Preeclampsia is often reported to be associated with exposure of the placenta to various stress conditions, in particular hypoxic stress 32
. The goal of this investigation was to compare the expression and distribution patterns of a significant stress response protein, PML, in preeclamptic placentae. Increased PML protein expression was identified via immunohistochemical analyses in the placentae of preeclamptic patients relative to normotensive term control placentae. Increase PML immunoreactivity was most noticeable within cells of the villus core (macrophages, endothelial cells, fibroblasts) rather than in trophoblast cells. Indeed, little PML reactivity could be detected within trophoblast cells in vivo or in vitro. Increased PML isoform expression was evident in nuclear extracts of preeclamptic placentae in spite of less PML mRNA being detected. In vitro, PML expression and nuclear organization can be altered in endothelial cells, but not trophoblast, by low oxygen or interferon-β.
Our study is consistent with previous reports 22
in that PML expression is detectable in normal placenta localizing within diffuse or speckled nuclear bodies throughout gestation. This heterogeneous morphology is limited to specific cell-types of the villus stroma, including capillary endothelial cells, villus stromal fibroblasts, and resident macrophages but not villus cytotrophoblast or syncytiotrophoblast 22
. Outside of the villi, PML protein expression had been described in amnion cells, trophoblast giant cells and intermediate trophoblast within the deciduas 22
. We did not have an opportunity to study these extravillous locations in our archived samples.
Temporally, PML protein expression in normal placentae has been reported to increase as gestation proceeds to term 22
. Our study extends these observations by including preeclamptic samples. Whereas PML protein levels were very low at less than 31 weeks gestational age in normal placentae22
, our PE samples at 28–34 weeks, demonstrated relatively greater nuclear PML protein expression than did control samples. Therefore, we anticipate that differences in PML expression could be larger than depicted here if normal placenta samples from 28–34 weeks gestation were available for study. Because stresses of labor may induce changes in PML expression, our study examined PML expression in preeclamptic placentae from unlabored patients. Thus, it was imperative to obtain control placentae from patients who underwent Cesarean delivery in the absence of labor. Ideally, normal placentae from scheduled Cesarean deliveries to match the gestational age as the preeclamptic patients would be preferable. However, there are very few, if any, circumstances in which normal pregnant unlabored patients are delivered by elective Cesarean delivery at the same average gestational age as the preeclamptic group. Larger studies are needed to determine if changes in PML expression correlate with severity of preeclampsia and/or whether other clinical parameters (IUGR, diabetes, smoking, etc) are associated with altered PML expression patterns in the villi.
We found a dissociation in the relative expression levels of PML protein and mRNA in the preeclamptic placenta samples. This is likely due to post-translational modifications which alter PML protein stability (see 33
). For example, PML protein can be phosphorylated and/or sumoylated and protected from degradation and exogenous stimuli have been shown to increase or decrease PML protein levels independent of mRNA levels in different cell types 34;35
. Interestingly, we did not detect prominent levels of PML protein in isolated primary trophoblast nor could we induce PML protein expression in trophoblast with interferon-β. Post-translational modifications may explain these results. SUMO-1 extends the half-life of PML by inhibiting ubiquitination 21
. Sumoylation is also required for nuclear body formation, as PML that is diffusely localized within the nucleoplasm lack SUMO-1 modification 36
. It has recently been shown that SUMO-1 is degraded by a highly expressed trophoblast-specific SUMO protease, SENP-2, a protein necessary for trophoblast differentiation and placentation 37
. Thus, we speculate that the relatively low and stable expression of PML in trophoblast may be due to SENP-2 activity. Whether post-translational modifications are responsible for altering PML protein expression and/or functional activity in the preeclamptic tissues is not known but suggest future investigations of sumoylation in preeclampsia should be considered.
Recently, it was shown that preeclamptic placentae not complicated by IUGR, are affected by increased endothelial progenitor cell senescence and reduced function 6
. Over expression of PML has been shown to cause increased fibroblast senescence 38
and can function to inhibit angiogenesis 18
. Thus, it could be that increased endothelial cell nuclear expression of PML during preeclampsia contributes to the relative senescence of these cells. Similarly, it is possible that increased expression of PML in placental macrophages (Hofbauer cells) could affect placental development and vascularity as these cells have been shown to co-localize near fetal stem vessels and express numerous growth factors, including angiogenic growth factors, that can regulate trophoblast and villus branching morphogenesis 39;40
. Expression of the angiogenesis related genes used in this study (PGF, sflt-1) were significantly different between normal and preeclamptic samples, as expected. Although these genes were used primarily to validate the PML mRNA findings, both can be expressed in vascular endothelial cells. The possible relationship between the expression levels of these genes and changes in PML expression in preeclampsia is unknown. Nonetheless, it is conceivable that differences in PML expression or function within several different cells of the villus core may contribute to the inadequate placentation of preeclampsia. Furthermore, such effects may be independent of trophoblast in that alterations in PML expression/localization were not readily evident in these cells.
PML function is highly studied in cancer and viral infection, but its significance in other disease conditions is relatively unknown. In the context of preeclampsia, PML nuclear bodies may interfere with angiogenic responses through inhibition of mTOR activation, a mechanism which limits angiogenic responses in both neoplastic and ischemic conditions 41
. Additionally, it may reduce hypoxia-induced angiogenesis through inhibition of hypoxia-inducible factor 1-alpha (HIF-1α) protein synthesis 18
. In correlative support of this, increased expression of HIF-1α protein is most notable in syncytiotrophoblast of preeclamptic placentae 42
, where there is relatively little PML expression (current study and 22
). Thus, ischemic stress may enable increased HIF-1α production but PML-mediated inhibition of HIF-1α protein translation may result in heterogenous expression of HIF-1α protein between different cell types. Since HIF-1α protein expression is not thought to be increased in IUGR 43
, PML regulatory pathways should also be investigated in other obstetrical complications associated with hypoxia.
In summary, our findings agree with previous studies showing PML expression within cells of the villus core, but not trophoblast, in normal placentae 22
. We extended these studies to show that nuclear PML expression is elevated within these cells, but not trophoblast, in preeclamptic placentae. Furthermore, PML expression is inducible by hypoxia or interferon-β treatments in vascular endothelial cells but not trophoblast,. Our results, coupled with others’ showing PML protein can adversely effect hypoxia-mediated angiogenic responses 18
, provide a novel suggestion that increased PML expression may contribute to inadequate placental vascularity associated with preeclampsia.