through downregulation of proapoptotic and proinßammatory factors and upregulation of Bcl-2-family antiapoptotic proteins, substantiating NPD1 as a vital regulator of key modulators of cell survival.

Oxidative stress enhances proinßammatory gene expression and is an important mechanism of RPE cell injury. The inducible enzyme COX-2 is the rate-limiting step in the synthesis of prostaglandins, and is involved in oxidative stress and normal cell function. COX-2 expression is regulated in RPE cells by photoreceptor outer-segment phagocytosis and by growth factors [85]. IL-1b activates expression of the proximal COX-2 promoter and NPD1 can potently counteract this induction

with an IC50 of <5 nM [69].

As described earlier, a consequence of RPE cell damage and apoptosis is impaired photoreceptor cell survival [86]. The pigment lipofuscin increases in the RPE during aging and further accumulates in AMD. The progressive assault brought on by photooxidative damage to the RPE effects photoreceptor survival. For example, in StargardtÕs disease, oxidative stress mediated by the lipofuscin ßuorophore N-retinylidene-N-retinylethanolamine (A2E) causes RPE damage. NPD1 downregulates A2E-mediated apoptosis induced by oxidative stress, restoring the integrity of the RPE and perhaps its relationship with the photoreceptor [87].

7.5Neurotrophins Trigger the Synthesis and Polarized Secretion of Neuroprotectin D1 from Human RPE Cells

Neurotrophins contribute to photoreceptor survival [88Ð91]. In hRPE cells grown to conßuence with a high degree of differentiation and apicalÐbasolateral polarization [92], neurotrophins (PEDF, BDNF, CNTF, FGF, GDNF, LIF, NT3, or persephin), promoters of neuronal and/or photoreceptor cell survival, are agonists of NPD1 synthesis [87]. More speciÞcally, neurotrophins trigger synthesis and release of NPD1 through the apical surface of the cell. When neurotrophins are added to the apical side of hRPE cells, they exert concentration-dependent increases of NPD1 release [87]. Pigment epithelium-derived factor (PEDF), a serine protease inhibitor of the serpin family [93], is by far the most potent stimulator of NPD1 synthesis in RPE cells.

The hRPE cells used for these studies may have limited DHA in their phospholipids available to synthesize NPD1 because they are in cell culture and are not undergoing photoreceptor membrane phagocytosis. To corroborate this, when DHA content in the media was increased, PEDF demonstrated a remarkable potentiation of NPD1 release into the apical media. In contrast, much less NPD1 was found in the media bathing the basolateral side of the cells. Regardless of the side of the cell where PEDF was added, the amount of NPD1 released through the basolateral side was similar. Addition of DHA to either side of the cell monolayer provoked PEDFinduced NPD1 release selectively on the apical side. Cytoprotection and enhanced NPD1 formation occurred synergistically when PEDF was added along with DHA

prolonged periods of omega-3 fatty acid deprivation [1Ð3, 36, 104Ð107]. In studies correlating periods of photoreceptor biogenesis and synapse formation with omega-3 fatty acid supply in mouse postnatal development, dietary linolenic acid (18:3, n-3) is actively elongated and desaturated in the liver prior to its distribution to the retina and brain [108]. The biosynthesis of DHA, its incorporation into liver phospholipids, and its trafÞcking through RPE and photoreceptors is depicted in Fig. 7.3. Several studies have shown that DHA is required for photoreceptor function and vision in both animals [109Ð114] and humans [115]. Moreover, the essentiality of DHA has been documented for vision and brain maturation in premature babies and newborns [115]. Although the RPE cell takes up DHA from the bloodstream through the choriocapillaris [3], the bulk of DHA in the RPE cell is a component of photoreceptor disc membrane phospholipids that, after shedding and phagocytosis, are recycled as part of outer segment renewal.

Although it is known that ROS phagocytosis in RPE cells is essential for photoreceptor cell function and survival, thus far, no speciÞc messengers, mediators or mechanisms that promote cell survival during this process have been identiÞed. Some studies have suggested that DHA-phospholipid peroxidation may be an ÒonÓ signal to initiate phagocytosis. Evidence suggests that protein adducts of DHA oxidation accumulate in drusen and are detectable in the blood stream as a potential biomarker for AMD [116, 117]. Recently, it has been shown that phagocytosis of oxidized ROS containing high oxidation products downregulated complement factor H in RPE cells [118]. The RPE complement regulatory system may be suppressed by proinßammatory conditions such as the phagocytosis of oxidized ROS [118].

In studying ROS phagocytosis we have been surprised to Þnd a remarkable but unexpected ability of RPE cells to resist oxidative stress-induced apoptosis [119]. This action is a speciÞc response to ROS since nonspeciÞc phagocytosis (polystyrene microspheres) by RPE cells did not lead to a protective response against oxidative stress. In relation to this, ROS, but not polystyrene microspheres, induce DHA

Fig. 7.2 (continued) depicted as released from the RPE cell or provided from another cell. The same is true for the other growth factors. Oxidative stress, protein misfolding or A2E(N-retinyl-N- retinylidene ethanolamine)/A2E oxiranes (epoxides) are activators of NPD1 synthesis as well (in red). DHA is shown to arrive to the RPE as part of the phagosome (DHA-phospholipids). After the phagolysosomal digestion, most of the DHA is recycled back to the inner segments of photoreceptors through the IPM (black arrows, Fig. 7.3). The precise phospholipid-DHA molecular species that is hydrolyzed to generate the free DHA pool precursor of NPD1 has not been identiÞed (NPD1 synthesis pathway, blue color). NPD1 is released through the apical cellular side and acts on a putative receptor. Intracellular signaling then inhibits proinßammatory gene expression. The proinßammatory genes illustrated here are IL-1b, COX-2 (cyclooxygenase 2), B94 (TNFa- inducible proinßammatory element), and CEX-1 (cytokine exodus protein-1, a marker for inßammatory and oxidative stress responses). As a consequence, a decrease in proinßammatory proteins takes place that, when available, plays a role in drusen development, choroidal neovascularization, and cell injury. In addition, NPD1-triggered signaling upregulates antiapoptotic Bcl-2 family protein expression and downregulates proapoptotic Bcl-2 family protein expression. As a result, caspase three activity is decreased and apoptosis reduced. (Republished from Bazan [150], with permission. The Association for Research in Vision and Ophthalmology is the copyright holder)

Fig. 7.3 Illustration depicting aspects of the interorgan, intercellular, and intracellular trafÞcking of DHA. Dietary 18:3, n-3 or 22:6, n-3 (DHA) are actively taken up by hepatocytes where elongation and desaturation of 18:3 to DHA takes place, catalyzed by enzymes located in the endoplasmic reticulum and peroxisomes. 22:6 then is activated (22:6-CoA) and acylated mainly into phospholipids that are secreted into the blood stream as lipoproteins. DHA from lipoproteins is taken up by RPE cells (green) through the choriocapillaris and then channeled through the IPM to the inner segments (IS) of photoreceptors (purple). It is not clear if free DHA (unesteriÞed) is the sole form of DHA moving through the IPM. In the IS, phospholipid biosynthesis utilizes DHA and in turn provides phospholipids containing DHA to the biogenesis of outer segment membranes. Within the IS, diacylglycerol and phosphatidic acid containing two DHAs on the same glycerol backbone have been proposed to be intermediaries in DHA-phospholipid metabolism. In the outer segments (OS), unique molecular species of phospholipids are included to highlight the fact that these membranes contain molecular species of phosphatidylcholine with 34:6, n-3 esteriÞed in sn-1, and DHA in sn-2; molecular species of phospholipids with two DHAs, and others with DHA in sn-2 and a non-omega-3 fatty acyl group in sn-1. The photoreceptor cell is connected to the RPE cell by red arrows that indicate the omega-3 fatty acids conservation route (short loop) during photoreceptor outer segment renewal. Thus, a phagosome is shown in the RPE from where red arrows follow this conservation route, while a blue arrow indicates DHA being used for NPD1 synthesis. This is consistent with the recent demonstration that ROS phagocytosis selectively increases NPD1 synthesis in the RPE cell. NPD1 is illustrated as acting on a putative receptor on the RPE cell. Signaling evolving from this receptor is shown in Fig. 7.2. PEDF secreted by the RPE or other cells is shown as an inducer of NPD1 synthesis. In the IS, biosynthesized phospholipids are also depicted to be utilized in the biogenesis of other photoreceptor cell membranes, including those of the synaptic terminals. (Republished from Bazan [150], with permission. The Association for Research in Vision and Ophthalmology is the copyright holder)

release and activate NPD1 synthesis. When the free DHA pool size is simultaneously measured in RPE cells and in incubation media by MS/MS, it increases as a function of time of exposure to oxidative stress in RPE cells [69]. Free DHA in cells shows a moderate increase after 6 h when cells are subjected to ROS phagocytosis alone (10.5-fold increase). Oxidative stress, however, strongly enhanced free DHA accumulation in a time-dependent manner, peaking at 16 h. Interestingly, although the overall increase reached tenfold, ROS phagocytosis kept the DHA pool size constant at a 2.4-fold level of increase. The implication here is that NPD1 synthesis reßects an event other than enhanced overall availability of free DHA upon phagocytosis. We do know that a correlation exists between increases in free DHA pool size and in NPD1 synthesis. During ROS phagocytosis, free DHA increases and continues to accumulate for up to 16 h. In addition, it stimulates NPD1 synthesis at 3Ð6 h, after the rise in free DHA occurs. The increase of NPD1 in media also persists for up to 16 h. To reiterate, microsphere phagocytosis does not cause changes in DHA and NPD1; therefore, a very speciÞc free DHA pool might be the precursor for NPD1.

Arachidonic acid is also an active precursor of several bioactive lipids including prostaglandins and lipoxygenase-products which have been correlated with photoreceptor phagocytosis [120, 121]. Since arachidonic acid is released under the aforementioned experimental conditions (data not shown) and during light exposure, the arachidonic acid cascade members Lipoxin A4, 12(S) HETE, and 15(S) HETE were studied and found to be unchanged during ROS phagocytosis [119].

Deuterium-labeled DHA (2H5-DHA) was used to ascertain if the enhanced availability of free DHA leads to the synthesis of NPD1 in RPE cells undergoing oxidative stress. We followed 2H5-NPD1 synthesis by tandem liquid chromatography-photodiode array-electrospray ionization-tandem mass spectrometry-based lipidomic analysis. This approach allowed us to speciÞcally assess DHA conversion because the deuterium is on the metabolically unaltered methylene carbons 21 and 22. Additionally, products are heavier by a mass unit of 1 in comparison to the same nondeuterated molecule and can be detected by tandem mass spectrometry. The characterization of 2H5- NPD1 (negative molecular ion m/z 364.2) and of endogenous nondeuterated NPD1 (negative molecular ion m/z 359.2) is also possible. Our results support the notion that as free DHA accumulates in RPE cells in culture during ROS phagocytosis, the fatty acid is used as a substrate for NPD1 synthesis. Thus, NPD1 may be a major endogenous promoter of RPE cell survival during photoreceptor ROS renewal. Further studies are being conducted to determine if other docosanoids are also being formed under these conditions. The enhanced synthesis of NPD1 after ROS phagocytosis is associated with ROS-induced attenuation of oxidative stress-mediated apoptosis. Although ARPE-19 cells also phagocytized biologically inert polystyrene microspheres, NPD1 content was not affected in RPE cells or in the incubation media. Again, nonspeciÞc, nonbiological microspheres, unlike ROS, did not promote early-response gene induction in the RPE [67], including COX-2 [85] and PPARg expression [122].

These results reveal that the supply of DHA and the induction of NPD1 synthesis during ROS phagocytosis represent a homeostatic regulatory event for RPE cell protection in conditions of oxidative stress challenge allowing the maintenance of photoreceptor cell integrity [93].

7.7Perspective: DHA Signalolipidomics in the Modulation of Oxidative Stress in Photoreceptor-RPE Cell Interactions

Mediator lipidomics is an evolving approach to the detailed identiÞcation of lipid classes and molecular species including both structural and bioactive lipids that are mediators of cell signaling. The lipidome is the complete characterization of the lipids of an entire cell or part of a cell. We are using lipidomic-based analyses to initiate the decoding of RPE, retina, and neural omega-3 fatty acids. Thus far, this approach has led to the discovery of NPD1 in the RPE cell [69] and the elucidation of its bioactivity.

As described earlier, NPD1 is a DHA-derived mediator synthesized by RPE cells that promotes photoreceptor-RPE cell homeostasis through modulation of multiple signaling pathways. NPD1 downregulates the expression of proinßammatory genes, including cytokine-induced COX-2 expression in RPE cells [69]. Similarly, in ischemiaÐreperfusion-injured hippocampus and IL-1b stressed neural progenitor cells, examples of in vivo and in vitro models, respectively, NPD1 inhibits COX-2 induction [69, 72]. In brain ischemiaÐreperfusion, NPD1 decreases infarct size and inhibits polymorphonuclear leukocyte inÞltration [72]. Figure 7.2 illustrates NPD1 bioactivity as a modulatory signal that counteracts proinßammatory injury to the RPE.

Excessive oxidative stress turns on multiple signaling pathways in the RPE, photoreceptors, and other cells. Several of these pathways, in turn, participate in the pathophysiology of retinal degenerative disease and lead to cell damage and, eventually, cell death [123]. An early response when homeostasis has been threatened is the active induction of NPD1 synthesis.

During successful aging, the homeostatic regulation between photoreceptors and RPE preserves RPE cell integrity. If eye pathology does not arise, RPE cell density is maintained over nine decades [64]. However, failure of homeostasis results in enhanced DHA peroxidation, drusen formation, lipid peroxide protein adduct accumulation, apoptosis, and pathoangiogenesis. Overall, it is apparent that a breakdown in the balance of protective and potentially cytotoxic factors is involved in various forms of retinal degeneration [3, 5, 17, 68]. NPD1 synthesis is induced under conditions where excessive oxidative stress threatens to disrupt homeostasis along with other rescue signals such as neurotrophins to protect cellular integrity (Fig. 7.2). Other triggers of the NPD1 response include A2E and A2E epoxides (oxiranes), which are compounds that are known to accumulate in aging RPE, StargardtÕs disease, and other retinal degenerative diseases [124].

NPD1 synthesis is dependent on DHA availability in certain pools of phospholipids [3]. DHA, or DHA precursors are supplied by the diet, packaged by the liver, and then sent to the retina and elsewhere. Once DHA is incorporated into disk membrane phospholipids and sloughed off to the RPE, DHA can be recycled back to photoreceptors for reuse, closing the short loop [3, 71, 84, 125] and conserving DHA. The long loop of omega-3 fatty acids is the connection between the liver and the RPE cell through the choriocapillaris (Fig. 7.3) [108]. Disruption of either the

long or short loop can result in impaired DHA supply to the photoreceptors, inducing detrimental changes to photoreceptor function. Dietary linolenic acid (18:3, n-3) is elongated and desaturated in the liver, followed by supply to the RPE through blood lipoproteins [126]. It has been suggested that the photoreceptors and cellular membranes in the brain, the second-richest tissue containing DHA, may release signals to evoke secretion from the liver when phospholipids rich in DHA are needed [108]. Photoreceptor biogenesis and synaptogenesis during postnatal development actively accrue DHA, which is why systemic alterations in the supply of DHA to the retina have been implicated in retinal degeneration [50, 58, 59, 71, 108]. Moreover, during slow photoreceptor cell demise, as in RP, the need to supply ÒDHA building blocksÓ for photoreceptors might be an early biological repair response [108].

The molecular bases for the tenacious retention of DHA in retina, photoreceptors, and brain have yet to be understood. The discovery of very-long-chain DHAs (e.g., 24:6 and 36:6) raises important questions regarding membrane organization, function and, more speciÞcally, the lipid environment of rhodopsin in the disc membrane [45]. As a result, candidate-gene approaches have been implemented to understand the genetic etiology of retinal degenerative diseases. For example, genetic studies of AMD have linked the gene encoding elongation of very-long- chain fatty acids-like 4 (ELOVL4) with StargardtÕs autosomal-dominant-like macular dystrophy [127]. The relationship of this gene with DHA elongation products and NPD1 remains to be studied.

Photoreceptor outer segment renewal in the context of well regulated oxidative stress responses may not elicit NPD1 synthesis. NPD1 cytoprotective signaling may be induced when oxidative stress surpasses a certain threshold. Thus, if the ROS and/or RPE are oxidatively challenged, some DHA may be used for NPD1 synthesis [119]. One theory is that when DHA is used as a precursor of NPD1 during protective responses to perturbations in retinal function, the balance of DHA retention and recycling is disrupted and requires an additional dietary supply of DHA. In support of this interpretation, studies have shown beneÞcial effects of dietary DHA in AMD [128Ð135] and RP [136].

The presence of additional bioactive docosanoids in retina and brain, and the precise natures of the PLA2(s) and 15-lipoxygenase(s) involved in docosanoid synthesis, have not been fully characterized. Because a calcium ionophore activates DHA release and NPD1 synthesis in RPE cells [69], the activity of a Ca2+-dependent PLA2 is implied but the speciÞc PLA2 involved remains a question. IdentiÞcation of NPD1 catabolism pathways will provide insight into what turns off NPD1 signaling pathways. Overall, deÞning selective DHA-delivery systems to the retina and an understanding of NPD1 and its cellular target(s) might enable the design of therapeutic approaches to manage RPE protection. Protection of RPE will subsequently enhance photoreceptor survival in both aging and retinal degeneration. One additional relevant question is whether NPD1 or a synthetic active analog can be therapeutically administered, or whether inducers of NPD1 synthesis can be used during early stages of retinal degeneration. Our laboratory has established the potential for this application in an experimental stroke model with intravenous administration of serum albumin complexed with DHA. In this model, albumin complexed with DHA attenuated infarct