Analysis of newly synthesized Bruch's membrane proteoglycans

Bruch's membrane may provide a selective filtration barrier for nutrients coming from the choriocapillaris to the outer retina. Because proteoglycans have been shown to have structural and filtration properties in other tissues, we have been investigating the deposition of newly synthesized proteoglycans into Bruch's membrane and how these may be affected by aging and pathology. Proteoglycans deposited in human Bruch's membrane were metabolically labelled with 35SO4 and 3H-glucosamine using a whole-eye organ culture system. Labeled proteoglycans were extracted from dissected Bruch's membranes with 4 M guanidine and isolated by ion-exchange column chromatography on DEAE cellulose using a linear salt gradient. These molecules were subsequently chromatographed on Sepharose CL-4B and glycosaminoglycan content characterized by enzymatic and chemical degradation. The elution profiles for proteoglycans remains relatively unchanged with age, although in eyes from donors over age 70 there is a small change in size distribution toward higher molecular weights. However, the proportions of newly synthesized glycosaminoglycans remain unchanged with age, being approximately 75% chondroitin sulfate/dermatan sulfate and 25% heparan sulfate. Bruch's membrane proteoglycans from donors with different retinal pathologies, however, exhibited an increased proportion of heparan sulfate. Considering the structural and filtration properties of proteoglycans, such alterations could result in abnormal functioning of Bruch's membrane that could ultimately affect the maintenance of the outer retina.