Regulable Vascular Endothelial Growth Factor165 Overexpression by Ex Vivo Expanded Keratinocyte Cultures Promotes Matrix Formation, Angiogenesis, and Healing in Porcine Full-Thickness Wounds

Abstract

The intricate wound repair process involves the interplay of numerous cells and proteins. Using a porcine full-thickness wound (FTW) healing model, we hypothesized that the ex vivo gene transfer of vascular endothelial growth factor (VEGF)-transfected basal keratinocyte (KC) cell suspensions may generate cross-talk and induce matrix formation, angiogenesis, and accelerated healing. Moreover, to regulate overexpression of isoform 165 of VEGF and its effect on healing, we introduced a tetracycline (TC)-inducible gene switch in the expression plasmid. Autologous basal KCs were cultivated from the porcine donor and transfected using cationic liposomes. A dose-response curve was established to determine optimal activation of the gene switch by TC. In vivo, FTWs were treated with VEGF-transfected KCs and controls. Wound fluids were collected daily and examined using enzyme-linked immunosorbent assay. Biopsies were evaluated using hematoxylin and eosin and immunostaining for fibronectin, CD144, and lectin BS-1. In vitro, highest regulable VEGF165-expression was obtained with 1 microg/mL of TCs. In vivo, after induction of the gene switch by adding 1 microg/mL of TCs to the FTW, we obtained upregulated VEGF165 levels and enhanced fibronectin deposition and found more endothelial cell tubular formations and higher rates of reepithelialization than in controls. This ex vivo gene transfer model may serve as a platform for vascular induction in full-thickness tissue repair.