I. Introduction
Cyclosporine (Cs) is an immunosuppressant used for preventing graft rejection and treating autoimmune diseases[1]. Gingival overgrowth (GO) is an adverse effect associated with Cs administration. It is frequent in adolescents and children as well as in adults. The prevalence of GO in young patients is 13 - 85%[2]. GO is a serious intraoral complication that is difficult to treat completely without discontinuing the drug; therefore, it presents difficulties in maintaining oral health and has a negative impact on the overall health of the patient. Since GO does not occur in all patients, identification of patients with an increased risk of GO is important when prescribing immu-nosuppressants. This should be a consideration especially for oral health of young patients. It's because it can have a greater impact on children and adolescents who suffer from such adverse effects from an early age. To date, several studies and clinical approaches have attempted to clarify the pathogenesis of Cs-induced GO, and extracellular matrix accumulation has been suggested as the most important event. Studies on type I collagen (CoL-I), which is the main extracellular matrix component, have focused on CoL-I metabolism and matrix metal-loproteinase, which are the main targets of Cs[3,4]. A limitation of these studies is that different individuals were assigned to the control group and test group for investigating the effect of Cs on CoL-I metabolism. Because these studies did not control the biological variation between individuals, the effects of factors other than CoL-I cannot be dismissed. Therefore, the study design needs to be modified to identify the effect of Cs on CoL-I metabolism accurately, with all the other factors controlled. The present study addressed the limitations of previous studies by using a novel approach, wherein the experiment was conducted on the same individuals to control variability between individuals
II. Material and methods
All of the animals and protocols used in this study were approved by the Institutional Animal Center and Use Com-mittee, Ajou University Medical Center (approval No.: IA-CUC-2013-0031).
1. Animals and gingival specimens
Five 5-week-old male Sprague-Dawley rats weighing 150 - 160 g were used (designated as A1 - A5). The animals received Cs (10 mg/kg body weight) in corn oil daily by gastric feeding for 6 weeks[5]. The Cs was solution (Sandimmune Neoral® Solution, Novartis Korea, Seoul, South Korea) (10 g/100 mL). The rats were anesthetized by intraperitoneal injection of a combination of tiletamine hypochloride and zolazepam hypochloride (Zoletil 50®, Virbac Korea, Seoul, South Korea) (25 mg/kg) and Xylazine (Rompun®, Bayer, Leverkusen, Germany) (10 mg/kg), and samples of their gingival tissues (3.0 mm × 2.0 mm × 0.5 mm, width×height×depth) were taken from the buccal area of mandibular first and second molars. Specimens harvested before the commencement of Cs treatment served as Controls. All gingival specimens were obtained 2, 4, and 6 weeks after the commencement of Cs treatment; these were designated as in vivo test groups Tv2, Tv4, and Tv6, respectively. All of the animals were sacrificed at week 6.
2. Preparation of gingival fibroblasts
Rat gingival fibroblasts were prepared according to a modified version of a previously described method[6]. The gingival specimens were placed in Hank's balanced salt solution (GIB-CO-BRL, Grand Island, NE, USA) containing penicillin/strepto-mycin. They were minced into smaller pieces and digested in a solution of 4 mg/mL type 1 collagenase, and 3 mg/mL dispase for 3 hours at 37°C. Single-cell suspensions were obtained by passing the solution through a 70-µm strainer. Monolayer cultures were prepared using conventional techniques in Dulbecco's modified Eagle's medium (WELGENE, Daegu, South Korea), supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin. The culture medium was refreshed every 2 - 3 days. Cells at passages 3 and 4 were used for further in vitro study.
3. In vitro Cs treatment of cultured cells
Some of the cells from Controls were separated for in vitro Cs treatment and assigned to the in vitro test group (designated as Tt). A stock solution of 1 mg/mL was prepared by dis-solving 1 mg of Cs in 45 µL of ethanol and 5 µL of Tween-20 as cosolvents[7]. In consideration of Cs being used clinically at concentrations ranging from 250 to 400 ng/mL, and at a maximum dose of 1000 ng for in vitro studies[8], a mid-range concentration of 700 ng/mL was chosen for the present study. The medium was then added to give a final volume of 1.0 mL. Subconfluent cultures were then maintained (24 hours) in serum-free medium before addition of Cs (700 ng/mL). When the gingival fibroblasts were close to confluence (i.e., at 80%), Cs was added to the cultures, which were then incubated at 37°C for 6 hours[8]. The cell cultures were trypsinized and the fibroblasts were collected by centrifugation at 1610 × g for 5 min. They were finally washed in phosphate-buffered saline (PBS) and harvested by further centrifugation (also 1610 × g for 5 min).
4. Immunofluorescence analysis
Immunofluorescence staining was conducted to enable observation of CoL-I. After culturing the Control, Tv2, Tv4 and Tv6 samples including Tt, the cellular samples were fixed with formaldehyde and dehydrated with ethanol. They were then washed with Dulbecco's phosphate-buffered saline (DPBS) (WELGENE) and incubated with the primary antibody (Poly-clonal goat CoL-I, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a concentration of 1 : 200 at 4°C. The cells were washed with DPBS and incubated in fluorescein isothiocyanate (FITC)-labeled mouse antigoat IgG and FITC-labeled goat an-timouse IgG secondary antibodies (Santa Cruz Biotechnology) (diluted to 1 : 5000) at 37°C in the dark. After washing with PBS, the cells were finally mounted and visualized with the aid of confocal microscopy (GeneAll, Seoul, South Korea).
5. Gene expression by quantitative real-time polymerase chain reaction (PCR)
The effect of Cs was monitored by examining differences in the differentiation marker CoL-I. Total RNA was extracted from each sample using a Ribospin device (GeneAll), and then converted to complementary DNA (cDNA) with a first-strand cDNA synthesis kit (PrimeScript RT reagent kit, Bioneer, Daejeon, South Korea). The reverse-transcriptase reaction was carried out using 2-µg aliquots of total RNA. The sense and antisense primers were designed according to published cDNA sequences available in GenBank (NIH genetic sequence database), and β-actin was used as a standard housekeeping gene against which to normalize mRNA expression (as presented in Table 1)[9,10]. Real-time PCR products were monitored and quantified using a Sensimix SYBR® kit (Bioline, London, UK) in a Rotor-Gene RG-3000A® system (Corbett Research, Mortlake, NSW, Australia). After the real-time PCR ran, the threshold cycle (Ct) was used to determine the efficiency of different genes relative to the internal control (β-actin). The amount of mRNA in each sample was calculated using the comparative ΔΔCt method[9]. Each measurement was assessed in triplicate.
6. Western blot analysis
After the harvested cells were cultured, the total cellular proteins were prepared using 0.4 mL of cell lysis buffer (RIPA buffer, Cell signaling technology, Danvers, MA, USA). Equal amounts of each lysate were used for electrophoresis through a sodium dodecyl sulfate (SDS)-polyacrylamide gel in trigly-cine-SDS running buffer. The proteins were transferred onto polyvinylidene difluoride (PVDF) membranes, which were then incubated in 5% bovine serum albumin blocking solution for 2 hours at room temperature and then overnight at 4°C in the presence of the CoL-I antibody (diluted to 1 : 200). After the membrane was washed with Tris-buffered saline (0.2% Tween-20) three times to remove antibodies, the horseradish-peroxidase-conjugated secondary antibody (1 : 10,000 dilution) was added to the membrane and incubated for 2 hours at 37°C[11]. After further washing in Tris-buffered saline (0.2% Tween-20), the PVDF membranes were visualized with an enhanced chemiluminescence reagent (Luminol, Santa Cruz Biotechnology) and exposed to MicroChemi 4.2 (DNR Bio-Imaging Systems, Jerusalem, Israel).
III. Results
1. Detection of CoL-I using direct immunofluorescence
Immunofluorescence staining revealed the presence of CoL-I in fibroblasts from the control, Tv2, Tv4, Tv6, and Tt samples (Fig. 1). CoL-I was much more prominent in the control, Tt, and Tv2 samples than in Tv4 and Tv6 samples.
2. Comparison of CoL-I mRNA expression
Real-time PCR analysis was performed to quantitatively assess the mRNA expression level of CoL-I in gingival fibroblasts, and the results are presented in Fig. 2. CoL-I mRNA expression appeared to increase in all animals, except one, following in vitro Cs treatment, and the changes were not statistically significant. When Cs was administered in vivo, two animals exhibited considerably decreased CoL-I mRNA levels after 6 weeks of Cs administration, while the other animals did not exhibit any noticeable change.
3. Analysis of CoL-I protein expression by western blotting
The expression levels of CoL-I protein were assessed by western blot analysis. The bands in Fig. 3 represent the expression levels of CoL-I protein and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). As shown in Fig. 4, western blot analysis revealed marked differences in the amount of CoL-I protein in the Cs-treated test groups and in untreated controls. This result was similar to that obtained in in vitro Cs treatment.
IV. Discussion
In patients with drug-induced GO, it is inevitable to dis-continue the drug to treat GO, because GO has a high recurrence rate even with surgical treatment. Therefore, treatment of GO remains a challenge. Considerable efforts have been made to elucidate the mechanism underlying the onset of GO following Cs administration. Although the use of Cs has decreased recently with the discovery of another immunosuppressant, FK506, it is still a widely used immunosuppressant along with FK506. Therefore, studies on GO, an intraoral complication of Cs, are necessary for the healthy life of patients. Many studies have attempted to determine the pathogenesis of drug-induced GO in both humans and other animal models[7,12-15]; however, the mechanism remains unclear. It has been suggested that disorders of collagen metabolism in gingival connective tissue are mostly related to the pathogenesis of GO[3,4,7,8,14,16]. In previous studies, the effects of Cs were analyzed using gingival fibroblasts harvested from different individuals in the control and test groups. Therefore, these studies did not consider the effects of factors other than CoL-I in different individuals. Since all individuals do not share the same biologic conditions, it is necessary to control all factors other than the factor selected for the study when evaluating drug effects. Tipton et al.[17] demonstrated variations among individuals and heterogeneity of human gingival fibroblasts and found that fibroblasts from different individuals varied markedly in collagenolysis and production of proteins in response to Cs treatment. Furthermore, the genetically de-termined capacity of the host to cope with the administered Cs, the responsiveness of gingival tissue to the drug, and periodontal conditions increase the difficulty of examining the etiology of Cs-induced GO. In the present study, cultures of gingival fibroblasts were harvested from the same rat before and after Cs treatment to exclude inter-individual variations. To overcome the limitations in the experimental design of previous studies, the present study analyzed the changes in CoL-I in the same individual by assigning specimens harvested from rats prior to Cs administration to the control group and specimens harvested from the same rats after Cs administration to the test group. Our data from gene and protein expression analyses showed a considerable gap among individuals, indi-cating variations. In addition, the study compared the results of in vivo Cs treatment against the results of in vitro Cs treatment applied on some gingival fibroblasts in the control group prior to in vivo Cs treatment. This step was designed to determine whether GO incidence could be predicted with only the results of laboratory test performed on gingival fibroblasts prior to drug administration. The results of the present study showed that CoL-I protein expression level increased after both in vitro and in vivo Cs treatment.
Some in vitro studies have revealed an increase or decrease in CoL-I mRNA expression levels[8,18]. This discrepancy may have originated from the culture conditions and indicate the need to concomitantly conduct in vivo studies. Our real-time PCR analysis clearly showed that the mRNA expression levels of CoL-I were inconsistent between in vitro and in vivo Cs administration, compared with the control. In contrast, CoL-I protein analysis in our study demonstrated upregulated CoL-I expression in all subjects in both in vitro and in vivo Cs treatment. A marked increase in CoL-I expression was observed approximately 2 weeks after in vivo treatment, and it corre-sponded to the increase observed after in vitro treatment. The data on CoL-I protein expression confirmed its tendency to increase after Cs treatment regardless of mRNA expression, as shown previously[18]. This result suggests that the regulation of CoL-I protein expression may primarily occur at the post-transcriptional level. During soft tissue fibrosis, CoL-I protein expression is highly upregulated both transcriptionally and post-transcriptionally[19-21]. Transcription alone is not suf-ficient to regulate the expression of CoL-I[22]. The production of CoL-I can be increased several hundred-fold in wound healing or in pathological fibrosis. This increase is principally due to post-transcriptional regulation, followed by increased half-life and stability of mRNAs, and transcription rate of collagen genes[23-25]. These studies are in agreement with our study showing that CoL-I protein expression considerably increased in two animals(Fig. 3), with significantly decreased mRNA expression levels 2 weeks after Cs administration(Fig. 2).
We found that Cs treatment had no specific effect after 4 weeks of administration. This finding may be supported by studies on the pathogenesis of drug-induced GO in a rat model[25-27]. When Cs was administered to 4-week-old rats, macroscopic GO was detected as early as 3 weeks, and the severity of overgrowth increased slightly, reaching a plateau by approximately 6 weeks[28]. In the present study, 5-week-old rats were used, and the induction of GO was maximal after 2 weeks of Cs administration. Longer treatment periods of up to 10 weeks did not result in further GO[26]. No noticeable bands were observed in western blot analysis for CoL-I protein after 4 and 6 weeks of Cs administration in the present study.
V. Conclusion
We performed an experiment that considered inter-individ-ual differences and controlled factors other than CoL-1. It cannot be concluded that the protein level of CoL-I is a marker for GO based on the results of this experiment alone. Never-theless, this experimental design that compared the results at 2, 4, and 6 weeks after in vitro and in vivo Cs administration is useful, because if the response of markers, such as CoL-I, in preoperative in vitro setting corresponds to the response after in vivo treatment in the same individual, then predictions on GO can be made prior to in vivo treatment. It is suggested that markers other than CoL-I, which were used in previous studies, should be investigated using the experimental design of the present study. In future, studies on human gingival fibroblast are warranted.