Endothelial Dysfunction in Severe Preeclampsia is Mediated by Soluble Factors, Rather than Extracellular Vesicles

Michelle O'Brien, Dora Baczyk, John C Kingdom, Michelle O'Brien, Dora Baczyk, John C Kingdom

Abstract

In severe early-onset preeclampsia (sPE) the placenta releases soluble angiogenesis-regulating proteins, trophoblast-derived fragments, and extracellular vesicles (EVs). Their relative importance in disease pathogenesis is not presently understood. We explanted placental villi from healthy and sPE women then separated the media into: total-conditioned, EV-depleted and EV-enriched media. Three fractions were compared for; angiogenic protein secretion by ELISA, angiogenic and inflammation gene mRNA expression and leukocyte adhesion assay. sPE placental villi secreted significantly less PlGF (70 ± 18 pg/mL) than preterm controls (338 ± 203; p = 0.03). sFlt-1:PlGF ratios in total-conditioned (115 ± 29) and EV-depleted media (136 ± 40) from sPE placental villi were significantly higher than in EV-enriched media (42 ± 12; p < 0.01) or any preterm or term media. Fluorescent-labeled EVs derived across normal gestation, but not from sPE, actively entered HUVECs. From sPE placental villi, the soluble fraction, but not EV-enriched fraction, significantly repressed angiogenesis (0.83 ± 0.05 fold, p = 0.02), induced HO-1 mRNA (15.3 ± 5.1 fold, p < 0.05) and induced leukocyte adhesion (2.2 ± 0.4 fold, p = 0.04). Soluble media (total-conditioned and EV-depleted media) from sPE placental villi induced endothelial dysfunction in HUVEC, while the corresponding EV-enriched fraction showed no such effects. Our data suggest that soluble factors including angiogenesis-regulating proteins, dominate the vascular pathology of this disease.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Purity of EVs was assessed using low magnification TEM. (A) EV preparation prior to and (B) following qEV exclusion column. Images taken at 19,000X magnification. Arrows = exosome, * = micro-vesicles. (C–F) Additional characterization of the EVs was performed by Western blotting for EV-specific markers, Flotilin-1 and Hsp70, and for placenta specific marker-PLAP. Purity of preparations was further validated with GM130 antibody. Densitometry analysis for (C) Flotilin-1, (D) Hsp70, (E) PLAP and (F) GM130 are presented.
Figure 2
Figure 2
PKH67 fluorescently labelled EVs generated from (A) first, (B) preterm, (C) term and (D) PE placental explants were incubated with HUVEC cells for 4 hrs. (E) Pre-eclamptic EVs were also incubated with MCF7 breast cancer cell line for 4 hrs. (F) PE-derived EVs are unable to enter HUVEC cells but are able to enter into cancer cells. (G) EVs generated from PE express fibronectin.
Figure 3
Figure 3
Media analysis by ELISA for (A–C) PlGF, (D–F) sFlt-1 and (G–I) Endoglin from preterm, term and PE placenta. PlGF and sFlt-1 levels were mainly detected in total and EV-free media. Endoglin was detected in total media but its levels were markedly reduced in EV-depleted media. All three angiogenic proteins showed minimal levels in EV-enriched media, microvesicle-enriched media and EV lysates. Large micro-vesicles = obtained after the 10,000 g spin, include micro-vesicle and apoptotic bodies. EV lysates = obtained by RIPA lysis of purified EV pellets.
Figure 4
Figure 4
sFLt-1:PlGF ratio in media preparations from preterm, term and sPE placentas. NS = not significant.
Figure 5
Figure 5
Angiogenic activity of HUVECs treated with (A) preterm or (B) term conditioned and enriched media was not altered. (C) HUVEC angiogenesis was moderately decreased when treated with PE conditioned media but not EV-enriched media. Representative images of (D) positive control, (E) total PE media, (F) EV-depleted media and (G) PE-EV enriched media.
Figure 6
Figure 6
HUVECs were treated with total, EV-depleted and EV-enriched media from (A,B) first trimester, (C,D) second trimester, (E,F) preterm, (G,H) term and (I,J) PE placentas for 24 hr.
Figure 7
Figure 7
Endothelial cells were treated with total and EV-enriched media from (A) first trimester, (B) second trimester, (C) preterm, (D) term and (E) PE placentas for 6 hrs prior to incubation with fluorescently labelled monocytes for 45 min. First trimester and PE total media but not the EV-enriched media elicited leukocyte activation.

References

    1. Mol BW, et al. Pre-eclampsia. Lancet. 2016;387:999–1011. doi: 10.1016/S0140-6736(15)00070-7.
    1. Karumanchi SA, Granger JP. Preeclampsia and Pregnancy-Related Hypertensive Disorders. Hypertension. 2016;67:238–242. doi: 10.1161/HYPERTENSIONAHA.116.06421.
    1. Walker MG, et al. Sex-specific basis of severe placental dysfunction leading to extreme preterm delivery. Placenta. 2012;33:568–571. doi: 10.1016/j.placenta.2012.03.011.
    1. Redline RW. Classification of placental lesions. American journal of obstetrics and gynecology. 2015;213:S21–28. doi: 10.1016/j.ajog.2015.05.056.
    1. Rajakumar A, et al. Transcriptionally active syncytial aggregates in the maternal circulation may contribute to circulating soluble fms-like tyrosine kinase 1 in preeclampsia. Hypertension. 2012;59:256–264. doi: 10.1161/HYPERTENSIONAHA.111.182170.
    1. Tache V, LaCoursiere DY, Saleemuddin A, Parast MM. Placental expression of vascular endothelial growth factor receptor-1/soluble vascular endothelial growth factor receptor-1 correlates with severity of clinical preeclampsia and villous hypermaturity. Human pathology. 2011;42:1283–1288. doi: 10.1016/j.humpath.2010.11.018.
    1. Rajakumar A, et al. Novel soluble Flt-1 isoforms in plasma and cultured placental explants from normotensive pregnant and preeclamptic women. Placenta. 2009;30:25–34. doi: 10.1016/j.placenta.2008.10.006.
    1. Chappell LC, et al. Diagnostic accuracy of placental growth factor in women with suspected preeclampsia: a prospective multicenter study. Circulation. 2013;128:2121–2131. doi: 10.1161/CIRCULATIONAHA.113.003215.
    1. Romero R, et al. A longitudinal study of angiogenic (placental growth factor) and anti-angiogenic (soluble endoglin and soluble vascular endothelial growth factor receptor-1) factors in normal pregnancy and patients destined to develop preeclampsia and deliver a small for gestational age neonate. The journal of maternal-fetal & neonatal medicine: the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstet. 2008;21:9–23. doi: 10.1080/14767050701830480.
    1. Chamley LW, Chen Q, Ding J, Stone PR, Abumaree M. Trophoblast deportation: just a waste disposal system or antigen sharing? Journal of reproductive immunology. 2011;88:99–105. doi: 10.1016/j.jri.2011.01.002.
    1. Escudero CA, et al. Role of Extracellular Vesicles and microRNAs on Dysfunctional Angiogenesis during Preeclamptic Pregnancies. Frontiers in physiology. 2016;7:98. doi: 10.3389/fphys.2016.00098.
    1. Tannetta D, Masliukaite I, Vatish M, Redman C, Sargent I. Update of syncytiotrophoblast derived extracellular vesicles in normal pregnancy and preeclampsia. Journal of reproductive immunology. 2016
    1. Tannetta D, Dragovic R, Alyahyaei Z, Southcombe J. Extracellular vesicles and reproduction-promotion of successful pregnancy. Cellular & molecular immunology. 2014;11:548–563. doi: 10.1038/cmi.2014.42.
    1. Buurma AJ, et al. Preeclampsia is associated with the presence of transcriptionally active placental fragments in the maternal lung. Hypertension. 2013;62:608–613. doi: 10.1161/HYPERTENSIONAHA.113.01505.
    1. Smarason AK, Sargent IL, Starkey PM, Redman CW. The effect of placental syncytiotrophoblast microvillous membranes from normal and pre-eclamptic women on the growth of endothelial cells in vitro. British journal of obstetrics and gynaecology. 1993;100:943–949. doi: 10.1111/j.1471-0528.1993.tb15114.x.
    1. Knight M, Redman CW, Linton EA, Sargent IL. Shedding of syncytiotrophoblast microvilli into the maternal circulation in pre-eclamptic pregnancies. British journal of obstetrics and gynaecology. 1998;105:632–640. doi: 10.1111/j.1471-0528.1998.tb10178.x.
    1. Andaloussi SEL, Mager I, Breakefield XO, Wood MJ. Extracellular vesicles: biology and emerging therapeutic opportunities. Nature reviews. Drug discovery. 2013;12:347–357. doi: 10.1038/nrd3978.
    1. Turturici G, Tinnirello R, Sconzo G, Geraci F. Extracellular membrane vesicles as a mechanism of cell-to-cell communication: advantages and disadvantages. American journal of physiology. Cell physiology. 2014;306:C621–633. doi: 10.1152/ajpcell.00228.2013.
    1. Goswami D, et al. Excess syncytiotrophoblast microparticle shedding is a feature of early-onset pre-eclampsia, but not normotensive intrauterine growth restriction. Placenta. 2006;27:56–61. doi: 10.1016/j.placenta.2004.11.007.
    1. Germain SJ, Sacks GP, Sooranna SR, Sargent IL, Redman CW. Systemic inflammatory priming in normal pregnancy and preeclampsia: the role of circulating syncytiotrophoblast microparticles. J Immunol. 2007;178:5949–5956. doi: 10.4049/jimmunol.178.9.5949.
    1. Dragovic RA, Southcombe JH, Tannetta DS, Redman CW, Sargent IL. Multicolor flow cytometry and nanoparticle tracking analysis of extracellular vesicles in the plasma of normal pregnant and pre-eclamptic women. Biology of reproduction. 2013;89:151. doi: 10.1095/biolreprod.113.113266.
    1. Shomer E, et al. Microvesicles of pregnant women receiving low molecular weight heparin improve trophoblast function. Thrombosis research. 2016;137:141–147. doi: 10.1016/j.thromres.2015.11.026.
    1. Chua S, Wilkins T, Sargent I, Redman C. Trophoblast deportation in pre-eclamptic pregnancy. British journal of obstetrics and gynaecology. 1991;98:973–979. doi: 10.1111/j.1471-0528.1991.tb15334.x.
    1. Tannetta DS, Dragovic RA, Gardiner C, Redman CW, Sargent IL. Characterisation of syncytiotrophoblast vesicles in normal pregnancy and pre-eclampsia: expression of Flt-1 and endoglin. PloS one. 2013;8:e56754. doi: 10.1371/journal.pone.0056754.
    1. Shen F, et al. Trophoblast debris extruded from preeclamptic placentae activates endothelial cells: a mechanism by which the placenta communicates with the maternal endothelium. Placenta. 2014;35:839–847. doi: 10.1016/j.placenta.2014.07.009.
    1. Yanez-Mo M, et al. Biological properties of extracellular vesicles and their physiological functions. Journal of extracellular vesicles. 2015;4:27066. doi: 10.3402/jev.v4.27066.
    1. Lowry MC, Gallagher WM, O’Driscoll L. The Role of Exosomes in Breast Cancer. Clinical chemistry. 2015;61:1457–1465. doi: 10.1373/clinchem.2015.240028.
    1. Wang Z, Chen JQ, Liu JL, Tian L. Exosomes in tumor microenvironment: novel transporters and biomarkers. Journal of translational medicine. 2016;14:297. doi: 10.1186/s12967-016-1056-9.
    1. Witwer, K. W. et al. Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. Journal of extracellular vesicles2, doi:10.3402/jev.v2i0.20360 (2013).
    1. Abramowicz A, Widlak P, Pietrowska M. Proteomic analysis of exosomal cargo: the challenge of high purity vesicle isolation. Molecular bioSystems. 2016;12:1407–1419. doi: 10.1039/C6MB00082G.
    1. Lotvall J, et al. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. Journal of extracellular vesicles. 2014;3:26913. doi: 10.3402/jev.v3.26913.
    1. Purushothaman A, et al. Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions. The Journal of biological chemistry. 2016;291:1652–1663. doi: 10.1074/jbc.M115.686295.
    1. Zeisler H, et al. Predictive Value of the sFlt-1:PlGF Ratio in Women with Suspected Preeclampsia. The New England journal of medicine. 2016;374:13–22. doi: 10.1056/NEJMoa1414838.
    1. Sobel ML, Kingdom J, Drewlo S. Angiogenic response of placental villi to heparin. Obstetrics and gynecology. 2011;117:1375–1383. doi: 10.1097/AOG.0b013e31821b5384.
    1. Herraiz I, et al. Characterization of the soluble fms-like tyrosine kinase-1 to placental growth factor ratio in pregnancies complicated by fetal growth restriction. Obstetrics and gynecology. 2014;124:265–273. doi: 10.1097/AOG.0000000000000367.
    1. Atay S, Gercel-Taylor C, Taylor DD. Human trophoblast-derived exosomal fibronectin induces pro-inflammatory IL-1beta production by macrophages. Am J Reprod Immunol. 2011;66:259–269. doi: 10.1111/j.1600-0897.2011.00995.x.
    1. Hoegh AM, et al. Effect of syncytiotrophoblast microvillous membrane treatment on gene expression in human umbilical vein endothelial cells. BJOG: an international journal of obstetrics and gynaecology. 2006;113:1270–1279. doi: 10.1111/j.1471-0528.2006.01061.x.
    1. Hung TH, Skepper JN, Charnock-Jones DS, Burton GJ. Hypoxia-reoxygenation: a potent inducer of apoptotic changes in the human placenta and possible etiological factor in preeclampsia. Circulation research. 2002;90:1274–1281. doi: 10.1161/01.RES.0000024411.22110.AA.
    1. Meah VL, Cockcroft JR, Backx K, Shave R, Stohr EJ. Cardiac output and related haemodynamics during pregnancy: a series of meta-analyses. Heart. 2016;102:518–526. doi: 10.1136/heartjnl-2015-308476.
    1. Salomon C, et al. A gestational profile of placental exosomes in maternal plasma and their effects on endothelial cell migration. PloS one. 2014;9:e98667. doi: 10.1371/journal.pone.0098667.
    1. Hayman R, Warren A, Brockelsby J, Johnson I, Baker P. Plasma from women with pre-eclampsia induces an in vitro alteration in the endothelium-dependent behaviour of myometrial resistance arteries. BJOG: an international journal of obstetrics and gynaecology. 2000;107:108–115. doi: 10.1111/j.1471-0528.2000.tb11586.x.
    1. Mincheva-Nilsson L, Baranov V. Placenta-derived exosomes and syncytiotrophoblast microparticles and their role in human reproduction: immune modulation for pregnancy success. Am J Reprod Immunol. 2014;72:440–457. doi: 10.1111/aji.12311.
    1. Leavey K, et al. Unsupervised Placental Gene Expression Profiling Identifies Clinically Relevant Subclasses of Human Preeclampsia. Hypertension. 2016;68:137–147. doi: 10.1161/HYPERTENSIONAHA.116.07293.
    1. Cockell AP, et al. Human placental syncytiotrophoblast microvillous membranes impair maternal vascular endothelial function. British journal of obstetrics and gynaecology. 1997;104:235–240. doi: 10.1111/j.1471-0528.1997.tb11052.x.
    1. American College of, O., Gynecologists & Task Force on Hypertension in, P. Hypertension in pregnancy. Report of the American College of Obstetricians and Gynecologists’ Task Force on Hypertension in Pregnancy. Obstetrics and gynecology122, 1122–1131, doi:10.1097/01.AOG.0000437382.03963.88 (2013).

Source: PubMed

Patrocinadores y Colaboradores

Condiciones médicas

Intervenciones de drogas

3
Suscribir