Low-molecular Weight Heparin Induces In Vitro Trophoblast Invasiveness: Role of Matrix Metalloproteinases and Tissue Inhibitors☆
Introduction
Heparin is used routinely to treat and prevent thrombotic disorders and to improve the pregnancy outcomes in women with previous miscarriages, intrauterine growth restriction, preeclampsia or sudden intrauterine death associated with thrombophilias [1], [2]. The most extensively investigated thrombophilia has been antiphospholipid syndrome (APS). The use of heparin to prevent pregnancy loss in this syndrome was based on the premise that some pregnancy losses were caused by a placental thrombosis and that thromboprophylaxis with heparin could prevent this process [2]. However, examination of placentas and first-trimester decidua from APS complicated pregnancies has found little evidence of specific thrombotic placental pathology [3], [4]. There is evidence that APS may be associated with direct inhibition of trophoblast invasion and this, rather than placental thrombosis, may be the reason for pregnancy loss [4]. Defective decidual endovascular trophoblast invasion, rather than excessive intervillous thrombosis, was the most frequent histological abnormality in APS associated early pregnancy loss [5], [6].
However, the mechanisms by which heparin exerts its beneficial effects have still to be ascertained. Investigators have considered the possibility that heparin prevents the binding of β2-glycoprotein I to negatively charged phospholipids, which in turn prevents the deposition of the anti-β2-glycoprotein I antibodies in tissues [7], [8]. Then, the rationale for the clinical use of heparin could be that, in addition to its anticoagulant action, it inhibits the binding of antiphospholipid antibodies (aPL) with phospholipids, thus protecting the trophoblast from injury.
It is still unknown if heparin can directly affect placental functions. The purpose of the present study was to investigate the in vitro ability of low-molecular weight heparin (LMWH) to affect trophoblast invasiveness. Although the mechanisms of placental invasion are not elucidated to date, it is reported that matrix metalloproteinases (MMPs) and the tissues inhibitors (TIMPs) are related to this invasion [9]. The present results suggested that the effect of heparin on placental invasion might be due to MMPs and their TIMPs. Our data indicate that heparin is able to modify, in a dose-dependent manner, the proteolytic phenotype of human trophoblast in culture.
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Cell culture
Placental tissues from four first-trimester (9–12 weeks) spontaneous abortions were obtained from the Department of Obstetrics and Gynecology of Catholic University of Sacred Heart in Rome. The gestational age was established by ultrasonography. Only fertile women who had no previous history of reproductive difficulties were included in the study. All patients were investigated for genetic defects, infections, autoimmune diseases, endocrine profiles and glucose intolerance. Only samples
Effect of LMWH on in vitro trophoblast invasiveness
Trophoblast differentiation is characterized by the development of EVTC that migrate into maternal myometrium. To explore whether the invasion activity was regulated in choriocarcinoma cells (JAR) and human EVCT in response to heparin, we examined invasion activity using membrane invasion culture system. We found that incubation with LMWH significantly increased both choriocarcinoma and EVCT invasiveness. Fig. 1 shows the results of a 24 h in vitro invasion assay. After LMWH treatment, the
Discussion
The functional role of heparin in the human placenta has not yet been clarified. In this study we demonstrated for the first time that heparin plays a role in trophoblast invasion probably with an enhancement of the activity of specific proteases, such as MMPs.
The MMPs are a group of degradative enzymes which are secreted as inactive zymogen and must be cleaved to become active [9], [10], [11], [12], [13], [14], [15]. Among the members of the MMP family, MMP-2 (72 kDa, gelatinase A) and MMP-9 (92
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Supported in part by research grants from the Catholic University of Rome (D1, year 2003) and by Istituto Scientifico Internazionale, Paolo VI Institute, Catholic University of Rome.