Akt/protein kinase B and endothelial nitric oxide synthase mediate muscular neovascularization induced by tissue kallikrein gene transfer

Costanza Emanueli, Maria B. Salis, Sophie Van Linthout, Marco Meloni, Elisa Desortes, Jean Sebastien Silvestre, Michel Clergue, Carlos D. Figueroa, Sergio Gadau, Gianluigi Condorelli, Paolo Madeddu

Research output: Contribution to journalArticle

Abstract

Background - Angiogenesis gene therapy with human tissue kallikrein (hTK) has shown promise for ischemic disease. The present study was undertaken to (1) assess an optimal gene transfer modality, (2) clarify hTK angiogenic pathways, and (3) discount possible side effects. Methods and Results - The hTK gene was transferred to murine adductors by increasing doses of an adenovirus (Ad.hTK). Heterologous protein production was evaluated by ELISA and immunohistochemistry. Structural and functional characteristics of hTK-induced neovascularization were assessed. Muscular endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF)-A mRNA and protein content were evaluated by real-time polymerase chain reaction and Western blotting. The ability of hTK to phosphorylate-activate Akt/protein kinase B (Akt-B) and VEGF receptor 2 (VEGF-R2) was also determined. Implication of the aforementioned mechanisms in Ad.hTK-induced neovascularization was challenged by blocking Akt-B with a dominant-negative Akt construct; NOS with NG-nitro-L-arginine methyl ester; and VEGF-A with neutralizing antibody, VEGF-R2 antagonist, or Ad carrying soluble VEGF-R1 gene. We found that 107 PFU Ad.hTK led to peak increases in capillary and arteriole density. Newly developed arterioles persisted for up to 8 weeks. Ad.hTK did not change microvascular permeability. Ad.hTK upregulated eNOS mRNA and protein and activated Akt-B through Ser-473 phosphorylation. Inhibitory studies documented that these biochemical events were instrumental to Ad.hTK-induced neovascularization. In contrast, Ad.hTK neither affected VEGF-A and VEGF-R2 levels nor increased VEGF-R2 phosphorylation. Consistently, Ad.hTK-induced neovascularization was not disturbed by any of the different approaches used to block VEGF-A. Conclusions - Our findings provide new information on the pathway involved in hTK-induced neoangiogenesis and represent an advancement toward clinical applications with Ad.hTK.

Original languageEnglish
Pages (from-to)1638-1644
Number of pages7
JournalCirculation
Volume110
Issue number12
DOIs
Publication statusPublished - Sep 21 2004

Keywords

  • Angiogenesis
  • Bradykinin
  • Gene therapy
  • Muscle
  • Nitric oxide synthase

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

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    Emanueli, C., Salis, M. B., Van Linthout, S., Meloni, M., Desortes, E., Silvestre, J. S., Clergue, M., Figueroa, C. D., Gadau, S., Condorelli, G., & Madeddu, P. (2004). Akt/protein kinase B and endothelial nitric oxide synthase mediate muscular neovascularization induced by tissue kallikrein gene transfer. Circulation, 110(12), 1638-1644. https://doi.org/10.1161/01.CIR.0000142051.36244.83