Abstract Cellular microvesicles and nanovesicles (exosomes) are involved in many disease processes and have major potential as biomarkers. However, developments in this area are constrained by ...limitations in the technology available for their measurement. Here we report on the use of fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles. In this system vesicles are visualized by light scattering using a light microscope. A video is taken, and the NTA software tracks the brownian motion of individual vesicles and calculates their size and total concentration. Using human placental vesicles and plasma, we have demonstrated that NTA can measure cellular vesicles as small as ∼50 nm and is far more sensitive than conventional flow cytometry (lower limit ∼300 nm). By combining NTA with fluorescence measurement we have demonstrated that vesicles can be labeled with specific antibody-conjugated quantum dots, allowing their phenotype to be determined. From the Clinical Editor The authors of this study utilized fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles, demonstrating that NTA is far more sensitive than conventional flow cytometry.
Latest Advances in Understanding Preeclampsia Redman, Christopher W; Sargent, Ian L
Science (American Association for the Advancement of Science),
06/2005, Letnik:
308, Številka:
5728
Journal Article
Recenzirano
Preeclampsia is a relatively common pregnancy disorder that originates in the placenta and causes variable maternal and fetal problems. In the worst cases, it may threaten the survival of both mother ...and baby. We summarize recent work on the causes of preeclampsia, which reveals a new mode of maternal immune recognition of the fetus, relevant to the condition. The circulating factors derived from the placenta, which contributes to the clinical syndrome, are now better understood. This brief review on preeclampsia does not cover all aspects of this intriguing condition but focuses on some new and interesting findings.
Abstract Alpha-synuclein aggregation plays a central role in Parkinson's disease pathology. Direct transmission of alpha-synuclein from pathologically affected to healthy unaffected neurons may be ...important in the anatomical spread of the disease through the nervous system. We have demonstrated that exosomes released from alpha-synuclein over-expressing SH-SY5Y cells contained alpha-synuclein and these exosomes were capable of efficiently transferring alpha-synuclein protein to normal SH-SY5Y cells. Moreover, the incubation of cells with ammonium chloride or bafilomycin A1 to produce the lysosomal dysfunction recently reported in Parkinson's disease led to an increase in the release of alpha-synuclein in exosomes and a concomitant increase in alpha-synuclein transmission to recipient cells. This study clearly demonstrates the importance of exosomes in both the release of alpha synuclein and its transmission between cells and suggests that factors associated with PD pathology accelerate this process. These mechanisms may play an important role in PD pathology and provide a suitable target for therapeutic intervention.
The placental syncytiotrophoblast releases micro and nanovesicles (STBM), into the maternal circulation in normal pregnancy and in increased amounts in pre-eclampsia (PE), which have proinflammatory ...and antiangiogenic activity and are implicated in PE pathophysiology. Better characterisation of STBM is essential to understand their role in PE.
STBM prepared by placental lobe dual perfusion (pSTBM) and mechanical disruption (mSTBM) were analysed by four colour flow cytometry (4CFC), nanoparticle tracking analysis (NTA) and Western blotting to determine vesicle size, purity and Flt-1 and endoglin (Eng) expression. Biological activity of STBM associated Flt-1 and endoglin was assessed by the ability of VEGF, PlGF and TGFβ to bind to mSTBM and inhibit mSTBM induced endothelial monolayer disruption. STBM content was consistently high (~87-95%) across the different preparations. However, surface antigen intensities differed, with significantly lower placental alkaline phosphatase (P<0.05) and Eng (P<0.05) expression on mSTBM, and Flt-1 (P<0.05) expression on pSTBM. For PE placenta derived preparations, pSTBM contained lower Eng positive STBM (P<0.05) and mSTBM Eng expression was increased (P<0.05). Western blotting revealed increased Flt-1/sFlt-1 (P<0.02) and decreased placental alkaline phosphatase (P = 0.0002) content of PE placenta pSTBM. Using NTA, perfused PE placentas released significantly larger MV (P<0.001). Finally, VEGF, PlGF and TGFβ bound to mSTBM at physiologically relevant concentrations and inhibited mSTBM induced endothelial disruption (P<0.05-P<0.001).
This study has found differences in physical and antigenic characteristics of normal and PE placenta STBM preparations produced by placental perfusion or mechanical disruption. We have also demonstrated that large quantities of biologically active STBM associated endoglin and Flt-1/sFlt-1 could contribute to the increased circulating levels measured in PE patients and add to the perturbation of the maternal vascular endothelium, normally attributed to non-membrane bound sFlt-1 and sEndoglin.
Objective The purpose of this study was to perform a comprehensive analysis of the microRNA expression profile in placentas from preeclamptic pregnancies vs normal placentas. Study Design Placentas ...were obtained from patients with (1) mild preeclampsia (n = 8) and (2) severe preeclampsia (n = 15) and (3) in a normal control group (n = 11) with elective cesarean delivery. The microRNA expression profile was assessed by microRNA microarray and real-time reverse transcriptase-polymerase chain reaction analysis. Results Thirty-four microRNAs were expressed differentially in preeclamptic placentas, compared with normal placentas. Of these, 11 microRNAs were overexpressed, and 23 microRNAs were underexpressed in preeclamptic pregnancies. Notably, several microRNA clusters on human chromosome 19q13.42, 13q31.3, Xq26.2, Xq26.3, and 14q32.31 (a human imprinted region) were expressed differentially in preeclamptic placentas. These results were confirmed with the use of real-time polymerase chain reaction for selected microRNAs (miR-210, -152, -411, and so on). Conclusion The results show that 34 microRNAs are deregulated in preeclamptic pregnancies, which suggests the involvement of these microRNAs in the pathogenesis of preeclampsia.
The use of small interfering RNAs (siRNAs) to induce gene silencing has opened a new avenue in drug discovery. However, their therapeutic potential is hampered by inadequate tissue-specific delivery. ...Exosomes are promising tools for drug delivery across different biological barriers. Here we show how exosomes derived from cultured cells can be harnessed for delivery of siRNA in vitro and in vivo. This protocol first describes the generation of targeted exosomes through transfection of an expression vector, comprising an exosomal protein fused with a peptide ligand. Next, we explain how to purify and characterize exosomes from transfected cell supernatant. Next, we detail crucial steps for loading siRNA into exosomes. Finally, we outline how to use exosomes to efficiently deliver siRNA in vitro and in vivo in mouse brain. Examples of anticipated results in which exosome-mediated siRNA delivery is evaluated by functional assays and imaging are also provided. The entire protocol takes ~3 weeks.
Disturbances in endoplasmic reticulum (ER) function lead to ER stress which, when severe or prolonged, may result in apoptosis. Severe ER stress has been implicated in several pathological conditions ...including pre-eclampsia, a multisystem disorder of pregnancy associated with the release of pro-inflammatory factors from the placenta into the maternal circulation. Here, we show that severe ER stress induced by two distinct mechanisms in BeWo choriocarcinoma cells leads to the release of extracellular vesicles (EVs) carrying pro-inflammatory damage-associated molecular pattern (DAMP) molecules. Co-treatment with the antioxidant pyrrolidine dithiocarbamate results in a reduction in ER stress-induced EV-associated DAMP release. We further demonstrate that severe ER stress is associated with changes in the expression of several stress-related proteins, notably Cited-2 and phosphorylated JNK. Together, these data indicate that severe ER stress-mediated release of EV-associated DAMPs may contribute to the heightened systemic maternal inflammatory response characteristic of pre-eclampsia and may also be relevant to other chronic inflammatory diseases which display elevated ER stress.
Preeclampsia, a multisystem hypertensive disorder of pregnancy, is associated with increased systemic vascular resistance. Placentae from patients with preeclampsia have reduced levels of endothelial ...nitric oxide synthase (eNOS) and, thus, less nitric oxide (NO). Syncytiotrophoblast extracellular vesicles (STBEV), comprising microvesicles (STBMV) and exosomes, carry signals from the syncytiotrophoblast to the mother. We hypothesized that STBEV-bound eNOS (STBEV-eNOS), capable of producing NO, are released into the maternal circulation. Dual-lobe ex vivo placental perfusion and differential centrifugation was used to isolate STBEV from preeclampsia (n=8) and normal pregnancies (NP; n=11). Plasma samples of gestational age-matched preeclampsia and NP (n=6) were used to isolate circulating STBMV. STBEV expressed placental alkaline phosphatase, confirming placental origin. STBEV coexpressed eNOS, but not inducible nitric oxide synthase, confirmed using Western blot, flow cytometry, and immunodepletion. STBEV-eNOS produced NO, which was significantly inhibited by
-nitro-l-arginine methyl ester (eNOS inhibitor;
<0.05) but not by
-(3-(aminomethyl) bezyl) acetamidine) (inducible nitric oxide synthase inhibitor). STBEV-eNOS catalytic activity was confirmed by visualizing eNOS dimerization. STBEV-eNOS was more abundant in uterine vein compared with peripheral blood, indicating placental origin. STBEV isolated from preeclampsia-perfused placentae had lower levels of STBEV-eNOS (STBMV;
<0.05) and overall lower NO activity (STBMV, not significant; syncytiotrophoblast extracellular exosomes,
<0.05) compared with those from NP. Circulating plasma STBMV from preeclampsia women had lower STBEV-eNOS expression compared with that from NP women (
<0.01). This is the first observation of functional eNOS expressed on STBEV from NP and preeclampsia placentae, as well as in plasma. The lower STBEV-eNOS NO production seen in preeclampsia may contribute to the decreased NO bioavailability in this disease.
Immunology of pre-eclampsia Redman, Christopher W G; Sargent, Ian L
American journal of reproductive immunology (1989),
06/2010, Letnik:
63, Številka:
6
Journal Article
Recenzirano
Pre-eclampsia develops in stages, only the last being the clinical illness. This is generated by a non-specific, systemic (vascular), inflammatory response, secondary to placental oxidative stress ...and not by reactivity to fetal alloantigens. However, maternal adaptation to fetal (paternal alloantigens) is crucial in the earlier stages. A pre-conceptual phase involves maternal tolerization to paternal antigens by seminal plasma. After conception, regulatory T cells, interacting with indoleamine 2,3-dioxygenase, together with decidual NK cell recognition of fetal HLA-C on extravillous trophoblast may facilitate placental growth by immunoregulation. Complete failure of this mechanism would cause miscarriage, while partial failure would cause poor placentation and dysfunctional uteroplacental perfusion. The first pregnancy preponderance and partner specificity of pre-eclampsia can be explained by this model. For the first time, the pathogenesis of pre-eclampsia can be related to defined immune mechanisms that are appropriate to the fetomaternal frontier. Now, the challenge is to prove the detail.
Pre-eclampsia (PE) complicates around 3% of all pregnancies and is one of the most common causes of maternal mortality worldwide. The pathophysiology of PE remains unclear however its underlying ...cause originates from the placenta and manifests as raised blood pressure, proteinuria, vascular or systemic inflammation and hypercoagulation in the mother. Women who develop PE are also at significantly higher risk of subsequently developing cardiovascular (CV) disease. In PE, the failing endoplasmic reticulum, oxidative and inflammatory stressed syncytiotrophoblast layer of the placenta sheds increased numbers of syncytiotrophoblast extracellular vesicles (STBEV) into the maternal circulation. Platelet reactivity, size and concentration are also known to be altered in some women who develop PE, although the underlying reasons for this have not been determined. In this study we show that STBEV from disease free placenta isolated ex vivo by dual placental perfusion associate rapidly with platelets. We provide evidence that STBEV isolated from normal placentas cause platelet activation and that this is increased with STBEV from PE pregnancies. Furthermore, treatment of platelets with aspirin, currently prescribed for women at high risk of PE to reduce platelet aggregation, also inhibits STBEV-induced reversible aggregation of washed platelets. Increased platelet reactivity as a result of exposure to PE placenta derived STBEVs correlates with increased thrombotic risk associated with PE. These observations establish a possible direct link between the clotting disturbances of PE and dysfunction of the placenta, as well as the known increased risk of thromboembolism associated with this condition.