S-18-5: ENDOTHELIAL GATA5 POSITIVELY REGULATES ANGIOGENESIS VIA CATHEPSIN S-MEDIATED ANGPT2/FLK1 AND MMP2/9 SIGNALING PATHWAYS

Abstract

Objective: Although GATA5 is vital in maintaining the function of endothelial cells, the relationship between GATA5 and angiogenesis, however, remains unclear. Our study aims to determine how endothelial GATA5 mediates angiogenesis. Design and method: 1. C57BL/6 mice were injected with endothelial cell-specific GATA5-overexpressing adeno-associated virus. One week after virus infection, the hindlimb ischemia model was established by ligating the femoral artery of mice, and the blood perfusion ratio of the operated limb/healthy limb was detected by a blood perfusion imager. CD31 staining of mouse gastrocnemius muscle showed the number of new capillaries per unit area. Western Blot and qPCR were used to detect the expression of GATA5 and related angiogenesis factors. 2. On human umbilical vein endothelial cells, transfected lentivirus to knock down the expression of GATA5 and/or administered vascular endothelial growth factor-165 intervention, through CCK-8 experiments were used to detect cell proliferation, scratch experiments to detect cell migration, and endothelial tube formation experiments to detect the ability of cells to form tubes. Western blot and qPCR assays were used to detect the protein and mRNA levels of GATA5, cathepsin S, Angpt2 and Flk1, and MMP2/MMP9. The direct binding of GATA5 to cathepsin S was verified by co-immunoprecipitation experiments. 3. GATA5 knockdown human umbilical vein endothelial cells were transfected with plasmids overexpressing GATA5 or cathepsin S to increase the expression levels of GATA5 or cathepsin S. Cell function and expression of related factors were detected. Results: 1. The blood perfusion ratio and CD31 immunofluorescence expression of the mice in the endothelium-specific overexpression GATA5 group were significantly higher than those of the negative control mice, and the mRNA and protein levels of angiogenesis factors Angpt2 and Flk1 were increased as well. 2. Cell experiments showed that the knockdown of GATA5 could inhibit cell proliferation, migration and endothelial tube formation stimulated by VEGF-165, and at the same time, reducing the expression of cathepsin S, Angpt2, Flk1 and MMP2/MMP2. Co-immunoprecipitation experiments showed that GATA5 had a direct binding relationship with cathepsin S. 3. Overexpression of GATA5 or cathepsin S reversed the decline in cell migration and tube formation caused by GATA5 knockdown, and up-regulated the expressions of Angpt2/Flk1 and MMP2/MMP9. Conclusion: Endothelial GATA5 regulates angiogenesis by regulating endothelial cell proliferation, migration and tube formation through the cathepsin S-Angpt2/Flk1 and MMP2/MMP9 signaling pathways.