Micro- and Nano-vesicles from First Trimester Human Placentae Carry Flt-1 and Levels Are Increased in Severe Preeclampsia

Abstract

Background/objectives: Preeclampsia is a life-threatening hypertensive disease affecting 3-5% of pregnancies. While the pathogenesis of preeclampsia remains unclear, it is known that placenta-derived factors trigger the disease by activating maternal endothelial cells prior to the onset of clinical symptoms. Extracellular vesicles (EVs) of different sizes extruded by the placenta may be one factor. The truncated/secreted form of Flt-1 (sFlt-1) has also been implicated in the pathogenesis of preeclampsia. We investigated whether placental EV production is altered in preeclampsia such that they induce endothelial cell activation, and whether (s)Flt-1 is involved.Methods: Macro-, micro-, and nano-vesicles were collected from normal and preeclamptic (PE) placental explants, and separated by differential centrifugation. The number and size of micro-and nano-vesicles was measured by nanoparticle tracking analysis and their ability to activate endothelial cells was quantified by endothelial cell intercellular adhesion molecule 1 expression and monocyte adhesion. The levels of Flt-1 were measured by western blots and ELISA.results: PE placentae extruded significantly more micro-and nano-vesicles than control placentae and the extruded micro-vesicles were larger than those from control placentae. Micro-and nano-vesicles from both first trimester and term human placentae carried Flt-1 and levels were significantly increased in EVs from severe, but not mild, PE compared to normotensive placentae. All fractions of EVs from PE placentae activated endothelial cells, and for micro-and nano-vesicles, activation was reduced in the presence of exogenous vascular endothelial growth factor (VEGF), a Flt-1 neutralizing antibody, or by pre-treatment with VEGF. While EV-bound VEGF constituted over 20% of the total detected VEGF secreted by PE and normotensive placentae, EV-bound Flt-1 did not significantly contribute to the total level of sFlt-1/Flt-1 released by human third trimester placentae.Discussion: Micro-and nano-vesicles extruded by human placentae carry Flt-1 across gestation and in severe preeclampsia, the levels of vesicle-bound Flt-1 are upregulated. All fractions of PE placental EVs activated endothelial cells and for micro-and nano-vesicles, this was in part due to the ability of EV-bound Flt-1 to sequester VEGF. That placental EVs can activate endothelial cells supports the contention that EVs are one placental toxin contributing to the pathogenesis of preeclampsia.

Immunological effects of placental extracellular vesicles

“…In addition to immunomodulatory factors, vasoactive factors, such as Flt‐1 and endoglin are also present on placental EVs . Furthermore, proteins involved in coagulation/hemostasis, such as tissue factor, are carried by placental EVs .…”

“…Uptake of EVs allows the delivery of the specific EV‐associated cargos, such as miRNA, into endothelial cells. However, placental EVs may also affect endothelial cell function without being internalized as we and others have shown that EVs derived from preeclamptic placentae carry increased levels of Flt‐1, which can sequester vascular endothelial growth factor and placental growth factor from the micro‐environment …”

“…Moreover, conditioned media derived from endothelial cells cultured with placental micro‐EVs could activate resting leukocytes, demonstrating a complex interplay between placental EVs and the maternal cardiovascular and immune systems . Micro‐EVs from preeclamptic placentae further reduced endothelial cell proliferation and increased endothelial cell activation . Placental micro‐EVs have also been reported to inhibit vasodilation of subcutaneous arteries in vitro while in vivo , placental nano‐EVs affected vasodilation of mesenteric arteries in a time‐dependent manner .…”

“…While some studies report that EV production is increased by preeclamptic placentae, circulating placental EV number alone does not seem to be predictive of preeclampsia in prospective studies . It is likely that qualitative changes in the placental EV cargo may be more important than quantitative differences, both for the pathogenesis and prediction of obstetric diseases . Furthermore standardization of the sample collection, isolation and characterization protocols will allow comparisons between studies to rapidly advance this field.…”

Extracellular vesicles generated by placental tissues ex vivo: A transport system for immune mediators and growth factors

“…A number of publications have addressed placental EVs and their potential role in pregnancy and its complications . Several ex vivo (eg, placental perfusion) and in vivo systems have been used as a source of EVs. Placental perfusion is a useful method for obtaining large numbers of EVs directly from the placenta; however, this technique is suitable only for a short period of time (2‐6 hours) after delivery .…”