Plasminogen activators are involved in keratinocyte and fibroblast migration in wounded cultures in vitro

During skin repair both keratinocytes and fibroblasts migrate into the wounded area. In the process of cell migration, controlled proteolytic degradation of the extracellular matrix occurs. It has been suggested that the plasminogen activator system is involved in such proteolytic processes occurring during wound healing.The role of plasmin, urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA) in keratinocyte and fibroblast migration in vitro was examined in the present study. Confluent cultures of normal human keratinocytes, human squamous cell carcinoma (SCC-4), SV40 transformed keratinocytes (SVK-14) and human fibroblasts were mechanically wounded and the repopulation of the denuded area was examined. Migration of cells into the denuded area could be inhibited by neutralizing antibodies against t-PA or u-PA, or the serine protease inhibitor Trasylol in all cell types studied. For detailed study on involvement of u-PA and t-PA in migration processes wounded cultures of SVK14 cells were used. Both t-PA and u-PA activity could be detected at the migrating edge of SVK14 cultures as revealed by zymography and by immunocytochemistry using polyclonal antibodies.Our results demonstrate the direct involvement of not only u-PA but also t-PA in migration of keratinocytes and fibroblasts in wounded cultures in vitro.     

Increased TIMP/MMP ratio in varicose veins: a possible explanation for extracellular matrix accumulation

Primary varicose veins are functionally characterized by venous back-flow and blood stagnation in the upright position. Dilatation and tortuosity provide evidence for progressive venous wall remodelling, with disturbance of smooth muscle cell/extracellular matrix organization. Affected areas are not uniformly distributed, some areas being hypertrophic, whereas others are atrophic or unaffected. In 12 varicose veins and ten control veins, the proteolytic enzyme/inhibitor balance which may participate in the remodelling of the venous wall was investigated. For this purpose, the presence and enzymatic activity of matrix metalloproteinases (MMP-2, MMP-9), tissue inhibitors of MMPs (TIMP-1, TIMP-2), urokinase-type (uPA) and tissue-type (tPA) plasminogen activators (PAs), and plasminogen activator inhibitor-1 (PAI-1) were quantified by western blot and gelatin or plasminogen-casein zymography. In addition, MMP-2, TIMP-1, TIMP-2, and PAI-1 levels were measured by ELISA. A high TIMP-1 level and a low MMP-2 level/activity were found in varicose veins (p<0.005), resulting in a three-fold increase in the TIMP-1/MMP-2 ratio in varicose versus control veins. Levels of PAs (uPA and tPA) as well as PAI-1 were both lower in varicose veins (p<0.005), with minimal change in the PAI/PA ratio. These results demonstrate that varicose veins are characterized by a higher than normal TIMP/MMP ratio, which may facilitate extracellular matrix accumulation in the diseased venous wall.     

Regulation of matrix metalloproteinases and their inhibitors in the left ventricular myocardium of patients with aortic stenosis

Aortic stenosis (AS) results in myocyte and extracellular matrix remodeling in the human left ventricle (LV). The myocardial renin-angiotensin system is activated and collagens I and III and fibronectin accumulate. We determined the yet unknown regulation of enzymes that control collagen turnover, i.e., LV matrix metalloproteinases (MMP) and their tissue inhibitors (TIMPs) in human AS. We compared LV samples from AS patients undergoing elective aortic valve replacement (n=19) with nonused donor hearts with normal LV function (controls, n=12). MMP-2, MMP-9, MT1-MMP, and extracellular matrix metalloproteinase inducer (EMMPRIN), TIMP-1, TIMP-2, TIMP-3, and TIMP-4 mRNA were quantitated by real-time RCR. MMP-1, MMP-2, MMP-3, TIMP-3, TIMP-4, and EMMPRIN protein were measured by immunoblotting and MMP-9 and TIMP-1 protein by ELISA. Gelatinolytic MMP-2 and MMP-9 activity was measured by zymography. MMP-2 was increased in AS at mRNA, protein, and activity levels (131%, 193%, and 138% of controls). MMP-3 protein (308%) and EMMPRIN mRNA and protein were also upregulated (171% and 200%). In contrast, MMP-1 (37%) and MMP-9 mRNA, protein, and activity (26%, 21%, and 52%) were downregulated. MMP-9 activity was inversely correlated with LV size. TIMP-1 mRNA and protein were decreased (55% and 73%). In contrast, TIMP-2 mRNA (358%), TIMP-3 mRNA and protein (145% and 249%) were increased. TIMP-4 mRNA was not altered, but TIMP-4 protein was upregulated to 350%. Changes were similar in AS patients with normal and impaired LV ejection fraction. The dysregulation of myocardial MMPs and TIMPs in human AS starts at an early disease stage when LV function is still normal. In spite of upregulation of some MMPs the balance between MMP and TIMP is shifted towards MMP inhibition in human AS and may contribute to collagen accumulation.     

脑源性神经营养因子对细胞外蛋白水解酶激活作用的研究

 目的：研究脑源性神经营养因子 (BDNF) 对细胞外蛋白水解酶表达和激活作用的影响。 方法：体外分离并培养人脐静脉内皮细胞（HUVEC），RT-PCR法检测HUVEC基质金属蛋白酶MMP-2 、MMP-9 和基质金属蛋白酶组织抑制剂TIMP-1、TIMP-2 mRNA的表达，明胶酶谱检测MMP-2和MMP-9蛋白酶活性，纤维蛋白酶谱检测尿激酶型纤溶酶原激活剂（uPA）蛋白酶活性，Western blotting检测uPA、纤溶酶原激活剂抑制剂（PAI）、TIMP-1及TIMP-2表达。 结果：在对HUVEC增殖无明显促进作用的浓度范围内，BDNF可促进无血清培养的HUVEC MMP-2和MMP-9 mRNA表达,并可促进MMP-2和MMP-9酶原的激活产生活性明胶酶，BDNF对TIMP-1和TIMP-2的表达无明显影响。BDNF以浓度和时间依赖性方式上调HUVEC uPA和PAI-1的表达，并可促进uPA的活性。 结论：BDNF可激活MMPs和uPA/PAI相关的蛋白级联。     

Compression and tension: differential effects on matrix accumulation by periodontal ligament fibroblasts in vitro

Human periodontal ligament fibroblasts were subjected to 10% cyclic equibiaxial tensional and compressive forces in vitro. Media supernatants were analyzed for changes in total protein, extracellular matrix proteins type I collagen and fibronectin, as well as MMP expression by gelatin zymography and Western blot. RNA analyses for changes in collagen, MMP-2, and TIMP-2 were carried out by either Real-time PCR and/or Northern blot. Application of compressional forces resulted in decreases in type I collagen and fibronectin protein, Col1A1 RNA, and increases in total protein, MMP-2 protein (latent and active), and MMP-2 RNA. TIMP-2 RNA was unchanged by compressive forces. In contrast, tensional forces increased total protein, type I collagen, Col1A1 RNA, as well as MMP-2 and TIMP-2 RNA. These studies show that cells can perceive two different forms of mechanical stimuli and respond in a differential manner relative to extracellular matrix synthesis and degradation.     

Loss or inhibition of uPA or MMP-9 attenuates LV remodeling and dysfunction after acute pressure overload in mice

Left ventricular (LV) hypertrophy is a natural response of the heart to increased pressure loading, but accompanying fibrosis and dilatation may result in irreversible life-threatening heart failure. Matrix metalloproteinases (MMPs) have been invoked in various cardiac diseases, however, direct genetic evidence for a role of the plasminogen activator (PA) and MMP systems in pressure overload-induced LV hypertrophy and in heart failure is lacking. Therefore, the consequences of transverse aortic banding (TAB) were analyzed in mice lacking tissue-type PA (t-PA(-/-)), urokinase-type PA (u-PA(-/-)), or gelatinase-B (MMP-9(-/-)), and in wild-type (WT) mice after adenoviral gene transfer of the PA-inhibitor PAI-1 or the MMP-inhibitor TIMP-1. TAB elevated LV pressure comparably in all genotypes. In WT and t-PA(-/-) mice, cardiomyocyte hypertrophy was associated with myocardial fibrosis, LV dilatation and dysfunction, and pump failure after 7 weeks. In contrast, in u-PA(-/-) mice or in WT mice after PAI-1- and TIMP-1-gene transfer, cardiomyocyte hypertrophy was moderate and only minimally associated with cardiac fibrosis and LV dilatation, resulting in better preservation of pump function. Deficiency of MMP-9 had an intermediate effect. These findings suggest that the use of u-PA- or MMP-inhibitors might preserve cardiac pump function in LV pressure overloading.     

EMMPRIN-mediated MMP regulation in tumor and endothelial cells

Tumor invasion and metastasis are multistep processes which require extracellular matrix remodeling by proteolytic enzymes such as matrix metalloproteinases (MMPs). The production of these enzymes is stimulated by many soluble or cell-bound factors. Among these factors, extracellular matrix metalloproteinase inducer (EMMPRIN) is known to increase in vitro stromal cell production of MMP-1, MMP-2 and MMP-3. In this study, we demonstrated that EMMPRIN-transfected MDA-MB-436 tumor cells displayed a more invasive capacity than vector-transfected cells in a modified Boyden chamber invasion assay. Using gelatin zymography and protein analyses, we showed that EMMPRIN-transfected cancer cells produced significantly more latent and active MMP-2 and MMP-3 than vector-transfected cancer cells. We found that EMMPRIN did not regulate MMP-1, MMP-9, membrane type-1 MMP (MT1-MMP) expression and had also no effect on the production of the specific tissue inhibitors of MMPs (TIMPs), TIMP-1 and TIMP-2. We also demonstrated that tumor-derived EMMPRIN stimulated MMP-1, -2, and -3 without modification of MMP-9, MT1-MMP, TIMP-1 and TIMP-2 production in human umbilical vein endothelial cells (HUVEC). These data provide support for the role of EMMPRIN in tumor invasion, metastasis, and neoangiogenesis by stimulating extracellular matrix remodeling around tumor cell clusters, stroma, and blood vessels. This revised version was published online in July 2006 with corrections to the Cover Date.     

Metalloproteinases in juvenile angiofibroma--a collagen rich tumor

Matrix metalloproteinases (MMPs) act in diverse physiological and pathological conditions such as tumor growth and angiogenesis by cleaving extracellular matrix and nonmatrix substrates. MMPs with gelatinase/collagenase activity have not yet been studied in juvenile angiofibroma, a unique fibrovascular tumor with prominent collagen expression. Quantitative real-time polymerase chain reaction studies, Western blot analysis, immunofluorescence studies, gel zymography, and in situ zymography were used to analyze MMP-1, MMP-2, MMP-9, MMP-13, MMP-14, TIMP-1, and TIMP-2 in 9 juvenile angiofibromas and 2 inferior nasal turbinate specimens. Quantitative real-time polymerase chain reaction found significantly elevated expression of MMP-2, MMP-9, and MMP-14 (P < .05) in tumor tissue compared with the inferior nasal turbinate specimens. Western blot analysis detected more prominent MMP-1, MMP-2, and MMP-9 protein levels in juvenile angiofibromas compared with inferior nasal turbinates, but not MMP-13, MMP-14, TIMP-1, and TIMP-2. Immunofluorescent staining proved a mainly stromal localization of the analyzed MMPs. Only MMP-9 and MMP-14 were also detected in vessel walls. MMP-1, MMP-2, and MMP-13 also stained mast cells. Gel zymography indicated increased MMP-2 and MMP-9 gelatinase activity in juvenile angiofibromas compared with inferior nasal turbinates. Finally, in situ zymography detected very high stromal gelatinase/collagenase activity. This study indicates significant expression of MMPs with gelatinase/collagenase activity in juvenile angiofibromas with evidence of a disturbed balance of MMPs to TIMPs toward enhanced MMP activity. These MMPs are assumed to be involved in tumor pathology with an influence on tumor growth and angiogenesis.     

Matrix metalloproteinase-2, -9, and tissue inhibitor of metalloproteinase-1 in patients with hypertension.

There are conflicting data in the literature regarding the expression pattern of the vascular matrix metalloproteinase (MMP) system and their inhibitors (TIMPs) in human hypertension. The authors hypothesized that MMP-2, MMP-9, and TIMP-1 would be abnormal in hypertension, reflecting alterations in extracellular matrix (ECM) turnover. The authors measured plasma levels and activities of MMP-2, MMP-9, and TIMP-1 in 44 hypertensive patients and 44 controls. MMP-2 levels and activity were significantly higher in hypertensive group (p < .0001). Significant increase was also observed for MMP-9 level and activity (p < .0001) and for TIMP-1 (p < .0001) in hypertensive patients. Plasma levels and activities of MMP-2, MMP-9, and TIMP-1 are increased in hypertensive patients, which may reflect abnormal ECM metabolism.     

MMP-2- and MMP-9-linked gelatinolytic activity in the sputum from patients with asthma and chronic obstructive pulmonary disease

BACKGROUND: The course of asthma and chronic obstructive pulmonary disease (COPD) is associated with bronchial morphological changes. Metalloproteinases are thought to play a role in these structural changes. METHODS: We studied the gelatinolytic activity present in the induced sputum from 20 patients with asthma, 20 with COPD and 19 healthy controls. The assessment of gelatinolytic activity was performed by quantitative zymography, and gelatinolytic species were identified by Western blot analysis. Tissue inhibitor of metalloproteinase-1 (TIMP-1) was detected by reverse zymography and ELISA. RESULTS: From zymography, we found significantly higher gelatinolytic activity linked to pro-matrix metalloproteinase-9 (pro-MMP-9) in the sputum from asthmatics (p < 0.0001) and COPD patients (p < 0.0001) compared to the control group. Furthermore, the activated form of MMP-9 (85 kD) was found in the sputum from 60% of asthmatics and 85% of COPD patients, but was absent in that of control subjects (p < 0.0001). Importantly, although less frequently detectable than pro-MMP-9, pro- MMP-2 (72 kD) was found more frequently in asthmatics (50%) than in control subjects (5%) (p < 0. 005). We also described two unusual gelatinolytic species of 45 and 120 kD and showed that they derived from MMP-9 according to their ability to bind gelatin and anti-MMP-9 antibody. Levels of TIMP-1 were higher in asthmatics (p < 0.05) and COPD patients (p < 0.05) than in controls. CONCLUSION: Asthmatics and COPD patients display an increased gelatinolytic activity linked to MMP-2 and MMP-9 and higher levels of TIMP-1 in their sputum.     

Plasminogen activators in bovine milk during mastitis,an inflammatory disease

We have investigated the content of plasminogen activators in bovine milk during mastitic inflammation induced by Staphylococcus aureus. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with fibrin agarose zymography and a coupled peptidyl anilide plasminogen activation assay of samples of whey prepared by acidification, we found that the level of tisue-type plasminogen activator (t-PA) in milk was increased immediately after infection and remained elevated during an experimental period of 42 days. The maximal increase was 10 to 20-fold. By zymography, we also demonstrated a strong increase in urokinase-type plasminogen activator (u-PA) associated with the bovine cells in the milk. By ligand blotting, we demonstrated an increase in the level of the urokinase-receptor (u-PAR) on the milk cells during inflammation. Plasma kallikrein was also detected as a plasminogen dependent proteolytic activity by zymography of whey samples. When analyzed in the presence of the t-PA in the milk, the plasma kallikrein lysis zone was strongly increased in mastitic whey, but when analyzed after separation from t-PA, its level was unaffected by mastitis; this could be ascribed to a t-PA dependent stimulation of plasma prekallikrein. These results suggest an important role for plasminogen activators in the inflammatory response during bovine mastitis. Using an enzyme-linked immunosorbent assay we measured the plasminogen/plasmin level during the inflammation, but found a less than 2-fold increase during the experimental period.     

Matrix metalloproteinase (MMP)-2 and MMP-9 and their inhibitor, TIMP-1, in human term decidua and fetal membranes: the effect of prostaglandin F(2alpha) and indomethacin.

Degradation and breakdown of gestational membranes and the adjacent decidua are essential processes for the advancement of labour. We have assessed the effect of prostaglandin (PG) synthesis on the expression and activity of matrix metalloproteinase (MMP)-2 and MMP-9 and tissue inhibitor of metalloproteinases (TIMP-1) in fetal membranes at the edge of the placenta and decidua, by using ex-vivo organ culture of the tissues in the absence or presence of PGF(2alpha) (0.1, 1.0 and 10 microg/ml) or a PG synthesis inhibitor, indomethacin (10(-4)-10(-6) mol/l). Conditioned media were assessed for MMP by zymography on gelatin containing sodium dodecyl sulphate-polyacrylamide gels and for TIMP-1 by Western blot analysis. Compared to the membranes, decidua produced significantly more MMP-2 and MMP-9 as well as TIMP-1. PGF(2alpha) caused a 2.4- and 1.9-fold increase in the production of MMP-2 and MMP-9 in the decidua, respectively (P < 0.05), and an 11.3-fold increase of the active form of MMP-2 (62 kDa) which could hardly be detected in basal culture conditions (P < 0.01). PGF(2alpha) decreased TIMP-1 production by 70% in the decidua. The production of MMP-2 and MMP-9 and TIMP-1 by the amniotic and chorionic membranes was not affected by PGF(2alpha). Indomethacin decreased the production of MMP-2 and MMP-9 by 78 and 35% in chorion, and by 70 and 58% in amnion, respectively (P < 0.05), but did not affect production in decidual tissue. Indomethacin increased the production of TIMP-1 in chorion and amnion [by 4.1- and 4.5-fold respectively (P < 0.01)], but had no effect on decidua. Cumulatively, PGF(2alpha) increases decidual gelatinolytic activity. Meanwhile the inhibition of PG production by indomethacin reduces total gelatinolytic activity in fetal membranes, possibly accounting for some of its labour-arresting property.     

Specific expression of matrix metalloproteinases 1, 3, 9 and 13 associated with invasiveness of breast cancer cells in vitro

Several matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) were studied in highly invasive (MDA-MB-231) and slightly invasive (MCF-7, T47D, BT-20) breast cancer cell lines. Investigations were carried out at the protein level and/or at the mRNA level, either in cells cultured as monolayers on plastic, or in cells seeded on a thin layer of Matrigel basement membrane matrix. Analysis of MMP expression by RT-PCR showed expression of MMP-1, MMP-3, and MMP-13 in highly invasive MDA-MB-231 cells, but not in slightly invasive cell lines. The extracellular secretion of MMP-1 and MMP-3 by MDA-MB 231 cells could be also shown by ELISA. TIMP-1 and TIMP-2 mRNAs were found in all cell lines, however, the extracellular secretion of both TIMPs was much higher in MDA-MB-231 cells than in the other cell lines. When the cells were cultured on Matrigel matrix, MMP-9 expression was induced in MDA-MB-231 cells only, as assessed by RT-PCR and zymography experiments. The invasive potential of MDA-MB-231 cells evaluated in vitro through Matrigel was significantly inhibited by the MMP inhibitor BB-2516, by 25% and 50% at the concentrations of 2 × 10⁻⁶M and 10⁻⁵M, respectively. In conclusion, our data show that highly invasive MDA-MB-231 cells but not slightly invasive T47D, MCF-7 and BT-20 cells express MMP-1, MMP-3, MMP-9 and MMP-13. MMP-9 which is specifically up-regulated by cell contact to Matrigel, may play a key role in the invasiveness of MDA-MB-231 cells through basement membranes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Interaction of tissue-type plasminogen activator with fibronectin and fibronectin fragments

In this paper we present data on the interaction, in solution, of the tissue-type plasminogen activator (t-PA) with fibronectin (FN) and its degradation products (FNdp). A cross radial caseinolytic assay (CRACA) was developed for the evaluation of the effect of the FN and/or FNdp on t-PA and urokinase plasminogen activator (u-PA) activity. A directional caseinolysis was observed when t-PA or u-PA were tested in the proximity of FNdp; no directionality was observed when intact FN or BSA were used. After incubation of t-PA, but not u-PA, with FNdp, PA activity at 170, 150, 100 and 30 kDa was detected by SDS-PAGE followed by zymography. The incubation of intact FN with t-PA gives rise to two forms of 500 and 150 kDa after prolonged incubation of the zymograms; no higher MW forms appear when u-PA substitutes t-PA.The immunoblotting analysis of the mixtures of t-PA and FN or FNdp with anti-t-PA or anti-FN sera showed that intact FN and some of its fragments interact with t-PA, giving rise to complexes recognized by both antisera and resistant to SDS-PAGE. Similar complexes are also evident, in vivo, in biological fluids like human plasma cryoprecipitates (cryos).     

Matrix metalloproteinase expression is related to angiogenesis and histologic grade in spindle cell soft tissue neoplasms of the extremities

We defined the immunocytochemical expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in benign soft tissue neoplasms, fibromatoses, and sarcomas, together with the activity of gelatinase MMPs and TIMPs measured by zymography and reverse zymography in a subset of cases. The most strongly expressed MMP in all tumors was MMP-1, with weaker expression of MMP-10, MMP-11, and MMP-14 in most tumors. Nuclear expression of MMP-1, MMP-8, and MMP-13 was an unusual feature. TIMP-2 was expressed in all tumors, with stronger expression in fibromatoses than in sarcomas. Fibromatoses and high-grade sarcomas showed greater MMP-1 expression than other groups, and endothelial MMP-2 expression was more extensive in sarcomas. Differences in MMP and TIMP expression might be linked to the biologic behavior of soft tissue neoplasms. The activation of endothelial MMP-2 linked to widespread MMP-14 expression provides a mechanism for sarcomas to modulate their matrix and facilitate angiogenesis.     

Immunohistochemical and gelatin zymography studies for matrix metalloproteinases in bleomycin-induced pulmonary fibrosis

The role of various matrix metalloproteinases (MMP) and tissue Inhibitor of metalloprotelnases-2 (TIMP-2), and the gelatholytic activities of MMP involved in the process of bleomycin-induced pulmonary fibrosis in rabbits were Investigated. Male Japanese white rabbits were intubated with tracheal tubes under anesthesia, and bleomycin hydrochloride in sterile saline or only sterile saline was administered through the tracheal tubes. The animals were killed 1, 3, 7, 14 and 28 days after the administration of bleomycln ( n = 3) or saline ( n = 2). Light microscopic lmmunohistochemlstry for MMP-1 (interstitial collagenase), MMPP (gelatinase A), MMP-9 (gelatinase B) and TIMP-2 was performed. The gelatinolytic activities of lung tissue homogenates were studied by gelatin zymography. In the early stages, the gelatholytic activity of MMP-9 was predominant. MYP-9 localized in the infiltrating neutrophils, macrophages, bronchial and bronchiolar epithelial cells. The alveolar epithelial basement membrane was frequently disrupted in the early stages, where MMP-9 possibly contributed to the disruption. In the late stages, the gelatinolytic activities of the latent and active forms of MMP-2 were predominant, and MMPP localized in the regenerated alveolar epithelial cells in addition to the bronchial epithelial cells. MMP-2, especially its active form, possibly plays a role in alveolar epithelial cell regeneration. The localization of MMP-1 was similar to that of MMP-9. TIMP-2 localized in the epithelial cells and in some fibroblasts in fibro tic lesions. TIMP-2 possibly plays a role in extracellular matrix deposition in balance with MMP.     

Expression profile of trophoblast invasion-associated genes in the pre-eclamptic placenta

The primary pathology of pre-eclampsia is thought be a defect in placentation due to failure of trophoblast invasion. Here, we aim to identify the expression profile of invasion-associated genes in the pre-eclamptic placenta. Messenger RNA (mRNA) expression levels of extracellular matrix molecule-related genes in five pre-eclamptic placentas and in five strictly matched normal placentas were assayed using complementary DNA (cDNA) microarrays representing over 220 human cytokine-associated or hormone-associated genes. Results demonstrated greater than two-fold higher expression of 18 extracellular matrix molecule genes, including cadherin, collagen, integrin and selectin, in the pre-eclamptic placenta. Extracellular matrix molecule degradation-related genes, including matrix metalloproteinase (MMP)-10, MMP-13, MMP-15, tissue inhibitor of metalloproteinase (TIMP)-2, TIMP-3, plasminogen and plasminogen activator, were also highly expressed in the pre-eclamptic placenta, compared to the normal placenta. Results suggest that the abnormal expression profiles of extracellular matrix molecules and degrading proteinases might be associated with the pathogenesis of pre-eclampsia.     

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.