The protective effects of GLP-1 receptor agonist lixisenatide on oxygen-glucose deprivation/reperfusion (OGD/R)-induced deregulation of endothelial tube formation

Figures

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  Fig. 1. Peptide sequence of native human GLP-1 and lixisenatide. (A) Native human GLP-1; (B) lixisenatide.

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  Fig. 2. Lixisenatide protects oxygen-glucose deprivation/reperfusion (OGD/R)-induced reduction of cell viability and release of LDH in HUVECs. Cells were treated with lixisenatide (5, 10, 20 nM) for 6 h, followed by exposure to oxygen-glucose deprivation (6 h)/reperfusion (24 h) (OGD/R). (A) Cell viability of HUVECs; (B) release of lactate dehydrogenase (LDH); (C) cell morphology of HUVECs (****, P < 0.0001 vs. control group; ##, ###, ####, P < 0.01, 0.001, 0.0001 vs. OGD/R treatment group, n = 5).

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  Fig. 3. Lixisenatide reduced OGD/R-induced generation of ROS in HUVECs. Cells were treated with lixisenatide (10, 20 nM) for 6 h, followed by exposure to oxygen-glucose deprivation (6 h)/reperfusion (24 h) (OGD/R). Generation of ROS was measured by dihydroethidium (DHE) staining (****, P < 0.0001 vs. control group; ###, ####, P < 0.001, 0.0001 vs. OGD/R treatment group, n = 5).

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  Fig. 4. Lixisenatide activated the expression of Nrf2 and HO-1 under OGD/R in HUVECs. Cells were treated with lixisenatide (10, 20 nM) for 6 h, followed by exposure to oxygen-glucose deprivation (6 h)/reperfusion (24 h) (OGD/R). Expression of Nrf2 and HO-1 was measured by western blot analysis (**, ***, ****, P < 0.01, 0.001, 0.0001 vs. control group, n = 4).

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  Fig. 5. Lixisenatide prevented OGD/R-induced reduced angiogenesis in HUVECs. Cells were treated with lixisenatide (20 nM) for 6 h, followed by exposure to oxygen-glucose deprivation (6 h)/reperfusion (24 h) (OGD/R). Microtubule formation was quantified by Matrigel assay (****, P < 0.0001 vs. control group; ####, P < 0.0001 vs. OGD/R treatment group, n = 5).

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  Fig. 6. Lixisenatide prevented OGD/R-induced dephosphorylation of PI3K and Akt in HUVECs. Cells were treated with lixisenatide (20 nM) for 6 h, followed by exposure to oxygen-glucose deprivation (6 h)/reperfusion (24 h) (OGD/R). Phosphorylated and total levels of PI3K and Akt were measured by western blot analysis (****, P < 0.0001 vs. control group; ####, P < 0.0001 vs. OGD/R treatment group, n = 4).

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  Fig. 7. Lixisenatide prevented OGD/R-induced dephosphorylation of eNOS and production of nitric oxide (NO) in HUVECs. Cells were treated with lixisenatide (20 nM) for 6 h, followed by exposure to oxygen-glucose deprivation (6 h)/reperfusion (24 h) (OGD/R). (A) Phosphorylated and total levels of eNOS were measured by western blot analysis. (B) Intracellular nitric oxide (NO) was measured by diaminofluorescein-FM diacetate (DAF-FM DA) (****, P < 0.0001 vs. control group; ###, ####, P < 0.01, 0.001, 0.0001 vs. OGD/R treatment group, n = 5).

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  Fig. 8. Blockage of the PI3K/Akt signaling pathway using LY294002 abolished the protective effects of lixisenatide on promoting angiogenesis in HUVECs. Cells were treated with lixisenatide (20 nM) in the presence or absence of LY294002 (20 μM) for 6 h, followed by exposed to oxygen-glucose deprivation (6 h)/reperfusion (24 h) (OGD/R). (A) Molecular structure of LY294002; (B) microtubule formation was quantified by Matrigel assay; (C) production of ROS (****, P < 0.0001, n = 5).

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  Fig. 9. A graphical representation of the underlying mechanism.