Study of plasminogen activation by human trophoblasts

Jojart, Istvan (1997) Study of plasminogen activation by human trophoblasts. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Successful implantation and placentation during pregnancy depend on the regulated expression of trophoblast-associated protease activity. One of the proteolytic mechanisms utilized by trophoblasts is plasmin generation which is thought to be involved in trophoblast invasion and control of placental fibrin deposition. In this study, the production and regulation of plasminogen activator (PA) activity by human villous trophoblasts were investigated. -- Primary trophoblast cultures were established from normal human term placentas. Highly purified villous cytotrophoblasts were isolated through sequential trypsin-DNase digestions of the selected villous tissue, followed by Percoll density gradient centrifugation and subsequent removal of contaminating cells using monoclonal antibodies to monomorphic determinants of HLA class I and class II antigens. When placed in culture, the isolated cells were characterized as being cytokeratin positive, vimentin, HLA-ABC, HLA-DR and CD68 negative. In about 48 hours the majority of the cytotrophoblasts transformed into syncytiotrophoblasts. The cultures were functionally viable as assessed by their capacity for hCG secretion and reductive cleavage of MTT. No significant cytotrophoblast cell proliferation could be demonstrated in ³H-thymidine uptake experiments, and trophoblasts at their syncytiotrophoblastic stage did not proliferate at all. -- When the expression of plasminogen activators and their specific inhibitors in vitro was compared with m vivo findings 7 distinct differences were found. Immunocytochemical studies in term placental tissue showed strong syncytial PAI-2 immunoreactivity and faint to moderate PAI-1 staining. No staining could be demonstrated for either u-P A or t-PA. In contrast, the majority of cultured trophoblasts expressed u-PA during the first 24 hours of culture. All forms of trophoblast (mononuclear trophoblasts, multinuclear aggregates and syncytiotrophoblasts) expressed PAI-1, but only occasional syncytiotrophoblasts stained positively for PAI-2 and no t-PA immunoreactive trophoblasts were observed. Trophoblast-secreted PAI-1 could also be detected in conditioned media by ELISA. -- Activation of plasminogen was measured by monitoring the conversion of exogenously added Glu-plasminogen to plasmin in serum-free trophoblast cultures, using a chromogenic plasmin substrate H-D-V al-Leu-Lys-pNA (S-2251 ). Plasminogen activation by trophoblasts followed Michaelis-Menten type kinetics and produced parabolic progress curves of pNA accumulation that could be transformed to linear progress curves of plasmin generation, the slopes of which yielded constant rates of plasmin generation (i.e. PA activity). Trophoblast-associated plasminogen activation was found to originate from u-PA but not t-PA, because plasmin generation was markedly reduced in the presence of a neutralizing antibody to u-PA but not appreciably in the presence of a neutralizing antibody to t-PA. In addition, trophoblasts enhanced plasminogen activation induced by exogenous pro-u-PA, suggesting that cell surface binding of pro-u-PA potentiates the generation of plasmin. The basal level of endogenous trophoblast PA activity was specifically augmented by a neutralizing antibody to PAI-1, indicating that trophoblast-secreted PAI-1 regulates PA activity in an autocrine manner. Consistent with this finding, physiological concentrations of exogenous PAI-1 and PAI-2 resulted in a dose-dependent inhibition of PA activity. -- In order to define modulators of trophoblast-associated PA activity, several types of molecules were tested Activated protein C (APC) increased, whereas thrombin decreased PA activity, demonstrating that the coagulation system has the potential to modulate trophoblast plasminogen activation. Among growth factors/cytokines, EGF and its structural analog TGF-α proved to be potent stimulators. A neutralizing antibody to EGF did not have an effect, suggesting that term trophoblast cultures do not produce a sufficient quantity of EGF to modulate PA activity. TGF-β, IGF-II and IL-1β did not change PA activity appreciably, nor did LPS, a known stimulator of IL-1 secretion. With respect to hormones, dexamethasone and hCG had an inhibitory effect. Agents that elevate intracellular cAMP levels (8-bromo-cAMP and forskolin) inhibited plasminogen activation, whereas PMA had no appreciable effect. -- Taken together, this study demonstrates that the human term villous trophoblast possesses a highly regulated plasminogen activation system, and various types of physiologically important molecules including enzymes, growth factors, steroid and peptide hormones modulate the generation of trophoblast-associated plasmin.

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