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.

Integration of Blood Outgrowth Endothelial Cells in Dermal Fibroblast Sheets Promotes Full Thickness Wound Healing

“…Apart from EPC, other (stem) cell sources have been considered to vascularize wounds and efforts have mostly been made in the field of mesenchymal stem cells (MSC) [22,23]. In addition to cell therapy, angiogenic growth factor therapy [e.g., vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), plateletderived growth factor (PDGF), and placental growth factor (PlGF)] has been explored to grow vessels in nonhealing or chronic wounds [24][25][26].…”

Experimental Study of Human Bone Marrow Mesenchymal Stem Cells Transfected with Vascular Endothelial Growth Factor Gene in Vitro

“…VEGF165 is a role to play in the main form which has the strongest activity and is most widely distributed [2]. Gene therapy with VEGF has been shown to promote therapeutic angiogenesis in preclinical models of tissue ischemia and in human clinical trials [3,4,5,6]. The present results indicate that VEGF165 is a promising candidate for gene therapy in wound healing applications.…”

A New Technique of Ex Vivo Gene Delivery of VEGF to Wounds Using Genetically Modified Skin Particles Promotes Wound Angiogenesis

“…Subsequently in 1990, they demonstrated in vivo hGH expression after transplantation of the transfected keratinocytes in nude mice, as first ex vivo gene delivery to wounds [19]. The potential application of this approach to promote wound healing has been explored by various researchers [20–24]. In particular, we have demonstrated that ex vivo delivery of hEGF can significantly improve the healing of porcine full-thickness wounds [4, 7].…”