Base de datos de flora y fauna en Galicia

Sección: NoticiasLa Biblioteca del Instituto de Investigaciones Marinas de Vigo recopila, desde 1984 aproximadamente, una base de datos sobre Galicia que nació a partir de unas citas bibliográficas seleccionadas por un investigador de este centro y se fue ampliando en vista de la enorme solicitud de esta información que nos iban haciendo nuestros usuarios, sobre todo los externos al centroPeer reviewe

Expression of the Metalloproteinase ADAM8 Is Upregulated in Liver Inflammation Models and Enhances Cytokine Release In Vitro

Acute and chronic liver inflammation is driven by cytokine and chemokine release from various cell types in the liver. Here, we report that the induction of inflammatory mediators is associated with a yet undescribed upregulation of the metalloproteinase ADAM8 in different murine hepatitis models. We further show the importance of ADAM8 expression for the production of inflammatory mediators in cultured liver cells. As a model of acute inflammation, we investigated liver tissue from lipopolysaccharide- (LPS-) treated mice in which ADAM8 expression was markedly upregulated compared to control mice. In vitro, stimulation with LPS enhanced ADAM8 expression in murine and human endothelial and hepatoma cell lines as well as in primary murine hepatocytes. The enhanced ADAM8 expression was associated with an upregulation of TNF-α and IL-6 expression and release. Inhibition studies indicate that the cytokine response of hepatoma cells to LPS depends on the activity of ADAM8 and that signalling by TNF-α can contribute to these ADAM8-dependent effects. The role of ADAM8 was further confirmed with primary hepatocytes from ADAM8 knockout mice in which TNF-α and IL-6 induction and release were considerably attenuated. As a model of chronic liver injury, we studied liver tissue from mice undergoing high-fat diet-induced steatohepatitis and again observed upregulation of ADAM8 mRNA expression compared to healthy controls. In vitro, ADAM8 expression was upregulated in hepatoma, endothelial, and stellate cell lines by various mediators of steatohepatitis including fatty acid (linoleic-oleic acid), IL-1β, TNF-α, IFN-γ, and TGF-β. Upregulation of ADAM8 was associated with the induction and release of proinflammatory cytokines (TNF-α and IL-6) and chemokines (CX3CL1). Finally, knockdown of ADAM8 expression in all tested cell types attenuated the release of these mediators. Thus, ADAM8 is upregulated in acute and chronic liver inflammation and is able to promote inflammation by enhancing expression and release of inflammatory mediators

Influence of laminar shear stress on transmembrane chemokines and proteases of the ADAM family in endothelial cells

Shear stress generated by blood flow is sensed by endothelial cells and influences the integrity and inflammatory state of the endothelium. This response involves transcriptional and post-transcriptional regulatory mechanisms. Physiological blood flow has a protective effect on the endothelium, while pathologically reduced blood flow can impair endothelial integrity and thus promote the development of vascular inflammatory lesions. The transmembrane chemokine CX3CL1 is one of the contributors, which lead to the formation of these vascular inflammatory lesions. This can be achieved by acting either as surface-expressed adhesion molecule on the endothelial cells or as a soluble chemoattractant for monocytic cells. Soluble CX3CL1 is generated by the cleavage of CX3CL1 by metalloproteinases of the ADAM family. ADAMs can cleave a number of signalling molecules including CX3CL1 from the cell surface of endothelial cells. By that, ADAMs can regulate a variety of endothelial processes such as survival or inflammatory responses. The aim of this PhD study was to investigate whether transmembrane chemokines and proteases of the ADAM family are regulated by shear stress and are thus involved in influencing endothelial functions. For this purpose, a flow culture system for primary endothelial cells from different vascular beds was established. The system was first validated by confirming upregulation of endothelial NO synthase, and the Krüppel like transcription factor 2. In addition, physiological shear stress was found to suppress both the basal and TNF-induced expression of the transmembrane chemokine CX3CL1 on the mRNA level. This led to a reduced surface expression of CX3CL1 on flow exposed endothelial cells. Adhesion experiments with monocytic cells carrying the CX3CL1 receptor CX3CR1 showed that the suppressed CX3CL1 expression on endothelial cells contributes to a reduced monocyte adhesion on endothelial cells cultured under physiological flow. An additionally performed transcriptomic analysis showed that several proteases from the ADAM family are regulated by flow conditions. Of particular interest was the enhanced expression of ADAM15 and ADAM17 under physiological flow conditions. It could be shown in this work that the induction of the ADAM15 expression is mediated by shear stress via the transcription factor KLF2 as shown by pharmacological manipulation, lentiviral overexpression and siRNA-mediated knockdown of KLF2 expression. Through shRNA-mediated knockdown of ADAM15 evidence could be provided that ADAM15 contributes to increased endothelial cell survival. For ADAM17, a more complex regulatory mechanism was observed. ADAM17 is not only regulated on the transcriptional level but especially on the post-transcriptional level. This is due to an altered maturation of ADAM17 caused by the transcriptional regulation of iRhoms. While the mRNA expression of iRhom1 is preferentially induced by shear stress via KLF2, iRhom2 is predominantly induced by the inflammatory stimulus TNF. This leads to increased surface expression of ADAM17 both under physiological flow conditions and under inflammatory conditions, such as stimulation with TNF. Finally, a microfluidic chamber was developed, which allows monitoring the effect on endothelial permeability under flow and over time by continuous impedance spectroscopic measurements. This chamber may be useful in further studies to investigate the function of ADAMs in endothelial permeability regulation under flow. Thus it could be shown in this work that shear stress can have protective effects on the endothelium by downregulation of inflammatory ADAM substrates such as CX3CL1 and by KLF2 mediated upregulation of protective ADAM15. Moreover, ADAM17, which cleaves inflammatory, as well as protective substrates on endothelial cells, can be upregulated on the cell surface by both inflammatory conditions via induction of iRhom2 and flow conditions via induction of iRhom1. The results all indicate that in vitro investigations of inflammatory and protective endothelial cell functions need to consider appropriate flow conditions

Shear Stress Counteracts Endothelial CX3CL1 Induction and Monocytic Cell Adhesion

Flow conditions critically regulate endothelial cell functions in the vasculature. Reduced shear stress resulting from disturbed blood flow can drive the development of vascular inflammatory lesions. On endothelial cells, the transmembrane chemokine CX3CL1/fractalkine promotes vascular inflammation by functioning as a surface-expressed adhesion molecule and by becoming released as soluble chemoattractant for monocytic cells expressing the receptor CX3CR1. Here, we report that endothelial cells from human artery, vein, or microvasculature constitutively express CX3CL1 when cultured under static conditions. Stimulation with TNFα under static or very low shear stress conditions strongly upregulates CX3CL1 expression. By contrast, CX3CL1 induction is profoundly reduced when cells are exposed to higher shear stress. When endothelial cells were grown and subsequently stimulated with TNFα under low shear stress, strong adhesion of monocytic THP-1 cells to endothelial cells was observed. This adhesion was in part mediated by transmembrane CX3CL1 as demonstrated with a neutralizing antibody. By contrast, no CX3CL1-dependent adhesion to stimulated endothelium was observed at high shear stress. Thus, during early stages of vascular inflammation, low shear stress typically seen at atherosclerosis-prone regions promotes the induction of endothelial CX3CL1 and monocytic cell recruitment, whereas physiological shear stress counteracts this inflammatory activation of endothelial cells

Differential Regulation of Lung Endothelial Permeability in Vitro and in Situ

In the lungs, increased vascular permeability can lead to acute lung injury. Because vascular permeability is regulated primarily by endothelial cells, many researchers have studied endothelial cell monolayers in culture, in order to understand the pathomechanisms of pulmonary edema. Such studies are based on the assumption that endothelial cells in culture behave like endothelial cells in situ. Here we show that this assumption is largely unfounded. Cultured endothelial cells show profound differences compared to their physiological counterparts, including a dysregulated calcium homeostasis. They fail to reproduce the pulmonary responses to agents such as platelet-activating factor. In contrast, they respond in a Rho-kinase depend fashion to thrombin, LPS or TNF. This is a striking finding for three reasons: (i) in the lungs, none of these agents increases vascular permeability by a direct interaction with endothelial cells; (ii) The endothelial Rho-kinase pathway seems to play little role in the development of pulmonary edema; (iii) This response pattern is similar for many endothelial cells in culture irrespective of their origin, which is in contrast to the stark heterogeneity of endothelial cells in situ. It appears that most endothelial in culture tend to develop a similar phenotyp that is not representative of any of the known endothelial cells of the lungs. We conclude that at present cultured endothelial cells are not useful to study the pathomechanisms of pulmonary edema

Additional file 1 of Functional changes in long-term incubated rat precision-cut lung slices

Additional file 1: Table S1. Concentration of components within the culture medium

Never Change a Flowing System? The Effects of Retrograde Flow on Isolated Perfused Lungs and Vessels

Retrograde perfusion may occur during disease, surgery or extracorporeal circulation. While it is clear that endothelial cells sense and respond to changes in blood flow, the consequences of retrograde perfusion are only poorly defined. Similar to shear stress or disturbed flow, retrograde perfusion might result in vasomotor responses, edema formation or inflammation in and around vessels. In this study we investigated in rats the effects of retrograde perfusion in isolated systemic vessels (IPV) and in pulmonary vessels of isolated perfused lungs (IPL). Anterograde and retrograde perfusion was performed for 480 min in IPV and for 180 min in the IPL. Perfusion pressure, cytokine levels in perfusate and bronchoalveolar lavage fluid (BALF), edema formation and mRNA expression were studied. In IPV, an increased perfusion pressure and initially also increased cytokine levels were observed during retrograde perfusion. In the IPL, increased edema formation occurred, while cytokine levels were not increased, though dilution of cytokines in BALF due to pulmonary edema cannot be excluded. In conclusion, effects of flow reversal were visible immediately after initiation of retrograde perfusion. Pulmonary edema formation was the only effect of the 3 h retrograde perfusion. Therefore, further research should focus on identification of possible long-term complications of flow reversal