Simvastatin Attenuates Glucocorticoid-Induced Human Trabecular Meshwork Cell Dysfunction via YAP/TAZ Inactivation

Abstract

Purpose: Impairment of the trabecular meshwork (TM) is the principal cause of increased outflow resistance in the glaucomatous eye. Yes-associated protein (YAP) and transcriptional coactivator with PDZ binding motif (TAZ) are emerging as potential mediators of TM cell/tissue dysfunction. Furthermore, YAP/TAZ activity was recently found to be controlled by the mevalonate pathway in non-ocular cells. Clinically used statins block the mevalonate cascade and were shown to improve TM cell pathobiology; yet, the link to YAP/TAZ signaling was not investigated. In this study, we hypothesized that simvastatin attenuates glucocorticoid-induced human TM (HTM) cell dysfunction via YAP/TAZ inactivation.

Methods: Primary HTM cells were seeded atop or encapsulated within bioengineered extracellular matrix (ECM) hydrogels. Dexamethasone was used to induce a pathologic phenotype in HTM cells in the absence or presence of simvastatin. Changes in YAP/TAZ activity, actin cytoskeletal organization, phospho-myosin light chain levels, hydrogel contraction/stiffness, and fibronectin deposition were assessed.

Results: Simvastatin potently blocked pathologic YAP/TAZ nuclear localization/activity, actin stress fiber formation, and myosin light chain phosphorylation in HTM cells. Importantly, simvastatin co-treatment significantly attenuated dexamethasone-induced ECM contraction/stiffening and fibronectin mRNA and protein levels. Sequential treatment was similarly effective but did not match clinically-used Rho kinase inhibition.

Conclusions: YAP/TAZ inactivation with simvastatin attenuates HTM cell pathobiology in a tissue-mimetic ECM microenvironment. Our data may help explain the association of statin use with a reduced risk of developing glaucoma via indirect YAP/TAZ inhibition as a proposed regulatory mechanism.

Keywords: Mechanotransduction; TM cell pathobiology; cell-ECM interaction; cholesterol; steroid-induced glaucoma.

Fig. 1.. Effects of simvastatin on YAP/TAZ nuclear localization and TGM2 levels in HTM cells.

(A,C,E) Representative fluorescence micrographs of YAP, TAZ, and TGM2 in HTM cells cultured atop ECM hydrogels subjected to vehicle control, dexamethasone (D; 100 nM), simvastatin (S; 10 μM), dexamethasone + simvastatin, and dexamethasone + simvastatin + mevalonate-5-phosphate (M; 500 μM) at 3 d. Arrows indicate YAP/TAZ nuclear localization. Scale bar, 20 μm. (B,D,F) Analysis of YAP/TAZ nuclear/cytoplasmic ratios and TGM2 fluorescence intensity (N = 27-34 images from 3 HTM cell strains with 3 experimental replicates per cell strain). Symbols with different colors represent different cell strains; dotted lines indicate control baselines. The bars and error bars indicate Mean ± SD. Significance was determined by two-way ANOVA using multiple comparisons tests; shared significance indicator letters = non-significant difference (p>0.05), distinct letters = significant difference (p<0.05). (G) Qualitative immunoblot of TGM2 with GAPDH serving as loading control (N = 1 per group [pooled from 3 experimental replicates] from 1 HTM cell strain).

Fig. 2.. Effects of simvastatin on F-actin and αSMA levels in HTM cells.

(A,C) Representative fluorescence micrographs of F-actin and αSMA in HTM cells cultured atop ECM hydrogels subjected to vehicle control, dexamethasone (D; 100 nM), simvastatin (S; 10 μM), dexamethasone + simvastatin, and dexamethasone + simvastatin + mevalonate-5-phosphate (M; 500 μM) at 3 d. Scale bar, 20 μm. (B,D) Analysis of F-actin and αSMA fluorescence intensities (N = 30-34 images from 3 HTM cell strains with 3 experimental replicates per cell strain). Symbols with different colors represent different cell strains; dotted lines indicate control baselines. The bars and error bars indicate Mean ± SD. Significance was determined by two-way ANOVA using multiple comparisons tests; shared significance indicator letters = non-significant difference (p>0.05), distinct letters = significant difference (p<0.05). (E) Qualitative immunoblot of αSMA with GAPDH serving as loading control (N = 1 per group [pooled from 3 experimental replicates] from 1 HTM cell strain).

Fig. 3.. Effect of simvastatin on pMLC levels in HTM cells.

(A) Representative fluorescence micrographs of p-MLC in HTM cells cultured atop ECM hydrogels subjected to vehicle control, dexamethasone (D; 100 nM), simvastatin (S; 10 μM), dexamethasone + simvastatin, and dexamethasone + simvastatin + mevalonate-5-phosphate (M; 500 μM) at 3 d. Scale bar, 20 μm. (B) Analysis of p-MLC fluorescence intensity (N = 29-33 images from 3 HTM cell strains with 3 experimental replicates per cell strain). Symbols with different colors represent different cell strains; dotted line indicates control baseline. The bars and error bars indicate Mean ± SD. Significance was determined by two-way ANOVA using multiple comparisons test; shared significance indicator letters = non-significant difference (p>0.05), distinct letters = significant difference (p<0.05).

Fig. 4.. Effects of simvastatin on HTM hydrogel contraction, stiffness, and FN expression/deposition.

(A) Representative brightfield images of HTM cell-encapsulated ECM hydrogels subjected to vehicle control, dexamethasone (D; 100 nM), simvastatin (S; 10 μM), dexamethasone + simvastatin, and dexamethasone + simvastatin + mevalonate-5-phosphate (M; 500 μM) at 10 d. Dashed lines outline original size of constructs at 0 d. Scale bar, 1 mm. (B) Analysis of HTM hydrogel construct size (N = 13-17 experimental replicates from 3 HTM cell strains). (C) Analysis of HTM hydrogel elastic modulus (N = 6-7 experimental replicates from 2 HTM cell strains). (D) Representative fluorescence micrographs of FN in HTM cell-encapsulated ECM hydrogels. Scale bar, 20 μm. (E) Analysis of FN fluorescence intensity (N = 9 images from 2 HTM cell strains with 2 experimental replicates per cell strain). (F) Analysis of FN mRNA levels (N = 8 experimental replicates from 2 HTM cell strains). Symbols with different colors represent different cell strains; dotted lines indicate control baselines. The bars and error bars indicate Mean ± SD. Significance was determined by two-way ANOVA using multiple comparisons tests; shared significance indicator letters = non-significant difference (p>0.05), distinct letters = significant difference (p<0.05).

Fig. 5.. Effect of simvastatin on HTM hydrogel contraction compared to clinically-used Rho kinase inhibitor.

(A) Representative brightfield images of HTM cell-encapsulated ECM hydrogels subjected to vehicle control, dexamethasone₅ (D₅; 100 nM [0-5 d]; vehicle [5-10 d]), dexamethasone₅ + simvastatin₅ (D₅; [0-5 d]; S₅; 10 μM [5-10 d]), and dexamethasone₅ + netarsudil₅ (D₅; [0-5 d]; N₅; 1.0 μM [5-10 d]) at 10 d. Dashed lines outline original size of constructs at 0 d. Scale bar, 1 mm. (B) Analysis of HTM hydrogel construct size (N = 24 experimental replicates from 3 HTM cell strains). Symbols with different colors represent different cell strains; dotted line indicates control baseline. The bars and error bars indicate Mean ± SD. Significance was determined by two-way ANOVA using multiple comparisons test; shared significance indicator letters = non-significant difference (p>0.05), distinct letters = significant difference (p<0.05).