Uttio Roy Chowdhury

Proteome analysis of human aqueous humor.

PURPOSE Human aqueous humor (hAH) provides nutrition and immunity within the anterior chamber of the eye. Characterization of the protein composition of hAH will identify molecules involved in maintaining a homeostatic environment for anterior segment tissues. The present study was conducted to analyze the proteome of hAH. METHODS hAH samples obtained during elective cataract surgery were divided into three matched groups and immunodepleted of albumin, IgG, IgA, haploglobin, antitrypsin, and transferrin. Reduced and denatured proteins (20 μg) from each group were separated by gel electrophoresis. Thirty-three gel slices were excised from each of three gel lanes (n = 99), digested with trypsin, and subjected to nanoflow liquid chromatography electrospray ionization tandem mass spectrometry (nano-LC-ESI-MS/MS). The protein component of hAH was also analyzed by antibody-based protein arrays, and selected proteins were quantified. RESULTS A total of 676 proteins were identified in hAH. Of the 355 proteins identified by nano-LC-ESI-MS/MS, 206 were found in all three groups. Most of the proteins identified by nano-LC-ESI-MS/MS had catalytic, enzymatic, and structural properties. Using antibody-based protein arrays, 328 cytokines, chemokines, and receptors were identified. Most of the quantified proteins had concentrations that ranged between 0.1 and 2.5 ng/mL. Ten proteins were identified by both nano-LC-ESI-MS/MS and antibody protein arrays. CONCLUSIONS Proteomic analysis of hAH identified 676 nonredundant proteins. More than 80% of these proteins are novel identifications. The elucidation of the aqueous proteome will establish a foundation for protein function analysis and identification of differentially expressed markers associated with diseases of the anterior segment.

Aqueous Humor Outflow: Dynamics and Disease

This review summarizes the goals and outcomes of the eighth annual ARVO/Pfizer Ophthalmic Research Institute conference that was held on May 4th and 5th, 2012, at the Embassy Suites Fort Lauderdale, Fort Lauderdale, Florida. This conference series has been funded by the ARVO Foundation for Eye Research with the help of a generous grant from Pfizer Ophthalmics. Funding from the ARVO/Pfizer Ophthalmics Research Institute has allowed a series of “think tanks” for the leading experts on various subspecialties of ophthalmology research. These meetings have helped to define various challenges and potential solutions to issues relevant to the field of ophthalmology. In 2012, the emphasis of the conference was on the physiological role of the conventional outflow pathway in aqueous humor dynamics in normal and glaucomatous eyes. In particular, clinical observations associated with aqueous humor outflow in health and disease, animal models in the understanding of aqueous outflow, role of mechanosensing in aqueous humor fluid flow, and new paradigms in cell and extracellular matrix crosstalk within the conventional outflow pathway were discussed. The primary goal of the conference was to bring together a diverse group of experts who are pioneers in conventional outflow biology and related nonocular areas of research with the hope of utilizing the groups knowledge and experience to evaluate the current understanding of aqueous outflow regulation and problems thereof. The invited group consisted of 29 investigators directly involved in conventional outflow research. Also present were three invited outside experts in cellular mechanics and cytoskeleton structures (Jeffrey Fredberg, PhD, Harvard School of Public Health, Boston, MA, USA), molecular mechanisms of cell adhesion and mechanosensing (Benjamin Geiger, PhD, Weizmann Institute of Science, Rehovot, Israel), and murine genetics (Richard Libby, PhD, University of Rochester Medical Center, Rochester, NY, USA). In addition, the conference was attended by 27 observers who participated in discussion of key topics at the end of each presentation. Together, the group was tasked with reviewing the current scientific dogma regarding aqueous outflow diseases and identifying the most pressing research questions and needs in the current funding environment. Above all, the group was asked to think “outside the box” to develop future research directions. The conference generated a list of highly relevant but currently unanswered questions with the hope that solutions to these issues would improve understanding of the role of the conventional outflow pathway in normal and glaucoma eyes. The meeting was organized into four sessions: (a) clinical insights into conventional outflow dysfunction, (b) the use of mice as model systems for conventional outflow, (c) mechanosensing within the conventional outflow pathway, and (d) the role of the extracellular matrix and signaling in conventional outflow dynamics. Speakers presented their thoughts on the preselected topics and provided key research goals that have been addressed or need to be addressed in the near future. The sessions were followed by discussion aimed at summarizing and interpreting the current knowledge as well as identifying unique research questions that were as yet unanswered. Anatomy and Physiology of the Conventional Aqueous Humor Outflow Pathway The conventional outflow pathway is mainly a pressure-driven system. Under homeostatic conditions, this pathway regulates the drainage of aqueous humor from the anterior chamber of the eye, thereby maintaining a constant intraocular pressure (IOP).1 Relevant tissues of the anterior segment that are anatomically involved in IOP control include the ciliary muscles, trabecular meshwork (TM), Schlemms canal (SC), collector channels, and aqueous veins. The ciliary muscle is composed of smooth muscle fibers that have a true elastic net of tendons that anchor into the choroid posteriorly and the scleral spur, TM, and inner wall of SC anteriorly.2,3 It is widely accepted that contraction of the ciliary muscle causes expansion of the TM and opening of SC, which subsequently increases the conductivity of aqueous humor (AH) through the TM. All these, along with reports of nerve innervation in the TM, indicate that the TM is a self-regulating tissue with both afferent and efferent nervous components responsible for controlling its functions.2,4

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.

Expression profile of the matricellular protein osteopontin in primary open-angle glaucoma and the normal human eye

PURPOSE. To characterize the role of osteopontin (OPN) in primary open-angle glaucoma (POAG) and normal eyes. METHODS. OPN quantification was performed by enzyme-linked immunosorbent assay in aqueous humor (AH) obtained from human donor eyes (POAG and normal) and surgical samples (POAG and elective cataract removal). OPN expression and localization in whole eye tissue sections and primary normal human trabecular meshwork (NTM) cells were studied by Western blot and immunohistochemistry. Latanoprost-free acid (LFA)-treated NTM cells were analyzed for OPN gene and protein expression. Intraocular pressure was measured by tonometry, and central corneal thickness was measured by optical coherence tomography in young OPN(-/-) and wild-type mice. RESULTS. OPN levels were significantly reduced in donor POAG AH compared with normal AH (0.54 ± 0.18 ng/μg [n = 8] vs. 0.77 ± 0.23 ng/μg [n = 9]; P = 0.039). A similar trend was observed in surgical AH (1.05 ± 0.31 ng/μg [n = 20] vs. 1.43 ± 0.88 ng/μg [n = 20]; P = 0.083). OPN was present in the trabecular meshwork, corneal epithelium and endothelium, iris, ciliary body, retina, vitreous humor, and optic nerve. LFA increased OPN gene expression, but minimal change in OPN protein expression was observed. No difference in intraocular pressure (17.5 ± 2.0 mm Hg [n = 56] vs. 17.3 ± 1.9 mm Hg [n = 68]) but thinner central corneal thickness (91.7 ± 3.6 μm [n = 50] vs. 99.2 ± 5.5 μm [n = 70]) was noted between OPN(-/-) and wild-type mice. CONCLUSIONS. OPN is widely distributed in the human eye and was found in lower concentrations in POAG AH. Reduction of OPN in young mice does not affect IOP.

A Novel Rat Model to Study the Role of Intracranial Pressure Modulation on Optic Neuropathies

Reduced intracranial pressure is considered a risk factor for glaucomatous optic neuropathies. All current data supporting intracranial pressure as a glaucoma risk factor comes from retrospective and prospective studies. Unfortunately, there are no relevant animal models for investigating this link experimentally. Here we report a novel rat model that can be used to study the role of intracranial pressure modulation on optic neuropathies. Stainless steel cannulae were inserted into the cisterna magna or the lateral ventricle of Sprague-Dawley and Brown Norway rats. The cannula was attached to a pressure transducer connected to a computer that recorded intracranial pressure in real-time. Intracranial pressure was modulated manually by adjusting the height of a column filled with artificial cerebrospinal fluid in relation to the animal’s head. After data collection the morphological appearance of the brain tissue was analyzed. Based on ease of surgery and ability to retain the cannula, Brown Norway rats with the cannula implanted in the lateral ventricle were selected for further studies. Baseline intracranial pressure for rats was 5.5±1.5 cm water (n=5). Lowering of the artificial cerebrospinal fluid column by 2 cm and 4 cm below head level reduced ICP to 3.7±1.0 cm water (n=5) and 1.5±0.6 cm water (n=4), a reduction of 33.0% and 72.7% below baseline. Raising the cerebrospinal fluid column by 4 cm increased ICP to 7.5±1.4 cm water (n=2) corresponding to a 38.3% increase in intracranial pressure. Histological studies confirmed correct cannula placement and indicated minimal invasive damage to brain tissues. Our data suggests that the intraventricular cannula model is a unique and viable model that can be used to study the effect of altered intracranial pressure on glaucomatous optic neuropathies.

MYOCILIN LEVELS IN PRIMARY OPEN-ANGLE GLAUCOMA AND PSEUDOEXFOLIATION GLAUCOMA HUMAN AQUEOUS HUMOR

PurposeTo determine the concentration of myocilin in primary open-angle glaucoma (POAG) and pseudoexfoliation glaucoma (PEXG) aqueous humor. MethodsAqueous humor was collected during surgery from patients with POAG, PEXG, and elective cataract removal (control). Volume-equivalent aqueous samples were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gradient gels. Quantification of myocilin levels was performed using Western blots probed with 2 independent N-terminal polyclonal anti-myocilin antibodies (AB1 and AB2) followed by densitometry. Myocilin levels in aqueous humor were quantified by plotting the densitometry readings of the aqueous samples against a recombinant myocilin standard curve. Total protein concentration was determined by Bradford protein assay. Transforming growth factor &bgr; 2 levels were assessed by enzyme-linked immunosorbent assay. ResultsMyocilin levels are significantly elevated in human POAG aqueous humor when compared with control aqueous humor (AB1: 0.66±0.53 ng/&mgr;L vs. 0.23±0.20 ng/&mgr;L, P<0.001; AB2: 0.98±0.59 ng/&mgr;L vs. 0.65±0.5 ng/&mgr;L, P<0.03; mean±SD). Myocilin makes up a larger percent of the total protein in POAG aqueous humor compared with control aqueous (AB1: 0.26±0.20% vs. 0.10±0.20%, P<0.001; AB2: 0.43±0.32% vs. 0.28±0.18%, P<0.05). In contrast to POAG, myocilin levels were not elevated in PEXG aqueous humor when compared with control aqueous humor. No correlation between myocilin and transforming growth factor &bgr; 2 levels was observed. ConclusionsMyocilin is elevated in POAG, but not in PEXG aqueous humor.

ATP-Sensitive Potassium (KATP) Channel Openers Diazoxide and Nicorandil Lower Intraocular Pressure In Vivo

PURPOSE To evaluate the expression of ATP-sensitive potassium (K(ATP)) channel subunits and study the effect of K(ATP) channel openers diazoxide and nicorandil on intraocular pressure (IOP) in an in vivo mouse model. METHODS Expression of K(ATP) channel subunits in normal C57BL/6 mouse eyes was studied by immunohistochemistry and confocal microscopy. Wild-type C57BL/6 mice were treated with K(ATP) channel openers diazoxide (n = 10) and nicorandil (n = 10) for 14 days. Similar treatments with diazoxide were performed on K(ir)6.2((-/-)) mice (n = 10). IOP was recorded with a handheld tonometer 1 hour, 4 hours, and 23 hours following daily treatment. Posttreatment histology was examined by light and transmission electron microscopy. RESULTS The K(ATP) channel subunits SUR2B, K(ir)6.1, and K(ir)6.2 were identified in all tissues within mouse eyes. Treatment with diazoxide in wild-type mice decreased IOP by 21.5 ± 3.2% with an absolute IOP reduction of 3.9 ± 0.6 mm Hg (P = 0.002). Nicorandil also decreased IOP (18.9 ± 1.8%) with an absolute IOP reduction of 3.4 ± 0.4 mm Hg (P = 0.002). Treatment with diazoxide in K(ir)6.2((-/-)) mice had no effect on IOP. No morphological abnormalities were observed in diazoxide- or nicorandil-treated eyes. CONCLUSIONS K(ATP) channel openers diazoxide and nicorandil are effective regulators of IOP in mouse eyes. K(ir)6.2 appears to be a major K(ATP) channel subunit through which IOP is lowered following treatment with diazoxide.

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.

ATP sensitive potassium channel openers: A new class of ocular hypotensive agents.

ABSTRACT ATP sensitive potassium (KATP) channels connect the metabolic and energetic state of cells due to their sensitivity to ATP and ADP concentrations. KATP channels have been identified in multiple tissues and organs of the body including heart, pancreas, vascular smooth muscles and skeletal muscles. These channels are obligatory hetero‐octamers and contain four sulfonylurea (SUR) and four potassium inward rectifier (Kir) subunits. Based on the particular type of SUR and Kir present, there are several tissue specific subtypes of KATP channels, each with their own unique set of functions. Recently, KATP channels have been reported in human and mouse ocular tissues. In ex vivo and in vivo model systems, KATP channel openers showed significant ocular hypotensive properties with no appearance of toxic side effects. Additionally, when used in conjunction with known intraocular pressure lowering drugs, an additive effect on IOP reduction was observed. These KATP channel openers have also been reported to protect the retinal ganglion cells during ischemic stress and glutamate induced toxicity suggesting a neuroprotective property for this drug class. Medications that are currently used for treating ocular hypertensive diseases like glaucoma do not directly protect the affected retinal cells, are sometimes ineffective and may show significant side effects. In light of this, KATP channel openers with both ocular hypotensive and neuroprotective properties, have the potential to develop into a new class of glaucoma therapeutics. HIGHLIGHTSATP sensitive potassium channels contain sulfonylurea receptor (SUR) and potassium inward rectifying (Kir) subunits.ATP sensitive potassium channels are found in many tissues of the body including the trabecular meshwork and retina.ATP sensitive potassium channel openers show ocular hypotensive activity in human anterior segment cultures and in mice.ATP sensitive potassium channels have direct neuroprotective properties on retinal ganglion cells.

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.