Tuula Salo

Matrix Metalloproteinase-8 Is Expressed in Rheumatoid Synovial Fibroblasts and Endothelial Cells REGULATION BY TUMOR NECROSIS FACTOR-α AND DOXYCYCLINE

Neutrophil collagenase (matrix metalloproteinase-8 or MMP-8) is regarded as being synthesized exclusively by polymorphonuclear neutrophils (PMN). However, in vivo MMP-8 expression was observed in mononuclear fibroblast-like cells in the rheumatoid synovial membrane. In addition, we detected MMP-8 mRNA expression in cultured rheumatoid synovial fibroblasts and human endothelial cells. Up-regulation of MMP-8 was observed after treatment of the cells with either tumor necrosis factor-α (10 ng/ml) or phorbol 12-myristate 13-acetate (10 nm). Western analysis showed a similar regulation at the protein level. The size of secreted MMP-8 was 50 kDa, which is about 30 kDa smaller than MMP-8 from PMN. Conditioned media from rheumatoid synovial fibroblasts contained both type I and II collagen degrading activity. However, degradation of type II collagen, but not that of type I collagen, was completely inhibited by 50 μm doxycycline, suggesting specific MMP-8 activity. In addition, doxycycline down-regulated MMP-8 induction, at both the mRNA and protein levels. Thus MMP-8 exerts markedly wider expression in human cells than had been thought previously, implying that PMN are not the only source of cartilage degrading activity at arthritic sites. The inhibition of both MMP-8 activity and synthesis by doxycycline provides an incentive for further studies on the clinical effects of doxycycline in the treatment of rheumatoid arthritis.

The Effects of MMP Inhibitors on Human Salivary MMP Activity and Caries Progression in Rats

Previous studies suggest that salivary and pulp-derived host enzymes, matrix metalloproteinases (MMPs), may be involved in dentin caries pathogenesis. To study the inhibition of acid-activated human salivary MMPs by non-antimicrobial chemically modified tetracyclines (CMTs), we used a functional activity assay with 125I-labeled gelatin as a substrate. To address the role of MMPs in the progression of fissure caries in vivo, we administered the MMP inhibitors CMT-3 and zoledronate to young rats per os for 7 weeks, 5 days a week. Caries lesions were visualized by Schiff reagent in sagittally sectioned mandibular molars. Marked reduction in gelatinolytic activity of human salivary MMPs was observed with CMT-3. CMT-3 and zoledronate, both alone and in combination, also reduced dentin caries progression in the rats. These results suggest that MMPs have an important role in dentin caries pathogenesis, and that MMP inhibitors may prove to be useful in the prevention of caries progression.

Expression and induction of collagenases (MMP-8 and -13) in plasma cells associated with bone-destructive lesions

Matrix metalloproteinases (MMPs) collectively degrade extracellular matrix and basement membrane proteins in chronic inflammation and bone‐destructive lesions. This study examined the ability of immunoglobulin‐producing plasma cells, typically present in sites of chronic inflammation, to express collagenases (MMP‐8 and ‐13) in vivo and in vitro. Phorbol‐12‐myristate‐13‐acetate, interleukin‐6, and tumour necrosis factor‐α and heparin with the tumour promoter or cytokines potently enhanced (up to nine‐fold) MMP‐8 and ‐13 expression by the RPMI 8226 myeloma cell line, as evidenced by western blotting and semi‐quantitative reverse transcriptase‐polymerase chain reaction. Immunohistochemical analysis and in situ hybridization revealed that plasma cells expressed MMP‐8 and ‐13 focally in periapical granulomas, odontogenic cysts, and malignant plasmacytomas. MMP‐8 and MMP‐13 from plasma cells can participate in bone organic matrix destruction at sites of chronic inflammation and neoplastic growth. Since MMP‐13 was more frequently expressed than MMP‐8 in plasma cells of strongly recurring keratocysts and malignant plasmacytomas, it is concluded that plasma cell MMP‐13 has a particularly important role in benign and malignant bone‐destructive lesions. Copyright

Prognostic factors in tongue cancer – relative importance of demographic, clinical and histopathological factors

The incidence of and mortality from squamous cell carcinoma (SCC) of the tongue have increased during the recent decades in the Western world. Much effort has been made to predict tumour behaviour, but we still lack specific prognostic indicators. The aim of our study was to evaluate the relative importance of the known demographic, clinical and histological factors in a homogeneous population-based group of patients with SCC of the mobile tongue. The demographic and clinical factors were reviewed retrospectively from primary and tertiary care patient files. Histological prognostic factors were determined from pre-treatment biopsies. The TNM stage was found to be the most important prognostic factor. In particular, local spread outside the tongue rather than spread to regional lymph nodes was related to poor prognosis. Several demographic and histopathological factors were closely related to TNM stage. When the cases were divided into stage I–II carcinomas and stage III–IV carcinomas, it appeared that the patient’s older age (> 65 years), a high malignancy score and an absence of overexpressed p53 protein were associated with a poorer prognosis in stage I–II carcinomas. Such cases may require more aggressive treatment. Among patients with stage III–IV carcinomas, heavy use of alcohol was significantly associated with a poor disease-specific survival time.

Angiogenesis is involved in the pathogenesis of nonrheumatic aortic valve stenosis

Angiogenesis is an essential biological process not only in embryogenesis, but also in the progression of several major diseases, including cancer, diabetes, and inflammation. Excessive vascularization can also contribute to some cardiovascular pathologies, such as atherosclerosis, but contradictory reports still prevail regarding its impact on aortic stenosis. Using immunohistochemical techniques, we assessed the vascular density and distribution of angiogenesis (FVIII) and vascular endothelial growth factor (VEGF) expression as well as the expression of 2 VEGF receptors, Flt-1 and Flk-1, in 55 nonrheumatic and 6 control aortic valves. In the light of the fact that the angiogenic effect of VEGF is mediated by sustained formation of nitric oxide, the samples were also immunostained with 3 nitric oxide synthase (eNOS, iNOS, and nNOS) antibodies. The immunohistochemical findings of VEGF and its receptors were verified by immunoblotting techniques. Vascular density was highest in the cases with moderate valve stenosis, and the mean number of FVIII-positive blood vessels was 1.7 +/- 1.9 vessels/mm(2) in the diseased valves, whereas the normal valves contained no blood vessels. Vascular density was significantly higher in the cases showing chronic inflammation (P = 0.007). Interestingly, the patients receiving statin therapy had significantly lower vascular densities than those not receiving such therapy (P = 0.001). Diseased valves showed distinct VEGF, Flt-1, Flk-1, and eNOS positivity of activated endothelial, stromal fusiform myofibroblastic, and histocytic cells. In contrast, immunoreactivity for iNOS and nNOS was seen only in nonendothelial stromal cells, and their expression was weaker. Enhanced vascular density was significantly associated with increased expression of Flk-1 (P = 0.028 for endothelial and P = 0.009 for stromal cells) and with endothelial eNOS expression (P = 0.024). A similar tendency was also observed for VEGF, but not for Flt-1. Our results show a distinct angiogenic response and the presence of angiogenic factors in nonrheumatic aortic valve stenosis, suggesting that angiogenesis may influence on the evolution of this disease.

Altered distribution and synthesis of laminin‐5 (kalinin) in oral lichen planus, epithelial dysplasias and squamous cell carcinomas

Laminin‐5 is a glycoprotein which mediates epithelial cell adhesion to the basement membrane. This study describes the distribution and synthesis of laminin‐5 in oral lichen planus, epithelial dysplasias, squamous cell carcinomas and a lymph node metastasis using immunohistochemistry and in situ hybridization. In normal oral mucosa and lichen planus, immunoreaction to the laminin‐5 was seen as a thin continuous, delicate line in the basement membrane region, although slight irregularities in the thickness and intensity of the immunoreaction could be detected in some cases with lichen planus. In epithelial dysplasias, the laminin‐5 staining was discontinuous and more diffuse compared to lichen planus and normal mucosa. The immunoreaction was generally extracellular, although in some cases with lichen planus and epithelial dysplasia there were a few basal epithelial cells showing cytoplasmic staining. The invasive carcinomas and the lymph node metastasis showed a striking, intense cytoplasmic, staining of the carcinoma cells along the invasive border of the neoplastic islands and in individual infiltrating carcinoma cells. Using in situ hybridization, the laminin‐5 γ2 chain mRNA expression could not be detected in normal oral mucosa whereas, in non‐dysplastic lichen planus and, more strongly, in dysplasias, there was a clear increase in the expression of laminin‐5 mRNA in the basal epithelial cells. The most intensive signal was detected in the invasive front of the oral squamous cell carcinomas and the lymph node metastasis. We conclude that, in oral squamous cell carcinoma, there is altered synthesis and secretion of laminin‐5 mRNA and protein. It is also evident that in dysplastic lesions of oral epithelium the synthesis and distribution of laminin‐5 is abnormal.

Expression of estrogen receptor (ER) in oral mucosa and salivary glands

OBJECTIVESnTo assess the expression of estrogen receptor (ER) in oral mucosa and salivary glands, buccal mucosal biopsies from ten postmenopausal women (taken before and during the hormone replacement therapy), as well as, single biopsies from 20 healthy 19-year-old women were analyzed for ER expression. Salivary gland biopsies were taken from the minor labial salivary glands (n=6), submandibular glands (n=5) and parotid gland (n=1) from women at different ages.nnnMETHODSntotal RNA extracted from the tissue samples was reverse-transcripted (RT) to single-stranded cDNA, and the RT-polymerase chain reaction (RT-PCR) product was subjected to nucleotide sequencing to confirm the match with ER cDNA. Immunohistochemistry (IHC) with a monoclonal ER antibody (ER-ICA, Abbott) and Western blot analysis with monoclonal antibody against ER-related antigen (ER-D5, Amersham) were performed on the biopsies taken from the postmenopausal women.nnnRESULTSnER mRNA was expressed in 18/20 (90%) and 20/20 (100%) of the mucosal biopsies in the postmenopausal and 19-year-old women, respectively. The expression of mRNA was detected in all the submandibular gland samples, in the single parotid gland, as well as, in 4/6 (67%) of the labial glands. ER expression could not be detected by IHC, indicating either a very low level of expressed protein or difficulties in recognizing the epitopes by IHC. However, Western blot demonstrated 8/8 (100%) of the mucosal biopsies of postmenopausal women positive for ER-related antigen.nnnCONCLUSIONSnthe presence of ER mRNA and immunoreactive ER protein suggests that estrogens have a biological role in oral mucosa and salivary glands.

Expression of claudins 1, 4, 5, and 7 and occludin, and relationship with prognosis in squamous cell carcinoma of the tongue

Claudins and occludin are tight junctional proteins known to play a role in normal tissues and epithelial tumors. We analyzed the distribution patterns of claudins 1, 4, 5, and 7 and occludin in the superficial and invasive front of squamous cell carcinoma of the tongue of 97 patients and their relationship to cause-specific (squamous cell carcinoma of the tongue) patient survival (median follow-up period of 33.5 months; range, 1-234 months). Claudins 1 and 7 were strongly expressed, claudin 4 had moderate expression, whereas claudin 5 was least expressed. Occludin staining was mostly negative or very weak. Western blot analysis of tongue carcinoma (HSC-3) cells showed that all these proteins are also expressed in vitro. In cause-specific survival analysis, strong and low immunoreactivity of claudin 7 tended to be associated with decreased survival compared with medium immunoreactivity. We suggest that analyzing the immunohistologic staining levels of claudin 7 could be used for the prognostic purposes in patients with tongue squamous cell carcinoma.

Human Odontoblast Culture Method: The Expression of Collagen and Matrix Metalloproteinases (MMPs)

Studies on mature human odontoblasts have suffered for the lack of in vitro models. We recently introduced a human odontoblast and pulp tissue organ culture method, in which the odontoblasts are cultured in the pulp chamber after removal of the pulp tissue, and the pulp tissue can be cultured separately (Tjaderhane et al., 1998a). With this method, we have studied the effects of growth factors on the expression of collagen and extracellular matrix (ECM)-degrading enzymes, matrix metalloproteinases (MMPs), in mature human odontoblasts. TGF-β1 was selected because of its ability to regulate the response of the dentin-pulp complex to external irritation. The effect of TGF-β1 (10 ng/mL) on proal(I) collagen mRNA was analyzed by quantitative PCR, and type I procollagen propeptide (PINP) was analyzed from conditioned culture media with RIA. Odontoblast media were also assayed for respective type III procollagen propeptide (PIIINP). TGF-β had a negligible effect on collagen mRNA expression or protein synthesis, indicating that TGF-β alone does not markedly induce dentin matrix formation per se in the human dentin-pulp complex (Palosaari et al., 2001). However, TGF-β1 seems to regulate MMP expression in mature human odontoblasts differentially. A strong down-regulation of MMP-8 (Palosaari et al., 2000), a modest down-regulation of MMP-20 (Tjaderhane et al., 2000), and considerable up-regulation of MMP-9, with no apparent effect on MMP-2 expression (Tjaderhane et al., 1998b), indicate that growth factors may affect the matrix synthesis by controlling the expression and activity of MMPs instead of collagen synthesis. The altered expression of MMPs may result in altered ECM formation, which in turn may contribute to the formation of atubular reparative dentin.

Increased expression of collagen types I and III in human skin as a consequence of radiotherapy

To study the mechanisms of irradiation-induced fibrosis, the expression of types I and III collagen was analysed in radiotherapy-treated human skin. The subjects were ten randomly chosen women who had been treated for breast cancer with surgery and radiotherapy. The subjects ranged in age from 42 to 68xa0years (mean 53xa0years) and the time from treatment ranged from 7 to 94xa0months. The irradiated skin was compared with a corresponding healthy skin area in the same subject. Suction blisters were induced on both skin areas. The aminoterminal propeptides of types I and III collagen (PINP and PIIINP), which reflect actual in vivo skin collagen synthesis, were determined in the suction blister fluid using radioimmunoassays. mRNA of types I and III collagen were determined in skin specimens using a nonradioactive in situ hybridization (ISH) technique. Immunohistochemical staining for PINP was also performed. The level of PINP in suction blister fluid was increased more than threefold and the level of PIIINP more than twofold in irradiated skin compared to control skin. The number of cells containing type I and type III collagen mRNA was increased in the upper dermis of irradiated skin. Immunohistochemical staining showed the amount of PINP-positive fibroblasts to be increased in irradiated skin. We conclude that skin collagen gene expression is increased as a result of irradiation and this leads to fibrosis and thickening of the dermis.