title: Endocytosis of tight junctions caveolin nitrosylation dependent is improved by cocoa via opioid receptor on RPE cells in diabetic conditions.
abstract: OBJECTIVE
  Retinal pigment epithelium cells, along with tight junction (TJ) proteins, constitute the outer blood retinal barrier (BRB). Contradictory findings suggest a role for the outer BRB in the pathogenesis of diabetic retinopathy (DR). The aim of this study was to investigate whether the mechanisms involved in these alterations are sensitive to nitrosative stress, and if cocoa or epicatechin (EC) protects from this damage under diabetic (DM) milieu conditions.
  
  
  METHODS
  Cells of a human RPE line (ARPE-19) were exposed to high-glucose (HG) conditions for 24 hours in the presence or absence of cocoa powder containing 0.5% or 60.5% polyphenol (low-polyphenol cocoa [LPC] and high-polyphenol cocoa [HPC], respectively).
  
  
  RESULTS
  Exposure to HG decreased claudin-1 and occludin TJ expressions and increased extracellular matrix accumulation (ECM), whereas levels of TNF-α and inducible nitric oxide synthase (iNOS) were upregulated, accompanied by increased nitric oxide levels. This nitrosative stress resulted in S-nitrosylation of caveolin-1 (CAV-1), which in turn increased CAV-1 traffic and its interactions with claudin-1 and occludin. This cascade was inhibited by treatment with HPC or EC through δ-opioid receptor (DOR) binding and stimulation, thereby decreasing TNF-α-induced iNOS upregulation and CAV-1 endocytosis. The TJ functions were restored, leading to prevention of paracellular permeability, restoration of resistance of the ARPE-19 monolayer, and decreased ECM accumulation.
  
  
  CONCLUSIONS
  The detrimental effects on TJs in ARPE-19 cells exposed to DM milieu occur through a CAV-1 S-nitrosylation-dependent endocytosis mechanism. High-polyphenol cocoa or EC exerts protective effects through DOR stimulation.
authors.affiliation: Renal Pathophysiology Laboratory and Investigation on Diabetes Complications, School of Medical Sciences, State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil.
authors.forename: Mariana Aparecida B
authors.initials: MA
authors.lastname: Rosales
authors.affiliation: Renal Pathophysiology Laboratory and Investigation on Diabetes Complications, School of Medical Sciences, State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil.
authors.forename: Kamila Cristina
authors.initials: KC
authors.lastname: Silva
authors.affiliation: Renal Pathophysiology Laboratory and Investigation on Diabetes Complications, School of Medical Sciences, State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil.
authors.forename: Diego A
authors.initials: DA
authors.lastname: Duarte
authors.affiliation: Bioenergetics Laboratory, Department of Clinical Pathology, School of Medical Sciences, State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil.
authors.forename: Franco Aparecido
authors.initials: FA
authors.lastname: Rossato
authors.affiliation: Renal Pathophysiology Laboratory and Investigation on Diabetes Complications, School of Medical Sciences, State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil.
authors.forename: José B
authors.initials: JB
authors.lastname: Lopes de Faria
authors.affiliation: Renal Pathophysiology Laboratory and Investigation on Diabetes Complications, School of Medical Sciences, State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil.
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: Lopes de Faria
mesh_terms: D000818:Animals; D001813:Blood-Retinal Barrier; D015153:Blotting, Western; D002099:Cacao / Q000737:chemistry*; D051242:Caveolin 1 / Q000378:metabolism*; D002460:Cell Line; D062445:Claudin-1 / Q000378:metabolism; D003911:Dextrans / Q000378:metabolism; D017097:Electric Impedance; D004705:Endocytosis / Q000502:physiology*; D019084:Fluorescent Antibody Technique, Indirect; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D015538:Nitrosation; D062793:Occludin / Q000378:metabolism; D010539:Permeability; D059808:Polyphenols / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D011957:Receptors, Opioid / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism; D013552:Swine; D019108:Tight Junctions / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: C495540:CAV1 protein, human; D051242:Caveolin 1; D062445:Claudin-1; D003911:Dextrans; C568985:OCLN protein, human; D062793:Occludin; D059808:Polyphenols; D017382:Reactive Oxygen Species; D011957:Receptors, Opioid; D014409:Tumor Necrosis Factor-alpha; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; D005947:Glucose
keywords: RPE; cocoa; high glucose; nitrosative stress; tight junction