J Korean Med Sci.  2014 Nov;29(Suppl 3):S228-S236. 10.3346/jkms.2014.29.S3.S228.

Gene Therapy Using Hepatocyte Growth Factor Expressing Adenovirus Improves Skin Flap Survival in a Rat Model

• Rah DK
• Yun IS
• Yun CO
• Lee SB
• Lee WJ

• KMID: 2151418
• DOI: http://doi.org/10.3346/jkms.2014.29.S3.S228

Abstract

Hepatocyte growth factor (HGF) is a potent angiogenic factor that can stimulate the production of blood vessels in ischemic tissue. We investigated whether gene therapy using HGF-expressing adenovirus could enhance skin flap survival. Sprague-Dawley rats were randomly divided into three groups. Rats were subdermally injected with HGF-expressing adenovirus (HGF virus group), recombinant HGF (rhHGF group), or phosphate buffered saline (PBS group) 2 days before and immediately after 3 x 9 cm caudal flap elevation. The survival area of the skin flap, the ratio of blood flow, CD31-positive vessels and, VEGF expression were examined. Skin flap viability was significantly increased in the HGF virus group compared to the rhHGF and PBS groups (71.4% +/- 5.9%, 63.8%+/- 6.4%, and 39.2% +/- 13.0%, respectively) (P = 0.025). Furthermore, the blood flow ratio was significantly increased in the HGF virus group. In the HGF virus group, the number of CD31-positive vessels and vascular endothelial growth factor (VEGF) expression were significantly increased. Gene therapy using HGF-expressing adenovirus increase VEGF expression, the number of viable capillaries, and blood flow to the flap, thereby improving skin flap survival.

Keyword

Hepatocyte Growth Factor; Adenovirus; Gene Therapy; Skin Flap

MeSH Terms

Adenoviridae/genetics
Animals
Genetic Therapy/*methods
Graft Survival/genetics
Hepatocyte Growth Factor/biosynthesis/*genetics
Male
Models, Animal
Neovascularization, Physiologic/*genetics
Random Allocation
Rats
Rats, Sprague-Dawley
Reconstructive Surgical Procedures
Skin Transplantation/*methods
Surgical Flaps/*surgery
Hepatocyte Growth Factor

Figure

• 

  Fig. 1 Schematic representations of the adenoviral vectors. dE1-RGD/lacZ has the entire E1 region deleted and expresses the reporter gene lacZ (β-galactosidase protein) under the control of the constitutive cytomegalovirus (CMV) promoter inserted into the E1 region. The replication-incompetent HGF-expressing adenovirus, dE1-RGD/lacZ/HGF, carries a HGF gene driven by the CMV promoter that was inserted into the E3 region. ΔE3 denotes the deletion of E3 gene. The RGD-incorporated adenovirus was generated by inserting RGD peptide between HI-loop of the fiber knob. ITR, inverted terminal repeat; Ψ, packaging signal; pA, polyA sequence; IX, protein IX; HGF, hepatocyte growth factor.

• 

  Fig. 2 Immunofluorescence staining of HGF 10 days after flap elevation. Sections (3 µm thick) of rat skin flap tissue (distal part) were stained with DAPI to label nuclei (A, D, G) and antibodies against HGF (B, E, H). Scale bars (50 µm) are indicated in all photomicrographs. Merged immunofluorescence images reveal that HGF is expressed in and around DAPI-stained cells (C, F, I).

• 

  Fig. 3 Representative photographs of skin flaps survival. Partial flap necrosis on postoperative day 10 (A) and the extent of skin flap viability (B). Skin flap viability in the HGF virus group was significantly greater than that of the other groups on postoperative days 7 and 10. Values are means ± S.D. (n = 10). *P = 0.034; †P = 0.025.

• 

  Fig. 4 Blood flow measurements in the skin flaps. In the mid-distal portion of skin flap, the ratio of the blood flow was significantly increased in the HGF virus group on postoperative days 7 and 10 compared with the other groups (A). In the distal portion of skin flap, there was significant increase in the ratio of blood flow in the HGF virus group on postoperative days 3 and 7 compared with the other groups (B). Values are means ± S.D. (n = 10). *P = 0.031; †P = 0.028; ‡P = 0.021; §P = 0.017.

• 

  Fig. 5 Representative photomicrographs of CD31 immunohistochemistry. In distal part of flaps 10 days after flap elevation (PBS group (A), rhHGF group (B), HGF virus group (C), arrow: CD31-positive vessels, ×200) and the comparative analysis of the number of CD31-positive vessels (D). One-way ANOVA adjusted by Bonferroni's correction revealed that the number of CD31-positive vessels was significantly higher in the HGF virus group than the other two groups. Values are means ± S.D. (n = 10). *P = 0.037.

• 

  Fig. 6 Representative photomicrographs of VEGF immunohistochemistry. In distal part of flaps 10 days after flap elevation (PBS group (A), rhHGF group (B), HGF virus group (C), ×200) and semi-quantitative analysis using MetaMorph® image analysis software (D). One-way ANOVA adjusted by Bonferroni's correction revealed that VEGF immunoreactivity was significantly higher in the HGF virus group than the other two groups. Values are means ± S.D. (n = 10). *P = 0.029.

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