Impact of Magnetic Resonance Lymphography on Lymphaticolvenular Anastomosis for Lower-Limb Lymphedema.

BACKGROUND: Although several investigations have described the safety, utility, and precision of magnetic resonance lymphography (MRL) as a preoperative examination for lymphaticovenular anastomosis (LVA), it is unclear how much MRL assistance impacts LVA results. The present study aimed to clarify the outcome of MRL-assisted LVA for leg lymphedema using body water measurements obtained by bioelectrical impedance analysis. METHODS: The water reductive effect of MRL-assisted LVA in female secondary leg lymphedema patients was compared with that of non-MRL-assisted controls in this retrospective study. In the MRL-assisted group, all LVA candidates underwent MRL prior to surgery, and the lymphatic vessels to be anastomosed were primarily determined by MRL findings. The body water composition of the treated legs was assessed before LVA and at 6 months postoperatively using a multi-frequency bioelectrical impedance analyzer. RESULTS: Twenty-three patients in the MRL-assisted study group and an equal number in the non-MRL-assisted control group were analyzed. Although mean leg water volume before LVA, mean excess water volume of the affected leg before LVA, and number of anastomoses created were comparable between the groups, the water volume reduction (1.02 L versus 0.49 L; 95% confidence interval [CI]: 0.03-1.03, p < 0.05) and edema reduction rate (46.7% versus 27.2%; 95% CI: 3.7-35.5%, p < 0.05) in the MRL-assisted group were significantly greater than in controls. CONCLUSION: Preoperative MRL-assisted lymph vessel visualization and selection appeared to significantly enhance the water reductive effect of LVA for International Society of Lymphology classification stage 2 leg lymphedema. MRL also helped to reliably identify lymphatic vessels for anastomosis. Without increasing the number of anastomoses, LVA could be performed more effectively by better detecting stagnant lymphatic vessels using MRL.

Extracellular Water Ratio as an Indicator of the Development and Severity of Leg Lymphedema Using Bioelectrical Impedance Analysis.

Background: This case-control retrospective study focused on the extracellular water ratio (%ECW) of lymphedemic limbs measured by bioelectrical impedance analysis (BIA) as a possible indicator of the development and severity of unilateral and bilateral leg lymphedema. Methods and Results: BIA was used to evaluate changes in %ECW due to lymphedema in female patients with unilateral secondary leg lymphedema and in healthy controls. Receiver operating characteristic (ROC) analysis was employed to assess the diagnostic ability of %ECW to distinguish leg lymphedema patients from controls. Thirty-eight female patients were eligible for inclusion along with an equal number of healthy control volunteers. The %ECW of the affected leg correlated with leg body water volume (R2 = 0.28) and the water volume difference between affected and unaffected legs (R2 = 0.58). The ROC analysis showed that %ECW had a high diagnostic ability as a screening tool for the development of leg lymphedema (area under the ROC curve = 0.96). A cutoff %ECW value of 40.0% could predict the presence of leg lymphedema with a sensitivity of 81.6% and specificity of 97.4%. Conclusions: %ECW value may be a simple and useful indicator of the development and severity of leg lymphedema. As a screening test, %ECW measurement can predict the presence of unilateral or bilateral leg lymphedema in a single measurement without the need for arm, contralateral leg, or previous measurements as controls.

Water Reductive Effect of Lymphaticovenular Anastomosis on Upper-Limb Lymphedema: Bioelectrical Impedance Analysis and Comparison with Lower-Limb Lymphedema.

BACKGROUND: We previously examined the water reductive effect of lymphaticovenular anastomosis (LVA) using bioelectrical impedance analysis (BIA) measurement on lower-limb lymphedema and revealed mean water volume reduction and edema reduction rate by leg LVA to be 0.86 L and 45.1%, respectively. This study aimed to clarify the water reductive effect of LVA on arm lymphedema and compare its results with those for leg lymphedema. PATIENTS AND METHODS: The efficacy of LVA for unilateral arm lymphedema was evaluated using BIA in a retrospective cohort. Limb circumference and arm body water volume (ABW) of the affected and unaffected arms were measured before and after LVA. Mean water volume reduction (ΔABW) and edema reduction rate by arm LVA were compared with values for leg LVA cited from our previous report as a historical control. RESULTS: Nineteen consecutive patients were enrolled. The mean ΔABW and edema reduction rate by BIA were 0.267 L and 46.0%, respectively. The decreasing rate of ABW by BIA was significantly larger than those of the upper extremity lymphedema index and sum of 5 circumferences measurement methods. ΔABW could be predicted by a regression line based on the preoperative water volume difference between affected and unaffected limbs. The mean edema reduction rates for arm and leg LVA were comparable. CONCLUSION: The water reductive effect of LVA on upper-limb lymphedema was demonstrated by BIA assessment. BIA can reflect the effect of LVA more sensitively than conventional objective measurements and may facilitate the interpretation of LVA results. Although water volume reduction by arm LVA was less than that by leg LVA, the edema reduction rates were comparable.

Proteomic analysis of exosomes reveals an association between cell invasiveness and exosomal bioactivity on endothelial and mesenchymal cell migration in vitro.

Ovarian cancer has resulted in over 140 000 deaths reported annually worldwide. This is often attributed to cellular changes in the microenvironment, including increased migration of mesenchymal stem cells (MSCs) and endothelial cells (ECs) to facilitate metastasis. Recently, the ability of exosomes to communicate signals between cells (and promote cancer progression) has been established. In the present study, we explored the effect of exosomes on cells present in the tumour microenvironment. Exosomes were isolated from ovarian cancer cells with different invasive capacity (high = SKOV-3 and low = OVCAR-3) by differential and buoyant density centrifugation and characterised using nanoparticle tracking analysis (NTA), Western blot, and EM. Exosome secretion was positively correlated with invasiveness of releasing cells. Proteomic analyses identified common and unique proteins between exosomes from SKOV-3 and OVCAR-3 with gene ontology analyses revealing that these exosomes are involved in the regulation of cell migration. Since the tumour microenvironment contains multiple cell types, including MSCs and ECs, we examined the effect of these exosomes on MSC and EC migration. Exosomes promoted MSC and EC migration in a time- and concentration-dependent manner. The effect of exosomes isolated from SKOV-3 on cell migration was significantly higher compared with exosomes from OVCAR-3. Thus, we suggest that exosomes from ovarian cancer cells contain a specific set of proteins that are representative of its cell of origin and the invasive capacity.

Gestational Diabetes Mellitus Is Associated With Changes in the Concentration and Bioactivity of Placenta-Derived Exosomes in Maternal Circulation Across Gestation.

Although there is significant interest in elucidating the role of placenta-derived exosomes (PdEs) during pregnancy, the exosomal profile in pregnancies complicated by gestational diabetes mellitus (GDM) remains to be established. The aim of this study was to compare the gestational-age profile of PdEs in maternal plasma of GDM with normal pregnancies and to determine the effect of exosomes on cytokine release from human umbilical vein endothelial cells. A prospective cohort of patients was sampled at three time points during pregnancy for each patient (i.e., 11-14, 22-24, and 32-36 weeks' gestation). A retrospective stratified study design was used to quantify exosomes present in maternal plasma of normal (n = 13) and GDM (n = 7) pregnancies. Gestational age and pregnancy status were identified as significant factors contributing to variation in plasma exosome concentration (ANOVA, P < 0.05). Post hoc analyses established that PdE concentration increased during gestation in both normal and GDM pregnancies; however, the increase was significantly greater in GDM (∼2.2-fold, ∼1.5-fold, and ∼1.8-fold greater at each gestational age compared with normal pregnancies). Exosomes isolated from GDM pregnancies significantly increased the release of proinflammatory cytokines from endothelial cells. Although the role of exosomes during GDM remains to be fully elucidated, exosome profiles may be of diagnostic utility for screening asymptomatic populations.

Placental exosomes in normal and complicated pregnancy.

While there is considerable contemporary interest in elucidating the role of placenta-derived extracellular vesicles in normal and complicated pregnancies and their utility as biomarkers and therapeutic interventions, progress in the field is hindered by a lack of standardized extracellular vesicle taxonomy and isolation protocols. The term "extracellular vesicle" is nonspecific and refers to all membrane-bound vesicles from nanometer to micrometer diameters and of different biogenic origins. To meaningfully ascribe biological function and/or diagnostic and therapeutic utility to extracellular vesicles, and in particular exosomes, greater specificity and vesicle characterization is required. The current literature relating to exosome biology must be interpreted in this context. Exosomes are a subtype of extracellular vesicle that are specifically defined by an endosomal biogenesis and particle size (40-120 nm) and density (1.13-1.19 g/mL(-1)). Exosomes are specifically package with signaling molecules (including protein, messenger RNA, microRNA, and noncoding RNA) and are released by exocytosis into biofluid compartments. Exosomes regulate the activity of both proximal and distal target cells, including translational activity, angiogenesis, proliferation, metabolism, and apoptosis. As such, exosomal signaling represents an integral pathway mediating intercellular communication. During pregnancy, the placenta releases exosomes into the maternal circulation from as early as 6 weeks of gestation. Release is regulated by factors that include both oxygen tension and glucose concentration and correlates with placental mass and perfusion. The concentration of placenta-derived exosomes in maternal plasma increases progressively during gestation. Exosomes isolated from maternal plasma are bioactive in vitro and are incorporated into target cells by endocytosis. While the functional significance of placental exosomes in pregnancy remains to be fully elucidated, available data support a role in normal placental development and maternal immunotolerance. Similarly, the role of exosomes in the etiology and progression of complications of pregnancy remains in a formative stage. Changes in the release of placenta- and nonplacenta-derived exosomes, their concentration in maternal plasma, composition, and bioactivity have been reported in association with pregnancies complicated by gestational diabetes and preeclampsia. The data, however, are confounded by the use of different isolation methodologies and vesicle subpopulations. The application of specific and well-characterized isolation methodologies is requisite to resolving the precise role of exosomes in complications of pregnancies and their ultimate clinical utility.

The Effect of Glucose on the Release and Bioactivity of Exosomes From First Trimester Trophoblast Cells.

CONTEXT: Hyperglycemia and hypoxia are risk factors of metabolic complication during pregnancy. The interactions between oxygen and glucose-sensing pathways that regulate exosome bioactivity from placental cells, however, have not been established. OBJECTIVE: The aim of this study was to test the hypothesis that exosomal signaling by placental cells (defined as the number of exosomes released per unit time and their bioactivity) is responsive to extracellular glucose concentration. METHODS: First-trimester primary trophoblast cells were incubated with D-glucose (5 mM or 25 mM) under 1%, 3%, or 8% O2 for 48 hours. Exosomes were isolated from cell-conditioned media by differential and buoyant density centrifugation. The total number of exosome vesicles was determined by quantifying immunoreactive exosomal CD63. The effect of exosomes on cytokine (granulocyte macrophage colony-stimulating factor, IL-2, IL-4, IL-6. IL-8, IL-10, interferon-γ, and TNF-α) release from endothelial cells was established by a protein solution array analysis. RESULTS: Glucose (25 mM) significantly increased the release of exosomes from trophoblast cells at all oxygen tensions tested (by approximately 2-fold when compared with controls, P < .001). Exosomes (100 µg/mL exosomal protein) released from trophoblast cells significantly increased (P < .05) the release of all cytokines from human umbilical vein endothelial cells when compared with the control (ie, cells without exosomes), with the exception of IL-2 and IL-10 (P > .05). CONCLUSIONS: The effects of high glucose on exosomes bioactivity may be recapitulated in vivo and is of clinical relevance in association with maternal insulin resistance (resulting in hyperglycemia) and preeclampsia (associated with placental insufficiency and hypoxia).

Extravillous trophoblast cells-derived exosomes promote vascular smooth muscle cell migration.

BACKGROUND: Vascular smooth muscle cells (VSMCs) migration is a critical process during human uterine spiral artery (SpA) remodeling and a successful pregnancy. Extravillous trophoblast cells (EVT) interact with VSMC and enhance their migration, however, the mechanisms by which EVT remodel SpA remain to be fully elucidated. We hypothesize that exosomes released from EVT promote VSMC migration. METHODS: JEG-3 and HTR-8/SVneo cell lines were used as models for EVT. Cells were cultured at 37°C and humidified under an atmosphere of 5% CO2-balanced N2 to obtain 8% O2. Cell-conditioned media were collected, and exosomes (exo-JEG-3 and exo- HTR-8/SVneo) isolated by differential and buoyant density centrifugation. The effects of exo-EVT on VSMC migration were established using a real-time, live-cell imaging system (Incucyte™). Exosomal proteins where identified by mass spectrometry and submitted to bioinformatic pathway analysis (Ingenuity software). RESULTS: HTR-8/SVneo cells were significantly more (~30%) invasive than JEG-3 cells. HTR-8/SVneo cells released 2.6-fold more exosomes (6.39 × 10(8) ± 2.5 × 10(8) particles/10(6) cells) compared to JEG-3 (2.86 × 10(8) ± 0.78 × 10(8) particles/10(6) cells). VSMC migration was significantly increased in the presence of exo-JEG-3 and exo-HTR-8/SVneo compared to control (-exosomes) (21.83 ± 0.49 h and 15.57 ± 0.32, respectively, vs. control 25.09 ± 0.58 h, p < 0.05). Sonication completely abolished the effect of exosomes on VSMC migration. Finally, mass spectrometry analysis identified unique exosomal proteins for each EVT cell line-derived exosomes. CONCLUSION: The data obtained in this study are consistent with the hypothesis that the release, content, and bioactivity of exosomes derived from EVT-like cell lines is cell origin-dependent and differentially regulates VSMC migration. Thus, an EVT exosomal signaling pathway may contribute to SpA remodeling by promoting the migration of VSMC out of the vessel walls.

A gestational profile of placental exosomes in maternal plasma and their effects on endothelial cell migration.

Studies completed to date provide persuasive evidence that placental cell-derived exosomes play a significant role in intercellular communication pathways that potentially contribute to placentation and development of materno-fetal vascular circulation. The aim of this study was to establish the gestational-age release profile and bioactivity of placental cell-derived exosome in maternal plasma. Plasma samples (n = 20 per pregnant group) were obtained from non-pregnant and pregnant women in the first (FT, 6-12 weeks), second (ST, 22-24 weeks) and third (TT, 32-38 weeks) trimester. The number of exosomes and placental exosome contribution were determined by quantifying immunoreactive exosomal CD63 and placenta-specific marker (PLAP), respectively. The effect of exosomes isolated from FT, ST and TT on endothelial cell migration were established using a real-time, live-cell imaging system (Incucyte). Exosome plasma concentration was more than 50-fold greater in pregnant women than in non-pregnant women (p<0.001). During normal healthy pregnancy, the number of exosomes present in maternal plasma increased significantly with gestational age by more that two-fold (p<0.001). Exosomes isolated from FT, ST and TT increased endothelial cell migration by 1.9±0.1, 1.6±0.2 and 1.3±0.1-fold, respectively compared to the control. Pregnancy is associated with a dramatic increase in the number of exosomes present in plasma and maternal plasma exosomes are bioactive. While the role of placental cell-derived exosome in regulating maternal and/or fetal vascular responses remains to be elucidated, changes in exosome profile may be of clinical utility in the diagnosis of placental dysfunction.

Ovarian cancer cell invasiveness is associated with discordant exosomal sequestration of Let-7 miRNA and miR-200.

BACKGROUND: The role of exosomes in the pathogenesis and metastatic spread of cancer remains to be fully elucidated. Recent studies support the hypothesis that the release of exosomes from cells modifies local extracellular conditions to promote cell growth and neovascularisation. In addition, exosomes may modify the phenotype of parent and/or target cell. For example, sequestration of signaling mediators into exosomes may reduce their intracellular bioavailability to the parent cell thereby altering cell phenotype and metastatic potential. The fusion of released exosomes with target cell and delivery may also modify cell function and activity. In this study, to further elucidate the role of exosomes in ovarian cancer, the release of exosomes from two ovarian cancer cell lines of different invasive capacity and their miRNA content of exosomes were compared. The hypothesis to be tested was that ovarian cancer cell invasiveness is associated with altered release of exosomes and discordant exosomal sequestration of miRNA. METHODS: High (SKOV-3) and low (OVCAR-3) invasive ovarian cancer cell lines were used to characterize their exosome release. SKOV-3 and OVCAR-3 cells were cultured (DMEM, 20% exosome-free FBS) under an atmosphere of 8% O2 for 24 hours. Cell-conditioned media were collected and exosomes were isolated by differential and buoyant density centrifugation and characterised by Western blot (CD63 and CD9). Exosomal microRNA (let-7a-f and miR-200a-c) content was established by real-time PCR. RESULTS: Exosomes were identified with by the presence of typical cup-shaped spherical vesicle and the expression of exosome markers: CD63, CD9. SKOV-3 cells released 2.7-fold more exosomes (1.22 ± 0.11 µg/106 cells) compared to OVCAR-3 (0.44 ± 0.05 µg/106 cells). The let-7 family miRNA transcripts were identified in both ovarian cancer cell lines and their exosomes. The let-7 family transcripts were more abundant in OVCAR-3 cell than SKOV-3 cells. In contrast, let-7 family transcripts were more abundant in exosomes from SKOV-3 than OVCAR-3. miR-200 family transcripts were only identified in OVCAR-3 cells and their exosomes. CONCLUSIONS: The data obtained in this study are consistent with the hypothesis that the releases of exosomes varies significantly between ovarian cancer cell lines and correlates with their invasive potential.

Hypoxia-induced changes in the bioactivity of cytotrophoblast-derived exosomes.

Migration of extravillous trophoblasts (EVT) into decidua and myometrium is a critical process in the conversion of maternal spiral arterioles and establishing placenta perfusion. EVT migration is affected by cell-to-cell communication and oxygen tension. While the release of exosomes from placental cells has been identified as a significant pathway in materno-fetal communication, the role of placental-derived exosomes in placentation has yet to be established. The aim of this study was to establish the effect of oxygen tension on the release and bioactivity of cytotrophoblast (CT)-derived exosomes on EVT invasion and proliferation. CT were isolated from first trimester fetal tissue (n = 12) using a trypsin-deoxyribonuclease-dispase/Percoll method. CT were cultured under 8%, 3% or 1% O2 for 48 h. Exosomes from CT-conditioned media were isolated by differential and buoyant density centrifugation. The effect of oxygen tension on exosome release (µg exosomal protein/10(6)cells/48 h) and bioactivity were established. HTR-8/SVneo (EVT) were used as target cells to establish the effect (bioactivity) of exosomes on invasion and proliferation as assessed by real-time, live-cell imaging (Incucyte™). The release and bioactivity of CT-derived exosomes were inversely correlated with oxygen tension (p<0.001). Under low oxygen tensions (i.e. 1% O2), CT-derived exosomes promoted EVT invasion and proliferation. Proteomic analysis of exosomes identified oxygen-dependent changes in protein content. We propose that in response to changes in oxygen tension, CTs modify the bioactivity of exosomes, thereby, regulating EVT phenotype. Exosomal induction of EVT migration may represent a normal process of placentation and/or an adaptive response to placental hypoxia.

Exosomal signaling during hypoxia mediates microvascular endothelial cell migration and vasculogenesis.

Vasculogenesis and angiogenesis are critical processes in fetal circulation and placental vasculature development. Placental mesenchymal stem cells (pMSC) are known to release paracrine factors (some of which are contained within exosomes) that promote angiogenesis and cell migration. The aims of this study were: to determine the effects of oxygen tension on the release of exosomes from pMSC; and to establish the effects of pMSC-derived exosomes on the migration and angiogenic tube formation of placental microvascular endothelial cells (hPMEC). pMSC were isolated from placental villi (8-12 weeks of gestation, n = 6) and cultured under an atmosphere of 1%, 3% or 8% O2. Cell-conditioned media were collected and exosomes (exo-pMSC) isolated by differential and buoyant density centrifugation. The dose effect (5-20 µg exosomal protein/ml) of pMSC-derived exosomes on hPMEC migration and tube formation were established using a real-time, live-cell imaging system (Incucyte™). The exosome pellet was resuspended in PBS and protein content was established by mass spectrometry (MS). Protein function and canonical pathways were identified using the PANTHER program and Ingenuity Pathway Analysis, respectively. Exo-pMSC were identified, by electron microscopy, as spherical vesicles, with a typical cup-shape and diameters around of 100 nm and positive for exosome markers: CD63, CD9 and CD81. Under hypoxic conditions (1% and 3% O2) exo-pMSC released increased by 3.3 and 6.7 folds, respectively, when compared to the controls (8% O2; p<0.01). Exo-pMSC increased hPMEC migration by 1.6 fold compared to the control (p<0.05) and increased hPMEC tube formation by 7.2 fold (p<0.05). MS analysis identified 390 different proteins involved in cytoskeleton organization, development, immunomodulatory, and cell-to-cell communication. The data obtained support the hypothesis that pMSC-derived exosomes may contribute to placental vascular adaptation to low oxygen tension under both physiological and pathological conditions.