PI3K in Retinal Angiogenesis Figure 7. LY294002 inhibits retinal angiogenesis without affecting retinal function. A–C) Electroretinography of control (n = 13) and LY294002-treated (n = 15) zebrafish at 5 dpf. Shown is representative data from control (A) and LY294002-treated (B) larval zebrafish in response to an intensity series of white light stimulation. The stimulus flash was attenuated from 3 log units to unattenuated. The black bars at the bottom represent stimulus duration. The two groups show similar intensity response curves for the b-wave (C). To generate the intensity response curve, the average response at the maximum flash intensity was set to 1 and the average response at attenuated flash intensities was normalised to this (average 6 SEM). D) Histological sections of zebrafish eyes from 5 dpf larvae treated with LY294002 from 1–5 dpf or 2–5 dpf. Retinal histology appears normal in fish treated from 2–5 dpf showing lamination, presence of optic nerve and similar size/shape to controls. In larvae treated from 1– 5 dpf sparse apoptotic nuclei (arrowheads) and tissue vacuoles are observed (Magnification 4006). doi:10.1371/journal.pone.0007867.g007 assay, fish with normal visual function track rotating black and white stripes via reproducible saccades. No difference is observed in the OKR response of fish injected intraocularly with vehicle alone or with LY294002 (Fig. 8B). These results indicate that intraocular administration of LY294002 can selectively block angiogenesis in the eye without detrimental effects on visual function. Discussion Summary We report here evidence demonstrating that the small molecule inhibitor LY294002 can be an effective and safe inhibitor of ocular angiogenesis in vivo. LY294002 inhibits normal and extraneous developmental angiogenesis in the zebrafish eye, in dose- and time-dependent manners. Systemic administration of LY294002 results in oedema and locomotor activity defects are indicated (data not shown). However, intraocular administration of LY294002 can selectively inhibit intraocular angiogenesis without affecting retinal morphology or visual function. LY294002- An Ocular Anti-Angiogenic Does LY294002 or its cellular target have potential for clinical use as an ocular anti-angiogenic? Our data demonstrates that LY294002 is an effective inhibitor of hyaloid vasculature angiogenesis in the zebrafish eye. In addition, recent studies report that intraocular administration of LY294002 reduces unwanted retinal and choroidal neovascularisation in murine models [15,16]. The mouse studies did not address potential effects of LY294002 on retinal function, but our study shows that short- term treatment with LY294002 can have no effect on ocular morphology or visual physiology. LY294002 is most likely exerting its anti-angiogenic activity by inhibiting the PI3K signalling pathway, which is known to mediate angiogenesis [30]. Indeed, LY294002 is a classic, broad-spectrum inhibitor of phosphoino- sitide 3-kinases (PI3K) [33]. The PI3K enzyme family act as second messengers during signal transduction events associated with diverse cellular activities, including migration, proliferation and differentiation [30]. Recent evidence suggests that LY294002 also inhibits casein kinase, GSK3b and mTOR (a PI3K-related kinase) [34]. LY294002, however, was previously excluded from PLoS ONE | www.plosone.org 7 November 2009 | Volume 4 | Issue 11 | e7867