Prevention of Phenytoin-Induced Gingival Overgrowth by Lovastatin in Mice
2015; Elsevier BV; Volume: 185; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2015.02.004
ISSN1525-2191
AutoresMohammad Assaggaf, Alpdoğan Kantarcı, Siddika Selva Sume, Philip C. Trackman,
Tópico(s)Peptidase Inhibition and Analysis
ResumoDrug-induced gingival overgrowth is caused by the antiseizure medication phenytoin, calcium channel blockers, and ciclosporin. Characteristics of these drug-induced gingival overgrowth lesions differ. We evaluate the ability of a mouse model to mimic human phenytoin-induced gingival overgrowth and assess the ability of a drug to prevent its development. Lovastatin was chosen based on previous analyses of tissue-specific regulation of CCN2 production in human gingival fibroblasts and the known roles of CCN2 in promoting fibrosis and epithelial to mesenchymal transition. Data indicate that anterior gingival tissue overgrowth occurred in phenytoin-treated mice based on gross tissue observations and histomorphometry of tissue sections. Molecular markers of epithelial plasticity and fibrosis were regulated by phenytoin in gingival epithelial tissues and in connective tissues similar to that seen in humans. Lovastatin attenuated epithelial gingival tissue growth in phenytoin-treated mice and altered the expressions of markers for epithelial to mesenchymal transition. Data indicate that phenytoin-induced gingival overgrowth in mice mimics molecular aspects of human gingival overgrowth and that lovastatin normalizes the tissue morphology and the expression of the molecular markers studied. Data are consistent with characterization of phenytoin-induced human gingival overgrowth in vivo and in vitro characteristics of cultured human gingival epithelial and connective tissue cells. Findings suggest that statins may serve to prevent or attenuate phenytoin-induced human gingival overgrowth, although specific human studies are required. Drug-induced gingival overgrowth is caused by the antiseizure medication phenytoin, calcium channel blockers, and ciclosporin. Characteristics of these drug-induced gingival overgrowth lesions differ. We evaluate the ability of a mouse model to mimic human phenytoin-induced gingival overgrowth and assess the ability of a drug to prevent its development. Lovastatin was chosen based on previous analyses of tissue-specific regulation of CCN2 production in human gingival fibroblasts and the known roles of CCN2 in promoting fibrosis and epithelial to mesenchymal transition. Data indicate that anterior gingival tissue overgrowth occurred in phenytoin-treated mice based on gross tissue observations and histomorphometry of tissue sections. Molecular markers of epithelial plasticity and fibrosis were regulated by phenytoin in gingival epithelial tissues and in connective tissues similar to that seen in humans. Lovastatin attenuated epithelial gingival tissue growth in phenytoin-treated mice and altered the expressions of markers for epithelial to mesenchymal transition. Data indicate that phenytoin-induced gingival overgrowth in mice mimics molecular aspects of human gingival overgrowth and that lovastatin normalizes the tissue morphology and the expression of the molecular markers studied. Data are consistent with characterization of phenytoin-induced human gingival overgrowth in vivo and in vitro characteristics of cultured human gingival epithelial and connective tissue cells. Findings suggest that statins may serve to prevent or attenuate phenytoin-induced human gingival overgrowth, although specific human studies are required. Gingival overgrowth is principally a tissue-specific adverse effect of the antiseizure drug phenytoin, antihypertensive calcium channel blockers, and the immunosuppressant ciclosporin (formerly cyclosporine A). Oral complications of gingival overgrowth include difficulty in maintaining adequate oral hygiene that can have systemic consequences related to excess inflammation. More than 3 million Americans have seizure disorders, 20% of whom continued to receive phenytoin principally because of grand mal epilepsy. Alternative medications can sometimes be prescribed, but this is not possible for patients with grand mal epileptic seizures, which are optimally treated with phenytoin. Some patients require the calcium channel blocker nifedipine to treat hypertension and cannot tolerate other antihypertensive drugs.1Ramon Y. Behar S. Kishon Y. Engelberg I.S. Gingival hyperplasia caused by nifedipine–a preliminary report.Int J Cardiol. 1984; 5: 195-206Abstract Full Text PDF PubMed Scopus (122) Google Scholar Similarly, although tacrolimus is now often substituted for cyclosporin, data indicate that gingival overgrowth still occurs, albeit with delayed onset and perhaps reduced severity.1Ramon Y. Behar S. Kishon Y. Engelberg I.S. Gingival hyperplasia caused by nifedipine–a preliminary report.Int J Cardiol. 1984; 5: 195-206Abstract Full Text PDF PubMed Scopus (122) Google Scholar, 2Rateitschak-Pluss E.M. Hefti A. Lortscher R. Thiel G. Initial observation that cyclosporin-A induces gingival enlargement in man.J Clin Periodontol. 1983; 10: 237-246Crossref PubMed Scopus (188) Google Scholar The current surgical therapy for drug-induced gingival overgrowth is gingivectomy. Gingivectomy is unsatisfactory because in many cases there is a need for repeated operations due to recurrence of the disease. In addition, the surgical approach leads to substantial patient discomfort and risk of infection. There remains, therefore, a need to investigate tissue-specific mechanisms and develop therapeutic approaches to treat this disfiguring and detrimental craniofacial disease. Analyses of tissue-specific characteristics of human gingival overgrowth clinical samples and in vitro pathway analyses using primary human gingival fibroblasts and epithelial cells have resulted in identification of specific pathological processes and pharmacological approaches to potentially prevent or alleviate gingival overgrowth.3Black Jr., S.A. Palamakumbura A.H. Stan M. Trackman P.C. Tissue-specific mechanisms for CCN2/CTGF persistence in fibrotic gingiva: interactions between cAMP and MAPK signaling pathways, and prostaglandin E2-EP3 receptor mediated activation of the c-JUN N-terminal kinase.J Biol Chem. 2007; 282: 15416-15429Crossref PubMed Scopus (65) Google Scholar, 4Black Jr., S.A. Trackman P.C. Transforming growth factor-beta1 (TGFbeta1) stimulates connective tissue growth factor (CCN2/CTGF) expression in human gingival fibroblasts through a RhoA-independent, Rac1/Cdc42-dependent mechanism: statins with forskolin block TGFbeta1-induced CCN2/CTGF expression.J Biol Chem. 2008; 283: 10835-10847Crossref PubMed Scopus (49) Google Scholar, 5Sume S.S. Kantarci A. Lee A. Hasturk H. Trackman P.C. Epithelial to mesenchymal transition in gingival overgrowth.Am J Pathol. 2010; 177: 208-218Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar These studies have focused on pathways that regulate CCN2, also known as connective tissue growth factor, which is highly elevated in phenytoin-induced gingival overgrowth and in idiopathic human gingival fibromatosis, which are both fibrotic, unlike some other forms of gingival overgrowth, which are not fibrotic.6Uzel M.I. Kantarci A. Hong H.H. Uygur C. Sheff M.C. Firatli E. Trackman P.C. Connective tissue growth factor in drug-induced gingival overgrowth.J Periodontol. 2001; 72: 921-931Crossref PubMed Scopus (130) Google Scholar CDC42 and RAC1 are small G-proteins that require lipid modification for activity and are critical mediators of transforming growth factor (TGF)-β1–induced CCN2 expression in a tissue-specific manner.4Black Jr., S.A. Trackman P.C. Transforming growth factor-beta1 (TGFbeta1) stimulates connective tissue growth factor (CCN2/CTGF) expression in human gingival fibroblasts through a RhoA-independent, Rac1/Cdc42-dependent mechanism: statins with forskolin block TGFbeta1-induced CCN2/CTGF expression.J Biol Chem. 2008; 283: 10835-10847Crossref PubMed Scopus (49) Google Scholar Lipid modifications of CDC42 and RAC1 are required for activation and are derived from the cholesterol biosynthetic pathway.7Alegret M. Silvestre J.S. Pleiotropic effects of statins and related pharmacological experimental approaches.Methods Find Exp Clin Pharmacol. 2006; 28: 627-656Crossref PubMed Scopus (50) Google Scholar Inhibition of the cholesterol biosynthetic pathway by the HMG-CoA reductase inhibitors lovastatin and simvastatin inhibits TGF-β1–induced CCN2 expression in primary human gingival fibroblasts.4Black Jr., S.A. Trackman P.C. Transforming growth factor-beta1 (TGFbeta1) stimulates connective tissue growth factor (CCN2/CTGF) expression in human gingival fibroblasts through a RhoA-independent, Rac1/Cdc42-dependent mechanism: statins with forskolin block TGFbeta1-induced CCN2/CTGF expression.J Biol Chem. 2008; 283: 10835-10847Crossref PubMed Scopus (49) Google Scholar Lovastatin is an approved drug in widespread use for the treatment of hypercholesteremia. Therefore, we wished to determine whether in vivo administration of lovastatin could prevent the development of gingival overgrowth in a mouse model. We report both the development of a mouse model of phenytoin-induced gingival overgrowth in mice and its use to determine the effectiveness of lovastatin to prevent the development of gingival overgrowth and to normalize the expression of Ccn2 and other proteins associated with phenytoin-induced gingival overgrowth. Data are consistent with earlier in vitro studies that identified molecular pathways that drive phenytoin-induced gingival overgrowth in humans in a tissue-specific manner.3Black Jr., S.A. Palamakumbura A.H. Stan M. Trackman P.C. Tissue-specific mechanisms for CCN2/CTGF persistence in fibrotic gingiva: interactions between cAMP and MAPK signaling pathways, and prostaglandin E2-EP3 receptor mediated activation of the c-JUN N-terminal kinase.J Biol Chem. 2007; 282: 15416-15429Crossref PubMed Scopus (65) Google Scholar, 4Black Jr., S.A. Trackman P.C. Transforming growth factor-beta1 (TGFbeta1) stimulates connective tissue growth factor (CCN2/CTGF) expression in human gingival fibroblasts through a RhoA-independent, Rac1/Cdc42-dependent mechanism: statins with forskolin block TGFbeta1-induced CCN2/CTGF expression.J Biol Chem. 2008; 283: 10835-10847Crossref PubMed Scopus (49) Google Scholar Phenytoin was purchased from Henry Schein Corporation (NDC 0641-0493; Roswell, GA). Lovastatin was purchased from EMD Millipore (Calbiochem, 438186; Billerica, MA). Physiological sterile saline was purchased from the Laboratory Animal Science Center at Boston University. Propylene glycol was purchased from MP Biomedical, (151957; Santa Ana, CA). Harris modified hematoxylin (SH-30), eosin B (E514), and eosin Y (E511) were purchased from Fisher Scientific (Hampton, NH). Rabbit polyclonal anti-Tgfβ1 IgG and blocking peptide were purchased from Santa Cruz Biotechnology (sc-146; Dallas, TX). Rabbit polyclonal anti-CTGF/CCN2 (ab6992) antibody was purchased from Abcam (Cambridge, MA). Rabbit polyclonal anti LOXL2 IgG (105085) was purchased from GeneTex (Irvine, CA) and mouse LOXL2 shRNA plasmids were purchased from Sigma (St Louis, MO). Mouse monoclonal anti E-cadherin IgG was purchased from BD Bioscience (610181; San Jose, CA). Immunoperoxidase detection kits and reagents were purchased from Vector Laboratories (Burlingame, CA). Human gingival samples were collected from patients with severe gingival overgrowth who were treated with phenytoin for epilepsy and subjected to gingivectomy and from systemically healthy individuals undergoing crown-lengthening surgery. Samples were collected at the Department of Periodontology and Oral Biology and the Clinical Research Center of Boston University at the Goldman School of Dental Medicine and the Franciscan Children's Hospital and Rehabilitation Center. Informed consent forms were signed by all donors. The consent forms and study protocols were approved by the Institutional Review Board of Boston University Medical Center. All excised tissues were fixed in 4% paraformaldehyde in phosphate-buffered saline at 4°C for 4 hours and then incubated in cold 30% sucrose overnight. Tissues were then stored in 2-methylbutane at −80°C. At least twenty 5-μm serial sections per sample were made on a cryostat and were stored at −80°C. Eight-week-old BALB/cByJ male mice were purchased from Jackson Laboratory (Cat# 001026; Bar Harbor, ME). Mice were housed for 1 week before any procedure for acclimation. Mice were then divided into groups. Control groups consisted of a negative control group receiving physiological saline (NaCl 0.9%, n = 8) and a control group receiving vehicle (50% water, 41% propylene glycol, and 9% ethanol) (n = 3). The treatment groups consisted of a group receiving phenytoin (NDC 0641-0493, 30 mg/kg; Henry Schein Corporation) (n = 10) and a group receiving phenytoin plus lovastatin (0.65 mg/kg) (n = 7). Phenytoin was supplied from the vendor as pharmaceutical-grade phenytoin sodium in liquid form with a concentration of 50 mg/mL in 50% water, 41% propylene glycol, and 9% ethanol. Lovastatin was supplied as the lovastatin sodium salt and was dissolved directly in either 4.2 mL of phenytoin sodium solution or 4.2 mL of the vehicle (50% water, 41% propylene glycol, and 9% ethanol) to give a concentration of 1.13 mg/mL of lovastatin. Phenytoin, control vehicle, or physiologic saline were delivered with miniosmotic pumps (ALZET Osmotic Pumps, 2002, Cupertino, CA) at a 0.5-μL/h delivery rate for a period of 2 weeks. Pumps were replaced every 2 weeks as needed for the duration of the experiment. Lovastatin, when present, was dissolved in the same vehicle as phenytoin and when administered together with phenytoin was dissolved in the same solution and applied simultaneously in a single osmotic minipump. Although a detailed time course for gingival overgrowth development was not performed, a pilot study indicated that only minimal levels of gingival overgrowth could be observed at an experimental period of 6 weeks. Therefore, the duration of the experimental period reported here was 12 weeks. The dose of lovastatin of 0.65 mg/kg is equivalent to an intermediate human dose, which ranges from 0.3 to 1.2 mg/kg.8Endo A. The discovery and development of HMG-CoA reductase inhibitors.J Lipid Res. 1992; 33: 1569-1582Abstract Full Text PDF PubMed Google Scholar Mice were preinjected with buprenorphine for analgesia 30 minutes before surgery. Then, mice were placed in an induction chamber to induce anesthesia with isoflurane. Fur was shaved from the mouse posterior to the shoulder, and skin was wiped with povidone-iodine and 70% ethanol. Using a scalpel blade 11, a small incision was made, and then blunt dissection was performed with a hemostat. A pump that contained physiological saline, vehicle, lovastatin, phenytoin, or phenytoin plus lovastatin was placed subcutaneously. Then, the wound was closed with autoclips (427631; Clay Adams, New York, NY). All pumps were primed in sterile physiological saline overnight at 37°C to ensure immediate release of the drugs on implantation. Pumps were replaced every 2 weeks throughout the 12-week experimental period. Mice were then euthanized and heads were immediately placed in freshly made 4% paraformaldehyde for 24 hours for fixation. After fixation, mouse heads were washed under running tap water to remove excess fixative for 3 minutes. Then, each head was immersed in 50 mL 0.5 mol/L EDTA at pH 8.0 for decalcification. The EDTA was changed every other day, and the heads were incubated with EDTA at 4°C on a shaker for a 6- to 8-week period. Serial X-rays assessed the degree of decalcification. Anterior maxillae and mandibles were separated from posterior dentition. Samples were trimmed so that each sample included either maxillary central incisors or maxillary molars or mandibular incisors or mandibular molars, with their respective surrounding gingival tissues. Gross morphology images were taken with a stereomicroscope (Carl Zeiss AG, Jena, Germany). To compare the gross morphological differences among control and treatment groups, papillary height and papillary area were analyzed on labial and buccal gingival tissue of anterior and posterior upper and lower jaws. All measurements were performed using the Olympus Microsuite Five software version 5 (Richmond Hill, ON, Canada). Means of these measurements were calculated and subjected to one-way analysis of variance statistical analysis with the Bonferroni post hoc test. For embedding, samples were dehydrated in serial ethanol concentrations: 50%, 70%, 95%, and 100% for half a day, overnight, half a day, and overnight, respectively. Samples were then placed in 100% acetone followed by 100% chloroform at 60°C for 45 minutes each. Samples were placed in paraffin for 3 successive hourly changes. Samples were then embedded in paraffin using metal molds, cooled down at room temperature, and kept at 4°C overnight. The orientation of the samples during embedding was set up for sagittal sectioning (Figure 1A), whereas posterior tissues were for transverse sectioning. Sectioning was performed using a microtome with stainless steel blades (Fisher Scientific) producing 5-μm- thick sections. Slides were baked at 60°C for 2 hours to adhere sections to slides. For each sample, each fifth slide (three sections per slide) was stained with hematoxylin-eosin to determine the range of the fine sections that were used for histomorphometric and immunohistochemistry (IHC) analyses. Slides were selected that contained buccal and lingual gingival tissue and the decalcified tooth structure that provided a landmark for orientation. Two different parameters were measured in three representative central sections from mandibles (lower jaw) and maxillae (upper jaw) from both the anterior and posterior teeth to perform a comparative histomorphometric analysis. The variables were the width of the epithelium and the area of the epithelium. For measuring the width of the epithelium, at least 10 arbitrary distances were measured from the outer layer of the epithelium to the basement membrane in buccal and lingual gingival tissue, separately. Then, the means of these measurements were calculated, and one-way analysis of variance with the Bonferroni post hoc test statistical analysis was performed. For measuring the area of the epithelium, two area measurements for each of the buccal and lingual sides from each representative section were obtained, and the means were calculated followed by one-way analysis of variance with the Bonferroni post hoc statistical analyses. All measurements were performed using the Olympus Microsuite software. Significance was declared at P < 0.05. The expression levels of different epithelial to mesenchymal transition (EMT) and fibrosis markers were assessed by IHC. Four sections per sample per marker were assayed. The IHC technique used heat-based antigen retrieval followed by immunoperoxidase staining. Sections were deparafinized in xylene for 10 minutes, followed by rehydration in serial concentrations of ethanol down to 75% and distilled water. Then, sections were subjected to high-temperature antigen retrieval in 10 mmol/L citric acid at pH 6.0. Endogenous peroxidase was inactivated with 3% hydrogen peroxide in methanol for 30 minutes. Endogenous immunoglobulin blocking was performed using 10% normal serum in phosphate-buffered saline from the same species in which the secondary antibody was raised for 30 minutes. Endogenous avidin and biotin were blocked for 20 minutes each to eliminate nonspecific binding and background. Rabbit polyclonal primary antibodies were used for detection of TGF-β1, CCN2, and LOXL2. A mouse monoclonal primary antibody was used for detection of E-cadherin. Sections stained with nonimmune rabbit and mouse IgG primary antibody were used as the negative control. Sections were incubated with primary antibody diluted in 5% normal serum in phosphate-buffered saline overnight at 4°C. Bound primary antibody was detected using Vectastain Elite kits (Vector Laboratories) with biotinylated secondary antibody for 30 minutes and immunoperoxidase staining as described by the manufacturer. For IHC analysis of LOXL2 expression in human tissues, frozen sections were fixed in 100% acetone for 10 minutes at 37°C and stained as previously described.9Hong H.H. Uzel M.I. Duan C. Sheff M.C. Trackman P.C. Regulation of lysyl oxidase, collagen, and connective tissue growth factor by TGF-beta1 and detection in human gingiva.Lab Invest. 1999; 79: 1655-1667PubMed Google Scholar Antibody specificity for CCN2 was established by Western blotting of nontreated and TGF-β1–treated gingival fibroblasts; for TGF-β1, this was established by probing mouse gingival tissue sections with anti–TGF-β1 alone or with anti–TGF-β preabsorbed with blocking peptide; for E-cadherin, by Western blotting of cultured gingival epithelial cells; and for LOXL2, by shRNA knockdown of LOXL2 and Western blotting of extracts of control and Ccn2 knockdown gingival fibroblasts (Supplemental Figure S1). Threshold analysis was used to quantify the percentage of stained area in two representative sections from each sample using ImageJ version 1.46 (NIH, Bethesda, MD). In this technique, the threshold of a digital image is composed of three different variables: hue, saturation, and brightness. By setting the hue and saturation to maximum and adjusting the brightness, the total area of tissue was determined. Next, by adjusting the hue and brightness again, the area of stained tissue in the section was determined.10Van Bruaene N.N. Derycke L.L. Perez-Novo C.A.C. Gevaert P.P. Holtappels G.G. De Ruyck N.N. Cuvelier C.C. Van Cauwenberge P.P. Bachert C.C. TGF-beta signaling and collagen deposition in chronic rhinosinusitis.J Allergy Clin Immunol. 2009; 124: 253-259Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar Then, the percentage of the stained area normalized to the total tissue area in each section was calculated. One-way analysis of variance statistical analyses with Bonferroni post hoc tests were finally performed. Gross gingival morphology differences between groups were investigated, and gingival overgrowth was apparent in the maxillary anterior gingival tissue of phenytoin-treated mice compared with control and phenytoin plus lovastatin–treated mice (Figure 1B). Quantitative analyses (Figure 1, C and D) revealed a trend toward an increase of papillary height in the phenytoin group compared with the control group (P = 0.094) and a significant reduction of the papillary height in the phenytoin plus lovastatin group when compared with phenytoin (P = 0.046). Area measurements revealed a significant increase in the phenytoin group when compared with the control group (P = 0.028). A comparison between the area measurements of the phenytoin group and the phenytoin plus lovastatin group revealed a trend toward reduction in the papillary area of the phenytoin plus lovastatin group (P = 0.094). No gingival overgrowth could be identified around the anterior mandibles or in the posterior mandible or maxillary molar areas (data not shown). Thus, data suggest that phenytoin successfully induced gingival overgrowth in anterior regions under these experimental conditions. Lovastatin appears to prevent gingival overgrowth development and is further analyzed below. Quantitative histomorphometric analysis of tissue sections from all mouse groups revealed increased epithelial thickness and area in the maxillary anterior zone of phenytoin-treated mice. In addition, lovastatin appeared to reduce the hyperplastic effect of phenytoin on gingival tissue as was observed in the phenytoin plus lovastatin–treated mouse group (Figure 2). The phenytoin-treated group had a 2.1-fold increase in labial epithelial width and a 2.7-fold increase in epithelial palatal width, implying an increased epithelial volume. In addition, the phenytoin group had higher values of epithelial area with a 1.9-fold labial increase and a 1.6-fold increase in palatal area compared with the control group. Interestingly, administration of both lovastatin and phenytoin together prevented the hyperplastic effect of phenytoin and resulted in the width and area of the epithelium essentially equivalent to control levels (Figure 2A). By contrast, maxillary and mandibular posterior gingival epithelia had either no differences or inconsistent results (data not shown). Mice treated with lovastatin alone had no alteration in histomorphometric parameters compared with saline or vehicle controls (Supplemental Figure S2). In summary, histomorphometric analysis revealed that phenytoin increases the gingival epithelial volume in the maxillary anterior zone and that lovastatin prevents development of the hyperplastic effect of phenytoin. These observations were greater on the palatal (lingual) side than on the labial side of the gingival tissue. Ccn2 was expressed mostly in the epithelium in all three groups, with some connective tissue expression in the phenytoin group, which was seen only in three phenytoin samples. Qualitatively, the phenytoin group had increased intensity of staining, suggesting overexpression of Ccn2 in all areas of the epithelium when compared with the control and the phenytoin plus lovastatin groups (Figure 3, A and B). Quantitative analysis of Ccn2 staining was consistent with these features except that the effect of lovastatin was significant for the palatal tissues and not for the labial tissues (Figure 3, C and D). Although Ccn2 was detected in the connective tissue stroma, no obvious regulation by phenytoin or lovastatin could be discerned under these conditions. We conclude that phenytoin causes overexpression of Ccn2 primarily in the epithelium of the phenytoin-treated mice and that lovastatin largely prevented the phenytoin-induced overexpression of Ccn2, especially in palatal anterior gingiva. TGF-β is a major inducer of CCN2. We therefore investigated expression of TGF-β1 by IHC in these mouse tissues. Data from mouse tissue sections assayed with anti–TGF-β1 antibody revealed that phenytoin alone increased TGF-β staining in both the gingival epithelium and connective tissue in anterior gingival tissues when compared with the control group (Figure 4, A and B). Interestingly, lovastatin in the phenytoin plus lovastatin group prevented or diminished phenytoin-induced TGF-β1 expression in gingival epithelium and connective tissues. The connective tissue expression of TGF-β1 observed was both extracellular and intracellular. The intensity of the staining was found to be higher on the palatal side than the labial side of the gingival tissue (Figure 4, A and B). Interestingly, connective tissue staining for Tgf-β was similarly regulated in these tissues. Quantitative analysis revealed that the phenytoin-treated mice exhibited 3.7-fold higher TGF-β1 levels in the labial gingiva and 2.3-fold higher levels in the palatal gingiva compared with the control group (Figure 4, C and D). Mice treated with phenytoin plus lovastatin had significantly reduced levels of TGF-β1 staining, even slightly lower than levels seen in the control group, confirming our qualitative findings. Data indicate that TGF-β1 was overexpressed in the phenytoin-treated mice in both the epithelium and connective tissue. This overexpression was significantly reduced in the phenytoin plus lovastatin group. Ccn2 and TGF-β are drivers of EMT in epithelial tissues. Therefore, we next stained tissues for the classic epithelial cell marker E-cadherin, which is down-regulated as cells undergo EMT. E-cadherin staining revealed a lower intensity in phenytoin-treated mice compared with both control and lovastatin-treated mice (Figure 5, A and B). Quantitative analyses of areas stained suggest a partial restoration by lovastatin of approximately 25% of E-cadherin staining on the palatal side compared with phenytoin alone, whereas there was no apparent effect of lovastatin on the labial side (Figure 5, C and D). Besides intense epithelial staining, there was some nonspecific immunoreactivity observed in the connective tissue and periodontal ligament area in all groups, including the nonimmune control. However, the nonimmune control exhibited no epithelial staining, which supports that epithelial E-cadherin staining is specific. Similar data were obtained with a polyclonal E-cadherin antibody (data not shown). It can be concluded that phenytoin strongly inhibits E-cadherin staining in gingival epithelia, whereas lovastatin has a limited ability to restore this expression on palatal tissues. Evidence has been presented that LOXL2 is a driver of EMT in the context of cancer.11Millanes-Romero A. Herranz N. Perrera V. Iturbide A. Loubat-Casanovas J. Gil J. Jenuwein T. Garcia de Herreros A. Peiro S. Regulation of heterochromatin transcription by Snail1/LOXL2 during epithelial-to-mesenchymal transition.Mol Cell. 2013; 52: 746-757Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 12Moreno-Bueno G. Salvador F. Martín A. Floristán A. Cuevas E.P. Santos V. Montes A. Morales S. Castilla M.A. Rojo-Sebastián A. Martínez A. Hardisson D. Csiszar K. Portillo F. Peinado H. Palacios J. Cano A. Lysyl oxidase-like 2 (LOXL2), a new regulator of cell polarity required for metastatic dissemination of basal-like breast carcinomas.EMBO Mol Med. 2011; 3: 528-544Crossref PubMed Scopus (136) Google Scholar Additional ongoing studies suggest that LOXL2 expression may contribute to human gingival overgrowth (D. Saxena and P.C. Trackman, unpublished data). We, therefore, wanted to determine whether Loxl2 was expressed in the epithelium of phenytoin-treated mice and whether lovastatin would modulate this expression. Staining of mouse tissue sections with anti-LOXL2 antibody revealed epithelial expression in all groups, and connective tissue expression was seen only in one phenytoin sample on the palatal side. The intensity of LOXL2 staining appeared to be slightly higher in phenytoin-treated mice compared with the control, whereas lovastatin appeared to lower the intensity of LOXL2 expression belo
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