Augmented Placental Protein 13 in Placental-Associated Extracellular Vesicles in Term and Preterm Preeclampsia Is Further Elevated by Corticosteroids

Abstract

Placental protein 13 (PP13) is a regulatory protein involved in remodeling the vascular system of the pregnancy and extending the immune tolerance of the mother to the growing fetus. PP13 is localized on the surface of the syncytiotrophoblast. An ex vivo placental model shows that the PP13 is released via placental-associated extracellular vesicles (PEVs) to the maternal uterine vein. This exploratory study aimed to determine PEV-associated PP13 in the maternal circulation as compared to the known soluble fraction since each has a specific communication pathway. Patients admitted to Bnai Zion Medical Center for delivery were recruited, and included 19 preeclampsia (PE) patients (7 preterm PE gestational age < 37 weeks' gestation), 16 preterm delivery (PTD, delivery at GA < 37 weeks' gestation), and 15 matched term delivery controls. Treatment by corticosteroids (Celestone), which is often given to patients with suspected preterm PE and PTD, was recorded. The PEV proteome was purified from the patients' plasma by size exclusion chromatography (SEC) to separate the soluble and PEV-associated PP13. The total level of PP13 (soluble and PEV-associated) was determined using mild detergent that depleted the PEV proteome. PP13 fractions were determined by ELISA with PP13 specific antibodies. ELISA with alkaline phosphatase (PLAP)- and cluster differentiation 63 (CD63)-specific antibodies served to verify the placental origin of the PEVs. SPSS was used for statistical analysis. The patients' medical, pregnancy, and delivery records in all groups were similar except, as expected, that a larger number of PE and PTD patients had smaller babies who were delivered earlier, and the PE patients had hypertension and proteinuria. The SEC analysis detected the presence of PP13 in the cargo of the PEVs and on their surface, in addition to the known soluble fraction. The median soluble PP13 was not significantly different across the PE, PTD, and term delivery control groups. However, after depleting the PEV of their proteome, the total PP13 (soluble and PEV-associated) was augmented in the cases of preterm PE, reaching 2153 pg/mL [IQR 1866-2838] but not in cases of PTD reaching 1576 pg/mL [1011-2014] or term delivery groups reaching 964 pg/mL [875-1636]), p < 0.01. On the surface of the circulating PEV from PTD patients, there was a decrease in PP13. Corticosteroid treatment was accompanied by a massive depletion of PP13 from the PEV, especially in preterm PE patients. This exploratory study is, thus, the first to determine PEV-associated PP13 in maternal circulation, providing a quantitative determination of the soluble and the PEV-associated fractions, and it shows that the latter is the larger. We found an increase in the amount of PP13 carried via the PEV-associated pathway in PE and PTD patients compared to term delivery cases, which was further augmented when the patients were treated with corticosteroids, especially in preterm PE. The signal conveyed by this novel communication pathway warrants further research to investigate these two differential pathways for the liberation of PP13.

Keywords: corticosteroids; extracellular vesicles; galectin 13; placental protein 13 (PP13); preeclampsia (PE); preterm delivery (PTD); size exclusion chromatography (SEC).

Figure 1

Study flow chart. We enrolled 50 patients—19 cases of PE, of whom 7 delivered at GA < 37 weeks’ gestation due to PE severity (PE < 37 wks), 16 PTD delivered at GA < 37 weeks’ gestation (PTD < 37 wks) unrelated to PE or other iatrogenic causes, and 15 term delivery controls. The number of women treated and untreated with corticosteroids (+/−) is shown.

Figure 2

Violin plot of PP13 levels as a function of the different fractions. The four panels on the right side are the violin plots of the level of PP13 in the total fraction (top), the solubilized fraction (below), the PEV-associated fraction (below), while the very bottom plot is the PP13 of the surface of PEV. The four panels on the left side are the violin plots of the same PP13 fractions for the groups of patients treated with (pink) and without (blue) corticosteroids. In each violin plot, the horizontal dashed line represents the lower quartile, the median, and the third quartile. Dots along the violin show different patients’ values within the violin. The longitudinal lines on both violin apexes represent the minimum and the maximum values. PE—preeclampsia, PTD—preterm delivery, PE < 37—preterm PE, PEV—placental-associated extracellular vesicles.

Figure 3

Sub-fractionation of maternal plasma. Starting from whole blood, the plasma was collected by EDTA-containing tubes. A–C: PP13 level was determined by ELISA in the soluble compartment (A). The total PP13 was determined by ELISA after treating the plasma with mild detergent to deplete PP13 from the PEV (B). Exo Spin mini-size exclusion chromatography (SEC) columns were used to exclusively isolate the PEV and determine PP13 on their surface (C). Note that in C, the level was limited by column capacity. To the right side, we describe the entire process of SEC while the actual SEC profile is shown in the Supplementary Figure S1. The schematic profile at the lower right side below the SEC large column tube indicates the zone of fractions 6–8 of the SEC includes the PEVs while the zone of fractions 12–24 include the soluble proteins (further details are demonstrated in Supplementary Figure S1).

Figure 4

PP13 pathways to the maternal circulation. The placenta syncytiotrophoblast (green to the right) releases PP13 through the uterine vein into the maternal circulation (the blue blood vessel to the left). The PEVs serve as the communication pathway. The microvesicles (pink) and the exosomes (turquoise) carry PP13 (purple) on their surface and inside. There is also a leakage of soluble PP13 (purple) either into the intracellular space or through necrotic vesicles (orange) that are too big to pass through the capillaries, thus, leaking their content into the soluble fraction of the maternal circulation. PP13 is, thus, liberated either as a soluble protein or in association with the PEVs. Clinical pathologies affect PP13 mainly via the PEV pathway. When reaching the maternal circulation, PP13 binds to the ABO antigen on the red blood cells (RBC, [47]), the white blood cells (WBC [3,26]), or the endothelial layer [27,28,48], Sammar et al. Elements of this figure were modified from our former review Sammar et al. [7] Int. J. Mol. Sci. 2019, 20, 3192. https://doi.org/10.3390/ijms20133192.