Purpose Oxidant- and inflammation-induced damage to retinal pigment epithelial (RPE) cells

Purpose Oxidant- and inflammation-induced damage to retinal pigment epithelial (RPE) cells is central to the pathogenesis of age-related macular degeneration (AMD). OTC, followed by analysis of IL-6 and Ccl2 expression with real-time quantitative polymerase chain reaction or enzyme-linked immunosorbent assay. Cellular and molecular markers of inflammation and oxidative stress (i.e., IL-1, TGF-, ABCG1, ABCA1, reduced glutathione, and dihydroethidium) were evaluated in double knockout mice on rd8 background (DKO rd8) treated with OTC (10 mg/ml) in drinking water for a period of 5 months. Results OTC treatment significantly inhibited the expression and secretion of IL-6 and Ccl2 in TNF–stimulated ARPE-19 cells. Studies conducted using DKO rd8 animals treated with OTC in drinking water confirmed these findings. Cellular and molecular markers of inflammation were significantly suppressed in the retinas of the OTC-treated DKO rd8 animals. Subsequent in vitro and in vivo studies of the possible mechanism(s) to explain these actions revealed that although OTC is an agonist of the anti-inflammatory G-protein coupled receptor GPR109A and L-Mimosine supplier a transportable substrate of the sodium-coupled monocarboxylate transporter SMCT1 (SLC5A8), these properties may play a role but do not explain entirely the anti-inflammatory effects this compound elicits in cultured RPE cells and the intact mouse retina. Conclusions This study represents, to our knowledge, the first report of the suppressive effects of OTC on inflammation in cultured RPE cells and on inflammation and oxidative stress in the retina in vivo. Introduction Age-related macular degeneration (AMD) is a leading cause of blindness worldwide [1-3]. The pathogenesis of the disease is multifactorial and complex; thus, the task of elucidating mechanisms and developing novel strategies Rabbit Polyclonal to TTF2 for treating and preventing AMD involves significant challenges. However, several major findings related to the disease, based upon an abundance of clinical and experimental evidence, are relatively indisputable. First, as the name implies, AMD is a disease of aging; clinical symptoms begin to appear only at relatively older ages (>60 years). Second, oxidative stress and inflammation are crucial players, both in disease development and progression. Last, retinal pigment epithelial (RPE) cells, cells crucial for normal retinal health and visual function, are highly susceptible to damage or dysfunction and therefore represent a primary site of pathology in the disease. Our focus in this study centers on the latter two points, as our aim is to explore a novel means of limiting oxidative stress and inflammation not only in cultured RPE cells but also in the eyes of the living animal. Specifically, we evaluate the efficacy of L-2-oxothiazolidine-4-carboxylic acid (OTC) as a dual antioxidant and anti-inflammatory agent in cultured RPE cells (ARPE-19 and primary mouse RPE cells), and in the eyes of the mouse, L-Mimosine supplier a murine model predisposed to increased oxidative stress and inflammation in the retina [4]. OTC is a prodrug of cysteine. Upon L-Mimosine supplier entering cells, the compound is cleaved by the ubiquitous intracellular enzyme 5-oxoprolinase, readily generating cysteine, the limiting amino acid in glutathione (GSH) biosynthesis [5]. The beneficial effects of OTC in terms of augmenting levels of this major cellular antioxidant have been documented in several cell and tissue types and confirmed by studies in animals and humans [6-13]. Congruent with this is our recent report demonstrating for the first time the robust antioxidant and cell-protective properties of this compound in cultured human RPE cells [14]. However, whether this benefit can be extrapolated to the intact retina in vivo is unknown. Regarding AMD pathogenesis, oxidative stress and inflammation go hand in hand; inflammation is a common consequence of increased oxidative stress in RPE cells and the retina, and once initiated, inflammation further potentiates reactive oxygen species (ROS) production in this cell and tissue type [15-17]. This may be reflective directly of the fact that L-Mimosine supplier RPE cells are exposed to considerable amounts of oxidative stress continuously, even in the absence of disease [18]. Additionally, RPE cells represent a major source of cytokines in the retina and therefore are a critical regulator of inflammation in this tissue [18-20]. Thus, in aging, when the antioxidant capacity of RPE cells decreases and the balance between anti- and pro-oxidant factors.