The role of MDM2 in angiogenesis: implications for endothelial tip cell formation

• Published in: In vitro cellular & developmental biology. Animal 2024-10, Vol.60 (9), p.983-995
• Main Authors: Yi, Yi, Suo, Lina, Ma, Haixiu, Ma, Ronghua, Zhao, Jing, Zhai, Shaoqian, Wang, Haiyan, Su, Zhanhai
• Format: Article
• Language: English
• Subjects: 1-Phosphatidylinositol 3-kinase; AKT protein; Angiogenesis; Animal Genetics and Genomics; Biomedical and Life Sciences; CD34 antigen; Cell Biology; Cell Culture; Cell Hypoxia; Cell Line, Tumor; Cell migration; Cell Movement; Culture Media, Conditioned - pharmacology; Developmental Biology; Down-regulation; Endothelial cells; Endothelial Cells - metabolism; Enzyme-linked immunosorbent assay; Filopodia; Gastric cancer; Gene expression; Humans; Hypoxia; Immunofluorescence; Life Sciences; MDM2 protein; mRNA; Neovascularization, Pathologic - genetics; Neovascularization, Pathologic - metabolism; Neovascularization, Physiologic; Phosphatidylinositol 3-Kinases - metabolism; Proteins; Proto-Oncogene Proteins c-akt - metabolism; Proto-Oncogene Proteins c-mdm2 - genetics; Proto-Oncogene Proteins c-mdm2 - metabolism; Scanning electron microscopy; Signal Transduction; siRNA; Stem Cells; Tight junctions; Vascular endothelial growth factor; Vascular Endothelial Growth Factor A - genetics; Vascular Endothelial Growth Factor A - metabolism; Wound Healing
• ISSN: 1071-2690; 1543-706X; 1543-706X
• DOI: 10.1007/s11626-024-00946-8

In the present study, we examined the role of MDM2 in the angiogenesis process and its potential association with the sprouting of endothelial tip cells. To address this, we performed hypoxia-treated gastric cancer cells (HGC-27) to quantitative RT-PCR and Western blot analysis to measure the levels of MDM2 and VEGF-A mRNA and protein expression. Subsequently, we employed siRNA to disrupt MDM2 expression, followed by hypoxia treatment. The expression levels of MDM2 and VEGF-A mRNA and protein were subsequently reassessed. Additionally, ELISA was utilized to quantify the secretion levels of VEGF-A in each experimental group. A conditioned medium derived from HGC-27 cells treated with different agents was employed to assess its influence on the formation of EA.hy926 endothelial tip cells, using various techniques including Transwell plates migration assays, wound healing experiments, vascular formation assays, scanning electron microscopy, and immunofluorescence staining. These findings demonstrated that the in vitro knockdown of MDM2 in the conditioned medium exhibited significant inhibitory effects on endothelial cell migration, wound healing, and vascular formation. Additionally, the intervention led to a reduction in the presence of CD34 + tip cells and the formation of filopodia in endothelial cells, while partially restoring the integrity of tight junctions. Subsequent examination utilizing RNA-seq revealed that the suppression of MDM2 in HGC-27 cells resulted in the downregulation of the PI3K/AKT signaling pathway. Consequently, this downregulation led to an elevation in angiogenic effects induced by hypoxia.