-Secretase Is A Critical Regulator Of Laser-induced CNV In The Mouse

Abstract

Purpose: : We have previously shown that pigment epithelial-derived factor (PEDF) inhibits VEGF-induced in vitro angiogenesis via a γ-secretase dependent pathway (Cai et al, J Biol Chem 2006). The aims of this study were to first determine if PEDF inhibition of CNV was dependent on γ-secretase and second to assess whether viral-mediated overexpression of presenilin-1 (PS-1) alone, a critical subunit of the γ-secretase complex, could also inhibit CNV.

Methods: : Mice received intravitreal PEDF in the presence or absence of the γ-secretase inhibitors (DAPT, L685485) immediately following laser induction of CNV. Animals were sacrificed 14 days post laser and the volume of CNV lesions was quantified. For viral-mediated overexpression, the mouse PS-1 gene driven by the VE-cadherin promoter was tagged with HA and inserted into a recombinant quadruple mutant adeno-associated virus serotype 2 (AAV2-VEc-PS-1). For in vitro studies, bovine retinal microvascular endothelial cells were infected with AAV2-VEc-PS-1 or control virus and 1) PS-1 expression was determined by PCR and Western blot and 2) the effect on in vitro angiogenesis was assessed in the Matrigel tube forming assay. The effect of PS-1 overexpression on CNV was assessed by subretinal injection of AAV2-VEc-PS-1 or control virus followed by laser induction of CNV 3 weeks later.

Results: : PEDF induced a greater than 60% (p<0.001) reduction in CNV which was completely blocked by either γ-secretase inhibitor confirming γ-secretase as a critical regulator of CNV. Transfection of cultured retinal endothelial cells with AAV2-VEc-PS-1resulted in a greater than 40% overexpression in PS-1 which reduced tubule formation in the Matrigel assay by 45% compared to control (p<0.05). After subretinal delivery of AAV2-VEc-PS-1, PS-1 expression was increased 1.5 fold (p<0.05) and PS-1 was localized to vessels of both the retinal and choroid. Subretinal AAV2-VEC-PS-1 injection resulted in a 65% reduction in CNV lesion size (p<0.001). This was associated with an increase in mRNA levels of the antioxidant enzymes (SOD1, SOD2, GST) and a decrease in proinflammatory cytokines (MCP1, Rantes, TNFα) in the posterior cups.

Conclusions: : Increasing the activity of γ-secretase or increasing the expression of the key component of γ-secretase, PS-1, may represent novel therapeutic strategies for treatment of CNV.

Keywords: choroid: neovascularization • growth factors/growth factor receptors • gene transfer/gene therapy