Elsevier

Radiotherapy and Oncology

Volume 72, Issue 3, September 2004, Pages 297-303
Radiotherapy and Oncology

Transforming growth factor beta 1 dependent regulation of Tenascin-C in radiation impaired wound healing

https://doi.org/10.1016/j.radonc.2004.07.011Get rights and content

Abstract

Background

Following preoperative radiotherapy prior to ablative surgery of squamous epithelial cell carcinomas of the head and neck region fibrocontractive wound healing disorders occur. Tenascin-C is significantly increased in fibrotic tissue conditions and can be stimulated by the transcription factor NFκB p65. Previous studies showed a reduction of irradiation induced fibrosis during the wound healing process by anti-TGFβ1-treatment. The aim of the study was to clarify the question whether Tenascin-C expression is elevated in radiation impaired wounds and whether anti-TGFβ1-treatment is capable to influence Tenascin-C and NFκB expression.

Material and methods

Wistar rats (male, weight 300–500 g) underwent preoperative irradiation of the head and neck region with 40 Gy, fractionated four times 10 Gy (16 animals), whereas 8 non-irradated animals served as a control. Four weeks after irradiation a free myocutaneous gracilis flap taken from the groin was transplanted to the neck. Eight animals additionally received 5 μg anti-TGFβ1 into the graft bed by intradermal injection prior to each fraction of irradiation and on days 1–7 post-operation. On day 14 and 28 following surgery immunohistochemistry (ABC–POX method) was performed assessing the cytoplasmic NFκB and Tenascin-C staining in the transition area between transplant and graft bed. For quantitative considerations the labeling index (ratio: positive cells/total cells) was determined.

Results

A significantly altered expression of Tenascin-C in the preirradiated tissue was observed following anti-TGFβ1-treatment. NFκB protein was upregulated in irradiated animals and was significantly reduced in the anti-TGFβ1 treated group on day 28 after transplantation.

Conclusions

Tenascin-C expression is prolonged in irradiated animals as compared to non-irradiated tissue. Tenascin-C seems to be regulated by TGFβ1 as the application of TGFβ1-neutralizing antibodies reduces Tenascin-C expression. Tenascin-C is a potentially useful marker for tissue remodeling due to its restricted distribution in adult and healthy tissue and a hallmark for developing fibrosis.

Introduction

Following reconstructive surgery procedures in irradiated, fibrotic graft beds performing free soft tissue transplantation chronical inflammatory wound healing disorders occur with a rate of 8–39% [2], [33], [39]. Reduced vascularization in the transition area between graft and irradiated graft bed and increased synthesis of extracellular matrix components (ECM) with consecutively developing fibrosis is mediated by radiation induced upregulation of profibrotic cytokines like TGFβ1 [6], [24], [44]. Radiation induced expression of TGFβ1 and activation of its receptor downstream signalling pathway result in deposition of collagenous matrix and scarring during the proliferative and remodeling phase of wound healing [41], [42], [45]. Irradiated fibrotic tissue is characterized by overabundant expression of collagens -I, -IV, an abnormous collagen cross-linking and by accumulation of ECM components [27], [28], [46].

Tenascin-C, an ECM protein modulating functions of integrins in cell adhesion and motility, has been shown to be induced in fibrotic skin diseases like psoriasis, Bowen's disease and sclerosis [10], [11], [40]. In normal tissue Tenascin-C is located predominantly around dermal papillae, tendons and ligaments subjected to high tensile stress. Expression of Tenascin-C is transiently upregulated during wound healing and inflammation [11], [18], [19]. It is downregulated following wound contraction but persists in hypertrophic scars and fibrotic tissue conditions [9]. Adhesion of fibroblasts by Tenascin-C is associated with the activation of intracellular signalling pathways which are normally stimulated by cell contact with adhesive ECM components [31]. Tenascin-C promoter region was shown to contain a NFκB p65 binding site suggesting that Tenascin-C is a NFκB-dependent gene p65 [50]. NFκB is activated in irradiated tissue [37]. Previous studies identified several inflammation related cytokines like IL-1, IL-4, IL-13, TNF-α and TGFβ as inducers of Tenascin-C expression [5], [21], [34]. However, reports on a direct stimulation of tenascin by TGFβ1 are contradictory. Whereas local administration of TGFβ1 to calvarial bone stimulated Tenascin-C production in osteoprogenitor cells, TGFβ showed more variable results on fibroblast mediating induction and suppression of Tenascin-C [4], [25], [36]. Overexpression of Tenascin-C following high dose irradiation (total reference dose of 160 Gy) has been reported[12]. However, no reports are available elucidating temporo-spatial expression profile of Tenascin-C following surgery in preirradiated tissues with a more therapeutical total dose of 40–70 Gy. With regard to the known stimulation of Tenascin-C by NFκB p65 and described NFκB-TGFβ1 interaction, the expression profile of NFκB p65 during the wound healing process in preirradiated tissue is also of interest [37,53].

Exogenous application of TGFβ1-neutralizing antibodies was shown to be capable to diminish TGFβ1-activity and its signalling intermediates (Smad-3 protein) in preirradiated surgical sites in parallel with a reduced collagen synthesis [41]. Aim of our study was to investigate whether the expression of Tenascin-C is significantly upregulated in radiation impaired dermal wound healing and whether exogenous suppression of TGFβ1 in early wound healing influences Tenascin-C expression profile during the remodeling phase. Because of the absence of Tenascin-C in normal tissue, Tenascin-C could be a more specific target of antifibrotic therapeutical approaches than blocking collagen synthesis or multifunctional cytokines. Furthermore, the influence of surgical procedures in preirradiated tissue and the influence of anti-TGFβ1 treatment on NFκB p65 expression profiles was assessed in this study.

Section snippets

Materials and methods

24 male Wistar rats (weight 220–350 g, age 2.5±1 months; Charles River Wiga, Sulzfeld, Germany) were used. The rats were kept in pairs in polycarbonate cages Type II (Techniplast, Varese, Italy) provided with standard research bedding (wood granulate, Altromin, Lage, Germany) in accordance with the requirements of the German Animal Welfare Act (animal experiment application 621-2531.31-3/99) at a temperature of 22±0.5 °C, 55% humidity and a 12-h light/dark cycle. No special housing or feeding

Qualitative analysis

Cytoplasmic Tenascin-C expression was observed in dermal fibroblasts. The extent and intensity of staining was dependent from timepoint of observation, radiation dose and antibody treatment. The strongest expression was observed on day 28 after transplantation in the only irradiated animals (group 2) (Fig. 1a). The staining increased from day 14 after transplantation until day 28 and showed a diffuse pattern throughout the dermis. Tenascin-C staining was associated to collagen bundles. In

Discussion

The results of this study showed an induction of Tenascin-C in rat skin exposed to therapeutical irradiation doses. The altered expression levels at 6 and 8 weeks following irradiation of the skin confirmed an influence of Tenascin-C on the development of fibrosis during the wound healing process. Regarding the previous shown inhibition of TGFβ1 by neutralizing antibody treatment, the results of the current study suggested a positive correlation between Tenascin-C and TGFβ1 and the suitability

Acknowledgements

This research project was supported by the Federal Ministry of Education and Research (BMBF) and the Interdisciplinary Center for Clinical Research (IZKF) at the University Hospital of the University of Erlangen-Nuremberg; Project No. B8 and by the Wilhelm-Sander-Foundation No. 2002.017.1.

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