14.1Introduction

Age-related macular degeneration (AMD) is the most common form of irreversible vision loss among the elderly in industrial countries [1Ð5]. The prevalence of AMD increases with age and currently about 1.75 million people have advanced AMD with associated vision loss. This number is expected to grow to almost three million by 2020 [4, 6]. There are two forms of AMD: dry (or atrophic, or nonexudative) and wet (neovascular or exudative). Dry AMD is the most common. However, wet AMD is associated with more sudden and severe vision loss. Approximately 10Ð15% of dry AMD cases progress to the wet form. Wet AMD is characterized by new blood vessel growth beneath the retina and is referred to clinically as choroidal neovascularization (CNV).

The pathogenesis of AMD is complex, involving a variety of genetic and environmental factors. Increasing experimental and clinical evidence indicates that pathogenic oxidative mechanisms contribute to the progression of AMD [7, 8]. The retina is particularly vulnerable to oxidative damage due to a number of factors: greater oxygen consumption than any other tissue, life-long and accumulative exposure to light (and thus prone to photooxidative damage), and enrichment in polyunsaturated fatty acids and photosensitizers in the retina and similar products in the adjacent retinal pigment epithelium (RPE) [7]. In addition, phagocytosis of photoreceptor outer segments by the RPE cells, a critical process in visual function, also results in the generation of reactive oxygen species (ROS) [9Ð11]. The role of oxidative stress in pathogenesis of AMD is also supported by clinical studies demonstrating a reduced risk for AMD with dietary supplements of antioxidants [12, 13]. Furthermore, oxidative free radicals modulate immune-inßammatory system, which also plays an important role in pathogenesis of AMD [14Ð17], at least in part due to an enhanced expression of proinßammatory genes [18]. Inßammation in turn, enhances oxidative stress, causing a self-perpetuating ampliÞcation loop of oxidative stress/inßammation and tissue damage [19].

ROS, including free radicals such as superoxide anion (O2−), nitric oxide (NO−), hydroxyl (OH−), and nonfree radicals such as H2O2 [20], can be generated physiologically as by-products of normal biological reactions as occurring in mitochondria, peroxisomes, and the endoplasmic reticulum [21Ð29]. NADPH oxidase, however, is one of the few enzymes that result in ROS generation not as a by-product, but rather as the primary function of the enzyme system [30Ð32], and it has been shown to be one of the main intracellular ROS sources in the vascular system [33, 34]. NADPH oxidases participate in a broad array of cellular functions including cell proliferation, differentiation, migration, growth, apoptosis, and cytoskeleton regulation. NADPH oxidase-derived ROS has been implicated in a variety of pathologies including pathologic angiogenesis, inßammation, hypertension, and diabetes [35Ð42].

In order to assess the importance of NADPH oxidase in retinal oxidative processes, we examined the expression of its p22phox subunit, an integral component of the NADPH oxidase multisubunit enzyme complex, in mouse retina, and found that p22phox is normally expressed in RPE cells, as well as in retinal neurons.

A sequence speciÞc siRNA against p22phox efÞciently reduced its expression when delivered as a small hairpin RNA under the control of an H1 promoter via recombinant adeno-associated virus (AAV). This AAV-siRNAp22phox vector inhibited CNV in the laser induced rodent model. These results suggest that NADPH oxidasemediated ROS production by RPE cells may play an important role in promoting the pathogenesis of AMD and this pathway may therefore represent a new target for AMD therapeutic intervention.

14.2NADPH Oxidase and Redox Signaling

NADPH oxidases are a family of multimeric enzymes that catalyze the transfer of electrons from NADPH to molecular oxygen via their catalytic subunits to generate superoxide (O2−). The active NADPH oxidase complex consists of two membranebound catalytic subunits, p22phox and gp91phox, the cytoplasmic proteins p40phox, p47phox, p67phox, and the GTP-binding protein, Rac1/Rac2. The enzyme converts NADPH to NADP and produces superoxide upon assembly of the active complex [43, 44]. NADPH oxidase was originally identiÞed in neutrophils as a key component of the innate immune response. It is now well established, however, that NADPH oxidase and related enzymes are also present in many nonphagocytotic cells and tissues. Six additional homologs of the prototype gp91phox (also known as NOX2) have been identiÞed in various tissues and cell types including neurons, cardiomyocytes, skeletal muscle myocytes, hepatocytes, endothelial cells, and hematopoietic cells [45]. The p22phox subunit forms heterodimers with various NOX enzymes and with the cytoplasmic components to form stable complexes that participate in many other important cellular processes, including signal transduction, cell proliferation, and apoptosis, and contribute to a multitude of physiological events [45]. The assembly and activity of the NADPH oxidase protein complex is regulated by cytosolic components. In phagocytes, NOX2 is localized in both internal membranes and in the plasma membrane closely associated with p22phox. In resting neutrophiles and endothelial cells [46], most NOX2 is localized within the intracellular membranes. Upon stimulation, the cytosolic components such as p47phox and p67phox translocate to the plasma membrane, where they associate with the membrane-bound subunits, catalyzing electron transfer from the enzyme complex to molecular O2 and thus generating superoxide [45, 47]. In nonphagocytotic cells, the subcellular distribution of NOX2 varies depending on the cell type. Low levels of ROS are produced in a highly regulated manner as signaling molecules in a wide variety of cells and under various conditions to mediate several types of responses, including cell proliferation, migration, differentiation, and gene expression [20, 39, 40, 48, 49]. ROS modulate signaling cascades and ultimately the physiological response of the cell by reversible oxidation of redox-sensitive target proteins that may lead to downstream receptor activation [50]. Critically, however, overproduction of ROS due to increased activation of NADPH-oxidase has been shown to be involved in a variety of pathologic conditions [41, 45, 51Ð53]. Furthermore, NADPH oxidase-derived ROS elevation often leads to activation of

other ROS generating systems, such as xanthine oxidase [54], mitochondria [55], and eNOS uncoupling [56], amplifying the initial responses. Moreover, ROS can also activate Nox enzymes and increase their own production in a positive feedback loop [56].

14.3Expression of NADPH Oxidase Subunit p22phox in the Retina

NADPH oxidase is produced in phagocytic cells including those of the RPE, a monolayer of cells whose functions include turnover of rod photoreceptor outer segments. Although NADPH oxidase activity has been detected in cultured human RPE cells [9, 10], this enzyme has not been precisely localized within the neural retina. To detect the expression of p22phox protein in the mouse retina, we dissected eyes from C57 Bl/6 mice and separated the neural retina from the RPE choroid complex. Western blotting using a polyclonal antibody against p22phox detected a protein of molecular weight 22kd in both retinal and RPE cell extracts (data not shown). To examine the cell types that express p22phox in more detail, we carried out indirect immunoßuorescence staining of retinal tissue sections using the same antibody. Positive staining was detected in the retinal ganglion cell layer and outer plexiform layer, a dense network of synapses between photoreceptor cells and the dendrites of secondary neurons (Fig. 14.1a). The majority of p22phox staining colocalizes with

Fig. 14.1 p22phox expression in the mouse eye. p22phox expression was detected by immunoßuorescence (aÐd), and in situ hybridization (eÐh). (aÐc) Cross sections of pigmented C57BL/6J mouse eye double labeled with antibody against p22phox (red) and NF-H (green); (d) cross sections of an albino mouse eye. (a) p22phox expression in retina of the pigmented C57BL/6J mouse (red). (b) The same Þeld as in (a) stained with NF-H antibody (green). (c) Merged image of (a, b). (d) p22phox expression in the albino mouse eye showing clear expression in RPE cells in addition to the RGC and the IPL. The mRNA p22phox in the retina was detected by in situ hybridization. (eÐf) Cross sections from the pigmented C57BL/6J mouse eye; (gÐh) cross sections from the albino mouse eye. (e, g) Antisense probe against p22phox cDNA; (f, h) sense probe to p22phox (negative controls). In situ signal from p22phox mRNA was intense in RGCs, but seen in outer nuclear layer. Signal in the RPE is most evident in the albino mouse eye (g). ONL outer nuclear layer; OPL outer plexiform layer; INL inner nuclear layer; IPL inner plexiform layer; RGC retinal ganglion cells