influx from discrete ion channels is functionally coupled to specific outcomes. Thus we conducted a systematic study in mouse lung to address whether the ␣1G T-type Ca 2ϩ channel and the transient receptor potential channel TRPV4 have discrete functional roles in pulmonary capillary endothelium. We used real-time fluorescence imaging for endothelial cytosolic Ca 2ϩ , immunohistochemistry to probe for surface expression of P-selectin, and the filtration coefficient to specifically measure lung endothelial permeability. We demonstrate that membrane depolarization via exposure of pulmonary vascular endothelium to a high-K ϩ perfusate induces Ca 2ϩ entry into alveolar septal endothelial cells and exclusively leads to the surface expression of P-selectin. In contrast, Ca 2ϩ entry in septal endothelium evoked by the selective TRPV4 activator 4␣-phorbol-12,13-didecanoate (4␣-PDD) specifically increases lung endothelial permeability without effect on P-selectin expression. Pharmacological blockade or knockout of ␣1G abolishes depolarization-induced Ca 2ϩ entry and surface expression of P-selectin but does not prevent 4␣-PDD-activated Ca 2ϩ entry and the resultant increase in permeability. Conversely, blockade or knockout of TRPV4 specifically abolishes 4␣-PDD-activated Ca 2ϩ entry and the increase in permeability, while not impacting depolarization-induced Ca 2ϩ entry and surface expression of P-selectin. We conclude that in alveolar septal capillaries Ca 2ϩ entry through ␣1G and TRPV4 channels differentially and specifically regulates the transition of endothelial procoagulant phenotype and barrier integrity, respectively.
Exogenously applied caveolin-1 scaffolding domain (CAV) has been shown to inhibit inflammatory mediator-induced nitric oxide (NO) production and NO-mediated increases in microvessel permeability. However, the effect of CAV on endothelial basal NO that prevents leukocyte adhesion remains unknown. This study aims to investigate the roles of exogenously applied CAV in endothelial basal NO production, leukocyte adhesion, and adhesion-induced changes in microvessel permeability. Experiments were conducted in individually perfused rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp). NO was quantified with fluorescence imaging in DAF-2-loaded vessels. Perfusing venules with CAV inhibited basal NO production without affecting basal Lp. Resuming blood flow in CAV-perfused vessels significantly increased leukocyte adhesion. The firmly adherent leukocytes altered neither basal Lp nor adherens junction integrity. Increases in Lp occurred only upon formyl-Met-Leu-Phe application that induces release of reactive oxygen species from the adherent leukocytes. The application of NO synthase inhibitor showed similar results to CAV, and NO donor abolished CAV-mediated leukocyte adhesion. Immunofluorescence staining showed increases in binding of ICAM-1 to an adhesion-blocking antibody concurrent with a Src-dependent ICAM-1 phosphorylation following CAV perfusion. Pre-perfusing vessels with anti-ICAM-1 blocking antibody or a Src kinase inhibitor attenuated CAV-induced leukocyte adhesion. These results indicate that the application of CAV, in addition to preventing excessive NO-mediated permeability increases, also causes reduction of basal NO and promotes ICAM-1-mediated leukocyte adhesion through Src activation-mediated ICAM-1 phosphorylation. CAV-induced leukocyte adhesion was uncoupled from leukocyte oxidative burst and microvessel barrier function, unless in the presence of a secondary stimulation.
Exosomes have been shown to effectively regulate the biological functions of target cells. Here, we investigated the protective effect and mechanism of hypoxia-induced renal tubular epithelial cells (TECs)-derived exosomes on acute tubular injury. We found that in vitro hypoxia-induced tubular exosomes (Hy-EXOs) were protective in acute tubular injury by promoting TECs proliferation and improving mitochondrial functions. By using exosome miRNA sequencing, we identified miR-20a-5p was abundant and was a key mechanism for the protective effect of Hy-EXOs on tubular injury as up-regulation of miR-20a-5p enhanced but down regulation of miR-20a-5p inhibited the protective effect of Hy-EXOs on tubular injury under hypoxia conditions. Further study in a mouse model of ischemia-reperfusion induced acute kidney injury (IRI-AKI) also confirmed this notion as pre-treating mice with the miR-20a-5p agomir 48 hours prior to AKI induction was capable of inhibiting IRI-AKI by lowering serum levels of creatinine and urea nitrogen, and attenuating the severity of tubular necrosis, F4/80(+) macrophages infiltration and vascular rarefaction. Mechanistically, the protective effect of miR-20a-5p on AKI was associated with inhibition of TECs mitochondrial injury and apoptosis in vitro and in vivo. In conclusion, miR-20a-5p is enriched in hypoxia-derived tubular exosomes and protects against acute tubular injury. Results from this study also reveal that miR-20a-5p may represent as a novel therapy for AKI.
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