May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Combined Antisense Oligonucleotide Strategy Against High Glucose-Induced Overexpression of Extracellular Matrix Components and Excess Endothelial Cell Permeability
Author Affiliations & Notes
  • M. Chadda
    Ophthalmology, Boston University School of Medicine, Boston, MA, United States
  • A. Li
    Ophthalmology, Boston University School of Medicine, Boston, MA, United States
  • S. Roy
    Ophthalmology, Boston University School of Medicine, Boston, MA, United States
  • Footnotes
    Commercial Relationships  M. Chadda, None; A. Li, None; S. Roy, None.
  • Footnotes
    Support  ADA, NEI EY11990
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 1079. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      M. Chadda, A. Li, S. Roy; Combined Antisense Oligonucleotide Strategy Against High Glucose-Induced Overexpression of Extracellular Matrix Components and Excess Endothelial Cell Permeability . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1079.

      Download citation file:


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

      ×
  • Supplements
Abstract

Abstract: : Purpose: To establish the efficacy of combined antisense oligonucleotide (oligo) strategy against excess synthesis of extracellular matrix (ECM) proteins, fibronectin (FN), collagen type IV (coll IV), and laminin (LM), and its effect on endothelial cell permeability. Methods: Rat microvascular endothelial cells (RMECs) were grown in normal (5mM) or high glucose (30mM) medium for 10 days. In addition, two separate groups of RMECs grown in high glucose medium for 7 days were transfected for 72 h with combined antisense phosphorothioate (PS) oligos against three ECM components, FN, coll IV, and LM at a concentration of 0.4 uM each or with 0.4 uM random PS oligos. In parallel, RMECs were grown on inserts of 24-well plates in normal or high glucose medium. In addition, cells grown in high glucose medium were transfected with combined or random oligos. In vitro permeability assay was performed in confluent monolayers by adding fluorescein isothiocyanate-dextran (FITC-dex) at a concentration of (1 mg/ml) to the upper chamber and monitoring its passage into the lower chamber of the transwells. Presence of FITC-dex in the lower chamber was determined by spectrophotometric readings performed at 492 nm. Results: Western blot analysis showed that cells grown in high glucose medium exhibited increased protein expression for FN, coll IV, and LM (174±31%, 149±25%, and 135±18% of control, respectively) compared to cells grown in normal medium. Cells transfected with combined antisense oligos showed reduced protein level for the three ECM components, FN, coll IV and LM (61±20%, 97±17%, and 95±23% of control, respectively). Cells grown in high glucose medium showed increased monolayer permeability compared to cells grown in normal medium (153±43% of control, P=0.001); transfection with combined antisense oligos significantly reduced monolayer permeability (91±28% of control, P=0.002). Cells transfected with random PS oligos had no effect on FN, coll IV and LM protein expression or endothelial cell monolayer permeability. Conclusions: Taken together, the results indicate that combined antisense oligo strategy is effective in reducing simultaneously three ECM protein expression and that altered ECM synthesis may be linked to increased vascular permeability associated with diabetic retinopathy. Potential additive effect of the combined antisense approach may be a powerful means to prevent the development of vascular BM thickening in diabetic retinopathy.

Keywords: retina • diabetic retinopathy • extracellular matrix 
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×