TGF-β1 and radiation fibrosis: a master switch and a specific therapeutic target?

Int. J. Radiation Oncology Biol. Phys., Vol. 47, No. 2, pp. 277–290, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0360-3016/00/$–see front matter

PII S0360-3016(00)00435-1

CRITICAL REVIEW

TGF-␤1 AND RADIATION FIBROSIS: A MASTER SWITCH AND A SPECIFIC THERAPEUTIC TARGET? MICHE` LE MARTIN, PH.D.,* JEAN-LOUIS LEFAIX, PH.D.,*

AND

SYLVIE DELANIAN, M.D., PH.D.†

*Laboratoire de Radiobiologie et d’Etude du Ge´nome, DRR, DSV, C.E.A. Saclay, France; †Service d’Oncologie- Radiothe´rapie, Hoˆpital Saint-Louis, Paris, France Radiation fibrosis is a frequent sequel of therapeutic or accidental radiation overexposure in normal human tissues. One of the main fundamental problems yet unsolved in fibrotic tissues is the origin of the chronic activation of myofibroblasts within these tissues. It has been postulated that this chronic activation results from a continuous production of activating factors. In this context, fibrosis could be defined as a wound where continuous signals for tissue repair are emitted. Cytokines and growth factors probably play a central role in this process. Among them, transforming growth factor-␤1 (TGF-␤1) is considered as a master switch for the fibrotic program. This review discusses recent evidence on the critical role played by TGF-␤ in the initiation, development, and persistence of radiation fibrosis. It summarizes the results concerning this factor after irradiation of various tissues and cells, with an emphasis on superficial fibrosis, including skin and subcutaneous tissues. Finally, recent data concerning the treatment of established fibrotic disorders of various etiology are presented, as well as the possible mechanisms involved in fibrosis regression, which show that the TGF-␤ pathway may constitute a specific target for antifibrotic agents. © 2000 Elsevier Science Inc. TGF-␤1, Ionizing radiation, Fibrosis, Myofibroblast, Treatment.

results obtained in skin models will be more particularly developed.

INTRODUCTION The research on radiation damage to normal tissues has gained in enthusiasm over the past few years both in experimental clinical oncology and fundamental radiobiology. One reason was the first publications demonstrating that such damage could be reversible. Another reason was that the research on predictors of patient radiosensitivity had progressed, and new tests were proposed, such as cell growth or DNA repair capacity assays, that showed correlations between in vitro radiosensitivity and the degree of late reactions in patients treated with radiotherapy. An additional reason was the appearance of new biological tools that allowed progress in fundamental radiobiology. In this context, it was important to further define late radiation damage and the mechanisms involved in their development. This review will focus on tissue fibrosis, which is a major late radiation damage. We will discuss recent evidence on the critical role of the TGF-␤ growth factor in radiation fibrosis and propose this factor as a major target for antifibrotic agents. As both early and late damage to the skin are often used as criteria of patient radiosensitivity, the

TGF-␤ growth factor Transforming growth factors are a family of cellular mediators present in mammals as three distinct isoforms of TGF-␤ called ␤1 to ␤3 (1–3). From gene knockout studies in mice, it has been shown that TGF-␤s are essential for survival, as the disruption of any one of the corresponding genes results in either embryonic or perinatal lethality (4). In this review, we will concentrate on TGF-␤1, which is the isoform most implicated in fibroproliferative diseases. TGF-␤1 was originally described as a peptide that caused reversible transformation of rodent fibroblasts (5, 6). It was first purified to homogeneity from human platelets (7) and was characterized as a homodimeric peptide with a molecular mass of 25 kDa. The cloning of human TGF-␤1 resulted in the elucidation of its precursor structure (8). TGF-␤1 is ubiquitously produced and generally secreted by the cells as a large latent complex (9). This complex includes the TGF-␤1 homodimer, the latency-associated pepFranc¸oise Crechet, Yves Tricaud, Jean-Jacques Leplat, Philippe Pinton, and Jean-Franc¸ois Dossin. They thank David Lawrence for careful reading of the manuscript, and Bernard Dubray and JeanMarc Cosset for helpful discussions. Studies developed in the Laboratoire de Radiobiologie et d’Etudes du Ge´nome were supported by EC Grant FI4P-CT95-0029 and by the Comite´ de Radioprotection d’Electricite´ de France. Accepted for publication 5 January 2000.

Reprint requests to: Dr. Miche`le Martin, Laboratoire de Radiobiologie et d’Etude du Ge´nome, Laboratoire Mixte CEA-INRA, Domaine de Vilvert, Jouy en Josas, 78352, cedex, France. E-mail: [email protected] Acknowledgments—The authors thank Marie-Catherine VozeninBrotons and Virginie Sivan for their active participation in the work on radiation skin damage performed in the laboratory over the last years, as well as Franc¸ois Daburon who initiated this work. They also thank for their technical assistance: Nathalie Gault, 277

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tide (LAP), and another high-molecular-weight protein, latent TGF-␤ binding protein (LTBP) responsible for the interactions with the components of the extracellular matrix. The latent TGF complex must be activated by dissociation for full activity, for example by protease treatment (10). The mature protein shows remarkable interspecies structural homology, as the processed TGF-␤1 molecules from human, simian, bovine, porcine, and murine species are almost identical. Signal transduction by TGF-␤ is mediated via two types of cell surface receptors that have been termed type I and II receptors and that exhibit serine/threonine kinase activities (11, 12). The T␤R-II first binds TGF-␤ and presents it to the type I receptor. These two receptors form a heterotetrameric receptor complex, which is the active form of the receptor. Activation of the T␤R-I kinase generates the first step of the TGF-␤ signaling pathway. The recent discovery of the new family of Smad transduction proteins involved in this pathway opened the black box of intracellular TGF-␤ signaling. The Smad proteins 1 to 8 are a new class of proteins that mediate responses to the TGF-␤ family and that can exhibit functional activities of both transduction proteins and transcription factors. By interacting with its receptors, TGF-␤ induces the phosphorylation and nuclear accumulation of Smad proteins, which can then act as transcription factors for various target genes (13, 14). TGF-␤1 is a multifunctional cytokine exerting in vivo three main biological activities. Firstly, it is a key cytokine in the regulation and general inhibition of cell growth. Secondly, it exerts immunosuppressive activities. Thirdly, it regulates the deposition of extracellular matrix components. As these functions are very specific, TGF-␤ may oppose the effects of various other cytokines. Another important characteristic of TGF-␤1 is that its activities vary according to the cell type studied and to the tissue context involved (15). TGF-␤1 elicits a number of rapid responses in cells, including alterations in their proliferation and apoptosis. It is a potent negative regulator of growth for most cell types, including epithelial, endothelial, and hematopoietic cells. The mechanisms of this effect have been more precisely studied in cell culture. TGF-␤1 can induce reversible G1 cell cycle arrest through signaling by its receptors, and it has a direct role on cell cycle–regulated genes. An important step in the antiproliferative action of TGF-␤1 is at the level of the cyclin kinase activities. TGF-␤1 was shown to inhibit the growth of Mv1Lu epithelial cells by preventing the formation of active cyclin E-cdk2 complexes (16), and by inducing the inhibitors of the cyclin-dependent kinases, such as p15, p21, and p27, in human HaCaT keratinocyte cell line (17, 18). Apart from regulating cell growth, TGF-␤1 can also elicit apoptosis in a variety of normal cells in primary cultures, including rat hepatocytes (19), rabbit endometrial cells (20), and human keratinocytes (21). The mechanisms of TGF-␤mediated cell death are poorly understood, but they may

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involve generation of reactive oxygen species. By these effects on cell growth and apoptosis, the TGF-␤ pathway generally acts as a tumor suppressor. Recently, mutations or deletions in the TGF-␤1 protein, receptor, or transduction proteins were found to be involved in the formation of various tumors. In mice heterozygous for deletion of the TGF-␤1 gene, enhanced tumorigenesis was found when compared with wild-type littermates (22) and in transgenic mice overexpressing a dominant negative type II TGF-␤ receptor in skin, increased carcinoma incidence was described (23). In human tumors, mutations of the type II TGF-␤ receptor are associated with carcinogenesis of sporadic gastrointestinal cancer (24, 25). Concerning signal transduction, Smad 4 is mutated or deleted in a large proportion of human pancreatic carcinomas (26), and Smad 2 is mutated in colorectal carcinoma (27). Although the main effect in vitro of TGF-␤ on cell proliferation is inhibition, TGF-␤1 can promote the proliferation of cultured mesenchymal cells such as fibroblasts and osteoblasts. In WI38 human fibroblasts, TGF-␤1 can downregulate p21 synthesis and activate the cyclinE-Cdk2 kinase (28). In 3T3 mouse fibroblasts, TGF-␤ leads to the rapid downregulation of p27, with kinetics consistent with the timing of cyclinE-cdk2 activation (29). Thus concerning cell proliferation, TGF-␤ can be a bifunctional cytokine. The second important activity of TGF-␤ is its anti-inflammatory action. The generation of TGF-␤1 null mice revealed the crucial role of this cytokine in modulating in vivo the immune system. These mice exhibit altered development, activation, and function of various immune cell populations, and they develop multiple manifestations of autoimmune-like processes (4). These activities were also assessed in cell culture. The growth of T- and B-cells can be suppressed by TGF-␤, as is cytotoxicity of natural killer cells and the production of immunoglobulins by B-cells, with the notable exception of IgA (1). TGF-␤ also modulates the cytotoxicity of macrophages by suppressing the production of nitric oxide (30). The last important function of TGF-␤ is to control the homeostasis of the extracellular matrix (1). TGF-␤1 causes the remodeling of extracellular matrix by simultaneously stimulating cells to increase the synthesis of most matrix proteins, decrease the production of matrix-degrading proteases, increase the production of inhibitors of these proteases, and modulate the expression of integrins. Through these functions, TGF-␤ has a central role in development and normal wound healing (31), and it was proposed that deregulations of the activities of this cytokine could be involved in the development of fibroproliferative diseases. Two lines of evidence point to a causal relation between elevated production of TGF-␤ and tissue fibrosis. First, in vivo administration of TGF-␤ in healthy animals produced tissue fibrosis. In newborn mice, subcutaneous injection of TGF-␤ caused the formation of granulation tissue within

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2–3 days at dose levels of less than 1 ␮g (32), and in rats, intravenous injections of TGF-␤ for 2 weeks produced kidney and liver fibrosis (33). In nude mice, generalized tissue fibrosis developed when TGF-␤1 was given intraperitoneally at doses exceeding 2 ␮g/day for 10 days (34). Second, the generation of transgenic mice that overexpressed TGF-␤1 in specific organs brought definitive demonstration of the causative role of TGF-␤ in tissue fibrosis. Thus, transgenic mice that overexpressed TGF-␤1 in the liver developed hepatic fibrosis as well as extrahepatic pathologies such as renal fibrosis, probably due to an increased level of circulating TGF-␤ in plasma (35). In another transgenic model, a constitutively active human TGF-␤1 gene was targeted to the liver, kidney, and adipose tissue. These mice developed severe fibrotic diseases in these tissues and a severe reduction in body fat (36). From these studies, it appears that TGF-␤1 is a key molecule and a master switch for the general fibrotic program.

Radiation fibrosis Fibrosis is a sequel of both radiotherapy and accidental overexposures. It is a complex tissue response whose predominant characteristics are massive deposition of extracellular matrix and excessive fibroblast proliferation. It has been described in vivo in many tissues, including skin (37, 38), lung (39), heart (40), and liver (41). Although radiation fibrosis has been reported for many years in histopathological studies, the mechanisms of its initiation and chronic extension still remain to be resolved (42). From a clinical point of view, established fibrotic tissues have long been described as irreversible dead scar tissues. However, a set of recent data obtained in our laboratory and in various other groups led us to support a very different view of radiation fibrosis (42– 45). Fibrosis is in fact a dynamic process, characterized by a constant remodeling and long-term fibroblast activation. The origin of fibroblast activation in chronic fibrosis has now become a major issue in this field of research. Most fibrotic lesions, whatever the type of stress that initially induced them, display common general features. In the constitutive phase, they contain infiltrating inflammatory cells and macrophages, numerous endothelial cells in neocapillaries, and abundant specific fibroblasts termed myofibroblasts (46 – 48). These cells are activated fibroblasts characterized by the appearance of cytoskeletal proteins involved in wound contraction, such as the smooth muscle actin. They play a central role in fibrogenesis as they deposit a fibrous matrix which is abnormal both in quantity and quality. All these general features suggest that common mechanisms of cellular activation may underlie many of the fibrotic disorders. In normal wound healing, fibroblasts are transiently activated into myofibroblasts to proliferate and deposit the collagen matrix. Then feedback mechanisms occur to downregulate cellular activities, and it has been proposed that

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myofibroblasts are terminally differentiated cells that finally disappear by apoptosis (49). In fibrosis, on the contrary, the feedback regulations are not observed, and chronic, longterm myofibroblast activation is sustained. One possible origin of the chronic cellular activation could be an abnormal production of stimulating factors such as cytokines and growth factors. In this context, fibrosis could be defined as a wound where continuous signals for tissue repair are emitted. Thus several growth factors were found dysregulated in fibrotic tissues not induced by radiation exposure, such as TGF-␤, platelet-derived growth factor (PDGF), tumor necrosis factor-alpha (TNF-␣), basic fibroblast growth factor (bFGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 (IL-1), interleukin-4 (IL-4), and connective tissue growth factor (CTGF) (50, 51). Deregulations of growth factors were also assessed in irradiated tissue, and particularly those of TGF-␤. TGF-␤ AND THE ONSET OF RADIATION FIBROSIS A new concept recently emerged concerning the initiation of radiation damage (43), which proposes that a cascade of cytokines is initiated immediately after irradiation, during the clinically silent period, persists for long periods of time and leads to the development of late damage. This concept stimulated research on the intercellular communications induced by radiation. The involvement of TGF-␤ in this early cascade has been addressed in various irradiated tissues mostly through in vivo experiments, including skin, intestine, mammary gland, and lung. TGF-␤1 and early cell response to radiation in skin Radiation overexposure frequently induces damage in skin and the underlying subcutaneous tissues, due to the superficial nature of these tissues (52–54). The early lesions that can result from such exposures are erythema, dry and moist desquamation, and ulceration. Possible late lesions include dermal atrophy, telangiectasia, late ulceration, fibrosis, and skin tumors. We have investigated the initiation of these processes during the first 24 hours after irradiation. For that purpose, early activation of the TGF-␤1 growth factor by ionizing radiation was studied in vivo in pig skin (55). Induction of TGF-␤ was detected in skin at 6 hours after ␥-irradiation at both the protein (55) and messenger RNA (mRNA) levels (Figs. 1 and 2), for single doses ranging from 16 to 64 Gy. Immunofluorescence staining on sections of irradiated skin revealed that fibroblasts, endothelial and epidermal cells secreted the TGF-␤ protein. mRNA induction was detected in skin samples from 2 hours, peaking at 6 hours and decreasing to basal values by 24 hours postirradiation; this induction had a mean threshold dose of 16 Gy. Interestingly, it was concomitant to the activation of other genes that are involved in the development of late

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Fig. 1. Early induction of TGF-␤1 and collagen type I [pro-␣1(I)] mRNA in irradiated skin. Northern blot analysis of pig skin isolated 6 hours after irradiation. The iridium source was sequentially applied on the flank of the animal to deliver from 8 to 64 Gy. Control skin was removed from the same flank. Five micrograms of mRNA was loaded on the gel. The 18S probe was used as a control of RNA loading (for detailed methods, see Ref. 55).

damage to the skin, particularly the collagen type I and ␤-actin, suggesting the rapid induction of tissue remodeling by irradiation. Induction of TGF-␤ also occurred after ␤-irradiation of the skin (56). In CBA and CD1 mice exposed to 50 Gy of 90 Sr/90Y ␤ radiation, an increased level of TGF-␤1 mRNA was found at 6 hours postirradiation, which returned to basal level within 48 hours, and increased again after 14 days. These observations of TGF-␤ induction in mice after ␤-irradiation and in pigs after ␥-irradiation lend support to the hypothesis that this is a general response of skin cells to ionizing irradiation. Next, we wanted to address the mechanism of this early activation. As the TGF-␤1 promoter contains activator protein-1 (AP-1) binding sites, the hypothesis that Fos and Jun proteins can regulate the TGF-␤1 promoter was tested (55). We found an induction of fos and jun mRNAs and proteins at 6 hours in irradiated skin (Fig. 2). To test the activity of these induced proteins, nuclear proteins were isolated from cultured normal human fibroblasts and their binding to oligonucleotides containing either a consensus AP-1 sequence or the -365 AP-1 sequence of the TGF-␤1 promoter was studied. Within 6 hours, 16 Gy (60Co ␥-rays) induced a significant increase in DNA binding both to the consensus AP-1 sequence and the TGF-␤1-specific AP-1 sequence. This increase was found in fibroblasts irradiated both in a confluent state and in the exponential phase of growth (Fig. 3). These results show that TGF-␤1, fos, and jun all take

Fig. 2. Early induction of c-fos, c-jun, and ␤-actin mRNAs in irradiated skin. Northern blot analysis of pig skin isolated 6 hours after irradiation. A volume of 10 ␮g of mRNA was loaded on the gel. The 18S probe was used as a control of RNA loading (for detailed methods, see Ref. 120).

part in the immediate early gene response of skin cells to high-dose ␥-irradiation. They suggest that stress-inducible TGF-␤1 expression is mediated by the activation of AP-1 transcription factor. Future transactivation studies will address more precisely the specific sequences of the TGF-␤1 promoter involved in this activation. TGF-␤ and early cell response in lung, small intestine, and mammary gland Rapid alterations of TGF-␤ expression have also been described in the rat small intestine and mouse mammary gland at the protein level. Increased TGF-␤1 protein was observed by immunohistochemistry in the rat small intestine after fractionated irradiation. This increase was observed at 1 day after irradiation, mainly in inflammatory cells, and persisted at 26 days (57). In mouse mammary glands, Barcellos-Hoff found that immunoreactivity was induced at 1 hour after whole-body irradiation and persisted for 7 days (58). Further, they addressed the process of TGF-␤ activation, which is an important step of regulation for this growth

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Fig. 3. Activation of DNA binding of AP-1 proteins after irradiation. Three hours after irradiation, nuclear proteins were isolated from human normal fibroblasts cultured in the exponential phase of growth. Their binding to the -365 AP-1 sequence of the human TGF-␤1 promoter was assayed. Five micrograms of nuclear proteins were incubated with 1 ng of the 32P-labeled oligonucleotide (for detailed Methods, see Ref. 55).

factor. Using antibodies that allows the detection of active versus latent TGF-␤, these authors showed that irradiation specifically generates the active protein (59). By studying immunoreactivity of TGF-␤ and LAP, which is the latency protein bound to TGF-␤ in its inactive form, they showed that the TGF-␤ and LAP immunoreactivity were modified in an opposite manner after irradiation. The shift from LAP to TGF-␤ was detected at 24 hours after doses as low as 0.1 Gy, and increased up to 5 Gy in the periepithelium of the mammary gland (60). So these authors suggested that low and moderate doses of radiation induced the activation of

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latent TGF-␤1 in the mouse mammary gland. Activation of the TGF-␤ complex has been poorly characterized in radiopathology, although it may be an essential step of regulation of the activity of TGF-␤s after irradiation. Thus, these studies are very important and should be extended to other models. Several hypotheses can be proposed concerning the function of such rapid inductions. During the first 24 hours, induction of TGF-␤ can slow cell growth in order to allow cell repair or induce apoptosis in lethally damaged cells. In a second step, it can favor tissue repair. In skin, for example, an increased TGF-␤ protein level could inhibit proliferation and induce spreading and migration of the epithelial cells, while inducing tissue remodeling and fibroblast proliferation in the deeper structures. In this context, it was shown in the mouse mammary gland that early radiation-induced collagen III staining in the adipose stroma was blocked by administration of a TGF-␤ panspecific monoclonal antibody to irradiated mice (60). Future studies of these processes should address the regulation of these activations, both in the TGF-␤ signaling pathway and in the modulations of specific promoter sequences. Finally, the search for target genes of the early alterations of TGF-␤ expression could allow a better understanding of the immediate cell response to ionizing radiation. However, TGF-␤ was not induced by irradiation in some other tissues. In the colorectum of mice irradiated with 30 Gy (single dose), no change was found at 6 hours for the TGF-␤1 mRNA level, both in a fibrosis-prone and a normal strain (45). In mouse bone marrow and spleen, no induction of mRNA level was observed from 2 to 14 days after 7.75 Gy in vivo exposure (61). There have even been examples of reduced TGF-␤ expression. In mouse lung, mRNAs for TGF-␤ and extracellular matrix genes were found decreased at 1 day after single exposures to 5–12 Gy of ␥-rays (62). In mouse jejunum, the alterations of the three TGF-␤ isoforms by 5 Gy were studied at the protein level. In the villi cells, all TGF-␤ isoforms decreased at 1 day and remained so at 6 days, whereas in the crypt cells, the TGF-␤s slightly increased (63). Most of the latter studies, however, used moderate doses of ionizing radiation, which can be under the threshold dose necessary to obtain a significant effect on mRNA or protein levels in these tissues. Another explanation can be that the occurrence and kinetics of the early radiation-induced alterations of TGF-␤ can differ according to the cell type and the tissue studied. Finally, regulations at the level of the activation of the TGF-␤ protein could occur in the absence of regulation at the level of transcription or translation, but this endpoint was not examined in most studies. However, latent TGF-␤ is stored in extracellular matrices and connective tissues, and activation of the protein may be a major stressinducible response in these tissues. TGF-␤ and early cell response in cultured cells Surprisingly, very few studies reported induction of TGF-␤ in irradiated cultured cells. Most available data of

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Table 1. Human and animal fibrotic disorders in which TGF-␤ has been implicated in the pathogenesis of radiation fibrosis Organ

Species

Irradiation

Reference

Skin

Human

Benyahia et al. (77)

Plasma

Human

Intestine

Human

Radiotherapy 55 Gy (TD) breast cancer Radiotherapy 30 Gy (TD) lung cancer Radiotherapy 50 Gy (TD) bowel tumours

␥ 192Ir 14 to 140 Gy (SD) ␤-irradiation 90 Str/90Y, 50 Gy (SD)

Martin et al. (70)

␥ 60Co 5 and 17.5 Gy (SD) ␥ 137Cs 5 and 12.5 Gy (SD) 5 and 12.5 Gy (SD) 5 and 12.5 Gy (SD) 300 kV X-rays 11 or 18 Gy (SD) 6–10 MV accelerator 15 and 30 Gy (SD) 250 kV X-rays 47 Gy (TD) 250 kV X-rays 50 Gy (TD) 250 kV X-rays 12 and 21 Gy (SD) 250 kV X-rays 5 Gy (SD) ␥ 137Cs 30 Gy (SD) 6 MeV electron source 15 to 75 Gy (TD) ␥ 137Cs 5–25 Gy (SD) 300 kV X-rays 19 or 25 Gy (SD) 200 kVp X-rays 18 Gy (SD) hemithorax

Rubin et al. (81)

Human diseases

Anscher et al. (80) Canney et al. (79)

Animal models Skin

Pig Mouse

Lung

Rabbit Mouse

Rat Intestine

Rat

Mouse

Liver

Rat

Bladder

Mouse

Plasma

Rat

Randall et al. (78)

Finkelstein et al. (62) Rubin et al. (43) Johnston et al. (90) Franko et al. (83) Yi et al. (82) Langberg et al. (57) Richter et al. (85) Hauer-Jensen et al. (86) Ruifrok et al. (63) Skwarchuk et al. (45) Anscher et al. (87) Geraci et al. (41)

Kraft et al. (88) Vujaskovic et al. (84)

Abbreviations: SD ⫽ single dose; TD ⫽ total dose.

early activations were obtained in situ, as if the TGF-␤ cell response was difficult to reproduce in the conditions of cell culture. For example, TGF-␤ mRNA was not induced up to 48 hours by 3 Gy in the KG1a cell line, which displays a primitive hematopoietic phenotype (64). The three-dimensional structure of the tissue and the interactions of cells with connective tissue as well as the differentiation of the cells are probably very important parameters in the control of TGF-␤ expression. In our laboratory, we found strong

induction of TGF-␤1 by ␥-rays both in the skin and in the in vitro 3-D model of reconstituted skin. In these conditions, keratinocytes and fibroblasts secreted increased amounts of TGF-␤ protein after irradiation. In monolayer cultures of the same normal skin cells, where both the epithelial/mesenchymal cell intercommunication and the interactions with the extracellular matrix are lost, induction of TGF-␤ protein by ionizing radiation was reduced. As a result of these difficulties, direct activation of the

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TGF-␤1 promoter by radiation has never published, and the regulatory sequences possibly involved are unknown. TGF-␤ AND THE DEVELOPMENT OF RADIATION FIBROSIS Several groups addressed the possibility that TGF-␤ is involved in the development of late radiation damage, and more specifically radiation fibrosis. Various types of fibrotic tissues have been studied, including skin, intestine, lung, bladder, and liver, and both in vivo and in vitro experiments have been developed. Table 1 lists the human disorders and animal models in which TGF-␤ has been implicated in the pathogenesis of radiation fibrosis. TGF-␤ and radiation fibrosis of the skin Superficial fibrosis is a sequel in humans after radiotherapy (37, 54, 65), which is characterized by induration of the dermis and the subcutaneous tissue. This induration is associated with telangiectasia and a hyalinization of the collagen of the reticular dermis. The epidermis may be hyperplastic or atrophic. All these lesions are now evaluated using the SOMA Lent Score. In cases of radiation accidents (38, 66 – 68), high doses of radiation can be delivered to the skin and the underlying subcutaneous tissues, and severe skin burns can be observed, resulting in extensive fibronecrotic tissues. In our laboratory, in vivo and in vitro pig models were developed in order to characterize radiation-induced lesions in skin, and more particularly necrosis and fibrosis (69, 70). Studies were also performed on human fibrosis samples, originating both from radiation therapy patients (71) and radiation accident patients (72). In these studies, we tested the hypothesis that an abnormal production of TGF-␤1 in irradiated skin results in continuous signals for tissue repair and long-term cell activation. TGF-␤1 in pig skin fibrosis. Daburon and coworkers developed an experimental model, in which acute localized ␥-irradiation of the skin resulted in extensive cutaneous and muscular fibrosis very similar to that observed in cases of human overexposure of the skin (38, 69). It was found in this model that TGF-␤1 was overexpressed throughout all phases of fibrosis development (Fig. 4). The mRNA level was increased 12-fold in the irradiated skin during the early erythematous phase, which started 3 weeks after irradiation. During the later phases of fibrosis, from 6 to 12 months after irradiation, it remained 10-fold elevated over the basal level in the repaired skin and the underlying muscular fibrotic tissue (70). At the protein level, immunostaining for TGF-␤1 revealed in the fibrotic tissue intense staining in myofibroblasts, in endothelial cells of capillaries and associated with collagenous matrix fibers. Moreover, cultured myofibroblasts retained this overexpression of the growth factor, exhibiting in primary culture a 2-fold increase of the secretion of the TGF-␤1 protein in the culture medium as compared to normal skin fibroblasts (Fig. 5). This result showed that this cell type was an important source of

Fig. 4. TGF- ␤1 mRNA was overexpressed at all stages of fibrosis development. Pig skin was removed at various times after irradiation. Five micrograms of mRNA was hybridized with the radiolabeled TGF-␤1 probe. The quantification of the Northern blots is shown as TGF-␤1 values corrected with the 18S values (for detailed methods, see Ref. 70).

TGF-␤1 within the fibrotic tissue and that chronic activation of myofibroblasts probably involved autocrine stimulation. TGF-␤1 in human skin fibrosis. Biopsies obtained from patients enabled a study of human fibrosis induced by radiotherapy. On fibrotic samples surgically removed from 6 months to 20 years after irradiation, gene expression studies for collagen type I and III and TGF-␤1 showed that the mRNAs specific for these three genes were significantly enhanced. Thus, long-term cellular activation and matrix remodeling seem to occur in human skin irradiated in the therapeutic range (60 –90 Gy total doses) (73). Human fibrotic tissues were also studied after radiation accidents (72). From a patient irradiated in Lilo, Georgia, a fibronecrotic block was surgically removed 18 months after the end

Fig. 5. Fibrosis myofibroblasts secreted high amounts of TGF-␤1 protein in culture. Enzyme-linked immunosorbent assay (ELISA) was performed using the kit from Promega on TGF-␤ secreted for 24 hours in the culture medium by pig fibroblasts isolated from normal skin and from fibrotic skin. Twenty-four hours before collection of the culture media, fibroblasts were cultured in UltraMEM supplemented with 0.2% insulin-transferrin-selenium. For detailed methods, see Ref. 72.

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radiation therapy for breast carcinoma, mammary skin biopsies were studied by immunohistochemistry. Irradiated skin exhibited positive intranuclear staining for TGF-␤ protein in fibroblasts and epidermal cells (77). In mouse skin irradiated with single doses of 50 Gy of ␤-radiation, it was found that TGF-␤ mRNA exhibited three distinct waves of expression, at 6 hours, 30 days, and 9 months. The second peak correlated with the macroscopic reaction of the skin and the last peak with the presence of skin fibrosis (78). Thus, long-term activation seems to be a hallmark of superficial radiation-induced fibrosis, both in humans and experimental models.

Fig. 6. TGF-␤1 protein was highly expressed in human fibrotic skin, both in keratinocytes of the scar epidermis (left side) and in myofibroblasts of the fibronecrotic dermis (right side).

of exposure. We found that TGF-␤1 protein was overexpressed in this fibronecrotic skin, both in the scar epidermis and in the fibrotic dermis (Fig. 6). Moreover, skin cells isolated from the scar retained their dysregulated secretion of TGF-␤ in culture, especially in scar keratinocytes. Thus, this cell type, although much less studied in the fibrotic process than the fibroblast, may be also very important in the long-standing activation process. Interactions between epithelial cells and fibroblasts certainly deserve more studies in the future concerning fibroproliferative disorders of the skin. These studies of human fibrotic tissues allowed us to distinguish more clearly two types of fibrosis, corresponding to two stages of development of the lesion: a young, active, and inflammatory fibrotic tissue versus a constituted, noninflammatory, and poorly cellularized fibrotic tissue. The pig fibrotic samples, the human postaccidental, and the young postradiotherapy samples that we studied all belonged to the first type, as do most of the experimental models described in the literature. Many postradiation therapy samples studied from 10 to 20 years after irradiation belonged, on the contrary, to the second type. Important differences were observed according to the type of fibrosis. For example, young type fibrosis samples contained numerous myofibroblasts that exhibited both a high proliferation rate (74) and a high secretion of TGF-␤1, whereas the old type contained fibroblasts that exhibited a senescent-like phenotype, reduced proliferation (71), and a low secretion of TGF-␤. The latter cells were similar to the terminally differentiated fibrocytes described by Rodeman et al. after irradiation of fibroblasts in culture (75). Nevertheless, common features still persisted in old fibrotic tissues, as both young and old fibrosis samples were able to respond to antifibrotic treatment (76), as described in a subsequent section of this paper (“TGF-␤1—A Target for Antifibrotic Agents?”). Similar long-term activation of TGF-␤ expression was found in other studies. Three months to 11 years following

TGF-␤ and radiation fibrosis in intestinal tract, lung, bladder, and liver The role of TGF-␤ in the development of radiation fibrosis has also been assessed in a variety of tissues, in a few human studies and in several animal studies, with the latter mainly performed in rodent models. The first historical observation of abnormal TGF-␤1 expression in irradiated tissues was described in the intestine of patients therapeutically irradiated with 50 Gy to the pelvis (79). In the hypertrophic, non–tumor-bearing irradiated colon, positive staining for TGF-␤ antibody was associated with epithelial cells, endothelial cells, macrophages, and lymphocytes, and appeared from 2 months after irradiation. Following thoracic radiotherapy, the development of pulmonary injury was associated with persistently elevated plasma TGF-␤ levels after the end of radiotherapy for lung cancer (80). Although it was not determined whether this TGF-␤ was active or latent, the authors proposed using the plasma level of TGF-␤ as a parameter to discriminate between the patients with low and high risk of developing radiation pneumonitis. In experimental models of radiation fibrosis, the lung has been the best studied organ. Depending on the species, the dose, and the delay, ionizing radiation can induce severe fibrosis in this tissue, which has been related to an abnormal TGF␤-1 expression in rabbit macrophages (81). In rats, it was demonstrated that TGF-␤ overexpression preceded histologically discernible pulmonary fibrosis, suggesting a role in the initiation of radiation fibrosis (82). In fibrosis-prone mice, TGF-␤ overexpression in macrophages and type II pneumocytes could also be related to the initiation of lung fibrosis, whereas positive cells were mainly fibroblasts in contracted, cellular fibrosis (83). In the irradiated intestine, TGF-␤ overexpression was observed at all assessment times. In a rat model of radiation enteropathy, a segment of small bowel was irradiated with 9 daily fractions of 5.2 Gy. Irradiated intestine was examined 24 h, 14 days, and 26 weeks after irradiation by immunohistochemistry. Increased TGF-␤ immunoreactivity was found at all times and correlated with fibrosis and inflammatory cell infiltrates (57, 85, 86). The involvement of TGF-␤1 in the development of radiation lesions was also found in the liver and the bladder. Thus, in the liver of rats irradiated with fractions of 2 Gy

TGF-␤ and radiation fibrosis

and total doses ranging from 15 to 75 Gy, immunostaining for TGF-␤1 was found to have increased in hepatocytes 9 months after irradiation at all doses, and this increase correlated with the severity of fibrosis (87). In the bladder of mice irradiated with 19 or 25 Gy, the extent of radiationinduced uroepithelial denudations, TGF-␤ staining intensity, and collagen I/III ratio were each correlated to bladder function. TGF staining intensity showed a progressive increase between 3 and 10 months after treatment, detected in the uroepithelium, the submucosa, and the muscle layers (88). Interestingly, deregulation of the TGF-␤ expression in myofibroblasts isolated from various fibrosis models was found maintained in cell culture for several passages. This persistence of the myofibroblastic phenotype in culture thus allows the development of mechanistic studies on radiation fibrosis. The complex role of TGF-␤ in fibrosis When comparing the general functions of TGF-␤1 and its role in fibrosis, several points fit well. Thus, its bimodal action on cell proliferation, with inhibition of epithelial cells and activation of fibroblasts, can certainly favor fibrosis and scar development. Similarly, the capacity of TGF-␤ to induce apoptosis in specific cell types can favor parenchymal damage and replacement by a fibrotic tissue. However, the important function of downregulation of the inflammatory processes which has been described for TGF-␤1 is difficult to reconcile with the development of tissue fibrosis, where inflammation is a chronic, long-term process, with phases of extremely high activities in some cases. We probably point here again to the fact that the activities of growth factors, and TGF-␤ in particular, are highly context-dependent in nature (15) and particularly after irradiation (44). For example, localized administration of TGF-␤1 enhances inflammation by increasing leukocyte adhesion and infiltration via chemoattraction of inflammatory cells, whereas systemic administration opposes this process probably because by this route TGF-␤1 initially encounters capillary endothelium and decreases endothelial cell expression of adhesion molecules (89). Although TGF-␤ is certainly a key cytokine, the fibrotic process cannot be explained by a single factor. It involves a complex network of interacting cytokines and growth factors, which include for radiation fibrosis PDGF, IL-1, insulin-like growth factor-1 (IGF-1), and TNF-␣. For example, TNF-␣ is part of the network in radiation lung fibrosis (90) and skin fibrosis (72). Interestingly, studies on cultured cells showed that TGF-␤1 also acts by modifying the expression and activity of genes encoding growth factors and their receptors. Thus, TGF-␤ regulates epidermal growth factor (EGF), PDGF, fibroblast growth factor (FGF), TNF, and IL-1 by stimulating or inhibiting their production in various cell types, including fibroblasts, endothelial cells, and smooth muscle cells (31). It induces CTGF mRNA and protein in cultured human skin fibroblasts through a specific promoter element (91). Although most of these data have

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been obtained on cultured cells, mice in which TGF-␤1 has been inactivated allowed the demonstration of in vivo alterations, such as a marked elevation of TNF-␣ and interleukin-1. All these interactions are probably very important in vivo in fibrotic tissues, where the whole intercellular communication network is altered. TGF-␤1—A TARGET FOR ANTIFIBROTIC AGENTS? Understanding that TGF-␤1 is a key factor in fibrogenesis offered a new possible target for therapeutic agents with potential antifibrotic effects. Until very recently, fibrotic tissue was considered as a dead, irreversible tissue which could not be cured. Drugs of several categories have been tried in the management of fibroproliferative disorders, including anti-inflammatory agents (steroids, colchicine, Dpenicillamine), drugs acting on blood flow (heparin, pentoxifylline), and interferons. Corticosteroids are still the first-line therapeutics, even though they essentially reduce symptoms associated with inflammatory reactions. Several drugs were effective in preventing the occurrence of fibrosis in experimental models, whereas few were able to reduce an established fibrotic tissue (Table 2, 92–108). Among the latter, several such as interferons produced significant clinical improvement in various fibrotic disorders, but were associated with toxicity and side effects. Recently, new data challenged the postulate of the irreversibility of established radiation fibrosis. Liposomal Cu/Zn superoxide dismutase (SOD) was shown to be the first agent effective in reducing long-standing radiation fibrosis in patients treated by radiotherapy (76). The treatment was short, as Lipsod was administered for 3 weeks in twice weekly i.m. injections of 5 mg, and it was well tolerated. This successful treatment could be reproduced in our pig fibrosis model both with bovine Cu/Zn SOD and human recombinant Mn SOD (102). At the Curie Institute (Paris), a clinical trial using topical applications of Cu/ZnSOD to treat breast fibrosis also reported an improvement at 6 months of treatment using twice-daily applications (101). These results were striking, as it was the first time that agents were shown to reverse the fibrotic process. However, the mechanisms of this therapeutic action were unclear. According to our data, a possible mechanism could be a SOD-induced downregulation of TGF-␤ secretion by myofibroblasts. We tested this hypothesis in our in vitro model of reconstituted fibrotic skin, where fibrosis myofibroblasts isolated from pig fibrotic tissue were cultured in a collagen gel and overlaid with scar keratinocytes. After a 1-week incubation with exogenous Cu/Zn SOD, we found that SOD reduced TGF-␤1 expression in myofibroblasts both at the mRNA and the protein levels (Vozenin-Brotons, submitted). Furthermore, the mRNA for the ␣1(I) collagen chain was also significantly downregulated by SOD incubation. The results on pig myofibroblasts correlated with those obtained on human myofibroblasts cultured in monolayers and incubated for 24 hours with Cu/Zn SOD (Delanian,

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Table 2. Molecules used in vivo in therapeutic strategies against established fibrotic disorders of various etiology

Molecule, availability

Therapeutic use

Colchicine, available Interferon-␥, non available

Experimental

Experimental Clinical Interferon-␣, available Experimental Clinical Clinical Glucocorticoids, available Experimental Essential fatty acids, non available Experimental SOD, non available Clinical Clinical Experimental Pentoxifylline, Clinical available Clinical Experimental Vitamin E, available Clinical Pentoxifylline ⫹ Clinical vitamin E, available Experimental Direct TNF-␣ antagonists Experimental Antibodies to integrins Experimental

Beneficial effect on

Inhibition of matrix Reduction of synthesis inflammation

Fibrosis

⫹

Fibrosis Fibrosis Fibrosis Hypertrophic scars Fibrosis

⫹

Fibrosis

⫹

⫹

⫹

⫹

⫹

⫹ ⫹ ⫹

⫹ ⫹

Fibrosis Fibrosis Fibrosis Fibrosis Pain Pain No effects on fibrosis Fibrosis Fibrosis Fibrosis

⫹ ⫹

⫹ ⫹

Fibrosis Fibrosis

⫹ ⫹

⫹

submitted). In this study, it was demonstrated that SOD entered the cells and reduced TGF-␤1 and tissue inhibitor of metalloproteinase (TIMP) gene expression. Consequently, we propose that exogenous SOD has an antifibrotic action, notably due to an anti-TGF-␤ effect in fibrotic myofibroblasts. This antifibrotic action may be generalized to other antioxidant factors, alone or in combination with other compounds, as it was more recently extended to the combination of ␣-tocopherol (vitamin E) plus pentoxifylline, both in patients and in the pig model (106, 104). The results in the pig model showed a striking regression of the subcutaneous fibrotic scar tissue, and a decreased immunostaining for TGF-␤1 in the residual fibrotic tissue. Interestingly, pentoxifylline alone had no therapeutic action in this model, whereas it seems to be an effective agent to heal superficial radiation necrosis (109, 110). According to several authors (111), a growing body of evidence supports a causative role of oxidative stress in fibrogenesis. They postulate that the effect of oxidative stress on cytokine gene expression is an important mechanism of fibrotic degeneration. Our overall results support this hypothesis. The importance of TGF-␤ as a target for antifibrotic strategies has been found in fibroproliferative disorders of various origin, including those in kidney, skin, lung, joint, and arterial wall (112–117). Various approaches have been tested to downregulate TGF-␤. Thus, administration of TGF-␤ neutralizing antibodies significantly reduced glomerulonephritis in rats when given at the time of induction by antithymocyte serum (112), cutaneous scarring when injected in rat wounds (113), intimal hyperplasia when

⫺

Growth factor antagonism

TGF-␤

Reference Dubrawsky et al. (92) Grossman et al. (93) Cale`s (94) Peter et al. (95) Moreno et al. (96) Tredget et al. (97) Dufour et al. (98) Cutroneo et al. (99)

TGF-␤ TNFR-␤ receptor

Hopewell et al. (100) Delanian et al. (76) Benyaha et al. (101) Lefaix et al. (102) Werner-Wasik et al. (103) Futran et al. (110) Lefaix et al. (104) Baillet (105) Delanian et al. (106) Lefaix et al. (104) Piguet et al. (107) Piguet et al. (108)

injected in a rat model of carotid artery injury (114), and lung fibrosis induced by bleomycin in mice (115). Similarly, antisense TGF-␤1 applied on dermal wounds resulted in markedly reduced scarring and fibrosis, associated with decreased TGF-␤ expression (116). Recombinant LAP, the latency-associated peptide of TGF-␤1, was shown to be a potent inhibitor of bioactive TGF-␤ both in vitro and in vivo, in transgenic mice with elevated TGF-␤ in liver (117). When interferon ␣-2b was used to treat hypertrophic scars, a significant clinical improvement was associated with normalization of the level of TGF-␤ in patient serum (97). The activation of the latent TGF-␤ has been poorly studied; however, it might be an important target of therapeutic agents, as shown for glucocorticoids that activated latent TGF-␤ in osteoblast cells (118). CONCLUSION Radiation fibrosis can no longer be seen as an irreversible, dead scar tissue. It can be better described as “a wound that does not heal”. We and others have demonstrated the continuous production of TGF-␤1 in irradiated tissues, and we support the view that this production has a causative role in the radiation-induced fibrotic process. Thus, important progress was achieved in the understanding of the initiation of fibroproliferative disorders. However, the mechanisms of feedback control in normal wound healing and their alterations in fibrotic tissues remain a complete “black box”. The recent findings on the TGF-␤ signaling pathway could provide new tools to address this important issue in future studies. The Smad family is a new class of proteins which

TGF-␤ and radiation fibrosis

exhibits activities of both transduction proteins and transcription factors. Interestingly, this system includes both proteins activating the TGF-␤ pathway, such as Smad 2 and 3, and inhibitory proteins, such as Smad 6 and 7. We postulate that deregulation of this TGF-␤ signaling pathway may occur in tissue fibrosis. Both overactivity of activating proteins and downregulation of inhibitory proteins may be linked to the chronic TGF-␤ overexpression in fibrosis; these points deserve further studies. Other important objectives of such studies can be to better understand the action mechanisms of antifibrotic agents on the TGF-␤ pathway as well as to define new therapeutic targets. In this connection, an important mechanism of action of interferon-␥ (IFN-␥)

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has recently been described, which supports our hypothesis. In osteosarcoma cells, IFN- ␥ can induce the expression of the inhibitory Smad 7 protein, thus preventing the cellular response to TGF-␤1 (119). If similar effects could be demonstrated on myofibroblasts, such mechanisms might be involved in the regression of tissue fibrosis. In conclusion, research recently developed both in clinical oncology and experimental radiobiology has led to important progress in the definition and understanding of late radiation damage, that may increase the possibilities of treatment of these lesions and result in new assays to select radiosensitive patients. These results should sustain the renewed scientific interest in this field of research in the future.

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