Abstract
Purpose :
Loss of endothelial cell integrity is a critical factor of ischaemic retinopathies. The pathogenesis is highly complex and multifaceted but the AT2 pathway has been implicated in vascular pathology in both the retina and brain. For example, in a murine model of cerebral malaria, deletion of AT2 augmented blood barrier breakdown and localized hemorrhage. This study tested the hypothesis that deletion of AT2 could lead to damaging effects in the retinal vasculature during ischaemic conditions.
Methods :
AT2 knockout (KO) and C57BL/6J wildtype (WT) mice were subjected to the OIR protocol. At postnatal days 13 and 17 (P13; P17) eyes were enucleated, flat-mounted and the vasculature stained using lectin and TER119 (red blood cell marker). Using ImageJ, levels of retinal vasobliteration, neovascularization and hemorrhage were assessed. In a parallel in vitro experiment, vasopermeability was induced in Human Retinal Microvascular Endothelial Cells (HRMECs) monolayers with VEGF (50ng/ml) and/or treated with an endogenous agonist for AT2 (EA)(10-10 µM) with barrier function being assessed via xCELLigence.
Results :
At P13 OIR, AT2KO mice (N=11) displayed an increase in retinal vasobliteration as compared to WT (N=12)(P<0.01). IBA1+ microglia, displayed regular and diffuse staining patterns within the WT retina coupled with highly ramified morphology, whereas the AT2KO mice, showed dense clustering of microglia along the avascular interface with the vasculature while displaying an amoeboid-like phenotype. At P17 OIR, AT2KO mice (N=13) had increased retinal neovascularization when compared to WT (N=12)(P<0.05). Retinal hemorrhage was macroscopically visible in AT2KO mice, but not in WT. This was further supported via TER119 staining in murine retinas, where AT2KO (N=4) had a greater TER119+ area than WT (N=5)(P<0.05). HRMECs exposed to VEGF showed a reduction in endothelial integrity (P<0.05), which was attenuated upon pre-treatment with EA (P<0.05).
Conclusions :
Genetic deletion of AT2 induced greater vasobliteration and neovascularization in OIR. AT2 is a key regulator of retinal vascularisation and barrier integrity with a lack of AT2 being detrimental and activation being protective. Taken together, our data suggest that activation of AT2, may provide a therapeutic option in strengthening tight junctions in diseases where endothelial integrity is compromised.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.