Obstetrics and Gynaecology - Theses

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    Proteolysis and human parturition
    Tsatas, Dina ( 1998)
    Intrinsic to successful parturition are processes that depend on extensive extracellular matrix remodelling. The plasminogen activation cascade represents a central enzyme pathway involved in peri-partal events, yet the mechanisms that regulate its activity remain largely unknown. In this regard, the focus of this thesis has been to characterise the expression and activity of selected components of the plasminogen activation cascade, namely urokinase (uPA), urokinase receptor (uPAR), plasminogen activator inhibitor type-2 (PAI-2), the relaxed conformation of PAI-2 (PAIr) and the low density lipoprotein receptor-related protein (LRP) in human gestational tissues at the time of labour. Initially, messenger RNA (mRNA) transcripts encoding for uPA, uPAR and PAI-2 were detected in human placenta, chorion and amnion tissue before, during and after spontaneous onset labour at term. Levels of uPAR and PAI-2 mRNA were significantly greater in amnion tissue during labour and delivery as compared to before labour. In contrast, the gene expression of uPA was not altered by labour status in any of the gestational tissues examined. The cellular expression of uPA, uPAR, PAI-2, PAIr and LRP was localised in tissue sections of placenta, chorion and amnion obtained from different labour states using conventional peroxidase-anti-peroxidase techniques. These results revealed that, whereas PAI-2 and uPA were present in all tissues examined, the relaxed form of PAI-2 was predominantly present in the epithelial cells of the amnion. Furthermore, while present in some of the other tissues examined, LRP was not immunolocalised to the amnion. uPAR expression was predominantly localised to cells of the amnion, underlying chorion and extravillous trophoblast cells. Significantly lower levels of immunoreactive uPAR were found in placental tissue when compared to either amnion or chorion. In addition, significantly higher levels of immunoreactive PAI-2 were detected in placenta when compared to tissues of the fetal membranes. No tissue-specific differences were observed with respect to uPA immunoreactivity in any of the gestational tissues examined. Immunoreactive uPAR and PAI-2 content was not altered with labour status in placenta, chorion or amnion tissues. In contrast, immunoreactive uPA content was significantly increased during and after labour onset in all tissues. Labour-associated changes in protease activity in human gestational tissues were assessed by gelatin-substrate SOS-PAGE zymography. Placenta, chorion and amnion displayed uPA activity that did not vary across labour. All tissues also exhibited matrix metalloprotease activity that increased during and after labour onset. Specific serine and metalloprotease inhibitors selectively blocked protease activity in zymograms, confirming the identity of these enzymes. To investigate the role of uPA in matrix degradation, choriocarcinoma cell lines were cultured on an isotopically labelled subendothelial basement membrane. Release of radiolabel in the conditioned medium increased in a time- and plasminogen dose-dependent manner. Addition of serine protease inhibitors and uPA antibodies significantly suppressed proteolysis of labelled matrices by all cell lines. In conclusion, the novel data presented in this thesis clearly establish that human placenta, chorion and amnion tissue are capable of synthesising uPAR, PAI-2 and uPA at both the gene and protein level. The differential expression of these components in human term gestational tissues with labour indicates an important role for the plasminogen activation cascade in peri-partal remodelling events such as fetal membrane rupture.