BACKGROUND: Endothelial barrier dysfunction is critical for the pathogenesis of sepsis-induced acute lung injury (ALI). Lipopolysaccharide (LPS)-stimulated human pulmonary microvascular endothelial cells (HPMECs) are widely used as the cell model of sepsis-associated ALI for exploration of endothelial barrier dysfunction. Dickkopf (DKK) family proteins were reported to mediate endothelial functions in various diseases. The present study explored the effect of Dickkopf-3 (DKK3) on endothelial barrier permeability, angiogenesis, and tight junctions in LPS-stimulated HPMECs.
METHODS: RT-qPCR was required for detecting DKK3 and miR-98-3p expression. The angiogenesis of HPMECs was evaluated by tube formation assays. Monolayer permeability of HPMECs was examined by Transwell rhodamine assays. The protein expression of DKK3 and tight junctions in HPMECs was measured via western blotting. Luciferase reporter assay was used to verify the interaction between miR-98-3p and DKK3.
RESULTS: LPS treatment inhibited angiogenetic ability while increasing the permeability of HPMECs. DKK3 expression was upregulated while miR-98-3p level was reduced in LPS-treated HPMECs. DKK3 knockdown alleviated HPMEC injury triggered by LPS stimulation. MiR-98-3p targeted DKK3 in HPMECs. Overexpression of miR-98-3p protects HPMECs from the LPS-induced endothelial barrier dysfunction, and the protective effect was reversed by DKK3 overexpression.
CONCLUSIONS: MiR-98-3p ameliorates LPS-evoked pulmonary microvascular endothelial barrier dysfunction in sepsis-associated ALI by targeting DKK3.

This study aimed to investigate whether HMGB1 (high mobility group box-1 protein) and receptor for advanced glycation end products (RAGE) were involved in the irradiation-induced endothelial barrier damage and their mechanism. We constructed the damage model of endothelium barrier model with bEnd.3 cells. The permeability of endothelial barrier was detected by sodium fluorescein (Na-F) permeation test, and the irradiation dose which could induce permeability transition was determined by being exposed to different irradiation doses (5, 10, 15, 20 Gy). MTT assay was applied to detect cell viability under different concentrations of HMGB1, glycyrrhizic acid (GA, a specific inhibitor of HMGB1), and FPS-ZM1 (a blood-brain-barrier permeant blocker of RAGE V domain-mediated ligand binding). The expression of HMGB1, RAGE, and related molecules involved in MAPK signaling pathway, MMP-2, MMP-9, ZO-1, and claudin 5 of differently treated groups were measured by qRT-PCR, western blot, and immunofluorescence. Cells possessed stable endothelial barrier function on 4-7 days after seeded on transwell plates. The permeability of endothelial barrier would change under at least 10 Gy radiation. Both radiation and HMGB1 treatment alone could improve the permeability. After irradiation, the expressions of HMGB1 and RAGE increased and MAPK signal pathway was activated. Meanwhile, MMP-2 and MMP-9 were overexpressed, while the expression of tight junction proteins ZO-1 and claudin 5 was decreased. Radiation could activate MAPK signaling pathway through promoting the expression of HMGB1 and RAGE, which further led to endothelial barrier injury and changed its permeability.