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.     

NAD(P)H oxidase in human fetal membrane chorion laeve trophoblasts with or without chorioamnionitis: ultrastructural enzyme histochemical study

We examined the subcellular localizations of NAD(P)H oxidase, a reactive oxygen species (ROS)-producing enzyme, in fetal membrane chorion laeve trophoblasts from preterm or term pregnant women with or without chorioamnionitis (CAM). Ultrastructural enzyme histochemistry for NAD(P)H oxidase was used. In fetal membranes without CAM, approximately one quarter of the chorion laeve trophoblasts (25.6%) showed NAD(P)H oxidase activity on their surface plasma membranes and microvillous membranes. In mild CAM, the proportion of these NAD(P)H oxidase-positive cells significantly increased, reaching about half (51.0%). Enzyme activity appeared on the plasma and microvillous membranes and also on both phagosomal membranes and intracellular vesico-tubular structures. Appearance of NAD(P)H oxidase on surface plasma membranes, phagosomal membranes, and vesico-tubular structures is strong cytochemical evidence of phagocytic cell activation. These observations indicate that chorion laeve trophoblasts possess NAD(P)H oxidase activity, and therefore that fetal membranes themselves have ROS-generating capacity. Further, in fetal membrane inflammation, chorion laeve trophoblasts exhibited enzyme distribution characteristic of activated professional phagocytes. Similar to phagocytes infiltrating to the intrauterine environment, chorion laeve trophoblast NAD(P)H oxidase may play a role both in the defence of chorioamnion against infection and in the pathogenesis or pathophysiology of CAM-related preterm delivery.     

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.     

Immunolocalization of the Inducible Nitric Oxide Synthase Isoform in Human Fetal Membranes

PROBLEM: Nitric oxide (NO) synthesized by fetal membranes may protect the fetus from maternal infection or immune challenge or have a tocolytic effect on myometrium. The sites of synthesis and enzymes responsible for NO production in human fetal membranes remain unidentified. METHOD OF STUDY: Fetal membranes were obtained from four groups of patients: term (>37 weeks gestation) or preterm (<37 weeks gestation), both either in labor or not in labor. Frozen sections of membrane rolls were immunostained for inducible (iNOS) and endothelial (eNOS) nitric oxide synthase isoforms and the monocyte/macrophage marker CD14. RESULTS: Positive iNOS immunostaining was found in fibroblasts of amnionic and chorionic mesenchyme and in decidual macrophages identified by CD14 from all four groups of tissues. No iNOS immunostaining was seen in amnion epithelium or chorion trophoblast. Very intense iNOS staining was seen with evidence of monocyte/macrophage invasion of membranes. eNOS immunostaining was only found in decidual vascular endothelium. CONCLUSIONS: Constitutive expression of iNOS in decidual macrophages and fetal membrane fibroblasts may form an immune barrier against maternal insult. In chorioamnionitis, macrophage recruitment and NO expression may be part of the maternal immune response.     

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.     

Differential activity of the gelatinases (matrix metalloproteinases 2 and 9) in the fetal membranes and decidua,associated with labour

Degradation of the extracellular matrix in fetal membranes has been implicated in the rupture of fetal membranes, the process of parturition and placental detachment from the decidua after parturition. In this study we assessed labour-associated changes in gelatinase activity in cultured human amnion, chorion and decidua, as well as in amniotic fluid. We found that in media conditioned by decidua, following the establishment of uterine contractions, matrix metalloproteinase-2 (MMP-2) activity is increased while the protein tissue inhibitors of matrix metalloproteinase-1 (TIMP-1) level is decreased. The formation of a 130 kDa gelatinase band was also significantly increased after contractions began. In media conditioned by chorion, the initiation of uterine contractions did not change MMP activity or TIMP-1 levels. However, an increase in MMP-9 activity and a decrease in TIMP-1 protein levels were observed following the establishment of uterine contractions in media conditioned by amnion. We suggest that this differential spatial regulation provides a form for modulatory hieratical activity of the MMPs in the onset of labour allowing rupture of the membranes while avoiding premature placental separation.     

II. Expression of TNF-α and TNFR p55 in Cultured Amniochorion

PROBLEM: Preterm labor and PROM are major complications of pregnancy. We have reported the possible role of amniochorionic membrane as it relates to the production of cytokines and the early onset of labor. Amniochorion is capable of responding to an infectious process with the production of IL-6 and IL-1β. Here we examine the expression of TNF-α and TNFR in amniochorion. METHOD: Amniochorionic membranes were collected and maintained in an organ explant system. Samples were frozen and/or fixed for RT-PCR, in situ hybridization, and immunocytochemistry. RESULTS: RT-PCR demonstrated mRNA for TNF-α and in situ hybridization localized mRNA in chorion and amnion. Immunocytochemistry demonstrated TNF-α peptide in amnion but not in chorion. Immunocytochemical localization of TNFR indicates presence of that peptide in both amnion and chorion. CONCLUSIONS: We conclude that the fetal membranes are sources of TNF-α and TNFR, supporting our previous work indicating that fetal membranes are active participants in the response to intraamniotic infection.     

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.     

Intercellular junctions in the human fetal membranes

Summary Freeze-fracture replicas of the human reflected and placental amnion and chorion laeve at term were studied in order to give a systematic survey of the nature and extension of the intercellular junctions in the fetal membranes. No differences could be detected between the reflected and placental amniotic epithelium. In both the replicas never displayed plasma membrane differentiations typical of occluding junctions, while communicating junctions were occasionally and desmosomes frequently seen. In the chorionic trophoblast maculae occludentes, communicating junctions and desmosomes were regularly encountered. It is assumed that the maculae occludentes are remnants of occluding junctions which early in gestation possibly seal off the chorionic cavity; it appears improbable that they contribute significantly to the permeability properties of the chorionic trophoblast, since it is known from previous ultrastructural studies that large open intercellular channels cross the chorionic trophoblast. Thus the absence of occluding junctions, which could act as effective permeability barriers, in both epithelial components of the fetal membranes suggests that the factors able to influence the amniotic fluid turnover or the paraplacental protein exchange are the geometrical relationships and physico-chemical properties of the intercellular channels in the amniotic epithelium and chorionic trophoblast. In addition, communicating junctions were present between fibroblasts in the chorion laeve but not in the amnion, possibly indicating differences in the functional state of these cells and/or their extracellular microenvironments.The results of this study have been presented in preliminary form at the 77. Versammlung der Anatomischen Gesellschaft, Hannover 31.5.-4.6.1982Supported by the DFG     

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.     

MRL/lpr狼疮小鼠肾脏明胶酶表达随增龄变化及意义

目的 观察不同周龄MBL/lpr狼疮小鼠肾脏明胶酶的表达及其随增龄而发生的活性变化及意义。方法 取8、16与24周龄RMRL/lpr狼疮小鼠的肾组织进行常规病理检测。利用含有放射自显影的成像乳胶对冷冻肾组织切片进行原位明胶酶活性的检测;利用免疫组织化学检测肾脏明胶酶A与明胶酶B的酶B的蛋白表达变化,十二氨基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PACE）明胶酶谱法检测肾脏明胶酶A、B的活性变化。结果 8周龄狼疮小鼠肾组织吵仅在血管处检测到明胶酶的活性;16与24周龄狼疮小鼠肾小球内明胶酶的净活性明显增加。在肾小球以及肾小管间质上也可检测到明胶酶的活性,乙二胺四乙酸（EDTA）能够抑制肾脏明胶酶的活性,免疫组织化学与SDS-PAGE明胶酶谱法结果显示其肾组织中明胶酶A与明胶酶B的蛋白质表达及活性均明显增加。结论 明胶酶A、B在自发性狼疮小鼠肾炎中的表达及活化随增龄均明显增加,活化态的明胶酶可能在狼疮性肾炎细胞外基质重塑中发挥重要的作用。     

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.     

Localization in Human Term Placental Bed and Amniochorion of Cells Bearing Trophoblast Antigens Identified by Monoclonal Antibodies*

ABSTRACT: The monoclonal antibodies (mAb) H315, H317, and OKT9 have been used in immunofluorescence to investigate the expression of fetal trophoblast membrane antigens by cells within human term amniochorionic membranes and the marginal area of term placental bed tissue. OKT9 reacted only with trophoblast of placental chorionic villi and did not react with any nonvillous cytotrophoblast population: this mAb is known to identify the cell surface receptor for transferrin. H315 identifies a trophoblast-specific cell-surface antigen and strongly stained both placental villous trophoblast and the cytotrophoblastic layer of amniochorion. This mAb also stained some extravillous cytotrophoblast in the term placental bed, notable interstitial cytotrophoblast within maternal decidua. H317, which identifies placental-type alkaline phosphatase, gave the same distribution pattern as H315.     

慢性肾病患者血清、尿液中明胶酶变化的临床意义

目的:探讨血、尿MMP-2、MMP-9的水平和活性变化与慢性肾脏病(CKD)患者肾功能及尿蛋白的关系.方法:用ELISA法及明胶酶谱法检测90例CKD患者血、尿明胶酶的水平,其中慢性肾小球肾炎(chronic glomerulonephritis,CGN)60例,慢性肾小管间质性肾炎(chronic tubuloint...     

Matrix metalloproteinases with gelatinolytic activity induced by Paracoccidioides brasiliensis infection

Matrix metalloproteinases (MMPs) modulate extracellular matrix turnover, inflammation and immunity. We studied MMP-9 and MMP-2 in experimental paracoccidioidomycosis. At 15 and 120 days after infection (DAI) with virulent Paracoccidioides brasiliensis , MMP-9 was positive by immunohistochemistry in multinucleated giant cells, in mononuclear cells with macrophage and lymphocyte morphologies and also in fungal cells in the lesions of susceptible and resistant mice. Using gelatin zymography, pro- and active MMP-9 and active MMP-2 were detected in all infected mice, but not in controls. Gelatinolytic activity was not observed in P. brasiliensis extracts. Semiquantitative analysis of gelatinolytic activities revealed weak or absent MMP-2 and strong MMP-9 activity in both mouse strains at 15 DAI, declining at 120 DAI. Avirulent P. brasiliensis -infected mice had residual lesions with MMP-9-positive pseudoxantomatous macrophages, but no gelatinase activity at 120 DAI. Our findings demonstrate the induction of MMPs, particularly MMP-9, in experimental paracoccidioidomycosis, suggesting a possible influence in the pattern of granulomas and in fungal dissemination.     

1141526807Expression of inducible microsomal prostaglandin E synthase in local endometriosis lesions

Objective:  Leukocytes arriving to choriodecidua during labor are capable to secrete cytokines and collagenases that may play a role in extracellular matrix degradation leading to the rupture of amniochorion. The aim of this work was to study the role of amniochorion in the active recruitment of leukocytes through production of specific chemokynes during labor.
Methods:  Amniochorion explants were obtained from women at term with spontaneous labor ( n  = 4) and subjected to cesarean section without labor ( n  = 4). Explant cultures were carried out during 24 hr and an homogenate including the culture media was made after this period. Cell free extracts were tested in Boyden chambers for chemotaxis using leukocytes from maternal blood obtained from women with ( n  = 2) and without labor ( n  = 2). Attracted cells were analyzed by flow cytometry for count and immunophenotype. Chemokynes were identified in the extracts using a commercial chemiarray.
Results:  All tested amniochorion extracts induced chemotaxis for leukocytes; however, those from labor tissues induced a higher chemotactic activity than the correspondent non-in-labor tissues ( P  = 0.003). Chemotactic effect was higher over leukocytes from labor women ( P  = 0.001). More than 80% of the attracted cells were polymorphonuclears in all cases. IL-8 was the main chemokyne found in all extracts; however its concentration was increased in extracts from labor.
Conclusions:  Fetal membranes induced chemotaxis for leukocytes and this condition is enhanced by the presence of labor. Amniochorion under active labor secretes IL-8 which induces a preferential chemotaxis for polymorphonuclears. Infiltration of these cells in choriodecidua during labor may play a role in the amniochorion degradation associated to rupture.     

1141753263Amniochorion secretes chemotactic signals for leukocytes during human labor

Objective:  Leukocytes arriving to choriodecidua during labor are capable to secrete cytokines and collagenases that may play a role in extracellular matrix degradation leading to the rupture of amniochorion. The aim of this work was to study the role of amniochorion in the active recruitment of leukocytes through production of specific chemokines during labor.
Methods:  Amniochorion explants were obtained from women at term with spontaneous labor ( n  = 4) and subjected to cesarean section without labor ( n  = 4). Explant cultures were carried out during 24 hr and a homogenate including the culture media was made after this period. Cell free extracts were tested in Boyden chambers for chemotaxis using leukocytes from maternal blood obtained from women with ( n  = 2) and without labor ( n  = 2). Attracted cells were analyzed by flow cytometry for count and immunophenotype. Chemokines were identified in the extracts using a commercial chemiarray.
Results:  All tested amniochorion extracts induced chemotaxis for leukocytes; however, those from labor tissues induced a higher chemotactic activity than the correspondent non-in-labor tissues ( P  = 0.003). Chemotactic effect was higher over leukocytes from labor women ( P  = 0.001). More than 80% of the attracted cells were polymorphonuclear cells in all cases. IL-8 was the main chemokine found in all extracts; however its concentration was increased in extracts from labor.
Conclusions:  Fetal membranes induced chemotaxis for leukocytes and this condition is enhanced by the presence of labor. Amniochorion under active labor secretes IL-8 which induces a preferential chemotaxis for polymorphonuclears. Infiltration of these cells in choriodecidua during labor may play a role in the amniochorion degradation associated to rupture.