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Oxidative Stress Protection by Exogenous Delivery of rhHsp70 Chaperone to the Retinal Pigment Epithelium (RPE), a Possible Therapeutic Strategy Against RPE Degeneration

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Abstract

Purpose

To measure the cytoprotective effects of rhHsp70 against oxidative stress and study its cellular uptake, intracellular and intraocular distribution in the retinal pigment epithelium.

Methods

Human retinal pigment epithelial cells (ARPE-19) were pre-treated with rhHsp70 for 24 h, 48 h, and 72 h before being exposed to 1.25 mM hydrogen peroxide. Non-treated cells served as control. We analysed interleukin 6 secretion, cell viability, and cytolysis. Uptake and intracellular distribution of fluorescently labelled rhHsp70 were investigated with flow cytometry and confocal microscopy, respectively. Ocular distribution of radioactively labelled rhHsp70 was followed ex vivo in porcine eyes by micro SPECT/CT.

Results

After exposure to hydrogen peroxide, IL-6 secretion decreased by 35–39% when ARPE-19 cells were pre-treated with rhHsp70. Cell viability increased by 17–32%, and cell lysis, measured by the release of lactate dehydrogenase, decreased by 6-43%. ARPE-19 cells endocytosed rhHsp70 added to the culture medium and the protein was localized in late endosomes and lysosomes. Following intravitreal injection into isolated porcine eyes, we found 20% rhHsp70 in the RPE.

Conclusions

Recombinant hHsp70 protein offers protection against oxidative stress. RPE cells take up the exogenously delivered rhHsp70 and localize it in late endosomes and lysosomes. This work provides the basis for a therapeutic strategy to target aggregate-associated neurodegeneration in AMD.

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Abbreviations

AMD:

Age-related macular degeneration

Hsp:

Heat shock protein

ROS:

Reactive oxygen species

RPE:

Retinal pigment epithelium

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Acknowledgments

We would like to thank Ms. Leena Pietilä at the Centre for Drug Research, University of Helsinki and Ms. Anne Seppänen at the Department of Ophthalmology, University of Eastern Finland for technical assistance, the Light Microscopy Unit of the Institute of Biotechnology at the University of Helsinki for help with confocal microscopy, Dr. Mari Raki for operating the micro SPECT/CT, and Dr. Shuang Wang for assistance in image analysis. We also acknowledge the Finnish Funding Agency for Innovation (Tekes), the Friends of the Blind foundation (Sokeain Ystävät ry), Evald and Hilda Nissi foundation, Orion-Farmos research foundation, and Academy of Finland (decision no. 136805) for financial support.

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Authors and Affiliations

  1. Centre for Drug Research, Division of Pharmaceutical Biosciences Faculty of Pharmacy, University of Helsinki, Helsinki, Finland

    Astrid Subrizi, Eva Ramsay & Arto Urtti

  2. Department of Ophthalmology, Institute of Clinical Medicine, University of Eastern Finland, Kuopio, Finland

    Elisa Toropainen & Kai Kaarniranta

  3. Laboratory of Radiochemistry, Department of Chemistry, University of Helsinki, Helsinki, Finland

    Anu J. Airaksinen

  4. Department of Ophthalmology, Kuopio University Hospital, Kuopio, Finland

    Kai Kaarniranta

  5. School of Pharmacy, University of Eastern Finland, Kuopio, Finland

    Arto Urtti

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  1. Astrid Subrizi
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  2. Elisa Toropainen
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Correspondence to Astrid Subrizi.

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Subrizi and Toropainen have contributed equally to this work

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Subrizi, A., Toropainen, E., Ramsay, E. et al. Oxidative Stress Protection by Exogenous Delivery of rhHsp70 Chaperone to the Retinal Pigment Epithelium (RPE), a Possible Therapeutic Strategy Against RPE Degeneration. Pharm Res 32, 211–221 (2015). https://doi.org/10.1007/s11095-014-1456-6

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  • DOI: https://doi.org/10.1007/s11095-014-1456-6

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