PURPOSE. To examine the effects of oxidized low-density lipoproteins (oxLDL) on phagocytosis and processing of photoreceptor outer segments (OS) by retinal pigment epithelial (RPE) cells. METHODS. Confluent cultures of RPE-J cells were pretreated with oxLDL or LDL, and the effects of such treatment on the processing of added OS was determined. Processing was determined either by the degradation of 125I-labeled OS to trichloroacetic acid-soluble label or by the cleavage of rhodopsin observed on Western blot analysis of cell lysates separated by sucrose density gradient fractionation. Binding to and uptake of OS by RPE-J cells was assessed by determining the fluorescence of FITC-labeled OS before and after quenching with trypan blue. RESULTS. OxLDL induced a significant decrease in the degradation of 125I-OS in RPE-J cells but no reductions in either binding or uptake, when a 24-hour recovery period was inserted between treatment with oxLDL and challenge with OS. Rhodopsin cleavage increased in a time-dependent manner after phagocytosis of OS by RPE-J cells. The small guanosine triphosphatase (GTPase), Rab5, was first found in phagosome fractions containing rhodopsin and its cleavage products, and at later times of challenge, in more dense fractions representing phagolysosomes. These were assessed by the colocalization of rhodopsin cleavage products in density fractions with cathepsin D, a marker of lysosomes. OxLDL induced a reduction in rhodopsin cleavage product formation and in phagosome-lysosome fusion (maturation). It also reduced the time-dependent shift of rhodopsin to higher density fractions containing more cathepsin D without any detectable reduction in the expression of cathepsin D or in acid protease activity. CONCLUSIONS. OxLDL induces a reduction in the processing of OS by RPE by perturbing the fusion of lysosomes with phagosomes.
|Original language||English (US)|
|Number of pages||7|
|Journal||Investigative Ophthalmology and Visual Science|
|State||Published - Oct 22 2001|
ASJC Scopus subject areas
- Sensory Systems
- Cellular and Molecular Neuroscience