title: Viability, apoptosis, proliferation, activation, and cytokine secretion of human keratoconus keratocytes after cross-linking.
abstract: OBJECTIVE
  The purpose of this study was to determine the impact of cross-linking (CXL) on viability, apoptosis, proliferation, activation, and cytokine secretion of human keratoconus (KC) keratocytes, in vitro.
  
  
  METHODS
  Primary KC keratocytes were cultured in DMEM/Ham's F12 medium supplemented with 10% FCS and underwent UVA illumination (370 nm, 2 J/cm(2)) during exposure to 0.1% riboflavin and 20% Dextran in PBS. Twenty-four hours after CXL, viability was assessed using Alamar blue assay; apoptosis using APO-DIRECT Kit; proliferation using ELISA-BrdU kit; and CD34 and alpha-smooth muscle actin (α-SMA) expression using flow cytometry. Five and 24 hours after CXL, FGFb, HGF, TGFβ1, VEGF, KGF, IL-1β, IL-6, and IL-8 secretion was measured using enzyme-linked-immunoabsorbent assay (ELISA).
  
  
  RESULTS
  Following CXL, cell viability and proliferation decreased (P < 0.05; P = 0.009), the percentage of apoptotic keratocytes increased (P < 0.05) significantly, and CD34 and α-SMA expression remained unchanged (P > 0.06). Five hours after CXL, FGFb secretion increased significantly (P = 0.037); however no other cytokine secretion differed significantly from controls after 5 or 24 hours (P > 0.12).
  
  
  CONCLUSIONS
  Cross-linking decreases viability, triggers apoptosis, and inhibits proliferation, without an impact on multipotent hematopoietic stem cell transformation and myofibroblastic transformation of KC keratocytes. CXL triggers FGFb secretion of KC keratocytes transiently (5 hours), normalizing after 24 hours.
authors.affiliation: Department of Ophthalmology, Saarland University Medical Center, Kirrberger Straße 100, 66424 Homburg, Germany.
authors.forename: Xuefei
authors.identifier: 0000-0003-4421-6017
authors.initials: X
authors.lastname: Song
authors.affiliation: Department of Ophthalmology, Saarland University Medical Center, Kirrberger Straße 100, 66424 Homburg, Germany.
authors.forename: Tanja
authors.initials: T
authors.lastname: Stachon
authors.affiliation: Department of Ophthalmology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China.
authors.forename: Jiong
authors.initials: J
authors.lastname: Wang
authors.affiliation: Experimental Ophthalmology, Saarland University, 66424 Homburg, Germany.
authors.forename: Achim
authors.identifier: 0000-0001-9175-6177
authors.initials: A
authors.lastname: Langenbucher
authors.affiliation: Department of Ophthalmology, Saarland University Medical Center, Kirrberger Straße 100, 66424 Homburg, Germany.
authors.forename: Berthold
authors.initials: B
authors.lastname: Seitz
authors.affiliation: Department of Ophthalmology, Saarland University Medical Center, Kirrberger Straße 100, 66424 Homburg, Germany.
authors.forename: Nóra
authors.initials: N
authors.lastname: Szentmáry
mesh_terms: D000199:Actins / Q000378:metabolism; D018952:Antigens, CD34 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D002352:Carrier Proteins / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects*; D002478:Cells, Cultured; D060527:Corneal Keratocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D016207:Cytokines / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D007640:Keratoconus / Q000378:metabolism / Q000473:pathology*; D017319:Photosensitizing Agents / Q000494:pharmacology; D012256:Riboflavin / Q000494:pharmacology; D014466:Ultraviolet Rays
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; D018952:Antigens, CD34; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D016207:Cytokines; D036341:Intercellular Signaling Peptides and Proteins; D017319:Photosensitizing Agents; C070699:FGFBP1 protein, human; D012256:Riboflavin