title: Oxidative stress protection by exogenous delivery of rhHsp70 chaperone to the retinal pigment epithelium (RPE), a possible therapeutic strategy against RPE degeneration.
abstract: OBJECTIVE
  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.
authors.affiliation: Centre for Drug Research, Division of Pharmaceutical Biosciences Faculty of Pharmacy, University of Helsinki, Helsinki, Finland, astrid.subrizi@helsinki.fi.
authors.forename: Astrid
authors.initials: A
authors.lastname: Subrizi
authors.forename: Elisa
authors.initials: E
authors.lastname: Toropainen
authors.forename: Eva
authors.initials: E
authors.lastname: Ramsay
authors.forename: Anu J
authors.initials: AJ
authors.lastname: Airaksinen
authors.forename: Kai
authors.initials: K
authors.lastname: Kaarniranta
authors.forename: Arto
authors.initials: A
authors.lastname: Urtti
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D018840:HSP70 Heat-Shock Proteins / Q000008:administration & dosage / Q000493:pharmacokinetics / Q000627:therapeutic use*; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D015850:Interleukin-6 / Q000378:metabolism; D058449:Intravitreal Injections; D008268:Macular Degeneration / Q000276:immunology / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D018384:Oxidative Stress / Q000187:drug effects*; D011994:Recombinant Proteins; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D013552:Swine; D014018:Tissue Distribution
chemical_list: D018840:HSP70 Heat-Shock Proteins; D015850:Interleukin-6; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide