γ-Secretase inhibition of murine choroidal neovascularization is associated with reduction of superoxide and proinflammatory cytokines.

Neovascularization is a major cause of vision loss in patients with age-related macular degeneration (AMD) that is characterized by abnormal, new vessel growth into the subretinal space from the underlying choroid resulting in choroidal neovascularization (CNV).1,2 It is now quite evident that there is a plethora of pro- and anti-angiogenic factors that regulate the ocular vasculature and are involved in the development and progression of aberrant neovascularization such as AMD. The collective evidence suggests that the VEGF family is critical for ocular angiogenesis and treatment of AMD patients with CNV with agents that neutralize extracellular VEGF significantly reduce CNV.3,4 Pigment epithelium-derived factor (PEDF) is a potent inhibitor of VEGF-induced angiogenesis5,6 and the RPE is a major source of PEDF in the retina. The decline in expression of PEDF, both with increasing age and in AMD, facilitates a proangiogenic subretinal environment.7–9 Although numerous studies have demonstrated that PEDF inhibits VEGF-induced CNV the mechanisms are poorly understood and no definitive receptor has yet been identified. Previously, we have reported that PEDF inhibits VEGF-induced angiogenesis in cultured microvascular endothelial cells. This inhibition is due to a γ-secretase dependent cleavage and intracellular translocation of VEGF receptor (VEGFR) 1 10 and others have reported that VEGFR2 is cleaved in a similar fashion in RPE cells.11 γ-Secretase is a complex composed of four different integral membrane proteins: presenilin (PS), nicastrin, Aph-1, and Pen-2.12–14 The most studied component of the γ-secretase complex is presenilin which is an integral enzyme in the cleavage of amyloid precursor protein and contributes to the accumulation of amyloid-β peptide in Alzheimer's disease. Activation of PS is dependent on its endoproteolysis of full length PS into an N-terminal fragment (NTF) and C-terminal fragment (CTF).10,12 Nicastrin appears to be necessary for substrate recognition by the γ-secretase complex and nicastrin binding to the substrate is required before presenilin can exert its proteolytic activity.15 Of the two remaining proteins, which constitute γ-secretase, Aph-1 is believed to be a scaffolding protein and Pen-2 appears to regulate PS activity. Assembly of the γ-secretase complex begins in the endoplasmic reticulum and is concluded after translocation of the four proteins to the cell membrane.12,14 Valine residue(s) followed with charged residues within the transmembrane domain serve as cleavage sites for γ-secretase16 and we have recently demonstrated that valine 767 is critical for VEGFR1 cleavage by γ-secretase.17 It is also evident that PS can regulate protein trafficking and protein-protein interactions independently of its protease activity and association with the γ-secretase complex.17–20 There is extensive evidence that oxidative stress is associated with both the wet and dry forms of AMD.1,2,21 Antioxidants such as N-acetyl-cysteine and siRNA against p22phox (an integral subunit of NAPDH oxidase) are potent inhibitors of laser-induced CNV in animal models.22,23 Furthermore, a combination of oxidative stress and vascular growth can lead to an upregulation of proinflammatory cytokines which further exacerbate the progression of CNV24 and anti-inflammatory strategies have been shown to ameliorate CNV.25–27 PEDF, which we have shown regulates γ-secretase activity in cultured cells,10,17 has been previously reported to reduce both oxidative stress and inflammation in the retina.28,29 We therefore decided to determine in this study whether increasing expression of the γ-secretase complex, or of PS alone, can inhibit laser-induced CNV in the mouse CNV model and if this is associated with protection of the retina from oxidative damage and inflammation.