Combined vascular endothelial growth factor-A and fibroblast growth factor 4 gene transfer improves wound healing in diabetic mice.
BACKGROUND
Impaired wound healing in diabetes is related to decreased production of growth factors. Hence, gene therapy is considered as promising treatment modality. So far, efforts concentrated on single gene therapy with particular emphasis on vascular endothelial growth factor-A (VEGF-A). However, as multiple proteins are involved in this process it is rational to test new approaches. Therefore, the aim of this study was to investigate whether single AAV vector-mediated simultaneous transfer of VEGF-A and fibroblast growth factor 4 (FGF4) coding sequences will improve the wound healing over the effect of VEGF-A in diabetic (db/db) mice.


METHODS
Leptin receptor-deficient db/db mice were randomized to receive intradermal injections of PBS or AAVs carrying β-galactosidase gene (AAV-LacZ), VEGF-A (AAV-VEGF-A), FGF-4 (AAV-FGF4-IRES-GFP) or both therapeutic genes (AAV-FGF4-IRES-VEGF-A). Wound healing kinetics was analyzed until day 21 when all animals were sacrificed for biochemical and histological examination.


RESULTS
Complete wound closure in animals treated with AAV-VEGF-A was achieved earlier (day 19) than in control mice or animals injected with AAV harboring FGF4 (both on day 21). However, the fastest healing was observed in mice injected with bicistronic AAV-FGF4-IRES-VEGF-A vector (day 17). This was paralleled by significantly increased granulation tissue formation, vascularity and dermal matrix deposition. Mechanistically, as shown in vitro, FGF4 stimulated matrix metalloproteinase-9 (MMP-9) and VEGF receptor-1 expression in mouse dermal fibroblasts and when delivered in combination with VEGF-A, enhanced their migration.


CONCLUSIONS
Combined gene transfer of VEGF-A and FGF4 can improve reparative processes in the wounded skin of diabetic mice better than single agent treatment.
Jazwa
Agnieszka
A
Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland. jozef.dulak@uj.edu.pl.
Kucharzewska
Paulina
P
Leja
Justyna
J
Zagorska
Anna
A
Sierpniowska
Aleksandra
A
Stepniewski
Jacek
J
Kozakowska
Magdalena
M
Taha
Hevidar
H
Ochiya
Takahiro
T
Derlacz
Rafal
R
Vahakangas
Elisa
E
Yla-Herttuala
Seppo
S
Jozkowicz
Alicja
A
Dulak
Jozef
J
Lab Invest. 1999 Dec;79(12):1479-87
10616199
Diabetes. 2001 Mar;50(3):667-74
11246889
Lancet. 2005 Nov 12;366(9498):1736-43
16291068
FASEB J. 2003 Jan;17(1):100-2
12475908
Life Sci. 1995;57(11):1111-23
7658918
Mol Ther. 2002 Feb;5(2):154-60
11829522
Wound Repair Regen. 2007 Sep-Oct;15(5):636-45
17971009
Am J Pathol. 2004 Jun;164(6):1935-47
15161630
Proc Natl Acad Sci U S A. 2008 Dec 9;105(49):19426-31
19057015
Microvasc Res. 1998 Jan;55(1):29-42
9473407
J Biol Chem. 2006 Jun 9;281(23):15694-700
16597617
Genome Biol. 2001;2(3):REVIEWS3005
11276432
Oncogene. 2000 Aug 3;19(33):3805-10
10949936
Mol Cell Biol. 1988 Jul;8(7):2933-41
3043199
Diabetologia. 2003 Apr;46(4):546-55
12677400
J Surg Res. 2007 Nov;143(1):35-42
17950070
FEBS Lett. 1994 Mar 28;342(1):71-5
8143853
Cell. 1993 Nov 5;75(3):579-87
8221896
Vasc Med. 2000;5(1):33-40
10737154
J Invest Dermatol. 1994 Oct;103(4):469-73
7930669
Wound Repair Regen. 1998 Mar-Apr;6(2):135-41
9776856
Biochem Biophys Res Commun. 2005 Dec 23;338(3):1542-6
16274668
Biochem Biophys Res Commun. 1991 Dec 16;181(2):902-6
1755866
Clin Sci (Lond). 2008 Jun;114(12):707-18
18078386
J Biol Chem. 1995 May 26;270(21):12607-13
7759509
Diabetes. 1982;31(Suppl 1 Pt 2):1-6
7160533
Cancer Res. 1997 Dec 15;57(24):5590-7
9407972
J Clin Invest. 1979 Mar;63(3):358-70
429558
Biochem Biophys Res Commun. 1999 Oct 5;263(3):621-6
10512728
J Invest Dermatol. 2009 Sep;129(9):2275-87
19282838
Mol Hum Reprod. 2004 Apr;10(4):229-35
14996996
Gene Ther. 2002 Oct;9(19):1271-7
12224009
Am J Pathol. 2003 Jan;162(1):303-12
12507913
Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12368-72
7809043
Biochem Cell Biol. 1997;75(6):669-85
9599656
J Exp Med. 1992 Nov 1;176(5):1375-9
1402682
Oncogene. 1991 Apr;6(4):659-63
1851551
Am J Physiol Cell Physiol. 2001 Jun;280(6):C1358-66
11350730
Science. 1997 Apr 4;276(5309):75-81
9082989
Science. 1995 Jan 13;267(5195):246-9
7809630
Circ Res. 1998 Oct 19;83(8):832-40
9776730
Prog Histochem Cytochem. 2007;42(3):115-70
17980716
Oncogene. 2001 May 10;20(21):2655-63
11420677