Abstract
Purpose :
Muller cells which comprise the primary glial cell type in the retina are known to secrete angiogenic factors under hypoxic conditions similar to those found in the mouse oxygen induced retinopathy (OIR) model and clinically in diabetic retinopathy. Previously, we constructed adeno-associated virus (AAV) vectors containing a hypoxia responsive domain together with the glial fibrillary acidic protein (GFAP) promoter to drive transgene expression specifically in Muller cells and astrocytes in culture and in the mouse OIR model.. In this study we tested a hypoxia regulated and glial cell specific vector for driving expression of the anti-angiogenic factor decorin in the retina.
Methods :
AAV vectors were constructed with the ubiquitously active CMV promoter, the GFAP promoter or a regulatory region containing the GFAP promoter plus a hypoxia responsive domain (HRE). Vectors contained the human decorin coding sequence. Vectors were used to infect rat brain astrocytes, PC12 cells (neuronal cell line) or ARPE-19 cells (retinal epithelium cell line). Cells were then exposed to hypoxia (72 h) or normoxia. Decorin mRNA levels were determined using real time RT-PCR.
Results :
In primary astrocytes infected with AAV-HRE-GFAP-decorin and exposed to hypoxia, an approximate 5 fold increase in decorin mRNA was observed relative to normoxic cells. Infection with AAV-GFAP-decorin resulted in high levels of decorin mRNA expression in both hypoxic and normoxic astrocytes. In the control ARPE19 cells and PC12 cells there was no significant decorin mRNA transcription in either hypoxia or normoxia. Infection with AAV-CMV-decorin resulted in high levels of expression of decorin mRNA in astrocytes, PC12 cells and ARPE19 cells in both normoxia and hypoxia.
Conclusions :
Our study indicates that AAV-HRE-GFAP-Decorin expresses decorin mRNA in a hypoxia inducible and glial cell dependent manner. The glial specific AAV-GFAP decorin vector expresses decorin mRNA in astrocytes in both normoxic and hypoxic conditions. A positive control vector, AAV-CMV-decorin, expresses decorin mRNA in astrocytes as well as in PC12 cells and ARPE19 cells in both hypoxia and normoxia. The AAV-HRE-GFAP decorin vector may be applicable for use as a hypoxia inducible and glial cell specific vector for preventing neovascularization in models of diabetic retinopathy.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.