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.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Anterior Eye Disease Study Section (AED)
Program Officer
Shen, Grace L
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Kansas State University
Schools of Arts and Sciences
United States
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Mao, Xiuli; Zhang, Yuntao; Schwend, Tyler et al. (2015) Effects of polysialic acid on sensory innervation of the cornea. Dev Biol 398:193-205
Zhang, Yuntao; Mao, Xiuli; Schwend, Tyler et al. (2013) Resistance of corneal RFUVA–cross-linked collagens and small leucine-rich proteoglycans to degradation by matrix metalloproteinases. Invest Ophthalmol Vis Sci 54:1014-25
Littlechild, Stacy L; Zhang, Yuntao; Tomich, John M et al. (2012) Fibrinogen, riboflavin, and UVA to immobilize a corneal flap--molecular mechanisms. Invest Ophthalmol Vis Sci 53:5991-6003
Zhang, Guodong; Boyle, Daniel L; Zhang, Yuntao et al. (2012) Development and mineralization of embryonic avian scleral ossicles. Mol Vis 18:348-61
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Zhang, Yuntao; Sukthankar, Pinakin; Tomich, John M et al. (2012) Effect of the synthetic NC-1059 peptide on diffusion of riboflavin across an intact corneal epithelium. Invest Ophthalmol Vis Sci 53:2620-9
Schwend, Tyler; Lwigale, Peter Y; Conrad, Gary W (2012) Nerve repulsion by the lens and cornea during cornea innervation is dependent on Robo-Slit signaling and diminishes with neuron age. Dev Biol 363:115-27
Schwend, Tyler; Deaton, Ryan J; Zhang, Yuntao et al. (2012) Corneal sulfated glycosaminoglycans and their effects on trigeminal nerve growth cone behavior in vitro: roles for ECM in cornea innervation. Invest Ophthalmol Vis Sci 53:8118-37
Brummer, Gage; Littlechild, Stacy; McCall, Scott et al. (2011) The role of nonenzymatic glycation and carbonyls in collagen cross-linking for the treatment of keratoconus. Invest Ophthalmol Vis Sci 52:6363-9
Zhang, Yuntao; Conrad, Abigail H; Conrad, Gary W (2011) Effects of ultraviolet-A and riboflavin on the interaction of collagen and proteoglycans during corneal cross-linking. J Biol Chem 286:13011-22

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