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With the ever-increasing quality and quantity of imaging data in biomedical research comes the demand for computational methodologies that enable efficient and reliable automated extraction of the quantitative information contained within these images. One of the challenges in providing such methodology is the need for tailoring algorithms to the specifics of the data, limiting their areas of application. Here we present a broadly applicable approach to quantification and classification of complex shapes and patterns in biological or other multi-component formations. This approach integrates the mapping of all shape boundaries within an image onto a global information-rich graph and machine learning on the multidimensional measures of the graph. We demonstrated the power of this method by (1) extracting subtle structural differences from visually indistinguishable images in our phenotype rescue experiments using the endothelial tube formations assay, (2) training the algorithm to identify biophysical parameters underlying the formation of different multicellular networks in our simulation model of collective cell behavior, and (3) analyzing the response of U2OS cell cultures to a broad array of small molecule perturbations.
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Biología Computacional/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Aprendizaje Automático , Reconocimiento de Normas Patrones Automatizadas/métodos , Algoritmos , Línea Celular Tumoral , Técnicas Citológicas , Árboles de Decisión , Técnicas de Silenciamiento del Gen , Células Endoteliales de la Vena Umbilical Humana , HumanosRESUMEN
von Willebrand factor (vWF) secretion by endothelial cells (ECs) is essential for hemostasis and thrombosis; however, the molecular mechanisms are poorly understood. Interestingly, we observed increased bleeding in EC-Gα13(-/-);Gα12(-/-) mice that could be normalized by infusion of human vWF. Blood from Gα12(-/-) mice exhibited significantly reduced vWF levels but normal vWF multimers and impaired laser-induced thrombus formation, indicating that Gα12 plays a prominent role in EC vWF secretion required for hemostasis and thrombosis. In isolated buffer-perfused mouse lungs, basal vWF levels were significantly reduced in Gα12(-/-), whereas thrombin-induced vWF secretion was defective in both EC-Gαq(-/-);Gα11(-/-) and Gα12(-/-) mice. Using siRNA in cultured human umbilical vein ECs and human pulmonary artery ECs, depletion of Gα12 and soluble N-ethylmaleimide-sensitive-fusion factor attachment protein α (α-SNAP), but not Gα13, inhibited both basal and thrombin-induced vWF secretion, whereas overexpression of activated Gα12 promoted vWF secretion. In Gαq, p115 RhoGEF, and RhoA-depleted human umbilical vein ECs, thrombin-induced vWF secretion was reduced by 40%, whereas basal secretion was unchanged. Finally, in vitro binding assays revealed that Gα12 N-terminal residues 10-15 mediated the binding of Gα12 to α-SNAP, and an engineered α-SNAP binding-domain minigene peptide blocked basal and evoked vWF secretion. Discovery of obligatory and complementary roles of Gα12 and Gαq/11 in basal vs evoked EC vWF secretion may provide promising new therapeutic strategies for treatment of thrombotic disease.
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Células Endoteliales/citología , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Factor de von Willebrand/metabolismo , Animales , Anticuerpos Monoclonales/química , Regulación de la Expresión Génica , Hemostasis , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Noqueados , Adhesividad Plaquetaria , Unión Proteica , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/química , TrombosisRESUMEN
The primary function of the mammalian lung is to facilitate diffusion of oxygen to venous blood and to ventilate carbon dioxide produced by catabolic reactions within cells. However, it is also responsible for a variety of other important functions, including host defense and production of vasoactive agents to regulate not only systemic blood pressure, but also water, electrolyte and acid-base balance. Caveolin-1 is highly expressed in the majority of cell types in the lung, including epithelial, endothelial, smooth muscle, connective tissue cells, and alveolar macrophages. Deletion of caveolin-1 in these cells results in major functional aberrations, suggesting that caveolin-1 may be crucial to lung homeostasis and development. Furthermore, generation of mutant mice that under-express caveolin-1 results in severe functional distortion with phenotypes covering practically the entire spectrum of known lung diseases, including pulmonary hypertension, fibrosis, increased endothelial permeability, and immune defects. In this Chapter, we outline the current state of knowledge regarding caveolin-1-dependent regulation of pulmonary cell functions and discuss recent research findings on the role of caveolin-1 in various pulmonary disease states, including obstructive and fibrotic pulmonary vascular and inflammatory diseases.
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Caveolinas , Pulmón/fisiología , Animales , Caveolinas/metabolismo , Endotelio/citología , Endotelio/metabolismo , Endotelio/patología , Humanos , Pulmón/crecimiento & desarrollo , Pulmón/metabolismo , Pulmón/fisiopatología , Enfermedades Pulmonares/metabolismo , Enfermedades Pulmonares/patología , Enfermedades Pulmonares/fisiopatología , Alveolos Pulmonares/citología , Alveolos Pulmonares/patología , Alveolos Pulmonares/fisiología , Alveolos Pulmonares/fisiopatologíaRESUMEN
Regulation of gene transcription in vascular smooth muscle cells (VSMCs) by serum response factor (SRF) plays a crucial role in vascular development and in the pathophysiology of vascular diseases. Nevertheless, the regulation of specific genes by SRF in vascular diseases is poorly understood. Therefore, we investigated the regulation of smooth muscle myosin light chain kinase (smMLCK) by using spontaneously hypertensive rats (SHR) as an experimental model. We found that smMLCK expression in blood vessels increases during the development of hypertension and is always greater in blood vessels from SHR compared with normotensive rats. Analysis of the DNA sequences of the promoters isolated from SHR and normotensive rats revealed that SHR contain a 12-base pair insertion adjacent to the CArG box. This insertion increases SRF binding to the CArG box and positively regulates SRF-dependent promoter activity. The increase in smMLCK expression was blocked by dominant-negative SRF, dominant-negative Ras, or antisense oligonucleotides to ERK. In vivo, inhibiting MEK decreased smMLCK expression and blood pressure in SHR partly by decreasing SRF binding to the smMLCK promoter. These data provide novel insight into the regulation of smMLCK expression at the molecular level and demonstrate the importance of SRF in regulating smMLCK promoter activity in SHR.
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Regulación Enzimológica de la Expresión Génica , Hipertensión/enzimología , Mutagénesis Insercional , Quinasa de Cadena Ligera de Miosina/genética , Quinasa de Cadena Ligera de Miosina/metabolismo , Regiones Promotoras Genéticas/genética , Factor de Respuesta Sérica/metabolismo , Animales , Secuencia de Bases , Presión Sanguínea/fisiología , Células COS , Células Cultivadas , Chlorocebus aethiops , Hipertensión/fisiopatología , Intrones/genética , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Datos de Secuencia Molecular , Músculo Liso Vascular/citología , Músculo Liso Vascular/enzimología , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Ratas , Ratas Endogámicas SHR , Transducción de SeñalRESUMEN
At early stages of organismal development, endothelial cells self-organize into complex networks subsequently giving rise to mature blood vessels. The compromised collective behavior of endothelial cells leads to the development of a number of vascular diseases, many of which can be life-threatening. Cerebral cavernous malformation is an example of vascular diseases caused by abnormal development of blood vessels in the brain. Despite numerous efforts to date, enlarged blood vessels (cavernomas) can be effectively treated only by risky and complex brain surgery. In this work, we use a comprehensive simulation model to dissect the mechanisms contributing to an emergent behavior of the multicellular system. By tightly integrating computational and experimental approaches we gain a systems-level understanding of the basic mechanisms of vascular tubule formation, its destabilization, and pharmacological rescue, which may facilitate the development of new strategies for manipulating collective endothelial cell behavior in the disease context.
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The existence and identity of multipotent stem cells in the adult lung is currently highly debated. At present, it remains unclear whether candidate stem/progenitor cells are located in the airways, alveoli, or throughout the epithelial lining of the lung. Here, we introduce a method of airway microdissection, which enabled us to study the progenitor behavior of pulmonary epithelial cells in region-specific contexts. The progenitor characteristics of epithelial cells isolated from the trachea, proximal and distal airways, and lung parenchyme were evaluated in vitro and in vivo. We identified a population of airway-derived basal-like epithelial cells with the potential to self-renew and differentiate into airway and alveolar lineages in culture and in vivo after subcutaneous transplantation. The multipotent candidate progenitors originated from a minor fraction of the airway epithelial cell population characterized by high expression of α6 integrin. Results of the current study provide new insights into the regenerative potential of region-specific integrin α6-positive pulmonary epithelial cells.
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Células Madre Adultas/fisiología , Células Epiteliales/fisiología , Alveolos Pulmonares/fisiología , Células Madre Adultas/metabolismo , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Células Epiteliales/metabolismo , Integrina alfa6/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Madre Multipotentes/metabolismo , Células Madre Multipotentes/fisiología , Alveolos Pulmonares/metabolismo , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/fisiologíaRESUMEN
Nitrate tolerance developed after persistent nitroglycerin (GTN) exposure limits its clinical utility. Previously, we have shown that the vasodilatory action of GTN is dependent on endothelial nitric oxide synthase (eNOS/NOS3) activity. Caveolin-1 (Cav-1) is known to interact with NOS3 on the cytoplasmic side of cholesterol-enriched plasma membrane microdomains (caveolae) and to inhibit NOS3 activity. Loss of Cav-1 expression results in NOS3 hyperactivation and uncoupling, converting NOS3 into a source of superoxide radicals, peroxynitrite, and oxidative stress. Therefore, we hypothesized that nitrate tolerance induced by persistent GTN treatment results from NOS3 dysfunction and vascular toxicity. Exposure to GTN for 48-72 h resulted in nitrosation and depletion (>50%) of Cav-1, NOS3 uncoupling as measured by an increase in peroxynitrite production (>100%), and endothelial toxicity in cultured cells. In the Cav-1 deficient mice, NOS3 dysfunction was accompanied by GTN tolerance (>50% dilation inhibition at low GTN concentrations). In conclusion, GTN tolerance results from Cav-1 modification and depletion by GTN that causes persistent NOS3 activation and uncoupling, preventing it from participating in GTN-medicated vasodilation.
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Caveolina 1/genética , Tolerancia a Medicamentos , Nitroglicerina/farmacología , Vasodilatadores/farmacología , Animales , Caveolina 1/metabolismo , Ratones , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo III/metabolismo , Estrés Oxidativo/efectos de los fármacos , Fosfohidrolasa PTEN/metabolismo , Proteolisis/efectos de los fármacosRESUMEN
In the present study, we tested the hypothesis that chronic inflammation and oxidative/nitrosative stress induce caveolin 1 (Cav-1) degradation, providing an underlying mechanism of endothelial cell activation/dysfunction and pulmonary vascular remodeling in patients with idiopathic pulmonary arterial hypertension (IPAH). We observed reduced Cav-1 protein despite increased Cav-1 messenger RNA expression and also endothelial nitric oxide synthase (eNOS) hyperphosphorylation in human pulmonary artery endothelial cells (PAECs) from patients with IPAH. In control human lung endothelial cell cultures, tumor necrosis factor α-induced nitric oxide (NO) production and S-nitrosation (SNO) of Cav-1 Cys-156 were associated with Src displacement and activation, Cav-1 Tyr-14 phosphorylation, and destabilization of Cav-1 oligomers within 5 minutes that could be blocked by eNOS or Src inhibition. Prolonged stimulation (72 hours) with NO donor DETANONOate reduced oligomerized and total Cav-1 levels by 40%-80%, similar to that observed in IPAH patient-derived PAECs. NO donor stimulation of endothelial cells for >72 hours, which was associated with sustained Src activation and Cav-1 phosphorylation, ubiquitination, and degradation, was blocked by NOS inhibitor L-NAME, Src inhibitor PP2, and proteosomal inhibitor MG132. Thus, chronic inflammation, sustained eNOS and Src signaling, and Cav-1 degradation may be important causal factors in the development of IPAH by promoting PAEC dysfunction/activation via sustained oxidative/nitrosative stress.
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Many tumors are stiffer than their surrounding tissue. This increase in stiffness has been attributed, in part, to a Rho-dependent elevation of myosin II light chain phosphorylation. To characterize this mechanism further, we studied myosin light chain kinase (MLCK), the main enzyme that phosphorylates myosin II light chains. We anticipated that increases in MLCK expression and activity would contribute to the increased stiffness of cancer cells. However, we find that MLCK mRNA and protein levels are substantially less in cancer cells and tissues than in normal cells. Consistent with this observation, cancer cells contract 3D collagen matrices much more slowly than normal cells. Interestingly, inhibiting MLCK or Rho kinase did not affect the 3D gel contractions while blebbistatin partially and cytochalasin D maximally inhibited contractions. Live cell imaging of cells in collagen gels showed that cytochalasin D inhibited filopodia-like projections that formed between cells while a MLCK inhibitor had no effect on these projections. These data suggest that myosin II phosphorylation is dispensable in regulating the mechanical properties of tumors.