γ-Secretase inhibition of murine choroidal neovascularization is associated with reduction of superoxide and proinflammatory cytokines.
OBJECTIVE
This study aimed to determine whether upregulation of γ-secretase could inhibit laser-induced choroidal neovascularization (CNV) and if this was associated with a reduction in both oxidative stress and proinflammatory cytokines.


METHODS
γ-Secretase, or its catalytic subunit presenilin 1 (PS1), were upregulated by exposure to either pigment epithelial derived factor (PEDF) or an AAV2 vector containing a PS1 gene driven by a vascular endothelial-cadherin promoter. Retinal endothelial cells were infected with AAV2 or exposed to PEDF in the presence or absence of VEGF and in vitro angiogenesis determined. Mouse eyes either received intravitreal injection of PEDF, DAPT (a γ-secretase inhibitor) or PEDF + DAPT at the time of laser injury, or AAV2 infection 3 weeks before receiving laser burns. Lesion volume was determined 14 days post laser injury. Superoxide generation, antioxidant activity and the production of proinflammatory mediators were assessed. Knockdown of γ-secretase was achieved using siRNA.


RESULTS
γ-Secretase upregulation and PS1 overexpression suppressed VEGF-induced in vitro angiogenesis and in vivo laser-induced CNV. This was associated with a reduction in the expression of VEGF and angiogenin 1 together with reduced superoxide anion generation and an increase in MnSOD compared with untreated CNV eyes. PS1 overexpression reduced proinflammatory factors and microglial activation in eyes with CNV compared with control. siRNA inhibition of γ-secretase resulted in increased angiogenesis.


CONCLUSIONS
γ-Secretase, and in particular PS1 alone, are potent regulators of angiogenesis and this is due in part to stabilizing endogenous superoxide generation and reducing proinflammatory cytokine expression during CNV.
Departments of Anatomy and Cell Biology, University of Florida, Gainesville, Florida 32610-0235, USA.
Xiaoping
X
Qi
Jun
J
Cai
Qing
Q
Ruan
Li
L
Liu
Sanford L
SL
Boye
Zhijuan
Z
Chen
William W
WW
Hauswirth
Renee C
RC
Ryals
Lynn
L
Shaw
Sergio
S
Caballero
Maria B
MB
Grant
Michael E
ME
Boulton
D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D020256:Choroidal Neovascularization / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D016207:Cytokines / Q000187:drug effects / Q000378:metabolism*; D004195:Disease Models, Animal; D005136:Eye Proteins / Q000008:administration & dosage; D005260:Female; D005451:Fluorescein Angiography; D005654:Fundus Oculi; D005786:Gene Expression Regulation / Q000187:drug effects; D007150:Immunohistochemistry; D058449:Intravitreal Injections; D051379:Mice; D008810:Mice, Inbred C57BL; D009414:Nerve Growth Factors / Q000008:administration & dosage; D018384:Oxidative Stress / Q000187:drug effects; D011480:Protease Inhibitors / Q000008:administration & dosage*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D015843:Serpins / Q000008:administration & dosage; D013481:Superoxides / Q000378:metabolism*; D000099174:Pigment Epithelium-Derived Factor
D053829:Amyloid Precursor Protein Secretases; D016207:Cytokines; D005136:Eye Proteins; D009414:Nerve Growth Factors; D011480:Protease Inhibitors; D012333:RNA, Messenger; D015843:Serpins; D013481:Superoxides; D000099174:Pigment Epithelium-Derived Factor