14.5Implication and Therapeutic Potential of NADPH Oxidase in Development of CNV

Accumulating evidence suggests that NAPDH oxidase plays an important role in redoxsignaling pathways and contributes to age-related increases in oxidative stress in many vascular and neural degenerative diseases [45]. In this study, we sought to characterize expression of the small, membrane-bound catalytic subunit of NADPH oxidase, p22phox, in the retina and test the effect of its downregulation by AAV-mediated delivery of siRNA against p22phox on development of laserinduced CNV in the mouse. We demonstrate that p22phox is expressed in the RPE cells and inner retinal neurons, and its downregulation in RPE cells in vivo efÞciently inhibits the development of CNV, suggesting that NADPH oxidase-derived ROS contributes to CNV development.

The RPE is a monolayer of pigmented cells that separates the neural retina from blood vessels of the choriocapillaris. This cell layer forms a part of the bloodÐretina barrier [62, 63] and provides multiple functions essential for normal visual functions including nourishing the adjacent photoreceptor cell layer and removing their continually shed outer segments [64]. Dysfunction of the RPE mediated by ROS has been suggested as a contributing factor in age related macular degeneration [7]. Although there are many other intracellular sources of ROS, NADPH oxidase is the only system that is ÒdedicatedÓ to generating superoxide in a regulated manner. For that reason it participates in a variety of redox-sensing cellular functions ranging from cell growth/survival and death, to immune cell signaling. The importance of these functions of NADPH oxidase is also attested to by the evolutionary conservation of members of NOX family in all multicellular organisms [65]. Constitutive expression of the enzymatic p22phox subunit in RPE cells suggests that this NADPH oxidase complex also plays an important role in many RPE cellular events that are redox-sensing dependent.

It has also been well documented that although low level ROS production by NADPH oxidase is essential for cellular functions, overproduction of ROS via NADPH oxidase under pathophysiologic conditions contributes to many pathological vascular complications and neural degenerative diseases [45]. NADPH oxidase activity in nonphagocytic cells is increased by diverse pathophysiological stimuli including growth factors such as VEGF; thrombin; insulin; G-protein-coupled receptor agonists such as angiotensin II and endothelin-1; cytokines such as TNFa (tumor necrosis factor a) and TGFb (transforming growth factor b); ÒmetabolicÓ factors such as oxidized low-density lipoprotein, nonesteriÞed (ÒfreeÓ) fatty acids and glycated proteins; and hypoxiaÐreoxygenation or ischaemiaÐreperfusion [49, 66, 67]. These conditions may also stimulate NADPH oxidase-derived ROS production in RPE cells and contribute to pathogenesis of ocular diseases involving RPE.

Having shown that downregulation of p22phox expression by AAV vector delivered siRNA in RPE cells reduced the development of CNV, it is worth considering possible mechanisms. It is well established that VEGF stimulates ROS production

through NADPH oxidase, and the resultant ROS stimulates diverse redox signaling pathways leading to angiogenesis-related gene induction as well as to endothelial cell migration and proliferation, both proangiogenic stimuli in vivo [68, 69]. Therefore, one possibility is that the inhibition of CNV we observe operates through a reduction in VEFG signaling mediated by NADPH oxidase-derived ROS. We showed that downregulation of p22phox signiÞcantly reduced the level of VEGF. It is known that VEGF is constitutively secreted from basal side of RPE cells to maintain the normal choriocapillaris. Increased VEGF levels have been observed in the RPE overlying the macula in both AMD patients [70] and in the animal models of CNV [71, 72]. It is also possible that alternative, nonmutually exclusive mechanisms are involved in the inhibition of CNV we observed. NADPH oxidase is also upregulated by several other growth factors and cytokines, many of which are secreted by RPE cells to maintain normal retina and choriocapillaris, and the immune suppressive environment that protects the retina from acute inßammatory responses (reviewed in [73]). Although not well understood, several other mechanisms have been suggested in CNV development, including local hypoxia, wound healing processes, and inßammatory processes [74Ð78]. Moreover, NADPH oxi- dase-derived ROS production has been implicated in these mechanisms in other tissues (see recent review [45], and references therein). Thus, NADPH oxidasederived ROS appears to be at the focus of multiple signaling pathways and cellular events that are redox-dependent, and mechanisms to inhibit its activity provide promising targets for AMD therapy.

We also report p22phox expression in the retinal ganglion cells and subset of other inner retinal neurons. This may have important implications for retinal degenerative disorders involving ganglion cell loss such as glaucoma. It has been shown that ischemiaÐreperfusion induces elevated production of ROS and results in neurotoxicity and neurodegeneration both in animal models and in humans [79Ð81]. In the central nervous system (CNS), components of NADPH oxidase are present in many cell types including microglia, astrocytes, oligodendrocytes, and neurons (reviewed in [45, 64]). Although NADPH oxidase-derived ROS function as signaling molecules in normal tissue [82Ð85], ROS overproduction by NOX enzymes has been implicated in a variety of diseases of the CNS, including ischemic stroke, AlzheimerÕs disease, and ParkinsonÕs disease (reviewed in [45, 64]). Thus it is conceivable that NADPH oxidase-derived ROS may play an equivalent role in retinal neuron pathology, for example, under physiological stress such as increased intraocular pressure or ischemia/reperfusion retinal ganglion cells may experience oxidative damage-induced cell death mediated by NADPH oxidase. Further information on speciÞc mechanisms of its activation and regulation should provide insights for designing effective general therapies to combat a range of vision-threatening including glaucoma, optic neuritis and retinitis pigmentosa.

Finally, endothelial NADPH oxidase is a major source of superoxide in blood vessels and has been implicated in the oxidative stress accompanying vascular diseases, including atherosclerosis, hypertension, diabetic retinopathy, and heart failure [86, 87]. All components of phagocytic NADPH oxidase including p22phox,

gp91phox, p47phox, p67phox, and the small G protein Rac1 are expressed in the cellular components of vascular systems (endothelial cells, vascular smooth muscle cells, adventitial and cardiac Þbroblasts, and cardiomyocytes) [45]. Interestingly, we did not detect p22phox expression in retinal vessels either by immunolabeling or in situ hybridization. This discrepancy from the well documented expression of p22phox in vascular system may be due to detection limitations inherent in methods used in this study. It is likely that p22phox is expressed at low levels in the retinal vessels but is elevated in response to pathological conditions such as hyperglycemia. In fact, an NADPH oxidase-like enzyme was found to be expressed in retinal pericytes [88, 89] and upregulated in response to Angiotensin II, high glucose [89] or the saturated free fatty acid palmitate [88]. NADPH oxidase activity is also increased in the retina of diabetic rats [90] and in ischemic retinopathy [59, 91]. Thus, low vascular expression levels coupled with the paucity of vascular cells available for antibody reaction in retinal sections likely explain our inability to detect p22phox in normal retinal tissue.

14.6Summary and Future Perspective

In summary, our Þndings that the p22phox, an indispensable component of NADPH oxidase complex, is expressed abundantly in the inner retinal neurons and RPE suggest that NADPH oxidase-mediated ROS generation may play an important role in normal retinal function. Given that NADPH oxidase-derived ROS participate in diverse signaling pathways critical to many important biological processes and the likely involvement of ROS in aging, vascular pathophysiology, and neurodegenerative diseases, it is likely that this ROS-production pathway contributes to many degenerative ocular diseases such glaucoma, diabetic retinopathy, and AMD. Supporting this view, as shown in this study, downregulation of p22phox by AAV-mediated delivery of siRNA dramatically inhibits the development of CNV, suggesting a direct role of NADPH oxidase in the progression to pathogenic CNV. Hence, targeting this pathway presents a new opportunity for therapeutic intervention against the CNV seen in patients with the exudative form of AMD. Since oxidative damage may also contribute to the pathogenesis of the atrophic form of AMD [92], targeting this pathway may also provide a novel strategy for treating nonexudative AMD as well.

Acknowledgments We acknowledge NIH grants EY13729, EY11123, EY08571, and grants from the Macular Vision Research Foundation, Foundation Fighting Blindness, Juvenile Diabetes Research Foundation, and Research to Prevent Blindness, Inc. American Diabetes Association for partial support of this work. W.W.H. and the University of Florida have a Þnancial interest in the use of AAV therapies, and own equity in a company (AGTC Inc.) that might, in the future, commercialize some aspects of this work. We thank Mike Daniel for help with statistic analysis, and Clay Smith for help with Western blot analysis.

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