Leukocyte-endothelial Cell Interactions Research Articles

(Homo-)harringtonine prevents endothelial inflammation through IRF-1 dependent downregulation of VCAM1 mRNA expression and inhibition of cell adhesion molecule protein biosynthesis

The plant alkaloid homoharringtonine (HHT) is a Food and Drug Administration (FDA)-approved drug for the treatment of hematologic malignancies. In addition to its well-established antitumor activity, accumulating evidence attributes anti-inflammatory effects to HHT, which have mainly been studied in leukocytes to date. However, a potential influence of HHT on inflammatory activation processes in endothelial cells, which are a key feature of inflammation and a prerequisite for the leukocyte-endothelial cell interaction and leukocyte extravasation, remains poorly understood. In this study, the anti-inflammatory potential of HHT and its derivative harringtonine (HT) on the TNF-induced leukocyte-endothelial cell interaction was assessed, and the underlying mechanistic basis of these effects was elucidated. HHT affected inflammation in vivo in a murine peritonitis model by reducing leukocyte infiltration and proinflammatory cytokine expression as well as ameliorating abdominal pain behavior. In vitro, HT and HHT impaired the leukocyte-endothelial cell interaction by decreasing the expression of the endothelial cell adhesion molecules intracellular adhesion molecule −1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). This effect was mediated by a bipartite mechanism. While HHT did not affect the prominent TNF-induced pro-inflammatory NF-ĸB signaling cascade, the compound downregulated the VCAM1 mRNA expression in an IRF-1-dependent manner and diminished active ICAM1 mRNA translation as determined by polysome profiling. This study highlights HHT as an anti-inflammatory compound that efficiently hampers the leukocyte-endothelial cell interaction by targeting endothelial activation processes.

HPLC- and NMR-Based Chemical Profiling, Wound-Healing Potential, Anti-Inflammatory and Antibacterial Activities of Satureja pilosa (Lamiaceae), a Neglected Medicinal-Aromatic Herb.

Satureja pilosa Velen. (Lamiaceae) is a perennial and melliferous aromatic-medicinal subshrub which is range-restricted in adjacent parts of Greece and Bulgaria and locally in Italy, known in Northern Greece as wild oregano ("agriorigani") and traditionally collected from the wild for culinary purposes. Since the ethnopharmacological data and modern biological activities of Satureja spp. suggest promising applications in skin conditions, the present study aimed to investigate the hitherto unknown phenolic content of cultivated S. pilosa and its potential biological activities, focusing mainly on wound-healing and anti-inflammatory effects. An HPLC-PDA-MS-targeted phytochemical investigation, along with NMR, allowed for the isolation and characterization of the main constituents, resulting in 18 compounds. Representative extracts and purified compounds were tested for wound-healing activity on NIH/3T3 fibroblasts. The butanol extract exhibited a significantly higher cell migration rate (73.4%) compared to aqueous (50.6%) and methanolic (49.6%) ones, enhancing the cell migration more rapidly at both concentration levels, whilst rosmarinic acid was the most potent among the isolated compounds, with a migration rate of 64.0% at the concentration level of 10-5 mg/mL, followed by 3,4-dihydrophenyllactic acid (54.7%). Moreover, potential effects on endothelial activation processes were explored, including the leukocyte-endothelial cell interaction during inflammatory processes and the migratory capacity during angiogenic actions, since these processes are commonly associated with skin diseases. Finally, extracts and purified compounds demonstrated weak antibacterial potential against two important pathogens (Staphylococcus aureus and Pseudomonas aeruginosa), suggesting that further investigation is warrented.

Removal of the endothelial surface layer via hyaluronidase does not modulate monocyte and neutrophil interactions with the glomerular endothelium.

The endothelial surface layer (ESL), a layer of macromolecules on the surface of endothelial cells, can both impede and facilitate leukocyte recruitment. However, its role in monocyte and neutrophil recruitment in glomerular capillaries is unknown. We used multiphoton intravital microscopy to examine monocyte and neutrophil behavior in the glomerulus following ESL disruption with hyaluronidase. Constitutive retention and migration of monocytes and neutrophils within the glomerular microvasculature was unaltered by hyaluronidase. Consistent with this, inhibition of the hyaluronan-binding molecule CD44 also failed to modulate glomerular trafficking of these immune cells. To investigate the contribution of the ESL during acute inflammation, we induced glomerulonephritis via in situ immune complex deposition. This resulted in increases in glomerular retention of monocytes and neutrophils but did not induce marked reduction in the glomerular ESL. Furthermore, hyaluronidase treatment did not modify the prolonged retention of monocytes and neutrophils in the acutely inflamed glomerular microvasculature. These observations indicate that, despite evidence that the ESL has the capacity to inhibit leukocyte-endothelial cell interactions while also containing adhesive ligands for immune cells, neither of these functions modulate trafficking of monocytes and neutrophils in steady-state or acutely-inflamed glomeruli.

Inhibiting leukocyte-endothelial cell interactions by Chinese medicine Tongxinluo capsule alleviates no-reflow after arterial recanalization in ischemic stroke.

Despite successful vascular recanalization in stroke, one-fourth of patients have an unfavorable outcome due to no-reflow. The pathogenesis of no-reflow is fully unclear, and therapeutic strategies are lacking. Upon traditional Chinese medicine, Tongxinluo capsule (TXL) is a potential therapeutic agent for no-reflow. Thus, this study is aimed to investigate the pathogenesis of no-reflow in stroke, and whether TXL could alleviate no-reflow as well as its potential mechanisms of action. Mice were orally administered with TXL (3.0 g/kg/d) after transient middle cerebral artery occlusion. We examined the following parameters: neurological function, no-reflow, leukocyte-endothelial cell interactions, HE staining, leukocyte subtypes, adhesion molecules, and chemokines. Our results showed stroke caused neurological deficits, neuron death, and no-reflow. Adherent and aggregated leukocytes obstructed microvessels as well as leukocyte infiltration in ischemic brain. Leukocyte subtypes changed after stroke mainly including neutrophils, lymphocytes, regulatory T cells, suppressor T cells, helper T type 1 (Th1) cells, Th2 cells, B cells, macrophages, natural killer cells, and dendritic cells. Stroke resulted in upregulated expression of adhesion molecules (P-selectin, E-selectin, and ICAM-1) and chemokines (CC-chemokine ligand (CCL)-2, CCL-3, CCL-4, CCL-5, and chemokine C-X-C ligand 1 (CXCL-1)). Notably, TXL improved neurological deficits, protected neurons, alleviated no-reflow and leukocyte-endothelial cell interactions, regulated multiple leukocyte subtypes, and inhibited the expression of various inflammatory mediators. Leukocyte-endothelial cell interactions mediated by multiple inflammatory factors are an important cause of no-reflow in stroke. Accordingly, TXL could alleviate no-reflow via suppressing the interactions through modulating various leukocyte subtypes and inhibiting the expression of multiple inflammatory mediators.

Methods for Imaging Inflammation and Transendothelial Migration in Vivo and ex Vivo.

Inflammation is the body's response to injury and harmful stimuli and contributes to a range of infectious and noninfectious diseases. Inflammation occurs through a series of well-defined leukocyte-endothelial cell interactions, including rolling, activation, adhesion, transmigration, and subsequent migration through the extracellular matrix. Being able to visualize the stages of inflammation is important for a better understanding of its role in diseases processes. Detailed in this article are protocols for imaging immune cell infiltration and transendothelial migration in vascular tissue beds, including those in the mouse ear, cremaster muscle, brain, lung, and retina. Also described are protocols for inducing inflammation and quantifying leukocytes with FIJI imaging software. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Induction of croton oil dermatitis Alternate Protocol 1: Induction of croton oil dermatitis using genetically fluorescent mice Basic Protocol 2: Intravital microscopy of the mouse cremaster muscle Support Protocol: Making a silicone stage Basic Protocol 3: Wide-field microscopy of the mouse brain Basic Protocol 4: Imaging the lungs (ex vivo) Alternate Protocol 2: Inflating the lungs without tracheostomy Basic Protocol 5: Inducing, imaging, and quantifying infiltration of leukocytes in mouse retina.

Effect of Roux-en-Y gastric bypass surgery on subclinical atherosclerosis, oxidative stress, and mitochondrial dynamics in leukocytes of obese patients: A one-year follow-up study

Background and Aims : Little is known about the mechanisms underlying the cardiovascular protective effect of Roux en-Y gastric bypass (RYGB) surgery. The present study aimed to investigate how RYGB influences the oxidative and mitochondrial status of leukocytes and subclinical atherosclerotic markers.Methods: This interventional study was carried out in 57 obese subjects who underwent RYGB surgery. Leukocytes were isolated at baseline and 12 months after the intervention. We evaluated oxidative stress markers by static cytometry and mitochondrial markers by western blot. Leukocyte-endothelial cell interactions - rolling flux, velocity, and adhesion - were evaluated in vitro in a parallel-plate flow chamber.Results: RYGB induced weight loss and an improvement of anthropometric clinical indicators. At the molecular level, a significant reduction of superoxide production (p<0.01) and mitochondrial membrane potential (p<0.05) was observed, while GPX1 content increased (p<0.05). In terms of mitochondria, RYGB induced upregulation of complexes (I-V) (p<0.05 and p<0.001), MIEAP (p<0.05), and Pink1 (p<0.05), and regulated the fusion-fission machinery through an increase in MFN1 (p<0.05). Likewise, a significant reduction of sICAM (p< 0.05) and sP-Selectin (p < 0.001) and of rolling flux (p< 0.05) and adhesion of leukocytes (p< 0.01) were reported at follow-up.Conclusions: Our results suggest that patients undergoing RYGB benefit from an amelioration of their prooxidant status and enhanced mitochondrial dynamics in leukocytes, which could be responsible for the reduction of subclinical markers of atherosclerosis after surgery Acknowledgements: PI19/00838, PI19/00437, FI17/00144, CD19/00180, CP19/00077 from ISCIII-ERDF (“A way to build Europe”), PROMETEO/2019/027; APOSTD/2020/145, ACIF/2020/371 from the Valencian Regional Government. Background and Aims : Little is known about the mechanisms underlying the cardiovascular protective effect of Roux en-Y gastric bypass (RYGB) surgery. The present study aimed to investigate how RYGB influences the oxidative and mitochondrial status of leukocytes and subclinical atherosclerotic markers. Methods: This interventional study was carried out in 57 obese subjects who underwent RYGB surgery. Leukocytes were isolated at baseline and 12 months after the intervention. We evaluated oxidative stress markers by static cytometry and mitochondrial markers by western blot. Leukocyte-endothelial cell interactions - rolling flux, velocity, and adhesion - were evaluated in vitro in a parallel-plate flow chamber. Results: RYGB induced weight loss and an improvement of anthropometric clinical indicators. At the molecular level, a significant reduction of superoxide production (p<0.01) and mitochondrial membrane potential (p<0.05) was observed, while GPX1 content increased (p<0.05). In terms of mitochondria, RYGB induced upregulation of complexes (I-V) (p<0.05 and p<0.001), MIEAP (p<0.05), and Pink1 (p<0.05), and regulated the fusion-fission machinery through an increase in MFN1 (p<0.05). Likewise, a significant reduction of sICAM (p< 0.05) and sP-Selectin (p < 0.001) and of rolling flux (p< 0.05) and adhesion of leukocytes (p< 0.01) were reported at follow-up. Conclusions: Our results suggest that patients undergoing RYGB benefit from an amelioration of their prooxidant status and enhanced mitochondrial dynamics in leukocytes, which could be responsible for the reduction of subclinical markers of atherosclerosis after surgery Acknowledgements: PI19/00838, PI19/00437, FI17/00144, CD19/00180, CP19/00077 from ISCIII-ERDF (“A way to build Europe”), PROMETEO/2019/027; APOSTD/2020/145, ACIF/2020/371 from the Valencian Regional Government.

HDL and the pathogenesis of delayed cerebral vasospasm following subarachnoid hemorrhage

Background and Aims : Delayed cerebral vasospasm (DCV) is the leading cause of mortality after subarachnoid hemorrhage (SAH). There is increasing evidence that inflammation, specifically leukocyte-endothelial cell interactions, plays a role in the pathogenesis of DCV. High-density lipoproteins (HDL) are biological molecules that prevent the expression of cell adhesion molecules induced by pro-inflammatory agents in endothelial cells, thus inhibiting leukocyte adhesion. In pathological conditions, HDL undergo quantitative and qualitative changes that have been associated with loss of physiological function.

Time‐of‐Day Influence on Central Nervous System Autoimmunity

Circadian rhythms are a characteristic of most living organisms and describe biological processes experiencing an oscillation of about 24h. These rhythms regulate multiple physiological systems, including innate and adaptive immune responses. Circadian disruption is an important risk factor in neuroinflammation such as multiple sclerosis (MS), a T‐cell mediated autoimmune disease affecting around 2.8 million people worldwide. In the central nervous system (CNS), autoimmune T cells attack oligodendrocytes leading to progressive demyelination and neurological impairment. Studies performed by our lab demonstrate that the circadian clock plays an important role in the severity of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis. However, the mechanisms responsible for this time‐of‐day difference are unknown. We investigated time‐of‐day differences in adhesion molecule expression and immune cell infiltration to the CNS that could explain differences in disease phenotype. Our results indicate a time‐of‐day‐dependent infiltration of cells into spinal cords during EAE development that correlates with expression of the adhesion molecules VCAM‐1 and ICAM‐1 in the CNS vasculature. Targeting leukocyte‐endothelial cell interactions in a time‐of‐day dependent manner with an anti‐VLA4 antibody that blocks VLA‐4/VCAM‐1 engagement reduces leukocyte infiltration and disease severity. This study shows the importance of circadian oscillations in adhesion molecule expression and cell infiltration in the CNS, and identifies an axis to target for chronotherapy purposes in MS patients.

Pericyte Loss Leads to Capillary Stalling Through Increased Leukocyte-Endothelial Cell Interaction in the Brain.

The neurovascular unit is a functional unit composed of neurons, glial cells, pericytes, and endothelial cells which sustain brain activity. While pericyte is a key component of the neurovascular unit, its role in cerebral blood flow regulation remains elusive. Recently, capillary stalling, which means the transient interruption of microcirculation in capillaries, has been shown to have an outsized impact on microcirculatory changes in several neurological diseases. In this study, we investigated capillary stalling and its possible causes, such as the cerebral endothelial glycocalyx and leukocyte adhesion molecules after depleting pericytes postnatally in mice. Moreover, we investigated hypoxia and gliosis as consequences of capillary stalling. Although there were no differences in the capillary structure and RBC flow, longitudinal optical coherence tomography angiography showed an increased number of stalled segments in capillaries after pericyte loss. Furthermore, the extent of the cerebral endothelial glycocalyx was decreased with increased expression of leukocyte adhesion molecules, suggesting enhanced interaction between leukocytes and endothelial cells. Finally, pericyte loss induced cerebral hypoxia and gliosis. Cumulatively, the results suggest that pericyte loss induces capillary stalling through increased interaction between leukocytes and endothelial cells in the brain.

Ginger Constituent 6-Shogaol Inhibits Inflammation- and Angiogenesis-Related Cell Functions in Primary Human Endothelial Cells.

Rhizomes from Zingiber officinale Roscoe are traditionally used for the treatment of a plethora of pathophysiological conditions such as diarrhea, nausea, or rheumatoid arthritis. While 6-gingerol is the pungent principle in fresh ginger, in dried rhizomes, 6-gingerol is dehydrated to 6-shogaol. 6-Shogaol has been demonstrated to exhibit anticancer, antioxidative, and anti-inflammatory actions more effectively than 6-gingerol due to the presence of an electrophilic Michael acceptor moiety. In vitro, 6-shogaol exhibits anti-inflammatory actions in a variety of cell types, including leukocytes. Our study focused on the effects of 6-shogaol on activated endothelial cells. We found that 6-shogaol significantly reduced the adhesion of leukocytes onto lipopolysaccharide (LPS)-activated human umbilical vein endothelial cells (HUVECs), resulting in a significantly reduced transmigration of THP-1 cells through an endothelial cell monolayer. Analyzing the mediators of endothelial cell–leukocyte interactions, we found that 30 µM of 6-shogaol blocked the LPS-triggered mRNA and protein expression of cell adhesion molecules. In concert with this, our study demonstrates that the LPS-induced nuclear factor κB (NFκB) promoter activity was significantly reduced upon treatment with 6-shogaol. Interestingly, the nuclear translocation of p65 was slightly decreased, and protein levels of the LPS receptor Toll-like receptor 4 remained unimpaired. Analyzing the impact of 6-shogaol on angiogenesis-related cell functions in vitro, we found that 6-shogaol attenuated the proliferation as well as the directed and undirected migration of HUVECs. Of note, 6-shogaol also strongly reduced the chemotactic migration of endothelial cells in the direction of a serum gradient. Moreover, 30 µM of 6-shogaol blocked the formation of vascular endothelial growth factor (VEGF)-induced endothelial sprouts from HUVEC spheroids and from murine aortic rings. Importantly, this study shows for the first time that 6-shogaol exhibits a vascular-disruptive impact on angiogenic sprouts from murine aortae. Our study demonstrates that the main bioactive ingredient in dried ginger, 6-shogaol, exhibits beneficial characteristics as an inhibitor of inflammation- and angiogenesis-related processes in vascular endothelial cells.

Quantification of Leukocyte-Endothelial Cell Interaction During In Vitro Models of Thrombosis at Arterial and Venous Shear Rates.

Upon injury, stable thrombi formation requires the recruitment of platelets, leukocytes, and various clotting factors, to provide sufficient inhibition of hemostasis. Classical models of thrombosis involve either ex vivo isolation of platelets and subsequent quantification of aggregation through light transmission aggregometry or in vivo murine intravital thrombosis models (laser injury, ferric chloride, or rose Bengal). Flow adhesion models allow for accurate quantification of the contribution of cell-types to thrombi formation. Here, we describe the use of flow chambers to flow human blood over activated endothelial cells to observe leukocyte-endothelial adhesion at arterial and venous shear rates.

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation.

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of activated leukocytes across the vascular endothelium into key organs can lead to organ failure. A physiologically relevant biomimetic microfluidic assay (bMFA) has been developed and validated using several experimental and computational techniques, which can reproduce the entire leukocyte rolling/adhesion/migration cascade to study leukocyte-endothelial cell interactions. Microvascular networks obtained from in vivo images in rodents were digitized using a Geographic Information System (GIS) approach and microfabricated with polydimethylsiloxane (PDMS) on a microscope slide. To study the effect of shear rate and vascular topology on leukocyte-endothelial cell interactions, a Computational Fluid Dynamics (CFD) model was developed to generate a corresponding map of shear rates and velocities throughout the network. The bMFA enables the quantification of leukocyte-endothelial cells interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, endothelial cell permeability, adhesion molecule expression and other important variables. Furthermore, by using human-related samples, such as human endothelial cells and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability.

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation

Bariatric surgery improves autophagy and leukocyte-endothelial cell interactions through AMPK activation at one year follow-up

Background and Aims: BACKGROUND: It has been reported that a Roux-en-Y gastric bypass (RYGB) results in amelioration of the pro-oxidant status of leukocytes and metabolic profile. Nevertheless, the molecular pathways involved and how they are related with reduced cardiovascular risk are poorly understood. Hereof, AMP-activated protein kinase (AMPK) has been shown to play a role in energy homeostasis, including autophagy. Therefore, the objective of this study was to explore whether AMPK and autophagic pathways in leukocytes are implicated in their interaction with the endothelium

The Clinical-Stage sGC Stimulator IW-1701 Prevents Increase of Plasma Biomarkers of Intravascular Inflammation and Suppresses Leukocyte-Endothelial Interactions in Tnfα-Treated Mice

Soluble guanylate cyclase (sGC) is a heme containing signaling enzyme that catalyzes the formation of cyclic guanosine 3‘,5‘-monophosphate (cGMP) from GTP in response to nitric oxide (NO) binding to heme. The second messenger cGMP modulates many physiological processes including inflammation. Treatment with NO donors or inhibitors of cGMP-selective phosphodiesterase 9 (PDE9) significantly attenuates TNFα-induced intravascular inflammation in C57BL/6 and sickle cell mice. The anti-inflammatory effect of PDE9 inhibitors was augmented by co-administration of hydroxyurea (HU), which is the current standard of care for sickle cell disease. It has been proposed that HU may exert its anti-inflammatory effect by acting as an NO donor. sGC stimulators are small molecules that bind to sGC and enhance NO-dependent catalytic activity of the enzyme. Here we evaluated the effect of the sGC stimulator IW-1701 in the absence and presence of HU on biomarkers of intravascular inflammation, leukocyte-endothelial cell interactions and neutrophil trafficking in C57BL/6 mice.Treatment of C57BL/6 mice with TNFα (50 ng/mouse, i.p.) increased the plasma levels of biomarkers of endothelial cell activation (sP-selectin, sE-selectin, sICAM-1) and leukocyte activation (MIP-2, sL-selectin). Pretreatment of mice with IW-1701 (10 mg/kg, p.o.) reduced the increase in plasma concentrations of MIP-2 and sL-selectin following the TNFα challenge by 81% and 58%, respectively. The TNFα-induced increase in plasma levels of sP-selectin, sE-selectin and sICAM-1 were also inhibited by 31%, 37% and 34%, respectively in IW-1701-treated mice. Co-treatment with HU augmented the effect of IW-1701 on plasma concentrations of sE-selectin and sICAM-1 in TNFα treated mice.The effects of IW-1701, HU and the combination on leukocyte endothelial cell interactions was assessed using intravital microscopy of post-capillary venules of the mouse cremaster muscle. TNFα treatment of C57BL/6 mice decreased leukocyte velocity from 26.6±3.1 μm/sec to 5.5±0.7 μm/sec and leukocyte rolling flux from 43.3±7.1 cells/min to 16.1±2.7 cells/min as compared to control mice. Pre-treatment of mice with IW-1701 (10 mg/kg) alone increased leukocyte velocity and rolling flux to 10.3±1.1 μm/sec and 24.1±2.3 cells/min, respectively. Similarly, pre-treatment of mice with HU (100 mg/kg, p.o.) alone increased leukocyte velocity to 15.5±1.7 μm/sec and leukocyte rolling flux to 50.2±5.9 cells/min. Co-administration of IW-1701 in combination with HU further increased leukocyte velocity (19.7±1.9 μm/sec) and leukocyte rolling flux (64.5±5.5 cells/min) in post-capillary venules of the mouse cremaster muscle. Treatment with either IW-1701, HU, or the combination did not change the number of adherent leukocytes to the vascular wall following TNFα stimulation. To further evaluate the effect of IW-1701 on neutrophil extravasation and trafficking we used a peritoneal recruitment model. Intraperitoneal injection of TNFα induced accumulation of Gr.1 positive neutrophils in the mouse peritoneal cavity (4±0.4×105 PMNs/peritoneal lavage). Trafficking of PMNs into mouse peritoneum was attenuated by treatment of mice with HU (2.7±0.5×105 PMNs/peritoneal lavage). Co-administration of HU and IW-1701 further decreased the number of neutrophils extravasated into the peritoneum of TNFα-challenged mice (1.4±0.3×105 PMNs/peritoneal lavage).In summary, prophylactic treatment with IW-1701 inhibited the TNFα-induced increase in plasma levels of biomarkers of intravascular inflammation and attenuated the effect of TNFα on transient adhesive interactions between leukocytes and endothelial cells in mice. Co-administration of HU significantly reduced neutrophil trafficking and augmented the effect of IW-1701 on biomarkers of endothelial cell activation and transient adhesive interactions in the mouse cremaster muscle. This study supports further evaluation of the sGC stimulator IW-1701 to reduce complications associated with intravascular inflammation such as vaso-occlusive crisis in patients with sickle cell disease. DisclosuresTchernychev:Ironwood Pharmaceuticals: Employment, Other: own stock options. Feil:Ironwood Pharmaceuticals: Other: This research was sponsored by Ironwood Pharmaceuticals. Germano:Ironwood Pharmaceuticals: Employment, Other: own stock options of Ironwood Pharmaceuticals . Warren:Ironwood Pharmaceuticals: Employment, Other: own stock options of Ironwood Pharmaceuticals . Lonie:Ironwood Pharmaceuticals: Employment, Other: own stock options of Ironwood Pharmaceuticals . Feil:Ironwood Pharmaceuticals: Other: This research was sponsored by Ironwood Pharmaceuticals. Milne:Ironwood Pharmaceuticals: Employment, Equity Ownership. Hadcock:Ironwood Pharmaceuticals: Employment, Other: own stock options of Ironwood Pharmaceuticals . Chien:Ironwood Pharmaceuticals: Employment, Equity Ownership. Currie:Ironwood Pharmaceuticals: Employment, Equity Ownership. Graul:Ironwood Pharmaceuticals: Employment, Other: own stock options of Ironwood Pharmaceuticals .

C81-evoked inhibition of the TNFR1-NFκB pathway during inflammatory processes for stabilization of the impaired vascular endothelial barrier for leukocytes.

Chronic inflammation-related diseases are characterized by persistent leukocyte infiltration into the underlying tissue. The vascular endothelium plays a major role in this pathophysiological condition. Only few therapeutic strategies focus on the vascular endothelium as a major target for an anti-inflammatory approach. In this study, we present the natural compound-derived carbazole derivative C81 as chemical modulator interfering with leukocyte-endothelial cell interactions. An in vivo assay employing intravital microscopy to monitor leukocyte trafficking after C81 treatment in postcapillary venules of a murine cremaster muscle was performed. Moreover, in vitro assays using HUVECs and monocytes were implemented. The impact of C81 on cell adhesion molecules and the NFκB signaling cascade was analyzed in vitro in endothelial cells. Effects of C81 on protein translation were determined by incorporation of a puromycin analog-based approach and polysome profiling. We found that C81 significantly reduced TNF-activated leukocyte trafficking in postcapillary venules. Similar results were obtained in vitro when C81 reduced leukocyte-endothelial cell interactions by down-regulating cell adhesion molecules. Focusing on the NFκB signaling cascade, we found that C81 reduced the activation on multiple levels of the cascade through promoted IκBα recovery by attenuation of IκBα ubiquitination and through reduced protein levels of TNFR1 caused by protein translation inhibition. We suggest that C81 might represent a promising lead compound for interfering with inflammation-related processes in endothelial cells by down-regulation of IκBα ubiquitination on the one hand and inhibition of translation on the other hand without exerting cytotoxic effects.

P043 Succinate receptor (SUCNR1) mediates leukocyte-endothelial cell interactions induced by TNFa

Abstract Background The Krebs cycle metabolite succinate contributes to inflammatory conditions like arthritis and colitis by activating its receptor SUCNR1. We aimed to analyze whether the succinate-SUCNR1 pathway contributes to the leukocyte-endothelial cell interactions that initiate the inflammatory response. Methods We evaluated leukocyte rolling and adhesion by intravital microscopy in cremaster venules of wild-type (WT) and Sucrn1−/− mice treated, 4h before, with succinate (1 Mm, intraescrotally), combined or not with the common pro-inflammatory cytokine TNFα (500 ng/mice, i.p.), or with their vehicles. We analyzed the activity of the NF-κB signaling pathway in endothelial cells (HUVEC) treated (4h) with succinate (0.5–1 mM) ± TNFα (20ng/ml), by detecting this transcription factor, its inhibitor iκBα, and their phosphorylated forms, by western blot. Results are expressed as mean±SEM (N≥6) and analyzed by ANOVA + Newman-Keuls test. Results In WT mice, succinate tended to induce leukocyte rolling and adhesion, and amplified the effects of TNFα on rolling. SUCNR1-deficient mice failed to increase leukocyte rolling in response to these treatments while showed an attenuated response to the effect of TNFα on leukocyte adhesion (Figure 1). Succinate treatment intensified the activation of the NF-κB pathway in HUVEC treated with TNFα, as shown by the increased IκBα phosphorylation, which allows its degradation, the reduced total IκBα, and the increased NF-κB/IκBα ratio (Figure 2). Conclusion Our results suggest that the endogenous release of succinate and the consequent stimulation of the SUCNR1 mediates the leukocyte-endothelial cell interactions induced by TNFα, and this pro-inflammatory activity may be related with an increased activation of the NF-κB signaling pathway in endothelial cells. Thus, the accumulation of succinate observed in various inflammatory diseases probably contributes to the initiation of the inflammatory focus by promoting the accumulation of leukocytes. Bibliography

Watching the Vessels Grow: Establishment of Intravital Microscopy in the Arteriovenous Loop Rat Model.

Tissue engineering in reconstructive surgery seeks to generate bioartificial tissue substitutes. The arteriovenous (AV) loop allows the generation of axially vascularized tissue constructs. Cellular mechanisms of this vascularization process are largely unclear. In this study, we developed two different chamber models for intravital microscopy and imaging of the AV loop in the rat. Multiple design variations were implanted and the stability of the chamber and AV loop patency was tested in vivo. Our novel chamber facilitates repetitive observation of the AV loop using fluorescence-enhanced intravital microscopy. This technique can be used for daily evaluation of leukocyte-endothelial cell interactions, vascularization, and tissue formation in the AV loop model on 14 consecutive days. Therefore, our newly developed model for intravital microscopy will provide better understanding of cellular and molecular processes in tissue engineering in the AV loop. Moreover, it supports initiation of the novel approaches for therapeutic applications. Impact statement In the Arteriovenous (AV) loop, axially vascularized tissue can be generated and modified using different tissue engineering approaches. Cellular mechanisms of this vascularization process are largely unclear. We managed to develop an intravital microscopy model for long-term observation of intravascular and perivascular events in the AV loop. Leukocyte-endothelial cell interactions, vascularization, and tissue formation in the AV loop can now be evaluated on a day-to-day basis. This will provide better understanding of cellular and molecular processes happening during tissue engineering within the AV loop.

Abacavir Increases Purinergic P2X7 Receptor Activation by ATP: Does a Pro-inflammatory Synergism Underlie Its Cardiovascular Toxicity?

The cardiovascular toxicity of Abacavir is related to its purinergic structure. Purinergic P2X7-receptors (P2X7R), characterized by activation by high concentrations of ATP and with high plasticity, seem implicated. We appraise the nature of the interplay between Abacavir and P2X7R in generating vascular inflammation. The effects of Abacavir on leukocyte-endothelium interactions were compared with those of its metabolite carbovir triphosphate (CBV-TP) or ATP in the presence of apyrase (ATP-ase) or A804598 (P2X7R-antagonist). CBV-TP and ATP levels were evaluated by HPLC, while binding of Abacavir, CBV-TP and ATP to P2X7R was assessed by radioligand and docking studies. Hypersensitivity studies explored a potential allosteric action of Abacavir. Clinical concentrations of Abacavir (20 µmol/L) induced leukocyte-endothelial cell interactions by specifically activating P2X7R, but the drug did not show affinity for the P2X7R ATP-binding site (site 1). CBV-TP levels were undetectable in Abacavir-treated cells, while those of ATP were unaltered. The effects of Abacavir were Apyrase-dependent, implying dependence on endogenous ATP. Exogenous ATP induced a profile of proinflammatory actions similar to Abacavir, but was not entirely P2X7R-dependent. Docking calculations suggested ATP-binding to sites 1 and 2, and Abacavir-binding only to allosteric site 2. A combination of concentrations of Abacavir (1 µmol/L) and ATP (0.1 µmol/L) that had no effect when administered separately induced leukocyte-endothelium interactions mediated by P2X7R and involving Connexin43 channels. Therefore, Abacavir acts as a positive allosteric modulator of P2X7R, turning low concentrations of endogenous ATP themselves incapable of stimulating P2X7R into a functional proinflammatory agonist of the receptor.

Deficiency of Dietary Fiber Modulates Gut Microbiota Composition, Neutrophil Recruitment and Worsens Experimental Colitis.

Ulcerative colitis is an inflammatory disease of the colon that is associated with colonic neutrophil accumulation. Recent evidence indicates that diet alters the composition of the gut microbiota and influences host–pathogen interactions. Specifically, bacterial fermentation of dietary fiber produces metabolites called short-chain fatty acids (SCFAs), which have been shown to protect against various inflammatory diseases. However, the effect of fiber deficiency on the key initial steps of inflammation, such as leukocyte–endothelial cell interactions, is unknown. Moreover, the impact of fiber deficiency on neutrophil recruitment under basal conditions and during inflammation in vivo is unknown. Herein, we hypothesized that a fiber-deficient diet promotes an inflammatory state in the colon at baseline and predisposes the host to more severe colitis pathology. Mice fed a no-fiber diet for 14 days showed significant changes in the gut microbiota and exhibited increased neutrophil-endothelial interactions in the colonic microvasculature. Although mice fed a no-fiber diet alone did not have observable colitis-associated symptoms, these animals were highly susceptible to low dose (0.5%) dextran sodium sulphate (DSS)-induced model of colitis. Supplementation of the most abundant SCFA, acetate, prevented no-fiber diet-mediated enrichment of colonic neutrophils and colitis pathology. Therefore, dietary fiber, possibly through the actions of acetate, plays an important role in regulating neutrophil recruitment and host protection against inflammatory colonic damage in an experimental model of colitis.