Linköping University Medical Dissertations
No. 557

The effects of reactive oxygen species and lipofuscin on the function and health of the retinal pigment epithelium
Possible pathophysiological mechanism behind the development of age-related macular degeneration

Ulf Wihlmark
Legitimerad läkare

Akademisk avhandling
som för avläggande av medicine doktorsexamen vid Universitetet i Linköping kommer att offentligt försvaras i aulan, administrationsbyggnaden, Hälsouniversitetet. Linköping, torsdagen den 28 maj 1998, kl 09.00


Age-related macular degeneration (ARMD) is a common cause of central vision loss in elderly people. Specific treatment is possible only for selected patients. A dysfunction of the retinal pigment epithelial (RPE) cells has been proposed to help explain the pathogenesis of ARMD. In the normal turnover of photoreceptor outer segments (POS), membranes rich in polyunsaturated fatty acids (PUFAs) are shed and phagocytised by the RPE. PUFAs are highly susceptible to free radical damage, causing peroxidation and subsequent formation of products with fluorescence similar to Schiff bases, a component of lipofuscin. With increasing age, lipofuscin accumulates in the RPE cells, and it has been suggested that lipofusein could be detrimental to RPE function through free radical generation or interference with the autophagocytic capacity of cells having lipofuscin-loaded lysosomes.

To study the effect of oxidative stress on lipofuscin accumulation, rabbit RPE cell cultures were kept at an ambient oxygen concentration of either 8 % or 40 %. To simulate the normal phagocytic function of RPE cells, bovine POS were added daily. The lipofuscin-specific autofluorescence was measured after 1, 2 and 3 weeks. RPE cells cultured under normobaric hyperoxic conditions (40 % oxygen) showed significantly higher levels of lipofuscin-like autofluorescence than those kept at normobaric and probably normooxic conditions (8 % oxygen) after 1, 2 as well as after 3 weeks. For both oxygen concentrations, the lipofuscin accumulation level was increased after 2 weeks of POS exposure and had increased even further after 3 weeks. The results suggest an involvement of oxidative mechanisms in the formation of lipofuscin from phagocytised POS by RPE cells.

In the second study, bovine POS were photo-oxidised, and turned into a lipofuscin-like material, by irradiation with UV light. Transmission electron microscopy of irradiated POS showed loss of the normal stacks of the disk membranes with conversion into an amorphous osmiophilic, electron dense mass. The formation of thiobarbituric acid reactive substances (TBARS), estimated during the irradiation process, indicated lipid peroxidation. The later decline in TBARS indicates fragmentation of the peroxides and conjugation of formed aldehydes with proteins under the formation of more stable Schiff bases and their secondary reaction products, e.g. lipofuscin. Irradiated POS also showed a strong granular yellow auto-fluorescence. RPE cell cultures, kept at 21 % ambient oxygen, were fed daily for 3, 5 or 7 days with either UV-peroxidised POS, native POS or culture medium only. RPE cells fed irradiated POS showed significantly higher levels of lipofuscin-specific autofluorescence compared to cells exposed to native POS after 3 days, 5 days and 7 days and to the non-exposed control cells. The lipofuscin content of cells exposed to irradiated POS increased significantly between days 3 and 7. Ultrastructural studies showed much more numerous and larger lipofuscin-like inclusions in RPE cells fed irradiated POS compared to cells exposed to native POS. In the control cells, lipofuscin-like granules were small and sparse. It appears that exposing RPE cells to previously peroxidised POS, thus artificially converted into lipofuscin-like material and obviously not digestible by the lysosomal enzymes, accelerates the formation of severely lipofuscin-loaded cells.

A well-known physical property of lipofuscin is its yellowish autofluorescence when irradiated by blue light. Such energy transformation is known to induce photo-oxidative processes since oxygen present in the immediate surroundings would be activated into reactive oxygen metabolises. RPE cells are constantly exposed to visible light during the time the subject is awake. Consequently, in RPE cells exposed to light, the membranes of the lysosomes surrounding enclosed lipofuscin would be subjected to oxidative stress, which may result in damage, with leakage to the cytosol of lysosomal hydrolytic enzymes and ensuing cellular degeneration.

To test this hypothesis, cultures of heavily lipofuscin~loaded RPE cells were blue-light. irradiated and compared to relevant controls. Following irradiation, lysosomal membrane stability was measured by vital staining with the lysosomotropic weak base, and metachromatic fluorochrome, acridine orange (AO). Quantifying red (high AO concentration within intact lysosomes with preserved proton gradient over their membranes) and green fluorescence (low AO concentration in nuclei, damaged lysosomes with decreased or lost proton gradients, and in the cytosol) allowed an estimation of the lysosomal membrane stability. Cellular viability was estimated with the delayed trypan blue dye exclusion test. Lipofuscin-loaded blue-light-exposed RPE cells showed a considerably enhanced loss of both lysosomal stability and viability when compared to control cells. It is concluded that the accumulation of lipofuscin within secondary lysosomes of RPE sensitizes these cells to blue light by inducing photo-oxidative alterations of their lysosomal membranes resulting in a presumed leakage of lysosomal contents to the cytosol with ensuing cellular degeneration of apoptotic type.

The aim of the last investigation was to study whether heavy loading with lipofuscin of RPE lysosomes would affect the further phagocytic ability of the cells.

In the first section of the investigation, cultures of rabbit RPE cells were exposed daily to bovine UV-irradiated POS for 4 weeks, resulting in a pronounced lipofuscin accumulation of the cells. Fluorescent latex beads (labelled with a red fluorophore) were added to unloaded control cultures at 0 and 4 weeks after their establishment, and to lipofuscin loaded cells after 4 weeks of feeding with artificial lipofuscin. Cellular amounts of lipofuscin, and their phagocytotic activity, were quantified by static fluorometry measuring lipofuscin-specific and red bead-specific fluorescence, respectively. Unloaded, and thus almost lipofuscin-free, control cells exposed to latex beads showed numerous cytoplasmic particles emitting reddish fluorescence, while few particles were taken up by cells initially loaded with artificial, POS-derived, lipofuscin. Measurement of the latex bead-specific fluorescence showed significantly higher levels in unloaded control cells than in lipofuscin-loaded ones.

In the second part of the investigation, unloaded control cultures and lipofuscin-loaded cultures were exposed to native bovine Texas Red-X-labelled POS 4 weeks after the establishment of the cultures. Unloaded control cells showed a large number of cytoplasmic POS emitting reddish fluorescence, while fewer POS were fagocytosed by cells loaded with artificial lipofuscin. Measurement of the Texas Red-X-specific fluorescence, thus quantifying the fagocytic ability of the cells, showed significantly higher levels in control cells than in lipofuscin-loaded ones. Severe lipofuscin accumulation of RPE cells appears to result in a greatly decreased phagocytic capacity.

The suggested mechanisms may be of relevance in the pathogenesis of ARMD.

Department of Ophthalmology
Linköping University, S-581 85 Linköping, Sweden

Linköping 1998