| Cataract and age-related macular degeneration (AMD) are the leading causes of blindness. In particular, cataract is the leading cause of blindness worldwide while AMD is the leading cause in the Western countries. Age is a major risk factor for both of these ocular diseases and they are also strongly associated with oxidative stress. Therefore, specific changes to the lens and Bruch's membrane (BM) due to oxidative stress are studied to understand and elucidate the pathology of cataract and AMD.;The transport of oxygen in the whole lens remains poorly understood. In this work, a novel method was developed using an Ocean Optics Oxygen Sensor to measure the viscosity of whole lenses. This method determined that the average viscosity for a whole bovine lens is 5.74+/-0.88 cP. Using the Stokes-Einstein equation at the slip boundary condition, the diffusion coefficient for oxygen was calculated to be 8.2 x 10-6 cm 2 s-1. The human donor lenses ranged from 14 to 80 years old with an average viscosity of 2.8 +/-1.01 cP and average mass of 0.212 +/-0.03 g. The diffusion coefficient of oxygen for the human donor lenses is calculated to be 1.7 x 10-5 cm2 s-1. Overall, these data allow a better understanding of oxygen transport within the lens.;AMD is characterized by photoreceptor degeneration as a result of damage to the retinal pigment epithelium (RPE) and BM. In this work, the changes to the structure of BM by age and inflammation are investigated. Specifically, blue light mediated A2E damage, non-enzymatic glycation and non-enzymatic nitration of a fibronectin (FN) peptide were used as models for ageing and inflammation. This synthetic FN peptide contains the RGD amino acid binding sequence to the alpha-5 beta-1 integrin region of RPE cells. Using liquid chromatography/mass spectrometry, the sites of modification were identified on the FN peptide. A2E, a component of lipofuscin, photochemically modifies FN preferentially at lysine and arginine residues. The reaction proceeds via A2E-derived aldehydes. Glycation of FN via glycolaldehyde generates aldoamine and Nepsilon-carboxymethyllysine adducts that attach to lysine residues. When FN was glycated with methygloxal, Nepsilon-carboxyethyllysine adducts form on lysine residues. Tetrapyrimidine and hydroimidazolone adducts attach on arginine residues. Non-enzymatic nitration of FN produces 3-nitrotyrosine at tyrosine residues. The lysine residues of FN undergo deamination and generate an alkene or alcohol group. The effects of modified FN protein on ARPE19-cells have also been studied. ARPE-19 cells did not attach to modified FN coated plates. Furthermore, modified FN enhanced the necrosis of RPE cells that were UV irradiated. These data enhance our understanding of the structural changes to BM and RPE cells during ageing and inflammation. These data also may help to explain underlying causes of AMD. |
