RESUMEN
BACKGROUND: Iron excess is a risk factor for cardiovascular diseases and it is important to understand the effect of iron on vascular permeability, particularly for the transport of large metabolic hormones such as adiponectin. METHODS: We used 2-dimensional monolayers of cultured human dermal microvascular endothelial cells (HDMEC) and human umbilical vein endothelial cells (HUVEC) as well as 3-dimensional microvascular networks to measure transendothelial flux. RESULTS: Iron supplementation reduced transendothelial electric resistance (TEER). Flux analysis indicated that under control conditions permeability of 70 kDa dextran and oligomeric forms of adiponectin were restricted in comparison with a 3 kDa dextran, however upon iron treatment permeability of the larger molecules was increased. The increased permeability and size-dependent trans-endothelial movement in response to iron was also observed in 3-dimensional microvascular networks. Mechanistically, the alteration in barrier functionality was associated with increased oxidative stress in response to iron since alterations in TEER and permeability were rescued when reactive oxygen species production was attenuated by pre-treatment with the antioxidant N-acetyl cysteine.]. CONCLUSIONS: Iron supplementation induced ROS production resulting in increased transendothelial permeability. GENERAL SIGNIFICANCE: Altogether, this suggests that the oxidative stress associated with iron excess could play an important role in the regulation of endothelial functionality, controlling hormone action in peripheral tissues by regulating the first rate-limiting step controlling hormone access to target tissues.
Asunto(s)
Adiponectina/metabolismo , Células Endoteliales/metabolismo , Hierro/metabolismo , Microvasos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Permeabilidad Capilar , Línea Celular , Impedancia Eléctrica , Células Endoteliales/citología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Dispositivos Laboratorio en un Chip , Microvasos/citología , PermeabilidadRESUMEN
Adiponectin, a highly abundant polypeptide hormone in plasma, plays an important role in the regulation of energy metabolism in a wide variety of tissues, as well as providing important beneficial effects in diabetes, inflammation, and cardiovascular disease. To act on target tissues, adiponectin must move from the circulation to the interstitial space, suggesting that vascular permeability plays an important role in regulating adiponectin action. To test this hypothesis, fluorescently labeled adiponectin was used to monitor its biodistribution in mice with streptozotocin-induced diabetes (STZD). Adiponectin was, indeed, found to have increased sequestration in the highly fenestrated liver and other tissues within 90 min in STZD mice. In addition, increased myocardial adiponectin was detected and confirmed using computed tomography (CT) coregistration. This provided support of adiponectin delivery to affected cardiac tissue as a cardioprotective mechanism. Higher adiponectin content in the STZD heart tissues was further examined by ex vivo fluorescence molecular tomography (FMT) imaging, immunohistochemistry, and Western blot analysis. In vitro mechanistic studies using an endothelial monolayer on inserts and three-dimensional microvascular networks on microfluidic chips further confirmed that adiponectin flux was increased by high glucose. However, in the in vitro model and mouse heart tissue, high glucose levels did not change adiponectin receptor levels. An examination of the tight junction (TJ) complex revealed a decrease in the TJ protein claudin (CLDN)-7 in high glucose-treated endothelial cells, and the functional significance of this change was underscored by increased endothelium permeability upon siRNA-mediated knockdown of CLDN-7. Our data support the idea that glucose-induced effects on permeability of the vascular endothelium contribute to the actions of adiponectin by regulating its transendothelial movement from blood to the interstitial space. These observations are physiologically significant and critical when considering ways to harness the therapeutic potential of adiponectin for diabetes.
Asunto(s)
Adiponectina/metabolismo , Permeabilidad Capilar , Diabetes Mellitus Experimental/metabolismo , Animales , Línea Celular , Diabetes Mellitus Experimental/patología , Células Endoteliales/metabolismo , Fluorescencia , Técnicas de Silenciamiento del Gen , Glucosa/farmacología , Humanos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Microcirculación , Miocardio/metabolismo , Ratas , Ratas Wistar , Distribución Tisular , Tomografía/métodos , Tomografía Computarizada por Rayos XRESUMEN
Altered permeability of the endothelial barrier in a variety of tissues has implications both in disease pathogenesis and treatment. Glucocorticoids are potent mediators of endothelial permeability, and this forms the basis for their heavily prescribed use as medications to treat ocular disease. However, the effect of glucocorticoids on endothelial barriers elsewhere in the body is less well studied. Here, we investigated glucocorticoid-mediated changes in endothelial flux of Adiponectin (Ad), a hormone with a critical role in diabetes. First, we used monolayers of endothelial cells in vitro and found that the glucocorticoid dexamethasone increased transendothelial electrical resistance and reduced permeability of polyethylene glycol (PEG, molecular weight 4000 Da). Dexamethasone reduced flux of Ad from the apical to basolateral side, measured both by ELISA and Western blotting. We then examined a diabetic rat model induced by treatment with exogenous corticosterone, which was characterized by glucose intolerance and hyperinsulinemia. There was no change in circulating Ad but less Ad protein in skeletal muscle homogenates, despite slightly higher mRNA levels, in diabetic vs control muscles. Dexamethasone-induced changes in Ad flux across endothelial monolayers were associated with alterations in the abundance of select claudin tight junction (TJ) proteins. shRNA-mediated knockdown of one such gene, claudin-7, in HUVEC resulted in decreased TEER and increased adiponectin flux, confirming the functional significance of Dex-induced changes in its expression. In conclusion, our study identifies glucocorticoid-mediated reductions in flux of Ad across endothelial monolayers in vivo and in vitro This suggests that impaired Ad action in target tissues, as a consequence of reduced transendothelial flux, may contribute to the glucocorticoid-induced diabetic phenotype.
Asunto(s)
Adiponectina/metabolismo , Dexametasona/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Adiponectina/genética , Animales , Diabetes Mellitus Experimental , Impedancia Eléctrica , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Humanos , Ratones , Músculo Esquelético , Miosinas/clasificación , Miosinas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas de Uniones Estrechas/genética , Proteínas de Uniones Estrechas/metabolismoRESUMEN
The vascular endothelium is a dynamic structure responsible for the separation and regulated movement of biological material between circulation and interstitial fluid. Hormones and nutrients can move across the endothelium either via a transcellular or paracellular route. Transcellular endothelial transport is well understood and broadly acknowledged to play an important role in the normal and abnormal physiology of endothelial function. However, less is known about the role of the paracellular route. Although the concept of endothelial dysfunction in diabetes is now widely accepted, we suggest that alterations in paracellular transport should be studied in greater detail and incorporated into this model. In this review we provide an overview of endothelial paracellular permeability and discuss its potential importance in contributing to the development of diabetes and associated complications. Accordingly, we also contend that if better understood, altered endothelial paracellular permeability could be considered as a potential therapeutic target for diabetes.