Age related macular degeneration: a molecular insight.

Age-related macular degeneration (AMD) is a progressive and degenerative disease affecting the macula lutea. Late stages include neovascular AMD (nAMD), which represents the most severe form of the disease and is characterized by the abnormal growth of newly formed blood vessels from the pre-existing choroidal vessels. Neovascularization breaks through Bruchʼs membrane reaching the retina where bleeding, fluids extravasation and a chronic inflammatory reaction eventually cause severe vision loss, up to blindness if left untreated. With the aim to better elucidate the neoangiogenic process underlining nAMD and to evaluate novel targets to treat it, we collected aqueous humour samples and choroidal neovascular membranes (CNVM) from nAMD patients, before and after antiangiogenic treatment, and analyzed them for the content of proteins known to be involved in angiogenesis. ELISA of the aqueous humour samples revealed the up-regulation of proteins such as VEGF-A, ATRA1, CD93 and TGF-β1 in naïve nAMD patientsʼ samples. While anti-VEGF treatment could efficiently reduce not only VEGF-A but also HTRA1 levels, it exerted little effect on CD93 aqueous concentration that was further confirmed by immunofluorescence of CNVM that displayed a up- regulation of the protein on the endothelium of proliferating vessels both from treated and untreated patients. Since anti-VEGF injections further increased TGF-β1 aqueous content, thus advocating for a possible targeting to treat nAMD, we investigated the concentrations of the active form of all the three TGF-βs, TGF-β1, TGF-β2 and TGF-β3, actually responsible for signalling activation. Using ELISA we could assess that, in the eye, TGF-β2 represents the main active form followed by TGF-β3. TGF-β1 accounted for only a 1,6% of the total active TGF-β. Interestingly, active TGF-β2 was found to be down regulated in naïve nAMD samples, with concentrations lower than the control group even after the second treatment. Since TGF-β signalling can be activated and modulated by several other molecules, the actual activation state of this pathway could not reflect active TGF-βs concentrations. We, therefore, set up a luciferase-based assay to evaluate the real activation state of the pathway and could demonstrate that a significant reduction of the anti- angiogenic SMAD2/3 signalling was evident in naïve nAMD samples and that the pathway activation was restored after the first anti-VEGF treatment. Taken together our data propose the possibility to evaluate HTRA1 and CD93 as novel targets to treat nAMD and, at the same time, suggest caution when considering TGF-β activity general inhibition. ! 4! Given the great impact oxidative stress exerts on AMD development, we also carried out in vitro and in vivo studies to evaluate NACET, a novel n-acetyl cysteine (NAC) derivative, as potential antioxidant molecule to prevent or delay AMD onset and progression. Our results proved NACET to be a safe and powerful antioxidant, able to counteract oxidative stress even in a preventive way. Thank to its improved bioavailability compared to NAC, NACET oral administration to rats led to a significant increase in the ocular glutathione content thus supporting its potential eligibility for ocular therapy. Further studies are needed to better evaluate NACET activity and toxicity in vivo but our in vitro and in vivo preliminary results advocate for a possible use of this molecule as prevention for AMD development.