Extracellular myocilin affects activity of human trabecular meshwork cells

The trabecular meshwork (TM), a specialized eye tissue, is a major site for regulation of the aqueous humor outflow. Malfunctioning of this tissue is believed to be responsible for development of glaucoma, a blinding disease. Myocilin is a gene linked to the most common form of glaucoma. The protein product has been localized to both intra and extracellular sites, but its function still remains unclear. This study was to determine whether extracellular myocilin presented in the matrix affects adhesion, morphology, and migratory and phagocytic activities of human TM cells in culture. Cell adhesion assays indicated that TM cells, while adhering readily on fibronectin, failed to attach on recombinant myocilin purified from bacterial cultures. Adhesion on fibronectin was also compromised by myocilin in a dose dependent manner. Myocilin in addition triggered TM cells to assume a stellate appearance with broad cell bodies and microspikes. Loss of actin stress fibers and focal adhesions was observed. TM cell migration on fibronectin/myocilin to scratched wounds was reduced compared to fibronectin controls. Myocilin, however, had little impact on phagocytic activities of TM cells. Cell attachment on fibronectin and migration of corneal fibroblasts, a control cell type, were not altered by myocilin. These results demonstrate that extracellular myocilin elicits anti-adhesive and counter-migratory effects on TM cells. Myocilin in the matrix of tissues could be exerting a similar influence on TM cells in vivo, impacting the flexibility and resilience required for maintenance of the normal aqueous outflow.     

Extracellular trafficking of myocilin in human trabecular meshwork cells

Myocilin (MYOC) is a protein with a broad expression pattern, but unknown function. MYOC associates with intracellular structures that are consistent with secretory vesicles, however, in most cell types studied, MYOC is limited to the intracellular compartment. In the trabecular meshwork, MYOC associates with intracellular vesicles, but is also found in the extracellular space. The purpose of the present study was to better understand the mechanism of extracellular transport of MYOC in trabecular meshwork cells. Using a biochemical approach, we found that MYOC localizes intracellularly to both the cytosolic and particulate fractions. When intracellular membranes were separated over a linear sucrose gradient, MYOC equilibrated in a fraction less dense than traditional secretory vesicles and lysosomes. In pulse-labeling experiments that followed nascent MYOC over time, the characteristic doublet observed for MYOC by SDS-PAGE did not change, even in the presence of brefeldin A; indicating that MYOC is not glycosylated and is not released via a traditional secretory mechanism. When conditioned media from human trabecular meshwork cells were examined, both native and recombinant MYOC associated with an extracellular membrane population having biochemical characteristics of exosomes, and containing the major histocompatibility complex class II antigen, HLA-DR. The association of MYOC with exosome-like membranes appeared to be specific, on the extracellular face, and reversible. Taken together, data suggest that MYOC appears in the extracellular space of trabecular meshwork cells by an unconventional mechanism, likely associated with exosome-like vesicles.     

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.     

Caveolin-1 regulates human trabecular meshwork cell adhesion,endocytosis, and autophagy

Impaired trabecular meshwork (TM) outflow is implicated in the pathogenesis of primary open-angle glaucoma (POAG). We previously identified the association of a caveolin-1 (CAV1) variant with POAG by genome-wide association study. Here we report a study of CAV1 knockout (KO) effect on human TM cell properties. We generated human CAV1-KO TM cells by CRISPR/Cas9 technology, and we found that the CAV1-KO TM cells less adhered to the surface coating than the wildtype TM cells by 69.34% ( P < 0.05), but showed no difference in apoptosis. Higher endocytosis ability of dextran and transferrin was also observed in the CAV1-KO TM cells (4.37 and 1.89-fold respectively, P < 0.001), compared to the wildtype TM cells. Moreover, the CAV1-KO TM cells had higher expression of extracellular matrix-degrading enzyme genes ( ADMTS13 and MMP14) as well as autophagy-related genes ( ATG7 and BECN1) and protein (LC3B-II) than the wildtype TM cells. In summary, results from this study showed that the CAV1-KO TM cells have reduced adhesion with higher extracellular matrix-degrading enzyme expression, but increased endocytosis and autophagy activities, indicating that CAV1 could be involved in the regulation of adhesion, endocytosis, and autophagy in human TM cells.     

Proteasome inhibition by chronic oxidative stress in human trabecular meshwork cells

The pathophysiologic mechanisms leading to the malfunction of the trabecular meshwork (TM)-Schlemm's canal (SC) outflow pathway in glaucoma are still unclear. We hypothesize that chronic oxidative stress may contribute to the malfunction of the outflow pathway by impairing the intracellular proteasome system of the cells, decreasing the ability of the tissue to modulate outflow resistance. To study the effects of chronic oxidative stress on proteasome function, primary cultures of human TM cells were incubated under 40% oxygen and proteasome activity was analyzed by measuring the accumulation of enhanced green fluorescent protein fused to a PEST motif. Changes in proteasome content, cellular senescence, and cell viability were also monitored. After 10 days of exposure to chronic oxidative stress, TM cells showed a marked decline in proteasome activity that was associated with premature senescence and decreased cell viability. These results suggest that proteasome failure may be involved in glaucoma pathophysiology.     

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.     

Ultrastructural localization of myocilin in human trabecular meshwork cells and tissues.

We examined ultrastructurally the localization of myocilin (formerly called trabecular meshwork inducible glucocorticoid response, or TIGR) protein in cultured human trabecular meshwork (TM) cells and in normal human TM tissues. The TM, a specialized tissue located at the chamber angle of the eye, is believed to be responsible for the development of glaucoma. The myocilin gene has been directly linked to both juvenile and primary open-angle glaucomas, and multiple mutations have been identified. Human TM cells were treated with 0.1 mM of dexamethasone (DEX) to induce myocilin expression. This protein was immunolocalized by colloidal gold electron microscopy using an anti-human myocilin polyclonal antibody. Double labeling with different sizes of gold particles was also performed with additional monoclonal antibodies specific for cell organelles and structures. In both DEX-treated and untreated cultured cells, myocilin was associated with mitochondria, cytoplasmic filaments, and vesicles. In TM tissues, myocilin was localized to mitochondria and cytoplasmic filaments of TM cells, elastic-like fibers in trabecular beams, and extracellular matrices in the juxtacanalicular region. These results indicate that myocilin is localized both intracellularly and extracellularly at multiple sites. This protein may exert diverse biological functions at different sites.     

Effects of donor age on proteasome activity and senescence in trabecular meshwork cells

The mechanisms involved in the progressive malfunction of the trabecular meshwork (TM) in glaucoma are not yet understood. To study age-related changes in human TM cells, we isolated primary TM cell cultures from young (ages 9, 14, and 25) and old (ages 66, 70, and 73) donors, and compared levels of oxidized proteins, autofluorescence, proteasome function, and markers for cellular senescence. TM cells from old donors showed a 3-fold increase in oxidized proteins and a 7.5-fold decrease of proteasome activity. Loss of proteasome function was not associated with decreased proteasome content but with partial replacement of the proteolytic subunit PSMB5 with the inducible subunit LMP7. Cells from old donors also demonstrated features characteristic of cellular senescence associated with phosphorylation of p38MAPK but only a modest increase in p53. These data suggest that age-related proteasome inhibition and cellular senescence could contribute to the pathophysiological alterations of the TM in glaucoma.     

Protein profile of exosomes from trabecular meshwork cells

To better understand the role of exosomes in the trabecular meshwork (TM), the site of intraocular pressure control, the exosome proteome from primary cultures of human TM cell monolayers was analyzed. Exosomes were purified from urine and conditioned media from primary cultures of human TM cell monolayers and subjected to a two dimensional HPLC separation and MS/MS analyses using the MudPIT strategy. Spectra were searched against a human protein database using Sequest. Protein profiles were compared to each other and the Exocarta database and the presence of specific protein markers confirmed by Western blot analyses of exosomes from aqueous humor and human TM cell strains (n=5) that were untreated, or exposed to dexamethasone and/or ionomycin. TM cell exosomes contained 108 of the 143 most represented exosome proteins in ExoCarta, including previously characterized markers such as membrane organizing and tetraspanin proteins. Several cell-specific proteins in TM exosomes were identified including myocilin, emilin-1 and neuropilin-1. All TM exosome proteins had flotation densities on sucrose gradients and release responses to ionomycin typical for exosomes. Taken together, TM exosomes have a characteristic exosome protein profile plus contain unique proteins, including the glaucoma-causing protein, myocilin; suggesting a role for exosomes in the control of intraocular pressure.     

Stimulation of Na-K-Cl cotransport in cultured vascular endothelial cells by atrial natriuretic peptide

Vascular endothelial cells have been shown to contain atrial natriuretic peptide (ANP)-sensitive Na-K-Cl cotransport system whose activity is regulated by intracellular cGMP levels. Addition of ANP to culture medium stimulated 86Rb+ uptake in bovine endothelial cells with a concomitant increase in cGMP contents. This action of ANP was mimicked by 8-bromo-cGMP and completely diminished by furosemide. These results indicate that ANP selectively activates the Na-K-Cl cotransporter in vascular endothelial cells via cGMP and offer new insight into the physiological significance of endothelial ANP receptors.