Cystatin a, a potential common link for mutant myocilin causative glaucoma

Myocilin (MYOC) is a 504 aa secreted glycoprotein induced by stress factors in the trabecular meshwork tissue of the eye, where it was discovered. Mutations in MYOC are linked to glaucoma. The glaucoma phenotype of each of the different MYOC mutation varies, but all of them cause elevated intraocular pressure (IOP). In cells, forty percent of wild-type MYOC is cleaved by calpain II, a cysteine protease. This proteolytic process is inhibited by MYOC mutants. In this study, we investigated the molecular mechanisms by which MYOC mutants cause glaucoma. We constructed adenoviral vectors with variants Q368X, R342K, D380N, K423E, and overexpressed them in human trabecular meshwork cells. We analyzed expression profiles with Affymetrix U133Plus2 GeneChips using wild-type and null viruses as controls. Analysis of trabecular meshwork relevant mechanisms showed that the unfolded protein response (UPR) was the most affected. Search for individual candidate genes revealed that genes that have been historically connected to trabecular meshwork physiology and pathology were altered by the MYOC mutants. Some of those had known MYOC associations (MMP1, PDIA4, CALR, SFPR1) while others did not (EDN1, MGP, IGF1, TAC1). Some, were top-changed in only one mutant (LOXL1, CYP1B1, FBN1), others followed a mutant group pattern. Some of the genes were new (RAB39B, STC1, CXCL12, CSTA). In particular, one selected gene, the cysteine protease inhibitor cystatin A (CSTA), was commonly induced by all mutants and not by the wild-type. Subsequent functional analysis of the selected gene showed that CSTA was able to reduce wild-type MYOC cleavage in primary trabecular meshwork cells while an inactive mutated CSTA was not. These findings provide a new molecular understanding of the mechanisms of MYOC-causative glaucoma and reveal CSTA, a serum biomarker for cancer, as a potential biomarker and drug for the treatment of MYOC-induced glaucoma.     

Src mediates TGF-β-induced intraocular pressure elevation in glaucoma

Glaucoma, a progressive and irreversible optic neuropathy, is one of the leading causes of vision impairment worldwide. Elevation of intraocular pressure (IOP) due to transforming growth factor-β (TGF-β)-induced dysfunction of the trabecular meshwork is a risk factor for glaucoma, but the underlying molecular mechanisms remain elusive. Here, we show that Src kinase is involved in TGF-β-induced IOP elevation. We observed that dasatinib, a potent Src inhibitor, suppressed TGF-β2-induced IOP in rat eyes. Mechanistic analyses in human trabecular meshwork cells showed that TGF-β2 activated Src signaling and concomitantly increased cytoskeletal remodeling, cell adhesion, and extracellular matrix (ECM) accumulation. Src was activated via TGF-β2-induced upregulation of the Src scaffolding protein CasL, which mediates the assembly of focal adhesions, cytoskeletal remodeling, and ECM deposition. Activation of Src suppressed the expression of tissue plasminogen activator, thereby attenuating ECM degradation. Furthermore, the Src inhibitor ameliorated TGF-β2-induced changes in the contractile and adhesive characteristics of trabecular meshwork cells, and ECM deposition. These findings underscore the crucial role of Src activity in TGF-β-induced IOP elevation and identify Src signaling as a potential therapeutic target in glaucoma.     

Adenovirus conducted connective tissue growth factor on extracellular matrix in trabecular meshwork and its role on aqueous humor outflow facility

Deposition of extracellular matrix (ECM) in trabecular meshwork, such as fibronectin, collagen IV, elastin. leads to increased resistance of trabecular meshwork in primary open angle glaucoma (POAG). Connective tissue growth factor (CTGF) is known to regulate the ECM deposits. In this study, we detect the effect of adenovirus conducted CTGF (Adv-CTGF) transfection on either the expression of ECM components or aqueous humor outflow facility. Adv-CTGF was used to transfect rat trabecular meshwork cells in vivo and in vitro. Aqueous humor outflow facility was test by microbeads perfusion. Protein expression of CTGF, fibronectin, and collagen IV was determined using Western blot. In the Adv-CTGF group, the outflow facility displayed a significant decrease from baseline. It appears as though the transfection with Adv-CTGF significantly affects the aqueous humor outflow pattern. A negative correlation between IOP and PEFL indicated that a decrease in the area of bead deposition corresponded to an overall decrease of outflow, leading to an elevated IOP. Adv-CTGF can enhance the expression of CTGF, fibronectin and collagen IV. CTGF is the novel target for treatment of POAG. It is necessary to further study to test inhibition of CTGF expression for treatment of POAG.     

Autotaxin-lysophosphatidic Acid axis is a novel molecular target for lowering intraocular pressure

Primary open-angle glaucoma is the second leading cause of blindness in the United States and is commonly associated with elevated intraocular pressure (IOP) resulting from diminished aqueous humor (AH) drainage through the trabecular pathway. Developing effective therapies for increased IOP in glaucoma patients requires identification and characterization of molecular mechanisms that regulate IOP and AH outflow. This study describes the identification and role of autotaxin (ATX), a secretory protein and a major source for extracellular lysophosphatidic acid (LPA), in regulation of IOP in a rabbit model. Quantitative proteomics analysis identified ATX as an abundant protein in both human AH derived from non-glaucoma subjects and in AH from different animal species. The lysophospholipase D (LysoPLD) activity of ATX was found to be significantly elevated (by ∼1.8 fold; n = 20) in AH derived from human primary open angle glaucoma patients as compared to AH derived from age-matched cataract control patients. Immunoblotting analysis of conditioned media derived from primary cultures of human trabecular meshwork (HTM) cells has confirmed secretion of ATX and the ability of cyclic mechanical stretch of TM cells to increase the levels of secreted ATX. Topical application of a small molecular chemical inhibitor of ATX (S32826), which inhibited AH LysoPLD activity in vitro (by >90%), led to a dose-dependent and significant decrease of IOP in Dutch-Belted rabbits. Single intracameral injection of S32826 (∼2 µM) led to significant reduction of IOP in rabbits, with the ocular hypotensive response lasting for more than 48 hrs. Suppression of ATX expression in HTM cells using small-interfering RNA (siRNA) caused a decrease in actin stress fibers and myosin light chain phosphorylation. Collectively, these observations indicate that the ATX-LPA axis represents a potential therapeutic target for lowering IOP in glaucoma patients.     

The role of TGF-β in the pathogenesis of primary open-angle glaucoma

Transforming growth factor-β2 (TGF-β2) is found in increasing amounts in aqueous humor and reactive optic nerve astrocytes of patients with primary open-angle glaucoma (POAG), a major cause of blindness worldwide. The available data strongly indicate that TGF-β2 is a key player contributing to the structural changes in the extracellular matrix (ECM) of the trabecular meshwork and optic nerve head as characteristically seen in POAG. The changes involve an induction in the expression of various ECM molecules and are remarkably similar in trabecular meshwork cells and optic nerve head astrocytes. The ECM changes in the trabecular meshwork most probably play a role in the increase of aqueous humor outflow resistance causing higher intraocular pressure (IOP). In the optic nerve head, TGF-β2-induced changes might contribute to deformation of the optic nerve axons causing impairment of axonal transport and neurotrophic supply and leading to their continuous degeneration. The increase in IOP further adds mechanical stress and strain to optic nerve axons and accelerates degenerative changes. In addition, high IOP might induce the expression of activated TGF-β1 in trabecular meshwork cells and optic nerve head astrocytes; this again might significantly lead to the progress of axonal degeneration. The action of TGF-β2 in POAG is largely mediated through the connective tissue growth factor, whereas the activities of TGF-β1 and -β2 are modulated by the blocking effects of bone morphogenetic protein-4 (BMP-4) and BMP-7, by gremlin that inhibits BMP signaling and by several species of microRNAs.     

Myocilin is associated with mitochondria in human trabecular meshwork cells.

The trabecular meshwork (TM) is a specialized tissue located at the chamber angle of the eye next to the cornea. This tissue is believed to be responsible for regulation of the aqueous humor outflow and control of the intraocular pressure (IOP). Alterations in functions of the TM may lead to IOP elevation and development of glaucoma, a major cause of blindness. The myocilin gene has recently been directly linked to open-angle glaucomas. The gene product was originally identified as a protein inducible in TM cells by treatment with glucocorticoids such as dexamethasone (DEX) and termed TIGR (TM inducible-glucocorticoid response). The exact nature and function of the myocilin protein so far still remain elusive. In this study, myocilin was localized to the perinuclear region of both DEX-treated and control TM cells. Its distribution overlapped considerably with that of mitochondria. Subcellular fractionation and Western blot analyses suggested a rather extensive association of myocilin with mitochondria. The DEX-treated TM cells were found to undergo apoptosis, when exposed to anti-Fas antibody, to a significantly higher degree than the untreated control cells. It appears that the TM cell integrity remains intact after DEX treatment. However, the induced myocilin or myocilin-mitochondria association seems to render the cells more susceptible to a second stress or challenge. This vulnerability may be the basis that ultimately leads to pathological consequences.     

Overexpression of myocilin in cultured human trabecular meshwork cells

The trabecular meshwork, a specialized eye tissue, is a major site for regulation of the aqueous humor outflow. Malfunctioning of the trabecular meshwork is believed to be responsible for development of glaucoma, a blinding disease. Myocilin is a gene linked to the most common form of glaucoma. Its expression is known to be upregulated by glucocorticoids in trabecular meshwork cells and the altered myocilin level may be the culprit for glaucomatous conditions such as corticosteroid-induced glaucoma. In this study, we examined the influence of myocilin overexpression on the adhesion, spreading, migration, phagocytosis, and apoptosis of human trabecular meshwork cells in culture. When the myocilin expression was increased by 3- to 4-fold, the transfectants showed a dramatic loss of actin stress fibers and focal adhesions. Cell adhesion to fibronectin and spreading were also compromised. Myocilin thus appeared to have a de-adhesive activity, similar to that reported extensively with matricellular proteins. The transfected cells in addition displayed an increased sensitivity to apoptosis. These results demonstrate that overexpression of myocilin renders trabecular meshwork cells in a de-adhesive and vulnerable state. This vulnerability may be the basis for pathologic consequences in subtypes of glaucoma.     

Schlemm’s Canal and Trabecular Meshwork in Eyes with Primary Open Angle Glaucoma: A Comparative Study Using High-Frequency Ultrasound Biomicroscopy

We investigated in vivo changes in Schlemm’s canal and the trabecular meshwork in eyes with primary open angle glaucoma (POAG). Relationships between Schlemm’s canal diameter, trabecular meshwork thickness, and intraocular pressure (IOP) were examined. Forty POAG patients and 40 normal individuals underwent 80-MHz Ultrasound Biomicroscopy examinations. The Schlemm’s canal and trabecular meshwork were imaged in superior, inferior, nasal and temporal regions. Normal individuals had an observable Schlemm’s canal in 80.3% of sections, a meridional canal diameter of 233.0±34.5 μm, a coronal diameter of 44.5±12.6 μm and a trabecular meshwork thickness of 103.9±11.1 μm, in POAG patients, Schlemm’s canal was observable in 53.1% of sections, a meridional canal diameter of 195.6±31.3 μm, a coronal diameter of 35.7±8.0 μm, and a trabecular meshwork thickness of 88.3±13.2 μm, which significantly differed from normal (both p <0.001). Coronal canal diameter (r = -0.623, p < 0.001) and trabecular meshwork thickness (r = -0.663, p < 0.001) were negatively correlated with IOP, but meridional canal diameter was not (r = -0.160, p = 0.156). Schlemm’s canal was observable in 50.5% and 56.6% of POAG patients with normal (<21 mmHg) and elevated (>21 mmHg) IOP, respectively (χ = 1.159, p = 0.282). Coronal canal diameter was significantly lower in the elevated IOP group (32.6±4.9 μm) than in the normal IOP group (35.7±8.0 μm, p < 0.001). This was also true of trabecular meshwork thickness (81.9±10.0 μm vs. 97.1±12.0 μm, p < 0.001). In conclusion, eyes with POAG had fewer sections with an observable Schlemm’s canal. Canal diameter and trabecular meshwork thickness were also lower than normal in POAG patients. Schlemm’s canal coronal diameter and trabecular meshwork thickness were negatively correlated with IOP.