Diabetes Metab J.  2011 Jun;35(3):226-235. 10.4093/dmj.2011.35.3.226.

Comparison of EGF with VEGF Non-Viral Gene Therapy for Cutaneous Wound Healing of Streptozotocin Diabetic Mice

• Ko J
• Jun H
• Chung H
• Yoon C
• Kim T
• Kwon M
• Lee S
• Jung S
• Kim M
• Park JH

• KMID: 2175423
• DOI: http://doi.org/10.4093/dmj.2011.35.3.226

Abstract

BACKGROUND
To accelerate the healing of diabetic wounds, various kinds of growth factors have been employed. It is the short half-life of administered growth factors in hostile wound beds that have limited wide-spread clinical usage. To overcome this limitation, growth factor gene therapy could be an attractive alternative rather than direct application of factors onto the wound beds. We administered two growth factor DNAs, epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) into a cutaneous wound on diabetic mice. We compared the different characteristics of the healing wounds.
METHODS
Streptozotocin was injected intraperitoneally to induce diabetes into C57BL/6J mice. The ultrasound micro-bubble destruction method with SonoVue as a bubbling agent was used for non-viral gene delivery of EGF828 and VEGF165 DNAs. Each gene was modified for increasing efficacy as FRM-EGF828 or minicircle VEGF165. The degree of neoangiogenesis was assessed using qualitative laser Doppler flowmetry. We compared wound size and histological findings of the skin wounds in each group.
RESULTS
In both groups, accelerated wound closure was observed in the mice receiving gene therapy compared with non treated diabetic control mice. Blood flow detected by laser doppler flowmetry was better in the VEGF group than in the EGF group. Wound healing rates and histological findings were more accelerated in the EGF gene therapy group than the VEGF group, but were not statistically significant.
CONCLUSION
Both non-viral EGF and VEGF gene therapy administrations could improve the speed and quality of skin wound healing. However, the detailed histological characteristics of the healing wounds were different.

Keyword

Epidermal growth factor; Gene therapy; Non-viral; Skin wound; Vascular endothelial growth factor

MeSH Terms

Animals
DNA
Epidermal Growth Factor
Genetic Therapy
Half-Life
Intercellular Signaling Peptides and Proteins
Laser-Doppler Flowmetry
Mice
Phospholipids
Skin
Streptozocin
Sulfur Hexafluoride
Vascular Endothelial Growth Factor A
Wound Healing
DNA
Epidermal Growth Factor
Intercellular Signaling Peptides and Proteins
Phospholipids
Streptozocin
Sulfur Hexafluoride
Vascular Endothelial Growth Factor A

Figure

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  Fig. 1 Plasmid constructions and their expressions by ultrasound mediated gene delivery in human embryonic kidney (HEK) 293 cells. (A) cDNA encoding human epidermal growth factor (EGF) with a N-terminal furin recognition sequence was inserted between EcoR I and Not I restriction sites under chicken β-actin promoter. (B) cDNA of human vascular endothelial growth factor 165 (VEGF165) was inserted to produce pβ-VEGF165. (C, D) Ultrasound mediated gene delivery in HEK 293 cells. The cells were exposed to 1 MHz US in the presence of 2 µg of each plasmid with a concentration of microbubble (10 mg/mL). Ultrasound intensity was 2.0 W/cm2; ultrasound exposure time was 30 seconds; duty cycle was 20%.

• 

  Fig. 2 Production of minicircle-vascular endothelial growth factor (VEGF) and the comparison of efficiency to a typical form of plasmid (pβ-VEGF) containing of bacterial backbone using branched polyethylenimine (BPEI) as a gene carrier in human embryonic kidney (HEK) 293 cells. (A) p2øC31-β-VEGF165 the expression cassette from pβ-VEGF165 was excised by restriction enzymes and bluntly ligated between attB and attP site of p2øC31 vector, which contains phi-C31 integrase and I-SceI homing endonuclease. (B) Minicircle-VEGF165. (C) Minicircle-VEGF165 showed 2 to 3 folds higher transfection efficiency in HEK 293 cells. 2×105 cells of HEK293 were treated for 4 hours with plasmid DNA (pβ-VEGF165 or Minicircle-VEGF165) complexed with branched polyethylenimine (BPEI, 25 kDa, N/P ratio 10:1). VEGF concentrations in the culture media were measured by ELISA. aP<0.05.

• 

  Fig. 3 Wound size comparison by ultrasound mediated gene delivery of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF). (A) Growth factor gene delivery by sonoporation enhanced the progress of wound closure in diabetic mice to non-diabetic normal control group. (B) Average areas (in pixels) of wound that received ultrasound mediated gene delivery of EGF and VEGF were significantly reduced to non-diabetic control mice by day 12 post operation. C, control; DC, Diabetic (DM) control; DE, DM+EGF; DV, DM+VEGF.

• 

  Fig. 4 Blood perfusion in the wound tissue was significantly increased by ultrasound mediated gene delivery of growth factors to diabetic mice. The laser doppler imager (PeriScan) used in this study was employed for the visualization of blood perfusion. Red color represents high blood perfusion in the wound site. At the 6th day post operation, a significant increase of blood perfusion was observed in diabetic (DM) mice group that received vascular endothelial growth factor (VEGF) gene delivery.

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  Fig. 5 Histology of wound tissues. (A) Histology of wound tissues at day 12 post operation (H&E stain, ×100). After the generation of skin wound, non-treated diabetic (DM) control showed severe edema, disorganized micro-architectures and the heavy infiltration of inflammatory cells. The wound tissues that received ultrasound mediated gene delivery of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) showed a highly restored well organized state of tissue compared to the non-diabetic normal control. (B) Tissue reorganization of wound tissues at day 12 post operation (M&T, ×100). In contrast to non-treated diabetic control, the wound tissues that received gene delivery of EGF showed more collagen accumulation and appeared as a organized wound matrix.

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