Extracellular matrix gene expression in human trabecular meshwork cells following mechanical fluid flow stimulation

Abstract

Aim: To investigate changes in extracellular matrix (ECM) gene expression in human trabecular meshwork (HTM) cells in response to mechanical fluid flow stimulation.

Methods: HTM cells were grown on a glass plate coated with 0.02% type I collagen (COL) and exposed to shear stress (0, 0.2, 1.0 dyne/cm²) for 12h. Changes in genes related to the ECM were evaluated by real-time reverse transcriptase-polymerase chain reaction. Phosphorylation of Smad2 protein was investigated by Western blotting.

Results: After mechanical stimulation, COL type 4 alpha 2, COL type 6 alpha 1, and fibronectin-1 mRNA were significantly higher than the static control (P<0.05, <0.05, and <0.01, respectively). The metalloproteinase-2 and plasminogen activator inhibitor-1 mRNA were significantly higher than the static control (P<0.05 and <0.01, respectively), while the differences in the tissue inhibitors of metalloproteinases-2 mRNA were not significant. The phosphorylation of Smad2 levels was significantly higher compared to the static control cells.

Conclusion: Changes in the expressions of genes associated ECM metabolism result in HTM cells after mechanical stimulation. The mechanical stimulation of the aqueous humor to the trabecular meshwork may promote ECM turnover and contribute to intraocular pressure homeostasis.

Keywords: aqueous humor; extracellular matrix; glaucoma; intraocular pressure; shear stress; trabecular meshwork.

Figure 1. Characterization of the primary human trabecular meshwork (HTM) cells

A: The HTM cells were treated with 500 nmol/L dexamethasone (DEX) for 7d. Cell lysates are subjected to Western blotting to detect polyclonal myocilin (MYOC) proteins. Quantitative assessment of the intensity of each band was performed by densitometry. The data indicate as the means±SDs (n=5). ^aP<0.01 using the Mann-Whitney U test. B: HTM cells are incubated with pHrodo Escherichia coli bioparticles for 3h (green). Cell nuclei are counterstained by Hoechst 33258 (blue) and observed by fluorescence microscope at 20× magnification (n=4). Scale bar: 100 µmol/L.

Figure 2. Effect of transforming growth factor (TGF)-β2 on fibronectin (FN) and collagen (COL) IV protein expression in human trabecular meshwork (HTM) cells

The HTM cells treated with 5 ng/mL TGF-β2 for 24h and/or 10 µmol/L Y-27632 pretreatment for 30min are stimulated with TGF-β2. Representative images of Western blot analysis of FN (A) and COL IV (B) expression. Quantitative assessment of the intensity of each band was performed by densitometry. The data indicate as the means±SDs (n=5). ^aP<0.05.

Figure 3. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis of collagen (COL) 1A2, COL4A2, COL6A1, and fibronectin (FN) 1 mRNA expression in human trabecular meshwork (HTM) cells

COL4A2, COL6A1, and FN1 mRNA levels in the HTM cells exposed to shear stress (0.2 and 1 dyne/cm²) for 12h. The data indicate as the means±SDs (n=5). ^aP<0.05, ^bP<0.01.

Figure 4. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis of matrix metalloproteinase (MMP)-2 mRNA, tissue inhibitors of metalloproteinases (TIMP)-2, and plasminogen activator inhibitor (PAI)-1 mRNA expression in human trabecular meshwork (HTM) cells

MMP2, TIMP2, and PAI-1 mRNA levels in the HTM cells exposed to shear stress (0.2 and 1 dyne/cm²) for 12h. The data indicate as the means±SDs (n=5). ^aP<0.05, ^bP<0.01.

Figure 5. Phosphorylation of Smad2 in human trabecular meshwork (HTM) cells exposed to shear stress

After 12h of exposure to shear stress (0.2 and 1 dyne/cm²), the phosphorylation of Smad2 expression was examined by Western blotting. Quantitative assessment of the intensity of each band was performed by densitometry. The blots were stripped and re-blotted with anti-Smad2 antibody to demonstrate similar loading of all samples. The data indicate as the means±SDs (n=4). ^aP<0.05.