Investigation Of Endogenous Gene Expression Changes After Vegf Gene Therapy Via Aav2 Double-stranded Vectors
Xiao Tian Wang, Vikram G. Mookerjee, William R. Miklavcic, Sherry Yq Tang, Paul Y. Liu.
Rhode Island Hospital, Providence, RI, USA.
Background: We previously reported that diabetic mouse (db/db) wound healing is significantly delayed with downregulated gene expression of multiple growth factors and their receptors including bFGF, PDGFb and VEGF. In this study, we aimed to investigate if adeno-associated double-stranded viral vector 2 (AAV2-ds) mediated VEGF gene therapy activates endogenous wound healing related genes. Methods: In 12 db/db mice, dorsal paired 8 mm-diameter wounds were created. 1011viral particles of AAV2-ds-VEGF or AAV2-ds-GFP diluted in saline were injected intradermally into the wound edge and the wound bed, 6 mice per group. At day 15 and 21 post-wounding, six wounds from each group were formalin-fixed, paraffin-embedded (FFPE) and sectioned. Immunohistochemistry was used to verify the success of VEGF gene transfer. Total RNA was extracted from FFPE sections, wounds and edges separated. RT-qPCR was performed to examine gene expression of bFGF, EGF, IGF1, FN, MMP9, PDGFb, TGFb1, TIMP1, and VEGFa. Results: We verified significantly increased VEGF staining in AAV2-ds-VEGF treated wound granulation tissue at day 15. At day 15, the expression of PDGFb and TIMP1 genes was significantly increased (p<0.05 and p<0.01) in AAV2-ds-VEGF group compared to that in AAV2-ds-GFP group; the EGF gene expression was significantly higher in AAV2-ds-VEGF treated wounds than that in AAV2-ds-GFP treated wounds. At day 21, the expression of EGF gene in AAV2-ds-VEGF group was significantly decreased than that at day 15 (p<0.05). There was no significant difference in other studied genes. Conclusions: VEGF gene was successfully transduced by AAV2-ds vectors. The exogenous VEGF gene significantly increased endogenous genes specific to wound healing after AAV2-ds-VEGF therapy. Further optimization of AAV2-ds vector mediated VEGF gene therapy could improve the diabetic wound healing.
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