Eyes with CNV showed persistent labeling of MAC at the level of the choriocapillaris even after degeneration of the endothelium was complete (Physique?5A). high-risk genotype had thinner choroids than low-risk homozygotes (and/or (recently reviewed by Khandhadia et?al4). One polymorphism in the gene (rs1061170) increases risk of AMD by approximately twofold to sevenfold, depending on the populace studied.5C8 This variant results in the substitution of histidine for tyrosine UAMC 00039 dihydrochloride at amino acid residue 402. The effect of this polymorphism in the human eye is not well comprehended, although adults harboring the Y402H polymorphism show increased choroidal C-reactive protein9 and increased membrane attack complex (MAC).10 Formation of the MAC is the final event in the terminal portion of the complement cascade and results from the binding of C5b to plasma complement proteins C6, C7, C8, and multiple molecules of C9. MAC forms transmembrane channels that lead to cell lysis and death. The MAC has been found in drusen of older eyes with AMD.11 However, the relative abundance and distribution of MAC in aging, early AMD, and advanced AMD have not been comprehensively studied. Inhibition of MAC components such as C6 can inhibit CNV,12 and other complement pathway inhibitors are in active clinical trials for the treatment of AMD.13 Because it is the ultimate downstream effector of the complement pathway, understanding the role of the MAC in the pathophysiology of AMD is important for the development of new therapies. We evaluated the MAC in a large series of donor eyes. MAC was present in Bruchs UAMC 00039 dihydrochloride membrane and choriocapillaris in very young eyes, but the concentration increased with age; we observed the highest levels in eyes with AMD. We further evaluated the MAC in a series of eyes from young and aged donors, and from donors with early UAMC 00039 dihydrochloride and advanced AMD. Although in early AMD the MAC is usually associated exclusively with the choriocapillaris, in advanced AMD the RPE may be exposed as well. Morphometric experiments suggest that high-risk genotypes may contribute to thinning or atrophy of the choroid. Overall, these studies suggest that choroidal endothelial cells are targets of the MAC and that approaches to prevent their injury from complement-mediated lysis may be useful in the treatment of AMD. Materials and Methods Human Donor Eyes Whole globes from human donors were obtained from the Iowa Lions Vision Bank (Iowa City, IA). Full consent for research was obtained from the donors next of kin in all cases, and all experiments were performed in accordance with the Declaration of Helsinki. Eyes were processed within 9.5 hours of death (range, 1 hour 42 minutes to 9 hours 15 minutes). For biochemical studies, a 6-mm juxtamacular, inferotemporal punch was acquired. Neural retina and RPECchoroid layers were collected separately and snap-frozen in liquid nitrogen, before long-term storage at ?80C. Macular punches and/or superotemporal wedges were collected from each vision and preserved in 4% paraformaldehyde in phosphate-buffered saline within 8 hours of death. After 2 hours of fixation, eyes were washed in phosphate-buffered saline and then were cryoprotected in sucrose and embedded in?sucroseCoptimal cutting temperature medium, as described by Barthel and Raymond.14 Quantification of Soluble C5b-9/MAC Samples were chosen for MAC quantification from a collection of frozen juxtamacular punches of RPECchoroid, centered approximately 7 mm temporal to the fovea. Ten RPECchoroid samples were selected from each of three groups: young (mean age, 39.6 years; range, 21 to 48 years); aged, with a clinical and/or histological diagnosis of dry AMD (mean Rabbit polyclonal to PPP1R10 age, 87.1 years; range, 77 to 99 years); and age-matched control, without AMD (mean age, 82.8 years; range, 71 to 96 years) (Table?1). Of the 30 samples studied, 2 samples in the AMD group were new punches from donor eyes reported previously.10 Samples were homogenized for 90 seconds using.