title: Corneal Endothelial Cell Migration and Proliferation Enhanced by Rho Kinase (ROCK) Inhibitors in In Vitro and In Vivo Models.
abstract: To explore the role of Rho-associated kinases (ROCK) in corneal physiology and regeneration, and the effects of suppressing its activity in stimulating corneal endothelial cell proliferation and migration in vitro and in vivo.
  
  
  
  Immunohistochemistry was performed to detect RhoA and ROCK-1 and ROCK-2 in human corneal tissue. Adult porcine corneal endothelial cells (CECs) were isolated, grown to confluence, and further characterized. Under the treatment of ROCK inhibitors, changes in the cellular distribution profile of ZO-1 and F-actin were examined by immunofluorescence staining. Corneal endothelial cells migration was evaluated by scratch assay and analyzed with Axiovision software. Cell proliferation was quantified using Click-iT EdU HCS Assay. In vivo, the corneal endothelia of rabbits were surgically injured and H-1152 was topically applied for 10 days. Progress of wound healing was evaluated daily by monitoring corneal edema, inflammation, and thickness using slit-lamp examination, photography, and pachymetry. Rabbits were euthanized and enucleated for further evaluation.
  
  
  
  H-1152 exhibited significant stimulatory effect on CEC migration and proliferation in vitro compared with both untreated and Y-27632-treated cells. Furthermore, topical administration of H-1152 led to marked reduction in corneal edema and formation of multinucleate CECs in vivo suggestive of proliferation associated with healing.
  
  
  
  H-1152 exhibited a better stimulatory effect on CEC migration and proliferation in vitro than Y-27632. Our findings suggest that topical administration of H-1152 promotes healing of injured corneal endothelium in vivo. These results demonstrate the efficacy of ROCK inhibitors as a potential topical therapy for patients with corneal endothelial disease.
authors.affiliation: Duke University Eye Center, Duke University Medical Center, Durham, North Carolina, United States.
authors.forename: Landon C
authors.initials: LC
authors.lastname: Meekins
authors.affiliation: Duke University Eye Center, Duke University Medical Center, Durham, North Carolina, United States.
authors.forename: Noel
authors.initials: N
authors.lastname: Rosado-Adames
authors.affiliation: Duke University Eye Center, Duke University Medical Center, Durham, North Carolina, United States.
authors.forename: Rupalatha
authors.initials: R
authors.lastname: Maddala
authors.affiliation: Shiley Eye Institute, University of California San Diego, La Jolla, California, United States.
authors.forename: Jiagang J
authors.initials: JJ
authors.lastname: Zhao
authors.affiliation: Duke University Eye Center, Duke University Medical Center, Durham, North Carolina, United States.
authors.forename: Ponugoti V
authors.initials: PV
authors.lastname: Rao
authors.affiliation: Shiley Eye Institute, University of California San Diego, La Jolla, California, United States.
authors.forename: Natalie A
authors.initials: NA
authors.lastname: Afshari
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D003316:Corneal Diseases / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D004728:Endothelium, Corneal / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D007150:Immunohistochemistry; D011817:Rabbits; D013552:Swine; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: C459092:2-methyl-1-((4-methyl-5-isoquinolinyl)sulfonyl)homopiperazine; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D054460:rho-Associated Kinases