Wound Healing/Plastic Surgery
Angiogenic effects of injected VEGF165 and sVEGFR-1 (sFLT-1) in a rat flap model

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Abstract

Background. Injections of single-dose vascular endothelial growth factor (VEGF)165 have been advocated as a therapeutic tool for angiogenesis in ischemic flaps. We challenged this thesis by employing both VEGF165 and vascular endothelial growth factor receptor-1 (VEGFR-1) (for competitive inhibition of VEGF signal transduction) in different experimental settings of an ischemic rat flap model.

Material and methods. 80 isogenic rats were divided in two groups of 40 animals (groups 1A–1D and 2A–2D). The ischemic target was a 7 × 7-cm epigastric island flap, based on the right inferior epigastric pedicle. Group 1 received flap treatment 1 week prior to flap elevation by test substance injection into its flap panniculus carnosus: 1 ml NaCl 0.9% (1A), 1 ml Dulbecco’s modified Eagle’s medium (1B), 1.0 μg VEGF165 (1C), and 10 μg sFLT-1 with 1.0 μg VEGF165 (1D). sFLT-1 is a soluble receptor for VEGF and is able to prevent VEGF signaling through the cell surface receptor. Group 2 had the same flap treatment at the day of flap elevation.

Results. In group 1C we found the most vital flap tissue, without reaching significance. Compared with group 1D, however, significantly more flap tissue maintained vital. In groups 2A–2D, no significant results were found with respect to flap survival.

Conclusions. Local application of single-dose VEGF165 1 week prior to ischemia dose not have significant clinical angiogenic effects. In this experimental setting, VEGF165-induced angiogenic effects can be significantly inhibited by adding sFLT1 in vivo. A single-dose of VEGF165 under ischemic conditions causes no significantly better flap survival in this model.

Introduction

Our previous studies have shown that functional angiogenesis can be induced in flap tissue by means of genetic modification and transplantation of isogenic cells 1, 2. Now the impact of local, direct application of vascular endothelial growth factor (VEGF165) to induce functional angiogenesis in the same flap model was examined without using gene transfer techniques. Several authors have recently reported that local application of VEGF165 may improve survival of ischemically challenged tissue 3, 4, 5, 6. However, none of the studies could prove that the induction of angiogenesis was directly VEGF165-dependent. Evidence for this hypothesis can be given only by selective blocking of applied VEGF165.

The soluble human receptor 1, VEGFR-1 (sFLT-1), represents a member of the VEGF-receptor (VEGFR) family and is able to prevent VEGF signaling through the cell surface receptor by several means 7, 8. Therefore, the aim of this study was to examine the angiogenic effects of VEGF165 and its inhibition by sVEGFR-1 in a rat flap model.

Section snippets

Purification of recombinant VEGF165 by heparin-affinity chromatography

For purification of VEGF165 (National Research Center for Biotechnology, Braunschweig, Germany) protein from insect-cell supernatant acid precipitable proteins were removed by acidification of the supernatant to pH 3.5 and centrifugation at 10.000g for 15 min at 4°C. The supernatant was vacuum filtered through a 0.45-μm nitrocellulose membrane. After adjusting the pH to 6.5, the NaCl concentration of the supernatant was increased to 0.4 M and VEGF165 was applied on to a heparin-Sepharose

Induction of DNA synthesis in human vascular endothelial cells by VEGF165

Concentrations between 2.5 and 5.0 ng/ml were sufficient to maximize stimulation of DNA-synthesis. Results are shown in Fig. 2.

Flap survival rate in groups 1 and 2

Flap survival was slightly higher in group 1C, compared with groups 1A and 1B without reaching statistical significance (P = 0.189 for 1C versus 1A, P = 0.341 for 1C versus 1B). Comparing group 1C with 1D, however, significantly more flap tissue survived in group 1C (P = 0.037 for 1C versus 1D). Flaps in group 2C showed slightly better survival rates, compared with all

Discussion

In this study, we tested the impact of direct application of VEGF165 to induce functional angiogenesis in a McFarlane rat flap model. Local application of single-dose VEGF165 1 week prior to ischemia had some, but no significant, angiogenic effects in group 1C clinically. By adding the 10-fold amount of VEGFR-1 in group 1D, flap survival was significantly reduced compared with group 1C (P = 0.037). A single-dose of VEGF165 (group 2C) or VEGF165 plus the 10-fold amount of VEGFR-1 (group 2D)

Acknowledgements

This study was supported by a grant from German Research Society (D.F.G) (Ma1951/2-1).

References (18)

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Hans-Günther Machens, Ph.D., and Jila Salehi, M.S. contributed equally to this paper.

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