Markers Of Endothelial Cell Activation Research Articles

Trauma promotes heparan sulfate modifications and cleavage that disrupt homeostatic gene expression in microvascular endothelial cells.

Introduction: Heparan sulfate (HS) in the vascular endothelial glycocalyx (eGC) is a critical regulator of blood vessel homeostasis. Trauma results in HS shedding from the eGC, but the impact of trauma on HS structural modifications that could influence mechanisms of vascular injury and repair has not been evaluated. Moreover, the effect of eGC HS shedding on endothelial cell (EC) homeostasis has not been fully elucidated. The objectives of this work were to characterize the impact of trauma on HS sulfation and determine the effect of eGC HS shedding on the transcriptional landscape of vascular ECs. Methods: Plasma was collected from 25 controls and 49 adults admitted to a level 1 trauma center at arrival and 24h after hospitalization. Total levels of HS and angiopoietin-2, a marker of pathologic EC activation, were measured at each time point. Enzymatic activity of heparanase, the enzyme responsible for HS shedding, was determined in plasma from hospital arrival. Liquid chromatography-tandem mass spectrometry was used to characterize HS di-/tetrasaccharides in plasma. In vitro work was performed using flow conditioned primary human lung microvascular ECs treated with vehicle or heparinase III to simulate human heparanase activity. Bulk RNA sequencing was performed to determine differentially expressed gene-enriched pathways following heparinase III treatment. Results: We found that heparanase activity was increased in trauma plasma relative to controls, and HS levels at arrival were elevated in a manner proportional to injury severity. Di-/tetrasaccharide analysis revealed lower levels of 3-O-sulfated tetramers with a concomitant increase in ΔIIIS and ΔIIS disaccharides following trauma. Admission levels of total HS and specific HS sulfation motifs correlated with 24-h angiopoietin-2 levels, suggesting an association between HS shedding and persistent, pathological EC activation. In vitro pathway analysis demonstrated downregulation of genes that support cell junction integrity, EC polarity, and EC senescence while upregulating genes that promote cell differentiation and proliferation following HS shedding. Discussion: Taken together, our findings suggest that HS cleavage associated with eGC injury may disrupt homeostatic EC signaling and influence biosynthetic mechanisms governing eGC repair. These results require validation in larger, multicenter trauma populations coupled with in vivo EC-targeted transcriptomic and proteomic analyses.

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 < 0.001). Psel-Crry resulted in 60% reduction in infarct volume at 3 days compare to vehicle (N = 8/group, p < 0.05), reduction in NS scores compared to vehicle (Median 3 vs. 1.5, p < 0.05), and improved 1-week survival (80% vs. 50%, p < 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.

Fibrillar tau induces blood brain barrier permeability, endothelial cell activation and bioenergetic alterations

AbstractBackgroundAlzheimer’s Disease, the most common form of dementia, is characterized by the accumulation of amyloid‐beta plaques and neurofibrillary tangles formed by hyperphosphorylated tau in the brain, leading to neuronal loss and cognitive decline. The neurovascular unit, a complex system of several cell types surrounding brain vessels, is extremely important for blood flow regulation, amyloid and tau clearance, and proper brain function. Tau aggregates produced by neurons and glial cells reach endothelial cells (EC) at vessel walls by tau spreading. Currently, the mechanisms responsible for the effects of tau on ECs remain to be understood. We hypothesize that aggregated tau fibrils mediate alterations in bioenergetics in these cells, associated to pro‐inflammatory EC activation, BBB permeability and eventually EC death.MethodImmortalized human brain microvascular endothelial cells (D3) and primary human cerebral endothelial cells (hCECs) were challenged with aggregated 1N4R tau fibrils (5nM‐25nM) for 3‐72h. Trans‐endothelial electrical resistance (TEER) was measured by the ECIS Zθ system as an in vitro model of the BBB. Cytokine production and release was assessed using a V‐Plex Neuroinflammatory Panel 1 (MSD). We performed immunocytochemical analysis of junction protein expression and western blotting for EC activation markers. EC bioenergetics were measured by Seahorse Extracellular Flux Analyzer and EC death was determined by Cell Death Elisa and LDH Release (Roche).ResultTEER was reduced following incubation with fibrillar 1N4R tau. VCAM‐1 expression significantly increased with 25nM tau (24h) and 5nM (48h), and ICAM‐1 followed similar trends. Junction protein (ZO‐1, VE‐Cadherin) expression was decreased. Aggregated tau (25nM) caused an increase in glycolysis at 24‐hours, which reverted at further timepoints, whereas mitochondrial ATP production increased (48h) and then decreased (72h). After 72h treatment, 25nM tau induced a significant increase in apoptotic cell death and LDH release, yet no significant changes in cell death were observed at previous timepoints.ConclusionOur results suggest that tau fibrils mediate a stress response with a metabolic shift in ECs, in association with pro‐inflammatory EC activation. These molecular changes can contribute to BBB permeability and eventually cell death, further exacerbating cerebrovascular dysfunction.

Inhibition of mitochondrial carbonic anhydrases prevents Amyloid β‐induced cell death and blood brain barrier permeability in cerebrovascular endothelial cells

AbstractBackgroundThe Neurovascular Unit (NVU) is an important multicellular structure of the central nervous system (CNS) which has been observed to become dysfunctional in Alzheimer Disease (AD) and cerebral amyloid angiopathy (CAA). Amyloidosis disrupts the function of cerebrovascular endothelial cells (ECs) through the activation of the intrinsic apoptotic pathway, and the induction of blood brain barrier (BBB) permeability. Previous findings in our lab demonstrate that Acetazolamide (ATZ) and methazolamide (MTZ), pan‐Carbonic Anhydrase (CA) inhibitors, prevent mitochondria mediated apoptosis in cerebrovascular ECs through the reduction of Aβ induced cytochrome C release from the mitochondria, caspase‐9 activation, and mitochondrial H2O2 production. Due to the reduction of mitochondria mediated cell death via pan‐CA inhibitors we hypothesize that the mitochondrial isoforms, CA‐VA/B, are involved in cell death and other deleterious mechanisms induced by Aβ. Thus, we anticipate using selective CA‐V inhibitors will reduce cerebrovascular EC apoptosis and prevent the loss of BBB resistance.MethodHuman cerebral microvascular ECs (hCMECs) were treated with Aβ40‐Q22 Dutch mutant alone and in combination with a CA‐V inhibitor. We measured outcomes of mitochondria mediated apoptosis and BBB integrity. Apoptosis was evaluated by measuring the changes in the protein expression of caspase‐9, BAX, and BCL‐2 with western blot. We used live‐cell imaging to measure active caspase‐3. The release of cytochrome C and the mitochondrial membrane potential was detected using immunocytochemistry (ICC). Mitochondria H2O2 production was measured by isolating mitochondria and using a commercially available kit, Amplex Red (Invitrogen). We assessed the permeability of hCMECs using the ECIS ZΘ technology. The changes in protein expression of EC activation markers, VCAM‐1 and ICAM‐1, were determined with western blot.ResultInhibition of CA‐VA/B prevented Aβ40‐Q22 induced DNA fragmentation, caspase‐9 and ‐3 activation, cytochrome C release from the mitochondria, loss of mitochondria membrane potential, and mitochondrial H2O2 production in human cerebrovascular ECs. The inhibition of CA‐VA/B also reduced Aβ40‐Q22 induced BBB permeability and increase in vascular adhesion protein VCAM‐1 in hCMECs.ConclusionThe inhibition of CA‐V is protective to cerebrovascular ECs in models of amyloidosis, through the prevention of mitochondria‐mediated cell death and loss of cerebral EC barrier function.

SGLT2 expression in the left ventricle of cardiac patients is correlated with low-grade inflammation involving the pro-oxidant AT1R/NADPH oxidases/SGLT2 crosstalk: potential role in heart failure

Abstract Introduction Clinical studies showed that sodium-glucose co-transporter2 inhibitors (SGLT2i) have beneficial effects in heart failure patients regardless of ejection fraction and diabetes. Such cardiac diseases are often characterized by low-grade inflammation and impaired endothelial coronary microcirculation function. Recently, angiotensin II and TNF-α were shown to induce SGLT2 expression in endothelial cells (ECs) to sustain oxidative stress leading to endothelial dysfunction. However, the role and function of SGLT2 in the human left ventricle (LV) remain unclear. Therefore, this study evaluated the expression of SGLT2 in the LV of patients with cardiac diseases and, if so, determined the cellular localization, the underlying mechanism and the functional role. Methods Human LV biopsies were collected from 20 patients subjected to valve surgery at the Nouvel Hôpital Civil, Strasbourg, France. Expression levels of targets were determined by RT-qPCR and Western blot analysis, the in situ tissue localization by immunofluorescence (IF) staining and oxidative stress by dihydroethidium staining. Results RT-qPCR analysis of LV revealed variable SGLT1 and SGLT2 mRNA levels with no significant correlation. However, SGLT2 mRNA expression showed a positive correlation with those of pro-inflammatory markers (IL-1ß, IL-6, TNF-α, MCP-1 and CD68), AT1R, and VCAM-1. Western blot analysis confirmed the heterogenous SGLT2 protein expression level in LV with a difference up to 3.5-fold among the specimens. SGLT2 protein levels showed a positive correlation with markers of oxidative stress (phospho-p65 NF-κB, AT1R, nitrotyrosine) and markers of ECs activation (VCAM-1, ICAM-1), and a negative correlation with levels of eNOS. Further, immunofluorescence analysis of LV cryosections showed SGLT2 staining in the endothelium of the coronary microcirculation, as indicated by its co-localization with the endothelial cell marker CD31, and throughout the heart tissue. SGLT2 signals were also observed in areas showing IF signals for TNF-α, phospho-p65 NF-κB, CD68 and VCAM-1 suggesting that SGLT2 expression is localized to sites of inflammation. LV specimens with high SGLT2 protein levels showed elevated levels of oxidative stress, which were inhibited by N-acetylcysteine, VAS-2870, perindoprilat, losartan, empagliflozin, and infliximab. Conclusions The findings indicate that endothelial and cardiac SGLT2 expression in the LV of cardiac patients is associated with low-grade inflammation and oxidative stress. They further indicate that the pro-inflammatory cytokines/AT1R/NAPDH oxidases/SGLT2 crosstalk contributes to sustain a pro-oxidant state. Thus, SGLT2 inhibitors might ameliorate HF by mitigating the low-grade inflammatory-related pro-oxidant state and, hence, preserve the crucial endothelial control of the cardiomyocyte function in the LV.

Abstract P3023: Endothelial Pparδ Deletion Exacerbates Endotoxemia-Induced Vascular Injury And Inflammation

Rationale and Background: Impairment of vascular endothelial integrity is one of the hallmarks of acute lung injury, which underlies leukocyte infiltration and edema formation and contributes to organ dysfunction. The peroxisome proliferator-activated receptor delta (PPARδ) is a transcription factor regulating vascular endothelial cell (EC) function. Our previous study revealed that selective deletion of PPARδ in the endothelium worsens ischemic vascular injury, disrupts endothelial integrity, and exacerbates vascular inflammation in a mouse model of hindlimb ischemia. Methods and Results: In this study, we investigated whether PPARδ is protective for EC function in acute vascular injury and inflammation in the mouse lung. We performed lipopolysaccharide (LPS)-induced endotoxemia in the EC-selective PPARδ knockout mice (Ppard EC-KO ) and their wild-type littermates (Ppard EC-WT ). The mortality of Ppard EC-KO mice was higher after LPS injection. The survived Ppard EC-KO mice showed more severe lung vascular inflammation suggested by sustained leukocyte infiltration in the lung tissues and in the bronchoalveolar fluid, more pro-inflammatory cytokine production, and higher expression of EC activation markers CCL2, ICAM1, VCAM1, and E-selectin. In addition, the lung endothelium of LPS-treated Ppard EC-KO mice has degraded endothelial glycocalyx and discontinuous EC junctions, resulting in increased vascular leakage. In vitro experiments using human lung microvascular endothelial cells (HLMVECs) revealed that silencing PPARδ downregulated the expressions of ZO-1, claudin-5, and VE-cadherin, and altered their membrane distribution, leading to hyperpermeability of EC monolayer, at basal level and also in the presence of LPS or TNFα. Silencing of PPARδ also induces more activation of pro-inflammatory transcription factors including STAT1, IRF3, and IRF7. Conclusion: In summary, our study showed the protective role of endothelial PPARδ against endotoxemia-induced disruption of vascular barrier function and vascular inflammation in the lung endothelium. (Supported by Hong Kong Research Grant Council GRF 14107822 and 14105321)

Natriuretic Peptides and Cardiac Troponins: Markers of Disease Progression and Risk in Light Chain Cardiac Amyloidosis.

Light chain (AL) amyloidosis can cause an infiltrative cardiomyopathy that can result in symptomatic heart failure. The vague, nonspecific onset of signs and symptoms may lead to a delay in diagnosis and treatment leading to poor outcomes. Cardiac biomarkers, such as troponins and natriuretic peptides, play a pivotal role in diagnosis, determining prognosis, and assessing treatment response in patients with AL amyloidosis. Because of the evolving landscape for both diagnosis and treatment of AL cardiac amyloidosis, we review the critical role these and other biomarkers play in the clinical management of this disease. A number of conventional cardiac and noncardiac serum biomarkers are commonly used in AL cardiac amyloidosis and may be surrogates for cardiac involvement and inform prognosis. These include typical heart failure biomarkers such as levels of circulating natriuretic peptides as well as cardiac troponins. Other noncardiac biomarkers frequently measured in AL cardiac amyloidosis included difference between the involved and uninvolved free light chains (dFLC) and markers of endothelial cell activation and damage such as von Willebrand factor antigen and matrix metalloproteinases. AL amyloidosis can lead to cardiac involvement which has been associated with poor outcomes, especially if not identified and treated early. Natriuretic peptides and cardiac troponins are cornerstones for the diagnosis and management of AL cardiac amyloidosis. Their levels may represent cardiac stress, injury, and possibly degree of cardiac involvement, and they play a key role in AL amyloidosis disease staging.

Procoagulant effect of extracellular vesicles in patients after transcatheter aortic valve replacement or transcatheter aortic valve replacement with percutaneous coronary intervention.

Patients with severe aortic stenosis (AS) after replacement of the transcatheter aortic valve (TAVR) are more likely to develop thrombotic complications such as cerebral embolism and artificial valve thrombosis. However, the mechanism is not yet well defined. We aimed to explore the plasma extracellular vesicles (EVs) levels and their role in the induction of procoagulant activity (PCA) in patients receiving TAVR alone or TAVR with percutaneous coronary intervention (PCI). EVs were analyzed with flow cytometer. Markers of platelet and endothelial cell activation were quantified using selective enzyme-linked immunosorbent assay (ELISA) kits. Procoagulant activity (PCA) was assessed by clotting time, purified clotting complex assays, and fibrin production assays. Our results confirmed that EVs with positive phosphatedylserin (PS+EV), platelet EVs (PEVs) and positive tissue factor EVs (TF+EVs) were higher in patients following TAVR than before TAVR, particularly in TAVR with PCI. Furthermore, endothelial-derived EVs (EEVs) were also higher in patients after TAVR with PCI than pre-TAVR, however, the EEVs levels in TAVR alone patients were gradually reduce than pre-TAVR. In addition, we further proved that total EVs contributed to dramatically shortened coagulation time, increased intrinsic/extrinsic factor Xa and thrombin generation in patients after TAVR, especially in TAVR with PCI. The PCA was markedly attenuated by approximately 80% with lactucin. Our study reveals a previously unrecognized link between plasma EV levels and hypercoagulability in patients after TAVR, especially TAVR with PCI. Blockade of PS+EVs may improve the hypercoagulable state and prognosis of patients.

Neuroprotection by Preconditioning in Mice is Dependent on MyD88-Mediated CXCL10 Expression in Endothelial Cells.

The central nervous system (CNS) can be preconditioned to resist damage by peripheral pretreatment with low-dose gram-negative bacterial endotoxin lipopolysaccharide (LPS). Underlying mechanisms associated with transient protection of the cerebral cortex against traumatic brain injury include increased neuronal production of antiapoptotic and neurotrophic molecules, microglial-mediated displacement of inhibitory presynaptic terminals innervating the soma of cortical projection neurons, and synchronized firing of cortical projection neurons. However, the cell types and signaling responsible for these neuronal and microglial changes are unknown. A fundamental question is whether LPS penetrates the CNS or acts on the luminal surface of brain endothelial cells, thereby triggering an indirect parenchymal neuroprotective response. The present study shows that a low-dose intraperitoneal LPS treatment increases brain endothelial cell activation markers CD54, but does not open the blood-brain barrier or alter brain endothelial cell tight junctions as assessed by electron microscopy. NanoString nCounter transcript analyses of CD31-positive brain endothelial cells further revealed significant upregulation of Cxcl10, C3, Ccl2, Il1β, Cxcl2, and Cxcl1, consistent with identification of myeloid differentiation primary response 88 (MyD88) as a regulator of these transcripts by pathway analysis. Conditional genetic endothelial cell gene ablation approaches demonstrated that both MyD88-dependent Toll-like receptor 4 (TLR4) signaling and Cxcl10 expression are essential for LPS-induced neuroprotection and microglial activation. These results suggest that C-X-C motif chemokine ligand 10 (CXCL10) production by endothelial cells in response to circulating TLR ligands may directly or indirectly signal to CXCR3 on neurons and/or microglia. Targeted activation of brain endothelial receptors may thus provide an attractive approach for inducing transient neuroprotection.

Circulating hyaluronic acid signature in CAP and ARDS – the role of pneumolysin in hyaluronic acid shedding

Shedding of hyaluronan (HA), the component of endothelial cell (EC) glycocalyx, has been associated with acute lung injury. HA degradation allows plasma proteins and fluid to penetrate across the vascular wall leading to lung edema formation and leukocyte recruitment. Here, we analyzed sHA levels and size in patients with community-acquired pneumonia (CAP) and acute respiratory distress syndrome (ARDS), correlated them to disease severity, and evaluated the impact of pneumolysin (PLY), the Streptococcus pneumoniae (S.p.) exotoxin, on HA shedding from human pulmonary microvascular EC (HPMVEC).sHA levels were elevated in CAP and ARDS and correlated with the CRB65 severity score and with markers of inflammation (interleukin-6), EC activation (E-selectin), and basement membrane destruction (collagen IV). Furthermore, sHA levels were associated with an increase in 28-day mortality. Small and large sHA fragments were detected in plasma of most severe CAP or ARDS patients, and the presence of large sHA fragments was accompanied by the elevated levels of circulating collagen IV. In vitro, PLY induced sHA release from HPMVEC. This effect was dependent on reactive oxygen species (ROS) production and was not associated with endothelial barrier dysfunction. Conversely, HA shedding was impaired following HPMVEC infection with a S.p. PLY-deficient mutant.Our study identifies association between the severity of CAP and ARDS and the levels and size of sHA in plasma. It links sHA levels with, inflammation, EC activation status and basement membrane disassembly in ARDS and provides insights into the mechanism of HA shedding during infection.

Blood Transfusion Ameliorates Free Haem-Induced Loss of Thrombomodulin and Endothelial Cell Activation in Paediatric Sickle Cell Anaemia

Background Sickle cell anaemia (SCA) is responsible for major paediatric morbidity including risk of stroke in up to 10% of children. Mainstays of therapy include hydroxyurea (HU) and chronic blood transfusion (BT), with BT considered gold standard for stroke prevention. However, the biological mechanisms through which BT is significantly superior to HU in reducing stroke risk have not been clearly defined. SCA complications are due in part to free haem-mediated damage, leading to endothelial cell (EC) activation, tissue factor (TF) expression and release of von Willebrand Factor (VWF), which binds platelets and sickle RBCs. Recently loss of EC Thrombomodulin (TM) has been shown to promote EC activation and VWF release and soluble TM (sTM) plays a role in disseminated intravascular coagulation and COVID-19. Hypothesis We hypothesize free haem alters EC TM expression, promoting EC activation and VWF release. Intravascular hemolysis and free haem may be reduced in children on chronic BT, maintaining EC quiescence and enhancing protection against stroke compared with HU. Methods Following ethical approval and informed consent 180 SCA children (median age 12 years, 49% male) were recruited. Steady-state plasma samples were collected prior to BT (n=84) or at outpatient clinic for HU (n=96). Paired crisis samples were obtained in a subset of HU (n=30). EC activation markers and Weibel Palade Body (WPB) proteins were evaluated by ELISA, VWF multimers were assessed by 1.8% agarose gels and ADAMTS13 activity assessed by FRET. Free haem levels were quantified and thrombin generation (TG) assays performed. Factor VIII:C was measured in the National Coagulation Centre at St James's Hospital. Human umbilical vein endothelial cells (HUVEC) were exposed to cell-free haem (Hemin), hydroxyurea, cytokines (IL-1β and TNFα) or patient plasma; VWF was measured in the supernatant and HUVEC TM expression assessed by flow cytometry. Results VWF:Ag and VWF propeptide (VWFpp) levels were significantly increased in HU compared to BT children, suggesting acute EC activation (median plasma VWF:Ag levels 1.29 v 1.03 IU/ml; p=0.0005, median VWFpp levels 1.63 v 1.17 IU/ml p=0.006). Furthermore, plasma levels of other WPB stored proteins were also significantly elevated in the HU cohort, including Angiopoietin-2 (Ang-2) (4214 v 2337 pg/ml, p<0.0001 and Osteoprotegerin (OPG) (1825 v 1507 pg/ml p=0.006). Abnormalities of high-, intermediate- and low molecular weight VWF multimers were noted in HU but not BT. While putative inhibitors of ADAMTS13 were increased in HU, including Platelet Factor 4 and Interleukin-6, ADAMTS13 activity did not differ between groups. FVIII:C levels were significantly elevated in 65% of the total cohort and were higher in HU than BT (1.62 IU/ml v 1.30 IU/ml p<0.0001). Free haem levels were increased in HU versus BT (12.5 v 9.5μM p=0.0003), as well as levels of LDH and reticulocyte %, pointing to increased intravascular hemolysis. EC markers and FVIII:C correlated strongly with hemolytic indices. In addition to increased EC activation, increased endogenous thrombin potential (ETP), peak thrombin and velocity index were observed in HU versus BT, pointing to enhanced TG, possibly driven by elevated circulating TF. TG parameters correlated with plasma FVIII:C levels. Exposure of HUVEC to free haem in vitro resulted in a significant decrease in surface TM expression and increased VWF secretion (p<0.05). Conversely exposure to hydroxyurea or cytokines did not alter TM expression. Exposure of HUVEC to plasma from crisis but not paired steady-state plasma induced EC activation and VWF release compared with control plasma. Importantly, free haem levels ≥20 μM were observed in 25% HU children in steady state and 40% in crisis, compared with 0% of BT. Furthermore, significantly elevated sTM levels in SCA children with a history of cerebrovascular disease compared to those without (5.7 v 4.6 ng/ml p=0.02), suggesting that sTM may predict end-organ dysfunction. Conclusions We have shown in a large cohort of SCA children that EC activation, TG and intravascular haemolysis are increased in HU-treated children compared with BT. Free haem induces loss of EC TM and VWF release in vitro, leading to EC activation. Elevated sTM levels were a biomarker of end-organ damage. These data suggest that reduction of free haem in children undergoing regular BT is a key mechanism through which EC quiescence is maintained in paediatric SCA.

Pretransplant serum levels of endothelial cell activation markers are associated with graft loss and mortality after kidney transplantation.

Long-term allograft survival remains a challenge in kidney transplantation. In this study, we aimed to identify biomarkers for potentially modifiable pathways involved in the outcome of kidney transplantation. We tested the hypothesis that a pre-existing systemic environment with endothelial cell activation in the recipient is associated with the outcome after kidney transplantation. In a retrospective study cohort of 611 kidney transplanted patients, we investigated associations between serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) before transplantation and delayed graft function, acute rejection, graft loss and mortality after transplantation. We adjusted associations for age, sex, preformed donor-specific antibodies (DSA), pretransplant diabetes, cardiovascular disease and dialysis. Additionally, we investigated if associations between endothelial cell activation markers and outcomes differed in recipients with and without preformed DSA. Serum levels of endothelial cell activation markers were associated with delayed graft function and mortality but not with rejection. Additionally, high levels of sICAM-1 were associated with graft loss. Associations were most pronounced in recipients without DSA, adjusted for potential confounders. Data suggest that endothelial cell activation at the time of transplantation is associated with graft loss and mortality after kidney transplantation, especially in transplant candidates without preformed DSA.

An Immunological Axis Involving Interleukin 1β and Leucine-Rich-α2-Glycoprotein Reflects Therapeutic Response of Children with Kawasaki Disease: Implications from the KAWAKINRA Trial

PurposeA recent phase II open-label study of the interleukin 1 (IL-1) receptor antagonist (IL-1Ra) anakinra in treating IVIG-resistant Kawasaki disease (KD) patients reported promising results. Here, we aimed to characterize the immunological impact of IL-1 blockade in this unique study population.MethodsPatients’ and control sera and supernatants of cells (whole blood, neutrophils, coronary artery endothelial cells) stimulated with recombinant IL-1β were analyzed for single or multiple marker (n = 22) expression by ELISA or multiplexed bead array assay. Data were analyzed using unsupervised hierarchical clustering, multiple correlation, and multi-comparison statistics and were compared to retrospective analyses of KD transcriptomics.ResultsInflammation in IVIG-resistant KD (n = 16) is hallmarked by over-expression of innate immune mediators (particularly IL-6 > CXCL10 > S100A12 > IL-1Ra). Those as well as levels of immune or endothelial cell activation markers (sICAM-1, sVCAM-1) declined most significantly in course of anakinra treatment. Prior as well as following IL-1R blockade, over-expression of leucine-rich-α2-glycoprotein 1 (LRG1) associated best with remnant inflammatory activity and the necessity to escalate anakinra dosage and separated inflammatory KD patients from sJIA-MAS (n = 13) and MIS-C (n = 4). Protein as well as retrospective gene expression analyses indicated tight association of LRG1 with IL-1β signaling and neutrophilia, while particularly neutrophil stimulation with recombinant IL-1β resulted in concentration-dependent LRG1 release.ConclusionOur study identifies LRG1 as known trigger of endothelial activation and cardiac re-modeling to associate with IL-1β signaling in KD. Besides a potential patho-mechanistic implication of these findings, our data suggest blood leukocyte and neutrophil counts to best predict response to IL-1Ra treatment in IVIG-resistant KD.

Serum Chemerin and Apelin Levels in Obese Children: Relation to Endothelial Function and Inflammation from a Cross-sectional Case–Control Study

BACKGROUND: Childhood obesity is a global threat with subsequent health problems among which and most important is cardiovascular problems. It is now claimed that adipokines secreted by adipose tissue are responsible for such consequences. Newly discovered adipokines chemerin and apelin are under investigation for their link with obesity related co-morbidites. AIM: The aim of the present study was to assess the serum levels of chemerin and apelin in obese children and to explore the correlation between these two biomarkers and the inflammatory as well as the endothelial cell activation markers PATIENTS AND METHODS: This study was a cross-sectional case control study that comprised 45 pre-pubertal obese children aged (6– &lt; 12) years old of both sexes (22 males and 23 females), in addition to 45 matched age and sex lean children serving as controls (21 males and 24 females). Serum levels of chemerin, apelin, ICAM-1, E-selectin and hs-CRP were measured for obese and controls. RESULTS: Obese children showed higher levels of chemerin, apelin, ICAM-1 and E-selectin than controls. Chemerin and apelin showed significant correlations with all parameters except for age. Anthropometric parameters with hs-CRP revealed significant correlation even after adjustment for age and sex while apelin only showed a significant correlation with age. Multiple regression analyses with hs -CR , E-selectin and ICAM-1 as dependent variables and BMI Z score ,age, sex, chemerin and apelin as independent variables showed the effect of chemerin and apelin on the increased levels of hs -CR , E-selectin and ICAM-1 . CONCLUSION: Elevated levels of chemerin and apelin may serve as indices of ongoing obesity-related disorders in obese children.

Markers of endothelial cell activation are associated with the severity of pulmonary disease in COVID-19.

Severe coronavirus disease-19 (COVID-19) is characterized by vascular inflammation and thrombosis. We and others have proposed that the inflammatory response to coronavirus infection activates endothelial cells, leading to endothelial release of pro-thrombotic proteins. These mediators can trigger obstruction of the pulmonary microvasculature, leading to worsening oxygenation, acute respiratory distress syndrome, and death. In the current study, we tested the hypothesis that higher levels of biomarkers released from endothelial cells are associated with worse oxygenation in patients with COVID-19. We studied 83 participants aged 18–84 years with COVID-19 admitted to a single center. The severity of pulmonary disease was classified by oxygen requirement, including no oxygen requirement, low-flow oxygen, high-flow nasal cannula oxygen, mechanical ventilation, and death. We measured plasma levels of two proteins released by activated endothelial cells, von Willebrand Factor (VWF) antigen and soluble P-Selectin (sP-Sel), and a biomarker of systemic thrombosis, D-dimer. Additionally, we explored the association of endothelial biomarker levels with the levels of pro-inflammatory cytokine and chemokines, and vascular inflammation biomarkers. We found that levels of VWF, sP-sel, and D-dimer were increased in individuals with more severe COVID-19 pulmonary disease. Biomarkers of endothelial cell activation were also correlated with proinflammatory cytokines and chemokines. Taken together, our data demonstrate increased levels of VWF and sP-selectin are linked to the severity of lung disease in COVID-19 and correlated with biomarkers of inflammation and vascular inflammation. Our data support the concept that COVID-19 is a vascular disease which involves endothelial injury in the context of an inflammatory state.

Caspase-8 in endothelial cells maintains gut homeostasis and prevents small bowel inflammation in mice.

The gut has a specific vascular barrier that controls trafficking of antigens and microbiota into the bloodstream. However, the molecular mechanisms regulating the maintenance of this vascular barrier remain elusive. Here, we identified Caspase‐8 as a pro‐survival factor in mature intestinal endothelial cells that is required to actively maintain vascular homeostasis in the small intestine in an organ‐specific manner. In particular, we find that deletion of Caspase‐8 in endothelial cells results in small intestinal hemorrhages and bowel inflammation, while all other organs remained unaffected. We also show that Caspase‐8 seems to be particularly needed in lymphatic endothelial cells to maintain gut homeostasis. Our work demonstrates that endothelial cell dysfunction, leading to the breakdown of the gut‐vascular barrier, is an active driver of chronic small intestinal inflammation, highlighting the role of the intestinal vasculature as a safeguard of organ function.

Cardiovascular signatures of COVID-19 predict mortality and identify barrier stabilizing therapies.

SummaryBackgroundEndothelial cell (EC) activation, endotheliitis, vascular permeability, and thrombosis have been observed in patients with severe coronavirus disease 2019 (COVID-19), indicating that the vasculature is affected during the acute stages of SARS-CoV-2 infection. It remains unknown whether circulating vascular markers are sufficient to predict clinical outcomes, are unique to COVID-19, and if vascular permeability can be therapeutically targeted.MethodsProspectively evaluating the prevalence of circulating inflammatory, cardiac, and EC activation markers as well as developing a microRNA atlas in 241 unvaccinated patients with suspected SARS-CoV-2 infection allowed for prognostic value assessment using a Random Forest model machine learning approach. Subsequent ex vivo experiments assessed EC permeability responses to patient plasma and were used to uncover modulated gene regulatory networks from which rational therapeutic design was inferred.FindingsMultiple inflammatory and EC activation biomarkers were associated with mortality in COVID-19 patients and in severity-matched SARS-CoV-2-negative patients, while dysregulation of specific microRNAs at presentation was specific for poor COVID-19-related outcomes and revealed disease-relevant pathways. Integrating the datasets using a machine learning approach further enhanced clinical risk prediction for in-hospital mortality. Exposure of ECs to COVID-19 patient plasma resulted in severity-specific gene expression responses and EC barrier dysfunction, which was ameliorated using angiopoietin-1 mimetic or recombinant Slit2-N.InterpretationIntegration of multi-omics data identified microRNA and vascular biomarkers prognostic of in-hospital mortality in COVID-19 patients and revealed that vascular stabilizing therapies should be explored as a treatment for endothelial dysfunction in COVID-19, and other severe diseases where endothelial dysfunction has a central role in pathogenesis.Funding InformationThis work was directly supported by grant funding from the Ted Rogers Center for Heart Research, Toronto, Ontario, Canada and the Peter Munk Cardiac Center, Toronto, Ontario, Canada.

The impact of anti-endothelial cell antibodies (AECAs) on the development of blood vessel damage in patients with systemic lupus erythematosus: the preliminary study.

Vascular injury represents one of the most frequent lesions in systemic lupus erythematosus (SLE). The aim of the study was to assess the influence of anti-endothelial cell antibodies (AECAs) on the development of endothelial cell (EC) activation, dysfunction and subsequent vasculitis in women with SLE. Fifty six women with SLE were divided into 2 subgroups, i.e. subjects with positive AECAs (+) and those with negative AECAs (-). The control group consisted of 25 healthy women. Clinical characteristics, routine laboratory tests and circulating markers of EC activation/dysfunction, i.e. monocyte-chemotactic protein-1 (MCP-1), soluble E- and P-selectin, vascular and intercellular adhesion molecule-1 (sVCAM-1, sICAM-1), von Willebrand factor (vWF), pentraxin 3 (the marker of vasculitis) the indicator of procoagulant activity i.e. prothrombin fragment 1 + 2 (F1 + 2) were detected using ELISA and compared between patients with AECA (+), AECA (-) and control subgroups. Serum concentrations of AECAs in AECA(+), AECA(-) and control groups were 4.58 ± 2.97, 0.92 ± 0.50 and 0.72 ± 0.28 AU/ml, respectively (p < 0.001). The study showed significant increases in EC activation markers, i.e. MCP-1, sE-selectin, sVCAM-1 and F1 + 2 in SLE AECA(+) compared to SLE AECA(-) and control groups. However, the indicator of vasculitis (PTX3) was significantly lower in SLE AECA(+). Moreover, multivariate analysis of variance showed a positive correlation between AECAs and sE-selectin and sVCAM-1 levels, but not with PTX3. AECAs were involved in the initial stages of vascular damage in SLE, i.e. in EC activation and dysfunction. However, they did not play a role in the development of vasculitis.

Endocan, a Soluble Marker of Endothelial Cell Activation Is a Molecular Marker of Disease Severity in Women with Preeclampsia.

Endocan is a proteoglycan secreted by activated endothelium that regulates angiogenesis via interaction with hepatocyte growth factor (HGF). We hypothesized that women diagnosed with preeclampsia (PE) and/or fetal growth restriction (FGR) have elevated circulating endocan concentrations in direct relationship with severity of clinical manifestations. Serum concentration of endocan and HGF were analyzed in 224 women grouped as healthy pregnant controls (P-CRL, n = 77), PE with severe features (sPE, n = 83), chronic hypertension (crHTN: n = 36), idiopathic FGR (n = 18), and healthy non-pregnant controls (NP-CRL, n = 7). Endocan and HGF measured by immunoassay were analyzed along with markers of inflammation, angiogenesis, and protein misfolding (urine congophilia). Endocan expression in the placenta and/or myometrium was studied by immunohistochemistry and real-time PCR. Compared to gestational age-matched P-CRL, women with early-onset sPE had higher circulating endocan concentrations. Among women with PE and/or FGR, endocan concentration correlated with soluble endoglin and urine congophilia but not with HGF or markers of inflammation or angiogenesis. In the placenta, endocan was expressed in villous and extravillous trophoblasts and endothelium. Intense endocan immunostaining was observed in plaque-like aggregations of sPE placentas complicated with FGR. In addition, thickened blood vessels in the myometrium of sPE patients stained positive for endocan. Women with early-onset sPE have elevated serum endocan likely reflecting chronic endothelial activation. Enhanced expression and/or deposition of endocan at the sites of placental injury and in remodeled maternal blood vessels supports a role for endocan in either vascular rescue or as a contributor to FGR and perhaps long-term cardiovascular morbidity.