Journal of Thrombosis and Haemostasis

Background. Balanced (1:1:1) transfusion of red blood cells (RBCs), plasma, and platelets is the standard of care in trauma-induced massive haemorrhage, where early coagulopathy is a defining feature. In gastrointestinal (GI) haemorrhage this physiology is non-prominent, and whether plasma and platelets provide benefit when [≥] 10 RBC units are required within 24 hours is unknown. Objective. To test whether a red-blood-cell-only (RBC-only) transfusion strategy is non-inferior to a balanced (Balanced) strategy for in-hospital mortality in adults meeting massive-transfusion criteria for GI haemorrhage. Design. Single-centre retrospective cohort of 559 adult massive-transfusion encounters (536 patients; 2021-2025) with a primary admitting diagnosis of upper, lower, or unspecified GI haemorrhage. Exposures were RBC-only versus Balanced (RBCs with any plasma and/or platelets). The primary outcome was in-hospital mortality, with a pre-specified 5-percentage-point (pp) non-inferiority margin on the absolute risk difference and a 3-pp sensitivity margin. Analysis used augmented inverse-probability-of-treatment weighting (AIPTW) with bootstrap inference (2,000 resamples by patient). Five pre-specified sensitivity analyses were performed. Results. 505 encounters (90.3%) received RBC-only and 54 (9.7%) received Balanced transfusion. The AIPTW risk difference for in-hospital mortality (RBC-only - Balanced) was -19.8 pp (95% CI -68.1 - -2.2 pp). Non-inferiority was demonstrated at both the primary 5-pp and the more stringent 3-pp margins. Five pre-specified sensitivity analyses, (1) a propensity-score matched cohort, (2) a complete-case model incorporating INR, (3) a broader GI diagnosis set (n = 749), (4) a first encounter per patient restriction, and (5) E-value bound analysis were concordant with the primary estimate. Conclusion. In this propensity-score-weighted cohort of adults with massive GI haemorrhage, an RBC-only transfusion strategy was non-inferior to a balanced strategy for in-hospital mortality at both 5-pp and 3-pp margins. The findings support individualized use of plasma and platelets in GI haemorrhage rather than reflexive application of the 1:1:1 trauma protocol; prospective confirmation is warranted.

BACKGROUNDMesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) exert neuroprotective effects in ischemic stroke largely through immunomodulatory mechanisms. Monocytes are first-line responders to MSC-EVs. Their contribution to MSC-EV-induced neuroprotection remains poorly understood. This study investigated the role of monocytes in shaping neuroprotective responses to MSC-EVs after ischemic stroke. METHODSMale C57BL/6J mice were exposed to transient middle cerebral artery occlusion (MCAO). Monocytes were depleted using pharmacological (clodronate liposomes), immunological (anti-CCR2), or genetic (Mrp8-Cre+/- Nr4a1fl/fl) approaches removing total, CCR2+, or Ly6Clow monocytes, respectively. In additional cohorts, neutrophils and T cells were simultaneously depleted by anti-Ly6G or anti-CD4/CD8 antibodies. Small EVs from clonally expanded immortalized MSCs were administered intravenously. Neurological deficits, ischemic injury, and immune responses were analyzed up to 72 hours post-MCAO. Complementary ex vivo studies were performed, in which MSC-EVs were administered to monocyte-depleted or non-depleted peripheral blood mononuclear cells (PBMCs) obtained from acute ischemic stroke patients. RESULTSIn ischemic mice with intact monocyte compartment, MSC-EVs reduced neurological deficits, infarct volume, neuronal injury, and brain leukocyte infiltrates. These protective effects were abolished in monocyte-depleted mice, particularly following CCR2+ monocyte depletion. Under these conditions, MSC-EV treatment exacerbated neurological deficits, ischemic injury, and leukocyte infiltration, accompanied by neutrophil and T cell expansion and overactivation. Depletion of neutrophils or T cells prevented the EV-induced worsening of stroke outcome in monocyte-deficient mice. Ly6Clow monocytes played a crucial role in orchestrating immune responses to MSC-EVs. Their depletion abolished EV-induced neuroprotection. In stroke patient PBMCs, MSC-EVs induced phenotypic reprogramming of monocytes, whereas they promoted CD4+ and CD8+ T cell activation in the absence of monocytes. CONCLUSIONSMonocytes shape the immunomodulatory actions of MSC-EVs. In their absence, MSC-EVs trigger neutrophil and T cell overactivation that worsens stroke outcome. These findings highlight the importance of monocyte- and T cell-related potency assays for the clinical translation of MSC-EV therapies.

Diabetes is associated with endothelial dysfunction, impaired wound healing, and increased thrombotic risk, yet the impact of diabetes on endothelial secretory organelles remains poorly understood. Weibel-Palade bodies (WPBs) are specialised endothelial granules that store and release von Willebrand factor (VWF) and other vasoactive cargo essential for haemostasis, inflammation, and vascular repair. Here, we investigated how diabetic environments influence WPB biogenesis and VWF structure under physiologically relevant flow conditions. Acute exposure of endothelial cells to constant or fluctuating high glucose concentrations, designed to model diabetic glycaemic conditions, did not alter WPB number or morphology under either static or high laminar shear stress conditions. In contrast, primary endothelial cells derived from a diabetic donor exhibited reduced Akt and eNOS signalling, significantly fewer WPBs, reduced intracellular VWF content, and shorter stimulus-evoked VWF strings compared with non-diabetic endothelial cells. Although total cellular VWF levels were reduced, high molecular weight (HMW) VWF content within endothelial lysates was not significantly altered. Plasma from diabetic patients demonstrated elevated circulating VWF levels together with marked inter-patient heterogeneity in VWF multimer composition. These findings suggest that chronic diabetes-associated endothelial dysfunction, rather than hyperglycaemia alone, alters WPB biology and VWF handling. We propose that dysregulated basal endothelial secretion may deplete endothelial VWF stores, limiting appropriate stimulus-coupled WPB release during vascular injury and contributing to defective vascular repair in diabetes.

BackgroundtPA is used for the acute treatment of ischaemic stroke because it converts plasminogen to active plasmin, which breaks down clots. Previous studies show that tPA-activated plasminogen impairs brain endothelial barrier function. However, it is unclear whether the plasmin product of this reaction directly contributes to brain endothelial barrier deterioration. ObjectiveDetermine whether plasmin directly influences the human brain endothelial barrier. MethodsWe developed a new serum-free hCMEC/D3 culture model with ECIS real-time monitoring to establish how plasmin in isolation influences the brain endothelial barrier. ResultsECIS monitoring demonstrated that plasmin caused a concentration-dependent decline in hCMEC/D3 barrier integrity, which was primarily mediated by a reduction in endothelial cell-to-cell interactions. Whilst a decrease in membrane capacitance and increase in basolateral adhesion were also observed, these changes were less marked. The inclusion of 2-antiplasmin ameliorated the changes in hCMEC/D3 barrier properties, suggesting this response is mediated by plasmins proteolytic activity. Quantitative immunocytochemistry confirmed that plasmin stimulated a decline in the key junctional molecules, Claudin-5, VE-Cadherin (CD144), {beta}-Catenin, ZO-1 and PECAM-1 (CD31), which likely contributed to the deterioration of paracellular cell-to-cell interactions. Interestingly, using this serum-free model, tPA alone didnt influence hCMEC/D3 barrier properties, whilst tPA with plasminogen did, implicating plasmins involvement. ConclusionPlasmin directly impaired the barrier function of hCMEC/D3 brain endothelial cell monolayers by stimulating a decline in key junctional molecules. This plasmin-mediated brain endothelial barrier deterioration has important implications for tPA use and should be considered whilst designing safer thrombolytic treatment options for patients experiencing acute ischemic stroke.

ObjectiveHereditary hemorrhagic telangiectasia (HHT) is a vascular genetic disorder caused by endothelial cell dysfunction and characterized by telangiectasias and arteriovenous malformations (AVMs). HHT results primarily from loss-of-function mutations affecting components of the BMP9-ALK1-ENG-SMAD signaling cascade, a pathway essential for endothelial quiescence and vascular homeostasis, and currently lacks a cure. Here, we investigated whether nitazoxanide, an orally bioavailable drug with extensive clinical use, can modulate endothelial signaling relevant to HHT. Approach and ResultsNitazoxanide treatment activated SMAD1/5/8 signaling and increased expression of the downstream target ID1 in endothelial cells, while concurrently inhibiting mTOR signaling, indicating a dual modulatory effect on pathways implicated in HHT pathogenesis. In vivo, nitazoxanide activated SMAD signaling in BMP9/10-immunoblocked mice and significantly reduced AVM formation and hypervascularization. Importantly, nitazoxanide restored SMAD1/5/8 activation and ID1 expression in patient-derived blood outgrowth endothelial cells harboring loss-of-function mutations in ALK1 or SMAD4, which exhibit impaired BMP signaling. ConclusionThese findings identify nitazoxanide as a pharmacological modulator capable of activating BMP-SMAD signaling while restraining mTOR activity, thereby overcoming key signaling defects in HHT endothelial cells. Collectively, our results highlight nitazoxanide as a promising therapeutic candidate to target endothelial dysfunction in HHT.

Background: Immune-mediated mechanisms are increasingly implicated in childhood arterial ischemic stroke (AIS), but the associated inflammatory pathways and how they differ by stroke subtype and outcome remain poorly understood. Understanding immune responses to AIS may identify subtype-specific mechanisms and inform targeted strategies to reduce ischemic injury. Methods: We conducted a prospective cohort study with cross-sectional transcriptomic analysis through the Vascular Effects of Infection in Pediatric Stroke Study Part II (VIPS II) at 22 academic centers in the United States, Canada, and Australia between December 2016 and January 2022. Children aged 28 days to 18 years with centrally confirmed AIS were enrolled within 72 hours of stroke onset, in addition to enrollment of stroke-free well children. Peripheral blood RNA sequencing was performed on samples collected within 72 hours of stroke or at enrollment for controls. Differential gene expression (DGE) and pathway analyses were performed comparing all AIS cases to stroke-free well children. Additional cross-sectional analyses stratified by stroke subtype and neurological outcomes were performed. Results: Transcriptomes were available in 190/205 AIS cases (median age 11.7 years) and 91/100 stroke-free children (11.8 years). Stroke subtypes included 67 definite arteriopathic, 74 probable arteriopathic, 23 cardioembolic, and 26 idiopathic, with similar demographics but smaller infarct size for idiopathic cases. 47 genes (false discovery rate (FDR) <0.05 and log2 fold-change (log2FC)>1) were differentially expressed in AIS versus stroke-free well children, with upregulated pathways reflecting innate immune responses. Stratification by subtype revealed these inflammatory responses occurred after arteriopathic and cardioembolic AIS, but not idiopathic AIS; in sensitivity analyses, these findings were not explained by infarct size. Four immune-related genes were differentially expressed in children with good versus poor neurological outcomes at hospital discharge or 12 months; upregulation of one (Joining Chain; JCHAIN) correlated with poor outcomes at both timepoints. Conclusions: Compared with stroke-free children, children with AIS, particularly arteriopathic and cardioembolic subtypes, have upregulated innate immune pathways, including neutrophil activation and interleukin-1 signaling. Differential expression of immune-related genes also correlated with neurological outcomes. These findings support immune dysregulation as a key feature of early pediatric AIS while highlighting differences across subtypes and clinical outcomes, with implications for targeted immunomodulatory therapies and future biomarker development.

Fetal hemoglobin (HbF) prevents the polymerization of sickle hemoglobin (HbS). HbF, measured usually as a percent of total hemoglobin (%HbF), is inversely associated with the severity of sickle cell disease (SCD) but fails to capture the distribution of HbF concentrations within red blood cells (RBCs). The relative proportion of HbF and HbS within a RBC is reflected by the HbF:HbS ratio whereas HbF/F-cell quantifies the absolute amount of HbF/RBC. While correlated, HbF:HbS ratio and HbF/F-cell are not interchangeable. In the context of mean corpuscular hemoglobin (MCH), HbF/F-cell approximates whether sufficient HbF is present to inhibit HbS polymerization. We examined the association of mean HbF/F-cell with sub-phenotypes of sickle cell disease in three independent cohorts. Both %HbF and HbF/F-cell were significantly associated with multiple clinical and laboratory features of SCD; however, HbF/F-cell demonstrated stronger associations with clinical severity measures across cohorts. Higher HbF/F-cell was associated with fewer clinical events, reduced hemolysis, and mortality. Changes in HbF/F-cell after hydroxyurea treatment were associated with ~11-13% reduction in acute events in patients with <1 pg increase and >60% reduction with a >5 pg increase in HbF/F-cell. For each pg increase in HbF/F-cell there was ~6% reduction in the rate of acute events. As a surrogate for the distribution of HbF concentrations among F-cells, HbF/F-cell adds physiologically relevant insights that could guide prognosis and treatment

BackgroundThe formation of a patent vascular lumen is fundamental to circulatory function, a process governed by cytoskeletal dynamics and mechanosensory signalling. Endothelial cilia are present during blood vessel lumen development, but their precise functional role remains poorly understood. Understanding how cilia coordinate with endothelial cytoskeletal and signalling pathways is critical for elucidating mechanisms of vascular morphogenesis. MethodsWe have established a microfluidic system that recapitulates endothelial tube formation under fluid flow, enabling pharmacological and genetic manipulation with real-time visualisation of tube behaviour. Cilia, cytoskeletal dynamics, and lumen development were analysed in vitro, and in vivo. ResultsEarly perfusion in the microfluidic system induced a hierarchical vascular network. Inhibiting Rho-kinase (ROCK) or knocking down ciliary components (IFT88 and RPGRIPL1) suppressed lumen formation. ROCK inhibition or genetic ablation disrupted cilia in endothelial and non-endothelial cells, associated with LIM-kinase inhibition. Crucially, ROCK2 genetic ablation caused endothelial cilia loss, misorientation, and abrogated lumen formation, leading to haemorrhages and compromised vascular integrity in vivo. ConclusionsOur findings unveil a previously unrecognised co-regulation between cilia and ROCK signalling essential in vascular lumen formation.

Pediatric platelet disorders are commonly classified according to specific structural or functional abnormalities, yet it remains unclear how well these diagnoses capture overall hemostatic phenotype. Here, we combined quantitative single-cell platelet measurements with spatially resolved plasma clotting analysis to characterize pediatric patients with dense granule deficiency, platelet function defects, immune thrombocytopenia, and other inherited platelet disorders. Quantitative fluorescence microscopy revealed reduced dense granule abundance not only in dense granule deficiency but also in several patients from other diagnostic groups. Measurements of platelet adhesion, spreading, and calcium signaling identified substantial functional diversity, with individual patients exhibiting distinct combinations of abnormalities that were not predicted by diagnostic category. Unexpectedly, plasma clotting analysis frequently revealed hypercoagulable behavior, including accelerated fibrin clot growth and spontaneous fibrin formation, despite clinical diagnoses associated with platelet-related bleeding disorders. Hypercoagulable phenotypes occurred across multiple diagnostic groups and did not show a simple relationship with platelet functional abnormalities. Together, these findings reveal previously unrecognized complexity in pediatric platelet disorders and suggest that platelet and plasma pathways contribute independently to hemostatic variability. These findings argue that pediatric platelet disorders are best viewed as multidimensional functional phenotypes rather than isolated platelet defects and motivate broader integration of platelet and coagulation measurements in future studies.

BackgroundPatients with systemic lupus erythematosus (SLE) face markedly increased cardiovascular disease (CVD) risk driven by mechanisms beyond traditional risk factors. Thoracic aortic perivascular adipose tissue (tPVAT) is dysfunctional in lupus and exacerbates endothelial dysfunction, yet the molecular basis of this dysfunction remains poorly defined. MethodsIntegrated multi-omics profiling, including bulk RNA-seq, untargeted proteomics, lipidomics, and high-dimensional spectral flow cytometry, was performed on tPVAT from 15-week-old MRL/lpr mice (active lupus, n = 4-6) and MRL control mice (n = 5-6). Adipogenic differentiation capacity of tPVAT adipose stromal and progenitor cells (ASPCs) from MRL/lpr was assessed by Oil Red O staining at 5 (pre-dieasea) and 15 weeks (active disease), with subcutaneous ASPCs used as depot controls. ResultsTranscriptomic profiling of tPVAT from MRL/lpr mice identified 2,742 upregulated and 1,494 downregulated genes (adjusted p < 0.001, |log2FC| > 1), with strong activation of interferon, IL6-JAK-STAT3, and TNFA signaling pathways together with suppression of fatty acid metabolism, oxidative phosphorylation, and adipogenic pathways. Proteomic and lipidomic analyses were concordant, revealing broad downregulation of mitochondrial bioenergetic machinery, depletion of cardiolipin and acylcarnitines, and enrichment of ceramide phosphoinositols and lysophosphatidylcholines. Cardiolipin strongly correlated with the mitochondrial/metabolic protein module (r = 0.95) and inversely with the immune/inflammatory protein module (r = -0.92). Spectral flow cytometry confirmed marked CD45+ leukocyte infiltration dominated by T cells, together with a significantly reduced Treg/CD4+ ratio indicating loss of local immunoregulatory balance. ASPCs derived from PVAT of 15-week-old MRL/lpr mice exhibited impaired white and beige adipogenic differentiation, while APCs from PVAT of 5-week-old MRL/lpr mice, and from subcutaneous adipose tissues of 15-week-old MRL/lpr mice, had normal white and beige differentiation, consistent with an acquired, depot-specific, disease-stage-dependent progenitor defect in PVAT of MRL/lpr mice. ConclusionsLupus tPVAT undergoes a concordant cross-platform molecular reprogramming of mitochondrial bioenergetic genes coupled with establishment of an interferon-dominant immune niche and acquired loss of ASPC adipogenic capacity. These findings provide a molecular framework for lupus PVAT dysfunction and identify restoration of mitochondrial function, suppression of interferon-driven inflammation, and renewal of progenitor differentiation as potential therapeutic strategies for lupus vasculopathy.

Background and Purpose: Hemorrhagic transformation (HT) is a serious complication of endovascular thrombectomy (EVT), yet dedicated prediction models for young adults are lacking. We aimed to develop and externally validate a simplified risk score for HT in young adults with acute ischemic stroke undergoing EVT. Methods: This multicenter retrospective study included patients aged 18 to 49 years with acute anterior circulation large vessel occlusion who underwent EVT. The primary outcome was any HT within 24 hours after EVT. Multivariable logistic regression was used to identify independent predictors of HT, from which the NO?PAIN Score was derived. External validation was performed in an independent cohort of 138 patients. Results: Among 598 patients in the derivation cohort, HT occurred in 176 (29.4%). Five independent predictors were identified: admission NIHSS, number of thrombectomy passes, atrial fibrillation, alcohol consumption, and mTICI grade. The mTICI grade demonstrated a non-linear, inverted U-shaped relationship with HT risk, peaking at partial recanalization. The NO-PAIN Score showed acceptable discrimination in both the derivation (C-index, 0.737; optimism-corrected C-index, 0.748) and external validation cohorts (C-index, 0.726), with satisfactory calibration. Conclusions: The NO-PAIN Score is a simple risk prediction tool for HT after EVT in young adults with acute anterior circulation large vessel occlusion. It may assist in individualized risk stratification in this population.

Sickle cell disease (SCD) is caused by a point mutation in the {beta}-globin gene that promotes hemoglobin polymerization, leading to chronic hemolytic anemia, vaso-occlusive episodes, and progressive organ damage. The most efficacious therapies focus on reactivating fetal hemoglobin (HbF) expression to mitigate the pathological effects of sickle hemoglobin (HbS) polymerization. However, the predominantly used HbF inducer, hydroxyurea (HU), exhibits substantial interpatient variability in efficacy, and curative approaches such as gene therapy remain inaccessible to the vast majority of patients. Although all SCD patients share the same causative HBB glu7val mutation, differences in genetic background significantly influence disease severity and therapeutic response. We describe a SCD-specific induced pluripotent stem cell (iPSC) platform as a renewable and scalable preclinical model to interrogate treatment responses across the genetically diverse SCD patient population. By generating patient-specific iPSC-derived erythroblasts (iEry) representing distinct SCD genetic backgrounds, we demonstrate that this system faithfully recapitulates the heterogeneous HbF induction observed clinically in response to HU. Moreover, this platform enables the identification and evaluation of alternative therapeutic agents for HU non-responders and provides sufficient resolution to dissect drug-specific effects on erythroid differentiation and cellular phenotypes. Together, these findings support the use of iPSC-derived erythroid models as a versatile tool to advance precision therapeutic strategies for SCD. KEY POINTS- SCD iPSC-derived erythroid cells (iEry) reflect the diversity in HU-mediated HbF induction seen in SCD patients - SCD iEry recapitulate patient-specific treatment responses and can be used to identify therapeutic alternatives for HU non-responders - iEry provide a versatile platform to study the impact of novel HbF inducers on erythroid cell characteristics and differentiation parameters

BackgroundThoracic aortic aneurysm (TAA) is a life-threatening condition with an unpredictable lisk of rupture. Current clinical parameters have limited ability to accurately predict imminent rupture. Osteopontin (OPN) has been implicated in aortic aneurysm pathology, however, it role as a marker of imminent rupture remains. unclear. We investigated the dynamics of OPN expression dynamics in a mouse model with predictable rupture timing and validated our findings in human TAA. MethodsOne-month-old fibrillin-1 hypomorphic (Fbn1mgR/mgR) mice were used as a TAA model; with wild-type (WT) mice served as controls. Angiotensin II (AngII) was administered to Fbn1mgR/mgR to induce acute aortic rupture. Single-section transcriptome analysis and immunofluorescence staining were performed on ascending aortic tissue at 24 and 72 hours after AngII infusion, with pre-treatment Fbn1mgR/mgR and WT mice serving as controls. To determine conservation in human disease, we reanalyzed publicly available single-cell RNA sequencing data from ascending thoracic aortic aneurysm (ATAA) patients. ResultsAngII infusion induced progressive mortality beginning at 24 hours, with approximately 60% survival at 72 hours and nearly no survival by 8 days in Fbn1mgR/mgR mice. At this pre-rupture time point, OPN showed prominent upregulation at both mRNA and protein levels in ascending aortic tissues compared to controls. Immunofluorescence staining revealed increased OPN expression in the aortic wall, particularly in regions exhibiting structural deterioration. Reanalysis of human ATAA single-cell data showed elevated OPN expression compared to controls, with enrichment in immune cell populations, especially macrophages. Within the macrophage compartment, subcluster analysis identified a stress-responsive subpopulation (MC1) that was markedly expanded and almost exclusively composed of ATAA-derived cells, representing the primary source of OPN upregulation. ConclusionsOPN upregulation represents a conserved molecular signature of the pre-rupture state in TAA across mice and humans. Our mode, which enables predictable rupture timing, allowed the capture of acute pre-rupture molecular changes, suggesting OPN as a potential biomarker for predicting imminent aortic rupture.

Sex-specific genetic effects on platelet aggregation may contribute to differences in thrombotic risk between women and men, yet the underlying genetic mechanisms remain poorly defined. We performed whole-genome sequencing-based genome-wide association studies (GWAS) of 19 harmonized platelet aggregation phenotypes in response to ADP, epinephrine (EPI), and collagen across three independent cohorts: GeneSTAR, the Framingham Heart Study and the Old Order Amish. Our meta-analysis identified a cluster of low-frequency variants within a 20 kb region on chromosome 10 showing strong sex-specific associations with platelet aggregation in response to low-dose EPI. The lead variant, rs116725046, exhibited a genome-wide significant sex interaction (p = 5.2e-9), with opposite phenotypic effects in women and men. Female carriers demonstrated substantially increased platelet aggregation, whereas male carriers showed decreased aggregation, consistent across cohorts. Several additional variants in tight linkage disequilibrium yielded comparable interaction signals for low-dose EPI, including five SNPs driving the lowest meta-analysis p-value (p = 8.3e-9). The associated variants reside within an intronic region of the long noncoding gene LINC00702, with FUMA annotations indicating regulatory chromatin states. Megakaryocyte epigenome data also indicates potential regulatory activity in platelet precursor cells near the lead variants. eQTL analyses suggested sex-differentiated genetic regulation of LINC00702 in multiple tissues, with reduced expression in male heterozygotes only. An ARE motif was identified upstream of LINC00702, supporting a potential hormone-responsive regulatory mechanism. Together, these findings identify a novel sex-specific regulatory locus influencing platelet reactivity and highlight LINC00702 as a biologically plausible mediator of sexually dimorphic platelet responses.

BackgroundHyaluronan (HA) is a major extracellular matrix glycosaminoglycan that regulates vascular integrity and immune signaling in the lung. Its biological effects are strongly size-dependent, with high-molecular-weight HA (HMW-HA) generally protective and low-molecular-weight HA (LMW-HA) pro-inflammatory. However, how different HA sizes and concentrations globally remodel endothelial cell signaling remains poorly understood. MethodsHuman lung microvascular endothelial cells (HULEC-5a) were treated with physiologic (200 ng/mL) or supraphysiologic (1 {micro}g/mL) concentrations of LMW-, medium-molecular-weight (MMW-), or HMW-HA. Cell viability was confirmed by LDH assay. Quantitative proteomics with downstream Ingenuity Pathway Analysis (IPA) was used to profile HA-induced signaling networks. ResultsProteomic analysis revealed a conserved HA-response signature across all conditions involving cell cycle regulation, senescence, and immune modulation, with distinct size-and dose-dependent differences. At supraphysiologic concentrations, HMW-HA suppressed proliferative and inflammatory pathways, consistent with a protective, quiescent phenotype. LMW-HA induced the broadest stress-associated proteomic changes, consistent with its role as a damage-associated molecular pattern. Unexpectedly, physiologic MMW-HA elicited the strongest responses, driving metabolic and cytoskeletal pathways including insulin signaling and Rho GTPase activity. Network analysis highlighted 176 overlapping pathways across HA treatments, with unique contributions of LMW- and HMW-HA to stress- versus barrier-stabilizing signaling, respectively. ConclusionHA is not a passive structural molecule but an active regulator of endothelial signaling, with effects shaped by both molecular weight and concentration. Our findings identify a distinct role for MMW-HA at physiologic levels and highlight how HA fragmentation and accumulation may contribute to endothelial dysfunction in lung injury, with implications for targeted HA-based therapies.

Endothelial cells (ECs) are key players in maintaining homeostasis and coordinating immune responses, activating during acute inflammation to recruit immune cells. Endothelial heterogeneity has been found to impact transcription level differences across EC sources, but how these differences drive downstream effects in inflammatory signaling and immune interactions remains unclear. Here, we employed multiplexed ELISA to quantify secretion for 19 inflammatory factors following tumor necrosis factor (TNF) or Pseudomonas aeruginosa activation of four primary human EC sources: umbilical artery (HUAEC), umbilical vein (HUVEC), dermal microvascular (HDMEC), and pulmonary microvascular (HPMEC) endothelial cells. We also quantified changes in neutrophil adhesion to each EC source and used partial least squares regression (PLSR) to identify key inflammatory proteins associated with changes in neutrophil adhesion. We found distinct inflammatory secretion profiles across all cell types, with veinous ECs showing the highest basal secretion of most inflammatory proteins and pulmonary ECs exhibiting the lowest. Arterial ECs exhibited the lowest sensitivity to inflammatory stimulus, while pulmonary ECs exhibited dynamic responses following activation. Furthermore, inflammatory stimulus caused large differences in expression across cell sources for six factors: GM-CSF, IL-1{beta}, IL-6, IP-10, E-selectin, and ICAM-1. We found endothelial heterogeneity also contributed to differences in neutrophil adhesion to unstimulated ECs. Our PLSR analysis revealed five secreted factors most indicative of changes in neutrophil adhesion: E-selectin, ICAM-1, PECAM1, IL-6, and IL-8. Collectively, our findings strengthen the emerging view that vascular-bed specific differences in EC phenotype can impact downstream immune responses.

Background: Circulating endothelial progenitor cells (cEPCs) contribute to vascular repair following an ischemic stroke. The aim of the study was to evaluate the association between cEPCs and functional outcomes in patients with acute ischemic stroke (AIS) due to large vessel occlusion (LVO) who received endovascular therapy (EVT). Methods: Prospective study of patients with LVO-AIS who received EVT. Blood samples were obtained within 24 +- 12 hours and on day 7+-1 from stroke onset. cEPCs were detected using flow cytometry (CD34+/VEGFR2+/CD133+). The primary endpoint was a favourable functional outcome (modified Rankin Scale 0-2) at three months of follow-up. Secondary endpoints include baseline to 24 hours/day 7 changes in the National Institutes of Health Stroke Scale (NIHSS) score and collateral circulation (CC) status. Bivariate and multivariable logistic regression analyses were performed. Results: Included were 90 patients (73.2+-12.7 years, 41.1% women) in 42 of whom (46.7%) cEPCs were detected at 24 hours. On day 7, cEPCs were detected in 27 (43.6%) of 62 patients for which this information was available. Atrial fibrillation, prior anticoagulant treatment and stroke onset-to-door time <6 hours were associated with lower cEPC counts, and intravenous fibrinolysis therapy was associated with a higher cEPC count on day 7. No association was found between cEPCs and functional outcomes at three months. Patients with the highest cEPC count (Q4) at 24 hours had a lower probability of good CC (46.2% vs 77.3%; p=0.031). Conclusion: cEPC count in patients with LVO-AIS who received EVT was not associated with functional outcomes.

Congenital heart defects (CHDs) are the most common form of fetal malformation however, our understanding of trophoblast health and communication throughout gestation in CHD pregnancies remains limited. The purpose of this study was to assess extracellular vesicles (EVs) and microRNA (miRNA) present in maternal and umbilical cord plasma from a spectrum of CHD subtypes during gestation and at time of delivery. We hypothesized that circulating placenta-derived EVs and miRNA will differ in CHD when compared to controls. Maternal plasma samples were collected between 16-24 weeks of gestation and at the time of delivery. Umbilical cord plasma was obtained following delivery. EVs were isolated from plasma samples using nanoscale flow cytometry, and total EV counts as well as counts by cellular origin were determined. MicroRNA was extracted from maternal plasma and levels quantified using qPCR. Maternal plasma from pregnancies complicated by fetal CHD exhibited higher total EV counts at delivery compared to control. Platelets derived extracellular vesicles (pdEVs) were significantly higher both in maternal and cord blood plasma at the time of delivery in CHD pregnancies compared to gestationally age-matched control pregnancies. Circulating miR22 and miR421 levels were reduced, while miR29c levels were increased in maternal plasma from CHD pregnancies between 16-24 weeks but no differences seen at time of delivery. Pregnancies complicated by CHD are associated with an altered in utero environment by changes in extracellular vesicles and miRNA profile in maternal serum. Circulating EVs and miRNA profiles may therefore serve as minimally invasive indicators of placental and maternal vascular dysfunction in CHD.

BackgroundAcute myeloid leukemia (AML) remains the most lethal acute leukemia in adults, with 5-year overall survival below 32% despite recent advances including venetoclax-, FLT3-, IDH1/2-, and Menin-targeted therapies. Clinical outcomes remain highly heterogeneous across patients, highlighting the need for robust molecular biomarkers capable of improving prognostic precision. MicroRNAs (miRNAs) are critical regulators of hematopoietic differentiation, apoptosis, and therapeutic resistance and are differentially expressed across AML subtypes. However, their clinical translation has been limited by high dimensionality, feature redundancy, and relatively small cohort sizes. MethodsWe developed and evaluated the AML Survival Estimator (AMLS), an inheritable bi-objective combinatorial genetic algorithm integrated with support vector regression (SVR), using TCGA-LAML miRNA expression profiles (n = 156). AMLS was benchmarked against ten widely used machine-learning approaches, including penalized regression, tree-based ensembles, support-vector regression, k-nearest neighbors, and multilayer perceptron models. Performance was assessed using stratified cross-validation with Pearson correlation (R), Harrells concordance index (C-index), and mean absolute error (MAE). Functional characterization of the derived miRNA signature was performed through consensus target integration followed by pathway enrichment, gene ontology analysis, network reconstruction, and Kaplan-Meier risk stratification. ResultsAMLS achieved superior prognostic performance with pooled out-of-fold metrics of Pearson R = 0.86, C-index = 0.788, and MAE = 7.49 months, substantially outperforming all comparator models. Restricting analyses to the AMLS-derived 28-miRNA signature improved all baseline learners by approximately 2-4-fold, with the multilayer perceptron achieving R = 0.674; however, none matched the native AMLS framework, indicating that the evolutionary optimization strategy contributes predictive information beyond feature selection alone. The prognostic signature included biologically established AML-associated miRNAs, including hsa-miR-191, hsa-miR-29c, hsa-miR-125b, hsa-miR-148a, hsa-miR-15b, hsa-miR-10b, and hsa-miR-30c, linked to DNA methylation, apoptosis, cell-cycle regulation, and oncogenic Wnt/MAPK signaling pathways. Functional analyses demonstrated significant enrichment of canonical AML-associated pathways, including p53, PI3K-AKT, TGF-{beta}, JAK-STAT, FoxO, and hematopoietic lineage signaling. ConclusionsOur findings demonstrate that evolutionary learning integrated with SVR can recover a compact and biologically interpretable miRNA prognostic signature that substantially outperforms conventional machine-learning approaches for AML survival prediction. The identified miRNA network converged on key leukemogenic pathways involved in apoptosis, cell-cycle regulation, and oncogenic signaling, supporting both the biological relevance and prognostic utility of the framework. Given the minimally invasive and quantitatively scalable nature of miRNA profiling, this approach may provide a practical molecular adjunct for improving prognostic assessment and precision medicine strategies in AML. Abstract FigureSchematic overview of the AMLS framework. Left: acute myeloid leukemia, a clonal hematological malignancy with persistent prognostic heterogeneity. Middle: AMLS couples an evolutionary learning-based feature selection algorithms to support vector regression for miRNA-based survival modeling. Right: AMLS recovers a 28-miRNA prognostic signature that predicts overall survival with Pearson R = 0.86 and MAE = 7.5 months. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/727196v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@11ead1org.highwire.dtl.DTLVardef@4f5c19org.highwire.dtl.DTLVardef@277de1org.highwire.dtl.DTLVardef@b95c9a_HPS_FORMAT_FIGEXP M_FIG C_FIG

In red blood cells (RBCs), the mechanosensitive channel PIEZO1 provides an upstream Ca2+ signal that activates TMEM16F, a Ca2+-activated phospholipid scramblase (CaPLSase) responsible for phosphatidylserine (PS) externalization. However, limited PIEZO1-mediated Ca{superscript 2} entry and the relatively low Ca2+ sensitivity of TMEM16F suggest the need for signal amplification. Here, we identify the Gardos (KCNN4) Ca2+-activated K channel as a critical amplifier of the PIEZO1-TMEM16F axis. Gardos activation induces membrane hyperpolarization, thereby increasing the driving force for Ca2+ entry and enhancing TMEM16F activation and phospholipid scrambling. Gardos-mediated amplification also contributes to excessive PS externalization in sickle cell disease (SCD) and hereditary xerocytosis (HX) RBCs, and functional disruption of Gardos-mediated K+ efflux attenuates this response. These findings demonstrate Gardos as a critical amplifier of RBC mechanotransduction and highlight Gardos as a potential therapeutic target for mitigating pathogenic PS exposure. HIGHLIGHTSO_LIGardos amplifies PIEZO1-mediated Ca{superscript 2} influx to promote TMEM16F-dependent phosphatidylserine exposure in healthy and diseased RBCs. C_LIO_LIUnexpected effects of some Gardos inhibitors may complicate the use in hematologic diseases. C_LI