Alterations in vascular function by syncytiotrophoblast extracellular vesicles via lectin-like oxidized low-density lipoprotein receptor-1 in mouse uterine arteries.

Syncytiotrophoblast extracellular vesicles (STBEVs), released into the maternal circulation during pregnancy, have been shown to affect vascular function; however, the mechanism remains unknown. In rats, STBEVs were shown to reduce endothelium-mediated vasodilation via lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), a multi-ligand scavenger receptor that has been associated with vascular dysfunction. Recently, LOX-1 was shown to interact with the angiotensin II type 1 receptor (AT-1). We hypothesized that, in pregnant mice, STBEVs would impair vascular function via LOX-1 and would specifically affect angiotensin II responses. Uterine arteries from pregnant control (C57BL/6) and LOX-1 knockout (LOX-1KO) mice were isolated on gestational day (GD) 18.5. Endothelium-dependent (methylcholine (MCh); ± N(G)-Nitro-L-arginine methyl ester to assess nitric oxide (NO) contribution), and -independent (sodium nitroprusside) vasodilation, and vasoconstriction (angiotensin II; ± AT-1 [candesartan] or angiotensin II type 2 receptor (AT-2) [PD123.319] receptor antagonists; high potassium salt solution) responses were assessed using wire myography. AT-1 and AT-2 expression was analyzed using fluorescence microscopy. Human umbilical vein endothelial cells (HUVECs) were stimulated with STBEVs ± LOX-1 blocking antibody, and superoxide and peroxynitrite production were analyzed. Although MCh-induced vasodilation was decreased (P=0.0012), NO contribution to vasodilation was greater in LOX-1KO mice (P=0.0055). STBEVs delayed angiotensin II tachyphylaxis in arteries from control but not LOX-1KO mice (P<0.0001), while AT-1 and AT-2 expression was unchanged. STBEVs increased peroxynitrite production in HUVECs via LOX-1 (P=0.0091). In summary, LOX-1 deletion altered endothelium-mediated vasodilation, suggesting that LOX-1 contributes to vascular adaptations in pregnancy. STBEVs increased angiotensin II responsiveness and oxidative stress levels via LOX-1, suggesting that increased LOX-1 expression/activation or STBEVs could adversely affect vascular function and contribute to vascular complications of pregnancy.

Extracellular microRNAs are dynamic non-vesicular biomarkers of muscle turnover.

Extracellular microRNAs (miRNAs) are promising biomarkers of the inherited muscle wasting condition Duchenne muscular dystrophy, as they allow non-invasive monitoring of either disease progression or response to therapy. In this study, serum miRNA profiling reveals a distinct extracellular miRNA signature in dystrophin-deficient mdx mice, which shows profound dose-responsive restoration following dystrophin rescue. Extracellular dystrophy-associated miRNAs (dystromiRs) show dynamic patterns of expression that mirror the progression of muscle pathology in mdx mice. Expression of the myogenic miRNA, miR-206 and the myogenic transcription factor myogenin in the tibialis anterior muscle were found to positively correlate with serum dystromiR levels, suggesting that extracellular miRNAs are indicators of the regenerative status of the musculature. Similarly, extracellular dystromiRs were elevated following experimentally-induced skeletal muscle injury and regeneration in non-dystrophic mice. Only a minority of serum dystromiRs were found in extracellular vesicles, whereas the majority were protected from serum nucleases by association with protein/lipoprotein complexes. In conclusion, extracellular miRNAs are dynamic indices of pathophysiological processes in skeletal muscle.

Systemically administered anti-TNF therapy ameliorates functional outcomes after focal cerebral ischemia.

BACKGROUND: The innate immune system contributes to the outcome after stroke, where neuroinflammation and post-stroke systemic immune depression are central features. Tumor necrosis factor (TNF), which exists in both a transmembrane (tm) and soluble (sol) form, is known to sustain complex inflammatory responses associated with stroke. We tested the effect of systemically blocking only solTNF versus blocking both tmTNF and solTNF on infarct volume, functional outcome and inflammation in focal cerebral ischemia. METHODS: We used XPro1595 (a dominant-negative inhibitor of solTNF) and etanercept (which blocks both solTNF and tmTNF) to test the effect of systemic administration on infarct volume, functional recovery and inflammation after focal cerebral ischemia in mice. Functional recovery was evaluated after one, three and five days, and infarct volumes at six hours, 24 hours and five days after ischemia. Brain inflammation, liver acute phase response (APR), spleen and blood leukocyte profiles, along with plasma microvesicle analysis, were evaluated. RESULTS: We found that both XPro1595 and etanercept significantly improved functional outcomes, altered microglial responses, and modified APR, spleen T cell and microvesicle numbers, but without affecting infarct volumes. CONCLUSIONS: Our data suggest that XPro1595 and etanercept improve functional outcome after focal cerebral ischemia by altering the peripheral immune response, changing blood and spleen cell populations and decreasing granulocyte infiltration into the brain. Blocking solTNF, using XPro1595, was just as efficient as blocking both solTNF and tmTNF using etanercept. Our findings may have implications for future treatments with anti-TNF drugs in TNF-dependent diseases.

Multicolor flow cytometry and nanoparticle tracking analysis of extracellular vesicles in the plasma of normal pregnant and pre-eclamptic women.

Excessive release of syncytiotrophoblast extracellular vesicles (STBMs) from the placenta into the maternal circulation may contribute to the systemic inflammation that is characteristic of pre-eclampsia (PE). Other intravascular cells types (platelets, leukocytes, red blood cells [RBCs], and endothelium) may also be activated and release extracellular vesicles (EVs). We developed a multicolor flow cytometry antibody panel to enumerate and phenotype STBMs in relation to other EVs in plasma from nonpregnant (NonP) and normal pregnant (NormP) women, and women with late-onset PE. Nanoparticle tracking analysis (NTA) was used to determine EV size and concentration. In vitro-derived STBMs and EVs from platelets, leukocytes, RBCs, and endothelial cells were examined to select suitable antibodies to analyze the corresponding plasma EVs. Flow cytometry analysis of plasma from NonP, NormP, and PE showed that STBMs comprised the smallest group of circulating EVs, whereas most were derived from platelets. The next most abundant group comprised unidentified orphan EVs (which did not label with any of the antibodies in the panel), followed by EVs from RBCs and leukocytes. NTA showed that the total number of EVs in plasma was significantly elevated in NormP and late-onset PE women compared to NonP controls, and that EVs were smaller in size. In general, EVs were elevated in pregnancy plasma apart from platelet EVs, which were reduced. These studies did not show any differences in EVs between NormP and PE, probably because late-onset PE was studied.

Placental disease and the maternal syndrome of preeclampsia: missing links?

Preeclampsia remains a significant obstetric risk worldwide. The pathophysiology of preeclampsia is complex, with multiple stages involving maladaptations in both placental and maternal physiology. The placenta links the pre-clinical stage of impaired remodeling of the uterine vasculature, occurring in early pregnancy, to the later clinical stages characterised by the maternal syndrome of hypertension and proteinuria. This review focuses on some of the recent candidates for the missing links in this process.

New mechanism for Notch signaling to endothelium at a distance by Delta-like 4 incorporation into exosomes.

Notch signaling is an evolutionary conserved pathway that is mediated by cell-cell contact. It is involved in a variety of developmental processes and has an essential role in vascular development and angiogenesis. Delta-like 4 (Dll4) is a Notch ligand that is up-regulated during angiogenesis. It is expressed in endothelial cells and regulates the differentiation between tip cells and stalk cells of neovasculature. Here, we present evidence that Dll4 is incorporated into endothelial exosomes. It can also be incorporated into the exosomes of tumor cells that overexpress Dll4. These exosomes can transfer the Dll4 protein to other endothelial cells and incorporate it into their cell membrane, which results in an inhibition of Notch signaling and a loss of Notch receptor. Transfer of Dll4 was also shown in vivo from tumor cells to host endothelium. Addition of Dll4 exosomes confers a tip cell phenotype on the endothelial cell, which results in a high Dll4/Notch-receptor ratio, low Notch signaling, and filopodia formation. This was further evidenced by increased branching in a tube-formation assay and in vivo. This reversal in phenotype appears to enhance vessel formation and is a new form of signaling for Notch ligands that expands their signaling potential beyond cell-cell contact.

Lysosomal dysfunction increases exosome-mediated alpha-synuclein release and transmission.

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 functions of microparticles in pre-eclampsia.

Pre-eclampsia (P-EC), a heterogenic multisystem disorder characterized by hypertension and proteinuria, usually develops in the second half of pregnancy. The incidence is 2 to 5%, and P-EC is therefore a major cause of maternal and perinatal morbidity and mortality. Although the exact etiology is unknown, placental factors released into the maternal circulation lead to systemic maternal inflammation and endothelial dysfunction. Growing evidence indicates that placenta-derived microparticles, best known as syncytiotrophoblast microparticles (STBM), are important among these factors. This review provides an overview of the presence and function(s) of STBM and other cell-derived microparticles and exosomes.

Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis.

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.

Role of Lectin-like Oxidized LDL Receptor-1 and Syncytiotrophoblast Extracellular Vesicles in the Vascular Reactivity of Mouse Uterine Arteries During Pregnancy.

Vascular complications in pregnancy (e.g. preeclampsia) are a major source of maternal and foetal morbidity and mortality, and may be due to excessive release of placental syncytiotrophoblast-derived extracellular vesicles (STBEVs) into the maternal circulation. Increased activity of the multi-ligand scavenger receptor Lectin-like Oxidized LDL Receptor-1 (LOX-1) is associated with vascular dysfunction, and LOX-1 has been shown to interact with angiotensin II receptor type 1 (AT1). We hypothesized that STBEVs contribute to vascular dysfunction via LOX-1 and AT1 receptors during pregnancy. Uterine arteries from late pregnant wildtype and LOX-1 overexpressing mice were incubated overnight with or without STBEVs and vascular function was assessed using wire myography. STBEV-incubation decreased angiotensin II responsiveness only in wildtype mice, which coincided with decreased AT1 contribution and expression. Thus, STBEVs reduced angiotensin II responsiveness in normal pregnancy, but not in conditions of increased LOX-1 expression, suggesting that STBEVs (via LOX-1) play a role in normal adaptations to pregnancy. Oxidized LDL (a LOX-1 ligand) increased angiotensin II-induced vasoconstriction in STBEV-incubated arteries from both mouse strains, suggesting that the LOX-1 pathway may be involved in complicated pregnancies with elevated STBEVs and oxidized LDL levels (such as preeclampsia). These data increase our understanding of vascular complications during pregnancy.

Soluble HLA-G in IVF/ICSI embryo culture supernatants does not always predict implantation success: a multicentre study.

Several reports have described an association between the presence of soluble human leukocyte antigen G (sHLA-G) in human embryo culture supernatants (ES) and implantation success. However, not all studies agree with these findings. To further document this debate, a multicentre blinded study was performed to investigate, on a large number of IVF ES and ICSI ES, whether sHLA-G is a useful criterion for embryo selection before transfer. A total of 1405 ES from 355 patients were collected from three assisted reproductive technique (ART) centres and evaluated for their sHLA-G content in a single laboratory, using a chemiluminescence enzyme-linked immunosorbent assay. In only one centre was a significant association between sHLA-G-positive ES and successful implantation established (P = 0.0379), whereas no such association was observed in the other centres. It was found that the percentages and concentrations of sHLA-G-positive ES varied between centres, depending on culture media and ART conditions. The percentage of sHLA-G-positive ES was significantly higher in IVF ES than ICSI ES (P < 0.001 and P < 0.01 for two centres). These data demonstrate that substantial variations of sHLA-G content in ES occur between different ART centres, highlighting the influence of several technical parameters that differ from one centre to another.

Differential expression profile of microRNAs in human placentas from preeclamptic pregnancies vs normal pregnancies.

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.

Placental extracellular vesicles express active dipeptidyl peptidase IV; levels are increased in gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) is the most common metabolic disorder in pregnancy and is characterized by insulin resistance and decreased circulating glucagon-like peptide-1 (GLP-1). GDM resolves rapidly after delivery implicating the placenta in the disease. This study examines the biological functions that cause this pathology. The placenta releases syncytiotrophoblast-derived extracellular vesicles (STB-EVs) into the maternal circulation, which is enhanced in GDM. Dipeptidyl peptidase IV (DPPIV) is known to play a role in type 2 diabetes by breaking down GLP-1, which in turn regulates glucose-dependent insulin secretion. STB-EVs from control and GDM women were analysed. We show that normal human placenta releases DPPIV-positive STB-EVs and that they are higher in uterine than paired peripheral blood, confirming placental origin. DPPIV-bound STB-EVs from normal perfused placentae are dose dependently inhibited with vildagliptin. DPPIV-bound STB-EVs from perfused placentae are able to breakdown GLP-1 in vitro. STB-EVs from GDM perfused placentae show greater DPPIV activity. Importantly, DPPIV-bound STB-EVs increase eightfold in the circulation of women with GDM. This is the first report of STB-EVs carrying a biologically active molecule that has the potential to regulate maternal insulin secretion.

In vitro placenta barrier model using primary human trophoblasts, underlying connective tissue and vascular endothelium.

Fetal development may be compromised by adverse events at the placental interface between mother and fetus. However, it is still unclear how the communication between mother and fetus occurs through the placenta. In vitro - models of the human placental barrier, which could help our understanding and which recreate three-dimensional (3D) structures with biological functionalities and vasculatures, have not been reported yet. Here we present a 3D-vascularized human primary placental barrier model which can be constructed in 1 day. We illustrate the similarity of our model to first trimester human placenta, both in its structure and in its ability to respond to altered oxygen and to secrete factors that cause damage cells across the barrier including embryonic cortical neurons. We use this model to highlight the possibility that both the trophoblast and the endothelium within the placenta might play a role in the fetomaternal dialogue.

Differential effect of cross-linking the CD98 heavy chain on fusion and amino acid transport in the human placental trophoblast (BeWo) cell line.

CD98 (otherwise known as 4F2) is an integral membrane protein with multiple functions including amino acid transport, integrin activation, cell fusion and cell activation. The molecular mechanisms coordinating these multiple functions remain unclear. We have studied CD98 heavy chain (hc) function in a human placental trophoblast cell line (BeWo). We show that cross-linking of CD98hc by incubation of cells in the presence of functional monoclonal antibodies causes cellular re-distribution of the protein from the cytoplasm to the plasma membrane as measured by flow cytometry, western blotting and quantitative immuno-electron microscopy. The latter technique also indicated that CD98hc is trafficked between cell surface and cytoplasmic pools in vesicles. Increased cell surface CD98 correlates with increased cellular fusion in BeWo cells. In addition, we show reduced LAT 1 surface expression and neutral amino acid transport in the presence of the CD98 mabs. The results thus suggest that the function of CD98 in cell fusion is distinct from its role in cellular nutrient delivery.

Endoplasmic reticulum stress stimulates the release of extracellular vesicles carrying danger-associated molecular pattern (DAMP) molecules.

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.

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.

INTRODUCTION: Placental syncytiotrophoblast (STB) release extracellular vesicles (STB-EVs) that communicate physiological and pathological placental signals to the maternal organs. STB-EV release also increases in preeclampsia (PE). Here we explored the cargo of PP13 in STB-EVs from PE versus control placentas. METHODS: Placentae were harvested following cesarean section deliveries, and dual placental lobe perfusion was used to harvest STB-EV. Maternal side perfusate was centrifuged at 10,000 × g to yield the STB microvesicles, and then at 150,000 × g to yield STB exosomes. Total STB-EVs (tSTB-EVs) were collected using a one step 150,000 × g centrifugation. Placental origin and size distribution were assessed by Western blotting and Nanoparticle Tracking Analysis, respectively. PP13 expression was determined by Western blot and ELISA. RESULTS: Placental alkaline phosphatase (PLAP; a STB specific marker) was present in all preparations. Total tSTB-EVs and STB-EXs also expressed the exosome markers such as the Apoptosis-Linked Gene 2-Interacting Protein X (Alix) and the cluster differentiation protein 9 (CD9). PP13 was localized to the outer surface and intra-vesicular compartments of all fractions. Surface to total PP13 ratios were ∼1:1 for all STB-EV preparations. In contrast to the previously reported higher circulating concentrations of soluble PP13 in PE, significantly lower levels of PP13, normalized to total vesicular protein, were observed in PE samples. PP13 reduction in all STB-EVs' sub-populations may be attributed to differences in gestational age (GA). A simple correction for GA suggested that PE may be an important influence. CONCLUSIONS: PP13 is located in and on all types of STB-EVs. Circulating PP13 may therefore be either soluble or associated with extracellular vesicles with different pathophysiological effects in the maternal circulation.

Pre-eclampsia affects procalcitonin production in placental tissue.

PROBLEM: Procalcitonin (PCT) is the prohormone of calcitonin which is usually released from neuroendocrine cells of the thyroid gland (parafollicular) and the lungs (K cells). PCT is synthesized by almost all cell types and tissues, including monocytes and parenchymal tissue, upon LPS stimulation. To date, there is no evidence for PCT expression in the placenta both in physiological and pathological conditions. METHOD: Circulating and placental PCT levels were analysed in pre-eclamptic (PE) and control patients. Placental cells and macrophages (PBDM), stimulated with PE sera, were analysed for PCT expression. The effect of anti-TNF-α antibody was analysed. RESULTS: Higher PCT levels were detected in PE sera and in PE placentae compared to healthy women. PE trophoblasts showed increased PCT expression compared to those isolated from healthy placentae. PE sera induced an upregulation of PCT production in macrophages and placental cells. The treatment of PBDM with PE sera in the presence of anti-TNF-α completely abrogated the effect induced by pathologic sera. CONCLUSION: Trophoblast cells are the main producer of PCT in PE placentae. TNF-α, in association with other circulating factors present in PE sera, upregulates PCT production in macrophages and normal placental cells, thus contributing to the observed increased in circulating PCT in PE sera.

Membrane trafficking of CD98 and its ligand galectin 3 in BeWo cells--implication for placental cell fusion.

CD98 heavy chain (CD98hc), expressed at high levels in developing human trophoblasts, is an integral membrane protein with multiple N-linked glycosylation sites and known to be important for cell fusion, adhesion, and amino acid transport. Western blotting and flow cytometry were used to study the effect of brefeldin A, an inhibitor of protein translocation through the Golgi, on CD98hc in the human placental trophoblast cell line BeWo. Although brefeldin A treatment caused increased cell surface expression of CD98hc, a novel partially glycosylated form of the protein was found and, concomitantly, cell fusion was reduced. Western blotting showed that CD98 and galectin 3, a proposed ligand for the glycosylated extracellular domain of CD98hc, co-immunoprecipitated, and double-label immuno-electron microscopy confirmed that CD98hc associated with galectin 3. Furthermore, cell fusion was reduced (specifically) by the disaccharide lactose, a known ligand for the carbohydrate recognition domain of galectin 3, suggesting that the association was functional. Taken together, the data suggest that N-glycosylation of CD98 and subsequent interaction with galectin 3 is critical for aspects of placental cell biology, and provides a rationale for the observation that, in the mouse, truncation of the CD98hc extracellular domain leads to early embryonic lethality [Tsumura H, Suzuki N, Saito H, Kawano M, Otake S, Kozuka Y, Komada H, Tsurudome M & Ito Y (2003) Biochem Biophys Res Commun 308, 847-851].

sHLA-G production by human IVF embryos: can it be measured reliably?

A number of reports have demonstrated that sHLA-G can be detected in the culture medium of human IVF embryos and that levels correlate with the potential of an embryo to implant. This has aroused considerable interest in the IVF field. If sHLA-G can be used as a non-invasive marker of embryo quality, it will facilitate selection of the best embryos to transfer to the mother and thereby increase IVF pregnancy rates. However, there have been concerns about some aspects of these studies, including the sensitivity of the sHLA-G ELISAs used, the IVF culture conditions and the levels of sHLA-G which have been reported. A recent study by Sageshima et al. [J. Reprod. Immunol. 75, 11-22, 2007] attempts to address some of these concerns. However, despite using a sensitive ELISA, they were unable to detect sHLA-G in 111 embryo culture supernatants, or sHLA-G secretion by less than 10,000 sHLA-G transfected cells. They concluded that it is not possible to measure sHLA-G production by human embryos. This study has highlighted technical differences between IVF culture techniques and sHLA-G ELISAs that are currently confounding the system. Further collaboration between the research groups involved is required to establish robust reproducible systems that function identically in all laboratories.