­PlGF Regulates Angiopoietin-1 and Tie-2 Expression in Human Retinal Endothelial Cell-Pericyte Cocultures and iPSC-Derived Vascular Organoids through VEGFR1

Abstract Placental growth factor (PlGF) and Angiopoietin (Ang)-1 are two angiogenic factors that play vital roles in vascular cell growth and stabilization. The present study's objective is to examine PlGF's regulation of Ang-1 and its Tie-2 receptor expression in human vascular cells and vasculature. We exploited the cocultures of human primary retinal endothelial cells (HREC) and pericytes (HRP) and the blood vessel or vascular organoids derived from human-induced pluripotent stem cells (iPSC) as experimental models. In the HREC-HRP cocultures, PlGF blockage upregulated the expressions of Ang-1 and Tie-2 in an antibody dose-dependent manner. Upregulation of Ang-1 and Tie-2 by PlGF blockade did not occur in HREC and HRP monocultures but only in HREC-HRP cocultures, indicating the interactions of the two cell types. VEGFR1 inhibition diminished Ang-1 and Tie-2 upregulation caused by PlGF blockade and reduced pericyte variability in high glucose conditions. In the iPSC-derived vascular organoids (VO), PlGF, Ang-1, and Ang-2 were expressed mainly by perivascular cells. Bioinformatics analysis of RNA sequencing data revealed that diabetes-mimicking conditions upregulated PlGF and Ang-2 expressions in the VO cultures. PlGF blockade upregulated Ang-1 and Tie-2 expression and promoted pericyte coverage and association with ECs in the VO. Together, the data suggest that PlGF regulates Ang-1 and Tie-2 expression in part through VEGFR1, which is involved in vascular cell function and stabilization. The findings may help design new therapeutic interventions for diabetic vasculopathy, such as diabetic macular edema and proliferative diabetic retinopathy, by targeting PlGF and Ang signaling pathways.