In view of the oxygen-rich environment in the eye, it is particularly noteworthy that phospholipids containing docosahexaenoic acid (DHA), which are exquisitely sensitive to oxidative damage (1), are abundant in photoreceptor cells. These cells contain a stack of DHA-rich membrane disks that are bathed in oxygen and light (see Figure). A molecule of rhodopsin, a retinylidene Schiff base of the protein opsin, is embedded in each disk. New disks are generated at the nuclear end of the stack and old disks are shed at the tip of the rod outer segment (ROS) where they are digested by phagocytic cells of the retinyl pigmented epithelium (RPE) (2). To protect against oxidative damage, the retina contains the antioxidant a-tocopherol (vitamin E) and antioxidant enzyme systems including glutathione peroxidase and superoxide dismutase. Nevertheless, the disks are replaced every twelve days, probably because they are routinely damaged by oxidative modifications spawned by photogenerated radicals. Previously, the lipid oxidation product malondialdehyde was found in the subretinal fluid from individuals with retinal detachment (3).
By acting as antigens that engender an immune response, oxidatively modified proteins can induce pathological processes. Some evidence suggests the involvement of the cellular immune system in retinitis pigmentosa (RP) (4-6), Usher’s syndrome (7), and cone dystrophy (8). Thus, retinal antigens, especially those localized in the ROS, foster immune reactivity in RP patients. Although autoimmunity may not initiate RP, it may contribute to damage of ocular tissues by perpetuating and maintaining the inflammatory state (9). Antibodies to human retinal antigens are present in blood serum from RP patients (10-12). One variant of age-related macular degeneration (ARMD) involves sprouting of new blood vessels (neovascularization) from the choriocapillaris (see Figure) into the subretinal space. Large amounts of immunoglobins and complement components were found in subretinal neovascular membranes from ARMD patients (13, 14). Very little is known about the molecular structures of retinal antigens. One possibility is that an immune response may result from a defect in maintaining tolerance for self-antigens, e. g., rhodopsin. However, another possibility is that the presence of an abnormally high level of oxidative protein modifications engenders an immune response.
Oxidative modification of photoreceptor
disk proteins or membrane lipids may also be involved in recognition of damaged
disks by RPE cell CD36 receptors that mediate endocytosis of ROS tips (15).
In analogy with the recognition of oxLDL by macrophage CD36 receptors, it
seems reasonable to expect that modifications of ROS protein or lipids by
products from the free radical-induced oxidation of DHA may be involved in
recognition of damaged ROS disks by CD36. We also recently demonstrated
that a family of oxidized phospholipids derived from linoleate or arachidonate
are CD36 receptor ligands. Structurally similar oxidized phospholipids
may contribute to CD36 receptor recognition of oxidatively damaged ROS.
In analogy with the formation of caboxypropylpyrroles
(CPPs) from arachidonyl phospholipids and LDL protein, we postulated the
formation of carboxyethylpyrroles (CEPs) from docosahexanoyl phospholipids
and retinyl protein. Remarkably selective anti-CEP polyclonal rabbit
antibodies were raised that show less than 1% crossreactivity with the analogous
CPPs. Since DHA is the only common polyunsaturated fatty acid whose
oxidation can lead to the production of CEPs, anti-CEP antibodies are a unique
tool for detecting oxidative damage of lipids containing DHA.
Immunostaining of mouse retina with anti-CEP antibodies (left panel) was prominant only for the rod outer segments (OS) and retinyl pigmented epithelium (RPE). As predicted, CEPs are only generated in DHA-rich regions of the retina. The antibody binding is specific since immunostaining was blocked if the antibodies were preincubated with a CEP-containing protein (right panel).
Ongoing studies are directed at identifying specific proteins in the ROS-RPE proteome that contain CEP modifications using MALDI-TOF mass spectroscopic analysis of immunoreactive proteins after separation by two dimensional gel electrophoresis. To further enhance specificity, monoclonal antibodies are being prepared. The possibility that oxidized lipids from the retina can be detected as CEP modifications of blood proteins is receiving special attention because this could provide a clinically useful tool for the early detection of oxidative injury of the retina.
A large proportion of the lipids in photoreceptor disk membranes are contained in ethanolamine phospholipids. In analogy with the oxidative cleavage of arachidonyl phosphatidylcholine, we expect that oxobutyryl (OB), succinyl (S) and hydroxy-7-oxoheptenoic acid (HOHA) phosphatidylethanolamine (PE) esters will be produced by oxidative cleavage of the docosahexaenoic acid (DHA) ester of PE. HOHA-PE is the putative precursor of CEP
|Return to Current Projects|