Disruption of cell-cell junctions and induction of pathological cytokines in the retinal pigment epithelium of light-exposed mice

Toshio Narimatsu, Yoko Ozawa, Seiji Miyake, Shunsuke Kubota, Manabu Hirasawa, Norihiro Nagai, Shigeto Shimmura, Kazuo Tsubota

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

PURPOSE. To elucidate the influences of light exposure on the retinal pigment epithelium (RPE) in vivo that may be involved in the pathogenesis of AMD. METHODS. Six- to 7-week-old BALB/c mice were exposed to light at 2000 lux for 3 hours. Flatmount RPE samples were immunostained with anti-ZO-1 antibody for evaluating tight junction, anti-N-cadherin, and anti-b-catenin antibodies for adherens junction, and stained with phalloidin for actin cytoskeleton. The reactive oxygen species (ROS) level was measured using DCFH-DA; Rho-associated coiled-coil forming kinase (ROCK) activity was by ELISA. Cytokine expression was analyzed by real-time RT-PCR and/or ELISA in the RPE-choroid, and macrophage recruitment was by real-time RT-PCR and immunohistochemistry. Either an antioxidant, N-Acetyl-L-cysteine (NAC), or a ROCK inhibitor, Y-27632, were administered to analyze the roles of ROS and ROCK activation, respectively. RESULTS. Light exposure disrupted staining patterns of tight junctions, adherens junctions, and actin cytoskeleton in the RPE, where ROS was elevated. However, NAC treatment avoided the RPE changes, reducing ROS. ROCK activity increased after light exposure was suppressed by NAC, and the structural disruptions were suppressed by Y-27632. The levels of MCP-1, CCL11, and IL-6 increased after light exposure were suppressed by NAC. Light-induced MCP-1 and IL- 6 were suppressed by Y-27632. Macrophage recruitment after light exposure was also suppressed either by NAC or Y-27632. CONCLUSIONS. Light exposure induced ROS and Rho/ROCK activation, which caused disruption of cell-cell junctions (tight junctions and adherens junctions) and actin cytoskeleton, the RPE's barrier structure, and induced AMD-associated pathological changes in the RPE-choroid.

Original languageEnglish
Pages (from-to)4555-4562
Number of pages8
JournalInvestigative Ophthalmology and Visual Science
Volume54
Issue number7
DOIs
Publication statusPublished - 2013

Fingerprint

Intercellular Junctions
Retinal Pigment Epithelium
rho-Associated Kinases
Cytokines
Acetylcysteine
Light
Reactive Oxygen Species
Adherens Junctions
Tight Junctions
Actin Cytoskeleton
Choroid
Real-Time Polymerase Chain Reaction
Interleukin-6
Enzyme-Linked Immunosorbent Assay
Macrophages
Phalloidine
Catenins
Antibodies
Cadherins
Antioxidants

Keywords

  • Age-related macular degeneration
  • Light damage
  • Oxidative damage
  • Retina
  • Retinal pigment epithelium

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Disruption of cell-cell junctions and induction of pathological cytokines in the retinal pigment epithelium of light-exposed mice. / Narimatsu, Toshio; Ozawa, Yoko; Miyake, Seiji; Kubota, Shunsuke; Hirasawa, Manabu; Nagai, Norihiro; Shimmura, Shigeto; Tsubota, Kazuo.

In: Investigative Ophthalmology and Visual Science, Vol. 54, No. 7, 2013, p. 4555-4562.

Research output: Contribution to journalArticle

@article{35c0bd5335c44c78a53e34f47e7d7432,
title = "Disruption of cell-cell junctions and induction of pathological cytokines in the retinal pigment epithelium of light-exposed mice",
abstract = "PURPOSE. To elucidate the influences of light exposure on the retinal pigment epithelium (RPE) in vivo that may be involved in the pathogenesis of AMD. METHODS. Six- to 7-week-old BALB/c mice were exposed to light at 2000 lux for 3 hours. Flatmount RPE samples were immunostained with anti-ZO-1 antibody for evaluating tight junction, anti-N-cadherin, and anti-b-catenin antibodies for adherens junction, and stained with phalloidin for actin cytoskeleton. The reactive oxygen species (ROS) level was measured using DCFH-DA; Rho-associated coiled-coil forming kinase (ROCK) activity was by ELISA. Cytokine expression was analyzed by real-time RT-PCR and/or ELISA in the RPE-choroid, and macrophage recruitment was by real-time RT-PCR and immunohistochemistry. Either an antioxidant, N-Acetyl-L-cysteine (NAC), or a ROCK inhibitor, Y-27632, were administered to analyze the roles of ROS and ROCK activation, respectively. RESULTS. Light exposure disrupted staining patterns of tight junctions, adherens junctions, and actin cytoskeleton in the RPE, where ROS was elevated. However, NAC treatment avoided the RPE changes, reducing ROS. ROCK activity increased after light exposure was suppressed by NAC, and the structural disruptions were suppressed by Y-27632. The levels of MCP-1, CCL11, and IL-6 increased after light exposure were suppressed by NAC. Light-induced MCP-1 and IL- 6 were suppressed by Y-27632. Macrophage recruitment after light exposure was also suppressed either by NAC or Y-27632. CONCLUSIONS. Light exposure induced ROS and Rho/ROCK activation, which caused disruption of cell-cell junctions (tight junctions and adherens junctions) and actin cytoskeleton, the RPE's barrier structure, and induced AMD-associated pathological changes in the RPE-choroid.",
keywords = "Age-related macular degeneration, Light damage, Oxidative damage, Retina, Retinal pigment epithelium",
author = "Toshio Narimatsu and Yoko Ozawa and Seiji Miyake and Shunsuke Kubota and Manabu Hirasawa and Norihiro Nagai and Shigeto Shimmura and Kazuo Tsubota",
year = "2013",
doi = "10.1167/iovs.12-11572",
language = "English",
volume = "54",
pages = "4555--4562",
journal = "Investigative Ophthalmology and Visual Science",
issn = "0146-0404",
publisher = "Association for Research in Vision and Ophthalmology Inc.",
number = "7",

}

TY - JOUR

T1 - Disruption of cell-cell junctions and induction of pathological cytokines in the retinal pigment epithelium of light-exposed mice

AU - Narimatsu, Toshio

AU - Ozawa, Yoko

AU - Miyake, Seiji

AU - Kubota, Shunsuke

AU - Hirasawa, Manabu

AU - Nagai, Norihiro

AU - Shimmura, Shigeto

AU - Tsubota, Kazuo

PY - 2013

Y1 - 2013

N2 - PURPOSE. To elucidate the influences of light exposure on the retinal pigment epithelium (RPE) in vivo that may be involved in the pathogenesis of AMD. METHODS. Six- to 7-week-old BALB/c mice were exposed to light at 2000 lux for 3 hours. Flatmount RPE samples were immunostained with anti-ZO-1 antibody for evaluating tight junction, anti-N-cadherin, and anti-b-catenin antibodies for adherens junction, and stained with phalloidin for actin cytoskeleton. The reactive oxygen species (ROS) level was measured using DCFH-DA; Rho-associated coiled-coil forming kinase (ROCK) activity was by ELISA. Cytokine expression was analyzed by real-time RT-PCR and/or ELISA in the RPE-choroid, and macrophage recruitment was by real-time RT-PCR and immunohistochemistry. Either an antioxidant, N-Acetyl-L-cysteine (NAC), or a ROCK inhibitor, Y-27632, were administered to analyze the roles of ROS and ROCK activation, respectively. RESULTS. Light exposure disrupted staining patterns of tight junctions, adherens junctions, and actin cytoskeleton in the RPE, where ROS was elevated. However, NAC treatment avoided the RPE changes, reducing ROS. ROCK activity increased after light exposure was suppressed by NAC, and the structural disruptions were suppressed by Y-27632. The levels of MCP-1, CCL11, and IL-6 increased after light exposure were suppressed by NAC. Light-induced MCP-1 and IL- 6 were suppressed by Y-27632. Macrophage recruitment after light exposure was also suppressed either by NAC or Y-27632. CONCLUSIONS. Light exposure induced ROS and Rho/ROCK activation, which caused disruption of cell-cell junctions (tight junctions and adherens junctions) and actin cytoskeleton, the RPE's barrier structure, and induced AMD-associated pathological changes in the RPE-choroid.

AB - PURPOSE. To elucidate the influences of light exposure on the retinal pigment epithelium (RPE) in vivo that may be involved in the pathogenesis of AMD. METHODS. Six- to 7-week-old BALB/c mice were exposed to light at 2000 lux for 3 hours. Flatmount RPE samples were immunostained with anti-ZO-1 antibody for evaluating tight junction, anti-N-cadherin, and anti-b-catenin antibodies for adherens junction, and stained with phalloidin for actin cytoskeleton. The reactive oxygen species (ROS) level was measured using DCFH-DA; Rho-associated coiled-coil forming kinase (ROCK) activity was by ELISA. Cytokine expression was analyzed by real-time RT-PCR and/or ELISA in the RPE-choroid, and macrophage recruitment was by real-time RT-PCR and immunohistochemistry. Either an antioxidant, N-Acetyl-L-cysteine (NAC), or a ROCK inhibitor, Y-27632, were administered to analyze the roles of ROS and ROCK activation, respectively. RESULTS. Light exposure disrupted staining patterns of tight junctions, adherens junctions, and actin cytoskeleton in the RPE, where ROS was elevated. However, NAC treatment avoided the RPE changes, reducing ROS. ROCK activity increased after light exposure was suppressed by NAC, and the structural disruptions were suppressed by Y-27632. The levels of MCP-1, CCL11, and IL-6 increased after light exposure were suppressed by NAC. Light-induced MCP-1 and IL- 6 were suppressed by Y-27632. Macrophage recruitment after light exposure was also suppressed either by NAC or Y-27632. CONCLUSIONS. Light exposure induced ROS and Rho/ROCK activation, which caused disruption of cell-cell junctions (tight junctions and adherens junctions) and actin cytoskeleton, the RPE's barrier structure, and induced AMD-associated pathological changes in the RPE-choroid.

KW - Age-related macular degeneration

KW - Light damage

KW - Oxidative damage

KW - Retina

KW - Retinal pigment epithelium

UR - http://www.scopus.com/inward/record.url?scp=84879971182&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84879971182&partnerID=8YFLogxK

U2 - 10.1167/iovs.12-11572

DO - 10.1167/iovs.12-11572

M3 - Article

VL - 54

SP - 4555

EP - 4562

JO - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

SN - 0146-0404

IS - 7

ER -