title: Effects of Cytokine Activation and Oxidative Stress on the Function of the Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells.
abstract: OBJECTIVE
  In several retinal complications, such as age-dependent macular degeneration (AMD), oxidative stress is increased and cytokine level is elevated. These are shown to alter the activation and expression of matrix metalloproteinase (MMP) both in human primary and immortalized retinal pigment epithelial (RPE) cells. However, the effects on human embryonic stem cell (hESC)-derived RPE cells remain to be elucidated.
  
  
  METHODS
  The mature hESC-RPE cells were exposed to inflammatory cytokines (IFN-γ or TNF-α) for 24 hours or oxidative stress (H2O2) for 1 hour. Effects on barrier properties were analyzed with transepithelial electrical resistance (TEER), the expression of MMP-1, MMP-2, MMP-3, MMP-9, collagen I, and collagen IV genes with quantitative RT-PCR, and the expression of MMP-1 and MMP-3 proteins with Western blot or ELISA, respectively. Also, activation and secretion of MMP-2 and -9 proteins were analyzed with zymography.
  
  
  RESULTS
  In normal state, mature hESC-RPE cells expressed MMP-1, -2, -3, and -9 genes in low levels, respectively. Tumor necrosis factor-α increased MMP-1 and -2 gene expression, and H2O2 increased MMP-3 and -9 gene expression. Zymography revealed IFN-γ- and TNF-α-induced secretion of MMP-2 and high-molecular-weight species of MMP (HMW MMP), but H2O2 decreased their secretion. Furthermore, TNF-α and H2O2 significantly decreased barrier properties.
  
  
  CONCLUSIONS
  Here, cytokines induced the MMP-1 and -2 gene and protein expression. Also, H2O2 induced MMP-3 and -9 gene expression, but not their protein secretion. These data propose that under oxidative stress and cytokine stimuli, mature hESC-RPE cells resemble their native counterpart in the human eye in regard to MMP secretion and expression and could be used to model retinal disorders involving alterations in MMP activity such as AMD, diabetic retinopathy, or proliferative vitreoretinopathy in vitro.
authors.affiliation: BioMediTech University of Tampere, Tampere, Finland.
authors.forename: Kati
authors.initials: K
authors.lastname: Juuti-Uusitalo
authors.affiliation: BioMediTech University of Tampere, Tampere, Finland.
authors.forename: Monica
authors.initials: M
authors.lastname: Nieminen
authors.affiliation: Department of Ophthalmology, University of Kiel, University Medical Center, Kiel, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Treumer
authors.affiliation: BioMediTech University of Tampere, Tampere, Finland 3FimLab Laboratories, Tampere, Finland.
authors.forename: Minna
authors.initials: M
authors.lastname: Ampuja
authors.affiliation: BioMediTech University of Tampere, Tampere, Finland 3FimLab Laboratories, Tampere, Finland.
authors.forename: Anne
authors.initials: A
authors.lastname: Kallioniemi
authors.affiliation: Department of Ophthalmology, University of Kiel, University Medical Center, Kiel, Germany.
authors.forename: Alexa
authors.initials: A
authors.lastname: Klettner
authors.affiliation: BioMediTech University of Tampere, Tampere, Finland.
authors.forename: Heli
authors.initials: H
authors.lastname: Skottman
mesh_terms: D015153:Blotting, Western; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D000066449:Human Embryonic Stem Cells / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D008268:Macular Degeneration / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D018384:Oxidative Stress; D012333:RNA, Messenger / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D055213:Retinal Pigment Epithelium / Q000378:metabolism* / Q000473:pathology
chemical_list: D016207:Cytokines; D012333:RNA, Messenger