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.     

Effects of endothelin-1 and flunarizine on human trabecular meshwork cell contraction

Trabecular meshwork (TM) cells are now considered to play an active role in the aqueous outflow mechanism because they exhibit smooth muscle-like contractile properties. Endothelin-1 (ET-1), a potent vasoconstrictor peptide, has been proposed to play a role in the local regulation of aqueous outflow and intraocular pressure (IOP) control. We propose an in vitro culture model as a method for the study of ET-1-induced human TM (HTM) cell contractility and for the study of whether pre-incubation with flunarizine, a calcium-channel blocker, can inhibit the action of ET-1. Experiments were performed on semiconfluent HTM cells (primary cultures established from normotensive human donor eyes) at the second passage, with phosphate-buffered saline (PBS) as a control. The contractile status of the cells was evaluated by a morphometric analysis of cell area, assuming that HTM cells in culture are able to reduce their area as a consequence of cytoskeletal contraction, rather than regulatory volume decrease. After incubation with 10 microM ET-1 for 5 mins, we observed a reduction of HTM cell area with respect to PBS-treated cells: 2425 +/- 876 microm2 versus 3125 +/- 987 microm2 (P < 0.001); and cells exhibited a retraction in shape and a reduction in number of indented profiles. Administration of ET-1 at progressively lower doses produced a corresponding lower reduction of HTM cell area, suggesting a dose-response effect of ET-1. Pre-incubation with 10 microM flunarizine strongly inhibited the ET-1 effect on HTM cell contraction: 2806 +/- 865 microm2 versus 2910 +/- 846 microm2 (P = not significant). Our data indicate that ET-1 induced a statistically significant reduction in the area of HTM cells versus controls, and that ET-1 can directly influence the aqueous outflow. Moreover, we observed that flunarizine inhibited the effect of ET-1 on the HTM cells.     

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.     

Mitochondrial association of myocilin, product of a glaucoma gene, in human trabecular meshwork cells

The trabecular meshwork (TM), an ocular tissue next to the cornea, 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 major blinding disease. Myocilin is a gene directly linked to the most common form of glaucoma. Its protein product has been localized to both intra- and extra-cellular sites in TM cells. This study was to investigate the association of myocilin with mitochondria in TM cells. In vitro mitochondrial import assays showed that myocilin was imported to the TM mitochondria, targeting to mitochondrial membranes and/or the intermembrane space. The targeting was mediated mostly via the amino-terminal region of myocilin. When myocilin expression was induced either by treatment with dexamethasone or transfection with a myocilin construct, the mitochondrial membrane potential in TM cells, as assessed by JC-1 staining, was lowered. Subcellular fractionation and Western blot analyses confirmed that a portion of myocilin sedimented with the mitochondrial fractions. Upon anti-Fas treatment to provoke apoptosis, an increase of myocilin distribution in cytosolic fraction was observed, suggesting that myocilin was partially released from mitochondrial compartments. These results confirmed the association of myocilin with TM cell mitochondria and indicated that myocilin may have a proapoptotic role in TM cells.     

MTOR-independent induction of autophagy in trabecular meshwork cells subjected to biaxial stretch

The trabecular meshwork (TM) is part of a complex tissue that controls the exit of aqueous humor from the anterior chamber of the eye, and therefore helps maintaining intraocular pressure (IOP). Because of variations in IOP with changing pressure gradients and fluid movement, the TM and its contained cells undergo morphological deformations, resulting in distention and stretching. It is therefore essential for TM cells to continuously detect and respond to these mechanical forces and adapt their physiology to maintain proper cellular function and protect against mechanical injury. Here we demonstrate the activation of autophagy, a pro-survival pathway responsible for the degradation of long-lived proteins and organelles, in TM cells when subjected to biaxial static stretch (20% elongation), as well as in high-pressure perfused eyes (30 mm Hg). Morphological and biochemical markers for autophagy found in the stretched cells include elevated LC3-II levels, increased autophagic flux, and the presence of autophagic figures in electron micrographs. Furthermore, our results indicate that the stretch-induced autophagy in TM cells occurs in an MTOR- and BAG3-independent manner. We hypothesize that activation of autophagy is part of the physiological response that allows TM cells to cope and adapt to mechanical forces.     

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.     

Senescent phenotype of trabecular meshwork cells displays biomarkers in primary open-angle glaucoma

Glaucoma is a major cause of irreversible blindness, affecting more than 70 million individuals worldwide. Elevated intraocular pressure (IOP) is a major risk factor in the development of glaucoma and in the progression of glaucomatous damage. High IOP usually occurs as a result of an increase in aqueous humor outflow resistance in trabecular meshwork (TM). Primary open angle glaucoma (POAG) is characterized by quantifiable parameters including the IOP, the aqueous outflow facility, and geometric measurements of the optic disc and visual defects. Morphological and biochemical analyses of the TM of POAG patients revealed loss of cells, increased accumulation of extracellular matrix (ECM), changes in the cytoskeleton, cellular senescence and the process of subclinical inflammation. Various biochemical and molecular biology biomarkers of TM cells senescence are considered in the article. Oxidative stress is becoming an important factor more likely to be involved in the pathogenesis of POAG. Treatment of TM cells with oxidative stress induced POAG-typical changes like ECM accumulation, cell death, disarrangement of the cytoskeleton, advanced senescence and the release of inflammatory markers. Oxidative stress is able to induce characteristic glaucomatous TM changes and these oxidative stress-induced TM changes can be minimized by the use of antioxidants, such as carnosine-related analogues and IOP-lowering substances. There is evidence demonstrating that carnosine related analogues may have antioxidative capacities, can prevent cellular senescence and the attrition of telomeres during the action of oxidative stress. Prevention of oxidative stress exposure to the TM with N-acetylcarnosine ophthalmic prodrug of carnosine and oral formulation of non-hydrolized carnosine may help to reduce the progression of POAG. The previous work has demonstrated that carnosine is able to reach the TM directly via the transcorneal and systemic pathways of administration with N-acetylcarnosine ophthalmic prodrug and oral formulation of non-hydrolized carnosine. We suggest in this article that dual therapy with N-acetylcarnosine lubricant eye drops, oral formulation of non-hydrolized carnosine combined with anti-glaucoma adrenergic drug may become the first-line therapy in glaucoma due to their efficiency in reducing IOP, prevention and reversal of oxidative stress-induced damages in TM and the low rate of severe side effects during combined treatment.     

Endothelia of Schlemm's canal and trabecular meshwork: distinct molecular, functional, and anatomic features

The purpose of this study was to compare human endothelial cells from Schlemm's canal (SCEs) and the trabecular meshwork (TMEs) in terms of ZO-1 isoform expression, hydraulic conductivity (HC) properties, and "giant" vacuole (GV) formation. The principal study methods were Western blot, RT-PCR, immunofluorescence, and perfusion chambers. Blot signals for ⁺-and ^--isoforms were similar in SCEs but less intense for the ⁺-relative to the ^--signal in TMEs. With the anti-⁺ antibody used at 1/50 dilution, binding occurred at cell borders of both cell types, but only to SCEs when used at a =" BORDER="0">1/200 dilution in vitro and in vivo. SCEs were more resistive than TMEs (HC = 0.66 vs. 1.32 µl·min^-1·mmHg^-1·cm^-2; P < 0.001) when perfused from apex to base. When perfused in the other direction, SCEs were again more resistive (5.23 vs. 9.04 µl·min^-1·mmHg^-1·cm^-2; P < 0.01). GV formation occurred only in SCEs as a function of flow direction, perfusion pressure, and time. We conclude that SCEs and TMEs have distinctive phenotypic properties involving their content of ZO-1 isoforms, barrier function, and GV formation. tight junctions; ZO-1; giant vacuoles; conductivity     

Cytokine profile and cytoskeletal changes after herpes simplex virus type 1 infection in human trabecular meshwork cells

Uveitis caused by herpes simplex virus (HSV)-1 is characterized by increased intraocular pressure (IOP) in the presence of anterior chamber inflammation. Despite their clinical significance, the pathogenic changes associated with HSV-1 infection in trabecular meshwork (TM) cells, the key cell type regulating IOP, have not been completely elucidated. In this study, cytokine array analyses showed a significant stepwise increase in monocyte chemoattractant protein (MCP)-1 expression upon HSV-1 infection in TM cells (p < 0.05). HSV-1 infection led to downregulation of fibrogenic molecules (fibronectin, α-smooth muscle actin, connective tissue growth factor and TGF-β1). Notably, HSV-1 infection caused a significant increase in actin stress fibres, with a twofold increase in active RhoA, which was enhanced by treatment with TGF-β1 and inhibited by treatment with the Rho-kinase inhibitor, Y-27632. TM cells treated with MCP-1 exhibited a dose-dependent increase in actin stress fibres compared to untreated TM cells. Our study suggests that HSV-1 infection in TM cells increases cell contractile activity rather than fibrotic changes in the extracellular matrix (ECM) components. Taken together, these observations demonstrate the enhanced expression of MCP-1 and TM cell contractile activity upon HSV-1 infection and events with potential implications for the pathobiology of abrupt IOP elevation in HSV-1 anterior uveitis.     

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.     

Common actions of adenosine receptor agonists in modulating human trabecular meshwork cell transport

A₁ adenosine receptors (ARs) reduce, and A₂ARs increase intraocular pressure, partly by differentially altering resistance to aqueous humor outflow. It is unknown whether the opposing effects of A₁AR and A₂AR agonists are mediated at different outflow-pathway cell targets or by opposing actions on a single cell target. We tested whether a major outflow-pathway cell, the trabecular meshwork (TM) cell might constitute the primary AR-agonist target and respond differentially to A₁, A_2A and A₃AR agonists. Receptor activation in human TM cells was identified by applying subtype-selective AR agonists: CPA and ADAC for A₁ARs, CGS 21680 and DPMA for A_2AARs, and Cl-IB-MECA and IB-MECA for A₃ARs. Stimulation of A₁, A_2A and A₃ARs elevated Ca²⁺, measured with fura-2. Whole-cell patch clamping indicated that AR agonists activated ion channels non-uniformly, possibly reflecting variability in magnitude of agonist-triggered second-messenger responses. A₁, A_2A and A₃AR agonists all reduced volume, determined by calcein cell imaging. The endogenous source of adenosine delivery to the outflow pathway could be the TM cells since these cells were stimulated to release ATP by hypotonic perfusion. We conclude that: (1) TM cells express functional A₁, A_2A and A₃ARs; and (2) the reported differential effects of AR agonists on aqueous humor outflow are not mediated by differential actions on TM-cell Ca²⁺ and volume, but likely by actions on separate cell targets. Reprint requests should be addressed to: Dr. Mortimer M. Civan, Dept. of Physiology, University of Pennsylvania, Richards Building, Philadelphia, PA 19104-6085. [Tel.: (215)-898-8773; Fax: (215)-573-5851]