Increased plasminogen activator inhibitor-1/tissue type plasminogen activator ratio in oral submucous fibrosis

OBJECTIVE: Plasminogen activators and their inhibitors are thought to be key participants in the balance of proteolytic and antiproteolytic activities that regulate extracellular matrix (ECM) turnover. However, little is known about the expression of plasminogen/plasmin system at the site of oral submucous fibrosis (OSF). METHODS: We compared the activities of tissue type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) between fibroblasts derived from normal buccal mucosa and OSF by using an enzyme-linked immunosorbent assay. Furthermore, arecoline, a major areca nut alkaloid, was challenged with normal buccal mucosal fibroblasts (BMFs) to elucidate whether the activities of t-PA and PAI-1 could be affected by arecoline. RESULTS: Both t-PA and PAI-1 were found to be increased in OSF than in BMFs (P < 0.01). In addition, there was a statistically significant difference in PAI-1/t-PA ratio between OSF and BMF (P < 0.01). The addition of arecoline upregulated not only PAI-1, but also t-PA in BMFs (P < 0.05). In addition, the ratio between PAI-1 and t-PA was found to be significantly increased by a linear regression assay (P < 0.01). CONCLUSION: These results suggest that OSF caused by areca quid chewing may be the result of an imbalance in the plasminogen/plasmin system, the net result of which is increased deposition of ECM.     

Elevated transglutaminase-2 expression mediates fibrosis in areca quid chewing-associated oral submucocal fibrosis via reactive oxygen species generation

Objectives

Transglutaminase-2 (TGM-2) protein is involved in the cross-linking of matrix proteins resulting in several fibrotic disorders and can be induced by reactive oxygen species (ROS). Little is known about its role in the development of oral submucocal fibrosis (OSF). Hence, we hypothesize that TGM-2 may have a role in the pathogenesis of areca quid chewing-associated OSF and arecoline, a major areca nut alkaloid, could regulate TGM-2 via ROS generation.

Materials

Forty OSF specimens from areca quid chewing-associated OSF and ten normal buccal mucosa biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The expression of TGM-2 from fibroblasts cultured from OSF and normal buccal mucosa was evaluated by Western blot. The effect of arecoline on normal buccal mucosa fibroblasts (BMFs) was used to elucidate whether TGM-2 expression could be affected by arecoline by using 2′, 7′-dichlorofluorescein diacetate assay and Western blot. In addition, glutathione precursor N-acetyl-l-cysteine (NAC) and epigallocatechin-3 gallate (EGCG) were added to find the possible regulatory mechanisms.

Results

TGM-2 expression was significantly higher in OSF specimens than normal specimens (p < 0.05). Fibroblasts derived from OSF were found to exhibit higher TGM-2 expression than BMFs in protein levels (p < 0.05). Arecoline significantly upregulated the intracellular ROS generation in a dose-dependent manner (p < 0.05). TGM-2 protein induced by arecoline was found in BMFs in a dose-dependent manner (p < 0.05). Treatment with NAC and EGCG markedly inhibited TGM-2 expression induced by arecoline (p < 0.05).

Conclusions

Our results suggest that TGM-2 expression is significantly upregulated in OSF tissues from areca quid chewers. Arecoline-upregulated TGM-2 expression may be mediated by ROS generation.

Clinical relevance

TGM-2 protein is upregulated in areca quid chewing-associated OSF. Using this in vitro model, antioxidants could inhibit arecoline-upregulated TGM-2 expression. NAC and EGCG may serve as a useful agent in controlling OSF.

     

Regulation of interleukin-6 expression by arecoline in human buccal mucosal fibroblasts is related to intracellular glutathione levels

OBJECTIVES: Cytokines play an important role in regulating fibroblast function and is likely to play a key role in regulating the initiation and progression of scarring in any fibrotic disease. Interleukin-6 (IL-6) has been implicated in the development of a variety of fibrotic diseases. The aim of this study was to compare IL-6 expression in fibroblasts cultured from normal human buccal mucosa and oral submucous fibrosis (OSF) specimens and further explore the potential mechanism that may lead to induce IL-6 expression. METHODS: mRNA level of IL-6 in fibroblasts from OSF was compared with normal buccal mucosa. The effects of arecoline, the major areca nut alkaloid, on IL-6 expression in normal human buccal mucosa fibroblasts (BMFs) were measured in vitro. mRNA was quantified with AlphaImager 2000. To determine whether glutathione (GSH) levels were important in the induction of IL-6 by arecoline, we pretreated cells with 2-oxothiazolidine-4-carboxylic acid (OTZ) to boost GSH levels or with buthionine sulfoximine (BSO) to deplete GSH. RESULTS: Fibroblasts derived from OSF exhibited higher IL-6 gene expression than BMF in mRNA levels (P < 0.05). The exposure of quiescent BMF to arecoline resulted in the elevation of IL-6 mRNA expression in a dose-dependent manner (P < 0.05). IL-6 gene regulated by arecoline correlated with intracellular GSH levels in BMF. Arecoline at a concentration of 129 muM induced about 2.7-fold IL-6 mRNA levels over the 6-h incubation period. However, BSO enhanced the IL-6 mRNA levels by 3.9-fold (P < 0.05). In addition, OTZ was found to marginally reduce the arecoline-induced IL-6 expression by about 1.7-fold (P < 0.05). CONCLUSIONS: Taken together, these results suggest that IL-6 expression is significantly upregulated in OSF fibroblasts in areca quid chewers and arecoline may be responsible for the enhanced IL-6 expression. In addition, the regulation of IL-6 expression induced by arecoline is critically dependent on the intracellular GSH concentrations.     

Therapeutic Interventions in Oral Submucous Fibrosis: An Experimental and Clinical Study

Introduction

Oral submucous fibrosis (OSF) is a chronic debilitating disease and premalignant condition of the oral cavity and is a serious public health issue in India and many parts of the world. The treatment is still elusive and empirical because of poorly understood etiopathogenesis, which is believed to be multifactorial including areca nut chewing, ingestion of chillies, genetic and immunologic processes, nutritional deficiencies, and many others. The present investigations was focused to understand the possible therapeutic interventions of anti-OSF agents in arecoline induced experimental in vitro model of OSF and clinical application of these anti-OSF agents in the restoration of various grade of the disease.

Materials and Methods

The 127 subjects were selected from patients who visited the OPD of Department of Oral and Maxillofacial Surgery, Faculty of Dental Sciences, K.G. Medical University, Lucknow. Further the subjects were divided in two groups on the basis of clinical examination. Group-1 subjects showed presence of fibrosis bands in the labial and/or buccal mucosa, loss of elasticity, difficulty to open the mouth and had a habit chewing areca-nut in some form. Group-2 subjects had no habit of chewing areca-nut, were apparently healthy with no mucosal disorder. The samples were collected and were immediately transported to Indian Institute of Toxicology Research, Lucknow, for isolation and cultivation of primary cultures of mucosal fibroblast cells. Then isolation and cultivation of oral mucosal fibroblast, identification of non-cytotoxic doses of arecoline, real time PCR, immunocytochemistry, cytokine determination in culture cells, western blot analyses, functional activity of collagenase, lysyl oxidase enzyme activity, collagen beads assay, cyclooxygenase (COX-2) expression analysis was done.

Results and Conclusions

This study, shows that the reduction of phagocytic cells was strongly related to the arecoline levels in fibroblast culture when we exposed arecoline to normal oral mucosal cells (NOMC) and cells from OSF patient. An enhancement of phagocytic cells was observed following the pre exposure of cells to 1 μM dexamethasone, a glucocorticoids, In this study, histologic evidence is presented which supports the finding that COX-2 expression is upregulated in OSF specimens compared to normal oral submucosal cells. Strong immunostaining for COX-2 was detected in arecoline exposed NOMC and cells from OSF patient. Areca nut extract up-regulates prostaglandin production, cyclooxygenase-2 mRNA and protein expression of human oral keratinocytes. The number of phagocytic cells and phagocytic activity in cultured human oral fibroblasts from OSF site was lower than the fibroblasts from the normal regions of the same person.     

Growth of oral and skin fibroblasts from patients with oral submucous fibrosis

The purpose of the investigation was to evaluate and compare the proliferation (growth) of mouth fibroblasts and skin fibroblasts from patients with oral submucous fibrosis (OSF). Material comprised fibroblasts from fibrous bands situated in the buccal mucosa and from the inner aspect of the forearm of 8 patients with classic features of OSF as well as fibroblasts from 6 buccal mucosa and 8 skin biopsy specimens from healthy non-areca nut chewing individuals. Cells were cultured for 8 days according to standard techniques. Their growth was monitored daily, under optimal conditions as well as exposure to concentrations of arecoline. The data were analyzed using regression analysis, analysis of variance and the Kruskal-Wallis test. We found no statistically significant differences between the proliferation patterns of oral and skin fibroblasts from patients or between those from patients and controls. The reaction of the cells exposed to concentrations of arecoline was similar; at low concentrations (0.1–10 μg/ml) normal growth was maintained, while 100 μg/ml inhibited growth. It is concluded that fibroblasts from mouths affected by OSF have proliferation patterns which fall within normal parameters, that the excessive collagen formation in established OSF is not due to increased fibroblast proliferation and that arecoline does not stimulate fibroblast proliferation.     

槟榔提取物抑制人类口腔黏膜成纤维细胞生长的实验研究

目的通过比较正常口腔黏膜和口腔黏膜下纤维性变(OSF)组织中成纤维细胞(FB)增殖差异、检测槟榔提取物(ANE)对成纤维细胞增殖的影响,来探讨OSF的发病机理.方法对人类口腔黏膜成纤维细胞进行分离培养,然后用四唑盐(MTT)比色试验法检测OSF患者和正常人口腔黏膜FB增殖状况,并且观察ANE对FB增殖的影响.结果表示增殖水平的OD值在OSF-FB为0.254±0.045,高于NM-FB的OD值0.236±0.012(P<0.05),ANE以浓度-效应依赖关系抑制FB增殖.结论 OSF-FB细胞增殖率较NM-FB高;ANE对口腔黏膜FB有细胞毒作用,提示槟榔及其有效成分不完全是通过直接刺激FB增殖而诱发OSF.     

The fibroblast population in oral submucous fibrosis

The purpose of the investigation was to compare the morphology of fibroblasts cultured from healthy oral mucosa and mucosa of patients with oral submucous fibrosis (OSF) and to collate the occurrence of cell types of similar morphology. Cells cultured from biopsy specimens from the buccal mucosa of six subjects who did not chew the areca nut and six patients with OSF who chewed areca nut were grown according to standard techniques. Ninety cells per cell line were recorded daily for 8 days, classified into types F1, F2 and F3 according to their morphology, and the results statistically analyzed. We found that there was a relative increase of F3 cells in relation to Fl cells in OSF resulting in the ratio of F3 to F1 cells being significantly larger in OSF than the ratio in the controls. As it has been reported that F3 cells m rat connective tissues produce significantly more collagen types I and III than F1 cells, we concluded that a change of fibroblast population has occurred in OSF and that this relative increase of F3 cells in humans, which could be committed to the production of large quantities of collagen, can be an explanation for the excessive collagen formation in OSF.     

口腔黏膜下纤维性变中MMP-2基因的表达及意义

目的：检测口腔黏膜下纤维性变中基质金属蛋白酶-2（MMP-2）的表达并探讨其病理意义.方法：抽提11例口腔黏膜下纤维化组织和10例正常口腔黏膜组织的总RNA,通过逆转录聚合酶链反应（PF-PCR）检测MMP-2mRNA在口腔黏膜下纤维性变患者颊黏膜中的表达并与正常口腔黏膜进行比较.结果：口腔黏膜下纤维性变患者颊黏膜组织中MMP-2 mRNA表达高于正常颊黏膜（p＜0.05）.结论：MMP-2基因的表达与口腔黏膜下纤维性变组织重塑过程密切相关.     

槟榔提取物抑制人类口腔粘膜角朊细胞生长的实验研究

目的：建立人类口腔粘膜上皮角朊细胞体外原代培养方法：探讨口腔粘膜下纤维性变患者上皮厚度改变的机理;方法;先对人类口腔粘膜上皮角朊细胞进行分离,培养和鉴定,然后用四唑盐比色试验检测ＯＳＦ患者和正常人口腔粘膜上皮ＫＣ增殖状况,并且观察槟榔提取物对ＫＣ增殖的影响。     

Deficiency in collagen and fibronectin phagocytosis by human buccal mucosa fibroblasts in vitro as a possible mechanism for oral submucous fibrosis

Oral submucous fibrosis (OSF), a chronic oral mucosal condition commonly found in south Asians, is a disorder characterized by a quantitative as well as a qualitative alteration of collagen deposition within the subepithelial layer of the oral mucosa. Since degradation of collagen by fibroblast phagocytosis is an important pathway for physiological remodelling of soft connective tissues, we have investigated phagocytosis of collagen- and fibronectin-coated latex beads by fibroblast cultures with an in vitro model system. Coated fluorescent latex beads were incubated with human oral mucosa fibroblasts and the fluorescence associated with internalized beads was measured by flow cytometry. Cells from normal tissues that had been incubated with beads for 16 h contained a mean of 75% collagen phagocytic cells and 70% fibronectin phagocytic cells; however, about 15% and 10% of phagocytic cells individually contained more than twice the mean number of beads per cell. In contrast, cells from OSF tissues exhibited a 40% reduction of the proportions of collagen phagocytic cells (mean=35%) and a 48% decrease of the proportions of fibronectin phagocytic cells (mean=22%), none of the cells having a high number of beads as compared to normal fibroblasts. OSF lesions appear to contain fibroblasts with marked deficiencies in collagen and fibronectin phagocytosis. To investigate if inhibition of phagocytosis could be demonstrated in vitro, normal fibroblast cultures were incubated with areca nut alkaloids (arecoline, arecaidine). The cultures had a dose-dependent reduction in the proportions of phagocytic cells. On the other hand, corticosteroid used in the treatment of OSF exhibited a dose-dependent enhancement in the proportion of phagocytic cells. Therefore, our hypothesis for OSF, although over-simplified, is that betel nut alkaloids (arecoline, arecaidine) inhibit fibroblast phagocytosis and this provides a mechanism for the development of OSF. The benefit of a local intralesional injection of corticosteroid is also possibly, at least in part, through an enhancement of fibroblast collagen phagocytosis.     

Stimulation of human buccal mucosa fibroblasts in vitro by betel-nut alkaloids

Oral submucous fibrosis (OSF) is characterized by excessive collagen production by mucosal fibroblasts and is associated with the habitual chewing of betel-nuts (Areca catechu); nut extracts stimulate fibroblast activity in vitro. The metabolism of arecoline, the major alkaloid in the nut, by human buccal mucosa fibroblasts in vitro was investigated; alkaloid metabolites extracted from culture media were analysed by gas chromatography and thin-layer chromatography. [3H]-arecoline was metabolized predominantly to [3H]-arecaidine and this was accompanied by a concentration-dependent stimulation of collagen synthesis and cell proliferation. Arecaidine was a more potent stimulator than arecoline. The rate of hydrolysis of a series of synthetic arecaidine esters (methyl, ethyl, butyl, propyl and pentyl) by fibroblasts was closely correlated with the extent of stimulation of collagen synthesis. Thus fibroblasts are responsive to the major metabolite of arecoline and hydrolysis of the ester group may be necessary for this action. Exposure of buccal mucosa fibroblasts to these alkaloids in vivo may contribute to the accumulation of collagen in OSF.     

Hypoxia inducible factor‐1α expression in areca quid chewing‐associated oral squamous cell carcinomas

Oral Diseases (2010) 16 , 696–701 Objectives: Hypoxia inducible factor (HIF)‐1α gene expression is mainly induced by tissue hypoxia. Overexpression of HIF‐1α has been demonstrated in a variety of cancers. The aim of this study was to compare HIF‐1α expression in normal human oral epithelium and areca quid chewing‐associated oral squamous cell carcinoma (OSCC) and further to explore the potential mechanisms that may lead to induce HIF‐1α expression. Methods: Twenty‐five OSCC from areca quid chewing‐associated OSCC and 10 normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The oral epithelial cell line GNM cells were challenged with arecoline, a major areca nut alkaloid, by using Western blot analysis. Furthermore, glutathione precursor N‐acetyl‐l ‐cysteine (NAC), AP‐1 inhibitor curcumin, extracellular signal‐regulated protein kinase inhibitor PD98059, and protein kinase C inhibitor staurosporine were added to find the possible regulatory mechanisms. Results: Hypoxia inducible factor‐1α expression was significantly higher in OSCC specimens than normal specimen (P < 0.05). Arecoline was found to elevate HIF‐1α expression in a dose‐ and time‐dependent manner (P < 0.05). The addition of NAC, curcumin, PD98059, and staurosporine markedly inhibited the arecoline‐induced HIF‐1α expression (P < 0.05). Conclusions: Hypoxia inducible factor‐1α expression is significantly upregulated in areca quid chewing‐associated OSCC and HIF‐1α expression induced by arecoline is downregulated by NAC, curcumin, PD98059, and staurosporine.     

Tyrosine sulphation of CXCR4 induces the migration of fibroblast in OSF

Oral submucous fibrosis (OSF) caused by areca nut chewing is a prevalent fibrotic disease in Asia-Pacific countries. Arecoline-induced migration of fibroblasts (FBs) plays a vital role in the development of OSF. However, the specific molecular mechanisms involved remain unclear. Many studies have shown that tyrosine sulphation of chemokines can influence cell migration. Herein, we demonstrated that arecoline stimulates tyrosine sulphation of the chemokine receptor 4 (CXCR4) through the tyrosylprotein sulphotransferase-1 (TPST-1) to enhance the migration ability of FBs. Moreover, by RNA-Seq analysis, we found that the most significantly altered pathway was the EGFR pathway after the arecoline stimulation for FBs. After the knockdown of arecoline-induced EGFR expression, the tyrosine sulphation of CXCR4 was significantly decreased by the inhibition of TPST-1 induction. Finally, in human OSF specimens, TPST-1 expression was directly correlated with the expression of CXCR4. These data indicate that the arecoline-induced tyrosine sulphation of CXCR4, which is regulated by TPST-1, might be a potential mechanism that contributes to FB migration in OSF.     

兜甲蛋白和细胞色素P4503A5在口腔黏膜下纤维化中的表达及意义

目的探讨兜甲蛋白（LOR）和细胞色素P450 3A5（CYP 3A5）在口腔黏膜下纤维化颊黏膜（OSF）和正常颊黏膜中的表达以及在上皮防御作用中的意义。方法首先采用免疫组化染色检测66例OSF颊黏膜和14例正常颊黏膜中LOR和CYP 3A5这2种蛋白的表达和定位,然后采用Western blot和RT-PCR方法分别在OSF患者颊黏膜和正常人颊黏膜中检测LOR和CYP 3A5的蛋白和mRNA表达。结果42例（63.6%）OSF呈LOR阳性表达,其在早、中期之间差异有统计学意义（P<0.05）,而中、晚期之间差异无统计学意义（P＞0.05）;正常颊黏膜中CYP 3A5均呈阳性表达,5例（7.6%）OSF出现棘细胞弱阳性表达,33例（50%）出现内皮细胞弱阳性表达,与病理分期呈负相关（P<0.05）。RTPCR显示LOR和CYP 3A5 mRNA在OSF与正常黏膜中表达的差异均有统计学意义（P<0.05）;而Western blot检测显示,只有CYP 3A5蛋白表达在两者间差异有统计学意义（P<0.05）。结论LOR和CYP 3A5的异常表达是OSF黏膜上皮防御能力改变的表现,它们在OSF发生发展和癌变过程中可能发挥了重要作用。     

Arecoline and oral keratinocytes may affect the collagen metabolism of fibroblasts

Background:  The characteristic of oral submucous fibrosis (OSF) is related with the disturbance of synthesis and degradation of extracellular matrix. Arecoline, the areca nut (betel nut) component of betel quid, plays a major role in pathogenesis of OSF. But the exact mechanism how arecoline influences the collagen metabolism is unclear.
Methods:  Oral keratinocytes and fibroblasts were cocultured and keratinocytes were pre-treated by arecoline. Fibroblasts alone, fibroblasts stimulated by arecoline, fibroblasts cocultured with keratinocytes and fibroblasts cocultured with keratinocyte pre-treated by arecoline were included as the four groups in the present study. The concentration of collagen, the content and activity of matrix metalloproteinase (MMP) and the concentration of tissue inhibitor of metalloproteinase (TIMP) were assessed.
Results:  The collagen production of fibroblasts decreased when cocultured with keratinocytes; when cocultured with keratinocytes pre-treated by arecoline, fibroblasts produced more soluble collagen than non-pretreated coculture group. MMP-9 was produced only in coculture groups. There was no significant difference in the two coculture groups. The activation ratio of pro-MMP-2 in arecoline pre-treated keratinocytes-fibroblasts coculture group was significantly higher than that of non-coculture groups, but no significant difference existed in the two coculture groups. TIMP-1 produced by arecoline pre-treated keratinocytes-fibroblasts coculture group was significantly higher than those by the other three groups.
Conclusion:  TIMP-1 and the interaction of oral keratinocytes and fibroblasts play important role in pathogenesis of OSF.     

The role of basic fibroblast growth factor in oral submucous fibrosis pathogenesis

Background:  Oral submucous fibrosis (OSF) is a chronic fibrotic disease of oral mucosa and oropharynx, induced by betel quid chewing often resulting in restricted mouth opening. The principal cells implicated as a source of extracellular matrix in areas of fibrosis are fibroblasts. Accumulation of connective tissue matrix is secondary to factors such as cytokines and growth factors. The contribution of basic fibroblast growth factor (bFGF) in disease progression and the consequent stromal changes with increase in the severity of OSF was studied.
Methods:  A case series analysis of 30 cases of OSF was carried out for bFGF expression using immunohistochemistry. Connective tissue changes in these cases were corroborated using aldehyde fuchsin and Verhoeff's hematoxylin special stains.
Results:  bFGF immunoreactivity was found to be increased in fibroblasts and in endothelial cells in early OSF cases, while the expression of bFGF in stroma increased notably in advanced fibrosis.
Conclusion:  Increased bFGF expression in early stages of the disease was explainable to an initial injury phase because of areca consumption, followed by cellular activation by chemotactic cytokines and other growth factors with eventual fibrosis occurring as a result of molecular alteration at the cellular level.     

槟榔碱对颊黏膜成纤维细胞增殖及迁移的影响

目的通过观察槟榔碱诱发人颊黏膜成纤维细胞增殖、迁移能力以及微丝形态的改变,探讨槟榔碱在口腔黏膜下纤维性变(OSF)的致病途径。方法使用甲噻唑四唑氮(MTT)比色法、划痕法、激光共聚焦扫描显微镜检测不同浓度槟榔碱(5、10、20、40、80μg·mL^-1)对成纤维细胞增殖、迁移、微丝形态的影响。结果5、10、20μg·mL^-1的槟榔碱可轻微提高颊黏膜成纤维细胞增殖迁移能力及微丝聚合(P<0.05);40、80μg·mL^-1的槟榔碱抑制颊黏膜成纤维细胞增殖、迁移能力及微丝解聚(P<0.05)。结论槟榔碱改变颊黏膜成纤维细胞增殖、迁移能力及微丝形态分布,可能在口腔黏膜下纤维性变病理过程中起着重要的作用。