title: Gene transfer of dominant-negative RhoA increases outflow facility in perfused human anterior segment cultures.
abstract: OBJECTIVE
  To investigate the regulation of expression and the role of the RhoA gene in the human trabecular meshwork (TM). To attempt to modulate outflow facility by gene transfer of the RhoA gene's dominant-negative mutant protein.
  
  
  METHODS
  Total RNA extracted from cultured human trabecular meshwork (HTM) cells treated with outflow facility drugs were analyzed by northern blot hybridization using an amplified human RhoA cDNA from plasmid pZip-RhoA wild type (wt) [1]. A dominant-negative form of RhoA (single amino acid substitution of Thr19 to Asn) was placed under the control of the CMV promoter and inserted into a replication-deficient adenoviral vector by overlapping recombination (AdhRhoA2). AdhRhoA2 was infected into perfused anterior segment cultures from post-mortem human donors and HTM and Schlemm's canal cells in culture. Changes in outflow facility (flow/pressure) were calculated as percent changes from baseline values (C0), pooled into treated and control groups and expressed as the mean plus minus standard error. HTM and Schlemm's Canal (SC) cells were fluorescently double-labeled for the RhoA protein and actin, paxillin, or ZO-1.
  
  
  RESULTS
  Transcription of RhoA in HTM cells was not considerably affected by treatment of the cells with cytoskeletal/outflow facility drugs. At 66 h post-injection, anterior segments treated with AdhRhoA2 (n=9) exhibited an increase in outflow facility of 32.5 +/- 7.7% while that of the vehicle-injected controls (n=6) was 5.1 +/- 4.0% (p=0.02). HTM cells treated with AdhRhoA2 showed a marked change in morphology with a reduction in actin stress fibers and of the focal adhesion-containing protein, paxillin. Confluent monolayers of SC cells infected with AdhRhoA2 were devoid of peripheral ZO-1 staining indicating a loss of intercellular junctions.
  
  
  CONCLUSIONS
  In the HTM cells, cytoskeletal/outflow facility drugs do not seem to affect the levels of RhoA mRNA, possibly suggesting the importance of mRNA availability to allow rapid turnover of its function. Gene transfer of inactive RhoA to the intact human TM results in an increase in outflow facility. This increase appears to be correlated with a loosening of the cell-substrate and cell-cell attachments in the cells of the outflow pathway. Adenoviral vectors carrying the dominant negative form of RhoA could potentially be utilized as a gene therapy to modulate outflow facility.
authors.affiliation: Dept. of Ophthalmology, Duke University Medical Center, Durham, NC 27710, USA. jason.vittitow@duke.edu
authors.forename: Jason L
authors.initials: JL
authors.lastname: Vittitow
authors.forename: Rahul
authors.initials: R
authors.lastname: Garg
authors.forename: Laura-Leigh S
authors.initials: LL
authors.lastname: Rowlette
authors.forename: David L
authors.initials: DL
authors.lastname: Epstein
authors.forename: Edward T
authors.initials: ET
authors.lastname: O'Brien
authors.forename: Teresa
authors.initials: T
authors.lastname: Borrás
mesh_terms: D000199:Actins / Q000378:metabolism; D000256:Adenoviridae / Q000235:genetics; D000368:Aged; D000369:Aged, 80 and over; D001082:Aqueous Humor / Q000378:metabolism*; D015152:Blotting, Northern; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D009924:Organ Culture Techniques; D051419:Paxillin; D010477:Perfusion; D010750:Phosphoproteins / Q000378:metabolism; D011312:Pressure; D012333:RNA, Messenger / Q000378:metabolism; D014129:Trabecular Meshwork / Q000187:drug effects / Q000378:metabolism*; D062826:Zonula Occludens-1 Protein; D020742:rhoA GTP-Binding Protein / Q000235:genetics* / Q000378:metabolism
chemical_list: D000199:Actins; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D012333:RNA, Messenger; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; D020742:rhoA GTP-Binding Protein