缺氧人胎盘的小细胞外囊泡破坏小鼠血脑屏障
1Vascular Physiology Laboratory, Department of Basic Sciences,Universidad del Bío-Bío, Chillan, 2Nursery School, Health Faculty,Universidad Santo Tomás, Los Angeles, 3Obstetrics and Gynecology Department,Herminda Martin Clinical Hospital, Chillan, 4Faculty of Medicine,Universidad Católica de la Santísima Concepción, Concepcion, 5Laboratory of Animal Biotechnology, Department of Animal Science, Faculty of Veterinary Sciences,Universidad de Concepción, Chillan, 6Group of Research and Innovation in Vascular Health (GRIVAS Health), Chillan
Summary
提出了一种方案来评估从缺氧条件下培养的胎盘外植体中分离出的小 EV （sEV）（模拟先兆子痫的一个方面）是否会破坏未怀孕成年雌性小鼠的血脑屏障。
Abstract
脑血管并发症，包括脑水肿、缺血性和出血性卒中，是先兆子痫导致孕产妇死亡的主要原因。这些脑血管并发症的潜在机制尚不清楚。然而，它们与胎盘功能障碍和血脑屏障 （BBB） 破坏有关。然而，这两个遥远器官之间的联系仍在确定中。越来越多的证据表明，胎盘将信号分子（包括细胞外囊泡）释放到母体循环中。细胞外囊泡根据其大小进行分类，小细胞外囊泡（直径小于 200 nm 的 sEV）在生理和病理条件下都被认为是关键信号转导颗粒。在先兆子痫中，母体循环中循环的sEV数量增加，其信号传导功能尚不清楚。在先兆子痫或暴露于缺氧的正常妊娠胎盘中释放的胎盘 sEV 会诱导脑内皮功能障碍和 BBB 破坏。在该方案中，我们评估了从缺氧条件下培养的胎盘外植体中分离的sEV（模拟先兆子痫的一个方面）是否会破坏 体内的BBB。
Introduction
大约70%的孕产妇死于先兆子痫，这是一种高血压妊娠综合征，其特征是胎盘过程受损，母亲全身性内皮功能障碍，严重时出现多器官衰竭1,2，与急性脑血管并发症有关3,4。大多数孕产妇死亡发生在低收入和中等收入国家5.然而，尽管与先兆子痫相关的脑血管并发症具有临床和流行病学相关性，但其潜在机制仍不清楚。
另一方面，细胞外囊泡 （EV）（直径 ~30-400 nm）是组织和器官之间细胞间通讯的重要介质，包括母胎相互作用6。除了外表面的蛋白质和脂质外，电动汽车还携带货物（蛋白质、RNA 和脂质）。EV 可分为 （1） 外泌体（直径 ~50-150 nm，也称为小型 EV （sEV））、（2） 中型/大型 EV 和 （3） 凋亡体，其大小、生物发生、含量和潜在的信号传导功能不同。EV 的组成由它们起源的细胞和疾病类型 7 决定。合体滋养层衍生的 EV 表达胎盘碱性磷酸酶 （PLAP）8,9，可检测妊娠期胎盘衍生的循环小 EV （PDsEV）。此外，PLAP 有助于辨别 PDsEV 货物的变化及其对子痫前期与血压正常妊娠的影响 10、11、12、13、14、15。
胎盘已被公认为先兆子痫病理生理学的必要组成部分16 或与该疾病相关的脑并发症 17,18,19。然而，这个远处的器官如何诱导脑循环的改变尚不清楚。由于 sEV 能够将生物活性成分从供体转移到受体细胞 6,20,21，因此越来越多的研究将胎盘 sEV 与母体内皮功能障碍的产生联系起来 21,22,23,24，包括脑内皮细胞25,26在患有先兆子痫的女性中。因此，脑内皮功能的损害可能导致血脑屏障 （BBB） 的破坏，血脑屏障是与子痫前期相关的脑血管并发症的关键组成部分 3,27。
然而，使用暴露于子痫前期女性血清的大鼠脑血管28 或暴露于子痫前期女性血浆的人脑内皮细胞29 的临床前研究结果报告说，循环因子诱导 BBB 的破坏。尽管有几种候选药物有可能损害先兆子痫期间母体循环中存在的血脑屏障，例如促炎细胞因子（即肿瘤坏死因子）18,28 或血管调节剂（即血管内皮生长因子 （VEGF））29,30,31 或氧化分子（如氧化脂蛋白 （oxo-LDL）32,33 等）水平升高34，它们都没有在胎盘和血脑屏障之间建立直接联系。最近，从缺氧胎盘中分离出的 sEV 显示出破坏未怀孕雌性小鼠 BBB 的能力25。由于胎盘 sEV 可能携带大多数列出的循环因子，具有破坏 BBB 的能力，因此 sEV 被认为是连接受损胎盘、成为有害循环因子的载体并破坏先兆子痫的 BBB 的合适候选者。
该协议使我们能够研究从在缺氧条件下培养的胎盘外植体中分离的sEV是否可以破坏未怀孕雌性小鼠的BBB，作为了解先兆子痫期间脑并发症的病理生理学的代表。
Protocol
该研究是按照《赫尔辛基宣言》中表达的原则进行的，并得到了各自伦理审查委员会的授权。如前所述，所有人类参与者在样本采集前都给予了知情同意25.此外，比奥比奥大学生物伦理学和生物安全委员会批准了该项目（Fondecyt 赠款1200250）。动物工作是根据实验35中使用动物的三个R的基本原则进行的，并根据美国国立卫生研究院发布的实验动物护理和使用指南…
Representative Results
该方案评估了来自在缺氧中培养的胎盘的sEV破坏非妊娠小鼠BBB的能力。这种方法可以更好地理解正常和病理条件下胎盘和大脑之间的潜在联系。特别是，该方法可以构成分析胎盘sEV参与先兆子痫脑并发症发作的代理。 与注射sEVs-Nor的小鼠相反，注射sEVs-Hyp的小鼠在24小时内显示神经评分逐渐下降（表1），这表明sEVs-Hyp损害大脑功能的能力。 此?…
Discussion
这项研究揭示了从缺氧条件下培养的胎盘外植体中分离出的 sEV 对啮齿动物血脑屏障破坏的潜在危害的新见解。病理机制涉及后脑区25中CLND-5的减少。
先前的研究表明，使用体外模型46,47，来自先兆子痫患者的血浆 sEV 会诱导各种器官的内皮功能障碍。这项研究特别仔细检查了血脑屏障，为从缺氧培养的胎盘?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢 GRIVAS Health 的研究人员的宝贵意见。此外，妇产科的助产士和临床工作人员属于智利奇兰医院。由 Fondecyt Regular 1200250 创立。
Materials
| Adult mice brain slecer matrice 3D printed | Open access file | Adult mice | Adult mice brain slicer. Printed in PLA filament. |
| Anti β-Actin primary antibody | Sigma-Aldrich | Clon AC-74 | Antibody for loading control (Western blot) |
| Anti-Claudin5 primary antibody | Santa cruz Biotechnology | sc-374221 | Primary antibody for tight junction protein CLDN5 of mice BBB (Western blot) |
| BCA protein kit | Thermo Scientific | 23225 | Kit for measuring protein concentration |
| Culture media #200 500 mL | Thermo Fisher Scientific | m200500 | Culture media for placental explants |
| D180 CO2 incubator | RWD Life science | D180 | Standard incubator to estabilize explants and culture sEVs-Nor |
| Evans blue dye > 75% 10 g | Sigma-Aldrich | E2129.10G | Dye to analize blood brain barrier disruption IN VIVO |
| Fetal bovine serum 500 mL | Thermo Fisher Scientific | 16000044 | Additive growth factor for culture media 200 |
| Himac Ultracentrifuge CP100NX | Himac eppendorf group | 5720410101 | Ultracentrifuge for condicioned media > 1,20,000 x g |
| ImageJ software | NIH | https://imagej.nih.gov/ij/download.html | |
| Isoflurane x 100 mL | USP Baxter | 212-094 | Volatile inhalated anaesthesia agent for mice |
| Kit CellTiter 96 Non-radioactive | Promega | 0000105232 | In vitro assay for placental explants viability |
| Mouse IgG Secondary antibody | Thermo Fisher Scientific | MO 63103 | Secondary antibody for CLDN5 (western blot) |
| NanoSight NS300 | Malvern Panalytical | 90278090 | Nanotracking analysis of particles from placental explants condicioned media |
| Paraformaldehide E 97% solution 500 mL | Thermo Fisher Scientific | A11313.22 | Fixative solution for brain tissue slices and intracardial perfusion (once diluted) |
| PBS 1 X pH 7.4 500 mL | Thermo Fisher Scientific | 10010023 | Wash solution for placenta explants |
| Peniciline-streptomicine 100x 20 mL | Thermo Fisher Scientific | 10378016 | Antiobiotics for placental explants culture media |
| ProOX C21 Cytocentric O2 and CO2 Subchamber Controller | BioSpherix | SCR_021131 | CO2 regulator to induce Hypoxia in sealed chamber for sEVs-Hyp |
| Sodium Thiopental 1 g | Chemie | 7061 | humanitarian euthanasia agent |
| Somnosuite low flow anesthesia system | Kent Scientifics | SS-01 | Isoflurane vaporizer for small rodents |
| Surgical Warming platform | Kent Scientifics | A41166 | Warming platform for mainteinance anesthesia in mice |
| Syringe Filters, Polytetrafluoroethylene (PTFE), Hydrophobic, 0.22 µm, Sterile, 25 mm | Southern labware | 10026 | Filtration of condicioned media harvested from placental explants |
| Tabletop High-Speed Micro Centrifuges HITACHI himac CT15E/CT15RE | Hitachi medical systems | 6020 | Serial centrifugations of condicioned media < 1,20, 000 x g |
| Trinocular stereomicroscope transmided and reflective light 10x-160x | Center Medical | 2597 | Stereomicroscope to register brain slices |
References
- Lisonkova, S., Joseph, K. S. Incidence of preeclampsia: risk factors and outcomes associated with early- versus late-onset disease. Am J Obstet Gynecol. 209 (544), 544.e1-544.e12 (2013).
- Sibai, B., Dekker, G., Kupferminc, M. Preeclampsia. Lancet. 365 (9461), 785-799 (2005).
- Hammer, E. S., Cipolla, M. J. Cerebrovascular dysfunction in preeclamptic pregnancies. Curr Hypertens Rep. 17 (8), 64 (2015).
- Okanloma, K. A., Moodley, J. Neurological complications associated with the preeclampsia/eclampsia syndrome. Int J Gynaecol Obstet. 71, 223-225 (2000).
- Frias, A. E., Belfort, M. A. Post magpie: how should we be managing severe preeclampsia. Curr Opin Gynecol Obstet. 15 (6), 489-495 (2003).
- Familari, M., Cronqvist, T., Masoumi, Z., Hansson, S. R. Placenta-derived extracellular vesicles: Their cargo and possible functions. Reprod Fertil Dev. 29 (3), 433-447 (2017).
- Montoro-Garcia, S., Shantsila, E., Marin, F., Blann, A., Lip, G. Y. Circulating microparticles: new insights into the biochemical basis of microparticle release and activity. Basic Res Cardiol. 106, 911-923 (2011).
- Germain, S. J., Sacks, G. P., Sooranna, S. R., Sargent, I. L., Redman, C. W. Systemic inflammatory priming in normal pregnancy and preeclampsia: the role of circulating syncytiotrophoblast microparticles. J Immunol. 178 (9), 5949-5956 (2007).
- Tannetta, D., Masliukaite, I., Vatish, M., Redman, C., Sargent, I. Update of syncytiotrophoblast derived extracellular vesicles in normal pregnancy and preeclampsia. J Reprod Immunol. 119, 98-106 (2017).
- Collett, G. P., Redman, C. W., Sargent, I. L., Vatish, M. Endoplasmic reticulum stress stimulates the release of extracellular vesicles carrying danger-associated molecular pattern (DAMP) molecules. Oncotarget. 9 (6), 6707-6717 (2018).
- Cooke, W. R., et al. Maternal circulating syncytiotrophoblast-derived extracellular vesicles contain biologically active 5′-tRNA halves. Biochem Biophys Res Commun. 518 (1), 107-113 (2019).
- Gill, M., et al. Placental syncytiotrophoblast-derived extracellular vesicles carry active nep (neprilysin) and are increased in preeclampsia. Hypertension. 73 (5), 1112-1119 (2019).
- Kandzija, N., et al. Placental extracellular vesicles express active dipeptidyl peptidase IV; levels are increased in gestational diabetes mellitus. J Extracell Vesicles. 8 (1), 1617000 (2019).
- Motta-Mejia, C., et al. Placental vesicles carry active endothelial nitric oxide synthase and their activity is reduced in preeclampsia. Hypertension. 70 (2), 372-381 (2017).
- Sammar, M., et al. Reduced placental protein 13 (PP13) in placental derived syncytiotrophoblast extracellular vesicles in preeclampsia – A novel tool to study the impaired cargo transmission of the placenta to the maternal organs. Placenta. 66, 17-25 (2018).
- Burton, G. J., Woods, A. W., Jauniaux, E., Kingdom, J. C. Rheological and physiological consequences of conversion of the maternal spiral arteries for uteroplacental blood flow during human pregnancy. Placenta. 30 (6), 473-482 (2009).
- Warrington, J. P., et al. Placental ischemia in pregnant rats impairs cerebral blood flow autoregulation and increases blood-brain barrier permeability. Physiological Reports. 2 (8), e12134-e12134 (2014).
- Warrington, J. P., Drummond, H. A., Granger, J. P., Ryan, M. J. Placental Ischemia-induced increases in brain water content and cerebrovascular permeability: Role of TNFα. Am J Physiol Regul Integr Comp Physiol. 309 (11), R1425-R1431 (2015).
- Johnson, A. C., et al. Magnesium sulfate treatment reverses seizure susceptibility and decreases neuroinflammation in a rat model of severe preeclampsia. PLoS ONE. 9 (11), e113670 (2014).
- Escudero, C. A., et al. Role of extracellular vesicles and microRNAs on dysfunctional angiogenesis during preeclamptic pregnancies. Front Physiol. 7, 1-17 (2016).
- Salomon, C., et al. Placental exosomes as early biomarker of preeclampsia: Potential role of exosomalmicrornas across gestation. J Clin Endocrinol Metab. 102 (9), 3182-3194 (2017).
- Knight, M., Redman, C. W., Linton, E. A., Sargent, I. L. Shedding of syncytiotrophoblast microvilli into the maternal circulation in pre-eclamptic pregnancies. Br J Obstet Gynaecol. 105 (6), 632-640 (1998).
- Gilani, S. I., Weissgerber, T. L., Garovic, V. D., Jayachandran, M. Preeclampsia and extracellular vesicles. Curr Hypertens Rep. 18 (9), 68 (2016).
- Dutta, S., et al. Hypoxia-induced small extracellular vesicle proteins regulate proinflammatory cytokines and systemic blood pressure in pregnant rats. Clin Sci (Lond). 134 (6), 593-607 (2020).
- Leon, J., et al. Disruption of the blood-brain barrier by extracellular vesicles from preeclampsia plasma and hypoxic placentae: attenuation by magnesium sulfate. Hypertension. 78 (5), 1423-1433 (2021).
- Han, C., et al. Placenta-derived extracellular vesicles induce preeclampsia in mouse models. Haematologica. 105 (6), 1686-1694 (2020).
- Amburgey, O. A., Chapman, A. C., May, V., Bernstein, I. M., Cipolla, M. J. Plasma from preeclamptic women increases blood-brain barrier permeability: role of vascular endothelial growth factor signaling. Hypertension. 56 (5), 1003-1008 (2010).
- Cipolla, M. J., et al. Pregnant serum induces neuroinflammation and seizure activity via TNFalpha. Exp Neurol. 234 (2), 398-404 (2012).
- Bergman, L., et al. Preeclampsia and increased permeability over the blood brain barrier – a role of vascular endothelial growth receptor 2. Am J Hypertens. 34 (1), 73-81 (2021).
- Torres-Vergara, P., et al. Dysregulation of vascular endothelial growth factor receptor 2 phosphorylation is associated with disruption of the blood-brain barrier and brain endothelial cell apoptosis induced by plasma from women with preeclampsia. Biochim Biophys Acta Mol Basis Dis. 1868 (9), 166451 (2022).
- Schreurs, M. P., Houston, E. M., May, V., Cipolla, M. J. The adaptation of the blood-brain barrier to vascular endothelial growth factor and placental growth factor during pregnancy. FASEB J. 26 (1), 355-362 (2012).
- Schreurs, M. P., Cipolla, M. J. Cerebrovascular dysfunction and blood-brain barrier permeability induced by oxidized LDL are prevented by apocynin and magnesium sulfate in female rats. J Cardiovasc Pharmacol. 63 (1), 33-39 (2014).
- Schreurs, M. P. H., et al. Increased oxidized low-density lipoprotein causes blood-brain barrier disruption in early-onset preeclampsia through LOX-1. FASEB J. 27 (3), 1254-1263 (2013).
- Escudero, C., et al. Brain vascular dysfunction in mothers and their children exposed to preeclampsia. Hypertension. 80 (2), 242-256 (2023).
- Russell, W. M. S., Burch, R. L. The principles of humane experimental technique. Universities Federation of Animal Welfare. , (1959).
- Miller, R. K., et al. Human placental explants in culture: approaches and assessments. Placenta. 26 (6), 439-448 (2005).
- Troncoso, F. A. J., Herlitz, K., Ruiz, F., Bertoglia, P., Escudero, C. Elevated pro-angiogenic phenotype in feto-placental tissue from gestational diabetes mellitus. Placenta. 36 (4), 2 (2015).
- Zhang, H. C., et al. Microvesicles derived from human umbilical cord mesenchymal stem cells stimulated by hypoxia promote angiogenesis both in vitro and in vivo. Stem Cells Dev. 21 (18), 3289-3297 (2012).
- Thery, C., Amigorena, S., Raposo, G., Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. Chapter 3 (Unit 3), 22 (2006).
- Carroll, R. W., et al. A rapid murine coma and behavior scale for quantitative assessment of murine cerebral malaria. PLoS One. 5 (10), e13124 (2010).
- Wu, J., et al. Transcardiac perfusion of the mouse for brain tissue dissection and fixation. Bio Protoc. 11 (5), e3988 (2021).
- Walchli, T., et al. Quantitative assessment of angiogenesis, perfused blood vessels and endothelial tip cells in the postnatal mouse brain. Nat Protoc. 10 (1), 53-74 (2015).
- Wang, H. L., Lai, T. W. Optimization of Evans blue quantitation in limited rat tissue samples. Sci Rep. 4, 6588 (2014).
- Morita, K., Sasaki, H., Furuse, M., Tsukita, S. Endothelial claudin: claudin-5/TMVCF constitutes tight junction strands in endothelial cells. J Cell Biol. 147 (1), 185-194 (1999).
- Lara, E., et al. Abnormal cerebral microvascular perfusion and reactivity in female offspring of reduced uterine perfusion pressure (RUPP) mice model. J Cereb Blood Flow Metab. 42 (12), 2318-2332 (2022).
- Chang, X., et al. Exosomes from women with preeclampsia induced vascular dysfunction by delivering sflt (soluble fms-like tyrosine kinase)-1 and seng (soluble endoglin) to endothelial cells. Hypertension. 72, 1381-1390 (2018).
- Smarason, A. K., Sargent, I. L., Starkey, P. M., Redman, C. W. The effect of placental syncytiotrophoblast microvillous membranes from normal and pre-eclamptic women on the growth of endothelial cells in vitro. BJOG. 100 (10), 943-949 (1993).
Cite This Article
Sandoval, H., León, J., Troncoso, F., de la Hoz, V., Cisterna, A., Contreras, M., Castro, F. O., Ibañez, B., Acurio, J., Escudero, C. Disruption of the Mouse Blood-Brain Barrier by Small Extracellular Vesicles from Hypoxic Human Placentas. J. Vis. Exp. (203), e65867, doi:10.3791/65867 (2024).