Aerosolized, Gas-Phase, Intravitreal Methotrexate Reduces Proliferative Vitreoretinopathy in a Randomized Trial in a Porcine Model.

Purpose: To determine the effectiveness of aerosol-delivered methotrexate (AD-MTx) in a large-animal (porcine) model of proliferative vitreoretinopathy (PVR). Design: Prospective, randomized, interventional, double-masked, controlled, large-animal study with predetermined clinical and histopathologic outcome criteria. Controls: Half of the pigs were randomly assigned to receive an identical volume of aerosol-delivered normal saline (AD-NS) using identical delivery systems and treatment intervals. Methods: Proliferative vitreoretinopathy was surgically induced in 16 pigs (8 males and 8 females), randomly assigned to receive 2 doses (group A) or 3 doses (group B) of either AD-MTx (1.6 mg/0.4 ml) or normal saline (AD-NS). Group A pigs were euthanized at week 2 (n = 8), and group B pigs were euthanized at week 3 (n = 8). Masked clinical PVR scores (0-6) by a vitreoretinal surgeon and histopathology PVR scores (0-8) by a masked ophthalmic pathologist were used to determine outcomes. Main Outcome Measures: The mean, combined clinical and histopathology scores (both anterior and posterior) were used to determine the overall treatment effect between the groups. Results: The mean masked score (± standard deviation) when all grading end points (clinical + histopathology) were combined was a mean of 8.0 ± 2.3 in the AD-MTx group versus a higher 9.9 ± 2.0 in the AD-NS control group (P = 0.05). The clinical score was 3.88 ± 1.2 in the AD-MTx group versus 4.63 ± 1.6 in the AD-NS group (P = 0.16). The histopathology score for anterior PVR was 2.5 ± 0.8 in the AD-MTx group versus 2.5 ± 0.5 in the AD-NS group (P = 0.50), and the posterior PVR was 1.63 ± 1.6 in the AD-MTx group versus 2.75 ± 1.3 in the AD-NS group (P = 0.07). When the frequency of methotrexate dosing in group A (2 doses) was compared with that in group B (3 doses), the mean score was 8.75 versus 9.13 (P = 0.38), respectively, suggesting an insignificant difference. Conclusions: After surgical induction of PVR in an aggressive, high-risk, large-animal model, AD-MTx reduced posterior PVR formation compared with AD-NS. Additional dosing at week 3 did not improve the outcomes. No difference in anterior PVR formation was noted with intervention. This novel drug delivery system has implications for PVR reduction and warrants further investigation. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Transgene expression of Stanniocalcin-1 provides sustained intraocular pressure reduction by increasing outflow facility.

Glaucoma is the leading cause of irreversible blindness worldwide. Therapies for glaucoma are directed toward reducing intraocular pressure (IOP), the leading risk factor and only reliable therapeutic target via topical medications or with procedural intervention including laser or surgery. Though topical therapeutics are typically first line, less than 50% of patients take drops as prescribed. Sustained release technologies that decrease IOP for extended periods of time are being examined for clinical use. We recently identified Stanniocalcin-1, a naturally occurring hormone, as an IOP-lowering agent. Here, we show that a single injection into the anterior chamber of mice with an adeno-associated viral vector containing the transgene of stanniocalcin-1 results in diffuse and sustained expression of the protein and produces IOP reduction for up to 6 months. As the treatment effect begins to wane, IOP-lowering can be rescued with a repeat injection. Aqueous humor dynamic studies revealed an increase in outflow facility as the mechanism of action. This first-in-class therapeutic approach has the potential to improve care and reduce the rates of vision loss in the 80 million people worldwide currently affected by glaucoma.

Evaluation of neural innervation in the human conventional outflow pathway distal to Schlemm's canal.

The distal outflow pathway of the human eye consists of the outer wall of Schlemm's canal, collector channels, and the deep-scleral, mid-scleral and episcleral vessels. It is the last region of transit for aqueous humor before returning to the venous system. While the trabecular meshwork, scleral spur, and inner wall of Schlemm's canal have been extensively analyzed to define their contributions to aqueous outflow, the role of the distal outflow pathway is not completely understood. Collector channels, emanating from Schlemm's canal were previously thought to be passive conduits for aqueous humor. However, recent studies have shown many collector channels contain flap-like appendages which move with changes in pressure. These findings, along with studies demonstrating innervation of episcleral vessels, have led to questions regarding whether other structures in the distal outflow pathway are under neural regulation and how this may influence aqueous humor outflow. This study evaluates the innervation of the outer wall of Schlemm's canal and collector channels, along with the deep-scleral, mid-scleral and episcleral vasculature with microcomputed tomography and 3-dimensional reconstruction, correlative light microscopy, immunohistochemistry, and transmission electron microscopy. Peripheral, autonomic, and sensory nerve fibers were found to be present adjacent to Schlemm's canal outer wall endothelium, collector channel endothelium, and in the different regions of the distal outflow vasculature. Nerves were more commonly identified in regions that contained collector channels when compared to regions without collector channels. These findings regarding the neural anatomy suggest an active neural regulation of aqueous humor outflow throughout the proximal and distal regions of the conventional outflow pathway.

Pharmacological Profile and Ocular Hypotensive Effects of Cromakalim Prodrug 1, a Novel ATP-Sensitive Potassium Channel Opener, in Normotensive Dogs and Nonhuman Primates.

Purpose: To evaluate pharmacokinetic parameters and ocular hypotensive effects of cromakalim prodrug 1 (CKLP1) in normotensive large animal models. Methods: Optimal CKLP1 concentration was determined by dose response and utilized in short- (5-8 days) and long-term (60 days) evaluation in hound dogs (n = 5) and African Green Monkeys (n = 5). Blood pressure was recorded 3-5 times per week with a tail cuff. Concentrations of CKLP1 and the parent compound levcromakalim were assessed in hound dog plasma and select tissues by LC-MS/MS after bilateral ocular treatment with CKLP1 for 8 days. Pharmacokinetic parameters were calculated from days 1, 4, and 8 data. After necropsy, histology was assessed in 43 tissue samples from each animal. Results: In hound dogs and African Green monkeys, 10 mM CKLP1 (optimal concentration) significantly lowered intraocular pressure (IOP) by 18.9% ± 1.1% and 16.7% ± 6.7%, respectively, compared with control eyes (P < 0.05). During treatment, no significant change in systolic or diastolic blood pressure was observed in either species (P > 0.1). Average values for half-life of CKLP1 was 295.3 ± 140.4 min, Cmax, 10.5 ± 1.6 ng/mL, and area under the concentration vs. time curve (AUClast) 5261.4 ± 918.9 ng·min/mL. For levcromakalim, average values of half-life were 96.2 ± 27 min, Cmax 1.2 ± 0.2 ng/mL, and AUClast 281.2 ± 110.8 ng·min/mL. No significant pathology was identified. Conclusions: CKLP1 lowered IOP in hound dogs and African green monkeys with no effect on systemic blood pressure. Ocular topical treatment of CKLP1 showed excellent tolerability even after extended treatment periods.

Isolation and characterization of novel primary cells from the human distal outflow pathway.

Ocular hypertension occurs due to increased resistance to aqueous humor removal through the conventional outflow pathway. Unlike the proximal region of the conventional outflow pathway, the distal region has not been well studied, mostly due to lack of model systems. Here we describe isolation and characterization of human primary vascular distal outflow pathway (VDOP) cells from the distal region of the conventional outflow pathway. Tissue from the distal region was isolated from human corneo-scleral rims, digested with collagenase type I (100 U/ml) and placed on gelatin coated plates to allow cellular growth in Dulbecco's Modified Eagle's Medium (low glucose) containing fetal bovine serum and antibiotic/antimycotic. VDOP cells showed consistent proliferation for up to 7 passages, retained endothelial-like nature of the parent tissues and showed a unique marker phenotype of Lectin+VEGFR2-CD34-NG2- that was distinct from neighboring trabecular meshwork (Lectin+VEGFR2-CD34-NG2+) and Schlemm's canal (Lectin+VEGFR2+CD34+NG2+) cells. Dexamethasone treated VDOP cells did not express myocilin and did not form cross-linked actin networks, in contrast to trabecular meshwork cells. These data show that VDOP cells are unique to the distal outflow region and can be used as a viable in vitro model system to understand the biology of the distal outflow pathway and intraocular pressure regulation.

Pharmacological and pharmacokinetic profile of the novel ocular hypotensive prodrug CKLP1 in Dutch-belted pigmented rabbits.

Elevated intraocular pressure is the only treatable risk factor for glaucoma, an eye disease that is the leading cause of irreversible blindness worldwide. We have identified cromakalim prodrug 1 (CKLP1), a novel water-soluble ATP-sensitive potassium channel opener, as a new ocular hypotensive agent. To evaluate the pharmacokinetic and safety profile of CKLP1 and its parent compound levcromakalim, Dutch-belted pigmented rabbits were treated intravenously (0.25 mg/kg) or topically (10 mM; 4.1 mg/ml) with CKLP1. Body fluids (blood, aqueous and vitreous humor) were collected at multiple time points and evaluated for the presence of CKLP1 and levcromakalim using a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) based assay. Histology of tissues isolated from Dutch-belted pigmented rabbits treated once daily for 90 days was evaluated in a masked manner by a certified veterinary pathologist. The estimated plasma parameters following intravenous administration of 0.25 mg/kg of CKLP1 showed CKLP1 had a terminal half-life of 61.8 ± 55.2 min, Tmax of 19.8 ± 23.0 min and Cmax of 1968.5 ± 831.0 ng/ml. Levcromakalim had a plasma terminal half-life of 85.0 ± 37.0 min, Tmax of 61.0 ± 32.0 min and Cmax of 10.6 ± 1.2 ng/ml. Topical CKLP1 treatment in the eye showed low levels (<0.3 ng/mL) of levcromakalim in aqueous and vitreous humor, and trace amounts of CKLP1 and levcromakalim in the plasma. No observable histological changes were noted in selected tissues that were examined following topical application of CKLP1 for 90 consecutive days. These results suggest that CKPL1 is converted to levcromakalim in the eye and likely to some extent in the systemic circulation.

Stanniocalcin-1 is a Modifier of Oxygen-Induced Retinopathy Severity.

Purpose/Aim: Abnormal activation of signaling pathways related to angiogenesis, inflammation, and oxidative stress has been implicated in the pathophysiology of retinopathy of prematurity (ROP), a leading cause of blindness in pre-term infants. Therapies for ROP include laser and anti-vascular endothelial growth factor agents. However, these therapies have side effects, and even with adequate treatment, visual acuity can be impaired. Novel therapeutic options are needed. Stanniocalcin-1 (STC-1) is a neuroprotective protein with anti-inflammatory and anti-oxidative stress properties. Rodent models of oxygen-induced retinopathy (OIR) were selected to determine whether STC-1 plays a role in the development of OIR.Materials and methods: STC-1 gene and protein expression was first evaluated in the Sprague Dawley rat OIR model that is most similar to human ROP. OIR was then induced in wild-type and Stc-1-/- mice. Retinas were isolated and evaluated for avascular and neovascular area on retinal flat mounts. Quantification of gene expression by quantitative real-time PCR was performed. VEGF was assayed by ELISA in media obtained from induced pluripotent stem-cell-derived retinal pigment epithelial (iPS-RPE) cells following treatment with recombinant STC-1.Results: STC-1 was significantly upregulated in a rat model of OIR compared to room air controls at the gene (P < .05) and protein (P < .001) level. Stc-1-/- OIR mice showed significantly worse ROP compared to wild-type mice as assessed by avascular (20.2 ± 2.4% vs 15.2 ± 2.5%; P = .02) and neovascular area (14.3 ± 2.7% vs 8.8 ± 3.7%; P < .05). Transcript levels of vascular endothelial growth factor-A were significantly higher in Stc-1-/- OIR mice compared to wild-type controls (P = .03). STC-1 reduced VEGF production in iPS-RPE cells (P = .01).Conclusions: STC-1 plays a role in the OIR stress response and development of pathologic vascular features in rodent OIR models by regulating VEGF levels.

Diet Mimicking "Fast Food" Causes Structural Changes to the Retina Relevant to Age-Related Macular Degeneration.

Introduction: Metabolic syndrome is a disorder characterized by a constellation of findings including truncal obesity, elevated blood pressure, abnormal cholesterol levels, and high blood glucose. Recent evidence suggests that metabolic syndrome may be associated with increased risk of age-related macular degeneration (AMD) and other eye diseases. Recently, C57BL/6J wild-type mice fed with a "fast food" diet consisting of high fat, cholesterol, and fructose-supplemented water showed unique systemic pathology consistent with metabolic syndrome and nonalcoholic steatohepatitis. Additionally, these mice showed higher levels of fibrosis, inflammation, endoplasmic reticulum stress, and mitochondrial dysfunction compared to mice fed with only a high-fat diet alone. Since similar pathways are activated in AMD, we sought to determine whether mice fed a "fast food" diet exhibited retinal changes.Methods: 3-month-old wild-type mice were randomized to a standard chow (n = 11) or a "fast food" (n = 18) diet and fed for 9 months. At 1 year of age, tissues were collected and retinas were analyzed using transmission electron microscopy. Quantitative measures of Bruch's membrane thickness and retinal pigment epithelium (RPE) cell counts were performed.Results: "Fast food" fed mice showed ocular pathology relevant to various stages of AMD including basal laminar deposits, focal thickening of Bruch's membrane, and a significant loss of RPE cells.Discussion/conclusion: A wild-type mouse model of metabolic syndrome fed a "fast food" diet developed changes to the retina similar to some of the pathologic features seen in AMD. Further investigations into this and similar animal models as well as further epidemiological studies are needed to more clearly define the association between metabolic syndrome and AMD.

Anatomical Variation of Human Collector Channel Orifices.

PURPOSE: To examine the anatomical variation of normal human collector channel orifices and their relationship with Schlemm's canal. METHODS: Ten human anterior segments fixed by immersion or perfusion were dissected radially and further divided by fine dissection into corresponding inner and outer wall segments. The tissues were dehydrated, critical-point dried, sputter coated, and examined by scanning electron microscopy. Images were obtained at magnifications from ×200 to ×10,000. Selected radial collector channel regions were processed for plastic embedding. RESULTS: Two classes of collector channel orifices were identified. Simple oval orifices (54.7 ± 4.6-µm diameter) were lined with endothelial cells and most often occurred on a planar region of Schlemm's canal outer wall. Complex orifices (62.7 ± 3.4-µm diameter) were often found associated with septal columns and bridges, and typically covered with flap-like structures (10-40 µm) that extended between the inner and outer wall and over the collector channel orifices. Both simple and complex orifices had complete or partial lip-like rims. In orifices with partial rims, a trough-like groove was often visible on the outer wall surface opposite the lip. Transected septa and inner and outer wall adhesion sites were often found in association with complex collector channel orifices. CONCLUSIONS: Collector channel orifice structure varied from simple ovals to complex tethered flaps and bridges. Collector channel orifices with complex flaps connect the inner and outer walls of Schlemm's canal, and may serve to enhance and regulate aqueous outflow in these regions.

Recent Developments in Understanding the Role of Aqueous Humor Outflow in Normal and Primary Open Angle Glaucoma.

Primary open angle glaucoma (POAG) is the second leading cause of blindness in the world's rapidly aging population. POAG is characterized by progressive degeneration of neural structures in the posterior segment, often associated with a concomitant elevation of intraocular pressure. Changes in IOP are believed to be caused by a disruption in the normal outflow of aqueous humor. This article reviews recent research associated with normal and POAG aqueous humor outflow. Novel findings elucidating biochemical and pathological changes in the ocular tissues affected in POAG are presented. Stem cell research, identification of lymphatic markers, and increased use of mouse models give researchers exciting new tools to understand aqueous humor outflow, changes associated with POAG and identify underlying causes of the disease.

Anatomic changes in Schlemm's canal and collector channels in normal and primary open-angle glaucoma eyes using low and high perfusion pressures.

PURPOSE: To examine the anatomy of Schlemm's canal (SC) and collector channels (CCs) in normal human and primary open-angle glaucoma (POAG) eyes under low and high perfusion pressure. METHODS: In normal (n = 3) and POAG (n = 3) eye pairs, one eye was perfused at 10 mm Hg while the fellow eye was perfused at 20 mm Hg for 2 hours. Eyes were perfusion fixed at like pressures, dissected into quadrants, embedded in Epon Araldite, and scanned by three-dimensional micro-computed tomography (3D micro-CT). Schlemm's canal volume, CC orifice area, diameter, and number were measured using ANALYZE software. RESULTS: Normal eyes showed a larger SC volume (3.3-fold) and CC orifice area (9962.8 vs. 8825.2 µm(2)) and a similar CC diameter (34.3 ± 17.8 vs. 32.7 ± 13.0 µm) at 10 mm Hg compared to 20 mm Hg. In POAG eyes, SC volume (2.0-fold), CC orifice area (8049.2 µm(2)-6468.4 µm(2)), and CC diameter (36.2 ± 19.1 vs. 29.0 ± 13.8 µm) were increased in 10 mm Hg compared to 20 mm Hg perfusion pressures. Partial and total CC occlusions were present in normal and POAG eyes, with a 3.7-fold increase in total occlusions in POAG eyes compared to normal eyes at 20 mm Hg. Visualization of CCs increased by 24% in normal and by 21% in POAG eyes at 20 mm Hg compared to 10 mm Hg. Schlemm's canal volume, CC area, and CC diameter were decreased in POAG eyes compared to normal eyes at like pressures. CONCLUSIONS: Compensatory mechanisms for transient and short periods of increased pressure appear to be diminished in POAG eyes. Variable response to pressure change in SC and CCs may be a contributing factor to outflow facility change in POAG eyes.

Second-generation trabecular meshwork bypass stent (iStent inject) increases outflow facility in cultured human anterior segments.

PURPOSE: To determine whether a second-generation trabecular meshwork (TM) bypass stent (iStent inject) influences outflow facility in cultured human anterior segments. DESIGN: Prospective laboratory investigation using normal human donor eyes. METHODS: Human anterior segments (n = 7) were placed in perfusion organ culture. One or 2 iStent inject stents were inserted into the TM within the nasal and/or superior quadrants using a specially designed injector. Anterior segments were returned to culture and perfused for an additional 24 hours. Morphology of the TM and Schlemm canal (SC) was assessed by scanning electron microscopy (SEM) and 3-dimensional micro-computed tomography (3D micro-CT). RESULTS: Insertion of 1 iStent inject into the nasal or superior quadrant of the TM increased outflow facility from 0.16 ± 0.05 µL/min/mm Hg to 0.38 ± 0.23 µL/min/mm Hg (P < .03, n = 7), with concurrent pressure reduction from 16.7 ± 5.4 mm Hg to 8.6 ± 4.4 mm Hg. Addition of a second iStent inject further increased outflow facility to 0.78 ± 0.66 µL/min/mm Hg (n = 2). SEM showed the iStent inject flange compressed against the uveal region of the TM, the thorax securely inserted within the TM, and the head located in the lumen of SC. Dilation of SC was noted around the iStent inject head and SC cell disruption was observed at the iStent inject insertion site. 3D micro-CT confirmed iStent inject placement. CONCLUSION: iStent inject, a second-generation bypass stent, increased outflow facility in human anterior segment culture. The iStent inject is a promising new device to lower intraocular pressure via TM bypass.

ATP-sensitive potassium (KATP) channel activation decreases intraocular pressure in the anterior chamber of the eye.

PURPOSE. ATP-sensitive potassium channel (K(ATP)) openers target key cellular events, many of which have been implicated in glaucoma. The authors sought to determine whether K(ATP) channel openers influence outflow facility in human anterior segment culture and intraocular pressure (IOP) in vivo. METHODS. Anterior segments from human eyes were placed in perfusion organ culture and treated with the K(ATP) channel openers diazoxide, nicorandil, and P1075 or the K(ATP) channel closer glyburide (glibenclamide). The presence, functionality, and specificity of K(ATP) channels were determined by RT-PCR, immunohistochemistry, and inside-out patch clamp in human trabecular meshwork (TM) tissue or primary cultures of normal human trabecular meshwork (NTM) cells. The effect of diazoxide on IOP in anesthetized Brown Norway rats was measured with a rebound tonometer. RESULTS. K(ATP) channel openers increased outflow facility in human anterior segments (0.14 ± 0.02 to 0.26 ± 0.09 µL/min/mm Hg; P < 0.001) compared with fellow control eyes (0.22 ± 0.11 to 0.21 ± 0.11 µL/min/mm Hg; P > 0.5). The effect was reversible, with outflow facility returning to baseline after drug removal. The addition of glyburide inhibited diazoxide from increasing outflow facility. Electrophysiology confirmed the presence and specificity of functional K(ATP) channels. K(ATP) channel subunits K(ir)6.1, K(ir)6.2, SUR2A, and SUR2B were expressed in TM and NTM cells. In vivo, diazoxide significantly lowered IOP in Brown Norway rats. CONCLUSIONS. Functional K(ATP) channels are present in the trabecular meshwork. When activated by K(ATP) channel openers, these channels increase outflow facility through the trabecular outflow pathway in human anterior segment organ culture and decrease IOP in Brown Norway rat eyes.

The elastin fiber system between and adjacent to collector channels in the human juxtacanalicular tissue.

PURPOSE: To determine the composition and investigate the elastin fiber system in the juxtacanalicular tissue adjacent to and between collector channel orifices in normal human eyes. METHODS: Normal human eyes (71.0 ± 8.6 years; mean ± SD; n = 4) were perfusion fixed at low (10 mm Hg) and high pressure (20 mm Hg) with 3% paraformaldehyde/0.1 M phosphate buffer. Frontal serial sections were cut from paraffin blocks, and regions with and without collector channels were selected. Sections were stained using Weigert's resorcin-fuchsin stain with oxidation. Immunohistochemistry was performed using antibodies against elastin, fibrillin-1, and microfibrillar-associated protein-1/2. RESULTS: Elastin, elaunin, and oxytalan fibers were identified within the juxtacanalicular tissue of the inner and outer walls in low- and high-pressure eyes. These fibers were found at collector channel orifices, between collector channels, and within collector channel walls. Fibrillin-1 was located at the base and lateral edges of Schlemm's canal endothelial cells. Microfibrillar-associated protein-1/2 was found with elastin-like fibers at the base of Schlemm's canal endothelium cells, in the juxtacanalicular tissue, and in the uveal region. CONCLUSIONS: Elastin, elaunin, oxytalan, and elastin-associated proteins fibrillin-1 and microfibrillar-associated protein-1/2 were identified within the juxtacanalicular tissue of the inner and outer walls and within collector channel walls of human eyes perfused at low and high pressure. No differences in labeling patterns for elastin, elaunin, and oxytalan were found in the juxtacanalicular tissue adjacent to or between collector channel orifices. The elastin fiber system appears to have a significant role in the support and distensibility of the juxtacanalicular region under collector channels.

Imaging the aqueous humor outflow pathway in human eyes by three-dimensional micro-computed tomography (3D micro-CT).

The site of outflow resistance leading to elevated intraocular pressure in primary open-angle glaucoma is believed to be located in the region of Schlemm's canal inner wall endothelium, its basement membrane and the adjacent juxtacanalicular tissue. Evidence also suggests collector channels and intrascleral vessels may have a role in intraocular pressure in both normal and glaucoma eyes. Traditional imaging modalities limit the ability to view both proximal and distal portions of the trabecular outflow pathway as a single unit. In this study, we examined the effectiveness of three-dimensional micro-computed tomography (3D micro-CT) as a potential method to view the trabecular outflow pathway. Two normal human eyes were used: one immersion fixed in 4% paraformaldehyde and one with anterior chamber perfusion at 10 mmHg followed by perfusion fixation in 4% paraformaldehyde/2% glutaraldehyde. Both eyes were postfixed in 1% osmium tetroxide and scanned with 3D micro-CT at 2 µm or 5 µm voxel resolution. In the immersion fixed eye, 24 collector channels were identified with an average orifice size of 27.5 ± 5 µm. In comparison, the perfusion fixed eye had 29 collector channels with a mean orifice size of 40.5 ± 13 µm. Collector channels were not evenly dispersed around the circumference of the eye. There was no significant difference in the length of Schlemm's canal in the immersed versus the perfused eye (33.2 versus 35.1 mm). Structures, locations and size measurements identified by 3D micro-CT were confirmed by correlative light microscopy. These findings confirm 3D micro-CT can be used effectively for the non-invasive examination of the trabecular meshwork, Schlemm's canal, collector channels and intrascleral vasculature that comprise the distal outflow pathway. This imaging modality will be useful for non-invasive study of the role of the trabecular outflow pathway as a whole unit.

Aqueous humor rapidly stimulates myocilin secretion from human trabecular meshwork cells.

Myocilin, a protein associated with the development of glaucoma, is expressed in most eye tissues with highest expression observed in trabecular meshwork cells. In culture, primary human trabecular meshwork cells incubated in 10% fetal bovine serum have reduced myocilin expression compared to in vivo, but incubation in human aqueous humor, their normal in vivo nutrient source, restores myocilin expression to near in vivo levels. To investigate the mechanism by which human aqueous humor stimulates myocilin accumulation in conditioned media from normal human trabecular meshwork cells, three independent trabecular meshwork cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing various supplements: fetal bovine serum (10%), human serum (0.2%), porcine aqueous humor (50%), bovine serum albumin (0.1%), dexamethasone (10(-7)M), human aqueous humor (50%) or heat-inactivated human aqueous humor (50%). Conditioned media from cultured primary trabecular meshwork cells following incubation in human aqueous humor showed significant accumulation of myocilin in a time- (15 min) and dose-dependent manner (half maximal effective concentration ∼ 30%) while intracellular myocilin levels decreased. Minimal myocilin accumulation was observed in conditioned media isolated from trabecular meshwork cells cultured in DMEM containing fetal bovine or human serum, bovine serum albumin, porcine aqueous humor, dexamethasone or DMEM alone. Heat inactivation of human aqueous humor nearly eliminated human aqueous humor-stimulated myocilin secretion. Inhibitors of new protein synthesis, gene transcription, the endoplasmic reticulum/Golgi system and endocytic/exocytic secretory pathways failed to inhibit human aqueous humor-stimulated myocilin secretion. Using immunolabeling and transmission electron microscopy, myocilin was found associated with 70-90 nm vesicle-like structures within the cytoplasm of human aqueous humor treated trabecular meshwork cells. These studies suggest that myocilin secretion from trabecular meshwork cells occurs in a Golgi-independent manner following human aqueous humor treatment. Heat-labile factors in human aqueous humor are responsible for the time- and dose-dependent release of myocilin from vesicle-like structures within the cytoplasm of trabecular meshwork cells.

Human corneal endothelial cell transplantation in a human ex vivo model.

PURPOSE: To determine the effects of incorporating superparamagnetic microspheres (SPMs) into cultured human corneal endothelial cells (HCECs) and to describe preliminary experiments of HCEC transplantation, facilitated by SPMs and an external magnetic field, in a human anterior segment ex vivo model. METHODS: HCECs were cultured as monolayers and incorporated with magnetite oxide SPMs (900, 300, and 100 nm) at different concentrations. Cell viability, migration toward a magnetic field, and light transmittance were measured after incorporation of the SPMs. HCEC transplantation into the eyes of human recipients was investigated by subjecting anterior segments in organ culture to an external magnetic field. Light and electron microscopy were used to assess HCEC attachment to corneal stroma. RESULTS: SPMs were incorporated into the cytoplasm of HCECs after overnight incubation. None of the SPMs affected the short-term viability of cultured HCECs (P > 0.14, n = 6) or their light transmittance (P > 0.06, n = 5), although there was a trend toward decreased transmittance with the higher concentration of 900-nm SPMs. Cell migration toward a magnetic field was higher for HCECs with incorporated SPMs than for HCECs without SPMs (P < or = 0.01, n = 6), with dose-response relationships evident for the 300- and 100-nm SPMs. SPMs facilitated the attachment of HCECs to the corneal stroma in the human anterior segment model with minimal change in intracameral (intraocular) pressure. CONCLUSIONS: SPMs facilitate migration of HCECs toward a magnetic source and attachment of cells to the corneal stroma without affecting cell viability or light transmittance. The human anterior segment model can be used to study HCEC transplantation.

Preferential fluid flow in the human trabecular meshwork near collector channels.

PURPOSE: To determine whether preferential pathways exist within the human trabecular meshwork, pigmented and nonpigmented regions adjacent to and between collector channels were examined, and the configuration of the juxtacanalicular tissue (JCT) was analyzed. METHODS: Healthy whole human eyes were perfused at 10 or 25 mm Hg with 0.5 mum fluorescent beads. Tissue wedges of pigmented and nonpigmented meshwork (with and without collector channels) were dissected from each eye and examined by confocal microscopy. Bead concentration adjacent to and between collector channels was quantified. The configuration of the JCT adjacent to collector channels from whole eyes perfused at 20 mm Hg was analyzed by light microscopy. RESULTS: Eyes perfused at 25 mm Hg had more beads adjacent to collector channels in pigmented than in nonpigmented regions (4.9%+/-3.5% vs. 1.1%+/-0.9%; P=0.02). In pigmented regions without collector channels, bead concentration was decreased by fivefold (4.9%+/-3.5% vs. 0.96%+/-0.88%; P=0.04). Perfusion at 25 mm Hg increased beads by threefold under pigmented collector channels compared with the same regions in eyes perfused at 10 mm Hg. Expansion of the JCT occurred more often under collector channels at 25 mm Hg than at 10 mm Hg (44% vs. 17%; P=0.01). The JCT region under collector channels was expanded compared with JCT regions between adjacent collector channels (1053+/-424 microm(2) vs. 571+/-216 microm(2); P<0.001). CONCLUSIONS: Increased levels of beads in pigmented trabecular meshwork adjacent to collector channels suggest preferential flow pathways are present in human trabecular meshwork. At elevated pressure, the JCT region under collector channels is expanded, possibly because of increased fluid flow.

Prostaglandins increase trabecular meshwork outflow facility in cultured human anterior segments.

PURPOSE: To determine the effect of latanoprost free acid and prostaglandin E1 (PGE1) on outflow facility in cultured human anterior segments. Clinical studies find prostaglandin treatment increases uveoscleral outflow, but do not agree whether trabecular outflow increases. Cultured anterior segments eliminate the uveoscleral pathway and enable a direct assessment of trabecular outflow. DESIGN: Laboratory investigation. METHODS: One anterior segment of an eye pair was placed in perfusion organ culture and received a continuous infusion of drug while the fellow anterior segment received vehicle. Histologic changes were assessed. Zymography and Western blots were used to analyze matrix metalloprotease (MMP) activity. Scleral hydraulic conductivities were measured. RESULTS: Latanoprost significantly increased outflow facility (67% +/- 11% vs control 6% +/- 10%, P < .001). Facility changes occurred within one hour of receiving drug, reaching a new baseline by 24 hours. Facility changes were reversible, requiring about 48 hours to return to pre-drug values. PGE1 caused less facility change (13% +/- 17% vs control 1% +/- 11%, P = .02). Prostaglandin treated anterior segments had regions of focal detachment and loss of Schlemm canal endothelial cells, with loss of extracellular matrix underlying some areas. MMPs were not consistently increased in Western blots, zymography, or immunohistochemistry. Scleral hydraulic conductivity increased, but not enough to account for total facility increase. CONCLUSIONS: Prostaglandins increase outflow facility in perfusion organ culture of human anterior segments. MMP activity was not consistently increased, and scleral hydraulic conductivity was not increased sufficiently to account for total facility increase. The histologic changes suggest a direct trabecular meshwork effect.