IJCRR - Vol 03 Issue 08, August, 2011
Date of Publication: 30-Nov--0001
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GINGIVAL MMP and TIMP LEVELS IN CYCLOSPORINE AND TACROLIMUS TREATED RATS - AN EXPERIMENTAL STUDY
Author: Sheeja S. Varghese, Rajesh A, Jayakumar ND, Sankari M, Padmalatha O, Dare BJ
Abstract:Objectives:Gingival overgrowth is an infrequent side effect with tacrolimus when compared to
cyclosporine. The exact mechanism underlying this is not fully understood. The study was aimed to
compare the collagen content, levels of MMP-1, MMP-2 and TIMP-1 in cyclosporine and tacrolimus
induced gingival overgrowth in rats.
Methods:A total of eighteen, 21 days old male wistar rats were assigned to three group of six each.
Group-1 (control) \? Administration of olive oil daily, Group-2 \? Administration of cyclosporine 30 mg /
kg body weight in olive oil, Group-3 \? administration of tacrolimus 1.5 mg / kg body weight in olive oil.
Gingival tissues were collected after 30 days from incisor region and total collagen content was calculated
from hydroxyproline estimation. SDS-PAGE zymography was used to obtain zymogram profiles of
MMP-1 and MMP-2. Western blot analysis was performed to confirm the expression MMPs and TIMP-1.
ELISA was used to quantify the expression of MMPs and TIMP-1. Mean values were compared by oneway
ANOVA followed by Tukey-HSD procedure.
Results:Collagen content was significantly higher in Group-2 with no significant difference between
Groups-1 and 3. MMP-1, MMP-2 were significantly reduced in Group-2 but was significantly higher in
Group-3 compared to Group-1. TIMP-1 was highest in Group-2 but Group-3 also showed higher level
compared to Group-1.
Conclusion:Cyclosporine increases the collagen content and reduces MMP-1 and MMP-2 whereas
tacrolimus doesn't alter collagen content but increases MMP-1 and MMP-2 and cyclosporine increases
TIMP-1 more than in rats.
Keywords: Cyclosporine, Tacrolimus, Gingival overgrowth, MMP, TIMP, Collagen.
Drug- induced gingival overgrowth is an adverse effect associated principally with 3 types of drugs such as immunosuppressive agents, antiepileptics, and calcium channel blockers. Cyclosporine is a cyclic hydrophobic polypeptide widely used as an immunosuppressive drug that is especially effective in cell – mediated immune response. The side effects associated with cyclosporine A (CsA) therapy includes nephropathy, hypertension, hepatotoxicity, thromboembolic GINGIVAL MMP and TIMP LEVELS IN CYCLOSPORINE AND TACROLIMUS TREATED RATS - AN EXPERIMENTAL STUDY Sheeja S. Varghese1 , Rajesh A1 , Jayakumar ND1 , Sankari M1 , Padmalatha O 1 , Dare BJ2 1Department of Periodontics, Saveetha Dental College, Saveetha University, Chennai 2Biomedical Research Unit and Laboratory Animal Centre (BRULAC) Saveetha University, Chennai E-mail of corresponding author: firstname.lastname@example.org International Journal of Current Research and Review www.ijcrr.com Vol. 03 issue 08 August 2011 130 complications, neurotoxicity, hypertrichosis, and gingival overgrowth (1-5). Gingival overgrowth is observed in 25% to 81% of patients undertaking CsA (6). Tacrolimus (formerly known as FK-506) was introduced as an immunosuppressive agent for use in organ transplantation in 1987. It has immunosuppressive capacity 100 times that of other immunosuppressive agents (7). Studies have reported that tacrolimus causes infrequently or lesser degree of gingival overgrowth when compared to cyclosporine (8- 12). CsA induced gingival overgrowth is caused by accumulation of extracellular matrix components, particularly collagen in the gingival connective tissue compartment. Experimental investigations indicate interstitial collagen as the main target of CsA, and altered collagen degradation pathway seems to be responsible for this pathology (13-15). Collagen turnover is, in fact, very tightly regulated. Under physiological conditions there is dynamic balance between collagen synthesis and degradation, fine tuned by matrix metalloproteinases (MMPs) a family of proteolytic enzymes responsible for the remodeling of extracellular matrix (16-17). MMP activity is closely controlled by tissue inhibitors of matrix metalloproteinases (TIMP). Any derangement of this mechanism will lead to gingival overgrowth. It has been reported that MMP-1, MMP-2 and MMP -3 were significantly reduced in CsA induced gingival overgrowth in rats (13). The levels of TIMP-1 were significantly lower in cyclosporine induced gingival overgrowth than in periodontaly healthy gingiva and there was no significant difference in the level of MMP-1 between the healthy gingiva and CsA induced gingival overgrowth (18). On the contrary another study reported that MMP-1 and MMP-2 were significantly reduced and TIMP-1 and TIMP-2 were increased but not at significant level in cultured fibroblast, when incubated with CsA (19). Gagliano et al. (20) analyzed the effect of tacrolimus (FK-506) on cultured human gingival fibroblasts and found that MMP-1 and MMP-2 mRNA levels rose significantly in tacrolimus treated fibroblasts compared to control and TIMP-1 levels were not significantly affected. The same author later reported that tacrolimus did not induce interstitial collagen over expression by cultured gingival fibroblast and induced upregulation of MMP protein levels (21). Only very few studies have compared the in vivo levels of MMPs between cyclosporine and tacrolimus induced gingival overgrowth. This experimental study was aimed to estimate and compare the levels of MMP-1, MMP-2 TIMP-1 and total collagen content between cyclosporine and tacrolimus induced gingival overgrowth in rats.
MATERIALS AND METHODS The study was conducted in the department of periodontics, Saveetha Dental College, Biomedical Research Unit and Laboratory Animal Centre (BRULAC), Saveetha University, and Central Leather Research Institute (CLRI), Chennai. Ethical clearance was obtained from IAEC (Institutional Animal Ethical Committee) New Delhi, India. (IAEC no: Perio.001/07)
Experimental animals and their diet The experiment was performed using eighteen, 21 days old male wistar rats weighing 55 – 60 gm. The rats were housed in plastic bottomed cages using husk as bedding. The animals were subjected to normal atmospheric conditions at 21° c and subjected to the same regimen of lighting feeding and handling. Rodent diet in the form of pellets and drinking water was provided.
Experimental protocol Experiment was carried out with the assistance of person trained to handle experimental animals. The rats were randomly assigned to experimental groups of 6 rats each prior to the commencement of the experiment. Group 1- Administration of olive oil daily (CONTROLS) Group 2- Administration of CYCLOSPORINE 30 mg/kg of body weight in olive oil daily Group 3- Administration of TACROLIMUS 1.5 mg/ kg of body weight in olive oil.
Drug dosage and administration At the beginning and during the course of the study the rats were weighed and drug dosages were adjusted accordingly. The drug solutions were freshly prepared each day and administered by means of infant feeding tube (gastric feeding). The amount of drug was calculated according to the measured body weight of the rat. The drugs were administered for 30 days.
Collection of Gingival tissues Gingival tissues were collected after 30 days from upper and lower central incisor regions after sedation with ketamine. Immediately following excision the tissue specimens were stored at - 70° C until use. From the sample tissues collagen content was estimated from hydroxyproline content. MMPs and TIMP were extracted and SDS-PAGE zymography was used to obtain zymogram profiles of MMP-1 and MMP-2. Western blot analysis was performed to specifically confirm the expression of MMPs and TIMP-1. Further ELISA was used to quantify the levels of MMPs and TIMP-1.
Collagen Estimation: Total collagen content was calculated from hydroproxyproline content (Neuman RE and Logan MA) (22) as given below; Total Collagen = Total Hydroxyproline x 7.46
Samples were taken in a test tube, Acetone was added and kept in refrigerator for 1 hr, and then acetone was allowed to evaporate and the dried tissue was weighed accurately. 2mg of dried tissue was then transferred to hydrolysis tubes containing 6N HCI, sealed and kept for hydrolysis at 110°C, for 24 hrs. The contents were then transferred to a petridish and kept in water bath for acid evaporation. This was repeated twice after addition of water and evaporated to dryness. To this water was added to about 4 ml and rinsed thoroughly and transferred to 5ml standard flask and made up to 5 ml. The reagents used were,
1. 0.01 M CuSO4 – 0.29 gms in 100 ml (Fresh)
2. 2.5 N NaOH – 20 gms in 200 ml
3. 6 % H2O2 – 15 ml in 75 ml (Fresh)
4. 3 N H2SO4 – 82 ml in 984 ml d. H2O 5. 5% PDAB –
5 g/100 ml of n-propanol (Fresh)
6. Stock Standard - 10mg – L hydroxyproline in 10 ml of 0.01 N HCI (mg/ml)
7. Standard – 1 ml of stock in 10 ml d. H20 (1 mg/10 ml) (use 2 µg – 50 µg
for standard curve)
Extraction of MMPs Frozen gingival tissues were allowed to thaw at room temperature. 30 mg (wet weight) of gingival tissues from each group were homogenized with Tris buffer (Saline – 0.9%, Tris – 0.05M, Triton X-100 – 0.25 % and CaCl2 - 0.02M) and centrifuged at 15000 rpm for 5 minutes at 4° C. The resultant supernatant was separated and used for analysis.
Gelatine zymography The presence of MMP-1 and MMP-2 and TIMP- 1 in the tissue extracts were studied by gelatine Zymography using 10% SDS – PAGE containing 1 mg / ml gelatine under nonreducing conditions without prior boiling. After electrophoresis, gels were washed in 2.5% Triton X-100 for 1 h to remove SDS and allow the protein to denture, and subsequently immersed in a mixture containing Tris-HCI 50 mM/L (pH 7.5) and CaCl2 20 mM/L for 16 h at 37° C. The gels were then stained with 0.25% Coomassie Brilliant Blue R250/40% ethanol/10%acetic acid, and destained in 25% ethanol/8% acetic acid. Enzymatic activities were detected as clear bands of gelatine lysis against blue background. To measure the relative levels of MMPs, clear zones were scanned and analyzed by Multi – Image Gel Documentation system.
SDS – PAGE and Immunoblotting MMPS in the tissue extracts were separated in parallel with appropriate molecular weight markers using SDS-PAGE in 10% (w/v) polyacrylamide gel with 5% (w/v) stacking gel, and stained with Coomassie Brilliant Blue R250. For immunoblotting the proteins were separated by SDS – PAGE and transferred to nitrocellulose membrane. The membranes were incubated with anti MMP-1, MMP-2 and TIMP- 1 antibody (1:1000 dilution) raised in rabbit in 0.05M Tris-HCI, 0.2M NaCI, 0.05% (v/v) Tween 20, pH 7.4, followed by goat anti-rabbit IgG alkaline phosphate conjugate (1:500 dilution in the same buffer) and stained with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate reagent, according to the manufacturer‘s instruction.
Procedure 50μ1 of each (MMP-1, MMP-2 and TIMP-1) antibody solution (20 μg/ml in phosphate buffer saline – PBS, pH 7.4) was loaded in the wells and incubated overnight at 4° C. After incubation the wells were washed twice with PBS. The wells were filled with blocking solution (3% BSA/ PBS) and incubated for 2 hours in humid atmosphere at room temperature. After incubation the wells were washed withPBS and 50μ1 of antigen solution was added to the wells. After 2 hours of incubation at humid atmosphere at room temperature, the wells were washed with PBS. 50μ1 of secondary antibody (1:1000) was added and incubated for 2 hours. After incubation the wells were washed with PBS and 200μ1of substrate solution was added and incubated overnight at 4° C. After incubation the plate was read at 492 nm.
Statistical Analysis Quantitative levels of collagen, MMP- 1 and 2 and TIMP-1 were statistically analyzed. Mean values were analyzed statistically by one way ANOVA to determine the existence of difference among the group and further pair wise comparisons of all the three groups were done by Tukey HSD procedure. P<0.05 was considered as the level of significance.
In this study we could observe gingival overgrowth in all the 6 rats in cyclosporine group and it was more prominent in the interdental papilla, which promoted a gradual tooth separation of incisors. Whereas tacrolimus group did not show any gingival overgrowth and gingival condition were similar to controls. No clinical or morphometric measurements were taken as it was not the aim of our study. Quantitative assessment showed that the collagen content was highest in cyclosporine group (638±6.60). In tacrolimus group it was 287±2.1 and control group it was 279.3±8.21 (Table 1). MMP-1 and MMP-2 levels were highest in tacrolimus group (297.54±7.48, 526.62±12.5 respectively) followed by control group (225.2±9.12, 405.24±9.99 respectively) and lowest in cyclosporine group (168.6±11.88, 360.53±8.55 respectively) (Table 1). TIMP-1 levels were highest in cyclosporine group (434.39±13.2) followed by tacrolimus group (293.43±10.3) and lowest in control group (225.49±7.43) (Table 1). Statistical analysis by one way ANOVA showed that mean values were significantly different among the three groups (P<0.001) (Table 1). Pair wise comparison between the study groups were done by Tukey HSD procedure. Mean collagen content in cyclosporine group was significantly higher than the tacrolimus group and in controls (P<0.001) (Table 2) and there was no statistically significant difference in collagen content between the tacrolimus and control group (P=0.115) (Table 2) The mean levels of MMP-1 and MMP-2 in cyclosporine group were significantly reduced when compared to tacrolimus (P<0.001, P<0.001) and control group (P=0.004, P<0.001) (Table 2). Further the mean value of MMP-1, MMP-2 in tacrolimus group was significantly increased when compared to cyclosporine (P<0.001, P<0.001) and control group (P=0.005, P<0.001) (Table 2).
The mean levels of TIMP-1 in cyclosporine group and tacrolimus group were significantly increased when compared to controls (P<0.001). And when cyclosporine and tacrolimus were compared cyclosporine group showed significantly higher level than tacrolimus group (P<0.001) (Table 2). Figure 1 shows the expression of MMP-1 and MMP-2 on gelatin zymogram where the band width is reduced in cyclosporine group compared to control group and tacrolimus group. Figure 2 and 3 illustrating the MMP-1 and expression by western blotting assay on nitrocellulose paper shows maximum band width for tacrolimus group and minimum for cyclosporine group. Figure 4 depicting the western blot analysis for TIMP-1 shows that band width is increased for both cyclosporine group and tacrolimus group with the highest in the cyclosporine group.
Various researchers(18-20) have studied the role of MMPs and TIMP in the pathogenesis of the drug induced gingival overgrowth and these studies have reported an imbalance in MMP-1, MMP-2 and TIMP-1 in cyclosporine induced gingival overgrowth. Effect of tacrolimus on MMPs was not completely understood. The present study was undertaken to quantitatively estimate and compare the levels of MMP-1, MMP-2, and TIMP-1 and total collagen content in cyclosporine and tacrolimus induced gingival overgrowth in rats. In this study we have used rat model since it is one of the most suitable animals for disease models with respect to availability, handling, and preparing an inbred population. It also bears a close resemblance to humans with respect to the periodontal anatomy of teeth, development and composition of dental plaque, and histopathology of periodontal lesions. Younger rats were chosen as they are more susceptible to gingival enlargement than older rats (23) and only male rats were included in order to avoid gender bias as female sex hormones may suppress gingival overgrowth (23). In the study by Fu et al. it was seen that cyclosporine induces gingival overgrowth in a dose dependent manner and among the different concentrations used (3, 10, 30 mg / kg body weight) the maximum enlargement was associated with 30 mg / kg body weight (24). Hence we have chosen the dosage of 30 mg / kg body weight. Tacrolimus was administered at 1.5 mg / kg of body weight as their therapeutic dosages falls in the range of 0.5 – 1.5 mg / kg of body weight (25). Our findings showed that mean collagen content in cyclosporine group was significantly higher than the collagen content in control group. These findings are in consistence with previous study by Spolidorio et al (26). By stereological estimation they found that cyclosporine treatment in rats resulted in increased volume density of fibroblast and collagen fibres. In vitro studies on human gingival fibroblast also indicated stimulatory effect of cyclosporine on collagen synthesis (27-28). When compared, the mean collagen content in tacrolimus group was not significantly different from the control group. This finding is also in agreement with other studies (20-21) where they found that COL-1 mRNA and protein were not significantly affected by FK-506 (tacrolimus). It has also been reported that FK-506 did not affect the basal expression of type 1 procollagen protein or alpha 2(1) collagen mRNA in normal fibroblast (29). In the present study MMP-1, 2 and TIMP-1 were analyzed as MMP-1 or interstitial collagenase has the unique capacity to cleave interstitial collagens into ¾ and ¼ degradation fragments, the so called gelatins (16). These cleavage products can be further degraded by other proteinases such as gelatinase or MMP-2, leading to complete digestion of fibrillary collagen. TIMP-1 controls gene expression and activation of MMP-1 and MMP-2 (30). We found that the levels of MMP-1 and MMP-2 in cyclosporine group were significantly reduced when compared to control group. These findings are in accordance with the previous studies (19, 13) where they found that MMP-1 and MMP-2, were significantly reduced in cultured gingival fibroblasts when incubated with CsA. Another study (31) also reported similar results where cyclosporine inhibited the expression of membrane type 1 MMP in gingiva of rats and reduced the activation of MMP-2. When the tacrolimus group was compared with the control we observed that MMP-1 and MMP-2 were significantly increased in the former. These in vivo findings agree with the in vitro study (20) where they found that MMP-1 and MMP-2 mRNA levels rose significantly in FK-506 treated fibroblasts compared to healthy controls. Same author later reported that tacrolimus induced upregulation of MMPs in cultured human fibroblast (21). Higher MMP-1 and MMP-2 levels in tacrolimus group when compared to cyclosporine group speculate that the mechanism activated by tacrolimus differs from CsA toxicity and also suggest that tacrolimus has an inducing effect on MMPs and thereby counteracting collagen accumulation in the connective tissue compartment. TIMPs form classical non-covalent bimolecular complexes with the active forms of matrix metalloproteinase and in some instances with latent matrix metalloproteinase precursors as well and specifically control matrix metalloproteinase activities. TIMPs regulate matrix degradation both by proteinase elimination and by blockage of autolytic matrix metalloproteinase activation, expressed by fibroblast, keratinocytes, monocytes and endothelial cells (32). TIMP-1 forms complexes with all active collagenase but not procollagenase (30). TIMP-1 levels in cyclosporine group were significantly increased when compared to control group in this study, which is in accordance with study by Cotrim et al. (19) who found that TIMP-1 and TIMP-2 were increased in primary cultures of human gingival fibroblasts incubated with increasing concentrations of CsA. In contrary a few studies (18, 33) showed that the levels of TIMP-1 were reduced in primary cultures of human fibroblasts incubated with CsA compared to controls. Similarly in our study TIMP-1 level in tacrolimus group were significantly increased when compared to control group whereas previous study (20) reported that TIMP-1 mRNA levels were not significantly affected by FK-506 but observed slight increase when human gingival fibroblasts were incubated with FK-506 for 42 hrs. Even though cyclosporine group and tacrolimus group showed higher TIMP-1 level when compared to control group, the tacrolimus group had significantly lesser TIMP-1 level than the cyclosporine group. It has been reported that cyclosporine and tacrolimus had different effects on TIMP-1 gene expression in transplant glomeruli and that cyclosporine treated patients had significantly higher TIMP-1 level than tacrolimus treated patients (34). Reduced MMP levels observed in cyclosporine group could also be correlated with increased TIMP-1 level in our study. Another interesting finding we could infer was in spite of the increased TIMP-1 level, the tacrolimus group showed an increase in MMP levels. This suggests that tacrolimus may have an inducing effect on MMPs. Initiation of the transcription of the most matrix metalloproteinases requires the building of the transcription factor AP-1, to a specific promoter sequence of the gene. There is considerable evidence that protein kinase C plays a significant role in regulating gene expression of metalloproteinases by its control of AP-1. Activation of protein kinase C is a Ca++ dependant process which may be directly influenced by lowering of intracellular free calcium within the fibroblast. Cyclosporine is known to have an effect on the intracellular Ca++ levels by inhibiting it‘s entry into the cell (35). This mechanism by reducing MMP synthesis and release, both directly and indirectly, may be implicated in the pathogenesis of gingival overgrowth. Reduced MMP-1 and MMP-2 levels and increased TIMP-1 level seen in our study would offer an explanation for the increased collagen content in cyclosporine treated rats. Even though the pharmacodynamics of tacrolimus and cyclosporine are similar (35) tacrolimus was acting differently on collagen, MMPs and TIMPs. In spite of having increase in TIMP-1 level there was increased MMP levels too counteracting the collagen accumulation in the ingival connective tissue compartment in tacrolimus treated rats. The limitation of this study was the drug administration period was limited for 30 days. It has been reported that deleterious side effect of tacrolimus on the gingival tissues of rats may be time related as they could observe gingival overgrowth only after 180 days of drug administration (36). Hence the long term effect could not be determined with our experimental results. Nevertheless within the limits of this study it can be concluded that cyclosporine increases the collagen content and reduces MMP-1 and MMP- 2 whereas tacrolimus does not alter collagen content but increases MMP-1 and MMP-2 and cyclosporine increases TIMP-1 more than tacrolimus in rats. More studies are required to establish the long term effects of tacrolimus on gingiva and also further studies are needed to find out the mechanism by which cyclosporine and tacrolimus act differently on MMPs and TIMP.
The authors thank Dr Senthil kumar M Sc, PhD, Dr.P.K. Sehgal, M.Sc., PhD, Deputy Director and Head, Department of Bioproducts, Central Leather Research Institute, Chennai, for their valuable technical guidance.
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