Release Of Angiopoietin-2 Research Articles

Thrombomodulin Resistance May be Involved in the Prothrombotic State of COVID-19

Introduction: Hypercoagulability and endothelial dysfunction seem to be involved in the worsening of the clinical condition of patients with COVID-19. In the most severe cases, it has been shown that the inflammatory process triggers the release of angiopoietin-2 (ANGPT2) through the endothelium, which could decrease levels of circulating thrombomodulin (TM), a key component in the activation of the protein C, one of the major regulatory mechanisms of thrombin generation. In fact, recent studies suggest ANGPT2 as a therapeutic target to improve the prothrombotic profile of patients with COVID-19 and other clinical conditions associated to a hypercoagulable state. However, there are no studies evaluating the role of resistance to TM in the hypercoagulability of COVID-19 so far. Objectives: To analyze the resistance to TM in hospitalized COVID-19 patients by thrombin generation (TG) test and its correlation with the severity of the disease. Methods: A total of 120 hospitalized COVID-19 patients were included, 47 of them with data for correlation with the clinical profile. Citrated blood samples were drawn at admission and centrifuged to obtain platelet-poor plasma and frozen at −80°C until analysis. TG test and clot formation kinetics were evaluated by GENESIA® and Quantra®, respectively. Severe adverse events (SAE) were considered non-invasive mechanical ventilation, ICU admission, encephalitis, thrombosis, or death. Results: Measurement of endogenous thrombin potential (ETP) with/without TM revealed that 39% of patients had % ETP inhibition <40%, suggesting TM resistance. In the correlation with the clinic, 23.4% of patients (n=11/47) presented an adverse event. TG at admission was higher in patients who developed SAE though differences between groups did not reach the statistical significance. TG with addition of TM showed important resistance to TM in the group who developed SAE. In this group, ETP dropped -24,3 ± 10.2 % vs no-SAE group who ETP dropped -47.3 ± 22.0 % (p< 0.001). Similar effect was observed in the Peak of TG after addition of TM. In the SAE group the Peak dropped -15.7 ± 7.9 % vs the no-SAE group who presented a Peak drop of -33.3 ± 19.6 % (p< 0.001). Both results suggest a marked resistance to TM in the group that developed SAE. Lower % inhibition of Peak of TG by TM addition and higher values of ETP were significantly correlated with higher clot stiffness (r= 0.339, p= 0.028 and r=0.322, p= 0.027, respectively). Both Peak and ETP drop by the addition of TM in TG test proved to be good predictors of SAE at admission with an AUC of 0.756 (95% CI: 0.615-0.897, p=0.015) and 0.803 (95% CI: 0.672-0.934, p= 0.004), respectively. Conclusions: Thrombin generation test can be a powerful tool for risk stratification of COVID-19 patients at admission. Increased resistance to thrombomodulin is associated with the development of severe adverse events and can be considered as a new independent prognostic marker of patients at risk of poor prognosis.

Oroxylin a Attenuates Limb Ischemia by Promoting Angiogenesis via Modulation of Endothelial Cell Migration.

Oroxylin A (OA) has been shown to simultaneously increase coronary flow and provide a strong anti-inflammatory effect. In this study, we described the angiogenic properties of OA. OA treatment accelerated perfusion recovery, reduced tissue injury, and promoted angiogenesis after hindlimb ischemia (HLI). In addition, OA regulated the secretion of multiple cytokines, including vascular endothelial growth factor A (VEGFA), angiopoietin-2 (ANG-2), fibroblast growth factor-basic (FGF-2), and platelet derived growth factor BB (PDGF-BB). Specifically, those multiple cytokines were involved in cell migration, cell population proliferation, and angiogenesis. These effects were observed at 3, 7, and 14 days after HLI. In skeletal muscle cells, OA promoted the release of VEGFA and ANG-2. After OA treatment, the conditioned medium derived from skeletal muscle cells was found to significantly induce endothelial cell (EC) proliferation. OA also induced EC migration by activating the Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil kinase 2 (ROCK-II) signaling pathway and the T-box20 (TBX20)/prokineticin 2 (PROK2) signaling pathway. In addition, OA was able to downregulate the number of macrophages and neutrophils, along with the secretion of interleukin-1β, at 3 days after HLI. These results expanded current knowledge about the beneficial effects of OA in angiogenesis and blood flow recovery. This research could open new directions for the development of novel therapeutic intervention for patients with peripheral artery disease (PAD).

Synaptotagmin-Like Protein 2a Regulates Angiogenic Lumen Formation via Weibel-Palade Body Apical Secretion of Angiopoietin-2.

[Figure: see text].

Thrombomodulin is essential for maintaining quiescence in vascular endothelial cells

Thrombomodulin (TM) is a thrombin receptor on endothelial cells that is involved in promoting activation of the anticoagulant protein C pathway during blood coagulation. TM also exerts protective anti-inflammatory properties through a poorly understood mechanism. In this study, we investigated the importance of TM signaling to cellular functions by deleting it from endothelial cells by CRISPR-Cas9 technology and analyzed the resultant phenotype of TM-deficient (TM-/- ) cells. Deficiency of TM in endothelial cells resulted in increased basal permeability and hyperpermeability when stimulated by thrombin and TNF-α. The loss of the basal barrier permeability function was accompanied by increased tyrosine phosphorylation of VE-cadherin and reduced polymerization of F-actin filaments at cellular junctions. A significant increase in basal NF-κB signaling and expression of inflammatory cell adhesion molecules was observed in TM-/- cells that resulted in enhanced adhesion of leukocytes to TM-/- cells in flow chamber experiments. There was also a marked increase in expression, storage, and release of the von Willebrand factor (VWF) and decreased storage and release of angiopoietin-2 in TM-/- cells. In a flow chamber assay, isolated platelets adhered to TM-/- cells, forming characteristic VWF-platelet strings. Increased VWF levels and inflammatory foci were also observed in the lungs of tamoxifen-treated ERcre-TMf/f mice. Reexpression of the TM construct in TM-/- cells, but not treatment with soluble TM, normalized the cellular phenotype. Based on these results, we postulate cell-bound TM endows a quiescent cellular phenotype by tightly regulating expression of procoagulant, proinflammatory, and angiogenic molecules in vascular endothelial cells.

Complement promotes endothelial von Willebrand factor and angiopoietin-2 release in obstructive sleep apnea.

Obstructive sleep apnea (OSA) is highly prevalent and triples vascular thromboembolic risk. Intermittent hypoxia (IH) during transient cessation of breathing in OSA impairs endothelial protection against complement. Complement activation stimulates the endothelial release of a pro-thrombotic von Willebrand factor (vWF). We investigated whether increased complement activity in OSA promotes the endothelial release of vWF and pro-inflammatory angiopoietin-2. We further investigated whether improving complement protection with statins reverses these changes. Using endothelial cells (ECs) and blood collected from OSA patients (n = 109) and controls (n = 67), we assessed whether altered cellular localization of complement inhibitor CD59 in OSA modulates exocytosis of Weibel-Palade bodies (WPB), secretory granules that store vWF and angiopoietin-2. These interactions were also assessed in vitro in ECs exposed to normoxia or IH with or without recombinant complement C9 and with or without atorvastatin. Circulating levels of angiopoietin-2 were greater in OSA than controls and levels of vWF cleavage products correlated with OSA severity. In cultured ECs, IH enhanced complement-stimulated angiopoietin-2 and vWF release by reducing EC surface and increasing intracellular expression of complement inhibitor CD59. Intracellular CD59 co-localized with WPB in OSA. IH increased binding of intracellular CD59 to syntaxin-3, which dissociated syntaxin-3 from voltage-sensitive calcium channel Cav1.2, and activated WPB exocytosis in a calcium-dependent manner. Atorvastatin reversed IH-enhanced endothelial release of vWF and angiopoietin-2. IH promotes the complement-mediated release of vWF and angiopoietin-2, which may contribute to pro-thrombotic and pro-inflammatory conditions in OSA. Statin reversed these effects, suggesting a potential approach to reduce cardiovascular risk in OSA.

Hyperinsulinemia promotes endothelial inflammation via increased expression and release of Angiopoietin-2

Background and aimsAngiopoietin-2 (ANG-2) mediates endothelial inflammation to initiate atherosclerosis and angiogenesis. Here we determined the serum levels of ANG-2 in hyperinsulinemic subjects and whether insulin increases its expression and release. MethodsHealthy male subjects were recruited from the D-CLIP and CURES studies and, based on their fasting insulin levels, were classified as normoinsulinemic (n = 228) and hyperinsulinemic (n = 32). Serum proteins were estimated by ELISA. Endothelial inflammation was scored as the number of THP-1 monocytes adhered to HUVEC monolayer. Gene expression was determined with promoter reporter assays and semi-quantitative RT-PCR. Western blotting was used to assess changes in protein expression and activation. Immunofluorescence imaging and ChIP assay were used for nuclear localization and promoter binding studies, respectively. ResultsANG-2 and sTIE2 levels were higher in hyperinsulinemic subjects. Hyperinsulinemic serum elicited endothelial inflammation, which was abrogated by an ANG-2 blocker antibody. Insulin (100 nM) increased mRNA and protein expression of ANG-2, and its release from HUVECs. It induced activation of p38 MAPK and an increase in protein levels and nuclear localization of cFOS. Binding of cFOS to the −640 to −494 promoter region mediated insulin dependent ANG-2 transcription. p38 MAPK inhibitor (25 μM) blocked insulin-induced nuclear localization of cFOS, expression of ANG-2 and ICAM-1, and release of ANG-2 into the culture medium. Spent medium collected from insulin treated cells enhanced endothelial inflammation, which was lost upon ANG-2 knockdown as well as upon p38 MAPK inhibition. ConclusionsANG-2 levels are high in hyperinsulinemic subjects and insulin induces expression and release of ANG-2 from HUVECs through p38 MAPK-cFOS pathway to elicit endothelial inflammation.

Correction for Ye et al., "Kaposi's Sarcoma-Associated Herpesvirus Induces Rapid Release of Angiopoietin-2 from Endothelial Cells".

Volume 87, no. 11, p. 6326–6335, 2013, https://doi.org/10.1128/JVI.03303-12. Page 6332, Fig. 4: Ang-2 protein bands shown in Fig. 4A were inadvertently duplicated in Fig. 4C. We have corrected this error.

Experimental Venous Congestion Causes Peripheral Release of Angiopoietin-2 and Tissue Necrosis Factor-α - A New Insight into the Pathophysiology of Gastrointestinal Bleeding in CF-LVAD Patients

Purpose Gastrointestinal bleeding (GIB) from angiodysplasia is the most frequent cause of readmission in LVAD patients. Angiogenesis, inflammation and coagulopathy as well as venous congestion (VC) are hypothesized to contribute to GIB pathophysiology. We used an established experimental model of peripheral VC to investigate whether increased venous pressure alters plasma levels of angiopoietin-2 (Ang-2) tumor necrosis factor-α (TNF-α) and vonWillebrand factor (vWF). Methods 42 ambulatory patients with NYHA functional class II or III, LVEF Results Patients were 54±12 years, 32% female, with mean LVEF=22±8% and 32% had an ischemic etiology. Pretest, baseline plasma levels of Ang-2, TNF-α and vWF were 6.73±0.70 ng/ml, 1.90±0.10 pg/ml, and 1.52±0.14 U/ml, respectively. Plasma Ang-2 and TNF-α increased in the congested (vs control arm) after 90 minutes of VC: 7.01±0.71 vs 6.69±0.67 ng/ml, p Conclusion We provide the first evidence that experimental peripheral VC is sufficient to increase circulating angiogenic and inflammatory biomarkers in HF patients. Future studies are warranted to investigate whether aggressive control of peripheral VC may prevent development and/or progression of angiodysplasia and thereby reduce the risk of GIB in CF-LVAD patients.

Validation of a human angiopoietin-2 ELISA for measurement of angiopoietin-2 concentrations in canine plasma samples and supernatant of primary canine aortic endothelial cell cultures.

OBJECTIVE To assess 2 human ELISA kits for measurement of angiopoietin-1 and -2 concentrations in canine plasma samples, determine whether plasma angiopoeitin-2 concentration differed between septic and healthy dogs, and determine the effect of tumor necrosis factor-α (TNF-α) stimulation on angiopoeitin-2 release from primary canine aortic endothelial cells (pCAECs) in vitro. ANIMALS 10 healthy dogs and 10 septic dogs. PROCEDURES Human angiopoietin-1 and -2 ELISAs were used to detect recombinant canine angiopoietins-1 and -2 in canine plasma samples. The angiopoietin-2 ELISA was further validated by use of plasma samples from healthy and septic dogs and supernatants of pCAEC cultures. Associations between plasma angiopoeitin-2 and C-reactive protein (CRP) concentrations were examined. RESULTS Angiopoeitin-2 but not angiopoeitin-1 was detected in canine plasma samples by the respective ELISAs. The angiopoeitin-2 ELISA had excellent dilutional linearity, parallelism, accuracy, precision, and reproducibility for measurements in canine plasma samples and pCAEC supernatants. Plasma angiopoeitin-2 concentration was significantly higher in septic dogs (median, 25.5 ng/mL) than in healthy dogs (median, 6.7 ng/mL) and was positively correlated with plasma CRP concentration (R2 = 0.60). Stimulation of pCAECs with TNF-α resulted in a significant increase in supernatant angiopoietin-2 concentration. CONCLUSIONS AND CLINICAL RELEVANCE The tested human angiopoietin-2 ELISA kit was useful for measuring angiopoietin-2 concentrations in canine plasma samples and pCAEC supernatants. Sepsis appeared to increase angiopoietin-2 concentration in dogs in vivo, whereas TNF-α stimulation caused release of angiopoietin-2 from pCAECs in vitro. These findings support the use of angiopoietin-2 as a marker of endothelial cell activation and inflammation in dogs.

Inorganic Polyphosphate Amplifies High Mobility Group Box 1-Mediated Von Willebrand Factor Release and Platelet String Formation on Endothelial Cells.

Objective- Inorganic polyphosphate (polyP) is known to modulate coagulation, inflammation, and metabolic pathways. It also amplifies inflammatory responses of HMGB1 (high mobility group box 1) in endothelial cells. The objective of this study was to evaluate the effect of polyP on von Willebrand factor (VWF) release from endothelial cells with or without HMGB1. Approach and Results- EA.hy926 endothelial cells were treated with different concentrations of polyP70 alone or in combination with different concentrations of HMGB1. VWF release was measured by an ELISA assay in the absence or presence of pharmacological inhibitors of the receptor for advanced glycation end products, P2Y1, and Ca2+. A flow chamber assay was used to monitor polyP70-mediated platelet recruitment and VWF-platelet string formation. PolyP70 and HMGB1 induced VWF release from endothelial cells by a concentration-dependent manner. PolyP70 amplified HMGB1-mediated VWF release from endothelial cells. This was also true if boiled platelet releasate was used as the source of polyP. Gene silencing or pharmacological inhibitors of receptor for advanced glycation end products, P2Y1, and Ca2+ significantly inhibited VWF release. PolyP70 and HMGB1 synergistically promoted VWF-platelet string formation in the flow chamber assay, which was inhibited by the anti-GPIbα (glycoprotein Ib alpha) antibody. VWF release by polyP70-HMGB1 complex required phosphorylation of Src and phospholipase C because inhibitors of Src, phospholipase C, and Ca2+ signaling significantly decreased VWF secretion. The polyP70-HMGB1 complex also increased angiopoietin-2 release, indicating that Weibel-Palade body exocytosis is involved in the VWF release. Conclusions- PolyP70 can promote thrombotic and inflammatory pathways by inducing VWF release and platelet string formation on endothelial cells.

Prognostic and Pathogenic Role of Angiopoietin-1 and -2 in Pneumonia.

During pneumonia, pathogen-host interaction evokes inflammation and lung barrier dysfunction. Tie2 activation by angiopoietin-1 reduces, whereas Tie2 blockade by angiopoietin-2 increases, inflammation and permeability during sepsis. The role of angiopoietin-1/-2 in pneumonia remains unidentified. To investigate the prognostic and pathogenic impact of angiopoietins in regulating pulmonary vascular barrier function and inflammation in bacterial pneumonia. Serum angiopoietin levels were quantified in pneumonia patients of two independent cohorts (n = 148, n = 395). Human postmortem lung tissue, pneumolysin- or angiopoietin-2-stimulated endothelial cells, isolated perfused and ventilated mouse lungs, and mice with pneumococcal pneumonia were investigated. In patients with pneumonia, decreased serum angiopoietin-1 and increased angiopoietin-2 levels were observed as compared with healthy subjects. Higher angiopoietin-2 serum levels were found in patients with community-acquired pneumonia who died within 28 days of diagnosis compared with survivors. Receiver operating characteristic analysis revealed improved prognostic accuracy of CURB-65 for 28-day survival, intensive care treatment, and length of hospital stay if combined with angiopoietin-2 serum levels. In vitro, pneumolysin enhanced endothelial angiopoietin-2 release, angiopoietin-2 increased endothelial permeability, and angiopoietin-1 reduced pneumolysin-evoked endothelial permeability. Ventilated and perfused lungs of mice with angiopoietin-2 knockdown showed reduced permeability on pneumolysin stimulation. Increased pulmonary angiopoietin-2 and reduced angiopoietin-1 mRNA expression were observed in Streptococcus pneumoniae-infected mice. Finally, angiopoietin-1 therapy reduced inflammation and permeability in murine pneumonia. These data suggest a central role of angiopoietin-1/-2 in pneumonia-evoked inflammation and permeability. Increased angiopoietin-2 serum levels predicted mortality and length of hospital stay, and angiopoietin-1 may provide a therapeutic target for severe pneumonia.

Myeloperoxidase‐derived 2‐Chlorofatty Acids: Accumulation in Rat and Human Sepsis, and Association with Acute Respiratory Distress Syndrome Mortality in Human Sepsis

Neutrophilic inflammation is a component of sepsis‐associated acute respiratory distress syndrome (ARDS). Neutrophil myeloperoxidase (MPO) activity produces HOCl, which leads to increased plasma 2‐chlorofatty acid (2‐ClFA) levels due to HOCl targeting plasmalogens. Thus, these studies were designed to investigate the role of 2‐ClFA in ARDS during sepsis. In patients with sepsis (n=198), free 2‐ClFA levels were significantly associated with ARDS (odds ratio (OR) 1.87 (95% CI 1.41, 2.49); p &lt; 0.001), and with 30‐day mortality (OR 1.38 (95% CI 1.11, 1.72); p=0.004), for each log increase in free 2‐chlorostearic acid. Of considerable interest, plasma 2‐ClFA levels correlated with plasma levels of angiopoietin‐2, E‐selectin, and soluble thrombomodulin suggesting 2‐ClFAs may mediate endothelial dysfunction in human sepsis. Further studies with human lung microvascular endothelial cells (HMVEC‐L) showed 2‐ClFA treatment resulted in increased adhesion molecule surface expression; increased angiopoietin‐2 release and dose‐dependent endothelial permeability. In contrast, these changes were not observed in endothelial cells treated with non‐chlorinated palmitic acid. Next, the cecal slurry model of rat sepsis was investigated. Injecting rats with 15ml of cecal slurry/kg resulted in mortality within 26h accompanied by significant increases in 2‐ClFA levels in the plasma and lung in comparison to rats injected with vehicle. The ultimate goal of these studies will be to test therapeutic agents that prevent chlorinated lipid‐caused organ injury including ARDS during sepsis. Collectively, these studies suggest that 2‐ClFAs derived from neutrophil MPO‐catalyzed oxidation contribute to pulmonary endothelial injury and ARDS, and have prognostic utility in sepsis‐associated ARDS.Support or Funding InformationThis study was supported by NIH grants R01GM115553 (to DAF and JM). and R01HL122474 (to NJM).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Circulating Angiogenic Mediators in Patients with Moderate and Severe von Willebrand Disease: A Multicentre Cross-Sectional Study.

Inhibition of von Willebrand factor (VWF) expression in endothelial cells results in enhanced, possible dysfunctional angiogenesis, consistent with observations of severe gastrointestinal bleedings caused by vascular malformations in patients with von Willebrand disease (VWD). VWF is stored in endothelial Weibel-Palade bodies (WPB) with several other mediators of angiogenesis, like angiopoietin-2, osteoprotegerin and galectin-3. Increased release of angiopoietin-2 has been observed in medium of endothelial cells lacking VWF, but data on circulating levels of angiogenic factors in patients with VWD are lacking. The aim of this study was therefore to investigate plasma levels of angiogenic factors in patients with various types of VWD to obtain more insight into the pathogenesis of vascular malformations in these patients. We hypothesized that VWF deficiency leads to increased circulating levels of other WPB components. We therefore measured plasma levels of the WPB components angiopoietin-2, osteoprotegerin and galectin-3 as well as two other angiogenic factors (angiopoietin-1 and vascular endothelial growth factor [VEGF]) that are not stored within WPB. We observed that various angiogenic mediators are significantly different between types of VWD patients. Type 2A VWD patients had higher angiopoietin-1 levels compared with type 2B patients. Patients who have increased VWF clearance had higher angiopoietin-2 levels, whereas patients who have impaired VWF synthesis had higher galectin-3 levels. VEGF levels were negatively associated with VWF levels as type 3 VWD patients had the highest VEGF levels. However, complete VWF deficiency did not lead to increased circulating levels of other WPB components.

2-Chlorofatty acids induce Weibel-Palade body mobilization

Endothelial dysfunction is a hallmark of multiple inflammatory diseases. Leukocyte interactions with the endothelium have significant effects on vascular wall biology and pathophysiology. Myeloperoxidase (MPO)-derived oxidant products released from leukocytes are potential mediators of inflammation and endothelial dysfunction. 2-Chlorofatty acids (2-ClFAs) are produced as a result of MPO-derived HOCl targeting plasmalogen phospholipids. Chlorinated lipids have been shown to be associated with multiple inflammatory diseases, but their impact on surrounding endothelial cells has not been examined. This study tested the biological properties of the 2-ClFA molecular species 2-chlorohexadecanoic acid (2-ClHA) on endothelial cells. A synthetic alkyne analog of 2-ClHA, 2-chlorohexadec-15-ynoic acid (2-ClHyA), was used to examine the subcellular localization of 2-ClFA in human coronary artery endothelial cells. Click chemistry experiments revealed that 2-ClHyA localizes to Weibel-Palade bodies. 2-ClHA and 2-ClHyA promote the release of P-selectin, von Willebrand factor, and angiopoietin-2 from endothelial cells. Functionally, 2-ClHA and 2-ClHyA cause neutrophils to adhere to and platelets to aggregate on the endothelium, as well as increase permeability of the endothelial barrier which has been tied to the release of angiopoietin-2. These findings suggest that 2-ClFAs promote endothelial cell dysfunction, which may lead to broad implications in inflammation, thrombosis, and blood vessel stability.

Myeloperoxidase-derived 2-chlorofatty acids contribute to human sepsis mortality via acute respiratory distress syndrome.

Sepsis-associated acute respiratory distress syndrome (ARDS) is characterized by neutrophilic inflammation and poor survival. Since neutrophil myeloperoxidase (MPO) activity leads to increased plasma 2-chlorofatty acid (2-ClFA) levels, we hypothesized that plasma concentrations of 2-ClFAs would associate with ARDS and mortality in subjects with sepsis. In sequential consenting patients with sepsis, free 2-ClFA levels were significantly associated with ARDS, and with 30-day mortality, for each log increase in free 2-chlorostearic acid. Plasma MPO was not associated with either ARDS or 30-day mortality but was correlated with 2-ClFA levels. Addition of plasma 2-ClFA levels to the APACHE III score improved prediction for ARDS. Plasma 2-ClFA levels correlated with plasma levels of angiopoietin-2, E selectin, and soluble thrombomodulin. Endothelial cells treated with 2-ClFA responded with increased adhesion molecule surface expression, increased angiopoietin-2 release, and dose-dependent endothelial permeability. Our results suggest that 2-ClFAs derived from neutrophil MPO-catalyzed oxidation contribute to pulmonary endothelial injury and have prognostic utility in sepsis-associated ARDS.

Pathogenesis-Based Development of Potential Mitigation Strategies for Blinatumomab-Associated Neurologic Events (NEs)

▪Introduction: Blinatumomab (blin), a CD19/CD3 bispecific T-cell engager (BiTE®) antibody construct, has shown clinical efficacy in patients with both B-precursor acute lymphoblastic leukemia (ALL) and B-cell non-Hodgkin lymphoma (NHL). NEs are known blin-associated adverse drug reactions; most NEs occur early during the first treatment cycle and are transient and fully reversible. While NEs are manageable in ALL (favorable risk/benefit profile at a maximum target dose of 28 µg/day), NEs increase in frequency and were dose-limiting in low-grade NHL. The maximum tolerated dose in NHL was 112 µg/day. Here we present translational and single-patient data for potential mitigation strategies for blin-associated NEs.Methods: Pharmacokinetic and pharmacodynamic data from blin clinical studies in NHL and ALL were combined to develop a pathogenetic model for blin-associated NEs. The mitigation potential of substances with antiadhesive properties was studied in vitro in a flow chamber system mimicking blin-induced T-cell redistribution. Three patients with low-grade NHL who had a higher risk of developing NEs due to their low peripheral blood B/T-cell ratios received concomitant pentosan polysulfate (PPS) at infusion start and dose step. ClinicalTrials.gov NCT00274742, NCT00560794, NCT01209286, NCT01471782.Results: T-cell redistribution occurred at infusion start and dose step of blin irrespective of circulating B cells. Concurrently, T cells upregulated the activation marker CD69 and the T-cell adhesion molecule LFA-1 switched from its low to intermediate affinity isoform as shown by binding of soluble ICAM-1. Blood vessel endothelial cells were activated as indicated by release of angiopoietin-2 and clinical signs of peripheral edema. Monocyte and thrombocyte redistribution were also observed. Both B and T cells were detected in cerebrospinal fluid (CSF) of some patients with NEs, and quantifiable blin concentrations were measured in CSF of some patients independent of blood-CSF barrier integrity. A low B/T-cell ratio in peripheral blood (NHL) and/or bone marrow (ALL) has been identified as a potential risk factor for developing NEs. In flow chamber experiments, addition of blin mimicked T-cell redistribution as indicated by reduced T-cell rolling velocity and increased T-cell adhesion to brain microvascular endothelial cells and by upregulation of P-selectin and ICAM-1 adhesion molecules on endothelial cells. Substances potentially interfering with interactions between T cells and endothelial cells (eg, PPS, minocycline) reversed the above-described effects to levels seen without the addition of blin. T-cell kinetics of three patients receiving concomitant PPS at infusion start and dose step showed delayed T-cell redistribution (without circulating B cells) compared with patients without PPS coadministration. These altered T-cell kinetics correlated with less severe NEs; no treatment interruptions or discontinuations were necessary. Finite PPS coadministration had no apparent negative impact on clinical response as two patients achieved an objective response, including one patient who has been in ongoing remission for more than six years.Conclusion: We propose an evidence-based model for the development of blin-associated NEs: 1) First exposure to blin or dose steps increase endothelial adhesiveness of circulating T cells. 2) Adhering T cells activate the endothelium and start extravasating; activated endothelium attracts other circulating leukocytes. 3) At low B/T-cell ratios, extravasated T cells first encountering (rare) B cells in the brain are activated by blin in the perivascular space to locally secret cytokines and chemokines that induce transient neuroinflammation including transmigration of monocytes. 4) Attracted non-T cells and other released factors enhance transient neurotoxicity. Based on this model, T-cell adhesion to blood vessel endothelial cells is the first necessary (but not sufficient) step in the pathogenesis of blin-associated NEs. Coadministration of substances with antiadhesive properties at infusion start and dose steps may offer opportunities to mitigate blin-associated NEs to further improve the safety and efficacy of blin therapy. DisclosuresKlinger:Amgen Research (Munich) GmbH: Employment, Equity Ownership, Patents & Royalties: Blinatumomab. Zugmaier:Amgen: Employment, Patents & Royalties. Naegele:Amgen Research (Munich) GmbH: Employment, Equity Ownership, Patents & Royalties. Goebeler:GEmoAb: Consultancy; Novartis: Consultancy; Roche: Consultancy. Brandl:Amgen: Employment. Kufer:AMGEN Research Munich: Employment, Equity Ownership, Patents & Royalties.

Autocrine release of angiopoietin-2 mediates cerebrovascular disintegration in Moyamoya disease

Moyamoya disease is a rare steno-occlusive cerebrovascular disorder often resulting in hemorrhagic and ischemic strokes. Although sharing the same ischemic stimulus with atherosclerotic cerebrovascular disease, Moyamoya disease is characterized by a highly instable cerebrovascular system which is prone to rupture due to pathological neovascularization. To understand the molecular mechanisms underlying this instability, angiopoietin-2 gene expression was analyzed in middle cerebral artery lesions obtained from Moyamoya disease and atherosclerotic cerebrovascular disease patients. Angiopoietin-2 was significantly up-regulated in Moyamoya vessels, while serum concentrations of soluble angiopoietins were not changed. For further evaluations, cerebral endothelial cells incubated with serum from these patients in vitro were applied. In contrast to atherosclerotic cerebrovascular disease serum, Moyamoya disease serum induced an angiopoietin-2 overexpression and secretion, accompanied by loss of endothelial integrity. These effects were absent or inverse in endothelial cells of non-brain origin suggesting brain endothelium specificity. The destabilizing effects on brain endothelial cells to Moyamoya disease serum were partially suppressed by the inhibition of angiopoietin-2. Our findings define brain endothelial cells as the potential source of vessel-destabilizing factors inducing the high plasticity state and disintegration in Moyamoya disease in an autocrine manner. We also provide new insights into Moyamoya disease pathophysiology that may be helpful for preventive treatment strategies in future.

Anti-atherogenic effects of statins: Impact on angiopoietin-2 release from endothelial cells

Beyond lipid lowering, statins are supposed to exert pleiotropic effects positively influencing the progression of atherosclerotic lesions. The development of such lesions is associated with increased release of angiopoietin-2 (Ang-2), an endothelial cell-specific protein growth factor stored in Weibel-Palade bodies (WPBs). The aim of our study was to examine whether statin pretreatment influences the release of Ang-2 from endothelial cells. Stimulation of HUVECs and HMVECs with PMA, thrombin or histamine resulted in significant release of Ang-2, as evidenced by ELISA. Pretreatment with simvastatin and mevastatin suppressed this release to basal level, while pravastatin had no effect. Simvastatin itself increased nitric oxide (NO, EC number 1.14.13.39) synthesis, measured by Griess reaction. Combining the statin pretreatment with the eNOS inhibitor L-NNA as well as bypassing the HMG-CoA reductase (EC number: 1.1.1.34) by adding mevalonic acid or geranyl pyrophosphate restored the exocytotic effect of PMA. Immunofluorescence microscopy showed that depletion of WPBs upon PMA stimulation ceased after pretreatment with simvastatin. This study demonstrates a potent suppressive effect of statins on the release of Ang-2 from endothelial cells. Regarding its harmful effects in the development of atherosclerotic lesions, our data provide further insight into the mechanisms of the anti-atherogenic potential of statins.

Renal Ischemia-Reperfusion Induces Release of Angiopoietin-2 From Human Grafts of Living and Deceased Donors

Recent insights suggest that endothelial cell (EC) activation plays a major role in renal ischemia-reperfusion (I/R) injury. Interactions between ECs and pericytes via signaling molecules, including angiopoietins, are involved in maintenance of the vascular integrity. Experimental data have shown that enhancement of Angiopoietin (Ang)-1 signaling might be beneficial in renal I/R injury. However, little is known about the role of angiopoietins in human renal I/R injury. In this study, EC activation and changes in angiopoeitins are assessed in human living-donor (LD) and deceased-donor (DD) kidney transplantation. Local release of angiopoietins was measured by unique, dynamic arteriovenous measurements over the reperfused kidney. Renal I/R is associated with acute EC activation shown by a vast Ang-2 release from both LD and DD shortly after reperfusion. Its counterpart Ang-1 was not released. Histologic analysis of kidney biopsies showed EC loss after reperfusion. Baseline protein and mRNA Ang-1 expression was significantly reduced in DD compared with LD and declined further after reperfusion. Human renal I/R injury induces EC activation after reperfusion reflected by Ang-2 release from the kidney. Interventions aimed at maintenance of vascular integrity by modulating angiopoietin signaling may be promising in human clinical kidney transplantation.

Modulation of the release of Ang-2 in experimental endotoxic shock by a species-specific circulating factor

ObjectivesTo study the modulation of the release of angiopoietin-2 (Ang-2) in experimental endotoxic shock. MethodsTwelve pigs were studied; eight became septic after the intravenous infusion of lipopolysaccharide (LPS) of Escherichia coli O55:B5. The concentrations of LPS, angiopoietin-2 (Ang-2), tumour necrosis factor-alpha (TNFα) and malondialdehyde (MDA) were measured soon after the LPS infusion in the serum samples from the pulmonary and systemic circulation. Peripheral blood mononuclear cells (PBMCs) were isolated from two healthy swine, from two healthy human donors and from four patients with septic shock. The PBMCs were cultured with the serum of the septic animals in the presence or absence of polymyxin B. Concentrations of Ang-2 and TNFα were measured in supernatants. ResultsSerum Ang-2 was higher in the systemic circulation than in the pulmonary circulation. Increased Ang-2 release was noted in swine PBMCs in the presence of polymyxin B. A reciprocal decrease in TNFα release was observed, typically after incubation with serum sampled from the pulmonary circulation. ConclusionThere is evidence for a circulating factor that primes Ang-2 release from blood monocytes in the event of septic shock. The finding indicates a possible site of interference within the septic shock cascade.