A proteomic analysis of the effect of radiation therapy on wound healing in women reconstructed with the TRAM flap

doi:10.4236/abb.2013.411134

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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

Email: [email protected]

Received 10 July 2013; revised 20 September 2013; accepted 13 October 2013

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

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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

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OPEN ACCESS

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

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(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

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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.

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