FIBROBLAST DIFFERENTIATION DURING EYE DEVELOPMENT

Summary

Principal Investigator: GARY CONRAD
Affiliation: Kansas State University
Country: USA
Abstract: The long-range goal of the proposed research is to understand the biological physical chemistry of the corneal extracellular matrix (ECM) during embryonic development and be able to repair that ECM in adults. This is of immediate medical importance for two reasons: 1) In the U.S. alone, several million patients have undergone LASIK. LASIK manipulations of the cornea create a transparent, but permanently non-attached stromal flap. LASIK also significantly reduces corneal innervation, and can greatly increase perceived Dry Eye. In the general population, Dry Eye affects 11% of people over 30 and 15% of people over 65, and has been linked to corneal neuropathy. Proteoglycans of the LASIK scar interface are abnormal compared with adjacent stroma. Existing non-attached LASIK flaps are of concern for their physical instability during facial trauma. Herein, we develop methods to reduce that instability by chemically crosslinking normal and abnormal stromal ECM components, and assess gene expression in adjacent corneal stroma fibroblasts (keratocytes), Schwann cells, and nerves. 2) Another clinically important physical manipulation of corneal ECM, combined riboflavin and ultraviolet light (UVA) administration, has begun trials in the U.S. as a treatment for keratoconus. This treatment alters corneal ECM by mechanisms assumed, but not yet proven, to involve collagen crosslinking, with proteoglycan involvement. Resulting regeneration and wound healing reactions of surrounding surviving corneal nerves and keratocytes involve cellular migrations within the stroma and changes in keratocyte and nerve differentiated states yet to be characterized. Here, we begin a study of the biological physical chemistry of the corneal ECM, characterizing the molecular biology of the ECM macromolecules synthesized normally and also in response to clinical treatments. Proteomic and glycomic microarrays will be used to determine physical binding specificities of ECM molecules. Laser Capture Microdissection, MALDI-TOF/TOF and other mass spectrometry techniques will be used for detailed analyses that can detect changes in very restricted corneal ECM regions and cell populations following LASIK and riboflavin+UVA treatments, as well as after other clinically important corneal procedures and conditions, e.g., LASEK, PRK, corneal transplantation, and Dry Eye. Specific Aims: 1) Determine chemical mechanisms by which riboflavin+UVA treatment cross-links the corneal stroma and affects corneal cells, and thereby devise chemical mechanisms for covalently immobilizing LASIK flaps. 2) Use glycomic and proteomic microarrays and surface plasmon resonance assays to determine binding preferences and association and dissociation kinetics of corneal proteoglycans for neurotrophins, growth factors, neurotransmitters, hormones, and structural macromolecules of the cornea. 3) Characterize gene expression in trigeminal neural crest-derived nerves and in their non-myelinating Schwann cells following neuronal growth cone contact with keratan sulfate (KS) and with KS-proteoglycan core proteins. PUBLIC HEALTH RELEVANCE: The cornea is the only living tissue in the body that is both transparent and highly innervated with sensory nerve endings, meaning that any medical treatment of the cornea, elective or required, has the potential for permanently damaging sight and producing great pain. The long-range goal of the proposed research is to understand and to learn to repair the connective tissue of the cornea of the eye, based upon lessons learned from studying the differentiation of the connective tissue fibroblasts of the corneal stroma (the "keratocytes") during embryonic development and in adults. This is of immediate medical public health importance, first, because the most currently popular elective eye surgery, LASIK, produces corneas that, while transparent in most patients, never truly heal and become problematic in 2-3% of patients, and, second, because an exciting new treatment involving riboflavin (vitamin B2) and long wavelength-ultraviolet light (UVA) has been devised whose primary mechanism and potential side effects remain unknown, but whose remarkable primary effect of "toughening" the cornea greatly slows the rate at which a very serious, progressive syndrome, keratoconus, makes corneas thin, cone-shaped, and perforated, and could provide a mechanism for "healing" LASIK corneas.
Funding Period: -------------------- - --------------------
more information: NIH RePORT

Top Publications

  1. pmc Focus on molecules: lumican
    Winston W Y Kao
    Department of Ophthalmology, University of Cincinnati, Cincinnati, OH, USA
    Exp Eye Res 82:3-4. 2006
  2. pmc Corneal sulfated glycosaminoglycans and their effects on trigeminal nerve growth cone behavior in vitro: roles for ECM in cornea innervation
    Tyler Schwend
    Division of Biology, Kansas State University, Manhattan, Kansas 66506, USA
    Invest Ophthalmol Vis Sci 53:8118-37. 2012
  3. pmc Fibrinogen, riboflavin, and UVA to immobilize a corneal flap--molecular mechanisms
    Stacy L Littlechild
    Division of Biology, Kansas State University, Manhattan, Kansas 66506 4901, USA
    Invest Ophthalmol Vis Sci 53:5991-6003. 2012
  4. doi Fibrinogen, riboflavin, and UVA to immobilize a corneal flap--conditions for tissue adhesion
    Stacy L Littlechild
    Division of Biology, Kansas State University, Manhattan, Kansas 66506 4901, USA
    Invest Ophthalmol Vis Sci 53:4011-20. 2012
  5. pmc The role of nonenzymatic glycation and carbonyls in collagen cross-linking for the treatment of keratoconus
    Gage Brummer
    Division of Biology, Kansas State University, Manhattan, Kansas 66506 4901, USA
    Invest Ophthalmol Vis Sci 52:6363-9. 2011
  6. doi Resistance of corneal RFUVA–cross-linked collagens and small leucine-rich proteoglycans to degradation by matrix metalloproteinases
    Yuntao Zhang
    Division of Biology, Kansas State University, Manhattan, Kansas 66506 4901, USA
    Invest Ophthalmol Vis Sci 54:1014-25. 2013
  7. pmc Integrated genomic approaches implicate osteoglycin (Ogn) in the regulation of left ventricular mass
    Enrico Petretto
    Medical Research Council Clinical Sciences Centre, Faculty of Medicine, Imperial College London, Hammersmith Hospital, Du Cane Road, London, W12 0NN, UK
    Nat Genet 40:546-52. 2008
  8. doi On-target derivatization of keratan sulfate oligosaccharides with pyrenebutyric acid hydrazide for MALDI-TOF/TOF-MS
    Yuntao Zhang
    Division of Biology, Kansas State University, Ackert Hall, Manhattan, Kansas 66506 4901, USA
    J Mass Spectrom 43:765-72. 2008
  9. ncbi Differentiation of diiodothyronines using electrospray ionization tandem mass spectrometry
    Yuntao Zhang
    Division of Biology, Ackert Hall, Kansas State University, Manhattan, Kansas 66506 4901, USA
    J Mass Spectrom 41:162-8. 2006
  10. ncbi Thyroxine affects expression of KSPG-related genes, the carbonic anhydrase II gene, and KS sulfation in the embryonic chicken cornea
    Abigail H Conrad
    Division of Biology, Kansas State University, Manhattan, Kansas 66506 4901, USA
    Invest Ophthalmol Vis Sci 47:120-32. 2006

Scientific Experts

  • Kiyoshi Suzuki
  • A Scott McCall
  • Tyler Schwend
  • Yuntao Zhang
  • Gary W Conrad
  • Abigail H Conrad
  • Stacy L Littlechild
  • Gage Brummer
  • John M Tomich
  • Stacy Littlechild
  • Guodong Zhang
  • Elena S Tasheva
  • Enrico Petretto
  • Yutaka Kariya
  • Rudy Thoma
  • Winston W Y Kao
  • Xiuli Mao
  • Pinakin Sukthankar
  • Austin R Rogers
  • Daniel L Boyle
  • Scott McCall
  • Maya Pettit-Scott
  • Michael Albrecht
  • Timothy J Aitman
  • Han Lu
  • Lolita M Corpuz
  • Matthew Benson
  • Takeo Iwamoto
  • Stuart A Cook
  • Theodore W Kurtz
  • Judith Fischer
  • Sabrina Conway
  • Ian Grieve
  • Rizwan Sarwar
  • Michal Pravenec
  • Gary Radke
  • Mande K Kumaran
  • Sanjay K Prasad
  • Blanche Schroen
  • Yigal M Pinto
  • Jessica M Strafuss
  • Megan D Webb
  • Maria D Wittman
  • Chris Kiesewetter
  • Dudley J Pennell
  • Vladimir Kren
  • Jonathan Mangion
  • Prakash P Punjabi
  • Norbert Hubner
  • Phillip J Muckett
  • Daniel G Dawson
  • Lisa A Olberding
  • Ying Xia
  • Raul Morffi
  • Hans E Grossniklaus
  • James L Funderburgh
  • Andrea J Zimmer
  • Henry F Edelhauser
  • Ann Hanzlick
  • Chia Yang Liu
  • Amy R Walker
  • Ingo Schmack

Detail Information

Publications20

  1. pmc Focus on molecules: lumican
    Winston W Y Kao
    Department of Ophthalmology, University of Cincinnati, Cincinnati, OH, USA
    Exp Eye Res 82:3-4. 2006
  2. pmc Corneal sulfated glycosaminoglycans and their effects on trigeminal nerve growth cone behavior in vitro: roles for ECM in cornea innervation
    Tyler Schwend
    Division of Biology, Kansas State University, Manhattan, Kansas 66506, USA
    Invest Ophthalmol Vis Sci 53:8118-37. 2012
    ....
  3. pmc Fibrinogen, riboflavin, and UVA to immobilize a corneal flap--molecular mechanisms
    Stacy L Littlechild
    Division of Biology, Kansas State University, Manhattan, Kansas 66506 4901, USA
    Invest Ophthalmol Vis Sci 53:5991-6003. 2012
    ..Here, we document several covalent and noncovalent interactions between these classes of macromolecules...
  4. doi Fibrinogen, riboflavin, and UVA to immobilize a corneal flap--conditions for tissue adhesion
    Stacy L Littlechild
    Division of Biology, Kansas State University, Manhattan, Kansas 66506 4901, USA
    Invest Ophthalmol Vis Sci 53:4011-20. 2012
    ..This lack of permanent adhesion is a liability. To immobilize a corneal flap, a protocol using fibrinogen (FIB), riboflavin (RF), and ultraviolet (UVA) light (FIB+RF+UVA) was devised to re-adhere the flap to the stroma...
  5. pmc The role of nonenzymatic glycation and carbonyls in collagen cross-linking for the treatment of keratoconus
    Gage Brummer
    Division of Biology, Kansas State University, Manhattan, Kansas 66506 4901, USA
    Invest Ophthalmol Vis Sci 52:6363-9. 2011
    ..Advanced glycation end products (AGEs) have been suggested by many studies as the causative strengthening agent during CXL, though no studies to date have directly tested this hypothesis...
  6. doi Resistance of corneal RFUVA–cross-linked collagens and small leucine-rich proteoglycans to degradation by matrix metalloproteinases
    Yuntao Zhang
    Division of Biology, Kansas State University, Manhattan, Kansas 66506 4901, USA
    Invest Ophthalmol Vis Sci 54:1014-25. 2013
    ..However, the extent to which MMPs digest cross-linked collagen and small leucine-rich proteoglycans (SLRPs) remains unknown. In this study, the resistance of RFUVA-cross-linked collagens and SLRPs to MMPs has been investigated...
  7. pmc Integrated genomic approaches implicate osteoglycin (Ogn) in the regulation of left ventricular mass
    Enrico Petretto
    Medical Research Council Clinical Sciences Centre, Faculty of Medicine, Imperial College London, Hammersmith Hospital, Du Cane Road, London, W12 0NN, UK
    Nat Genet 40:546-52. 2008
    ..Taken together, these data implicate Ogn as a key regulator of LVM in rats, mice and humans, and suggest that Ogn modifies the hypertrophic response to extrinsic factors such as hypertension and aortic stenosis...
  8. doi On-target derivatization of keratan sulfate oligosaccharides with pyrenebutyric acid hydrazide for MALDI-TOF/TOF-MS
    Yuntao Zhang
    Division of Biology, Kansas State University, Ackert Hall, Manhattan, Kansas 66506 4901, USA
    J Mass Spectrom 43:765-72. 2008
    ..This one-step on-target derivatization method makes MALDI-TOF/TOF-MS identification of KS fast, simple and highly throughput for trace amounts of biological samples...
  9. ncbi Differentiation of diiodothyronines using electrospray ionization tandem mass spectrometry
    Yuntao Zhang
    Division of Biology, Ackert Hall, Kansas State University, Manhattan, Kansas 66506 4901, USA
    J Mass Spectrom 41:162-8. 2006
    ..On the basis of the relative abundance of fragment ions in MS2 spectra under the positive ionization mode, quantification of the 3,5-T2, 3',5'-T2 and 3,3'-T2 isomers in mixtures is also achieved without prior separation...
  10. ncbi Thyroxine affects expression of KSPG-related genes, the carbonic anhydrase II gene, and KS sulfation in the embryonic chicken cornea
    Abigail H Conrad
    Division of Biology, Kansas State University, Manhattan, Kansas 66506 4901, USA
    Invest Ophthalmol Vis Sci 47:120-32. 2006
    ....
  11. doi Thyroxine increases the rate but does not alter the pattern of innervation during embryonic chick corneal development
    Abigail H Conrad
    Division of Biology, Kansas State University, Manhattan, KS 66506, USA
    Invest Ophthalmol Vis Sci 49:139-53. 2008
    ..The hypothesis for this study was that precocious T4 exposure increases corneal innervation similarly...
  12. ncbi Differentiation of monoiodothyronines using electrospray ionization tandem mass spectrometry
    Yuntao Zhang
    Division of Biology, Kansas State University, Manhattan, KS 66506 4901, USA
    Rapid Commun Mass Spectrom 20:481-6. 2006
    ..The detection limits for 3-T1 and 3'-T1 were 0.5 and 0.7 pg/microL, respectively...
  13. ncbi Keratan sulfate and chondroitin/dermatan sulfate in maximally recovered hypocellular stromal interface scars of postmortem human LASIK corneas
    Yuntao Zhang
    Division of Biology, Kansas State University, Manhattan, Kansas 66506 4901, USA
    Invest Ophthalmol Vis Sci 47:2390-6. 2006
    ....
  14. pmc Effect of the synthetic NC-1059 peptide on diffusion of riboflavin across an intact corneal epithelium
    Yuntao Zhang
    Division of Biology, Kansas State University, Manhattan, KS 66506 4901, USA
    Invest Ophthalmol Vis Sci 53:2620-9. 2012
    ..To investigate the effect of the peptide NC-1059 on riboflavin (RF) diffusion across an intact corneal epithelium into the stroma...
  15. pmc Development and mineralization of embryonic avian scleral ossicles
    Guodong Zhang
    Division of Biology, Kansas State University, Manhattan, KS 66506 4901, USA
    Mol Vis 18:348-61. 2012
    ..To investigate the development and mineralization of avian scleral ossicles using fluorescence microscopy in combination with field emission scanning electron microscopy (FESEM) and energy dispersive spectroscopy (EDS)...
  16. pmc Nerve repulsion by the lens and cornea during cornea innervation is dependent on Robo-Slit signaling and diminishes with neuron age
    Tyler Schwend
    Division of Biology, Ackert Hall, Kansas State University, Manhattan, KS 66506, USA
    Dev Biol 363:115-27. 2012
    ..Later, a shift in nerve guidance behavior occurs, in part due to molecular changes in trigeminal neurons, including Robo1 downregulation, thus allowing nerves to find the Slit-expressing cornea permissive for growth cones...
  17. pmc Effects of ultraviolet-A and riboflavin on the interaction of collagen and proteoglycans during corneal cross-linking
    Yuntao Zhang
    Division of Biology, Kansas State University, Manhattan, Kansas 66506 4901, USA
    J Biol Chem 286:13011-22. 2011
    ..RFUVA as a diagnostic tool reveals that keratocan and lumican core proteins interact with collagen very differently than do mimecan and decorin...
  18. pmc Mechanisms of corneal tissue cross-linking in response to treatment with topical riboflavin and long-wavelength ultraviolet radiation (UVA)
    A Scott McCall
    Division of Biology, Kansas State University, Manhattan, Kansas 66506 4901, USA
    Invest Ophthalmol Vis Sci 51:129-38. 2010
    ..RFUVA treatment retards the progression of keratoconus, perhaps by cross-linking of collagen molecules, but exact molecular mechanisms remain unknown. Research described here tested possible chemical mechanisms of cross-linking...
  19. pmc Embryonic corneal Schwann cells express some Schwann cell marker mRNAs, but no mature Schwann cell marker proteins
    Abigail H Conrad
    Division of Biology, Kansas State University, Manhattan, Kansas 66506 4901, USA
    Invest Ophthalmol Vis Sci 50:4173-84. 2009
    ..Adult corneal nerves, cut during transplantation or LASIK, never fully regenerate. Schwann cells (SCs) protect nerve fibers and augment nerve repair. This study evaluates SC differentiation in embryonic chick corneas...
  20. pmc Proteomic analysis of potential keratan sulfate, chondroitin sulfate A, and hyaluronic acid molecular interactions
    Abigail H Conrad
    Division of Biology, Kansas State University, Manhattan, Kansas 66506 4901, USA
    Invest Ophthalmol Vis Sci 51:4500-15. 2010
    ..This study asked which of the proteins that may regulate keratocytes or corneal nerve growth cone immigration interact with corneal GAGs...