Basic fibroblast growth factor (FGF-2) influences the differentiation and survival of retinal photoreceptors in vivo and in vitro, but it is not known whether it acts directly on photoreceptor FGF receptors or indirectly through activation of surrounding cells. To clarify the effects of FGF-2 on photoreceptor survival, we developed a purified photoreceptor culture system. The outer nuclear layers of postnatal day 5–15 rat retinas were isolated by vibratome sectioning, and the photoreceptor fractions obtained were enzymatically dissociated. Photoreceptors were maintained in monolayer culture for 1 week in a chemically defined medium. Immunocytochemical labeling showed that >99.5% of cells were photoreceptors, and glial contamination represented ∼0.2%. Photoreceptors from postnatal day 5–9 retinas survived for at least 24 hr in vitro, whereas cells from postnatal day 10–15 retinas died rapidly. Subsequent studies performed with postnatal day 5 photoreceptors showed that their survival was increased in a dose-dependent manner after the addition of FGF-2. In control cultures, 36% of originally seeded photoreceptors were alive after 5 din vitro, and in the presence of 20 ng/ml FGF-2 this number was doubled to 62%. This increase was not caused by proliferation of photoreceptor precursors. Denaturing or blocking FGF-2 prevented enhancement of survival. Conversely, only 25.5% of photoreceptors survived in the presence of epidermal growth factor (EGF). FGF- and EGF-receptor mRNA and proteins were detected in purified photoreceptors in vitro, and addition of FGF-2 or EGF led to tyrosine phosphorylation of photoreceptor proteins. These data support a direct mechanism of action for FGF-2 stimulation of photoreceptor survival.

The accumulation of N-retinylidene-N-retinylethanolamine (A2E, a toxic by-product of the visual pigment cycle) in the retinal pigment epithelium (RPE) is a major cause of visual impairment in the elderly. Photooxidation of A2E results in retinal pigment epithelium degeneration followed by that of associated photoreceptors. Present treatments rely on nutrient supplementation with antioxidants. 9’-cis-Norbixin (a natural diapocarotenoid, 97% purity) was prepared from Bixa orellana seeds. It was first evaluated in primary cultures of porcine retinal pigment epithelium cells challenged with A2E and illuminated with blue light, and it provided an improved photo-protection as compared with lutein or zeaxanthin. In Abca4^-/- Rdh8^-/- mice (a model of dry AMD), intravitreally-injected norbixin maintained the electroretinogram and protected photoreceptors against light damage. In a standard rat blue-light model of photodamage, norbixin was at least equally as active as phenyl-N-tert-butylnitrone, a free radical spin-trap. Chronic experiments performed with Abca4^-/- Rdh8^-/- mice treated orally for 3 months with norbixin showed a reduced A2E accumulation in the retina. Norbixin appears promising for developing an oral treatment of macular degeneration. A drug candidate (BIO201) with 9’-cis-norbixin as the active principle ingredient is under development, and its potential will be assessed in a forthcoming clinical trial.

N-retinylidene-N-retinylethanolamine (A2E) plays a central role in age-related macular degeneration (AMD) by inducing angiogenesis and inflammation. A2E effects are mediated at least partly via the retinoic acid receptor (RAR)-α. Here we show that A2E binds and transactivates also peroxisome proliferator-activated receptors (PPAR) and retinoid X receptors (RXR). 9’-cis-norbixin, a di-apocarotenoid is also a ligand of these nuclear receptors (NR). Norbixin inhibits PPAR and RXR transactivation induced by A2E. Moreover, norbixin reduces protein kinase B (AKT) phosphorylation, NF-κB and AP-1 transactivation and mRNA expression of the inflammatory interleukins (IL) -6 and -8 and of vascular endothelial growth factor (VEGF) enhanced by A2E. By contrast, norbixin increases matrix metalloproteinase 9 (MMP9) and C-C motif chemokine ligand 2 (CCL2) mRNA expression in response to A2E. Selective PPAR-α, -β/δ and –γ antagonists inhibit the expression of IL-6 and IL-8 while only the antagonist of PPAR-γ inhibits the transactivation of NF-κB following A2E exposure. In addition, a cocktail of all three PPARs antagonists and also HX531, an antagonist of RXR reproduce norbixin effects on inflammation. Altogether, A2E’s deleterious biological effects could be inhibited through PPAR and RXR regulation. Moreover, the modulation of these NR by norbixin may open new avenues for the treatment of AMD.

9′-cis-norbixin (norbixin/BIO201) protects RPE cells against phototoxicity induced by blue light and N-retinylidene-N-retinylethanolamine (A2E) in vitro and preserves visual functions in animal models of age-related macular degeneration (AMD) in vivo. The purpose of this study was to examine the mode of action and the in vitro and in vivo effects of BIO203, a novel norbixin amide conjugate. Compared to norbixin, BIO203 displays improved stability at all temperatures tested for up to 18 months. In vitro, BIO203 and norbixin share a similar mode of action involving the inhibition of PPARs, NF-κB, and AP-1 transactivations. The two compounds also reduce IL-6, IL-8, and VEGF expression induced by A2E. In vivo, ocular maximal concentration and BIO203 plasma exposure are increased compared to those of norbixin. Moreover, BIO203 administered systemically protects visual functions and retinal structure in albino rats subjected to blue-light illumination and in the retinal degeneration model of Abca4^−/− Rdh8^−/− double knock-out mice following 6 months of oral complementation. In conclusion, we report here that BIO203 and norbixin share similar modes of action and protective effects in vitro and in vivo. BIO203, with its improved pharmacokinetic and stability properties, could be developed for the treatment of retinal degenerative diseases such as AMD.

N-retinylidene-N-retinylethanolamine (A2E) plays a central role in age-related macular degeneration (AMD) by inducing apoptosis, angiogenesis and inflammation. It has been proposed that A2E effects are mediated at least partly via the retinoic acid receptor (RAR)-α. Here we show that A2E binds and transactivates not only RARs, but also peroxisome proliferator-activated receptors (PPARs) and retinoid X receptors (RXRs). Norbixin, which protects retinal pigment epithelium (RPE) cells against apoptosis induced by combined blue light illumination and A2E exposure, is also a ligand of these nuclear receptors (NRs) but does not induce their transactivation. Norbixin inhibits RXRs and PPARs but enhances RARs transactivation induced by A2E. Norbixin also inhibits PPAR-γ transactivation induced by its high affinity ligand troglitazone. Photoprotection of RPE cells by norbixin correlates with maintained levels of the antiapoptotic B-cell lymphoma 2 (Bcl2) protein. Moreover, norbixin reduces protein kinase B (AKT) phosphorylation, NF-κB and activator protein 1 (AP-1) transactivation, and the mRNA expression of the inflammatory interleukins (IL) 6 and 8 and of vascular endothelial growth factor (VEGF) that are enhanced by A2E. By contrast, norbixin increases matrix metalloproteinase 9 (MMP9) and C-C motif chemokine ligand 2 (CCL2) mRNA expression but has neither effect on extracellular signal-regulated kinase (ERK) phosphorylation, nor on IL-18 mRNA expression in response to A2E. Altogether, we show for the first time that A2E deleterious biological effects appear to be mediated through RARs, PPARs and RXRs. Moreover, we report that the modulation of these NRs by norbixin may open new avenues for the treatment of AMD.

N-retinylidene-N-retinylethanolamine (A2E) has been associated with age-related macular degeneration (AMD) physiopathology by inducing cell death, angiogenesis and inflammation in retinal pigmented epithelial (RPE) cells. It was previously thought that the A2E effects were solely mediated via the retinoic acid receptor (RAR)-α activation. However, this conclusion was based on experiments using the RAR “specific” antagonist RO-41-5253, which was found to also be a ligand and partial agonist of the peroxisome proliferator-activated receptor (PPAR)-γ. Moreover, we previously reported that inhibiting PPAR and retinoid X receptor (RXR) transactivation with norbixin also modulated inflammation and angiogenesis in RPE cells challenged in the presence of A2E. Here, using several RAR inhibitors, we deciphered the respective roles of RAR, PPAR and RXR transactivations in an in vitro model of AMD. We showed that BMS 195614 (a selective RAR-α antagonist) displayed photoprotective properties against toxic blue light exposure in the presence of A2E. BMS 195614 also significantly reduced the AP-1 transactivation and mRNA expression of the inflammatory interleukin (IL)-6 and vascular endothelial growth factor (VEGF) induced by A2E in RPE cells in vitro, suggesting a major role of RAR in these processes. Surprisingly, however, we showed that (1) Norbixin increased the RAR transactivation and (2) AGN 193109 (a high affinity pan-RAR antagonist) and BMS 493 (a pan-RAR inverse agonist), which are photoprotective against toxic blue light exposure in the presence of A2E, also inhibited PPARs transactivation and RXR transactivation, respectively. Therefore, in our in vitro model of AMD, several commercialized RAR inhibitors appear to be non-specific, and we propose that the phototoxicity and expression of IL-6 and VEGF induced by A2E in RPE cells operates through the activation of PPAR or RXR rather than by RAR transactivation.