Purpose: Rho-associated protein kinase (ROCK) inhibitors lower intraocular pressure (IOP) by increasing aqueous outflow through the trabecular meshwork (TM). The preclinical characterization of netarsudil, a new ROCK/norepinephrine transporter (NET) inhibitor currently in clinical development, is presented herein. Methods: inhibitory activity netarsudil was compared to its esterase metabolite, netarsudil-M1, and 3 other ROCK using commercially available assay kit. Disruption actin stress...

Elevated intraocular pressure arising from impaired aqueous humor drainage through the trabecular pathway is a major risk factor for glaucoma. To understand molecular basis Rho GTPase-mediated resistance to drainage, we investigated possible interrelationship between actomyosin contractile properties and extracellular matrix (ECM) synthesis in human meshwork (TM) cells expressing constitutively active form of RhoA (RhoAV14). TM RhoAV14 exhibited significant increases fibronectin, tenascin C,...

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 TM tissue contraction and stiffness in association accumulation of extracellular matrix (ECM) are believed be partly responsible for resistance AH outflow, cues intracellular mechanisms regulating cell ECM production not well defined. This study tested hypothesis that sustained activation Rho GTPase...

Purpose: To explore the role of Rho-associated kinases (ROCK) in corneal physiology and regeneration, effects suppressing its activity stimulating endothelial cell proliferation migration vitro vivo. Methods: Immunohistochemistry was performed to detect RhoA ROCK-1 ROCK-2 human tissue. Adult porcine cells (CECs) were isolated, grown confluence, further characterized. Under treatment ROCK inhibitors, changes cellular distribution profile ZO-1 F-actin examined by immunofluorescence staining....

Impaired drainage of aqueous humor through the trabecular meshwork (TM) culminating in increased intraocular pressure is a major risk factor for glaucoma, leading cause blindness worldwide. Regulation TM, however, poorly understood. The role RhoA GTPase-mediated actomyosin organization, cell adhesive interactions, and gene expression regulation outflow was investigated using adenoviral vector-driven constitutively active mutant (RhoAV14). Organ-cultured anterior segments from porcine eyes...

purpose. To investigate the role of lysophospholipid growth factors in regulation aqueous humor outflow trabecular meshwork (TM). methods. The expression profile endothelial differentiation gene (Edg) family G-protein coupled receptors was determined by RT-PCR human TM (HTM) cell–derived total RNA and PCR amplification HTM tissue–derived cDNA libraries. effects lysophosphatidic acid (LPA) sphingosine-1-phosphate (S1P) on actin cytoskeleton focal adhesions myosin light-chain (MLC)...

To investigate the effects of cholesterol-lowering statin drugs on trabecular meshwork cellular properties and aqueous humor outflow.Primary cell cultures porcine (PTM) ciliary body (PCB) were treated with either lovastatin or compactin, to determine statins shape, actin cytoskeletal organization, cell-extracellular matrix interactions (focal adhesions) by immunofluorescence staining. Changes in myosin light-chain (MLC) phosphorylation evaluated Western blot analysis. Rho GTPase content...

To determine changes in the expression profile of RhoA and Rho kinase (ROCK-1 ROCK-2) aqueous humor outflow pathway optic nerve head (ONH) human eyes with or without glaucoma to explore their potential involvement pathophysiology.Age-matched paraffin-embedded postmortem from patients were stained immunohistochemically using polyclonal antibodies raised against RhoA, ROCK-1, ROCK-2. The intensity immunostaining ONH was graded by 4 individuals who masked concerning whether normal those...

Ocular hypertension arising from increased resistance to aqueous humor (AH) outflow through the trabecular meshwork is a primary risk factor for open-angle glaucoma, leading cause of blindness. Ongoing efforts have found little about molecular and cellular bases AH in ocular patients. To test hypothesis that dysregulated Rho GTPase signaling resulting fibrotic activity within may result hypertension, we investigated effects expressing constitutively active RhoA (RhoAV14) pathway...

Purpose: Lysophosphatidic acid (LPA), a bioactive lipid, has been shown to increase resistance aqueous humor outflow (AH) through the trabecular meshwork (TM). The molecular basis for this response of TM LPA, however, is not completely understood. In study, we explored possible involvement mechanosensitive Yes-associated protein (YAP) and its paralog, transcriptional coactivator with PDZ-binding domain (TAZ), activation in extracellular matrix (ECM) production by LPA-induced contractile...

To investigate the specific role of myosin II, a critical biochemical determinant cellular contraction, in modulation aqueous humor outflow facility through trabecular meshwork (TM) pathway.Expression nonmuscle II heavy chains (IIA, IIB, and IIC) human TM ciliary body (CB) cells was determined by RT-PCR analyses. The effects inhibition on cell morphology, actomyosin organization, adhesions were evaluated porcine CB treated with blebbistatin, cell-permeable, inhibitor adenosine triphosphatase...

The goal of this study was to investigate the possible link between actin cytoskeletal integrity and activation matrix metalloproteinases (MMPs) in trabecular meshwork (TM) cells.Primary human TM (HTM) cells treated with different cytoskeleton-interfering agents, including cytochalasin D, latrunculin A, ethacrynic acid (ECA), a Rho kinase inhibitor (Y-27632), H-7 (serine/threonine inhibitor), were examined for changes organization by phalloidin staining, MMP-2 gelatin zymography, expression...

Connective tissue growth factor (CTGF) is a matricellular protein presumed to be involved in the pathobiology of various fibrotic diseases, including glaucoma. We investigated effects Rho GTPase-dependent actin cytoskeletal integrity on CTGF expression and CTGF-induced changes gene profile human trabecular meshwork (HTM) cells.CTGF levels were quantified by immunoblotting ELISA. expression, cytoskeleton, myosin light chain (MLC) phosphorylation, extracellular matrix (ECM) proteins evaluated...

Multidrug, resistance-associated protein-4 (MRP4) is a membrane transporter that regulates the cellular efflux of cyclic nucleotides (cAMP and cGMP) involved in various physiologic responses. This study examined expression distribution MRP4 trabecular meshwork (TM) cells its role homeostasis IOP.

To determine the role and regulation of growth differentiation factor-15 (GDF-15), a TGF-β-related cytokine in human trabecular meshwork (TM) cells context aqueous humor (AH) outflow IOP.Regulation expression by external cues, distribution secretion GDF-15 TM primary cell cultures, effects recombinant (r) on contractile characteristics, actin cytoskeleton, adhesion, extracellular matrix (ECM), α-smooth muscle (αSMA), SMAD signaling, gene were determined immunoblot, immunofluorescence, mass...

Purpose: To explore the role of inducible focal adhesion (FA) protein Hic-5 in actin cytoskeletal reorganization, FA formation, fibrogenic activity, and expression myocilin trabecular meshwork (TM) cells. Methods: Using primary cultures human TM (HTM) cells, effects various external factors on levels, as well recombinant small interfering RNA (siRNA) cytoskeleton, FAs, myocilin, α-smooth muscle (αSMA), collagen-1 were determined by immunofluorescence immunoblot analyses. Results: distributes...