Markers Of Endothelial Activation Research Articles

Imbalance in the vWF – ADAMTS13 axis exists early in acute pancreatitis and predicts persistent organ failure and pancreatic necrosis-a prospective study

BackgroundThe pathophysiology of Acute Pancreatitis (AP) may be complicated by endothelial activation. von Willebrand Factor (vWF)- ADAMTS13 axis is a marker of endothelial activation. The study aimed to investigate the axis in AP, comparing it in patients with and without persistent organ failure (OF), with and without pancreatic necrosis, and correlating it with the standard severity scores (CRP, APACHE II, BISAP, SOFA, and qSOFA) MethodsvWF-Antigen (vWF:Ag), vWF-Collagen-Binding-Assay (vWF:CBA), and ADAMTS13 activity (ADAMTS13:act) levels were measured within 5 days of symptom onset in consecutive patients (n = 98), who were admitted with a first episode of AP (Dec 2021–May 2023). ResultsOf the 98 patients admitted with AP, 78(79.6 %) had no or transient OF; 20(20.4 %) had persistent OF. Age was comparable (43.73 ± 15.36 vs 38.65 ± 13.69) [mean ± SD](years), and males were predominant in both groups (70.5 % vs 80 %). Patientswith persistent OF had higher vWF:CBA(%)[323(279–486.5) vs 199.5(159.1–295.75)] and lower ADAMTS13:act(%)[35.4(23.8–56.85) vs 56.35(44.1–71.9)][median (25th – 75th percentile)](P = 0.001) than those with no or transient OF. Patients with pancreatic necrosis (n = 19) had lower ADAMTS13:act(%)[42.79 ± 18.69] than those without pancreatic necrosis (n = 18) [62.49 ± 22.64] (P < 0.01). ADAMTS13:act had a negative correlation(r = −0.2), whereas vWF:Ag and vWF:CBA had a positive correlation (r = 0.2) with the standard severity scores (P < 0.05). ADAMTS13:act could predict pancreatic necrosis [AUROC-0.737, P < 0.05] and persistent OF [AUROC–0.746, P < 0.001], while vWF:CBA could predict persistent OF [AUROC– 0.73, P < 0.001]. ConclusionvWF-ADAMTS13 axis helps to predict severe disease and is associated with poor outcomes in acute pancreatitis.

#435 Endothelial activation and inflammation in chronic kidney disease (CKD): role of neutrophil extracellular traps (NETs)

Abstract Background and Aims Advanced chronic kidney disease (CKD), especially in stages 4-5, amplifies cardiovascular disease (CVD) risk by around 50%. This heightened susceptibility is linked to a 1.5 to 3-fold increase in venous thromboembolism incidence, driven by coagulation dysregulation and heightened inflammation. During inflammation, neutrophils can release extracellular traps (NETs), that was described contribute to endothelial damage, through adhesion molecule elevation, and tissue factor (TF) secretion, exacerbating thrombosis. Additionally, NETs interact with von Willebrand Factor (vWF) and factor XII, further amplifying thrombus formation and the activation of the intrinsic coagulation pathway. The aim of this study was to observe whether NETs may contribute to increased CVD risk in CKD patients by inducing endothelial activation and thromboinflammatory responses to a larger extent compared to NETs from healthy donors. Method For that, neutrophils were isolated from healthy donors’ and CKD patients’ (stages 3-5 HD) peripheral blood, and NETs were collected by stimulating neutrophils with PMA for 3.5 hours. Human aortic endothelial cells (HAoECs) were subsequently exposed to NETs for 3 to 24 hours, followed by gene expression analysis with a focus on inflammation, endothelial activation, and prothrombotic markers. Additionally, protein analysis of the supernatant was conducted using ELISA. All experiments were performed under ethical protocol EK 225-21 from the University Hospital of Aachen. Results Exposure to both healthy NETs as well as the ones isolated from CKD patients resulted in a notable upregulation of endothelial activation (VCAM-1), prothrombotic (TF), and inflammatory (IL-6) gene expression within 3 hours, without difference between healthy and CKD conditions. On the other hand, when examining the cellular supernatant, a significantly higher increase in vWF secretion was observed upon treatment with CKD-NETs compared to healthy NETs. Furthermore, while secretion of the inflammatory cytokine IL-6 presented a comparable increase upon treatment with NETs derived from healthy and CKD neutrophils, IL-1β secretion was markedly enhanced after CKD-NET treatment in comparison to healthy NETs. Conclusion In summary, our findings suggest that NETs exert a significant influence on endothelial cells, contributing to increased thromboinflammatory activation. Notably, CKD-NETs evoked a more rapid impact on the endothelium, accompanied by increased secretion of IL-1β and vWF, and underlying mechanisms will be further investigated.

Whole-blood transcriptome analysis reveals distinct gene expression signatures in paediatric patients with sickle cell anaemia before and after exercise.

Sickle cell anaemia (SCA) patients display elevated levels of circulating pro-inflammatory cytokines and endothelial activation markers compared to healthy peers. The impact of exercise on the pro-inflammatory state in SCA remains unclear. This study aimed to characterize the whole-blood transcriptome profile in response to an acute bout of exercise in paediatric SCA patients. Twenty-three SCA participants (13 ± 3 years, 52% girls) and 17 healthy controls (14 ± 3 years, 29% girls) performed eight 2-min bouts of cycle ergometry interspersed with 1-min rest intervals. Whole-blood transcriptome profile (RNA-seq) was performed before and after exercise. At baseline, gene pathways associated with gas transport in erythrocytes were up-regulated in SCA patients compared to controls. Following exercise, gene pathways associated with innate immunity were altered in both groups. Interaction analyses revealed 160 annotated genes (101 up- and 59 down-regulated) that differentially altered by exercise in SCA patients. Moreover, genes that exhibited a blunted response to exercise in SCA patients were enriched in the IL-17 signalling pathway, suggesting an impaired innate immune response to exercise. This data will contribute to the development of evidence-based exercise prescription guidelines for this patient population.

Activated factor X stimulates atrial endothelial cells and tissues to promote remodeling responses through AT1R/NADPH oxidases/SGLT1/2.

Atrial fibrillation (AF), the most common cardiac arrhythmia favoring ischemic stroke and heart failure involves left atrial remodeling, fibrosis and a complex interplay between cardiovascular risk factors. This study examined whether activated factor X (FXa) induces pro-remodeling and pro-fibrotic responses in atrial endothelial cells (AECs) and human atrial tissues and determined the underlying mechanisms. AECs were from porcine hearts and human right atrial appendages (RAA) from patients undergoing heart surgery. Protein expression levels were assessed by Western blot and immunofluorescence staining, mRNA levels by RT-qPCR, formation of reactive oxygen species (ROS) and NO using fluorescent probes, thrombin and angiotensin II generation by specific assays, fibrosis by Sirius red staining and senescence by senescence-associated beta-galactosidase (SA-β-gal) activity.In AECs, FXa increased ROS formation, senescence (SA-β-gal activity, p53, p21), angiotensin II generation and the expression of pro-inflammatory (VCAM-1, MCP-1), pro-thrombotic (tissue factor), pro-fibrotic (TGF-β and collagen-1/3a) and pro-remodeling (MMP-2/9) markers whereas eNOS levels and NO formation were reduced. These effects were prevented by inhibitors of FXa but not thrombin, protease-activated receptors antagonists (PAR-1/2) and inhibitors of NADPH oxidases, ACE, AT1R, SGLT1/SGLT2. FXa also increased expression levels of ACE1, AT1R, SGLT1/2 proteins which was prevented by SGLT1/2 inhibitors. Human RAA showed tissue factor mRNA levels that correlated with markers of endothelial activation, pro-remodeling and pro-fibrotic responses and SGLT1/2 mRNA levels. They also showed protein expression levels of ACE1, AT1R, p22phox, SGLT1/2, and immunofluorescence signals of nitrotyrosine and SGLT1/2 colocalized with those of CD31. FXa increased oxidative stress levels which were prevented by inhibitors of the AT1R/NADPH oxidases/SGLT1/2 pathway. FXa promotes oxidative stress triggering premature endothelial senescence and dysfunction associated with pro-thrombotic, pro-remodeling and pro-fibrotic responses in AECs and in human RAA involving the AT1R/NADPH oxidases/SGLT1/2 pro-oxidant pathway. Targeting this pathway may be of interest to prevent atrial remodeling and the progression of atrial fibrillation substrate.

Idiopathic multicentric Castleman disease - TAFRO results in high levels of mTOR activator SVEP1, tissue factor, and endotheliopathy

AbstractIdiopathic multicentric Castleman disease (iMCD) is an inflammatory disease associated with a cytokine storm, activation of the PI3K/AKT/mTOR pathway, coagulopathy, and increased risk of thrombosis. The mechanisms underlying these pathologic processes remain elusive. We studied novel markers of mTOR activation and thrombosis in 1 patient with typical features of iMCD with TAFRO (thrombocytopenia, anasarca, fevers, reticulin myelofibrosis, and organomegaly) syndrome (iMCD-TAFRO). Plasma levels of SVEP1 (Sushi, von Willebrand factor type A, epidermal growth factor, and pentraxin domain–containing 1 protein), a newly identified mTOR activator associated with cardiovascular diseases and dementia, in addition to cytokines, chemokines and components of the coagulation cascade and complement system were evaluated by enzyme-linked immunosorbent assay (ELISA) and arrays. Compared with healthy controls, a 15-fold increase in SVEP1 was observed. High levels of factor VIIa/antithrombin and microparticles expressing functional tissue factor (TF) were detected. The anticoagulants thrombomodulin and soluble endothelial protein C receptor were elevated, indicating shedding from endothelial cells. Plasminogen activator inhibitor 1 was increased, consistent with hypofibrinolysis, whereas high levels of C3b and C5a are in keeping with complement activation. Furthermore, markers of endothelial cell activation (e.g. von Willebrand factor, angiopoietin-2), cell adhesion molecules, and angiogenesis mediators were upregulated. SVEP1 emerges as a potential mechanism of mTOR activation in iMCD-TAFRO, while multiple pathways influence coagulopathy. Immunothrombosis emerges as a potential therapeutic target for iMCD.

108 A Translational Approach to Suppress Local Inflammation in the Ischemic Penumbra and Deliver Local Anti-platelet Activity After Stroke

INTRODUCTION: Mechanical thrombectomy is the standard of care for ischemic stroke, but there is a significant mismatch between successful recanalization and clinical outcomes. Inflammation in the ischemic penumbra remains a target for neuroprotective interventions even in recanalized and non-recanalized patients. P-selectin (Psel) is a marker of endothelial cell activation and could be used to deliver therapeutics locally to the site of ischemic injury. METHODS: The fusion protein Pselectin-Crry was constructed using single chain antibody sequence linked to the extracellular region of mouse Crry. In-vivo testing using surface plasmon resonance for binding affinity to Psel, and zymosan assay for complement inhibitory activity. In-vivo studies were performed in C57bl/6 mice subjected to transient middle cerebral artery occlusion for 1 hour followed by administration of Psel-Crry 2 hours after reperfusion. Psel-Crry was labelled using far-red fluorescence and live-animal imaging was used to study brain-specific targeting. The modified Neurological Severity (NS) score was used for functional outcome along with survival, infarct volume and histological outcomes. RESULTS: Psel-Crry demonstrated dose-dependent complement inhibitory activity, and localized specifically to the stroke brain on in-vivo live imaging. Ex-vivo imaging of explanted brain showed Psel-Crry binding specifically to the injured hemisphere without off-target binding (Stroke vs. Sham, p &lt; 0.001). Psel-Crry resulted in 60% reduction in infarct volume at 3 days compare to vehicle (N = 8/group, p &lt; 0.05), reduction in NS scores compared to vehicle (Median 3 vs. 1.5, p &lt; 0.05), and improved 1-week survival (80% vs. 50%, p &lt; 0.05). Histological analyses demonstrated preservation of dendritic arborization in the ischemic penumbra with Psel-Crry treatment with concurrent significant reduction in microglial activation. CONCLUSIONS: Psel-Crry is a translational agent that can achieve local complement inhibitory effect and antiplatelet activity after stroke and has promise for potential use as ajuvant therapy in stroke.

Oxidative stress, microparticles, and E-selectin do not depend on HIV suppression.

Oxidative stress and inflammation are considered predictors of diseases associated with aging. Markers of oxidative stress, inflammation, and endothelial activation were investigated in people with HIV on antiretroviral treatment to determine whether they had an immunosenescent phenotype that might predispose to the development of premature age-related diseases. This study was conducted on 213 subjects with HIV. The control groups consisted of healthy HIV-negative adults. The level of oxidative stress was measured by assessing the production of malondialdehyde levels, which were detected by thiobarbituric acid reactive substance (TBARS) assay. The level of microparticles indicated the presence of inflammation and endothelial activation was measured by E-selectin levels. Significant differences were determined by appropriate statistical tests, depending on the distribution of variables. Relationships between continuous variables were quantified using Spearman's rank correlation coefficient. TBARS, and microparticle and E-selectin levels were significantly higher in untreated and treated subjects with HIV compared with HIV-negative controls (P<0.001). The levels of the investigated markers were not significantly different between untreated and treated patients and no significant correlation of these markers was found with CD4+ count, CD4+/CD8+ ratio, and the number of HIV-1 RNA copies. Elevated markers of oxidative stress, inflammatory and endothelial activation were independent of the virologic and immunologic status of people with HIV. These results support the hypothesis that residual viremia in cellular reservoirs of various tissues is a key factor related to the premature aging of the immune system and predisposition to the premature development of diseases associated with aging.

Pathophysiological profile of non-ventilated lung injury in healthy female pigs undergoing mechanical ventilation

BackgroundLung regions excluded from mechanical insufflation are traditionally assumed to be spared from ventilation-associated lung injury. However, preliminary data showed activation of potential mechanisms of injury within these non-ventilated regions (e.g., hypoperfusion, inflammation).MethodsIn the present study, we hypothesized that non-ventilated lung injury (NVLI) may develop within 24 h of unilateral mechanical ventilation in previously healthy pigs, and we performed extended pathophysiological measures to profile NVLI. We included two experimental groups undergoing exclusion of the left lung from the ventilation with two different tidal volumes (15 vs 7.5 ml/kg) and a control group on bilateral ventilation. Pathophysiological alteration including lung collapse, changes in lung perfusion, lung stress and inflammation were measured. Lung injury was quantified by histological score.ResultsHistological injury score of the non-ventilated lung is significantly higher than normally expanded lung from control animals. The histological score showed lower intermediate values (but still higher than controls) when the tidal volume distending the ventilated lung was reduced by 50%. Main pathophysiological alterations associated with NVLI were: extensive lung collapse; very low pulmonary perfusion; high inspiratory airways pressure; and higher concentrations of acute-phase inflammatory cytokines IL-6, IL-1β and TNF-α and of Angiopoietin-2 (a marker of endothelial activation) in the broncho-alveolar lavage. Only the last two alterations were mitigated by reducing tidal volume, potentially explaining partial protection.ConclusionsNon-ventilated lung injury develops within 24 h of controlled mechanical ventilation due to multiple pathophysiological alterations, which are only partially prevented by low tidal volume.

Angiopoietin-2 is associated with sickle cell complications, including stroke risk, and decreases with hydroxyurea therapy

AbstractHydroxyurea reduces morbidity and mortality in children with sickle cell anemia (SCA). The endothelium is central to SCA-related complications including stroke. However, hydroxyurea’s impact on the endothelium is not well described. To address this gap, we measured plasma levels of endothelial activation markers (angiopoietin-2, P-selectin, soluble endothelial selectin [sE-selectin], soluble intercellular cellular adhesion molecule 1, and soluble vascular endothelial cellular adhesion molecule) by enzyme-linked immunosorbent assay after initiation of hydroxyurea therapy. Samples were collected from Ugandan children with SCA enrolled in a clinical trial evaluating hydroxyurea vs placebo (NOHARM trial). Samples were collected at enrollment; and then after 2, 4, and 12 months of follow-up. Longitudinal changes in biomarker levels were evaluated using linear mixed effects models. Transcranial Doppler (TCD) velocities were measured at 10 to 12 months follow-up to assess cerebral blood flow and primary stroke risk. Mediation analysis was used to explore causal pathways of hydroxyurea-mediated effects on TCD velocities. In total, 798 plasma samples were tested from 205 children (mean enrollment age, 2.2 years). At enrollment, higher levels of angiopoietin-2 were associated with a previous medical history of dactylitis, vaso-occlusive crises, acute chest syndrome, and transfusion (P < .05 for all). Hydroxyurea therapy at a fixed dose of 20 mg/kg per day decreased plasma angiopoietin-2, P-selectin, and sE-selectin levels over the study period (P < .05 for all). Angiopoietin-2 and sE-selectin were associated with higher TCD velocities. Mediation analysis suggests that hydroxyurea decreases TCD velocities through an increase in fetal and total hemoglobin. Increased fetal and total hemoglobin, and decreased white blood cell count may decrease TCD velocity, in part, through an angiopoiten-2–mediated pathway. This trial was registered at www.ClinicalTrials.gov as #NCT01976416.

Biomarkers of coagulation, endothelial, platelet function, and fibrinolysis in patients with COVID-19: a prospective study

Prospective and sequential evaluation of homeostatic changes leading to thrombosis across COVID 19 disease severity spectrum are limited. In this prospective observational study, haemostasis was evaluated in patients with mild, moderate-severe, and critical COVID-19 infection. Markers of endothelial activation [Soluble thrombomodulin (sTM), von Willebrand Factor (VWF)], platelet activation [Soluble P-selectin, beta-thromboglobulin (BTG)] and global haemostasis [Rotational thromboelastometry (ROTEM)] were evaluated on days 1 and 5 after admission. The study cohort comprised of 100 adult patients (mild = 20, moderate-severe = 22, critical = 58). Sixty-five patients received anticoagulation for 10 (7–14) days. Thrombotic events were seen in 9 patients. In-hospital mortality was 21%. Endothelial activation markers were elevated at baseline in all subgroups, with levels in moderate-severe (sTM = 4.92 ng/ml, VWF = 295 U/dl) [reference-ranges: sTM = 2.26–4.55 ng/ml; Soluble P-selectin = 13.5–31.5 ng/ml; BTG = 0.034–1.99 ng/ml] and critical patients (sTM = 6.07 ng/ml, VWF = 294 U/dl) being significantly higher than in the mild group (sTM = 4.18 ng/ml, VWF = 206 U/dl). In contrast, platelet activation markers were elevated only in critically ill patients at baseline (Soluble P-selectin = 37.3 ng/ml, BTG = 2.51 ng/ml). The critical group had significantly lower fibrinolysis on days 1 and 5 when compared with the moderate-severe arm. COVID-19 infection was associated with graded endothelial activation and lower fibrinolysis that correlated with illness severity.

Pneumonia in the first week after polytrauma is associated with reduced blood levels of soluble herpes virus entry mediator.

Pneumonia develops frequently after major surgery and polytrauma and thus in the presence of systemic inflammatory response syndrome (SIRS) and organ dysfunction. Immune checkpoints balance self-tolerance and immune activation. Altered checkpoint blood levels were reported for sepsis. We analyzed associations of pneumonia incidence in the presence of SIRS during the first week of critical illness and trends in checkpoint blood levels. Patients were studied from day two to six after admission to a surgical intensive care unit (ICU). Blood was sampled and physician experts retrospectively adjudicated upon the presence of SIRS and Sepsis-1/2 every eight hours. We measured the daily levels of immune checkpoints and inflammatory markers by bead arrays for polytrauma patients developing pneumonia. Immune checkpoint time series were additionally determined for clinically highly similar polytrauma controls remaining infection-free during follow-up. We performed cluster analyses. Immune checkpoint time trends in cases and controls were compared with hierarchical linear models. For patients with surgical trauma and with and without sepsis, selected immune checkpoints were determined in study baseline samples. In polytrauma patients with post-injury pneumonia, eleven immune checkpoints dominated subcluster 3 that separated subclusters 1 and 2 of myeloid markers from subcluster 4 of endothelial activation, tissue inflammation, and adaptive immunity markers. Immune checkpoint blood levels were more stable in polytrauma cases than controls, where they trended towards an increase in subcluster A and a decrease in subcluster B. Herpes virus entry mediator (HVEM) levels (subcluster A) were lower in cases throughout. In unselected surgical patients, sepsis was not associated with altered HVEM levels at the study baseline. Pneumonia development after polytrauma until ICU-day six was associated with decreased blood levels of HVEM. HVEM signaling may reduce pneumonia risk by strengthening myeloid antimicrobial defense and dampening lymphoid-mediated tissue damage. Future investigations into the role of HVEM in pneumonia and sepsis development and as a predictive biomarker should consider the etiology of critical illness and the site of infection.

Damage-associated cellular markers in the clinical and pathogenic profile of vaccine-induced immune thrombotic thrombocytopenia

BackgroundAdenoviral vector-based COVID-19 vaccine-induced immune thrombotic thrombocytopenia (VITT) is rare but carries significant risks of mortality and long-term morbidity. The underlying pathophysiology of severe disease is still not fully understood. The objectives were to explore the pathophysiological profile and examine for clinically informative biomarkers in patients with severe VITT. MethodsTwenty-two hospitalized patients with VITT, 9 pre- and 21 post-ChAdOx1 vaccine controls, were recruited across England, United Kingdom. Admission blood samples were analyzed for cytokine profiles, cell death markers (lactate dehydrogenase and circulating histones), neutrophil extracellular traps, and coagulation parameters. Tissue specimens from deceased patients were analyzed. ResultsThere were strong immune responses characterized by significant elevations in proinflammatory cytokines and T helper 1 and 2 cell activation in patients with VITT. Markers of systemic endothelial activation and coagulation activation in both circulation and organ sections were also significantly elevated. About 70% (n = 15/22) of patients met the International Society for Thrombosis and Haemostasis criteria for disseminated intravascular coagulation despite negligible changes in the prothrombin time. The increased neutrophil extracellular trap formation, in conjunction with marked lymphopenia, elevated lactate dehydrogenase, and circulating histone levels, indicates systemic immune cell injury or death. Both lymphopenia and circulating histone levels independently predicted 28-day mortality in patients with VITT. ConclusionThe coupling of systemic cell damage and death with strong immune-inflammatory and coagulant responses are pathophysiologically dominant and clinically relevant in severe VITT.

Resolvin-D1 (RvD1) and Its Association with Markers of Inflammation, Endothelial Activation, Hemolysis and Clinical Correlates in Children with Sickle Cell Disease (SCD)

Introduction: Sickle Cell Disease (SCD) is an inflammatory and vaso-occlusive pathology. Multiple lipid mediators: resolvins, protectins and maresins, collectively known as Specialized Pro-resolving Mediators (SPMs), that are biosynthesized from Omega-3-PolyUnsaturated Fatty Acids (O3PUFAs), docosahexaenoic acid and eicosapentaenoic acid, appear to play a role in the resolution of inflammation. In a recent study, we have documented the presence of SPMs including Resolvin-D1 (RvD1), 17R-RvD3, and their pathway markers in plasma from both children with SCD and age-and race-matched controls with a significant increase in RvD1 levels noted in plasma from children with SCD. In an SCD mouse model exposed to hypoxia/reoxygenation, a previous study has shown that oral administration of 17R-RvD1, the longer acting isomer of RvD1, promotes resolution of SCD-associated inflammation and tissue damage. Objectives: We assessed whether there were any associations in children with SCD between baseline plasma levels of RvD1 and either markers of inflammation, endothelial activation and hemolysis or SCD-related clinical correlates. Methods: Multiplex assays were employed to assess endothelial activation markers (soluble adhesion molecules VCAM-1, ICAM-1 and E-selectin), and inflammatory cytokines (IL-1β, IL-4, IL-6, IL-10, TNF-α and CRP) in plasma. A 3-year retrospective review of individual SCD patient records was performed, and the number of painful episodes of vaso-occlusion or VOC, including those complicated by the occurrence of acute chest syndrome (ACS) were recorded. Total cumulative painful episodes over a 3-year period were included in the analysis. Spearman Rank Correlation was employed to assess the association between two variables. Results: Consistent with the published results, levels of endothelial activation markers (soluble VCAM-1 and soluble E-selectin), cytokines (IL-4, IL-6 and TNF-α), CRP and LDH were increased in SCD plasma compared to controls (Table-1). No significant associations were found in SCD between RvD1 or other lipid mediators and markers of endothelial activation or inflammation (Table-2). However, there were two significant associations noted between 5-HEPE and IL-1β, and between 12-HETE and IL-6. Plasma levels of the hemolytic marker LDH positively correlated with PGE2, TxB2, 7-HDHA, 5-HEPE and 5-HETE (Table-2). Review of medical history revealed 33 of 45 subjects (73%) had at least one VOC episode, and 17 (38%) had at least one ACS episode in the past 3 years. Subjects who had at least one ACS episode in the past 3 years also reported at least one VOC episode during the same period. There was no statistically significant difference in the RvD1 level by VOC [median RvD1 (ng/ml) of 0.056 in VOC -ve sub-group (n=12) vs 0.084 in VOC +ve sub-group (n=33), p=0.748] or ACS history [median RvD1 (ng/ml) of 0.060 in ACS -ve sub-group (n=28) vs 0.104 in ACS +ve sub-group (n=17), p=0.256]. There was no statistically significant association between levels of RvD1 and the total number of VOC/ACS in the past 3 years (correlation coefficient = -0.06, p=0.694). Summary and Conclusions: Because of the documented pro-resolving and anti-inflammatory effects of orally administered 17R-RvD1 in a mouse model of SCD, and elevated basal plasma levels of RvD1 in children with SCD, we investigated whether endogenous RvD1 provides protection against inflammation and clinical complications in SCD children. Other lipid mediators, which were synthesized from O3PUFAs and arachidonic acid, present in increased levels in SCD plasma and potentially involved in modulation of inflammation were also included in the analysis. Unlike in SCD mouse model in which exogenous RvD1 exerted anti-inflammatory effect in ex-vivo experiments and orally administered 17R-RvD1 promoted resolution of inflammation and down-regulated adhesion molecule expression and cytokine production, endogenous RvD1 did not show any significant association with markers of inflammation and endothelial activation or clinical correlates in a small group of SCD children under healthy condition. No significant findings were noted with other lipid mediators. Whether these lipid mediators modulate inflammation during crisis, pre- or post-crisis clinical conditions and whether their production is different during these clinical conditions compared to steady state healthy condition require further investigation.

Blood-Brain Barrier and Neuronal Damage during Icans in Patients Treated with Anti-CD19 CAR-T Cells

Background Immune effector cell-associated neurotoxicity syndrome (ICANS) is the most frequent neurological toxicity occurring after treatment with chimeric antigen receptor T-cells (CAR-T), with a reported incidence of around 30-50% according to different settings and products. Clinical manifestations may widely range from dizziness to seizures, and tend to achieve response with high-dose steroids. The pathogenesis of ICANS is unclear; despite some data suggest a role for endothelial activation and impairment of the blood-brain barrier (BBB). Glial fibrillary acidic protein (GFAP) and neurofilament light chain (NFL) are reliable astroglial and axonal neuronal damage biomarkers, respectively. NFL provides structural support to neurons, while GFAP is crucial for astrocyte function and BBB maintenance. Elevated levels of NFL and GFAP in cerebrospinal fluid indicate BBB damage, aiding the diagnosis and monitoring of neurological conditions like multiple sclerosis and traumatic brain injury. Recently, preliminary studies reported that NFL serum levels correlate with the severity of neurotoxicity after CAR T-cell treatment. Aim and Methods In our study, we measured GFAP and NFL both at baseline and at day 7 in 34 patients with R/R B-cell malignancies treated with CD19 CAR-T cells (15 Axi-cel, 12 Tisa-cel, 7 Brexu-cel), and explored their association with clinically relevant neurological toxicities and markers of endothelial impairment. We have collected peripheral blood samples from patients treated with CD19-directed CAR-T cells at baseline and at every other day up to 14 days after CAR-T infusion. We have measured GFAP and NFL levels and compared them with clinical neurotoxicity and serum markers of endothelial damage or inflammatory activity. Results Elderly patients tended to have higher values of GFAP at day 0 and to raise those levels after CAR-T infusion. When measured after infusion, GFAP positively correlated with endothelial biomarkers such as von Willebrand factor (p=0.041) and sST2 (p=0.015) and got some trends with IL-2R (p=0.050) and SUPAR (p=0.074). NFL positively correlated with the vWF (p=0.021) and with the endothelial activation score mEASIX (p=0.015) (Figure 1A). Overall, 11/34 patients (33%) developed ICANS. Baseline GFAP and NFL values did not impact the subsequent development of CRS or ICANS. When compared to the baseline, an increase of NFL was observed in 50% of cases. Patients experiencing an increase in NFL values after CAR-T infusion were more likely to develop ICANS of higher grades (p=0.046, OR 4.14) and to receive treatment with steroids (p=0.023). The median increase of NFL in patients with ICANS was of 25% of baseline levels. When representing values measured after CAR-T infusion in a heatmap, it emerges that a profile with higher GFAP and NFL may describe patients with ICANS of a higher grade (Figure 1B). Conclusion To sum up, our data confirm a correlation between severe ICANS and elevated NFL and G-FAP serum levels. Interestingly, these data also correlate with markers of endothelial activation, suggesting an active role of BBB impairment - considered in both its components- and concomitant neuronal damage in the pathogenesis of ICANS.

Unraveling the Mechanisms of P-Selectin in Sickle Cell Disease Using a Microvascular-on-a-Chip Model

Individuals who suffer from Sickle Cell Disease (SCD) is known to have multifaceted complications one such being vaso-occlusive crises, which result from blockages in their blood vessels. Recently, progress has been made to get drugs to the marketplace that address this issue. Several of these drugs target p-selectin, a transmembrane protein that facilitates the adhesion of platelets, leukocytes, and activated endothelial cells. P-selectin Glycoprotein Ligand-1 (PSGL-1), a P-selectin antagonist, has been extensively researched in vitro for its ability to mediate Microvascular obstruction through leukocyte interactions, particularly leukocyte rolling. Particularly, in human patients rather than animal models, the precise role of P-selectin in the interactions between activated platelets and endothelial cells remains unclear. This study delves into this interaction using a 3D vein-on-a-chip model that can capture microvascular obstruction formation at a similar scale to the microvascular. Our data shows that P Selectin in SCD inhibition is the attenuation mainly driven by platelets with contributions from WBC rather than driven by the activated endothelium. Through the creation of a PDMS branching 32-channel microfluidic, the cell interactions and obstruction of the microvascular can be quantified. Additionally, this model serves as an analog to test the various intercellular PSGL-1 interactions. Endothelial cells are seeded into the channels and cultured under flow to create 3D structures. Once confluent, healthy, and SCD patient blood samples collected in sodium citrate can be recalcified and perfused into the endothelial network to simulate physiological conditions then endothelial activation markers like P Selectin can be tagged, imaged, and quantified. Microvascular obstruction can be imaged in live time and quantified primarily through area and accumulation. Finally, various PSGL-1 analogs can be doped into the blood before and after perfusion to simulate prophylaxis or treatment. This system allows for the study of PSGL-1's effect on the microvascular obstruction process and its effect on individual cell types through tagging [Figure 1A]. When investigating PSGL1 analogs, the system does indicate a significant reduction in microvascular obstruction, quantified by the amount of occlusion within the channels [Figure 1B &amp; 1C]. With a cohort of n=10 SS patients on Hydroxyurea, there is a downstream effect on the amount of occlusion, specifically the percentage of the area of the channel that is occluded. Each patient is compared against itself to serve as its control, and each patient is perfused into three near-identical chips of treated and three untreated [Figure 1D]. When targeting CD41 and CD45 within the model, occlusions seem to be primarily driven by activated platelets, an indication that PSGL-1's interactions may be more involved in platelets than leukocyte dynamics. Work is ongoing to target this specific interaction. PSGL1's interaction with the activated endothelium is driving microvascular obstruction to be dominated by activated platelet leukocyte interactions. Current data shows that attenuation of P Selectin decreases vascular obstruction. PSGL-1 cell interactions and PSGL-1's effect on occlusion mitigation are quantified. By quantifying PSGL-1's interactions through a vein-on-a-chip model, SCD patient-specific drug interactions can be identified. Additionally, a greater understanding of how these drugs affect vaso-occlusive crisis and SCD.

Survival Advantage Associated with Heterozygous Factor V Leiden Mutation in Sars-Cov-2-Infected K18-hACE2 Mice

The pathogenesis of coronavirus disease 2019 (COVID-19) complications in critically ill patients involves virus-mediated cellular damage in infected tissue and secondary organ damage associated with a hyperinflammatory response. The proinflammatory milieu can result in endothelial dysfunction, predisposing patients to thrombosis leading to a life-threatening condition known as COVID-19-associated coagulopathy (CAC) which is driven by cellular and molecular mechanisms that are still unclear. We developed a mouse model based on the K18-hACE2 transgenic mouse recapitulating many of the CAC-associated pathological findings observed in human patients offering a reliable animal model for the study of SARS-CoV-2 pathogenesis. Plasma analysis revealed virus-induced upregulation of endothelial activation markers and alterations of all the measured coagulation factors' activities, resulting first in increased vascular leakage in the lung on day 5 post-infection, followed by the appearance of focal hemorrhagic lesions potentially associated with local thrombotic events on day 7. The infection-associated procoagulant phenotype appeared as early as day 3 post-infection (p.i.). Prothrombin time showed a 1/3 reduction in the normal clotting time in males. The overall procoagulant state was also reflected in the alteration of Factor(F) VIII and FX activity increasing to ~150% compared to uninfected controls. FIX showed the most significant alteration and was ~200% of the normal activity on day 3 p.i. and gradually increased over time, reaching ~350%. We also observed an increase in the activity of FV starting 3 days p.i. at ~210% of the normal level and persisting unchanged up to day 7 p.i. As reported during human infections, FV activity increased without significant changes in protein concentration or FVa levels. Natural anticoagulant and fibrinolytic mechanisms were dysregulated during virus infection resulting in a profound imbalance in thrombus formation and resolution. Infected K18-hACE2 mice showed downregulation of plasma protein C concentration which was not associated with any alteration of liver function, while plasma protein S concentration remained unaltered. Given the procoagulant environment generated after SARS-CoV-2 infection, reducing activated protein C (APC) anticoagulant and cytoprotective activities by blocking APC's active site with the SPC-54 monoclonal antibody worsened disease outcome and brought the mortality rate from 50% to 90%. In this observed procoagulant pathological context, where we observe increased activity of FV and where a deficiency of APC functions resulted in increased mortality, it wouldn't be surprising to observe an even more severe phenotype with the addition of APC resistance. To test this idea, we generated K18-hACE2 mice harboring the homologous human FV Leiden (FV L) mutation (R504Q). The Leiden mutation in FV eliminates one of several sites in FVa that are substrates for proteolysis by APC, resulting in diminished inactivation of FVa by APC and preventing the formation of the anticoagulant form of FV that serves as a cofactor for protein S-dependent inactivation of FVIIIa by APC. Upon SARS-CoV-2 infection, the presence of the homozygous FV L mutation in K18-hACE2 mice didn't affect the severity of the disease as measured by body weight loss during the course of the infection, and homozygous mutated mice had the same mortality rate as FV WT mice. Surprisingly, K18-hACE2 male mice carrying a heterozygous FV L mutation were extremely protected from SARS-CoV-2 infection, as their mortality dropped from 50% to only 15%. Histological analysis reveals how heterozygous FV L mice displayed a great reduction in immune infiltrate-associated fibrin deposition, suggesting an unexpected protective role of this prothrombotic mutation in the development of CAC. The protective effect of FV L heterozygosity may be attributable to enhanced thrombin formation which boosts inflammation and enhanced APC generation controlling proinflammatory and prothrombotic signaling, thus showing a delicate balance achieved by FV L heterozygosity that was lost for FV L homozygosity. As seen also in mouse models of bacterial sepsis, such favorable effects associated with FV L support the somewhat counterintuitive speculation that a partial increase in thrombin production in the early phase of the infection may support the resolution of COVID-19 complications.

High-fat diet promotes coagulation and endothelial activation in Sprague Dawley rats: Short-term effects of combined oral contraceptives

BackgroundCombined oral contraceptives (COCs), use in individuals are associated with increased risk of thrombotic events. This highlights the significance of assessing the impact of COC on promoting coagulation and endothelial activation in high-fat diet (HFD)-fed Sprague Dawley rats. MethodsTwenty (20) five-weeks-old female Sprague Dawley rats weighing between 150 and 200g were subjected to both LFD and HFD-feeding for 8-weeks to determine its influence on basic metabolic status, hemostatic profile, hemodynamic parameters (blood pressure and heart rate), as well as selected biomarkers of coagulation (tissue factor and D-dimer) and endothelial activation (Von Willebrand factor and nitric oxide). Thereafter HFD-fed animals were treated with receive high dose combined oral contraceptive (HCOC) and low dose combine oral contraceptive (LCOC) for 6 weeks. ResultsOur results showed that beyond weight gain, HFD-feeding was associated with hyperglycemia, increased mean arterial pressure, and reduced nitric oxide levels when compared with LFD group (p<0.05). Interestingly, treatment with high dose of COC for 6-weeks did not significantly alter atherothrombotic markers (p>0.05). However, this study is not without limitation as regulation of these markers remains to be confirmed within the cardiac tissues or endothelial cells of these animals. ConclusionHFD-feeding orchestrate the concomitant release of pro-coagulants and endothelial activation markers in rats leading to haemostatic imbalance and endothelial dysfunction. Short-term treatment with COC shows no detrimental effects in these HFD-fed rats. Although in terms of clinical relevance, our findings depict the notion that the risk of CVD in association with COC may depend on the dosage and duration of use among other factors especially in certain conditions. However, additional studies are required to confirm these findings, especially long-term effects of this treatment within the cardiac tissues or endothelial cells of these animals in certain conditions relating to postmenopausal state.

Evaluation of Circulating Endothelial Cells (CECs) As Marker of Endothelial Damage in Allo-Transplanted Patients at High Risk of Hepatic Veno-Occlusive Disease/Sinusoidal Obstruction Syndrome (VOD/SOS): The Cecinvod Study

INTRODUCTION Sinusoidal obstruction syndrome (SOS), also known as veno-occlusive disease (VOD), is a potentially fatal complication after allogeneic stem cell transplantation (alloSCT). Identifying a predictive biomarker for VOD has been challenging. Since endothelial injury is considered one of the main pathogenic factors for VOD onset, with the CECinVOD prospective study we aimed to evaluate Circulating Endothelial Cells (CEC) in allo-transplanted patients (pts) at higher risk to develop VOD. METHODS From October 2020 to November 2022, 150 pts have been enrolled in the CECinVOD study from 11 Italian Bone Marrow Transplantation Units. All pts must be older than 18 years and undergoing myeloablative alloSCT. CECs were detected using the CellSearch system, the FDA-approved immunomagnetic selection approach incorporating ferrofluid nanoparticles (anti CD146) and fluorophore-labelled antibodies (anti CD105, CD45 and DAPI). CEC were defined as CD146+, CD105+, DAPI+ and CD45-. CEC were collected at the following timepoints: before conditioning regimen (T0), at the end of conditioning regimen and before alloSCT (T1), at the time of neutrophils engraftment (T2), and 7-10 days after engraftment (T3). In pts who developed VOD, additional timepoints were collected as follows: at any time of suspected or proven VOD onset (T4), and then weekly during Defibrotide treatment (T5-T8). SOS/VOD was defined according to the 2016 European Group for Blood and Marrow Transplantation criteria. RESULTS Pts' main characteristics are summarized in Table 1. Six out of 150 pts (4%) developed VOD during the follow up (4 “severe”, and 2 “very severe”). All pts were treated with Defibrotide, obtaining a complete remission in 5 of them, while 1 pt died due to VOD complications. Pts receiving TBI-based regimen were more likely to develop VOD compared to those receiving Treosulfan (10 to 14 g/m2) or Busulfan ev (9.6 to 12.8 mg/kg) (p 0.08). Similarly, higher baseline levels of bilirubin were associated with a higher incidence of VOD (p 0.08). Considering the CECs analysis, 615 samples were evaluated. At the enrollment, CECs levels were not related with any of clinical characteristics analyzed, except for the number of previous treatments. Indeed, those pts with 2 or more previous lines of treatments had higher levels of CECs (OR=0.53; p&amp;lt;0.001). Considering the different timepoints, conditioning regimen and alloSCT result in higher levels of CECs. Thus, CEC were higher at T1 than at T0 (p 0.02), as well as they were even higher at T2 than at T1 (p&amp;lt;0.0001) (Fig.1). Conversely, no significant differences have been observed between T3 and T2. No other clinical characteristic was correlated with CEC counts at the different timepoints. Pts who developed VOD had higher median levels of CEC at all the timepoints analyzed, but this difference has not achieved statistical significance, probably due to the low number of pts in the VOD group. At VOD onset, the pts always had an increase in CEC levels compared with the previous timepoint. After defibrotide treatment, the CEC levels increased in the first week, while they progressively decreased during the VOD treatment (T6 and T7, -50,7% and -71,5%, respectively). Recently, another endothelial activation marker has been considered: the Easix score. We didn't find any relationship between the Easix score at transplant and the VOD onset, even if a trend may be observed. We also investigated whether the CEC levels may be related with Easix score, but we didn't find any relationship at each timepoint. Interestingly, in a subgroup of pts CECs have been observed as a cluster of multiple cells. This data is firstly described in allo-SCT. Its functional significate remains unclear, but we found a relationship between CECs cluster and the number of CECs (p &amp;lt; 0.001). CONCLUSIONS The incidence of VOD in our prospective study was low with respect to the one reported in the literature. This can be explained by the changes in alloSCT from the past (better selection of the pts, lower use of TBI, higher use of reduced intensity regimen) and the retrospective nature of most of the previous reports. We show that CECs can be considered reliable marker of endothelial damage in alloSCT pts, highlighting the impact of previous treatments, the conditioning regimen, and allo-SCT itself. Increased CEC level may be helpful to confirm VOD diagnosis, as well as their monitoring may be useful to evaluate the response to the treatment for VOD.

SERUM HUMANIN IN PEDIATRIC SEPTIC SHOCK-ASSOCIATED MULTIPLE-ORGAN DYSFUNCTION SYNDROME.

Background: Multiple-organ dysfunction syndrome disproportionately contributes to pediatric sepsis morbidity. Humanin (HN) is a small peptide encoded by mitochondrial DNA and thought to exert cytoprotective effects in endothelial cells and platelets. We sought to test the association between serum HN (sHN) concentrations and multiple-organ dysfunction syndrome in a prospectively enrolled cohort of pediatric septic shock. Methods: Human MT-RNR2 ELISA was used to determine sHN concentrations on days 1 and 3. The primary outcome was thrombocytopenia-associated multiorgan failure (TAMOF). Secondary outcomes included individual organ dysfunctions on day 7. Associations across pediatric sepsis biomarker (PERSEVERE)-based mortality risk strata and correlation with platelet and markers of endothelial activation were tested. Results: One hundred forty subjects were included in this cohort, of whom 39 had TAMOF. The concentration of sHN was higher on day 1 relative to day 3 and among those with TAMOF phenotype in comparison to those without. However, the association between sHN and TAMOF phenotype was not significant after adjusting for age and illness severity in multivariate models. In secondary analyses, sHN was associated with presence of day 7 sepsis-associated acute kidney injury ( P = 0.049). Furthermore, sHN was higher among those with high PERSEVERE-mortality risk strata and correlated with platelet counts and several markers of endothelial activation. Conclusion: Future investigation is necessary to validate the association between sHN and sepsis-associated acute kidney injury among children with septic shock. Furthermore, mechanistic studies that elucidate the role of HN may lead to therapies that promote organ recovery through restoration of mitochondrial homeostasis among those critically ill.

Increased ferroptosis in leukocytes from preeclamptic women involving the long non-coding taurine upregulated gene 1 (TUG1).

Ferroptosis plays a key role in placental development and physiology, and abnormal ferroptosis has been implicated in trophoblast injury leading to preeclampsia (PE). We hypothesize that leukocytes isolated from PE exhibit increased ferroptosis and that extracellular vesicles contain long non-coding (lnc) RNA/mRNAs that modulate oxidative stress and iron toxicity in vascular endothelial cells. We measured the expression of key regulators of ferroptosis in leukocytes and extracellular vesicles as well as circulating biomarkers of iron homeostasis and oxidative stress in plasma from women with/without PE at different timepoints during pregnancy. For markers that were dysregulated, we assessed their temporal correlation with established markers of disease activity and marker of endothelial activation. For markers dysregulated in early pregnancy, we assessed their ability to predict the development of PE. We found decreased lncRNA/mRNAs in leukocytes, but not extracellular vesicles, in PE that may modulate oxidative stress and iron toxicity. This decrease in anti-ferroptotic markers does not appear to be related to maternal disease activity or plasma oxidative stress status but rather to attenuated anti-inflammatory expression in these cells. Circulating ferritin was elevated in PE, supporting the hypothesis that PE represents a disbalance in iron homeostasis. Low lncRNA taurine upregulated gene 1 RNA levels in leukocytes at 22-24 weeks were strongly associated with the development of PE. Our findings suggest that maternal leukocytes in PE show decreased anti-ferroptotic activity that correlates with anti-inflammatory expression. Moreover, some of these changes in ferroptotic activity appear to precede the development of PE.