Abstract
Angiogenesis is a complex phenomenon regulated by both pro- and antiangiogenic factors such as the vascular endothelial growth factor (VEGF), and inflammation may be involved in the process. Although antagonizing VEGF has been proposed as a therapeutic approach to limit corneal angiogenesis, alternative targets are needed. In this study, we demonstrate that, under proangiogenic experimental conditions, human endothelial cells (hECs) express more insulin receptor substrate (IRS)-1 proteins relative to quiescent cells. The antisense oligonucleotide, GS-101 (5′-TATCCGGAGGGCTCGCCATGCTGCT-3′), targeting IRS-1 mRNA, dose-dependently inhibited (p < 0.01) both IRS-1 expression and in vitro angiogenesis (hEC tube-like structure formation) with IC50 of 8.51 ± 3.01 μM (mean ± S.E.M.) and 2.47 ± 0.56 μM, respectively, demonstrating that partial IRS-1 down-regulation interferes with angiogenesis. The antiangiogenic effects of GS-101 were associated with a decrease in protein kinase B (Akt) activation but not mitogen-activated protein kinase-1/2 and a dose-dependent reduction in vascular endothelial growth factor-A (IC50 = 5.59 ± 2.76 μM) and the proinflammatory cytokine interleukin-1β (IC50 = 2.19 ± 1.07 μM) mRNA expression. In accordance, once daily topical application of GS-101 dose-dependently inhibited injury-dependent corneal angiogenesis in vivo (p < 0.05). GS-101 in vivo efficacy was achieved at final tissue concentrations within in vitro EC50 for IRS-1 down-regulation. In conclusion, these results suggest that IRS-1 is important for angiogenesis and that GS-101 could become a novel therapeutic tool against corneal angiogenesis.
Footnotes
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This work was supported by an unrestricted educational grant from Gene Signal Laboratories.
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doi:10.1124/jpet.108.147496.
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ABBREVIATIONS: IRS, insulin receptor substrate; IGF, insulin-like growth factor; VEGF-A, vascular endothelial growth factor-A; IL, interleukin; hEC, human endothelial cell; FGF2, fibroblast growth factor-2; PBS, physiological buffer solution; RT, reverse transcriptase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; ERK, extracellular signal-regulated kinase; mAb, monoclonal antibody; GS-101, 5′-TATCCGGAGGGCTCGCCATGCTGCT-3′.
- Received October 15, 2008.
- Accepted February 9, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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