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Oxidative stress-related damage of retinal pigment epithelial cells: possible protective properties of autophagocytosed iron-binding proteins
Linköping University, Department of Clinical and Experimental Medicine, Division of Neuroscience. Linköping University, Faculty of Health Sciences.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2014. , 93 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1426
National Category
Clinical Medicine Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:liu:diva-111558DOI: 10.3384/diss.diva-111558ISBN: 978-91-7519-209-3 (print)OAI: oai:DiVA.org:liu-111558DiVA: diva2:758005
Public defence
2014-11-28, Nils-Holgersalen, Campus US, Linköpings universitet, Linköping, 13:00 (Swedish)
Opponent
Supervisors
Available from: 2014-10-24 Created: 2014-10-24 Last updated: 2015-09-29Bibliographically approved
List of papers
1. ARPE-19 retinal pigment epithelial cells are highly resistant to oxidative stress and exercise strict control over their lysosomal redox-active iron
Open this publication in new window or tab >>ARPE-19 retinal pigment epithelial cells are highly resistant to oxidative stress and exercise strict control over their lysosomal redox-active iron
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2009 (English)In: AUTOPHAGY, ISSN 1554-8627, Vol. 5, no 4, 494-501 p.Article in journal (Refereed) 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.

Keyword
age-related macular degeneration, hydrogen peroxide, iron, iron chelation, lipofuscin, lysosomal stability, lysosomes, macrophage, oxidative stress, retinal pigment epithelial cells
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-18569 (URN)10.4161/auto.5.4.7961 (DOI)
Available from: 2009-06-01 Created: 2009-06-01 Last updated: 2014-10-24
2. What does the commonly used DCF test for oxidative stress really show?
Open this publication in new window or tab >>What does the commonly used DCF test for oxidative stress really show?
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2010 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 428, no 2, 183-90 p.Article in journal (Refereed) Published
Abstract [en]

H(2)DCF-DA (dihydrodichlorofluorescein diacetate) is widely used to evaluate 'cellular oxidative stress'. After passing through the plasma membrane, this lipophilic and non-fluorescent compound is de-esterified to a hydrophilic alcohol [H(2)DCF (dihydrodichlorofluorescein)] that may be oxidized to fluorescent DCF (2',7'-dichlorofluorescein) by a process usually considered to involve ROS (reactive oxygen species). It is, however, not always recognized that, being a hydrophilic molecule, H(2)DCF does not cross membranes, except for the outer fenestrated mitochondrial ones. It is also not generally realized that oxidation of H(2)DCF is dependent either on Fenton-type reactions or on unspecific enzymatic oxidation by cytochrome c, for neither superoxide, nor H(2)O(2), directly oxidizes H(2)DCF. Consequently, oxidation of H(2)DCF requires the presence of either cytochrome c or of both redox-active transition metals and H(2)O(2). Redox-active metals exist mainly within lysosomes, whereas cytochrome c resides bound to the outer side of the inner mitochondrial membrane. Following exposure to H(2)DCF-DA, weak mitochondrial fluorescence was found in both the oxidation-resistant ARPE-19 cells and the much more sensitive J774 cells. This fluorescence was only marginally enhanced following short exposure to H(2)O(2), showing that by itself it is unable to oxidize H(2)DCF. Cells that were either exposed to the lysosomotropic detergent MSDH (O-methylserine dodecylamide hydrochloride), exposed to prolonged oxidative stress, or spontaneously apoptotic showed lysosomal permeabilization and strong DCF-induced fluorescence. The results suggest that DCF-dependent fluorescence largely reflects relocation to the cytosol of lysosomal iron and/or mitochondrial cytochrome c.

Keyword
age-related macular degeneration (AMD), 2', 7'-dichlorofluorescein (DCF), lysosome, oxidative stress, reactive oxygen species (ROS), transition metal.
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-56466 (URN)10.1042/BJ20100208 (DOI)20331437 (PubMedID)
Available from: 2010-05-17 Created: 2010-05-17 Last updated: 2017-12-12
3. Autophagy of iron-binding proteins may contribute to the oxidative stress resistance of ARPE-19 cells
Open this publication in new window or tab >>Autophagy of iron-binding proteins may contribute to the oxidative stress resistance of ARPE-19 cells
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2013 (English)In: Experimental Eye Research, ISSN 0014-4835, E-ISSN 1096-0007, Vol. 116, 359-365 p.Article in journal (Refereed) Published
Abstract [en]

The objective of this study was to elucidate possible reasons for the remarkable resistance of human retinal pigment epithelial (RPE) cells to oxidative stress. Much oxidative damage is due to hydrogen peroxide meeting redox-active iron in the acidic and reducing lysosomal environment, resulting in the production of toxic hydroxyl radicals that may oxidize intralysosomal content, leading to lipofuscin (LF) formation or, if more extensive, to permeabilization of lysosomal membranes. Formation of LF is a risk factor for age-related macular degeneration (AMD) and known to jeopardize normal autophagic rejuvenation of vital cellular biomolecules. Lysosomal membrane permeabilization causes release of lysosomal content (redox-active iron, lytic enzymes), which may then cause cell death. Total cellular and lysosomal low-mass iron of cultured, immortalized human RPE (ARPE-19) cells was compared to that of another professional scavenger cell line, J774, using atomic absorption spectroscopy and the cytochemical sulfide-silver method (SSM). It was found that both cell lines contained comparable levels of total as well as intralysosomal iron, suggesting that the latter is mainly kept in a non-redox-active state in ARPE-19 cells. Basal levels and capacity for upregulation of the iron-binding proteins ferritin, metallothionein and heat shock protein 70 were tested in both cell lines using immunoblotting. Compared to J774 cells, ARPE-19 cells were found to contain very high basal levels of all these proteins, which could be even further upregulated following appropriate stimulation. These findings suggest that a high basal expression of iron-binding stress proteins, which during their normal autophagic turnover in lysosomes may temporarily bind iron prior to their degradation, could contribute to the unusual oxidative stress-resistance of ARPE-19 cells. A high steady state influx of such proteins into lysosomes would keep the level of lysosomal redox-active iron permanently low. This, in turn, should delay intralysosomal accumulation of LF in RPE cells, which is known to reduce autophagic turnover as well as uptake and degradation of worn out photoreceptor tips. This may explain why severe LF accumulation and AMD normally do not develop until fairly late in life, in spite of RPE cells being continuously exposed to high levels of oxygen and light, as well as large amounts of lipid-rich material.

Place, publisher, year, edition, pages
Elsevier, 2013
Keyword
oxidative stress, ARPE-19, retinal pigment epithelium, iron, metallothionein, HSP70, ferritin, age-related macular degeneration
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-102718 (URN)10.1016/j.exer.2013.10.014 (DOI)000327562500041 ()
Note

Funding Agencies|Crown Princess Margaretas Foundation for the Visually Handicapped||Edvin Jordan Foundation for Ophthalmological Research||Linkoping University Hospital Research Fund (ALF)||

Available from: 2013-12-19 Created: 2013-12-19 Last updated: 2017-12-06
4. Attenuation of iron-binding proteins in ARPE-19 cells reduces their resistance to oxidative stress
Open this publication in new window or tab >>Attenuation of iron-binding proteins in ARPE-19 cells reduces their resistance to oxidative stress
2016 (English)In: Acta Ophthalmologica, ISSN 1755-375X, E-ISSN 1755-3768, Vol. 94, no 6, 556-565 p.Article in journal (Refereed) Published
Abstract [en]

Purpose

Oxidative stress-related damage to retinal pigment epithelial (RPE) cells is an important feature in the development of age-related macular degeneration. Iron-catalysed intralysosomal production of hydroxyl radicals is considered a major pathogenic factor, leading to lipofuscin formation with ensuing depressed cellular autophagic capacity, lysosomal membrane permeabilization and apoptosis. Previously, we have shown that cultured immortalized human RPE (ARPE-19) cells are extremely resistant to exposure to bolus doses of hydrogen peroxide and contain considerable amounts of the iron-binding proteins metallothionein (MT), heat-shock protein 70 (HSP70) and ferritin (FT). According to previous findings, autophagy of these proteins depresses lysosomal redox-active iron. The aim of this study was to investigate whether up- or downregulation of these proteins would affect the resistance of ARPE-19 cells to oxidative stress.

Methods

The sensitivity of ARPE-19 cells to H2O2 exposure was tested following upregulation of MT, HSP70 and/or FT by pretreatment with ZnSO4, heat shock or FeCl3, as well as siRNA-mediated downregulation of the same proteins.

Results

Upregulation of MT, HSP70 and FT did not improve survival following exposure to H2O2. This was interpreted as existence of an already maximal protection. Combined siRNA-mediated attenuation of both FT chains (H and L), or simultaneous downregulation of all three proteins, made the cells significantly more susceptible to oxidative stress confirming the importance of iron-binding proteins.

Conclusion

The findings support our hypothesis that the oxidative stress resistance exhibited by RPE cells may be explained by a high autophagic influx of iron-binding proteins that would keep levels of redox-active lysosomal iron low.

Place, publisher, year, edition, pages
Wiley-Blackwell Publishing Inc., 2016
Keyword
age-related macular degeneration, ARPE-19, ferritin, HSP70, iron metallothionein, oxidative stress, retinal pigment epithelium
National Category
Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-111557 (URN)10.1111/aos.13076 (DOI)000383520800034 ()
Note

At the time for thesis presentation publication was in status: Manuscript

Funding agencies:  Crown Princess Margaretas Foundation for the Visually Handicapped; Edvin Jordan Foundation for Ophthalmological Research; Linkoping University Hospital Research Fund (ALF)

Available from: 2014-10-24 Created: 2014-10-24 Last updated: 2017-12-05Bibliographically approved

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