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

Emanueli, Costanza and Salis, Maria Bonaria and Linthout, Sophie Van and Meloni, Marco and Desortes, Elisa and Silvestre, Jean Sebastien and Clergue, Michel and Figueroa, Carlos D. and Gadau, Sergio Domenico and Condorelli, Gianluigi and Madeddu, Paolo Roberto (2004) Akt/protein kinase B and endothelial nitric oxide synthase mediate muscular neovascularization induced by tissue kallikrein gene transfer. Circulation, Vol. 110 (12), p. 1638-1644. eISSN 1524-4539. Article.

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DOI: 10.1161/01.CIR.0000142051.36244.83

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 N(G)-nitro-L-arginine methyl ester; and VEGF-A with neutralizing antibody, VEGF-R2 antagonist, or Ad carrying soluble VEGF-R1 gene. We found that 10(7) 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.

Item Type:Article
ID Code:7749
Status:Published
Refereed:Yes
Uncontrolled Keywords:Gene therapy, nitric oxide synthase, angiogenesis, muscle, bradykinin
Subjects:Area 07 - Scienze agrarie e veterinarie > VET/01 Anatomia degli animali domestici
Divisions:001 Università di Sassari > 01 Dipartimenti > Biologia animale
002 Altri enti e centri di ricerca del Nord Sardegna > Istituto nazionale biostrutture e biosistemi, Unità di ricerca, Sassari
Publisher:Lippincott Williams & Wilkins
eISSN:1524-4539
Copyright Holders:© 2004 American Heart Association
Deposited On:06 Jul 2012 09:14

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