c-Jun-mediated miR-19b expression induces endothelial barrier dysfunction in an in vitro model of hemorrhagic shock

Abstract

Background Our previous data demonstrated that miR-19b expression was increased in human lung microvascular endothelial cells in-vitro-, in-vivo and in patients with hemorrhagic shock, leading to a decrease in syndecan-1 mRNA and protein and resulting in loss of endothelial barrier function. However, the mechanism underlying increased miR-19b expression remains unclear. The objective of the current study was to determine if c-Jun mediates the early responsive microRNA, miR-19b, to cause endothelial barrier dysfunction. Method Human lung microvascular endothelial cells (HLMEC) or HEK293T cells were transfected with c-Jun overexpressing vector, c-Jun siRNA, miR-19b promoter vector, miR-19b mutated promoter vector, miR-19b oligo inhibitor, then subjected to hypoxia/reoxygenation as in-vitro model of hemorrhagic shock. Levels of protein, miRNA, and luciferase activity were measured. Transwell permeability of endothelial monolayers were also determined. Plasma levels of c-Jun were measured in injured patients with hemorrhagic shock. Result Hypoxia/reoxygenation induced primary (pri-)miR-19b, mature miR-19b, and c-Jun expression over time in a comparable timeframe. c-Jun silencing by transfection with its specific siRNA reduced both pri-miR-19b and mature miR-19b levels. Conversely, c-Jun overexpression enhanced H/R-induced pri-miR-19b. Studies using a luciferase reporter assay revealed that in cells transfected with vectors containing the wild-type miR-19b promoter and luciferase reporter, c-Jun overexpression or hypoxia/ reoxygenation significantly increased luciferase activity. c-Jun knockdown reduced the luciferase activity in these cells, suggesting that the miR-19b promoter is directly activated by c-Jun. Further, chromatin immunoprecipitation assay confirmed that c-Jun directly bound to the promoter DNA of miR-19b and hypoxia/reoxygenation significantly increased this interaction. Additionally, c-Jun silencing prevented cell surface syndecan-1 loss and endothelial barrier dysfunction in HLMECs after hypoxia/reoxygenation. Lastly, c-Jun was significantly elevated in patients with hemorrhagic shock compared to healthy controls. Conclusion Transcription factor c-Jun is inducible by hypoxia/reoxygenation, binds to and activates the miR-19b promoter. Using an in-vitro model of hemorrhagic shock, our findings identified a novel cellular mechanism whereby hypoxia/ reoxygenation increases miR-19b transcription by inducing c-Jun and leads to syndecan-1 decrease and endothelial cell barrier dysfunction. This finding supports that miR-19b could be a potential therapeutic target for hemorrhage shock.

Succinate metabolism and membrane reorganization drives the endotheliopathy and coagulopathy of traumatic hemorrhage

“…Persistent damage to the EG, likely due to both ongoing shedding as well as transcriptional down-regulation of glycocalyx components, is reported in trauma and other critical illness states (45,(75)(76)(77)(78). An increase in succinate metabolism has been reported to drive transcriptional alterations (31,56) in several cell types.…”

“…This linkage of succinate-induced mitoROS with transcriptional changes suggests that the mechanism we describe here, which mediates acute glycocalyx damage, could potentially cascade into longer-term, transcriptionally mediated effects as well. Transcriptional changes in trauma have been linked to c-Jun activity ( 75 ), and succinate has been shown to stabilize HIF-1α ( 31 , 56 ). As HIF-1α and c-Jun have been shown to cooperatively regulate transcription ( 79 ), these factors may act as the interface between short-term and long-term endotheliopathy.…”

Epigenome-Wide Changes in the Cell Layers of the Vein Wall When Exposing the Venous Endothelium to Oscillatory Shear Stress

“…JUN (Jun proto-oncogene, AP-1 transcription factor subunit) is a member of the Jun family of proteins, which are primary components of the activating protein transcription factor [ 31 ]. It is inducible by hypoxia related to endothelial cell barrier dysfunction [ 32 ]. CDX2 (caudal-type homeobox-2) is a member of the caudal-related homeobox transcription factor gene family.…”