Improved survival of rat ischemic cutaneous and musculocutaneous flaps after VEGF gene transfer

IMPROVED SURVIVAL OF RAT ISCHEMIC CUTANEOUS ANDMUSCULOCUTANEOUS FLAPS AFTER VEGF GENE TRANSFER

ANDREA ANTONINI, M.D.,1* SERENA ZACCHIGNA, M.D.,2 GIOVANNI PAPA, M.D.,1 FEDERICO NOVATI, M.D.,1

MICHELE PASCONE, M.D.,1 and MAURO GIACCA, M.D.2

When harvesting microsurgical flaps, the main goals are to obtain as much tissue as possible based on a single vascular pedicle and areliable vascularization of the entire flap. These aims being in contrast to each other, microsurgeons have been looking for an effectiveway to enhance skin and muscle perfusion in order to avoid partial flap loss in reconstructive surgery. In this study we demonstrate the ef-ficacy of VEGF 165 delivered by an Adeno-Associated Virus (AAV) vector in two widely recognized rat flap models. In the rectus abdominismiocutaneous flap, intramuscular injection of AAV-VEGF reduced flap necrosis by 50%, while cutaneous delivery of the same amount ofvector put down the epigastric flap’s ischemia by >40%. Histological evidence of neoangiogenesis (enhanced presence of CD31-positivecapillaries and a-Smooth Muscle Actin-positive arteriolae) confirmed the therapeutic effect of AAV-VEGF on flap perfusion.VVC 2007 Wiley-Liss, Inc. Microsurgery 27:439–445, 2007.

Microsurgical flap harvesting is subjected to very strict

rules on flap size and quantity of tissue which can be

safely elevated. The main goal of a microsurgical recon-

structive procedure is often that of providing highly per-

fused tissue in an injured and infected region. Transfering

tissue with unreliable viability may cause the exact oppo-

site effect.1 Therefore flap vascularization is of primary

importance when planning such a surgical procedure. At

the same time, we often need to cover very large losses

of substance in one single operation. Large ‘‘extended’’

flaps do not always grant a sufficient blood flow, espe-

cially in peripheral areas. Vascular Endothelial Growth

Factor is a family of molecules proved to play an impor-

tant role in the angiogenic response to tissue ischemia.

The predominant form, produced by all tissues and cells

subject to hypoxia is VEGF 165. Its role is to stimulate

migration and proliferation of endothelial cells, building a

primitive capillary network, which then leads to the for-

mation of mature vessels.2,3

Several authors have described the possibility of pro-

moting skin flap neovascularization by using recombinant

VEGF proteins. In most of these studies, recombinant

VEGF provided a beneficial effect on flap survival.3–12

Despite these encouraging findings, the use of recombi-

nant proteins in a clinical setting is hampered by several

factors, such as their short half-lives, poor bioavailability,

and consequent need for frequent administrations to sus-

tain long-lasting effects. The solution to most of these

problems may be achieved through gene therapy, which

obtains local overexpression of VEGF produced by local

cells.13–18

A variety of techniques allow to insert coding DNA

in host cells. Many of them have been used for gene

delivery to muscle and skin, including the use of naked

plasmid DNA and viral vectors.19–27 The use of viral

vectors provides a higher rate of transduction and expres-

sion, in respect to nonviral techniques. Vectors based on

the adeno-associated virus (AAV), a nonpathogenic and

widespread parvovirus, incapable of autonomous replica-

tion, are able to transduce both dividing and nondividing

cells and show a specific tropism for postmitotic cells,

including skeletal and cardiac muscle,28 neurons,29 and

liver.30,31 Because these vectors do not contain any viral

genes—which are transiently transfected in trans for the

packaging process—they elicit virtually no inflammatory

or immune response.32,33 As a consequence, transgene

expression from these vectors persists for several months

in a variety of animal tissues in vivo.34

The subcutaneous delivery of AAV vectors results in

the efficient transduction of hair follicles, sweat gland

ducts,35 and the panniculus carnosus (the skeletal muscle

layer within the dermal sheet in rodents).36,37

MATERIALS AND METHODS

Two recombinant AAV vectors were obtained in this

study, expressing the LacZ reporter gene and the cDNA

for the 165 amino acid isoform of VEGF (VEGF165)

under the control of the constitutive cytomegalovirus im-

mediate early promoter. Infectious vector stocks were

generated in 293 cells and titrated by a competitive poly-

merase chain reaction procedure, as already described.38

Animal care and treatment were conducted in confor-

mity with institutional guidelines in compliance with

national and international laws and policies (European

1Plastic Surgery Unit, Faculty of Medicine and Surgery, University of Trieste,Italy2Molecular Medicine Laboratory, International Centre for Genetic Engineer-ing and Biotechnology, Trieste, Italy

*Correspondence to: Andrea Antonini, Plastic Surgery Unit - Ospedale diCattinara, Strada Di Fiume, 447, 34100 Trieste, Italy. E-mail: [email protected]

Received 28 February 2007; Accepted 5 March 2007

Published online 27 June 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/micr.20378

VVC 2007 Wiley-Liss, Inc.

Economic Community Council Directive 86/609, OJL

358, December 12th, 1987). A total of 48 male Wistar

rats weighing 260–290 g were used for this study. All

rats were anesthetized with 10 ml/kg of intraperitoneal

tribromoethanol (Avertin) 2%.

The rats were divided in 12 groups (Table 1). On the

first 6 groups were performed the Epigastric Flap, elevat-

ing an 8 3 5 cm cutaneous flap (Fig. 1A) extending

caudally from the xiphoid and bilaterally from the ab-

dominal mieline, based on the right superficial inferior

epigastric artery (Fig. 1B). Group 1 received 150 ll of

AAV-VEGF in 10 subcutaneous spots on both sides of

the midline at the time of operation, group 2 received the

vector in the same locations 7 days before surgery, while

group 3 underwent vector subministration 14 days prior

to flap elevation. Groups 4–6 (control groups) received

placebo (AAV-LacZ or saline) subcutaneous injection on

the same timings as groups 1–3.

On the last 6 groups, we harvested the same skin pad-

dle as the previous ones, but it was based on the four

constant type III perforator arteries arising from the right

rectus abdominis muscle (Fig. 1C), in order to consider

the behavior of a composite musculocutaneous flap,

although the muscle was not elevated. Group 7 received

subministration of 150 ll of intramuscular AAV-VEGFin four spots of the rectus abdominis muscle near each

perforator artery. Groups 8 and 9 were treated with the

same amount of vector through a small incision per-

formed 4 cm to the right from the midline (the flap’s

right border), respectively, 7 and 14 days before the sur-

gical procedure. Groups 10 to 12 were the control groups

and received an equal amount of placebo in the same

timing and manner.

Necrotic area was always assessed on day 7 after sur-

gery, by analyzing digital images of the dissected flap

using the UTHSCSA Image Tool software. At the same

time all animals were sacrificed, and two specimen of

flap tissue were taken. Sample A in the area of vector

injection, and sample B on the cutaneous portion of the

flap where the border between viable and necrotic areas

could be seen.

Fixed samples were dehydrated with graded ethanol

and embedded in paraffin. Five-lm sections were stained

with hematoxylin for morphological analysis of tissues.

To visualize blood vessels by immunohistochemistry,

rehydrated serial paraffin sections were subjected to anti-

gen retrieval procedures. After inactivation of endogenous

peroxidase with 3% hydrogen peroxide, samples were

rinsed in phosphate-buffered saline (PBS) and blocked

with nonimmune horse serum followed by incubation

Table 1. Summary of the 12 Groups

Epigastric flap

Group 01 Injection of AAV-VEGF at the time of flap harvest

Group 02 Flap harvest 7 days after AAV-VEGF injection


Group 04 Injection of placebo at the time of flap harvest

Group 05 Flap harvest 7 days after placebo injection


Musculocutaneous flap

Group 07 Injection of AAV-VEGF at the time of flap harvest



Group 10 Injection of placebo at the time of flap harvest



Figure 1. Schematic representation of the skin flaps and their vascu-

lar components, with the indication of the vector injection sites. A: The

surgical models of skin flap used in this study are based on a rectangu-

lar skin paddle measuring 53 8 cm, drawn on the abdomen of the ani-

mals. The predictable vascular system of the flap is symmetrically

composed of the lateral thoracic arteries, the inferior epigastric

arteries, and the musculocutaneous perforator arteries arising from

the rectus abdominis muscle (usually four vessels on each side).

B and C: The pictures schematically show the vascular component

providing the blood supply to each flap and the injection sites. In partic-

ular, the skin flap (B) was based on the inferior epigastric artery, and

the vector injected at 10 equally spaced subcutaneous sites along the

midline (inset). The rectus abdominis musculocutaneous flap (C) was

raised on a plane between the panniculus carnosus and the abdomi-

nal fascia, with the rectus abdominis as the only source of blood supply

through the perforator arteries; in this model, the vector was adminis-

tered by intramuscular injection in the region where each perforator ar-

tery arises from the rectus sheet (inset). [Color figure can be viewed in

the online issue, which is available at www.interscience.wiley.com.]

440 Antonini et al.

Microsurgery DOI 10.1002/micr

with an anti-CD31 antibody (Santa Cruz) or an anti-a-

smooth muscle actin (a-SMA) antibody (Sigma-Aldrich,

St. Louis, MO). Slides were rinsed in PBS and then incu-

bated with biotinylated horse secondary antibody (Vector

Laboratories, Burlingame, CA). After an additional wash-

ing in PBS, slides were incubated in the presence of an

avidin-biotin complex and developed with 3,30-diamino-

benzidine (Lab Vision Corporation, Fremont, CA).

RESULTS

The percentage of flap subject to necrosis was notably

reduced in all AAV-VEGF treated groups. The area

undergoing necrosis in LacZ or saline treated control

groups was predictable and constant [37.4 6 1.8% for

the epigastric flap, and 24.4 6 2.3% for the rectus ab-

dominis flap]. The epigastric flap showed a notable

reduction of the necrotic area also for flaps that received

the vector during the surgical procedure, but the action of

VEGF was proved to be increasingly effective in groups

injected 7 and 14 days preoperatively (Table 2, Fig. 2).

On the contrary, there seemed to be no difference in cu-

taneous necrosis of the rectus abdominis flap’s skin pad-

dle, when AAV-VEGF delivery was concomitant to the

surgical procedure. Nevertheless, the best results were

obtained on this musculocutaneous flap when the vector

was inoculated 7 or 14 days before the surgical procedure

(Table 3, Fig. 3).

Marked differences between AAV-VEGF treated and

untreated specimen were noticed during histological anal-

ysis. Sample A (vector inoculation site) showed massive

cellular infiltration between rectus abdominis or pannicu-

lus carnosus muscle fibers. In sample B (healthy þ ne-

crotic skin) there also was a substantial difference in

terms of tissue viability. Specimen from the control

groups showed a thin and immature epithelial layer, with

evidence of acute inflammation, adipose substitution, and

myonecrosis (almost complete disappearance of the pan-

niculus carnosus); the infiltrating inflammatory cells,

mostly monocytes and neutrophils, were dispersed

throughout the skin layers, concomitant with a severe dis-

ruption of the tissue architecture. The viable area of

treated flaps, instead, showed a preserved histology, with

an intact epithelial layer, only a mild local inflammatory

response, and minor accumulation of adipose tissue (Fig. 4).

By using an antibody specific for CD31 endothelial

marker, we were able to demonstrate that most of the

cellular infiltration in treated muscle and skin was made

up of endothelial cells. Specimens from rodents treated

14 days preoperatively, showed these CD31-positive cells

were already organizing into a newly formed capillary

Table 2. Results Showing the Percentage of Flap Skin Subject to

Necrosis in the Epigastric Flap

Placebo AAV-VEGF Difference

Harvest and injection 36.1 6 0.6 27.8 6 1.6 �23.0%

Harvest 7 days

after injection 38.9 6 2.2 23.3 6 1.5 �40.1%

Harvest 14 days

after injection 37.2 6 1.4 21.7 6 2.8 �41.7%

Figure 2. Graph comparing skin necrosis in treated and control

groups for the epigastric flap. [Color figure can be viewed in the

online issue, which is available at www.interscience.wiley.com.]

Table 3. Results Showing the Percentage of Flap Skin Subject to

Necrosis in the Rectus Abdominis Musculocutaneous Flap

Placebo AAV-VEGF Difference

Harvest and injection 22.7 6 1.2 22.4 6 1.1 –

Harvest 7 days

after injection 26.0 6 3.0 16.1 6 1.7 �38.1%

Harvest 14 days

after injection 24.6 6 0.8 12.3 6 0.8 �50.0%

Figure 3. Graph comparing skin necrosis in treated and control

groups for the rectus abdominis flap. [Color figure can be viewed in

the online issue, which is available at www.interscience.wiley.com.]

VEGF Gene Therapy 441


network (Fig. 5), confirming a property already described

in literature.6,13,14,22,39,40 Immunostaining the same sam-

ples with an antibody specific for a-Smooth Muscle

Actin, emphasized the neoangiogenetic process taking

place in AAV-VEGF-treated tissues, by demonstrating the

massive presence of arterial vessels (Fig. 6), particularly

in samples from group 9 (vector inoculation 14 days

before surgery).

DISCUSSION

The employment of VEGF to enhance tissutal perfu-

sion in microsurgical flaps has recently become one of

the most innovative fields of research in reconstructive

surgery. This family of molecules and its use in the

struggle against ischemia in a variety of different tissues

and diseases can be effectively applied to many recon-

structive procedures, where ischemia seriously threatens

the final surgical result. Many authors have already stated

the efficacy of such therapies. Our contribution to re-

search in this field mainly concerns AAV vectors, which

show important advantages when compared to other gene

delivery techniques. Plasmids have a very low rate of

transduction, while adenoviral vectors produce a strong

immunologic response in the host, which has been proven

to cause a significant loss of transgene expression. On the

contrary, AAVs show a good rate of expression through-

out the first weeks after inoculation, property confirmed

by our study, since we obtained best results when vector

inoculation was done 14 days prior to the operation.

Cutaneous necrosis reduction in flaps that received

AAV-VEGF in the muscular portion, could be explained

through secretion of the overexpressed VEGF in the ves-

sels, leading to high concentrations of the molecule in

the skin perfused by the perforators.

Histological findings showed a newly formed vascular

network only in specimen from rodents treated 7 and 14

days before surgery. This is apparently in contrast with

Figure 4. Shown are representative sections of samples A and B from AAV-VEGF-treated (right) and control (left) animals. At the injec-

tion site (sample A), a massive cellular infiltration appeared as a consequence of AAV-VEGF treatment (top). More notably, sample B

of VEGF-treated flaps showed an intact and viable epithelial layer with conserved tissue architecture, whereas, in control flaps, the epi-

thelium was thin and discontinuous, with massive inflammation and adipose substitution. Myonecrosis was detected only in LacZ-

treated flaps, as indicated by the disappearance of the panniculus carnosus (shown by asterisks in the VEGF sample). Note the pres-

ence of circulating inflammatory cells in the arterial lumen in the insets, more abundant in the LacZ-treated as compared to the VEGF-

treated samples. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

442 Antonini et al.


clinical results, since the epigastric flap received a nota-

ble reduction in skin necrosis also in cases treated con-

comitantly to flap elevation. The VEGF-mediated vasodi-

lator effect and capillary permeability enhancement4,41–44

seem the most reasonable explanation for these early

effects that are apparently not supported by anatomical

modifications.

Considering the importance of VEGF in neoangiogen-

esis and improvement of tissutal perfusion, and the

advantages of AAVs in comparison to other vectors,

Figure 5. The presence of endothelial cells in control (left) and VEGF-treated (right) flaps was detected by immunohistochemistry using an

anti-CD31 antibody. AAV-VEGF induced the proliferation of endothelial cells at the injection site (sample A), in which several CD31-positive

cells infiltrated the interstitial spaces between the fibers of the rectus abdominis muscle (inset on the right), as well as in the more distal

sample B. This endothelial cell proliferation was paralleled by the formation of a great number of new capillaries, most evident at the level

of the panniculus carnosus (p.c.), as shown in the bottom panels at a higher magnification. [Color figure can be viewed in the online issue,

which is available at www.interscience.wiley.com.]



AAV-VEGF seems to be a promising option for future

clinical application in reconstructive microsurgery.

ACKNOWLEDGMENTS

We thank Marina Dapas and Maria Elena Lopez for

technical support in AAV vector production, Marco Ste-

bel for pre- and postoperative animal care.

REFERENCES

1. Hallock GG. Physiological studies using laser Doppler flowmetry tocompare blood flow to the zones of the free TRAM flap. Ann PlastSurg 2001;47:229–233.

2. Lineaweaver WC, Lei MP, Mustain W, Oswald TM, Cui D, ZhangF. Vascular endothelium growth factor, surgical delay, and skin

flap survival. Ann Surg 2004;239(6):866–873; discussion 873–875.

3. Zhang F, Oswald T, Lin S, Kai Z, Lei M, Jones M, Angel MF, Line-aweaver WC. Vascular endothelial growth factor (VEGF) expressionand the effect of exogenous VEGF on survival of a random flap inthe rat. Br J Plast Surg 2003;56(7):653–659.

4. Padubidri A, Browne E Jr. Effect of vascular endothelial growth fac-tor (VEGF) on survival of random extension of axial pattern skinflaps in the rat. Ann Plast Surg 1996;37:604–611.

5. Machens HG, Salehi J, Weich H, Munch S, Siemers F, Krapohl BD,Herter KH, Kruger S, Reichert B, Berger A, Vogt P, Mailander P.Angiogenic effects of injected VEGF165 and sVEGFR-1 (sFLT-1)in a rat flap model. J Surg Res 2003;111:136–142.

6. Li QF, Reis ED, Zhang WX, Silver L, Fallon JT, Weinberg H.Accelerated flap prefabrication with vascular endothelial growth fac-tor. J Reconstr Microsurg 2000;16:45–49.

7. Kryger Z, Dogan T, Zhang F, Komorowska-Timek E, Shi DY,Cheng C, Lineaweaver WC, Buncke HJ. Effects of VEGF adminis-

Figure 6. The property of VEGF to sustain the formation of arterial vessels was assessed by immunohistochemistry using an antibody

against the a-actin isoform specific for the smooth muscle cells (a-SMA). A modest effect was detected in sample A, with a greater reac-

tivity in the AAV-VEGF-injected muscle (inset on the right), whereas a remarkable increase in the number of arteriolae was observed after

VEGF treatment in the more distal sample B (bottom), approximately corresponding to the border between viable and necrotic skin. [Color

figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

444 Antonini et al.


tration following ischemia on survival of the gracilis muscle flap inthe rat. Ann Plast Surg 1999;43:172–178.

8. Kryger Z, Zhang F, Dogan T, Cheng C, Lineaweaver WC, BunckeHJ. The effects of VEGF on survival of a random flap in the rat: Ex-amination of various routes of administration. Br J Plast Surg 2000;53:234–239.

9. Banbury J, Siemionow M, Porvasnik S, Petras S, Browne E.Improved perfusion after subcritical ischemia in muscle flaps treatedwith vascular endothelial growth factor. Plast Reconstr Surg2000;106:1541–1546.

10. Zhang F, Fischer K, Komorowska-Timek E, Guo M, Cui D, Dorsett-Martin W, Buncke HJ, Lineaweaver WC. Improvement of skin pad-dle survival by application of vascular endothelial growth factor in arat TRAM flap model. Ann Plast Surg 2001;46:314–319.

11. Zhang F, Richards L, Angel MF, Zhang J, Liu H, Dorsett-Martin W,Lineaweaver WC. Accelerating flap maturation by vascular endothe-lium growth factor in a rat tube flap model. Br J Plast Surg 2002;55:59–63.

12. Seify H, Bilkay U, Jones G. Improvement of TRAM flap viabilityusing human VEGF-induced angiogenesis: a comparative study ofdelay techniques. Plast Reconstr Surg 2003;112(4):1032–1039.

13. Zhang F, Yang F, Hu EC, Sones W, Lei M, Lineaweaver WC. Vas-cular endothelial growth factor gene therapy in improvement of skinpaddle survival in a rat TRAM flap model. J Reconstr Microsurg2005;21(6):391–396.

14. Gurunluoglu R, Meirer R, Shafighi M, Huemer GM, Yilmaz B, Piza-Katzer H. Gene therapy with adenovirus-mediated VEGF enhancesskin flap prefabrication. Microsurgery 2005;25(5):339–347.

15. Giunta RE, Holzbach T, Taskov C, Holm PS, Konerding MA,Schams D, Biemer E, Gansbacher B. Ad VEGF165 gene transferincrease survival in overdimensioned skin flaps. J Gene Med 2005;7(3):297–306.

16. Taub PJ, Marmur JD, Zhang WX, Senderoff D, Urken ML, Silver L,Wienberg H. Effect of time on the viability of ischemic skin flapstreated with vascular endothelial growth factor (VEGF) cDNA.J Reconstr Microsurg 1998;14:387–390.

17. Taub PJ, Marmur JD, Zhang WX, Senderoff D, Nhat PD, Phelps R,Urken ML, Silver L, Wienberg H. Locally administered vascular en-dothelial growth factor cDNA increases survival of ischemic experi-mental skin flaps. Plast Reconstr Surg 1998;102:2033–2039.

18. Zacchigna S, Papa G, Antonini A, Novati F, Moimas S, Carter A,Arsic N, Zentilin L, Visintini V, Pascone M, Giacca M. Improvedsurvival of ischemic cutaneous and muscolocutaneous flaps after vas-cular endothelial growth factor gene transfer using adeno-associatedvirus vectors. Am J Pathol 2005;167(4):981–991.

19. Snyder RO, Spratt SK, Lagarde C, Bohl D, Kaspar B, Sloan B,Cohen LK, Danos O. Efficient and stable adeno-associated virus-mediated transduction in the skeletal muscle of adult immunocompe-tent mice. Hum Gene Ther 1997;8:1891–1900.

20. Greenhalgh DA, Rothnagel JA, Roop DR. Epidermis: An attractivetarget tissue for gene therapy. J Invest Dermatol 1994;103:63S–69S.

21. Cui L, Li FC, Zhang Q, Qian YL, Guan WX. Effect of adenovirus-mediated gene transfection of vascular endothelial growth factor onsurvival of random flaps in rats. Chin J Traumatol 2003;6:199–204.

22. Gurunluoglu R, Ozer K, Skugor B, Lubiatowski P, Carnevale K, Sie-mionow M. Effect of transfection time on the survival of epigastricskin flaps pretreated with adenovirus encoding the VEGF gene. AnnPlast Surg 2002;49:161–169.

23. Ghazizadeh S, Taichman LB. Virus-mediated gene transfer for cuta-neous gene therapy. Hum Gene Ther 2000;11:2247–2251.

24. O’Toole G, MacKenzie D, Lindeman R, Buckley MF, Marucci D,McCarthy N, Poole M. Vascular endothelial growth factor gene ther-apy in ischaemic rat skin flaps. Br J Plast Surg 2002;55:55–58.

25. Neumeister MW, Song YH, Mowlavi A, Suchy H, Mathur A. Effectsof liposome-mediated gene transfer of VEGF in ischemic rat gracilismuscle. Microsurgery 2001;21:58–62.

26. Lubiatowski P, Goldman CK, Gurunluoglu R, Carnevale K, SiemionowM. Enhancement of epigastric skin flap survival by adenovirus-medi-ated VEGF gene therapy. Plast Reconstr Surg 2002;109:1986–1993.

27. Liu PY, Tong W, Liu K, Han SH, Wang XT, Badiavas E, Rieger-Christ K, Summerhayes I. Liposome-mediated transfer of vascularendothelial growth factor cDNA augments survival of random-pat-tern skin flaps in the rat. Wound Repair Regen 2004;12:80–85.

28. Su H, Lu R, Kan YW. Adeno-associated viral vector-mediated vas-cular endothelial growth factor gene transfer induces neovascularformation in ischemic heart. Proc Natl Acad Sci USA 2000;97:13801–13806.

29. Kaplitt MG, Leone P, Samulski RJ, Xiao X, Pfaff DW, O’MalleyKL, During MJ. Long-term gene expression and phenotypic correc-tion using adeno-associated virus vectors in the mammalian brain.Nat Genet 1994;8:148–154.

30. Nakai H, Herzog RW, Hagstrom JN, Walter J, Kung SH, Yang EY, TaiSJ, Iwaki Y, Kurtzman GJ, Fisher KJ, Colosi P, Couto LB, High KA.Adeno-associated viral vector-mediated gene transfer of human bloodcoagulation factor IX into mouse liver. Blood 1998;91:4600–4607.

31. Xiao W, Berta SC, Lu MM, Moscioni AD, Tazelaar J, Wilson JM.Adeno-associated virus as a vector for liver-directed gene therapy.J Virol 1998;72:10222–10226.

32. Chirmule N, Propert K, Magosin S, Qian Y, Qian R, Wilson J.Immune responses to adenovirus and adeno-associated virus inhumans. Gene Ther 1999;6:1574–1583.

33. Kay MA, Manno CS, Ragni MV, Larson PJ, Couto LB, McClellandA, Glader B, Chew AJ, Tai SJ, Herzog RW, Arruda V, Johnson F,Scallan C, Skarsgard E, Flake AW, High KA. Evidence for genetransfer and expression of factor IX in haemophilia B patientstreated with an AAV vector. Nat Genet 2000;24:257–261.

34. Monahan PE, Samulski RJ. AAV vectors: Is clinical success on thehorizon? Gene Ther 2000;7:24–30.

35. Hengge UR, Mirmohammadsadegh A. Adeno-associated virusexpresses transgenes in hair follicles and epidermis. Mol Ther 2000;2:188–194.

36. Donahue BA, McArthur JG, Spratt SK, Bohl D, Lagarde C, SanchezL, Kaspar BA, Sloan BA, Lee YL, Danos O, Snyder RO. Selectiveuptake and sustained expression of AAV vectors following subcuta-neous delivery. J Gene Med 1999;1:31–42.

37. Deodato B, Arsic N, Zentilin L, Galeano M, Santoro D, Torre V,Altavilla D, Valdembri D, Bussolino F, Squadrito F, Giacca M.Recombinant AAV vector encoding human VEGF165 enhanceswound healing. Gene Ther 2002;9:777–785.

38. Arsic N, Zentilin L, Zacchigna S, Santoro D, Stanta G, Salvi A,Sinagra G, Giacca M. Induction of functional neovascularization bycombined VEGF and angiopoietin-1 gene transfer using AAV vec-tors. Mol Ther 2003;7:450–459.

39. Zhang F, Lei MP, Oswald TM, Pang Y, Blain B, Cai ZW, LineaweaverWC. Defect of vascular endothelial growth factor on the healing ofischaemic skin wounds. Br J Plast Surg 2003;56(4):334–341.

40. Lubiatowski P, Gurunluoglu R, Goldman CK, Skugor B, CarnevaleK, Siemionow M. Gene therapy by adenovirus-mediated vascular en-dothelial growth factor and angiopoietin-1 promotes perfusion ofmuscle flaps. Plast Reconstr Surg 2002;110:149–159.

41. Komorowska Timek E, Timek TA, Brevetti LS, Zhang F, Lineawea-ver WC, Buncke HJ. The effect of single administration of vascularendothelial growth factor or L-arginine on necrosis and vasculatureof the epigastric flap in the rat model. Br J Plast Surg 2004;57(4):317–325.

42. Ashrafpour H, Huang N, Neligan PC, Forrest CR, Addison PD,Moses MA, Levine RH, Pang CY. Vasodilator effect and mechanismof action of vascular endothelial growth factor in skin vasculature.Am J Physiol Heart Circ Physiol 2004;286(3):946–954.

43. Pang Y, Lineawaver WC, Lei MP, Oswald T, Shamburger S, Cai Z,Zhang F. Evaluation of the mechanism of vascular endothelialgrowth factor improvement of ischemic flap survival in rats. PlastReconstr Surg 2003;112(2):556–564.

44. Dafni H, Landsman L, Schechter B, Kohen F, Neeman M. MRI andfluorescence microscopy of the acute vascular response toVEGF165: Vasodilation, hyper-permeability and lymphatic uptake,followed by rapid inactivation of the growth factor. NMR Biomed2002;15:120–131.



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Improved survival of rat ischemic cutaneous and musculocutaneous flaps after VEGF gene transfer