Inhibitory effect of gamma-secretase inhibitor on retinal neovascularization and regulative mechanism of Notch1 signal pathway

Objective

To investigate the inhibitory effect and underlying mechanism of gamma-secretase inhibitor blocking Notch1 signaling on retinal neovascularization caused by oxygen-induced retinopathy (OIR) in mice.

Methods

To establish the OIR model, 7-day-old pups of C57BL/6J mice were exposed to 75% oxygen together with their mother until postnatal day (P)12.On P12, the mice were transferred to room air.All the mice were randomly divided into three groups, OIR group as control group, OIR+ DAPT group and OIR+ DMSO group receiving 1 μl intravitreal injection of gamma-secretase inhibitor (DAPT, 10 mmol/L) and 1∶20 DMSO dilution respectively.The right eye was taken as experimental eye.The mice were euthanized on P17 and the eyes were harvested to obtain retinas for further investigation.The total proteins were extracted from the retinas.The relative expression levels of Notch1 signal pathway and its downstream Hes1, the markers of M1 phenotype inducible nitric oxide synthase (iNOS) and M2 phenotype arginase-1 (Arg-1) microglia were measured by western blot.Retinal flat mounts were made and the retinal vessels were stained with isolectin B4 (IB4) to investigate the relative retinal neovascularization areas which was calculated as the ratio of neovascularization area/retinal area.The mumber of the neovascular endothelium cells beyond the inner limiting membrane was observed by hematoxylin-eosin staining.The use and care of animals complied with ARVO statement.This study protocol was approved by the Animal Ethics Committee of Guangdong Provincial People’s Hospital (No.KY-Z-2021-2015-01).

Results

The relative protein expression levels of Notch1 and Hes1 in OIR+ DAPT group, OIR group, and OIR+ DMSO group were 0.68±0.06 and 0.70±0.08, 1.00±0.00 and 1.00±0.00, 1.03±0.08 and 1.02±0.07, respectively, with statistically significant differences among them (F=70.62, 53.65; both at P<0.01). Compared with the OIR group and OIR+ DMSO group, the expressions of Notch1 and Hes1 were significantly reduced in OIR+ DAPT group (all at P<0.01). The relative protein expression levels of iNOS and Arg-1 in OIR+ DAPT group, OIR group, and OIR+ DMSO group were 0.74±0.07 and 1.49±0.12, 1.00±0.00 and 1.00±0.00, 1.04±0.10 and 0.94±0.07, respectively, showing statistically significant differences (F=31.63, 89.32; both at P<0.01). Compared with OIR group and OIR+ DMSO group, the expression of iNOS in OIR+ DAPT group was significantly reduced, and the expression of Arg-1 was significantly increased (all at P<0.01). The relative neovascularization area and the number of neovascular endothelium cells beyond the inner limiting membrane in OIR+ DAPT group, OIR group, and OIR+ DMSO group were (8.82±2.71)% and 38.17±3.29, (22.32±5.34)% and 60.83±5.11, (20.27±3.36)% and 58.67±4.75, respectively, showing statistically significant differences (F=33.72, 39.44; both at P<0.01). The relative neovascularization area and the number of neovascular endothelium cells in OIR+ DAPT group were significantly reduced in comparison with OIR group and OIR+ DMSO group (all at P<0.01).

Conclusions

Intravitreal injection of DAPT can inhibit the retinal neovascularization in OIR mice through blocking Notch1 signaling activation and promoting retinal microglia polarization from M1 to M2 phenotype.

Microglia; Retinal neovascularization; Gamma-secretase inhibitor; Notch1 signaling; Oxygen-induced retinopathy; Mice