Screening and Characterization of Drugs That Protect Corneal Endothelial Cells Against Unfolded Protein Response and Oxidative Stress.
To screen for and characterize compounds that protect corneal endothelial cells against unfolded protein response (UPR) and oxidative stress.



Bovine corneal endothelial cells (BCECs) were treated for 48 hours with 640 compounds from a Food and Drug Administration (FDA)-approved drug library and then challenged with thapsigargin or H2O2 to induce UPR or oxidative stress, respectively. Cell viability was measured using the CellTiter-Glo survival assay. Selected "hits" were subjected to further dose-response testing, and their ability to modulate expression of UPR and oxidative stress markers was assessed by RT-PCR, Western blot, and measurement of protein carbonyl and 8-hydroxydeoxyguanosine (8-OHdG) adducts in immortalized human corneal endothelial cells (iHCECs).



Forty-one drugs at 20 μM and 55 drugs at 100 μM increased survival of H2O2-challenged cells, and 8 drugs at 20 μM and 2 drugs at 100 μM increased survival of thapsigargin-challenged cells, compared with untreated control cells. Nicergoline, ergothioneine, nimesulide, oxotremorine, and mefenamic acid increased survival of both H2O2- and thapsigargin-challenged cells. Oxotremorine altered DNA damage inducible 3 (CHOP) gene expression, glucose-regulated protein 78 kDa (GRP78) and activating transcription factor 4 (ATF4) protein expression, and protein carbonyl and 8-OHdG levels. Mefenamic acid altered GRP78 protein expression and protein carbonyl and 8-OHdG levels.



Oxotremorine and mefenamic acid are potential survival factors for corneal endothelial cells under UPR and oxidative stress. The described assay can be further expanded to screen additional drugs for potential therapeutic effect in corneal endothelial diseases such as Fuchs' endothelial corneal dystrophy.
Wilmer Eye Institute, Johns Hopkins Medical Institutions, Baltimore, Maryland, United States 2Department of Ophthalmology & Visual Science, Catholic University of Korea, Seoul, Korea.
Eun Chul
EC
Kim
Wilmer Eye Institute, Johns Hopkins Medical Institutions, Baltimore, Maryland, United States 3Department of Ophthalmology, University of Tokyo, Tokyo, Japan.
Tetsuya
T
Toyono
Wilmer Eye Institute, Johns Hopkins Medical Institutions, Baltimore, Maryland, United States.
Cynthia A
CA
Berlinicke
Wilmer Eye Institute, Johns Hopkins Medical Institutions, Baltimore, Maryland, United States 4Departments of Molecular Biology and Genetics, Neuroscience, and Institute of Genetic Medicine, Johns Hopkins Medical Institutions, Baltimore, Maryland, United States.
Donald J
DJ
Zack
Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, United States.
Ula
U
Jurkunas
Department of Ophthalmology, University of Tokyo, Tokyo, Japan.
Tomohiko
T
Usui
Wilmer Eye Institute, Johns Hopkins Medical Institutions, Baltimore, Maryland, United States.
Albert S
AS
Jun
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000975:Antioxidants / Q000494:pharmacology*; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D000091342:Endoplasmic Reticulum Chaperone BiP; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D004728:Endothelium, Corneal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008528:Mefenamic Acid / Q000494:pharmacology; D018721:Muscarinic Agonists / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects*; D010095:Oxotremorine / Q000494:pharmacology; D050050:Protein Carbonylation / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019284:Thapsigargin / Q000494:pharmacology; D056811:Unfolded Protein Response / Q000187:drug effects*
D000975:Antioxidants; D016861:Cyclooxygenase Inhibitors; D000091342:Endoplasmic Reticulum Chaperone BiP; D004791:Enzyme Inhibitors; C000717247:HSPA5 protein, human; D018721:Muscarinic Agonists; D008528:Mefenamic Acid; D010095:Oxotremorine; D019284:Thapsigargin; D000080242:8-Hydroxy-2'-Deoxyguanosine; D006861:Hydrogen Peroxide; D003849:Deoxyguanosine