- Email: [email protected]
Growth regulation of skin fibroblasts
Journal of Dermatological Science 24 Suppl. 1 (2000) S70 – S77 www.elsevier.com/locate/jdermsci
Growth regulation of skin fibroblasts Kazuhiko Takehara * Department of Dermatology, Kanazawa Uni6ersity School of Medicine, 13 -1 Takara-machl, Kanazawa 920 -8641, Japan
Abstract The growth of skin fibroblasts is regulated in a complex manner by various growth factors. Representative growth factors are platelet-derived growth factor (PDGF), basic fibroblast growth factor (b-FGF), transforming growth factor-b (TGF-b), and connective tissue growth factor (CTGF). These growth factors have various biological activities besides growth regulation of skin fibroblasts, and are involved in wound healing and in the pathogenesis of various disorders. For example, PDGF and CTGF stimulate chemotaxis of skin fibroblasts, b-FGF stimulates angiogenesis, and TGF-b stimulates production of matrix proteins. First, the properties of these growth factors are reviewed briefly. Our skin fibrosis model in newborn mice are also described here. In 1986, Roberts et al. reported that subcutaneous injection of TGF-b in newborn mice caused granulation tissue formation followed by fibrosis (Roberts et al. Proc Natl Acad Sci USA 1986;83:4167 – 71). We conducted similar experiments, and found that TGF-b1, b2 or b3 caused skin fibrosis after 3 consecutive days of injection; this change was transient and disappeared after 7 consecutive days of injection. In contrast, irreversible fibrosis was observed upon stimultaneous injection of TGF-b and b-FGF or TGF-b and CTGF, or TGF-b injection for the first 3 days and b-FGF or CTGF injection for the next 4 days (Shinozaki et al. Biochem Biophys Res Commun 1997;237:292 – 7; Mori et al. J Cell Physiol 1999;181:153–9). These observations suggest that TGF-b induces skin fibrosis and b-FGF or CTGF maintains it in various skin fibrotic disorders. In the 21st century, we speculate that cocktails of various growth factors may permit subtle growth regulation of skin fibroblasts; such technology would have applications in the treatment of many skin diseases. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Skin fibroblasts; Growth regulation; Growth factors
1. Introduction The abnormal growth or function of skin fibroblasts causes fibrotic disorders such as scleroderma, keloid and hypertrophic scars. We have been studying the role of growth factors in the
* Tel.: +81-76-2652343; fax: + 81-76-2344270. E-mail address: [email protected] (K. Takehara).
pathogenesis of scleroderma and our results suggest that certain growth factors and cytokines play key roles in the occurrence of scleroderma [1–10]. In other words, fibrotic disorders of various organs, including skin, can be considered to be a result of excessive healing (overhealing). As shown in Figs. 1 and 2, the early sclerotic skin of scleroderma clinically shows a shiny appearance, and histologically there is an abundant accumulation of collagen bundles. If we could clarify in detail the mechanisms of skin fibrosis, it might be
0923-1811/00/$ - see front matter © 224 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 9 2 3 - 1 8 1 1 ( 0 0 ) 0 0 1 4 4 - 4
K. Takehara / Journal of Dermatological Science 24 (2000) S70–S77
possible to modulate the growth regulation and function of skin fibroblasts in such a way as to prevent skin aging and to maintain young-appearing skin over a long period. In the 21st century, we speculate that the use of
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cocktails of various growth factors may enable subtle regulation of the growth of skin fibroblasts, which will be applicable to the treatment of various skin diseases, as well as to the prevention, or at least delay, of skin aging.
Fig. 1. Clinical features of typical early scleroderma. Chest skin has a shiny appearance. Fig. 2. The left panel shows histological change of early edematous scleroderma. The right panel shows histological change of advanced sclerotic stage of scleroderma.
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Table 1 Bioactive peptides and the fields in which they were discovered Peptide hormones (classical endocrinology) Cytokines Interleukins (immunology) Interferons (virology) Colony stimulating factors (hematology) Tumor necrosis factors (oncology) Growth factor (cell biology) Others Endothelin, etc.
2. Cytokines and growth factors Cytokines are a group of regulatory molecules that function as mediators of cell communication in both normal and pathologic conditions. Cytokines are low-molecular-weight (glyco)proteins that are produced by a variety of cells. Via interaction with specific cell surface receptors, they exert multiple biological functions. Mediators in the cytokine family are interleukins, hematopoietic growth factors, interferons, tumor necrosis factors, and growth factors, as shown in Table 1. These regulatory proteins appear to participate in the mediation of inflammatory as well as immune reactions, and some may influence tumor growth. Growth factors were originally regarded only as regulators of cell proliferation. The family of growth factors includes epidermal growth factor (EGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (b-FGF), transforming growth factor-b (TGF-b) and connective tissue growth factor (CTGF). In addition to controlling the growth of normal and malignant cells, as well as matrix protein formation, some growth factors are important mediators of cell surface alteration and immunoregulation. Several growth factors are essential for normal wound healing and clinical application of such growth factors for impaired wound healing has already been tried [11 – 13].
origins of these growth factors and their target cells are shown in Table 2.
3.1. PDGF In 1974, it was discovered that platelets were the main source of mitogenic activity found in whole blood serum and missing in plasma. The growth factor with this mitogenic activity was called PDGF [14]. PDGF has a molecular weight of 28 77–35 000. It is made up of two chains, which share 60% homology. The A chain has a molecular weight of 14 000 and the B chain has a molecular weight of 17 000. The B chain has a high degree of homology with v-sis oncogene. The PDGF receptor is found on cells that show a mitogenic response to PDGF. Thus, the receptor is present on fibroblasts, glial cells and vascular smooth-muscle cells. The PDGF receptor has an extracellular ligand-binding domain, and an intracellular effector domain with tyrosine kinase activity, and the peptide-receptor complex is internalized by endocytosis. The receptor is downgraded by PDGF binding. PDGF is chemotactic for fibroblasts and smooth-muscle cells, at low concentrations [15]. Recently, PDGF B chain products were approved by the FDA as a wound repair drug for Table 2 Characteristics of growth factors relevant to wound healing Factor
Producing cells
Target cells
PDGF
Platelets
Fibroblasts (mitogen, chemotaxis)
b-FGF TGF-b
Fibroblasts
3. Major growth factors in wound healing The major growth factors in wound healing are PDGF, b-FGF, TGF-b and CTGF. The cellular
Endothelial cells Macrophages Smooth muscle cells Keratinocytes Macrophages Endothelial cells Platelets Macrophages
CTGF
Endothelial cells Fibroblasts
Fibroblasts (mitogen) Endothelial (angiogenesis) Fibroblasts (collagen synthesis) Endothelial (growth inhibition, matrix synthesis) Fibroblasts (mitogen) Endothelial (angiogenesis)
K. Takehara / Journal of Dermatological Science 24 (2000) S70–S77
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Fig. 3. The effects of various growth factors in the serum-free culture system of skin fibroblasts. b-FGF had the most potent growth stimulatory effect among these growth factors.
diabetic ulcers in the United States. Clinical trials for the treatment of impaired wound healing, including diabetic ulcers, are being planned in Japan.
3.2. b-FGF b-FGF, by stimulating division and migration of fibroblasts, endothelial cells and epithelial cells, promotes formation of new capillaries and granulation tissue. In our serum-free experimental systems, b-FGF is the most potent growth stimulator among the various growth factors (Fig. 3). b-FGF has a strong homology with hst-1 oncogene. Clinical trials of b-FGF for treatment of impaired wound healing in Japan have shown that this agent is very effective for skin ulcers, and this product is expected to be approved for clinical usage by the Japanese FDA.
produced as a latent form. The detailed mechanisms of activation of TGF-b in vivo have not been clarified. In general, the biological activities of TGF-b are very strong, and therefore the molecule is probably produced as an inactive form in order to block excessive TGF-b action [18]. Serine I threonine kinase is involved in TGF-b signal transduction, which is quite different from
3.3. TGF-b TGF-b is a 25 kd homodimer. Five distinct isoforms were found, of which three are present in humans. TGF-b1, b2 and b3 exhibit similar, but not identical biological activities in vitro. TGF-b acts as a growth inhibitor for most cell types (Fig. 4) [16] and stimulates extracellular matrix production, including type I collagen, type III collagen and fibronectin, in fibroblasts [17]. This stimulatory effect on ECM production is much stronger than those of other cytokines. One of the most important characteristics of TGF-b is that it is
Fig. 4. Effects of various growth factors on endothelial cell growth. Confluent RHECs were trypsinized, and 1 ×104 cells were suspended in DMEM containing 10% FCS and plated in 24-well plates. Following cell attachment overnight, the medium was changed to DMEM containing 2.5% FCS without growth factor ( ), with 1 ng/ml TGF-b () with 0.25 ng/ml TGF-b (), with 10 ng/ml EGF ( ), with 10 ng/ml FGF (), or with both 1 ng/ml TGF-b and 10 ng/ml EGF ( × ) (day 0). Cell numbers were determined on days 3, 6 and 9. (Takehara et al. [16]).
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Fig. 5. CTGF is an autocrine growth factor and is produced by skin fibroblasts only when they are stimulated with TGF-b.
the case of other growth factors such as PDGF and EGF, which use tyrosine kinase. TGF-b has attracted much attention as a key factor in various fibrotic disorders [19], because it potently increases ECM production by many cells.
3.4. CTGF CTGF was identified originally from cultured human umbilical vein endothelial cells by Grotendorst and his group at Miami University [20]. This factor is also produced by skin fibroblasts, but only when they are stimulated with TGF-b [21]. CTGF is recognized by anti-PDGF polyclonal antibody and CTGF c-DNA was cloned using anti-PDGF antibody. This factor exhibits PDGF-like chemotactic and mitogenic activities and binds to PDGF receptors, though CTGF has little peptide sequence homology to PDQF A or B chains. Fig. 5 summarizes the relationship between TGF-b and CTGF. CTGF acts as an autocrine growth factor when skin fibroblasts are stimulated with TGF-b.
4. An animal model of skin fibrosis by exogenous injection of TGF-b and other growth factors We recently established an animal model of skin fibrosis by exogeneous injection of TGF-b and other growth factors [22,23]. In 1986, Roberts et al. reported that TGF-b injection into newborn mice caused granulation tissue
formation and skin fibrosis [24]. We initially tried to establish an animal model of skin fibrosis by TGF-b injection using a similar method to that described by Roberts et al. [24]. We injected 800 ng of TGF-b1, b2 or b3 into subcutaneous tissue of newborn mice for 7 days. The details were described elsewhere [22,23]. The results of these experiments are summarized in Table 3.
4.1. Response to application of single growth factors Three days of TGF-b injection resulted in a firm infiltrated lesion at the injection site. Histologic examination revealed granulated tissue formation (Fig. 6A), consisting of lymphocytes, histiocytes and fibroblasts. After 7 days of consecutive injections, however, this response was not detectable either macroscopically or microscopically (Fig. 6B). CTGF injection caused slight edema and some cell infiltration. Similarly, b-FGF injection caused slight edematous granulated tissue formation.
4.2. Response to simultaneous and serial applications of two growth factors Simultaneous injection of TGF-b plus CTGF or TGF-b plus b-FGF resulted in fibrotic tissue formation (Fig. 6C), consisting of fibroblast aggregation and ECM deposition, which persisted for up to 14 days, even though the injections were discontinued on day 7. To examine further the tissue response, two different growth factors were injected serially, viz. TGF-b on days 1–3 followed by CTGF on days 4–7. Serial injections of CTGF after TGF-b caused fibrotic tissue formation. This response was relatively weak but persisted for at least 14 days, even though we stopped the injection of growth factors on day 7. Injecting b-FGF after TGF-b caused increased edematous granulated tissue formation that remained for 10 days. Injection of CTGF or b-FGF before TGF-b did not cause any significant change compared with TGF-b alone.
K. Takehara / Journal of Dermatological Science 24 (2000) S70–S77
4.3. Meaning of our results The results of our study clearly demonstrate that single application of any growth factor is not sufficient to induce persistent fibrosis, despite continuous injections. Instead, interaction of plural growth factors seems to be necessary for the induction of persistent fibrosis in this animal model. Our observations on serial application of different growth factors suggest that TGF-b plays an important role in inducing granulation and fibrotic tissue formation. The other growth factors, CTGF and b-FGF, are important in maintaining fibrosis. In contrast, injections in the reverse order, viz. CTGF followed by TGF-b or b-FGF followed by TGF-b did not produce any significant response. Hence, we conclude that induction of persistent fibrosis most likely requires two factors: an induction factor such as TGF-b and a maintenance factor such as CTGF. B-FGF may act synergistically by inducing CTGF, because our results suggest that either CTGF mRNA expression or the CTGF protein itself is required to develop persistent fibrotic changes.
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We then focused on the action of CTGF as a candidate mediator to maintain skin fibrosis, because TGF-b selectively stimulates CTGF expression of fibroblasts. CTGF, which is known to increase fibroblast proliferation and ECM synthesis, has recently been proposed to be a TGF-bspecific downstream mediator. We previously reported that CTGF mRNA expression is strongly correlated with tissue fibrosis in systemic sclerosis and other skin fibrotic disorders [7,8,25]. 5. Two-step fibrosis hypothesis in systemic sclerosis Based on the results with TGF-b and CTGF described above, we hypothesized that a two-step process of fibrosis occurs in scleroderma.We think that TGF-b induces fibrosis in the early stage of systemic sclerosis and then CTGF acts to maintain tissue fibrosis. TGF-b induces CTGF mRNA, but some additional factor is required for continuous CTGF mRNA expression.
Table 3 Histological responses to single, simultaneous and serial injections of different growth factors into newborn micea Injection schedule
Histological change
Days 1–3
Days 4–7
Days 8–10
Day 4
Day 8
Day 11
Single injection TGF-b CTGF b-FGF PBS
TGF-b CTGF b-FGF PBS
− − − −
++ +− + −
− − +− −
− − − −
TGF-b+CTGF TGF-b+b-FGF
− −
+++ +++
+++* +++*
+++* +++*
CTGF b-FGF TGF-b TGF-b TGF-b TGF-b b-FGF CTGF
− − − TGF-b − TGF-b − −
++ ++ +− +− + + +− +
+++* +++* ++ ++ ++ ++ + −
++ ++ − + − + − −
Simultaneous injection TGF-b+CTGF TGF-b+b-FGF Serial injection TGF-b TGF-b CTGF CTGF b-FGF b-FGF CTGF b-FGF a
−, no change; +−, slight edema and some cell infiltration; +, edematous granulation tissue; ++, granulation tissue consisting of lymphocytes, histiocytes, and fibroblasts; +++, fibrotic tissue consisting of fibroblast aggregation and extracellular matrix deposition, and asterisks denotes marked fibrosis. See the text for the concentrations of different growth factors injected.
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K. Takehara / Journal of Dermatological Science 24 (2000) S70–S77
Acknowledgements This article is based on a presentation given at the Shiseido Science Symposium 277, held on April 15, 2000, Tokyo. The major part of these investigations were conducted by collaborators at Tokyo University (Drs Atsuyuki Igarashi, Yoshinao Soma, Nobukazu Hayashi and Takashi Kakinuma) and at Kanazawa University (Drs Mikio Shinozaki, Toshifumi Mori, Shigeru Kawara and Shinichi Sato).
References
Fig. 6. An animal model of skin fibrosis induced by exogenous growth factor injections. (A) After 3 consecutive days of injections of TGF-b2. (B) After 7 consecutive days of injections of TGF-b2. (C) Combination of TGF-b 3 and b-FGF for 7 consecutive injections.
In the future, substances that block CTGF activities, such as anti-CTGF neutralizing antibody or CTGF antisense molecules are likely to be candidates for therapeutic use to treat systemic sclerosis. Some preliminary experiments using our animal model are in progress in our laboratory.
[1] Igarashi A, Takehara K, Soma Y, Kikuchi K, Ishibashi Y. Mitogenic activities for skin fibroblasts in the sera from patients at the early stage. Arch Dermatol Res 1989;281:383 – 6. [2] Etoh T, Igarashi A, Ishibashi Y, Takehara K. The effects of scleroderma sera on endothelial cell survival in vitro. Arch Dermatol Res 1990;282:516 – 9. [3] Takehara K, Soma Y, Igarashi A, Kikuchi K, Moro A, Ishibashi Y. Response of scleroderma fibroblasts to various growth factors. Arch Dermatol Res 1991;283:461 – 4. [4] Flanga V, Gerehardt CO, Dasch JR, Takehara K, Ksander GA. Skin distribution and differential expression of transforming growth factor b1 and b2. J Dermatol Sci 1992;3:131 – 6. [5] Kikuchi K, Kadono T, Sato S, Tamaki K, Takehara K. Impaired growth response to endothelin-1 in scleroderma fibroblasts. Biochem Biophys Res Commun 1995;207:829 – 38. [6] Kikuchi K, Kadono T, Ihn H, Sato S, Igarashi A, Nakagawa H, Tamaki K, Takehara K. Growth regulation in scleroderma fibroblasts: increased response to transforming growth factor-bI. J Invest Dermatol 1995;105:128 – 32. [7] IgarashIi A, Nashiro K, Kikuchi K, Sato S, Ihn H, Grotendrost G, Takehara K. Significant correlation between connective tissue growth factor gene expression and skin sclerosis in tissue sections from patients with systemic sclerosis. J Invest Dermatol 1995;105:280 – 4. [8] Igarashi A, Nashiro K, Kikuchi K, Sato S, Ihn H, Fujimoto M, Grotendrost G, Takehara K. Connective tissue growth factor gene expression in tissue sections from localized scleroderma, keloid and other fibrotic skin disorders. J Invest Dermatol 1996;106:729 – 33. [9] Hasegawa M, Fujimoto M, Kikuchi L, Takehara K. Elevated serum tumor necrosis factor-alpha level in patients with systemic sclerosis: association with pulmonary fibrosis. J Rheumatol 1997;24:663 – 5. [10] Kadono T, Kikuchi K, Ihn H, Takehara K, Tamaki K. Increased production of interleukin 6 and interleukin 8 in scleroderma fibroblasts. J Reumatol 1998;25:296 – 301.
K. Takehara / Journal of Dermatological Science 24 (2000) S70–S77 [11] Rothe M, Flanga V. Growth factors: their biological and promise in dermatologic diseases and tissue repair. Arch Dermatol 1989;125:1390–8. [12] Luger TA, Schwarz T. Therapeutic use of cytokines in dermatology. J Am Acad Dermatol 1991;24:915–26. [13] Sporn MB, Roberts AB. A major advance in the use of growth factors to enhance wound healing. J Clin Invest 1993;92:2565 – 6. [14] Ross R, Vogel A. The platelet-derived growth factor. Cell 1978;14:203 – 10. [15] Grotendorst GR. Alteration of the chemotactic response of NIH/3T3 cells to PDGF by growth factors, transformation and tumor promoters. Cell 1984;36:279–85. [16] Takehara K, LeRoy EC, Grotendorst GR. TGF-13 inhibition of endothelial cell proliferation: Alteration of EGF binding and EGF-induced growth regulatory (competence) gene expression. Cell 1987;49:415–22. [17] Varge J, Rosenbloom J, Jimenez SA. Transforming growth factor b (TGF-b) causes a persistent increase in steady-state amounts of type I and type III collagen and fibronectin mRNAs in normal human dermal fibroblasts. Biochemistry 1987;297:597–604. [18] Roberts AB, Sporn MB. Physiological actions and clinical applications of transforming growth factor-b (TGF-b). Growth Factors 1993;8:1–9. [19] LeRoy EC, Edwin A, Smith M, Kahaieh B, Trojanowska M, Silver RM. A strategy for determining the pathogenesis of systemic sclerosis: is transforming growth factor b the answer? Arthritis Rheum 1989;32:817–25.
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[20] Bradham DM, Igarashi A, Potter RL, Grotendorst GR. Connective tissue growth factor: a cystine-rich mitogen secreted by human vascular endothelial cells is related to the SRC induced immediate early gene product CEF-lO. J Cell Biol 1991;114:1285– 94. [21] Igarashi A, Okachi H, Bradham DM, Grotendorst GR. Regulation of connective tissue growth factor gene expression in human skin fibroblasts and during wound repair. Mol Cell Biol 1993;4:637 – 45. [22] Shinozaki M, Kawara S, Ilayashi N, Kakinuma T, Igarashi A, Takehara K. Induction of subcutaneous tissue fibrosis in newborn mice by transforming growth factorbeta — simultaneous application with basic fibroblast growth factor causes persistent fibrosis. Biochem Biophys Res Commun 1997;237:292 – 7. [23] Mori T, Kawara S, Shinozaki M, Hayashi N, Kakinuma T, Igarashi A, Takigawa M, Nakanishi T, Takehara K. Role and interaction of connective tissue growth factor with transforming growth factor-13 in persistent fibrosis: a mouse fibrosis model. J Cell Physiol 1999;181:153 – 9. [24] Roberts AB, Sporn MB, Assoian JK, Smith JM, Roche NS, Wakefield LM, Heine UI, Liotta LA, Falanga V, Kehrl JH, Fauci AS. Transforming growth factor type b: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Natl Acad Sci USA 1986;83:4167 – 71. [25] Igarashi A, Hayashi N, Nashiro K, Takehara K. Differential expression of connective tissue growth factor gene in cutaneous fibrohistiocytic and vascular tumor. J Dermatol Sci 1998;16:152 – 7.
Growth regulation of skin fibroblasts Kazuhiko Takehara * Department of Dermatology, Kanazawa Uni6ersity School of Medicine, 13 -1 Takara-machl, Kanazawa 920 -8641, Japan
Abstract The growth of skin fibroblasts is regulated in a complex manner by various growth factors. Representative growth factors are platelet-derived growth factor (PDGF), basic fibroblast growth factor (b-FGF), transforming growth factor-b (TGF-b), and connective tissue growth factor (CTGF). These growth factors have various biological activities besides growth regulation of skin fibroblasts, and are involved in wound healing and in the pathogenesis of various disorders. For example, PDGF and CTGF stimulate chemotaxis of skin fibroblasts, b-FGF stimulates angiogenesis, and TGF-b stimulates production of matrix proteins. First, the properties of these growth factors are reviewed briefly. Our skin fibrosis model in newborn mice are also described here. In 1986, Roberts et al. reported that subcutaneous injection of TGF-b in newborn mice caused granulation tissue formation followed by fibrosis (Roberts et al. Proc Natl Acad Sci USA 1986;83:4167 – 71). We conducted similar experiments, and found that TGF-b1, b2 or b3 caused skin fibrosis after 3 consecutive days of injection; this change was transient and disappeared after 7 consecutive days of injection. In contrast, irreversible fibrosis was observed upon stimultaneous injection of TGF-b and b-FGF or TGF-b and CTGF, or TGF-b injection for the first 3 days and b-FGF or CTGF injection for the next 4 days (Shinozaki et al. Biochem Biophys Res Commun 1997;237:292 – 7; Mori et al. J Cell Physiol 1999;181:153–9). These observations suggest that TGF-b induces skin fibrosis and b-FGF or CTGF maintains it in various skin fibrotic disorders. In the 21st century, we speculate that cocktails of various growth factors may permit subtle growth regulation of skin fibroblasts; such technology would have applications in the treatment of many skin diseases. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Skin fibroblasts; Growth regulation; Growth factors
1. Introduction The abnormal growth or function of skin fibroblasts causes fibrotic disorders such as scleroderma, keloid and hypertrophic scars. We have been studying the role of growth factors in the
* Tel.: +81-76-2652343; fax: + 81-76-2344270. E-mail address: [email protected] (K. Takehara).
pathogenesis of scleroderma and our results suggest that certain growth factors and cytokines play key roles in the occurrence of scleroderma [1–10]. In other words, fibrotic disorders of various organs, including skin, can be considered to be a result of excessive healing (overhealing). As shown in Figs. 1 and 2, the early sclerotic skin of scleroderma clinically shows a shiny appearance, and histologically there is an abundant accumulation of collagen bundles. If we could clarify in detail the mechanisms of skin fibrosis, it might be
0923-1811/00/$ - see front matter © 224 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 9 2 3 - 1 8 1 1 ( 0 0 ) 0 0 1 4 4 - 4
K. Takehara / Journal of Dermatological Science 24 (2000) S70–S77
possible to modulate the growth regulation and function of skin fibroblasts in such a way as to prevent skin aging and to maintain young-appearing skin over a long period. In the 21st century, we speculate that the use of
S71
cocktails of various growth factors may enable subtle regulation of the growth of skin fibroblasts, which will be applicable to the treatment of various skin diseases, as well as to the prevention, or at least delay, of skin aging.
Fig. 1. Clinical features of typical early scleroderma. Chest skin has a shiny appearance. Fig. 2. The left panel shows histological change of early edematous scleroderma. The right panel shows histological change of advanced sclerotic stage of scleroderma.
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Table 1 Bioactive peptides and the fields in which they were discovered Peptide hormones (classical endocrinology) Cytokines Interleukins (immunology) Interferons (virology) Colony stimulating factors (hematology) Tumor necrosis factors (oncology) Growth factor (cell biology) Others Endothelin, etc.
2. Cytokines and growth factors Cytokines are a group of regulatory molecules that function as mediators of cell communication in both normal and pathologic conditions. Cytokines are low-molecular-weight (glyco)proteins that are produced by a variety of cells. Via interaction with specific cell surface receptors, they exert multiple biological functions. Mediators in the cytokine family are interleukins, hematopoietic growth factors, interferons, tumor necrosis factors, and growth factors, as shown in Table 1. These regulatory proteins appear to participate in the mediation of inflammatory as well as immune reactions, and some may influence tumor growth. Growth factors were originally regarded only as regulators of cell proliferation. The family of growth factors includes epidermal growth factor (EGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (b-FGF), transforming growth factor-b (TGF-b) and connective tissue growth factor (CTGF). In addition to controlling the growth of normal and malignant cells, as well as matrix protein formation, some growth factors are important mediators of cell surface alteration and immunoregulation. Several growth factors are essential for normal wound healing and clinical application of such growth factors for impaired wound healing has already been tried [11 – 13].
origins of these growth factors and their target cells are shown in Table 2.
3.1. PDGF In 1974, it was discovered that platelets were the main source of mitogenic activity found in whole blood serum and missing in plasma. The growth factor with this mitogenic activity was called PDGF [14]. PDGF has a molecular weight of 28 77–35 000. It is made up of two chains, which share 60% homology. The A chain has a molecular weight of 14 000 and the B chain has a molecular weight of 17 000. The B chain has a high degree of homology with v-sis oncogene. The PDGF receptor is found on cells that show a mitogenic response to PDGF. Thus, the receptor is present on fibroblasts, glial cells and vascular smooth-muscle cells. The PDGF receptor has an extracellular ligand-binding domain, and an intracellular effector domain with tyrosine kinase activity, and the peptide-receptor complex is internalized by endocytosis. The receptor is downgraded by PDGF binding. PDGF is chemotactic for fibroblasts and smooth-muscle cells, at low concentrations [15]. Recently, PDGF B chain products were approved by the FDA as a wound repair drug for Table 2 Characteristics of growth factors relevant to wound healing Factor
Producing cells
Target cells
PDGF
Platelets
Fibroblasts (mitogen, chemotaxis)
b-FGF TGF-b
Fibroblasts
3. Major growth factors in wound healing The major growth factors in wound healing are PDGF, b-FGF, TGF-b and CTGF. The cellular
Endothelial cells Macrophages Smooth muscle cells Keratinocytes Macrophages Endothelial cells Platelets Macrophages
CTGF
Endothelial cells Fibroblasts
Fibroblasts (mitogen) Endothelial (angiogenesis) Fibroblasts (collagen synthesis) Endothelial (growth inhibition, matrix synthesis) Fibroblasts (mitogen) Endothelial (angiogenesis)
K. Takehara / Journal of Dermatological Science 24 (2000) S70–S77
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Fig. 3. The effects of various growth factors in the serum-free culture system of skin fibroblasts. b-FGF had the most potent growth stimulatory effect among these growth factors.
diabetic ulcers in the United States. Clinical trials for the treatment of impaired wound healing, including diabetic ulcers, are being planned in Japan.
3.2. b-FGF b-FGF, by stimulating division and migration of fibroblasts, endothelial cells and epithelial cells, promotes formation of new capillaries and granulation tissue. In our serum-free experimental systems, b-FGF is the most potent growth stimulator among the various growth factors (Fig. 3). b-FGF has a strong homology with hst-1 oncogene. Clinical trials of b-FGF for treatment of impaired wound healing in Japan have shown that this agent is very effective for skin ulcers, and this product is expected to be approved for clinical usage by the Japanese FDA.
produced as a latent form. The detailed mechanisms of activation of TGF-b in vivo have not been clarified. In general, the biological activities of TGF-b are very strong, and therefore the molecule is probably produced as an inactive form in order to block excessive TGF-b action [18]. Serine I threonine kinase is involved in TGF-b signal transduction, which is quite different from
3.3. TGF-b TGF-b is a 25 kd homodimer. Five distinct isoforms were found, of which three are present in humans. TGF-b1, b2 and b3 exhibit similar, but not identical biological activities in vitro. TGF-b acts as a growth inhibitor for most cell types (Fig. 4) [16] and stimulates extracellular matrix production, including type I collagen, type III collagen and fibronectin, in fibroblasts [17]. This stimulatory effect on ECM production is much stronger than those of other cytokines. One of the most important characteristics of TGF-b is that it is
Fig. 4. Effects of various growth factors on endothelial cell growth. Confluent RHECs were trypsinized, and 1 ×104 cells were suspended in DMEM containing 10% FCS and plated in 24-well plates. Following cell attachment overnight, the medium was changed to DMEM containing 2.5% FCS without growth factor ( ), with 1 ng/ml TGF-b () with 0.25 ng/ml TGF-b (), with 10 ng/ml EGF ( ), with 10 ng/ml FGF (), or with both 1 ng/ml TGF-b and 10 ng/ml EGF ( × ) (day 0). Cell numbers were determined on days 3, 6 and 9. (Takehara et al. [16]).
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Fig. 5. CTGF is an autocrine growth factor and is produced by skin fibroblasts only when they are stimulated with TGF-b.
the case of other growth factors such as PDGF and EGF, which use tyrosine kinase. TGF-b has attracted much attention as a key factor in various fibrotic disorders [19], because it potently increases ECM production by many cells.
3.4. CTGF CTGF was identified originally from cultured human umbilical vein endothelial cells by Grotendorst and his group at Miami University [20]. This factor is also produced by skin fibroblasts, but only when they are stimulated with TGF-b [21]. CTGF is recognized by anti-PDGF polyclonal antibody and CTGF c-DNA was cloned using anti-PDGF antibody. This factor exhibits PDGF-like chemotactic and mitogenic activities and binds to PDGF receptors, though CTGF has little peptide sequence homology to PDQF A or B chains. Fig. 5 summarizes the relationship between TGF-b and CTGF. CTGF acts as an autocrine growth factor when skin fibroblasts are stimulated with TGF-b.
4. An animal model of skin fibrosis by exogenous injection of TGF-b and other growth factors We recently established an animal model of skin fibrosis by exogeneous injection of TGF-b and other growth factors [22,23]. In 1986, Roberts et al. reported that TGF-b injection into newborn mice caused granulation tissue
formation and skin fibrosis [24]. We initially tried to establish an animal model of skin fibrosis by TGF-b injection using a similar method to that described by Roberts et al. [24]. We injected 800 ng of TGF-b1, b2 or b3 into subcutaneous tissue of newborn mice for 7 days. The details were described elsewhere [22,23]. The results of these experiments are summarized in Table 3.
4.1. Response to application of single growth factors Three days of TGF-b injection resulted in a firm infiltrated lesion at the injection site. Histologic examination revealed granulated tissue formation (Fig. 6A), consisting of lymphocytes, histiocytes and fibroblasts. After 7 days of consecutive injections, however, this response was not detectable either macroscopically or microscopically (Fig. 6B). CTGF injection caused slight edema and some cell infiltration. Similarly, b-FGF injection caused slight edematous granulated tissue formation.
4.2. Response to simultaneous and serial applications of two growth factors Simultaneous injection of TGF-b plus CTGF or TGF-b plus b-FGF resulted in fibrotic tissue formation (Fig. 6C), consisting of fibroblast aggregation and ECM deposition, which persisted for up to 14 days, even though the injections were discontinued on day 7. To examine further the tissue response, two different growth factors were injected serially, viz. TGF-b on days 1–3 followed by CTGF on days 4–7. Serial injections of CTGF after TGF-b caused fibrotic tissue formation. This response was relatively weak but persisted for at least 14 days, even though we stopped the injection of growth factors on day 7. Injecting b-FGF after TGF-b caused increased edematous granulated tissue formation that remained for 10 days. Injection of CTGF or b-FGF before TGF-b did not cause any significant change compared with TGF-b alone.
K. Takehara / Journal of Dermatological Science 24 (2000) S70–S77
4.3. Meaning of our results The results of our study clearly demonstrate that single application of any growth factor is not sufficient to induce persistent fibrosis, despite continuous injections. Instead, interaction of plural growth factors seems to be necessary for the induction of persistent fibrosis in this animal model. Our observations on serial application of different growth factors suggest that TGF-b plays an important role in inducing granulation and fibrotic tissue formation. The other growth factors, CTGF and b-FGF, are important in maintaining fibrosis. In contrast, injections in the reverse order, viz. CTGF followed by TGF-b or b-FGF followed by TGF-b did not produce any significant response. Hence, we conclude that induction of persistent fibrosis most likely requires two factors: an induction factor such as TGF-b and a maintenance factor such as CTGF. B-FGF may act synergistically by inducing CTGF, because our results suggest that either CTGF mRNA expression or the CTGF protein itself is required to develop persistent fibrotic changes.
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We then focused on the action of CTGF as a candidate mediator to maintain skin fibrosis, because TGF-b selectively stimulates CTGF expression of fibroblasts. CTGF, which is known to increase fibroblast proliferation and ECM synthesis, has recently been proposed to be a TGF-bspecific downstream mediator. We previously reported that CTGF mRNA expression is strongly correlated with tissue fibrosis in systemic sclerosis and other skin fibrotic disorders [7,8,25]. 5. Two-step fibrosis hypothesis in systemic sclerosis Based on the results with TGF-b and CTGF described above, we hypothesized that a two-step process of fibrosis occurs in scleroderma.We think that TGF-b induces fibrosis in the early stage of systemic sclerosis and then CTGF acts to maintain tissue fibrosis. TGF-b induces CTGF mRNA, but some additional factor is required for continuous CTGF mRNA expression.
Table 3 Histological responses to single, simultaneous and serial injections of different growth factors into newborn micea Injection schedule
Histological change
Days 1–3
Days 4–7
Days 8–10
Day 4
Day 8
Day 11
Single injection TGF-b CTGF b-FGF PBS
TGF-b CTGF b-FGF PBS
− − − −
++ +− + −
− − +− −
− − − −
TGF-b+CTGF TGF-b+b-FGF
− −
+++ +++
+++* +++*
+++* +++*
CTGF b-FGF TGF-b TGF-b TGF-b TGF-b b-FGF CTGF
− − − TGF-b − TGF-b − −
++ ++ +− +− + + +− +
+++* +++* ++ ++ ++ ++ + −
++ ++ − + − + − −
Simultaneous injection TGF-b+CTGF TGF-b+b-FGF Serial injection TGF-b TGF-b CTGF CTGF b-FGF b-FGF CTGF b-FGF a
−, no change; +−, slight edema and some cell infiltration; +, edematous granulation tissue; ++, granulation tissue consisting of lymphocytes, histiocytes, and fibroblasts; +++, fibrotic tissue consisting of fibroblast aggregation and extracellular matrix deposition, and asterisks denotes marked fibrosis. See the text for the concentrations of different growth factors injected.
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Acknowledgements This article is based on a presentation given at the Shiseido Science Symposium 277, held on April 15, 2000, Tokyo. The major part of these investigations were conducted by collaborators at Tokyo University (Drs Atsuyuki Igarashi, Yoshinao Soma, Nobukazu Hayashi and Takashi Kakinuma) and at Kanazawa University (Drs Mikio Shinozaki, Toshifumi Mori, Shigeru Kawara and Shinichi Sato).
References
Fig. 6. An animal model of skin fibrosis induced by exogenous growth factor injections. (A) After 3 consecutive days of injections of TGF-b2. (B) After 7 consecutive days of injections of TGF-b2. (C) Combination of TGF-b 3 and b-FGF for 7 consecutive injections.
In the future, substances that block CTGF activities, such as anti-CTGF neutralizing antibody or CTGF antisense molecules are likely to be candidates for therapeutic use to treat systemic sclerosis. Some preliminary experiments using our animal model are in progress in our laboratory.
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