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ARPE-19 retinal pigment epithelial cells are highly resistant to oxidative stress and exercise strict control over their lysosomal redox-active iron
Linköpings universitet, Institutionen för medicin och hälsa, Farmakologi. Linköpings universitet, Hälsouniversitetet.
Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet.
Linköpings universitet, Institutionen för medicin och hälsa, Farmakologi. Linköpings universitet, Hälsouniversitetet.
Linköpings universitet, Institutionen för klinisk och experimentell medicin, Oftalmiatrik. Linköpings universitet, Hälsouniversitetet.
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2009 (Engelska)Ingår i: AUTOPHAGY, ISSN 1554-8627, Vol. 5, nr 4, s. 494-501Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Normal retinal pigment epithelial (RPE) cells are postmitotic, long-lived and basically not replaced. Daily, they phagocytose substantial amounts of lipid-rich material (photoreceptor outer segment discs), and they do so in the most oxygenated part of the body-the retina. One would imagine that this state of affairs should be associated with a rapid formation of the age pigment lipofuscin (LF). However, LF accumulation is slow and reaches significant amounts only late in life when, if substantial, it often coincides with or causes age-related macular degeneration. LF formation occurs inside the lysosomal compartment as a result of iron-catalyzed peroxidation and polymerization. This process requires phagocytosed or autophagocytosed material under degradation, but also the presence of redox-active low mass iron and hydrogen peroxide. To gain some information on how RPE cells are able to evade LF formation, we investigated the response of immortalized human RPE cells (ARPE-19) to oxidative stress with/without the protection of a strong iron-chelator. The cells were found to be extremely resistant to hydrogen peroxide-induced lysosomal rupture and ensuing cell death. This marked resistance to oxidative stress was not explained by enhanced degradation of hydrogen peroxide, but to a certain extent further increased by the potent lipophilic iron chelator STH. The cells were also able to survive, and even replicate, at high concentrations of SIH and showed a high degree of basal autophagic flux. We hypothesize that RPE cells have a highly developed capacity to keep lysosomal iron in a nonredox-active form, perhaps by pronounced autophagy of iron-binding proteins in combination with an ability to rapidly relocate low mass iron from the lysosomal compartment.

Ort, förlag, år, upplaga, sidor
2009. Vol. 5, nr 4, s. 494-501
Nyckelord [en]
age-related macular degeneration, hydrogen peroxide, iron, iron chelation, lipofuscin, lysosomal stability, lysosomes, macrophage, oxidative stress, retinal pigment epithelial cells
Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
URN: urn:nbn:se:liu:diva-18569DOI: 10.4161/auto.5.4.7961OAI: oai:DiVA.org:liu-18569DiVA, id: diva2:220604
Tillgänglig från: 2009-06-01 Skapad: 2009-06-01 Senast uppdaterad: 2014-10-24
Ingår i avhandling
1. Oxidative stress-related damage of retinal pigment epithelial cells: possible protective properties of autophagocytosed iron-binding proteins
Öppna denna publikation i ny flik eller fönster >>Oxidative stress-related damage of retinal pigment epithelial cells: possible protective properties of autophagocytosed iron-binding proteins
2014 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Oxidative stress is a major pathogenic factor in the development of age-related macular degeneration (AMD), which is the most common cause of severe central visual impairment in the elderly population in the western world.

It is believed that the degenerative process starts in the retinal pigment epithelium (RPE). The post-mitotic RPE is a single layer of pigmented cells located behind the photoreceptors – rods and cones – of the retina. Daily, the RPE cells phagocytose and recycle the expended tips of the photoreceptor outer segments. This heavy phagocytic burden leads to substantial oxidative stress in the cells, which is further enhanced by intense illumination and a high oxygen tension. A hallmark of early AMD is a progressive build-up of the non-degradable age pigment lipofuscin (LF) in lysosomes of the RPE. LF accumulation hampers phagocytosis and autophagy in the RPE, resulting in increased amounts of cellular debris in and around the cells. This decreases the function and viability of both RPE cells and photoreceptors.

Iron is known to accumulate in the retina with increasing age, particularly in AMDaffected eyes, and amplifies oxidative stress by acting as a potent catalyst in the generation of hydroxyl radicals. These highly reactive radicals contribute to LF formation and may, if abundantly present, also directly damage lysosomal membranes. The subsequent leakage of degrading enzymes to the cytosol initiates cell death via apoptosis or necrosis.

In this thesis, we have investigated the oxidative stress response of human RPE (ARPE-19) cells compared to murine J774 cells, another type of lysosome-rich cells with a high phagocytic capacity. The ARPE-19 cells were found to be extremely resistant to oxidative stress and tolerated exposure to single doses of H2O2 in concentrations up to 150 times higher than the J774 cells before lysosomal rupture and ensuing cell death occurred. This resistance was increased even further when the cells were protected with a potent iron chelator that prevents redox-active iron to participate in hydroxyl radical generation. Both cell lines were shown to be equally effective in degrading H2O2 and seem to contain comparable amounts of total as well as intralysosomal iron. Therefore, we reasoned that the insensitivity of ARPE-19 cells to H2O2 exposure might be related to a mechanism which keeps their intralysosomal iron bound in a non redox-active form. This theory was supported by our finding of very high basal expression levels of metallothionein (MT), heat shock-protein 70 (HSP70) and ferritin (FT) in ARPE-19 cells compared to J774 cells. All of these proteins have previously been shown to possess potent iron-binding properties. The ARPE-19 cells were also shown to have a higher basal rate of autophagy. SiRNA-mediated attenuation of MT, HSP70 and FT levels in the ARPE-19 cells resulted, to some degree, in an increased sensitivity to H2O2 treatment. Furthermore, a human cell stress array showed several other stress-related proteins to be up-regulated in ARPE-19 cells.

Additionally, we evaluated the commonly used, but frequently misinterpreted, H2DCF test for oxidative stress. It was demonstrated that oxidation of H2DCF into fluorescent DCF mainly reflects relocation to the cytosol of lysosomal iron and mitochondrial cytochrome c, rather than being the result of some poorly defined “general” oxidative stress.

In conclusion, our results indicate that the extreme resistance to oxidative stress exhibited by the ARPE-19 cells might be related to a high continuous autophagic influx of iron-binding proteins into the lysosomal compartment. Before being degraded, such proteins will temporarily keep intralysosomal iron bound in a non redox-active form, thereby inhibiting hydroxyl radical formation. This may partly explain why RPE cells, in spite of their exposed location and heavy burden of phagocytosis, usually manage to survive and evade significant LF accumulation until late in life.

Ort, förlag, år, upplaga, sidor
Linköping: Linköping University Electronic Press, 2014. s. 93
Serie
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1426
Nationell ämneskategori
Klinisk medicin Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:liu:diva-111558 (URN)10.3384/diss.diva-111558 (DOI)978-91-7519-209-3 (ISBN)
Disputation
2014-11-28, Nils-Holgersalen, Campus US, Linköpings universitet, Linköping, 13:00 (Svenska)
Opponent
Handledare
Tillgänglig från: 2014-10-24 Skapad: 2014-10-24 Senast uppdaterad: 2019-11-19Bibliografiskt granskad

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Kurz, TinoKarlsson, MarkusBrunk, UlfErik Nilsson, SvenFrennesson, Christina

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