Inducible adeno-associated virus vectors promote functional angiogenesis in adult organisms via regulated vascular endothelial growth factor expression.
OBJECTIVE
Members of the vascular endothelial growth factor (VEGF) family are among the most promising cytokines to induce neovascularization of ischaemic tissues; however, their unregulated expression often results in major undesired effects. Here, we describe the properties of inducible vectors based on the adeno-associated virus (AAV), allowing precise control of VEGF expression, and exploit these vectors to define the kinetics of the angiogenic response elicited by the factor.


RESULTS
Based on a tetracycline-inducible transactivator, we designed an AAV vector system allowing the pharmacological regulation of VEGF production in vivo and tested its efficacy in inducing functional neoangiogenesis in both normoperfused and ischaemic skeletal muscle in mice by a combination of histological, immunofluorescent, and molecular imaging techniques. We observed that a prolonged expression of VEGF was required to determine the formation of stable vessels, able to persist upon withdrawal of the angiogenic stimulus. However, the vessels formed in the presence of continuous VEGF expression consisted mainly of dilated and leaky capillaries. As determined after pinhole scintigraphy, this abnormal vasculature accounted for a significant drop in functional tissue perfusion. In contrast, transient VEGF expression, followed by a period of VEGF withdrawal, allowed maintenance of functional perfusion under resting conditions and during exercise. This VEGF-inducible system was highly effective in improving vascularization and function in a hind-limb ischaemia model.


CONCLUSIONS
Together, these results clearly indicate that the fine tuning of VEGF expression is required to achieve the formation of a stable vasculature able to sustain functional neovascularization.
Tafuro
Sabrina
S
Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology, Padriciano, 99, 34012 Trieste, Italy.
Ayuso
Eduard
E
Zacchigna
Serena
S
Zentilin
Lorena
L
Moimas
Silvia
S
Dore
Franca
F
Giacca
Mauro
M
D000818:Animals; D000229:Dependovirus / Q000235:genetics*; D004318:Doxycycline / Q000008:administration & dosage; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors; D006801:Humans; D007511:Ischemia / Q000378:metabolism / Q000473:pathology / Q000628:therapy; D051379:Mice; D018482:Muscle, Skeletal / Q000098:blood supply / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000235:genetics* / Q000627:therapeutic use
D012333:RNA, Messenger; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D004318:Doxycycline