Cyclosporine A: Novel concepts in its role in drug-induced gingival overgrowth.

Cyclosporine is a selective immunosuppressant that has a variety of applications in medical practice. Like phenytoin and the calcium channel blockers, the drug is associated with gingival overgrowth. This review considers the pharmacokinetics, pharmacodynamics, and unwanted effects of cyclosporine, in particular the action of the drug on the gingival tissues. In addition, elucidates the current concepts in mechanisms of cyclosporine-induced gingival overgrowth. Clinical and cell culture studies suggest that the mechanism of gingival overgrowth is a result of the interaction between the drug and its metabolites with susceptible gingival fibroblasts. Plaque-induced gingival inflammation appears to enhance this interaction. However, understanding of the pathogenesis of gingival overgrowth is incomplete at best. Hence, it would be pertinent to identify and explore possible risk factors relating to both prevalence and severity of drug-induced gingival overgrowth. Newer molecular approaches are needed to clearly establish the pathogenesis of gingival overgrowth and to provide novel information for the design of future preventive and therapeutic modalities.

INTRODUCTION

The story of cyclosporine A (CsA) began in soil samples from Wisconsin (US) which was propagated and studied in the laboratories of the Sandoz Company in Switzerland. It yielded the fungus Tolypocladium inflatum gams which was used to synthesize cyclosporine.[1] The fungus is still used today for the production of CsA on a commercial scale via fermentation with remarkable immunosuppressive effect and extremely low toxicity. CsA a hydrophobic fungal metabolite is a potent immunosuppressant used as the first choice for counteracting the rejection phenomena in organ transplantation patients and for the treatment of various autoimmune diseases.[23]

The use of CsA, however, is associated with various side effects including neurotoxicity,[4] hepatotoxicity,[5] nephrotoxicity,[6] hypertension,[7] and gingival hyperplasia.[8] CsA-induced gingival hyperplasia was first reported by Seymour et al. in 1983 occurring in 25-81% of patients taking CsA.[9] A wide range of prevalence rate has been reported estimated between 8% and 70% for CsA.[10] The lesion usually develops in the first 6 months of CsA administration and initially appears as a papillary enlargement and is mostly restricted to the keratinized gingiva, but it may grow in size with time and cover the crowns of teeth causing difficulties in mastication, speech, and profound esthetic and psychological problems.[11] Despite the intensive studies investigating the cellular and molecular basis of the development of CsA-induced gingival hyperplasia, the exact mechanism underlying this condition is still unclear.[12] Various risk factors have been described including age, sex, genetic predisposition, duration of therapy, concomitant medication such as calcium channel blockers and gingival inflammation.[13] A recent study suggested that the imbalance between cell proliferation and apoptosis may contribute to the pathogenesis of the hypercellularity observed in CsA-induced gingival hyperplasia.[14]

The effect of CsA on proliferation of human gingival fibroblast (HGF) is still controversial ranging from absent to an increase or a decrease.[15] However, these discrepancies in studies have been attributed to heterogeneity of the HGF strain used,[16] or to differences in the experimental conditions applied.[17] Moreover, recent studies suggested that the accumulation of gingival fibroblasts observed in drug-associated gingival hyperplasia resulted from the inhibition of apoptosis.[1418] Furthermore, recent attention has been drawn to the gingival keratinocytes as a potential part of the pathogenesis of CsA-associated gingival hyperplasia. The effect of CsA on gingival keratinocytes is still inconclusive ranging from being pro-proliferative, antiproliferative,[19] pro-apoptotic, antiapoptotic,[20] or no effect. However, all of these in vitro studies were conducted in monolayer cultures that lack the multilayer structure of the in vivo epithelium. It has been shown that the pathogenesis of CsA-induced gingival hyperplasia is associated with up-regulated level of salivary contents of proinflammatory cytokines including interleukin (IL) 1α, IL-8, and IL-6 compared to healthy controls.[21] However, there have been no studies on release of these inflammatory cytokines in gingival keratinocytes following treatment with CsA. The clinical observations of the pathogenesis of CsA-induced gingival hyperplasia indicate a potential role for periodontal bacteria in the pathogenesis. However, most of these studies have been undertaken in vivo, and the findings are still inconclusive. It is not clear whether accumulation of dental plaque is a consequence of gingival changes caused by the drug itself, or it is an essential factor for initiating the pathogenesis.[8] This difficulty in determining the exact role of gingival inflammation might be related to the complexity of the in vivo situation.

Seymour and Smith reported that maintaining adequate oral hygiene in humans markedly reduced the severity of CsA-induced gingival hyperplasia, but it did not prevent the development of the condition.[22] Recently, there has been evidence that CsA treatment can modulate the local expression of angiotensin II (Ang II) and up-regulation of angiotensin type 1 (AT1) receptors in gingival fibroblasts and contribute to the role of renin angiotensin axis being stimulated by CsA contributing to the pathogenesis of drug-induced gingival overgrowth.[23] However, understanding of the pathogenesis of gingival overgrowth is incomplete at best. Hence, it would be pertinent to identify and explore various possible pathogenesis of drug-induced gingival overgrowth for devising prevention and treatment strategies in susceptible patients.

The present review analyzes the current concepts in pathogenesis of CsA-induced gingival overgrowth.

CYCLOSPORINE A PHARMACOLOGY

Cyclosporine A [Figure 1] is a neutral, hydrophobic cyclic peptide composed of 11 amino acids (mol. wt. 1202.6), all having the S-configuration of the L-amino acids, with the sole exception of the D-alanine in position 8, which has the R-configuration.[1] CsA taken orally in a vehicle such as olive oil enters the circulation where it is distributed and bound to blood components.[3] CsA rapidly equilibrates between plasma and red cells, with the establishment of a steady state within 20 min.[3] Erythrocytes account for 50% of binding and lymphocytes 4-9%. Of the total amount of CsA in blood, about 5% is free drug, and 25% is bound to blood lipoproteins.

Figure 1

Depicting molecular structure of cyclosporine A.

ROLE OF CYCLOSPORINE IN INHIBITION OF GRAFT REJECTION

Cyclosporine is a selective immunosuppressant with a weak antimicrobial activity[24] and can be administered orally, intramuscularly or intravenously.[25] The main use of cyclosporine is to prevent graft rejection in organ transplantation. Several studies have shown that cyclosporine selectively acts on the T-lymphocyte response, with little or no action on B lymphocytes.[26] The pharmacodynamics of cyclosporine mainly involves the T-cell response. Hence, it is essential to know the role of T-lymphocytes in graft rejection.[9] The mechanisms by which the T-lymphocytes reject the graft are shown in Figure 2.

Figure 2

Role of cyclosporine in inhibition of graft rejection: Mechanisms by which the T-lymphocytes reject the graft.

Cyclosporine inhibits many of the stages shown in Figure 2, acting at both the cellular and molecular level.[27] At low concentrations (10-20 ng/ml), it inhibits IL-2 synthesis, thereby limiting clonal amplification of cytotoxic T-lymphocytes. At a higher concentration (100 ng/ml), cyclosporine inhibits the ability of cytotoxic T-lymphocytes to respond to IL-2. The mechanism of this inhibition is uncertain but may be due to the drug blocking the induction of IL-2 receptors on these cells. By contrast, cyclosporine has a sparing effect on suppressor T-lymphocytes[25] to be resistant to cyclosporine, whereas cytotoxic T-lymphocytes and T-helper cells are sensitive to the drug.[25] Within the T-lymphocyte, cyclosporine binds to several proteins, in particular calmodulin and cyclophilin. It has been postulated that the resistance or sensitivity of T-lymphocytes to cyclosporine may be related to the proportionate intracellular concentrations of calmodulin (involved in activation of T-lymphocytes) and cyclophilin.[28] An increase in cyclophilin will increase cyclosporine binding, and thus prevent the drug from interacting with calmodulin. Conversely, low levels of cyclophilin will allow cyclosporine to bind to calmodulin and inhibit its formation and subsequent T-lymphocyte activation.

CYCLOSPORINE AND GINGIVAL OVERGROWTH

The precise mechanism of cyclosporine-induced gingival overgrowth is uncertain. Various investigations for pathogenesis of gingival overgrowth support the hypothesis that it is multifactorial.[29] A possible model is shown in Figure 3.[3031] Cell culture studies have shown that both cyclosporine and cyclosporine metabolites have direct effects on gingival fibroblast proliferation, protein synthesis and collagen production.[9] Furthermore, there exists a genetically determined subpopulation of cyclosporine sensitive gingival fibroblasts. Indeed, the terms “responders” and “nonresponders” have appeared in the literature to identify those who show or do not show drug-induced gingival changes.[32] Both findings are important in the pathogenesis of cyclosporine-induced gingival overgrowth.

Figure 3

Pathogenesis of cyclosporine A-induced gingival overgrowth: Model depicting the various mechanisms by which cyclosporine A causes gingival enlargement.

The results from clinical studies suggest that the incidence and severity of gingival overgrowth in cyclosporine-treated patients are dependent upon the interaction of several factors. These include plaque control, the level of gingival inflammation, extent of periodontal destruction, the dosage and duration of cyclosporine therapy, plasma and tissue concentrations of the drug and metabolites, age of the patient, and perhaps the underlying medical condition.[9]

Cyclosporine-induced gingival overgrowth commences as a papillary enlargement which is more pronounced on the labial aspects of the gingiva than the palatal or lingual surfaces.[28] The papillary enlargement increases and adjacent papillae appear to coalesce. This gives the gingival tissues a lobulated appearance [Figure 4].[13] Cyclosporine-induced gingival overgrowth has not been reported in edentulous subjects.[29] The hyperplastic gingival tissues often show marked inflammatory changes, bleed readily on probing and are generally more hyperemic than the gingival tissue from phenytoin-induced gingival overgrowth.[9]

Figure 4

Clinical presentation of cyclosporine A-induced gingival overgrowth.

PATHOGENSIS OF CYCLOSPORINE A-INDUCED GINGIVAL OVERGROWTH

Some authors suggest that there is a significant relationship between amount of time without plaque control and development of gingival overgrowth,[8] whereas others believe there is no significant relation between cyclosporine-induced gingival overgrowth and plaque scores.[22] It is well-known that the lipopolysaccharide is an important component of bacterial cell wall which is known to be cytotoxic for fibroblasts. There are various concepts highlighting the cause of gingival enlargement due to CsA.

Up-regulation of salivary pro-inflammatory cytokines interleukin (IL-1α), IL-8 and IL-6

Cytokines and chemokines such as IL-1α, IL-1 β, IL-6, and IL-8 play a significant role in periodontal diseases[33] and growing attention has been paid to their potential role in the pathogenesis of CsA-induced gingival hyperplasia with conflicting results. It has been observed that incubation of normal and overgrown gingival fibroblasts for 24 h in or with different concentrations of CsA released no detectable amount of IL-1β compared to control, whereas production of IL-6 in all fibroblast strains was suppressed in a dose-dependent manner with the maximal inhibition being reported at 2000 ng/ml CsA.[34] This was in contrast to a recent observation which demonstrated that 500 ng/ml CsA up-regulated production of IL-6 and transforming growth factor (TGF) β1 in HGFs in a time-dependent manner with the maximal release being reported at 24 h.[35] But, it has been observed that neutralization of TGF-β1 resulted in a significant reduction in expression of CsA-induced IL-6 while release of CsA-induced TGF-β1 was not affected by blocking IL-6.[36] These findings suggest that TGF-β1 is an essential regulator of CsA-induced IL-6 release in HGFs. In addition, it has been reported that HGFs treated with IL-6 or TGF-β1 exhibited an increased rate of proliferation compared to control.[37] Furthermore, neutralizing IL-6 and TGF-β1 caused down-regulation of CsA-induced HGF proliferation rate.[36] The effect of CsA on IL-1α and IL-8 has received little direct attention, although it has been suggested that the pathogenesis of CsA-induced gingival hyperplasia may involve alteration in the expression levels of various inflammatory cytokines in the gingiva.[21] It has been observed that an increase in the salivary levels of IL-1α, IL-6, and IL-8 in patients with CsA-induced gingival hyperplasia occurs.[2134] Up-regulation of these cytokines thus have a direct effect in causing gingival hyperplasia.

Increase in proliferation of human gingival fibroblasts

Long-term exposure to CsA may have a stimulatory effect on gingival fibroblast proliferation.[10] Bartold[15] observed that 10-9 g/ml CsA up-regulated DNA synthesis and the proliferation rate of HGF with most noticeable stimulation being reported in the presence of 10% fetal bovine serum (FBS), and that this capacity was retained even in absence of FBS or in the presence of lipopolysaccharide that usually inhibit proliferation of these cells. In the agreement with Bartold's findings, Chae et al.[35] also showed that treatment of serum starved HGFs with CsA (100 and 500 ng/ml) for 6 days significantly activated the cell cycle along with an increase in the cell viability and DNA incorporation. These findings were also in agreement with a flow cytometric study that showed an increase in the in vitro proliferative activity of CsA-treated HGF illustrated by a promoted progression of cell cycle and an increase in the expression level of cyclin B1.[37] It was recently observed that CsA up-regulated cell viability in HGF cell lines with a maximal increase being found in HGF cells obtained from CsA-induced overgrown gingival tissues.[38] Interestingly, a bimodal effect of CsA on proliferation of HGFs has been observed when cultured for 24 h; low doses of CsA (≤200 ng/ml) stimulated HGF proliferation, whilst higher doses of CsA (400-800 ng/ml) inhibited proliferation of these cells.[36] In contrast to the previous findings, it was observed that long-term exposure to a low dose of CsA (200 ng/ml) had no effect on viability and proliferation of HGFs.[39] While extremely high doses of CsA (5-10 g/ml) induced a significant decrease in HGF proliferation.[37] Furthermore, in vitro 24 h stimulation with CsA failed to induce an increase in HGF proliferation.[18] However, these discrepancies between studies could be attributed to heterogeneity of cell line strains or to differences in the experimental conditions.

Effect on human gingival keratinocytes

It has been reported that the release of both keratinocyte growth factor and scatter factor (SF) was increased in both normal and overgrowth gingival fibroblast strains treated with 2000 ng/ml CsA. It was observed that normal gingival epithelial cells incubated with 500 ng/ml CsA for 3 days expressed higher levels of keratinocyte growth factor receptor (KGFR) compared to control. Thus, increased level in KGFR is consistent with the increase in proliferation rate of epithelial cells observed in CsA-induced gingival hyperplasia.[40]

Yoshida et al. assessed proliferation of cultured rat gingival cells after incubation for 3 days and showed that CsA at concentration of 200-800 ng/ml induced hyperproliferation of gingival cells by 23-25% compared to control.[41] These findings were supported by an animal study that showed that treatment with CsA caused buccal epithelial hyperplasia associated with an increase in proliferating cell nuclear antigen (PCNA) expression. It was suggested that CsA-induced gingival hyperplasia might be mediated by an increase in the proliferative activity of epithelium cells and that dental plaque might aggravate the condition.[37] Similarly, it was demonstrated immunohistochemically that the epithelial hyperplasia reported in CsA-induced gingival hyperplasia was associated with hyperproliferation in both sulcular and oral gingival epithelium with a maximal proliferative activity being observed in the oral gingival epithelium.[40] However, conflicting data were also found through an animal study that showed the increase in the epithelial thickness observed in CsA-induced gingival hyperplasia was associated with an increase in cyclin D1 and PCNA expression.[41] Flow cytometric study on human oral epidermoid carcinoma cell lines showed that CsA caused a cell cycle stasis.[1920] Interestingly, both the inhibitory and stimulatory effect of CsA on human gingival keratinocyte proliferation has been observed where CsA significantly reduced their proliferation rate from day 3 to day 6 and then stimulated their proliferation from day 6 to day 9 with maximal increase being observed on day 9 at a concentration of 1,000 ng/ml.[40]

Overexpression of noncollagenous extracellular matrix proteins like heparan sulfate proteoglycans

There has been considerable interest on the effect of CsA on the extracellular matrix of gingival tissues. It has been observed that there are alterations in gene expression of extracellular matrix proteoglycans in CsA-induced gingival tissue alterations. CsA-induced gingival overgrowth seems to be associated with increased expression of perlecan, a typical basement membrane proteoglycan but not decorin, biglycan or versican.[42] Recently, the gene expression of cell surface heparin sulfate proteoglycans syndecan-2, syndecan-4, and betaglycan was observed in gingival tissues of transplant patients exposed to CsA. It was observed there was a high expression of noncollagenous extracellular matrix proteoglycans syndecan-4 and syndecan-2.[42]

Stimulation of the renin angiotensin axis

The renin angiotensin system (RAS) is considered as a hormonal circulatory system involved in maintaining blood pressure, electrolyte, and fluid homeostasis. RAS components can be synthesized in local tissues and are found to play a role in gingival overgrowth. Recently, its role has been implicated in causing drug-induced gingival overgrowth. The expression of Ang II and its receptors was observed in gingival fibroblast cells treated with cyclosporine. Ang II mRNA and protein expression was significantly higher in patients with drug-induced gingival enlargement than in patients with periodontitis and healthy subjects. The angiotensin II receptor type I (AT1) mRNA was expressed more than AT2 in all examined tissues. In gingival fibroblasts, Ang II and AT1 expressions were increased with cyclosporine incorporation compared to controls.[23] It was thus suggested that cyclosporine can modulate local expression of RAS components such as the angiotensinogen, Ang II, and its receptors in gingival tissues and gingival fibroblast cells.

Effect of cyclosporine A on gingival cell apoptosis

Apoptosis, programmed cell death, is stimulated mainly by two characteristic pathways: An extrinsic pathway that is mediated by death receptor (tumor necrosis factor-alpha) and an intrinsic or mitochondrial pathway that is regulated by pro-apoptotic proteins such as Bax and Bid and antiapoptotic proteins like Bcl-2. Both pathways are executed by a key component referred to as caspase-3 that is activated after being cleaved, leading in the end to cell apoptosis.[43] It has been observed that the increased number in HGF in CsA-induced gingival hyperplasia was the result of reduced apoptosis coupled with increased proliferative activity.[14] Further, in vitro studies revealed that stimulation of HGF with CsA for 24 h caused a significant decrease in apoptosis rate through both the intrinsic and extrinsic pathways suggesting that the decreased level of apoptosis might play a more significant effect than the increase in cell proliferation in increasing the HGF number.[151820] Recently, it was reported that CsA increased the cell viability of HGF cultures in vitro and this increase was resulted from a reduced apoptosis, illustrated by reduced expression Bax/Bcl-2 ratio, rather than increased cell proliferation.[18]

PREVENTION AND TREATMENT OF DRUG-INDUCED GINGIVAL ENLARGEMENT

Prevention

In the susceptible patient, drug-associated gingival enlargement may be ameliorated but not prevented by elimination of local factors, meticulous plaque control, and regular periodontal maintenance therapy. The 3-6 month interval for periodontal maintenance therapy has been recommended for patients taking drugs associated with gingival enlargement.[30] Each recall appointment should include detailed oral hygiene instructions and thorough prophylaxis with supra and subgingival calculus removal as needed. It is important that the dental professional encourage improved tooth cleaning in a supportive and positive manner, as well as providing information about the role of dental plaque in promoting gingival overgrowth. Mild gingival enlargement will often diminish with removal of plaque and calculus deposits. Even moderate gingival enlargement may reduce enough to avoid surgical intervention. Attempts at improving oral hygiene are of limited benefit in severe gingival enlargement, as surgical gingival excision is generally indicated.[44]

TREATMENT

Drug substitution or withdrawal

The most effective treatment of drug-related gingival enlargement is withdrawal or substitution of medication. Many cases of gingival enlargement will respond to local treatment, but consideration should be given to altering the medication if gingival enlargement covers more than about a third of the tooth surface. When possible, reducing the dose or changing to another drug may bring about partial or complete regression of the lesion. Most patients will observe an alteration in the soft tissues within a few days.[31] When this treatment approach is taken as suggested by another case report, it may take from 1 to 8 weeks for resolution of gingival lesions.[32] Unfortunately, not all patients respond to this mode of treatment, especially those with long-standing gingival overgrowth.[28]

Nonsurgical treatment

Mild gingival enlargement may only require local management as improvement in oral hygiene, together with professional cleaning of the teeth, can lead to resolution of inflammation and reduction in gingival enlargement. Professional debridement with scaling and root planing as needed has been shown to offer some relief in gingival overgrowth patients.[30] Treatment planning becomes more complex where there is periodontitis associated with gingival enlargement, which poses a cosmetic or functional problem. Periodontitis alone can be treated using conventional clinical care, but when associated with the gingival enlargement, may require changes in the medication regimen, periodontal surgery (flap technique) pocket elimination along with removal of excess tissue, or a combination of the two.[44]

Surgical periodontal treatment

Since the anterior labial gingiva is frequently involved, surgery is commonly performed for esthetic reasons before any functional consequences are present. The classical surgical approach has been the external bevel gingivectomy.[31] However, the total or partial internal gingivectomy approach has been suggested as an alternative.[44] This is a more technically demanding approach, which has the benefit of limiting a large denuded connective tissue wound that results from the external gingivectomy, thereby minimizing postoperative inflammation, pain and bleeding. The use of electrocautery and carbon dioxide lasers has shown some utility for reducing gingival enlargement, an approach which provides rapid postoperative hemostasis.[31] Consultation with the patient's physician prior to surgical treatment regarding antibiotic and steroid coverage should take place in the immune suppressed patient.[44]

CONCLUSION

Gingival overgrowth is a side effect of the long-term administration of cyclosporine and may also be influenced by other predisposing factors, like bacterial plaque. It needs to be emphasized that elimination of local factors followed by thorough maintenance has a definitive significant inhibitory effect on gingival overgrowth. Surgical treatment to excise and remodel the gingival contour should be considered whenever gingival overgrowth causes esthetic and functional problems.

Understanding the various mechanisms, underlying the pathogenesis of cyclosporine-induced gingival overgrowth, will help the clinician devise better prevention strategies in patients susceptible to gingival overgrowth. Hence, it would be pertinent to identify and explore possible risk factors relating to both prevalence and severity of drug-induced gingival overgrowth. Newer molecular approaches are needed to clearly establish the pathogenesis of gingival overgrowth and to provide novel information for the design of future preventive and therapeutic modalities.

Financial support and sponsorship

Nil.

Conflicts of interest

The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non-financial in this article.