Human red blood cells release microvesicles with distinct sizes and protein composition that alter neutrophil phagocytosis.

Extracellular vesicles (EVs) are membrane-bound structures released by cells and tissues into biofluids, involved in cell-cell communication. In humans, circulating red blood cells (RBCs), represent the most common cell-type in the body, generating daily large numbers of microvesicles. In vitro, RBC vesiculation can be mimicked by stimulating RBCs with calcium ionophores, such as ionomycin and A23187. The fate of microvesicles released during in vivo aging of RBCs and their interactions with circulating cells is hitherto unknown. Using SEC plus DEG isolation methods, we have found that human RBCs generate microvesicles with two distinct sizes, densities, and protein composition, identified by flow cytometry, and MRPS, and further validated by immune TEM. Furthermore, proteomic analysis revealed that RBC-derived microvesicles (RBC-MVs) are enriched in proteins with important functions in ion channel regulation, calcium homeostasis, and vesicular transport, such as of sorcin, stomatin, annexin A7, and RAB proteins. Cryo-electron microscopy identified two separate pathways of RBC-MV-neutrophil interaction, direct fusion with the plasma membrane and internalization, respectively. Functionally, RBC-MVs decrease neutrophil ability to phagocytose E. coli but do not affect their survival at 24 hrs. This work brings new insights regarding the complexity of the RBC-MVs biogenesis, as well as their possible role in circulation.

2D versus 3D comparison of angiographic imaging biomarkers using computational fluid dynamics simulations of contrast injections.

Quantitative angiography (QAngio) may provide hemodynamic information during neurointerventional procedures through imaging biomarkers related to contrast flow. The standard clinical implementation of QAngio is limited by projection imaging: analysis of contrast motion within complex 3D geometries is restricted to 1-2 projection views, truncating the potential wealth of imaging biomarkers related to disease progression or efficacy of treatment. To understand the limitations of 2D biomarkers, we propose the use of in-silico contrast distributions to investigate the potential benefits of 3D-QAngio within the context of neurovascular hemodynamics. Ground-truth in-silico contrast distributions were generated in two patient-specific intracranial aneurysm models, accounting for the physical interactions of contrast media and blood. A short bolus of contrast was utilized to obtain full a wash-in/ wash-out cycle within the aneurysm ROI. Simulated angiograms mimicking clinical cone-beam CT (CBCT) acquisitions were then generated, and volumetric contrast distributions were reconstructed to analyze bulk contrast flow. The ground-truth 3D-CFD, reconstructed 3D-CBCT-DSA, and 2D-DSA projections were used to extract QAngio parameters related to contrast time dilution curves, such as area under the curve (AUC), peak height (PH), mean-transit-time (MTT), time-to-peak (TTP), and time to arrival (TTA). An initial comparison of quantitative flow parameters in both 2D and 3D, in a smaller and larger aneurysm, indicated that 3D-QAngio can provide a good description of bulk flow characteristics (TTA, TTP, MTT), but recovery of integral parameters (PH, AUC) aneurysms is limited. Nonetheless, incorporation of 3D-QAngio methods may provide additional insight into our understanding of abnormal vascular flow patterns.

Multi-angled simultaneous biplane High-Speed Angiography (HSA) of patient-specific 3D-printed aneurysm phantoms using 1000 fps CdTe Photon-Counting Detectors (PCD's).

1000 fps HSA enables visualization of flow details, which may be important in accurately guiding interventional procedures; however, single-plane imaging may lack clear visualization of vessel geometry and flow detail. The previously presented high-speed orthogonal biplane imaging may overcome these limitations but may still result in foreshortening of vessel morphology. In certain morphologies, acquiring two non-orthogonal biplane projections at multiple angles can provide better flow detail rather than a standard orthogonal biplane acquisition. Flow studies of aneurysm models were performed, where simultaneous biplane acquisitions at various angles separating the two detector views allowed for better evaluation of morphology and flow. 3D-printed, patient-specific internal carotid artery aneurysm models were imaged with various non-orthogonal angles between the two high-speed photon-counting detectors (7.5 cm x 5 cm FOV) to provide frame-correlated simultaneous 1000-fps image sequences. Fluid dynamics were visualized in multi-angled planes of each model using automated injections of iodine contrast media. The resulting dual simultaneous frame-correlated 1000-fps acquisitions from multiple planes of each aneurysm model provided improved visualization of complex aneurysm geometries and flow streamlines. Multi-angled biplane acquisitions with frame correlation allows for further understanding of aneurysm morphology and flow details: additionally, the ability to recover fluid dynamics at depth enables accurate analysis of 3D flow streamlines, and it is expected that multiple-planar views will enable better volumetric flow visualization and quantification. Such better visualization has the potential to improve interventional procedures.

Comparison of pulsatile flow dynamics before and after endovascular intervention in 3D-printed patient-specific internal carotid artery aneurysm models using 1000 fps photon-counting detectors for Simultaneous Biplane High Speed Angiography (SB-HSA).

A significant challenge regarding the treatment of aneurysms is the variability in morphology and analysis of abnormal flow. With conventional DSA, low frame rates limit the flow information available to clinicians at the time of the vascular intervention. With 1000 fps High-Speed Angiography (HSA), high frame rates enable flow details to be better resolved for endovascular interventional guidance. The purpose of this work is to demonstrate how 1000 fps biplane-HSA can be used to differentiate flow features, such as vortex formation and endoleaks, amongst patient-specific internal carotid artery aneurysm phantoms pre- and post-endovascular intervention using an in-vitro flow setup. The aneurysm phantoms were attached to a flow loop configured to a carotid waveform, with automated injections of contrast media. Simultaneous Biplane High-Speed Angiographic (SB- HSA) acquisitions were obtained at 1000 fps using two photon-counting detectors with the respective aneurysm and inflow/ outflow vasculature in the FOV. After x-rays were turned on, the detector acquisitions occurred simultaneously, during which iodine contrast was injected at a continuous rate. A pipeline stent was then deployed to divert flow from the aneurysm, and image sequences were once again acquired using the same parameters. Optical Flow, an algorithm that calculates velocity based on spatial-temporal intensity changes between pixels, was used to derive velocity distributions from HSA image sequences. Both the image sequences and velocity distributions indicate detailed changes in flow features amongst the aneurysms before and after deployment of the interventional device. SB-HSA can provide detailed flow analysis, including streamline and velocity changes, which may be beneficial for interventional guidance.

Investigating Angiographic Injection Parameters for Cerebral Aneurysm Hemodynamic Characterization Using Patient-Specific Simulated Angiograms.

Cerebral aneurysm (CA) rupture is one of the major causes of hemorrhagic stroke. During endovascular therapy (ET), neurointerventionalists rely on qualitative image sequences and do not have access to crucial quantitative hemodynamic information. Quantifying angiographic image sequences can provide vital information, but it is not possible to perform this in a controlled manner in vivo. Computational fluid dynamics (CFD) is a valuable tool capable of providing high fidelity quantitative data by replicating the blood flow physics within the cerebrovasculature. In this work, we use simulated angiograms (SA) to quantify the hemodynamic interaction with a clinically utilized contrast agent. SA enables extraction of time density curves (TDC) within the desired region of interest to analyze hemodynamic parameters such as time to peak (TTP) and mean transit time (MTT) within the aneurysm. We present on the quantification of several hemodynamic parameters of interest for multiple, clinically-relevant scenarios such as variable contrast injection duration and bolus volumes for 7 patient-specific CA geometries. Results indicate that utilizing these analyses provides valuable hemodynamic information relating vascular and aneurysm morphology, contrast flow conditions and injection variability. The injected contrast circulates for multiple cardiac cycles within the aneurysmal region, especially for larger aneurysms and tortuous vasculature. The SA approach enables determination of angiographic parameters for each scenario. Together, these have the potential to overcome the existing barriers in quantifying angiographic procedures in vitro or in vivo, and can provide clinically valuable hemodynamic insights for CA treatment.

Determining 3D Distributions of Pulsatile Blood Flow Using Orthogonal Simultaneous Biplane High-Speed Angiography (SB-HSA) with 1000 fps CdTe Photon Counting Detectors for 3D X-ray Particle Image Velocimetry (3D-XPIV) compared to Results Using Computational Fluid Dynamics (CFD).

3D hemodynamic distributions are useful for the diagnosis and treatment of aneurysms. Detailed blood-flow patterns and derived velocity maps can be obtained using 1000 fps High Speed Angiography (HSA). The novel orthogonal Simultaneous Biplane High-Speed Angiography (SB-HSA) system enables flow information to be quantified in multiple planes, and with additional components of flow at depth, accurate 3D flow distributions are available. Computational Fluid Dynamics (CFD) is the current standard for derivation of volumetric flow distributions, but obtaining solution convergence is computationally expensive and time intensive. More importantly, matching in-vivo boundary conditions is non-trivial. Therefore, an experimentally derived 3D flow distribution method could offer realistic results with less computation time. Using SB-HSA image sequences, 3D X-Ray Particle Image Velocimetry (3D-XPIV) was explored as a new method for assessing 3D flow. 3D-XPIV was demonstrated using an in-vitro setup, where a patient-specific internal carotid artery aneurysm model was attached to a flow loop, and an automated injection of iodinated microspheres was used as a flow tracer. Two 1000 fps photon-counting detectors were placed orthogonally with the aneurysm model in the FOV of both planes. Frame-synchronization of the two detectors made correlation of single-particle velocity components at a given timepoint possible. With frame-rates of 1000 fps, small particle displacements between frames resolved realistic time varying flow, where accurate velocity distributions depended on near-instantaneous velocities. 3D-XPIV velocity distributions were compared to CFD velocity distributions, where the simulation boundary conditions matched the in-vitro setup. Results showed similar velocity distributions between CFD and 3D-XPIV.

Current challenges and future directions for engineering extracellular vesicles for heart, lung, blood and sleep diseases.

Extracellular vesicles (EVs) carry diverse bioactive components including nucleic acids, proteins, lipids and metabolites that play versatile roles in intercellular and interorgan communication. The capability to modulate their stability, tissue-specific targeting and cargo render EVs as promising nanotherapeutics for treating heart, lung, blood and sleep (HLBS) diseases. However, current limitations in large-scale manufacturing of therapeutic-grade EVs, and knowledge gaps in EV biogenesis and heterogeneity pose significant challenges in their clinical application as diagnostics or therapeutics for HLBS diseases. To address these challenges, a strategic workshop with multidisciplinary experts in EV biology and U.S. Food and Drug Administration (USFDA) officials was convened by the National Heart, Lung and Blood Institute. The presentations and discussions were focused on summarizing the current state of science and technology for engineering therapeutic EVs for HLBS diseases, identifying critical knowledge gaps and regulatory challenges and suggesting potential solutions to promulgate translation of therapeutic EVs to the clinic. Benchmarks to meet the critical quality attributes set by the USFDA for other cell-based therapeutics were discussed. Development of novel strategies and approaches for scaling-up EV production and the quality control/quality analysis (QC/QA) of EV-based therapeutics were recognized as the necessary milestones for future investigations.

Use of 1000fps High Speed X-ray Angiography (HSAngio) to quantify differences in flow diversion effects of three stents with different coverage densities in a cerebral aneurysm invitro model.

High temporal resolution images acquired using 1000fps HSAngio can be used to visualize blood flow patterns and derive flow velocities during neurointerventional procedures. In this work we use this technology to quantify the changes in the blood flow velocities inside a cerebral aneurysm after treatment with three different stents with varying degrees of metal coverage density; stent A : <2%, stent B: 23% and stent C: 40%. A 3D printed in-vitro model of internal carotid artery aneurysm was connected to a flow loop (60% water, 40% glycerol solution used as circulation fluid, circulation flow rate 8 L/s). An automatic programmable injector (KD Scientific Legato 110) was used to inject iodine contrast agent at a rate of 88 mL/min in 3secs. 1000 fps HSAngio sequences of the contrast injection were acquired using an Aries single photon counting detector (Direct Conversion Inc., Stockholm). From these images blood flow velocities were calculated using an optical flow algorithm. As expected the biggest reduction in blood flow velocity inside the aneurysm was 32.4% after deployment of stent C. However, the velocity profile distribution indicated there was still a significant inflow jet into the aneurysm which could be caused by a endoluminal leak between the stent and the vessel wall. The average reduction was only 14% after placement of stent B and 3% after placement of stent A. Blood velocity distribution maps derived using 1000fps HSAngiography technology can be used to evaluate the quality of flow diversion within the aneurysm after placement of stent. Critical information such as endo luminal leakage which can cause treatment failure can also be detected.

Simultaneous Biplane High Speed 1000 fps X-ray Angiography (HSAngio).

High-speed 1000-fps x-ray Angiography (HSAngio) images can be used to visualize blood-flow patterns and derive flow velocities during neurointerventional procedures. In this work, we present for the very first-time, orthogonal views of contrast injection in an aneurysm model acquired simultaneously using biplane HSAngio imaging. 3-D printed in-vitro models A and B of two different internal carotid-artery aneurysms were connected to a flow loop (circulation fluid: 60% water, 40% glycerol solution, circulation flow rate: 8 L/s). An automatic programmable injector (KD Scientific Legato 110) injected iodine contrast agent at a rate of 88 mL/min for a duration of 3 sec. With an RQA5 spectrum, 1000 fps HSAngio sequences of the contrast injection were acquired simultaneously on the frontal plane using the Actaeon detector (Direct Conversion, Stockholm) and on the lateral plane using the Aries (Direct Conversion, Stockholm) detector. The start of contrast injection and simultaneous biplane x-ray exposures and detector image acquisitions were manually synchronized to capture the initial inflow of contrast into the aneurysm region. For model A the frontal plane images gave a better visualization of the flow streamlines in the parent artery in the inflow (average velocity 28 cm/s) and outflow (average velocity 24 cm/s) region of the aneurysm. The vortices within the aneurysm region especially within the aneurysm dome were better visualized in the lateral plane images (average velocity 27 cm/s). Biplane HSAngio imaging techniques can give more accurate representations of 3-D blood flow within the complex vascular pathology of the human brain, compared to single-plane imaging.

Initial evaluation of 2D and 3D simulated high-speed 1000 fps vascular contrast-flow image sequences using computational fluid dynamics (CFD).

Digital subtraction angiography (DSA) remains the clinical standard for detailed visualization of the neurovasculature due to its high-spatial resolution; however, detailed blood-flow quantification is impaired by its low-temporal resolution. Advances in photon-counting detector technology have led us to develop High-Speed Angiography (HSA), where x-ray images are acquired at 1000 fps for more accurate visualization and quantification of blood flow. We have implemented a physics-based optical flow method to extract such information from HSA, but validation of the angiography-derived velocity distributions is not straightforward. Computational fluid dynamics (CFD) is widely regarded as the benchmark for hemodynamic analysis, as it provides a multitude of quantitative flow parameters throughout the volume of interest. However, there are several limitations with this method related to over-simplification of boundary conditions and suboptimal meshing (spatial resolution), that make CFD simulation results an inexact criterion for validation. To overcome this issue for HSA validation, CFD was used to generate both simulated high-speed angiograms and the corresponding ground-truth 3D flow fields to better understand the relationship between the 3D volumetric-flow distribution and the 2D projected-flow distribution as is obtained with angiography, and the subsequent 2D approximation of flow velocity. Several geometries were investigated, ranging from simple pipe models to complex patient-specific aneurysms. Simulated datasets were analyzed with the optical flow algorithm, and the effects of flow divergence, quantum mottle, and intensity gradient on the calculation were evaluated. From these simulations, we can evaluate whether flow fields reconstructed from HSA are representative of significant flow patterns in the 3D vasculature.

Leveraging Patient-Specific Simulated Angiograms to Characterize Cerebral Aneurysm Hemodynamics using Computational Fluid Dynamics.

Cerebral aneurysms (CA) affect nearly 6% of the US population and its rupture is one of the major causes of hemorrhagic stroke. Neurointerventionalists performing endovascular therapy (ET) to treat CA rely on qualitative image sequences obtained under fluoroscopy guidance alone, and do not have access to crucial quantitative information regarding blood flow before, during and after treatment - partially contributing to a failure rate of up to 30%. Computational fluid dynamics (CFD) is a powerful tool that can provide a wealth of quantitative data; however, CFD has found limited utility in the clinic due to the challenges in obtaining hemodynamic boundary conditions for each patient. In this work, we present a novel CFD-based simulated angiogram approach (SAA) that resolves the blood flow physics and interaction between blood and injected contrast agent to extract quantitative hemodynamic parameters which can be used to design real-time parametric imaging analysis. The SAA enables correlating contrast agent transport to the underlying hemodynamic conditions via time-density curves (TDC) obtained at several points in the region of interest. The ability of the TDC and the SAA to provide critical hemodynamic parameters in and around CA anatomies, such as washout and local flow changes is explored and presented. This provides invaluable quantitative data to the clinician at the time of intervention, since it incorporates the physics of blood flow and correlates the contrast transport to hemodynamic parameters quantitatively - thereby enabling the clinician to take informed decisions that improve treatment outcomes.

Derivation of vascular wall shear stress from 1000 fps high-speed angiography (HSA) velocity distributions.

Pathological changes in blood flow lead to altered hemodynamic forces, which are responsible for a number of conditions related to the remodeling and regeneration of the vasculature. More specifically, wall shear stress (WSS) has been shown to be a significant hemodynamic parameter with respect to aneurysm growth and rupture, as well as plaque activation leading to increased risk of stroke. In-vivo measurement of shear stress is difficult due to the stringent requirements on spatial resolution near the wall boundaries, as well as the deviation from the commonly assumed parabolic flow behavior at the wall. In this work, we propose an experimental method of in-vitro WSS calculations from high-temporal resolution velocity distributions, which are derived from 1000 fps high-speed angiography (HSA). The high-spatial and temporal resolution of our HSA detector makes such high-resolution velocity gradient measurements feasible. Presented here is the methodology for calculation of WSS in the imaging plane, as well as initial results for a variety of vascular geometries at physiologically realistic flow rates. Further, the effect of spatial resolution on the gradient calculation is explored using CFD-derived velocity data. Such angiographic-based analysis with HSA has the potential to provide critical hemodynamic feedback in an interventional setting, with the overarching objective of supporting clinical decision-making and improving patient outcomes.

Electrophoretic mobility shift as a molecular beacon-based readout for miRNA detection.

MicroRNAs are short, non-coding RNA sequences involved in gene expression regulation. Quantification of miRNAs in biological fluids involves time consuming and laborious methods such as Northern blotting or PCR-based techniques. Molecular beacons (MB) are an attractive means for rapid detection of miRNAs, although the need for sophisticated readout methods limits their use in research and clinical settings. Here, we introduce a novel method based on delayed electrophoretic mobility, as a quantitative means for detection of miRNAs-MB hybridization. Upon hybridization with the target miRNAs, MB form a fluorescent duplex with reduced electrophoretic mobility, thus bypassing the need for additional staining. In addition to emission of light, the location of the fluorescent band on the gel acts as an orthogonal validation of the target identity, further confirming the specificity of binding. The limit of detection of this approach is approximately 100 pM, depending on the MB sequence. The method is sensitive enough to detect specific red blood cell miRNAs molecules in total RNA, with single nucleotide specificity. Altogether, we describe a rapid and affordable method that offers sensitive detection of single-stranded small DNA and RNA sequences.

Evaluation of methods to derive blood flow velocity from 1000 fps high-speed angiographic sequences (HSA) using optical flow (OF) and computational fluid dynamics (CFD).

Digital Subtraction Angiography (DSA) is considered the gold standard for imaging and guiding treatment of neurovascular lesions, such as cerebral aneurysms and carotid stenoses. Though DSA can show high-resolution morphology, it remains difficult to extract temporal physiological information, because higher frame-rates are necessary to accurately quantify neurovascular flow details. Recent advances in photon-counting detector technology have led us to develop High-Speed Angiography (HSA), where X-ray images are acquired at 1000 fps for more accurate visualization and quantification of blood flow. Blood flow was imaged using HSA under constant flow conditions within various 3D printed patient-specific phantoms. Blood velocity was quantified using an open source Optical Flow algorithm, OpenOpticalFlow, to perform velocity estimation based on the spatio-temporal intensity changes of iodinated contrast wavefronts. The results of these algorithms are then compared with Computational Fluid Dynamics (CFD) simulations, using the same inlet boundary conditions and model geometries. The performance of these algorithms at lower temporal resolution was then also assessed by simulating lower frame rates from the acquired 1000 fps data. It is important to ascertain the hemodynamic effect of abnormal neurovascular conditions, as well as their effect on treatment of such conditions during the actual clinical interventional procedure. While theoretical CFD results requiring considerable computer capability are delayed for hours or more, it is expected that clinical results from multiple HSA sequences will be available almost immediately while the patient is still under treatment, and even right after flow conditions are changed beneficially by the intervention.

The NIH Somatic Cell Genome Editing program.

The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium's plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled-along with validated datasets-into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit-and the knowledge generated by its applications-as a means to accelerate the clinical development of new therapies for a wide range of conditions.

Characterization of velocity patterns produced by pulsatile and constant flows using 1000 fps high-speed angiography (HSA).

In order to accurately quantify rapidly changing blood flow velocities, as typically seen in the neurovasculature, high temporal resolution is necessary. Current methods to extract velocity data from angiographic image sequences are generally limited to 30 fps or less. High-speed angiography (HSA) with a maximal frame rate of 1000 fps can be used to evaluate time-dependent flow details normally averaged out with lower frame rates. For new HSA image sequences, two different quantitative methods were utilized to extract high-temporal resolution velocity changes: X-Ray Particle Image Velocimetry (X-PIV) and optical flow (OF). A variety of flow conditions were examined in a range of patient-specific 3D-printed phantoms. Both pulsatile and constant flow settings were investigated. X-PIV was performed using radiopaque sub-millimeter microspheres, which were tracked throughout the image sequence to provide accurate, but limited sampling of the velocity field within the 3D-printed models. Also, an open source optical flow algorithm, OpenOpticalFlow, was used to perform velocity estimation based on the spatio-temporal intensity changes of iodinated contrast wavefronts. Periodic changes in velocity within each phantom ROI can be illustrated throughout the pulsatile cycle capture by the high-speed detector. In the constant flow sequences, changes in velocity across the phantom geometry can be seen. The ability to accurately measure detailed velocity distributions and velocity changes throughout various flow conditions at high temporal resolution enables further insight into the evaluation and treatment of neurovascular disease states.

Measurement and standardization challenges for extracellular vesicle therapeutic delivery vectors.

Extracellular vesicles (EVs), such as exosomes and microvesicles, are nonreplicating lipid bilayer particles shed by most cell types which have the potential to revolutionize the development and efficient delivery of clinical therapeutics. This article provides an introduction to the landscape of EV-based vectors under development for the delivery of protein- and nucleic acid-based therapeutics. We highlight some of the most pressing measurement and standardization challenges that limit the translation of EVs to the clinic. Current challenges limiting development of EVs for drug delivery are the lack of: standardized cell-based platforms for the production of EV-based therapeutics; EV reference materials that allow researchers/manufacturers to validate EV measurements and standardized measurement systems for determining the molecular composition of EVs.

VWF maturation and release are controlled by 2 regulators of Weibel-Palade body biogenesis: exocyst and BLOC-2.

von Willebrand factor (VWF) is an essential hemostatic protein that is synthesized in endothelial cells and stored in Weibel-Palade bodies (WPBs). Understanding the mechanisms underlying WPB biogenesis and exocytosis could enable therapeutic modulation of endogenous VWF, yet optimal targets for modulating VWF release have not been established. Because biogenesis of lysosomal related organelle-2 (BLOC-2) functions in the biogenesis of platelet dense granules and melanosomes, which like WPBs are lysosome-related organelles, we hypothesized that BLOC-2-dependent endolysosomal trafficking is essential for WPB biogenesis and sought to identify BLOC-2-interacting proteins. Depletion of BLOC-2 caused misdirection of cargo-carrying transport tubules from endosomes, resulting in immature WPBs that lack endosomal input. Immunoprecipitation of BLOC-2 identified the exocyst complex as a binding partner. Depletion of the exocyst complex phenocopied BLOC-2 depletion, resulting in immature WPBs. Furthermore, releasates of immature WPBs from either BLOC-2 or exocyst-depleted endothelial cells lacked high-molecular weight (HMW) forms of VWF, demonstrating the importance of BLOC-2/exocyst-mediated endosomal input during VWF maturation. However, BLOC-2 and exocyst showed very different effects on VWF release. Although BLOC-2 depletion impaired exocytosis, exocyst depletion augmented WPB exocytosis, indicating that it acts as a clamp. Exposure of endothelial cells to a small molecule inhibitor of exocyst, Endosidin2, reversibly augmented secretion of mature WPBs containing HMW forms of VWF. These studies show that, although BLOC-2 and exocyst cooperate in WPB formation, only exocyst serves to clamp WPB release. Exocyst function in VWF maturation and release are separable, a feature that can be exploited to enhance VWF release.

Assessment of a Bayesian Vitrea CT Perfusion Analysis to Predict Final Infarct and Penumbra Volumes in Patients with Acute Ischemic Stroke: A Comparison with RAPID.

BACKGROUND AND PURPOSE: Brain CTP is used to estimate infarct and penumbra volumes to determine endovascular treatment eligibility for patients with acute ischemic stroke. We aimed to assess the accuracy of a Bayesian CTP algorithm in determining penumbra and final infarct volumes. MATERIALS AND METHODS: Data were retrospectively collected for 105 patients with acute ischemic stroke (55 patients with successful recanalization [TICI 2b/2c/3] and large-vessel occlusions and 50 patients without interventions). Final infarct volumes were calculated using DWI and FLAIR 24 hours following CTP imaging. RAPID and the Vitrea Bayesian CTP algorithm (with 3 different settings) predicted infarct and penumbra volumes for comparison with final infarct volumes to assess software performance. Vitrea settings used different combinations of perfusion maps (MTT, TTP, CBV, CBF, delay time) for infarct and penumbra quantification. Patients with and without interventions were included for assessment of predicted infarct and penumbra volumes, respectively. RESULTS: RAPID and Vitrea default setting had the most accurate final infarct volume prediction in patients with interventions ([Spearman correlation coefficient, mean infarct difference] default versus FLAIR: [0.77, 4.1 mL], default versus DWI: [0.72, 4.7 mL], RAPID versus FLAIR: [0.75, 7.5 mL], RAPID versus DWI: [0.75, 6.9 mL]). Default Vitrea and RAPID were the most and least accurate in determining final infarct volume for patients without an intervention, respectively (default versus FLAIR: [0.76, -0.4 mL], default versus DWI: [0.71, -2.6 mL], RAPID versus FLAIR: [0.68, -49.3 mL], RAPID versus DWI: [0.65, -51.5 mL]). CONCLUSIONS: Compared with RAPID, the Vitrea default setting was noninferior for patients with interventions and superior in penumbra estimation for patients without interventions as indicated by mean infarct differences and correlations with final infarct volumes.

Rational Design of a Bifunctional AND-Gate Ligand To Modulate Cell-Cell Interactions.

Protein "AND-gate" systems, in which a ligand acts only on cells with two different receptors, direct signaling activity to a particular cell type and avoid action on other cells. In a bifunctional AND-gate protein, the molecular geometry of the protein domains is crucial. Here we constructed a tissue-targeted erythropoietin (EPO) that stimulates red blood cell (RBC) production without triggering thrombosis. The EPO was directed to RBC precursors and mature RBCs by fusion to an anti-glycophorin A antibody V region. Many such constructs activated EPO receptors in vitro and stimulated RBC and not platelet production in mice but nonetheless enhanced thrombosis in mice and caused adhesion between RBCs and EPO-receptor-bearing cells. On the basis of a protein-structural model of the RBC surface, we rationally designed an anti-glycophorin-EPO fusion that does not induce cell adhesion in vitro or enhance thrombosis in vivo. Thus, mesoscale geometry can inform the design of synthetic-biological systems.