Treatment of Mouse Limb Ischemia with an Integrative Hypoxia-Responsive Vector Expressing the Vascular Endothelial Growth Factor Gene

Eduardo Gallatti Yasumura,Leonardo Pinto De Carvalho,Roberta Sessa Stilhano,Vívian Yochiko Samoto,Valderez Bastos Valero Lapchik,Sang Won Han,Priscila Keiko Matsumoto,Holger K Eltzschig

doi:10.1371/journal.pone.0033944

Eduardo Gallatti Yasumura, Leonardo Pinto De Carvalho + Show 6 more

Open Access

https://doi.org/10.1371/journal.pone.0033944

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Journal: PLoS ONE	Publication Date: Mar 21, 2012
Citations: 31	License type: CC BY 4.0

Affiliation: Universidade Federal de São Paulo

Abstract

Constitutive vascular endothelial growth factor (VEGF) gene expression systems have been extensively used to treat peripheral arterial diseases, but most of the results have not been satisfactory. In this study, we designed a plasmid vector with a hypoxia-responsive element sequence incorporated into it with the phiC31 integrative system (pVHAVI) to allow long-term VEGF gene expression and to be activated under hypoxia. Repeated activations of VEGF gene expression under hypoxia were confirmed in HEK293 and C2C12 cells transfected with pVHAVI. In limb ischemic mice, the local administration of pVHAVI promoted gastrocnemius mass and force recovery and ameliorated limb necrosis much better than the group treated with hypoxia-insensitive vector, even this last group had produced more VEGF in muscle. Histological analyses carried out after four weeks of gene therapy showed increased capillary density and matured vessels, and reduced number of necrotic cells and fibrosis in pVHAVI treated group. By our study, we demonstrate that the presence of high concentration of VEGF in ischemic tissue is not beneficial or is less beneficial than maintaining a lower but sufficient and long-term concentration of VEGF locally.

Highlights

Peripheral arterial disease (PAD) is characterized by arterial narrowing that reduces oxygen supply to the extremities resulting in severe pain, non-healing ulcers and possible loss of the affected limb
In vitro evaluation of vascular endothelial growth factor (VEGF) gene expression To assess the functionality of the constructed vectors (Fig. 1), HEK293T cells were transfected with these vectors, and VEGF gene expression was monitored for more than 90 days (Fig. 2)
All groups showed a peak of VEGF gene expression at 48 hours post-transfection, which is a wellknown transient gene expression pattern, but by the 14th day, VEGF expression in all groups had returned to basal levels (Fig. 2)

Summary

Introduction

Peripheral arterial disease (PAD) is characterized by arterial narrowing that reduces oxygen supply to the extremities resulting in severe pain, non-healing ulcers and possible loss of the affected limb. Among several growth factors used for angiogenic therapy, vascular endothelial growth factor (VEGF) has been the most extensively studied because it is a potent mitogenic factor that has anti-apoptotic and vessel dilation activities [5,6]. This factor acts primarily on endothelial cells through the VEGFR1 and VEGFR2 receptors, but it promotes chemotaxis of smooth muscle cells, monocytes and bone marrow progenitor cells [6,7,8]. Many of these studies have advanced to clinical trials, but most of the results have not been satisfactory

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