Endocytosis of Advanced Glycation End Products in Bovine Choriocapillary Endothelial Cells

Abstract

Abstract: : Advanced glycation end products (AGEs) are associated with a number of pathological conditions, such as diabetes mellitus, Alzheimer’s disease, uremia, as well as with normal ageing. Age–related retinal disease correlates with changes in Bruch’s membrane, including accumulation of AGEs and other modified macromolecules. Specialized scavenger cells are important for uptake and degradation of these macromolecules both locally and at distant sites. Our hypothesis is that choriocapillary endothelial cells (CCE), being located closely to Bruch’s membrane, have a local scavenger function in the eye. Purpose: The aim of the study was to examine endocytosis of AGEs in CCE. Methods: Long–term cultures of CCE from bovine eyes were established as described (Liu and Li, Exp. Eye Res. 57:37–44, 1993). Cultures were incubated with unlabeled, radiolabeled or fluorescently labeled AGE–modified bovine serum albumin (AGE–BSA) or formaldehyde–treated bovine serum albumin (FSA) under serum free conditions. Specificity, capacity and rate of endocytosis were measured. Immunocytochemistry and western blot analysis were done using an anti–AGE monoclonal antibody and a polyclonal antibody against the rat liver hyaluronan receptor, Stabilin 2, (McCourt et al., Hepatology, 30:1276–1286,1999; Politz et al. Biochem. J., 362:155–164, 2002) which is the major scavenger receptor for AGE–BSA in the sinusoidal scavenger endothelial cells of liver. Results: Radioiodinated AGE–BSA and FSA were actively taken up and degraded by the CCE cultures. Uptake was dose–dependent and Ca²⁺–independent and was blocked by excess amounts of non–labeled AGE–BSA, FSA, and fluorescently labeled FSA, respectively, but not by mannan. Fluorescently labeled FSA was seen in vesicles both in primary cells and subcultured cells. Immunoelectron microscopy of cells fed with AGE–BSA for 2h also showed distribution in vesicles throughout the cells. Immunostaining of the CCE cultures showed positive staining with the anti–Stabilin 2 antibodies. The expression of Stabilin 2 was confirmed by western blot analysis. Endocytosis of AGE–BSA was partly inhibited (25%) by co–incubation with the Stabilin 2 antibody (1 mg/ml), but not with non–immune IgG. Conclusions:We conclude that bovine CCE actively endocytose AGE–BSA. The cells express the scavenger receptor Stabilin 2, which may be partly responsible for the uptake.

Keywords: aging • vascular cells • receptors: pharmacology/physiology