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Introduction: The type I interferon (IFN) response is an innate immune program that mediates anti-viral, anti-cancer, auto-immune, auto-inflammatory, and sterile injury responses. Bone marrow derived macrophages (BMDMs) are commonly used to model macrophage type I IFN responses, but the use of bulk measurement techniques obscures underlying cellular heterogeneity. This is particularly important for the IFN response to immune stimulatory double-stranded DNA (dsDNA) because it elicits overlapping direct and indirect responses, the latter of which depend on type I IFN cytokines signaling via the IFN alpha receptor (IFNAR) to upregulate expression of interferon stimulated genes (ISGs). Single cell transcriptomics has emerged as a powerful tool for revealing functional variability within cell populations. Methods: Here, we use single cell RNA-Seq to examine BMDM heterogeneity at steady state and after immune-stimulatory DNA stimulation, with or without IFNAR-dependent amplification. Results: We find that many macrophages express ISGs after DNA stimulation. We also find that a subset of macrophages express ISGs even if IFNAR is inhibited, suggesting that they are direct responders. Analysis of this subset reveals Ccl5 to be an IFNAR-independent marker gene of direct DNA sensing cells. Discussion: Our studies provide a method for studying direct responders to IFN-inducing stimuli and demonstrate the importance of characterizing BMDM models of innate immune responses with single cell resolution.
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Interferón Tipo I , Transcriptoma , Biomarcadores , ADN , Interferón Tipo I/metabolismo , Interferón-alfa , Macrófagos , Animales , RatonesRESUMEN
Background Neutrophils are thought to be short-lived first responders to tissue injuries such as myocardial infarction (MI), but little is known about their diversification or dynamics. Methods and Results We permanently ligated the left anterior descending coronary arteries of mice and performed single-cell RNA sequencing and analysis of >28 000 neutrophil transcriptomes isolated from the heart, peripheral blood, and bone marrow of mice on days 1 to 4 after MI or at steady-state. Unsupervised clustering of cardiac neutrophils revealed 5 major subsets, 3 of which originated in the bone marrow, including a late-emerging granulocyte expressing SiglecF, a marker classically used to define eosinophils. SiglecFHI neutrophils represented ≈25% of neutrophils on day 1 and grew to account for >50% of neutrophils by day 4 post-MI. Validation studies using quantitative polymerase chain reaction of fluorescent-activated cell sorter sorted Ly6G+SiglecFHI and Ly6G+SiglecFLO neutrophils confirmed the distinct nature of these populations. To confirm that the cells were neutrophils rather than eosinophils, we infarcted GATA-deficient mice (∆dblGATA) and observed similar quantities of infiltrating Ly6G+SiglecFHI cells despite marked reductions of conventional eosinophils. In contrast to other neutrophil subsets, Ly6G+SiglecFHI neutrophils expressed high levels of Myc-regulated genes, which are associated with longevity and are consistent with the persistence of this population on day 4 after MI. Conclusions Overall, our data provide a spatial and temporal atlas of neutrophil specialization in response to MI and reveal a dynamic proinflammatory cardiac Ly6G+SigF+(Myc+NFÏ°B+) neutrophil that has been overlooked because of negative selection.
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Infarto del Miocardio/genética , Miocardio/metabolismo , Neutrófilos/patología , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Análisis de la Célula Individual/métodos , Transcriptoma , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Miocardio/patología , Neutrófilos/metabolismo , Análisis de Secuencia de ARN , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/genéticaRESUMEN
Prenatal alcohol exposure (PAE) is often associated with congenital heart defects, most commonly septal, valvular, and great vessel defects. However, there have been no known studies on whether PAE affects the resulting fibroblast population after development, and whether this has any consequences in the postnatal period. Our previous study focused on the effects of PAE on the postnatal fibroblast population, which translated into changes in cardiac extracellular matrix (ECM) composition and cardiac function in the neonatal heart. Moreover, our lab has previously demonstrated that alcohol-induced fibrosis is mediated by oxidative stress mechanisms in adult rat hearts following chronic alcohol exposure. Thus, we hypothesize that PAE alters cardiac ECM composition that persists into the postnatal period, leading to cardiac dysfunction, and these effects are prevented by antioxidant treatment. To investigate these effects, pregnant mice were intraperitoneally injected with 2.9â¯g EtOH/kg body weight on gestation days 6.75 and 7.25. Controls were injected with vehicle saline. Randomly selected dams in both groups were then treated with 100â¯mg/kg body weight of the antioxidant N-acetylcysteine (NAC) immediately after EtOH or vehicle administration. Left ventricular (LV) chamber dimension and function were assessed in sedated animals on neonatal day 5 using echocardiography. Ejection fraction decreased in the PAE group. NAC treatment prevented this depression of systolic function in PAE neonates. Hearts were analyzed for expression of fibroblast activation markers. Alpha smooth muscle actin (α-SMA) increased in PAE neonatal hearts, and this increase was prevented by NAC treatment. In PAE pups, collagen I decreased, but collagen III expression increased compared to saline animals; the overall collagen I/III ratio significantly decreased. When PAE mice were treated with NAC, collagen I/III ratio did not change. Overall, our data demonstrate that prenatal alcohol exposure produces changes in collagen subtype in neonatal cardiac ECM and a decline in systolic function, and these adverse effects were prevented by NAC treatment.
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Acetilcisteína/farmacología , Alcoholismo/fisiopatología , Proliferación Celular/efectos de los fármacos , Colágeno Tipo I/metabolismo , Vasos Coronarios/química , Etanol/toxicidad , Fibroblastos/efectos de los fármacos , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Femenino , Ratones , EmbarazoRESUMEN
Fetal alcohol syndrome (FAS) is the most severe condition of fetal alcohol spectrum disorders (FASD) and is associated with congenital heart defects. However, more subtle defects such as ventricular wall thinning and cardiac compliance may be overlooked in FASD. Our studies focus on the role of cardiac fibroblasts in the neonatal heart, and how they are affected by prenatal alcohol exposure (PAE). We hypothesize that PAE affects fibroblast function contributing to dysregulated collagen synthesis, which leads to cardiac dysfunction. To investigate these effects, pregnant C57/BL6 mice were intraperitoneally injected with 2.9 g EtOH/kg dose to achieve a blood alcohol content of approximately 0.35 on gestation days 6.75 and 7.25. Pups were sacrificed on neonatal day 5 following echocardiography measurements of left ventricular (LV) chamber dimension and function. Hearts were used for primary cardiac fibroblast isolation or protein expression analysis. PAE animals had thinner ventricular walls than saline exposed animals, which was associated with increased LV wall stress and decreased ejection fraction. In isolated fibroblasts, PAE decreased collagen I/III ratio and increased gene expression of profibrotic markers, including α-smooth muscle actin and lysyl oxidase. Notch1 signaling was assessed as a possible mechanism for fibroblast activation, and indicated that gene expression of Notch1 receptor and downstream Hey1 transcription factor were increased. Cardiac tissue analysis revealed decreased collagen I/III ratio and increased protein expression of α-smooth muscle actin and lysyl oxidase. However, Notch1 signaling components decreased in whole heart tissue. Our study demonstrates that PAE caused adverse changes in the cardiac collagen profile and a decline in cardiac function in the neonatal heart.
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Cardiomiopatías/metabolismo , Matriz Extracelular/metabolismo , Trastornos del Espectro Alcohólico Fetal/metabolismo , Fibroblastos/metabolismo , Miocardio/metabolismo , Función Ventricular Izquierda , Remodelación Ventricular , Animales , Animales Recién Nacidos , Cardiomiopatías/genética , Cardiomiopatías/patología , Cardiomiopatías/fisiopatología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Modelos Animales de Enfermedad , Matriz Extracelular/patología , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Trastornos del Espectro Alcohólico Fetal/genética , Trastornos del Espectro Alcohólico Fetal/patología , Trastornos del Espectro Alcohólico Fetal/fisiopatología , Fibroblastos/patología , Ratones Endogámicos C57BL , Miocardio/patología , Embarazo , Proteína-Lisina 6-Oxidasa/genética , Proteína-Lisina 6-Oxidasa/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Transducción de SeñalRESUMEN
The cardiac extracellular matrix is a complex architectural network that serves many functions, including providing structural and biochemical support to surrounding cells and regulating intercellular signaling pathways. Cardiac function is directly affected by extracellular matrix (ECM) composition, and alterations of the ECM contribute to the progression of heart failure. Initially, collagen deposition is an adaptive response that aims to preserve tissue integrity and maintain normal ventricular function. However, the synergistic effects of proinflammatory and profibrotic responses induce a vicious cycle, which causes excess activation of myofibroblasts, significantly increasing collagen deposition and accumulation in the matrix. Furthermore, excess synthesis and activation of the enzyme lysyl oxidase (LOX) during disease increases collagen cross-linking, which significantly increases collagen resistance to degradation by matrix metalloproteinases (MMPs). In the present study, the aortocaval fistula model of volume overload (VO) was used to determine whether LOX inhibition could prevent adverse changes in the ECM and subsequent cardiac dysfunction. The major findings from this study were that LOX inhibition 1) prevented VO-induced increases in left ventricular wall stress; 2) partially attenuated VO-induced ventricular hypertrophy; 3) completely blocked the increases in fibrotic proteins, including collagens, MMPs, and their tissue inhibitors; and 4) prevented the VO-induced decline in cardiac function. It remains unclear whether a direct interaction between LOX and MMPs exists; however, our experiments suggest a potential link between the two because LOX inhibition completely attenuated VO-induced increases in MMPs. Overall, our study demonstrated key cardioprotective effects of LOX inhibition against adverse cardiac remodeling due to chronic VO. NEW & NOTEWORTHY Although the primary role of lysyl oxidase (LOX) is to cross-link collagens, we found that elevated LOX during cardiac disease plays a key role in the progression of heart failure. Here, we show that inhibition of LOX in volume-overloaded rats prevented the development of cardiac dysfunction and improved ventricular collagen and matrix metalloproteinase/tissue inhibitor of metalloproteinase profiles.
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Matriz Extracelular/efectos de los fármacos , Hipertrofia Ventricular Izquierda/tratamiento farmacológico , Miocardio/metabolismo , Proteína-Lisina 6-Oxidasa/antagonistas & inhibidores , Aminopropionitrilo/farmacología , Aminopropionitrilo/uso terapéutico , Animales , Fístula Arteriovenosa/complicaciones , Colágeno/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Matriz Extracelular/metabolismo , Hipertrofia Ventricular Izquierda/etiología , Masculino , Metaloproteinasas de la Matriz/metabolismo , Proteína-Lisina 6-Oxidasa/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Alcohol is among the most commonly abused drugs worldwide and affects many organ systems, including the heart. Alcoholic cardiomyopathy is characterized by a dilated cardiac phenotype with extensive hypertrophy and extracellular matrix (ECM) remodeling. We have previously shown that chronic ethanol (EtOH) administration accelerates the progression to heart failure in a rat model of volume overload. However, the mechanism by which this decompensation occurs is unknown. For this study, we hypothesized that chronic EtOH administration would prevent compensatory hypertrophy and cardiac remodeling in a rodent model of pressure overload (PO). METHODS: Abdominal aortic constriction was used to create PO in 8-week-old male Wistar rats. Alcohol administration was performed via chronic intermittent EtOH vapor inhalation for 2 weeks prior to surgery and for the duration of the 8-week study. Echocardiography measurements were taken to assess ventricular functional and structural changes. RESULTS: PO increased posterior wall thickness and the hypertrophic markers, atrial and B-type natriuretic peptides (ANP and BNP). With the added stressor of EtOH, wall thickness, ANP, and BNP decreased in PO animals. The combination of PO and EtOH resulted in increased wall stress compared to PO alone. PO also caused increased expression of collagen I and III, whereas EtOH alone only increased collagen III. The combined stresses of PO and EtOH led to an increase in collagen I expression, but collagen III did not change, resulting in an increased collagen I/III ratio in the PO rats treated with EtOH. Lastly, Notch1 expression was significantly increased only in the PO rats treated with EtOH. CONCLUSIONS: Our data indicate that chronic EtOH may limit the cardiac hypertrophy induced by PO which may be associated with a Notch1 mechanism, resulting in increased wall stress and altered ECM profile.
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A key feature of heart failure is adverse extracellular matrix (ECM) remodeling, which is associated with increases in the collagen cross-linking enzyme, lysyl oxidase (LOX). In this study, we assess the progression of cardiovascular remodeling from the compensatory to decompensatory phase, with a focus on the change in LOX expression and activity as it relates to alterations in ECM composition and changes in cardiac function. Adult male Sprague-Dawley rats were studied after 4, 14, or 21weeks of aortocaval fistula-induced volume overload (VO). Progressive increases in the left and right ventricular mass indicated biventricular hypertrophy. Echocardiography revealed significant increases in the posterior wall thickness and internal diameter of the left ventricle as early as 3weeks, which persisted until the 21week endpoint. There were also significant decreases in eccentric index and fractional shortening in VO animals. Hemodynamic measurements showed progressive decreases in contractility, indicative of systolic dysfunction. There were progressive VO-induced increases in LOX expression and activity, collagen, and collagen cross-linking during the course of these experiments. We observed a negative correlation between LOX activity and cardiac function. Additional rats were treated with an inhibitor of LOX activity starting at 2weeks post-surgery and continued to 14weeks. LOX inhibition prevented the cardiac dysfunction and collagen accumulation caused by VO. Overall these data suggest a detrimental role for the chronic increase of cardiac LOX expression and activity in the transition from compensated remodeling to decompensated failure.
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Regulación Enzimológica de la Expresión Génica , Insuficiencia Cardíaca/enzimología , Contracción Miocárdica , Miocardio/enzimología , Proteína-Lisina 6-Oxidasa/metabolismo , Remodelación Ventricular , Animales , Ecocardiografía , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Masculino , Miocardio/patología , Ratas , Ratas Sprague-DawleyRESUMEN
Chronic alcohol abuse is one of the leading causes of dilated cardiomyopathy (DCM) in the United States. Volume overload (VO) also produces DCM characterized by left ventricular (LV) dilatation and reduced systolic and diastolic function, eventually progressing to congestive heart failure. For this study, we hypothesized that chronic alcohol exposure would exacerbate cardiac dysfunction and remodeling due to VO. Aortocaval fistula surgery was used to induce VO, and compensatory cardiac remodeling was allowed to progress for either 3days (acute) or 8weeks (chronic). Alcohol was administered via chronic intermittent ethanol vapor (EtOH) for 2weeks before the acute study and for the duration of the 8week chronic study. Temporal alterations in LV function were assessed by echocardiography. At the 8week end point, pressure-volume loop analysis was performed by LV catheterization and cardiac tissue collected. EtOH did not exacerbate LV dilatation (end-systolic and diastolic diameter) or systolic dysfunction (fractional shortening, ejection fraction) due to VO. The combined stress of EtOH and VO decreased the eccentric index (posterior wall thickness to end-diastolic diameter ratio), increased end-diastolic pressure (EDP), and elevated diastolic wall stress. VO also led to increases in posterior wall thickness, which was not observed in the VO+EtOH group, and wall thickness significantly correlated with LV BNP expression. VO alone led to increases in interstitial collagen staining (picrosirius red), which while not statistically significant, tended to be decreased by EtOH. VO increased LV collagen I protein expression, whereas in rats with VO+EtOH, LV collagen I was not elevated relative to Sham. The combination of VO and EtOH also led to increases in LV collagen III expression relative to Sham. Rats with VO+EtOH had significantly lower collagen I/III ratio than rats with VO alone. During the acute remodeling phase of VO (3days), VO significantly increased collagen III expression, whereas this effect was not observed in rats with VO+EtOH. In conclusion, chronic EtOH accelerates the development of elevated wall stress and promotes early eccentric remodeling in rats with VO. Our data indicate that these effects may be due to disruptions in compensatory hypertrophy and extracellular matrix remodeling in response to volume overload.
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Alcoholes/efectos adversos , Miocardio/metabolismo , Miocardio/patología , Remodelación Ventricular , Consumo de Bebidas Alcohólicas , Alcoholes/administración & dosificación , Animales , Biomarcadores , Cardiomiopatías/diagnóstico , Cardiomiopatías/etiología , Cardiomiopatías/metabolismo , Cardiomiopatías/mortalidad , Colágeno/genética , Colágeno/metabolismo , Modelos Animales de Enfermedad , Ecocardiografía , Matriz Extracelular/metabolismo , Expresión Génica , Masculino , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Isoformas de Proteínas , Proteína-Lisina 6-Oxidasa/genética , Proteína-Lisina 6-Oxidasa/metabolismo , Ratas , Función Ventricular IzquierdaRESUMEN
A hallmark of heart failure (HF) is adverse extracellular matrix (ECM) remodeling, which is regulated by the collagen cross-linking enzyme, lysyl oxidase (LOX). In this study, we evaluate the efficacy of LOX inhibition to prevent adverse left ventricular (LV) remodeling and dysfunction using an experimental model of HF. Sprague-Dawley rats were subjected to surgically induced volume overload (VO) by creation of aortocaval fistula (ACF). A LOX inhibitor, beta-aminopropionitrile (BAPN; 100 mg/kg/day), was administered to rats with ACF or sham surgery at eight weeks postsurgery. Echocardiography was used to assess progressive alterations in cardiac ventricular structure and function. Left ventricular (LV) catheterization was used to assess alterations in contractility, stiffness, LV pressure and volume, and other indices of cardiac function. The LV ECM alterations were assessed by: (a) histological staining of collagen, (b) protein expression of collagen types I and III, (c) hydroxyproline assay, and (d) cross-linking assay. LOX inhibition attenuated VO-induced increases in cardiac stress, and attenuated increases in interstitial myocardial collagen, total collagen, and protein levels of collagens I and III. Both echocardiography and catheterization measurements indicated improved cardiac function post-VO in BAPN treated rats vs. untreated. Inhibition of LOX attenuated VO-induced decreases in LV stiffness and cardiac function. Overall, our data indicate that LOX inhibition was cardioprotective in the volume overloaded heart.