Our results show that 3rd
trimester placentae from preeclamptic women have more syncytial knots that are more heavily loaded with sFlt1 protein compared to those from normal pregnancies. Gentle flushing of these placentae selectively releases more trophoblast mass in the form of syncytial aggregates from the preeclamptic placentae than their normal counterparts. Liberation of syncytial aggregates could not be attributed to aggressive handling as preeclamptic placentae in organ culture spontaneously released aggregates of identical size and multinuclear composition into the medium. This shed material contained both sFlt1 protein and mRNA. Placement of 3rd
trimester placental explants over a 500 μm mesh in organ culture not only enabled efficient isolation of syncytial aggregates, but also suggested that this shed material may arise by syncytial sprouting and fission from the underlying syncytium. Isolated aggregates were multinuclear, rich in cytoplasmic organelles, and capable of de novo gene expression, demonstrating that these structures were not only viable, but also biologically active. Finally, we returned to the 3rd
trimester maternal circulation, where we could demonstrate that at least 25% of plasma sFlt1 was associated with microparticles. While placental heparanase upregulation has been recently implicated as one factor that may contribute to the release of sFlt1 into systemic circulation16
, our data suggests that release of syncytial microparticles may be an important additional factor that contributes to the elevated sFlt1 in human preeclampsia.
Based on these results, we speculate that 3rd-trimester placentae spontaneously form living syncytial spouts/knots () that detach from placental villi through fission (), liberating membrane-bound multinuclear structures ( and ) that we called syncytial aggregates that possess critical biological capacities (), including the ability to synthesize sFlt1 protein from endogenous stores of mRNA. Since each phase of this sequence is exaggerated in preeclampsia—in situ knots () followed by liberated sFlt1-expressing syncytial aggregates () that then perhaps further disaggregate to sFlt1 associated microparticles ()—we also speculate that accelerated knot/sprout formation within the placenta may be an early event in preeclampsia that enhances the delivery of sFlt1 into the maternal circulation.
Normal pregnancy is characterized by trophoblast turnover and shedding, as evidenced by the detection of trophoblastic microparticles in the maternal circulation throughout pregnancy28–32
and by the appearance of detached syncytial aggregates with euchromatic nuclei—labeled “syncytial sprouts”—in term human placentae33
. Based on the current results, we speculate that 3rd
-trimester placentae spontaneously form syncytial sprouts/knots () that detach from placental villi through fission (), liberating membrane-bound multinuclear structures ( and ), termed syncytial aggregates, that possess critical biological capacities (), including the ability to synthesize sFlt1 protein from endogenous stores of mRNA. Therefore, while apoptotic, necrotic, or “aponecrotic” placental material34
loaded with sFlt1 protein may well be released into the maternal circulation, our data suggest that term placentae deport or shed biologically active syncytial aggregates that, in turn, may release smaller microparticles with similar biological capacities into the maternal circulation, akin to the release of platelets from megakaryocytes residing in the bone marrow.
In addition to demonstrating the biological capacity of 3rd
trimester shed trophoblast material, the current findings also add to the existing literature in other ways. First, deportation of living placental material, followed by de novo translation of pre-existing mRNA, may be a new mechanism by which sFlt1 is delivered into the maternal circulation. Second, while sFlt1 protein has been identified on circulating placental particles35
and an increase in circulating particles has been associated with PE 18
, no explanation has been proposed for how these particles are formed. This is perhaps because late-pregnancy particles in the maternal circulation have been assumed to be dead material. To our knowledge, ours are the first data that physically connect circulating microparticles to syncytial knots by suggesting that shed syncytial aggregates are the intermediary form. Third, if syncytial knots give rise to circulating sFlt1-expressing microparticles through the shed aggregates, and if these phenomena are quantitatively stronger in preeclampsia, our data suggest that accelerated syncytial knot formation is a proximal event in the pathogenesis of preeclampsia, in agreement with prior reports 16
. Regardless, it is unlikely that syncytial knots within the intact placenta simply represent an artifact of tangential sectioning, as has also been proposed 36
Important questions remain for future investigation. Metabolically active microparticles appear to begin forming in the first trimester 24
. Does PE, therefore, represent the same process, but accelerated? If so, what fraction of peripheral sFlt1 protein is contributed by viable sFlt1 expressing microparticles versus dead/inactive microparticles that are already pre-loaded with sFlt1 protein? Also, what PE-specific mechanisms drive the induction of knot formation, deportation of syncytial aggregates, and microparticle generation? Conversely, could enhanced sFlt1 production somehow trigger syncytial knot formation? Second, we present novel evidence that living syncytial aggregates arise from syncytial sprouting and fission, but the molecular apparatus of nuclear aggregation and cytokinesis remain to be described. Third, different mechanisms for sFlt1 export from the placenta have been described, including the shedding of dead syncytial material 37
and the release of matrix-bound sFlt1 by matrix-dissolving enzymes such as heparanase16
. While upregulation or accumulation of heparanase has not been shown to occur in the preeclamptic human placenta16
, it will be of interest to determine the relative contributions of these processes to maternal sFlt1 exposure. Fourth, we have established some of the biological capacity of syncytial aggregates by demonstrating de novo
gene expression, but other functions, including regulation of inflammation and immunity, may also be important 38
. It would also be important to determine whether pro-inflammatory stimuli and other factors such as angiotensin autoantibodies that have been linked with preeclampsia pathogenesis may induce syncytial knot and microparticle formation. Finally, it has been suggested that shed syncytial aggregates get trapped in the capillary beds of lung tissue, where they further undergo disaggregation or apoptosis/necrosis to release the smaller microparticles into the systemic circulation39–41
. The relative contribution of these processes to the formation of trophoblast microparticles within the maternal circulation remains to be determined.