Abstract
Purpose :
To assessed the function of mTOR complex-1 (mTORC1) and mTORC2 as well as the effect of rapamycin (sirolimus) on CNV using an experimental laser-induced mouse model.
Methods :
CNV was induced by laser photocoagulation of C57/BL6 mice (8 weeks of age) eyes and confirmed by fundus fluorescein angiography (FFA). In experiment A, the natural course of CNV development was observed during the 12-days after the laser injury. The expression of mTOR-related proteins was evaluated using Western blot (WB) and immunofluorescence staing (IF). In experiment B, rapamycin (3mg/kg) was injected intraperitoneally daily for 5 days (5d+Rapa) – prevention arm, 7 days after the 5th day of laser photocoagulation (5d+Rapa7d) and 12-days (12d+Rapa) – treatment arm. WB and IF were performed to evaluate the mode of action and the effect of mTOR inhibition on CNV development.
Results :
. In experiment A, the mTORC1 and mTORC2 activity were observed during the 5th – 12th day of laser injury by WB. Immunofluorescence imaging of cryosections of mice sacrificed on day-7 revealed greater co-immunoreactivity of inflammatory cell markers (CD11b and F4/80) with p-mTOR S2448 positive cells, while markers of endothelial cells (ECs, CD31), pericytes (α-SMA) and the retinal pigment epithelium cells (RPE, cytokeratin) were colocalized with p-mTORC S2481 positive cells. In experiment B, the expression of HIF1-α, VEGF-A, VEGF-R2, and mTORC1 activity, as well as mTORC2, were downregulated in all rapamycin-treated groups. WB analysis demonstrated rapamycin administration for 7 days after the 5th day of CNV induction increased LC3-II/LC3I ratio. Autophagy activation was also confirmed by immunofluorescence staining, which showed the presence of ATG9 positive cells in the CNV lesion. Human neovascular tufts showed that an abundant amount of CD31 positive cells have a high immunoreactivity to p-mTOR S2481.
Conclusions :
mTOR is a critical player during the subacute stage of the CNV development in laser-induced mouse model through differentially acting with the mTORC1 and mTORC2 axes. mTORC1 activity was high predominantly in inflammatory cells in CNV lesion, while mTORC2 activity was higher in vascular components and the RPE.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.