Heparin-binding epidermal growth factor–like growth factor (HB-EGF) is a member of the EGF family (1–3). It is synthesized as a transmembrane protein of 208 amino acids. A small part of the membrane-anchored HB-EGF form, or pro form of HB-EGF (proHB-EGF), is cleaved from the cell surface to yield a soluble growth factor of 75–86 amino acids, while most of the molecule remains uncleaved on the cell surface (1). ProHB-EGF is not merely a precursor of the soluble form. It is also a biologically active molecule that is complexed with both CD9 and α3β1 integrin. In this form, it actually has several biologic effects on different cell types (fibroblasts, endothelial cells, and smooth muscle cells) by increasing cell growth rate, migration, colony-forming ability, vascular endothelial growth factor (VEGF) expression, and activation of cyclin D1 promoter (2, 4). Recently, a heparin-binding domain in the N-terminal portion of the EGF-like domain was identified as a sequence able to modulate EGF-like biologic activity (1, 5).
Endometrial tissue expresses HB-EGF in response to sex steroids during the endometrial cycle and during early pregnancy in several species, suggesting a critical role in blastocyst implantation (6). HB-EGF is expressed in the human placenta during the first trimester, primarily within the villous trophoblast, but it also accumulates in the extravillous cytotrophoblast, in the basal plate, predominantly at the sites of cytotrophoblast extravillous invasion (7). Women with preeclampsia and infants who are small for gestational age display decreased expression of HB-EGF in placentae delivered as early as week 20 (8). This finding strongly suggests an association between HB-EGF down-regulation, poor trophoblast invasion, and failed physiologic transformation of the spiral arteries occurring in these disorders. Such a view is further supported by the evidence that HB-EGF favors trophoblast differentiation by inducing the invasive phenotype and stimulating cell motility (6).
Antiphospholipid syndrome (APS) is defined by the persistent presence of antiphospholipid antibodies (aPL) and by recurrent thrombotic events and/or fetal loss. The poor obstetric outcome in pregnant women with APS is also characterized by the occurrence of growth retardation and early and severe preeclampsia (9). There is evidence that aPL are pathogenic, but placental thrombosis cannot explain all the fetal losses, and aPL-mediated inhibition of trophoblast invasion has been suggested as one of the pathogenic mechanisms for APS pregnancy complications (10).
Owing to the putative physiologic role of HB-EGF during regular trophoblast development (6) and its alterations in pathologic conditions similar to APS (e.g., preeclampsia), we investigated whether HB-EGF expression is also affected in APS. To this end, we investigated HB-EGF expression in placental tissue obtained from women with APS and whether incubation with aPL may modulate its expression in human cytotrophoblast cells in vitro. Furthermore, we performed experiments to determine whether HB-EGF itself may interfere with the aPL-mediated effects on human trophoblast cell cultures, such as impairment of matrix metalloproteinases (MMPs).
Low molecular weight heparin (LMWH) together with low-dose aspirin is now widely accepted as the standard therapy for preventing recurrent fetal loss in APS (10). However, the fine pharmacologic mechanisms responsible for the effects of this treatment are still a matter for research (10). In this regard, we investigated whether the therapeutic activity of LMWH may be related to its ability to bind β2-glycoprotein I (β2GPI) with high affinity, thus competing with β2GPI for binding to cytotrophoblast cell membranes and eventually protecting them from the in vitro autoantibody-induced effects.
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This pilot study is the first to show decreased placental expression of HB-EGF in patients with obstetric manifestations of APS. Although a limited number of samples have been collected, a homogenous reduction of HB-EGF expression was found in all the samples, both at the protein and the mRNA levels. Accordingly, in vitro studies suggest that aPL are responsible for a comparable reduction in HB-EGF trophoblast expression.
Consistent with the findings of previous studies, we confirmed that β2GPI-dependent aPL bind human trophoblasts and affect in vitro invasiveness (14, 17). We extended this observation to show that the inhibition is at least partly related to an aPL-mediated inhibitory effect on trophoblast MMP-2 activity. Addition of exogenous recombinant HB-EGF to the cultures restored both the MMP-2 activity and the impaired trophoblast in vitro invasiveness.
As a whole, these findings suggest that decreased expression of HB-EGF at the level of the placenta may represent an important pathogenic mechanism in the defective placentation mediated by aPL.
HB-EGF expression is induced by sex steroids during the secretory phase of the endometrial cycle, and it persists during normal pregnancy. It has been suggested that accumulation of HB-EGF at sites of implantation may activate the downstream signaling of its receptors (human epidermal growth factor receptor 1 [HER-1] and HER-4) for cytotrophoblast survival (20). Accordingly, HB-EGF down-regulation was reported in patients with preeclampsia, in association with impaired extravillous trophoblast invasion, spiral artery remodeling, and increased apoptosis (20).
HB-EGF cell invasion–promoting and antiapoptotic activities support its potential role in APS-associated trophoblast abnormalities characterized by increased cell death and inadequate invasion. In fact, there is evidence that sera from APS patients inhibit trophoblast proliferation and increase cell apoptosis (21, 22). We also demonstrated that both human polyclonal and monoclonal β2GPI-dependent aPL may inhibit in vitro models of trophoblast invasion (17).
This study is the first to demonstrate that the pathogenic effect of aPL on trophoblasts may be mediated by a decrease in the expression of HB-EGF and by a reduction in the activity of specific proteases, such as MMP-2.
MMPs are a group of degradative enzymes that are secreted as inactive zymogen and become active after cleavage (23–26). Among the members of the MMP family, MMP-2 (also known as gelatinase A, type IV collagenase, or 72-kd gelatinase) was reported to be involved in trophoblast invasion of endometrial tissue (24, 25). MMP up-regulation results in proteolytic degradation of the ECM and of the basement membrane, promoting cellular growth, angiogenesis, and cellular migration (26). We found reduced MMP-2 levels in trophoblast cell cultures incubated in the presence of aPL. The addition of HB-EGF to the cultures restored the MMP-2 levels. It is possible that the addition of EGF favors trophoblast syncytialization with subsequent release of MMP-2, rather than being responsible for a direct effect on MMP-2 as was previously suggested (27). Whatever the fine mechanism is, this finding suggests that aPL may induce defective placentation by inhibiting MMPs and that such inhibition is at least partly related to the decreased expression of HB-EGF.
There is sound clinical evidence that LMWH is effective in preventing fetal loss in women with APS (28). However, its mechanisms of action are still a matter for research. Since thrombotic events can explain only part of the aPL-mediated fetal loss, pharmacologic mechanisms in addition to the well-known anticoagulant activity have been suggested (29).
We report that LMWH decreases aPL binding to human trophoblast monolayers and, as a consequence, prevents the antibody-mediated biologic effects. Beta2-glycoprotein I displays high affinity for heparin, and the heparin-binding site was shown to be located in the so-called phospholipid-binding site in the fifth domain of the molecule (30). The same part of the molecule is also involved in the binding to the syncytiotrophoblast surface membrane (14). We speculate that LMWH interacts with the phospholipid-binding site, thus inhibiting the adherence of β2GPI to the cell membranes. Such an event may explain the ability of LMWH to decrease antibody binding and, eventually, the protection from aPL-mediated functional effects. This activity may represent an additional pharmacologic mechanism to explain its protective effect on aPL-mediated recurrent fetal loss.
In conclusion, our preliminary ex vivo finding suggests that there is impaired expression of HB-EGF in placental tissue from women with APS that, apparently, is aPL-mediated, as supported by the results of the in vitro experiments with trophoblast cultures. The ability of exogenous recombinant HB-EGF to reduce the aPL-mediated effects on trophoblast cells supports a key pathogenic role of this molecule. The experimental conditions do not involve complement activation, indicating that aPL may also affect placental tissue through direct, complement-independent effects, as previously suggested (10). On the other hand, the ability of heparin to inhibit the in vitro aPL binding to human trophoblast cell monolayers, as well as the impaired invasiveness, represent an additional explanation for the therapeutic effect of heparin.
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All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Meroni had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Di Simone, Marana, D'Alessio, Raschi, Borghi, Chen, Caruso, Meroni.
Acquisition of data. Castellani, Di Nicuolo.
Analysis and interpretation of data. Raschi, Borghi, Sanguinetti, Meroni.