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.     

92-kd type IV collagenase (matrix metalloproteinase-9) activity in human amniochorion increases with labor.

To determine whether specific collagenolytic enzymes are expressed in human fetal membranes with labor, we examined gelatinase activity in extracts of amniochorion by zymography. The 92-kd gelatinase (MMP-9) was barely detectable in extracts of fetal membranes before the onset of labor but was readily demonstrable in extracts prepared from membranes isolated from laboring women or membranes collected immediately after delivery. In contrast, the 72-kd gelatinase (MMP-2) was detectable in extracts from pre- and post-labor membranes. Ethylenediaminetetracetic acid and the tissue inhibitor of metalloproteinases, TIMP-1, inhibited the gelatinase activities detected by zymography, confirming that the enzymes are metalloproteinase. Assay of amniochorion gelatinase activity using a radiolabeled denatured collagen substrate revealed a more than twofold increase in activity comparing pre-labor with post-labor fetal membrane extracts. A function-blocking anti-MMP-9 monoclonal antibody inhibited pre-labor membrane gelatinase activity by approximately 11.5%, which was only slightly greater inhibition than observed with irrelevant monoclonal antibodies. However, post-labor membrane gelatinase activity was reduced by 53% by the function-blocking antibody, indicating that MMP-9 is a major contributor to the increased gelatinase activity extractable from post-labor membranes. Western blot analyses demonstrated increased MMP-9 protein in amniochorion extracts after onset of labor. MMP-9 protein and mRNA were co-localized in amnion epithelium, underlying macrophages and chorion laeve trophoblast and decidual cells after labor. We conclude that 1) MMP-9 activity and protein in human amniochorion increases with labor and 2) MMP-9 is expressed by amnion epithelium, macrophages and chorion laeve trophoblast and decidual cells. The increased expression of MMP-9 may result in degradation of the extracellular matrix of the fetal membranes and facilitate their rupture under both physiological and pathological conditions.     

Matrix metalloproteinases-2, -3 and -9 in human term placenta

Matrix metalloproteinases (MMPs) are zinc-dependent enzymes that degrade the components of the extracellular matrix (ECM) and are known to be the main mediators of human placentation and parturition. Although there are many studies on the roles and distribution of MMPs in human term placenta, so far none of the studies has investigated the distribution of MMP-2, -3 and -9 in different cells of various placental sites. In this study, we aimed to determine the distribution and enzymatic activities of MMP-2, -3 and -9 with regard to different regions of term human placenta, such as amnion, basal plate, chorionic plate, decidua, chorion laeve, Nitabuch's stria, umbilical cord and placental villi. Eighteen normal human term placentas were obtained after vaginal deliveries. Immunohistochemistry and zymography were performed for MMP-2, -3 and -9 on placental tissue sections and protein extracts, respectively. Nearly all tissues showed immunoreactivity for MMPs. The strongest enzymatic activity for MMP-2 was seen in areas where invasive trophoblast cells invaded maternal tissues. MMP-9 had the highest enzymatic activity at the contact region of fetal and maternal parts, suggesting the importance of MMP-9 in separation of the placenta from the uterine wall during labor. MMP-3 had a similar localization to MMP-9, suggesting that besides gelatinases like MMP-2 and -9, MMP-3 (stromelysin-1) may also have important roles during labor. This study describes the site-specific distribution and activities of MMPs and therefore might help in elucidating the molecular mechanisms in pathologies such as premature rupture of membranes.     

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.     

Cytokine secretion by human fetal membranes, decidua and placenta at term

Cytokines such as monocyte chemotactic peptide-1 (MCP-1), interleukin-8 (IL-8), RANTES (Regulated on Activation and Normally T-cells Expressed and presumably Secreted) and interleukin-10 (IL-10) are thought to play pivotal roles in immune recognition, acceptance of the fetal allograft, maintenance of pregnancy and parturition. Their secretion and regulation within the third trimester uterus is, however, less well defined. We therefore investigated the release of these cytokines by third trimester amnion, chorion, placenta and decidua, and studied the influence of prostaglandin E2 (PGE2) infusion on their release in a dynamic placental cotyledon perfusion system. MCP-1 was released predominately by the chorion (78.2 +/- 7.3 pg/mg wet tissue weight; mean +/- SEM), decidua (112.4 +/- 5.2 pg/mg) and placenta (101.8 +/- 5.0 pg/mg) with low amounts from the amnion (1.3 +/- 0.4 pg/mg). High concentrations of IL-8 were released by the amnion (39.9 +/- 5.3 pg/mg), chorion (52.8 +/- 1.9 pg/mg), decidua (42.2 +/- 1.5 pg/mg) and placenta (45 +/- 1.3 pg/mg). Release of RANTES was not detectable from the amnion but was detected in moderate amounts from the chorion (6.0 +/- 1.2 pg/mg), decidua (15.2 +/- 1.4 pg/mg) and placenta (26.9 +/- 1.6 pg/mg). Low concentrations of IL-10 were secreted by the chorion (6.8 +/- 0.8 pg/mg), decidua (9.0 +/- 0.9 pg/mg) and placenta (3.3 +/- 0.3 pg/mg) with none detectable from the amnion. MCP-1, IL-8, RANTES and IL- 10 were all released by perfused placental cotyledons. PGE2 stimulated release of MCP-1, IL-8 and IL-10 into the maternal and of MCP-1 and IL- 8 into the fetal circulation of the placenta but had no effect on RANTES release. It is suggested that MCP-1 and IL-8 may be involved in the inflammatory process of parturition and IL-10 in the protection of the fetal allograft. In addition, PGE2 may have an important immunomodulatory role within the uterus at term.      

Presence of pro-opiomelanocortin peptides and corticotropin-releasing factor in human placenta

Immunoreactive adrenocorticotropin (ACTH), beta-endorphin (BEP) and corticotropin-releasing factor (CRF) were detected in human term placenta obtained from elective Caesarian surgery. The concentrations of ACTH, BEP and CRF in placenta detected by radioimmunoassay (RIA) were 2.83 +/- 0.36, 0.52 +/- 0.05 and 0.56 +/- 0.15 ng/g wet weight of tissue respectively. Pro-opiomelanocortin (POMC) peptides were also detected in the amnion and chorion membranes and in the decidua. The concentrations of ACTH were 1.72 +/- 0.20, 4.43 +/- 0.39 and 5.80 +/- 0.17 ng/g and the levels of BEP were 0.42 +/- 0.18, 0.65 +/- 0.20 and 3.66 +/- 1.10 ng/g in the amnion, chorion and decidua respectively. In contrast to placenta, immunoreactive CRF was not detected in the amnion, chorion and decidua. Immunoreactive N-acetylated BEP was also not detected in all the placental subfractions. Comparison of the amounts of both ACTH and BEP in the various placental components indicated the following distribution: decidua > chorion > placenta > amnion. In decidua, POMC peptides were present in an equi-molar ratio but in the other three placental fractions, ACTH levels were three to five-fold higher than BEP. In immunohistochemical studies, only a positive staining for ACTH was obtained for decidua. Our results confirm the presence of POMC peptides and CRF in placenta and their physiological roles in pregnancy and parturition.     

Expression and localization of alphavbeta6 integrin in extraplacental fetal membranes: possible role in human parturition

Successful outcome of human parturition is dependent upon extensive remodelling of the extracellular matrix (ECM) of the cervix, uterus and fetal membranes, a process that involves adhesion molecules and is also common in tumour invasion and metastasis. To elucidate the role of integrins in human parturition, this study characterizes the expression of the tumour-associated alpha(v)beta(6) integrin in human placenta and extraplacental membranes. Immunohistochemical analysis of the placenta and fetal membranes from normal vaginal deliveries (NVD) (n = 10) exhibited strong intensity of staining for alpha(v)beta(6) integrin (3 = dark brown) in the epithelial layer of the amnion. Weak immunohistochemical staining of alpha(v)beta(6) integrin (1 = pale brown) was detected in the chorion and at the decidual edge. These results were consistent with the immunodetection of alpha(v)beta(6) integrin by western blot analysis that showed 4-fold enhanced expression in the amnion compared to chorion of both NVD and term elective caesarean section (CS) deliveries. Even though there was no difference in the extent of immunohistochemical staining of alpha(v)beta(6) integrin between the amnion of NVD and CS groups, significantly higher intensity of staining was observed in the NVD amniotic epithelium compared to that of CS (n = 10) (chi(2) = 10.25, P = 0.0059). Western blot analysis of the fetal membranes showed no differences in the expression of alpha(v)beta(6) integrin between the NVD and CS groups. Gelatin zymography demonstrated the presence of pro-matrix metalloprotein-9 (MMP-9) and pro-MMP-2 in the amnion and chorion of NVD, whereas in CS only the presence of pro-MMP-2 was observed. These results suggest that in term pregnancy, human fetal membranes express alpha(v)beta(6) integrin and that the expression is significantly higher in amnion compared to chorion. The fact that enhanced expression of alpha(v)beta(6) integrin in fetal membranes correlates with the expression of pro-MMP-9 in NVD is consistent with the invasive role of the integrin in cancer and suggests that the molecule may have a proteolytic role in the initiation and progression of labour.     

Chorion releases a factor that inhibits oxytocin-stimulated myometrial contractility in the pregnant guinea pig

It was postulated that chorion releases a substance necessary for the maintenance of uterine quiescence during pregnancy. A decrease in the release of this substance at the end of the pregnancy would be necessary for normal myometrial activation. This hypothesis was tested by demonstrating the ability of chorion to inhibit oxytocin-stimulated myometrial contractility in vitro. Tissues were obtained from timed pregnant Duncan-Hartley guinea pigs either at pre-term or near-term gestation. Myometrial strips were placed in organ baths for isometric tension measurement and contractions stimulated by oxytocin (10(-8) mol/l). Fetal membranes or conditioned medium from chorion were added directly to the organ bath. Near-term chorion and chorion conditioned-medium decreased oxytocin-stimulated contractile activity to 39% and 49% respectively. Neither pre-term nor near-term amnion reduced oxytocin-stimulated myometrial contractile activity. Relaxation induced by pre-term chorion was greater than near-term chorion (23% and 41% of the oxytocin-induced basal level respectively; P < 0.05). Further, chorion-induced relaxation was independent of the gestational age of the myometrium. Human chorion from a term, not-in-labour woman also inhibited oxytocin-stimulated guinea pig myometrial contractility. It was concluded that the chorion releases a substance or substances that reduce oxytocin-stimulated myometrial contractility and may be involved in the maintenance of uterine quiescence during pregnancy.     

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.     

Evidence of in vitro differential secretion of 72 and 92 kDa type IV collagenases after selective exposure to lipopolysaccharide in human fetal membranes

Premature rupture of chorioamniotic membranes complicated with intrauterine infection has been associated to degradation of extracellular matrix (ECM), which could explain local morphological changes. We used a culture system in which the chorioamniotic membranes form two independent chambers, allowing for the selective stimulation of either the amnion (AMN) and/or the choriodecidua (CHD) regions. Lipopolysaccharide (500 ng/ml) was added to the AMN and/or the CHD; secretions and gelatinolytic activity of matrix metalloproteinase (MMP)-2 and MMP-9 were measured in both compartments by enzyme-linked immunosorbent assay (ELISA) and zymography. Secretions of TIMP-1, TIMP-2 and TIMP-4 were measured by ELISA. Both metalloproteinases were immunolocalized in tissue sections. All stimulation modalities induced a similar proMMP-2 and proMMP-9 secretion pattern in the CHD with concentrations of 2.49 ng/ml and 90.91 pg/ml, respectively; the AMN showed no significant changes. The active forms of both enzymes did not change with any stimulation modality. TIMP-1, TIMP-2 and TIMP-4 secretions remained without significant changes (P = 0.41). ECM degradation and structural disarrangement were evident after stimulation. Secretion of proMMP-2 and proMMP-9 mainly in the CHD, presence of active forms associated to the tissue and minor changes in TIMPs secretion could favor ECM degradation and explain the weakening and thinning associated with the pathological rupture of chorioamniotic membranes.     

蜕膜组织MMP-9/TIMP-3水平与自然流产关系

目的研究蜕膜组织中MMP-9／TIMP-3之间的平衡与自然流产发生的关系。方法用免疫组化S-P法测定30例自然流产患者与20例正常妊娠者蜕膜组织中MMP-9／TIMP-3的表达。结果研究组蜕膜细胞MMP-9表达阳性率为76．7％，高于对照组(55．0％，P-0．02)，两组蜕膜细胞TIMP-3的表达差异无显著性。结论自然流产患者蜕膜组织中MMP-9的表达增高，TIMP-3表达正常所导致的MMP-9／TIMP-3比例升高，在自然流产的发生中起重要作用。     

Endometrial TIMP-4 mRNA is expressed in the stroma, while TIMP-4 protein accumulates in the epithelium and is released to the uterine fluid

We have previously reported that endometrial mRNA expression of both tissue inhibitors of metalloproteinase-4 (TIMP-4) and matrix metalloproteinase-26 (MMP-26) peaks in the early secretory phase, which implies a role in implantation. The objective of this study was to compare the distribution of TIMP-4 and MMP-26 in endometrial tissue and uterine fluid over the menstrual cycle. Endometrial tissue was analysed with in situ hybridization and immunohistochemistry to localize mRNA and protein for TIMP-4 and MMP-26 in the same set of samples. TIMP-4 mRNA was quantified in separated stromal and epithelial cells using real-time PCR. Uterine fluid was analysed with western blotting. TIMP-4 mRNA was exclusively localized to the stroma, whereas MMP-26 mRNA was expressed by epithelial cells. TIMP-4 protein was only occasionally found in the stroma but was consistently present in granules of the apical part of luminal and glandular epithelial cells. TIMP-4, but not MMP-26, was demonstrated in uterine fluid. Thus, TIMP-4 is produced in the stroma only, secreted by stromal cells, taken up by epithelial cells, accumulated in apical granules and finally secreted to the uterine fluid. Maximal expression of MMP-26, and its strongest inhibitor TIMP-4, in the early and mid-secretory phase suggests a role during implantation. MMP-26 is stored in epithelial cells in its active form, is not released spontaneously and is controlled by TIMP-4 in both stroma and uterine fluid.     

Leukocyte density and pro-inflammatory cytokine expression in human fetal membranes,decidua, cervix and myometrium before and during labour at term

Accumulating evidence suggests that human parturition represents an inflammatory process. Leukocytes are known to infiltrate uterine tissues but the exact timing, nature and quantity of these cells has not been formally characterized. We have previously demonstrated an apparent increase in pro-inflammatory cytokines within tissues of the labouring uterus. The aims of this study were to quantify and compare the leukocyte subpopulations before and during labour in fetal membranes, decidua and cervix and to quantify and compare mRNA expression of interleukin-1beta (IL-1beta), IL-6, IL-8 and tumour necrosis factor-alpha in myometrium, cervix, chorio-decidua and amnion. Biopsies of each of these tissues were obtained from pregnant women delivered by Caesarean section before and after the onset of spontaneous labour at term. Subpopulations of leukocytes were identified using immunohistochemistry and cytokine mRNA expression was quantified using Northern analysis. We found that parturition was associated with a significant increase in IL-1beta, IL-6 and IL-8 mRNA expression in cervix and myometrium, IL-6 and IL-8 mRNA expression in chorio-decidua and IL-1beta and IL-8 mRNA expression in amnion. Histological analysis demonstrated that leukocytes (predominantly neutrophils and macrophages) infiltrate the uterine cervix coincident with the onset of labour. These data lend further support to the hypothesis that labour is an inflammatory process.     

Expression of superoxide dismutase and xanthine oxidase in myometrium, fetal membranes and placenta during normal human pregnancy and parturition

Superoxide, an agent which attenuates the half-life of nitric oxide, is metabolized and synthesized by superoxide dismutase (SOD) and xanthine oxidase, respectively. Over the last few years much work has focused on the role of nitric oxide in human parturition. The aim of this study was to determine whether the onset of human parturition is associated with a change in the expression of copper/zinc superoxide dismutase (Cu/Zn SOD), manganese superoxide dismutase (Mn SOD) or xanthine oxidase within the uterus. Samples of myometrium, placenta, decidua and fetal membranes were obtained from women before and after the onset of labour at term. Immunocytochemistry was used to localize Cu/Zn SOD, Mn SOD and xanthine oxidase and measure SOD enzyme activity. Cu/Zn and Mn SOD-like immunoreactivity was detected in syncytiotrophoblast cells, villous stromal cells and endothelial cells of blood vessels in the placenta. In the myometrium Cu/Zn and Mn SOD were localized to myocytes and endothelial cells and to some vascular smooth muscle cells. In the fetal membranes we observed staining for Cu/Zn SOD and Mn SOD in the amnion, chorion, extravillous trophoblast and decidua. There was no difference in SOD enzyme activity or staining intensity for SOD between different cell types before and during labour. Xanthine oxidase immunoreactivity was identified in each of the tissues examined and again there was no difference in immunostaining in tissues obtained from women delivered before or after the onset of labour. These results show that the pregnant uterus is capable of both synthesizing and degrading superoxide and suggest that superoxide dismutase and xanthine oxidase may play a role in the maintenance of uterine quiescence during pregnancy, but not in the initiation of parturition.      

Progesterone receptor expression in human decidua and fetal membranes before and after contractions: possible mechanism for functional progesterone withdrawal

In humans, progesterone levels are sustained before the onset of labour. Therefore, the mechanism for parturition that has been proposed for humans is 'functional' progesterone withdrawal. Immunohistochemical staining for the progesterone receptor (PR) was positive in the decidua with a decline after contractions began. Western blot analysis revealed a number of PR isoforms expressed in the decidua, with the PR-B form being dominant. After contractions began, all PR isoforms decreased sharply. PR-B and PR-A decreased by 85.8% +/- 6.7 and 78.2% +/- 7.1, respectively (P < 0.001). Incubation of decidua with Prostaglandin F2alpha 1.0 microg/ml decreased the expression of all forms of PR isoforms. PR-B was reduced by 64% +/- 6.09 (P < 0.01); PR-A was reduced by 77% +/- 5.9 (P < 0.05), while PR-C was reduced by 80% +/- 7.24 (P < 0.05). Progesterone (80 microg/ml) increased the PR-B, PR-C the 45 and 36 kDa isoforms to 150% +/- 7.89, 210% +/- 12.4, 270% +/- 9.7 and 216% +/- 13.5, respectively (P < 0.05). In immunohistochemical studies, the PR was not identified in the amnion or in the chorion, regardless of the presence or absence of contractions. Western blot analysis demonstrated that PR-C (60 kDa) and the 36 kDa isoforms were dominant in the amnion. After contractions began, PR-A decreased significantly by 61.9% +/- 7.1 (P < 0.001).     

活性氧对平滑肌细胞合成基质金属蛋白酶-1, 3及其抑制物的影响

目的:探讨活性氧(ROS)对血管平滑肌细胞合成基质金属蛋白酶-1,3(MMP-1,3)和基质金属蛋白酶抑制物-1(TIMP-1)的影响,从而推测其是否促进动脉粥样硬化斑块的破裂。方法:体外培养胎儿主动脉平滑肌细胞,加入含100μmol/L黄嘌呤,5U/L黄嘌呤氧化酶的无血清培养液,孵育24h,收集细胞上清液。用Westernblotting方法检测浓缩后的细胞上清液中MMP-1,3和TIMP-1的含量。结果:黄嘌呤/黄嘌呤氧化酶组细胞上清液中的MMP-1含量明显少于正常对照组,并且转化成活性形式;MMP-3的含量明显多于正常对照组,并且转化成活性形式;TIMP-1的含量明显少于正常对照组。结论:ROS对MMPs-TIMPs平衡的影响很复杂,可能对动脉粥样硬化斑块的破裂起一定作用。     

In vivo inhibition of human neutrophil collagenase (MMP-8) activity during long-term combination therapy of doxycycline and non-steroidal anti-inflammatory drugs (NSAID) in acute reactive arthritis.

We studied the in vivo effect of long-term doxycycline treatment combined with NSAID on human interstitial collagenases, other matrix metalloproteinases, serine proteinases, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and lactoferrin from saliva and serum during the course of acute reactive arthritis (ReA). Collagenase activity and serine proteases (elastase-like, cathepsin G-like and trypsin-like activities) of saliva (n = 10) and gelatinase, lactoferrin and TIMP-1 of saliva (n = 10) and serum (n = 10) samples before and after 2 months doxycycline treatment, combined with NSAID, were studied by quantitative SDS-PAGE assay, ELISA assay and by spectrophotometric assay. The cellular source and molecular forms of salivary collagenase were characterized by immunoblotting using specific antisera. We found that activities of total and endogenously active interstitial collagenase reduced significantly. The salivary collagenase was found to originate from neutrophils. No fragmentation of either pro 75-kD and active 65-kD MMP-8 was detected after 2 months doxycycline treatment. However, during 2 months doxycycline and NSAID treatment no reduction of salivary and serum gelatinase, lactoferrin and TIMP-1-levels and salivary serine protease activities were detected. The in vivo inhibition of collagenase (MMP-8) activity during long-term doxycycline therapy in human saliva containing inflammatory exudate of ReA patients may contribute to the reduced tissue destruction observed in recent clinical and animal model studies in arthritides during long-term doxycycline/tetracycline treatment.     

Prostaglandin E(2)-dependent production of latent matrix metalloproteinase-9 in cultures of human fetal membranes

Studies in our laboratory have shown that structural changes in cervical biopsied fetal membranes, prior to labour, coincide with differences in the expression of the gelatinase enzyme, latent matrix metalloproteinase-9 (MMP-9). Concurrently, in vivo, there is an increase in the expression of prostaglandins, notably prostaglandin E(2) (PGE(2)), which has been shown to regulate the expression of MMPs in other systems. The aim of this study was to test the hypothesis (using an in-vitro culture model) that endogenously produced PGE(2) has a role in the elevation of MMP-9 described in vivo. Non-infected fetal membranes sampled from women undergoing elective Caesarean section were stimulated with 10% (v/v) fetal bovine serum (FBS), a known inducer of prostaglandins. This activation resulted in a time-dependent increase in the secretion of PGE(2) into the media, as determined by enzyme-linked immunosorbent assay (day 1: 19 +/- 9 pg/ml/24 h to 358 +/- 54 pg/ml/24 h by day 4). A similar pattern of secretion of latent MMP-9 was observed in parallel with the increase in PGE(2) in the same culture media (day 1: 1.63 +/- 0.17 ng/ml/24 h to 4.2 +/- 1.4 ng/ml/24 h by day 4). When both molecules were compared, a significant (P: < 0.01) positive correlation (r = 0.623) was observed. Secretion of the tissue inhibitor of MMPs-9 (TIMP-1) was not significantly different between untreated (3.07 +/- 0.266 microg/ml/24 h) and FBS-treated (3. 85 +/- 0.24 microg/ml/24 h) cultures during the first 4 days in culture. Prostaglandin synthesis inhibition studies using indomethacin (100 micromol/l) resulted in a 70-80% reduction in the activated secretion of latent MMP-9. Direct PGE(2) stimulation of cultures resulted in the bell shaped dose-response curve with concentrations of 1-100 nmol/l (which are within the range secreted in culture in response to FBS), stimulating significant latent MMP-9 secretion. These results suggest a link between endogenous PGE(2) and latent MMP-9 production in human fetal membranes, raising the possibility that PGE(2) has a role in the mechanism of fetal membrane structural changes and, hence, in parturition-associated membrane rupture.     

Down-regulation of tissue inhibitor of matrix metalloprotease-1 (TIMP-1) in aged human skin contributes to matrix degradation and impaired cell growth and survival

Up regulation of matrix metalloproteinases (MMPs), particularly collagenase-1 (MMP-1), stromelysin-1 (MMP-3) and gelatinase A (MMP-2) is responsible for the lysis of dermal collagen and elastin fibers during chronological skin aging. Tissue inhibitor of metalloproteinase-1 (TIMP-1) is one representative of the natural MMP inhibitor family, encompassing four members. Its expression is decreased with fibroblast senescence, both ex vivo and in vivo, thus contributing to increased catabolic activity within dermis. TIMP-1 displays multiple biological functions. It inhibits most MMPs, except membrane-type MMP subfamily, with Ki in the subnanomolar range, but also interacts with the hemopexin-like (PEX) domain of pro MMP-9. Besides, it exhibits keratinocyte and fibroblast growth factor-like activity and has been described as a cell survival factor.