Substratum stiffness and latrunculin B regulate matrix gene and protein expression in human trabecular meshwork cells

Research output: Contribution to journalArticle

37 Scopus citations

Abstract

PURPOSE. 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.

Original languageEnglish (US)
Pages (from-to)952-958
Number of pages7
JournalInvestigative Ophthalmology and Visual Science
Volume53
Issue number2
DOIs
StatePublished - Feb 2012

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience
  • Medicine(all)

Fingerprint Dive into the research topics of 'Substratum stiffness and latrunculin B regulate matrix gene and protein expression in human trabecular meshwork cells'. Together they form a unique fingerprint.

  • Cite this