Cindy K. Bahler

Genetic Modification of Human Trabecular Meshwork with Lentiviral Vectors

Glaucoma, a group of optic neuropathies, is the leading cause of irreversible blindness. Neuronal apoptosis in glaucoma is primarily associated with high intraocular pressure caused by chronically impaired outflow of aqueous humor through the trabecular meshwork, a reticulum of mitotically inactive endothelial-like cells located in the angle of the anterior chamber. Anatomic, genetic, and expression profiling data suggest the possibility of using gene transfer to treat glaucomatous intraocular pressure dysregulation, but this approach will require stable genetic modification of the differentiated aqueous outflow tract. We injected transducing unit-normalized preparations of either of two lentiviral vectors or an oncoretroviral vector as a single bolus into the aqueous circulation of cultured human donor eyes, under perfusion conditions that mimicked natural anterior chamber flow and maintained viability ex vivo. Reporter gene expression was assessed in trabecular meshwork from 3 to 16 days after infusion of 1.0 x 10(8) transducing units of each vector. The oncoretroviral vector failed to transduce the trabecular meshwork. In contrast, feline immunodeficiency virus and human immunodeficiency virus vectors produced efficient, localized transduction of the trabecular meshwork in situ. The results demonstrate that lentiviral vectors permit efficient genetic modification of the human trabecular meshwork when delivered via the afferent aqueous circulation, a clinically accessible route. In addition, controlled comparisons in this study establish that feline and human immunodeficiency virus vectors are equivalently efficacious in delivering genes to this terminally differentiated human tissue.

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.

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.

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.

Characterization of monoclonal antibodies against the glaucoma-associated protein myocilin

Although the glaucoma-associated protein myocilin has been the focus of intensive research, its biological function is still unknown. One of the limiting factors has been the lack of well-characterized antibodies, particularly monoclonal antibodies. We describe the development of six monoclonal antibodies specific to myocilin and characterize their suitability in Western blot and immunohistochemical applications. Three of the six monoclonal antibodies recognize the N-terminus of myocilin (amino acids 33-214), two antibodies recognize the middle third of the protein (amino acids 215-368), and one antibody recognizes the C-terminus (amino acids 369-504). Isotyping revealed that all antibodies are of the IgG1 kappa class except one, which is IgG2b kappa. Purified myocilin monoclonal antibodies were able to recognize myocilin in human aqueous humor separated on denatured/reduced and native gels, and human trabecular meshwork lysate by Western blot. Myocilin was also detected by immunohistochemistry in trabecular meshwork, ciliary body, iris, cornea, sclera, choroid, and retinal pigment epithelial cells.

Ocular Hypotensive Effects of the ATP-Sensitive Potassium Channel Opener Cromakalim in Human and Murine Experimental Model Systems

Elevated intraocular pressure (IOP) is the most prevalent and only treatable risk factor for glaucoma, a leading cause of irreversible blindness worldwide. Unfortunately, all current therapeutics used to treat elevated IOP and glaucoma have significant and sometimes irreversible side effects necessitating the development of novel compounds. We evaluated the IOP lowering ability of the broad spectrum KATP channel opener cromakalim. Cultured human anterior segments when treated with 2 μM cromakalim showed a decrease in pressure (19.33 ± 2.78 mmHg at 0 hours to 13.22 ± 2.64 mmHg at 24 hours; p<0.001) when compared to vehicle treated controls (15.89 ± 5.33 mmHg at 0 h to 15.56 ± 4.88 mmHg at 24 hours; p = 0.89). In wild-type C57BL/6 mice, cromakalim reduced IOP by 18.75 ± 2.22% compared to vehicle treated contralateral eyes (17.01 ± 0.32 mmHg at 0 hours to 13.82 ± 0.37 mmHg at 24 hours; n = 10, p = 0.002). Cromakalim demonstrated an additive effect when used in conjunction with latanoprost free acid, a common ocular hypotensive drug prescribed to patients with elevated IOP. To examine KATP channel subunit specificity, Kir6.2(-/-) mice were treated with cromakalim, but unlike wild-type animals, no change in IOP was noted. Histologic analysis of treated and control eyes in cultured human anterior segments and in mice showed similar cell numbers and extracellular matrix integrity within the trabecular meshwork, with no disruptions in the inner and outer walls of Schlemm’s canal. Together, these studies suggest that cromakalim is a potent ocular hypotensive agent that lowers IOP via activation of Kir6.2 containing KATP channels, its effect is additive when used in combination with the commonly used glaucoma drug latanoprost, and is not toxic to cells and tissues of the aqueous humor outflow pathway, making it a candidate for future therapeutic development.

Stanniocalcin-1 Is an Ocular Hypotensive Agent and a Downstream Effector Molecule That Is Necessary for the Intraocular Pressure–Lowering Effects of Latanoprost

Purpose To identify downstream signaling molecules through which intraocular pressure (IOP) is lowered following treatment with the prostaglandin analog latanoprost. Methods Total RNA and protein isolated from primary human Schlemms canal cells (n = 3) treated with latanoprost (free acid; 100 nM) were processed for quantitative PCR and Western blot analysis. IOP was evaluated in stanniocalcin-1 (STC-1−/−) and wild-type mice following treatment with latanoprost or Rho kinase inhibitor Y27632. Human anterior segment pairs (n = 8) were treated with recombinant STC-1 (5, 50, or 500 ng/mL) and pressure was recorded using custom-designed software. The effect of recombinant STC-1 (0.5 mg/mL) on IOP was evaluated in wild-type mice. Tissue morphology was evaluated by light and transmission electron microscopy. Results Increased STC-1 mRNA (4.0- to 25.2-fold) and protein expression (1.9- to 5.1-fold) was observed within 12 hours following latanoprost treatment. Latanoprost reduced IOP in wild-type mice (22.0% ± 1.9%), but had no effect on STC-1−/− mice (0.5% ± 0.7%). In contrast, Y27632 reduced IOP in both wild-type (12.5% ± 1.2%) and in STC-1−/− mice (13.1% ± 2.8%). Human anterior segments treated with STC-1 (500 ng/mL) showed an increase in outflow facility (0.15 ± 0.03 to 0.27 ± 0.09 μL/min/mm Hg) while no change was observed in paired vehicle-treated controls. Recombinant STC-1 reduced IOP in wild-type mice by 15.2% ± 3.0%. No observable morphologic changes were identified between treatment groups when evaluated by microscopy. Conclusions Latanoprost-induced reduction of IOP is mediated through the downstream signaling molecule STC-1. When used by itself, STC-1 exhibits ocular hypotensive properties.

ATP-sensitive potassium (KATP) channel openers diazoxide and nicorandil lower intraocular pressure by activating the Erk1/2 signaling pathway

Elevated intraocular pressure is the most prevalent and only treatable risk factor for glaucoma, a degenerative disease of the optic nerve. While treatment options to slow disease progression are available, all current therapeutic and surgical treatments have unwanted side effects or limited efficacy, resulting in the need to identify new options. Previous reports from our laboratory have established a novel ocular hypotensive effect of ATP-sensitive potassium channel (KATP) openers including diazoxide (DZ) and nicorandil (NCD). In the current study, we evaluated the role of Erk1/2 signaling pathway in KATP channel opener mediated reduction of intraocular pressure (IOP). Western blot analysis of DZ and NCD treated primary normal trabecular meshwork (NTM) cells, human TM (isolated from perfusion cultures of human anterior segments) and mouse eyes showed increased phosphorylation of Erk1/2 when compared to vehicle treated controls. DZ and NCD mediated pressure reduction (p<0.02) in human anterior segments (n = 7 for DZ, n = 4 for NCD) was abrogated by U0126 (DZ + U0126: -9.7 ± 11.5%, p = 0.11; NCD + U0126: -0.1 ± 11.5%, p = 1.0). In contrast, U0126 had no effect on latanoprostfree acid-induced pressure reduction (-52.5 ± 6.8%, n = 4, p = 0.001). In mice, DZ and NCD reduced IOP (DZ, 14.9 ± 3.8%, NCD, 16.9 ± 2.5%, n = 10, p<0.001), but the pressure reduction was inhibited by U0126 (DZ + U0126, 0.7 ± 3.0%; NCD + U0126, 0.9 ± 2.2%, n = 10, p>0.1). Histologic evaluation of transmission electron micrographs from DZ + U0126 and NCD + U0126 treated eyes revealed no observable morphological changes in the ultrastructure of the conventional outflow pathway. Taken together, the results indicate that the Erk1/2 pathway is necessary for IOP reduction by KATP channel openers DZ and NCD.

Effect of Cromakalim Prodrug 1 (CKLP1) on Aqueous Humor Dynamics and Feasibility of Combination Therapy With Existing Ocular Hypotensive Agents

Purpose Cromakalim prodrug 1 (CKLP1) is a water-soluble ATP-sensitive potassium channel opener that has shown ocular hypotensive properties in ex vivo and in vivo experimental models. To determine its mechanism of action, we assessed the effect of CKLP1 on aqueous humor dynamics and in combination therapy with existing ocular hypotensive agents. Methods Outflow facility was assessed in C57BL/6 mice by ex vivo eye perfusions and by in vivo constant flow infusion following CKLP1 treatment. Human anterior segments with no trabecular meshwork were evaluated for effect on pressure following CKLP1 treatment. CKLP1 alone and in combination with latanoprost, timolol, and Rho kinase inhibitor Y27632 were evaluated for effect on intraocular pressure in C57BL/6 mice and Dutch-belted pigmented rabbits. Results CKLP1 lowered episcleral venous pressure (control: 8.9 ± 0.1 mm Hg versus treated: 6.2 ± 0.1 mm Hg, P < 0.0001) but had no detectable effect on outflow facility, aqueous humor flow rate, or uveoscleral outflow. Treatment with CKLP1 in human anterior segments without the trabecular meshwork resulted in a 50% ± 9% decrease in pressure, suggesting an effect on the distal portion of the conventional outflow pathway. CKLP1 worked additively with latanoprost, timolol, and Y27632 to lower IOP, presumably owing to combined effects on different aspects of aqueous humor dynamics. Conclusions CKLP1 lowered intraocular pressure by reducing episcleral venous pressure and lowering distal outflow resistance in the conventional outflow pathway. Owing to this unique mechanism of action, CKLP1 works in an additive manner to lower intraocular pressure with latanoprost, timolol, and Rho kinase inhibitor Y27632.