A Novel Selectin Antagonist, GMI-1070, Prevents Vaso-Occlusion in Sickle Cell Mice by Inhibiting Leukocyte Adhesion and Activation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2245-2245
Author(s):  
Jungshan Chang ◽  
John Patton ◽  
Arun Sarkar ◽  
John L. Magnani ◽  
Paul S. Frenette
Keyword(s):  
Sickle Cell ◽  
Intravital Microscopy ◽  
Competitive Inhibition ◽  
Leukocyte Adhesion ◽  
Fold Increase ◽  
Surgical Trauma ◽  
Mouse Bone Marrow ◽  
Leukocyte Rolling ◽  
Tnf Α ◽  
Adherent Leukocytes

Abstract Previous studies using intravital microscopy in a sickle cell disease (SCD) mouse model (Berkeley) suggest that adherent leukocytes (WBCs) play a key role in vaso-occlusion by capturing circulating erythrocytes (RBCs) in venules. In addition, mice deficient in both P-and E-selectins are protected from vaso-occlusion (VOC) induced by surgical trauma and TNF-α stimulation, suggesting that targeting selectins or their ligands represents a potentially useful strategy. Selectins bind to specific sialylated and fucosylated carbohydrate structures presented by glycoprotein or glycolipid ligands. Here, we tested the effect of novel small glycomimetic selectin inhibitors, GMI-1070 and GMI-1077, on leukocyte behavior and sickle cell VOC. Berkeley SCD mouse bone marrow was transplantated into lethally irradiated C57BL/6 animals to generate age- and gender-matched genetically identical cohorts of SCD mice. Fully engrafted male SCD mice were treated with TNF-α and prepared for intravital microscopy examination of the cremaster muscle 90 min later. GMI-1070, GMI-1077 (both 20 mg/kg) or vehicle (PBS) were administered immediately prior to cytokine stimulation (t=0 min), and an additional dose was given at t=70min. Another group of mice was injected with antibodies against P-and E-selectins (PES, 1 mg/kg) as positive control. Several post-capillary and collecting venules were examined between t= 90min and t= 150min. Antibody blockade of endothelial selectins completely ablated leukocyte rolling, whereas GMI-1070 and GMI-1077 significantly increased the rolling flux fractions (PBS: 5.0±1.2 GMI-1070: 10.6±1.3%%; GMI-1077: 9.9±1.0%; p< 0.001). Furthermore GMI-1070 and GMI-1077 significantly reduced the recruitment of adherent leukocytes (914±172 and 1433±119 cells/mm2, respectively) compared to sickle mice injected with PBS control (2400±392 cells/mm2, p< 0.001). Although the reduction in leukocyte adhesion was not as marked as with anti-P and E-selectins (61±25 cells/mm2, p< 0.001), GMI-1070, in particular, dramatically inhibited the capture of sickle RBCs by adherent leukocytes (PBS: 0.9±0.4, GMI-1077: 0.6±0.2, GMI-1070: 0.07±0.05 and PES: 0.01±0.01 RBC interactions/WBC/min, p< 0.05) and markedly improved the blood flow in venules (PBS: 312±24, GMI-1077: 398±41, GMI-1070: 710±68 and PES: 683±75 nL/s, p< 0.001), to levels observed in non-sickle mice. The increased leukocyte rolling fluxes by these glycomimetics suggest that they inhibit E-selectin > P-selectin. Since the hallmark of E-selectin-mediated adhesion is the slow leukocyte rolling, we analyzed leukocyte rolling velocities in the various group and indeed found a 2-fold increase in rolling velocities in sickle mice treated with GMI-1070 compared to PBS control (PBS: 21±1 μm/s, GMI-1070: 38±1 μm/s, p<0.001). Consistent with these results, other studies using a parallel plate flow chamber (0.9 dynes/cm2) revealed that GMI-1070 was much more potent (1000-fold difference) in inhibiting the binding of human PMNs to TNF-α-stimulated (to induce E-selectin) endothelial cells (HUVEC) than with IL-4 and histamine stimulated HUVECs (to induce P-selectin). Further, competitive inhibition assays revealed that the IC50 of GMI-1070, relative to the standard glycyrrhizin, was much lower for E-selectin than P-selectin. These studies suggest that E-selectin-mediated adhesion/signaling may play a more important role than previously appreciated in the pathophysiology of SCD, and suggest that GMI-1070 may be beneficial for the treatment of sickle cell vaso-occlusion.

Effects of Pan-Selectin Antagonist GMI-1070 on the Treatment of Vaso-Occlusion in Sickle Cell Mice

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 535-535 ◽  
Author(s):  
Jungshan Chang ◽  
John T Patton ◽  
Paul S. Frenette ◽  
John L. Magnani
Keyword(s):  
Blood Flow ◽  
Red Blood Cells ◽  
Sickle Cell ◽  
Small Molecule ◽  
Vascular Endothelium ◽  
Intravital Microscopy ◽  
Blood Cells ◽  
Tnf Α ◽  
Sickle Red Blood Cells ◽  
Adherent Leukocytes

Abstract Acute vaso-occlusion (VOC) in patients with sickle cell disease (SCD) induces intense pain arising from organ damage and is the major cause of morbidity and mortality. Hypoxia and abnormal sickle red blood cells (RBC) induce inflammatory mediators and activation of the vascular endothelium leading to the recruitment of adherent leukocytes and sickle RBC followed by aggregates that eventually occlude blood flow. Previous studies have implicated the critical roles of cell adhesion molecules E- and P-selectins by using intravital microscopy in SCD mice (Berkeley strain) with altered genetic backgrounds (SCD transplanted in recipients lacking E-and P-selectins), or antibodies against endothelial selectins, or small molecules directed against the selectins. Here, we designed a treatment protocol for this SCD mouse model, in which a small molecule pan-selectin antagonist (GMI-1070) is administered to sickle cell mice late in the process of established vaso-occlusion in order to test the effects of GMI-1070 in a more clinically relevant model. GMI-1070 is a small molecule pan-selectin antagonist designed on the bioactive conformation of the carbohydrate ligand and inhibits leukocyte adhesion to activated endothelium in vitro with particularly strong activity against E-selectin (IC50 = 3.4 μM). Berkeley SCD mice were generated by bone marrow transplantation into lethally irradiated C57BL/6 male mice and the fully engrafted (100% donor RBC chimerism) mice were used for intravital microscopy experiments. VOC events were induced by injection with TNF-α at time 0 and the formation of occlusions were allowed to proceed as long as possible just prior to the death of the control mice. GMI-1070 (20 mg/kg) or vehicle (PBS pH 7.4) were administered at t = 110 min. Post-capillary and collecting venules in the cremaster muscle were analyzed for effects on an established VOC event. Under these conditions, GMI-1070 significantly increased the microcirculatory blood flow to levels observed in non-sickle cell mice (vehicle: 237 ± 15 nL/sec; GMI-1070: 533 ± 58 nL/sec; p<0.0001). The recruitment of adherent leukocytes to the vascular endothelium was also significantly reduced (vehicle: 2235 ± 156; GMI-1070: 1270 ± 203 cells/mm2; p=0.0013), and there were significant and dramatic reductions in the capture of sickle red blood cells to adherent leukocytes (vehicle: 0.68 ± 0.27; GMI-1070: 0.03 ± 0.01 interactions/WBC, min, 100ml; p=0.0003). Mice began to succumb to VOC within 2.5 hours after injection of TNF-α and surgical trauma which continued until all of the control SCD mice died. Administration of GMI-1070 prevented the death of half of the treated mice within the timeframe of the experiment and extended the median survival of mice from 5 hours (control, vehicle-treated) to greater than 9 hours for the GMI-1070- treated SCD mice (p = 0.0067). These studies show that GMI-1070 can significantly and dramatically improve the condition and survival of the animals with a severe VOC even when dosed well after the initiating challenge. Thus these data strongly support the use of GMI-1070 for the treatment of patients in acute vaso-occlusive crisis. GMI-1070 is currently in a Phase I clinical trial.

Sickle Cell Vaso-Occlusion Is Triggered by E-Selectin Ligand Signaling and Propagated by the Leukocyte Integrin Mac-1.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 145-145 ◽  
Author(s):  
Andres Hidalgo ◽  
Jungshan Chang ◽  
Anna J. Peired ◽  
Elaine Y. Chiang ◽  
Paul S. Frenette
Keyword(s):  
Sickle Cell ◽  
Intravital Microscopy ◽  
High Speed ◽  
Molecular Mechanisms ◽  
Leading Edge ◽  
Tnf Α ◽  
Selectin Ligand ◽  
Adherent Leukocytes

Abstract Vasoocclusion (VOC) is the leading cause of morbidity and mortality in patients with sickle cell disease (SCD). Intravital microscopy studies in a murine model of SCD have revealed that capture of sickle red blood cells (RBC) by intravascular adherent leukocytes (WBC) plays an important role in VOC, and that deficiency in both P-and E-selectins protect from VOC. Here, we have investigated the cellular and molecular mechanisms leading to sickle RBCs interactions with adherent WBCs. Intravital microscopy analyses of the individual role of P- or E-selectin revealed, unexpectedly, a profound reduction in RBC-WBC interactions in Berkeley sickle mice lacking E-selectin (Sele−/−; >97% reduction), whereas the protection was only partial in the absence of endothelial P-selectin. Since E-selectin is expressed exclusively on the endothelium, and its deficiency does not alter WBC recruitment, we hypothesized that E-selectin might provide activation signals to neutrophils that allow them to capture RBCs. During our studies, we observed that RBC-WBC interactions are not exclusive of sickle animals but are also present in wild-type B6 mice treated with TNF-α, suggesting that this phenomenon accompanies a physiological inflammatory response. We found that RBC-WBC interactions in B6 mice occur at a lower frequency than in sickle mice and that these interactions are also reduced in Sele−/− mice (60% reduction; p<0.05). We thus reasoned that B6 mice might provide a convenient model to gain molecular insight into RBC-WBC interactions in vivo. Since PSGL-1, CD44 and ESL-1 harbor the entire E-selectin ligand activity on neutrophils in vivo (Hidalgo et al., Immunity 2007), we investigated which of these glycoproteins mediates the signals allowing RBC capture. High speed digital multichannel fluorescence intravital microscopy analyses revealed that RBC-WBC interactions were only markedly reduced in the absence of ESL-1 (63% reduction, p<0.001), but not in the absence of PSGL-1 or CD44. Further detailed image analyses mapped RBC captures at the leading edge of adherent neutrophils, an area where chemokine receptors and integrins may accumulate. Since selectin-mediated signaling is known to activate β2 integrins, we tested the role of Mac-1, whose expression and affinity are elevated in neutrophils from SCD patients. We found that RBC-WBC interactions were virtually absent in mice deficient in Mac-1 (97% reduction; p<0.0001). To determine whether E-selectin/ESL-1-mediated signaling promoted Mac-1 activation, we developed an assay to assess Mac-1 activity in real time on adherent WBCs in vivo. Albumin-coated fluorobeads bound to subsets of adherent leukocytes in TNF-α-stimulated venules of B6 mice. These interactions were Mac-1-dependent since they were ablated in Itgam−/− mice. Absence of E-selectin or ESL-1, but not P-selectin, PSGL-1 or CD44, significantly reduced Mac-1 activity (by 36% and 52%, respectively; p<0.05). In contrast, Mac-1 activation on adherent leukocytes was dramatically increased in inflamed venules of sickle mice (2.5-fold; p<0.001) and was restricted to a subset of adherent neutrophils. Preliminary experiments in which Mac-1 function is blocked in sickle animals with a monoclonal antibody revealed a reduction of RBC-WBC interactions (by 57%) compared to an isotype control antibody. Our results indicate that the binding of E-selectin to neutrophil ESL-1 promotes Mac-1 activation, which in turn mediates the capture of sickle RBCs. These findings provide attractive therapeutic targets to alleviate this devastating disease.

fMLP-stimulated release of reactive oxygen species from adherent leukocytes increases microvessel permeability

2006 ◽  
Vol 290 (1) ◽  
pp. H365-H372 ◽  
Author(s):  
Longkun Zhu ◽  
Pingnian He
Keyword(s):  
Reactive Oxygen Species ◽  
Respiratory Burst ◽  
Leukocyte Adhesion ◽  
Autologous Blood ◽  
Blood Perfusion ◽  
Oxygen Species ◽  
Tnf Α ◽  
Microvessel Permeability ◽  
Adherent Leukocytes ◽  
Neutrophil Respiratory Burst

Our previous study ( Am J Physiol Heart Circ Physiol 288: H1331–H1338, 2005) demonstrated that TNF-α induced significant leukocyte adhesion without causing increases in microvessel permeability, and that formyl-Met-Leu-Phe-OH (fMLP)-stimulated neutrophils in the absence of adhesion increased microvessel permeability via released reactive oxygen species (ROS). The objective of our present study is to investigate the mechanisms that regulate neutrophil respiratory burst and the roles of fMLP-stimulated ROS release from adherent leukocytes in microvessel permeability. A technique that combines single-microvessel perfusion with autologous blood perfusion was employed in venular microvessels of rat mesenteries. Leukocyte adhesion was induced by systemic application of TNF-α. Microvessel permeability was assessed by measuring hydraulic conductivity ( Lp). The 2-h autologous blood perfusion after TNF-α application increased leukocyte adhesion from 1.2 ± 0.2 to 13.3 ± 1.6 per 100 μm of vessel length without causing increases in Lp. When fMLP (10 μM) was applied to either perfusate ( n = 5) or superfusate ( n = 8) in the presence of adherent leukocytes, Lptransiently increased to 4.9 ± 0.9 and 4.4 ± 0.3 times the control value, respectively. Application of superoxide dismutase or an iron chelator, deferoxamine mesylate, after fMLP application prevented or attenuated the Lpincrease. Chemiluminescence measurements in isolated neutrophils demonstrated that TNF-α alone did not induce ROS release but that preexposure of neutrophils to TNF-α in vivo or in vitro potentiated fMLP-stimulated ROS release. These results suggest a priming role of TNF-α in fMLP-stimulated neutrophil respiratory burst and indicate that the released ROS play a key role in leukocyte-mediated permeability increases during acute inflammation.

scholarly journals TNF-α activation of arterioles and venules alters distribution and levels of ICAM-1 and affects leukocyte-endothelial cell interactions

2006 ◽  
Vol 291 (5) ◽  
pp. H2116-H2125 ◽  
Author(s):  
Ronen Sumagin ◽  
Ingrid H. Sarelius
Keyword(s):  
Spatial Distribution ◽  
Endothelial Cell ◽  
Inflammatory Responses ◽  
Leukocyte Adhesion ◽  
Leukocyte Rolling ◽  
Cremaster Muscle ◽  
Rolling Velocity ◽  
Tnf Α ◽  
Arteriolar Wall ◽  
Microvessel Wall

The observation that leukocyte-endothelial cell (EC) interactions are localized to specific regions on the microvessel wall suggests that adhesion molecule distribution is not uniform. We investigated ICAM-1 distribution and leukocyte-EC interactions in blood-perfused microvessels (<80 μm) in cremaster muscle of anesthetized mice, using intravital confocal microscopy and immunofluorescent labeling. Variability of ICAM-1 expression directly determines leukocyte adhesion distribution within the venular microcirculation and contributes to leukocyte rolling in arterioles during inflammation. The number of rolling interactions increased with ICAM-1 intensity ( r2 = 0.69, P < 0.05), and rolling velocity was lower in regions of higher ICAM-1 intensity. In controls, venular ICAM-1 expression was approximately twofold higher than in arterioles. After TNF-α treatment, ICAM-1 expression was significantly increased, 2.8 ± 0.2-fold in arterioles and 1.7 ± 0.2-fold in venules ( P < 0.05). ICAM-1 expression on activated arteriolar ECs only reached the level of control venular ICAM-1. Arteriolar but not venular ECs underwent redistribution of ICAM-1 among cells; some cells increased and some decreased ICAM-1 expression, magnifying the variability of ICAM-1. TNF-α treatment increased the length of bright fluorescent regions per unit vessel length (42%, control; 70%, TNF-α) along the arteriolar wall, whereas no significant change was observed in venules (60%, control; 63%, TNF-α). The spatial distribution and expression levels of adhesion molecules in the microcirculation determine the timing and placement of leukocyte interactions and hence significantly impact the inflammatory response. That arteriolar ECs respond to TNF-α by upregulation of ICAM-1, although in a different way compared with venules, suggests an explicit role for arterioles in inflammatory responses.

scholarly journals Mn Porphyrin-Based Redox Active Drugs As Novel Anti-Adhesive and Anti-Inflammatory Agents Targeting Sickle Red Blood Cell Oxidative Damage In Vivo

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 270-270
Author(s):  
Thamilarasan Madhan ◽  
Rodolfo Estupinan ◽  
Rahima Zennadi
Keyword(s):  
Cell Adhesion ◽  
Blood Cell ◽  
Red Blood Cell ◽  
Intravital Microscopy ◽  
Leukocyte Adhesion ◽  
Venous Blood ◽  
Blood Gases ◽  
Leukocyte Rolling ◽  
Sickle Cells

Abstract In sickle cell disease (SCD), painful vaso-occlusive crises and end-organ damage are caused by occlusion of the vessels due largely to sickle red blood cell (RBC) adhesion to both the endothelium and adherent leukocytes. RBC oxidative damage caused by continuous endogenous and exogenous oxidative stress may participate in the occurrence of vaso-occlusive crises. We have evaluated the effects of scavenging reactive oxygen species (ROS) in sickle RBCs on cell adhesion and vaso-occlusion in a humanized mouse model of vaso-occlusion in vivo analyzed by intravital microscopy. To scavenge RBC ROS, we used our manganese porphyrin-based superoxide dismutase (SOD) mimics MnTnBuOE-2-PyP5+ (MnBuOE) and MnTE-2-PyP5+ (MnE), powerful catalysts of superoxide dismutation, and reductants of peroxynitrite, peroxide and hypochlorite. Intravital microscopy observations of enflamed vessels visible through dorsal skin-fold window chamber implants was performed after the inflammatory trigger of tumor necrosis factor alpha (TNFα) to induce vaso-occlusion in transgenic sickle mice followed by subcutaneous injection of MnBuOE at 0.1, 0.2 or 2 mg/kg, or MnE at 0.5 or 2 mg/kg. Treatment of sickle mice with only one dose of 0.1, 0.2 and 2 mg/kg MnBuOE decreased dose-dependently adhesion of both sickle cells and leukocytes in enflamed vessels by 68±4% (p<0.01), 85±2.3% (p<0.01) and 89±4.3% (p<0.01), respectively, compared with vehicle-treated sickle mice. MnBuOE at 0.1, 0.2 and 2 mg/kg also caused significant and dose-dependent reduction in leukocyte rolling flux (p<0.05). Similar inhibitory benefits were obtained when MnE was administered to TNFa-treated sickle mice. MnE at 0.5 and 2 mg/kg significantly decreased the number of adherent sickle cells and leukocytes by 76±8.6% (p<0.01) and 92±2.5% (p<0.01), respectively, and leukocyte rolling flux (p<0.01) compared to vehicle-treated animals. The effect of these two SOD mimics on sickle RBCs and leukocyte adhesion, and leukocyte rolling flux was rapid, because a decline in cell adhesion and leukocyte rolling flux were already detectable within the first 15 minutes after injection of the compounds. In contrast, cell adhesion and leukocyte rolling flux were already pronounced 15 minutes following vehicle injection. Reduced cell adhesion to the endothelium by the SOD mimics resulted in improved microcirculatory blood flow in sickle mice. These favorable effects on cell adhesion and vaso-occlusion following SOD mimic treatment were indeed due at least to the significant decrease in sickle RBC ROS levels compared to vehicle-treated mice (p<0.001). The long-term anti-adhesive and anti-inflammatory effects of MnBuOE and MnE in sickle mice were next examined. Subcutaneous administration for 28 days of MnBuOE at 0.1 and 0.5 mg/kg inhibited significantly adhesion of RBCs and leukocytes in enflamed venules by 34±13% (p<0.05) and 69±3.5% (p<0.001), respectively, and leukocyte rolling flux (p<0.001) compared to vehicle-treated sickle mice. Subcutaneous injection of MnE at 0.5 and 1 mg/kg for 28 days also had significant effect on sickle cell and leukocyte adhesion (p<0.01), and leukocyte rolling flux (p<0.01). In addition, venous blood gases were significantly improved by the SOD mimics. The levels of partial pressure of Carbon dioxide (pCO2), partial pressure of oxygen (pO2), base excess of the extracellular fluid (BEecf), bicarbonate (HCO3-) concentration, total CO2 (TCO2) concentration, and the indicators of hypoxia, hemoglobin saturation of oxygen (sO2) and lactate, became close to or within the normal ranges (p<0.05) in sickle mice treated with 1 mg/kg MnE. MnBuOE at 0.1 mg/kg showed only a trend toward an increase in venous blood gases, with a significant decrease in lactate (p<0.05). Leukocytosis in sickle mice treated with the SOD mimics was also alleviated. A significant drop in leukocyte (p<0.05), neutrophil (p<0.01), lymphocyte (p<0.05) and monocyte (p<0.05) counts was detected in sickle mice treated with either 0.1 mg/kg MnBuOE or 1 mg/kg MnE. These beneficial therapeutic outcomes induced by the SOD mimics were due at least in part to a decline in RBC ROS levels (p<0.001) and RBC phosphatidylserine surface exposure (p<0.05), an eryptosis marker. These results suggest that our SOD mimics may represent a valuable novel therapeutic intervention for not only vaso-occlusive crises, but inflammation as well, that should be further evaluated in patients with SCD. Disclosures No relevant conflicts of interest to declare.

Deficiency of the VWF-Cleaving Protease ADAMTS13 Results in Increased Leukocyte Rolling and Adhesion in Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 290-290 ◽  
Author(s):  
Anil K. Chauhan ◽  
Janka Kiucka ◽  
Alexander Brill ◽  
Meghan T. Walsh ◽  
Denisa D. Wagner
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Leukocyte Adhesion ◽  
Surface Expression ◽  
Leukocyte Rolling ◽  
Type I ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Adherent Leukocytes

Abstract von Willebrand factor (VWF) is synthesized in megakaryocytes and endothelial cells and stored in a-granules and Weibel-Palade bodies, respectively. VWF levels are elevated in both chronic and acute inflammation. ADAMTS13 (A D isintegrin-like A nd M etalloprotease with T hrombo s pondin type I repeats-13) is a metalloprotease that cleaves ultra large von Willebrand factor (ULVWF) multimers quickly after its release from endothelium. Recent studies have found that VWF promotes leukocyte adhesion in vitro and that ADAMTS13 activity is reduced in inflammation and sepsis. We hypothesized that by cleaving ULVWF multimers, ADAMTS13 not only inhibits thrombosis, but also attenuates leukocyte rolling and adhesion. Using intravital microscopy, we found more leukocyte rolling/min on the unstimulated veins in Adamts13-/- mice (Mean ± SE: 98 ± 16) compared to WT (Mean ± SE: 35 ± 6, P<0.001), n=18–20 from 10–11 mice per group. This process was dependent on VWF because the number of leukocytes rolling in Adamts13-/-/Vwf-/- veins was similar to that in Vwf-/-. Significantly increased soluble P-selectin and VWF concentrations were found in the plasma of Adamts13-/- compared to WT mice as quantitated by ELISA. In addition, endothelial P-selectin surface expression was increased in Adamts13-/- mice compared to WT. These results suggest elevated release of Weibel-Palade bodies in Adamts13-/- mice. Notably, circulating platelets were not activated in the absence of ADAMTS13. Upon stimulation of the mesentery with histamine, leukocyte rolling was slower in Adamts13-/- veins compared to WT. Furthermore, upon stimulation with the inflammatory cytokine TNF-alpha (i.v) 3.5 h prior to surgery, the number of leukocytes adhering/250 um was significantly increased in microvenules (diameter of 25–30 um) of Adamts13-/- mice (Mean ± SD: 21 ± 6) compared to WT (Mean ± SD: 12 ± 5, P<0.001), n=10–11 mice per group. This firm adhesion was also dependent on VWF because the number of adherent leukocytes in veins of Adamts13-/-/Vwf-/- was similar to Vwf-/-. Our studies indicate a crucial role for ADAMTS13 in preventing excessive spontaneous Weibel-Palade secretion and in attenuating leukocyte rolling and adhesion to ultra large VWF presented by endothelial cells during inflammation.

Simvastatin Reduces the in Vitro Adhesion of Sickle Cell Disease Neutrophils to Endothelial Layers

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2489-2489 ◽  
Author(s):  
Andreia A Canalli ◽  
Renata P. Ferreira ◽  
Sara T.O. Saad ◽  
Nicola Conran ◽  
Fernando F. Costa
Keyword(s):  
Cell Adhesion ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Leukocyte Adhesion ◽  
Cell Disease ◽  
Tnf Α ◽  
Cell Layers ◽  
Anti Inflammatory ◽  
Vitro Effect

Abstract Leukocytes may have a propagating and, possibly, initiating role in sickle cell disease (SCD) vaso-occlusion. Endothelial dysfunction contributes to the vaso-occlusion process and leads to inflammation, leukocyte and red cell adhesion. Markers of neutrophil activation are also increased in SCD, in association with increased levels of circulating cytokines and increased leukocyte adhesion. In animal models, vaso-occlusion causes hypoxia/reperfusion, leading to vascular endothelium damage and an inflammatory response. We postulate that anti-inflammatory agents may reduce the participation of activated endothelium in the vaso-occlusive process. Statins are commonly used to treat arteriosclerosis and have anti-inflammatory effects that include a regulatory action on endothelial function, reduced oxidative stress and inflammation. The objective of this study was to investigate the in vitro effect of simvastatin on the adhesion of sickle neutrophils to activated endothelial cell layers (HUVEC). Neutrophils (Neu) were isolated from the peripheral blood of healthy controls (ConNeu) and SCD (SCDNeu) individuals in steady state over ficoll-paque gradients. Cell adhesion (2×106 cell/ml in Ham’s F12 K) to cultured human umbilical vein endothelial cells (HUVEC) grown to confluence was assessed using static adhesion assays. HUVEC cells were treated with or without 1 μg/ml simvastatin for 6 hours in the absence or presence of a 10nM TNF-α activating stimulus (3 hours) before allowing adhesion of Neu to the cell layers (30 min, 37°C, 5%CO2). Neu from SCD patients demonstrated a significantly greater adhesion to HUVEC than ConNeu (20.5 ± 1.9% compared to 13.8 ± 1.7 %; n=15; p&lt;0.02; Mann Whitney test). Subsequently, Neu from patients and controls were allowed to adhere to endothelial layers previously treated with simvastatin; adhesion was not significantly different to the adhesion of Neu to nonsimvastatin treated HUVEC (16.7 ± 3.2% for ConNeu; n=8, p&gt;0.05 and 19.8 ±2.7% for SCDNeu; n=11, p&gt;0.05, paired t test). Pre-treatment of HUVEC with the cytokine TNF-α increased the adhesion of SCD and Con Neu to HUVEC (40.9 ± 5.4%; 28.9 ± 5.0%, respect, N&gt;8, P&lt;0.01 compared to adhesion to non-activated HUVEC). Interestingly, when the endothelium layer was protected with simvastatin and then stimulated with TNF-α, SCDNeu adhesion was significantly diminished (reduced to 31.3% ± 3.6%; n=11, p&lt;0.005 comp. to adhesion to non-simvastatin-treated HUVEC); in contrast, no difference in the adhesion of ConNeu to HUVEC treated with TNF-α and simvastatin was observed (31.9 ± 5.8%, n=8, p&gt;0.05 for ConNeu). In conclusion, data indicate that under in vitro inflammatory conditions, simvastatin appears to protect endothelium layers and reduces SCD leukocyte adhesion. We speculate that statins may have anti-inflammatory properties and, as such, may be useful for diminishing endothelial activation and, in turn, preventing the adhesion of leukocytes adhesion to the vascular wall in SCD, a mechanism that is essential to the vaso-occlusive process.

Control of leukocyte rolling velocity in TNF-α–induced inflammation by LFA-1 and Mac-1

Blood ◽  
2002 ◽  
Vol 99 (1) ◽  
pp. 336-341 ◽  
Author(s):  
Jessica L. Dunne ◽  
Christie M. Ballantyne ◽  
Arthur L. Beaudet ◽  
Klaus Ley
Keyword(s):  
Leukocyte Adhesion ◽  
Leukocyte Rolling ◽  
Rolling Velocity ◽  
Slowing Down ◽  
Tnf Α ◽  
Adhesion Efficiency ◽  
Factor Α ◽  
Rolling Leukocytes ◽  
Necrosis Factor ◽  
Β2 Integrins

Previously it was shown that β2-integrins are necessary for slow leukocyte rolling in inflamed venules. In this study, mice that are deficient for either one of the β2-integrins, αLβ2 (LFA-1) or αMβ2 (Mac-1), were used to determine which of the β2-integrins are responsible for slowing rolling leukocytes. The cremaster muscles of these mice were treated with tumor necrosis factor-α and prepared for intravital microscopy. The average rolling velocities in venules were elevated in LFA-1−/−mice (11.0 ± 0.7 μm/s) and Mac-1−/− mice (10.1 ± 1.1 μm/s) compared to wild-type mice (4.8 ± 0.3 μm/s;P &lt; .05), but were lower than in CD18−/−mice (28.5 ± 2.1 μm/s). When both LFA-1 and Mac-1 were absent or blocked, rolling velocity became dependent on shear rate and approached that of CD18−/− mice. In addition, leukocyte adhesion efficiency was decreased in LFA-1−/− mice to near CD18−/− levels, but decreased only slightly in Mac-1−/− mice. Thus, both LFA-1 and Mac-1 contribute to slowing down rolling leukocytes, although LFA-1 is more important than Mac-1 in efficiently inducing firm adhesion.

Molecular mechanisms involved in superoxide-induced leukocyte-endothelial cell interactions in vivo

1994 ◽  
Vol 266 (2) ◽  
pp. H637-H642 ◽  
Author(s):  
J. P. Gaboury ◽  
D. C. Anderson ◽  
P. Kubes
Keyword(s):  
Molecular Mechanisms ◽  
Leukocyte Adhesion ◽  
Platelet Activating Factor ◽  
Leukocyte Rolling ◽  
Rolling Velocity ◽  
Paf Receptor ◽  
Rolling Leukocytes ◽  
Adherent Leukocytes ◽  
Web 2086

Intravital microscopy was used to monitor leukocyte adherence, flux, rolling velocity, and number of rolling leukocytes (flux/velocity) in venules 25–40 microns in diameter. The superoxide-generating system, hypoxanthine and xanthine oxidase (HX/XO), was infused into the mesenteric circulation in untreated animals or in animals pretreated with either catalase (a hydrogen peroxide scavenger), WEB-2086 [a platelet-activating factor (PAF) receptor antagonist], or monoclonal antibodies directed against adhesion molecules CD18 (CL26) or P-selectin (PB1.3). HX/XO infusion caused a decrease in leukocyte rolling velocity and an increase in the number of rolling and adherent leukocytes. WEB-2086 prevented the increase in leukocyte adhesion and markedly increased leukocyte rolling velocity. PB1.3 abolished the HX/XO-associated rise in the flux of rolling leukocytes and proportionally decreased the number of adherent leukocytes. CL26 abolished HX/XO-induced leukocyte adhesion and also reduced the number of rolling leukocytes. In conclusion, P-selectin mediates the increased leukocyte flux induced by superoxide, whereas PAF and CD18 modulate leukocyte adhesion. PAF also reduces leukocyte rolling velocity, possibly as a result of CD18, but not P-selectin.

Low-dose inhaled carbon monoxide attenuates the remote intestinal inflammatory response elicited by hindlimb ischemia-reperfusion

2009 ◽  
Vol 296 (1) ◽  
pp. G9-G14 ◽  
Author(s):  
Jeffrey R. Scott ◽  
Mark A. Cukiernik ◽  
Michael C. Ott ◽  
Aurelia Bihari ◽  
Amit Badhwar ◽  
...  
Keyword(s):  
Carbon Monoxide ◽  
Inflammatory Response ◽  
Proinflammatory Cytokine ◽  
Low Dose ◽  
Ischemia Reperfusion ◽  
Fold Increase ◽  
Leukocyte Recruitment ◽  
Leukocyte Rolling ◽  
Hindlimb Ischemia ◽  
Tnf Α

Heme oxygenase (HO) represents the rate-limiting enzyme in the degradation of heme into carbon monoxide (CO), iron, and biliverdin. Recent evidence suggests that several of the beneficial properties of HO, may be linked to CO. The objectives of this study were to determine if low-dose inhaled CO reduces remote intestinal leukocyte recruitment, proinflammatory cytokine expression, and oxidative stress elicited by hindlimb ischemia-reperfusion (I/R). Male mice underwent 1 h of hindlimb ischemia, followed by 3 h of reperfusion. Throughout reperfusion, mice were exposed to AIR or AIR + CO (250 ppm). Following reperfusion, the distal ileum was exteriorized to assess the intestinal inflammatory response by quantifying leukocyte rolling and adhesion in submucosal postcapillary venules with the use of intravital microscopy. Ileum samples were also analyzed for proinflammatory cytokine expression [tumor necrosis factor (TNF)-α and interleukin (IL)-1β] and malondialdehyde (MDA) with the use of enzyme-linked immunosorbent assay and thiobarbituric acid reactive substances assays, respectively. I/R + AIR led to a significant decrease in leukocyte rolling velocity and a sevenfold increase in leukocyte adhesion. This was also accompanied by a significant 1.3-fold increase in ileum MDA and 2.3-fold increase in TNF-α expression. Treatment with AIR + CO led to a significant reduction in leukocyte recruitment and TNF-α expression elicited by I/R; however, MDA levels remained unchanged. Our data suggest that low-dose inhaled CO selectively attenuates the remote intestinal inflammatory response elicited by hindlimb I/R, yet does not provide protection against intestinal lipid peroxidation. CO may represent a novel anti-inflammatory therapeutic treatment to target remote organs following acute trauma and/or I/R injury.

Sign in / Sign up

Export Citation Format

Share Document