Integrated transcriptomic and proteomic profiling reveals the key molecular signatures of brain endothelial reperfusion injury

BackgroundReperfusion therapy after ischemic stroke often causes brain microvascular injury. However, the underlying mechanisms are unclear.MethodsTranscriptomic and proteomic analyses were performed on human cerebral microvascular endothelial cells following oxygen-glucose deprivation (OGD) or OGD plus recovery (OGD/R) to identify molecules and signaling pathways dysregulated by reperfusion. Major findings were further validated in a mouse model of cerebral ischemia and reperfusion.ResultsTranscriptomic analysis identified 390 differentially expressed genes (DEGs) between the OGD/R and OGD group. Pathway analysis indicated that these genes were mostly associated with inflammation, including the TNF signaling pathway, TGF-beta signaling pathway, cytokine-cytokine receptor interaction, NOD-like receptor signaling pathway, and NF-kappa B signaling pathway. Proteomic analysis identified 201 differentially expressed proteins (DEPs), which were primarily associated with extracellular matrix destruction and remodeling, impairment of endothelial transport function, and inflammatory responses. Six genes (DUSP1, JUNB, NFKBIA, NR4A1, SERPINE1, and THBS1) were upregulated by OGD/R at both the mRNA and protein levels. In mice with cerebral ischemia and reperfusion, brain TNF signaling pathway was activated by reperfusion, and inhibiting TNF-alpha with adalimumab significantly attenuated reperfusion-induced brain endothelial inflammation. In addition, the protein level of THBS1 was substantially upregulated upon reperfusion in brain endothelial cells and the peri-endothelial area in mice receiving cerebral ischemia.ConclusionOur study reveals the key molecular signatures of brain endothelial reperfusion injury and provides potential therapeutic targets for the treatment of brain microvascular injury after reperfusion therapy in ischemic stroke. Transcriptomic analysis of human cerebral microvascular endothelial cell (HCMEC)/D3 cells subjected to OGD/R showed that the top significantly changed signaling pathways were mostly associated with inflammatory responses, especially the TNF signaling pathway. Reperfusion-induced differentially expressed proteins were primarily involved in extracellular matrix destruction and remodeling, impairment of endothelial transport function, and inflammatory responses.image