title: Substratum stiffness and latrunculin B regulate matrix gene and protein expression in human trabecular meshwork cells.
abstract: OBJECTIVE
  To determine the impact of substratum stiffness and latrunculin-B (Lat-B), on the expression of several matrix proteins that are associated with glaucoma.
  
  
  METHODS
  Human trabecular meshwork (HTM) cells were cultured on hydrogels possessing stiffness values mimicking those found in normal (5 kPa) and glaucomatous meshworks (75 kPa), or tissue culture polystyrene (TCP; >1 GPa). Cells were treated with 2.0 μM Lat-B in dimethyl sulfoxide (DMSO) or DMSO alone. RT-PCR was used to determine the impact of substratum stiffness and/or Lat-B treatment on the expression of secreted protein, acidic, cysteine rich (SPARC), myocilin, angiopoietin-like factor (ANGPTL)-7, and transglutaminase (TGM)-2. Immunofluorescence was used to assess changes in protein expression.
  
  
  RESULTS
  SPARC and myocilin mRNA expression were dramatically increased on the 75 kPa hydrogels and decreased on the 5 kPa hydrogels in comparison to TCP. In contrast, ANGPTL-7 mRNA and TGM-2 mRNA was decreased on the 75 kPa and 5 kPa hydrogels, respectively, in comparison with TCP. Treatment with Lat-B dramatically downregulated both SPARC and myocilin on 75 kPa hydrogels. In contrast, cells grown on TCP produced greater or similar amounts of SPARC and myocilin mRNA after Lat-B treatment. SPARC and myocilin protein expression paralleled changes in mRNA expression.
  
  
  CONCLUSIONS
  Substratum stiffness impacts HTM matrix gene and protein expression and modulates the impact of Lat-B treatment on the expression of these matrix proteins. Integrating the use of biologically relevant substratum stiffness in the conduction of in vitro experiments gives important insights into HTM cell response to drugs that may more accurately predict responses observed in vivo.
authors.affiliation: Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California-Davis, CA 95616, USA.
authors.forename: Sara M
authors.initials: SM
authors.lastname: Thomasy
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Wood
authors.forename: Philip H
authors.initials: PH
authors.lastname: Kass
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Murphy
authors.forename: Paul
authors.initials: P
authors.lastname: Russell
mesh_terms: D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology*; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000096:biosynthesis / Q000235:genetics*; D005136:Eye Proteins / Q000096:biosynthesis / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects*; D005901:Glaucoma / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D008387:Marine Toxins; D015676:Osteonectin / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053778:Thiazolidines / Q000494:pharmacology*; D014129:Trabecular Meshwork / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D000099179:Myocilin
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D003598:Cytoskeletal Proteins; D005136:Eye Proteins; D006023:Glycoproteins; D008387:Marine Toxins; D015676:Osteonectin; D012333:RNA, Messenger; D053778:Thiazolidines; D000099179:Myocilin; C037068:latrunculin B