Validation of Artificial Intelligence (AI)-Assisted Flow Cytometry Analysis for Immunological Disorders.

Flow cytometry is a vital diagnostic tool for hematologic and immunologic disorders, but manual analysis is prone to variation and time-consuming. Over the last decade, artificial intelligence (AI) has advanced significantly. In this study, we developed and validated an AI-assisted flow cytometry workflow using 379 clinical cases from 2021, employing a 3-tube, 10-color flow panel with 21 antibodies for primary immunodeficiency diseases and related immunological disorders. The AI software (DeepFlow™, version 2.1.1) is fully automated, reducing analysis time to under 5 min per case. It interacts with hematopatholoists for manual gating adjustments when necessary. Using proprietary multidimensional density-phenotype coupling algorithm, the AI model accurately classifies and enumerates T, B, and NK cells, along with important immune cell subsets, including CD4+ helper T cells, CD8+ cytotoxic T cells, CD3+/CD4-/CD8- double-negative T cells, and class-switched or non-switched B cells. Compared to manual analysis with hematopathologist-determined lymphocyte subset percentages as the gold standard, the AI model exhibited a strong correlation (r > 0.9) across lymphocyte subsets. This study highlights the accuracy and efficiency of AI-assisted flow cytometry in diagnosing immunological disorders in a clinical setting, providing a transformative approach within a concise timeframe.

Analysis of Cation Composition in Dolomites on the Intact Particles Sampled from Asteroid Ryugu.

Characterization of the elemental distribution of samples with rough surfaces has been strongly desired for the analysis of various natural and artificial materials. Particularly for pristine and rare analytes with micrometer sizes embedded on specimen surfaces, non-invasive and matrix effect-free analysis is required without surface polishing treatment. To satisfy these requirements, we proposed a new method employing the sequential combination of two imaging modalities, i.e., microenergy-dispersive X-ray fluorescence (micro-XRF) and Raman micro-spectroscopy. The applicability of the developed method is tested by the quantitative analysis of cation composition in micrometer-sized carbonate grains on the surfaces of intact particles sampled directly from the asteroid Ryugu. The first step of micro-XRF imaging enabled a quick search for the sparsely scattered and micrometer-sized carbonates by the codistributions of Ca2+ and Mn2+ on the Mg2+- and Fe2+-rich phyllosilicate matrix. The following step of Raman micro-spectroscopy probed the carbonate grains and analyzed their cation composition (Ca2+, Mg2+, and Fe2+ + Mn2+) in a matrix effect-free manner via the systematic Raman shifts of the lattice modes. The carbonates were basically assigned to ferroan dolomite bearing a considerable amount of Fe2+ + Mn2+ at around 10 atom %. These results are in good accordance with the assignments reported by scanning electron microscopy-energy-dispersive X-ray spectroscopy, where the thin-sectioned and surface-polished Ryugu particles were applicable. The proposed method requires neither sectioning nor surface polishing; hence, it can be applied to the remote sensing apparatus on spacecrafts and planetary rovers. Furthermore, the non-invasive and matrix effect-free characterization will provide a reliable analytical tool for quantitative analysis of the elemental distribution on the samples with surface roughness and chemical heterogeneity at a micrometer scale, such as art paintings, traditional crafts with decorated shapes, as well as sands and rocks with complex morphologies in nature.

Water circulation in Ryugu asteroid affected the distribution of nucleosynthetic isotope anomalies in returned sample.

Studies of material returned from Cb asteroid Ryugu have revealed considerable mineralogical and chemical heterogeneity, stemming primarily from brecciation and aqueous alteration. Isotopic anomalies could have also been affected by delivery of exogenous clasts and aqueous mobilization of soluble elements. Here, we show that isotopic anomalies for mildly soluble Cr are highly variable in Ryugu and CI chondrites, whereas those of Ti are relatively uniform. This variation in Cr isotope ratios is most likely due to physicochemical fractionation between 54Cr-rich presolar nanoparticles and Cr-bearing secondary minerals at the millimeter-scale in the bulk samples, likely due to extensive aqueous alteration in their parent bodies that occurred [Formula: see text] after Solar System birth. In contrast, Ti isotopes were marginally affected by this process. Our results show that isotopic heterogeneities in asteroids are not all nebular or accretionary in nature but can also reflect element redistribution by water.

CO formation from glucose and cellulose as treated in ionic liquids.

The formation of carbon monoxide (CO) from glucose and cellulose by the treatment with various ionic liquids was studied. Ionic liquids with an imidazolium structure as cation and the chloride or acetate as anion were used. Additionally, 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU) was employed as an additive. CO was generated from glucose with a maximum yield of 0.57 mol% after 90 min of treatment at 120 °C in the reaction system in which DBU was added to the ionic liquid. Pyrolysis above 600 °C has been commonly employed for the gasification of lignocellullosics to produce useful gases such as CO. However, this study has revealed that gasification of lignocellullosics to produce CO can occur at significantly lower temperature, specifically at 120 °C.

Abundant presolar grains and primordial organics preserved in carbon-rich exogenous clasts in asteroid Ryugu.

Preliminary analyses of asteroid Ryugu samples show kinship to aqueously altered CI (Ivuna-type) chondrites, suggesting similar origins. We report identification of C-rich, particularly primitive clasts in Ryugu samples that contain preserved presolar silicate grains and exceptional abundances of presolar SiC and isotopically anomalous organic matter. The high presolar silicate abundance (104 ppm) indicates that the clast escaped extensive alteration. The 5 to 10 times higher abundances of presolar SiC (~235 ppm), N-rich organic matter, organics with N isotopic anomalies (1.2%), and organics with C isotopic anomalies (0.2%) in the primitive clasts compared to bulk Ryugu suggest that the clasts formed in a unique part of the protoplanetary disk enriched in presolar materials. These clasts likely represent previously unsampled outer solar system material that accreted onto Ryugu after aqueous alteration ceased, consistent with Ryugu's rubble pile origin.

Extracellular vesicles in systemic juvenile idiopathic arthritis.

Systemic juvenile idiopathic arthritis is a chronic pediatric inflammatory disease of unknown etiology, characterized by fever, rash, hepatosplenomegaly, serositis, and arthritis. We hypothesized that intercellular communication, mediated by extracellular vesicles, contributes to systemic juvenile idiopathic arthritis pathogenesis and that the number and cellular sources of extracellular vesicles would differ between inactive and active states of systemic juvenile idiopathic arthritis and healthy controls. We evaluated plasma from healthy pediatric controls and patients with systemic juvenile idiopathic arthritis with active systemic flare or inactive disease. We isolated extracellular vesicles by size exclusion chromatography and determined total extracellular vesicle abundance and size distribution using microfluidic resistive pulse sensing. Cell-specific extracellular vesicle subpopulations were measured by nanoscale flow cytometry. Isolated extracellular vesicles were validated using a variety of ways, including nanotracking and cryo-electron microscopy. Extracellular vesicle protein content was analyzed in pooled samples using mass spectrometry. Total extracellular vesicle concentration did not significantly differ between controls and patients with systemic juvenile idiopathic arthritis. Extracellular vesicles with diameters <200 nm were the most abundant, including the majority of cell-specific extracellular vesicle subpopulations. Patients with systemic juvenile idiopathic arthritis had significantly higher levels of extracellular vesicles from activated platelets, intermediate monocytes, and chronically activated endothelial cells, with the latter significantly more elevated in active systemic juvenile idiopathic arthritis relative to inactive disease and controls. Protein analysis of isolated extracellular vesicles from active patients showed a proinflammatory profile, uniquely expressing heat shock protein 47, a stress-inducible protein. Our findings indicate that multiple cell types contribute to altered extracellular vesicle profiles in systemic juvenile idiopathic arthritis. The extracellular vesicle differences between systemic juvenile idiopathic arthritis disease states and healthy controls implicate extracellular vesicle-mediated cellular crosstalk as a potential driver of systemic juvenile idiopathic arthritis disease activity.

New analytical method for the evaluation of heterogeneity in cation compositions of dolomites by micro-XRF and Raman spectroscopies.

Dolomite (CaMg(CO3)2) is an abundant carbonate mineral contained in sedimentary rocks and plays significant roles in water and carbon cycle in geo/cosmochemical environments. Since the cation compositions of carbonates are sensitive to the aqueous environment where they were precipitated and persisted, quantitative analysis of their cation compositions provides valuable information on the aqueous environments and their changes. The difficulty for the analysis of natural dolomite is that Mg2+ is continuously substituted by Fe2+ or Mn2+, and hence they sometimes possess micrometer-scale heterogeneity. Such heterogeneity carries quite important information on the gradual changes in aqueous environments due to changes in thermodynamic conditions and/or aqueous chemical compositions. In the present study, we explored a new quantitative scale to assess such heterogeneity of cation composition in natural dolomite and ferroan dolomite by combining X-ray fluorescence (XRF) and Raman spectroscopy. While the Fe + Mn content differed spot-by-spot, it was found that the Raman wavenumber and Fe + Mn content linearly correlated with each other. Since the spatial resolution of micro-Raman spectroscopy is as high as 1 µm, it does not require vacuum conditions, and is free from so-called matrix effect faced in other methods utilizing X-Rays and electron beams, the proposed qualitative analytical scale can provide a useful tool to assess the cation compositions in dolomites found in nature.

Non-destructive estimation of the cation composition of natural carbonates by micro-Raman spectroscopy.

Carbonates play a crucial role in the water and carbon cycles of both geochemical and cosmochemical environments. As carbonates do not exist homogeneously with other minerals in rocks and sands of various sizes, an analytical method that simultaneously satisfies non-destructivity and high spatial resolution has been desired. Further, the ability of semi-quantitative analysis with carbonates-selectivity and without any pre-treatments is added, for its applicability would be extended to remote sensing for deep sea and outer spaces. Here, we focused on the application of micro-Raman spectroscopy, where the vibrational wavenumbers of the translational (T) and librational (L) modes of carbonates are sensitively related to their cation composition. By comparing the semi-quantitative information obtained by X-ray fluorescence spectroscopy, it was found that these vibrational wavenumbers are approximately linearly related to the cation composition. Consequently, a conversion matrix was proposed to estimate the cation composition from the T and L mode vibrational wavenumbers. This method is universally applicable to any cation composition in carbonates, with no background information on the analyte required. To improve the accuracy, conversion matrices were further optimized to three solid-solution series of carbonates. It is worth noting that the proposed conversion matrices are free from matrix effects and do not depend on the total amount of carbonate in a sample. Therefore, the proposed method provides a useful analytical basis for remote sensing of the cation composition of carbonates, both in terrestrial and extra-terrestrial environments.

Samples returned from the asteroid Ryugu are similar to Ivuna-type carbonaceous meteorites.

Carbonaceous meteorites are thought to be fragments of C-type (carbonaceous) asteroids. Samples of the C-type asteroid (162173) Ryugu were retrieved by the Hayabusa2 spacecraft. We measured the mineralogy and bulk chemical and isotopic compositions of Ryugu samples. The samples are mainly composed of materials similar to those of carbonaceous chondrite meteorites, particularly the CI (Ivuna-type) group. The samples consist predominantly of minerals formed in aqueous fluid on a parent planetesimal. The primary minerals were altered by fluids at a temperature of 37° ± 10°C, about [Formula: see text] million (statistical) or [Formula: see text] million (systematic) years after the formation of the first solids in the Solar System. After aqueous alteration, the Ryugu samples were likely never heated above ~100°C. The samples have a chemical composition that more closely resembles that of the Sun's photosphere than other natural samples do.

Oxygen isotopes of anhydrous primary minerals show kinship between asteroid Ryugu and comet 81P/Wild2.

The extraterrestrial materials returned from asteroid (162173) Ryugu consist predominantly of low-temperature aqueously formed secondary minerals and are chemically and mineralogically similar to CI (Ivuna-type) carbonaceous chondrites. Here, we show that high-temperature anhydrous primary minerals in Ryugu and CI chondrites exhibit a bimodal distribution of oxygen isotopic compositions: 16O-rich (associated with refractory inclusions) and 16O-poor (associated with chondrules). Both the 16O-rich and 16O-poor minerals probably formed in the inner solar protoplanetary disk and were subsequently transported outward. The abundance ratios of the 16O-rich to 16O-poor minerals in Ryugu and CI chondrites are higher than in other carbonaceous chondrite groups but are similar to that of comet 81P/Wild2, suggesting that Ryugu and CI chondrites accreted in the outer Solar System closer to the accretion region of comets.

An ionic silver coating prevents implant-associated infection by anaerobic bacteria in vitro and in vivo in mice.

Currently, implants are utilized clinically for bone transplant procedures. However, if infectious osteomyelitis occurs at implant sites, removal of bacteria can be challenging. Moreover, altered blood flow at peri-implant infectious sites can create an anaerobic environment, making it more difficult to treat infection with antibiotics. Thus, it would be beneficial if implants could be modified to exhibit antibacterial activity, even in anaerobic conditions. Here, we show antibacterial activity of silver ions coated on titanium rods, even against the anaerobic bacteria Porphyromonas gingivalis (P. gingivalis), both in vitro and in vivo. Specifically, we implanted silver-coated or control uncoated titanium rods along with P. gingivalis in mouse femoral bone BM cavities and observed significantly inhibited P. gingivalis infection with silver-coated compared with non-coated rods, based on in vivo bio-imaging. Osteonecrosis by infectious osteomyelitis and elevation of the inflammatory factors C-reactive protein and IL-6 promoted by P. gingivalis s were also significantly reduced in the presence of silver-coated rods. Overall, our study indicates that silver ion coating of an implant represents a therapeutic option to prevent associated infection, even in anaerobic conditions or against anaerobic bacteria.

Ryugu's nucleosynthetic heritage from the outskirts of the Solar System.

Little is known about the origin of the spectral diversity of asteroids and what it says about conditions in the protoplanetary disk. Here, we show that samples returned from Cb-type asteroid Ryugu have Fe isotopic anomalies indistinguishable from Ivuna-type (CI) chondrites, which are distinct from all other carbonaceous chondrites. Iron isotopes, therefore, demonstrate that Ryugu and CI chondrites formed in a reservoir that was different from the source regions of other carbonaceous asteroids. Growth and migration of the giant planets destabilized nearby planetesimals and ejected some inward to be implanted into the Main Belt. In this framework, most carbonaceous chondrites may have originated from regions around the birthplaces of Jupiter and Saturn, while the distinct isotopic composition of CI chondrites and Ryugu may reflect their formation further away in the disk, owing their presence in the inner Solar System to excitation by Uranus and Neptune.

IMP1/IGF2BP1 in human colorectal cancer extracellular vesicles.

Colorectal cancer (CRC) is a leading cause of cancer-related death. There is an urgent need for new methods of early CRC detection and monitoring to improve patient outcomes. Extracellular vesicles (EVs) are secreted, lipid-bilayer bound, nanoparticles that carry biological cargo throughout the body and in turn exhibit cancer-related biomarker potential. RNA binding proteins (RBPs) are posttranscriptional regulators of gene expression that may provide a link between host cell gene expression and EV phenotypes. Insulin-like growth factor 2 RNA binding protein 1 (IGF2BP1/IMP1) is an RBP that is highly expressed in CRC with higher levels of expression correlating with poor prognosis. IMP1 binds and potently regulates tumor-associated transcripts that may impact CRC EV phenotypes. Our objective was to test whether IMP1 expression levels impact EV secretion and/or cargo. We used RNA sequencing, in vitro CRC cell lines, ex vivo colonoid models, and xenograft mice to test the hypothesis that IMP1 influences EV secretion and/or cargo in human CRC. Our data demonstrate that IMP1 modulates the RNA expression of transcripts associated with extracellular vesicle pathway regulation, but it has no effect on EV secretion levels in vitro or in vivo. Rather, IMP1 appears to affect EV regulation by directly entering EVs in a transformation-dependent manner. These findings suggest that IMP1 has the ability to shape EV cargo in human CRC, which could serve as a diagnostic/prognostic circulating tumor biomarker.NEW & NOTEWORTHY This work demonstrates that the RNA binding protein IGF2BP1/IMP1 alters the transcript profile of colorectal cancer cell (CRC) mRNAs from extracellular vesicle (EV) pathways. IMP1 does not alter EV production or secretion in vitro or in vivo, but rather enters CRC cells where it may further impact EV cargo. Our work shows that IMP1 has the ability to shape EV cargo in human CRC, which could serve as a diagnostic/prognostic circulating tumor biomarker.

Effect of Electrostatic Interactions on the Interfacial Energy between Thermoplastic Polymers and Graphene Oxide: A Molecular Dynamics Study.

In this study, the atomistic-scale mechanisms affecting the interfacial stability of a thermoplastic polymer/graphene oxide interface are investigated using molecular dynamics simulations. Different combinations of thermoplastic polymers (polyethersulfone (PES) and polyetherimide (PEI)) and graphene oxides modified with -O-, -OH, and -COOH are prepared. PES is found to be more strongly stabilized with modified/functionalized graphene oxide in the order of -COOH, -OH, -O-, which is opposite to the stability order of PEI. Our results suggest that these orders of stability are governed by a balance between the following two factors resulting from electrostatic interactions: (1) atoms with a strong charge bias attract each other, thereby stabilizing the interface; (2) the excluded-volume effect of the functional groups on graphene oxide destabilizes the interface by preventing π-π stacking of aromatic rings.

Irreversible alteration of extracellular vesicle and cell-free messenger RNA profiles in human plasma associated with blood processing and storage.

The discovery and utility of clinically relevant circulating biomarkers depend on standardized methods that minimize preanalytical errors. Despite growing interest in studying extracellular vesicles (EVs) and cell-free messenger RNA (cf-mRNA) as potential biomarkers, how blood processing and freeze/thaw impacts the profiles of these analytes in plasma was not thoroughly understood. We utilized flow cytometric analysis to examine the effect of differential centrifugation and a freeze/thaw cycle on EV profiles. Utilizing flow cytometry postacquisition analysis software (FCMpass) to calibrate light scattering and fluorescence, we revealed how differential centrifugation and post-freeze/thaw processing removes and retains EV subpopulations. Additionally, cf-mRNA levels measured by RT-qPCR profiles from a panel of housekeeping, platelet, and tissue-specific genes were preferentially affected by differential centrifugation and post-freeze/thaw processing. Critically, freezing plasma containing residual platelets yielded irreversible ex vivo generation of EV subpopulations and cf-mRNA transcripts, which were not removable by additional processing after freeze/thaw. Our findings suggest the importance of minimizing confounding variation attributed to plasma processing and platelet contamination.

Osteonecrosis development by tooth extraction in zoledronate treated mice is inhibited by active vitamin D analogues, anti-inflammatory agents or antibiotics.

Invasive dental treatment such as tooth extraction following treatment with strong anti-bone resorptive agents, including bisphosphonates and denosumab, reportedly promotes osteonecrosis of the jaw (ONJ) at the extraction site, but strategies to prevent ONJ remain unclear. Here we show that in mice, administration of either active vitamin D analogues, antibiotics or anti-inflammatory agents can prevent ONJ development induced by tooth extraction during treatment with the bisphosphonate zoledronate. Specifically, tooth extraction during treatment with zoledronate induced osteonecrosis in mice, but administration of either 1,25(OH)2D3 or ED71, both active vitamin D analogues, significantly antagonized osteonecrosis development, even under continuous zoledronate treatment. 1,25(OH)2D3 or ED71 administration also significantly inhibited osteocyte apoptosis induced by tooth extraction and bisphosphonate treatment. Administration of either active vitamin D analogue significantly inhibited elevation of serum inflammatory cytokine levels in mice in response to injection of lipopolysaccharide, an infection mimetic. Furthermore, administration of either anti-inflammatory or antibiotic reagents significantly blocked ONJ development following tooth extraction and zoledronate treatment. These findings suggest that administration of active vitamin D, anti-inflammatory agents or antibiotics could prevent ONJ development induced by tooth extraction in patients treated with zoledronate.

An Enriched Environment Alters DNA Repair and Inflammatory Responses After Radiation Exposure.

After the Fukushima Daiichi Nuclear Power Plant accident, there is growing concern about radiation-induced carcinogenesis. In addition, living in a long-term shelter or temporary housing due to disasters might cause unpleasant stress, which adversely affects physical and mental health. It's been experimentally demonstrated that "eustress", which is rich and comfortable, has beneficial effects for health using mouse models. In a previous study, mice raised in the enriched environment (EE) has shown effects such as suppression of tumor growth and enhancement of drug sensitivity during cancer treatment. However, it's not yet been evaluated whether EE affects radiation-induced carcinogenesis. Therefore, to evaluate whether EE suppresses a radiation-induced carcinogenesis after radiation exposure, in this study, we assessed the serum leptin levels, radiation-induced DNA damage response and inflammatory response using the mouse model. In brief, serum and tissues were collected and analyzed over time in irradiated mice after manipulating the raising environment during the juvenile or adult stage. To assess the radiation-induced DNA damage response, we performed immunostaining for phosphorylated H2AX which is a marker of DNA double-strand break. Focusing on the polarization of macrophages in the inflammatory reaction that has an important role in carcinogenesis, we performed analysis using tissue immunofluorescence staining and RT-qPCR. Our data confirmed that EE breeding before radiation exposure improved the responsiveness to radiation-induced DNA damage and basal immunity, further suppressing the chronic inflammatory response, and that might lead to a reduction of the risk of radiation-induced carcinogenesis.

Male fetal sex affects uteroplacental angiogenesis in growth restriction mouse model†.

Abnormally increased angiotensin II activity related to maternal angiotensinogen (AGT) genetic variants, or aberrant receptor activation, is associated with small-for-gestational-age babies and abnormal uterine spiral artery remodeling in humans. Our group studies a murine AGT gene titration transgenic (TG; 3-copies of the AGT gene) model, which has a 20% increase in AGT expression mimicking a common human AGT genetic variant (A[-6]G) associated with intrauterine growth restriction (IUGR) and spiral artery pathology. We hypothesized that aberrant maternal AGT expression impacts pregnancy-induced uterine spiral artery angiogenesis in this mouse model leading to IUGR. We controlled for fetal sex and fetal genotype (e.g., only 2-copy wild-type [WT] progeny from WT and TG dams were included). Uteroplacental samples from WT and TG dams from early (days 6.5 and 8.5), mid (d12.5), and late (d16.5) gestation were studied to assess uterine natural killer (uNK) cell phenotypes, decidual metrial triangle angiogenic factors, placental growth and capillary density, placental transcriptomics, and placental nutrient transport. Spiral artery architecture was evaluated at day 16.5 by contrast-perfused three-dimensional microcomputed tomography (3D microCT). Our results suggest that uteroplacental angiogenesis is significantly reduced in TG dams at day 16.5. Males from TG dams are associated with significantly reduced uteroplacental angiogenesis from early to late gestation compared with their female littermates and WT controls. Angiogenesis was not different between fetal sexes from WT dams. We conclude that male fetal sex compounds the pathologic impact of maternal genotype in this mouse model of growth restriction.

Fetal Renal DNA Methylation and Developmental Programming of Stress-Induced Hypertension in Growth-Restricted Male Mice.

Fetal growth restriction (FGR) is associated with developmental programming of adult onset hypertension, which may be related to differences in nephron development. Prior studies showed that maternal nutrient restriction is associated with reduced nephrogenesis in rodents, especially in male progeny. We hypothesized that maternal genetic risk for FGR may similarly affect fetal kidney development, leading to adult onset hypertension. We employed an angiotensinogen (AGT) gene titration transgenic (TG) construct with 3 copies of the mouse AGT gene that mimics a common human genotype (AGT A[-6]G) associated with FGR. We investigated whether FGR in 2-copy (wild type, [WT]) progeny from 3-copy TG dams leads to developmental programming differences in kidney development and adult blood pressure compared with age- and sex-matched controls. Progeny were tested in the late fetal period (e17.5), neonatal period (2 weeks of age), and as young adults (12 weeks). We measured weights, tested for renal oxidative stress, compared renal DNA methylation profiles, counted the number of glomeruli, and measured adult blood pressure ± stress. Progeny from TG dams were growth restricted with evidence of renal oxidative stress, males showed fetal renal DNA hypermethylation, they had fewer glomeruli, and they developed stress-induced hypertension as adults. Their female siblings did not share this pathology and instead resembled progeny from WT dams. Surprisingly, glomerular counts in the neonatal period were not different between sexes or maternal genotypes. In turn, we suspect that differences in fetal renal DNA methylation may affect the long-term viability of glomeruli, rather than reducing nephrogenesis.

Adenoviral transduction of EGFR into pregnancy-adapted uterine artery endothelial cells remaps growth factor induction of endothelial dysfunction.

During pregnancy, uterine vascular vasodilation is enhanced through adapted Ca2+ signaling, facilitated through increased endothelial connexin 43 (Cx43) gap junctional communication (GJC). In preeclampsia (PE), this adaptive response is missing. Of note, the angiogenic factor VEGF can also act via Src and ERK to close Cx43 gap junctions. While VEGFR2 is necessary for such closure, a role VEGFR1 is less clear. We reasoned if VEGFR2 is acting alone, then substituting another growth factor receptor with VEGFR2-like signaling should have the same effect. In uterine artery endothelial cells derived from pregnant sheep (P-UAEC), endogenous EGFR expression is very low. When we used adenovirus to raise EGFR, we also dose-dependently induced EGF-sensitive Cx43 phosphorylation mainly via ERK, and corresponding loss of Ca2+ bursts, but eliminated VEGF effects on phosphorylation of Cx43 or loss of Ca2+ bursting. This surprising observation suggests that while activated EGFR may indeed substitute for VEGFR2, it also sequesters a limited pool of effector molecules needed for VEGFR2 to phosphorylate Cx43. Thus, low endogenous EGFR expression in P-UAEC may be a necessary strategy to allow VEGFR-2 control of GJC, a first step in initiating angiogenesis in healthy pregnancy. Of further note, trophoblasts are rich in EGFR, and we have demonstrated shed PLAP+/EGFR + extracellular vesicles in maternal circulation in first trimester plasma samples using nanoscale high resolution flow cytometry. Collectively our data suggest that placenta derived exosomes positive for EGFR should be further considered as a possible cause of endothelial dysfunction in women with PE.