Hypoxic human adipose mesenchymal stem cells-derived extracellular vesicles induce P311 expression and inhibit activation and injury of human brain microvascular endothelial cells

Abstract

ObjectiveStem cell therapy can modify angiogenic pathways. Neural protein 3.1 (P311) possesses the pro-angiogenic property. This study strived to explore the action and mechanism of human adipose mesenchymal stem cells (hADSCs) in human brain microvascular endothelial cell (hBMEC) injury by regulating P311.MethodsThe hADSCs of the 3^rd passage were stained with oil red O, Alizarin red, and Alcian blue to assess adipogenic, osteogenic, and chondrogenic differentiation, followed by an analysis of immune phenotype via flow cytometry. After culturing hADSCs in hypoxic (5% oxygen) and normoxic (20% oxygen) conditions, extracellular vesicles (EVs) were extracted via ultracentrifugation, followed by morphology observation by microscopy, size distribution analysis via Nanoparticle tracking analysis, and surface marker determination by Western blot. hBMECs were treated with lipopolysaccharide (LPS) and cultured with normoxia or hypoxic hADSC-EVs. The effects of normoxia and hypoxic hADSC-EVs on proliferation, migration, and tube formation of hBMECs were assessed via CCK-8, Transwell, and tube formation assays. hBMECs were transfected with pcDNA3.0-P311 or P311 siRNA to evaluate the action of P311 on hBMEC injury.ResultsHypoxic hADSC-EVs had a larger mean diameter, a wider diameter distribution range, and a higher particle concentration than normoxic hADSC-EVs. Hypoxia and normoxic hADSC-EVs were internalized by hBMECs, and hypoxic hADSC-EVs were more internalized. LPS suppressed hBMEC proliferation, migration, and tube formation and induced hBMEC injury. Hypoxia and normoxic hADSC-EVs ameliorated hBMEC injury, and hypoxic hADSC-EVs were superior to normoxic hADSC-EVs. P311 overexpression mitigated hBMEC injury, whereas P311 knockdown partly averted hypoxic hADSC-EV-exerted suppression on hBMEC injury.ConclusionHypoxic hADSC-EVs can protect against LPS-induced hBMEC injury by upregulating P311.

Keywords: angiogenesis; extracellular vesicles; human adipose mesenchymal stem cells; human brain microvascular endothelial cells; hypoxic microenvironment; lipopolysaccharide; migration; proliferation.