High glucose alters Cx43 expression and gap junction intercellular communication in retinal Müller cells

Promotes Müller cell and pericyte apoptosis

Tetsuya Muto, Thomas Tien, Dongjoon Kim, Vijay P Sarthy, Sayon Roy*

*Corresponding author for this work

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

Purpose. To investigate whether high glucose (HG) alters connexin 43 (Cx43) expression and gap junction intercellular communication (GJIC) activity in retinal M̈uller cells, and promotes Müller cell and pericyte loss. Methods. Retinal M̈uller cells (rMC-1) and cocultures of rMC-1 and retinal pericytes were grown in normal (N) or HG (30 mM glucose) medium. Additionally, rMC-1 transfected with Cx43 small interfering RNA (siRNA) were grown as cocultures with pericytes, and rMC-1 transfected with Cx43 plasmid were grown in HG. Expression of Cx43 was determined by Western blotting and immunostaining and GJIC was assessed by scrape-loading dye transfer (SLDT) technique. Apoptosis was analyzed by TUNEL or differential staining assay, and Akt activation by assessing Akt phosphorylation. Results. In monocultures of rMC-1 and cocultures of rMC-1 and pericytes, Cx43 protein level, number of Cx43 plaques, GJIC, and Akt phosphorylation were significantly reduced in HG medium. Number of TUNEL-positive cells was also significantly increased in rMC-1 monocultures and in rMC-1 and pericyte cocultures grown in HG medium. Importantly, when rMC-1 transfected with Cx43 siRNA were grown as cocultures with pericytes, a significant decrease in GJIC, and increase in TUNEL-positive cells was observed, concomitant with decreased Akt phosphorylation. Upregulation of Cx43 rescued rMC-1 from HG-induced apoptosis. Conclusions. Gap junction communication between Müller cells and pericytes is essential for their survival. Downregulation of Cx43 that is HG induced and impairment of GJIC activity in Müller cells contributes to loss of glial and vascular cells associated with the pathogenesis of diabetic retinopathy.

Original languageEnglish (US)
Pages (from-to)4327-4337
Number of pages11
JournalInvestigative Ophthalmology and Visual Science
Volume55
Issue number7
DOIs
StatePublished - Jan 1 2014

Fingerprint

Pericytes
Connexin 43
Gap Junctions
Apoptosis
Glucose
Coculture Techniques
In Situ Nick-End Labeling
Ependymoglial Cells
Phosphorylation
Small Interfering RNA
Diabetic Retinopathy
Neuroglia
Blood Vessels
Plasmids
Coloring Agents
Up-Regulation
Down-Regulation
Western Blotting
Staining and Labeling

Keywords

  • Connexin 43
  • Gap junctions
  • High glucose
  • Müller cells
  • Pericytes

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

@article{63307413d29e4a7ebfc0c8da72d98d0f,
title = "High glucose alters Cx43 expression and gap junction intercellular communication in retinal M{\"u}ller cells: Promotes M{\"u}ller cell and pericyte apoptosis",
abstract = "Purpose. To investigate whether high glucose (HG) alters connexin 43 (Cx43) expression and gap junction intercellular communication (GJIC) activity in retinal M̈uller cells, and promotes M{\"u}ller cell and pericyte loss. Methods. Retinal M̈uller cells (rMC-1) and cocultures of rMC-1 and retinal pericytes were grown in normal (N) or HG (30 mM glucose) medium. Additionally, rMC-1 transfected with Cx43 small interfering RNA (siRNA) were grown as cocultures with pericytes, and rMC-1 transfected with Cx43 plasmid were grown in HG. Expression of Cx43 was determined by Western blotting and immunostaining and GJIC was assessed by scrape-loading dye transfer (SLDT) technique. Apoptosis was analyzed by TUNEL or differential staining assay, and Akt activation by assessing Akt phosphorylation. Results. In monocultures of rMC-1 and cocultures of rMC-1 and pericytes, Cx43 protein level, number of Cx43 plaques, GJIC, and Akt phosphorylation were significantly reduced in HG medium. Number of TUNEL-positive cells was also significantly increased in rMC-1 monocultures and in rMC-1 and pericyte cocultures grown in HG medium. Importantly, when rMC-1 transfected with Cx43 siRNA were grown as cocultures with pericytes, a significant decrease in GJIC, and increase in TUNEL-positive cells was observed, concomitant with decreased Akt phosphorylation. Upregulation of Cx43 rescued rMC-1 from HG-induced apoptosis. Conclusions. Gap junction communication between M{\"u}ller cells and pericytes is essential for their survival. Downregulation of Cx43 that is HG induced and impairment of GJIC activity in M{\"u}ller cells contributes to loss of glial and vascular cells associated with the pathogenesis of diabetic retinopathy.",
keywords = "Connexin 43, Gap junctions, High glucose, M{\"u}ller cells, Pericytes",
author = "Tetsuya Muto and Thomas Tien and Dongjoon Kim and Sarthy, {Vijay P} and Sayon Roy",
year = "2014",
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doi = "10.1167/iovs.14-14606",
language = "English (US)",
volume = "55",
pages = "4327--4337",
journal = "Investigative Ophthalmology and Visual Science",
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number = "7",

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High glucose alters Cx43 expression and gap junction intercellular communication in retinal Müller cells : Promotes Müller cell and pericyte apoptosis. / Muto, Tetsuya; Tien, Thomas; Kim, Dongjoon; Sarthy, Vijay P; Roy, Sayon.

In: Investigative Ophthalmology and Visual Science, Vol. 55, No. 7, 01.01.2014, p. 4327-4337.

Research output: Contribution to journalArticle

TY - JOUR

T1 - High glucose alters Cx43 expression and gap junction intercellular communication in retinal Müller cells

T2 - Promotes Müller cell and pericyte apoptosis

AU - Muto, Tetsuya

AU - Tien, Thomas

AU - Kim, Dongjoon

AU - Sarthy, Vijay P

AU - Roy, Sayon

PY - 2014/1/1

Y1 - 2014/1/1

N2 - Purpose. To investigate whether high glucose (HG) alters connexin 43 (Cx43) expression and gap junction intercellular communication (GJIC) activity in retinal M̈uller cells, and promotes Müller cell and pericyte loss. Methods. Retinal M̈uller cells (rMC-1) and cocultures of rMC-1 and retinal pericytes were grown in normal (N) or HG (30 mM glucose) medium. Additionally, rMC-1 transfected with Cx43 small interfering RNA (siRNA) were grown as cocultures with pericytes, and rMC-1 transfected with Cx43 plasmid were grown in HG. Expression of Cx43 was determined by Western blotting and immunostaining and GJIC was assessed by scrape-loading dye transfer (SLDT) technique. Apoptosis was analyzed by TUNEL or differential staining assay, and Akt activation by assessing Akt phosphorylation. Results. In monocultures of rMC-1 and cocultures of rMC-1 and pericytes, Cx43 protein level, number of Cx43 plaques, GJIC, and Akt phosphorylation were significantly reduced in HG medium. Number of TUNEL-positive cells was also significantly increased in rMC-1 monocultures and in rMC-1 and pericyte cocultures grown in HG medium. Importantly, when rMC-1 transfected with Cx43 siRNA were grown as cocultures with pericytes, a significant decrease in GJIC, and increase in TUNEL-positive cells was observed, concomitant with decreased Akt phosphorylation. Upregulation of Cx43 rescued rMC-1 from HG-induced apoptosis. Conclusions. Gap junction communication between Müller cells and pericytes is essential for their survival. Downregulation of Cx43 that is HG induced and impairment of GJIC activity in Müller cells contributes to loss of glial and vascular cells associated with the pathogenesis of diabetic retinopathy.

AB - Purpose. To investigate whether high glucose (HG) alters connexin 43 (Cx43) expression and gap junction intercellular communication (GJIC) activity in retinal M̈uller cells, and promotes Müller cell and pericyte loss. Methods. Retinal M̈uller cells (rMC-1) and cocultures of rMC-1 and retinal pericytes were grown in normal (N) or HG (30 mM glucose) medium. Additionally, rMC-1 transfected with Cx43 small interfering RNA (siRNA) were grown as cocultures with pericytes, and rMC-1 transfected with Cx43 plasmid were grown in HG. Expression of Cx43 was determined by Western blotting and immunostaining and GJIC was assessed by scrape-loading dye transfer (SLDT) technique. Apoptosis was analyzed by TUNEL or differential staining assay, and Akt activation by assessing Akt phosphorylation. Results. In monocultures of rMC-1 and cocultures of rMC-1 and pericytes, Cx43 protein level, number of Cx43 plaques, GJIC, and Akt phosphorylation were significantly reduced in HG medium. Number of TUNEL-positive cells was also significantly increased in rMC-1 monocultures and in rMC-1 and pericyte cocultures grown in HG medium. Importantly, when rMC-1 transfected with Cx43 siRNA were grown as cocultures with pericytes, a significant decrease in GJIC, and increase in TUNEL-positive cells was observed, concomitant with decreased Akt phosphorylation. Upregulation of Cx43 rescued rMC-1 from HG-induced apoptosis. Conclusions. Gap junction communication between Müller cells and pericytes is essential for their survival. Downregulation of Cx43 that is HG induced and impairment of GJIC activity in Müller cells contributes to loss of glial and vascular cells associated with the pathogenesis of diabetic retinopathy.

KW - Connexin 43

KW - Gap junctions

KW - High glucose

KW - Müller cells

KW - Pericytes

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U2 - 10.1167/iovs.14-14606

DO - 10.1167/iovs.14-14606

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