title: Retinal O-linked N-acetylglucosamine protein modifications: implications for postnatal retinal vascularization and the pathogenesis of diabetic retinopathy.
abstract: OBJECTIVE
  Hyperglycemia activates several metabolic pathways, including the hexosamine biosynthetic pathway. Uridine diphosphate N-acetylglucosamine (GlcNAc) is the product of the hexosamine biosynthetic pathway and the substrate for O-linked GlcNAc (O-GlcNAc) modification. This modification affects a wide range of proteins by altering their activity, cellular localization, and/or protein interactions. However, the role O-GlcNAcylation may play in normal postnatal retinal vascular development and in the ocular complications of diabetes, including diabetic retinopathy, requires further investigation.
  
  
  METHODS
  The total levels of O-GlcNAc-modified proteins were evaluated by western blot analysis of lysates prepared from retinas obtained at different days during postnatal retinal vascularization and oxygen-induced ischemic retinopathy. Similar experiments were performed with retinal lysate prepared from diabetic Ins2(Akita/+) mice with different durations of diabetes and retinal vascular cells cultured under various glucose conditions. The localization of O-GlcNAc-modified proteins in the retinal vasculature was confirmed by immunofluorescence staining. The impact of altered O-GlcNAcylation on the migration of retinal vascular cells was determined using scratch wound and transwell migration assays.
  
  
  RESULTS
  We detected an increase in protein O-GlcNAcylation during mouse postnatal retinal vascularization and aging, in part through the regulation of the enzymes that control this modification. The study of the diabetic Ins2(Akita/+) mouse retina showed an increase in the O-GlcNAc modification of retinal proteins. We also observed an increase in retinal O-GlcNAcylated protein levels during the neovascularization phase of oxygen-induced ischemic retinopathy. Our fluorescence microscopy data confirmed that the alterations in retinal O-GlcNAcylation are similarly represented in the retinal vasculature and in retinal pericytes and endothelial cells. Particularly, the migration of retinal pericytes, but not retinal endothelial cells, was attenuated by increased O-GlcNAc modification.
  
  
  CONCLUSIONS
  The O-GlcNAc modification pattern changes during postnatal retinal vascular development and neovascularization, and its dysregulation under hyperglycemia and/or ischemia may contribute to the pathogenesis of the diabetic retinopathy and retinal neovascularization.
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Wisconsin School of Medicine and Public Health, Madison, WI 53792-4673, USA. zgurel@wisc.edu
authors.forename: Zafer
authors.initials: Z
authors.lastname: Gurel
authors.forename: Kelsey M
authors.initials: KM
authors.lastname: Sieg
authors.forename: Keegan D
authors.initials: KD
authors.lastname: Shallow
authors.forename: Christine M
authors.initials: CM
authors.lastname: Sorenson
authors.forename: Nader
authors.initials: N
authors.lastname: Sheibani
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D000375:Aging / Q000473:pathology; D000818:Animals; D000831:Animals, Newborn; D002465:Cell Movement / Q000187:drug effects; D003930:Diabetic Retinopathy / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D005136:Eye Proteins / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology; D006031:Glycosylation / Q000187:drug effects; D007328:Insulin / Q000235:genetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018919:Neovascularization, Physiologic / Q000187:drug effects; D020286:Pericytes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012160:Retina / Q000254:growth & development / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D012171:Retinal Vessels / Q000187:drug effects / Q000254:growth & development / Q000473:pathology / Q000503:physiopathology*
chemical_list: D005136:Eye Proteins; C561967:Ins2 protein, mouse; D007328:Insulin; D005947:Glucose; D000117:Acetylglucosamine