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Daniela F. Santos, Diogo B. Bitoque, Raquel L. Sequeira, Brigite Cabrita, Sónia Simão, Gabriela A. Silva; ACE2-overexpression protects RPE cells from inflammation and oxidative stress. Invest. Ophthalmol. Vis. Sci. 2021;62(8):1472.
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Treatment options for Diabetic Retinopathy (DR) are limited making research into alternative therapeutics of utmost need. The finding that the deregulation of the retinal Renin-Angiotensin System (RAS) triggers hallmarks of DR, support a role for RAS in this pathology.We have previously characterized the ACE/AngII/AT1R axis and found it to be over-activated in retinal pigmented cells (RPE) cells (Simão et al., 2016; 2017). We aim to investigate the potential benefit for DR of a gene therapy approach promoting the activation of the protective axis of the RAS, the ACE2/Ang (1-7)/Mas.
A plasmid containing the human gene of ACE2 (pEPito-hCMV-ACE2) was used to transfect RPE cells (D407), with pEPito-hCMV-eGFP used as control. The cellular localization and expression of ACE2 in RPE cells was analyzed by immunocytochemistry and Western blot. The expression of components from the protective axis of RAS, PEDF, VEGF, and pro-inflammatory markers TNF-α and IL1-β were evaluated by Western blot or ELISA, and the levels of ROS were assessed using an oxidative stress indicator.
The transfection of RPE cells with pEPito-hCMV-ACE2 significantly increased the expression of ACE2 and Mas receptor. Moreover, ACE2 localized in both the perinuclear and cytoplasmatic compartments of RPE cells. We found no differences in the levels of Ang (1-7) or expression of PEDF, VEGF or IL1-β when ACE2 is overexpressed in RPE cells. However, the levels of TNF-α in RPE cells transfected with pEPito-hCMV-ACE2 are much lower than those observed in cells transfected with the control (pEPito-hCMV-eGFP), which can be attributed to a protective effect from the ACE2 overexpression. A similar result has been observed for oxidative stress, with a significantly decrease in the levels of ROS in cells overexpressing ACE2.
Overexpression of ACE2 significantly increases the expression of the Mas receptor in RPE cells, with no effect on the levels of Ang (1-7). TNF-α and ROS levels are decreased in RPE cells overexpressing ACE2. Altogether these results point to a protective effect regarding inflammation and oxidative stress by ACE2 overexpression in RPE cells, and suggest a promising gene therapy approach towards the halting progression of retinal damage.
This is a 2021 ARVO Annual Meeting abstract.
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