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Rheumatology Advance Access published online on November 20, 2008
Rheumatology, doi:10.1093/rheumatology/ken405
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Review

Pathogenesis and therapeutic approaches for improved topical treatment in localized scleroderma and systemic sclerosis

I. Badea1, M. Taylor1, A. Rosenberg2 and M. Foldvari3

1College of Pharmacy and Nutrition, 2Department of Pediatrics, College of Medicine, University of Saskatchewan, Saskatoon and 3School of Pharmacy, University of Waterloo, Waterloo, Canada.

Correspondence to: M. Foldvari, School of Pharmacy, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1. E-mail: foldvari{at}uwaterloo.ca


   Abstract
Top
 Abstract
Introduction
 Pathogenesis of scleroderma
 Current treatment options for...
 New directions in the...
 Conclusion
 Acknowledgements
 References
 
SSc is a chronic progressive disorder of unknown aetiology characterized by excess synthesis and deposition of collagen and other extracellular matrix components in a variety of tissues and organs. Localized scleroderma (LS) differs from SSc in that with LS only skin and occasionally subcutaneous tissues are involved. Although rarely life threatening, LS can be disfiguring and disabling and, consequently, can adversely affect quality of life. There is no known effective treatment for LS, and various options, including, as examples, corticosteroids and other immunomodulatory agents, ultraviolet radiation and vitamin D analogues, are of unproven efficacy. Clinical trials evaluating combination therapy such as corticosteroids with MTX or UVA1 exposure with psoralens have not been established as consistently effective. New immunomodulators such as tacrolimus and thalidomide are also being evaluated. A better understanding of the molecular and cellular mechanisms of LS has led to evaluation of new treatments that modulate profibrotic cytokines such as TGF-β and IL-4, regulate assembly and deposition of extracellular matrix components, and restore Th1/Th2 immune balance by administering IL-12 or IFN-{gamma}. IFN-{gamma} acts by directly inhibiting collagen synthesis and by restoring immune balance. In this review, we evaluate current and future treatment options for LS and cutaneous involvement in SSc. Recent advances in therapy focus mainly on anti-fibrotic agents. Delivery of these drugs into the skin as the target tissue might be a key factor in developing more effective and safer therapy.

KEY WORDS: Localized scleroderma, Systemic sclerosis, Cutaneous fibrosis, Autoimmunity, Skin, Collagen, Extracellular matrix, Th1/Th2 type immune response, Drug delivery


   Introduction
Top
 Abstract
 Introduction
Pathogenesis of scleroderma
 Current treatment options for...
 New directions in the...
 Conclusion
 Acknowledgements
 References
 
SSc is a chronic disorder manifested by excess synthesis and deposition of collagen (both Types I and III) in skin and connective tissue, vascular abnormalities and autoimmunity. Localized scleroderma (LS) differs from SSc in that LS does not involve the internal organs and patient prognosis is more favourable. Skin involvement in SSc presents clinical signs similar to that of LS, and the two conditions are histopathologically identical, thus treatment approaches for LS could also be applicable to sclerotic skin in SSc. Clinical signs of fibrosis are caused by sclerotic fibroblasts (myofibroblasts) in the dermis, which are capable of multiple passages, thus their accumulation might be responsible for the fibrosis characteristic of the disorder [1]. These fibroblasts produce excessive collagen even without an immune stimulus, suggesting the dysfunction of some regulatory genes associated with phenotypic selection (i.e. the subpopulation of fibroblasts producing excessive collagen is amplified at the expense of the normal fibroblasts). Although generally not considered as serious a condition as SSc, LS still lowers a patient's quality of life. Those affected often develop lesions on their trunk, arms, face or legs. The number and size of lesions varies from person to person, with some showing limited involvement and others having more extensive pathology. The skin in affected areas, which may take the form of patches or linear bands, becomes thick, hard and discoloured (Fig. 1). In some cases, disfiguring and debilitating deformities can occur on the face and limbs, and mobility, as a result of the sclerotic lesions across joints, may become restricted [2]. LS has an estimated prevalence of 50 per 100 000 before the age of 18 yrs and 220 per 100 000 by the age of 80 yrs [3].


Figure 1
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  		FIG. 1. Clinical presentation of morphoea and linear scleroderma. The lesions can appear on any area of the body. Morphoea plaques are indurated, hyper- or hypopigmented with erythematous and/or violaceous halos (A). The size of the lesions can vary considerably. (B) Representation of linear scleroderma.

 
Treatment options for both LS and SSc are currently limited, thus there is a need to develop more rationally conceived, universally applicable and effective therapies. The clinical presentation of LS has multiple components, so researchers and clinicians are evaluating pathological markers as possible treatment targets. This review provides the latest information on the pathogenesis of LS, cutaneous involvement in SSc and the most widely used and effective treatments for the condition. We focus on novel treatment approaches in the context of the recently evaluated pathways, including signal transducers and activators of transcription (Stat) and Smad signalling pathways. In addition, improvement of drug delivery systems could have a major impact on the therapeutic outcome in LS and sclerotic skin in SSc.


   Pathogenesis of scleroderma
Top
 Abstract
 Introduction
 Pathogenesis of scleroderma
Current treatment options for...
 New directions in the...
 Conclusion
 Acknowledgements
 References
 
The identification of potential drug targets and treatment methods for scleroderma need to be considered in the context of the complex aetiology and pathways involved in the disorder's progression. A schematic diagram of the possible pathways of inflammation, the cytokine cascade and the skin extracellular matrix component synthesis in the pathogenesis of LS is shown in Fig. 2 [4–6].


Figure 2
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  		FIG. 2. Pathogenesis of scleroderma. IFN-{gamma} gene therapy may have an effect on four groups of pathophysiological markers of scleroderma: (i) collagen and extracellular matrix components, (ii) cell proliferation, (iii) cell adhesion molecules and (iv) cytokines. C: complement; CTL: cytotoxic T cell; LDL: low-density lipoprotein; Tx: thrombin.

 
In the early stages of LS, a large influx of mononuclear cells infiltrate the skin and surrounding blood vessels. In early studies, perivascular infiltration of the skin was shown to occur in 84% of the affected patients (n = 43) [7]. No definitive cause for this abrupt invasion has been determined, although several studies now point to LS as having a strong autoimmune component. A recent review by Takehara and Sato [8], in which the authors gathered and synthesized all previously reported information on the subject, supports this hypothesis. It appears that in patients with LS, ANAs, cytokines and soluble cell adhesion molecules are consistently elevated. In addition, soluble CD23, CD8 and CD4, which are serological indicators of immune activation, have all been shown to be elevated at levels of 20% (10/49), 20% (10/49) and 18% (9/49), respectively [9, 10]. Almost all infiltrating cells in sclerotic skin biopsies have also been shown to be activated T-lymphocytes, another indication that autoimmunity is pathogenically important in LS [1]. Other possible causes that have been suggested include genetic factors and several environmental agents [11, 12].

After mononuclear cell infiltration, functional and structural changes occur to the microvascular system. Small vessels underlying the epidermis are severely affected. Not only is blood flow to these vessels and capillaries reduced, but intense endothelial damage as a result of the invading cells also appears to occur [13]. A review by Cutolo and colleagues [14] summarizes all studies conducted on the use of nail-fold videocapillaroscopic patterns to visualize the damage that occurs in the microvasculature of the finger tips in patients with SSc. Periungual capillary changes observed in patients include haemorrhages and enlarged capillaries in the early stages of the disorder and loss of capillaries, ramified capillaries, vascular disorganization and the development of avascular areas in the later stages [15].

The inflammatory phase in the vascular endothelium is paralleled by an up-regulation of several adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in response to various cytokines and cellular mediators such as IFN-{gamma}, IL-1 and TNFs [16]. These adhesion molecules are important in the recruitment of monocytes to the area of inflammation, as they facilitate the processes of rolling, adhesion and transmigration. E-selectin, one of these molecules that mediates the monocyte's initial ‘rolling’ contact to the walls of the vessels, has consistently shown to be increased in the sera of patients with LS (20%, 12/59) [17]. Also, the intensity of mononuclear cell infiltrates and the number of sclerotic lesions are significantly greater in patients with elevated levels of soluble E-selectin than in healthy controls [17]. Both ICAM-1 and VCAM-1 are involved in the adherence of monocytes to the endothelium. ICAM-1 is located on the cell surfaces of both endothelial cells and fibroblasts and is elevated by 25% (12/48) in patients with LS. VCAM-1 is located only on endothelial cells and has also been shown to be elevated by 19% (11/59) in patients with LS [17, 18]. E-selectin, ICAM-1 and VCAM-1 are all up-regulated by IL-1, -4 and TNFs.

One of the most complex and least understood pathological processes involved with LS is the release of a large number of cytokines by the lymphocytes before and after cellular activation and invasion. The cytokines determine the path that an immune response will take and ultimately control the outcome. IL-4, -6 and -8 have been shown to be increased in affected sclerotic skin samples [19]. Specifically, IL-4 and -6 were increased by 17 and 47%, respectively, in the serum of patients with LS, while the levels of these cytokines [20] were not elevated in healthy controls. What makes these two cytokines important is that they both increase collagen synthesis. IL-4 also increases fibroblast proliferation [21].

Hardening of the skin, from excessive proliferation and deposition of collagen and other extracellular matrix components, is the last and most pronounced phase of the condition. This stage in the progression of LS causes the most deleterious effects and therefore could be a main target of future treatments. In-vitro experiments have shown that IL-4 regulates the levels of TGF-β and that the down-regulation of TGF-β prevents cutaneous fibrosis in the tight-skin (Tsk) mouse, an animal model for LS. Even more exciting is the discovery that disruption of one or both of the IL-4 alleles rescues mice homozygous for the Tsk mutation from certain death. Normally, these homozygous mouse embryos die within 7–8 days of conception. However, disruption of one allele caused 27% survival, while disruption of both alleles caused 47% survival [22].

In-vivo tissue fibrosis is caused by excessive TGF-β and IL-4 activity. TGF-β selectively induces connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF) and metalloproteinase-3, all of which increase mitogenic activity in fibroblasts [23]. TGF-β also stimulates the synthesis of several extracellular matrix proteins, such as collagens, fibronectin, tenascin, tissue inhibitor of metalloproteinase-1 and plasminogen inhibitor-1, and induces TGF-β secretion in fibroblasts (autoinduction). The fibrosis is induced through the Smad pathway (Fig. 3); the intracellular Smads (Smads 2 and 3) are activated by the binding of TGF-β to its serine–threonine kinase receptor, ALK-5, on the surface of the fibroblast [24]. The Smads then translocate into the nucleus, where they activate or repress gene transcription (e.g. activation of procollagen type I {alpha} gene, COL1A2) via the p300/CBP cofactor [25]. The profibrotic activity of IL-4 results in increased production of extracellular matrix via the Stat6 protein. This protein binds a Stat-responsive element facilitated by the p300/CBP cofactor (Fig. 3). Stat6 might also activate transcription of the TGFB gene [26]. Both IL-4 and -6 increase the production of collagen [21]. Additionally, TGF-β not only increases collagen synthesis and fibroblast proliferation in vivo and in vitro, but it also reduces collagenase synthesis [27]. The overall effect of the increases in the mentioned cytokines and growth factors (IL-4, TGF-β, TNF-{alpha}) results in the impairment of IFN-{gamma}, a cytokine that is secreted by activated T cells and has been clearly shown to be an inhibitor of procollagen synthesis in fibroblasts [28]. IFN-{gamma} directly suppresses collagen synthesis in two ways (Fig. 3): through Janus kinase (Jak)/Stat1 signalling and the Y-box binding protein (YB-1) pathway. IFN-{gamma} blocks COL1A2 promoter activity by Jak/Stat1{alpha} activation. The activated Stat1{alpha} competes with the TGF-β-activated Smad3 [29] for the limited amount of the cellular cofactor p300/CBP, blocking the transcription of COL1A2 [30]. The second pathway involves the activation of YB-1, which directly interacts with the promoter region of COL1A2, blocking its transcription. Additionally, YB-1 binds mRNA in the cytoplasm, which could constitute yet another way to block collagen synthesis at the translational level [31]. Moreover, IFN-{gamma} directly stimulates prostaglandin production (another fibroblast growth inhibitor) in monocytes. Monocytes play an important role in fibrosis; on their surface they express antigens characteristic of activated cells and they produce reduced amounts of IL-1, as demonstrated by monocytes removed from patients suffering from SSc [6]. Depending on which cytokines are predominant, up-regulation or suppression of tissue fibrosis is controlled by the immune system; presence of IL-4 and -10 inhibit secretion of IFN-{gamma} by Th1 cells, thus fibrosis is favoured. Conversely, if IFN-{gamma} is present, Th2 cells are inhibited, and production of IL-4 and -10 is blocked [4]. Recent evaluation of the cytokine balance in scleroderma patients revealed the presence of IL-4, -10 and TGF-β (induced by IL-4), which are characteristic of Th2 immune responses, agents that induce tissue fibrosis. Altered B-cell activity might also contribute to the response shift, by activation of Th2 lymphocytes [32]. IFN-{gamma} interferes with fibroblast proliferation via the Stat1 pathway, by inhibiting epidermal growth factor- and PGDF-induced cell growth [33]. Since the clinical presentation of the disease has multiple components, research groups are focusing on the evaluation of the pathological markers as possible treatment targets.


Figure 3
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  		FIG. 3. Schematic representation of the role of IFN-{gamma}, IL-4 and TGF-β in the regulation of collagen synthesis. IFN-{gamma} suppresses gene transcription directly via the YB-1 pathway and indirectly by inhibition of the p300/CBF protein. IL-4 and TGF-β both stimulate COL1A2 transcription via STAT6 and Smad3 pathways, respectively. Additionally, TGF-β autoinduces additional TGF-β production by fibroblasts through stimulation of the TGFB gene. ALK: activin-like kinase receptor; COL1A2: collagen, type I, {alpha}2 gene; IFNr: IFN receptor; ILr: IL receptor; Jak: Janus kinase; p300/CBP: E1A binding protein p300 and CREB binding protein; Smad: mothers against decapentaplegic homologue; Stat: signal transducers and activator of transcription; TGFB: TGF-β gene; Th1: type 1 helper T cell; YB: Y box binding protein.

 

   Current treatment options for LS and cutaneous involvement in SSc
Top
 Abstract
 Introduction
 Pathogenesis of scleroderma
 Current treatment options for...
New directions in the...
 Conclusion
 Acknowledgements
 References
 
Currently, there are no treatments for LS of proven benefit. Evaluating the efficacy of pharmacotherapeutic agents in LS is challenging because of the rarity of the condition, the heterogeneity of disease severity within study populations, the difficulty in establishing controls, and because LS in most patients eventually remits spontaneously making it difficult to discern the role of treatment. Table 1 summarizes recently completed clinical studies on treatment of LS or cutaneous involvement of SSc. Table 2 summarizes ongoing clinical studies on treatment of LS or cutaneous involvement of SSc.


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  		TABLE 1. Latest studies on widely used treatment options for and recently completed clinical trials on patients with LS and cutaneous involvement of SSc

 

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  		TABLE 2. Latest studies on ongoing, recently completed and terminated clinical trials on patients with LS and cutaneous involvement of SSc

 
Certain treatment strategies focus on controlling different pathological phases of LS. Corticosteroids, UVA1, vitamin D analogues and MTX have all been used to treat the inflammatory, immune or fibrotic processes associated with the disease. Recently, there has been a trend to combine treatments to improve outcome. A combination of MTX and corticosteroids has shown encouraging results. In a study of paediatric patients with LS, 9/10 improved after receiving MTX combined with pulse intravenous methylprednisolone [34]. Similar results were seen in a study of 15 patients with severe LS [35].

Targeting inflammation and an unbalanced immune system with drugs may have some benefit, especially in severe forms of LS. Yet, the most effective treatments for all other forms of the disease tend to focus on the cutaneous fibrosis typifying LS. Experimental treatments include the use of vitamin D analogues and drugs such as halofuginone. Vitamin D derivatives such as calcitriol and calcipotriol have been used with limited success [41, 45, 46]. Their anti-proliferative effect on fibroblasts helps impede progression of LS. Similarly, halofuginone has shown promise as an inhibitor of fibrosis. It has been shown to decrease collagen type I synthesis in Tsk mice and reduce skin scores in pilot studies on patients with SSc [47].

UVA therapy is associated with improvement in most patients and its relative safety makes this therapy an attractive treatment option. The treatment consists of several rounds of UVA radiation. The pre-application of topical psoralens followed by UVA exposure (PUVA) is a variant of the UV protocol thought to enhance the effectiveness of phototherapy [48]. Although the precise underlying mechanisms of action of UVA are unclear, it appears that UVA therapy decreases COL I, COL III and TGBF gene expression and increases IFNG gene expression [49]. This would suggest a direct effect on the excessive overproduction and deposition of connective tissue.

It also appears that the level of dosing of UVA therapy does not change the outcome. Low-, medium- and high-dose UVA therapies are all effective. In a low-dose UVA study, 20 patients with LS received 20 J/cm2 broadband UVA for 12 weeks for 30 sessions. Clinical improvements were noted, including skin softening and reduction in mean collagen content [36]. In another study with medium-dose UVA therapy (30 J/cm2), patients were treated three times a week for 10 weeks. All patients showed improvement in skin score and cutometer [37]. In a high-dose study, 8/8 patients improved after 20 sessions of high-dose UVA treatment (48 J/cm2) over a period of 5 weeks [38]. A recent comprehensive three-armed study used low-dose UVA1 (20 J/cm2 each session; 27 patients completed the study), medium-dose UVA1 (50 J/cm2 each session; 17 patients competed the study) and narrow-band UVB (maximum dose of 1.3 J/cm2 for type II skin and 1.5 J/cm2 for type III skin; 18 patients completed the study) five times per week for 8 weeks. Clinical evaluation of skin thickness showed significant improvement in all treatment groups compared with the baseline, with significantly better outcome for the medium-dose UVA1 therapy compared with the narrow-band UVB therapy. A comparison of biopsy specimens from 36 patients before and after treatment indicated a decrease of the histological score in all treatment groups. However, statistical significance was demonstrated only in the narrow-band UVB-treated group [39].


   New directions in the treatment of scleroderma
Top
 Abstract
 Introduction
 Pathogenesis of scleroderma
 Current treatment options for...
 New directions in the...
Conclusion
 Acknowledgements
 References
 
Novel approaches for decreasing skin fibrosis include modulation of cytokine expression in the sclerotic tissue. TGF-β, IFN-{gamma}, IL-4, endothelin and CTGF are targets that have been studied [42].

Recombinant human relaxin was used in a multicentre, parallel-group, randomized, double-blind, placebo-controlled trial [42]. This protein is secreted by the corpus luteum and placenta during pregnancy and plays a role in the growth of the uterus. Additionally, it has an anti-fibrotic effect. Patients with dcSSc receiving 25 µg relaxin per day for 24 weeks in continuous subcutaneous injection showed significant improvement in skin score by week 4, and global assessment indicated amelioration of the condition. Interestingly, a 4-fold higher dose did not produce significantly different results from the placebo treatment. The most common side-effect was menorrhagia in 35% of patients treated with low-dose relaxin, compared with 11% in the placebo-treated group.

Systemic administration of the anti-TGF-β antibody, CAT-192, did not show evidence of efficacy in a study on the treatment of early-stage dcSSc [44]. Of the 45 patients enrolled, 11 patients received placebo treatment and, within the treatment groups, 11 patients received 0.5 mg/kg, 11 patients received 5 mg/kg and 10 patients received the 10 mg/kg dose of CAT-192. Morbidity and mortality were both higher in the treatment groups, with four deaths in total and a higher proportion of adverse effects, including gastrointestinal, cardiac and pulmonary disorders and haemorrhage. Although there was a significant correlation between the clinical outcome (skin score) and the duration of disease at baseline, there was no improvement in any of the treatment groups compared with the placebo-treated subjects.

Thalidomide appears to induce immune stimulation in SSc; studies of thalidomide in LS patients are pending [50]. Thalidomide increases IL-12 and TFN-{alpha} levels in LS patients and causes a Th1-type immune response (IL-2, -3, GM-CSF and IFN-{gamma} production) in peripheral blood mononuclear cells isolated from SSc patients. Tacrolimus ointment was applied to seven patients with LS, inducing improvement in sclerotic and inflammatory lesions in all patients [40]. Although it may be a promising new therapy, tacrolimus may be a potential carcinogen thus obviating its routine use [51, 52]. Topical imiquimod, an IFN-{gamma} inducer, has been reported to induce regression of morphoea plaques [53]. Imiquimod was applied topically in a 5% cream (AldaraTM) to 12 LS patients. All patients showed reduction of induration of the skin after 6 months and improvement of dyspigmentation and inflammation after 3 months [54]. Controlled clinical trials of imiquimod are in progress. Long-term effects that would indicate a sustained response after discontinuation of the drug have not been reported.

Animal studies in Tsk mice, the genetic model for scleroderma, indicate that administration of IL-12-coding plasmid significantly reduces skin thickness and production of IL-4, and increases IFN-{gamma} levels at a dosing regimen of 100 µg plasmid injected once every 3 weeks for 21 weeks [55]. The same effect can be achieved by blocking endogenous TGF-β signalling with anti-TGF-β antibodies or TGF-β1 anti-sense oligonucleotide [56]. Both treatments involve invasive administration. To specifically treat LS, topical delivery systems should be developed to increase local concentration of the anti-fibrotic agent while minimizing systemic exposure, hence reducing undesired side-effects. Intravenous human immunoglobulin G (IgG) administered to Tsk mice reduced excessive collagen production in the skin by inhibiting IL-4 and TGF-β secretion by splenocytes [57]. IgG modulates MMPs and chemokine expression, also contributing to reduced fibrosis. Human trials using IgG have not been conducted. Systemic administration of plasmid coding for hepatocyte growth factor (HGF) prevented excessive fibrosis in mice injected with bleomycin and improved the clinical signs of bleomycin-induced skin sclerosis [58]. The mechanism by which transgene HGF prevented fibrosis was reduction of TGF-β mRNA levels, thus protein expression. As in the case of IgG treatment, HGF has not been evaluated in clinical trials. In both cases, systemic administration will affect TGF-β expression not only in the sclerotic tissue, but also in blood vessels. Although systemic administration of these anti-fibrotic drugs reduces excessive collagen synthesis in connective tissue, effects on wound healing and angiogenesis could conceivably adversely affect these therapies.

Imatinib mesylate is a tyrosine kinase inhibitor that interferes with TGF-β signalling via c-Abl, blocking extracellular matrix synthesis both in vitro and in vivo. Fibrosis was significantly suppressed in bleomycin-induced skin sclerosis in experimental animals. At the same time, no toxic effects were observed, indicating good tolerability of the drug [59].

Halofuginone, another potential treatment for LS, acts by blocking the activation of Smad3 protein, which interferes with the synthesis of TGF-β [26]. By blocking TGF-β, collagen synthesis by fibroblasts could be significantly reduced. Phase I clinical studies of topical 0.1% halofuginone ointment indicated that the drug was well tolerated and safe [47]. In Phase II trials, patients suffering from chronic graft-vs-host (GVHD) disease were topically treated with 0.03% halofuginone ointment. GVHD is a condition characterized by excessive collagen synthesis similar to LS. A 6-month treatment regimen resulted in significant reduction of collagen {alpha}1(I) in affected skin [60].

In-vitro evaluation of simvastatin, another potential anti-fibrotic agent, indicated significant down-regulation of collagen type I gene expression in fibroblasts isolated from patients with diffuse SSc [61]. Although simvastatin concentrations in vitro (1.25–10 µM) were 50- to 100-fold higher compared with the optimal plasma levels recommended for cholesterol lowering, the approved therapeutic indication for the drug, an appropriate dosage regimen with increased doses and tissue-targeting (e.g. topical delivery) could lead to effective treatment.

Another form of treatment has been administration of the cytokine IFN-{gamma} to patients with LS and SSc. IFN-{gamma} has potential as an effective treatment because it is a potent inhibitor of procollagen synthesis in fibroblasts [28]. There is also speculation, based on the pathogenesis model (Fig. 2) and gene transcription model (Fig. 3), that administering IFN-{gamma} could have an indirect inhibitory effect on TGF-β and an immunomodulatory effect on T cells, to switch the Th1/Th2 balance towards Th1. However, IFN-{gamma} as a therapeutic option for scleroderma has not been shown to have significant therapeutic efficacy.

IFN-{gamma} decreases collagen production in vitro in fibroblasts from hypertrophic scars and improves the clinical signs of overproliferative skin conditions such as scleroderma, morphoea and keloids in vivo [62, 63]. The mechanism by which reduction of collagen synthesis occurs is complex, involving direct and indirect pathways (Fig. 3) [Badea et al., submitted]. In vivo studies on experimentally induced scleroderma in mice demonstrate that after injecting the sclerotic agent (bleomycin), subcutaneous administration of 5 x 105 U/ml IFN-{gamma} for 3 weeks reduces the hydroxyproline content of the skin significantly by up to 72% [58]. Delivery of IFN-{gamma} to the skin could provide the required amount of drug at the target site without systemic exposure. After injection of gene expression vectors of eight human cytokines (IL-4, -6, -10, TGF-β1, TNF-{alpha}, MACAF, GM-CSF and IFN-{gamma}) into rat skin, transgenic cytokine expression in keratinocytes and serum were assessed. All cytokines were expressed in the keratinocytes (20–200 pg/µg protein). IL-4, -6 and -10 and TGF-β, but not the other cytokines, were detected in the sera of the animals [64]. These findings demonstrate that keratinocytes in the epidermis can be used to express genes introduced via plasmid vectors.

Clinical studies using IFN-{gamma} have indicated mixed results and side-effects were common, mainly flu-like symptoms [65]. In the most comprehensive controlled trial of IFN-{gamma}, no significant difference was seen in the size of fibrosis of the sclerotic lesions between patients with LS treated with subcutaneous IFN-{gamma} compared with untreated controls [43]. In another study, on 18 patients with rapidly evolving SSc, three patients developed renal crisis after undergoing intramuscular treatment with IFN-{gamma} [66, 67]. The poor outcome might be attributed to the low levels of IFN-{gamma} in the tissues affected by sclerosis after intramuscular or subcutaneous administration because of IFN-{gamma}'s short half-life of only 1–3 min (at low doses) to 30 min (at high doses) [68, 69], and the presence of side-effects might be associated with systemic administration of the protein-based treatment. Injection of 100 µg IFN-{gamma} subcutaneously in the periphery of localized lesions did not produce the desired effect of reducing the size of fibrosis in the existing lesions, but it was able to prevent the appearance of new lesions. The reason for this observation of preventing new lesions without degrading existing ones might be that IFN-{gamma} inhibits the synthesis of collagen, but has no effect on collagen-degrading enzymes [43]. The invasive administration of the therapeutic protein and the resultant tissue damage might also have triggered an unwanted cytokine cascade. Despite poor efficacy and complications, it is possible that in situ synthesis of IFN-{gamma} in sclerotic lesions would be an advantageous therapeutic approach.

Varga [70] has reviewed the therapeutic applications of IFN-{gamma}. Although IFN-{gamma} has been approved only to reduce severe infections in chronic granulomatous disease and to delay the progression of osteopetrosis, it was used in clinical trials for infectious diseases (genital warts, AIDS, Mycobacterium avium infection) and cancer (disseminated tumours, melanoma). These applications derive from the various biological functions of IFN-{gamma}, which include up-regulation of more than 20 genes, such as cell surface antigens, enzymes, transcription factors and cytokines. When using IFN-{gamma} as a therapeutic agent, some, but not all, of these activities are targeted. For treatment of scleroderma, its effect on collagen synthesis is exploited. Unfortunately, when the cytokine is directly injected into the tissue, in addition to its undeniable beneficial effect on fibrosis, it activates TNF-{alpha} and up-regulates the expression of ICAM-1 and VCAM-1. This leads to inflammation and adhesion of leucocytes to the blood vessels, causing flu-like symptoms and severe adverse effects, such as digital infarction and renal failure in SSc patients [70]. However, generating IFN-{gamma} in situ using gene delivery vectors could greatly increase the half-life of this protein. In a study by Jaffe and colleagues [71], adenoviral vector-derived murine IFN-{gamma} was measured at high levels in vitro for 7 days and caused significant and selective inhibition of collagen gene expression and collagen synthesis in fibroblasts. In light of these findings, cutaneous application of a DNA-based IFN-{gamma} preparation might be advantageous for several reasons: (i) it is non-invasive; (ii) therapeutic levels of the protein; generated in situ, could be maintained at the site of action for a longer time; and (iii) gene expression could be limited to the skin, reducing adverse effects from systemic exposure.

Based on the latest research, the novel treatment options for LS are directed towards inhibition of fibrosis through modulation of profibrotic cytokines. This can be accomplished by directly blocking the effect of TGF-β and IL-4 using antibodies, or by inhibiting their synthesis with chemical agents or anti-sense oligonucleotides. The disadvantage of these approaches would be that they do not address the underlying mechanism of the disease, and once treatment is stopped development of excessive fibrosis could resume, as indicated in cGVHD patients treated with halofuginone, where 3 months after termination of treatment, collagen levels returned to the pre-treatment level [60]. Moreover, effective delivery systems have to be developed for these agents to increase their local effect and reduce adverse reactions from systemic exposure. Another treatment strategy is restoration of Th1/Th2 immune balance. Administration of IL-12 or IFN-{gamma} can block Th2 immune responses and inhibit production of the fibrotic cytokines. Further, IFN-{gamma} directly blocks collagen synthesis at the transcriptional level. Additionally, IFN-{gamma} could play a role in the expansion of the population of healthy fibroblasts at the expense of the overproductive myofibroblasts in the dermis. The advantage of specific immunomodulation would be that a restored immune system could control extracellular matrix production, and thus the effect of the treatment could be long-lasting and terminated at that point.

Our group demonstrated in a preliminary study [72] that high levels of protein expression (480 pg/cm2 skin) could be achieved in the skin of normal CD1 mice after non-invasive application of plasmid DNA coding for IFN-{gamma}, using a gemini cationic lipid-based delivery system. After preliminary studies in CD1 mice, where background levels of IFN-{gamma} can contribute to an overestimation of the therapeutic IFN-{gamma} gene, we evaluated gene delivery in an IFN-{gamma}-deficient mouse model. Compared with normal CD1 mice, where IFN-{gamma} secretion can be easily triggered by physical injury or chemical irritation, the IFN-{gamma} levels in the IFN-{gamma}-deficient strain are minimal. Topical treatment with gemini cationic surfactant-based nanoparticles in the IFN-{gamma}-deficient mice resulted in significantly higher levels of IFN-{gamma} expression in the skin (278 pg/cm2) compared with the naked DNA-treated or untreated animals (IFN-{gamma} levels of 157 and 30 pg/cm2, respectively) [73]. Similarly, high levels of IFN-{gamma} were achieved in the mouse model of scleroderma (Tsk1/+) with gemini nanoparticles. Both non-invasive topical treatment and intradermal injection increased IFN-{gamma} expression in the skin of Tsk1/+ mice (345 and 1069 pg/cm2 skin, respectively) after a 20-day regimen. Moreover, a 70–72% reduction of procollagen type I {alpha}1 mRNA levels for the topical and injected treatments was observed, along with histological evidence of reduced skin thickness compared with that of the untreated animals [63].


   Conclusion
Top
 Abstract
 Introduction
 Pathogenesis of scleroderma
 Current treatment options for...
 New directions in the...
 Conclusion
Acknowledgements
 References
 
The causes of LS, like SSc, are unknown, although environmental stimuli and/or genetic factors might contribute to triggering the abnormally exuberant production of collagen. Clinical research for better treatment is hindered by the condition's wide variability in severity among patients, to recruit large numbers of patients with similar symptoms and stages of the disease. Moreover, the course of the disease varies from rapidly progressive to prompt spontaneous remission. Current treatment options, none of which is of proven efficacy, tend to target inflammation, immune imbalance and fibrosis. The rationale for the use of corticosteroids is based on anti-inflammatory effects, while modulation of the immune system is achieved by treatment with immunoglobulins, MTX, tacrolimus, imiquimod and thalidomide. Anti-fibrotic agents, such as UVB radiation, vitamin D derivatives, relaxin, halofuginone, IFN-{gamma} and anti-TGF-β are also used to treat scleroderma.

Novel approaches for treating LS include combination therapies: anti-inflammatory drugs and immunomodulators or combinations of anti-fibrotic agents. A better understanding of the molecular mechanism of excessive fibrosis has led to novel approaches using agents that can act at multiple levels. IFN-{gamma} blocks excessive collagen synthesis by direct and indirect pathways. In addition, it can restore the immune balance by stimulation of the Th1 type response. Moreover, its effect on fibroblast proliferation can expand a healthy fibroblast population at the expense of the myofibroblasts responsible for the excessive collagen and extracellular matrix synthesis. By creating an efficient protein delivery system or gene therapy vector and by delivering the active ingredient to the target tissue without systemic exposure, we believe that topical IFN-{gamma} could become a suitable dermal treatment for LS and SSc.

Formula


   Acknowledgements
Top
 Abstract
 Introduction
 Pathogenesis of scleroderma
 Current treatment options for...
 New directions in the...
 Conclusion
 Acknowledgements
References
 
The authors thank Joe Petrik for preparing the illustrations and for editorial assistance.

Funding: This review article was supported by a grant from the Canadian Institutes of Health Research.

Disclosure statement: The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Pathogenesis of scleroderma
 Current treatment options for...
 New directions in the...
 Conclusion
 Acknowledgements
 References
 

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Submitted 17 March 2008; revised version accepted 18 September 2008.
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