Protein composition of microparticles shed from human placenta during placental perfusion: Potential role in angiogenesis and fibrinolysis in preeclampsia

Abstract

Shedding of syncytiotrophoblast microparticles (MPs) from placenta to maternal blood occurs in normal pregnancy and is enhanced during preeclampsia (PE). The syncytiotrophoblast synthesizes plasminogen activator inhibitors (PAIs) which regulate fibrinolysis, as well as soluble forms of the fms-like tyrosine kinase (sFlt-1) and endoglin, which exert anti-angiogenic actions. An increase in the ratio of PAI-1/PAI-2 and elevated levels of sFlt-1 and sEng in maternal serum are linked to placental damage and maternal endothelial cell dysfunction in PE. The goal of the current study was to determine whether MPs released to maternal perfusate during dual perfusion contain these factors associated with placental pathophysiology in PE. Initially, high levels of alkaline phosphatase activity and Annexin V binding were found in MPs isolated by sequential centrifugation of maternal perfusates at 10,000 and 150,000×g(10 K and 150 K MPs), indicating their plasma membrane origin. ELISA revealed the presence of these factors at the following relative levels: Eng>PAI-2⋙PAI-1>sFlt-1. Based on comparisons of their concentration in perfusates, MPs, and MP-free 150 K supernatants, we determined that MPs constitute a significant portion of Eng released by placenta. Flow cytometric analysis of 10 K MPs supported the levels of expression found by ELISA and indicated that Eng and PAI-2 were almost exclusively localized to the surface of MPs, a site with biological potential. These results indicate that MPs shed from the syncytial surface express factors which may alter the fibrinolytic and angiogenic balance at the maternal-fetal interface and play a role in the pathophysiology of PE.

Fig. 1

Characteristics of MPs isolated from maternal perfusate. In the Left Panel alkaline phosphatase activity was measured in whole placental homogenates before and after perfusion (2 Left Bars), and in 10 K (3 Middle Bars) and 150 K (3 Right Bars) MPs obtained from the maternal perfusate at the indicated time of perfusion. Results are presented in log scale as a mean ± SE, and the level of statistical significance is indicated by the connecting lines at the top of the figure. Flow cytometric analysis of 10 K MPs, isolated from a 3 h perfusion sample, in the absence (broken line) and presence (solid line) of FITC-labeled Annexin V is shown in the Right Panel. In the Bottom Panel PAGE analysis was carried out for the following samples; Lane 1, 10 K MPs; Lane 2, 150 K MPs; Lane 3, whole placental tissue before perfusion; Lane 4, tissue after perfusion; Lane 5, protein standards of molecular weights indicated at the right of the panel. The arrow refers to a protein band of approximately 60 kDa. MPs from 5 different perfusions were used for alkaline phosphatase analysis, and a representative experiment of 3 identically conducted ones is shown for Annexin V binding and PAGE.

Fig. 2

Analysis of PAI-1 and PAI-2 levels in maternal perfusate, supernatant, and MPs. Levels of PAI-1 and PAI-2 in maternal perfusate, the final MP-free 150K supernatant, and in 10 K and 150 K MPs, were determined by ELISA. Results are expressed as a mean + SE obtained from 5 independent perfusions, and levels of statistical significance are shown be interconnecting lines at the top of the individual panels.

Fig. 3

Levels of sFlt-1 and Eng in maternal perfusate, supernatant, and MPs. The concentration of sFlt-1 and Eng in maternal perfusate, the MP-free supernatant, and in 10 K and 150 K MPs, was quantitated by ELISA. Results are expressed as a mean + SE obtained from 5 independent experiments, and the level of statistical significance is shown by interconnecting lines at the top of the individual panels.

Fig. 4

Flow cytometric analysis of PAI-1 and PAI-2 expression in 10 K MPs. Flow cytometric analysis of PAI-1 and PAI-2 was carried out in permeabilized (Panels A, C) or non-permeabilized (Panels B, D) 10 K MPs. Experiments were performed in the presence of protein-specific antibody (solid line) or isotype-matched control antibody (broken line). Representative results from 1 of 3 independent experiments are shown.

Fig. 5

Flow cytometric analysis of Flt-1 and Eng in 10 K MPs. Flow cytometry was carried out to assess levels of Flt-1 and Eng in permeabilized (Panels A, C) or non-permeabilized (Panels B, D) 10 K MPs. Experiments were performed in the presence of protein-specific antibody (solid line) or isotype-matched control antibody (broken line). Representative results from 1 of 3 independent experiments are shown.