Shock-Driven Endotheliopathy in Trauma Patients Is Associated with Leucocyte Derived Extracellular Vesicles.

Endotheliopathy following trauma is associated with poor outcome, but the underlying mechanisms are unknown. This study hypothesized that an increased extracellular vesicle (EV) concentration is associated with endotheliopathy after trauma and that red blood cell (RBC) transfusion could further enhance endotheliopathy. In this post hoc sub study of a multicentre observational trial, 75 trauma patients were stratified into three groups based on injury severity score or shock. In patient plasma obtained at hospital admission and after transfusion of four RBC transfusions, markers for endotheliopathy were measured and EVs were labelled with anti CD41 (platelet EVs), anti CD235a (red blood cell EVs), anti CD45 (leucocyte EVs), anti CD144 (endothelial EVs) or anti CD62e (activated endothelial EVs) and EV concentrations were measured with flow cytometry. Statistical analysis was performed by a Kruskall Wallis test with Bonferroni correction or Wilcoxon rank test for paired data. In patients with shock, syndecan-1 and von Willebrand Factor (vWF) were increased compared to patients without shock. Additionally, patients with shock had increased red blood cell EV and leucocyte EV concentrations compared to patients without shock. Endotheliopathy markers correlated with leucocyte EVs (ρ = 0.263, p = 0.023), but not with EVs derived from other cells. Injury severity score had no relation with EV release. RBC transfusion increased circulating red blood cell EVs but did not impact endotheliopathy. In conclusion, shock is (weakly) associated with EVs from leucocytes, suggesting an immune driven pathway mediated (at least in part) by shock.

Protocol for Measuring Concentrations of Extracellular Vesicles in Human Blood Plasma with Flow Cytometry.

Extracellular vesicles (EVs) are lipid membrane enclosed particles that are released from cells into body fluids, such as blood. EVs offer potential new biomarkers of diseases, because the cellular origin, composition, concentration, and function of EVs change in health and disease. The concentration of EVs from specific cell types in blood can be determined with flow cytometry. A flow cytometer measures fluorescence and light scattering signals from single EVs, but only if these signals are sufficiently bright to be detected. Measured concentrations of EVs are therefore only reproducible and comparable if the detection ranges are known and reported in standard units, such as molecules of equivalent soluble fluorophore (MESF) for fluorescence signals and the diameter in nm for scatter signals. The goal of this protocol is to discuss all steps needed to derive the concentration of cell-type specific EVs within a known diameter range and fluorescence range. More specifically, this protocol describes how to determine the concentration of CD61+ (Integrin beta-3, platelet marker), CD235a+ (Glycophorin A, erythrocyte marker), and CD45+ (leukocyte common antigen) EVs in human blood plasma with an Apogee A60-Micro flow cytometer using scatter-based triggering. The principles behind this protocol could lay a firm basis for the design of a protocol suitable for other flow cytometers and body fluids.

Prostacyclin Analogues Inhibit Platelet Reactivity, Extracellular Vesicle Release and Thrombus Formation in Patients with Pulmonary Arterial Hypertension.

(1) Background: Prostacyclin analogues (epoprostenol, treprostinil, and iloprost) induce vasodilation in pulmonary arterial hypertension (PAH) but also inhibit platelet function. (2) Objectives: We assessed platelet function in PAH patients treated with prostacyclin analogues and not receiving prostacyclin analogues. (3) Methods: Venous blood was collected from 42 patients treated with prostacyclin analogues (49.5 ± 15.9 years, 81% female) and 38 patients not receiving prostacyclin analogues (55.5 ± 15.6 years, 74% female). Platelet reactivity was analyzed by impedance aggregometry using arachidonic acid (AA; 0.5 mM), adenosine diphosphate (ADP; 6.5 µM), and thrombin receptor-activating peptide (TRAP; 32 µM) as agonists. In a subset of patients, concentrations of extracellular vesicles (EVs) from all platelets (CD61+), activated platelets (CD61+/CD62P+), leukocytes (CD45+), and endothelial cells (CD146+) were analyzed by flow cytometry. Platelet-rich thrombus formation was measured using a whole blood perfusion system. (4) Results: Compared to controls, PAH patients treated with prostacyclin analogues had lower platelet reactivity in response to AA and ADP (p = 0.01 for both), lower concentrations of platelet and leukocyte EVs (p ≤ 0.04), delayed thrombus formation (p ≤ 0.003), and decreased thrombus size (p = 0.008). Epoprostenol did not affect platelet reactivity but decreased the concentrations of platelet and leukocyte EVs (p ≤ 0.04). Treprostinil decreased platelet reactivity in response to AA and ADP (p ≤ 0.02) but had no effect on the concentrations of EVs. All prostacyclin analogues delayed thrombus formation and decreased thrombus size (p ≤ 0.04). (5) Conclusions: PAH patients treated with prostacyclin analogues had impaired platelet reactivity, EV release, and thrombus formation, compared to patients not receiving prostacyclin analogues.

Human milk triggers coagulation via tissue factor-exposing extracellular vesicles.

Almost a century ago, it was discovered that human milk activates the coagulation system, but the milk component that triggers coagulation had until now been unidentified. In the present study, we identify this component and demonstrate that extracellular vesicles (EVs) present in normal human milk expose coagulant tissue factor (TF). This coagulant activity withstands digestive conditions, mimicking those of breastfed infants, but is sensitive to pasteurization of pooled donor milk, which is routinely used in neonatal intensive care units. In contrast to human milk, bovine milk, the basis of most infant formulas, lacks coagulant activity. Currently, the physiological function of TF-exposing vesicles in human milk is unknown, but we speculate that these vesicles may be protective for infants. Another explanation could be nipple skin damage, which occurs in most breastfeeding women. Milk-derived TF-exposing EVs may seal the wound and thereby reduce bleeding and breast inflammation.

The P4-ATPase ATP9A is a novel determinant of exosome release.

Extracellular vesicles (EVs) released by cells have a role in intercellular communication to regulate a wide range of biological processes. Two types of EVs can be recognized. Exosomes, which are released from multi-vesicular bodies upon fusion with the plasma membrane, and ectosomes, which directly bud from the plasma membrane. How cells regulate the quantity of EV release is largely unknown. One of the initiating events in vesicle biogenesis is the regulated transport of phospholipids from the exoplasmic to the cytosolic leaflet of biological membranes. This process is catalyzed by P4-ATPases. The role of these phospholipid transporters in intracellular vesicle transport has been established in lower eukaryotes and is slowly emerging in mammalian cells. In Caenorhabditis elegans (C. elegans), deficiency of the P4-ATPase member TAT-5 resulted in enhanced EV shedding, indicating a role in the regulation of EV release. In this study, we investigated whether the mammalian ortholog of TAT-5, ATP9A, has a similar function in mammalian cells. We show that knockdown of ATP9A expression in human hepatoma cells resulted in a significant increase in EV release that was independent of caspase-3 activation. Pharmacological blocking of exosome release in ATP9A knockdown cells did significantly reduce the total number of EVs. Our data support a role for ATP9A in the regulation of exosome release from human cells.

P2Y12 antagonist ticagrelor inhibits the release of procoagulant extracellular vesicles from activated platelets.

BACKGROUND: Activated platelets release platelet extracellular vesicles (PEVs). Adenosine diphosphate (ADP) receptors P2Y1 and P2Y12 both play a role in platelet activation, The present hypothesis herein is that the inhibition of these receptors may affect the release of PEVs. METHODS: Platelet-rich plasma from 10 healthy subjects was incubated with saline, P2Y1 antagonist MRS2179 (100 µM), P2Y12 antagonist ticagrelor (1 µM), and a combination of both antagonists. Platelets were activated by ADP (10 µM) under stirring conditions at 37°C. Platelet reactivity was assessed by impedance aggregometry. Concentrations of PEVs- (positive for CD61 but negative for P-selectin and phosphatidylserine) and PEVs+ (positive for all) were determined by a state-of-the-art flow cytometer. Procoagulant activity of PEVs was measured by a fibrin generation test. RESULTS: ADP-induced aggregation (57 ± 13 area under curve {AUC] units) was inhibited 73% by the P2Y1 antagonist, 86% by the P2Y12 antagonist, and 95% when combined (p < 0.001 for all). The release of PEVs- (2.9 E ± 0.8 × 108/mL) was inhibited 48% in the presence of both antagonists (p = 0.015), whereas antagonists alone were ineffective. The release of PEVs+ (2.4 ± 1.6 × 107/mL) was unaffected by the P2Y1 antagonist, but was 62% inhibited by the P2Y12 antagonist (p = 0.035), and 72% by both antagonists (p = 0.022). PEVs promoted coagulation in presence of tissue factor. CONCLUSIONS: Inhibition of P2Y1 and P2Y12 receptors reduces platelet aggregation and affects the release of distinct subpopulations of PEVs. Ticagrelor decreases the release of procoagulant PEVs from activated platelets, which may contribute to the observed clinical benefits in patients treated with ticagrelor.

Tissue Factor Coagulant Activity is Regulated by the Plasma Membrane Microenvironment.

BACKGROUND: Tissue factor (TF) can be present in a non-coagulant and coagulant form. Whether the coagulant activity is affected by the plasma membrane microenvironment is unexplored. OBJECTIVE: This article studies the presence and coagulant activity of human TF in plasma membrane micro-domains. METHODS: Plasma membranes were isolated from human MIA PaCa2 cells, MDA-MB-231 cells and human vascular smooth muscle cells by Percoll gradient ultracentrifugation after cell disruption. Plasma membranes were fractionated by OptiPrep gradient ultracentrifugation, and the presence of TF, flotillin, caveolin, clathrin, protein disulphide isomerase (PDI), TF pathway inhibitor (TFPI) and phosphatidylserine (PS) were determined. RESULTS: Plasma membranes contain two detergent-resistant membrane (DRM) compartments differing in density and biochemical composition. High-density DRMs (DRM-H) have a density (ρ) of 1.15 to 1.20 g/mL and contain clathrin, whereas low-density DRMs (DRM-L) have a density between 1.09 and 1.13 g/mL and do not contain clathrin. Both DRMs contain TF, flotillin and caveolin. PDI is detectable in DRM-H, TFPI is not detectable in either DMR-H or DRM-L and PS is detectable in DRM-L. The DRM-H-associated TF (> 95% of the TF antigen) lacks detectable coagulant activity, whereas the DRM-L-associated TF triggers coagulation. This coagulant activity is inhibited by lactadherin and thus PS-dependent, but seemed insensitive to 16F16, an inhibitor of PDI. CONCLUSION: Non-coagulant and coagulant TF are present within different types of DRMs in the plasma membrane, and the composition of these DRMs may affect the TF coagulant activity.

A standardized method to determine the concentration of extracellular vesicles using tunable resistive pulse sensing.

BACKGROUND: Understanding the pathogenic role of extracellular vesicles (EVs) in disease and their potential diagnostic and therapeutic utility is extremely reliant on in-depth quantification, measurement and identification of EV sub-populations. Quantification of EVs has presented several challenges, predominantly due to the small size of vesicles such as exosomes and the availability of various technologies to measure nanosized particles, each technology having its own limitations. MATERIALS AND METHODS: A standardized methodology to measure the concentration of extracellular vesicles (EVs) has been developed and tested. The method is based on measuring the EV concentration as a function of a defined size range. Blood plasma EVs are isolated and purified using size exclusion columns (qEV) and consecutively measured with tunable resistive pulse sensing (TRPS). Six independent research groups measured liposome and EV samples with the aim to evaluate the developed methodology. Each group measured identical samples using up to 5 nanopores with 3 repeat measurements per pore. Descriptive statistics and unsupervised multivariate data analysis with principal component analysis (PCA) were used to evaluate reproducibility across the groups and to explore and visualise possible patterns and outliers in EV and liposome data sets. RESULTS: PCA revealed good reproducibility within and between laboratories, with few minor outlying samples. Measured mean liposome (not filtered with qEV) and EV (filtered with qEV) concentrations had coefficients of variance of 23.9% and 52.5%, respectively. The increased variance of the EV concentration measurements could be attributed to the use of qEVs and the polydisperse nature of EVs. CONCLUSION: The results of this study demonstrate the feasibility of this standardized methodology to facilitate comparable and reproducible EV concentration measurements.

Reproducible extracellular vesicle size and concentration determination with tunable resistive pulse sensing.

INTRODUCTION: The size of extracellular vesicles (EVs) can be determined with a tunable resistive pulse sensor (TRPS). Because the sensing pore diameter varies from pore to pore, the minimum detectable diameter also varies. The aim of this study is to determine and improve the reproducibility of TRPS measurements. METHODS: Experiments were performed with the qNano system (Izon) using beads and a standard urine vesicle sample. With a combination of voltage and stretch that yields a high blockade height, we investigate whether the minimum detected diameter is more reproducible when we configure the instrument targeting (a) fixed stretch and voltage, or (b) fixed blockade height. RESULTS: Daily measurements with a fixed stretch and voltage (n=102) on a standard urine sample show a minimum detected vesicle diameter of 128±19 nm [mean±standard deviation; coefficient of variation (CV) 14.8%]. The vesicle concentration was 2.4·109±3.8·109 vesicles/mL (range 1.4·108-1.8·1010). When we compared setting a fixed stretch and voltage to setting a fixed blockade height on 3 different pores, we found a minimum detected vesicle diameter of 118 nm (CV 15.5%, stretch), and 123 nm (CV 4.5%, blockade height). The detected vesicle concentration was 3.2-8.2·108 vesicles/mL with fixed stretch and 6.4-7.8·108 vesicles/mL with fixed blockade height. Summary/conclusion: Pore-to-pore variability is the cause of the variation in minimum detected size when setting a fixed stretch and voltage. The reproducibility of the minimum detectable diameter is much improved by setting a fixed blockade height.

Effects of muscarinic receptor stimulation on Ca2+ transient, cAMP production and pacemaker frequency of rabbit sinoatrial node cells.

We investigated the contribution of the intracellular calcium (Ca (i) (2+) ) transient to acetylcholine (ACh)-mediated reduction of pacemaker frequency and cAMP content in rabbit sinoatrial nodal (SAN) cells. Action potentials (whole cell perforated patch clamp) and Ca (i) (2+) transients (Indo-1 fluorescence) were recorded from single isolated rabbit SAN cells, whereas intracellular cAMP content was measured in SAN cell suspensions using a cAMP assay (LANCE((R))). Our data show that the Ca (i) (2+) transient, like the hyperpolarization-activated "funny current" (I (f)) and the ACh-sensitive potassium current (I (K,ACh)), is an important determinant of ACh-mediated pacemaker slowing. When I (f) and I (K,ACh) were both inhibited, by cesium (2 mM) and tertiapin (100 nM), respectively, 1 micro M ACh was still able to reduce pacemaker frequency by 72%. In these I (f) and I (K,ACh)-inhibited SAN cells, good correlations were found between the ACh-mediated change in interbeat interval and the ACh-mediated change in Ca (i) (2+) transient decay (r (2) = 0.98) and slow diastolic Ca (i) (2+) rise (r (2) = 0.73). Inhibition of the Ca (i) (2+) transient by ryanodine (3 microM) or BAPTA-AM (5 microM) facilitated ACh-mediated pacemaker slowing. Furthermore, ACh depressed the Ca (i) (2+) transient and reduced the sarcoplasmic reticulum (SR) Ca(2+) content, all in a concentration-dependent fashion. At 1 microM ACh, the spontaneous activity and Ca (i) (2+) transient were abolished, but completely recovered when cAMP production was stimulated by forskolin (10 microM) and I (K,ACh) was inhibited by tertiapin (100 nM). Also, inhibition of the Ca (i) (2+) transient by ryanodine (3 microM) or BAPTA-AM (25 microM) exaggerated the ACh-mediated inhibition of cAMP content, indicating that Ca (i) (2+) affects cAMP production in SAN cells. In conclusion, muscarinic receptor stimulation inhibits the Ca (i) (2+) transient via a cAMP-dependent signaling pathway. Inhibition of the Ca (i) (2+) transient contributes to pacemaker slowing and inhibits Ca (i) (2+) -stimulated cAMP production. Thus, we provide functional evidence for the contribution of the Ca (i) (2+) transient to ACh-induced inhibition of pacemaker activity and cAMP content in rabbit SAN cells.

Sphingosine-1-phosphate regulates RGS2 and RGS16 mRNA expression in vascular smooth muscle cells.

Regulator of G protein signalling (RGS) protein expression is altered under growth promoting conditions in vascular smooth muscle cells (VSMCs). Since sphingosine-1-phosphate (S1P) is an important growth stimulatory factor, we investigated whether stimulation of VSMCs with S1P results in alterations in mRNA expression levels of several RGS proteins and which signalling components are involved. VSMCs were stimulated with S1P and mRNA expression levels of RGS2, RGS3, RGS4, RGS5 and RGS16 were measured by real-time polymerase chain reaction. S1P caused a time-dependent up-regulation of RGS2 and RGS16 mRNA expression. FTY720-P, a S1P(1)/S1P(3-5) agonist, did not regulate RGS2 mRNA levels although it did up-regulate RGS16 mRNA expression. Pertussis toxin treatment revealed that the S1P-induced RGS16 expression was G(i/o)-dependent whereas up-regulation of RGS2 mRNA was not. Phosphatidylinositol 3-kinase, protein kinase C and mitogen-activated protein kinase kinase apparently were not involved in the S1P-induced up-regulation of both RGS proteins. The present study demonstrates that S1P induces RGS2 and RGS16 mRNA expression but uses distinct S1P receptor subtypes and signalling pathways to regulate expression of these RGS proteins.

Activation of sphingosine kinase by muscarinic receptors enhances NO-mediated and attenuates EDHF-mediated vasorelaxation.

Local formation of the sphingomyelin metabolite sphingosine-1-phosphate (S1P) within the vascular wall has been shown to modulate vascular reactivity. In this study we investigated whether sphingosine kinase, the enzyme responsible for S1P synthesis, plays a role in muscarinic receptor-mediated NO production and vascular relaxation in different blood vessel types. For this purpose, sphingosine kinase translocation and sphingolipid-dependent NO-production after muscarinic receptor stimulation were assessed in an endothelial cell line. Furthermore, we used the sphingosine kinase inhibitor N,N-dimethylsphingosine (DMS) to investigate the role of sphingosine kinase in the relaxant responses to the muscarinic agonist methacholine (MCh) in isolated rat aorta and mesenteric arteries. Activation of M(3)-receptors in an endothelial cell line induced a fast translocation of YFP-tagged sphingosine kinase-1 from the cytosol to the plasma membrane. Concomitant NO-production in this cell line was partially inhibited by DMS. Accordingly, in rat aorta the relaxant responses to MCh were attenuated in the presence of DMS, while the responses to the NO-donor sodium nitroprusside were unaltered. In contrast, DMS enhanced the relaxant responses to MCh in mesenteric artery preparations. This effect could also be observed in the presence of NO synthase and cyclooxygenase inhibitors, indicating that sphingosine kinase inhibition specifically enhanced endothelium-derived hyperpolarizing factor-mediated (i.e. non-NO and non-prostacyclin-dependent) relaxation. We conclude that sphingosine kinase differentially regulates vascular tone in different vessel types, enhancing NO-dependent vasorelaxation but counteracting EDHF-dependent vasorelaxation. This observation enhances our understanding of the complex mechanisms by which sphingolipids regulate vascular homeostasis. Moreover, a disturbed regulation of sphingolipid metabolism in the vascular wall may therefore play a role in the aetiology/pathology of disease states characterized by endothelial dysfunction.

S1P receptor signalling and RGS proteins; expression and function in vascular smooth muscle cells and transfected CHO cells.

Sphingosine-1-phosphate (S1P) signalling via G protein-coupled receptors is important for the regulation of cell function and differentiation. Specific Regulators of G protein Signalling (RGS) proteins modulate the function of these receptors in many cell types including vascular smooth muscle cells (VSMCs). Therefore, we investigated the role of altered expression levels of RGS proteins in S1P receptor function in VSMCs and transfected CHO cells. The mRNA expression of the S1P(1) receptor, RGS4 and RGS16 were down-regulated in VSMCs during phenotypic modulation induced by culturing, whereas mRNA levels of RGS2, RGS3, S1P(2) and S1P(3) receptors were unchanged. Interestingly, the expression level of RGS5 was transiently up-regulated. Despite major alterations in RGS levels, S1P-induced calcium elevation in VSMCs was not altered. Co-transfection of RGS2, RGS3, RGS4, RGS5 and RGS16 into CHO-Flp-In cells stably expressing the S1P(1) or S1P(3) receptor did not modify S1P-induced inhibition of cAMP accumulation to a major extent. Similar results were obtained with SEW2871, a selective S1P(1) receptor agonist. However, the inhibition of cAMP accumulation by the agonist FTY720-P via the S1P(1) receptor was significantly decreased by co-transfection with RGS5. These results indicate that mRNA of the S1P(1) receptor, RGS4, RGS5 and RGS16 is differentially regulated during phenotypic modulation. However, major alterations in RGS protein expression have only limited effect on S1P receptor function.

Cyclosporin A induces peritoneal fibrosis and angiogenesis during chronic peritoneal exposure to a glucose-based, lactate-buffered dialysis solution in the rat.

BACKGROUND/AIMS: Cyclosporin A (CsA) stimulates the development of fibrosis. We investigated whether CsA contributes to peritoneal alterations induced by long-term exposure to dialysis solutions. METHODS: Ten rats received peritoneal infusion of dialysis solution and oral CsA for 8 weeks. Eight received only the dialysis solution (controls). Peritoneal function was assessed at 8 weeks followed by sacrifice. The number of vessels was counted, fibrosis was assessed and hydroxyproline was determined. PCR was performed for vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF) and transforming growth factor-beta (TGF-beta). RESULTS: Histology revealed more fibrosis, hydroxyproline and vessels (thick walled) in CsA-exposed animals. Peritoneal transport was not different. The mRNA content of TGF-beta, CTGF and VEGF was higher in CsA. CONCLUSION: CsA combined with exposure to dialysis solutions was associated with increased peritoneal fibrosis and angiogenesis.

Noncanonical NF-kappaB signaling in dendritic cells is required for indoleamine 2,3-dioxygenase (IDO) induction and immune regulation.

Ligation of CD40 on dendritic cells (DCs) induces early production of inflammatory mediators via canonical NF-kappaB signaling, as well as late expression of the anti-inflammatory enzyme indoleamine 2,3-dioxygenase (IDO) via unknown signal transduction. By selective blocking of either the canonical NF-kappaB pathway using the NEMO-binding domain peptide or the noncanonical NF-kappaB pathway by small interfering RNA, we demonstrate that IDO expression requires noncanonical NF-kappaB signaling. Also, noncanonical NF-kappaB signaling down-regulates proinflammatory cytokine production in DCs. In addition, selective activation of the noncanonical NF-kappaB pathway results in noninflammatory DCs that suppress T-cell activation and promote the development of T cells with regulatory properties. These findings reveal an important role of the noncanonical NF-kappaB pathway in the regulation of immunity.

Amelioration of arthritis by intraarticular dominant negative Ikk beta gene therapy using adeno-associated virus type 5.

Nuclear factor (NF)-kappaB is highly activated in the synovium of rheumatoid arthritis (RA) patients, and can induce transcription of many proinflammatory molecules. Phosphorylation of inhibitor of kappaB (IkappaB) proteins is an important step in NF-kappaB activation and under inflammatory conditions is regulated predominantly by IkappaB kinase (IKK)beta. Consequently, specific targeting of IKK beta in the joint, using gene therapy, presents a sophisticated treatment option for arthritis. In the present study we investigated the effect of inhibiting IKK beta in adjuvant arthritis (AA) in rats, using recombinant adeno-associated virus (rAAV)-mediated intraarticular gene therapy. For this purpose rAAV5 carrying the dominant negative IKK beta gene (AAV5.IKK beta dn) or control AAV5.eGFP was injected into the right ankle joint. Rats treated with AAV5.IKK beta dn in early arthritis exhibited significantly reduced paw swelling (p < 0.05). Immunohistochemical analysis of synovial tissue revealed reduced levels of interleukin (IL)-6 (p = 0.005) and tumor necrosis factor-alpha (TNF-alpha) (p = 0.03), whereas IL-10 levels were not affected. No significant effect was found on cartilage and bone destruction, or on matrix metalloproteinase-3 and tissue inhibitor of matrix metalloproteinase-1 expression. Injection of AAV5.IKK beta dn in the preclinical phase showed only a marginal effect on arthritis. Importantly, in this study we also demonstrate for the first time that our vector is capable of transducing human RA whole synovial tissue biopsies ex vivo, resulting in reduced IL-6 production after TNF-alpha stimulation (p = 0.03). In conclusion, we are the first to demonstrate that rAAV5 can be used to successfully deliver a therapeutic gene (IKK beta dn) to the synovium, resulting in reduced severity of inflammation in AA in vivo and proinflammatory cytokine production in human RA synovial tissue ex vivo. This translational research represents a crucial next step toward the development of gene therapy for application in humans.

Local treatment with the selective IkappaB kinase beta inhibitor NEMO-binding domain peptide ameliorates synovial inflammation.

Nuclear factor (NF)-kappaB is a key regulator of synovial inflammation. We investigated the effect of local NF-kappaB inhibition in rat adjuvant arthritis (AA), using the specific IkappaB kinase (IKK)-beta blocking NF-kappaB essential modulator-binding domain (NBD) peptide. The effects of the NBD peptide on human fibroblast-like synoviocytes (FLS) and macrophages, as well as rheumatoid arthritis (RA) whole-tissue biopsies, were also evaluated. First, we investigated the effects of the NBD peptide on RA FLS in vitro. Subsequently, NBD peptides were administered intra-articularly into the right ankle joint of rats at the onset of disease. The severity of arthritis was monitored over time, rats were sacrificed on day 20, and tissue specimens were collected for routine histology and x-rays of the ankle joints. Human macrophages or RA synovial tissues were cultured ex vivo in the presence or absence of NBD peptides, and cytokine production was measured in the supernatant by enzyme-linked immunosorbent assay. The NBD peptide blocked interleukin (IL)-1-beta-induced IkappaB alpha phosphorylation and IL-6 production in RA FLS. Intra-articular injection of the NBD peptide led to significantly reduced severity of arthritis (p < 0.0001) and reduced radiological damage (p = 0.04). This was associated with decreased synovial cellularity and reduced expression of tumor necrosis factor (TNF)-alpha and IL-1-beta in the synovium. Incubation of human macrophages with NBD peptides resulted in 50% inhibition of IL-1-beta-induced TNF-alpha production in the supernatant (p < 0.01). In addition, the NBD peptide decreased TNF-alpha-induced IL-6 production by human RA synovial tissue biopsies by approximately 42% (p < 0.01). Specific NF-kappaB blockade using a small peptide inhibitor of IKK-beta has anti-inflammatory effects in AA and human RA synovial tissue as well as in two important cell types in the pathogenesis of RA: macrophages and FLS. These results indicate that IKK-beta-targeted NF-kappaB blockade using the NBD peptide could offer a new approach for the local treatment of arthritis.

Selective inhibition of NF-kappaB in dendritic cells by the NEMO-binding domain peptide blocks maturation and prevents T cell proliferation and polarization.

Dendritic cells (DC) are the only antigen-presenting cells for naive T cells and, therefore, they are crucial players in the initiation of immune responses. Because DC maturation and cytokine production are NF-kappaB dependent, we hypothesized that blocking NF-kappaB activity in DC by selectively targeting the inhibitor of kappaB (IkappaB) kinase (IKK) complex using the novel NF-kappaB inhibitor NEMO-binding domain (NBD) peptide could inhibit DC maturation and other functional characteristics, resulting in modulation of the immune response. We used human monocyte-derived DC to test the biological effects of the NBD peptide in vitro. NF-kappaB inhibition by the NBD peptide resulted in blockade of IKK-mediated IkappaBalpha phosphorylation and subsequent nuclear translocation and DNA binding of NF-kappaB p65 in DC. In addition, IL-6, IL-12, and TNF-alpha production was dose-dependently blocked and NBD peptide treatment also led to a strong reduction of LPS-induced maturation. Functional analysis of these DC showed marked inhibition of T cell proliferation in the allogeneic mixed lymphocyte reaction, accompanied by less Th1 and Th2 polarization. The current study reveals for the first time the unique properties of this novel, highly specific NF-kappaB inhibitor in DC. Also, these data indicate that the NBD peptide could be used as an elegant tool in DC based immunotherapy for unwanted cellular immune responses.