PI3K in Retinal Angiogenesis Figure 8. Intraocular injection of LY294002 inhibits angiogenesis but not visual function. A) The hyaloid vasculature of 5 dpf larvae intraocularly injected with 10 mM LY294002 at 2 dpf shows abnormal patterning and reduced numbers of primary branches compared with controls injected with 1% DMSO (left images are lateral views, and right images are dorsal views of lenses. OD: optic disc; scale bars: 50 mm) B) Graph of the percentage of 5 dpf larvae with optokinetic responses (OKR) following intraocular injection of LY294002 at 1 or 2 dpf. The Fisher test shows no statistically significant difference in visual function when LY294002 is injected intraocularly at 1 or 2 dpf (p values shown on table) C) Graph showing percentage of 5 dpf larvae with normal hyaloid vasculature after injection of LY294002 at 1 or 2 dpf. Intraocular LY294002 at 1 or 2 dpf, significantly inhibits hyaloid angiogenesis. Data graphed from 3 independent experiments; LY294002 at 1 dpf, n = 17; 1% DMSO at 1 dpf, n = 9; LY294002 at 2 dpf, n = 26; 1% DMSO at 2 dpf, n = 13 (***p,0.001, Chi-Square test). doi:10.1371/journal.pone.0007867.g008 clinical development due to toxicity issues. These may arise from the broad-spectrum of PI3Ks inhibited, off-target effects or systemic administration. Refined Targeting of PI3K Pathways Our data suggests that LY294002 can have selective anti- angiogenic effects in the eye following intraocular administration. No perturbations of retinal function or morphology are observed. Future studies will need to determine if the lack of toxicity is due to local administration to the eye, species difference or short-term treatment. However, instead of using a broad-spectrum PI3K inhibitor like LY294002, it should also be possible to selectively interfere with the subset of PI3K signals associated with pathological angiogen- esis in the eye. The development of isoform-selective PI3K inhibitors has already led to their approval for clinical trials in cancer [35,36]. The PI3K family is divided into several classes based on protein structure and substrate targets. The signal transduction mechanism is best understood for Class I PI3Ks [30]. These are heterodimers, comprising of one of four p110 catalytic subunits (a, b, d or c) plus a regulatory subunit. Growth factor or G protein-coupled receptor activation recruits the regulatory subunit of PI3K resulting in activation of the catalytic subunit. Subsequent conversion of PI(4,5)P2 to PI(3,4,5)P3 promotes the recruitment of proteins exemplified by AKT to the plasma membrane. AKT is phosphorylated by phosphoinositide-dependent protein kinases and targets proteins involved in angiogenesis, apoptosis, cell growth and glucose uptake [37,38]. Analysis of p110d and c reveals specific roles for these isoforms in the immune system. Indeed, IC87114, an inhibitor selective for p110d, was subsequently found to inhibit a form of leukaemia and is now in clinical trial [39]. Similarly, genetic analyses demonstrate that the p110a isoform is selectively required for angiogenesis [40]. To our knowledge no p110a-selective inhibitor has gone on to clinical development. However, several potential p110a selective inhibitors have been reported [35,39]. Our data with LY294002 strongly support testing the efficacy and safety of p110a-selective PI3K inhibitors in models of ocular neovascularisation. An alternative approach to isoform-selective PI3K inhibitors is the use of a modified version of LY294002. Until recently, it was generally considered that broad-spectrum PI3K inhibitors would not have clinical use due to toxicity arising from inhibition of physiological signalling. However, SF1126 is a modified version of LY294002 in clinical development for cancer. It consists of LY294002 linked to a peptide sequence that targets it to the vasculature [41]. Future studies are warranted to test the efficacy and safety of this prodrug in models of ocular angiogenesis. Screens for Ocular Anti-Angiogenics in Zebrafish Here we provide further proof-of-principle that chemical screens in zebrafish can uncover novel anti-angiogenic drugs for the eye. The screens are relatively simple with 5 larvae per well incubated from 1–5 days post-fertilisation with an experimental drug. Assessment of ocular angiogenesis is facilitated by the using the Tg(fli1:EGFP) line and a fluorescent dissecting microscope. The limiting step is the labour-intensive dissection of lenses but this may be overcome with improvements in imaging in vivo. Kitambi et al. recently identified several drugs that perturb intraocular angiogenesis in vivo [21] and our study demonstrates how such drugs can be further characterised to address specificity and safety. Finally, our screens PLoS ONE | www.plosone.org 8 November 2009 | Volume 4 | Issue 11 | e7867