Abstract
AIM: To investigate the role of pyruvate kinase M2 (PKM2) in high glucose (HG)-stimulated retinal endothelial cells and its underlying molecular mechanisms and signaling pathways in retinal angiogenesis. METHODS: Human retinal microvascular endothelial cells (HRMECs) were cultured and divided into the following groups: normal glucose (NG, 5.5 mmol/L), HG (30 mmol/L), HG with PKM2 knockdown (HG+shPKM2), and HG treated with the pharmacological activator TEPP-46 (HG+TEPP-46). Cellular viability, proliferation, migration, and tube-forming ability were assessed using CCK-8, EdU, wound healing/Transwell, and Matrigel assays, respectively. The expression levels of PKM2, phosphorylated PKM2 (p-PKM2, Y105), hypoxia-inducible factor-1α (HIF-1α), and vascular endothelial growth factor A (VEGFA) were detected by Western blotting. The oligomerization status of PKM2 was analyzed via native gel electrophoresis. The subcellular localization of PKM2 was examined by immunofluorescence and nuclear-cytoplasmic fractionation. RESULTS: Under HG stimulation, the expression level of PKM2 was significantly increased (P<0.05). Knockdown of PKM2 was found to markedly suppress cell viability, proliferation, migration, and tube formation in HRMECs (P<0.05). Mechanistic studies revealed that phosphorylation of PKM2 at the Y105 site was promoted by HG treatment, which induced its dissociation from a tetramer to a dimer, thereby driving its nuclear translocation. Upon entering the nucleus, PKM2 was shown to exert critical non-metabolic functions; it was physically bound to HIF-1α and acted as its co-activator, leading to significant upregulation of VEGFA expression (P<0.05). In contrast, the PKM2 activator TEPP-46 effectively prevented dimerization and nuclear translocation of PKM2 by promoting its tetramerization. Consequently, the PKM2/HIF-1α axis-mediated upregulation of VEGFA was blocked, ultimately resulting in the reversal of HG-induced angiogenesis. CONCLUSION: HG influences retinal endothelial cell function by inducing PKM2 phosphorylation, dimerization, and nuclear translocation. The shift in PKM2 phosphorylation and oligomerization status represents a key mechanism through which TEPP-46 reverses HG-induced angiogenesis.