Age-related macular degeneration (AMD) is caused by death of photoreceptor cells and their support tissue, retinal pigmented cells (RPEs) and consequently macula breakdown. Progression of AMD is linked to byproducts accumulation of cellular stresses such as oxidative stress, proteotoxic stress, inflammation and hypoxia. These conditions can trigger stress in endoplasmic reticulum (ER), which controls protein quality in cells via unfolded protein response (UPR). A large number of studies suggest that induction of 78 kDa glucose-regulated protein (GRP78), is a marker for ER stress due to its role in controlling the activation of transmembrane ER stress sensors (IRE1, PERK, and ATF6). In the present study the linkage between ER stress and inflammatory responses was explored through over-expression of GRP78 in adult RPE cells by using adeno-associated virus (AAV) helper free system as a prominent gene delivery system.

Validated GRP78 coding sequence was cloned into the expression pAAV-MCS plasmid vector. Afterwards the recombinant plasmid was co-transfected into the HEK239T cells conjointly with pHelper plasmid and pAAV-RC plasmid. pAAV-LacZ plasmid was used as control in a parallel co-transfection experiments. Recombinant AAV-2 viral particles were prepared from infected HEK293T cells and used to infect adult RPE cells according to spin infection method. Infected RPE cells examined for over-expression of GRP78 and the amount of CRP, CFH, C3, C5, MCP1, DAF and Caspase4 transcripts by Real-Time PCR.

Restriction digestion and sequencing of constructs confirmed the accuracy of GRP78 coding sequence cloned in pAAV-MCS plasmid vector. Recombinant AAV-2 viral particles production and infection into RPE cells were monitored by beta-galactosidase expression system. Real-Time PCR verified over-expression of GRP78 and significant changes in expression of C3 and CRP factors in transduced RPE cells.

The Results verified that ER stress may assist pathogenesis of AMD by activation of complement system. So progression of AMD may control by; decline of complement system components, elimination of ER stress and finally its main marker, GRP78.