RESUMEN
Mesenchymal stromal cells from rat adipose tissue were transduced with adeno-associated viral (AAV) vector encoding stem cell factor SCF that stimulates proliferation of cardiac c-kit+ cells and improved cardiac function and survival of animals after myocardial infarction. Extracellular vesicles isolated from the medium conditioned by mesenchymal stromal cells by ultracentrifugation were characterized by Western blotting, transmission electron microscopy, nanoparticle tracking analysis, immunostaining, and mass spectrometry analysis. Using proteomic analysis, we identified transgenic SCF in extracellular vesicles released by AAV-modified mesenchymal stromal cells and detected some proteins specific of extracellular vesicles secreted by transduced cells. Extracellular vesicles from AAV-transduced mesenchymal stromal cells could be used for delivery of transgenic proteins as they were readily endocytosed by both cardiosphere-derived cells and cardiac-progenitor cells.
Asunto(s)
Dependovirus/genética , Vesículas Extracelulares/metabolismo , Células Madre Mesenquimatosas/metabolismo , Factor de Células Madre/metabolismo , Tejido Adiposo/citología , Animales , Células Cultivadas , Espectrometría de Masas , Proteómica/métodos , RatasRESUMEN
We showed the possibility of generating combined tissue-engineered cell consisting of layers of rat cardiac stem cells and mesenchymal stromal cells from the adipose tissue. Cell-cell interaction within the cell sheet promoted proliferation of cardiac stem cells, expression of endothelial differentiation marker ETS1, and Notch signaling activation. The obtained results provide new insights into possible mechanisms of stimulation of endogenous regeneration processes after myocardial damage and demonstrate potential of cell-based cardiomyoplasty with the use of these combined cell sheets.
Asunto(s)
Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Miocitos Cardíacos/citología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/fisiología , Proliferación Celular/genética , Proliferación Celular/fisiología , Células Cultivadas , Infarto del Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Ratas , Proteínas Represoras/metabolismo , Transducción de Señal/fisiología , Ingeniería de Tejidos , Factor de Transcripción HES-1/metabolismoRESUMEN
We studied the effect of SIRT1 deacetylase and PPARγ receptor activators on proinflammatory (M1), anti-inflammatory (M2) polarization of RAW264.7 macrophages and their modulating effects on insulin sensitivity of adipocytes. In M1 macrophages, the expression of TNFα and CXCL9, secretion of CXCL11, ROS generation, and content of dendritic-like cells were elevated. In M2 macrophages, expression of IGF-1 and ALOX15 factors was enhanced. SIRT1 activator (DCHC) and PPARγ receptor ligand (rosiglitazone) reduced expression of inflammatory markers TNFα and CXCL9 and increased expression of IGF-1 and ALOX15. SIRT1 inhibitor Ex527 increased the proportion of dendritic cells in macrophage populations. The paracrine effect of M1-macrophage-conditioned media attenuated insulin-dependent phosphorylation of threonine (Thr308) in Akt kinase and enhanced phosphorylation of serine (Ser473). This effect was attenuated by DCHC and rosiglitazone.
Asunto(s)
Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Inflamación/metabolismo , Insulina/farmacología , Macrófagos/metabolismo , Animales , Araquidonato 15-Lipooxigenasa/metabolismo , Carbazoles/farmacología , Quimiocina CXCL9/metabolismo , Células Dendríticas/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , PPAR gamma/metabolismo , Células RAW 264.7 , Rosiglitazona , Tiazolidinedionas/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We studied the effect of urokinase, its recombinant forms, and domain fragments on migration and proliferation of adipose tissue mesenchymal stromal cells (MSCs) and MMP secretion by these cells. Urokinase, but not its recombinant forms, slightly induced directed migration of MSCs. Spontaneous migration of MSCs increased under the action of urokinase or its isolated kringle domain. Migration induced by platelet-derived growth factor was inhibited by proteolytically inactive form of urokinase, the kringle domain, and blocking antibody to urokinase receptor. Urokinase, its proteolytically inactive form, and kringle domain produced no effect on MSC proliferation. In contrast to platelet-derived growth factor, all urokinase forms induced secretion of MMP-9 by MSCs.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Células Madre Mesenquimatosas/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/farmacología , Tejido Adiposo/citología , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Proliferación Celular/efectos de los fármacos , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Humanos , Isoenzimas/farmacología , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Cultivo Primario de Células , Dominios Proteicos , Proteínas Recombinantes/farmacología , Transducción de SeñalRESUMEN
In cultured fibroblasts, urokinase stimulated expression of MMP-9 and generation of ROS, while antioxidant ebselen abolished the stimulating effect of urokinase on MMP-9 expression. sTNF-α produced similar and more pronounced stimulating effect. The data showed that urokinase could regulate MMP-9 expression via ROS generation in fibroblasts, which can play an important role in stimulation of their migration and development of constrictor (negative) vascular remodeling due to thickening of the adventitia.
Asunto(s)
Fibroblastos/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/biosíntesis , Especies Reactivas de Oxígeno/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/farmacología , Animales , Antioxidantes/farmacología , Azoles/farmacología , Fibroblastos/citología , Fibroblastos/metabolismo , Expresión Génica/efectos de los fármacos , Isoindoles , Metaloproteinasa 9 de la Matriz/genética , Ratones , Células 3T3 NIH , Compuestos de Organoselenio/farmacología , Especies Reactivas de Oxígeno/agonistas , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Proteolytically inactive recombinant forms of urokinase (uPAHQ and amino-terminal fragment) inhibit spontaneous migration of endothelial cells; amino-terminal fragment also suppresses angiogenesis stimulated by basic fibroblast growth factor in vitro. These findings suggest the possibility of using synthesized proteolytically inactive recombinant forms of urokinase for the regulation of endothelial cell migration and suppression of neoangiogenesis.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Células Endoteliales/metabolismo , Neovascularización Patológica/prevención & control , Proteínas Recombinantes/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/farmacología , Células Cultivadas , Endotelio Vascular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , HumanosRESUMEN
Urokinase stimulates the production of superoxide radical in cultured aortal smooth muscle cells simultaneously with activation of the expression of NAD(F)H-oxidases nox1, nox4, and phox22. Antioxidant ebselen abolishes the stimulating effect of urokinase on smooth muscle cell proliferation. The data showed that urokinase can potentiate oxidative stress in the arterial wall and can play an important role in the development of adverse arterial remodeling.