Sheldon S Miller

National Institutes of Health, Maryland, United States

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Publications (23)81.41 Total impact

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    ABSTRACT: Biomedical advances in vision research have been greatly facilitated by the clinical accessibility of the visual system, its ease of experimental manipulation, and its ability to be functionally monitored in real time with noninvasive imaging techniques at the level of single cells and with quantitative end-point measures. A recent example is the development of stem cell-based therapies for degenerative eye diseases including AMD. Two phase I clinical trials using embryonic stem cell-derived RPE are already underway and several others using both pluripotent and multipotent adult stem cells are in earlier stages of development. These clinical trials will use a variety of cell types, including embryonic or induced pluripotent stem cell-derived RPE, bone marrow- or umbilical cord-derived mesenchymal stem cells, fetal neural or retinal progenitor cells, and adult RPE stem cells-derived RPE. Although quite distinct, these approaches, share common principles, concerns and issues across the clinical development pipeline. These considerations were a central part of the discussions at a recent National Eye Institute meeting on the development of cellular therapies for retinal degenerative disease. At this meeting, emphasis was placed on the general value of identifying and sharing information in the so-called "precompetitive space." The utility of this behavior was described in terms of how it could allow us to remove road blocks in the clinical development pipeline, and more efficiently and economically move stem cell-based therapies for retinal degenerative diseases toward the clinic. Many of the ocular stem cell approaches we discuss are also being used more broadly, for nonocular conditions and therefore the model we develop here, using the precompetitive space, should benefit the entire scientific community.
    Investigative ophthalmology & visual science 01/2014; 55(2):1191-202. · 3.43 Impact Factor
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    Kapil Bharti, Sheldon S Miller, Heinz Arnheiter
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    ABSTRACT: Compared with neural crest-derived melanocytes, retinal pigment epithelium (RPE) cells in the back of the eye are pigment cells of a different kind. They are a part of the brain, form an epithelial monolayer, respond to distinct extracellular signals, and provide functions that far exceed those of a light-absorbing screen. For instance, they control nutrient and metabolite flow to and from the retina, replenish 11-cis-retinal by re-isomerizing all-trans-retinal generated during photoconversion, phagocytose daily a portion of the photoreceptors' outer segments, and secrete cytokines that locally control the innate and adaptive immune systems. Not surprisingly, RPE cell damage is a major cause of human blindness worldwide, with age-related macular degeneration a prevalent example. RPE replacement therapies using RPE cells generated from embryonic or induced pluripotent stem cells provide a novel approach to a rational treatment of such forms of blindness. In fact, RPE-like cells can be obtained relatively easily when stem cells are subjected to a two-step induction protocol, a first step that leads to a neuroectodermal fate and a second to RPE differentiation. Here, we discuss the characteristics of such cells, propose criteria they should fulfill in order to be considered authentic RPE cells, and point out the challenges one faces when using such cells in attempts to restore vision.
    Pigment Cell & Melanoma Research 02/2011; 24(1):21-34. · 5.84 Impact Factor
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    ABSTRACT: Ciliary neurotrophic factor (CNTF) protects photoreceptors and regulates their phototransduction machinery, but little is known about CNTF's effects on retinal pigment epithelial (RPE) physiology. Therefore, we determined the expression and localization of CNTF receptors and the physiological consequence of their activation in primary cultures of human fetal RPE (hfRPE). Cultured hfRPE express CNTF, CT1, and OsM and their receptors, including CNTFRα, LIFRβ, gp130, and OsMRβ, all localized mainly at the apical membrane. Exogenous CNTF, CT1, or OsM induces STAT3 phosphorylation, and OsM also induces the phosphorylation of ERK1/2 (p44/42 MAP kinase). CNTF increases RPE survivability, but not rates of phagocytosis. CNTF increases secretion of NT3 to the apical bath and decreases that of VEGF, IL8, and TGFβ2. It also significantly increases fluid absorption (J(V)) across intact monolayers of hfRPE by activating CFTR chloride channels at the basolateral membrane. CNTF induces profound changes in RPE cell biology, biochemistry, and physiology, including the increase in cell survival, polarized secretion of cytokines/neurotrophic factors, and the increase in steady-state fluid absorption mediated by JAK/STAT3 signaling. In vivo, these changes, taken together, could serve to regulate the microenvironment around the distal retinal/RPE/Bruch's membrane complex and provide protection against neurodegenerative disease.
    PLoS ONE 01/2011; 6(9):e23148. · 3.73 Impact Factor
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    ABSTRACT: MicroRNA (miRNA) expression in fetal human retinal pigment epithelium (hfRPE), retina, and choroid were pairwise compared to determine those miRNAs that are enriched by 10-fold or more in each tissue compared with both of its neighbors. miRs-184, 187, 200a/200b, 204/211, and 221/222 are enriched in hfRPE by 10- to 754-fold compared with neuroretina or choroid (P<0.05). Five of these miRNAs are enriched in RPE compared with 20 tissues throughout the body and are 10- to 20,000-fold more highly expressed (P<0.005). miR-204 and 211 are the most highly expressed in the RPE. In addition, expression of miR-204/211 is significantly lower in the NCI60 tumor cell line panel compared with that in 13 normal tissues, suggesting the progressive disruption of epithelial barriers and increased proliferation. We demonstrated that TGF-beta receptor 2 (TGF-betaR2) and SNAIL2 are direct targets of miR-204 and that a reduction in miR-204 expression leads to reduced expression of claudins 10, 16, and 19 (message/protein) consistent with our observation that anti-miR-204/211 decreased transepithelial resistance by 80% and reduced cell membrane voltage and conductance. The anti-miR-204-induced decrease in Kir7.1 protein levels suggests a signaling pathway that connects TGF-betaR2 and maintenance of potassium homeostasis. Overall, these data indicate a critical role for miR-204/211 in maintaining epithelial barrier function and cell physiology.
    The FASEB Journal 05/2010; 24(5):1552-71. · 5.70 Impact Factor
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    Arvydas Maminishkis, Sheldon S Miller
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    ABSTRACT: We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.
    Journal of Visualized Experiments 01/2010;
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    ABSTRACT: The present experiments show that IFNgamma receptors are mainly localized to the basolateral membrane of human retinal pigment epithelium (RPE). Activation of these receptors in primary cultures of human fetal RPE inhibited cell proliferation and migration, decreased RPE mitochondrial membrane potential, altered transepithelial potential and resistance, and significantly increased transepithelial fluid absorption. These effects are mediated through JAK-STAT and p38 MAPK signaling pathways. Second messenger signaling through cAMP-PKA pathway- and interferon regulatory factor-1-dependent production of nitric oxide/cGMP stimulated the CFTR at the basolateral membrane and increased transepithelial fluid absorption. In vivo experiments using a rat model of retinal reattachment showed that IFNgamma applied to the anterior surface of the eye can remove extra fluid deposited in the extracellular or subretinal space between the retinal photoreceptors and RPE. Removal of this extra fluid was blocked by a combination of PKA and JAK-STAT pathway inhibitors injected into the subretinal space. These results demonstrate a protective role for IFNgamma in regulating retinal hydration across the outer blood-retinal barrier in inflammatory disease processes and provide the basis for possible therapeutic interventions.
    AJP Cell Physiology 10/2009; 297(6):C1452-65. · 3.71 Impact Factor
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    ABSTRACT: The aim of this study was to determine the expression and localization of integrin alpha5beta1 in human retinal pigment epithelium (RPE) and its ability to modulate RPE cell attachment, proliferation, migration, and F-actin cytoskeleton distribution. Expression and localization of alpha5beta1 were analyzed on human RPE by immunoblot/immunofluorescence. Polarized secretion of fibronectin was measured. RPE attachments to different substrates were determined using cell attachment screening kits. BrdU incorporation and wound-healing assays were used to test hfRPE proliferation and migration. F-actin cytoskeleton was visualized with phalloidin. Integrin alpha5beta1 was detected in native adult and fetal human RPE. The alpha5-subunit is predominantly localized at the apical membrane of hfRPE, whereas the beta1-subunit is uniformly detected at the apical/basolateral membranes. The authors also found that hfRPE cultures secrete significant amounts of fibronectin to the apical bath. JSM6427, a specific integrin alpha5beta1 antagonist, significantly inhibited hfRPE cell attachment to fibronectin, but not laminin, or collagen I or IV. JSM6427 also showed a strong inhibitory effect on bFGF, PDGF-BB, and serum-induced cell migration and proliferation. Furthermore, JSM6427 induced significant disruption of the F-actin cytoskeleton of dividing RPE cells but had no effect on quiescent cells. The apical localization of alpha5beta1 and the secretion of fibronectin to the apical bath suggest the presence of an autocrine loop that can guide the migration of RPE. The strong inhibitory effects of JSM6427 on human RPE cell attachment, proliferation, and migration is probably mediated by F-actin cytoskeletal disruption in proliferating cells and suggests a potential clinical use of this compound in proliferative retinopathies.
    Investigative ophthalmology & visual science 08/2009; 50(12):5988-96. · 3.43 Impact Factor
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    ABSTRACT: In the intact eye, the transition from light to dark alters pH, [Ca2+], and [K] in the subretinal space (SRS) separating the photoreceptor outer segments and the apical membrane of the retinal pigment epithelium (RPE). In addition to these changes, oxygen consumption in the retina increases with a concomitant release of CO2 and H2O into the SRS. The RPE maintains SRS pH and volume homeostasis by transporting these metabolic byproducts to the choroidal blood supply. In vitro, we mimicked the transition from light to dark by increasing apical bath CO2 from 5 to 13%; this maneuver decreased cell pH from 7.37 +/- 0.05 to 7.14 +/- 0.06 (n = 13). Our analysis of native and cultured fetal human RPE shows that the apical membrane is significantly more permeable (approximately 10-fold; n = 7) to CO2 than the basolateral membrane, perhaps due to its larger exposed surface area. The limited CO2 diffusion at the basolateral membrane promotes carbonic anhydrase-mediated HCO3 transport by a basolateral membrane Na/nHCO3 cotransporter. The activity of this transporter was increased by elevating apical bath CO2 and was reduced by dorzolamide. Increasing apical bath CO2 also increased intracellular Na from 15.7 +/- 3.3 to 24.0 +/- 5.3 mM (n = 6; P < 0.05) by increasing apical membrane Na uptake. The CO2-induced acidification also inhibited the basolateral membrane Cl/HCO3 exchanger and increased net steady-state fluid absorption from 2.8 +/- 1.6 to 6.7 +/- 2.3 microl x cm(-2) x hr(-1) (n = 5; P < 0.05). The present experiments show how the RPE can accommodate the increased retinal production of CO2 and H(2)O in the dark, thus preventing acidosis in the SRS. This homeostatic process would preserve the close anatomical relationship between photoreceptor outer segments and RPE in the dark and light, thus protecting the health of the photoreceptors.
    The Journal of General Physiology 06/2009; 133(6):603-22. · 4.73 Impact Factor
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    ABSTRACT: To determine the localization of JAM-C in human RPE and characterize its functions. Immunofluorescence, Western blot, and PCR was used to identify the localization and expression of JAM-C, ZO-1, N-cadherin, and ezrin in cultures of human fetal RPE (hfRPE) with or without si-RNA mediated JAM-C knockdown and in adult native RPE wholemounts. A transepithelial migration assay was used to study the migration of leukocytes through the hfRPE monolayer. JAM-C localized at the tight junctions of cultured hfRPE cells and adult native RPE. During initial junction formation JAM-C was recruited to the primordial cell-cell contacts and after JAM-C knockdown, the organization of N-cadherin and ZO-1 at those contacts was disrupted. JAM-C knockdown caused a delay in the hfRPE cell polarization, as shown by reduced apical staining of ezrin. JAM-C inhibition significantly decreased the chemokine-induced transmigration of granulocytes but not monocytes through the hfRPE monolayer. JAM-C localizes specifically in the tight junctions of hfRPE and adult native RPE. It is important for tight junction formation in hfRPE, possibly by regulating the recruitment of N-cadherin and ZO-1 at the cell-cell contacts, and has a role in the polarization of hfRPE cells. Finally, JAM-C promotes the basal-to-apical transmigration of granulocytes but not monocytes through the hfRPE monolayer.
    Investigative ophthalmology & visual science 01/2009; 50(3):1454-63. · 3.43 Impact Factor
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    ABSTRACT: Noninfectious uveitis is a predominantly T cell-mediated autoimmune, intraocular inflammatory disease. To characterize the gene expression profile from patients with noninfectious uveitis, PBMCs were isolated from 50 patients with clinically characterized noninfectious uveitis syndrome. A pathway-specific cDNA microarray was used for gene expression profiling and real-time PCR array for further confirmation. Sixty-seven inflammation- and autoimmune-associated genes were found differentially expressed in uveitis patients, with 28 of those genes being validated by real-time PCR. Several genes previously unknown for autoimmune uveitis, including IL-22, IL-19, IL-20, and IL-25/IL-17E, were found to be highly expressed among uveitis patients compared with the normal subjects with IL-22 expression highly variable among the patients. Furthermore, we show that IL-22 can affect primary human retinal pigment epithelial cells by decreasing total tissue resistance and inducing apoptosis possibly by decreasing phospho-Bad level. In addition, the microarray data identified a possible uveitis-associated gene expression pattern, showed distinct gene expression profiles in patients during periods of clinical activity and quiescence, and demonstrated similar expression patterns in related patients with similar clinical phenotypes. Our data provide the first evidence that a subset of IL-10 family genes are implicated in noninfectious uveitis and that IL-22 can affect human retinal pigment epithelial cells. The results may facilitate further understanding of the molecular mechanisms of autoimmune uveitis and other autoimmune originated inflammatory diseases.
    The Journal of Immunology 11/2008; 181(7):5147-57. · 5.52 Impact Factor
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    ABSTRACT: Activation of an innate immune response in airway epithelia by the human pathogen Pseudomonas aeruginosa requires bacterial expression of flagellin. Addition of flagellin (10(-7) M) to airway epithelial cell monolayers (Calu-3, airway serous cell-like) increased Cl(-) secretion (I(Cl)) beginning after 3-10 min, reaching a plateau after 20-45 min at DeltaI(Cl) = 15-50 microA/cm(2). Similar, although 10-fold smaller, responses were observed in well-differentiated bronchial epithelial cultures. Flagellin stimulated I(Cl) in the presence of maximally stimulating doses of the purinergic agonist ATP, but had no effects following forskolin. IL-1beta (produced by both epithelia and neutrophils during infections) stimulated I(Cl) similar to flagellin. Flagellin-, IL-1beta-, ATP-, and forskolin-stimulated I(Cl) were inhibited by cystic fibrosis transmembrane conductance regulator (CFTR) blockers GlyH101, CFTRinh172, and glibenclamide. Neither flagellin nor IL-1beta altered transepithelial fluxes of membrane-impermeant dextran (10 kDa) or lucifer yellow (mol wt = 457), but both activated p38, NF-kappaB, and IL-8 secretion. Blockers of p38 (SB-202190 and SB-203580) reduced flagellin- and IL-1beta-stimulated I(Cl) by 33-50% but had smaller effects on IL-8 and NF-kappaB. It is concluded that: 1) flagellin and IL-1beta activated p38, NF-kappaB, IL-8, and CFTR-dependent anion secretion without altering tight junction permeability; 2) p38 played a role in regulating I(Cl) and IL-8 but not NF-kappaB; and 3) p38 was more important in flagellin- than IL-1beta-stimulated responses. During P. aeruginosa infections, flagellin and IL-1beta are expected to increase CFTR-dependent ion and fluid flow into and bacterial clearance from the airways. In cystic fibrosis, the secretory response would be absent, but activation of p38, NF-kappaB, and IL-8 would persist.
    AJP Lung Cellular and Molecular Physiology 10/2008; 295(4):L531-42. · 3.52 Impact Factor
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    ABSTRACT: Proinflammatory cytokines in degenerative diseases can lead to the loss of normal physiology and the destruction of surrounding tissues. In the present study, the physiological responses of human fetal retinal pigment epithelia (hfRPE) were examined in vitro after polarized activation of proinflammatory cytokine receptors. Primary cultures of hfRPE were stimulated with an inflammatory cytokine mixture (ICM): interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma. Western blot analysis and immunofluorescence were used to determine the expression/localization of the cytokine receptors on hfRPE. Polarized secretion of cytokines was measured. A capacitance probe technique was used to measure transepithelial fluid flow (J(V)) and resistance (R(T)). IL-1R1 was mainly localized to the apical membrane and TNFR1 to the basal membrane, whereas IFN-gammaR1 was detected on both membranes. Activation by apical ICM induced a significant secretion of angiogenic and angiostatic chemokines, mainly across the hfRPE apical membrane. Addition of the ICM to the basal but not the apical bath significantly increased net fluid absorption (J(V)) across the hfRPE within 20 minutes. Similar increases in J(V) were produced by a 24-hour exposure to ICM, which significantly decreased total R(T). Chemokine gradients across the RPE can be altered (1) through an ICM-induced change in polarized chemokine secretion and (2) through an increase in ICM-induced net fluid absorption. In vivo, both of these factors could contribute to the development of chemokine gradients that help mediate the progression of inflammation/angiogenesis at the retina/RPE/choroid complex.
    Investigative ophthalmology & visual science 05/2008; 49(10):4620-30. · 3.43 Impact Factor
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    Rong Li, Arvydas Maminishkis, Fei E Wang, Sheldon S Miller
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    ABSTRACT: The role of growth factors and inflammation in regulating retinal pigment epithelial (RPE) function is complex and still poorly understood. The present study investigated human RPE cell proliferation and migration mediated by platelet-derived growth factor (PDGF) and inflammatory cytokines. Human fetal RPE (hfRPE) cells were obtained as previously described. Gene expressions of PDGF isoforms and their receptors were detected using real-time PCR. Protein expression, activity, and localization of PDGFR-alpha and -beta were analyzed by Western blot and immunohistochemistry. BrdU incorporation and wound healing assays were used to test the effects of different PDGF isoforms and inflammatory cytokines on hfRPE proliferation and migration. Annexin-V and phalloidin staining were used to detect apoptosis and the actin cytoskeleton, respectively. PDGF-C and PDGF-D proteins are expressed in native human adult RPE, and mRNA levels are up to 100-fold higher than PDGF-A and -B. PDGFR-alpha and -beta proteins are expressed in native adult RPE and hfRPE (mainly localized to the apical membrane). In hfRPE, these receptors can be activated by PDGF-CC and -DD. PDGF-CC, -DD, and -BB significantly increased hfRPE proliferation, whereas PDGF-DD, -BB, and -AB significantly increased cell migration. An inflammatory cytokine mixture (TNF-alpha/IL-1beta/IFN-gamma) completely inhibited the stimulatory effect of PDGF-BB, -CC, and -DD; in contrast, this mixture stimulated the proliferation of choroidal cells. This inflammatory cytokine mixture also induced apoptosis, significant disruption of actin filaments and zonula occludens (ZO-1), and a decrease in transepithelial resistance. These results suggest that proinflammatory cytokines in vivo can inhibit the proliferative effect of PDGF on human RPE and, at the same time, stimulate the proliferation of choroidal cells. They also suggest an important role of proinflammatory cytokines in overcoming local proliferative/wound-healing responses, thereby controlling the development of disease processes at the retina/RPE/choroid interface.
    Investigative Ophthalmology &amp Visual Science 01/2008; 48(12):5722-32. · 3.44 Impact Factor
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    ABSTRACT: To understand better the cell and molecular basis for the epidemiologic association between cigarette smoke, oxidant injury, and age-associated macular degeneration, the authors examined the effects of acrolein, a major toxicant in cigarette smoke, on oxidative mitochondrial damage in retinal pigment epithelial (RPE) cells and the reduction of this damage by lipoic acid. Cultured human ARPE19 cells and primary cultures of human fetal (hf)RPE were treated with acrolein. The toxicity of acrolein and the protective effects of R-alpha-lipoic acid were examined with a variety of previously described techniques. Acute acrolein exposure exceeding 50 microM (24 hours) in ARPR19 cells caused toxicity, including decreases in cell viability, mitochondrial potential, GSH, antioxidant capacity, Nrf2 expression, enzyme activity (mitochondrial complexes I, II, III; superoxide dismutase; and glutathione peroxidase). Acute exposure also increased oxidant levels, protein carbonyls, and calcium. Continuous acrolein exposure over 8 or 32 days caused similar toxicity but from 10- to 100-fold lower doses (0.1-5 microM). Pretreatment with R-alpha-lipoic acid effectively protected ARPE-19 cells from acrolein toxicity. Primary hfRPE cells were comparable to the ARPE-19 cells in sensitivity to acrolein toxicity and lipoic acid protection. These results show that acrolein is a mitochondrial toxicant in RPE cells and that acrolein-induced oxidative mitochondrial dysfunction is reduced by lipoic acid. The similar sensitivity of the ARPE-19 and hfRPE cells suggests that both models are useful for studying RPE toxicity and protection. These experiments indicate that mitochondria-targeted antioxidants such as lipoic acid may be an effective strategy for reducing or preventing chronic oxidant-induced RPE degeneration in vivo from a variety of sources, including cigarette smoke.
    Investigative Ophthalmology &amp Visual Science 02/2007; 48(1):339-48. · 3.44 Impact Factor
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    ABSTRACT: Provide a reproducible method for culturing confluent monolayers of hfRPE cells that exhibit morphology, physiology, polarity, and protein expression patterns similar to native tissue. Human fetal eyes were dissected on arrival, and RPE cell sheets were mechanically separated from the choroid and cultured in a specifically designed medium comprised entirely of commercially available components. Physiology experiments were performed with previously described techniques. Standard techniques were used for immunohistochemistry, electron microscopy, and cytokine measurement by ELISA. Confluent monolayers of RPE cell cultures exhibited epithelial morphology and heavy pigmentation, and electron microscopy showed extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. The mean transepithelial potential (TEP) was 2.6 +/- 0.8 mV, apical positive, and the mean transepithelial resistance (R(T)) was 501 +/- 138 Omega . cm(2) (mean +/- SD; n = 35). Addition of 100 microM adenosine triphosphate (ATP) to the apical bath increased net fluid absorption from 13.6 +/- 2.6 to 18.8 +/- 4.6 microL . cm(-2) per hour (mean +/- SD; n = 4). In other experiments, VEGF was mainly secreted into the basal bath (n = 10), whereas PEDF was mainly secreted into the apical bath (n = 10). A new cell culture procedure has been developed that produces confluent primary hfRPE cultures with morphological and physiological characteristics of the native tissue. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied native human fetal and bovine RPE-choroid explants.
    Investigative Ophthalmology &amp Visual Science 09/2006; 47(8):3612-24. · 3.44 Impact Factor
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    ABSTRACT: Molecular Therapy (2006) 13, S341|[ndash]|S341; doi: 10.1016/j.ymthe.2006.08.975 886. AAV-Mediated Somatic Gene Transfer as an Approach To Delineate Pathogenic Mechanisms in an Autosomal Dominant Blindness Disorder Resembling Age-Related Macular Degeneration (AMD) Nicholas W. Keiser1, Arvydas Maminishkis2, Jeannette L. Bennicelli1, Sheldon S. Miller2 and Jean Bennett11Department of Ophthalmology, F.M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA2National Eye Institute, National Institutes of Health, Bethesda, MD
    Molecular Therapy 04/2006; · 7.04 Impact Factor
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    ABSTRACT: To determine whether (R)-alpha-lipoic acid (LA) protects cultured human fetal retinal pigment epithelial (hfRPE) cells against oxidative injury and identify the pathways that may mediate protection. Cultured hfRPE cells were pretreated with various concentrations of LA for 14 to 16 hours followed by treatment with a chemical oxidant, tert-butylhydroperoxide (t-BuOOH; 0.8 mM, 3 hours). Reactive oxygen species (ROS) production and cell viability were measured using H(2)DCF and MTT assays, respectively. RPE cells were evaluated with fluorescent dyes (SYTOX Orange and SYTO Green; Molecular Probes, Eugene, OR), which differentiate between live and dead cells. Apoptosis was visualized by using the TUNEL assay. Changes in mitochondrial membrane potential were detected by JC-1 dye. Intracellular levels of reduced glutathione (GSH) and oxidized glutathione (GSSG) were measured by HPLC. Regulation of gamma-glutamylcysteine ligase (GCL), the rate-controlling enzyme of GSH production, was assayed by RT-PCR. Pretreatment of hfRPE cells with LA, 0.2 mM and 0.5 mM, significantly reduced the levels of t-BuOOH-induced intracellular ROS, by 23% and 49%, respectively. LA (0.5 mM) prevented oxidant-induced cell death and apoptosis and also increased the viability of oxidant-treated hfRPE cells from 38% to 90% of control. LA upregulated the mRNA expression of GCL, and was protective against t-BuOOH-induced decreases in both mitochondrial membrane potential and intracellular levels of GSH and GSH/GSSG. The present study suggests that the protective effect of LA involves multiple pathways and that LA could be effective against age-associated increase in oxidative stress and mitochondrial dysfunction in RPE cells.
    Investigative Ophthalmology &amp Visual Science 12/2005; 46(11):4302-10. · 3.44 Impact Factor
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    ABSTRACT: Hypertrophy and hyperplasia of the retinal pigment epithelium (RPE) is associated with an inherited predisposition to human familial adenomatous polyposis coli, suggesting that expression of the adenomatous polyposis coli (APC) tumor suppressor may regulate RPE proliferation/differentiation. Distinctive APC isoforms exist in different cell types due to alternative splicing of the APC transcripts. We hypothesize that differences in expression patterns of APC protein isoforms are critical to RPE proliferation/differentiation. To investigate these relationships, APC gene expression was characterized in the retinas and RPE from fetal and adult human and mouse, and in the epiretinal membranes (ERM) from 5 patients with proliferative vitreoretinopathy (PVR). Expression patterns of alternative splice-forms of APC transcripts were evaluated by comparative quantitative RT-PCR. Exon 1 of APC encodes a heptad repeat that confers the ability of APC to homodimerize. APC protein isoforms containing or lacking this heptad were characterized by western blot analysis and immunohistochemistry. Comparative quantitative RT-PCR demonstrated a predominant exon 1 containing, conventional APC splice-form in the early developing fetal RPE and retina, and in all the tested ERM samples from patients with PVR. This method also demonstrated an increased level of exon 1 lacking APC splice-form in the mature RPE and retina. Western blot analysis and immunofluorescence microscopy demonstrated the conventional APC only in the RPE, and the APC isoform without the first heptad repeat in both the retina and RPE. Immunofluorescence microscopy also demonstrated only the conventional APC in the ERM samples tested. These results suggest that alternative splicing of APC leads to differential APC expression with potentially unique functions. APC isoform without the first heptad repeat may play a role in cell cycle cessation in the adult retina and RPE, and the down regulation of this APC isoform may contribute to the potential of RPE to migrate and proliferate.
    Molecular vision 07/2004; 10:383-91. · 1.99 Impact Factor
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    ABSTRACT: It has been reported that secretory mammary epithelial cells (MEC) release ATP, UTP, and UDP upon mechanical stimulation. Here we examined the physiological changes caused by ATP/UTP in nontransformed, clonal mouse mammary epithelia (31EG4 cells). In control conditions, transepithelial potential (apical side negative) and resistance were -4.4 +/- 1.3 mV (mean +/- SD, n = 12) and 517.7 +/- 39.4 Omega. cm(2), respectively. The apical membrane potential was -43.9 +/- 1.7 mV, and the ratio of apical to basolateral membrane resistance (R(A)/R(B)) was 3.5 +/- 0.2. Addition of ATP or UTP to the apical or basolateral membranes caused large voltage and resistance changes with an EC(50) of approximately 24 microM (apical) and approximately 30 microM (basal). Apical ATP/UTP (100 microM) depolarized apical membrane potential by 17.6 +/- 0.8 mV (n = 7) and decreased R(A)/R(B) by a factor of approximately 3. The addition of adenosine to either side (100 microM) had no effect on any of these parameters. The ATP/UTP responses were partially inhibited by DIDS and suramin and mediated by a transient increase in free intracellular Ca(2+) concentration (427 +/- 206 nM; 15-25 microM ATP, apical; n = 6). This Ca(2+) increase was blocked by cyclopiazonic acid, by BAPTA, or by xestospongin C. 31EG4 MEC monolayers also secreted or absorbed fluid in the resting state, and ATP or UTP increased fluid secretion by 5.6 +/- 3 microl x cm(-2) x h(-1) (n = 10). Pharmacology experiments indicate that 31EG4 epithelia contain P2Y(2) purinoceptors on the apical and basolateral membranes, which upon activation stimulate apical Ca(2+)-dependent Cl channels and cause fluid secretion across the monolayer. This suggests that extracellular nucleotides could play a fundamental role in mammary gland paracrine signaling and the regulation of milk composition in vivo.
    AJP Cell Physiology 05/2003; 284(4):C897-909. · 3.71 Impact Factor
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    ABSTRACT: To develop a small-animal model of choroidal neovascularization (CNV) by injecting adeno-associated virus (AAV)-VEGF into the subretinal space (SRS) of rats. An adeno-associated viral vector encoding human VEGF(165) was injected into the subretinal space (SRS) of Sprague-Dawley or Long Evans rats. Expression of VEGF was identified by RT-PCR and immunohistochemistry. Physiological and pathologic changes in the retina and choroid were evaluated by electroretinography, fluorescein angiography, light microscopy, and three-dimensional reconstruction of serial sections. Green fluorescent protein (GFP) and VEGF were expressed for at least 20 months in the retina and retinal pigment epithelium (RPE). Histologic sections showed extensive subretinal neovascularization, degenerating photoreceptors, and proliferating RPE at 5 weeks to 20 months after injection of AAV-VEGF. At 2 to 12 months after injection, leaking blood vessels were detected by fluorescein angiography. Electroretinogram a- and b-wave amplitudes were significantly decreased during this time. Three-dimensional reconstruction of serial sections demonstrated that choroidal blood vessels penetrated Bruch's membrane, one of them splitting into three branches in the SRS. In the current model, CNV was produced in 95% of the animals tested (19/20). It persisted for more than 20 months, a necessary requirement for modeling the development of CNV in age-related macular degeneration (AMD). In this study, a highly reproducible animal model of long-lasting CNV was developed. This model is being used to test antiangiogenic molecules to reduce or inhibit CNV and could be extended to primates.
    Investigative Ophthalmology &amp Visual Science 03/2003; 44(2):781-90. · 3.44 Impact Factor

Publication Stats

579 Citations
81.41 Total Impact Points

Institutions

  • 2005–2010
    • National Institutes of Health
      • Section on Epithelial and Retinal Physiology
      Maryland, United States
  • 2009
    • University of Maryland, College Park
      • Department of Chemical and Biomolecular Engineering
      College Park, MD, United States
  • 2002–2005
    • University of California, Berkeley
      • • Biophysics Graduate Group
      • • School of Optometry
      Berkeley, MO, United States