staining by an antibody against the NF-H, a neuronal marker (Fig. 14.1b, c), suggesting that retinal ganglion cells are the major site of p22phox expression in the retina. Fluorescent staining was also observed in the RPE of pigmented (C57 Bl/6) mice, but may have been quenched somewhat by the presence of pigment granules (Fig. 14.1a); it was more evident in the RPE layer of albino mice (Fig. 14.1d).

To establish that p22phox is synthesized in the retinal cells in which we detected its accumulation, we employed in situ hybridization using an antisense probe to its mRNA. Hybridization conÞrmed p22phox expression in retinal ganglion cells and other neurons (Fig. 14.1e), as well as in RPE cells, which, again, was more evident in the eyes of albino mice (Fig. 14.1g).

14.4NADPH Oxidase and Choroidal Neovascularization

14.4.1Ocular Injection of siRNA-p22phox-AAV Leads to Reduced p22phox Protein Level

In order to investigate the role of p22phox in ocular pathology, we designed a synthetic cDNA encoding an siRNA speciÞc for the p22phox mRNA and cloned it in an AAV2 vector under the control of the H1 promoter (Fig. 14.2a). This siRNA was 19 bp, too short to compete with processing of endogenous micro RNAs when expressed as an shRNA [57]. A GFP reporter gene under the control of the CBA promoter was cloned upstream of the siRNA expression cassette in order to directly visualize expression from the vector after delivery. The recombinant virus was injected into the subretinal space of mouse eyes, and p22phox protein levels were analyzed 4Ð6 weeks after injection by Western blotting using protein extracts isolated from whole eyecups with the lens and cornea removed. As shown in Fig. 14.2, the level of p22phox protein was reduced by more than 80% in the retinas that received injection of AAV2-p22phox-siRNA.

14.4.2Subretinal Injection of AAV2-p22phox-siRNA Leads to Reduced CNV in the Mouse Model

To investigate the role of p22phox in CNV, the major late complication of AMD, the aforementioned AAV2-p22phox-siRNA and a control virus containing a scrambled siRNA under the control of the same promoter were tested in a murine model of CNV. In this model, BruchÕs membrane, separating the RPE layer from the choroid layer, is disrupted at discrete sites using a laser. The subsequent proliferation of blood vessels from the wound as detected by ßuorescein angiography is taken as a measure of CNV. In our experiment, right eyes were injected with AAV expressing the active or the scrambled siRNA and left eyes were uninjected. Because intravitreal injection with AAV2 leads to robust transduction of retinal ganglion cells but

Fig. 14.2 Effects of subretinal AAV2-CBA-p22phox-siRNA vector. (a) Map of AAV vectors used. GFP reporter gene is under the control of a chicken-b-actin promoter with a CMV enhancer (CBA), and the siRNA (either p22phox-speciÞc or scrambled sequence), expressed as small hairpin RNA (shRNA) is under the control of the H1 promoter. (b) Western blots of retinal protein using antibody against p22phox. Protein was analyzed from eyes in which right eyes received AAV2-CBA-p22phox-siRNA, while left eyes were untreated to show that p22phox protein is reduced in eyes receiving vector. (c) Quantitative analysis of Western blot data from extracts treated with siRNA-p22phox (n = 7). The protein levels were normalized to those of alpha-tubulin and the densitometric value from each of the shRNA-p22phox-treated eyes was expressed as a percentage of corresponding untreated contralateral controls (set at 100%). The p-value was calculated based on Wilcoxon signed rank test. (dÐf) siRNA-p22phox AAV treatment reduced CNV lesions. Representative CNV images from untreated eye (d) and eye received subretinal injections of shRNA-p22phox-AAV2 (e). (f) CNV area measurements in treated and untreated eyes expressed as mm2. Standard deviation is shown on each bar graph. IV intravitreous injection; SR subretinal injection; Ctrl control (scrambled siRNA)

does not lead to gene transfer to the outer layers of the retina or to the RPE [58] we also compared the effects on CNV of subretinal vs. intravitreal vector. Four to six weeks after vector injection both eyes were then treated with the laser and the resultant CNV evaluated 2 weeks later. Representative CNV images from untreated and treated eyes are shown in Fig. 14.2d, e. The extent of CNV is estimated by measuring the diameter of the vasculature emanating from the laser lesions (Fig. 14.2f).

CNV was inhibited in the eyes receiving subretinal injection of the siRNAp22phox vector, but not in the eyes receiving intravitreal injection of the same vector

Fig. 14.3 VEGF levels in the eyes received different route of ocular administration of siRNA-p22phox-AAV2 assayed by ELISA. The average levels in uninjected eyes are normalized as 100%. IV intravitreal injection;

SR subretinal injection

or in eyes injected with vector expressing the scrambled siRNA vector via either subretinal or intravitreal injection (Fig. 14.2f).

In a separate experiment, we also tested the same vector packaged into serotype 5 AAV capsids (AAV5-p22phox-siRNA). Serotype 5 vectors have a faster onset of transgene expression than serotype 2 and better tropism for RPE cells (unpublished observations). Unlike the original experiment, both eyes of test animals were lasertreated Þrst, and then the vector was subretinally injected into one of the eye of each animal 24 h later. The development of CNV was evaluated 2 weeks after the laser injury (13 days after subretinal injection). Again, the treated eyes showed signiÞcant reduction in CNV comparing to untreated eyes (Fig. 14.2f).

14.4.3Subretinal Injection of siRNA-p22phox Leads to a Decrease in the VEGF Level

Since NADPH oxidase activation is known to stimulate angiogenesis by triggering VEGF expression [59], we measured VEGF levels in eyes that received either intravitreal or subretinal injections of siRNA-p22phox-AAV. Eyes receiving subretinal vector had signiÞcantly reduced levels of VEGF compared to control eyes, whereas intravitreal vector did not change ocular VEGF levels (Fig. 14.3).

To further determine the oxidative status of the retina following downregulation of p22phox, we also examined the level of 4-Hydroxy-2-nonenal (HNE) and nitrotyrosine modiÞed proteins markers for oxidative stress [60, 61] in eyes that received either intravitreal or subretinal injections of siRNA-p22phox-AAV. By Western blot analysis, no difference was found in the eyes receiving siRNA-p22phox vector via either route of injection (data not shown).