The combined effects of vitamin D and sulforaphane in the treatment of an in vitro model of macular degeneration.

Age-related macular degeneration (AMD) is a progressive eye disease affecting the central area of the retina, known as the macula, and is the leading cause of vision impairment worldwide in the elderly. The etiology of AMD is poorly understood although it has been linked with several pathological factors, including chronic oxidative stress, autophagy decline, and inflammation. An increased level of oxidative stress significantly influences the pathophysiology of AMD with an imbalance of protective and toxic factors of homeostatic conditions of the retina, leading to tissue dysfunction and chronic inflammation of RPE cells. Several therapeutic strategies targeting the inflammatory pathways have been shown to slow the progression of AMD to prevent or delay vision loss, but the only treatment currently used in clinics targets VEGF with anti-VEGF intravitreal injections. For this reason, the study aims to mitigate the inflammation that is associated to the AMD disease through the use of two molecules, the vitamin D (VD) and the sulforaphane (SFN), administered alone or in combination, to test their efficacy for future supplement treatment to intravitreal injections in vivo. An in vitro model of retinal pigment epithelial cell line ARPE-19, treated with TGF-β as a pro-inflammatory stimulus, was used to investigate the activity of SNF and VD in reducing TGF-β-induced effects, including oxidative stress, inflammation, angiogenesis, and epithelial markers’ transcription. The administration of SFN and VD lowered ROS levels greatly induced by TGF-β, and reduced the correlated expression of the pro-inflammatory cytokines, IL-6 and IL-8. Because the mitochondrial respiration is a source of ROS production, we evaluated the transcription of respiratory elements and we found that the increase triggered by TGF-β was prevented by VD and SFN treatment. Given that angiogenesis is frequently associated to inflammation, and VEGF is a key mediator of pathological new blood vessel formation, VEGF secretion was evaluated. The treatment with SFN and VD reduced the release of VEGF in the extracellular medium of TGF-β-stimulated ARPE-19 cells, and consequently, the markers of epithelial and tight junction integrity appeared restored. Remarkably, all the described biological effects were potentiated by the co-stimulation with the two compounds. Altogether, the results of this study highlight the potential synergistic activity of sulforaphane and vitamin D in abating the TGFβ-induced oxidative stress, angiogenesis, and inflammation. A wider knowledge of this complex scenario is necessary to develop a novel supplement strategy targeting the chronic inflammation in AMD, and to potentiate the therapeutic effect of anti-VEGF intravitreal injections. Keywords: vitamin D (VD), sulforaphane (SFN), age-related macular degeneration (AMD), retinal pigment epithelial cells (ARPE-19), inflammation, reactive oxygen species (ROS), vascular endothelial growth factor (VEGF).