Consensus Recommendation for Mouse Models of Ocular Hypertension to Study Aqueous Humor Outflow and Its Mechanisms.

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.

GLIS1 regulates trabecular meshwork function and intraocular pressure and is associated with glaucoma in humans.

Chronically elevated intraocular pressure (IOP) is the major risk factor of primary open-angle glaucoma, a leading cause of blindness. Dysfunction of the trabecular meshwork (TM), which controls the outflow of aqueous humor (AqH) from the anterior chamber, is the major cause of elevated IOP. Here, we demonstrate that mice deficient in the Krüppel-like zinc finger transcriptional factor GLI-similar-1 (GLIS1) develop chronically elevated IOP. Magnetic resonance imaging and histopathological analysis reveal that deficiency in GLIS1 expression induces progressive degeneration of the TM, leading to inefficient AqH drainage from the anterior chamber and elevated IOP. Transcriptome and cistrome analyses identified several glaucoma- and extracellular matrix-associated genes as direct transcriptional targets of GLIS1. We also identified a significant association between GLIS1 variant rs941125 and glaucoma in humans (P = 4.73 × 10-6), further supporting a role for GLIS1 into glaucoma etiology. Our study identifies GLIS1 as a critical regulator of TM function and maintenance, AqH dynamics, and IOP.

High-resolution ocular imaging: combining advanced optics and microtechnology.

Recent developments in imaging technologies offer great potential for the assessment of retinal ganglion cell disorders, with particular relevance to glaucoma. In particular, advances in this field have allowed unprecedented in vivo access to the retinal layers, using many different properties of light to differentiate cellular structures. This article is a summary of currently available and investigational advanced, high-resolution imaging technologies and their potential applications to glaucoma. It represents the topics of discussion at the annual Optic Nerve Rescue and Restoration Think Tank, sponsored by The Glaucoma Foundation, entitled "High Resolution Imaging of the Eye: Advanced Optics, Microtechnology and Nanotechnology" and held in New York, New York, September 28-29, 2007.

Accuracy of Tonopen Versus iCare in Human Cadaveric Eyes With Edematous Corneas Over a Wide Range of Intraocular Pressures.

PURPOSE: The purpose of this study was to compare the accuracy of 2 portable tonometers, Tonopen and iCare, in extreme corneal edema, while also accounting for the location of pressure measurement over a wide range of intraocular pressure (IOP). METHODS: Utilizing an experimental laboratory investigation, IOP measurements were obtained with iCare and Tonopen against an internal digital manometer in 2 enucleated human cadaveric eyes with edematous corneas. Pachymetry was used to measure central corneal thickness in each eye. Each eye was then mounted on an upright-positioned mannequin, and a 30-G needle connected to a digital manometer was introduced into the anterior chamber at the limbus. Pressures were maintained at 0, 10, 20, 30, 40, and 50 mmHg using the digital manometer. Three measurements were taken with the tonometers at the central cornea, limbal cornea, and sclera, for a total of 9 measurements per device per eye at each manometric pressure reading. RESULTS: Comparing the mean absolute error of Tonopen versus iCare measurements at the central cornea for each manometric pressure reading, both devices underestimated the control at pressures >10 mmHg, with mean error increasing as IOP increased [mean absolute error (MAE) ranged from 1.67 to 13.33 with Tonopen and 3.67 to 21.00 with iCare]. Tonopen more accurately estimated pressure with an average MAE of 5.72 at the central cornea, compared with MAE of 10.83 at the central cornea for iCare (P<0.03). Comparing MAE between the devices at the limbus, there was no significant difference between the 2 (P=0.91), with average MAE for the Tonopen 5.47 and average MAE for the iCare 5.22. There was no significant difference between average MAE for Tonopen measurements at the central cornea versus the limbus (P=0.89), while the iCare was more accurate at the limbus with average MAE of 5.22 than the central cornea with average MAE of 10.83 (P<0.03). When measuring at the sclera, there was no statistically significant difference between average MAE for Tonopen (18.03) versus iCare (10.39) (P=0.24). DISCUSSION: Tonopen was more accurate than iCare when measuring at the central cornea, but consistently underestimated the control. MAE for both devices increased linearly with increasing true IOP. With limbus measurements in an edematous cornea, Tonopen and iCare performed similarly. The iCare performed better at the limbus than at the central cornea. Scleral measurements for both Tonopen and iCare were poor. In this experimental model, Tonopen performed superiorly to iCare with central cornea measurements in a grossly edematous cornea. Both devices underestimated true IOP.

Exfoliation syndrome: assembling the puzzle pieces.

PURPOSE: To summarize various topics and the cutting edge approaches to refine XFS pathogenesis that were discussed at the 21st annual Glaucoma Foundation Think Tank meeting in New York City, Sept. 19-20, 2014. METHODS: The highlights of three categories of talks on cutting edge research in the field were summarized. RESULTS: Exfoliation syndrome (XFS) is a systemic disorder with a substantial ocular burden, including high rates of cataract, cataract surgery complications, glaucoma and retinal vein occlusion. New information about XFS is akin to puzzle pieces that do not quite join together to reveal a clear picture regarding how exfoliation material (XFM) forms. CONCLUSION: Meeting participants concluded that it is unclear how the mild homocysteinemia seen in XFS might contribute to the disarrayed extracellular aggregates characteristic of this syndrome. Lysyl oxidase-like 1 (LOXL1) variants are unequivocally genetic risk factors for XFS but exactly how these variants contribute to the assembly of exfoliation material (XFM) remains unclear. Variants in a new genomic region, CACNA1A associated with XFS, may alter calcium concentrations at the cell surface and facilitate XFM formation but much more work is needed before we can place this new finding in proper context. It is hoped that various animal model and ex vivo systems will emerge that will allow for proper assembly of the puzzle pieces into a coherent picture of XFS pathogenesis. A clear understanding of XFS pathogenesis may lead to 'upstream solutions' to reduce the ocular morbidity produced by XFS.

The cellular and molecular biology of the iris, an overlooked tissue: the iris and pseudoexfoliation glaucoma.

Located between the cornea and the lens, the Iris is fully immersed in aqueous humor. During exfoliation syndrome, a disorder of the elastic fibers, an abnormal fibrillar material (XFM) is deposited on the anterior lens capsule underneath the pigment epithelium of the Iris. Release of this material to the aqueous humor reaches the trabecular meshwork where its presence is associated with elevated intraocular pressure. Ultrastructural studies suggest that the XFM material is produced by the lens capsule, lens epithelial and iris pigment epithelial cells (IPE). The involvement of the IPE in pseudoexfoliation glaucoma has not been extensively addressed. Immunohistochemistry studies have shown higher levels of LOXL1 and clusterin in the IPE extracellular space of specimens from exfoliation patients. But studies using IPE cells to understand the formation of the XFM in vitro and/or in vivo are scarce. A focus on the Iris and its IPE cells would be key for the elucidation of XFM and the understanding of the development of pseudoexfoliation glaucoma.

Caldesmon effects on the actin cytoskeleton and cell adhesion in cultured HTM cells.

Caldesmon is a multifunctional ubiquitous regulator of the actin cytoskeleton, which can affect both actomyosin contractility and actin polymerization. Previous studies showed that caldesmon over-expression in cultured fibroblasts produces effects that resemble those of chemical inhibitors of cellular contractility. Since these inhibitors (H-7, Y-27632, etc.) have been shown to lower intraocular pressure and increase outflow facility from the anterior chamber of the eye, we proposed that caldesmon might be used for gene therapy of glaucoma. In the present study we examined the effects of expression of adenovirus-delivered rat non-muscle caldesmon fused with green fluorescent protein (AdCaldGFP) on the actin cytoskeleton and matrix adhesions in cultured human trabecular meshwork (HTM) cells. In addition, we assessed the effect of caldesmon on the stability of cell-cell junctions in kidney epithelial MDCK cells. Cultured HTM cells demonstrate a well-developed actin cytoskeleton, comprising mainly arrays of parallel actomyosin bundles (stress fibers). Lamellipodial protrusions containing dense actin networks are also observed. Cell-matrix adhesions are dominated by focal adhesions (FAs) associated with the ends of the stress fibers, focal complexes in lamellipodia, and fibrillar adhesions in the central part of the spread cells. Treatment of HTM cells with AdCaldGFP resulted in dose-dependent morphological changes within 24-48 hr post-infection. Cells expressing moderate levels of caldesmon exhibited straight bundles containing actin and myosin II, which were considerably shorter than those in control cells. Short filament bundles in caldesmon over-expressing cells formed arrays consisting of triangular actin structures with small vinculin-positive FAs at their vertices. In addition, the fraction of cells displaying large lamellipodia increased. About 40-50% of the population of caldesmon-expressing cells demonstrated high levels of GFP-caldesmon expression and severe changes in the actin cytoskeleton, manifested by the disappearance of stress fibers and the formation of curved actin- and myosin-containing bundles. These bundles formed together a dynamic network consisting of pulsating loops filling the entire cytoplasm. Addition of thapsigargin, which increases intracellular Ca++ concentration, resulted in a straightening of the curved bundles. Another type of novel actin structures induced by caldesmon over-expression were highly dynamic circular waves that propagated over the affected cells with a velocity about 10 microm min. In cells with disrupted stress fibers, vinculin-containing FAs and tensin-rich fibrillar adhesions had also essentially vanished. However, phosphotyrosine-positive focal complexes were still prominent throughout the lamellipodia of these cells. Over-expression of caldesmon in MDCK cells reduced, in a dose dependent manner, the beta-catenin content at cell-cell adherens junctions and in some cases led to physical disruption of adherens junctions. Thus, caldesmon over-expression induces unique reorganization of the actin cytoskeleton in affected cells, accompanied by disruption of focal and fibrillar cell-matrix adhesions, and destabilization of cell-cell adherens junctions. Inducing such changes in the contractility and actin cytoskeleton of HTM cells in glaucomatous eyes in vivo could produce a therapeutically useful increase in outflow facility.

Caldesmon transgene expression disrupts focal adhesions in HTM cells and increases outflow facility in organ-cultured human and monkey anterior segments.

Cytoskeleton modulating compounds have been shown to lower intraocular pressure (IOP) and increase outflow facility. Caldesmon is one protein that is involved in the regulation of actin stress fiber formation. The effects of rat non-muscle caldesmon (Cald) gene over-expression on focal adhesions in human trabecular meshwork (HTM) cells and on outflow facility in organ-cultured human and monkey anterior segments were determined. Treatment of HTM cells with adenovirus-delivered caldesmon (AdCaldGFP) resulted in characteristic changes in the actin cytoskeleton and matrix adhesions within 24-48 hr post-transduction. Stress fibers gradually disappeared and novel actin structures were formed (see manuscript by Grosheva et al., this issue). In cells with disrupted stress fibers, vinculin-containing focal adhesions were also disrupted. In organ-cultured anterior segments, baseline outflow facility (microl min-1 mmHg-1) for all anterior segments averaged (mean+/-sem): human, 0.19+/-0.03 (n=12); monkey, 0.36+/-0.02 (n=19). In human anterior segments, transduction with 10(7) plaque forming units of AdGFPCald increased outflow facility by 43+/-21% (p

Processing and transport of matrix gamma-carboxyglutamic acid protein and bone morphogenetic protein-2 in cultured human vascular smooth muscle cells: evidence for an uptake mechanism for serum fetuin.

Matrix gamma-carboxyglutamic acid protein (MGP) is a member of the vitamin K-dependent protein family with unique structural and physical properties. MGP has been shown to be an inhibitor of arterial wall and cartilage calcification. One inhibitory mechanism is thought to be binding of bone morphogenetic protein-2. Binding has been shown to be dependent upon the vitamin K-dependent gamma-carboxylation modification of MGP. Since MGP is an insoluble matrix protein, this work has focused on intracellular processing and transport of MGP to become an extracellular binding protein for bone morphogenetic protein-2. Human vascular smooth muscle cells (VSMCs) were infected with an adenovirus carrying the MGP construct, which produced non-gamma-carboxylated MGP and fully gamma-carboxylated MGP. Both forms of MGP were found in the cytosolic and microsomal fractions obtained from the cells by differential centrifugation. The crude microsomal fraction was shown to contain an additional, more acidic Ser-phosphorylated form of MGP believed to be the product of Golgi casein kinase. The data suggest that phosphorylation of MGP dictates different transport routes for MGP in VSMCs. A proteomic approach failed to identify a larger soluble precursor of MGP or an intracellular carrier protein for MGP. Evidence is presented for a receptor-mediated uptake mechanism for fetuin by cultured human VSMCs. Fetuin, shown by mass spectrometry not to contain MGP, was found to be recognized by anti-MGP antibodies. Fetuin uptake and secretion by proliferating and differentiating cells at sites of calcification in the arterial wall may represent an additional protective mechanism against arterial calcification.