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Complications

Effect of Combined Antisense Oligonucleotides Against High-Glucose–and Diabetes-Induced Overexpression of Extracellular Matrix Components and Increased Vascular Permeability

  1. Toshiyuki Oshitari12,
  2. Peter Polewski1,
  3. Manish Chadda1,
  4. An-Fei Li13,
  5. Tsuyoshi Sato14 and
  6. Sayon Roy1
  1. 1Departments of Medicine and Ophthalmology, Boston University School of Medicine, Boston, Massachusetts
  2. 2Department of Ophthalmology, Center for Sensory Organ Diseases, Sannoh Medical Center, Chiba, Japan
  3. 3Department of Ophthalmology, National Yang-Ming University, Taipei, Taiwan
  4. 4Eye Research Laboratory, Shin-Yahashiradai Hospital, Chiba, Japan
  1. Address correspondence and reprint requests to Sayon Roy, Department of Medicine, Boston University School of Medicine, 715 Albany St., Boston, MA 02118. E-mail: sayon{at}bu.edu
Diabetes 2006 Jan; 55(1): 86-92. https://doi.org/10.2337/diabetes.55.01.06.db05-0186
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  • FIG. 1.
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    FIG. 1.

    Western blot analysis of fibronectin, collagen IV, and laminin protein levels in cells grown in normal, high glucose, high glucose transfected with AS-oligos, and high glucose transfected with Ran-oligos mediums. Cells grown in high glucose medium showed increased fibronectin, collagen IV, and laminin expression compared with those grown in normal medium. Cells grown in high glucose medium and transfected with AS-oligos showed simultaneous reduction in fibronectin, collagen IV, and laminin expression compared with cells grown in high glucose medium.

  • FIG. 2.
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    FIG. 2.

    Graphical illustration of aggregate results of in vitro permeability assays showing a dose-dependent effect of AS-oligos on RMEC survival and proliferation. A: Cells grown in high glucose medium showed increased monolayer permeability compared with cells grown in normal medium. Cells grown in high glucose medium and transfected with combined AS-oligos showed reduced monolayer permeability compared with cells grown in high glucose medium. Cells grown in high glucose medium and transfected with Ran-oligos showed no reduction in monolayer permeability compared with cells grown in high glucose medium only. B: Dose ranging from 0.12 to 3.0 μmol/l of AS-oligos, which encompasses the dose of 0.4 μmol/l used in the study, did not affect cell survival or proliferation of RMECs grown in high glucose medium. *P < 0.05. HG, high glucose; AS, AS-oligos; Ran, Random; NS, not significant.

  • FIG. 3.
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    FIG. 3.

    Western blot analysis of fibronectin, collagen IV, and laminin protein levels in normal and diabetic retinas, diabetic retinas treated with combined AS-oligos, and diabetic retinas treated with Ran-oligos. In retinas of rats with 2 months of diabetes, fibronectin, collagen IV, and laminin protein expression was significantly increased compared with normal retinas. In diabetic retinas treated with combined AS-oligos, fibronectin, collagen IV, and laminin protein expression was significantly decreased compared with those in diabetic retinas. Retinas treated with Ran-oligos showed no effect. All densitometric values for Western blot analysis were normalized with β-actin signals.

  • FIG. 4.
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    FIG. 4.

    Representative areas showing FITC fluorescence in vessels of whole-mount retinas. Areas of intense FITC leakage from extravasation were observed in the diabetic (DM) retinas compared with control nondiabetic retinas. Also, in the diabetic retinas, some areas showed diffused “patches” at the site of intense vascular leakage (arrows). In the diabetic rat retinas, combined AS-oligos (AS) treatment partially but significantly reduced areas of extravasation associated with excess vascular permeability. Ran-oligos (Ran) treatment showed no effect on retinal vascular permeability. Nonperfused vessel (arrowhead). Magnification 200×.

  • FIG. 5.
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    FIG. 5.

    Representative images of retinal cryosections from nondiabetic control rats, diabetic (DM) rats, diabetic rats injected with combined AS-oligos (AS), and diabetic rats injected with Ran-oligos (Ran). In diabetic retinas, extravasation of FITC in and around the vessels was significantly increased compared with those in the nondiabetic control retinas. Combined AS-oligos–treated retinas showed reduction in vascular leakage compared with those in untreated diabetic retinas or Ran-oligos–treated diabetic retinas. Magnification 200×.

  • FIG. 6.
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    FIG. 6.

    Graphical illustration of aggregate results of in vivo permeability assays. A: In retinas of 2-month diabetic rats, the areas of FITC extravasation were significantly increased compared with those in control retinas. Combined AS-oligos–treated retinas showed partially but significantly reduced vascular leakage compared with the untreated diabetic retinas. Ran-oligo treatment showed no effect on retinal vascular permeability. Data shown are means ± SE. B: Reference points for the evaluation of excess permeability in relation to increased synthesis of ECM components. *P < 0.05. P, permeability; F, fibronectin; C, collagen IV; L, laminin; NS, not significant.

Tables

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  • TABLE 1

    Effect of combined AS-oligos against expression of ECM components, fibronectin, collegen IV, and laminin in cells grown in high glucose medium compared with cells grown in normal medium or cells transfected with Ran-oligos

    ECM geneNormalHigh glucoseHigh glucose + antisenseHigh glucose + random
    Fibronectin (n = 4) (%)100 ± 29174 ± 32*60 ± 21†167 ± 31
    Collagen IV (n = 7) (%)100 ± 15156 ± 23*86 ± 24†124 ± 17
    Laminin (n = 7) (%)100 ± 16128 ± 18*96 ± 29†123 ± 17
    • Date are means ± SD (% of control).

    • *

      * P < 0.05 compared with normal;

    • †

      † P < 0.05 compared with high glucose.

  • TABLE 2

    Effect of combined AS-oligos against expression of ECM components, fibronectin, collegen IV, and laminin in retinas of diabetic rats

    NormalDiabetesAntisenseRandom
    1 month
        Fibronectin100 ± 7124 ± 5494 ± 16109 ± 22
        Collagen IV100 ± 21155 ± 87101 ± 38163 ± 108
        Laminin100 ± 31127 ± 5996 ± 21114 ± 18
    2 months
        Fibronectin100 ± 6195 ± 38*147 ± 29†201 ± 46
        Collagen IV100 ± 14191 ± 48*146 ± 28†202 ± 41
        Laminin100 ± 10183 ± 54*128 ± 47†184 ± 31
    • Data are means ± SD (% of control).

    • *

      * P < 0.05 compared with control retinas;

    • †

      † P < 0.05 compared with diabetic retinas.

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Effect of Combined Antisense Oligonucleotides Against High-Glucose–and Diabetes-Induced Overexpression of Extracellular Matrix Components and Increased Vascular Permeability
Toshiyuki Oshitari, Peter Polewski, Manish Chadda, An-Fei Li, Tsuyoshi Sato, Sayon Roy
Diabetes Jan 2006, 55 (1) 86-92; DOI: 10.2337/diabetes.55.01.06.db05-0186

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Effect of Combined Antisense Oligonucleotides Against High-Glucose–and Diabetes-Induced Overexpression of Extracellular Matrix Components and Increased Vascular Permeability
Toshiyuki Oshitari, Peter Polewski, Manish Chadda, An-Fei Li, Tsuyoshi Sato, Sayon Roy
Diabetes Jan 2006, 55 (1) 86-92; DOI: 10.2337/diabetes.55.01.06.db05-0186
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