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Retinal pigment epithelial cells, oxidative stress and lipofuscin: relation to age-related macular degeneration
Linköping University, Department of Neuroscience and Locomotion, Ophthalmology. Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In developed countries, age-related macular degeneration (AMD) is the most common cause of loss of central vision in people over the age of 65. The retinal pigment epithelium (RPE) appears to be the site of early pathological changes in AMD. Thus, age related structural changes in the RPE, such as the accumulation of lipofuscin 'age pigment', decrease in melanin content, and accumulation of deposits beneath the RPE or -within Bruch's membrane, appear to be strongiy associated with the pathogenesis of AMD. The posunitotic RPE provides essential support for the photoreceptors through daily phagocytosis of the shed photoreceptor outer segment (POS) tips. The area comprised of photoreceptors, RPE, Bruch' s membrane, and the choroid represents a unique environment. The relatively high oxygen tension, and the exposure to short-wavelength light, promotes the generation of reactive oxygen species (ROS) and free radicals. The POS has a high concentration of polyunsatorated fatty acids (PUFA's) and are thus highly susceptible to attack from ROS and free radicals. The resultant peroxides may transform into aldehydes, cross-liok to protein fragments undergoing intralysosomal degradation and form fluorophores, similar to Schiff bases, known components of lipofuscin. Over time, lipofuscin accumulation in the RPE can be substantial and interfere with cellular functions. RPE deterioration and ensning photoreceptor degeneration may contribute to the development of AMD.

The present study focuses on the formation of lipofuscin and on different ways of op- and down-regolating its formation in cultored RPE cells. By developing an experimental model we have shown that the highly metabolically active, posunitotic RPE cells, when cultured nnder hyperoxia (40% O2) with addition of POS, fulfil the prerequisites for massive lipofuscin accumulation. Using this model, the present study shows: (1) that heavily lipofuscin loaded, cultored rabbit RPE cells, have a reduced capacity of phagocytizing both Texas Red labeled latex beads, and Texas Red labeled POS compared to uuloaded control cells after both 2 and 4 weeks of culturing; (2) that melanin-rich RPE cells from pigmented rabbit and calf were better protected against lipofuscin formation than albino rabbit RPE cells and melanin-poor calf RPE cells after 4 weeks of cultoring; (3) that RPE cells from rabbit and calf, supplemented with the antioxidant substances a-tocopherol (vitamin E), lutein, zeaxanthin or lycopene accumulated less lipofuscin compared to control cells after 18 days of culturing; ( 4) that hydroxychloroquine induced less intensive vacuolization of the cytoplasm in calf RPE cells at both 24 h and 2 weeks, and less intensive periodic acid Schiff staining (as an indication of lipofuscin content) at 2 weeks, than did chloroquine.

We conclude that the mvolvement of oxidative processes seems highly plausible to be involved in the formation of RPE lesions associated with aging and AMD. Furthermore, lipofuscin-engorged RPE cells have a reduced capacity for further phagocytosis, in turn resulting in degeneration of photoreceptors and the development of AMD. Melanin possesses aoti-oxidative properties, perhaps through the sequestration of heavy metals, especially traosition metals such as iron aod copper. Apart from quenching of siuglet oxygen and protection against blue light, the carotenoids lutein zeaxanthine and lycopene seem, as a-tocopherol, to have chain breaking abilities in peroxidation reactions, reducing lipofuscin formation. The lysosomotropic weak bases chloroquine, hydroxychloroquine and ammonium chloride compromise the function of lysosomes. An estimated difference in lysosomotropism between the two drugs would correspond to the difference in toxicity reported between chloroquiue and hydroxychloroquine.

Thus, this study has revealed several mechanisms involved in the formation of lipofuscin m RPE, mechanisms that may be iroplicated in the pathogenesis of AMD.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2002. , 43 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 717
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-28079Local ID: 12843ISBN: 91-7373-158-7 (print)OAI: oai:DiVA.org:liu-28079DiVA: diva2:248630
Public defence
2002-02-08, Berzeliussalen, Universitetssjukhuset, Linköping, 13:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-09-17Bibliographically approved
List of papers
1. Lipofuscin accumulation in cultured retinal pigment epithelial cells reduces their phagocytic capacity
Open this publication in new window or tab >>Lipofuscin accumulation in cultured retinal pigment epithelial cells reduces their phagocytic capacity
1998 (English)In: Current Eye Research, ISSN 0271-3683, E-ISSN 1460-2202, Vol. 17, no 8, 851-857 p.Article in journal (Refereed) Published
Abstract [en]

Purpose. Retinal pigment epithelial (RPE) cells slowly accumulate lipofuscin pigment within their acidic vacuolar apparatus as a result of extra- and/or intralysosomal oxidative alterations of phagocytosed photoreceptor outer segments (POS) with consequent imperfect degradation of these structures. In old age, lipofuscin accumulation may become quite substantial. It has been suggested that pronounced accumulation of lipofuscin is related to decreased RPE function and, possibly, to age-related macular degeneration. The aim of the present investigation was to study whether heavy loading with lipofuscin of RPE acidic lysosomes would affect the further phagocytic ability of the cells.

Methods. In the first section of the investigation, cultures of rabbit RPE cells were exposed daily to bovine UV-irradiated POS (artificial lipofuscin) 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. The intracellular location of the beads was verified by confocal laser scanning microscopy.

Results. 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 (p < 0.001) 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 cytopiasmic POS emitting reddish fluorescence, while fewer POS were phagocytosed by cells loaded with artificial lipofuscin. Measurement of the Texas Red-X-specific fluorescence, thus quantifying the phagocytic ability of the cells, showed significantly (p < 0.001) higher levels in control cells than in lipofuscin-loaded ones.

Conclusions. Severe lipofuscin accumulation of RPE cells appears to result in a greatly decreased phagocytic capacity. The resulting reduction in ability to cope with the needs of the overlying photoreceptor cells, in order to eliminate the obsolete tips of their POS, may well be of significance in the development of age-related macular degeneration.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-81490 (URN)10.1080/02713689808951268 (DOI)
Available from: 2012-09-17 Created: 2012-09-17 Last updated: 2017-12-07Bibliographically approved
2. Lipofuscin-formation in cultured retinal pigment epithelial cells is related to their melanin content
Open this publication in new window or tab >>Lipofuscin-formation in cultured retinal pigment epithelial cells is related to their melanin content
2001 (English)In: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 30, no 1, 74-81 p.Article in journal (Refereed) Published
Abstract [en]

Age-related macular degeneration (AMD), the leading cause of blindness in the developed world, is accompanied by degeneration of the retinal pigment epithelial (RPE) cells. There is an inverse correlation between the melanin content of the eye and the incidence of AMD. Lipofuscin (LF)-accumulation in RPE cells accompanies the process of aging, and may also be related to AMD. This study was designed to evaluate the effect of melanin/melanosomes on the rate of LF formation in cultured rabbit and bovine RPE cells subjected to oxidative stress (40% normobaric O2) and daily supplementation with photoreceptor outer segments for 4 weeks. The LF content was measured at 0, 2, and 4 weeks in RPE cells from pigmented and albino rabbits, as well as in pigment-rich and pigment-poor bovine cells. Albino rabbit and pigment-poor bovine cells accumulated significantly higher amounts of LF than pigmented rabbit cells and pigment-rich bovine RPE cells after both 2 and 4 weeks of exposure. Autometallography of melanin-containing cells, without previous exposure to ammonium sulfide, showed a positive outcome, indicating either the occurrence of pre-existing iron-sulphur clusters or an extremely high intrinsic reducing capacity. These results suggest that melanin acts as an efficient antioxidant, perhaps by interacting with transition metals.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27704 (URN)10.1016/S0891-5849(00)00444-5 (DOI)12442 (Local ID)12442 (Archive number)12442 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-09-17Bibliographically approved
3. Lipofuscin-formation in retinal pigment epithelial cells is reduced by antioxidants
Open this publication in new window or tab >>Lipofuscin-formation in retinal pigment epithelial cells is reduced by antioxidants
2001 (English)In: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 31, no 2, 217-225 p.Article in journal (Refereed) Published
Abstract [en]

The accumulation of lipofuscin by retinal pigment epithelium may be an important feature in the pathogenesis of age-related macular degeneration, suggesting the possibility that this common cause of blindness might be prevented or delayed by antioxidants. In support of this idea, we now report significantly reduced formation of lipofuscin when the antioxidant substances lutein, zeaxanthin, lycopene (carotenoids), or α-tocopherol were added to rabbit and bovine (calf) retinal pigment epithelial (RPE) cells exposed to normobaric hyperoxia (40%) and photoreceptor outer segments. Rabbit and calf RPE cells were grown for 2 weeks with addition of one of the test substances every 48 h. The cellular uptake of carotenoids and α-tocopherol was assayed by HPLC after 2 weeks. The lipofuscin-content was measured by static fluorometry (rabbit cells) or by image analysis (calf cells). Both rabbit and calf RPE showed similar results with significantly lower amounts of lipofuscin in antioxidant-treated cells. The effect of carotenoids is especially interesting, since the result is not dependent on their protective effect against photo-oxidative reactions. The chain-breaking abilities of these antioxidants in peroxidative reactions of lipid membranes and quenching of free radicals seem to be of importance for inhibition of lipofuscin formation.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27901 (URN)10.1016/S0891-5849(01)00573-1 (DOI)12661 (Local ID)12661 (Archive number)12661 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-09-17Bibliographically approved
4. Different effects of chloroquine and hydroxychloroquine on lysosomal function in cultured retinal pigment epithelial cells
Open this publication in new window or tab >>Different effects of chloroquine and hydroxychloroquine on lysosomal function in cultured retinal pigment epithelial cells
2002 (English)In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 110, no 6, 481-489 p.Article in journal (Refereed) Published
Abstract [en]

Although relatively rare, retinopathy based on a disturbed metabolism of the retinal pigment epithelium (RPE), with ensuing degeneration of photoreceptors, is a known complication of treatment with the 4-aminoquinolones, chloroquine (CQ) and hydroxychloroquine (HCQ), in autoimmune diseases. The reported frequency of retinopathy, however, is much lower for HCQ than for CQ (less than 0.08% versus 1–2%). To test whether the difference in toxicity between the two lysosomotropic drugs is related to different lysosomal influence, we exposed confluent RPE cell cultures to CQ or HCQ for 2 weeks. To induce lipofuscin (LF) formation, known to be accelerated by increased lysosomal pH and intra-lysosomal oxidation during degradation of auto-/heterophagocytosed material, such treatment was combined with feeding of cells with photoreceptor outer segments (POS) and hyperoxia (40% ambient oxygen). HCQ was found to be a less potent enhancer of lipofuscinogenesis compared to CQ, apparently due to its less effective inhibition of lysosomal degradative capacity (evaluated by vital staining of lysosomes with Lyso Tracker Red, and periodic acid-Schiff reaction). This conclusion is supported by the fact that NH4Cl, a non-fluorescent substance which acts similarly to 4-aminoquinolones, induced an increase in LF fluorescence paralleled by increased periodic acid-Schiff reactivity of RPE cells.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27938 (URN)10.1034/j.1600-0463.2002.100606.x (DOI)12699 (Local ID)12699 (Archive number)12699 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-09-17Bibliographically approved

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