Regulation of oxidative stress and inflammatory responses in human retinal pigment epithelial cells.

Age-related macular degeneration (AMD) is an eye disease, which causes impaired vision that can lead to blindness. The incidence of AMD increases with age. Retinal pigment epithelial (RPE) cells maintain retinal homeostasis and support the functionality of photoreceptors. In the pathogenesis of AMD, the degeneration of the RPE cells precedes photoreceptor cell death. RPE cells are susceptible to oxidative stress, and chronic inflammation involving nucleotide-binding domain, leucine-rich repeat and pyrin domain 3 (NLRP3) inflammasome activation and impaired autophagy are challenges faced by aged RPE cells in AMD. There are two types of AMD, dry (85-90%) and wet (10-15%) disease forms. Choroidal neovascularization is typical for wet AMD, and anti-vascular endothelial growth factor (anti-VEGF) injections are used to prevent the progression of the disease but there is no curative treatment. There is no cure for the dry disease form, but antioxidants have been proposed as a potential treatment option. Ageing is the most important risk factor of AMD, and tobacco smoke is the most important environmental risk factor that can be controlled. Hydroquinone is a cytotoxic, immunotoxic, carcinogenic and pro-oxidative component of tobacco smoke. The aim of this PhD thesis was to study hydroquinone-induced oxidative stress and NLRP3 inflammasome activation in human RPE cells (ARPE-19 cells). An age-related eye disease study (AREDS) formulation (incl. omega-3 fatty acids, vitamin C and E, copper, zinc, lutein and zeaxanthin), which is clinically investigated p.o. dosing combination of dietary supplements for AMD patients, has been evaluated as a possible treatment and restraining option for AMD. Resvega (4.1.1, Table 2) is a similar kind of product to AREDS with added resveratrol, and many of the components incorporated within Resvega can be considered as belonging to the normal antioxidative defence system of the retina. Another aim was to evaluate the effects of Resvega on hydroquinone-induced oxidative stress or NLRP3 inflammasome activation induced by impaired protein clearance. The results of this study reveal that hydroquinone elevated the activity of NADPH oxidase which subsequently mediated the production of reactive oxygen species (ROS) and predisposed RPE cells to degeneration by reducing levels of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). Hydroquinone induced an NLRP3-independent IL-18 release and NLRP3 accumulation inside the IL-1α-primed cells. Resvega treatment reduced the extent of hydroquinone-induced ROS production and NLRP3 inflammasome activation evoked by impaired protein clearance. Thus, Resvega alleviated hydroquinone- and impaired protein clearance-induced stress in human RPE cells, but more studies are needed, for example, to reveal the most optimal route of administration for targeting the cells in the retina, since both oxidative stress and NLRP3 inflammasome activation are important contributors to the development of AMD and represent significant treatment targets.

Unravelling the therapeutic potential of IL-33 for atrophic AMD.

Age-related macular degeneration (AMD), a degenerative disease affecting the retinal pigment epithelium (RPE) and photoreceptors in the macula, is the leading cause of central blindness in the elderly. AMD progresses to advanced stages of the disease, atrophic AMD (aAMD), or in 15% of cases "wet" or neovascular AMD (nAMD), associated with substantial vision loss. Whilst there has been advancement in therapies treating nAMD, to date, there are no licenced effective treatments for the 85% affected by aAMD, with disease managed by changes to diet, vitamin supplements, and regular monitoring. AMD has a complex pathogenesis, involving highly integrated and common age-related disease pathways, including dysregulated complement/inflammation, impaired autophagy, and oxidative stress. The intricacy of AMD pathogenesis makes therapeutic development challenging and identifying a target that combats the converging disease pathways is essential to provide a globally effective treatment. Interleukin-33 is a cytokine, classically known for the proinflammatory role it plays in allergic disease. Recent evidence across degenerative and inflammatory disease conditions reveals a diverse immune-modulatory role for IL-33, with promising therapeutic potential. Here, we will review IL-33 function in disease and discuss the future potential for this homeostatic cytokine in treating AMD.

Characterizing the effect of supplements on the phenotype of cultured macrophages from patients with age-related macular degeneration.

Purpose: Oral vitamin and mineral supplements reduce the risk of visual loss in age-related macular degeneration (AMD). However, the pathways that mediate this beneficial effect are poorly understood. Macrophages may exert oxidative, inflammatory, and angiogenic effects in the context of AMD. We aim to assess if oral supplements can modulate the macrophage phenotype in this disease. Methods: Monocytes were isolated from patients with neovascular AMD (nvAMD), cultured, matured to macrophages, and polarized to classical [M1 (stimulated by IFNγ and lipopolysaccharide (LPS))] and alternative [M2 (stimulated with IL-4 and IL-13)] phenotypes. Combinations of antioxidants including lutein+zeaxanthin (1 µM; 0.2 µM), zinc (10 µM), carnosic acid (2 µM), beta-carotene (2 µM), and standardized tomato extract containing lycopene and other tomato phytonutrients were added to the culture media. Levels of anti-inflammatory, antioxidant, and pro-angiogenic gene and protein expression were then evaluated. Results: Combinations of lutein and carnosic acid with zinc and standardized tomato extract or with beta-carotene yielded an antioxidative, anti-inflammatory, and antiangiogenic effect in M1 and M2 macrophages. These effects manifested in the upregulation of antioxidative genes (HMOX1, SOD1) and the downregulation of pro-angiogenic genes and pro-inflammatory genes (SDF-1, TNF-alpha, IL-6, MCP-1). Lutein monotherapy or a combination of lutein and zinc had less effect on the expression of these genes. Conclusions: Combinations of supplements can modify the expression of genes and proteins that may be relevant for the involvement of macrophages in the pathogenesis of AMD. Further studies are required to evaluate if the modulation of the macrophage phenotype partially accounts for the beneficial effect of oral supplements in AMD and if modification of the AREDS formula can improve its effect on macrophages.

Effect of Dietary Supplementation With Lutein, Zeaxanthin, and ω-3 on Macular Pigment: A Randomized Clinical Trial.

Importance: Nutritional uptake of lutein, zeaxanthin, and ω-3 polyunsaturated fatty acids may increase macular pigment optical density (MPOD) and thereby protect against the development of age-related macular degeneration (AMD). Objectives: To estimate the efficiency of dietary supplementation containing lutein, zeaxanthin, ω-3 polyunsaturated fatty acids, and vitamins to increase the density of macular pigment in first-generation offspring of parents with neovascular AMD. Design, Setting, and Participants: This study was a randomized clinical trial (Lutein Influence on Macula of Persons Issued From AMD Parents [LIMPIA]) with a 6-month treatment period, followed by a 6-month follow-up period. Analyses were based on the intent-to-treat principle. The setting was 2 university hospitals in France (at Bordeaux and Dijon) from January 2011 (first participant first visit) to February 2013 (last participant last visit). The analysis was conducted from January to November 2016. Participants were 120 individuals free of any retinal ocular disease. They were first-generation offspring of parents with neovascular AMD. Interventions: Participants were randomized in a 1:1 ratio to receive either 2 daily dietary supplementation capsules or placebo for 6 months. Main Outcomes and Measures: The primary assessment criterion was the evolution of MPOD after 6 months of supplementation (value of both eligible eyes) measured using the modified MPD-Visucam 200 (Carl Zeiss Meditec) and the modified Heidelberg Retina Angiograph (Heidelberg Engineering) (HRA) at 0.98° eccentricity. The statistical analysis was adjusted for hospital and for risk factors. Results: Overall, 120 participants (60 in each group) were included, and 239 eyes were analyzed (119 in the lutein plus zeaxanthin [L + Z] group and 120 in the placebo group). Their mean (SD) age was 56.7 (6.6) years, and 71.7% (n = 86) were female. A statistically significant increase in plasma lutein and zeaxanthin was shown in the L + Z group after 3 months and 6 months of treatment compared with the placebo group. However, the difference between groups in the evolution of MPOD measured by HRA 0.98° eccentricity between 6 months and baseline was 0.036 (95% CI, -0.037 to 0.110) (P = .33). Conclusions and Relevance: Among first-generation offspring of parents with neovascular AMD in the LIMPIA trial, MPOD as measured with the modified HRA and the MPD-Visucam was not modified after 6 months of lutein and zeaxanthin dietary supplementation despite plasma levels showing continuous exposure to lutein and zeaxanthin. Further research is necessary to understand the mechanism of absorption and metabolism of these nutrients in the macula, the best way to measure MPOD, and the clinical benefit for the patients. Trial Registration: clinicaltrials.gov Identifier: NCT01269697.

Angiostatic kinase inhibitors to sustain photodynamic angio-occlusion.

Targeted angiostatic therapy receives major attention for the treatment of cancer and exudative age-related macular degeneration (AMD). Photodynamic therapy (PDT) has been used as an effective clinical approach for these diseases. As PDT can cause an angiogenic response in the treated tissue, combination of PDT with anti-angiogenic compounds should lead to improved therapy. This study was undertaken to test the clinically used small molecule kinase inhibitors Nexavar® (sorafenib), Tarceva® (erlotinib) and Sutent® (sunitinib) for this purpose, and to compare the results to the combination of Visudyne®-PDT with Avastin® (bevacizumab) treatment. When topically applied to the chicken chorioallantoic membrane at embryo development day (EDD) 7, a clear inhibition of blood vessel development was observed, with sorafenib being most efficient. To investigate the combination with phototherapy, Visudyne®-PDT was first applied on EDD11 to close all <100 µm vessels. Application of angiostatics after PDT resulted in a significant decrease in vessel regrowth in terms of reduced vessel density and number of branching points/mm(2) . As the 50% effective dose (ED50) for all compounds was approximately 10-fold lower, Sorafenib outperformed the other compounds. In vitro, all kinase inhibitors decreased the viability of human umbilical vein endothelial cells. Sunitinib convincingly inhibited the in vitro migration of endothelial cells. These results suggest the therapeutic potential of these compounds for application in combination with PDT in anti-cancer approaches, and possibly also in the treatment of other diseases where angiogenesis plays an important role.

Assessment of macular pigment optical density (MPOD) in patients with unilateral wet age-related macular degeneration (AMD).

PURPOSE: To compare the macular pigment optical density (MPOD) of patients with unilateral wet age-related macular degeneration (AMD) with the MPOD of bilateral dry AMD patients and healthy elderly individuals. METHODS: The MPOD of 34 patients with unilateral wet AMD was measured in their fellow eye that had the dry form of the disease (study group). The MPOD of the study group was compared with the MPOD of 33 patients with bilateral dry AMD (patients' control group) and 35 elderly subjects without any signs of retinal disease (control group). None of the subjects was under carotenoid supplementation. The MPOD was measured with Heterochromatic Flicker Photometry [QuantifEYE™- MPS 9000 (ZeaVision(©))]. The statistical package SPSS v 17.0 was used for the analysis. RESULTS: The overall mean MPOD was 0.52 (SD 0.15). Patients with unilateral wet AMD have significantly higher levels of MPOD in their fellow eye compared with patients with bilateral dry AMD (0.58 versus 0.48, p = 0.026). Mean MPOD of patients with bilateral dry AMD does not differ significantly from that of healthy elderly subjects (0.48 versus 0.50, p = 0.865). In this population sample, no correlation with age was observed, while women have slightly but significantly higher levels of MPOD (0.55 versus 0.49, p = 0.029). CONCLUSION: In the present study, the mean MPOD at the fellow eye of patients with unilateral wet AMD was found to be significantly higher than that of patients with bilateral dry AMD, while no other significant difference emerged between groups. Further investigation is demanded to clarify the role of macular pigment in AMD progression.

Clinical evidence of intravitreal triamcinolone acetonide in the management of age-related macular degeneration.

Triamcinolone acetonide (TA) is one of the first pharmacologic compounds evaluated for the treatment of choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD). The most important effects of TA consist in the stabilisation of the blood-retinal barrier and the down-regulation of inflammation. TA also has anti-angiogenic and anti-fibrotic properties. The peculiar characteristic of being well tolerated by ocular tissues and the capability to remain active for many months after a single intravitreal injection, make this drug a safe and effective alternative. In the past decade, intravitreal injection of TA (IVTA) has emerged as a useful treatment of several ocular diseases such as uveitis, macular edema secondary to retinal vasculature disease, neovascularisation and vitreoretinopathy. In this paper, we review all the available evidence of its use in AMD as mono-therapy or in combination with other treatments, and we discuss which role TA will play in the treatment of AMD in the future. The first experiences with IVTA as monotherapy for the treatment of exudative AMD reported a positive outcome in transiently reducing the leakage from CNV. However, in the long-term follow-up, IVTA as monotherapy had no effect on the risk of severe visual acuity loss, despite a significant anti-angiogenic effect found 3 months after the treatment. Consequently, studies using the combination of IVTA and photodynamic therapy (PDT), which acts synergistically, were performed. They reported to improve vision and to reduce the number of re-treatments with PDT. A large number of publications confirmed the positive synergic role of combining TA and PDT (therapies) for the treatment of all types of CNV: classic or predominantly classic, occult or minimally classic and RAP (Retinal Angiomatous Proliferation) lesions. The advantages registered with the use of IVTA plus PDT compared to PDT alone were partially limited by the side effects, such as the rapid evolution of cataract. Nevertheless, cataract surgery may stimulate the development of CNV (result in stimulating CNV). However, in large, randomized, clinical trials on combination therapy of TA and PDT, visual acuity failed to show an improvement, even though the lesion size and subretinal fluid had decreased, compared to controls treated with PDT alone. Some authors reported an increased risk of developing macular atrophy after the combination therapy with IVTA and PDT. Reduction of the PDT fluence rate in association with the use of steroids resulted in reducing the risk of macular atrophy and in a better visual acuity outcome. The introduction of anti-VEGF-based drugs has revolutionized the treatment of AMD and has replaced all the previous therapies used for CNV. Visual improvement becomes an expectation in a higher proportion of patients, previously limited to minimizing vision loss. Anti-VEGF therapy also resulted in superior visual improvement compared to all types of combination therapy with IVT and PDT. Nevertheless, anti-VEGF monotherapy also has many limitations due to the need of repetitive treatments, increased costs and tachyphylaxis. Treatment regimens involving TA in combination therapy with anti-VEGF and PDT may preserve benefits for substantially longer periods. A question remains open on whether a combination treatment with anti-VEGF, triamcinolone and/or PDT may be a treatment option in patients with exudative AMD, by offering, with one cycle of therapy, functional VA benefits comparable to those observed with continued monthly anti-VEGF therapy. Further trials, of higher scientific significance, are needed to study the potential of these treatment options.