title: Analysis of sphingolipids in human corneal fibroblasts from normal and keratoconus patients.
abstract: The pathophysiology of human keratoconus (KC), a bilateral progressive corneal disease leading to protrusion of the cornea, stromal thinning, and scarring, is not well-understood. In this study, we investigated a novel sphingolipid (SPL) signaling pathway through which KC may be regulated. Using human corneal fibroblasts (HCFs) and human KC cells (HKCs), we examined the SPL pathway modulation. Both cell types were stimulated by the three transforming growth factor (TGF)-β isoforms: TGF-β1 (T1), TGF-β2 (T2), and TGF-β3 (T3). All samples were analyzed using lipidomics and real-time PCR. Our data showed that HKCs have increased levels of signaling SPLs, ceramide (Cer), and sphingosine 1-phosphate (S1P). Treatment with T1 reversed the increase in Cer in HKCs and treatment with T3 reversed the increase in S1P.S1P3receptor mRNA levels were also significantly upregulated in HKCs, but were reduced to normal levels following T3 treatment. Furthermore, stimulation with Cer and S1P led to significant upregulation of fibrotic markers in HCFs, but not in HKCs. Additionally, stimulation with a Cer synthesis inhibitor (FTY720) led to significant downregulation of specific fibrotic markers in HKCs (TGF-β1, collagen type III, and α smooth muscle actin) without an effect on healthy HCFs, suggesting a causative role of Cer and S1P in fibrogenesis. Overall, this study suggests an association of the SPL signaling pathway in KC disease and its relation with the TGF-β pathway.
authors.affiliation: Departments of Ophthalmology, University of Oklahoma Health Sciences Center (OUHSC), Oklahoma City, OK 73104; Dean McGee Eye Institute, Oklahoma City, OK 73104.
authors.forename: Hui
authors.initials: H
authors.lastname: Qi
authors.affiliation: Departments of Ophthalmology, University of Oklahoma Health Sciences Center (OUHSC), Oklahoma City, OK 73104; Dean McGee Eye Institute, Oklahoma City, OK 73104.
authors.forename: Shrestha
authors.initials: S
authors.lastname: Priyadarsini
authors.affiliation: Departments of Ophthalmology, University of Oklahoma Health Sciences Center (OUHSC), Oklahoma City, OK 73104; Dean McGee Eye Institute, Oklahoma City, OK 73104.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Nicholas
authors.affiliation: Departments of Ophthalmology, University of Oklahoma Health Sciences Center (OUHSC), Oklahoma City, OK 73104; Dean McGee Eye Institute, Oklahoma City, OK 73104.
authors.forename: Akhee
authors.initials: A
authors.lastname: Sarker-Nag
authors.affiliation: Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, VA 23249.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Allegood
authors.affiliation: Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, VA 23249; Research and Development, Hunter Holmes McGuire Veterans Administration Medical Center, Richmond, VA 23249 and VCU Massey Cancer Center, VCU Institute of Molecular Medicine, and VCU Johnson Center, Virginia Commonwealth University, Richmond, VA 23298.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Chalfant
authors.affiliation: Departments of Ophthalmology, University of Oklahoma Health Sciences Center (OUHSC), Oklahoma City, OK 73104; Dean McGee Eye Institute, Oklahoma City, OK 73104; Physiology, University of Oklahoma Health Sciences Center (OUHSC), Oklahoma City, OK 73104; Cell Biology, University of Oklahoma Health Sciences Center (OUHSC), Oklahoma City, OK 73104. Electronic address: nmandal@uthsc.edu.
authors.forename: Nawajes A
authors.initials: NA
authors.lastname: Mandal
authors.affiliation: Departments of Ophthalmology, University of Oklahoma Health Sciences Center (OUHSC), Oklahoma City, OK 73104; Dean McGee Eye Institute, Oklahoma City, OK 73104; Physiology, University of Oklahoma Health Sciences Center (OUHSC), Oklahoma City, OK 73104; Cell Biology, University of Oklahoma Health Sciences Center (OUHSC), Oklahoma City, OK 73104. Electronic address: Dimitrios-Karamichos@ouhsc.edu.
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Karamichos
mesh_terms: D002460:Cell Line; D002518:Ceramides / Q000008:administration & dosage / Q000235:genetics*; D003315:Cornea / Q000378:metabolism / Q000473:pathology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D000068876:Fingolimod Hydrochloride / Q000008:administration & dosage; D006801:Humans; D007640:Keratoconus / Q000235:genetics* / Q000473:pathology; D008246:Lysophospholipids / Q000008:administration & dosage / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000235:genetics; D015398:Signal Transduction; D013107:Sphingolipids / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013110:Sphingosine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000096:biosynthesis; D053773:Transforming Growth Factor beta1 / Q000008:administration & dosage; D053781:Transforming Growth Factor beta2 / Q000008:administration & dosage; D053782:Transforming Growth Factor beta3 / Q000008:administration & dosage
chemical_list: D002518:Ceramides; D008246:Lysophospholipids; D012333:RNA, Messenger; D013107:Sphingolipids; D053773:Transforming Growth Factor beta1; D053781:Transforming Growth Factor beta2; D053782:Transforming Growth Factor beta3; C060506:sphingosine 1-phosphate; D000068876:Fingolimod Hydrochloride; D013110:Sphingosine
keywords: cornea; lipidomics; transforming growth factor-β