title: Targeted suppression of HO-2 gene expression impairs the innate anti-inflammatory and repair responses of the cornea to injury.
abstract: OBJECTIVE
  Heme oxygenase (HO)-2 is highly expressed in the corneal epithelium and is a component of the heme oxygenase system that represents an intrinsic cytoprotective and anti-inflammatory system based on its ability to modulate leukocyte migration and to inhibit expression of inflammatory cytokines and proteins via its products biliverdin/bilirubin and carbon monoxide (CO). We have shown that in HO-2 null mice epithelial injury leads to unresolved corneal inflammation and chronic inflammatory complications including ulceration, perforation and neovascularization. In this study, we explore whether a localized corneal suppression of HO-2 is sufficient for disrupting the innate anti-inflammatory and repair capability of the cornea.
  
  
  METHODS
  Silencing hairpin RNA (shRNA) against HO-2 was administered subconjunctivally (100 ng/eye) as well as topically (100 ng/eye) starting one day before corneal epithelial debridement and once daily, thereafter. The corneal epithelium was removed using an Alger Brush in anesthetized mice. Re-epithelialization was assessed by fluorescein staining using a dissecting microscope and image analysis. Inflammatory response was quantified by myeloperoxidase activity. Levels of mRNA were measured by RT-PCR.
  
  
  RESULTS
  Local injection of HO-2-specific shRNA led to a 50% reduction in corneal HO-2 mRNA. Administration of HO-2-specific shRNA delayed corneal re-epithelialization when compared with the control shRNA-treated group by 14%, 20%, and 12% at days 3, 4, and 7 after injury, respectively (n=18-24). The observed delay in the wound repair process in HO-2 shRNA treated mice was accompanied by a threefold and 3.5 fold increase in the neovascular response at days 4 and 7 after injury. Further, local knockdown of HO-2 lead to an aberrant chronic inflammatory response, as shown by presence of high numbers of inflammatory cells still present in the cornea at day 7 after injury; 1.04±0.45×10(6) in HO-2 knockdown mice versus 0.14±0.03×10(6) inflammatory cells in control mice. Matrix metalloproteinase-2 (MMP-2) but not MMP-9 increased following injury and remained elevated in the injured corneas of the HO-2 shRNA-treated eyes.
  
  
  CONCLUSIONS
  Corneal knockdown of HO-2 via local administration of HO-2-specific shRNA leads to delayed re-epithelialization, increased neovascularization and an aberrant inflammatory response similar to what is observed in the HO-2 null mouse. The elevated MMP-2 expression may contribute to the increase in neovascularization in corneas in which HO-2 expression is suppressed.
authors.affiliation: Department of Pharmacology, New York Medical College, Valhalla, NY 10595, USA. Lars_Bellner@nymc.edu
authors.forename: Lars
authors.initials: L
authors.lastname: Bellner
authors.forename: Kiran A
authors.initials: KA
authors.lastname: Patil
authors.forename: Kirkland
authors.initials: K
authors.lastname: Castellano
authors.forename: Adna
authors.initials: A
authors.lastname: Halilovic
authors.forename: Michael W
authors.initials: MW
authors.lastname: Dunn
authors.forename: Michal Laniado
authors.initials: ML
authors.lastname: Schwartzman
mesh_terms: D000287:Administration, Topical; D000818:Animals; D003315:Cornea / Q000187:drug effects / Q000378:metabolism*; D065306:Corneal Injuries; D019573:Epithelium, Corneal / Q000187:drug effects / Q000293:injuries / Q000378:metabolism*; D019793:Fluorescein / Q000032:analysis; D015870:Gene Expression / Q000187:drug effects*; D006419:Heme Oxygenase (Decyclizing) / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D007249:Inflammation / Q000235:genetics / Q000378:metabolism; D056965:Injections, Intraocular; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009389:Neovascularization, Pathologic / Q000235:genetics / Q000378:metabolism; D009195:Peroxidase / Q000032:analysis / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D034741:RNA, Small Interfering / Q000378:metabolism / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation; D014945:Wound Healing / Q000187:drug effects / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; D009195:Peroxidase; D006419:Heme Oxygenase (Decyclizing); C105245:heme oxygenase-2; D020778:Matrix Metalloproteinase 2; D019793:Fluorescein