title: sCD44 overexpression increases intraocular pressure and aqueous outflow resistance.
abstract: OBJECTIVE
  CD44 plays major roles in multiple physiologic processes. The ectodomain concentration of the CD44 receptor, soluble CD44 (sCD44), is significantly increased in the aqueous humor of primary open-angle glaucoma (POAG). The purpose of this study was to determine if adenoviral constructs of CD44 and isolated 32-kDa sCD44 change intraocular pressure (IOP) in vivo and aqueous outflow resistance in vitro.
  
  
  METHODS
  Adenoviral constructs of human standard CD44 (Ad-CD44S), soluble CD44 (Ad-sCD44), and empty viral cDNA were injected into the vitreous of BALB/cJ mice, followed by serial IOP measurements. Overexpression of CD44S and sCD44 was verified in vitro by enzyme-linked immunosorbent assay (ELISA) and western blot analysis. Anterior segments of porcine eyes were perfused with the isolated sCD44. sCD44-treated human trabecular meshwork (TM) cells and microdissected porcine TM were examined by confocal microscopy and Optiprep density gradient with western blot analysis to determine changes in lipid raft components.
  
  
  RESULTS
  Intravitreous injection of adenoviral constructs with either Ad-CD44S or Ad-sCD44 vectors caused prolonged ocular hypertension in mice. Eight days after vector injection, Ad-CD44S significantly elevated IOP to 28.3±1.2 mmHg (mean±SEM, n=8; p<0.001); Ad-sCD44 increased IOP to 18.5±2.6 mmHg (n=8; p<0.01), whereas the IOP of uninjected eyes was 12.7±0.2 mmHg (n=16). The IOP elevation lasted more than 50 days. Topical administration of a γ-secretase inhibitor normalized Ad-sCD44-induced elevated IOP. sCD44 levels were significantly elevated in the aqueous humor of Ad-CD44S and Ad-sCD44 eyes versus contralateral uninjected eyes (p<0.01). Anterior segment perfusion of isolated 32-kDa sCD44 significantly decreased aqueous outflow rates. Co-administration of isolated sCD44 and CD44 neutralizing antibody or of γ-secretase inhibitor significantly enhanced flow rates. sCD44-treated human TM cells displayed cross-linked actin network formation. Optiprep density gradient and western blot analysis of human TM cells treated with sCD44 showed decreased annexin 2 expression and increased phosphorylated annexin 2 and caveolin 1 expression.
  
  
  CONCLUSIONS
  Our data suggest that sCD44 increases outflow resistance in vivo and in vitro. Viral overexpression of both CD44S and sCD44 is sufficient to cause ocular hypertension. Infusion of sCD44 in porcine anterior segment eyes significantly decreased flow rates. Notably, sCD44 enhanced cross-linked actin network formation. The elevated sCD44 levels seen in POAG aqueous humor may play an important causative role in POAG pathogenesis.
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois.
authors.forename: Michael
authors.initials: M
authors.lastname: Giovingo
authors.forename: Michael
authors.initials: M
authors.lastname: Nolan
authors.forename: Ryan
authors.initials: R
authors.lastname: McCarty
authors.forename: Iok-Hou
authors.initials: IH
authors.lastname: Pang
authors.forename: Abbot F
authors.initials: AF
authors.lastname: Clark
authors.forename: Rachel M
authors.initials: RM
authors.lastname: Beverley
authors.forename: Steven
authors.initials: S
authors.lastname: Schwartz
authors.forename: W Daniel
authors.initials: WD
authors.lastname: Stamer
authors.forename: Loyal
authors.initials: L
authors.lastname: Walker
authors.forename: Algis
authors.initials: A
authors.lastname: Grybauskas
authors.forename: Kevin
authors.initials: K
authors.lastname: Skuran
authors.forename: Paulius V
authors.initials: PV
authors.lastname: Kuprys
authors.forename: Beatrice Y J T
authors.initials: BY
authors.lastname: Yue
authors.forename: Paul A
authors.initials: PA
authors.lastname: Knepper
mesh_terms: D000199:Actins / Q000378:metabolism; D000256:Adenoviridae / Q000235:genetics; D000328:Adult; D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D000869:Anterior Eye Segment / Q000187:drug effects / Q000473:pathology; D057134:Antibodies, Neutralizing / Q000494:pharmacology; D001082:Aqueous Humor / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism*; D015151:Immunoblotting; D007429:Intraocular Pressure / Q000187:drug effects; D019169:Jurkat Cells; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008875:Middle Aged; D010477:Perfusion; D012995:Solubility; D034421:Sus scrofa; D014129:Trabecular Meshwork / Q000378:metabolism / Q000473:pathology; D014161:Transduction, Genetic; D055815:Young Adult
chemical_list: D000199:Actins; D057134:Antibodies, Neutralizing; C497483:CD44 protein, human; D004791:Enzyme Inhibitors; D018960:Hyaluronan Receptors; D053829:Amyloid Precursor Protein Secretases