The Experts below are selected from a list of 321 Experts worldwide ranked by ideXlab platform

David L. Epstein - One of the best experts on this subject based on the ideXlab platform.

  • Endogenous Production of Extracellular Adenosine by Trabecular Meshwork Cells: Potential Role in Outflow Regulation
    Investigative ophthalmology & visual science, 2012
    Co-Authors: Coralia Luna, David L. Epstein, Ivan Spasojevic, Pedro Gonzalez
    Abstract:

    Purpose. To investigate the mechanisms for endogenous production of extracellular adenosine in trabecular meshwork (TM) cells and evaluate its physiological relevance to the regulation of Aqueous Humor Outflow facility.

  • Rho GTPase-mediated cytoskeletal organization in Schlemm's canal cells play a critical role in the regulation of Aqueous Humor Outflow facility.
    Journal of cellular biochemistry, 2011
    Co-Authors: Janardan Kumar, David L. Epstein
    Abstract:

    The increased intraocular pressure (IOP) has been considered to be an increased resistance of the Aqueous Humor Outflow through the inner wall of Schlemm's canal (SC) and/or the juxtacanalicular tissue (JCT). The Rho GTPase-regulated actomyosin organization appears to be an important mechanistic determinant of Aqueous Humor Outflow facility. Therefore, in this study, we have evaluated the effects of modulating Rho GTPase activity on actomyosin cytoskeletal organization, monolayer permeability/barrier function of human SC cells, and Aqueous Humor Outflow facility in enucleated porcine eyes ex vivo. Human SC cells, isolated from cadaver eyes, were treated with either Rho GTPase activators such as thrombin and lysophosphatidic acid (LPA), or a specific inhibitor (C3-exoenzyme) of Rho GTPases. Treatment of SC cells with thrombin and LPA led to increased formation of stress fibers, focal adhesion, and increased myosin light chain phosphorylation, whereas treatment with C3-exoenzyme showed the opposite effects like H-7 and ECA, known for increasing the Outflow facility in porcine eyes. The findings presented here suggest that LPA and thrombin, presumably through activation of Rho GTPase-mediated actomyosin cytoskeletal reorganization in SC cells, cause a decrease in monolayer permeability of SC cells as well as a decrease in Outflow facility of porcine eyes in ex vivo. Our results suggest that decrease in Aqueous Humor Outflow may be correlated better with the changes in cytoskeletal organizations of SC, which could be the prime locus of the Outflow resistance. J. Cell. Biochem. 112: 600–606, 2011. © 2010 Wiley-Liss, Inc.

  • Characterization of cytoskeleton-enriched protein fraction of the trabecular meshwork and ciliary muscle cells.
    Investigative ophthalmology & visual science, 2010
    Co-Authors: Toshihiro Inoue, Padmanabhan P. Pattabiraman, David L. Epstein, Rupalatha Maddala, Paula E. Pecen, Nikolai P. Skiba, P. Vasantha Rao
    Abstract:

    Homeostasis of Aqueous Humor Outflow through the conventional pathway is critical for the regulation of intraocular pressure (IOP). Any abnormality or blockage of Aqueous Humor Outflow through the conventional pathway consisting of trabecular meshwork, juxtacanalicular tissue, and Schlemm's canal can lead to increased IOP, a major risk factor for glaucoma.1 Glaucoma is the second leading cause of blindness in the United States, and lowering IOP by medical and surgical treatment is the primary avenue for the treatment of glaucoma.2 Therefore, there is a great deal of interest in understanding the regulation of Aqueous Humor drainage through the TM tissue and finding novel avenues for lowering IOP in glaucoma patients.3 The conventional Aqueous Humor Outflow pathway is pressure sensitive, and changes in IOP influence Aqueous Humor drainage through the TM.4–8 Further, the perfusion of various physiological compounds (e.g., cytokines, lipid growth factors, steroids, agonists, and peptide growth factors) is known to influence Aqueous Humor Outflow facility.1,3 Importantly, changes in TM and CM contractile and relaxation properties,9 morphology of the Outflow pathway,10 and extracellular matrix organization and synthesis11 are presumed to influence the resistance of Aqueous Humor drainage, suggesting a role for cell adhesive interactions, cytoskeletal organization, and mechanotransduction in homeostasis of Aqueous Humor Outflow.11–14 This conclusion/hypothesis is supported by several ex vivo and in vivo studies in which various cytoskeleton-interacting agents were documented to alter Aqueous Humor Outflow facility through the conventional pathway.3,12,15 In addition to agents that directly disrupt cytoskeletal (both microfilament and microtubule) integrity, inhibitors of various kinases that regulate actomyosin assembly, contraction, cell shape, and cell-ECM and cell-cell interactions, including Rho kinase, protein kinase C, and myosin light chain kinase, influence Aqueous Humor Outflow facility and IOP in animals and humans.12,14,16–19 Thus, multiple and consistent observations collectively support the inference that cytoskeletal proteins and their organization play a crucial role in the modulation of Aqueous Humor Outflow resistance and eventually IOP. Despite ample evidence for the importance of cytoskeletal proteins in Aqueous Humor Outflow pathway function, little is known about the composition of major cytoskeletal and cytoskeletal-associated proteins in the cells and tissues of the Aqueous Humor Outflow pathway. Additionally, although the TM and SC cells are thought to be derived from the neural crest and endothelia,20,21 respectively, these two cell types express smooth muscle properties and exhibit many characteristics similar to those of vascular endothelial cells. On the other hand, the CM and TM tissue, which originate from the differentiation of the neural crest,20 are reported to exhibit distinct contractile and relaxation properties.9 Moreover, while contraction of the CM increases Aqueous Humor Outflow facility, contraction of the TM is thought to decrease Aqueous Humor Outflow facility.9,22,23 These observations indicate possible differences between the CM and TM in either the cytoskeletal protein profile or the signaling mechanisms that control contraction and relaxation between these two types of tissue.9 Thus, unraveling the molecular basis for the differences in contractile and relaxation properties of TM and CM might provide novel insights for targeted therapy for glaucoma. As part of an effort to understand the contractile and relaxation properties and the mechanobiology of TM, SC, and CM tissues and the potential role of these events in modulation of Aqueous Humor Outflow facility, in this study we identified and characterized the major cytoskeletal and cytoskeletal-associated proteins of the TM and CM cells by mass spectrometry (MS). Further, the cytoskeletal protein profiles of the TM and CM were compared with and contrasted to those of vascular endothelial cells (because SC cells are endothelial in origin) to understand the differences and similarities among these three different cell types with respect to their smooth muscle-like and mechanical properties.

  • Effects of chemical inhibition of N-WASP, a critical regulator of actin polymerization on Aqueous Humor Outflow through the conventional pathway
    Experimental eye research, 2009
    Co-Authors: Toshihiro Inoue, Padmanabhan P. Pattabiraman, David L. Epstein, P. Vasantha Rao
    Abstract:

    The integrity of actin cytoskeletal organization in Aqueous Humor Outflow pathway is thought to play a critical role in modulation of Aqueous Humor Outflow through the trabecular meshwork. Our understanding of the regulation of actin cytoskeletal dynamics in Outflow pathway, however, is very limited. To explore the potential importance of Neural Wiskott-Aldrich syndrome protein (N-WASP), a critical regulator of actin polymerization/nucleation in Aqueous Humor Outflow pathway, the effects of Wiskostatin, a selective pharmacological inhibitor of N-WASP, on Aqueous Humor Outflow facility were evaluated using enucleated porcine eyes and a constant pressure perfusion system. Further, drug induced effects on actin cytoskeletal organization, cell adhesions, myosin II phosphorylation, matrix metalloproteinase (MMP) activity, and cytoskeletal protein profile in porcine trabecular meshwork (TM) cells were determined by immunofluorescence, zymography, and mass spectrometry. Aqueous Humor Outflow facility was increased significantly and progressively in the Wiskostatin perfused porcine eyes. The Wiskostatin perfused eyes appear to exhibit increased giant vacuoles in the inner wall of Aqueous plexi and deformation of Aqueous plexi. The Wiskostatin treated TM cells demonstrated extensive vacuoles in their cytosol, and both actin stress fibers and focal adhesions were decreased in a reversible manner. The drug-treated TM cells also revealed decreased myosin II and actin in the cytoskeletal enriched triton insoluble fraction but did not affect myosin II phosphorylation or MMP-2 activity. These data demonstrate that the chemical inhibition of N-WASP increases Aqueous Humor Outflow facility in association with decreased actomyosin interaction and cell adhesive interactions revealing the importance of N-WASP in homeostasis of Aqueous Humor Outflow.

  • Regulation of connective tissue growth factor expression in the Aqueous Humor Outflow pathway.
    Molecular vision, 2006
    Co-Authors: Saumil M. Chudgar, David L. Epstein, Pei-feng Deng, Rupalatha Maddala, P. Vasantha Rao
    Abstract:

    PURPOSE Connective Tissue Growth Factor (CTGF) is an inducible secretory protein known to regulate proliferation and extracellular matrix production in various cell types. We hypothesize that CTGF plays a critical role in the physiological regulation of Aqueous Humor Outflow through the trabecular meshwork (TM) by influencing extracellular matrix synthesis and organization. METHODS To determine the expression of CTGF in tissues of the Aqueous Outflow pathway, cells obtained from human TM and Schlemm's Canal (SC) were analyzed by PCR and western blot analysis. To understand the regulation of CTGF expression in TM cells, TM cells were either treated with various physiologic factors or subjected to cyclical stretch prior to analysis of CTGF expression by RT-PCR and western blot analysis. To study the effect of increased intraocular pressure on CTGF production, we perfused porcine eyes at high pressure (50 mm Hg) for 5 h, followed by analysis of CTGF expression by RT-PCR and western blotting. RESULTS Treatment of human TM cells treated with either serum or transforming growth factor-beta 1 led to a robust stimulation, compared to thrombin, lysophosphatidic acid (LPA), and dexamethasone, which elicited a relatively moderate induction of CTGF expression. Both high pressure perfusion and mechanical stretch were associated with increases in the levels of CTGF at the protein and transcript levels. CONCLUSIONS This study demonstrates that CTGF expression in TM cells is modulated by several physiological agonists and by increased ocular pressure and mechanical stretch. These results suggest that the regulation of CTGF expression within tissues of the Outflow pathway may play a role in the homeostasis of intraocular pressure, possibly by modulation of ECM production in these tissues.

P. Vasantha Rao - One of the best experts on this subject based on the ideXlab platform.

  • Characterization of cytoskeleton-enriched protein fraction of the trabecular meshwork and ciliary muscle cells.
    Investigative ophthalmology & visual science, 2010
    Co-Authors: Toshihiro Inoue, Padmanabhan P. Pattabiraman, David L. Epstein, Rupalatha Maddala, Paula E. Pecen, Nikolai P. Skiba, P. Vasantha Rao
    Abstract:

    Homeostasis of Aqueous Humor Outflow through the conventional pathway is critical for the regulation of intraocular pressure (IOP). Any abnormality or blockage of Aqueous Humor Outflow through the conventional pathway consisting of trabecular meshwork, juxtacanalicular tissue, and Schlemm's canal can lead to increased IOP, a major risk factor for glaucoma.1 Glaucoma is the second leading cause of blindness in the United States, and lowering IOP by medical and surgical treatment is the primary avenue for the treatment of glaucoma.2 Therefore, there is a great deal of interest in understanding the regulation of Aqueous Humor drainage through the TM tissue and finding novel avenues for lowering IOP in glaucoma patients.3 The conventional Aqueous Humor Outflow pathway is pressure sensitive, and changes in IOP influence Aqueous Humor drainage through the TM.4–8 Further, the perfusion of various physiological compounds (e.g., cytokines, lipid growth factors, steroids, agonists, and peptide growth factors) is known to influence Aqueous Humor Outflow facility.1,3 Importantly, changes in TM and CM contractile and relaxation properties,9 morphology of the Outflow pathway,10 and extracellular matrix organization and synthesis11 are presumed to influence the resistance of Aqueous Humor drainage, suggesting a role for cell adhesive interactions, cytoskeletal organization, and mechanotransduction in homeostasis of Aqueous Humor Outflow.11–14 This conclusion/hypothesis is supported by several ex vivo and in vivo studies in which various cytoskeleton-interacting agents were documented to alter Aqueous Humor Outflow facility through the conventional pathway.3,12,15 In addition to agents that directly disrupt cytoskeletal (both microfilament and microtubule) integrity, inhibitors of various kinases that regulate actomyosin assembly, contraction, cell shape, and cell-ECM and cell-cell interactions, including Rho kinase, protein kinase C, and myosin light chain kinase, influence Aqueous Humor Outflow facility and IOP in animals and humans.12,14,16–19 Thus, multiple and consistent observations collectively support the inference that cytoskeletal proteins and their organization play a crucial role in the modulation of Aqueous Humor Outflow resistance and eventually IOP. Despite ample evidence for the importance of cytoskeletal proteins in Aqueous Humor Outflow pathway function, little is known about the composition of major cytoskeletal and cytoskeletal-associated proteins in the cells and tissues of the Aqueous Humor Outflow pathway. Additionally, although the TM and SC cells are thought to be derived from the neural crest and endothelia,20,21 respectively, these two cell types express smooth muscle properties and exhibit many characteristics similar to those of vascular endothelial cells. On the other hand, the CM and TM tissue, which originate from the differentiation of the neural crest,20 are reported to exhibit distinct contractile and relaxation properties.9 Moreover, while contraction of the CM increases Aqueous Humor Outflow facility, contraction of the TM is thought to decrease Aqueous Humor Outflow facility.9,22,23 These observations indicate possible differences between the CM and TM in either the cytoskeletal protein profile or the signaling mechanisms that control contraction and relaxation between these two types of tissue.9 Thus, unraveling the molecular basis for the differences in contractile and relaxation properties of TM and CM might provide novel insights for targeted therapy for glaucoma. As part of an effort to understand the contractile and relaxation properties and the mechanobiology of TM, SC, and CM tissues and the potential role of these events in modulation of Aqueous Humor Outflow facility, in this study we identified and characterized the major cytoskeletal and cytoskeletal-associated proteins of the TM and CM cells by mass spectrometry (MS). Further, the cytoskeletal protein profiles of the TM and CM were compared with and contrasted to those of vascular endothelial cells (because SC cells are endothelial in origin) to understand the differences and similarities among these three different cell types with respect to their smooth muscle-like and mechanical properties.

  • Effects of chemical inhibition of N-WASP, a critical regulator of actin polymerization on Aqueous Humor Outflow through the conventional pathway
    Experimental eye research, 2009
    Co-Authors: Toshihiro Inoue, Padmanabhan P. Pattabiraman, David L. Epstein, P. Vasantha Rao
    Abstract:

    The integrity of actin cytoskeletal organization in Aqueous Humor Outflow pathway is thought to play a critical role in modulation of Aqueous Humor Outflow through the trabecular meshwork. Our understanding of the regulation of actin cytoskeletal dynamics in Outflow pathway, however, is very limited. To explore the potential importance of Neural Wiskott-Aldrich syndrome protein (N-WASP), a critical regulator of actin polymerization/nucleation in Aqueous Humor Outflow pathway, the effects of Wiskostatin, a selective pharmacological inhibitor of N-WASP, on Aqueous Humor Outflow facility were evaluated using enucleated porcine eyes and a constant pressure perfusion system. Further, drug induced effects on actin cytoskeletal organization, cell adhesions, myosin II phosphorylation, matrix metalloproteinase (MMP) activity, and cytoskeletal protein profile in porcine trabecular meshwork (TM) cells were determined by immunofluorescence, zymography, and mass spectrometry. Aqueous Humor Outflow facility was increased significantly and progressively in the Wiskostatin perfused porcine eyes. The Wiskostatin perfused eyes appear to exhibit increased giant vacuoles in the inner wall of Aqueous plexi and deformation of Aqueous plexi. The Wiskostatin treated TM cells demonstrated extensive vacuoles in their cytosol, and both actin stress fibers and focal adhesions were decreased in a reversible manner. The drug-treated TM cells also revealed decreased myosin II and actin in the cytoskeletal enriched triton insoluble fraction but did not affect myosin II phosphorylation or MMP-2 activity. These data demonstrate that the chemical inhibition of N-WASP increases Aqueous Humor Outflow facility in association with decreased actomyosin interaction and cell adhesive interactions revealing the importance of N-WASP in homeostasis of Aqueous Humor Outflow.

  • Effects of Pharmacologic Inhibition of Protein Geranylgeranyltransferase Type I on Aqueous Humor Outflow through the Trabecular Meshwork
    Investigative ophthalmology & visual science, 2008
    Co-Authors: P. Vasantha Rao, Toshihiro Inoue, Yuri K. Peterson, Patrick J. Casey
    Abstract:

    Glaucoma, a leading cause of blindness characterized by optic nerve degeneration and progressive visual field loss, is commonly associated with elevated intraocular pressure (IOP). In primary open-angle glaucoma (POAG), the most common form of the disease, elevated IOP occurs as a result of pathologically increased resistance to drainage of Aqueous Humor through the pressure-dependent trabecular or conventional Outflow system.1 Therefore, elevated IOP is considered a major risk factor for glaucoma, and lowering IOP is the only modality available for the treatment of POAG.1 The conventional Outflow pathway is composed of the trabecular mesh-work (TM), juxtacanalicular region (JCT), and Schlemm canal. In humans this pathway represents a predominant route of Aqueous Humor drainage.2,3 Aqueous Humor is secreted by nonpigmented epithelial cells that line the iris and ciliary body and flows into the anterior chamber, which then drains through the TM into Schlemm canal and the episcleral veins on a continuous basis.4 Glaucoma is an age-related disease, and increased contractile activity and cell adhesive interactions of the cells of Aqueous Outflow pathway are believed to be partly responsible for the elevated IOP and POAG.4-7 Perfusion studies using cytoskeleton-interfering agents such as actin-depolymerizing agents, inhibitors of myosin light chain kinase, myosin II, protein kinase C, Rho GTPase, and Rho kinase, in different model systems, have indicated a link between cytoskeletal integrity within the TM and Aqueous Outflow through the TM.2,4,5,7,8 Agents that increase actin depolymerization and decrease cell– extracellular matrix interactions and myosin II phosphorylation in the TM increase Aqueous Outflow, presumably by cellular relaxation, altering the geometry of Outflow pathway and fluid flow through the inner wall of the Schlemm canal.2,7 In contrast to the effects of inhibiting Rho, Rho-kinase, myosin II, and myosin light-chain kinase, physiological agonists including endothelin-1, lysophosphatidic acid, thrombin, and TGF-β (which are known to activate Rho/Rho kinase signaling) have been shown to reduce Aqueous Humor Outflow in the perfused model systems.6,9-11 These different observations collectively implicate the activation status of the Rho/Rho-kinase pathway, heterotrimeric G-proteins, and the contractile force of the TM in the regulation of Aqueous Humor Outflow and homeostasis of IOP. GTP-binding proteins, including small GTP-binding proteins such as Rho and Rac and the heterotrimeric G-proteins G12/13 and Gq, regulate tissue contractile and relaxation properties and actin cytoskeletal organization and cell adhesive interactions in smooth muscle and nonmuscle cells.12-15 The activity of these G proteins (both monotrimeric and heterotrimeric) requires the addition of an isoprenoid lipid at the carboxyl terminal through enzyme-mediated isoprenylation posttranslational modification. This lipid modification aids the G-proteins and Rho GTPases to localize to the plasma membrane, where they interact with other regulatory proteins and participate in signaling activity to control various cellular processes.16,17 The CaaX prenyltransferases protein farnesyltransferase and protein geranylgeranyltransferase type I (GGTase-I) attach either a 15-carbon farnesyl group or a 20-carbon geranylgeranyl group, respectively, to the cysteine residue found within the CaaX motif at the C-terminus of protein substrates.16 GGTase-I substrates include Rho and Rac GTPases and the gamma subunit of most heterotrimeric G-proteins, and the inhibition of isoprenylation has been considered one of the rational and promising approaches to modulate the activity of oncogenic small GTP-binding proteins and heterotrimeric G-proteins with therapeutic importance for the treatment of cancers.16,18 Recently, a novel small molecular pyrazole-derived compound, GGTI-DU40 (Fig. 1), has been identified and characterized as a specific and potent inhibitor of GGTase-I. GGTI-DU40 has been shown to impair membrane localization of various geranylgeranylated G proteins, including Rho and Rap GTPases, by inhibiting the isoprenylation modification in different cell types.19 Figure 1 Structure of GGTI-DU40. Reprinted with permission from Peterson YK, Kelly P, Weinbaum CA, Casey PJ. A novel protein geranylgeranyltransferase-I inhibitor with high potency, selectivity, and cellular activity. J Biol Chem. 2006;281:12445–12450. ... Rho GTPases and G-protein–coupled receptors regulate cell shape, actin cytoskeletal organization, cell adhesive interactions, and contractile activity, which have now been well recognized to participate in the modulation of Aqueous Humor Outflow.2,6,7 Indeed, experiments using a peptidomimetic GGTI, GGTI-298, showed blocking geranylgeranylation can decrease leukocyte adhesion.20 In this study, we have explored the effects of GGTI-DU40 on Aqueous Humor Outflow and TM cell cytoskeletal and adhesive characteristics. The results of this study reveal the promising effect of GGTI-DU40 on Aqueous Outflow through the TM pathway and identify GGTase-I as a potential molecular target for lowering IOP in glaucoma patients.

  • Regulation of connective tissue growth factor expression in the Aqueous Humor Outflow pathway.
    Molecular vision, 2006
    Co-Authors: Saumil M. Chudgar, David L. Epstein, Pei-feng Deng, Rupalatha Maddala, P. Vasantha Rao
    Abstract:

    PURPOSE Connective Tissue Growth Factor (CTGF) is an inducible secretory protein known to regulate proliferation and extracellular matrix production in various cell types. We hypothesize that CTGF plays a critical role in the physiological regulation of Aqueous Humor Outflow through the trabecular meshwork (TM) by influencing extracellular matrix synthesis and organization. METHODS To determine the expression of CTGF in tissues of the Aqueous Outflow pathway, cells obtained from human TM and Schlemm's Canal (SC) were analyzed by PCR and western blot analysis. To understand the regulation of CTGF expression in TM cells, TM cells were either treated with various physiologic factors or subjected to cyclical stretch prior to analysis of CTGF expression by RT-PCR and western blot analysis. To study the effect of increased intraocular pressure on CTGF production, we perfused porcine eyes at high pressure (50 mm Hg) for 5 h, followed by analysis of CTGF expression by RT-PCR and western blotting. RESULTS Treatment of human TM cells treated with either serum or transforming growth factor-beta 1 led to a robust stimulation, compared to thrombin, lysophosphatidic acid (LPA), and dexamethasone, which elicited a relatively moderate induction of CTGF expression. Both high pressure perfusion and mechanical stretch were associated with increases in the levels of CTGF at the protein and transcript levels. CONCLUSIONS This study demonstrates that CTGF expression in TM cells is modulated by several physiological agonists and by increased ocular pressure and mechanical stretch. These results suggest that the regulation of CTGF expression within tissues of the Outflow pathway may play a role in the homeostasis of intraocular pressure, possibly by modulation of ECM production in these tissues.

  • Blebbistatin, a Novel Inhibitor of Myosin II ATPase Activity, Increases Aqueous Humor Outflow Facility in Perfused Enucleated Porcine Eyes
    Investigative ophthalmology & visual science, 2005
    Co-Authors: Min Zhang, P. Vasantha Rao
    Abstract:

    PURPOSE To investigate the specific role of myosin II, a critical biochemical determinant of cellular contraction, in modulation of Aqueous Humor Outflow facility through the trabecular meshwork (TM) pathway. METHODS Expression of the nonmuscle myosin II heavy chains (IIA, IIB, and IIC) in human TM and ciliary body (CB) cells was determined by RT-PCR analyses. The effects of inhibition of myosin II on cell morphology, actomyosin organization, and cell adhesions were evaluated in porcine TM and CB cells treated with blebbistatin, a cell-permeable, specific inhibitor of myosin II adenosine triphosphatase (ATPase) activity. Changes in Aqueous Humor Outflow facility were determined in enucleated porcine eyes by using a constant-pressure Grant perfusion model system. Ultrastructural integrity of the Outflow pathway in drug-perfused eyes was analyzed by transmission electron microscopy. RESULTS Expression of nonmuscle myosin IIA and IIB was confirmed in both human TM and CB cells. Confluent cultures of primary porcine TM and CB cells treated with blebbistatin in the presence of serum revealed dose (10-200 microM)-dependent changes in cell morphology, decreases in actin stress fiber content and in focal adhesions and adherens junctions. These changes were found to be reversible within 24 hours of drug withdrawal from the cell culture media. Blebbistatin did not affect the status of myosin light chain phosphorylation in TM cells. Perfusion of enucleated porcine eyes for 5 hours with 100 and 200 microM blebbistatin produced a significant increase (P < 0.01, n = 7) in Aqueous Outflow facility (53% and 64%, respectively) from the baseline facility, compared with a 21% facility increase in sham control specimens. The integrity of the inner wall of Aqueous plexi in drug-perfused porcine eyes was found to be intact, and TM cell morphology appeared to be similar to that noted in sham-treated eyes. CONCLUSIONS These data demonstrate that selective inhibition of myosin II in the Aqueous Humor Outflow pathway leads to increased Aqueous Outflow facility, suggesting a critical role for myosin II in the regulation of Aqueous Humor Outflow facility. This study also suggests myosin II as a potential therapeutic target for lowering intraocular pressure in patients with glaucoma.

Robert N. Weinreb - One of the best experts on this subject based on the ideXlab platform.

  • automated circumferential construction of first order Aqueous Humor Outflow pathways using spectral domain optical coherence tomography
    Journal of Biomedical Optics, 2017
    Co-Authors: Alex S. Huang, Akram Belghith, Anna Dastiridou, Vikas Chopra, Linda M. Zangwill, Robert N. Weinreb
    Abstract:

    The purpose was to create a three-dimensional (3-D) model of circumferential Aqueous Humor Outflow (AHO) in a living human eye with an automated detection algorithm for Schlemm’s canal (SC) and first-order collector channels (CC) applied to spectral-domain optical coherence tomography (SD-OCT). Anterior segment SD-OCT scans from a subject were acquired circumferentially around the limbus. A Bayesian Ridge method was used to approximate the location of the SC on infrared confocal laser scanning ophthalmoscopic images with a cross multiplication tool developed to initiate SC/CC detection automated through a fuzzy hidden Markov Chain approach. Automatic segmentation of SC and initial CC’s was manually confirmed by two masked graders. Outflow pathways detected by the segmentation algorithm were reconstructed into a 3-D representation of AHO. Overall, only <1% of images (5114 total B-scans) were ungradable. Automatic segmentation algorithm performed well with SC detection 98.3% of the time and <0.1% false positive detection compared to expert grader consensus. CC was detected 84.2% of the time with 1.4% false positive detection. 3-D representation of AHO pathways demonstrated variably thicker and thinner SC with some clear CC roots. Circumferential (360 deg), automated, and validated AHO detection of angle structures in the living human eye with reconstruction was possible.

  • Automated circumferential construction of first-order Aqueous Humor Outflow pathways using spectral-domain optical coherence tomography.
    Journal of biomedical optics, 2017
    Co-Authors: Alex S. Huang, Akram Belghith, Anna Dastiridou, Vikas Chopra, Linda M. Zangwill, Robert N. Weinreb
    Abstract:

    The purpose was to create a three-dimensional (3-D) model of circumferential Aqueous Humor Outflow (AHO) in a living human eye with an automated detection algorithm for Schlemm’s canal (SC) and first-order collector channels (CC) applied to spectral-domain optical coherence tomography (SD-OCT). Anterior segment SD-OCT scans from a subject were acquired circumferentially around the limbus. A Bayesian Ridge method was used to approximate the location of the SC on infrared confocal laser scanning ophthalmoscopic images with a cross multiplication tool developed to initiate SC/CC detection automated through a fuzzy hidden Markov Chain approach. Automatic segmentation of SC and initial CC’s was manually confirmed by two masked graders. Outflow pathways detected by the segmentation algorithm were reconstructed into a 3-D representation of AHO. Overall, only

  • Aqueous Angiography in Living Nonhuman Primates Shows Segmental, Pulsatile, and Dynamic Angiographic Aqueous Humor Outflow.
    Ophthalmology, 2017
    Co-Authors: Alex S. Huang, Diya Yang, Huaizhou Wang, Ningli Wang, Robert N. Weinreb
    Abstract:

    Purpose To evaluate the feasibility of safely performing Aqueous angiography in intact eyes of living nonhuman primates (NHPs) for evaluating Aqueous Humor Outflow and segmental patterns. Design Cross-sectional, observational study. Subjects Six nonhuman primates. Methods Aqueous angiography was performed in 6 nonhuman primates. After anesthesia, an anterior chamber (AC) maintainer was placed through a temporal 1-mm side-port wound. Indocyanine green (ICG; 0.4%) or 2.5% fluorescein was introduced (individually or in sequence) into the eye with a gravity-driven constant-pressure system. Aqueous angiography images were obtained with a Spectralis HRA+OCT (Heidelberg Engineering GmbH, Heidelberg, Germany) suspended over the NHP eye using a custom-designed surgical boom arm. Concurrent anterior segment optical coherence tomography (OCT) was performed on distally angiographically positive and negative regions. Main Outcome Measures Angiographic patterns described by location, time-course, choice of tracer, and anterior-segment OCT. Results Aqueous angiography in the living NHP eye demonstrated mostly stable angiographic patterns. With multimodal imaging, angiographically positive signal co-localized with episcleral veins as identified by infrared imaging and intrascleral lumens, as demonstrated by anterior segment OCT. Sequential Aqueous angiography in individual eyes with ICG followed by fluorescein showed similar angiographic patterns. A pulsatile nature of Aqueous angiographic Outflow was sometimes observed. Aqueous angiographic patterns could also dynamically change. In some instances, positive angiographic flow suddenly arose in regions previously without an angiographic signal. Alternatively, an angiographic signal could suddenly disappear from regions in which an angiographic signal was initially documented. Conclusions Aqueous angiography in living NHPs demonstrated segmental and pulsatile patterns with a newly described ability to dynamically shift. These characteristics further the understanding of live Aqueous Humor Outflow biology and may be useful in improving glaucoma surgeries aimed at trabecular meshwork bypass.

  • Aqueous Humor Outflow Structure and Function Imaging At the Bench and Bedside: A Review
    Journal of clinical & experimental ophthalmology, 2016
    Co-Authors: Alex S. Huang, Chirayu Mohindroo, Robert N. Weinreb
    Abstract:

    Anterior segment glaucoma clinical care and research has recently gained new focus because of novel imaging modalities and the advent of angle-based surgical treatments. Traditional investigation drawn to the trabecular meshwork now emphasizes the entire conventional Aqueous Humor Outflow (AHO) pathway from the anterior chamber to the episcleral vein. AHO investigation can be divided into structural and functional assessments using different methods. The historical basis for studying the anterior segment of the eye and AHO in glaucoma is discussed. Structural studies of AHO are reviewed and include traditional pathological approaches to modern tools such as multi-model two-photon microscopy and optical coherence tomography. Functional assessment focuses on visualizing AHO itself through a variety of non-real-time and real-time techniques such as Aqueous angiography. Implications of distal Outflow resistance and segmental AHO are discussed with an emphasis on melding bench-side research to viable clinical applications. Through the development of an improved structure: function relationship for AHO in the anterior segment of the normal and diseased eye, a better understanding of the eye with improved therapeutics may be developed.

  • Aqueous Angiography: Real-Time and Physiologic Aqueous Humor Outflow Imaging
    PloS one, 2016
    Co-Authors: Sindhu Saraswathy, Robert N. Weinreb, James C. Tan, Brian A. Francis, David R. Hinton, Alex S. Huang
    Abstract:

    Trabecular meshwork (TM) bypass surgeries attempt to enhance Aqueous Humor Outflow (AHO) to lower intraocular pressure (IOP). While TM bypass results are promising, inconsistent success is seen. One hypothesis for this variability rests upon segmental (non-360 degrees uniform) AHO. We describe Aqueous angiography as a real-time and physiologic AHO imaging technique in model eyes as a way to simulate live AHO imaging.Pig (n = 46) and human (n = 6) enucleated eyes were obtained, orientated based upon inferior oblique insertion, and pre-perfused with balanced salt solution via a Lewicky AC maintainer through a 1mm side-port. Fluorescein (2.5%) was introduced intracamerally at 10 or 30 mm Hg. With an angiographer, infrared and fluorescent (486 nm) images were acquired. Image processing allowed for collection of pixel information based on intensity or location for statistical analyses. Concurrent OCT was performed, and fixable fluorescent dextrans were introduced into the eye for histological analysis of angiographically active areas.Aqueous angiography yielded high quality images with segmental patterns (p

Alex S. Huang - One of the best experts on this subject based on the ideXlab platform.

  • Visual Assessment of Aqueous Humor Outflow.
    Asia-Pacific journal of ophthalmology (Philadelphia Pa.), 2019
    Co-Authors: Xiaobin Xie, Goichi Akiyama, Thania Bogarin, Sindhu Saraswathy, Alex S. Huang
    Abstract:

    In the past decade, many new pharmacological and surgical treatments have become available to lower intraocular pressure (IOP) for glaucoma. The majority of these options have targeted improving Aqueous Humor Outflow (AHO). At the same time, in addition to new treatments, research advances in AHO assessment have led to the development of new tools to structurally assess AHO pathways and to visualize where Aqueous is flowing in the eye. These new imaging modalities have uncovered novel AHO observations that challenge traditional AHO concepts. New behaviors including segmental, pulsatile, and dynamic AHO may have relevance to the disease and the level of therapeutic response for IOP-lowering treatments. By better understanding the regulation of segmental, pulsatile, and dynamic AHO, it may be possible to find new and innovative treatments for glaucoma aiming at these new AHO behaviors.

  • Automated circumferential construction of first-order Aqueous Humor Outflow pathways using spectral-domain optical coherence tomography.
    Journal of biomedical optics, 2017
    Co-Authors: Alex S. Huang, Akram Belghith, Anna Dastiridou, Vikas Chopra, Linda M. Zangwill, Robert N. Weinreb
    Abstract:

    The purpose was to create a three-dimensional (3-D) model of circumferential Aqueous Humor Outflow (AHO) in a living human eye with an automated detection algorithm for Schlemm’s canal (SC) and first-order collector channels (CC) applied to spectral-domain optical coherence tomography (SD-OCT). Anterior segment SD-OCT scans from a subject were acquired circumferentially around the limbus. A Bayesian Ridge method was used to approximate the location of the SC on infrared confocal laser scanning ophthalmoscopic images with a cross multiplication tool developed to initiate SC/CC detection automated through a fuzzy hidden Markov Chain approach. Automatic segmentation of SC and initial CC’s was manually confirmed by two masked graders. Outflow pathways detected by the segmentation algorithm were reconstructed into a 3-D representation of AHO. Overall, only

  • automated circumferential construction of first order Aqueous Humor Outflow pathways using spectral domain optical coherence tomography
    Journal of Biomedical Optics, 2017
    Co-Authors: Alex S. Huang, Akram Belghith, Anna Dastiridou, Vikas Chopra, Linda M. Zangwill, Robert N. Weinreb
    Abstract:

    The purpose was to create a three-dimensional (3-D) model of circumferential Aqueous Humor Outflow (AHO) in a living human eye with an automated detection algorithm for Schlemm’s canal (SC) and first-order collector channels (CC) applied to spectral-domain optical coherence tomography (SD-OCT). Anterior segment SD-OCT scans from a subject were acquired circumferentially around the limbus. A Bayesian Ridge method was used to approximate the location of the SC on infrared confocal laser scanning ophthalmoscopic images with a cross multiplication tool developed to initiate SC/CC detection automated through a fuzzy hidden Markov Chain approach. Automatic segmentation of SC and initial CC’s was manually confirmed by two masked graders. Outflow pathways detected by the segmentation algorithm were reconstructed into a 3-D representation of AHO. Overall, only <1% of images (5114 total B-scans) were ungradable. Automatic segmentation algorithm performed well with SC detection 98.3% of the time and <0.1% false positive detection compared to expert grader consensus. CC was detected 84.2% of the time with 1.4% false positive detection. 3-D representation of AHO pathways demonstrated variably thicker and thinner SC with some clear CC roots. Circumferential (360 deg), automated, and validated AHO detection of angle structures in the living human eye with reconstruction was possible.

  • Aqueous Angiography in Living Nonhuman Primates Shows Segmental, Pulsatile, and Dynamic Angiographic Aqueous Humor Outflow.
    Ophthalmology, 2017
    Co-Authors: Alex S. Huang, Diya Yang, Huaizhou Wang, Ningli Wang, Robert N. Weinreb
    Abstract:

    Purpose To evaluate the feasibility of safely performing Aqueous angiography in intact eyes of living nonhuman primates (NHPs) for evaluating Aqueous Humor Outflow and segmental patterns. Design Cross-sectional, observational study. Subjects Six nonhuman primates. Methods Aqueous angiography was performed in 6 nonhuman primates. After anesthesia, an anterior chamber (AC) maintainer was placed through a temporal 1-mm side-port wound. Indocyanine green (ICG; 0.4%) or 2.5% fluorescein was introduced (individually or in sequence) into the eye with a gravity-driven constant-pressure system. Aqueous angiography images were obtained with a Spectralis HRA+OCT (Heidelberg Engineering GmbH, Heidelberg, Germany) suspended over the NHP eye using a custom-designed surgical boom arm. Concurrent anterior segment optical coherence tomography (OCT) was performed on distally angiographically positive and negative regions. Main Outcome Measures Angiographic patterns described by location, time-course, choice of tracer, and anterior-segment OCT. Results Aqueous angiography in the living NHP eye demonstrated mostly stable angiographic patterns. With multimodal imaging, angiographically positive signal co-localized with episcleral veins as identified by infrared imaging and intrascleral lumens, as demonstrated by anterior segment OCT. Sequential Aqueous angiography in individual eyes with ICG followed by fluorescein showed similar angiographic patterns. A pulsatile nature of Aqueous angiographic Outflow was sometimes observed. Aqueous angiographic patterns could also dynamically change. In some instances, positive angiographic flow suddenly arose in regions previously without an angiographic signal. Alternatively, an angiographic signal could suddenly disappear from regions in which an angiographic signal was initially documented. Conclusions Aqueous angiography in living NHPs demonstrated segmental and pulsatile patterns with a newly described ability to dynamically shift. These characteristics further the understanding of live Aqueous Humor Outflow biology and may be useful in improving glaucoma surgeries aimed at trabecular meshwork bypass.

  • Aqueous Humor Outflow Structure and Function Imaging At the Bench and Bedside: A Review
    Journal of clinical & experimental ophthalmology, 2016
    Co-Authors: Alex S. Huang, Chirayu Mohindroo, Robert N. Weinreb
    Abstract:

    Anterior segment glaucoma clinical care and research has recently gained new focus because of novel imaging modalities and the advent of angle-based surgical treatments. Traditional investigation drawn to the trabecular meshwork now emphasizes the entire conventional Aqueous Humor Outflow (AHO) pathway from the anterior chamber to the episcleral vein. AHO investigation can be divided into structural and functional assessments using different methods. The historical basis for studying the anterior segment of the eye and AHO in glaucoma is discussed. Structural studies of AHO are reviewed and include traditional pathological approaches to modern tools such as multi-model two-photon microscopy and optical coherence tomography. Functional assessment focuses on visualizing AHO itself through a variety of non-real-time and real-time techniques such as Aqueous angiography. Implications of distal Outflow resistance and segmental AHO are discussed with an emphasis on melding bench-side research to viable clinical applications. Through the development of an improved structure: function relationship for AHO in the anterior segment of the normal and diseased eye, a better understanding of the eye with improved therapeutics may be developed.

Pei-feng Deng - One of the best experts on this subject based on the ideXlab platform.

  • Regulation of connective tissue growth factor expression in the Aqueous Humor Outflow pathway.
    Molecular vision, 2006
    Co-Authors: Saumil M. Chudgar, David L. Epstein, Pei-feng Deng, Rupalatha Maddala, P. Vasantha Rao
    Abstract:

    PURPOSE Connective Tissue Growth Factor (CTGF) is an inducible secretory protein known to regulate proliferation and extracellular matrix production in various cell types. We hypothesize that CTGF plays a critical role in the physiological regulation of Aqueous Humor Outflow through the trabecular meshwork (TM) by influencing extracellular matrix synthesis and organization. METHODS To determine the expression of CTGF in tissues of the Aqueous Outflow pathway, cells obtained from human TM and Schlemm's Canal (SC) were analyzed by PCR and western blot analysis. To understand the regulation of CTGF expression in TM cells, TM cells were either treated with various physiologic factors or subjected to cyclical stretch prior to analysis of CTGF expression by RT-PCR and western blot analysis. To study the effect of increased intraocular pressure on CTGF production, we perfused porcine eyes at high pressure (50 mm Hg) for 5 h, followed by analysis of CTGF expression by RT-PCR and western blotting. RESULTS Treatment of human TM cells treated with either serum or transforming growth factor-beta 1 led to a robust stimulation, compared to thrombin, lysophosphatidic acid (LPA), and dexamethasone, which elicited a relatively moderate induction of CTGF expression. Both high pressure perfusion and mechanical stretch were associated with increases in the levels of CTGF at the protein and transcript levels. CONCLUSIONS This study demonstrates that CTGF expression in TM cells is modulated by several physiological agonists and by increased ocular pressure and mechanical stretch. These results suggest that the regulation of CTGF expression within tissues of the Outflow pathway may play a role in the homeostasis of intraocular pressure, possibly by modulation of ECM production in these tissues.

  • Effects of Cholesterol-Lowering Statins on the Aqueous Humor Outflow Pathway
    Investigative ophthalmology & visual science, 2005
    Co-Authors: Julia Song, David L. Epstein, Pei-feng Deng, Sandra S. Stinnett, P. Vasantha Rao
    Abstract:

    PURPOSE. To investigate the effects of cholesterol-lowering statin drugs on trabecular meshwork cellular properties and Aqueous Humor Outflow. METHODS. Primary cell cultures of porcine trabecular meshwork (PTM) and ciliary body (PCB) were treated with either lovastatin or compactin, to determine the effects of statins on cell shape, actin cytoskeletal organization, and cell- extracellular matrix interactions (focal adhesions) by immunofluorescence staining. Changes in myosin light-chain (MLC) phosphorylation were evaluated by Western blot analysis. Changes in Rho GTPase content of membrane fractions from lovastatin-treated PTM cells were assessed by Western blot analysis. A constant-flow, organ-culture perfusion system was used to measure the effects of statins on Aqueous Humor Outflow facility in the anterior segments of porcine eyes. RESULTS. PTM and PCB cells treated with lovastatin or compactin exhibited dramatic changes in cell shape and cytoskeletal organization within 24 hours, consisting of cell rounding, actin depolymerization, and decreased focal adhesions. These effects were found to be reversible on supplementation with geranylgeranyl pyrophosphate. Both lovastatin and compactin decreased MLC phosphorylation in PTM and PCB cells. PTM cells treated with lovastatin exhibited marked decreases in membrane-bound Rho GTPase. In addition, perfusion of organ-cultured porcine eye anterior segments with 100 μM lovastatin for 96 hours caused a significant increase in Aqueous Humor Outflow facility (110%) compared with control eyes, in a reversible manner. CONCLUSIONS. This study demonstrates that the statin drugs lovastatin and compactin induce changes in cell shape and actin cytoskeletal organization and decrease MLC phosphorylation in PTM and PCB cells, all of which are events that are likely to lead to cellular and tissue relaxation. In addition, these effects of the statins appear to be mediated by inhibition of isoprenylation of the small GTP-binding proteins such as Rho GTPase. An important finding is that statins exert an ocular hypotensive response in an organ-culture perfusion model, indicating the potential for this class of drugs in glaucoma therapy.

  • Expression of dominant negative Rho-binding domain of Rho-kinase in organ cultured human eye anterior segments increases Aqueous Humor Outflow.
    Molecular vision, 2005
    Co-Authors: Ponugoti Vasantha Rao, David L. Epstein, Pei-feng Deng, Rupalatha Maddala, Hiroaki Shimokawa
    Abstract:

    Purpose: Based on pharmacological inhibition of activity, a role has been proposed for Rho-kinase in the modulation of Aqueous Outflow and intraocular pressure (IOP). This study employed a molecular approach to specifically address the role of Rho-kinase in the modulation of Aqueous Humor Outflow. Methods: Adenoviral vectors expressing green fluorescent protein alone (Ad-GFP) or the dominant negative Rho-binding domain of Rho-kinase and GFP (Ad-DNRK-GFP) were utilized in these experiments. Human and porcine primary trabecular meshwork (TM) cells were infected with 30 MOI (multiplicity of infection) of Ad-GFP alone or with AdDNRK-GFP. Changes in cell shape, actomyosin cytoskeletal integrity, and the status of myosin light chain (MLC) phosphorylation were evaluated. The Aqueous Outflow facility was determined in organ cultured anterior segments of human cadaver eyes infected with 10 7 pfu adenoviral vectors (Ad-GFP or Ad-DNRK-GFP) using a constant flow perfusion system. Results: Expression of DNRK resulted in changes in cell shape (cell rounding, cell-cell detachment) and decreased actin stress fiber and focal adhesion staining in TM cells. These cellular changes were associated with substantially reduced myosin light chain phosphorylation. Additionally, organ cultured human eye anterior segments infected with Ad-DNRKGFP exhibited a significant increase in the Outflow facility (80%, n=9) compared to control anterior segments infected with Ad-GFP (5%). Conclusions: This study demonstrated that specific inhibition of Rho-kinase activity in trabecular meshwork cells led to alterations in cell shape and presumed contractile properties, and we hypothesize that these morphological and contractile events underlie the observed effects of dominant negative Rho-kinase on the Aqueous Humor Outflow facility. These data provide molecular evidence for the hypothesis of Rho-kinase being a potential cellular target involved in the regulation of Aqueous Humor Outflow resistance, with implications for novel glaucoma therapy.

  • Role of lysophospholipid growth factors in the modulation of Aqueous Humor Outflow facility.
    Investigative ophthalmology & visual science, 2004
    Co-Authors: Priyatham S. Mettu, David L. Epstein, Pei-feng Deng, Uma K. Misra, Govind Gawdi, P. Vasantha Rao
    Abstract:

    Purpose To investigate the role of lysophospholipid growth factors in the regulation of Aqueous Humor Outflow in the trabecular meshwork (TM). Methods The expression profile of the endothelial differentiation gene (Edg) family of G-protein coupled receptors was determined by RT-PCR of human TM (HTM) cell-derived total RNA and by PCR amplification of HTM cell-derived and tissue-derived cDNA libraries. The effects of lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) on actin cytoskeleton and focal adhesions and on myosin light-chain (MLC) phosphorylation in HTM cells were evaluated by immunofluorescence microscopy and Western blot analysis, respectively. Activation of Rho GTPase in HTM cells was quantified by "pull-down" assays. Mobilization of intracellular calcium in HTM cells was determined using spectrofluorometric digital-imaging microscopy. The effects of LPA and S1P on Aqueous Humor Outflow facility were evaluated by perfusion of enucleated porcine eyes. Results Each of the receptor isoforms Edg1, -2, -3, and -4 was readily detectable in three of four HTM cell-derived libraries, whereas Edg2 was detectable in the HTM tissue library. LPA (20 microM) and S1P (1 microM) stimulated actin stress fiber and focal adhesion formation, increased MLC phosphorylation, and induced marked activation of Rho GTPase in HTM cells. Both LPA (20 microM) and S1P (10 microM) also stimulated increases in intracellular calcium concentration in HTM cells. LPA- and S1P-induced effects on MLC phosphorylation in HTM cells were markedly inhibited by pretreatment with the Rho kinase-specific inhibitor Y-27632 (5 microM). Perfusion of LPA (50 microM) and S1P (5 microM) in enucleated porcine eyes produced a significant decrease in Aqueous Humor Outflow facility from baseline of 37% (n = 6) and 31% (n = 5), respectively. Conclusions These studies demonstrate that LPA and S1P, the physiological agonists of Edg receptors, decrease Outflow facility in perfused porcine eyes in association with increased MLC phosphorylation and Rho guanosine triphosphatase (GTPase) activation. These data provide evidence for a novel mechanism for negative regulation of Outflow facility, which may contribute to overall physiological homeostasis of Aqueous Humor Outflow facility.

  • The role of protein kinase C in modulation of Aqueous Humor Outflow facility.
    Experimental eye research, 2003
    Co-Authors: Rahul N Khurana, David L. Epstein, Pei-feng Deng, P. Vasantha Rao
    Abstract:

    Abstract The elevated intraocular pressure that is commonly associated with glaucoma is believed to arise due to impairment of trabecular meshwork (TM) function. Although the TM and Schlemm's canal (SC) comprise the major route for Aqueous Humor Outflow, little is known about the potential signaling mechanisms involved in the regulation of Aqueous Outflow. Based on knowledge regarding the role of protein kinase C (PKC) in vascular biology, we sought to understand the contribution of the PKC pathway towards Outflow function by studying the modulation of contractile and morphological characteristics of TM and SC cells. We investigated the involvement of PKC in regulation of myosin light chain (MLC) phosphorylation, formation of actin stress fibers and integrin–ECM adhesions (focal adhesions) in human TM and SC cells and correlated these changes with Aqueous Outflow facility measured in an enucleated porcine whole eye perfusion model. Expression and distribution of PKC isoforms (α and e) in TM and SC cells and tissues was confirmed by Western blot and immunohistochemical analysis, respectively. Both, pharmacological activators (phorbol-12-myristate 13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu)) and inhibitors (staurosporine and GF109203X) of PKC were found to induce changes in cell shape (retraction and rounding up) and cytoskeletal organization in human TM and SC cells. While PMA and PDBu produced an increase in formation of actin stress fibers and focal adhesions and in MLC phosphorylation, PKC inhibitors were observed to induce contrasting effects in these cells. Intriguingly, both PDBU and GF109203X caused increases in Aqueous Outflow facility in the perfusion model. The PKC inhibitor (GF109203X) increased Outflow by 46% while the PKC activator (PDBu) only increased Outflow by 27%. These results suggest that PKC might play an important role in modulation of Aqueous Outflow facility by regulating MLC phosphorylation and thereby, the morphological and cytoskeletal characteristics of TM and SC cells.