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The origins and developmental mechanisms of coronary arteries are incompletely understood. We show here by fate mapping, clonal analysis, and immunohistochemistry that endocardial cells generate the endothelium of coronary arteries. Dye tracking, live imaging, and tissue transplantation also revealed that ventricular endocardial cells are not terminally differentiated; instead, they are angiogenic and form coronary endothelial networks. Myocardial Vegf-a or endocardial Vegfr-2 deletion inhibited coronary angiogenesis and arterial formation by ventricular endocardial cells. In contrast, lineage and knockout studies showed that endocardial cells make a small contribution to the coronary veins, the formation of which is independent of myocardial-to-endocardial Vegf signaling. Thus, contrary to the current view of a common source for the coronary vessels, our findings indicate that the coronary arteries and veins have distinct origins and are formed by different mechanisms. This information may help develop better cell therapies for coronary artery disease.
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Vasos Coronarios/embriología , Células Endoteliales/citología , Miocardio/citología , Neovascularización Fisiológica , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Diferenciación Celular , Vasos Coronarios/citología , Vasos Coronarios/metabolismo , Células Endoteliales/metabolismo , Ratones , Miocardio/metabolismo , Factores de Transcripción NFATC/metabolismoRESUMEN
Although periostin plays a significant role in adult cardiac remodeling diseases, the focus of this review is on periostin as a valvulogenic gene. Periostin is expressed throughout valvular development, initially being expressed in endocardial endothelial cells that have been activated to transform into prevalvular mesenchyme termed "cushion tissues" that sustain expression of periostin throughout their morphogenesis into mature (compacted) valve leaflets. The phenotype of periostin null indicates that periostin is not required for endocardial transformation nor the proliferation of its mesenchymal progeny but rather promotes cellular behaviors that promote migration, survival (anti-apoptotic), differentiation into fibroblastic lineages, collagen secretion and postnatal remodeling/maturation. These morphogenetic activities are promoted or coordinated by periostin signaling through integrin receptors activating downstream kinases in cushion cells that activate hyaluronan synthetase II (Akt/PI3K), collagen synthesis (Erk/MapK) and changes in cytoskeletal organization (Pak1) which regulate postnatal remodeling of cells and associated collagenous matrix into a trilaminar (zonal) histoarchitecture. Pak1 binding to filamin A is proposed as one mechanism by which periostin supports remodeling. The failure to properly remodel cushions sets up a trajectory of degenerative (myxomatous-like) changes that over time reduce biomechanical properties and increase chances for prolapse, regurgitation or calcification of the leaflets. Included in the review are considerations of lineage diversity and the role of periostin as a determinant of mesenchymal cell fate.
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Moléculas de Adhesión Celular/fisiología , Válvulas Cardíacas/crecimiento & desarrollo , Organogénesis , Diferenciación Celular , Células Endoteliales/citología , Humanos , Integrinas , Mesodermo/citologíaRESUMEN
Periostin (PN), a novel fasciclin-related matricellular protein, has been implicated in cardiac development and postnatal remodeling, but the mechanism remains unknown. We examined the role of PN in mediating intracellular kinase activation for atrioventricular valve morphogenesis using well defined explant cultures, gene transfection systems, and Western blotting. The results show that valve progenitor (cushion) cells secrete PN into the extracellular matrix, where it can bind to INTEGRINs and activate INTEGRIN/focal adhesion kinase signaling pathways and downstream kinases, PI3K/AKT and ERK. Functional assays with prevalvular progenitor cells showed that activating these signaling pathways promoted adhesion, migration, and anti-apoptosis. Through activation of PI3K/ERK, PN directly enhanced collagen expression. Comparing PN-null to WT mice also revealed that expression of hyaluronan (HA) and activation of hyaluronan synthase-2 (Has2) are also enhanced upon PN/INTEGRIN/focal adhesion kinase-mediated activation of PI3K and/or ERK, an effect confirmed by the reduction of HA synthase-2 in PN-null mice. We also identified in valve progenitor cells a potential autocrine signaling feedback loop between PN and HA through PI3K and/or ERK. Finally, in a three-dimensional assay to simulate normal valve maturation in vitro, PN promoted collagen compaction in a kinase-dependent fashion. In summary, this study provides the first direct evidence that PN can act to stimulate a valvulogenic signaling pathway.
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Moléculas de Adhesión Celular/metabolismo , Válvulas Cardíacas/embriología , Ácido Hialurónico/metabolismo , Transducción de Señal , Animales , Adhesión Celular , Moléculas de Adhesión Celular/genética , Movimiento Celular , Proliferación Celular , Células Cultivadas , Embrión de Pollo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Eliminación de Gen , Válvulas Cardíacas/citología , Válvulas Cardíacas/metabolismo , Integrinas/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , OvinosRESUMEN
The aim of this study was to assess structural and biochemical differences in the extracellular matrix of the fetal and adult porcine mitral heart valves in relation to their mechanical characteristics. Using tensile tests it was demonstrated that the material properties of porcine mitral heart valves progressively change with age. The collagen content of the adult heart valve, as estimated by hydroxyproline assay, increases three times as compared with fetal heart valves. Transmission electron microscopy demonstrated that the diameter of collagen fibrils increased in adult heart valves compared with fetal heart valves. The level of collagen cross-linking is lower in the fetal heart valve than the adult heart valve. The reported age differences in the material properties of fetal and adult porcine heart valves were associated with increases in collagen content, the diameter of collagen fibrils and the level of collagen cross-linking. These data lay a foundation for systematic elucidation of the structural determinants of material properties of heart valves during embryonic and postnatal valvulogenesis. They are also essential to define the desirable level of tissue maturation in heart valve tissue engineering.
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Envejecimiento/fisiología , Válvula Mitral/anatomía & histología , Válvula Mitral/fisiología , Animales , Fenómenos Biomecánicos , Colágeno/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , Feto/anatomía & histología , Feto/fisiología , Válvula Mitral/embriología , Válvula Mitral/ultraestructura , Sus scrofaRESUMEN
Agenda 2030 expresses, through the Sustainable Development Goals (SDG), and in particular through No. 4, the need to ensure an inclusive and equitable education, which promotes learning opportunities for all. At the university level, all students are urged to acquire the necessary theoretical and practical knowledge to promote sustainable development, so that they become graduates capable of facing the challenges of the future and the real demands of a society marked by heterogeneity, including the needs of people with some kind of disability. In this sense, the present work analyzed the impact of a transversal training program in Design for All on university degree students. For this purpose, a descriptive and comparative ex post facto study was developed in which the impact of an online training program was quantified by establishing comparative pre- and post-training. The results indicate that the approach, through the delivery of a training xplain eon Design for All, contributed to a change in the perceptions of students regarding disability, its role in the university and in the future workplace. Furthermore, it increased the knowledge of institutional action undertaken in terms of awareness and approach to human disability.
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COVID-19 , Diseño Universal , Humanos , SARS-CoV-2 , Estudiantes , UniversidadesRESUMEN
BACKGROUND: Discoveries in the identification of transcription factors, growth factors and extracellular signaling molecules have led to the detection of downstream targets that modulate valvular tissue organization that occurs during development, aging, or disease. Among these, matricellular protein, periostin, and cytoskeletal protein filamin A are highly expressed in developing heart valves. The phenotype of periostin null indicates that periostin promotes migration, survival, and differentiation of valve interstitial cushion cells into fibroblastic lineages necessary for postnatal valve remodeling/maturation. Genetically inhibiting filamin A expression in valve interstitial cushion cells mirrored the phenotype of periostin nulls, suggesting a molecular interaction between these two proteins resulted in poorly remodeled valve leaflets that might be prone to myxomatous over time. We examined whether filamin A has a cross-talk with periostin/signaling that promotes remodeling of postnatal heart valves into mature leaflets. RESULTS: We have previously shown that periostin/integrin-ß1 regulates Pak1 activation; here, we revealed that the strong interaction between Pak1 and filamin A proteins was only observed after stimulation of VICs with periostin; suggesting that periostin/integrin-ß-mediated interaction between FLNA and Pak1 may have a functional role in vivo. We found that FLNA phosphorylation (S2152) is activated by Pak1, and this interaction was observed after stimulation with periostin/integrin-ß1/Cdc42/Rac1 signaling; consequently, FLNA binding to Pak1 stimulates its kinase activity. Patients with floppy and/or prolapsed mitral valves, when genetically screened, were found to have point mutations in the filamin A gene at P637Q and G288R. Expression of either of these filamin A mutants failed to increase the magnitude of filamin A (S2152) expression, Pak1-kinase activity, actin polymerization, and differentiation of VICs into mature mitral valve leaflets in response to periostin signaling. CONCLUSION: PN-stimulated bidirectional interaction between activated FLNA and Pak1 is essential for actin cytoskeletal reorganization and the differentiation of immature VICs into mature valve leaflets.
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Here we show the temporal-spatial orchestration of early heart morphogenesis at cellular level resolution, in vivo, and reconcile conflicting positional fate mapping data regarding the primary heart-forming field(s). We determined the positional fates of precardiac cells using a precision electroporation approach in combination with wide-field time-lapse microscopy in the quail embryo, a warm-blooded vertebrate (HH Stages 4 through 10). Contrary to previous studies, the results demonstrate the existence of a "continuous" circle-shaped heart field that spans the midline, appearing at HH Stage 4, which then expands to form a wide arc of progenitors at HH Stages 5-7. Our time-resolved image data show that a subset of these cardiac progenitor cells do not overlap with the expression of common cardiogenic factors, Nkx-2.5 and Bmp-2, until HH Stage 10, when a tubular heart has formed, calling into question when cardiac fate is specified and by which key factors. Sub-groups and anatomical bands (cohorts) of heart precursor cells dramatically change their relative positions in a process largely driven by endodermal folding and other large-scale tissue deformations. Thus, our novel dynamic positional fate maps resolve the origin of cardiac progenitor cells in amniotes. The data also establish the concept that tissue motion contributes significantly to cellular position fate - i.e., much of the cellular displacement that occurs during assembly of a midline heart tube (HH Stage 9) is NOT due to "migration" (autonomous motility), a commonly held belief. Computational analysis of our time-resolved data lays the foundation for more precise analyses of how cardiac gene regulatory networks correlate with early heart tissue morphogenesis in birds and mammals.
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Proteínas Aviares/metabolismo , Embrión de Pollo/anatomía & histología , Coturnix/embriología , Corazón/embriología , Morfogénesis/fisiología , Animales , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Embrión de Pollo/fisiología , Coturnix/anatomía & histología , Edad Gestacional , Corazón/anatomía & histología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Hibridación in Situ , Células Madre/citología , Células Madre/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The secreted periostin protein, which marks mesenchymal cells in endocardial cushions following epithelial-mesenchymal transformation and in mature valves following remodeling, is a putative valvulogenesis target molecule. Indeed, periostin is expressed throughout cardiovascular morphogenesis and in all 4 adult mice valves (annulus and leaflets). Additionally, periostin is expressed throughout the fibrous cardiac skeleton and endocardial cushions in the developing heart but is absent from both normal and/or pathological mouse cardiomyocytes. Periostin (peri(lacZ)) knockout mice exhibit viable valve disease, with neonatal lethality in a minority and latent disease with leaflet abnormalities in the viable majority. Surviving peri(lacZ)-null leaflets are truncated, contain ectopic cardiomyocytes and smooth muscle, misexpress the cartilage proteoglycan aggrecan, demonstrate disorganized matrix stratification, and exhibit reduced transforming growth factor-beta signaling. Neonatal peri(lacZ) nulls that die (14%) display additional defects, including leaflet discontinuities, delamination defects, and deposition of acellular extracellular matrix. Assessment of collagen production, 3D lattice formation ability, and transforming growth factor-beta responsiveness indicate periostin-deficient fibroblasts are unable to support normal valvular remodeling and establishment of a mature cardiac skeleton. Furthermore, pediatric stenotic bicuspid aortic valves that have lost normal extracellular matrix trilaminar stratification have greatly reduced periostin. This suggests that loss of periostin results in inappropriate differentiation of mesenchymal cushion cells and valvular abnormalities via a transforming growth factor-beta-dependent pathway during establishment of the mature heart. Thus, peri(lacZ) knockouts provide a new model of viable latent valve disease.
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Moléculas de Adhesión Celular/metabolismo , Matriz Extracelular/metabolismo , Mesodermo/metabolismo , Mesodermo/patología , Miocardio/metabolismo , Miocardio/patología , Animales , Diferenciación Celular/fisiología , Proliferación Celular , Citoesqueleto/metabolismo , Citoesqueleto/patología , Modelos Animales de Enfermedad , Endocardio/metabolismo , Endocardio/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Enfermedades de las Válvulas Cardíacas/metabolismo , Enfermedades de las Válvulas Cardíacas/patología , Ratones , Ratones Noqueados , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Cardiac valve leaflets develop from rudimentary structures termed endocardial cushions. These pre-valve tissues arise from a complex interplay of signals between the myocardium and endocardium whereby secreted cues induce the endothelial cells to transform into migratory mesenchyme through an endothelial to mesenchymal transformation (EMT). Even though much is currently known regarding the initial EMT process, the mechanisms by which these undifferentiated cushion mesenchymal tissues are remodeled "post-EMT" into mature fibrous valve leaflets remains one of the major, unsolved questions in heart development. Expression analyses, presented in this report, demonstrate that periostin, a component of the extracellular matrix, is predominantly expressed in post-EMT valve tissues and their supporting apparatus from embryonic to adult life. Analyses of periostin gene targeted mice demonstrate that it is within these regions that significant defects are observed. Periostin null mice exhibit atrial septal defects, structural abnormalities of the AV valves and their supporting tensile apparatus, and aberrant differentiation of AV cushion mesenchyme. Rescue experiments further demonstrate that periostin functions as a hierarchical molecular switch that can promote the differentiation of mesenchymal cells into a fibroblastic lineage while repressing their transformation into other mesodermal cell lineages (e.g. myocytes). This is the first report of an extracellular matrix protein directly regulating post-EMT AV valve differentiation, a process foundational and indispensable for the morphogenesis of a cushion into a leaflet.
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Nodo Atrioventricular/embriología , Moléculas de Adhesión Celular/genética , Regulación del Desarrollo de la Expresión Génica , Válvulas Cardíacas/embriología , Corazón/embriología , Corazón/fisiología , Animales , Nodo Atrioventricular/ultraestructura , Moléculas de Adhesión Celular/deficiencia , Desarrollo Embrionario , Válvulas Cardíacas/ultraestructura , Ratones , Ratones Noqueados , Microscopía de Fuerza AtómicaRESUMEN
The Postn gene encodes protein periostin. During embryonic development, it is highly expressed in the outflow tract (OFT) endocardial cushions of the developing heart, which give rise to several structures of the mature heart including the aortic valve. Periostin was previously implicated in osteoblast differentiation, cancer metastasis, and tooth and bone development, but its role in cardiac OFT development is unclear. To elucidate the role that periostin plays in the developing heart we analyzed cardiac OFT phenotype in mice after deletion of the Postn gene. We found that lack of periostin in the embryonic OFT leads to ectopic expression of the proosteogenic growth factor pleiotrophin (Ptn) and overexpression of delta-like 1 homolog (Dlk1), a negative regulator of Notch1, in the distal (prevalvular) cushions of the OFT. This resulted in suppression of Notch1 signaling, strong induction of the central transcriptional regulator of osteoblast cell fate Runx2, upregulation of osteopontin and osteocalcin expression, and subsequent calcification of the aortic valve. Our data suggest that periostin represses a default osteogenic program in the OFT cushion mesenchyme and promotes differentiation along a fibrogenic lineage. Lack of periostin causes derepression of the osteogenic potential of OFT mesenchymal cells, calcium deposition, and calcific aortic valve disease. These results establish periostin as a key regulator of OFT endocardial cushion mesenchymal cell fate during embryonic development.
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Moléculas de Adhesión Celular/genética , Enfermedades de las Válvulas Cardíacas/genética , Receptor Notch1/genética , Transducción de Señal/genética , Animales , Válvula Aórtica/metabolismo , Válvula Aórtica/patología , Calcinosis , Proteínas de Unión al Calcio , Proteínas Portadoras/genética , Moléculas de Adhesión Celular/fisiología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Citocinas/genética , Ecocardiografía , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Enfermedades de las Válvulas Cardíacas/fisiopatología , Hibridación Fluorescente in Situ , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/metabolismo , Miocardio/patología , Osteopontina/genética , Receptor Notch1/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiología , Factores de TiempoRESUMEN
Cell migration, which is central to a wide variety of life processes, involves integration of the extracellular matrix (ECM) with the internal cytoskeleton and motor proteins via receptors spanning the plasma membrane. Cell migration can be induced by a variety of signals, including gradients of external soluble molecules, differences in ECM composition, or electrical gradients. Current in vitro methods to study cell migration only test one substrate at a time. Here, we present a method for assessing cell adhesion, migration, and differentiation in up to 20 different test conditions simultaneously, using only minute amounts of target substrate. Our system, which we call the linear array of multi-substrate cell migration assay (LAMA), has two configurations for direct comparison of one or two cell types in response to an array of ECM constituents under the same culture conditions. This culture model utilizes only nanogram amounts of test substrates and a minimal number of cells, which maximizes the use of limited and expensive test reagents. Moreover, LAMA can also be used for high-throughput screening of potential pharmaceuticals that target ECM-dependent cell behavior and differentiation.
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Fenómenos Fisiológicos Celulares/fisiología , Técnicas Citológicas/métodos , Matriz Extracelular/metabolismo , Animales , Línea Celular , Embrión de Pollo , Células Madre Embrionarias/citología , Matriz Extracelular/química , Células PC12 , RatasRESUMEN
This paper presents the Nursing Motives for Helping Scale (N-MHS), an instrument designed for the evaluation of three of the four motives for helping derived from Batson's helping pathway theory. Dimensionality was analyzed by means of principal component analysis (n = 113), followed by confirmatory factor analysis. A 3-factor structure (corresponding to Batson's differentiation among altruistic motivation, reward-seeking motivation, and punishment-avoidance motivation, respectively), with 9 items distributed in three latent variables, revealed an acceptable fit to the data. Alpha values (.60 - .74) showed that internal consistency was acceptable for a newly developed subscale with a small number of items. Convergence validity was evaluated with correlations between N-MHS subscales scores and scores on the Professional Expectations Scale (Garrosa, Moreno-Jiménez, Rodríguez-Carvajal, and Morante, 2005). The three resulting subscales are a promising instrument for the evaluation of three nursing motives for helping that can contribute to reduce the potential risks and to improve the potential benefits both for the nurse and the patient.
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Conducta de Ayuda , Motivación , Enfermeras y Enfermeros/psicología , Encuestas y Cuestionarios , Adulto , Análisis Factorial , Femenino , Humanos , Masculino , Psicometría , Reproducibilidad de los Resultados , EspañaRESUMEN
The outcome of patients with cancer has improved significantly in the past decade with the incorporation of drugs targeting cell surface adhesive receptors, receptor tyrosine kinases, and modulation of several molecules of extracellular matrices (ECMs), the complex composite of collagens, glycoproteins, proteoglycans, and glycosaminoglycans that dictates tissue architecture. Cancer tissue invasive processes progress by various oncogenic strategies, including interfering with ECM molecules and their interactions with invasive cells. In this review, we describe how the ECM components, proteoglycans and glycosaminoglycans, influence tumor cell signaling. In particular this review describes how the glycosaminoglycan hyaluronan (HA) and its major receptor CD44 impact invasive behavior of tumor cells, and provides useful insight when designing new therapeutic strategies in the treatment of cancer.
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A wound is a type of injury that damages living tissues. In this review, we will be referring mainly to healing responses in the organs including skin and the lungs. Fibrosis is a process of dysregulated extracellular matrix (ECM) production that leads to a dense and functionally abnormal connective tissue compartment (dermis). In tissues such as the skin, the repair of the dermis after wounding requires not only the fibroblasts that produce the ECM molecules, but also the overlying epithelial layer (keratinocytes), the endothelial cells, and smooth muscle cells of the blood vessel and white blood cells such as neutrophils and macrophages, which together orchestrate the cytokine-mediated signaling and paracrine interactions that are required to regulate the proper extent and timing of the repair process. This review will focus on the importance of extracellular molecules in the microenvironment, primarily the proteoglycans and glycosaminoglycan hyaluronan, and their roles in wound healing. First, we will briefly summarize the physiological, cellular, and biochemical elements of wound healing, including the importance of cytokine cross-talk between cell types. Second, we will discuss the role of proteoglycans and hyaluronan in regulating these processes. Finally, approaches that utilize these concepts as potential therapies for fibrosis are discussed.
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In this chapter, we review the working hypothesis that the roots of adult valvular heart disease (VHD) lie in embryonic development. Valvulogenesis is a complex process in which growth factors signal the process of endocardium-to-mesenchyme transformation (EMT) resulting in formation of prevalvular "cushions." The post-EMT processes, whereby cushions are morphogenetically remolded into valve leaflets, are less well understood, but they require periostin. Mice with targeted deletion of periostin develop degenerative changes similar to human forms of VHD. Mitral valves are also abnormally elongated in hypertrophic cardiomyopathy (HCM), which plays an important role in clinical disease expression. However, the mechanism for this is unclear, but correlates with enhanced expression of periostin in a specific population of ventricular cells derived from the embryonic proepicardial organ, which accumulate at sites where valvular endocardial EMT is reactivated. Collectively, these findings suggest that developmental mechanisms underlie adult valve responses to genetic mutations in degenerative VHD and HCM.
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Envejecimiento , Enfermedades de las Válvulas Cardíacas/embriología , Animales , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/metabolismo , Diferenciación Celular , Enfermedades de las Válvulas Cardíacas/metabolismo , Enfermedades de las Válvulas Cardíacas/patología , Humanos , Modelos Cardiovasculares , MutaciónRESUMEN
An epithelial-mesenchymal transformation (EMT) involves alterations in cell-cell and cell-matrix adhesion, the detachment of epithelial cells from their neighbors, the degradation of the basal lamina and acquisition of mesenchymal phenotype. Here we present Monte Carlo simulations for a specific EMT in early heart development: the formation of cardiac cushions. Cell rearrangements are described in accordance with Steinberg's differential adhesion hypothesis, which states that cells possess a type-dependent adhesion apparatus and are sufficiently motile to give rise to the tissue conformation with the largest number of strong bonds. We also implement epithelial and mesenchymal cell proliferation, cell type change and extracellular matrix production by mesenchymal cells. Our results show that an EMT is promoted more efficiently by an increase in cell-substrate adhesion than by a decrease in cell-cell adhesion. In addition to cushion tissue formation, the model also accounts for the phenomena of matrix invasion and mesenchymal condensation. We conclude that in order to maintain epithelial integrity during EMT the number of epithelial cells must increase at a controlled rate. Our model predictions are in qualitative agreement with available experimental data.
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Simulación por Computador , Células Epiteliales/citología , Mesodermo/citología , Corazón/embriología , Método de MontecarloRESUMEN
Utilization of MALDI-MS (matrix-assisted laser desorption/ionization mass spectrometry) for tissue imaging is a relatively new proteomic technique that simultaneously maps the spatial distribution of multiple proteins directly within a single frozen tissue section. Here, we report the development of a methodology to apply MALDI tissue imaging to chick heart tissue sections acquired from fixed and paraffin-embedded samples. This protocol produces molecular images that can be related to the high-quality histological tissue sections. Perfused term chick hearts were fixed in acidic ethanol and embedded in paraffin wax. Tissue sections (15 microm) were collected onto conductive slides, deparaffinized with xylene, and transitioned into water with graded ethanol washes and allowed to air dry. In separate experiments, three different MALDI matrices were applied to chick heart tissue sections through repeated cycles from a glass nebulizer. Tissue sections were then analyzed by MALDI mass spectrometry using a raster step-size of 75-100 microm, and molecular images for specific m/z ratios reconstituted. MALDI tissue imaging revealed spatially resolved protein signals within single heart sections that are specific to structures or regions of the heart, for example, vessels, valves, endocardium, myocardium, or septa. Moreover, no prior knowledge of protein expression is required as is the case for immunohistochemistry and in situ hybridization methodologies. The ability to simultaneously localize a large number of unique protein signals within a single tissue section, with good preservation of histological features, provides cardiovascular researchers a new tool to give insight into the molecular mechanisms underlying normal and pathological conditions.
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Corazón/crecimiento & desarrollo , Procesamiento de Imagen Asistido por Computador/métodos , Proteínas/metabolismo , Proteómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Bioquímica/métodos , Biomarcadores/metabolismo , Pollos/anatomía & histología , Pollos/crecimiento & desarrollo , Pollos/metabolismo , Vasos Coronarios/anatomía & histología , Vasos Coronarios/crecimiento & desarrollo , Vasos Coronarios/metabolismo , Endocardio/anatomía & histología , Endocardio/crecimiento & desarrollo , Endocardio/metabolismo , Corazón/anatomía & histología , Tabiques Cardíacos/anatomía & histología , Tabiques Cardíacos/crecimiento & desarrollo , Tabiques Cardíacos/metabolismo , Válvulas Cardíacas/anatomía & histología , Válvulas Cardíacas/crecimiento & desarrollo , Válvulas Cardíacas/metabolismo , Procesamiento de Imagen Asistido por Computador/instrumentación , Miocardio/citología , Miocardio/metabolismo , Adhesión en Parafina/métodos , Proteínas/análisis , Proteómica/instrumentación , Especificidad de la Especie , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/instrumentación , Fijación del Tejido/métodosRESUMEN
Periostin is a member of a growing family of matricellular proteins, defined by their ability to interact with components of the extracellular milieu, and with receptors at the cell surface. Through these interactions, periostin has been shown to play a crucial role as a profibrogenic molecule during tissue morphogenesis. Tissues destined to become fibrous structures are dependent on cooperative interactions between periostin and its binding partners, whereas in its absence, these structures either totally or partially fail to become mature fibrous entities. Within the heart, fibrogenic differentiation is required for normal tissue maturation, remodeling and function, as well as in response to a pathological myocardial insult. In this review, aspects related to the function of periostin during cardiac morphogenesis, remodeling and pathology are summarized.
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Periostin is predominantly expressed in collagen-rich fibrous connective tissues that are subjected to constant mechanical stresses including: heart valves, tendons, perichondrium, cornea, and the periodontal ligament (PDL). Based on these data we hypothesize that periostin can regulate collagen I fibrillogenesis and thereby affect the biomechanical properties of connective tissues. Immunoprecipitation and immunogold transmission electron microscopy experiments demonstrate that periostin is capable of directly interacting with collagen I. To analyze the potential role of periostin in collagen I fibrillogenesis, gene targeted mice were generated. Transmission electron microscopy and morphometric analyses demonstrated reduced collagen fibril diameters in skin dermis of periostin knockout mice, an indication of aberrant collagen I fibrillogenesis. In addition, differential scanning calorimetry (DSC) demonstrated a lower collagen denaturing temperature in periostin knockout mice, reflecting a reduced level of collagen cross-linking. Functional biomechanical properties of periostin null skin specimens and atrioventricular (AV) valve explant experiments provided direct evidence of the role that periostin plays in regulating the viscoelastic properties of connective tissues. Collectively, these data demonstrate for the first time that periostin can regulate collagen I fibrillogenesis and thereby serves as an important mediator of the biomechanical properties of fibrous connective tissues.