Uncontrolled activation of the alternative pathway of complement is thought to be associated with age-related macular degeneration (AMD). The alternative pathway is continuously activated in the fluid phase, and tissue surfaces require continuous complement inhibition to prevent spontaneous autologous tissue injury. Here, we examined the effects of oxidative stress on the ability of immortalized human retinal pigment epithelial cells (ARPE-19) to regulate complement activation on their cell surface. Combined treatment with H(2)O(2) (to induce oxidative stress) and complement-sufficient serum was found to disrupt the barrier function of stable ARPE-19 monolayers as determined by transepithelial resistance (TER) measurements. Neither treatment alone had any effect. TER reduction was correlated with increased cell surface deposition of C3, and could be prevented by using C7-depleted serum, an essential component of the terminal complement pathway. Treatment with H(2)O(2) reduced surface expression of the complement inhibitors DAF, CD55, and CD59, and impaired regulation at the cell surface by factor H present within the serum. Combined treatment of the monolayers with H(2)O(2) and serum elicited polarized secretion of vascular epidermal growth factor (VEGF). Both, secretion of VEGF and TER reduction could be attenuated using either an alternative pathway inhibitor or by blocking VEGF receptor-1/2 signaling. Regarded together, these studies demonstrate that oxidative stress reduces regulation of complement on the surface of ARPE-19 cells, increasing complement activation. This sublytic activation results in VEGF release, which mediates disruption of the cell monolayer. These findings link oxidative stress, complement activation, and apical VEGF release, which have all been associated with the pathogenesis of AMD.

Age-related macular degeneration (AMD) is a complex disease caused by genetic and environmental factors, including genetic variants in complement components and smoking. Smoke exposure leads to oxidative stress, complement activation, endoplasmic reticulum (ER) stress, and lipid dysregulation, which have all been proposed to be associated with AMD pathogenesis. Here we examine the effects of smoke exposure on the retinal pigment epithelium (RPE). Mice were exposed to cigarette smoke or filtered air for 6 months. RPE cells grown as stable monolayers were exposed to 5% cigarette smoke extract (CSE). Effects of smoke were determined by biochemical, molecular, and histological measures. Effects of the alternative pathway (AP) of complement and complement C3a anaphylatoxin receptor signaling were analyzed using knock-out mice or specific inhibitors. ER stress markers were elevated after smoke exposure in RPE of intact mice, which was eliminated in AP-deficient mice. To examine this relationship further, RPE monolayers were exposed to CSE. Short term smoke exposure resulted in production and release of complement C3, the generation of C3a, oxidative stress, complement activation on the cell membrane, and ER stress. Long term exposure to CSE resulted in lipid accumulation, and secretion. All measures were reversed by blocking C3a complement receptor (C3aR), alternative complement pathway signaling, and antioxidant therapy. Taken together, our results provide clear evidence that smoke exposure results in oxidative stress and complement activation via the AP, resulting in ER stress-mediated lipid accumulation, and further suggesting that oxidative stress and complement act synergistically in the pathogenesis of AMD.

To determine whether the photoreceptors change structurally and functionally during aging, and to analyze whether pigmentation in the retinal pigment epithelium might be a contributing factor. Young, adult, and aged C57BL/6 and Balb/c mice (1, 4, and 17 months of age) were housed under a 12-h light/12-h dark cycle, with an ambient light intensity at the eye level of the mice of 85 ± 18 lux. Scotopic single-flash and photopic-flicker electroretinograms (ERGs) after complete dark adaptation were used to assess rod and cone function, respectively. Numbers of rod photoreceptors were counted in plastic sections, and rhodopsin levels were measured using absorption difference spectrophotometry. Numbers and types of cones were determined using lectin staining in retinal flatmounts and cone-specific antibodies in radial frozen sections. Young pigmented C57BL/6 and nonpigmented Balb/c mice had similar numbers of rods. In both mouse strains, there was an overall decline in rod photoreceptor number during aging, which was more pronounced in albino mice. Rod cell numbers correlated with a drop in the overall amount of rhodopsin and a reduction in the maximum a-wave of the rod ERG. The number of short-wavelength cones was unaffected by age and pigmentation, whereas an age-related decline was observed in mid-wavelength (MWL) cones in albino, but not in pigmented mice. In contrast, MWL cone function was reduced during aging in both strains.

Purpose. Polymorphisms in factor H (fH), an inhibitor of the alternative pathway (AP) of complement activation, are associated with increased risk for age-related macular degeneration (AMD). The authors investigated the therapeutic use of a novel recombinant form of fH, CR2-fH, which is targeted to sites of complement activation, in mouse choroidal neovascularization (CNV). CR2-fH consists of the N terminus of mouse fH, which contains the AP-inhibitory domain, linked to a complement receptor 2 (CR2) targeting fragment that binds complement activation products. methods. Laser-induced CNV was analyzed in factor-B–deficient mice or in mice treated with CR2-fH, soluble CR2 (targeting domain), or PBS. CNV progression was analyzed by molecular, histologic, and electrophysiological readouts. results. Intravenously administered CR2-fH reduced CNV size, preserved retina function, and abrogated the injury-associated expression of C3 and VEGF mRNA. CR2 and PBS treatment was without effect. In therapeutically relevant paradigms involving delayed treatment after injury, CR2-fH was effective in reducing CNV and provided approximately 60% of the amount of protection of that seen in factor B–deficient mice that lacked functional AP. After intravenous injection, CR2-fH localized to sites of C3 deposition in RPE-choroid. conclusions. Specific inhibition of the AP reduces angiogenesis in mouse CNV. Of note, intravenous injection of C3d-targeted CR2-fH is protective even though endogenous fH is present in serum at a higher relative concentration, and serum fH contains native C3d and cell surface binding domains that target it to cell surfaces. The most common AMD-associated variant of fH resides within a native cell-binding region of fH (Tyr402His). These data may open new avenues for AMD treatment strategies.

Purpose. In retinal degenerative diseases, rod photoreceptors typically deteriorate more rapidly than cone photoreceptors. In the Rpe65^−/− mouse, a model for Leber’s congenital amaurosis, cones degenerate much more rapidly than rods. In this model, the retinoid processing pathway in the retinal pigment epithelium is disrupted, and 11-cis retinal is not generated. This study was designed to investigate the feasibility of restoring functional cones with exogenous 11-cis retinal.

Purpose. Genetic risk factors such as variations in complement factors H (CFH) and B (CFB) have been implicated in the etiology of age-related macular degeneration. It has been hypothesized that inadequate control of complement-driven inflammation may be a major factor in disease pathogenesis. The authors tested the involvement of the complement system in an experimental model for oxidative stress-mediated photoreceptor degeneration, the light-damage mouse model. methods. Changes in gene expression were assessed in BALB/c retinas in response to constant-light (CL) exposure using microarrays and real-time PCR. Susceptibility to CL exposure was tested in CFD ^−/− mice on a BALB/c background. Eyes were analyzed using electrophysiologic and histologic techniques. results. Genes encoding for proteins involved in complement activation were significantly upregulated after CL. The altered gene profiles were similar to proteins accumulated in drusen and to genes identified in the retina and RPE/choroid of patients with age-related macular degeneration. Cyclic-light reared CFD ^−/− and CFD ^+/+ mice had indistinguishable rod function and number; however, after CL challenge, CFD ^−/− photoreceptors were significantly protected. conclusions. These results suggest that rod degeneration in the CL-damaged retina involves the activity of the alternative complement pathway and that eliminating the alternative pathway is neuroprotective. Thus, the light damage albino mouse model may be a good model to study complement-mediated photoreceptor degeneration.