Advances in Bioscience and Biotechnology, 2013, 4, 1007-1012 ABB
http://dx.doi.org/10.4236/abb.2013.411134 Published Online November 2013 (http://www.scirp.org/journal/abb/)
A proteomic analysis of the effect of radiation therapy on
wound healing in women reconstructed with the TRAM
flap
Bekka O. Christensen1,2, Jens Overgaard2, Henrik Vorum4, Bent Honore3, Tine E. Damsgaard1
1Department of Plastic Surgery, Aarhus University Hospital, Aarhus, Denmark
2Department of Experimental Clinical Oncology, Aarhus University Hospital, Aarhus, Denmark,
3Department of Medical Biochemistry, University of Aarhus, Aarhus, Denmark
4Department of Ophthalmology, Aalborg University Hospital, Aalborg, Denmark
Received 10 July 2013; revised 20 September 2013; accepted 13 October 2013
Copyright © 2013 Bekka O. Christensen et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The incidence of breast cancer is still increasing, and
with improved cancer treatment, more women live
longer with the side effects of their treatment. The
response of normal tissue to radiation continues for
years after the treatment is completed. The influence
of radiotherapy on the outcome of breast reconstruct-
tive surgery remains unpredictable. The combination
of two surgical sites of which o ne is previously irradi-
ated, is rarely encountered in humans and thus com-
piles a unique opportunity to study the implications
of irradiation followed by surgery. The aim of this
study was to examine the long-term effect of radiation
therapy on the proteins expressed in the wound tissue
after a breast reconstruction. Ten patients were in-
cluded in the study, all treated with radiotherapy af-
ter a mastectomy and breast reconstruction with a
contralateral pedicled TRAM flap. Expanded poly-
tetrafluoretylene polymer tubes were implanted for
10 days, subcutaneously, below the inframammary
fold and below the donor site. The protein from the
newly synthesized granulation tissue in the tubes was
extracted and analyzed for differences in protein ex-
pression with 2D gel electrophoresis and mass spec-
trometry. A total of 676 proteins were detected; of
these, 4 proteins changed significantly and were suc-
cessfully identified. TPM4 and APOA4 from the ra-
diation treated tissue were shown to be significantly
decreased, whereas IGKC and VDAC1 were found to
be significantly increased. The proteomic technique
combined with the ePTFE tube wound model can
elucidate some of the molecular alterations in the
wound healing induced by radiation therapy. The
protein modifications of TPM4, APOA4, IGKC and
VDAC1 may influence the cell proliferation, apop-
tosis and the inflammation of the tissue repair proc-
ess.
Keywords: TRAM Flap; Radiation Therapy;
Reconstructive Surgery; Human; Proteomics; TPM;
VDAC; APOA4
1. INTRODUCTION
The healing process is the basis of all surgeries. There-
fore, it is crucial to gain knowledge of the biology of the
tissue repair process and the factors influencing it: how
to optimize the healing process, diminish the postopera-
tive complications.
The incidence of breast cancer is still increasing, and
with improved cancer treatment, more women live lon-
ger with the side-effects of their cancer treatment. The
destruction or damage of healthy tissue/cells outside the
tumour has both acute and long-term side-effects. Late
sequelae from radiation therapy (RT) include skin atro-
phy, pigmentation changes, soft tissue fibrosis and mi-
cro-vascular damage [1].
The combination of RT and breast reconstruction has
generated significant discussion. Several studies have
retrospectively evaluated aesthetic outcome and compli-
cations after mastectomy, radiation therapy and subse-
quent breast reconstruction. The overall conclusion is
that radiation therapy increases morbidity and decreases
the aesthetic outcome; patients will contract more com-
plications and an inferior outcome is unpredictable [2-6].
In 1982, Hartrampf et al. introduced the pedicled
transverse abdominal muscle flap (TRAM flap) for re-
construction of the breast. Variations of this versatile flap

B. O. Christensen et al. / Advances in Bioscience and Biotechnology 4 (2013) 1007-1012 1008
are common methods for autologous breast reconstruct-
tion, and often recommended for women previously
treated with radiation therapy, as it transposes non radi-
ated tissue into the irradiated area.
Radiation therapy is confined to the irradiated tissue
volume and therefore, enables examination of both irra-
diated and non irradiated tissue from the same individual.
The combination of two surgical sites, one of which is
previously irradiated, is rarely encountered in humans.
Thus, the TRAM flap procedure compiles a unique op-
portunity to study the implications of irradiation fol-
lowed by surgery.
Previous studies have reported impaired wound heal-
ing and radiation induced tissue changes [1,7-9]. The
pathogenesis of the radiation induced tissue alterations
are complex, involving a cascade of molecular and cel-
lular events. The time span in which such alterations
occur is uncertain.
The proteome is the entire complement of proteins, in-
cluding modifications that vary with time and require-
ments. It is well known that mRNA is not always trans-
lated into protein; the amount of protein produced for a
given amount of mRNA depends on both the gene. It is
transcribed from the current physiological state of the
cell. These (post-translational) modifications affect the
function of the proteins, and are therefore important in
the understanding of the patophysiological process caus-
ing the changes in the tissue repair process. Proteo- mic
analysis can determine the expression levels of pro- teins,
including the modifications [10].
Proteomic analysis, based on two dimensional elec-
trophoresis (2-DE), has primarily been conducted to
study carcinogenesis, signal pathway profiling, and mo-
lecular markers [11]. To our knowledge, no study has
used proteomics to examine the changes in the tissue
repair response, induced by previous radiation therapy, in
newly synthesized granulation tissue from humans. Most
studies working with radiation therapy and proteins use
techniques involving specific protein antibodies. They
require a beforehand knowledge of which proteins are in-
fluenced by RT. Other studies focus on cell cultures or
wound fluids [12,13].
The expanded polytetrafluorethylene polymer tube
(ePTFE tube) is a model for studying wound healing in
humans [14].
The proteomic analysis provides the possibility of
examining proteins without a priori knowledge of which
protein properties are altered by radiation therapy and
subsequent surgery.
2. MATERIALS AND METHODS
2.1. Patient
After a modified radical mastectomy for breast cancer,
ten women were consecutively included in the study. The
women were reconstructed with a contralateral pedicled
TRAM flap at the Department of Plastic Surgery, Aarhus
University Hospital, Denmark. All had been treated with
radiation therapy according to the national guidelines
from the Danish Breast Cancer Cooperative Group [15].
Data were obtained from personal communication,
physical examination and charts including information
regarding BMI (body mass index), tobacco smoking and
adjuvant therapy.
2.2. Surgery
The TRAM flap was harvested with a horizontal ellipse
of tissue, incorporating the full width and a variable
length of the rectus abdominis muscle. The musculocu-
taneous flap was tensionlessly transposed into the recre-
ated mastectomy defect, supplied by the superior epigas-
tric vessels. The transposed tissue was partly de-epithel-
ized and shaped into a new breast mound. Supra umbili-
cal fascial sheath defects were closed primarily and in-
fraubillical fascial defects were reconstructed using a 15
× 15 cm on-lay polypropylene mesh. The umbilicus was
brought out through a central opening in the midline of
the abdominal flap and sutured in place. Closure was
performed using a layered closure of Scarpa’s fascia,
subcutaneous tissue and skin.
2.3. The ePTFE Tube
The ePTFE tubes (International Polymer Enginering,
Tempe, Arizona, USA.) have a 0.12 cm inner diameter, a
0.25 cm outer diameter and a pore size of 90 - 120 µm.
The 9 cm ePTFE tube was threaded on a 3-0 non-ab-
sorbable nylon suture and sterilized before implantation.
The ePTFE tubes were implanted during general an-
esthesia, in the subcutaneous tissue at the intrammamary
fold and at the donor site, using the Seldinger technique.
Approximately 1cm of the ePTFE tube was left protrude-
ing outside the skin at the lateral perforation. The ePTFE
tube was sutured to the skin with a single nylon 4-0 su-
ture and covered with Tegaderm®. The ePTFE tubes
were removed on postoperative day 10 - 12. The external
part of the ePTFE tube was discarded and the former
subcutaneous parts were immediately divided and snap
frozen in liquid nitrogen and stored at −140 ˚C.
2.4. Proteomic Analysis
The ePTFE tubes were homogenized in lysis buffer and
the supernatants were removed. Two-dimensional gel
electrophoresis, image analysis and identification of dif-
ferentially expressed protein spots by mass spectrometry
were essentially performed as previously described [16].
The dry and transparent gels were scanned in the
transmissive mode on a GS-710 Imaging Densitometer

B. O. Christensen et al. / Advances in Bioscience and Biotechnology 4 (2013) 1007-1012 1009
from Bio-Rad (Hercules, CA, USA) using the Quantity
One software package. Gel images were exported as
16-bit gray scale TIFF files that were imported into the
PDQuest 8.0 2D Analysis Software (Bio-Rad). After
background subtraction, the protein spots were auto-
matically defined and quantified. Spot intensities were
expressed as optical densities (OD) and were normalized
against the total density in the gel image. One of the gels
used in the analysis comparison was selected as a refer-
ence gel to which the other gels used in the analysis was
aligned and matched using landmarks, as described in the
manual. In the reference gel each spot (feature) was as-
signed a number. The quality of the match made by the
computer was critically evaluated in each case, and nec-
essary editions and corrections were done manually. Ini-
tially, spots were selected for analysis that contained an
average relative intensity in the RT treated tissue that
differed by more than a factor 2 from the not treated
wound tissue. The significance of the differences was
calculated using the Student’s t-test.
Searches were performed in the SwissProt database
release 56.0 or 2012-07. Searches were performed with a
peptide mass tolerance of 20 or 50 ppm, a fragment mass
tolerance of 0.05 Da, max missed cleavages of 1 or 2 and
Carbamidomethyl (C) as fixed modification.
3. ETHICS
The study followed the Helsinki Declaration and was
approved by the Danish Regional Committee on Bio-
medical Research Ethics, M-20070039. All patients gave
their written informed consent.
4. RESULTS
The mean BMI was 25.4 and the mean age 57.2 years.
Seven women were non smokers and 3 reported to have
stopped a minimum of 4 weeks prior to surgery (Table
1).
Nine women participated in a follow-up visit. Seven
(7/9) of the women had a good overall result of the breast
reconstruction when assessed objectively. Three of the
10 women had flap necrosis, 2 required minor surgical
revision, one lost part of the reconstruction. The two
women with objectively inferior results also had necrosis
of the flap and small wound defects at the donor sites.
10 gels from the radiation treated tissue and 10 gels
from non radiated tissue were analyzed; representative
gels are presented in Figures 1(a) and (b). The overall
protein expression profiles were very similar. The spots
were distributed throughout the gels, ranging from pI 3
to 10 and with Mr between 10 - 160 kDa.
A total of 676 silver stained spots common for all gels
were detected, ten proteins were selected that were up-or
down-regulated in RT treated tissue compared to not RT
treated tissue from the same individual (Figures 1(a) and
(b)).
These 10 proteins were excised from the gels and 6
were identified (Table 2).
Tropomyosin alpha-4 (TPM4) and Apolipoprotein
A-IV (APOA4) from the RT treated tissue were signify-
cantly decreased, whereas Ig kappa chain C (IGKC) and
Voltage-dependent anion-selective channel protein 1
(VDAC1) were significantly increased. 5 excised spots
were not identified due to lack of protein and the spots
containing albumin were not included.
Table 1. Patient demographic.
Patient Age (years) BMI Time from RT to
reconstruction (years)
290 60 26.26 1.82
TNM stage Systemic treatment Total Dose
296 53 24.34 2.87 (Gy/fractions)
309 54 26.54 1.08 T1N1N0 CH 48/24
351 45 28.09 7.13 T1N1M0 C 48/24
362 64 26.61 1.44 T3N1Mx CH 48/24
367 56 26.99 4.07 T2N1M0 H 48/24
372 42 23.51 10.54 TisN0M0 48/24
412 52 22.23 3.34 T2N2bM0 CH 48/24
494 77 21.05 1.49 T1aN0M0 48/24
708 65 28.69 4.41 T2N1M0 CH 48/24
H = hormone therapy T3N2M0 CH 48/24
C = chemo therapy T2N2M0 CH 48/24

B. O. Christensen et al. / Advances in Bioscience and Biotechnology 4 (2013) 1007-1012 1010
(a)
(b)
Figure 1. (a) Proteins expressed in tissue collected from below the donor site (no irradiation)
by means of 2-Dimensional Electrophoresis. (b) Proteins expressed in tissue collected from
below the reconstructed breast (irradiated tissue, by means of 2-Dimensional Electrophoresis.
5. DISCUSSION
The expanded polytetrafluorethylene polymer tube
(ePTFE tube) wound model was combined with proteo-
mic analysis of the long-term changes in tissue repair
after breast reconstruction with a contralateral pedicled
TRAM flap in previously irradiated tissue. TPM4,
APOA4, IGKC and VDAC1 were identified as different-
tially expressed in irradiated tissue. In agreement with
our results, Menard et al. concluded that the composition
of proteins in tissue changes with radiation exposure
[11].
The identification of spot 5105 as both IGKC and al-
bumin introduces the possibility of either one of the pro-
teins expressions being altered, or both the proteins ex-
pression that were altered. The protein IGKC forms part
of the immune response of the tissues.
TPMs are present in virtually all eukaryotic cells,
where they bind actin filaments and stabilize their cell
structure. Changes in the expression of TPMs are com-
monly found in malignantly transformed cells [17]. In
humans, tropomyosins are a cytoskeletal family of pro-
teins that range in size from 35 to 45 kDa, encoded by
TPM1, TPM2, TPM3 and TPM4 genes.
Isoforms of tropomyosin are important determinants
of actin cytoskeletal functions such as intracellular vesi-
cle movement, cell migration, cytokinesis, cell prolifera-
tion and apoptosis.
Backovic et al. found that the expression of TPM4
protein was significantly correlated with the presence of
lymph node metastasis and the clinical stage in breast
cancer patients. Their results demonstrated that over ex-
pression of TPM4 in breast cancer cells is related to me-
tastatic behavior [17]. Da-Qiang Li et al. used pro-
eomics to analyze tumors of the breast and found that t

B. O. Christensen et al. / Advances in Bioscience and Biotechnology 4 (2013) 1007-1012 1011
Table 2. Identified proteins.
Protein spot no. Identity SWISS-PROT (primary
accession number) Gene Functional association
0302 Tropomyosin alpha-4 chain P67936 TPM4 Actin binding and cell movement.
Stabilizing of the cytoskeleton
1602 Apolipoprotein A-IV P06727 APOA4 Lipidtransport Antioxidant activity
Inflammation
4806 Serum albumin ALBU
5105 Serum albunin ALBU
5105 Ig kappa chain C region P06727 IGKC Immune response antigen
and proteinbinding
8310 Voltage-dependent
anion-selective channel protein 1 P21796 VDAC1 Apoptosis Ion transport
1402 Not identified
3705 Not identified
5606 Not identified
6006 Not identified
7003 Not identified
differently expressed TPM4 was associated to lymph
node metastasis and clinical stage [18].
Several molecules associated with neoplastic growth
are also nonspecific participants in the transient type of
benign proliferation seen in the normal wound healing
response. The decrease in TPM4, determined in our
study, is possibly part of the reason why healing after RT
treatment and surgery is sluggish and can result in a
suboptimal outcome. The normal increase in TPM4 does
not seem to occur or is delayed.
The voltage-dependent anion channel (VDAC) was
first identified in 1976. The VDAC proteins consist of 3
isoforms, VDAC1, VDAC2 and VDAC3, all located on
the outer mitochondrial membrane. VDAC forms the
main interface between the mitochondrial and the cellu-
lar metabolisms; it regulates the energy balance of the
mitochondria and the entire cell.
The mitochondria also have an important role in cell
signaling events, inter-organelle communication, aging,
many diseases, cell proliferation and cell death. The mi-
tochondria function as a center of apoptotic regulation.
VDAC has an important function in the regulation of
mitochondria-mediated apoptosis, and over expression
induces apoptotic cell death. The underlying mechanism
is unknown [19]. In our study VDAC1 was identified
and significantly increased, which possibly causes pre-
mature cell death. Corresponding to our results Voe-
hringer et al used DNA microarray in a B cell Lym-
phoma model and reported that up-regulation of VDAC
was induced by RT [20].
The reduced level of APOA4 protein measured in RT
tissue could indicate inflammation of the wound which
would be in agreement with Ebrini et al., who found that
reduced serum APOA4 expression in arthritis mouse was
an indicator of inflammation [21]. There exist only a few
non human studies focusing on the connection between
APOA4 and inflammation.
No growth factors or cytokines were detected in the
current studies perhaps because their high molecular
weight and solubility excluded them from the initial iso-
lates. Proteins bound to membranes or with extreme
acidic/basic properties that are highly hydrophobic or
outside the 10-200kDa interval are not easily identified
using 2D-PAGE. Likewise, protein samples can be dif-
ficult to prepare, and if the sample has large albumin
content, it can cloud smaller proteins.
All the women were previously treated with RT and
two had a suboptimal result of their breast reconstruct-
tion.
In conclusion, the understanding of the molecular
mechanisms involved in healing after treatment with RT
followed by surgery would be expanded with the possi-
bility of appropriate samples and models for further in-
vestigation. The present paper contributes with new
knowledge of the altered protein properties in previously
radiation treated healing tissue. The unique situation of
two surgical wounds, of which one was previously irra-
diated, and the proteomic technique combined with the
ePTFE tube wound model, can help elucidate part of the
molecular changes in radiation treated tissue. The
changes in expression levels of TPM4, APOA4, IGKC
and VDAC1 may influence the cell proliferation, apop-
tosis and inflammation in the wound healing process
after reconstructive breast surgery with the pedicled
TRAM flap.
6. ACKNOWLEDGEMENTS
The authors would like to thank the following for their financial sup-

B. O. Christensen et al. / Advances in Bioscience and Biotechnology 4 (2013) 1007-1012 1012
nport. None of the foundations had any involvement in the study.
The study was financially funded by the Faculty of Health Sciences,
Aarhus University, “Snedkermester Sophus Jacobsen og hustru Astrid
Jacobsens Fond”, The A. P. Møller Foundation for the advancement of
medical Science and The Danish Cancer Society.
REFERENCES
[1] Dormand, E.L., Banwell, P.E. and Goodacre, T.E. (2005)
Radiotherapy and wound healing. International Wound
Journal, 2, 112-127.
http://dx.doi.org/10.1111/j.1742-4801.2005.00079.x
[2] Christensen, B., Overgaard, J., Kettner, L.O. and Dams-
gaard, T.E. (2013) Long-term evaluation of postmastec-
tomy breast reconstruction with the TRAM flap. Journal
of Plastic Surgery and Hand Surgery.
[3] Bristol, S.G., Lennox, P.A. and Clugston, P.A. (2006) A
comparison of ipsilateral pedicled TRAM flap with and
without previous irradiation. Annals of Plastic Surgery,
56, 589-592.
http://dx.doi.org/10.1097/01.sap.0000205057.23543.48
[4] Spear, S.L., Ducic, I., Low, M. and Cuoco F. (2005) The
effect of radiation on pedicled TRAM flap breast recon-
struction: Outcomes and implications. Plastic and Recon-
stractive Surgery, 115, 84-95.
[5] Carlson, G.W., Page, A.L., Peters, K., Ashinoff, R.,
Schaefer, T. and Losken, A. (2008) Effects of radiation
therapy on pedicled transverse rectus abdominis myocu-
taneous flap breast reconstruction. Annals of Plastic Sur-
gery May, 60, 568-572.
http://dx.doi.org/10.1097/SAP.0b013e31815b6ced
[6] Williams, J.K., Bostwick, J. III, Bried, J.T., Mackay, G.,
Landry, J. and Benton, J. (1995) TRAM flap breast re-
construction after radiation treatment. Annals of Surgery,
221, 756-764.
http://dx.doi.org/10.1097/00000658-199506000-00014
[7] Denham, J.W. and Hauer-Jensen, M. (2002) The radio-
therapeutic injury—A complex “wound”. Radiotherapy
& Oncology, 63, 129-145.
http://dx.doi.org/10.1016/S0167-8140(02)00060-9
[8] Devalia, H.L. and Mansfield, L. (2008) Radiotherapy and
wound healing. International Wound Journal, 5, 40-44.
http://dx.doi.org/10.1111/j.1742-481X.2007.00351.x
[9] Johnson L.B., Adawi D., Agren M.S., Jorgensen L.N.,
Wittgren L., Mattsson S., et al. (2006) Combination of
pre-operative radiotherapy and surgery suppresses local
accumulation of collagen and TGF-β1 in rats. Journal of
Surgical Research, 133, 136-142.
http://dx.doi.org/10.1016/j.jss.2005.12.012
[10] Blumenberg, M. (2005) Skinomics. Journal of Investiga-
tive Dermatology, 124, viii-viix.
http://dx.doi.org/10.1111/j.0022-202X.2004.23639.x
[11] Menard, C., Johann, D., Lowenthal, M., Muanza, T.,
Sproull, M., Ross, S., et al. (2006) Discovering clinical
biomarkers of ionizing radiation exposure with serum
proteomic analysis. Cancer Research, 66, 1844-1850.
http://dx.doi.org/10.1158/0008-5472.CAN-05-3466
[12] Bernstein, E.F., Harisiadis, L., Salomon, G., Norton, J.,
Sollberg, S., Uitto, J., et al. (1991) Transforming growth
factor-β improves healing of radiation-impaired wounds.
Journal of Investigative Dermatology, 97, 430-434.
http://dx.doi.org/10.1111/1523-1747.ep12481258
[13] Schultze-Mosgau, S., Wehrhan, F., Amann, K., Rades-
piel-Troger, M., Rodel, F. and Grabenbauer, G.G. (2003)
In Vivo TGF-β3 expression during wound healing in irra-
diated tissue. An experimental study. Strahlentherapie
und Onkologie, 179, 410-416.
[14] Jorgensen, L.N. (2003) Collagen deposition in the subcu-
taneous tissue during wound healing in humans: A model
evaluation. APMIS. Supplementum, 115, 1-56.
[15] Overgaard, M. and Christensen, J.J. (2008) Postoperative
radiotherapy in DBCG during 30 years. Techniques, in-
dications and clinical radiobiological experience. Acta
Oncologica, 47, 639-653.
http://dx.doi.org/10.1080/02841860802078085
[16] Mandal, N., Lewis, G.P., Fisher, S.K., Heegaard, S.,
Prause, J.U., la, C.M., et al. (2011) Protein changes in the
retina following experimental retinal detachment in rab-
bits. Molecular Vision, 17, 2634-2648.
[17] Backovic, A., Huang, H.L., Del, F.B., Piza, H., Huber,
L.A. and Wick, G. (2007) Identification and dynamics of
proteins adhering to the surface of medical silicones in
vivo and in vitro. Journal of Proteome Research, 6,
376-381.
http://dx.doi.org/10.1021/pr0603755
[18] Li, D.Q., Wang, L., Fei, F., Hou, Y.F., Luo, J.M., Zeng,
R., et al. (2006) Identification of breast cancer metasta-
sis-associated proteins in an isogenic tumor metastasis mo-
del using two-dimensional gel electrophoresis and liquid
chromatography-ion trap-mass spectrometry. Proteomics,
6, 3352-3368. http://dx.doi.org/10.1002/pmic.200500617
[19] Shoshan-Barmatz, V., Keinan, N. and Zaid, H. (2008) Un-
covering the role of VDAC in the regulation of cell life and
death. Journal of Bioenergetics and Biomembranes, 40,
183-191. http://dx.doi.org/10.1007/s10863-008-9147-9
[20] Voehringer, D.W., Hirschberg, D.L., Xiao, J., Lu, Q.,
Roederer, M., Lock, C.B., et al. (2000) Gene microarray
identi- fication of redox and mitochondrial elements that
control resistance or sensitivity to apoptosis. Proceedings
of the National Academy of Science of the United States
of America, Vol. 97, No. 6, 2000, pp. 2680-2685.
http://dx.doi.org/10.1073/pnas.97.6.2680
[21] Ebrini, I., Agnello, D., Miller, I., Villa, P., Fratelli, M.,
Ghezzi, P., et al. (2000) Proteins of rat serum V: Adju-
vant arthritis and its modulation by nonsteroidal anti-in-
flammatory drugs. Electrophoresis, 21, 2170-2179.
http://dx.doi.org/10.1002/1522-2683(20000601)21:11<21
70::AID-ELPS2170>3.0.CO;2-1