Matrix Mechanotransduction via Yes-Associated Protein in Human Lamina Cribrosa Cells in Glaucoma

Abstract

Purpose Extracellular matrix stiffening is characteristic of both aging and glaucoma, and acts as a promoter and perpetuator of pathological fibrotic remodeling. Here, we investigate the role of a mechanosensitive transcriptional coactivator, Yes-associated protein (YAP), a downstream effector of multiple signaling pathways, in lamina cribrosa (LC) cell activation to a profibrotic, glaucomatous state. Methods LC cells isolated from glaucomatous human donor eyes (GLC; n = 3) were compared to LC cells from age-matched nonglaucomatous controls (NLC; n = 3) to determine differential YAP expression, protein levels, and proliferation rates. NLC cells were then cultured on soft (4 kPa), and stiff (100 kPa), collagen-1 coated polyacrylamide hydrogel substrates. Quantitative real-time RT-PCR, immunoblotting, and immunofluorescence microscopy were used to measure the expression, activity, and subcellular location of YAP and its downstream targets, respectively. Proliferation rates were examined in NLC and GLC cells by methyl thiazolyl tetrazolium salt assays, across a range of incrementally increased substrate stiffness. Endpoints were examined in the presence or absence of a YAP inhibitor, verteporfin (2 µM). Results GLC cells show significantly ( P < 0.05) increased YAP gene expression and total-YAP protein compared to NLC cells, with significantly increased proliferation. YAP regulation is mechanosensitive, because NLC cells cultured on pathomimetic, stiff substrates (100 kPa) show significantly upregulated YAP gene and protein expression, increased YAP phosphorylation at tyrosine 357, reduced YAP phosphorylation at serine 127, increased nuclear pooling, and increased transcriptional target, connective tissue growth factor. Accordingly, myofibroblastic markers, α-smooth muscle actin (α-SMA) and collagen type I, alpha 1 (Col1A1) are increased. Proliferation rates are elevated on 50 kPa substrates and tissue culture plastic. Verteporfin treatment significantly inhibits YAP-mediated cellular activation and proliferation despite a stiffened microenvironment. Conclusions These data demonstrate how YAP plays a pivotal role in LC cells adopting a profibrotic and proliferative phenotype in response to the stiffened LC present in aging and glaucoma. YAP provides an attractive and novel therapeutic target, and its inhibition via verteporfin warrants further clinical investigation.

YAP/TAZ mediate TGFβ2-induced Schlemm’s canal cell dysfunction

“…By contrast, Lat B-mediated actin depolymerization failed to reduce αSMA-positive cells. Importantly, we found that TGFβ2-induced EndMT in HSC cells was potently blocked by pharmacologic YAP/TAZ inhibition with verteporfin in absence of light stimulation consistent with previous reports in non-ocular 65–67 and ocular cells 51,68,69 ; significantly decreased fibronectin and actin stress fibers were noted ( Fig. 5A-D ).…”

“…Consistent with this, we showed that TGFβ2 enhanced EndMT of HSC cells cultured atop tissue-mimetic ECM hydrogels, as indicated by increased formation of fibronectin and αSMA (Fig. 2D,E Importantly, we found that TGFβ2-induced EndMT in HSC cells was potently blocked by pharmacologic YAP/TAZ inhibition with verteporfin in absence of light stimulation consistent with previous reports in non-ocular [65][66][67] and ocular cells 51,68,69 ; significantly decreased fibronectin and actin stress fibers were noted (Fig. 5A-D).…”

YAP/TAZ Mediate TGFβ2-Induced Schlemm's Canal Cell Dysfunction

“…By contrast, Lat B-mediated actin depolymerization failed to reduce αSMA-positive cells. Importantly, we found that TGFβ2-induced EndMT in HSC cells was potently blocked by pharmacological YAP/TAZ inhibition with verteporfin in absence of light stimulation consistent with previous reports in nonocular 65 – 67 and ocular cells 51 , 68 , 69 ; significantly decreased fibronectin and actin stress fibers were noted ( Figs. 5 A– 5 D).…”

Genome-Wide RNA Sequencing of Human Trabecular Meshwork Cells Treated with TGF-β1: Relevance to Pseudoexfoliation Glaucoma

“…Hippo signalling is a therapeutic target in cancer biology and inhibitors of YAP/TAZ, the downstream effectors of the Hippo pathway are attractive targets [ 103 , 104 ]. The inhibition of YAP with verteporfin in lamina cribrosa cells showed therapeutic benefits [ 105 ]. ER stress and the unfolded protein response (UPR) have also gained interest in glaucoma and ophthalmology as therapeutic targets [ 106 , 107 , 108 ].…”