• 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2020-03
  • 2020-07
  • 2020-08
  • 2021-03
  • In this study we aimed to establish an in


    In this study we aimed to establish an in vitro model of inflammatory barrier breakdown in the microcirculation that is applicable to endothelial cell lines of various origins (species and tissue) in the electric cell-substrate impedance sensing (ECIS™) system. In addition to human microvascular endothelial Oxidopamine hydrochloride (HMEC-1), we employed murine glomerular endothelial cells (GENCs), as the kidney is very sensitive to inflammation-induced organ failure [30] and the glomerular endothelium plays a key role in the progression of renal dysfunction [31]. S1P can phosphorylate AMPK in cells of the human [26] and bovine [25] macrovasculature as well as in kidney fibroblasts [27]. However, data on the S1P-AMPK interrelation in the microvasculature have not been reported. Therefore, we investigated the time courses of AMPK phosphorylation by S1P in HMEC-1 and GENCs. Subsequently, we evaluated the physiological relevance of this phosphorylation by siRNA-mediated knockdown of AMPK α1/2 in HMEC-1, which was implemented in the ECIS™ system by a reverse transfection protocol. Finally, we assessed effects of FTY720 on inflammatory barrier breakdown in HMEC-1 and GENCs using the in vitro model established in this study.
    Results and discussion
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    Introduction Mitochondria are responsible for the production of ATP through oxidative phosphorylation (OXPHOS) and also influence several other facets of cell biology, including intra- and inter-cellular metabolic fluxes, redox and Ca2+ homeostasis, production of physiological levels of reactive oxygen species (ROS) for cell signaling, and modulation of nuclear gene expression through genetic and epigenetic mechanisms [1,2]. Not surprisingly, dysfunction of these organelles is linked to the development of numerous diseases through a variety of mechanisms that include energetic failure, dysregulation of Ca2+ flux, oxidative damage, genetic reprogramming, and triggering of cell death [[2], [3], [4]]. Moreover, stressed mitochondria can also release pro-inflammatory bacterial-like alarmins and self-antigens that can lead to potentially adverse immune responses [[5], [6], [7]]. Considering the central roles played by these organelles in health and disease, cells have evolved sophisticated mechanisms to prevent the accumulation of abnormalities within the mitochondrial population. These mechanisms involve the continuous replacement of mitochondrial biomass through biogenesis, which is a consequence of the coordinated induction of key transcription factors and co-activators that collectively regulate the expression of nuclear- and mitochondria-encoded genes [[8], [9], [10], [11], [12], [13]]. In parallel, a variety of mitochondrial quality control processes (mQC) insure the repair or removal of various constituents [8,10,12,14,15], thus completing the mitochondrial life cycle. Over the recent years, studies have led to the discovery of multiple mQC processes that operate at various scales, ranging from the degradation of proteins mediated by intra-mitochondrial proteases to the delivery of selected cargos or entire organelles to lysosomes for degradation. While the basic molecular mechanisms governing these mQC processes are progressively being delineated, their role, level of interdependence, and physiopathological importance still remain unclear. There is increasing consensus that in vivo maintenance of mitochondrial health results from the coordinated action of multiple complimentary mechanisms that likely vary according to the cell type, the physiological state, and the type and impact of pathological stress. For this reason, knowledge gained from basic model systems of mQC need to be integrated within organ-specific (patho)physiological frameworks. Building on this notion, this article focuses on mQC in the heart, where developmental metabolic reprogramming, sustained contractile activity, and multiple pathophysiological conditions pose broadly different constraints on mitochondrial biogenesis and quality control. The intention is to provide an overview of current knowledge of mitochondrial quality control systems gained from various model systems and, when data is available, to specifically discuss their potential implication in cardiac mQC under normal and diseased conditions.