June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
High Glucose-Induced Connexin 43 (Cx43) Downregulation Modulates Rho/Rho kinase (ROCK) Signaling and Promotes Apoptosis in Retinal Endothelial Cells
Author Affiliations & Notes
  • Tetsuya Muto
    Medicine and Ophthalmology, Boston University School of Medicine, Boston, MA
  • Sayon Roy
    Medicine and Ophthalmology, Boston University School of Medicine, Boston, MA
  • Footnotes
    Commercial Relationships Tetsuya Muto, None; Sayon Roy, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 2687. doi:
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      Tetsuya Muto, Sayon Roy; High Glucose-Induced Connexin 43 (Cx43) Downregulation Modulates Rho/Rho kinase (ROCK) Signaling and Promotes Apoptosis in Retinal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2687.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: To evaluate whether HG-induced Cx43 downregulation and gap junction intercellular communication (GJIC) promote apoptosis in rat retinal endothelial cells (RRECs) by activating ROCK signaling.

Methods: RRECs were grown in normal medium (N; 5 mM glucose) or HG medium (30mM) for 7 days. In parallel, cells grown in HG were exposed to 10μM fasudil, a selective ROCK inhibitor. In addition, cells grown in HG and exposed to fasudil were transfected with Cx43-siRNA or scrambled siRNA. To determine ROCK signaling, phosphorylation of myosin phosphatase target protein (MYPT)-1 and Cx43 protein levels were determined by Western blot analysis. In parallel, GJIC activity was assessed by scrape-load dye transfer (SLDT) technique and apoptosis by TUNEL assay, respectively.

Results: HG significantly upregulated ROCK activity as determined from Western blot analysis showing increased MYPT-1 phosphorylation (170±28% of N, p<0.05), and significantly reduced Cx43 protein level in cells grown in HG (63±14% of N, p<0.01). When cells grown in HG were exposed to fasudil, ROCK activity was significantly inhibited (134±30% of N) with concomitant upregulation in Cx43 (92.0±14% of N), GJIC activity (96±12% vs 61±11% of N) and significant reduction in apoptosis (115±19% vs 180±29% of N). Interestingly, when these cells grown in HG and exposed to fasudil were transfected with Cx43-siRNA, ROCK activity and the number of apoptotic cells increased (165±43% vs 134±30% of N;155±16% vs 115±19% of N, p<0.05, respectively). These results suggest that HG-induced Cx43 downregulation is a critical step upstream of ROCK activity.

Conclusions: Apoptosis is mediated through Cx43 downregulation and increased activity of ROCK signaling in RRECs exposed to HG. Modulation of Cx43 is a critical step in ROCK-induced apoptotic cell death, a hallmark of diabetic retinopathy.

Keywords: 499 diabetic retinopathy • 426 apoptosis/cell death • 447 cell-cell communication  
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