotic nature of cardiomyocytes. The neonatal mouse heart can regenerate, but only during the first week of life. Here we show that changes in the composition of the extracellular matrix during this week can affect cardiomyocyte growth and differentiation in mice. We identify agrin, a component of neonatal extracellular matrix, as required for the full regenerative capacity of neonatal mouse hearts. In vitro, recombinant agrin promotes the division of cardiomyocytes that are derived from mouse and human induced pluripotent stem cells through a mechanism that involves the disassembly of the dystrophin-glycoprotein complex, and Yap- and ERK-mediated signalling. In vivo, a single administration of agrin promotes cardiac regeneration in adult mice after myocardial infarction, although the degree of cardiomyocyte proliferation observed in this model suggests that there are additional therapeutic mechanisms. Together, our results uncover a new inducer of mammalian heart regeneration and highlight fundamental roles of the extracellular matrix in cardiac repair.
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Agrina/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Corazón/fisiología , Regeneración , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Animales Recién Nacidos , Proteínas de Ciclo Celular , Proliferación Celular , Distroglicanos/metabolismo , Femenino , Ratones , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Miocardio/citología , Miocardio/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Señalizadoras YAPRESUMEN
BACKGROUND: The immune system plays a pivotal role in myocardial homeostasis and response to injury. Interleukins-4 and -13 are anti-inflammatory type-2 cytokines, signaling via the common interleukin-13 receptor α1 chain and the type-2 interleukin-4 receptor. The role of interleukin-13 receptor α1 in the heart is unknown. METHODS AND RESULTS: We analyzed myocardial samples from human donors (n=136) and patients with end-stage heart failure (n=177). We found that the interleukin-13 receptor α1 is present in the myocardium and, together with the complementary type-2 interleukin-4 receptor chain Il4ra, is significantly downregulated in the hearts of patients with heart failure. Next, we showed that Il13ra1-deficient mice develop severe myocardial dysfunction and dyssynchrony compared to wild-type mice (left ventricular ejection fraction 29.7±9.9 versus 45.0±8.0; P=0.004, left ventricular end-diastolic diameter 4.2±0.2 versus 3.92±0.3; P=0.03). A bioinformatic analysis of mouse hearts indicated that interleukin-13 receptor α1 regulates critical pathways in the heart other than the immune system, such as extracellular matrix (normalized enrichment score=1.90; false discovery rate q=0.005) and glucose metabolism (normalized enrichment score=-2.36; false discovery rate q=0). Deficiency of Il13ra1 was associated with reduced collagen deposition under normal and pressure-overload conditions. CONCLUSIONS: The results of our studies in humans and mice indicate, for the first time, a role of interleukin-13 receptor α1 in myocardial homeostasis and heart failure and suggests a new therapeutic target to treat heart disease.
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Regulación de la Expresión Génica , Insuficiencia Cardíaca/genética , Homeostasis , Subunidad alfa1 del Receptor de Interleucina-13/genética , Miocardio/metabolismo , ARN/genética , Animales , Western Blotting , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Humanos , Subunidad alfa1 del Receptor de Interleucina-13/biosíntesis , Ratones , Miocardio/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Remodelación VentricularRESUMEN
BACKGROUND: Little is known about the potentially unfavorable effects of mesenchymal stromal cell (MSC) activation on the heart. MSCs can respond to tissue injury by anti- or proinflammatory activation. We aimed to study the potential negative interaction between left ventricular dysfunction (LVD) and MSC activation. METHODS: We isolated MSCs from cardiac and subcutaneous fat tissues of mice with LVD 28 days after myocardial infarction or sham operation. To evaluate the effect of LVD on MSCs, we characterized cardiac MSCs and subcutaneous MSCs in vitro. Subsequently, we injected MSCs or saline into the infarcted myocardium of mice and evaluated LV remodeling and function 28 days after myocardial infarction. To test the hypothesis that toll-like receptor 4 (TLR4) mediates proinflammatory polarization of MSCs, we characterized cardiac MSCs from TLR4-/- and wild-type (WT) mice after inflammatory stimulation in vitro. Next, we transplanted cardiac MSCs from TLR4-/- and WT male mice into the infarcted myocardium of female WT mice and evaluated infarct size, MSC retention, inflammation, remodeling, and function after 7 days. RESULTS: LVD switched cardiac MSCs toward an inflammatory phenotype, with increased secretion of inflammatory cytokines as well as chemokines. The effect of LVD on subcutaneous MSCs was less remarkable. Although transplantation of cardiac MSCs and subcutaneous MSCs from LVD and sham hearts did not improve LV remodeling and function, cardiac MSCs from LVD exacerbated anterior wall thinning 28 days after myocardial infarction. The inflammatory polarization of cardiac MSCs by LVD was mediated by TLR4, as we found less secretion of inflammatory cytokines and higher secretion of anti-inflammatory cytokines from activated cardiac MSCs of TLR4-deficient mice, compared with WT cardiac MSCs. Significantly, TLR4 deficiency preserved the expression of CD47 (don't eat me signal) on cardiac MSCs after both TLR4 stimulation in vitro and transplantation into the infarcted heart. Compared with WT cardiac MSCs and saline, TLR4-/- cardiac MSCs survived in the cardiac tissue and maintained their reparative properties, reduced infarct size, increased scar thickness, and attenuated LV dilatation 7 days after myocardial infarction. CONCLUSIONS: The environment of the failing and infarcted myocardium drives resident and transplanted MSCs toward a proinflammatory phenotype and restricts their survival and reparative effects in a mechanism mediated by TLR4.
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Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Fenotipo , Receptor Toll-Like 4/deficiencia , Disfunción Ventricular Izquierda/patología , Animales , Células Cultivadas , Femenino , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones TransgénicosRESUMEN
BACKGROUND: Macrophages and Wnt proteins (Wnts) are independently involved in cardiac development, response to cardiac injury, and repair. However, the role of macrophage-derived Wnts in the healing and repair of myocardial infarction (MI) is unknown. We sought to determine the role of macrophage Wnts in infarct repair. METHODS AND RESULTS: We show that the Wnt pathway is activated after MI in mice. Furthermore, we demonstrate that isolated infarct macrophages express distinct Wnt pathway components and are a source of noncanonical Wnts after MI. To determine the effect of macrophage Wnts on cardiac repair, we evaluated mice lacking the essential Wnt transporter Wntless (Wls) in myeloid cells. Significantly, Wntless-deficient macrophages presented a unique subset of M2-like macrophages with anti-inflammatory, reparative, and angiogenic properties. Serial echocardiography studies revealed that mice lacking macrophage Wnt secretion showed improved function and less remodeling 30 days after MI. Finally, mice lacking macrophage-Wntless had increased vascularization near the infarct site compared with controls. CONCLUSIONS: Macrophage-derived Wnts are implicated in adverse cardiac remodeling and dysfunction after MI. Together, macrophage Wnts could be a new therapeutic target to improve infarct healing and repair.
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Corazón/diagnóstico por imagen , Péptidos y Proteínas de Señalización Intracelular/genética , Macrófagos/metabolismo , Infarto del Miocardio/diagnóstico por imagen , Neovascularización Fisiológica/genética , Receptores Acoplados a Proteínas G/genética , Remodelación Ventricular/genética , Proteínas Wnt/metabolismo , Animales , Modelos Animales de Enfermedad , Ecocardiografía , Femenino , Macrófagos/inmunología , Ratones , Infarto del Miocardio/inmunología , Neovascularización Fisiológica/inmunología , Remodelación Ventricular/inmunología , Vía de Señalización WntRESUMEN
Uncontrolled activation of pro-inflammatory macrophages after myocardial infarction (MI) accelerates adverse left ventricular (LV) remodeling and dysfunction. Hemin, an iron-containing porphyrin, activates heme oxygenase-1 (HO-1), an enzyme with anti-inflammatory and cytoprotective properties. We sought to determine the effects of hemin formulated in a macrophage-targeted lipid-based carrier (denoted HA-LP) on LV remodeling and function after MI. Hemin encapsulation efficiency was ~100% at therapeutic dose levels. In vitro, hemin/HA-LP abolished TNF-α secretion from macrophages, whereas the same doses of free hemin and drug free HA-LP had no effect. Hemin/HA-LP polarized peritoneal and splenic macrophages toward M2 anti-inflammatory phenotype. We next induced MI in mice and allocated them to IV treatment with hemin/HA-LP (10mg/kg), drug free HA-LP, free hemin (10mg/kg) or saline, one day after MI. Active in vivo targeting to infarct macrophages was confirmed with HA-LP doped with PE-rhodamine. LV remodeling and function were assessed by echocardiography before, 7, and 30days after treatment. Significantly, hemin/HA-LP effectively and specifically targets infarct macrophages, switches infarct macrophages toward M2 anti-inflammatory phenotype, improves angiogenesis, reduces scar expansion and improves infarct-related regional function. In conclusion, macrophage-targeted lipid-based drug carriers with hemin switch macrophages into an anti-inflammatory phenotype, and improve infarct healing and repair. Our approach presents a novel strategy to modulate inflammation and improve infarct repair.
Asunto(s)
Antiinflamatorios/uso terapéutico , Sistemas de Liberación de Medicamentos , Hemina/uso terapéutico , Macrófagos/efectos de los fármacos , Infarto del Miocardio/tratamiento farmacológico , Remodelación Ventricular/efectos de los fármacos , Animales , Antiinflamatorios/administración & dosificación , Portadores de Fármacos/química , Femenino , Corazón/efectos de los fármacos , Corazón/fisiopatología , Hemina/administración & dosificación , Lípidos/química , Macrófagos/inmunología , Macrófagos/patología , Ratones , Ratones Endogámicos BALB C , Infarto del Miocardio/inmunología , Infarto del Miocardio/fisiopatologíaRESUMEN
BACKGROUND: Irreversible electroporation (IRE) is a non-thermal cell ablation approach that induces selective damage to cell membranes only. The purpose of the current study was to evaluate and optimize its use for in-vivo myocardial decellularization. METHODS: Forty-two Sprague-Dawley rats were used to compare myocardial damage of seven different IRE protocols with anterior myocardial infarction damage. An in-vivo open thoracotomy model was used, with two-needle electrodes in the anterior ventricular wall. IRE protocols included different combinations of pulse lengths (70 vs. 100 µseconds), frequency (1, 2, 4 Hz), and number (10 vs. 20 pulses), as well as voltage intensity (50, 250 and 500 Volts). All animals underwent baseline echocardiographic evaluation. Degree of myocardial ablation was determined using repeated echocardiography measurements (days 7 and 28) as well as histologic and morphometric analysis at 28 days. RESULTS: All animals survived 28 days of follow-up. Compared with 50V and 250V, electroporation with 500V was associated with significantly increased myocardial scar and reduction in ejection fraction (67.4%±4% at baseline vs. 34.6%±20% at 28 days; p <0.01). Also, compared with pulse duration of 70 µsec, pulses of 100 µsec were associated with markedly reduced left ventricular function and markedly increased relative scar area ratio (28%±9% vs. 16%±3%, p = 0.02). Decreasing electroporation pulse frequency (1Hz vs. 2Hz, 2Hz vs. 4Hz) was associated with a significant increase in myocardial damage. Electroporation protocols with a greater number of pulses (20 vs. 10) correlated with more profound tissue damage (p<0.05). When compared with myocardial infarction damage, electroporation demonstrated a considerable likeness regarding the extent of the inflammatory process, but with relatively higher levels of extra-cellular preservation. CONCLUSIONS: IRE has a graded effect on the myocardium. The extent of ablation can be controlled by changing pulse length, frequency and number, as well as by changing electric field intensity.
Asunto(s)
Procedimientos Quirúrgicos Cardíacos/métodos , Electroporación/métodos , Miocardio/citología , Animales , Procedimientos Quirúrgicos Cardíacos/efectos adversos , Electrocardiografía , Electrodos , Electroporación/instrumentación , Femenino , Corazón/fisiopatología , Isquemia Miocárdica/fisiopatología , Ratas Sprague-DawleyRESUMEN
In Alzheimer's disease (AD), astrocytes undergo morphological changes ranging from atrophy to hypertrophy, but the effect of such changes at the functional level is still largely unknown. Here, we aimed to investigate whether alterations in astrocyte activity in AD are transient and depend on their microenvironment, or whether they are irreversible. We established and characterized a new protocol for the isolation of adult astrocytes and discovered that astrocytes isolated from old 5xFAD mice have higher GFAP expression than astrocytes derived from WT mice, as observed in vivo. We found high C1q levels in brain sections from old 5xFAD mice in close vicinity to amyloid plaques and astrocyte processes. Interestingly, while old 5xFAD astrocytes are impaired in uptake of soluble Aß42, this effect was reversed upon an addition of exogenous C1q, suggesting a potential role for C1q in astrocyte-mediated Aß clearance. Our results suggest that scavenger receptor B1 plays a role in C1q-facilitated Aß uptake by astrocytes and that expression of scavenger receptor B1 is reduced in adult old 5xFAD astrocytes. Furthermore, old 5xFAD astrocytes show impairment in support of neuronal growth in co-culture and neurotoxicity concomitant with an elevation in IL-6 expression. Further understanding of the impact of astrocyte impairment on AD pathology may provide insights into the etiology of AD.
Asunto(s)
Envejecimiento , Enfermedad de Alzheimer , Péptidos beta-Amiloides/metabolismo , Astrocitos/metabolismo , Encéfalo/patología , Regulación de la Expresión Génica/genética , Fármacos Neuroprotectores/uso terapéutico , Fragmentos de Péptidos/metabolismo , Envejecimiento/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/terapia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Antígeno CD11b/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Proteína Ácida Fibrilar de la Glía/metabolismo , Interleucina-6/metabolismo , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Transgénicos , Mutación/genética , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo , Receptores de Complemento/metabolismoRESUMEN
BACKGROUND: Inflammation has been implicated in the initiation, progression and manifestation of hypertensive heart disease. We sought to determine the role of monocytes/macrophages in hypertension and pressure overload induced left ventricular (LV) remodeling. METHODS AND RESULTS: We used two models of LV hypertrophy (LVH). First, to induce hypertension and LVH, we fed Sabra salt-sensitive rats with a high-salt diet. The number of macrophages increased in the hypertensive hearts, peaking at 10 weeks after a high-salt diet. Surprisingly, macrophage depletion, by IV clodronate (CL) liposomes, inhibited the development of hypertension. Moreover, macrophage depletion reduced LVH by 17% (p<0.05), and reduced cardiac fibrosis by 75%, compared with controls (p=0.001). Second, to determine the role of macrophages in the development and progression of LVH, independent of high-salt diet, we depleted macrophages in mice subjected to transverse aortic constriction and pressure overload. Significantly, macrophage depletion, for 3 weeks, attenuated LVH: a 12% decrease in diastolic and 20% in systolic wall thickness (p<0.05), and a 13% in LV mass (p=0.04), compared with controls. Additionally, macrophage depletion reduced cardiac fibrosis by 80% (p=0.006). Finally, macrophage depletion down-regulated the expression of genes associated with cardiac remodeling and fibrosis: transforming growth factor beta-1 (by 80%) collagen type III alpha-1 (by 71%) and atrial natriuretic factor (by 86%). CONCLUSIONS: Macrophages mediate the development of hypertension, LVH, adverse cardiac remodeling, and fibrosis. Macrophages, therefore, should be considered as a therapeutic target to reduce the adverse consequences of hypertensive heart disease.