A rat model of common bile duct ligation (BDL)-induced hepatic fibrosis was used to assess the expression and activities of collagen-degrading proteinases and their inhibitors during the progression of fibrosis. Expression of four members of the matrix metalloproteinase (MMP) family (MMP-2/gelatinase A, MMP-3, MMP-9/gelatinase B, and MMP-13) and three tissue inhibitors of metalloproteinases-1, -2, and -3 (TIMP-1, TIMP-2, and TIMP-3) were evaluated by Northern blot analysis of RNA from liver tissue isolated at 0, 2, 5, 10, 20, and 30 days after either a BDL or sham operation. In addition, we analyzed free gelatinase and TIMP activities by zymography and reverse zymography, respectively. We found that the proteolytic activities of MMP-2 and MMP-9 increased by 2 days after ligation, reached maximal levels at day 10, and remained high through the study period, whereas the gelatinolytic activities in plasma were unchanged. The increase in gelatinase activities was accompanied by an increase in the TIMP mRNA transcripts. TIMP-1 transcripts appeared at day 2, increased until day 10, and remained elevated throughout the study period. TIMP-2 and TIMP-3 transcripts become detectable on day 10 and remained stable afterwards. No corresponding increase in TIMP protein activity was detected by reverse zymography. This appears to result from the formation of TIMP/MMP complexes. These findings indicate a likely surplus in the BDL model of fibrosis of free gelatinases as compared with the TIMPs. Thus, excessive TIMP production is not a sufficient explanation for the observed extracellular matrix accumulation, but complex changes in the local MMP/TIMP balance may underlie the pathomechanisms of fibrosis.

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We compared the expression of matrix metalloproteinases (MMP-2 and MMP-9) and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) in bone marrow acute myelogenous leukaemia (AML) blasts and leukaemic cell lines (HEL, HL-60, K-562 and KG-1) with their expression in normal bone marrow cells. All AML samples and leukaemic cell lines tested expressed MMP-9 and/or MMP-2 mRNA and, accordingly, these gelatinases were secreted into media. Moreover, TIMP-1 and TIMP-2 mRNA and secreted proteins were demonstrated in all the AML samples. Although all the leukaemic cell lines expressed TIMP-1, the HL-60 cells also expressed TIMP-2. In contrast, normal steady-state bone marrow immature progenitor cells (CD34+ cells) did not express or secrete either MMP-2 or MMP-9, but more mature mononuclear cells from normal bone marrow expressed and secreted MMP-9. Also, normal bone marrow CD34+ cells and mononuclear cells expressed TIMP-1 and TIMP-2 mRNA, but these proteins were not detectable by reverse zymography. Furthermore, whereas bone marrow fibroblasts and endothelial cells secreted only latent MMP-2, the activated form of this enzyme was found in media conditioned by cells obtained from long-term cultures of normal and AML bone marrow adherent layers. Our finding of up-regulated production of gelatinases, TIMP-1 and TIMP-2 by leukaemic cells suggests that these proteins may be implicated in the invasive phenotype of AML.

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Summary Matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of metalloproteinases, TIMPs) play essential roles in the remodelling of the extracellular matrix (ECM). Results of in vivo and in vitro studies suggest that the balance between MMPs and TIMPs is altered in neoplasia, contributing to the invasive and metastatic properties of malignant tumours. In this study we have analysed the expression of five MMP genes and TIMP-1 and TIMP-2 in 37 benign and malignant lesions of human breast using Northern blot analysis. MMP-9 (92 kDa gelatinase) and MMP-l (stromelysin 3) were most consistently expressed by carcinomas. Based on detection of either MMP-9 or MMP-l mRNAs, we were able to distinguish between malignant and benign disease with a predictive accuracy of 90% with 94% sensitivity and 85% specificity. Subsequently, these results were compared with results for carcinomas of colon and lung and malignant nonHodgkin's lymphomas (NHL). Elevated MMP-9 and TIMP-1 expression was observed in all four systems. MMP-l characterised all carcinomas as well as carcinomas in situ but was not detectable in NHL. Our data therefore argue that there are remarkably similar patterns of specific functions involved in ECM remodelling that correlate with malignancy in different human tumours of different histogenesis. However, MMP-l expression is a characteristic of tumours of epithelial origin that is not found in lymphoid neoplasia. Thus it suggests that MMP-l may play a regulatory role in the invasion and metastasis of carcinomas.

Oxidative stress plays a critical role in the pathogenesis of hypertension; however, there are no data on salivary redox homeostasis and salivary gland function in children with hypertension. A total of 53 children with hypertension and age-and sex-matched controls were classified for the study. The antioxidant barrier and oxidative/nitrosative stress were evaluated in non-stimulated (NWS) and stimulated (SWS) whole saliva, plasma, and erythrocytes, with Student's t-test and Mann-Whitney U-test used for statistical analysis. We demonstrated that the activities of superoxide dismutase, catalase, and peroxidase were significantly higher in NWS, SWS, and erythrocytes of children with hypertension, similar to oxidative damage in proteins (advanced glycation end products) and lipids (malondialdehyde) as well as nitrosative stress markers (peroxynitrite and nitrotyrosine). The level of uric acid (UA) was significantly higher in NWS, SWS, and plasma of children with hypertension. UA concentration in SWS correlated positively with systolic and diastolic blood pressure and UA content in plasma. This parameter differentiates children with hypertension from healthy controls (AUC = 0.98) with a high degree of sensitivity (94%) and specificity (94%). Stimulated salivary flow was significantly lower in the hypertension group, similar to total protein content and salivary amylase activity. In summary, childhood hypertension is associated with hyposalivation as well as disturbances in antioxidant defense and enhanced oxidative/nitrosative damage both in the plasma/erythrocytes as well as saliva. Salivary UA may be a potential biomarker of hypertension in children.

Obesity is inseparably connected with oxidative stress. This process may disturb the functioning of the oral cavity, although the effect of oxidative stress on salivary gland function and changes in the qualitative composition of saliva are still unknown. Our study is the first to evaluate salivary redox homeostasis in 40 overweight and obese adolescents and in the age- and gender-matched control group. We demonstrated strengthening of the antioxidant barrier (superoxide dismutase, catalase, peroxidase, uric acid, total antioxidant capacity (TAC)) with a simultaneous decrease in reduced glutathione concentration in saliva (non-stimulated/stimulated) in overweight and obese teenagers compared to the controls. The concentration of the products of oxidative damage to proteins (advanced glycation end products), lipids (malondialdehyde, 4-hydroxynonenal) and DNA (8-hydroxydeoxyguanosine) as well as total oxidative status were significantly higher in both non-stimulated and stimulated saliva as well as plasma of overweight and obese adolescents. Importantly, we observed more severe salivary and plasma redox alterations in obese adolescents compared to overweight individuals. In the study group, we also noted a drop in stimulated salivary secretion and a decrease in total protein content. Interestingly, dysfunction of parotid glands in overweight and obese teenagers intensified with the increase of BMI. We also showed that the measurement of salivary catalase and TAC could be used to assess the central antioxidant status of overweight and obese adolescents.