Effect of GAG Removal & LDL Concentration on Hydraulic Conductivity of and LDL Transport Across Bruch's Membrane

Abstract

Purpose: : Lipids accumulate with age in Bruch's membrane (BrM) and are associated with AMD. In atherosclerosis, lipid accumulation in the vessel wall has been associated with both the presence (Skalen et al., Nature, 2002) and absence (Pillarisetti et al., JCI, 1997) of glycosaminoglycans. We used GAGases to remove GAGs from bovine BrM and determined the effects on hydraulic conductivity (Lp) of the tissue and on low density lipoprotein (LDL) transport across the tissue. We also separately determined the effect of LDL concentration on its transport through BrM.

Methods: : 2x2 cm tissue squares of BrM and choroid from non-tapetal regions were gently separated from post-mortem bovine eyes. The tissue was treated with chondroitinase ABC, heparinase III, both enzymes or neither for 6 to 24 hours. Enzymatic activity was confirmed morphologically in control studies. The tissue was then placed into a Ussing chamber and perfused with buffer or buffer with fluorescent 3,3'-dioctadecylindocarbocyanine low-density lipoprotein (DiI-LDL: 0.005 or 0.05 mg/ml). The concentrations of DiI-LDL in the upstream and downstream compartments were determined at the experiment's conclusion. Lp and the reflection coefficient of BrM/choroid to DiI-LDL were then calculated.

Results: : BrM had a significantly higher (p=0.035) reflection coefficient (0.61 ± 0.25, n=15) to the higher concentration of perfused LDL as compared to the lower concentration (0.39 ± 0.28, n=12). Use of either chondroitinase ABC (n=10) or heparinase III alone did not significantly alter either L_p of BrM or its reflection coefficient to LDL. However, use of both enzymes (n=4 in experimental group) together led to a marginally significant increase in hydraulic conductivity (1.05 ± 0.31 x 10^-8 cm²s/g to 1.45 ± 0.54 x 10^-8 cm²s/g, p=0.052) and decrease in the reflection coefficient (0.61 ± 0.26 to 0.34 ± 0.32, p=0.078).

Conclusions: : Increased LDL concentration hindered transport of these particles through BrM. GAGase treatment appeared to facilitate transport of LDL through BrM, consistent with findings in arterial walls that GAGs bind lipids. These results are consistent with a hypothesis that an increased level of lipoprotein secreted by the RPE and/or an increased concentration of GAGs in BrM contribute to the age-related accumulation of lipids in BrM.

Keywords: Bruch's membrane • lipids • extracellular matrix