Pigment Epithelium-Derived Factor From Müller Cells Is Involved in Angiopoietin-Mediated Survival Control of Retinal Neurons

Purpose: This study was performed to determine whether angiopoietin (Ang)-1 and Ang-2 influence the survival of retinal neurons via Müller cell-mediated mechanisms. Methods: Expression of Ang-1, Ang-2, and Tie-2 in retinal cells and their hypoxia-dependent regulation were investigated using (quantitative) reverse transcription (RT)-PCR (qPCR), immunofluorescence staining, and Western blotting. Survival of Ang-stimulated R28 cells was assessed in homotypic cultures and co-cultures with rat Müller cells (RMCs). Ang-1, Ang-2, PEDF in RMCs and PEDF receptor (PEDF-R) in R28 cells were knocked down by siRNA. Tie-2-PI3 kinase (PI3K)/Akt activation was demonstrated using signaling blockers and by detecting Tie-2-Akt phosphorylation. PEDF expression and secretion were determined by qPCR and ELISA. Results: Ang-1, Ang-2, and Tie-2 are expressed in primary retinal cells, in particular, RMCs and human Müller cells of gliosis tissue. Under hypoxia, Ang-1 and Ang-2 expression declined in RMCs and R28 cell viability was reduced. Although both angiopoietins did not significantly influence R28 cell survival in homotypic cultures, co-cultures with Ang-deficient RMCs revealed that Müller cell-derived Ang-1 promoted, whereas Ang-2 impaired, R28 cell survival. Ang-1 induced Tie-2 and PI3K/Akt activation in RMCs and prevented hypoxia-induced PEDF downregulation. Ang-2 suppressed Tie-2-Akt phosphorylation and PEDF expression. Impaired R28 cell survival under hypoxia or caused by Ang-1 knockdown in co-cultured RMCs was mitigated by Ang-2-deficient RMCs or rescued by supplemental Ang-1 in a PEDF-/PEDF-R-dependent fashion. Conclusions: Ang-regulated signaling and PEDF expression constitute a significant pathway in Müller cells to control retinal neuronal survival. This has implications for effector functions of the therapeutic antibody, Faricimab.