Sabine Kling1,2, Arthur Hammer2,3, Emilio A Torres Netto1,4, Farhad Hafezi1,2,5,6. 1. Laboratory of Ocular Cell Biology, Center of Applied Biotechnology and Molecular Medicine, University of Zurich, Switzerland. 2. Laboratory of Ocular Cell Biology, University of Geneva, Switzerland. 3. Hoptial ophtalmique Jules-Gonin, Fondation Asile des aveugles, Lausanne, Switzerland. 4. Department of Ophthalmology, Paulista School of Medicine, Federal University of Sao Paulo, Sao Paulo, Brazil. 5. ELZA Institute AG, Dietikon/Zurich, Switzerland. 6. University of Southern California, CA, USA.
Abstract
PURPOSE: We studied changes in gene transcription after corneal crosslinking (CXL) in the rabbit cornea in vivo and identified potential molecular signaling pathways. METHODS: A total of 15 corneas of eight male New-Zealand-White rabbits were de-epithelialized and equally divided into five groups. Group 1 served as an untreated control. Groups 2 to 5 were soaked with 0.1% riboflavin for 20 minutes, which in Groups 3 to 5 was followed by UV-A irradiation at a fluence of 5.4 J/cm2. Ultraviolet A (UVA) irradiation was delivered at 3 mW/cm2 for 30 minutes (Group 3, standard CXL protocol), 9 mW/cm2 for 10 minutes (Group 4, accelerated), and 18 mW/cm2 for 5 minutes (Group 5, accelerated). At 1 week after treatment, corneal buttons were obtained; mRNA was extracted and subjected to cDNA sequencing (RNA-seq). RESULTS: A total of 297 differentially transcribed genes were identified after CXL treatment. CXL downregulated extracellular matrix components (collagen types 1A1, 1A2, 6A2, 11A1, keratocan, fibromodulin) and upregulated glycan biosynthesis and proteoglycan glycosylation (GALNT 3, 7, and 8, B3GALT2). Also, CXL activated pathways related to protein crosslinking (transglutaminase 2 and 6). In 9.1% of the significantly different genes, CXL at 3 mW/cm2 (Group 1) induced a more distinct change in gene transcription than the accelerated CXL protocols, which induced a lower biomechanical stiffening effect. CONCLUSIONS: Several target genes have been identified that might be related to the biomechanical stability and shape of the cornea. Stiffening-dependent differential gene transcription suggests the activation of mechano-sensitive pathways. TRANSLATIONAL RELEVANCE: A better understanding of the molecular mechanisms behind CXL will permit an optimization and individualization of the clinical treatment protocol.
PURPOSE: We studied changes in gene transcription after corneal crosslinking (CXL) in the rabbit cornea in vivo and identified potential molecular signaling pathways. METHODS: A total of 15 corneas of eight male New-Zealand-White rabbits were de-epithelialized and equally divided into five groups. Group 1 served as an untreated control. Groups 2 to 5 were soaked with 0.1% riboflavin for 20 minutes, which in Groups 3 to 5 was followed by UV-A irradiation at a fluence of 5.4 J/cm2. Ultraviolet A (UVA) irradiation was delivered at 3 mW/cm2 for 30 minutes (Group 3, standard CXL protocol), 9 mW/cm2 for 10 minutes (Group 4, accelerated), and 18 mW/cm2 for 5 minutes (Group 5, accelerated). At 1 week after treatment, corneal buttons were obtained; mRNA was extracted and subjected to cDNA sequencing (RNA-seq). RESULTS: A total of 297 differentially transcribed genes were identified after CXL treatment. CXL downregulated extracellular matrix components (collagen types 1A1, 1A2, 6A2, 11A1, keratocan, fibromodulin) and upregulated glycan biosynthesis and proteoglycan glycosylation (GALNT 3, 7, and 8, B3GALT2). Also, CXL activated pathways related to protein crosslinking (transglutaminase 2 and 6). In 9.1% of the significantly different genes, CXL at 3 mW/cm2 (Group 1) induced a more distinct change in gene transcription than the accelerated CXL protocols, which induced a lower biomechanical stiffening effect. CONCLUSIONS: Several target genes have been identified that might be related to the biomechanical stability and shape of the cornea. Stiffening-dependent differential gene transcription suggests the activation of mechano-sensitive pathways. TRANSLATIONAL RELEVANCE: A better understanding of the molecular mechanisms behind CXL will permit an optimization and individualization of the clinical treatment protocol.
Authors: Zhijun Liu; John L Tan; Daniel M Cohen; Michael T Yang; Nathan J Sniadecki; Sami Alom Ruiz; Celeste M Nelson; Christopher S Chen Journal: Proc Natl Acad Sci U S A Date: 2010-05-12 Impact factor: 11.205