Cyclosporin A inhibits production and activity of matrix metalloproteinases by gingival fibroblasts

Cyclosporin A (CyA) is a potent immunosuppressor used in organ transplantation and in the management of various autoimmune diseases. Gingival overgrowth is one of the side-effects of the CyA-treatment, affecting the attached gingiva of 25-81% of treated patients. To investigate the production and activity of matrix metalloproteinases (MMPs) in the CyA-induced gingival overgrowth, 2 well-documented models were utilized: the in vivo CyA-induced rat gingival overgrowth and primary cultures of human gingival fibroblasts treated with CyA. Our results obtained from the Western blot assays demonstrated clearly that the production of MMP-1 and MMP-3 was significantly inhibited by CyA at similar concentrations found in the serum of patients undergoing CyA-treatment. Moreover, the gelatinolytic activity of MMP-2 was also reduced both in cultured fibroblasts and in the rat CyA-induced gingival overgrowth. Taken together, the data presented here suggest that these inhibitory effects may contribute to the extracellular matrix (ECM) components accumulation in the CyA-induced gingival overgrowth.     

Regulation of matrix metalloproteinase production by cytokines,pharmacological agents and periodontal pathogens in human periodontal ligament fibroblast cultures

Matrix metalloproteinases (MMPs). produced by both infiltrating and resident cells of the periodontium, play a role in physiologic and pathologic events. It is recognized that an imbalance between activated MMPs and their endogenous inhibitors leads to pathologic breakdown of the extracellular matrix during periodontitis. To date, little is known about the regulation of MMP synthesis and secretion in human periodontal ligament fibroblasts (PDLFs). The purpose of this study was to examine the effects of cytokines, pharmacological agents (protein synthesis inhibitor and protein kinase C inhibitors) and predominant periodontal pathogens (Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis) on MMP production in human PDLFs using gelatin zymography. The gelatin zymograms revealed that the main gelatinase secreted by human PDLFs migrated at 72 kDa and represents MMP-2. Minor gelatinolytic bands were also observed at 92 kDa regions that correspond to MMP-9. We found that A. actinomycetemcomitans, P. gingivalis and IL-1alpha can elevate MMP-2 secretion in human PDLFs. These results indicate that periodontal pathogens and inflammatory cytokines play an important role in tissue destruction and disintegration of extracellular matrix in periodontal diseases. Thus, activation of MMPs may be one of the distinct host degradative pathways in the pathogenesis of periodontitis. In addition, H7, staurosporine, cycloheximide and TGF-beta could suppress MMP-2 production. Agents that target protein synthesis or the protein kinase C pathway in human PDLFs inhibit MMP-2 production, and such inhibition may contribute to the pathogenesis of periodontal inflammation. Taken together, these findings suggest a possible new therapeutic approach, involving the use of drugs that modify host-response mechanisms to suppress or inhibit MMP-mediated tissue destruction.     

缺氧对人牙周膜成纤维细胞基质金属蛋白酶和组织金属蛋白酶抑制物mRNA表达的影响

目的 探讨缺氧对人牙周膜成纤维细胞基质金属蛋白酶(matrix metalloproteinase,MMP)和组织金属蛋白酶抑制物(tissue inhibitors of matrix metalloproteinase,TIMP) mRNA表达的影响,以期为牙周组织修复再生机制研究提供依据.方法 组织块法培养人牙周膜成纤维细胞,分为缺氧组和常氧组(对照组).缺氧组细胞分别于1％O2、5％ CO2、94％N2的37℃培养箱中培养12、24和48 h(分别为12、24、48 h缺氧组);常氧组细胞在20％O2、5％ CO2、75％N2的37℃培养箱中培养.采用反转录聚合酶链反应技术检测MMP和TIMP相关基因的表达.缺氧组与常氧组间均数比较采用t检验,多组间比较采用单因素方差分析,两两比较采用LSD检验.结果 12、24、48 h缺氧组MMP-2 mRNA的表达呈明显递增趋势(分别为0.769±0.038、0.793±0.043、0.865±0.047),均显著高于常氧组(0.294±0.117),P＜0.01;12h缺氧组TIMP-1,2 mRNA的表达(分别为1.870±0.645、0.862±0.043)均有一过性升高,均分别显著高于常氧组TIMP-1,2 mRNA的表达(分别为0.816±0.043、0.426±0.097),P＜0.05.12h缺氧组MMP-1/TIMP-1 mRNA的比值(0.392±0.206)显著低于常氧组(0.859 ±0.126),P＜0.05;24、48 h缺氧组MMP-2/TIMP-2 mRNA的比值(分别为1.091±0.207、1.264±0.377)均显著高于常氧组(0.695±0.255),P＜0.05.结论 缺氧可以导致MMP-1、MMP-2、TIMP-1、TIMP-2 mRNA的表达,并使MMP-2 mRNA与TIMP-2 mRNA的比值失衡,这可能是缺氧导致牙周炎患者牙周组织破坏加重的机制之一.     

Endoplasmic reticulum stress modulates nicotine-induced extracellular matrix degradation in human periodontal ligament cells

Lee S‐I, Kang K‐L, Shin S‐I, Herr Y, Lee Y‐M, Kim E‐C. Endoplasmic reticulum stress modulates nicotine‐induced extracellular matrix degradation in human periodontal ligament cells. J Periodont Res 2012; 47: 299–308. © 2012 John Wiley & Sons A/S Background and Objective: Tobacco smoking is considered to be one of the major risk factors for periodontitis. For example, about half the risk of periodontitis can be attributable to smoking in the USA. It is evident that smokers have greater bone loss, greater attachment loss and deeper periodontal pockets than nonsmoking patients. It has recently been reported that endoplasmic reticulum (ER) stress markers are upregulated in periodontitis patients; however, the direct effects of nicotine on ER stress in regard to extracellular matrix (ECM) degradation are unclear. The purpose of this study was to examine the effects of nicotine on cytotoxicity and expression of ER stress markers, selected ECM molecules and MMPs, and to identify the underlying mechanisms in human periodontal ligament cells. We also examined whether ER stress was responsible for the nicotine‐induced cytotoxicity and ECM degradation. Material and Methods: Cytotoxicity and cell death were measured by 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5 diphenyltetrazolium bromide assay and flow cytometric annexin V and propidium iodide staining. The mRNA and protein expressions of MMPs and ER markers were examined by RT‐PCR and western blot analysis. Results: Treatment with nicotine reduced cell viability and increased the proportion of annexin V‐negative, propidium iodide‐positive cells, an indication of cell death. Nicotine induced ER stress, as evidenced by survival molecules, such as phosphorylated protein kinase‐like ER‐resident kinase, phosphorylated eukaryotic initiation factor‐2α and glucose‐regulated protein‐78, and apoptotic molecules, such as CAAT/enhancer binding protein homologous protein (CHOP). Nicotine treatment led to the downregulation of ECM molecules, including collagen type I, elastin and fibronectin, and upregulation of MMPs (MMP‐1, MMP‐2, MMP‐8 and MMP‐9). Inhibition of ER stress by salubrinal and transfection of CHOP small interfering RNA attenuated the nicotine‐induced cell death, ECM degradation and production of MMPs. Salubrinal and CHOP small interfering RNA inhibited the effects of nicotine on the activation of Akt, JNK and nuclear factor‐κB. Conclusion: These results indicate that nicotine‐induced cell death is mediated by the ER stress pathway, involving ECM degradation by MMPs, in human periodontal ligament cells.     

Effects of nicotine on periodontal ligament fibroblasts in vitro

Abstract . Cigarette smoking is associated with increased incidence of periodontal disease and poor response to therapy. In the present study, we examined the effects of nicotine on several functions of periodontal ligament fibroblasts (PDLF): proliferation, attachment, alkaline phosphatase production and chemotaxis. Nicotine concentrations varying from 5 ng/ml to 250 μg7sol;ml were tested. Proliferation of cells was studied by the incorporation of 3H-thymidine, and a dose-dependent inhibition was observed with concentrations ≥100 ng/ml. Similar results were observed when studying the attachment of the cells on plastic surfaces, using a colorimetric method. The inhibition of attachment was even more evident after 6 h incubation of the cells with nicotine. The activity of alkaline phosphatase, as determined with the substrate p-nitrophenyl phosphate, in both conditioned medium (CM) and cellular extract (CE), was also significantly decreased in a concentration-related fashion. Finally, the chemotaxis of PDLF, as examined by a modification of the Boyden's blind-well chamber technique, was inhibited in a dose-dependent manner. The degree of inhibition varied from 15% with the lowest concentration of nicotine (50 ng/ml), to almost 90% with the highest (5 μg/ml). The results show that nicotine can have direct adverse effects on various functions of the periodontal cells.     

机械应力对人牙周膜成纤维细胞整合素β1mRNA表达的调节

目的观察机械力刺激对人牙周膜成纤维细胞整合素β1 mRNA表达的影响。方法用Forcel四点弯曲加载装置通过对体外培养的人牙周膜成纤维细胞分别施加不同动态张、压应力（强度为1 000、2 000、4 000 μstrain，加力时间为0、0.5、1、4、8、12 h），采用实时荧光定量PCR法研究机械力对人牙周膜成纤维细胞整合素β1 mRNA表达的影响。结果施加动态的张、压应力后，人牙周膜成纤维细胞整合素β1 mRNA表达量下调，这种下调变化与所施加应力的性质、大小和作用持续时间相关。人牙周膜成纤维细胞整合素β1对张、压应力刺激的感应不完全一致，机械力刺激越强，整合素β1表达越明显。结论动态张、压应力刺激在本实验提供的微应力范围内可以促进目的基因mRNA表达改变，不同的机械力对细胞整合素β1效应不同。     

Matrix metalloproteinases and periodontal diseases

Periodontitis is a chronic inflammatory disorder characterized a complex interaction between periodontopathic bacteria and the host inflammatory response resulting in release of pro‐inflammatory cytokines leading to the destruction of periodontal tissues and alveolar bone. One of the important host factors involved in periodontal diseases is matrix metalloproteinases (MMPs), which is responsible for collagen and extracellular matrix (ECM) degradation of the periodontal tissues. MMPs comprise a family of around 25 members broadly categorized into six groups, which are involved in various physiological and pathological conditions. The activities of MMP are generally balanced by endogenous inhibitors such as tissue inhibitors of metalloproteinase (TIMP), and any imbalance between MMP and TIMP levels plays an important role in the disease progression. Assessment of MMP in tissues, GCF, and saliva may serve as an important biomarker in diagnosis of periodontal diseases and also for prognostic follow‐up. Targeted therapy aimed at reducing effects of MMP may serve as a useful adjunct for treatment of periodontitis. This review provides an overview of MMP and its role in various physiological and pathological conditions with emphasis on its association with periodontal diseases. A note on its inhibitors and therapeutic importance is also provided.     

Nicotine increases the collagen-degrading ability of human gingival fibroblasts

BACKGROUND AND OBJECTIVE: The objective of this study was to determine the effects that nicotine and the combination of nicotine and Porphyromonas gingivalis supernatant have on human gingival fibroblast-mediated collagen degradation. MATERIAL AND METHODS: Human gingival fibroblasts were cultured with 25-500 microg/ml of nicotine in collagen-coated six-well plates. On days 1-5, the conditioned media was collected for zymography and western blot analyses of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). The cells were then removed and the collagen cleavage visualized by Coomassie blue staining. To examine the combined effect, 250 microg/ml of nicotine and 10% v/v culture supernatant of P. gingivalis ATCC 33277 were added to the human gingival fibroblasts. The mRNA levels of multiple MMPs and TIMPs were monitored. RESULTS: Nicotine increased the human gingival fibroblast-mediated collagen cleavage. The MMP-14 and MMP-2 produced by the nicotine-treated human gingival fibroblasts more readily underwent zymogen activation. Nicotine treatment resulted in TIMP-2 redistribution to the cell surface. The mRNAs of multiple MMPs and TIMPs were unaltered by nicotine. An additive collagen cleavage effect was observed when the human gingival fibroblasts were treated with both nicotine and P. gingivalis. CONCLUSION: Nicotine increased human gingival fibroblast-mediated collagen degradation, in part through the activation of membrane-associated MMPs. Nicotine and P. gingivalis had an additive effect on human gingival fibroblast-mediated collagen degradation.     

Keratinocyte growth factor mRNA expression in periodontal ligament fibroblasts

Keratinocyte growth factor (KGF) is a fibroblast growth factor which mediates epithelial growth and differentiation. K.GF is expressed in subepithelial fibroblasts, but generally not in fibroblasts of deep connective tissue, such as fascia and ligaments. Here we demonstrate that KGF mRNA is expressed in periodontal ligament fibroblasts, and that the expression is increased upon serum stimulation. Fibroblasts from human periodontal ligament, from buccal mucosa. from gingival, and from skin were established from explants. Alkaline phosphatase activity was used as an indicator of the periodontal nature of fibroblasts. Cells were first cultured in DMEM with 0.5% fetal calf serum (PCS) and then incubated for 8 h, and 72 h in fresh DMEM with 10% PCS. Total RNA was isolated and used for Northern blotting with a P³²-labeled KGP cDNA. probe. Total RNA from cultured keratinocytes was used as negative controls. KGF mRNA was found in all cultured fibroblasts. Upon addition of 10% PCS to the cell cultures, an increase in KGF mRNA levels was noticed especially after 72 h. Furthermore. RT-PCR analysis of material scraped from the tooth root surface indicated the presence of KGF mRNA even in non cultured periodontal ligament cells.     

Cell-bound and extracellular matrix-associated alkaline phosphatase activity in rat periodontal ligament

In previous studies it was noted that alkaline phosphatase (ALP) activity in periodontal ligament does not only seem to be related to cells but may also be associated with the extracellular matrix. In an attempt to clarify this we studied the distribution of the enzyme at the electron microscopic level. In addition, ALPactivity was assessed biochemically following extraction of the ligament with (i) agents dissolving the membrane or splitting the phosphatidylinositol anchor (Triton X-100 or phosphatidylinositol-phospholipase C, respectively), and (ii) a matrix-degrading enzyme cocktail (collagenase, hyaluronidase and elastase). Histochemical observations revealed (a) a heterogeneous distribution of ALP-activity, with highest activity adjacent to the alveolar bone and (b) two pools of activity; one bound to cells and one associated with the collagenous extracelluJar matrix. In line with this were the biochemical data indicating that approximately 10% of the enzyme activity was firmly bound to the extracellular matrix and 90% to plasma membranes. Isoelectric focusing did not reveal differences between the two fractions, both samples yielding a single broad band corresponding with an isoelectric point of about 4.4.     

Adverse effects of arecoline and nicotine on human periodontal ligament fibroblasts in vitro

BACKGROUND, aims: The habit of betel nut chewing impinges on the daily lives of approximately 200 million people. Betel quid chewers have a higher prevalence of periodontal diseases than non-chewers. This study examined the pathobiological effects of arecoline, a major component of the betel nut alkaloids, on human periodontal ligament fibroblasts (PDLF) in vitro. METHOD: Cell viability, proliferation, protein synthesis, and cellular thiol levels were used to investigate the effects of human PDLF exposed to arecoline levels of 0 to 200 microg/ml. In addition, nicotine was added to test how it modulated the effects of arecoline. RESULTS: Arecoline significantly inhibited cell proliferation in a dose-dependent manner. At concentrations of 10 and 30 microg/ml, arecoline suppressed the growth of PDLF by 20% and 50% (p < 0.05), respectively. Arecoline also decreased protein synthesis in a dose-dependent manner during a 24-h culture period. A 100 microg/ ml concentration level of arecoline significantly inhibited protein synthesis to only 50% of that in the untreated control (p < 0.05). Moreover, arecoline significantly depleted intracellular thiols in a dose-dependent manner. At concentrations of 25 microg/ml and 100 microg/ml, arecoline depleted about 18% and 56% of thiols (p < 0.05), respectively. This suggests that arecoline itself might augment the destruction of periodontium associated with betel nut use. Furthermore, the addition of nicotine acted with a synergistic effect on the arecoline-induced cytotoxicity. At a concentration of 60 microg/ml, arecoline suppressed the growth of PDLF by about 33% and 5 mM nicotine enhanced the arecoline-induced cytotoxic response to cause about 66% cell death. CONCLUSION: During thiol depletion, arecoline may render human PDLF more vulnerable to reactive agents within cigarettes. Taken together, people who combine habits of betel nut chewing with cigarette smoking could be more susceptible to periodontium damage than betel nut chewing alone.     

Cigarette smoke condensate affects the collagen-degrading ability of human gingival fibroblasts

Background and Objective: Cigarette smoke condensate, the particulate matter of cigarette smoke, is composed of thousands of chemicals, including nicotine. Cigarette smoking is a risk factor for periodontal disease. This study investigated the influence of cigarette smoke condensate on the collagen‐degrading ability of human gingival fibroblasts and its mechanism. Material and Methods: Human gingival fibroblasts were exposed for 72 h to various concentrations of total particulate matter cigarette smoke condensate. Cell proliferation and cytotoxicity were evaluated using water‐soluble tetrazolium‐1 and lactate dehydrogenase, respectively. The collagen‐degrading ability of human gingival fibroblasts was evaluated in collagen‐coated six‐well plates. Conditioned media and membrane extracts were collected for zymography and western blot analyses of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Results: Cell proliferation decreased and cytotoxicity increased in human gingival fibroblasts with increasing concentrations of cigarette smoke condensate. Cell proliferation decreased by more than 50% (p < 0.05) when the concentrations of total particulate matter cigarette smoke condensate were above 200 μg/mL, and cytotoxicity increased to more than 30% (p < 0.05) when the concentrations of total particulate matter cigarette smoke condensate were above 400 μg/mL. Cigarette smoke condensate increased the collagen‐degrading ability of human gingival fibroblasts, especially at a concentration of 100 μg/mL (1.5‐fold increase, p < 0.05) compared with the control. Cigarette smoke condensate increased the production of proMMP‐1, proMMP‐2, MMP‐14 and TIMP‐1, and decreased the production of TIMP‐2, in conditioned media. Furthermore, compared with the control group, cigarette smoke condensate increased the production of MMP‐2, MMP‐14 and TIMP‐2 in membrane extracts, especially at concentrations of 50–100 μg/mL. Conclusion: Cigarette smoke condensate affects human gingival fibroblast proliferation and is toxic at total particulate matter cigarette smoke condensate concentrations of ≥ 400 μg/mL. Cigarette smoke condensate can increase the collagen‐degrading ability of human gingival fibroblasts by altering the production and localization of MMPs and TIMPs.     

Effects of enamel matrix derivative and transforming growth factor-beta1 on human periodontal ligament fibroblasts

AIM: The objective of this study was to evaluate the effects of enamel matrix derivative (EMD), transforming growth factor-beta1 (TGF-beta1), and a combination of both factors (EMD+TGF-beta1) on periodontal ligament (PDL) fibroblasts. MATERIAL AND METHODS: Human PDL fibroblasts were obtained from three adult patients with a clinically healthy periodontium, using the explant technique. The effects of EMD, TGF-beta1, or a combination of both were analysed on PDL cell proliferation, adhesion, wound healing, and total protein synthesis, and on alkaline phosphatase (ALP) activity and bone-like nodule formation. RESULTS: Treatment with EMD for 4, 7, and 10 days increased cell proliferation significantly compared with the negative control (p<0.05). At day 10, EMD and EMD+TGF-beta1 showed a higher cell proliferation compared with TGF-beta1 (p<0.01). Cell adhesion was significantly up-regulated by TGF-beta1 compared with EMD and EMD+TGF-beta1 (p<0.01). EMD enhanced in vitro wound healing of PDL cells compared with the other treatments. Total protein synthesis was significantly increased in PDL cells cultured with EMD compared with PDL cells treated with TGF-beta1 or EMD+TGF-beta1 (p<0.05). EMD induced ALP activity in PDL fibroblasts, which was associated with an increase of bone-like nodules. CONCLUSION: These findings support the hypothesis that EMD and TGF-beta1 may play an important role in periodontal regeneration. EMD induced PDL fibroblast proliferation and migration, total protein synthesis, ALP activity, and mineralization, while TGF-beta1 increased cellular adhesion. However, the combination of both factors did not positively alter PDL fibroblast behaviour.