title: Perfusion of his-tagged eukaryotic myocilin increases outflow resistance in human anterior segments in the presence of aqueous humor.
abstract: OBJECTIVE
  A previous study by the authors has shown that recombinant myocilin purified from a prokaryotic expression system increases outflow resistance in cultured human anterior segments. The present study was performed to determine whether full-length myocilin purified from a human trabecular meshwork cell expression system alters outflow resistance after infusion into human anterior segments.
  
  
  METHODS
  A feline immunodeficiency virus vector encoding both full-length myocilin (amino acids 1-503 fused to C-terminal V5 and six-histidine epitopes) and puromycin resistance was used to transduce a transformed trabecular meshwork cell line (TM5). Stably expressing cells were selected with puromycin. Recombinant myocilin was purified from the media using nickel ion affinity chromatography. Control purifications were performed on media from parental TM5 cells. Anterior segments of human eyes were placed in organ culture and perfused with either Dulbecco's modified Eagle's medium (DMEM) or DMEM supplemented with 50% porcine aqueous humor. One eye received an anterior chamber exchange with recombinant myocilin (2 microg/mL), whereas the fellow eye received an equal volume of control. Immunohistochemistry was performed with anti-myocilin and anti-V5 antibodies. Native polyacrylamide gel electrophoresis was used to analyze myocilin complex formation in porcine aqueous humor.
  
  
  RESULTS
  Recombinant myocilin in porcine aqueous humor increased outflow resistance in cultured human anterior segments (91% +/- 68% [mean +/- SD] versus 18% +/- 31% in fellow control eye; n = 9, P = 0.004). Maximum outflow resistance was obtained 5 to 17 hours after infusion and remained above baseline for >3 days. Recombinant myocilin also increased outflow resistance in eyes incubated in DMEM, but only if myocilin was preincubated with porcine aqueous humor (78% +/- 77% when preincubated in DMEM containing porcine aqueous humor versus 13% +/- 15% when preincubated with DMEM alone, n = 6, P = 0.03). Recombinant myocilin appears to form a complex in porcine aqueous humor with a heat-labile protein(s). Immunohistochemistry revealed the presence of myocilin in the juxtacanalicular region of the trabecular meshwork.
  
  
  CONCLUSIONS
  Myocilin purified from human trabecular meshwork cells increased outflow resistance in cultured human anterior segments, but only after incubation with porcine aqueous humor. Recombinant myocilin appears to form a complex in porcine aqueous humor that enables it to bind specifically within the trabecular meshwork.
authors.affiliation: Department of Ophthalmology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA. fautsch.michael@mayo.edu
authors.forename: Michael P
authors.initials: MP
authors.lastname: Fautsch
authors.forename: Cindy K
authors.initials: CK
authors.lastname: Bahler
authors.forename: Anne M
authors.initials: AM
authors.lastname: Vrabel
authors.forename: Kyle G
authors.initials: KG
authors.lastname: Howell
authors.forename: Nils
authors.initials: N
authors.lastname: Loewen
authors.forename: Wulin L
authors.initials: WL
authors.lastname: Teo
authors.forename: Eric M
authors.initials: EM
authors.lastname: Poeschla
authors.forename: Douglas H
authors.initials: DH
authors.lastname: Johnson
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D001082:Aqueous Humor / Q000502:physiology*; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D005136:Eye Proteins / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D006023:Glycoproteins / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009924:Organ Culture Techniques; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013552:Swine; D014129:Trabecular Meshwork / Q000166:cytology / Q000378:metabolism*; D014162:Transfection; D000099179:Myocilin
chemical_list: D003598:Cytoskeletal Proteins; D005136:Eye Proteins; D006023:Glycoproteins; D006639:Histidine; D011993:Recombinant Fusion Proteins; D000099179:Myocilin