Phenotypic and Functional Alterations in Tunneling Nanotubes Formed by Glaucomatous Trabecular Meshwork Cells.

Purpose: Trabecular meshwork (TM) cells detect and coordinate responses to intraocular pressure (IOP) in the eye. TM cells become dysfunctional in glaucoma where IOP is often elevated. Recently, we showed that normal TM (NTM) cells communicate by forming tubular connections called tunneling nanotubes (TNTs). Here, we investigated TNTs in glaucomatous TM (GTM) cells. Methods: Primary GTM and NTM cells were established from cadaver eyes. Transfer of Vybrant DiO and DiD-labeled vesicles via TNT connections was measured. Imaris software measured the number and length of cell protrusions from immunofluorescent confocal images. Live-cell imaging of the actin cytoskeleton was performed. The distribution of myosin-X, a regulator of TNTs/filopodia, was investigated in TM cells and tissue. Results: GTM cells contained significantly more transferred fluorescent vesicles than NTM cells (49.6% vs. 35%). Although NTM cells had more protrusions at the cell surface than GTM cells (7.61 vs. 4.65 protrusions/cell), GTM protrusions were significantly longer (12.1 µm vs. 9.76 µm). Live-cell imaging demonstrated that the GTM actin cytoskeleton was less dynamic, and vesicle transfer between cells was significantly slower than NTM cells. Furthermore, rearrangement of the actin cortex adjacent to the TNT may influence TNT formation. Myosin-X immunostaining was punctate and disorganized in GTM cells and tissue compared to age-matched NTM controls. Conclusions: Together, our data demonstrate that GTM cells have phenotypic and functional differences in their TNTs. Significantly slower vesicle transfer via TNTs in GTM cells may delay the timely propagation of cellular signals when pressures become elevated in glaucoma.

Tunneling Nanotubes are Novel Cellular Structures That Communicate Signals Between Trabecular Meshwork Cells.

Purpose: The actin cytoskeleton of trabecular meshwork (TM) cells plays a role in regulating aqueous humor outflow. Many studies have investigated stress fibers, but F-actin also assembles into other supramolecular structures including filopodia. Recently, specialized filopodia called tunneling nanotubes (TNTs) have been described, which communicate molecular signals and organelles directly between cells. Here, we investigate TNT formation by TM cells. Methods: Human TM cells were labeled separately with the fluorescent dyes, DiO and DiD, or with mitochondrial dye. Fixed or live TM cells were imaged using confocal microscopy. Image analysis software was used to track fluorescent vesicles and count the number and length of filopodia. The number of fluorescently labeled vesicles transferred between cells was counted in response to specific inhibitors of the actin cytoskeleton. Human TM tissue was stained with phalloidin. Results: Live-cell confocal imaging of cultured TM cells showed transfer of fluorescently labeled vesicles and mitochondria via TNTs. In TM tissue, a long (160 µm) actin-rich cell process bridged an intertrabecular space and did not adhere to the substratum. Treatment of TM cells with CK-666, an Arp2/3 inhibitor, significantly decreased the number and length of filopodia, decreased transfer of fluorescently labeled vesicles and induced thick stress fibers compared to vehicle control. Conversely, inhibiting stress fibers using Y27632 increased transfer of vesicles and induced long cell processes. Conclusions: Identification of TNTs provides a means by which TM cells can directly communicate with each other over long distances. This may be particularly important to overcome limitations of diffusion-based signaling in the aqueous humor fluid environment.

Mapping molecular differences and extracellular matrix gene expression in segmental outflow pathways of the human ocular trabecular meshwork.

Elevated intraocular pressure (IOP) is the primary risk factor for glaucoma, and lowering IOP remains the only effective treatment for glaucoma. The trabecular meshwork (TM) in the anterior chamber of the eye regulates IOP by generating resistance to aqueous humor outflow. Aqueous humor outflow is segmental, but molecular differences between high and low outflow regions of the TM are poorly understood. In this study, flow regions of the TM were characterized using fluorescent tracers and PCR arrays. Anterior segments from human donor eyes were perfused at physiological pressure in an ex vivo organ culture system. Fluorescently-labeled microspheres of various sizes were perfused into anterior segments to label flow regions. Actively perfused microspheres were segmentally distributed, whereas microspheres soaked passively into anterior segments uniformly labeled the TM and surrounding tissues with no apparent segmentation. Cell-tracker quantum dots (20 nm) were localized to the outer uveal and corneoscleral TM, whereas larger, modified microspheres (200 nm) localized throughout the TM layers and Schlemm's canal. Distribution of fluorescent tracers demonstrated a variable labeling pattern on both a macro- and micro-scale. Quantitative PCR arrays allowed identification of a variety of extracellular matrix genes differentially expressed in high and low flow regions of the TM. Several collagen genes (COL16A1, COL4A2, COL6A1 and 2) and MMPs (1, 2, 3) were enriched in high, whereas COL15A1, and MMP16 were enriched in low flow regions. Matrix metalloproteinase activity was similar in high and low regions using a quantitative FRET peptide assay, whereas protein levels in tissues showed modest regional differences. These gene and protein differences across regions of the TM provide further evidence for a molecular basis of segmental flow routes within the aqueous outflow pathway. New insight into the molecular mechanisms of segmental aqueous outflow may aid in the design and delivery of improved treatments for glaucoma patients.

Differential effects of caveolin-1 and -2 knockdown on aqueous outflow and altered extracellular matrix turnover in caveolin-silenced trabecular meshwork cells.

PURPOSE: A single nucleotide polymorphism (SNP) identified between caveolin-1 (CAV1) and caveolin-2 (CAV2) on chromosome 7 is associated with glaucoma. One function of CAVs is endocytosis and recycling of extracellular matrix (ECM) components. Here, we generated CAV-silencing lentivirus to evaluate the effects on ECM turnover by trabecular meshwork (TM) cells and to measure the effect on outflow facility in anterior segment perfusion culture. METHODS: Short hairpin CAV1 and CAV2 silencing and control lentivirus were generated, characterized, and applied to anterior segments in perfusion culture. Colocalization of CAVs with various ECM molecules in TM cells was investigated using immunofluorescence and confocal microscopy. Western immunoblotting and fluorogenic-based enzyme activity assays were used to investigate ECM protein levels and degradation, respectively. RESULTS: Endogenous CAVs colocalized with cortactin at podosome- or invadopodia-like structures (PILS), which are areas of focal ECM degradation. In perfusion culture, outflow rates increased significantly in CAV1-silenced anterior segments, whereas outflow significantly decreased in CAV2-silenced anterior segments. Matrix metalloproteinase (MMP)2 and MMP14, and a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS4) colocalized with both CAVs in TM cells. Protein levels and enzyme activities of MMP/ADAMTS4, fibronectin protein levels, actin stress fibers, and α-smooth muscle actin were all increased in CAV-silenced cells. CONCLUSIONS: Caveolin-mediated endocytosis is one mechanism by which TM cells can alter the physiological catabolism of ECM in order to change the composition of the outflow channels in the TM to regulate aqueous outflow resistance. Dysregulation of CAV function could contribute to the pathological changes in ECM that are observed in glaucoma.

Intraocular pressure homeostasis: maintaining balance in a high-pressure environment.

Although glaucoma is a relatively common blinding disease, most people do not develop glaucoma. A robust intraocular pressure (IOP) homeostatic mechanism keeps ocular pressures within relatively narrow acceptable bounds throughout most peoples' lives. The trabecular meshwork and/or Schlemm's canal inner wall cells respond to sustained IOP elevation and adjust the aqueous humor outflow resistance to restore IOP to acceptable levels. It appears that the cells sense IOP elevations as mechanical stretch or distortion of the actual outflow resistance and respond by initiating a complex extracellular matrix (ECM) turnover process that takes several days to complete. Although considerable information pertinent to this process is available, many aspects of the IOP homeostatic process remain to be elucidated. Components and mechanisms beyond ECM turnover could also be relevant to IOP homeostasis, but will not be addressed in detail here. Known aspects of the IOP homeostasis process as well as possible ways that it might function and impact glaucoma are discussed.

Perturbation of hyaluronan synthesis in the trabecular meshwork and the effects on outflow facility.

PURPOSE: Hyaluronan (HA) is a major component of the aqueous outflow pathway. However, the contribution of HA to human outflow resistance remains unclear. Three HA synthase genes (HAS1-3) have been identified. Here, we evaluate the contribution of each of the HAS proteins to outflow facility in anterior segment perfusion culture. METHODS: Two methods were used to reduce HA synthesis: 1 mM 4-methylumbelliferone (4MU) was used to inhibit all HAS synthases and shRNA silencing lentivirus was generated to knock down expression of each HAS individually. Quantitative RT-PCR, Western immunoblotting and an HA ELISA assay were used to assess HAS mRNA and protein levels and HA concentration, respectively. The effects of 4MU treatment and HAS gene silencing on outflow facility were assessed in human and porcine perfusion culture. RESULTS: Quantitative RT-PCR and Western immunoblotting showed a reduction of each HAS in response to their respective silencing and 4MU treatment. HA concentration was concomitantly reduced. Treatment with 4MU decreased outflow facility in human anterior segments but increased outflow facility in porcine eyes. Lentiviral delivery of HAS1 and HAS2 silencing vectors caused similar opposite effects on outflow facility. Silencing of HAS3 did not significantly affect outflow resistance in either species. CONCLUSIONS: This is the first conclusive evidence for a significant role of HA in the human outflow pathway. HA chains synthesized by HAS1 and HAS2 contribute to outflow resistance, while hyaluronan produced by HAS3 does not appear to play a significant role.

Molecular chaperone function for myocilin.

PURPOSE: Myocilin is thought to be a stress response protein, but its exact molecular functions have not been established. Studies were conducted to see whether myocilin can act as a general molecular chaperone. METHODS: Myocilin was isolated and purified from porcine trabecular meshwork (TM) cell culture media. Its ability to protect citrate synthase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the restriction endonuclease DrdI from thermal inactivation was evaluated. Light scattering was used to evaluate thermally induced aggregation of citrate synthase. Myocilin induction was assessed after exposure of TM cells to several types of stress treatments. RESULTS: Levels of extracellular myocilin expressed by TM cells were increased in response to mechanical stretch, heat shock, TNFα, or IL-1α. Myocilin protected citrate synthase activity against thermal inactivation for 5 minutes at 55°C in a concentration-dependent manner, with nearly full protection of 1.5 µM citrate synthase in the presence of 650 nM myocilin. Myocilin significantly reduced thermal aggregation of citrate synthase to levels 36% to 44% of control levels. Myocilin also protected GAPDH from thermal inactivation for 10 minutes at 45°C. Myocilin at 18 nM was more effective than 1 µM bovine serum albumin at protecting DrdI from thermal inactivation. CONCLUSIONS: Myocilin is induced in response to several cellular stresses and displays general molecular chaperone activity by protecting DrdI, citrate synthase, and GAPDH from thermal inactivation. Myocilin also suppresses the thermal aggregation of citrate synthase. One function of myocilin may be to serve as a molecular chaperone.

Segmental versican expression in the trabecular meshwork and involvement in outflow facility.

PURPOSE: Versican is a large proteoglycan with numerous chondroitin sulfate (CS) glycosaminoglycan (GAG) side chains attached. To assess versican's potential contributions to aqueous humor outflow resistance, its segmental distribution in the trabecular meshwork (TM) and the effect on outflow facility of silencing the versican gene were evaluated. METHODS: Fluorescent quantum dots (Qdots) were perfused to label outflow pathways of anterior segments. Immunofluorescence with confocal microscopy and quantitative RT-PCR were used to determine versican protein and mRNA distribution relative to Qdot-labeled regions. Lentiviral delivery of shRNA-silencing cassettes to TM cells in perfused anterior segment cultures was used to evaluate the involvement of versican and CS GAG chains in outflow facility. RESULTS: Qdot uptake by TM cells showed considerable segmental variability in both human and porcine outflow pathways. Regional levels of Qdot labeling were inversely related to versican protein and mRNA levels; versican levels were relatively high in sparsely Qdot-labeled regions and low in densely labeled regions. Versican silencing decreased outflow facility in human and increased facility in porcine anterior segments. However, RNAi silencing of ChGn, an enzyme unique to CS GAG biosynthesis, increased outflow facility in both species. The fibrillar pattern of versican immunostaining in the TM juxtacanalicular region was disrupted after versican silencing in perfusion culture. CONCLUSIONS: Versican appears to be a central component of the outflow resistance, where it may organize GAGs and other ECM components to facilitate and control open flow channels in the TM. However, the exact molecular organization of this resistance appears to differ between human and porcine eyes.

Differential effects of ADAMTS-1, -4, and -5 in the trabecular meshwork.

PURPOSE: Matrix metalloproteinases (MMPs) degrade extracellular matrix (ECM) and increase outflow facility in anterior segment perfusion culture. One group is the ADAMTSs (a disintegrin and metalloproteinase with thrombospondin type 1 motifs). In this study, the authors examined the effects of ADAMTS-1, -4, and -5 on outflow facility and investigated their mRNA levels and protein expression in the trabecular meshwork (TM). METHODS: ADAMTS mRNA was quantitated by qRT-PCR in TM cells exposed to TNFalpha, IL-1alpha, TGFbeta2, or mechanical stretch. ADAMTS-4 mRNA was assessed in normal and glaucomatous human anterior segments perfused at physiological or elevated pressure. Immunofluorescence was used to localize ADAMTSs in human TM cells and tissue. Anterior segments in perfusion culture were treated with recombinant ADAMTSs to determine effects on outflow facility. RESULTS: Cytokine treatment increased mRNA of all three ADAMTSs. Mechanical stretch increased ADAMTS-4 mRNA but conversely decreased ADAMTS-1 and -5 mRNA. ADAMTS-4 mRNA levels increased in response to pressure elevation in normal eyes and to higher levels in glaucomatous eyes. ADAMTS-4 protein was highly increased in the juxtacanalicular region of the TM in anterior segments perfused at increased pressure. In human TM cells, ADAMTS-4 colocalized with cortactin in podosome- or invadopodia-like structures, but ADAMTS-1 and -5 did not. Recombinant ADAMTS-4 increased outflow facility in human and porcine anterior segments, whereas recombinant ADAMTSs-1 and -5 did not. CONCLUSIONS: These results show differential responses and expression of ADAMTS-1, -4, and -5 in human TM cells. Combined, these results suggest that ADAMTS-4 is a potential modifier of outflow facility.

Extracellular matrix turnover and outflow resistance.

Normal homeostatic adjustment of elevated intraocular pressure (IOP) involves remodeling the extracellular matrix (ECM) of the trabecular meshwork (TM). This entails sensing elevated IOP, releasing numerous activated proteinases to degrade existing ECM and concurrent biosynthesis of replacement ECM components. To increase or decrease IOP, the quantity, physical properties and/or organization of new components should be somewhat different from those replaced in order to modify outflow resistance. ECM degradation and replacement biosynthesis in the outflow pathway must be tightly controlled and focused to retain the complex structural organization of the tissue. Recently identified podosome- or invadopodia-like structures (PILS) may aid in the focal degradation of ECM and organization of replacement components.

Specialized podosome- or invadopodia-like structures (PILS) for focal trabecular meshwork extracellular matrix turnover.

PURPOSE: There are distinctive areas of colocalization of matrix metalloproteinase (MMP)-2 and -14 on trabecular meshwork (TM) cells that resemble podosomes or invadopodia. Studies were conducted to determine whether TM cells exhibit podosome- or invadopodia-like structures (PILS) and whether they produce focal extracellular matrix (ECM) turnover. METHODS: Porcine and human TM cells and perfused anterior segment organ cultures were studied. Localization of PILS components on TM cells and in sections from anterior segments was determined by immunohistochemistry and confocal microscopy. Cells were grown on type I collagen labeled with fluorescein isothiocyanate (FITC) for degradation analysis. Confocal time lapse images were taken of labeled TM cells on FITC-collagen. RESULTS: Immunostaining for MMP-2, MMP-14, and the typical PILS components cortactin, caldesmon, alpha-actinin, N-WASP, Arp-3, and cdc42 colocalized on these distinctive structures. Integrin-alphaV and -beta1, fibronectin, and versican colocalized with PILS components. TM cells on FITC-conjugated collagen developed focal regions of degradation. Time-lapse imaging showed dramatic and controlled movement of TM cell processes during this ECM degradation and fragment internalization. MMP-2, MMP-14, and cortactin colocalized at regions that appear to be PILS on cells within the outflow pathway in sections of human anterior segments. CONCLUSIONS: TM cells exhibit areas where PILS components colocalize with MMP-2 and -14. Similar structures are found in sections, suggesting that PILS occur in situ in the outflow pathway. The collagen degradation suggests that PILS may serve as focal sites for targeted ECM turnover, an event linked to modifications of aqueous outflow resistance and intraocular pressure homeostasis.

Effects of modifiers of glycosaminoglycan biosynthesis on outflow facility in perfusion culture.

PURPOSE: Glycosaminoglycans (GAGs) have been implicated in the regulation of outflow resistance of aqueous humor flow through the trabecular meshwork (TM). Their role was further investigated by assessment of the effects of chlorate, an inhibitor of sulfation, and beta-xyloside, which provides a competitive nucleation point for addition of disaccharide units, in anterior segment perfusion culture. METHODS: Outflow facility was measured in perfused porcine and human anterior organ cultures treated with 20 or 50 mM sodium chlorate, or 1 mM beta-xyloside. Perturbation of extracellular matrix (ECM) components was assessed in paraffin-embedded sections by immunofluorescence and confocal microscopy. Parallel experiments were conducted on cultured TM cells. RESULTS: Outflow facility increased in porcine eyes with chlorate (3-fold) and beta-xyloside (3.5-fold) treatments. In human eyes, outflow increased approximately 1.5-fold and took longer (>48 hours) to occur. By confocal microscopy, immunostaining for chondroitin and heparan sulfates was observed on edges of human TM beams in nontreated eyes, with intense staining in the juxtacanalicular tissue (JCT) region. In treated eyes, staining of beam edges was severely reduced and was instead found in plaques. Chlorate treatment resulted in a striated pattern of GAG staining in the human JCT region. Fibronectin immunostaining was altered in beta-xyloside-treated eyes, whereas in cell culture, chlorate induced formation of thick fibronectin fibrils, to which tenascin C colocalized. CONCLUSIONS: Disrupting GAG chain biosynthesis increased outflow facility in perfusion culture and induced atypical ECM molecule interactions in cell culture. This study provides direct evidence of the critical role of GAG chains in regulating outflow resistance in human TM.

Synergism of TNF and IL-1 in the induction of matrix metalloproteinase-3 in trabecular meshwork.

PURPOSE: TNF and IL-1 increase matrix metalloproteinase-3 (MMP-3) expression in the trabecular meshwork (TM). TNF-alpha, in combination with IL-1alpha or IL-1beta, produces highly synergistic MMP-3 increases. Possible mechanisms for this synergism in TM cells were investigated. METHODS: Porcine and human TM cells were treated with TNF-alpha, IL-1alpha, IL-1beta and their combinations. Western immunoblots were used to evaluate MMP-3, MMP-9, MMP-12, TNF-alpha, IL-1alpha, IL-1beta, IL-6, TNF receptor I (RI), IL-1 RI, and IL-1 RII levels and the phosphorylation of Erk, JNK, and p38 MAP kinases. Dose-response effects for TNF-alpha, IL-1alpha and IL-1beta on MMP-3 were evaluated. Microarray and quantitative RT-PCR were used to determine mRNA levels. MMP-3 transcription rate was assessed by transfecting TM cells with an MMP-3 promoter/reporter construct. Combined cytokine effects on outflow facility were appraised in perfused anterior segment organ culture. RESULTS: TNF-alpha, IL-1alpha, and IL-1beta each individually increased MMP-3 levels, whereas TNF-alpha in combination with IL-1alpha or IL-1beta produced highly synergistic increases. MMP-9 and MMP-12 levels were also elevated, but only MMP-12 showed synergism. IL-1alpha, IL-1beta, and IL-6, but not TNF-alpha mRNA or protein level, were elevated by these cytokines. Maximum MMP-3 production for individual cytokines, even at high doses, was far less than with dual cytokine doses. Erk 1 and 2, JNK 1 and 2, and p38 alpha and beta phosphorylation increased, but not synergistically. However, phosphorylation of novel isoforms of JNK and p38 delta and gamma did show synergism. MMP-3 mRNA levels and transcription rates also demonstrated synergism. TNF-alpha significantly increased IL-1 RI levels. Synergism in outflow facility was observed with TNF-alpha and IL-1alpha. CONCLUSIONS: TNF-alpha, in combination with IL-1alpha or IL-1beta, produced intense synergistic increases in MMP-3 and MMP-12 but not in MMP-9. Induction of IL-1 RI by TNF-alpha partially explains the synergism. Responses of novel JNK and p38 MAP kinase delta and gamma isoforms also partially account for the synergism. Understanding this strong synergistic effect may provide useful insight into optimizing therapeutic regulation of intraocular pressure in glaucoma.

Signaling pathways used in trabecular matrix metalloproteinase response to mechanical stretch.

PURPOSE: Trabecular meshwork (TM) matrix metalloproteinase (MMP), and tissue inhibitor (TIMP) changes in response to mechanical stretching appear to be central to intraocular pressure (IOP) homeostasis. Studies were conducted to define the signal transduction pathway responsible for the increases in MMP-2 and -14 that occur in response to mechanical stretching of TM cells. METHODS: Porcine TM cells were subjected to mechanical stretching, and changes in MMP-2 and -14 levels were determined by gelatin zymography and Western immunoblot analysis. Effects of signal transduction pathway inhibitors on MMP levels were analyzed. Phosphospecific antibodies were used to identify phosphorylation changes in select pathway intermediates. In silico secondary structure analysis was conducted on the 5' untranslated regions (UTRs) of MMP-2 and -14 mRNAs. RESULTS: The increases in MMP-2 and -14 that occur 24 hours after sustained mechanical stretching of TM cells were blocked by rapamycin. Wortmannin blocked the MMP-2 but not the MMP-14 increase. Protein kinase B (PKB) phosphorylation on S473 and T308 was increased significantly by stretching. Rapamycin-sensitive phosphorylation of T389 in p70/p85 S6 kinase was also increased. The phosphorylations of the translation initiation factor eIF-4E on S209 and of its inhibitory binding protein 4E-BP1 on T70 were both increased by stretch. The calculated free energies of secondary structures of the 5' UTRs of the mRNAs for MMP-2 and -14 were negative and relatively large. MMP-2 also had pyrimidine tracts in the extreme 5' region of its UTR. CONCLUSIONS: The increases in TM MMP-2 and -14 protein levels in response to mechanical stretching appear to be transduced at least in part by mTOR, the mammalian target of rapamycin (mTOR). The wortmannin sensitivity implicates phosphoinositide 3-kinase as a modulator of the MMP-2 but not the MMP-14 increase. Integrin-linked kinase (ILK), phosphoinositide-dependent kinase (PDK-1), and PKB are implicated in the MMP-2 increase. Translational initiation involving eIF-4E and its inhibitory binding protein 4E-BP1 appear to be involved in both the MMP-2 and -14 increases with stretching and are normally regulated by mTOR. The high degree of secondary structure in the 5' UTRs of these transcripts is typically an indicator of genes specifically sensitive to regulation through this pathway. P70/p85 S6 kinase is probably involved downstream from mTOR and PKB in regulating translation of MMP-2, which has pyrimidine tracts in its 5' UTR. Manipulation of these transduction pathways may provide new approaches to therapeutic IOP regulation.