Abstract
Purpose :
Angiogenesis is the growth of new blood vessels from the existing vasculature. Disruption of this process can result in pathological angiogenesis and eventual loss of vision. Retinal angiogenesis is a metabolically demanding process and metabolic rhythms within any cell are known to be regulated by the circadian clock. We tested the hypothesis that endothelial Bmal1 and per2, which are the core circadian clock genes are required to regulate endothelial cell growth in both retinal development and diseased conditions.
Methods :
To test this hypothesis, we conditionally deleted the circadian clock genes Bmal1 and Per2 from the VECs, using inducible Pdgf-iCreER transgenic animals. For developmental angiogenesis, retinas were stained for endothelial cells using Isolectin IB4 to analyze vascular density and migration. RNASeq was performed to identify the molecular mediators of Bmal1 in the endothelial cells. For pathological neovascularization, adult mouse RPE-Bruch’s membrane was ruptured using a diode laser, and CNV lesions were imaged using OCT and FA on days 7, 14, and 21 post-laser injury.
Results :
In animals with Bmal1 and Per2 deletion, VEC proliferation is drastically reduced in the superficial layer, resulting in reduced vessel density and delayed vascular angiogenic migration. Deletion of Bmal1 and Per2 after the vascular layers are established results in opposite phenotypes, this suggests a functional role for the circadian clock in the adult retinal vasculature. RNA sequencing data from the enriched endothelial cells suggests that pathways primarily related to cell cycle and circadian pathways are altered by the loss of Bmal1. Using a laser-induced CNV model, we show that loss of Bmal1 results in a significant decrease in both lesion volume and vascular leakage as shown by OCT and FA respectively, indicating reduced neovascularization. In contrast, Per2 mutants show an increase in lesion volume and vessel leakage.
Conclusions :
The data presented here raise an exciting question about the interrelationship between the circadian system in development and aging conditions. It is well known that the robustness of circadian rhythms decreases with age. Our results demonstrate that during development, the circadian system is co-opted to regulate angiogenesis by interacting with the cellular components that control cell cycle and proliferation while the same system plays an entirely different role in adults.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.