Leukocyte Rolling Flux Research Articles

Activation of the Constitutive Androstane Receptor Inhibits Leukocyte Adhesiveness to Dysfunctional Endothelium.

Leukocyte cell recruitment into the vascular subendothelium constitutes an early event in the atherogenic process. As the effect of the constitutive androstane receptor (CAR) on leukocyte recruitment and endothelial dysfunction is poorly understood, this study investigated whether the role of CAR activation can affect this response and the underlying mechanisms involved. Under physiological flow conditions, TNFα-induced endothelial adhesion of human leukocyte cells was concentration-dependently inhibited by preincubation of human umbilical arterial endothelial cells with the selective human CAR ligand CITCO. CAR agonism also prevented TNFα induced VCAM-1 expression, as well as MCP-1/CCL-2 and RANTES/CCL-5 release in endothelial cells. Suppression of CAR expression with a small interfering RNA abrogated the inhibitory effects of CITCO on these responses. Furthermore, CITCO increased interaction of CAR with Retinoid X Receptor (RXR) and reduced TNFα-induced p38-MAPK/NF-κB activation. In vivo, using intravital microscopy in the mouse cremasteric microcirculation treatment with the selective mouse CAR ligand TCPOBOP inhibited TNFα-induced leukocyte rolling flux, adhesion, and emigration and decreased VCAM-1 in endothelium. These results reveal that CAR agonists can inhibit the initial inflammatory response that precedes the atherogenic process by targeting different steps in the leukocyte recruitment cascade. Therefore, CAR agonists may constitute a new therapeutic tool in controlling cardiovascular disease-associated inflammatory processes.

Does Empagliflozin Modulate Leukocyte-Endothelium Interactions, Oxidative Stress, and Inflammation in Type 2 Diabetes?

Sodium-glucose co-transporter 2 inhibitors (iSGLT2) have been linked to cardiovascular risk reduction in patients with type 2 diabetes (T2D). However, their underlying molecular mechanisms remain unclear. This study aimed to evaluate the effects of empagliflozin, a novel potent and selective iSGLT-2, on anthropometric and endocrine parameters, leukocyte–endothelium interactions, adhesion molecules, ROS production, and NFkB-p65 transcription factor expression. According to standard clinical protocols, sixteen T2D patients receiving 10 mg/day of empagliflozin were followed-up for 24 weeks. Anthropometric and analytical measurements were performed at baseline, 12 weeks, and 24 weeks. Interactions between polymorphonuclear leukocytes and human umbilical vein endothelial cells (HUVECs), serum levels of adhesion molecules (P-Selectin, VCAM-1 and ICAM-1) and pro-inflammatory cytokines (TNF-α, IL-1β and IL-6), mitochondrial ROS levels, antioxidant enzymes (SOD1 and GPX1), and NFkB-p65 were measured. We observed a decrease in body weight, BMI, and HbA1C levels from 12 weeks of treatment, which became more pronounced at 24 weeks and was accompanied by a significant reduction in waist circumference and glucose. Leukocyte–endothelium interactions were reduced due to an enhancement in the leukocyte rolling velocity from 12 weeks onwards, together with a significant decrease in leukocyte rolling flux and adhesion at 24 weeks. Accordingly, a significant decrease in ICAM-1 levels, mitochondrial ROS levels, and IL-6 and NFkB-p65 expression was observed, as well as an increase in SOD1. This pilot study provides evidence of the anti-inflammatory and antioxidant properties of empagliflozin treatment in humans, properties which may underlie its beneficial cardiovascular effects.

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 .

Inhibition of Protein Disulfide Isomerase Ameliorates Vaso-Occlusion and Thrombosis in a Mouse Model of Sickle Cell Disease

IntroductionSickle cell disease (SCD) results from a single mutation in the β-globin gene. Altered erythrocyte physiology in SCD results in severe vascular complications, including vaso-occlusion (VOC) and painful crises along with various other manifestations, such as thrombosis and end organ damage. Extracellular protein disulfide isomerase (PDI) plays a critical role in the regulation of neutrophil recruitment, platelet activation and fibrin formation, processes implicated in the thrombo-inflammatory vasculopathy in SCD. We investigated the effect of a PDI inhibitor, isoquercetin, in combination with hydroxyurea, in TNFα-induced VOC and thrombosis in a mouse model of SCD. In addition, the effect of chronic PDI inhibition by isoquercetin on thrombo-inflammatory vasculopathy was also examined.MethodsHumanized TOWNES knock-in mice, containing human normal (βA/βA, control) or sickle (βS/βS, SCD) hemoglobin β chains, were used as a mouse model of SCD. PDI inhibition in the plasma was achieved by oral gavage or daily diet feeding of isoquercetin. Intravital microscopy was performed to determine vaso-occlusive events in the venules and thrombus formation in the arterioles in TNFα-stimulated mice. Plasma levels of PDI and parameters for coagulation and vascular inflammation were determined by ELISA. Neutrophil accumulation in the organs was determined as the myeloperoxidase (MPO) levels in tissue homogenates.ResultsCompared to control mice, SCD mice have elevated plasma PDI levels. Gavage of isoquercetin resulted in reduced PDI activity in the plasma of treated mice (38±10% inhibition, p <0.05). Isoquercetin ingestion increased leukocyte rolling flux fraction in TNFα-treated SCD mice as monitored using intravital microscopy, whereas the influence on leukocyte adhesion and shear rate was minimal. When a reversal agent for PDI inhibitor, b'x (the isolated substrate binding domain of PDI) was co-infused, the effect of isoquercetin on leukocyte rolling was reversed, suggesting this effect of isoquercetin is mediated by PDI inhibition. We also determined the effect of isoquercetin in combination with hydroxyurea, a treatment with established efficacy in SCD, on the vaso-occlusive events in our model. We confirmed that hydroxyurea treatment elevated the plasma nitric oxide level as determined by plasma nitrite/nitrate content (11.5±1.9 μM vs 5.0±1.9 μM, p <0.05). Consistent with previous studies, hydroxyurea decreased leukocyte adhesion while the rolling flux fraction was not changed. However, combined treatment with isoquercetin and hydroxyurea in SCD mice increased the rolling flux fraction (15±3% vs 6±1%, p <0.05), decreased leukocyte adhesion (158±13 mm-2 vs 289±24 mm-2, p <0.05), and increased vessel shear rate (302±18 s-1 vs 122±11 s-1, p <0.05). We investigated in vivo thrombus formation in the arterioles of TNFα-treated SCD mice using intravital microscopy following laser injury. Compared to the control mice, SCD mice have significantly enhanced fibrin generation but normal platelet accumulation. Both isoquercetin and hydroxyurea inhibited TNFα-induced platelet accumulation (62±36% AUC-Area Under the Curve with isoquercetin and 48±17% AUC with hydroxyurea of untreated SCD) and fibrin generation (30±6% AUC with isoquercetin and 44±9% AUC with hydroxyurea of untreated SCD). To study the effect of chronic isoquercetin treatment in SCD, control and SCD mice were fed isoquercetin for 4 weeks. Daily consumption of isoquercetin resulted in reduction of plasma PDI activity (18±5% inhibition, p <0.05). Consistent with the intravital microscopic data, chronic PDI inhibition reduced neutrophil migration and accumulation into the lung, as determined by the MPO level (481±85 ng/mg vs 829±140 ng/mg, p <0.05) in the tissue homogenate. Chronic treatment with isoquercetin also reduced plasma fibrinogen degradation products (FDP) (102±15 μg/ml vs 183±25 μg/ml, p <0.05) in SCD mice. In addition, a correlation (Pearson r=0.651) was observed between reducing plasma FDP and lung MPO levels.ConclusionsAcute and chronic PDI inhibition by isoquercetin may provide beneficial effect on vascular complications in a mouse model of SCD. PDI inhibition has the potential to ameliorate thrombo-inflammatory syndromes associated with SCD. These studies justify a longitudinal trial of PDI inhibitors in SCD. DisclosuresNo relevant conflicts of interest to declare.

Leukocyte–Endothelium Interaction Is Associated with Fat Mass in Children

To study leukocyte-endothelium interaction, a measure of the initial phase of atheromatosis, in children with overweight or obesity. A prospective study was conducted in 77 children aged 7-16years; 47 were children with overweight/obesity and 30 were normal weight. Polymorphonuclear neutrophils (PMNs) and peripheral blood mononuclear cells were isolated from venous blood samples and the interaction of leukocytes over a monolayer of human umbilical vein endothelial cells was analyzed using flow chamber microscopy. The variables studied included leukocyte rolling velocity, rolling flux, and adhesion to endothelial cells. These were compared between children with overweight/obesity and control children. Correlation between the measures of leukocyte-endothelium interaction and anthropometric and biochemical variables was evaluated. In comparison with normal weight children, the PMNs and peripheral blood mononuclear cells of the overweight/obesity group showed a reduction in rolling velocity (P=.000 and P=.001, respectively) and an increase in rolling flux (P=.001 and P=.004), and adhesion (P=.003 and P=.002). The homeostasis model of insulin resistance was correlated inversely with rolling velocity and positively with rolling flux in PMNs. C-reactive protein was correlated positively with rolling flux and adhesion in both types of leucocytes. Fat mass index was correlated with all measures of leukocyte-endothelial interaction and proved to be the main predictor of leukocyte adhesion in the multiple regression analysis (P=.001 for PMNs and P=.006 for peripheral blood mononuclear cells). Excess fat mass in children is related to the activation of the leukocyte-endothelium interaction, potentially contributing to the development of atherosclerosis.

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

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. DisclosuresNo relevant conflicts of interest to declare.

Inhibiting focal adhesion kinase (FAK) blocks IL-4 induced VCAM-1 expression and eosinophil recruitment in vitro and in vivo.

Leukocyte recruitment plays a critical role during both normal inflammation and chronic inflammatory diseases, and ongoing studies endeavor to better understand the complexities of this process. Focal adhesion kinase (FAK) is well known for its role in cancer, yet it also has been shown to regulate aspects of neutrophil and B16 melanoma cell recruitment by rapidly influencing endothelial cell focal adhesion dynamics and junctional opening. Recently, we found that FAK related non-kinase (FRNK), a protein that is often used as a FAK dominant negative, blocked eosinophil transmigration by preventing the transcription of vascular cell adhesion molecule-1 (VCAM-1) and eotaxin-3 (CCL26). Surprisingly, the blocking occurred even in the absence of endogenous FAK. To better understand the role of FAK in leukocyte recruitment, we used a FAK-specific inhibitor (PF-573228) and determined the effect on IL-4 induced eosinophil recruitment in vitro and in vivo. PF-573228 prevented the expression of VCAM-1 and CCL26 expression in IL-4-stimulated human endothelial cells in vitro. As a result, eosinophil adhesion and transmigration were blocked. PF-572338 also prevented IL-4-induced VCAM-1 expression in vivo. Using brightfield intravital microscopy, we found that PF-573228 decreased leukocyte rolling flux, adhesion, and emigration. We specifically examined eosinophil recruitment in vivo by using an eosinophil-GFP reporter mouse and found PF-573228 attenuated eosinophil emigration. This study reveals that a FAK inhibitor influences inflammation through its action on eosinophil recruitment.

Low testosterone levels are related to oxidative stress, mitochondrial dysfunction and altered subclinical atherosclerotic markers in type 2 diabetic male patients

IntroductionLow testosterone levels in men are associated with type 2 diabetes and cardiovascular risk. However, the role of testosterone in mitochondrial function and leukocyte-endothelium interactions is unknown. Our aim was to evaluate the relationship between testosterone levels, metabolic parameters, oxidative stress, mitochondrial function, inflammation and leukocyte-endothelium interactions in type 2 diabetic patients. Materials and methodsThe study was performed in 280 male type 2 diabetic patients and 50 control subjects. Anthropometric and metabolic parameters, testosterone levels, reactive oxygen species (ROS) production, mitochondrial membrane potential, TNFα, adhesion molecules and leukocyte-endothelium cell interactions were evaluated. ResultsTestosterone levels were lower in diabetic patients. Total and mitochondrial ROS were increased and mitochondrial membrane potential, SOD and GSR expression levels were reduced in diabetic patients. TNFα, ICAM-1 and VCAM-1 levels, leukocyte rolling flux and adhesion were all enhanced in diabetic patients, while rolling velocity was reduced. Testosterone levels correlated negatively with glucose, HOMA-IR, HbA1c, triglycerides, nonHDL-c, ApoB, hs-CRP and AIP, and positively with HDL-c and ApoA1. The multivariable regression model showed that HDL-c, HOMA-IR and age were independently associated with testosterone. Furthermore, testosterone levels correlated positively with membrane potential and rolling velocity and negatively with ROS production, VCAM-1, rolling flux and adhesion. ConclusionsOur data highlight that low testosterone levels in diabetic men are related to impaired metabolic profile and mitochondrial function and enhanced inflammation and leukocyte-endothelium cell interaction, which leaves said patients at risk of cardiovascular events.

Metabolic syndrome enhances endoplasmic reticulum, oxidative stress and leukocyte–endothelium interactions in PCOS

ObjectivePolycystic ovary syndrome (PCOS) is associated with insulin resistance, which can lead to metabolic syndrome (MetS). Oxidative stress and leukocyte–endothelium interactions are related to PCOS. Our aim was to evaluate whether the presence of MetS in PCOS patients can influence endoplasmic reticulum (ER) and oxidative stress and leukocyte–endothelium interactions. Material and MethodsThis was a prospective controlled study conducted in an academic medical center. The study population consisted of 148 PCOS women (116 without/32 with MetS) and 112 control subjects (87 without / 25 with MetS). Metabolic parameters, reactive oxygen species (ROS) production, ER stress markers (GRP78, sXBP1, ATF6), leukocyte–endothelium interactions, adhesion molecules (VCAM-1, ICAM-1, E-Selectin), TNF-α and IL-6 were determined. ResultsTotal ROS, inflammatory parameters and adhesion molecules were enhanced in the presence of MetS (p<0.05), and the PCOS+MetS group showed higher levels of IL-6 and ICAM-1 than controls (p<0.05). Increased adhesion and leukocyte rolling flux were observed in PCOS and PCOS+MetS groups vs their respective controls (p<0.05). GRP78 protein expression was higher in the PCOS groups (p<0.05 vs controls) and sXBP1 was associated with the presence of MetS (p<0.05 vs controls without MetS). Furthermore, PCOS+MetS patients exhibited higher GRP78 and ATF6 levels than controls and PCOS patients without MetS (p<0.05). In PCOS women, HOMA-IR was positively correlated with ICAM-1 (r=0.501; p<0.01), ROS (r=0.604; p<0.01), rolling flux (r=0.455;p<0.05) and GRP78 (r=0.574; p<0.001). ConclusionOur findings support the hypothesis of an association between altered metabolic status, increased ROS production, ER stress and leukocyte–endothelium interactions in PCOS, all of which are related to vascular complications.

Anti-Thrombin III Inhibits Adhesion of Sickle Red Blood Cells and Leukocytes to the Endothelium in a Sickle Cell Mouse Model: Potential Therapeutic Application for Vaso-Occlusive Episodes

Painful vaso-occlusive episodes are a hallmark of sickle cell disease (SCD), an inherited blood disorder caused by a point mutation in the β-globin gene. Vaso-occlusive events account for the majority of SCD-related hospital admissions and contribute importantly to major organ damage and decreased survival. Therapies to prevent and treat acute vaso-occlusive episodes are limited and there is a need for new treatment options. Adhesive interactions between circulating sickle red blood cells (sRBC), leukocytes, and endothelial cells have been implicated as critical pathologic events for the development of vaso-occlusive episodes. These cell adhesive interactions further manifest with increased local and systemic inflammation and activated coagulation evidenced in patients and mouse models of SCD.Anti-thrombin III (ATIII) is an endogenous inhibitor of thrombin and other serine proteases in the coagulation pathway. Apart from the role in hemostasis, ATIII exerts strong anti-inflammatory properties. Plasma levels of ATIII and ATIII activity are decreased in SCD and further decline during vaso-occlusive crisis. These findings indicate that ATIII deficiency may contribute by multiple mechanisms to the pathophysiology of acute vaso-occlusion and that repletion therapy with ATIII may provide a novel, multi-targeted therapeutic approach to this difficult-to-treat complication.In this study, we examined whether ATIII supplementation is able to reduce adhesion of sRBC and leukocytes to vascular endothelial cells in the calvarial bone marrow microvasculature in a mouse knockout-transgenic mouse model of SCD (Ryan, 1997). Homozygous SCD mice were injected intravenously with 230 U/kg ATIII (Thrombate III; Grifols, USA) or vehicle (saline). After 48 hours, the mice were subjected to 3 hours of hypoxia (8% O2 in medical air) followed by 3 hours of reoxygenation at room air (21% O2). Intravital microscopy analysis of the bone marrow microcirculation was performed to monitor adhesive interactions between blood cells and vascular endothelium. Leukocytes were labeled in vivo with phycoerythrin-conjugated anti-CD45 antibody which was infused via the carotid artery. Sickle RBC were obtained from donor mice, fluorescently labeled in vitro with 2,7-bis-(carboxyethyl)-5-(and-6) carboxyfluorescein, and infused through the carotid artery to the recipient mice. Effects of ATIII on sRBC and leukocyte adhesive interactions were compared to vehicle. Administration of ATIII to SCD mice resulted in more than 5 fold decrease in sRBC adhesion (p<0.01) compared to vehicle. Next the effects of ATIII on leukocyte flow dynamics were analyzed quantitatively. Mice pretreated with ATIII, but not those treated with vehicle, showed two-fold decrease in leukocyte rolling flux (p<0.01) and a 3-fold decrease in adhesion (p<0.001) compared with vehicle-treated mice. Furthermore, ATIII improved blood circulation compared to vehicle-treated mice as shown by increased RBC velocity and calculated wall shear rates. The pretreatment with ATIII was associated with decreased plasma levels of IL-1β and a trend to decreased soluble levels of vascular cell adhesion molecule 1 and thrombin-antithrombin complexes.In conclusion, our study demonstrates significant anti-adhesive activities of ATIII, which reduces the adhesion of sRBC and leukocytes to the vascular endothelium in a mouse model of SCD. ATIII may represent a novel and effective therapeutic intervention in the treatment of acute vaso-occlusive crisis in patients with SCD. DisclosuresGutsaeva:Grifols: Research Funding.

A Chemical APC Mimetic Protects Endothelium from Thromboinflammatory Injury

Stimulation of protease-activated receptor 1 (PAR1) on endothelium by activated protein C (APC) is protective in animal models of inflammation and APC has been used clinically in sepsis and wound healing. Clinical use of APC in sepsis, however, was terminated as it was compromised by APC’s anticoagulant activity, which is associated with bleeding and limits its dosing in patients. We used a small molecule approach to circumvent this problem. With support from the Molecular Libraries Program, we screened 302,457 compounds to identify small molecules that modulated PAR1-mediated platelet activation. One class of PAR1-targeted compounds, which we termed parmodulins, was found to act at the cytosolic face of PAR1, at the G-protein binding sites. When evaluated in endothelial cell cultures,parmodulin 1 (PM1) and parmodulin 2 (PM2) inhibited apoptosis induced by thrombin, TNF-α, or staurosporine, in a manner similar to APC. PAR1 knockdown using siRNA abolished these protective effects demonstrating that parmodulins elicit a cytoprotective pathway by acting through PAR1. To assess the mechanism of action of parmodulin cytoprotection, we first evaluated proximal signaling mechanisms. Parmodulins stimulated phosphorylation of PI3-kinase and Akt in endothelium. Inhibition of Gβγ blocked parmodulin-induced phosphorylation of Akt, indicating that parmodulins act at the cytosolic face of PAR1 by releasing Gβγ. Transcriptional profiling of over 30,000 genes and specific evaluation of NF-kB transcriptional activation showed that exposure to PM2 blocked TNF-α-induced transcriptional activation. In addition to interfering with inflammatory signaling, parmodulins also stimulated the upregulation of cytoprotective proteins such as stanniocalcin-1. Since our premise was that parmodulins could achieve cytoprotective effects without anticoagulant effects, we compared dose curves of APC versus PM2 in both apoptosis assays and standard clotting assays. APC prolonged the aPTT at concentrations lower than those required to achieve cytoprotection of the endothelium. The low APC concentration used in our study was similar to plasma concentrations measured in clinical studies. Hence, these data were consistent with the fact that clinical bleeding was observed at doses of APC used for sepsis. In contrast, despite inhibiting apoptosis as effectively as APC, PM2 had no effect on plasma aPTT at any concentration. Nonetheless, PM2 was able to inhibit LPS- and TNF-α-induced thrombin generation and FXa activation on endothelium owing to its cytoprotective effect. PM2 also prevented TNF-α-induced accumulation of platelets on endothelium in bioengineered microvessels. These data demonstrate that PM2 can reduce inflammation-induced endothelial pro-thrombotic phenotype even without directly inhibiting coagulation factors. To assess the endothelial protective effects of PM2 in vivo we evaluated leukocyte rolling in cremaster venules of mice. Infusion of PM2 significantly reduced surgery-induced leukocyte rolling flux compared to vehicle-treated mice. As selectins are critically involved in leukocyte rolling we monitored soluble E-selectin levels in LPS-induced inflammation. Treatment of mice with PM2 significantly reduced the LPS-induced release of soluble E-selectin. Previously, we demonstrated that PM2 blocks platelet accumulation in a mouse laser injury model of thrombus formation. We here show that infusion of PM2 also significantly reduces fibrin accumulation to 25% of control (p<0.001) at the site of laser injury. Together our data show that PM2 exerts endothelial-mediated anti-inflammatory, anti-coagulant and anti-thrombotic effects in vitro and in vivo. These results demonstrate that modulating PAR1 at the cytosolic face could represent an alternative approach to APC in the treatment of thromboinflammatory disorders like sepsis. DisclosuresNo relevant conflicts of interest to declare.

Are Mitochondrial Fusion and Fission Impaired in Leukocytes of Type 2 Diabetic Patients?

Mitochondrial fusion/fission alterations have been evaluated in different tissues of type 2 diabetic (T2D) patients. However, it is not known whether mitochondrial dynamics is disturbed in the leukocytes of T2D patients and whether glycemic control affects its regulation. Anthropometric and metabolic parameters in 91 T2D patients (48 with glycated hemoglobin [HbA1c] <6.5% and 43 with HbA1c >6.5%) were characteristic of the disease when compared with 78 control subjects. We observed increased reactive oxygen species production in leukocytes from diabetic patients, together with a reduced mitochondrial oxygen consumption rate, especially in poorly controlled patients. Mitochondrial fusion was reduced and fission was increased in diabetic patients, and both features were accentuated in patients with poor glycemic control. Furthermore, leukocyte rolling flux rose in parallel to HbA1c levels. The induction of leukocyte-endothelial interactions in diabetic patients was related to reduced mitochondrial fusion and higher mitochondrial fission. Our findings suggest that mitochondrial dynamics could be influenced by glycemic control in leukocytes of diabetic patients, in which there is decreased mitochondrial fusion and elevated fission related to enhanced leukocyte-endothelial interactions. These findings lead to the hypothesis that poor glycemic control during T2D may alter mitochondrial dynamics and could eventually promote leukocyte-endothelial interactions and the onset of cardiovascular diseases. Antioxid. Redox Signal. 25, 108-115.

Insulin Resistance in PCOS Patients Enhances Oxidative Stress and Leukocyte Adhesion: Role of Myeloperoxidase.

Cardiovascular diseases and oxidative stress are related to polycystic ovary syndrome (PCOS) and insulin resistance (IR). We have evaluated the relationship between myeloperoxidase (MPO) and leukocyte activation in PCOS patients according to homeostatic model assessment of IR (HOMA-IR), and have explored a possible correlation between these factors and endocrine and inflammatory parameters. This was a prospective controlled study conducted in an academic medical center. The study population consisted of 101 PCOS subjects and 105 control subjects. We divided PCOS subjects into PCOS non-IR (HOMA-IR<2.5) and PCOS IR (HOMA-IR>2.5). Metabolic and anthropometric parameters, total and mitochondrial reactive oxygen species (ROS) production, MPO levels, interactions between human umbilical vein endothelial cells and leukocytes, adhesion molecules (E-selectin, ICAM-1 and VCAM-1) and proinflammatory cytokines (IL-6 and TNF-α) were evaluated. Oxidative stress was observed in PCOS patients, in whom there was an increase in total and mitochondrial ROS production and MPO levels. Enhanced rolling flux and adhesion, and a decrease in polymorphonuclear cell rolling velocity were also detected in PCOS subjects. Increases in IL-6 and TNF-α and adhesion molecules (E-selectin, ICAM-1 and VCAM-1) were also observed, particularly in the PCOS IR group, providing evidence that inflammation and oxidative stress are related in PCOS patients. HOMA-IR was positively correlated with hsCRP (p<0.001, r = 0.304), ROS production (p<0.01, r = 0.593), leukocyte rolling flux (p<0.05, r = 0.446), E-selectin (p<0.01, r = 0.436) and IL-6 (p<0.001, r = 0.443). The results show an increase in the rate of ROS and MPO levels in PCOS patients in general, and particularly in those with IR. Inflammation in PCOS induces leukocyte-endothelium interactions and a simultaneous increase in IL-6, TNF-α, E-selectin, ICAM-1 and VCAM-1. These conditions are aggravated by the presence of IR.

CAMP‐signaling via EPAC1 mediates vascular endothelial cell adaptation to fluid‐shear stress

Herein, we report a novel role for cAMP‐signaling in human aortic endothelial cell (HAEC) adaptation to fluid‐mediated shear stress (FSS). Our results showed that increases in cAMP enhanced elongation and alignment of HAECs cultured under LSS for 48hrs and that this effect was largely mediated by the cAMP‐effector molecule, exchange protein activated by cAMP (EPAC)‐1, but not Protein Kinase A (PKA). Additionally, major HAEC cAMP‐hydrolyzing enzyme, phosphodiesterase (PDE)4D, was also shown to play a crucial role in integrating FSS signals in these cells.Our results demonstrated that flow‐mediated increases in atheroprotective genes, Kruppel‐like factor, endothelial nitric oxide synthase and thrombomodulin mRNA expression were reduced in EPAC‐1 and PDE4D knockdown HAECs exposed to both LSS and HSS. Furthermore, EPAC‐1 knockdown increased leukocyte extravasation across HAEC monolayers in vitro and activation of EPAC significantly reduced LPS‐induced leukocyte rolling flux in mice.VECs have numerous mechanosensors which facilitate the integration of FSS signals from the flowing blood. One such sensor consists of platelet‐endothelial cell adhesion molecule (PECAM)‐1, vascular endothelial‐cadherin (VECAD) and vascular endothelial growth factor receptor (VEFGR)‐2. Our current data suggests that EPAC1 and PDE4D can additionally mediate flow‐sensing through this mechanosensory complex. All together, this data provides important mechanistic insight providing therapeutic targets in endothelial dysfunction and atherosclerosis.

Abstract 199: Anticoagulant Heparan Sulfate Conveys Anti-Inflammatory Activity of Antithrombin

Antithrombin (AT) therapy reduces morbidity/mortality in systemic inflammatory response syndromes, such as septic shock. These effects stem from the ability of AT to directly initiate cell signaling, which requires AT engagement with heparan sulfate proteoglycans. It is presently unclear if this process involves anticoagulant heparan sulfate (HS AT+ ), a specific motif with high affinity for AT that is produced by endothelial cells. If HS AT+ regulates anti-inflammatory signaling, then Hs3st1 -/- mice, which lack endothelial HS AT+ , should exhibit a proinflammatory state and should not benefit from AT treatment. We tested these hypotheses using a mouse model of lipopolysaccharide (LPS)-induced septic shock. Hemodynamics were monitored with a left ventricle pressure-volume catheter. Both Hs3st1 +/+ and Hs3st1 -/- mice die of cardiogenic shock. However, Hs3st1 -/- , compared to Hs3st1 +/+ , mice exhibited a much more rapid onset of death. Left ventricular gene expression profiles of mice with systemic LPS challenge showed enhanced expression of leukocyte recruitment genes (P-selectin, Cxcl10) in Hs3st1 -/- mice. Consistent with these data, LPS-treated Hs3st1 -/- mice, compared to Hs3st1 +/+ , exhibited enhanced leukocyte rolling flux within cremaster muscle venules. Thus, mice lacking HS AT+ exhibit a proinflammatory phenotype. AT treatment of Hs3st1 +/+ mice reduced both LPS-induced lethality and leukocyte-endothelial interactions. Conversely AT treatment of Hs3st1 -/- mice enhanced both LPS-induced lethality and leukocyte-endothelial interactions. Thus, AT failed to elicit anti-inflammatory effects in mice lacking HS AT+ . Our results indicate that endothelial HS AT+ is one component that mediates AT’s anti-inflammatory activity. These findings provide an initial step towards elucidating the role of HS AT+ and AT in a novel natural anti-inflammatory pathway of the blood vessel wall. This pathway is an ideal target for developing therapeutics to combat the devastating outcomes of septic shock.

Abstract 26: Novel Role of Anticoagulant Heparan Sulfate in Antithrombin Anti-inflammatory Signaling

Antithrombin (AT) therapy reduces morbidity/mortality in systemic inflammatory response syndromes, such as septic shock. These effects stem from the ability of AT to directly initiate cell signaling, which requires AT engagement with heparan sulfate proteoglycans. It is presently unclear if this process involves anticoagulant heparan sulfate (HS AT+ ), a specific motif with high affinity for AT that is produced by endothelial cells. If HS AT+ regulates anti-inflammatory signaling, then Hs3st1 -/- mice, which lack endothelial HS AT+ , should exhibit a proinflammatory state and should not benefit from AT treatment. We tested these hypotheses using a mouse model of lipopolysaccharide (LPS)-induced septic shock. Hemodynamics were monitored with a left ventricle pressure-volume catheter. Both Hs3st1 +/+ and Hs3st1 -/- mice die of cardiogenic shock. However, Hs3st1 -/- , compared to Hs3st1 +/+ , mice exhibited a much more rapid onset of death. Left ventricular gene expression profiles of mice with systemic LPS challenge showed enhanced expression of leukocyte recruitment genes (P-selectin, Cxcl10) in Hs3st1 -/- mice. Consistent with these data, LPS-treated Hs3st1 -/- mice, compared to Hs3st1 +/+ , exhibited enhanced leukocyte rolling flux within cremaster muscle venules. Thus, mice lacking HS AT+ exhibit a proinflammatory phenotype. AT treatment of Hs3st1 +/+ mice reduced both LPS-induced lethality and leukocyte-endothelial interactions. Conversely AT treatment of Hs3st1 -/- mice enhanced both LPS-induced lethality and leukocyte-endothelial interactions. Thus, AT failed to elicit anti-inflammatory effects in mice lacking HS AT+ . Our results indicate that endothelial HS AT+ is one component that mediates AT’s anti-inflammatory activity. These findings provide an initial step towards elucidating the role of HS AT+ and AT in a novel natural anti-inflammatory pathway of the blood vessel wall. This pathway is an ideal target for developing therapeutics to combat the devastating outcomes of septic shock.

Abstract 9926: Molecular Imaging of the Pro-angiogenic Paracrine Effects of Stem Cell Therapy

Background: Pro-angiogenic stem cells are a promising therapy for patients with severe coronary or peripheral vascular disease. Mechanisms by which cell therapy promotes vascular remodeling remain unclear. We applied contrast-enhanced ultrasound (CEU) molecular imaging to test whether cell therapy acts in a paracrine fashion by promoting recruitment of pro-angiogenic (CX3CR-1 hi ) monocytes. Methods: Unilateral hindlimb ischemia was produced in 52 mice by iliac artery ligation. One day later, the proximal hindlimb received either: (1) IM injection of 1×10 6 multipotent adult progenitor cells (MAPC), (2) sham saline injection, or (3) no therapy. At Day3, 7, and 21 CEU perfusion imaging and molecular imaging of CX3CR-1 and P-selectin were performed. In 8 separate mice, intravital microscopy of TNF-a-treated cremaster muscle was performed 3 days after injection of either 5×10 5 MAPCs or saline. Results: Arterial ligation resulted in a 70% reduction in limb muscle blood flow to 0.20±0.06 ml/min/g. MAPC therapy produced a more rapid and complete recovery of flow (0.61±0.06 ml/min/g at day 21, ≈25% higher than both control groups, p&lt;0.05). Signal for CX3CR-1 was significantly (p&lt;0.05) higher vs control groups at all 3 study intervals (Figure),corroborated by a marked increase in tissue monocytes on Mac-2 immunohistology. P-selectin signal was also significantly higher in the MAPC-treated mice at days 3 and 7 (Figure). Intravital microscopy which showed that MAPCs migrated to a perivascular location and produced a 50% increase in leukocyte rolling flux fraction. Conclusions: In chronic limb ischemia, MAPCs migrate to a perivascular location and promote the sustained recruitment of pro-angiogenic monocytes, in part through effects on endothelial adhesion molecule expression. These results suggest that cell therapy can act to promote vascular remodeling through inflammatory pathways

Inhibition of cell adhesion by anti–P-selectin aptamer: a new potential therapeutic agent for sickle cell disease

AbstractAdhesive interactions between circulating sickle red blood cells (RBCs), leukocytes, and endothelial cells are major pathophysiologic events in sickle cell disease (SCD). To develop new therapeutics that efficiently inhibit adhesive interactions, we generated an anti–P-selectin aptamer and examined its effects on cell adhesion using knockout-transgenic SCD model mice. Aptamers, single-stranded oligonucleotides that bind molecular targets with high affinity and specificity, are emerging as new therapeutics for cardiovascular and hematologic disorders. In vitro studies found that the anti–P-selectin aptamer exhibits high specificity to mouse P-selectin but not other selectins. SCD mice were injected with the anti–P-selectin aptamer, and cell adhesion was observed under hypoxia. The anti–P-selectin aptamer inhibited the adhesion of sickle RBCs and leukocytes to endothelial cells by 90% and 80%, respectively. The anti–P-selectin aptamer also increased microvascular flow velocities and reduced the leukocyte rolling flux. SCD mice treated with the anti–P-selectin aptamer demonstrated a reduced mortality rate associated with the experimental procedures compared with control mice. These results demonstrate that anti–P-selectin aptamer efficiently inhibits the adhesion of both sickle RBCs and leukocytes to endothelial cells in SCD model mice, suggesting a critical role for P-selectin in cell adhesion. Anti–P-selectin aptamer may be useful as a novel therapeutic agent for SCD.

Inflammation Determines the Pro-Adhesive Properties of High Extracellular D-Glucose in Human Endothelial Cells In Vitro and Rat Microvessels In Vivo

BackgroundHyperglycemia is acknowledged as an independent risk factor for developing diabetes-associated atherosclerosis. At present, most therapeutic approaches are targeted at a tight glycemic control in diabetic patients, although this fails to prevent macrovascular complications of the disease. Indeed, it remains highly controversial whether or not the mere elevation of extracellular D-glucose can directly promote vascular inflammation, which favors early pro-atherosclerotic events.Methods and FindingsIn the present work, increasing extracellular D-glucose from 5.5 to 22 mmol/L was neither sufficient to induce intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression, analyzed by flow cytometry, nor to promote leukocyte adhesion to human umbilical vein endothelial cells (HUVEC) in vitro, measured by flow chamber assays. Interestingly, the elevation of D-glucose levels potentiated ICAM-1 and VCAM-1 expression and leukocyte adhesion induced by a pro-inflammatory stimulus, such as interleukin (IL)-1β (5 ng/mL). In HUVEC, high D-glucose augmented the activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and nuclear transcription factor-κB (NF-κB) elicited by IL-1β, measured by Western blot and electromobility shift assay (EMSA), respectively, but had no effect by itself. Both ERK 1/2 and NF-κB were necessary for VCAM-1 expression, but not for ICAM-1 expression. In vivo, leukocyte trafficking was evaluated in the rat mesenteric microcirculation by intravital microscopy. In accordance with the in vitro data, the acute intraperitoneal injection of D-glucose increased leukocyte rolling flux, adhesion and migration, but only when IL-1β was co-administered.ConclusionsThese results indicate that the elevation of extracellular D-glucose levels is not sufficient to promote vascular inflammation, and they highlight the pivotal role of a pro-inflammatory environment in diabetes, as a critical factor conditioning the early pro-atherosclerotic actions of hyperglycemia.

Response of the Murine Urinary Bladder Microcirculation to Lipopolysaccharide from Escherichia coli and Pseudomonas aeruginosa

There is in vitro evidence demonstrating the inflammatory response to lipopolysaccharide (LPS) differs depending on the bacterial source. The objective of our study was to examine the in vivo inflammatory response to LPS from two common uropathogens, Escherichia coli (Ec) and Pseudomonas aeruginosa (Pa).LPS or saline was injected transurethrally into bladders of female C57Bl/6 mice at varying doses. The bladder was exteriorized, and the bladder microcirculation examined by intravital microscopy. Leukocyte adhesion at 4 hrs post challenge with 5 mg/kg Pa LPS was 2.9±0.6 cells/field of view and at 7 mg/kg it was 3.9±0.8 cells/field of view, a significant increase from saline control. In contrast, leukocyte adhesion 4 hours post 5 mg/kg Ec LPS challenge was 17.4±3.9 and with 7 mg/kg 19.7±2.9 cells/field of view. However, at 4.5 hours adhesion was similar with either LPS (5 mg/kg) with 18.4±3.0 and 21.2±4.0 cells/field of view, respectively. The flux of rolling leukocytes was significantly increased above baseline levels only after Ec LPS challenge. There was also a significant increase in the number of neutrophils with Ec LPS stimulation.In support of the in vitro observations, these in vivo results demonstrate that Ec LPS is more potent and induces a more rapid inflammatory response than Pa LPS.Supported by a Collaborative Health Research Projects grant from CIHR and NSERC.