Mary J. Kelley

Extracellular matrix in the trabecular meshwork

The extracellular matrix (ECM) of the trabecular meshwork (TM) is thought to be important in regulating intraocular pressure (IOP) in both normal and glaucomatous eyes. IOP is regulated primarily by a fluid resistance to aqueous humor outflow. However, neither the exact site nor the identity of the normal resistance to aqueous humor outflow has been established. Whether the site and nature of the increased outflow resistance, which is associated with open-angle glaucoma, is the same or different from the normal resistance is also unclear. The ECMs of the TM beams, juxtacanalicular region (JCT) and Schlemms canal (SC) inner wall are comprised of fibrillar and non-fibrillar collagens, elastin-containing microfibrils, matricellular and structural organizing proteins, glycosaminoglycans (GAGs) and proteoglycans. Both basement membranes and stromal ECM are present in the TM beams and JCT region. Cell adhesion proteins, cell surface ECM receptors and associated binding proteins are also present in the beams, JCT and SC inner wall region. The outflow pathway ECM is relatively dynamic, undergoing constant turnover and remodeling. Regulated changes in enzymes responsible for ECM degradation and biosynthetic replacement are observed. IOP homeostasis, triggered by pressure changes or mechanical stretching of the TM, appears to involve ECM turnover. Several cytokines, growth factors and drugs, which affect the outflow resistance, change ECM component expression, mRNA alternative splicing, cellular cytoskeletal organization or all of these. Changes in ECM associated with open-angle glaucoma have been identified.

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.

Extracellular matrix in the trabecular meshwork: intraocular pressure regulation and dysregulation in glaucoma.

The trabecular meshwork (TM) is located in the anterior segment of the eye and is responsible for regulating the outflow of aqueous humor. Increased resistance to aqueous outflow causes intraocular pressure to increase, which is the primary risk factor for glaucoma. TM cells reside on a series of fenestrated beams and sheets through which the aqueous humor flows to exit the anterior chamber via Schlemms canal. The outer trabecular cells are phagocytic and are thought to function as a pre-filter. However, most of the outflow resistance is thought to be from the extracellular matrix (ECM) of the juxtacanalicular region, the deepest portion of the TM, and from the inner wall basement membrane of Schlemms canal. It is becoming increasingly evident that the extracellular milieu is important in maintaining the integrity of the TM. In glaucoma, not only have ultrastructural changes been observed in the ECM of the TM, and a significant number of mutations in ECM genes been noted, but the stiffness of glaucomatous TM appears to be greater than that of normal tissue. Additionally, TGFβ2 has been found to be elevated in the aqueous humor of glaucoma patients and is assumed to be involved in ECM changes deep with the juxtacanalicular region of the TM. This review summarizes the current literature on trabecular ECM as well as the development and function of the TM. Animal models and organ culture models targeting specific ECM molecules to investigate the mechanisms of glaucoma are described. Finally, the growing number of mutations that have been identified in ECM genes and genes that modulate ECM in humans with glaucoma are documented.

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.

Stem cells in the trabecular meshwork: Present and future promises

Primary open-angle glaucoma is recognized as a disease of aging, and studies show a relationship between aging and trabecular meshwork (TM) cell density. Human TM cell division occurs primarily in the anterior, non-filtering region. A commonly used glaucoma treatment, laser trabeculoplasty (LTP), triggers and increases cell division, as well as cell migration of these anterior TM cells. These freshly-divided migrating cells repopulate the burned laser sites, suggesting that they are stem cells. Several studies concerning this putative TM stem cell will be discussed.

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.

Induced Pluripotent Stem Cells Restore Function in a Human Cell Loss Model of Open‐Angle Glaucoma

Normally, trabecular meshwork (TM) and Schlemms canal inner wall endothelial cells within the aqueous humor outflow pathway maintain intraocular pressure within a narrow safe range. Elevation in intraocular pressure, because of the loss of homeostatic regulation by these outflow pathway cells, is the primary risk factor for vision loss due to glaucomatous optic neuropathy. A notable feature associated with glaucoma is outflow pathway cell loss. Using controlled cell loss in ex vivo perfused human outflow pathway organ culture, we developed compelling experimental evidence that this level of cell loss compromises intraocular pressure homeostatic function. This function was restored by repopulation of the model with fresh TM cells. We then differentiated induced pluripotent stem cells (iPSCs) and used them to repopulate this cell depletion model. These differentiated cells (TM‐like iPSCs) became similar to TM cells in both morphology and expression patterns. When transplanted, they were able to fully restore intraocular pressure homeostatic function. This successful transplantation of TM‐like iPSCs establishes the conceptual feasibility of using autologous stem cells to restore intraocular pressure regulatory function in open‐angle glaucoma patients, providing a novel alternative treatment option. Stem Cells 2015;33:751–761

The effects of tenascin C knockdown on trabecular meshwork outflow resistance

PURPOSE Tenascin C (TNC) is a matricellular glycoprotein whose expression in adult tissue is indicative of tissue remodeling. The purpose of the current study was to determine the localization of TNC in trabecular meshwork (TM) tissue and to analyze the effects of TNC on intraocular pressure (IOP). METHODS Human TM frontal sections were immunostained with anti-TNC and imaged by confocal microscopy. TNC mRNA and protein levels were quantitated in anterior segments perfused at physiological and elevated pressure. Short, hairpin RNA (shRNA) silencing lentivirus targeting full-length TNC (shTNC) was applied to anterior segment perfusion organ cultures. The IOPs and central corneal thickness (CCT) of wild-type, TNC(-/-), and tenascin X (TNX(-/-)) knockout mice were measured. RESULTS TNC was distributed in the juxtacanalicular (JCT) region of adult human TM, predominantly in the basement membrane underlying the inner wall of Schlemms canal. Application of shTNC lentivirus to human and porcine anterior segments in perfusion culture did not significantly affect outflow rate. Although TNC was upregulated in response to pressure, there was no difference in outflow rate when shTNC-silenced anterior segments were subjected to elevated pressure. Furthermore, IOPs and CCTs were not significantly different between TNC(-/-) or TNX(-/-) and wild-type mice. CONCLUSIONS TNC does not appear to contribute directly to outflow resistance. However, TNC immunolocalization in the JCT of adult human eyes suggests that certain areas of the TM are being continuously remodeled with or without an IOP increase.

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.

Induced Pluripotent Stem Cells Restore Function in a Human Cell Loss Model of Open-Angle Glaucoma

The possibilities of novel stem cell therapies to curtail treat, or even cure diseases are enticing, yet safety and functional questions remain prior to clinical use. Currently the stem cell transplantation field for eye diseases is generally defined as being at the pre-clinical or early clinical trials stages. Pre-clinical studies can hopefully eliminate or determine possible transplantation complications prior to human trials. In our manuscript in Stem Cells, we investigated the possibilities of using a personalized cell therapy to treat glaucoma, and in this paper reported two crucial breakthroughs towards this end.