Tissue Inhibitors Of Metalloproteinases Activity Research Articles

Increased levels and activity of matrix metalloproteinase-9 in obstructive sleep apnea syndrome.

Matrix metalloproteinases (MMPs) are involved in the pathogenesis of cardiovascular diseases. We examined serum levels of MMP-9 and its inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), activity of MMP-9, and the effect of nasal continuous positive airway pressure (nCPAP) in patients with obstructive sleep apnea syndrome (OSAS). After polysomnography, venous blood was collected at 5:00 A.M. from 44 patients with OSAS and 18 control subjects who were obese, and serum levels of MMP-9, TIMP-1, and enzymatic activity of MMP-9 were measured. In addition, the effects of 1 month of treatment with nCPAP were studied in patients with moderate to severe OSAS. Although serum levels of MMP-9 (p < 0.03) and MMP-9 activity (p < 0.01) were higher in patients with OSAS than in control subjects who were obese, TIMP-1 levels did not differ significantly. In patients with OSAS, the severity of OSAS was the primary factor influencing levels (p < 0.01) and activity (p < 0.01) of MMP-9. nCPAP significantly decreased serum levels (p < 0.01) and activity (p < 0.001) of MMP-9 but did not affect TIMP-1 levels. Therefore, OSAS may increase risks of cardiovascular morbidity, and nCPAP might be useful for decreasing these risks.

The antioxidant and antifibrogenic effects of the glycosaminoglycans hyaluronic acid and chondroitin-4-sulphate in a subchronic rat model of carbon tetrachloride-induced liver fibrogenesis

Hepatic fibrosis involves the interplay of many factors including reactive oxygen species. Recent reports described antioxidant properties of glycosaminoglycans (GAGs). Since several findings have shown that hyaluronic acid (HYA) and chondroitin-4-sulphate (C4S) may act as antioxidant molecules, the aim of this research was to evaluate the antioxidant effects of HYA and C4S treatment in a rat model of liver fibrosis. The effect on tissue inhibitors of metalloproteinases (TIMPs) was also studied.Liver fibrosis was induced in rats by eight intraperitoneal injections of CCl4, twice a week for 6 weeks. HYA or C4S alone (25mg/kg) or HYA and C4S in combination (12.5 + 12.5mg/kg) were administered daily by the same route during the 6 weeks. At the end of the 6-week treatment period (24h after the last dose of GAGs), the following parameters were evaluated: (1) serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, as index of hepatic cell disruption; (2) hepatic conjugated dienes (CD), as index of lipid peroxidation; (3) hepatic TIMPs activity and expression; (4) hepatic superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity, as index of endogenous defences; (5) hepatic hydroxyproline, as index of collagen deposition.CCl4-induced liver fibrosis enhanced lipid peroxidation and TIMPs activation, increased ALT and AST, depleted antioxidants SOD and GPx, and caused collagen deposition in liver tissue. Treatment with GAGs, especially when in combination, successfully reduced ALT and AST rise, lipid peroxidation by evaluating conjugated dienes, TIMPs activation and mRNA expression, partially restored SOD and GPx activities, and limited collagen deposition in the hepatic tissue.The data obtained showed that these molecules were able to limit hepatic injury induced by chronic CCl4 intoxication and especially limited liver fibrosis. They also confirm that HYA and C4S may exert antioxidant mechanism, while reduction of TIMPs expression suggests that GAGs may influence MMPs and TIMPs imbalance in liver fibrosis.

CHANGES IN SERUM MATRIX METALLOPROTEINASES AND THEIR INHIBITORS FOLLOWING ECCENTRIC MUSCLE CONTRACTIONS IN HUMANS

Matrix metalloproteinase (MMP)-2 and −9 degrade collagen IV, the principal component of skeletal muscle basement membrane, while tissue inhibitors of metalloproteinases (TIMP) inhibit these enzymes. Studies in animals have shown remodelling of connective tissue and changes in MMP and TIMP activity in response to exercise. PURPOSE To investigate the effects of a single bout of high force eccentric contractions on concentrations of MMPs and their inhibitors in serum. METHODS Nine volunteers (5 male, 4 female, mean age 23±SD 4 yrs) each performed a single bout of 100 maximal eccentric contractions of the knee extensors of one randomly selected leg on an isokinetic dynamometer. Venepuncture blood samples were collected from the volunteers pre-exercise and on days 1, 2, 3, 4, 8, 11 and 14 post exercise. Maximum voluntary contractile (MVC) force was also recorded on these days. Serum creatine kinase (CK) activity was measured and concentrations of MMP-9, TIMP-1, TIMP-2 and the MMP-2/TIMP-2 complex were determined by enzyme linked immunosorbent assay (ELISA). Data were analysed for statistical significance using repeated measures ANOVA. RESULTS The largest decline in MVC force (39.2±23.4\%, mean±SD) was observed on day 2 post exercise. Serum CK activity (mean±SD) increased from 216±149 U • L−1 to 11, 121±17,124 U • L−1 on day 4 post exercise (P<0.01). Serum MMP-9 increased from 111±42ng• ml−1 to 188± 49ng• ml−1 on day 8 post exercise (P<0.01). When compared with the pre-exercise value, serum TIMP-1 was significantly elevated on days 1, 2, 3, 4 and 14 post exercise (P<0.05). There were no changes in serum TIMP-2 or the MMP-2/TIMP-2 complex. CONCLUSION Eccentric contraction-induced muscle damage is associated with delayed and prolonged increases in serum concentrations of MMP-9 and TIMP-1, suggestive of muscle connective tissue remodelling.

Regulation of Tissue Inhibitors of Metalloproteinase-1 Gene Expression by Cytokines in Human Gingival Fibroblasts

Tissue inhibitors of metalloproteinase (TIMP) are important participants in various physiological processes that involve tissues remodeling. They help maintain a delicate balance between physiological degradation and synthesis of the extracellular matrix. A better understanding of TIMP activity will be helpful in understanding the etiology of periapical lesions and their means of treatment. The fibroblast is a prominent cellular component of the periapical tissues. The potential implications of cytokine-mediated tissue destruction still remain to be elucidated. The purpose of this study was to determine the effects of interleukin (IL)-1alpha and transforming growth factor (TGF)-beta on the expressing of TIMP-1 by primary gingival fibroblast cultures. After exposure to cytokines for 8 h, total RNA in gingival fibroblasts was isolated and evaluated by reverse-transcriptase polymerase chain reaction. Densitometric analysis of the TIMP-1 mRNA gene expression, after normalization by beta-actin, demonstrated that exposure to IL-1alpha resulted in a decreased level of TIMP-1 mRNA compared with the control groups. However, the TIMP-1 mRNA was up-regulated by TGF-beta. In addition, when the cells were cultured in combination with TGF-beta (1 ng/ml) and IL-1alpha for 8 h, the level of TIMP-1 mRNA was dramatically reduced. These results demonstrated that in human periapical tissue cytokines differentially and specifically regulate expression of TIMP-1 mRNA. An understanding of the actions of cytokines on gingival fibroblasts may result in new therapies to augment current treatment of periapical lesions.

Nitric Oxide Inhibition Increases Aortic Wall Matrix Metalloproteinase-9 Expression

Objective. Nitric oxide (NO) may mediate vessel wall remodeling by regulating expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). This study tested the hypothesis that nitric oxide synthase (NOS) inhibition in whole aortic wall causes increases in cytokine-stimulated MMP and TIMP expression.Methods. Cultured infrarenal aortic segments from Sprague-Dawley rats were exposed to increasing concentrations (0, 0.1, 0.5, 1, and 5 mM; n = 6 per concentration) of NG-monomethyl-l-arginine (L-NMMA), a known inhibitor of NOS. This was in the presence of 2 ng/ml of interleukin-1β, a known inducer of NOS, MMP, and TIMP expression. Media nitrate and nitrite (NOx) were measured at 72 h using the Saville method. Media MMP activity was measured using gelatin zymography. MMP-2 and -9 protein and mRNA levels were determined by Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR). TIMP activity and mRNA levels were evaluated by reverse zymography and RT-PCR. Data were analyzed using ANOVA.Results. Increasing concentrations of L-NMMA produced a dose-dependent decrease in NOx (2214 ± 405 to 347 ± 37 ng/mg, P < 0.001). Zymography demonstrated a dose-dependent increase in 92-kDa MMP (pro-MMP-9) activity (P < 0.001) with corresponding increases in pro-MMP-9 protein (P = 0.03) and mRNA levels (P = 0.004). While there was a dose-dependent increase in 72-kDa MMP (pro-MMP-2) activity (P = 0.001), pro-MMP-2 protein and mRNA levels were unchanged. Reverse zymography demonstrated a dose-dependent increase in 29-kDa TIMP-1 activity (P = 0.01), but there was no change in TIMP-1 mRNA levels.Conclusions. NOS inhibition in ex vivo aortic tissue causes a dose-dependent increase in MMP-9 expression and activity. It is speculated that deficiencies of NO in vivo alter MMP and TIMP homeostasis, favoring matrix degradation.

The role of matrix metalloproteinases in wound healing.

The structure, classification, function, and regulation of matrix metalloproteinases in normal and abnormal wound healing is discussed. Results from key studies suggest that neutrophil-derived matrix metalloproteinase 8 (MMP-8) is the predominant collagenase present in normal healing wounds, and that overexpression and activation of this collagenase may be involved in the pathogenesis of nonhealing chronic leg ulcers. Excessive collagenolytic activity in these chronic wounds is possible because of the reduced levels of tissue inhibitor metalloproteinase 1 (TIMP-1). However, until recently, there have been no studies evaluating levels of matrix metalloproteinase or tissue inhibitors of metalloproteinase activity in chronic diabetic foot wounds. Improving basic knowledge and pharmaceutical intervention in this area ultimately may help clinicians identify and proactively intervene in an effort to prevent normal wounds from becoming chronic. This may prevent the high prevalence of morbidity associated with this significant health problem.

Relaxin increases secretion of tissue inhibitor of matrix metalloproteinase-1 and -2 during uterine and cervical growth and remodeling in the pig.

Remodeling of reproductive organs during pregnancy requires degradation and resynthesis of structural barriers to cell invasion. Matrix metalloproteinases (MMPs) are enzymes that break down components of the extracellular matrix (ECM) and are essential for tissue remodeling processes. Tissue inhibitors of metalloproteinases (TIMPs) are important regulators of MMP activity. In the pig, relaxin stimulates growth and remodeling of the uterus and cervix during pregnancy, effects that include the ability to alter elements of the ECM. Therefore, the objective of this study was to determine whether relaxin alters the production and/or activity of TIMP-1 and TIMP-2 in the porcine uterus or cervix. The growth-promoting effects of relaxin were elicited by administering relaxin to prepubertal gilts every 6 h for 54 h. Expression of TIMP-1 and TIMP-2 was characterized by immunoblotting. Total enzyme activity was measured using an MMP-specific fluorescent substrate assay. TIMP-1 and TIMP-2 proteins were present in the uterus and cervix of control and relaxin-treated pigs, and both proteins were increased by relaxin in the uterine flushes and tissues (P < 0.05). Inhibitor activity in uterine tissue extracts and uterine flushes from relaxin-treated animals was greater than that in controls; however, this activity was restricted to inhibition of MMP-2. In the uterine cervix, relaxin enhanced expression of TIMP-1 and TIMP-2 (P < 0.05), whereas expression of both TIMP proteins was similar in the vaginal cervix of control and relaxin-treated animals. Likewise, inhibitor activity against MMP-2 in the uterine cervix was enhanced in response to relaxin (P < 0.05). In contrast, inhibitor activity was attenuated in extracts from the vaginal cervix (P < 0.05). This study highlights the complex nature of MMP/TIMP regulation during reproductive tissue growth and suggests that TIMP-1 and TIMP-2 may be involved in other aspects of the growth process. These data support a role for relaxin in regulating the activity of TIMPs during growth and remodeling of reproductive connective tissue.

Left ventricular remodeling in transgenic mice with cardiac restricted overexpression of tumor necrosis factor.

The mechanisms responsible for tumor necrosis factor (TNF)-induced LV structural remodeling in the adult heart are not known. We generated a line of transgenic mice (MHCsTNF) with cardiac restricted overexpression of TNF that develop progressive LV dilation/remodeling from 4 to 12 weeks of age. During the early phases of LV structural remodeling, there was a significant increase in total matrix metalloproteinase (MMP) activity that corresponded to a decrease in total myocardial fibrillar collagen content. As the MHCsTNF mice aged, there was a significant decrease in total MMP zymographic activity that was accompanied by an increase in total fibrillar collagen content. The changes in total MMP activity and myocardial fibrillar collagen content were related to a time- dependent increase in myocardial tissue inhibitor of metalloproteinases (TIMP)-1 levels, resulting in a significant time-dependent decrease in the MMP activity/TIMP level ratio in the MHCsTNF mice. To determine a possible mechanism for the increase in myocardial fibrosis, we also measured levels of TGF-beta(1) and TGF-beta(2) protein levels, which were shown to be significantly elevated in the hearts of the MHCsTNF mice. Our results suggest that progressive time-dependent changes in the balance between MMP activity and TIMP activity are responsible, at least in part, for the spectrum of TNF-induced changes in the myofibrillar collagen content that occur during LV structural remodeling in the MHCsTNF mice.

Skin and oral fibroblasts exhibit phenotypic differences in extracellular matrix reorganization and matrix metalloproteinase activity.

Oral mucosal wounds are characterized by rapid re-epithelialization and remodelling. In vitro, oral mucosal fibroblasts exhibit a fetal phenotype with increased extracellular matrix reorganizational ability, migration and experimental wound repopulation when compared with skin fibroblasts. To investigate whether phenotypic differences in the expression and production of matrix metalloproteinase (MMP) -2 and tissue inhibitors of metalloproteinases (TIMPs) could play an important part in mediating these in vitro differences. Skin and oral mucosal fibroblast MMP-2, TIMP-1 and TIMP-2 mRNA expression and protein production were studied in three-dimensional collagen lattices using quantitative competitive reverse transcriptase-polymerase chain reaction (QCRT-PCR), enzyme-linked immunosorbent assay (ELISA), zymography and reverse zymography. Oral mucosal fibroblasts exhibited increased levels of the 62-kDa active form of MMP-2 and lattice contraction when compared with skin fibroblasts. Oral mucosal and skin fibroblast MMP-2 gene expression and synthesis of the 72-kDa pro-MMP-2 was similar as assessed by QCRT-PCR, zymography and ELISA. Differential MMP-2 activation was, however, related to phenotypic differences in TIMP activity between the skin and oral mucosal fibroblasts, as assessed by reverse zymography. These studies propose a mechanism by which fibroblast phenotype may contribute directly to the observed preferential remodelling of oral wounds.

Thioredoxin alters the matrix metalloproteinase/tissue inhibitors of metalloproteinase balance and stimulates human SK‐N‐SH neuroblastoma cell invasion

Thioredoxin (Trx) inhibited tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 activity with an approximate IC50 of 0.3 microM, matrix metalloproteinase (MMP)-2 activity with an approximate IC50 of 2 microM but did not inhibit MMP-9 activity. This differential capacity of Trx to inhibit TIMP and MMP activity resulted in the promotion of MMP-2 and MMP-9 activity in the presence of molar TIMP excess. Inhibition of TIMP and MMP-2 activity by Trx was dependent upon thioredoxin reductase (TrxR), was abolished by Trx catalytic site mutation and did not result from TIMP or MMP-2 degradation. HepG2 hepatocellular carcinoma cells induced to secrete Trx inhibited TIMP activity in the presence of TrxR. SK-N-SH neuroblastoma cells secreted TrxR, which inhibited TIMP and MMP-2 activity in the presence of Trx. Trx stimulated SK-N-SH invasive capacity in vitro in the absence of exogenous TrxR. This study therefore identifies a novel extracellular role for the thioredoxin/thioredoxin reductase redox system in the differential inhibition of TIMP and MMP activity and provides a novel mechanism for altering the TIMP/MMP balance that is of potential relevance to tumor invasion.

Peritonitis Increases MMP-9 Activity in Peritoneal Effluent from CAPD Patients

Patients undergoing long-term continuous ambulatory peritoneal dialysis (CAPD) sometimes experience ultrafiltration failure. Mesothelial basement membrane thickening and the accumulation of submesothelial fibrotic tissue are common features of the diseased peritoneum. Peritonitis can lead to ultrafiltration failure, but the precise mechanism is not clear. The key enzymes in extracellular matrix (ECM) remodeling, namely matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), are produced by human peritoneal mesothelial cells. Using peritoneal effluent from 13 CAPD patients with peritonitis and 7 noninfected CAPD control individuals, we examined MMP and TIMP activities by gelatin and reverse zymography. Latent and activated types of MMP-2 and -9, and TIMP-1 and -2 were identified in peritoneal effluent (from all CAPD patients). Levels of latent and activated type MMP-9, as well as of TIMP-1 activities were higher at the onset of peritonitis than either during the recovery phase of peritonitis and/ or in control individuals. Activated MMP-9 activity positively correlated with leukocyte numbers and IL-6 levels in peritoneal effluent. Activities of MMP-2 and TIMP-2 in peritoneal effluent did not change between the onset of peritonitis and recovery. We concluded that increased MMP-9 and TIMP-1 levels might be associated with peritoneal ECM remodeling during peritonitis.

TRANSFORMING GROWTH FACTOR-β1 BLOCKS THE RELEASE OF COLLAGEN FRAGMENTS FROM BOVINE NASAL CARTILAGE STIMULATED BY ONCOSTATIN M IN COMBINATION WITH IL-1α

Oncostatin M in combination with interleukin-1 (IL-1) induced a rapid and reproducible release of collagen from bovine nasal cartilage in culture. This release was accompanied by a high collagenolytic activity and low or absent tissue inhibitor of metalloproteinase-1 activity in the culture medium. Transforming growth factor-β1 was able to block this release of collagen from the tissue, and reduce the expression and secretion of collagenases whilst maintaining TIMP-1 levels from bovine nasal chondrocytes. This study shows for the first time that TGF-β1 can protect cartilage collagen from destruction.

Matrix metalloproteinases -2 and -9 and their endogenous tissue inhibitors in fetal membrane repair following fetoscopy in a rabbit model.

The cellular mechanisms underlying fetal membrane repair are poorly understood. Matrix metalloproteinases (MMP) and the endogenous tissue inhibitors of metalloproteinases (TIMP) play a key role in the control of turnover of extracellular matrix in fetal membranes at normal parturition and preterm prelabour rupture of the fetal membranes (PPROM). The time course of secretion of MMP-2 (72 kDa, gelatinase A) and MMP-9 (92 kDa, gelatinase B) and TIMP into extra-embryonic coelomic, allantoic and amniotic fluids in a rabbit model was examined. Furthermore, to evaluate their role in fetal membrane repair, the changes induced by fetoscopy at mid-gestation (23 days; gestation length is 32 days) were investigated. Zymography showed predominantly secretion of latent MMP-2 at 18, 23 and 30 days of gestation in all gestational compartments. Reverse zymography detected a broad range of TIMP activity with molecular weights of 27-30 kDa (TIMP-1, glycosylated TIMP-3 and TIMP-4), 24 kDa (unglycosylated TIMP-3) and 21 kDa (TIMP-2). Following fetoscopy, both MMP-2 and TIMP increased significantly in amniotic fluid and extra-embryonic coelomic fluid, but not in allantoic fluid, as demonstrated by densitometric analyses. These findings indicate a modulating role for MMP and TIMP in the repair processes following a surgically induced fetal membrane defect.

FETAL PARTIAL URETHRAL OBSTRUCTION CAUSES RENAL FIBROSIS AND IS ASSOCIATED WITH PROTEOLYTIC IMBALANCE

Purpose We determine whether fetal bladder outlet obstruction induces renal fibrosis, and is associated with an alteration in the regulation of connective tissue degradation and the presence of fibrogenic interstitial cells. Materials and Methods Partial bladder outlet obstruction was surgically induced in 33 fetal sheep at 95 days of gestation. These animals and 24 normal age matched controls were sacrificed at 109, 116 and 135 (term) days of gestation, and the kidneys were rapidly retrieved, drained and weighed. Representative whole kidney samples were snap frozen for assessment of deoxyribonucleic acid, protein and collagen content. Morphometric analysis and alpha-smooth muscle actin immunohistochemistry were performed on histological specimens from formalin fixed kidneys. Tissue extract from fresh kidney specimens were analyzed for metalloproteinase and tissue inhibitor of metalloproteinase activity. Urine samples obtained at the time of sacrifice were analyzed for electrolyte, creatinine and N-acetyl glucosaminidase excretion. Results All obstructed kidneys were hydronephrotic and larger than age matched controls. Obstructed kidneys at term showed interstitial fibrosis, as measured by increased extracellular matrix volume fraction (45% in male obstructed kidneys versus 2.5% in normal male kidneys, p = 0.0004), increased total collagen content (120 mg./kidney in male obstructed versus 20 mg. in normal male animals, p = 0.016) and collagen/deoxyribonucleic acid content per kidney (2.78 versus 0.53 mg./mg., p = 0.016). Metalloproteinase-1 activity was significantly lower in obstructed kidneys (210 versus 380 U./mg. protein in normal kidneys). Tissue inhibitor of metalloproteinase activity was undetectable in both groups. The presence of an increased population of myofibroblasts often associated with fibrotic processes was seen by alpha-smooth muscle actin staining which was localized to interstitial cells throughout the cortex in obstructed kidneys. Conclusions Fetal partial bladder outlet obstruction induces renal interstitial fibrosis as early as 2 weeks after obstruction. A possible mechanism for this process is a shift in proteolytic activity to reduce matrix degradation in obstructed kidneys. These changes might be mediated by the increased number of fibrogenic interstitial cells. The observations suggest several potential approaches to developing an understanding of congenital obstructive uropathy.

IGF-1 decreases collagen degradation in diabetic NOD mesangial cells: implications for diabetic nephropathy.

Nonobese diabetic (NOD) mice develop glomerulosclerosis shortly after the onset of diabetes. We showed that mesangial cells (MCs) from diabetic mice exhibited a stable phenotypic switch, consisting of both increased IGF-1 synthesis and proliferation (Elliot SJ, Striker LJ, Hattori M, Yang CW, He CJ, Peten EP, Striker GE: Mesangial cells from diabetic NOD mice constitutively secrete increased amounts of insulin-like growth factor-I. Endocrinology 133:1783-1788, 1993). Because the extracellular matrix (ECM) accumulation in diabetic glomerulosclerosis may be partly due to decreased degradation, we examined the effect of excess IGF-1 on collagen turnover and the activity of metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) in diabetic and nondiabetic NOD-MC. Total collagen degradation was reduced by 58 +/- 18% in diabetic NOD-MCs, which correlated with a constitutive decrease in MMP-2 activity and mRNA levels, and nearly undetectable MMP-9 activity and mRNA. TIMP levels were slightly decreased in diabetic NOD-MC. The addition of recombinant IGF-1 to nondiabetic NOD-MC resulted in a decrease in MMP-2 and TIMP activity. Furthermore, treatment of diabetic NOD-MC with a neutralizing antibody against IGF-1 increased the latent form, and restored the active form, of MMP-2. In conclusion, the excessive production of IGF-1 contributes to the altered ECM turnover in diabetic NOD-MC, largely through a reduction of MMP-2 activity. These data suggest that IGF-1 could be a major contributor to the development of diabetic glomerulosclerosis.

Secretion of matrix metalloproteinase-2, matrix metalloproteinase-9 and tissue inhibitor of metalloproteinases into the intrauterine compartments during early pregnancy.

Matrix metalloproteinases (MMPs) are important enzymes in tissue remodelling, a key event for the development of the fetal membranes and placenta and establishing the feto-maternal interface during early pregnancy. This study has examined the secretion of the gelatinases, MMP-2 (72 kDa) and MMP-9 (92 kDa), and the endogenous tissue inhibitors of metalloproteinases (TIMPs) into extra-embryonic coelomic and amniotic fluids, the two principal intra-uterine compartments of the first trimester, and compared them to amniotic fluid collected later in gestation. In extra-embryonic coelomic fluid, gelatin zymography demonstrated that MMP-2 (72 kDa) was the predominant gelatinase, with some MMP-9 present. A broad range of TIMPs corresponding to TIMP-1 and TIMP-2, glycosylated and unglycosylated TIMP-3 and TIMP-4 was detected in this compartment by reverse zymography and immunoblot analyses. There was little gelatinase or TIMP activity in amniotic fluid in the first trimester. In amniotic fluid from the second trimester after fusion of the membranes obliterating the extra-embryonic coelom, and at term elective caesarean section, MMP-2 is the predominant gelatinase present, with a broad spectrum of TIMPs. These findings demonstrate that predominantly MMP-2 and also MMP-9, regulated by a range of TIMPs, are involved in intra-uterine tissue remodelling during the establishment of pregnancy.

The Oxidative Inactivation of Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) by Hypochlorous Acid (HOCl) is Suppressed by Anti-Rheumatic Drugs

Tissue inhibitors of metalloproteinases (TIMPs) prevent uncontrolled connective tissue destruction by limiting the activity of matrix metalloproteinases (MMPs). That TIMPs should be susceptible to oxidative inactivation is suggested by their complex tertiary structure which is dependent upon 6 disulphide bonds. We examined the oxidative inactivation of human recombinant TIMP-1 (hr TIMP-1) by HOCl and the inhibition of this process by anti-rheumatic agents.TIMP-1 was exposed to HOCl in the presence of a variety of disease modifying anti-rheumatic drugs. TIMP-1 activity was measured by its ability to inhibit BC1 collagenase activity as measured by a fluorimetric assay using the synthetic pEptide substrate (DNP-Pro-Leu-Ala-Leu-Trp-Ala-Arg), best cleaved by MMP-1.The neutrophil derived oxidant HOCl, but not the derived oxidant N-chlorotaurine, can inactivate TIMP-1 at concentrations achieved at sites of inflammation. Anti-rheumatic drugs have the ability to protect hrTIMP-1 from inactivation by HOCl. For D-penicil-lamine, this effect occurs at plasma levels achieved with patients taking the drug but for other anti-rheumatic drugs tested this occurs at relatively high concentrations that are unlikely to be achieved in vivo, except possibly in a microenvironment. These results are in keeping with the concept that biologically derived oxidants can potentiate tissue damage by inactivating key but susceptible protein inhibitors such as TIMP-1 which form the major local defence against MMP induced tissue breakdown.

Positive correlation between inhibitors of matrix metalloproteinase 1 and matrix metalloproteinases in malignant ovarian tumor tissues

To investigate the relation between matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in malignant ovarian tumor, MMP and TIMP activities in conditioned media of 16 malignant ovarian tumor tissues and six normal ovaries were detected by zymography and reverse zymography, respectively and were quantitated with a densitometer. TIMP-1 and TIMP-2 were detected in all normal and malignant ovarian tumor tissues by reverse zymography. In normal ovaries, the intensity of TIMP-2 bands was stronger than TIMP-1, but in malignant tumor tissues those of TIMP-1 were stronger. The ratios of TIMP-1 to TIMP-2 and MMP-9 to MMP-2 were significantly higher in malignant tumor tissues than in normal ovaries ( P<0.001). TIMP activity consisting of TIMP-1 and TIMP-2 correlated significantly to MMP activity of MMP-2 and MMP-9 ( r=0.67, P<0.005). There was a significant correlation between TIMP-1 activity and MMP activity ( r=0.72, P<0.001), but no correlation was observed between TIMP-2 activity and MMP activity. The high level of TIMP-1 appeared to be related to malignant phenotype in ovaries as well as the high level of MMP-9.

Co-expression of tissue inhibitor and matrix metalloproteinase in myocardium

Matrix metalloproteinases (MMP) are present in the latent form in normal myocardium. To examine the stringent balance between MMP and tissue inhibitor of metalloproteinase (TIMP) and to determine whether MMP are secreted simultaneously and in co-ordination with their inhibitors, we analysed MMP and TIMP by immunological, isolation by gel-permeation and affinity chromatography, and enzymatic assays in tissues and extracts. Using antibodies to MMP-1 and TIMP-1, we found strong in situ staining of MMP-1 and TIMP-1 in tissues. The staining was uniform in the endo- and subendomyocardium as well as in the interstitial space. TIMP-1 was present wherever MMP-1 was localized. From the tissue extract, proteins were separated on a gel-filtration column (Sephacryl S-200) and analysed for MMP and TIMP activity by zymography as well as by using succinyl-Gly-Pro-Leu-Gly-Pro-4-amido-7-methyl coumarin (Suc-GPLGP-AMC) as a selective fluorogenic substrate for collagenase. TIMP and MMP were further purified on collagen-Sepharose affinity column. The results indicated that MMP activity was co-eluted with TIMP activity. MMP-1, MMP-2 and TIMP-1 were further analysed by Northern blot for mRNA levels in the heart, skin, lung, liver and kidney. Results suggested co-expression of MMP-1 and TIMP-1 at the transcription level in all tissues. The level of MMP-2 mRNA was specifically higher in the heart tissue, which suggests a role of MMP-2 in the integrity of cardiovascular structure. The study indicated that myocardium as well as other tissue have an endogenous inhibitory system, suggesting that the MMPs activity is co-ordinated by their inhibitors at both the gene and protein levels. Furthermore, MMP and TIMP were co-expressed and were tightly regulated in maintaining the architecture of the interstitial tissue.

Human shoulder tendon biopsy samples in organ culture produce procollagenase and tissue inhibitor of metalloproteinases.

To investigate the production of the matrix metalloproteinase (MMP), collagenase (MMP-1), and its natural inhibitor, the tissue inhibitor of metalloproteinases (TIMP) by diseased human tendon samples in organ culture. Portions of tendons were excised from the shoulders of patients undergoing shoulder surgery, classified as either proximal to the lesion (abnormal) or distal to the lesion (normal) according to their macroscopic appearance at surgery, and placed in organ culture for periods of up to 28 days. The release of collagenase and TIMP activity in the conditioned culture medium was measured. Procollagenase and TIMP were both produced by all the tendon samples for an extended period of time. The levels of enzyme and inhibitor varied between patients, but in most of them TIMP levels were greater than collagenase levels. In one sample of calcified tendon, procollagenase levels were greater than those of TIMP. The mean level of collagenase produced by tendon proximal to the lesion and tendon distal to the lesion were not significantly different (95.2 (SD 106.8) U/g and 34.0 (45.3) U/g, respectively), while the corresponding figures for TIMP were 109.7 (62.3) U/g and 53.0 (27.9) U/g (p = < 0.05), although there was considerable variation in some samples. Western blotting and collagen fragment analysis confirmed that the collagenolytic activity detected was attributable to the metalloproteinase fibroblast collagenase (MMP-1). Tendon tissue can actively secrete procollagenase, an enzyme that, once activated, is capable of remodelling collagen, the major connective tissue component of tendon. Collagenase is produced even in unstimulated cultures, although the concentrations of TIMP are usually greater than that of collagenase in most samples. Some activation of collagenase appeared to have occurred. These results indicate that tendon tissue cells are capable of producing a remodelling response, even in end stage tendon disease.