Substratum stiffness and latrunculin B regulate matrix gene and protein expression in human trabecular meshwork cells,” Investigative Ophthalmology

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; Ͼ1 GPa). Cells were treated with 2.0 M 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. (Invest Ophthalmol Vis Sci. 2012;53:952-958) DOI:10.1167/iovs.11-8526 G laucoma is a common cause of blindness worldwide and is associated with irreversible degeneration of the optic nerve head. Although the mechanisms contributing to the onset of most glaucomas are multifactorial, a primary risk factor is increased intraocular pressure (IOP). 1 IOP is likely elevated because of an abnormal increase in resistance to aqueous outflow in the human trabecular meshwork (HTM). 1-3 The HTM is an intricate, three-dimensional structure comprised of extracellular matrix (ECM) possessing intrinsic complex topography and stiffness that supports the overlying trabecular meshwork cells. Many ECM proteins have been implicated in glaucoma including myocilin, transglutaminase (TGM)-2, fibronectin, and angiopoietin-like factor (ANGPTL)-7. 6 Mechanically stretching HTM cells, which occurs with increased IOP, has also been shown to upregulate more than 100 genes including secreted protein, acidic, cysteine rich (SPARC), fibronectin, and myocilin. 3 Substratum elastic modulus has been shown to modulate a variety of fundamental cell behaviors. 3,9 -13 Hydrogels mimicking the elastic modulus of normal and glaucomatous HTM have been shown to dramatically alter cytoskeletal structure and dynamics, protein expression patterns, cell stiffness, cell behavior, and the cellular response to therapeutic agents such as latrunculin-B (Lat-B). 16 Lat-B is thought to decrease IOP by decreasing the resistance to AH outflow through the HTM. 17-21 Recently, it has been demonstrated that HTM cells adhered to stiffer substrates were significantly more responsive to Lat-B due to the increased number of actin stress fibers suggesting that the effects of Lat-B treatment would be most pronounced in glaucomatous eyes with a stiffer HTM. 2 In addition, Lat-B treatment significantly diminished HTM cell migration on stiffer substrates