RESUMEN
Cardiac fibrosis is a pathological process characterized by excessive tissue deposition, matrix remodeling, and tissue stiffening, which eventually leads to organ failure. On a cellular level, the development of fibrosis is associated with the activation of cardiac fibroblasts into myofibroblasts, a highly contractile and secretory phenotype. Myofibroblasts are commonly identified in vitro by the de novo assembly of alpha-smooth muscle actin stress fibers; however, there are few methods to automate stress fiber identification, which can lead to subjectivity and tedium in the process. To address this limitation, we present a computer vision model to classify and segment cells containing alpha-smooth muscle actin stress fibers into 2 classes (α-SMA SF+ and α-SMA SF-), with a high degree of accuracy (cell accuracy: 77%, F1 score 0.79). The model combines standard image processing methods with deep learning techniques to achieve semantic segmentation of the different cell phenotypes. We apply this model to cardiac fibroblasts cultured on hyaluronic acid-based hydrogels of various moduli to induce alpha-smooth muscle actin stress fiber formation. The model successfully predicts the same trends in stress fiber identification as obtained with a manual analysis. Taken together, this work demonstrates a process to automate stress fiber identification in in vitro fibrotic models, thereby increasing reproducibility in fibroblast phenotypic characterization.
Asunto(s)
Actinas/metabolismo , Aprendizaje Profundo , Miocardio/citología , Miocardio/metabolismo , Fibras de Estrés/metabolismo , Inteligencia Artificial , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Técnicas de Cultivo de Célula , Células Cultivadas , Elasticidad , Fibroblastos/metabolismo , Humanos , Hidrogeles , Procesamiento de Imagen Asistido por Computador , Modelos Cardiovasculares , Miofibroblastos/clasificación , Miofibroblastos/metabolismo , Miofibroblastos/patología , Fibras de Estrés/clasificación , Fibras de Estrés/patología , Propiedades de SuperficieRESUMEN
Lung fibroblasts play a key role in postnatal lung development, namely, the formation of the alveolar gas exchange units, through the process of secondary septation. Although evidence initially highlighted roles for fibroblasts in the production and remodeling of the lung extracellular matrix, more recent studies have described the presence of different fibroblast subsets in the developing lung. These subsets include myofibroblasts and lipofibroblasts and their precursors. These cells are believed to play different roles in alveologenesis and are localized to different regions of the developing septa. The precise roles played by these different fibroblast subsets remain unclear. Understanding the signaling pathways that control the discrete functions of these fibroblast subsets would help to clarify the roles and the regulation of lung fibroblasts during lung development. Here, we critically evaluate a recent report that described divergent fibroblast growth factor (FGF) signaling pathways in two different subsets of lung fibroblasts that express different levels of green fluorescent protein (GFP) driven by the platelet-derived growth factor receptor-α promoter. The GFP expression was used as a surrogate for lipofibroblasts (GFP(low)) and myofibroblasts (GFP(high)). It was suggested that Fgf10/Fgf1 and Fgf18/Fgfr3 autocrine pathways may be operative in GFP(low) and GFP(high) cells, respectively, and that these pathways might regulate the proliferation and migration of different fibroblast subsets during alveologenesis. These observations lay important groundwork for the further exploration of FGF function during normal lung development, as well as in aberrant lung development associated with bronchopulmonary dysplasia.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Pulmón/citología , Pulmón/metabolismo , Animales , Factores de Crecimiento de Fibroblastos/genética , Fibroblastos/clasificación , Fibroblastos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Pulmón/crecimiento & desarrollo , Modelos Biológicos , Miofibroblastos/clasificación , Miofibroblastos/metabolismo , Organogénesis , Alveolos Pulmonares/citología , Alveolos Pulmonares/crecimiento & desarrollo , Alveolos Pulmonares/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de SeñalRESUMEN
The main objective of our research was to examine the role and immunophenotypic characteristics of myofibroblasts in sheep liver naturally infected by the lancet liver fluke (Dicrocoelium dendriticum). In the reported study we analyzed liver samples from 20 adult sheep, 14 infected animals and 6 controls. The liver samples were fixed in 10% buffered formalin, and routinely processed and stained using hematoxylin eosin, the periodic acid-Schiff and Masson-Goldner trichrome methods. The immunohistochemical examination was carried out by the streptavidin biotin (LSAB2) method, using antibodies for α-smooth muscle actin (α-SMA), desmin and vimentin. The histopathological examination revealed liver fibrosis in 6 out of 14 (42.9%) analyzed samples, while different forms of cholangitis were observed in the remaining 8 out of 14 (57.1%). The expression of α-SMA was proven in perisinusoidal hepatic stellate cells, portal/septal myofibroblasts, and interface myofibroblasts. The degree of α-SMA expression and the number of α-SMA immunopositive cells were the most intensive in the liver with fibrosis. Desmin expression in all liver samples of infected sheep was confirmed in hepatic stellate cells and smooth muscle cells. The hepatic stellate cells, portal/septal myofibroblasts, and interface myofibroblasts reacted as vimentin positive cells. In the liver without fibrotic changes hepatic stellate cells and smooth muscle cells were desmin positive. The obtained results suggest that all populations of myofibroblasts, especially hepatic stellate cells, play an important role in the increased extracellular matrix formation during parasitic liver fibrosis in sheep naturally infected with D. dendriticum.
Asunto(s)
Dicroceliasis/veterinaria , Dicrocoelium , Hígado/parasitología , Miofibroblastos/clasificación , Enfermedades de las Ovejas/parasitología , Animales , Dicroceliasis/inmunología , Dicroceliasis/patología , Inmunofenotipificación , Hígado/citología , Hígado/patología , Miofibroblastos/inmunología , Miofibroblastos/fisiología , Ovinos , Enfermedades de las Ovejas/patologíaRESUMEN
Cardiac myofibroblasts are key players in chronic remodeling of the cardiac extracellular matrix, which is mediated in part by elevated transforming growth factor-ß1 (TGF-ß1). The c-Ski proto-oncoprotein has been shown to modify TGF-ß1 post-receptor signaling through receptor-activated Smads (R-Smads); however, little is known about how c-Ski regulates fibroblast phenotype and function. We sought to elucidate the function of c-Ski in primary cardiac myofibroblasts using a c-Ski overexpression system. Cardiac myofibroblasts expressed three forms of c-Ski with the predominant band at 105 kDa, and adenoviral c-Ski treatment resulted in overexpression of 95-kDa c-Ski in cellular nuclei. Exogenous c-Ski led to significant inhibition of type I collagen secretion and myofibroblast contractility using two-dimensional semifloating gel contraction assay in both basal and with TGF-ß1 (10 ng/ml for 24 h) stimulation. Overexpressed c-Ski did not inhibit nuclear translocation of phosphorylated R-Smad2, despite their binding, as demonstrated by immunoprecipitation. Acute treatment of primary myofibroblasts with TGF-ß1 in vitro revealed a marked nuclear shuttling of c-Ski at 24 and 48 h following stimulation. Remarkably, overexpression of c-Ski led to a stepwise reduction of the myofibroblast marker α-smooth muscle actin with increasing multiplicity of infection, and these results indicate that 95-kDa c-Ski overexpression may effect a loss of the myofibroblastic phenotype. Furthermore, adenovirus (Ad) for hemagglutinin-tagged c-Ski infection led to a reduction in the number of myofibroblasts versus Ad-LacZ-infected and uninfected controls, due to induction of apoptosis. Finally, we observed a significant increase in 105-kDa c-Ski in the cytosolic fraction of cells of the infarct scar and adjacent remnant myocardium vs. noninfarcted controls.