[LPS-induced endothelial cytoskeleton remodeling in human lung vessels and related miRNAs-profiling].

Objective To investigate the effects of lipopolysaccharide (LPS) on human pulmonary vascular endothelial cells (HPVECs) cytoskeleton and perform biological analysis of the microRNA (miRNA) spectrum. Methods The morphology of HPVECs was observed by microscope, the cytoskeleton by FITC-phalloidin staining, and the expression of VE-cadherin was detected by immunofluorescence cytochemical staining; the tube formation assay was conducted to examine the angiogenesis, along with cell migration test to detect the migration, and JC-1 mitochondrial membrane potential to detect the apoptosis. Illumina small-RNA sequencing was used to identify differentially expressed miRNAs in NC and LPS group. The target genes of differentially expressed miRNAs were predicted by miRanda and TargetScan, and the functional and pathway enrichment analysis was performed on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Further biological analysis of related miRNAs was carried out. Results After the LPS got induced, the cells became round and the integrity of cytoskeleton was destroyed. The decreased expression of VE-cadherin was also observed, along with the decreased ability of angiogenesis and migration, and increased apoptosis. Sequencing results showed a total of 229 differential miRNAs, of which 84 miRNA were up-regulated and 145 miRNA were down-regulated. The target gene prediction and functional enrichment analysis of these differential miRNA showed that they were mainly concentrated in pathways related to cell connection and cytoskeleton regulation, cell adhesion process and inflammation. Conclusion In vitro model of lung injury, multiple miRNAs are involved in the process of HPVECs cytoskeleton remodeling, the reduction of barrier function, angiogenesis, migration and apoptosis.

[Exosomes derived from human placental mesenchymal stem cells reduce human lung microvascular endothelial cell injury induced by lipopolysaccharide via enhancing autophagy].

Objective To investigate the effect of exosomes derived from human placental mesenchymal stem cells (hPMSC-exs) on lipopolysaccharide (LPS)-induced injury of human pulmonary microvascular endothelial cells (HPMECs) and its possible mechanism. Methods hPMSCs were expanded and cultured in vitro and the cell culture supernatant was collected. The hPMSC-exs in the supernatant was separated and purified by ExoQuick exosomes extraction and purification kit. The morphological characteristics of exosomes were observed by transmission electron microscopy, and the expression of specific markers CD9 and CD63 on the surface of exosomes was detected by Western blotting. A non-contact co-culture system of hPMSCs and HPMECs was constructed. The experiment included a control group, an LPS injury group, an hPMSC group and an hPMSC-exs group. After 12 hours of co-cultivation, the fluorescence intensity of FITC-dextran from the upper chamber into the lower chamber was detected to reflect the permeability of single-layer pulmonary vascular endothelium; DiI-labeled hPMSC-exs was observed under a fluorescence microscope to judge the migration effect of hPMSC-exs on the damaged endothelium; CCK-8 assay was used to detect the proliferation level of the upper chamber cells in each group; JC-1 staining was used to detect the mitochondrial membrane potential of upper ventricular cells in each group; Western blotting was performed to detect the expression of autophagy-related protein LC3 and beclin-1 of HPMECs in the lower chamber. Results The diameter of hPMSC-exs was 30-120 nm, and the expression of CD9 and CD63 was positive, which matched the phenotypic characteristics of hPMSC-exs. Compared with the LPS injury group, the fluorescence intensity of FITC-dextran decreased; LC3II/I ratio and cell proliferation rate increased; beclin-1 protein expression was up-regulated in the hPMSC group and the hPMSC-exs group. Compared with the hPMSC group, the structure of endothelial cells was roughly intact under the fluorescence microscope; the FITC-dextran fluorescence intensity, endothelial cell proliferation rate, mitochondrial membrane potential, expression levels of LC3-II/I and beclin-1 did not change significantly in the hPMSC-exs group. Conclusion hPMSC-exs can alleviate the damage of HPMECs induced by LPS and improves mitochondrial function in the cells. Its mechanism may be related to enhance the autophagy of HPMECs.