Regulation of Plasticity and Fibrogenic Activity of Trabecular Meshwork Cells by Rho GTPase Signaling

Abstract

Glaucoma, a prevalent blinding disease is commonly associated with increased intraocular pressure due to impaired aqueous humor (AH) drainage through the trabecular meshwork (TM). Although increased TM tissue contraction and stiffness in association with accumulation of extracellular matrix (ECM) are believed to be partly responsible for increased resistance to AH outflow, the extracellular cues and intracellular mechanisms regulating TM cell contraction and ECM production are not well defined. This study tested the hypothesis that sustained activation of Rho GTPase signaling induced by lysophosphatidic acid (LPA), TGF-β and connective tissue growth factor (CTGF) influences TM cell plasticity and fibrogenic activity which may eventually impact resistance to AH outflow. Various experiments performed using human TM cells revealed that constitutively active RhoA (RhoAV14), TGF-β2, LPA and CTGF significantly increase the levels and expression of Fibroblast Specific Protein-1 (FSP-1), α-smooth muscle actin (αSMA), collagen-1A1 and secretory total collagen, as determined by q-RT-PCR, immunofluorescence, immunoblot, flow cytometry and the Sircol assay. Significantly, these changes appear to be mediated by Serum Response Factor (SRF), myocardin-related transcription factor (MRTF-A), Slug and Twist-1, which are transcriptional regulators known to control cell plasticity, myofibroblast generation/activation and fibrogenic activity. Additionally, the Rho kinase inhibitor-Y27632 and anti-fibrotic agent-pirfenidone were both found to suppress the TGF-β2-induced expression of αSMA, FSP-1 and collagen-1A1. Taken together, these observations demonstrate the significance of RhoA/Rho kinase signaling in regulation of TM cell plasticity, fibrogenic activity and myofibroblast activation, events with potential implications for the pathobiology of elevated intraocular pressure in glaucoma patients.

FK506-Binding Protein 10, a Potential Novel Drug Target for Idiopathic Pulmonary Fibrosis

“…ctrl = control; siRNA = small interfering RNA. *P , 0.05, **P , 0.01. cells, where collagen secretion was significantly reduced at a concentration of 2.5 mM (37). These concentrations are hardly physiologically relevant, indicating that the well-known antifibrotic effects of pirfenidone in vivo (38) differ substantially in underlying mechanisms or involve metabolic activation.…”

Activation of Prostaglandin FP and EP2 Receptors Differently Modulates Myofibroblast Transition in a Model of Adult Primary Human Trabecular Meshwork Cells

“…1e) in accordance with the TGF-b2-dependent myofibroblastic transition of TM cells. 46,47 In order to assess the acquisition of a contractile phenotype by hTM cells and the effects of PG analogues, the cells were embedded in a collagen type I solution. After gelling, the generated three-dimensional (3D) model of hTM cells, called hTM CPCG, was used as a force-sensing device as previously described.…”

MicroRNA-483-3p Inhibits Extracellular Matrix Production by Targeting Smad4 in Human Trabecular Meshwork Cells

“…Additionally, other pathways can react to TGF-b signaling and induce ECM synthesis through Smad-dependent or -independent pathways, such as PI3K/Akt 35 and p38 MAPK. 33 Transforming growth factor/Smad signaling also plays important role in TM, 5,36 which frequently is overactive in glaucoma. Both Smad and non-Smad signaling pathways participated in TGFb-induced LOX (lysyl oxidase) production, which is partially responsible for elevated IOP.…”