Extracellular matrix molecules in chronic obstructive sialadenitis: an immunocytochemical and Western blot investigation

The exact pathomechanism of inflammation progress and fibrosis in chronic sialadenitis is unknown. Connective tissue growth factor (CTGF), matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have been implicated in the pathogenesis of various fibrotic conditions. These factors are thought to be essential in the regulation of extracellular matrix turnover and the development of tissue fibrosis. In the present study, the expression of CTGF, MMP-2, -3, -9, -13 and TIMP-3 was examined in chronic obstructive sialadenitis. Tissue samples of 13 patients with chronic sialadenitis of the submandibular gland associated with sialolithiasis and 4 normal tissue samples of the submandibular gland were analyzed immunohistochemically and by Western blot analysis. An intense CTGF immunoreactivity was observed in the ductal system of inflamed salivary glands, whereas in normal glands no reactivity or a very low CTGF immunoreactivity was present. Immunohistochemical studies revealed a low to strong reactivity of MMP-2, -3, -9, -13, and TIMP-3 in the ductal system, in acinar cells and in lymphomonocytic infiltrates in normal and inflamed tissues. The expression of MMP-2, -3, -9, -13, and TIMP-3 was confirmed by Western blotting in all cases. Over-expression of CTGF in chronic obstructive sialadenitis suggests that this factor may play a role in glandular fibrosis. However, the physiological role of MMP-2, -3, -9, -13, and TIMP-3 in normal glands, as well as their possible role in inflammation progress and fibrosis in chronic obstructive sialadenitis, remains to be elucidated.     

Characterization of lymphoid infiltrates in chronic obstructive sialadenitis associated with sialolithiasis

BACKGROUND: Chronic obstructive sialadenitis is characterized by acinar atrophy, lymphocytic infiltrates and progressive fibrosis. The immunological mechanisms involved in the pathogenesis of this disease are, for the most part, unknown. The aim of the present study was to characterize the lymphocytic infiltrates in chronic obstructive sialadenitis associated with sialolithiasis. METHODS: Paraffin-embedded tissue samples from 23 affected submandibular glands were immunostained for T-cells (CD3, CD4, CD8), cytotoxic T-cells (granzyme B), B-cells (CD20), plasma cells (CD38) and macrophages (Ki-M1P). RESULTS: CD4-positive subsets were the predominant cells, and they were located mainly periductally. Isolated intraepithelial CD8-positive cytotoxic T-cells associated with ductal epithelial cell destruction were observed in all cases. B lymphocytes were restricted to lymphoid follicles located periductally and around intralobular ducts. In early stages of the disease, a large number of CD38-positive plasma cells were distributed diffusely in the periacinar area. With progression of the disease, conspicuous clusters of plasma cells were located especially between atrophic acini adjacent to fibrotic tissue. An intimate relation between the lymphocytic infiltrates and the ductal epithelium, the target of the inflammatory process, was observed. CONCLUSION: The composition and distribution of inflammatory cells suggest that intraepithelial infectious agents may be the cause of the inflammatory reaction and the progressive fibrosis in this disease.     

Differential uPA expression by TGF-beta1 in gingival fibroblasts

Transforming Growth Factor-beta1 (TGF-beta1) plays a key role in connective tissue remodeling and inflammation. Under pathological conditions, like periodontal disease, fibroblasts may display an altered response to this growth factor. To investigate this question, we have studied whether TGF-beta1 may differentially regulate the expression of urokinase at the protein level in primary cultures of fibroblasts derived from healthy gingiva, granulation tissue from gingival wounds, and chronic periodontal disease. We observed that TGF-beta1 may repress urokinase expression in healthy gingival fibroblasts and promote its production in granulation-tissue fibroblasts. A significant correlation was found between expression of the myofibroblast marker alpha-smooth-muscle actin and stimulation of urokinase production by TGF-beta1. Immunostaining of gingival wounds showed that myofibroblasts were involved in urokinase production. TGF-beta1-stimulated urokinase expression was blocked after inhibition of the c-jun-NH2 terminal kinase signaling pathway. We propose that stimulation of urokinase production by TGF-beta1 is involved in the responses of activated fibroblasts to tissue injury.     

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.     

Inhibition of growth of human gingival fibroblasts by chimeric DNA-RNA hammerhead ribozyme targeting transforming growth factor-beta 1

BACKGROUND: Transforming growth factor (TGF)-beta1 is involved in the pathogenesis of both drug-induced gingival overgrowth and hereditary gingival fibromatosis. Ribozymes enzymatically cleave target mRNAs and are expected to be utilized as the basis of novel nucleic acid-based therapies. We designed a chimeric DNA-RNA ribozyme targeting TGF-beta1 mRNA and examined its effect on growth of gingival fibroblasts in culture. METHODS: Chimeric DNA-RNA hammerhead ribozyme with sequence complementary to the loop structure of human TGF-beta1 mRNA was used. We evaluated transfer of the chimeric ribozyme by hemagglutinating virus of Japan (HVJ)-envelope into cultured human gingival fibroblasts in vitro and rat gingival tissues in vivo. We then examined effects of the chimeric ribozyme to TGF-beta1 on proliferation and DNA synthesis in human gingival fibroblasts. We also examined effects of the chimeric ribozyme to TGF-beta1 on expression of TGF-beta1, type IV collagens, and fibronectin mRNAs and expression of TGF-beta1 protein in human gingival fibroblasts. RESULTS: Chimeric ribozyme was sufficiently distributed into human fibroblasts in vitro and rat gingivae in vivo. Chimeric ribozyme to TGF-beta1 significantly inhibited expression of TGF-beta1, type IV collagen, and fibronectin mRNAs and TGF-beta1 protein in human gingival fibroblasts. Mismatch ribozyme had no effect on expression of these molecules. Chimeric ribozyme to TGF-beta1 also significantly inhibited proliferation and DNA synthesis in gingival fibroblasts. CONCLUSION: Chimeric DNA-RNA ribozyme targeting TGF-beta1 may be a useful gene therapy agent for treatment of gingival hyperplasia.     

转化生长因子β亚型在口腔鳞癌中的表达和意义

目的:探讨TGF-B与口腔鳞癌发生和发展的关系。方法:采用SABC免疫组化法检测了40例口腔鳞癌术后标本和20例正常口腔粘膜中TGF-B的表达。结果:口腔鳞癌和正常口腔粘膜中均表达TGF-B1、TGF-B2、TGF-B3,但程度不同。TGF-B1、TGF-B2在口腔鳞癌中呈过表达。口腔鳞癌临床分期、有无颈淋巴结转移及病理分级与TGF-B1、 TGF-B2过表达有关,与TGF-B3的表达无关。结论:TGF-B1和TGF-B2可能参与了口腔鳞癌的发生发展及颈淋巴结转移过程,是口腔鳞癌发生发展及预后的一个指标。     

Immunohistochemical study of fibroblasts and mast cells in chronic submandibular sialadenitis

Aim:  To further our understanding of the processes involved in fibrosis that occurs in chronic submandibular sialadenitis by investigating the distribution of myofibroblasts, CD34-positive fibroblasts and tryptase-containing mast cells.
Materials and methods:  Thirty specimens of chronic submandibular sialadenitis with varying degrees of fibrosis and five normal submandibular glands were examined immunohistochemically for the presence of CD34, α -smooth-muscle-actin, desmin and tryptase.
Results:  Myofibroblasts were not demonstrated by the techniques for α -smooth-muscle-actin or desmin. CD34-positive fibroblasts were found around normal and moderately atrophic acini, but were not found around extremely atrophic acini and duct-like structures or in periductal and interlobular fibrous tissue. Tryptase-containing mast cells were found around vessels in normal submandibular glands. They were found in increased numbers in chronic submandibular sialadenitis, particularly in glands with widespread fibrosis, in which they were found in the fibrous tissue, and in which the increase was statistically significant.
Conclusions:  The results of this investigation suggest that tryptase-containing mast cells are likely to be involved in the fibrosis of chronic submandibular sialadenitis, but myofibroblasts and CD34-positive fibroblasts are not.     

Growth factor expression following clinical mandibular distraction osteogenesis in humans and its comparison with existing animal studies

AIM: Lengthening the mandible by distraction osteogenesis (DO) is nowadays a well recognized technique in maxillofacial surgery. In this study growth factor expression profiles were examined in biopsies taken from six patients undergoing mandibular DO and compared with findings from a sheep model for mandibular DO. STUDY DESIGN: In all patients (and sheep), the ascending ramus was distracted 10-15 mm at a rate of 1mm/day using an intraoral device. Biopsies were taken from the centre of the distraction zone 21 days after completion of distraction. Using standard immunohistochemical techniques, samples were stained for platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta), basic fibroblast growth factor (bFGF) and bone morphogenetic proteins-2, -4 and -7 (BMP-2, -4, -7), matrix metalloproteinases-1 and -3 (MMP-1, -3), the vascular endothelial growth factor (VEGF), a marker for endothelial cells (CD-31) and type IV collagen (Col IV). RESULTS: Positive staining for PDGF, bFGF, TGF-beta, BMP-2, -4, and -7 was noted in cells and matrix components. There was intense staining for MMP-1. Strong staining for CD-31 and COL IV was observed adjacent to vessels. VEGF staining was less specific. Similar findings were noted in the sheep model. CONCLUSION: Growth factor expression in the human distraction site is similar to that in the sheep model.     

The expression of transforming growth factor beta (TGF-beta) in the synovial membrane of human temporomandibular joint with internal derangement: a comparison with tenascin expression

We examined the expression of the transforming growth factor beta (TGF-beta) in 28 human temporomandibular joint (TMJ) samples (internal derangement of TMJ and control specimens) by an immunohistological method using paraffin-embedded tissues and a polyclonal antibody specific to human TGF-beta. The resulting reaction of TGF-beta expression divided into three types as follows. The first type, around the fibrocyte and in the lacunae of chondrocytes in the disc. The second type, at the stroma of the mildly hypertrophic synovial membrane and severely hypertrophic synovial membrane. The first type was observed in all the cases including the control cases. The second type showed only in the internal derangement of TMJ, and its expression pattern resembled that of tenascin (TN) within the stroma of hypertrophic synovial membranes. In conclusion, TGF-beta and TN were distributed in the affected synovial membrane of TMJ with internal derangement. These findings suggested that TGF-beta and TN might have a close relationship with synovitis, followed by tissue repair.     

Increased expression of Smad proteins,and in particular Smad3, in oral lichen planus compared to normal oral mucosa

J Oral Pathol Med (2010) 39 : 639–644 Backgound: Oral lichen planus (OLP) is a chronic inflammatory disease of the oral mucosa which the World Health Organisation (WHO) considers a premalignant condition. One step in malignant development is so called epithelial mesenchymal transition (EMT), a process whereby epithelial cells acquire mesenchymal characteristics. EMT occurs during embryogenesis and wound healing but also in some human diseases such as cancer and fibrosis. A factor known to induce EMT is transforming growth factor‐β (TGF‐β), which uses the Smad proteins as mediators for its signalling. TGF‐β is also often over‐expressed in squamous cell carcinoma of the head and neck (SCCHN). Methods: In the present study we mapped expression of Smad proteins in OLP lesions by immunohistochemistry, and compared to expression in normal and sensitive oral mucosa. The latter group of patients had developed SCCHN after shorter or longer periods of diffuse oral symptoms. The aim was to see if there were any signs of EMT related changes in the OLP lesions, as judged by changes in the TGF‐β pathway. Conclusion: Changes in the TGF‐β pathway related to EMT are seen in the very earliest stages of oral malignancy and become more severe as lesions progress.     

Transforming growth factor-beta1 autocrine stimulation regulates fibroblast proliferation in hereditary gingival fibromatosis.

BACKGROUND: Hereditary gingival fibromatosis (HGF) is a rare oral disease characterized by a slow and progressive enlargement of both the maxilla and mandible gingiva. Increased proliferation, elevated synthesis of extracellular matrix, particularly collagen, and reduced levels of matrix metalloproteinases seem to contribute to the pathogenesis of gingival overgrowth in HGF patients. Transforming growth factor-beta1 (TGF-beta1) is an important cytokine thought to play a major role in fibrotic disorders such as HGF due to its ability to stimulate the synthesis and reduce the degradation of extracellular matrix. In HGF fibroblasts, TGF-beta1 autocrine stimulation reduces expression and production of matrix metalloproteinases. However, the role of TGF-beta1 in fibroblast growth modulation has not been established in this disease. METHODS: The aim of this study was to confirm the increased proliferation rate of HGF fibroblast cell lines and to explore a possible autocrine role of TGF-beta1 as a cell growth stimulator by blocking production of this endogenous cytokine using 2 well-established systems: antisense oligonucleotides and neutralizing antibodies. RESULTS: Four different cellular proliferation assays, bromodeoxyuridine labeling, argyrophilic nucleolar organizing region staining, proliferating cell nuclear antigen, and mitotic indexes, confirmed that fibroblasts from HGF proliferate significantly faster than those from normal gingiva. Antisense oligonucleotides reduced TGF-beta1 production as demonstrated by capture enzyme-linked immunosorbent assay, whereas TGF-beta1 expression levels were not significantly modified. Blocking TGF-beta1 synthesis with oligonucleotides or its activity with specific antibodies resulted in a decreased magnitude of HGF fibroblast proliferation. CONCLUSION: These results are consistent with the existence of an autocrine role of TGF-beta1 as a stimulator of HGF fibroblast proliferation.     

Expression of secretory leukocyte proteinase inhibitor in the submandibular glands of AIDS patients

OBJECTIVE: Secretory leukocyte proteinase inhibitor (SLPI) is an endogenous proteinase inhibitor present in mucosal secretions. It also displays antimicrobial activity including anti-human immunodeficiency virus activity. This protease inhibitor is also expressed in submandibular glands (SMG), but there are few data on its expression in AIDS patients with infectious conditions. METHODS: We analyzed the expression of SLPI using immunohistochemistry in submandibular gland samples of 36 AIDS patients [10 with normal histology, 10 with chronic nonspecific sialadenitis, eight with mycobacteriosis, and eight with cytomegalovirus (CMV) infection] and 10 HIV-negative controls. The proteinase inhibitor was quantified using image analysis and expressed as % of positively stained area. RESULTS: There was a higher expression of SLPI in AIDS patients with CMV infection (% of stained area, mean+/-SD: 37.37+/-14.45) when compared with all other groups (P=0.009). There were no significant differences between control subjects (22.70+/-9.42%) and AIDS patients without histologic alterations (18.10+/-7.58%), with chronic nonspecific sialadenitis (17.13+/-5.36%), or mycobacterial infection (21.09+/-4.66%). CONCLUSION: Cytomegalovirus infection increases SLPI expression in the SMG of AIDS patients. Our results reveal new insights into the pathogenic association between HIV and CMV in AIDS patients.     

Transforming growth factor beta2 regulates growth and differentiation of pulp cells via ALK5/Smad2/3

Transforming growth factor beta (TGF-beta) may regulate the biological activities of dental pulp cells. We found that human dental pulp cells expressed TGF-beta1, TGF-beta2, and a little amount of TGF-beta3 messenger RNA (mRNA). The exposure of pulp cells to TGF-beta2 induced the phosphorylation of Smad2/3, Smad1/5/8, and extracellular regulated-kinase 1/2 (ERK1/2) as observed by Western blotting. Exposure to TGF-beta2 decreased the alkaline phosphatase (ALP) mRNA expression and enzyme activity. Pretreatment of pulp cells with SB431542 (an inhibitor of TGF-beta ALK-4, ALK-5, and ALK-7 receptors) but not U0126 (a MEK1 inhibitor) prevented the inhibition of viable cell number, ALP activity, and mRNA expression by TGF-beta2 in dental pulp cells. These results suggest that TGF-beta may affect the growth and differentiation of dental pulp cells via an autocrine fashion by activation of the ALK/Smad2/3-signal transduction pathways. TGF-beta2 possibly regulates the differentiation of pulp cell at specific stages synergistically with other factors.     

Induction of the myofibroblastic phenotype in human gingival fibroblasts by transforming growth factor-beta1: role of RhoA-ROCK and c-Jun N-terminal kinase signaling pathways

BACKGROUND AND OBJECTIVES: Myofibroblastic differentiation is an important event in gingival wound healing and chronic inflammation. Transforming growth factor-beta 1 (TGF-beta1) is a potent growth factor that has been implicated in this process. Gingival myofibroblasts have an increased ability to remodel the extracellular matrix and this feature has been associated with changes in the distribution of F-actin and the expression of the myofibroblast marker alpha-smooth muscle actin. In the present study we have analyzed the role of TGF-beta1 and the signaling routes activated by this factor in the cytoskeletal changes that characterize the myofibroblastic differentiation process in human gingival fibroblasts. MATERIALS AND METHODS: The signalling pathways involved in myofibroblastic differentiation were studied in primary cultures of human gingival fibroblasts using several signal transduction inhibitors. RhoA activation was analyzed through a pull-down assay. Distribution of focal adhesions and actin cytoskeleton was assessed by means of immunofluorescence and western blot. A cell adhesion assay was performed in TGF-beta1-stimulated cells. Smooth muscle actin expression was studied through western blot and immunofluorescence. c-Jun N-terminal kinase phosphorylation was assessed through western blot. RESULTS: Our observations show that TGF-beta1 activated the GTPase RhoA, a key regulator of the actin cytoskeleton. As a consequence of this event, this growth factor stimulated the generation of actin stress fibers and the reinforcement of vinculin-enriched focal adhesions. These responses were blocked after inhibiting ROCK, the main target of RhoA activation. TGF-beta1 also stimulated the adhesion of fibroblasts over fibronectin, an extracellular matrix molecule involved in myofibroblastic differentiation. Finally, induction of the myofibroblast marker alpha-smooth muscle actin by TGF-beta1 was abolished by the c-Jun N-terminal protein kinase inhibitor SP600125, suggesting a role for this signaling pathway during the induction of this phenotype. CONCLUSIONS: We propose that TGF-beta1 may promote the differentiation of myofibroblasts through the stimulation of cell spreading and adhesion, the reinforcement of focal adhesions, the maturation of the actin cytoskeleton, and the induction of alpha-smooth muscle actin. Activity of RhoA-ROCK and c-Jun N-terminal protein kinase signaling pathways are probably involved in these cellular events.     

生长因子对人髁突软骨细胞Ⅰ、Ⅱ、Ⅲ型前胶原基因表达的调控研究

目的：研究低血清培养条件下转化生长因子－β（ＴＧＦ－β）,胰岛素生长因子－Ⅰ（ＩＧＦ－Ⅰ）,碱性成纤维生长因子（ｂＦＧＦ）对人髁突软骨细胞Ⅰ,Ⅱ,Ⅲ型前胶原基因表达的调控作用。方法：采用体外细胞培养及ｍＲＮＡ狭缝杂交的方法。结果：ＩＧＦ－Ⅰ对３种前胶原的ｍＲＮＡ水平无显著影响。ｂＧＦＧ,ＴＧＦ－β均呈不同程度地抑制Ⅱ型胶原的基因表达,分别为对照组的０．３５２及０．６５８倍同时ＴＧＦ－β显著增加了Ⅰ     

Changes in transforming growth factor-beta1 in gingival crevicular fluid following periodontal surgery

OBJECTIVES: Growth factors play a major part in wound healing, including in the periodontium. However, the presence of growth factors in gingival crevicular fluid (GCF) in humans during periodontal wound healing has not yet been determined. Our hypothesis is that such factors are present in GCF and that changes in their levels might be of value as a prognostic marker of wound-healing activity and therapeutic progress following periodontal surgery. The aim of this study was therefore to measure transforming growth factor-beta1 (TGF-beta1) in GCF collected from sites that have undergone guided tissue regeneration (GTR) and conventional flap (CF) surgery and to compare these with GCF collected from unaffected healthy sites. MATERIALS AND METHODS: GCF samples were collected, using filter paper strips, at baseline (pre-surgical) and then at intervals up to 26 weeks from 16 patients undergoing GTR and from 11 patients undergoing CF surgery. After elution and acid treatment, TGF-beta1 levels were measured by ELISA. RESULTS: Treatment of periodontal defect sites significantly reduced the mean probing pocket depth (PPD) and improved the mean lifetime cumulative attachment loss (LCAL). Average GCF volumes also significantly increased at all sites at 2 weeks post-surgery and thereafter declined to baseline levels, except at the GTR test sites that were still elevated at 7 weeks. TGF-beta1 could be detected in almost all GCF samples, and 2 weeks after surgery, the average levels increased two-fold at the surgically treated but not at the control sites, which remained unchanged. CONCLUSION: TGF-beta1 is readily detectable in GCF and increases transiently following periodontal surgery. This suggests that changes in the levels of this growth factor in GCF might be useful for monitoring the progress of periodontal repair and regeneration.     

The regional blood circulation in the parotid gland area in Sj?gren's disease (syndrome)]

Rheographic investigations of the parotid gland area in 103 patients with Sjogren's disease (syndrome) that developed in the presence of chronic nonspecific sialadenitis during its various stages and inflammation phases have demonstrated that the blood stream volumic rate directly depended on the inflammation phase and intensity of this phase. During remission the circulation in the salivary glands was close to normal.