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Inhibition of Gene Expression In Vivo Using Multiplex siRNA

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RNA Silencing

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 309))

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Abstract

Angiogenesis, also known as new blood vessel formation, is a critical process during physiological and pathological conditions (1,2). During normal development, the physiological wound healing process, and the female menstrual cycle, blood vessel growth is coordinated concomitantly with tissue growth. However, during pathological processes such as solid cancer development, diabetic retinopathy, rheumatoid arthritis, and psoriasis, abnormal angiogenesis contributes to disease progression. Thus, much effort is directed toward understanding of and the control of the angiogenesis process (3). Angiogenesis involves the orderly migration, proliferation, and differentiation of vascular endothelial cells into nascent blood vessel sprouts. This is followed by vascular stabilization, which entails the coverage with mural cells (pericytes or vascular smooth muscle cells) (4). Numerous factors regulate the various phases of angiogenesis. A major regulator of angiogenesis, vascular endothelial growth factor (VEGF), was upregulated in tumor angiogenesis, and inhibition of this factor with a specific monoclonal antibody was recently approved for clinical use in colorectal cancer (3). However, angiogenesis is regulated by multiple redundant pathways; therefore, multiple approaches might be needed to effectively control angiogenesis.

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References

  1. Risau, W. (1997) Mechanisms of angiogenesis. Nature 386, 671–674.

    Article  PubMed  CAS  Google Scholar 

  2. Ferrara, N., Gerber, H. P., and LeCouter, J. (2003) The biology of VEGF and its receptors. Nat. Med. 9, 669–676.

    Article  PubMed  CAS  Google Scholar 

  3. Ferrara, N., Hillan, K. J., Gerber, H. P., and Novotny, W. (2004) Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat. Rev. Drug Discov. 3, 391–400.

    Article  PubMed  CAS  Google Scholar 

  4. Jain, R. K. (2003) Molecular regulation of vessel maturation. Nat. Med. 9, 685–693.

    Article  PubMed  CAS  Google Scholar 

  5. Hla, T. (2001) Sphingosine 1-phosphate receptors. Prostaglandins 64, 135–142.

    PubMed  CAS  Google Scholar 

  6. Hla, T. and Maciag, T. (1990) An abundant transcript induced in differentiating human endothelial cells encodes a polypeptide with structural similarities to Gprotein-coupled receptors. J. Biol. Chem. 265, 9308–9313.

    PubMed  CAS  Google Scholar 

  7. Allende, M. L., Yamashita, T., and Proia, R. L. (2003) G-Protein-coupled receptor S1P acts within endothelial cells to regulate vascular maturation. Blood 102, 3665–3667.

    Article  PubMed  CAS  Google Scholar 

  8. Chae, S. S., Paik, J-H., Furneaux, H., and Hla, T. (2004) Requirement for sphingosine 1-phosphate receptor-1 in tumor angiogenesis demonstrated by in vivo RNA interference. J. Clin. Invest., in press.

    Google Scholar 

  9. Lee, M. J., Thangada, S., Paik, J. H., et al. (2001) Akt-Mediated phosphorylation of the G protein-coupled receptor EDG-1 is required for endothelial cell chemotaxis. Mol. Cell 8, 693–704.

    Article  PubMed  CAS  Google Scholar 

  10. Hannon, G. J. (2002) RNA interference. Nature 418, 244–251.

    Article  PubMed  CAS  Google Scholar 

  11. Elbashir, S. M., Lendeckel, W., and Tuschl, T. (2001) RNA interference is mediated by 21-and 22-nucleotide RNAs. Genes Dev. 15, 188–200.

    Article  PubMed  CAS  Google Scholar 

  12. Song, E., Lee, S. K., Wang, J., et al. (2003) RNA interference targeting Fas protects mice from fulminant hepatitis. Nat. Med. 9, 347–351.

    Article  PubMed  CAS  Google Scholar 

  13. Rubinson, D. A., Dillon, C. P., Kwiatkowski, A. V., et al. (2003) A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat. Genet. 33, 401–406.

    Article  PubMed  CAS  Google Scholar 

  14. Dave, R. S. and Pomerantz, R. J. (2003) RNA interference: on the road to an alternate therapeutic strategy! Rev. Med. Virol. 13, 373–385.

    Article  PubMed  CAS  Google Scholar 

  15. Amarasinghe, A. K., Calin-Jageman, I., Harmouch, A., Sun, W., and Nicholson, A. W. (2001) Escherichia coli ribonuclease III: affinity purification of hexahistidinetagged enzyme and assays for substrate binding and cleavage. Methods Enzymol. 342, 143–158.

    Article  PubMed  CAS  Google Scholar 

  16. Yang, D., Buchholz, F., Huang, Z., et al. (2002) Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells. Proc. Natl. Acad. Sci. USA 99, 9942–9947.

    Article  PubMed  CAS  Google Scholar 

  17. Liu, C. H. and Hla, T. (1997) The mouse gene for the inducible G-protein-coupled receptor edg-1. Genomics 43, 15–24.

    Article  PubMed  CAS  Google Scholar 

  18. Paik, J. H., Chae, S., Lee, M. J., Thangada, S., and Hla, T. (2001) Sphingosine 1-phosphate-induced endothelial cell migration requires the expression of EDG-1 and EDG-3 receptors and Rho-dependent activation of alpha vbeta3-and beta1-containing integrins. J. Biol. Chem. 276, 11,830–11,837.

    Article  PubMed  CAS  Google Scholar 

  19. Ancellin, N., Colmont, C., Su, J., et al. (2002) Extracellular export of sphingosine kinase-1 enzyme. Sphingosine 1-phosphate generation and the induction of angiogenic vascular maturation. J. Biol. Chem. 277, 6667–6675.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The authors thank Dr. Allan Nicholson for the gift of the RNase III plasmid, and Dr. Henry Furneaux and Dr. Ji-Hye Paik for helpful discussions.

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© 2005 Humana Press Inc.

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Chae, SS., Hla, T. (2005). Inhibition of Gene Expression In Vivo Using Multiplex siRNA. In: Carmichael, G.G. (eds) RNA Silencing. Methods in Molecular Biology™, vol 309. Humana Press. https://doi.org/10.1385/1-59259-935-4:197

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  • DOI: https://doi.org/10.1385/1-59259-935-4:197

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-436-4

  • Online ISBN: 978-1-59259-935-6

  • eBook Packages: Springer Protocols

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