Post-translational regulated and hypoxia-responsible VEGF plasmid for efficient secretion

Young Wook Won, Minhyung Lee, Hyun Ah Kim, David A. Bull, Sung Wan Kim

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Gene therapy using angiogenic genes has emerged as a potentially viable alternative treatment strategy for myocardial ischemia. Non-specific expression of angiogenic genes, however, may result in side effects, including the growth of occult tumors. Regulation of gene expression may help to avoid the occurrence of these side effects. In this study, a plasmid expressing vascular endothelial growth factor (VEGF) was constructed with an oxygen dependent degradation (ODD) domain and a secretion signal peptide (SP) in order to stabilize the VEGF protein and facilitate the secretion of VEGF protein, specifically under hypoxic conditions. We found that this plasmid, pβ-SP-ODD-VEGF, expresses the SP-ODD-VEGF protein at increased levels under hypoxic conditions compared to normoxic conditions. Since the size of the ODD domain is almost the same as that of VEGF, the ODD-VEGF fusion protein may have lower secretion efficiency. To address this issue, a furin recognition site was located between the ODD domain and the VEGF site to facilitate elimination of the SP-ODD domain from the fusion protein before its secretion. This optimizes the likelihood that the VEGF secreted from the target cells will be wild-type VEGF. Treatment with a furin inhibitor reduced the secretion efficiency of the VEGF, indicating that furin digestion increases the secretion of VEGF. The secreted wild-type VEGF facilitated the growth of endothelial cells more efficiently under hypoxic conditions than normoxic conditions. These results suggest that this plasmid, pβ-SP-ODD-VEGF, warrants further study as a more efficient form of hypoxia-inducible gene therapy for the treatment of myocardial ischemia.

Original languageEnglish
Pages (from-to)525-531
Number of pages7
JournalJournal of Controlled Release
Volume160
Issue number3
DOIs
StatePublished - 2012 Jun 28

Fingerprint

Vascular Endothelial Growth Factor A
Plasmids
Oxygen
Protein Sorting Signals
Furin
Hypoxia
Genetic Therapy
Myocardial Ischemia
Proteins
Gene Expression Regulation
Growth
Digestion
Therapeutics
Endothelial Cells
Gene Expression

Keywords

  • Gene therapy
  • Hypoxia
  • Myocardial ischemia
  • VEGF

Cite this

Won, Young Wook ; Lee, Minhyung ; Kim, Hyun Ah ; Bull, David A. ; Kim, Sung Wan. / Post-translational regulated and hypoxia-responsible VEGF plasmid for efficient secretion. In: Journal of Controlled Release. 2012 ; Vol. 160, No. 3. pp. 525-531.
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Post-translational regulated and hypoxia-responsible VEGF plasmid for efficient secretion. / Won, Young Wook; Lee, Minhyung; Kim, Hyun Ah; Bull, David A.; Kim, Sung Wan.

In: Journal of Controlled Release, Vol. 160, No. 3, 28.06.2012, p. 525-531.

Research output: Contribution to journalArticle

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AB - Gene therapy using angiogenic genes has emerged as a potentially viable alternative treatment strategy for myocardial ischemia. Non-specific expression of angiogenic genes, however, may result in side effects, including the growth of occult tumors. Regulation of gene expression may help to avoid the occurrence of these side effects. In this study, a plasmid expressing vascular endothelial growth factor (VEGF) was constructed with an oxygen dependent degradation (ODD) domain and a secretion signal peptide (SP) in order to stabilize the VEGF protein and facilitate the secretion of VEGF protein, specifically under hypoxic conditions. We found that this plasmid, pβ-SP-ODD-VEGF, expresses the SP-ODD-VEGF protein at increased levels under hypoxic conditions compared to normoxic conditions. Since the size of the ODD domain is almost the same as that of VEGF, the ODD-VEGF fusion protein may have lower secretion efficiency. To address this issue, a furin recognition site was located between the ODD domain and the VEGF site to facilitate elimination of the SP-ODD domain from the fusion protein before its secretion. This optimizes the likelihood that the VEGF secreted from the target cells will be wild-type VEGF. Treatment with a furin inhibitor reduced the secretion efficiency of the VEGF, indicating that furin digestion increases the secretion of VEGF. The secreted wild-type VEGF facilitated the growth of endothelial cells more efficiently under hypoxic conditions than normoxic conditions. These results suggest that this plasmid, pβ-SP-ODD-VEGF, warrants further study as a more efficient form of hypoxia-inducible gene therapy for the treatment of myocardial ischemia.

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