Recombinant P-selectin glycoprotein-ligand-1 delays thrombin-induced platelet aggregation: a new role for P-selectin in early aggregation

PubMed Central

Théorêt, Jean-François; Chahrour, Wissam; Yacoub, Daniel; Merhi, Yahye

2006-01-01

P-selectin is involved, with P-selectin glycoprotein (GP)-ligand-1 (PSGL-1), in platelet/leukocyte interactions during thrombo-inflammatory reactions; it also stabilizes platelet aggregates. Its antagonism accelerates thrombolysis and enhances the anti-aggregatory effects of GPIIb–IIIa inhibitors. This study was designed to investigate the mechanisms of P-selectin-mediated platelet aggregation. In freshly isolated human platelets, P-selectin translocation after thrombin stimulation increased rapidly to 48, 72, and 86% positive platelets after 60, 120, and 300 s, respectively. Platelet aggregation at 60 s post-stimulation averaged 46.7±1.9% and its extent followed closely the kinetics of P-selectin translocation. Pre-treatment of platelets with P-selectin antagonists, a recombinant PSGL-1 (rPSGL-Ig) or a blocking monoclonal antibody, significantly delayed platelet aggregation in a dose-dependent manner. At 100 μg ml−1 of rPSGL-Ig, platelet aggregation was completely inhibited up to 60 s post-stimulation and increased thereafter to reach maximal aggregation at 5 min. The second phase of platelet aggregation, in the presence of rPSGL-Ig, was completely prevented by the addition of a GPIIb–IIIa antagonist (Reopro) at 60 s, whereas its addition in the absence of rPSGL-Ig was without any significant effect. Combination of rPSGL-Ig with Reopro or with an inhibitor of Pi3K (LY294002), which reduces GPIIb–IIIa activation, showed to be more effective in inhibiting platelet aggregation, in comparison to the effects observed individually. rPSGL-Ig blocks P-selectin, whereas Reopro and LY294002 block GPIIb–IIIa and its activation, respectively, without a major effect on the percentage of platelets expressing P-selectin. In summary, platelet P-selectin participates with GPIIb–IIIa in the initiation of platelet aggregation. Its inhibition, with rPSGL-Ig, delays the aggregation process and increases the anti-aggregatory potency of Reopro. Thus, combination of P-selectin and GPIIb–IIIa antagonism may constitute a promising therapeutic option in the management of thrombotic disorders. PMID:16633357

Recombinant P-selectin glycoprotein-ligand-1 delays thrombin-induced platelet aggregation: a new role for P-selectin in early aggregation.

PubMed

Théorêt, Jean-François; Chahrour, Wissam; Yacoub, Daniel; Merhi, Yahye

2006-06-01

1. P-selectin is involved, with P-selectin glycoprotein (GP)-ligand-1 (PSGL-1), in platelet/leukocyte interactions during thrombo-inflammatory reactions; it also stabilizes platelet aggregates. Its antagonism accelerates thrombolysis and enhances the anti-aggregatory effects of GPIIb-IIIa inhibitors. This study was designed to investigate the mechanisms of P-selectin-mediated platelet aggregation. 2. In freshly isolated human platelets, P-selectin translocation after thrombin stimulation increased rapidly to 48, 72, and 86% positive platelets after 60, 120, and 300 s, respectively. Platelet aggregation at 60 s post-stimulation averaged 46.7 +/- 1.9% and its extent followed closely the kinetics of P-selectin translocation. 3. Pre-treatment of platelets with P-selectin antagonists, a recombinant PSGL-1 (rPSGL-Ig) or a blocking monoclonal antibody, significantly delayed platelet aggregation in a dose-dependent manner. At 100 microg ml(-1) of rPSGL-Ig, platelet aggregation was completely inhibited up to 60 s post-stimulation and increased thereafter to reach maximal aggregation at 5 min. The second phase of platelet aggregation, in the presence of rPSGL-Ig, was completely prevented by the addition of a GPIIb-IIIa antagonist (Reopro) at 60 s, whereas its addition in the absence of rPSGL-Ig was without any significant effect. 4. Combination of rPSGL-Ig with Reopro or with an inhibitor of Pi3K (LY294002), which reduces GPIIb-IIIa activation, showed to be more effective in inhibiting platelet aggregation, in comparison to the effects observed individually. 5. rPSGL-Ig blocks P-selectin, whereas Reopro and LY294002 block GPIIb-IIIa and its activation, respectively, without a major effect on the percentage of platelets expressing P-selectin. 6. In summary, platelet P-selectin participates with GPIIb-IIIa in the initiation of platelet aggregation. Its inhibition, with rPSGL-Ig, delays the aggregation process and increases the anti-aggregatory potency of Reopro. Thus, combination of P-selectin and GPIIb-IIIa antagonism may constitute a promising therapeutic option in the management of thrombotic disorders.

P-selectin ligation induces platelet activation and enhances microaggregate and thrombus formation.

PubMed

Théorêt, Jean-François; Yacoub, Daniel; Hachem, Ahmed; Gillis, Marc-Antoine; Merhi, Yahye

2011-09-01

Platelet P-selectin is a thrombo-inflammatory molecule involved in platelet activation and aggregation. This may occur via the adhesive function of P-selectin and its potential capacity to trigger intracellular signaling. However, its impact on platelet function remains elusive. This study was therefore designed to investigate the relationship between the signaling potential of platelet P-selectin and its function in platelet physiology. Human and mouse platelets were freshly isolated from whole blood. Platelet activation was assessed using flow cytometry and western blot analysis, while platelet physiological responses were evaluated through aggregation, microaggregate formation and in a thrombosis model in wild-type and P-selectin-deficient (CD62P(-/-)) mice. Interaction of P-selectin with its high-affinity ligand, a recombinant soluble form of P-Selectin Glycoprotein Ligand-1 (rPSGL-1), enhances platelet activation, adhesion and microaggregate formation. This augmented platelet microaggregates requires an intact cytoskeleton, but occurs independently of platelet α(IIb)β(3). Thrombus formation and microaggregate were both enhanced by rPSGL-1 in wild-type, but not in CD62P(-/-) mice. In addition, CD62P(-/-) mice exhibited thrombosis abnormalities without an α(IIb)β(3) activation defect. This study demonstrates that the role of platelet P-selectin is not solely adhesive; its binding to PSGL-1 induces platelet activation that enhances platelet aggregation and thrombus formation. Therefore, targeting platelet P-selectin or its ligand PSGL-1 could provide a potential therapeutic approach in the management of thrombotic disorders. Copyright © 2011 Elsevier Ltd. All rights reserved.

O-glycans direct selectin ligands to lipid rafts on leukocytes.

PubMed

Shao, Bojing; Yago, Tadayuki; Setiadi, Hendra; Wang, Ying; Mehta-D'souza, Padmaja; Fu, Jianxin; Crocker, Paul R; Rodgers, William; Xia, Lijun; McEver, Rodger P

2015-07-14

Palmitoylated cysteines typically target transmembrane proteins to domains enriched in cholesterol and sphingolipids (lipid rafts). P-selectin glycoprotein ligand-1 (PSGL-1), CD43, and CD44 are O-glycosylated proteins on leukocytes that associate with lipid rafts. During inflammation, they transduce signals by engaging selectins as leukocytes roll in venules, and they move to the raft-enriched uropods of polarized cells upon chemokine stimulation. It is not known how these glycoproteins associate with lipid rafts or whether this association is required for signaling or for translocation to uropods. Here, we found that loss of core 1-derived O-glycans in murine C1galt1(-/-) neutrophils blocked raft targeting of PSGL-1, CD43, and CD44, but not of other glycosylated proteins, as measured by resistance to solubilization in nonionic detergent and by copatching with a raft-resident sphingolipid on intact cells. Neuraminidase removal of sialic acids from wild-type neutrophils also blocked raft targeting. C1galt1(-/-) neutrophils or neuraminidase-treated neutrophils failed to activate tyrosine kinases when plated on immobilized anti-PSGL-1 or anti-CD44 F(ab')2. Furthermore, C1galt1(-/-) neutrophils incubated with anti-PSGL-1 F(ab')2 did not generate microparticles. In marked contrast, PSGL-1, CD43, and CD44 moved normally to the uropods of chemokine-stimulated C1galt1(-/-) neutrophils. These data define a role for core 1-derived O-glycans and terminal sialic acids in targeting glycoprotein ligands for selectins to lipid rafts of leukocytes. Preassociation of these glycoproteins with rafts is required for signaling but not for movement to uropods.

Genome-Wide Association Study in African Americans with Acute Respiratory Distress Syndrome Identifies the Selectin P Ligand Gene as a Risk Factor.

PubMed

Bime, Christian; Pouladi, Nima; Sammani, Saad; Batai, Ken; Casanova, Nancy; Zhou, Tong; Kempf, Carrie L; Sun, Xiaoguang; Camp, Sara M; Wang, Ting; Kittles, Rick A; Lussier, Yves A; Jones, Tiffanie K; Reilly, John P; Meyer, Nuala J; Christie, Jason D; Karnes, Jason H; Gonzalez-Garay, Manuel; Christiani, David C; Yates, Charles R; Wurfel, Mark M; Meduri, Gianfranco U; Garcia, Joe G N

2018-06-01

Genetic factors are involved in acute respiratory distress syndrome (ARDS) susceptibility. Identification of novel candidate genes associated with increased risk and severity will improve our understanding of ARDS pathophysiology and enhance efforts to develop novel preventive and therapeutic approaches. To identify genetic susceptibility targets for ARDS. A genome-wide association study was performed on 232 African American patients with ARDS and 162 at-risk control subjects. The Identify Candidate Causal SNPs and Pathways platform was used to infer the association of known gene sets with the top prioritized intragenic SNPs. Preclinical validation of SELPLG (selectin P ligand gene) was performed using mouse models of LPS- and ventilator-induced lung injury. Exonic variation within SELPLG distinguishing patients with ARDS from sepsis control subjects was confirmed in an independent cohort. Pathway prioritization analysis identified a nonsynonymous coding SNP (rs2228315) within SELPLG, encoding P-selectin glycoprotein ligand 1, to be associated with increased susceptibility. In an independent cohort, two exonic SELPLG SNPs were significantly associated with ARDS susceptibility. Additional support for SELPLG as an ARDS candidate gene was derived from preclinical ARDS models where SELPLG gene expression in lung tissues was significantly increased in both ventilator-induced (twofold increase) and LPS-induced (5.7-fold increase) murine lung injury models compared with controls. Furthermore, Selplg -/- mice exhibited significantly reduced LPS-induced inflammatory lung injury compared with wild-type C57/B6 mice. Finally, an antibody that neutralizes P-selectin glycoprotein ligand 1 significantly attenuated LPS-induced lung inflammation. These findings identify SELPLG as a novel ARDS susceptibility gene among individuals of European and African descent.

O-glycans direct selectin ligands to lipid rafts on leukocytes

PubMed Central

Shao, Bojing; Yago, Tadayuki; Setiadi, Hendra; Wang, Ying; Mehta-D’souza, Padmaja; Fu, Jianxin; Crocker, Paul R.; Rodgers, William; Xia, Lijun; McEver, Rodger P.

2015-01-01

Palmitoylated cysteines typically target transmembrane proteins to domains enriched in cholesterol and sphingolipids (lipid rafts). P-selectin glycoprotein ligand-1 (PSGL-1), CD43, and CD44 are O-glycosylated proteins on leukocytes that associate with lipid rafts. During inflammation, they transduce signals by engaging selectins as leukocytes roll in venules, and they move to the raft-enriched uropods of polarized cells upon chemokine stimulation. It is not known how these glycoproteins associate with lipid rafts or whether this association is required for signaling or for translocation to uropods. Here, we found that loss of core 1-derived O-glycans in murine C1galt1−/− neutrophils blocked raft targeting of PSGL-1, CD43, and CD44, but not of other glycosylated proteins, as measured by resistance to solubilization in nonionic detergent and by copatching with a raft-resident sphingolipid on intact cells. Neuraminidase removal of sialic acids from wild-type neutrophils also blocked raft targeting. C1galt1−/− neutrophils or neuraminidase-treated neutrophils failed to activate tyrosine kinases when plated on immobilized anti–PSGL-1 or anti-CD44 F(ab′)2. Furthermore, C1galt1−/− neutrophils incubated with anti–PSGL-1 F(ab′)2 did not generate microparticles. In marked contrast, PSGL-1, CD43, and CD44 moved normally to the uropods of chemokine-stimulated C1galt1−/− neutrophils. These data define a role for core 1-derived O-glycans and terminal sialic acids in targeting glycoprotein ligands for selectins to lipid rafts of leukocytes. Preassociation of these glycoproteins with rafts is required for signaling but not for movement to uropods. PMID:26124096

Regulation of Catch Bonds by Rate of Force Application*

PubMed Central

Sarangapani, Krishna K.; Qian, Jin; Chen, Wei; Zarnitsyna, Veronika I.; Mehta, Padmaja; Yago, Tadayuki; McEver, Rodger P.; Zhu, Cheng

2011-01-01

The current paradigm for receptor-ligand dissociation kinetics assumes off-rates as functions of instantaneous force without impact from its prior history. This a priori assumption is the foundation for predicting dissociation from a given initial state using kinetic equations. Here we have invalidated this assumption by demonstrating the impact of force history with single-bond kinetic experiments involving selectins and their ligands that mediate leukocyte tethering and rolling on vascular surfaces during inflammation. Dissociation of bonds between L-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) loaded at a constant ramp rate to a constant hold force behaved as catch-slip bonds at low ramp rates that transformed to slip-only bonds at high ramp rates. Strikingly, bonds between L-selectin and 6-sulfo-sialyl Lewis X were impervious to ramp rate changes. This ligand-specific force history effect resembled the effect of a point mutation at the L-selectin surface (L-selectinA108H) predicted to contact the former but not the latter ligand, suggesting that the high ramp rate induced similar structural changes as the mutation. Although the A108H substitution in L-selectin eliminated the ramp rate responsiveness of its dissociation from PSGL-1, the inverse mutation H108A in P-selectin acquired the ramp rate responsiveness. Our data are well explained by the sliding-rebinding model for catch-slip bonds extended to incorporate the additional force history dependence, with Ala-108 playing a pivotal role in this structural mechanism. These results call for a paradigm shift in modeling the mechanical regulation of receptor-ligand bond dissociation, which includes conformational coupling between binding pocket and remote regions of the interacting molecules. PMID:21775439

Affinity of low molecular weight fucoidan for P-selectin triggers its binding to activated human platelets.

PubMed

Bachelet, Laure; Bertholon, Isabelle; Lavigne, Damien; Vassy, Roger; Jandrot-Perrus, Martine; Chaubet, Frédéric; Letourneur, Didier

2009-02-01

P-selectin is an adhesion receptor expressed on activated platelets and endothelial cells. Its natural ligand, P-selectin glycoprotein ligand-1, is expressed on leucocytes and the P-selectin/PSGL-1 interaction is involved in leukocyte rolling. We have compared the interaction of P-selectin with several low molecular weight polysaccharides: fucoidan, heparin and dextran sulfate. Binding assays were obtained from the interaction of the polysaccharides with Sialyl Lewis X and PSGL-1 based constructs onto microtiter plates coated with P-selectin. SELDI TOF mass spectrometry was performed with anionic chips arrays coated with P-selectin in the absence or in the presence of polysaccharides. Kd were obtained from surface plasmon resonance experiments with immobilized P-selectin constructs, polysaccharides being injected in the mobile phase. Human whole blood flow cytometry experiments were performed with fluorescein isothiocyanate labelled polysaccharides with or without platelets activators. The fucoidan prevented P-selectin binding to Sialyl Lewis X with an IC(50) of 20 nM as compared to 400 nM for heparin and <25000 nM for dextran sulfate. It exhibited the highest affinity for immobilized P-selectin with a KD of 1.2 nM, two orders of magnitude greater than the K(D) of the other polysaccharides. Mass spectrometry evidenced the formation of a complex between P-selectin and fucoidan. The intensity of the fucoidan binding to platelets was dependent on the level of platelet activation. Competition between fucoidan and an anti P-selectin antibody demonstrated the specificity of the interaction. Low molecular weight fucoidan is a promising therapeutic agent of natural origin for biomedical applications.

P-selectin deficiency attenuates tumor growth and metastasis

PubMed Central

Kim, Young J.; Borsig, Lubor; Varki, Nissi M.; Varki, Ajit

1998-01-01

Selectins are adhesion receptors that normally recognize certain vascular mucin-type glycoproteins bearing the carbohydrate structure sialyl-Lewisx. The clinical prognosis and metastatic progression of many epithelial carcinomas has been correlated independently with production of tumor mucins and with enhanced expression of sialyl-Lewisx. Metastasis is thought to involve the formation of tumor-platelet-leukocyte emboli and their interactions with the endothelium of distant organs. We provide a link between these observations by showing that P-selectin, which normally binds leukocyte ligands, can promote tumor growth and facilitate the metastatic seeding of a mucin-producing carcinoma. P-selectin-deficient mice showed significantly slower growth of subcutaneously implanted human colon carcinoma cells and generated fewer lung metastases from intravenously injected cells. Three potential pathophysiological mechanisms are demonstrated: first, intravenously injected tumor cells home to the lungs of P-selectin deficient mice at a lower rate; second, P-selectin-deficient mouse platelets fail to adhere to tumor cell-surface mucins; and third, tumor cells lodged in lung vasculature after intravenous injection often are decorated with platelet clumps, and these are markedly diminished in P-selectin-deficient animals. PMID:9689079

Cancer cell–derived microparticles bearing P-selectin glycoprotein ligand 1 accelerate thrombus formation in vivo

PubMed Central

Thomas, Grace M.; Panicot-Dubois, Laurence; Lacroix, Romaric; Dignat-George, Françoise; Lombardo, Dominique

2009-01-01

Recent publications have demonstrated the presence of tissue factor (TF)–bearing microparticles (MPs) in the blood of patients suffering from cancer. However, whether these MPs are involved in thrombosis remains unknown. We show that pancreatic and lung cancer cells produce MPs that express active TF and P-selectin glycoprotein ligand 1 (PSGL-1). Cancer cell–derived MPs aggregate platelets via a TF-dependent pathway. In vivo, cancer cell–derived MPs, but not their parent cells, infused into a living mouse accumulate at the site of injury and reduce tail bleeding time and the time to occlusion of venules and arterioles. This thrombotic state is also observed in mice developing tumors. In such mice, the amount of circulating platelet-, endothelial cell–, and cancer cell–derived MPs is increased. Endogenous cancer cell–derived MPs shed from the growing tumor are able to accumulate at the site of injury. Infusion of a blocking P-selectin antibody abolishes the thrombotic state observed after injection of MPs or in mice developing a tumor. Collectively, our results indicate that cancer cell–derived MPs bearing PSGL-1 and TF play a key role in thrombus formation in vivo. Targeting these MPs could be of clinical interest in the prevention of thrombosis and to limit formation of metastasis in cancer patients. PMID:19667060

P-selectin mediates Ca(2+)-dependent adhesion of activated platelets to many different types of leukocytes: detection by flow cytometry.

PubMed

de Bruijne-Admiraal, L G; Modderman, P W; Von dem Borne, A E; Sonnenberg, A

1992-07-01

Previous studies have shown that thrombin-activated platelets interact through the P-selectin with neutrophils and monocytes. To identify other types of leukocytes capable of such an interaction, eosinophils, basophils, and lymphocytes were isolated from whole blood. Binding of these cells to activated platelets was examined in a double immunofluorescence assay and the results show that activated platelets not only bind to neutrophils and monocytes, but also to eosinophils, basophils, and subpopulations of T lymphocytes. Using monoclonal antibodies (MoAbs) specific for subsets of T cells, we could further demonstrate that the T cells which bind activated platelets are natural killer (NK) cells and an undefined subpopulation of CD4+ and CD8+ cells. All these interactions were dependent on divalent cations and were completely inhibited by an MoAb against P-selectin. Thus, P-selectin mediates the binding of activated platelets to many different types of leukocytes. Studies with leukocytes treated with proteases or neuraminidase have shown that the structures recognized by P-selectin are glycoproteins carrying sialic acid residues. Because the loss of binding of activated platelets to neuraminidase-treated neutrophils was almost complete, but only partial to treated eosinophils, basophils, and monocytes, the latter cell types may have different P-selectin ligands in addition to those present on neutrophils. We found that two previously identified ligands for P-selectin, the oligosaccharides Le(x) and sialyl-Le(x), had little or no inhibitory effect on adhesion of activated platelets to leukocytes and that binding was not inhibited by MoAbs against these oligosaccharides. In addition, there was no correlation between the expression of Le(x) on several cell types and their capacity to bind activated platelets. In contrast, the expression of sialyl-Le(x) on cells was almost perfectly correlated with their ability to bind activated platelets. Thus, while Le(x) cannot be a major ligand for P-selectin, a possible role for sialyl-Le(x) in P-selectin-mediated adhesion processes cannot be dismissed. Finally, activated platelets were found to bind normally to monocytes and neutrophils of patients with paroxysmal nocturnal hemoglobulinuria (PNH) and to neutrophils from which phosphatidyl inositol (PI)-linked proteins had been removed by glycosylphosphatidyl inositol-specific phospholipase C (GPI-PLC) digestion. This suggests that at least part of the P-selectin ligands on these cells are not GPI-anchored.

Catch bonds govern adhesion through L-selectin at threshold shear.

PubMed

Yago, Tadayuki; Wu, Jianhua; Wey, C Diana; Klopocki, Arkadiusz G; Zhu, Cheng; McEver, Rodger P

2004-09-13

Flow-enhanced cell adhesion is an unexplained phenomenon that might result from a transport-dependent increase in on-rates or a force-dependent decrease in off-rates of adhesive bonds. L-selectin requires a threshold shear to support leukocyte rolling on P-selectin glycoprotein ligand-1 (PSGL-1) and other vascular ligands. Low forces decrease L-selectin-PSGL-1 off-rates (catch bonds), whereas higher forces increase off-rates (slip bonds). We determined that a force-dependent decrease in off-rates dictated flow-enhanced rolling of L-selectin-bearing microspheres or neutrophils on PSGL-1. Catch bonds enabled increasing force to convert short-lived tethers into longer-lived tethers, which decreased rolling velocities and increased the regularity of rolling steps as shear rose from the threshold to an optimal value. As shear increased above the optimum, transitions to slip bonds shortened tether lifetimes, which increased rolling velocities and decreased rolling regularity. Thus, force-dependent alterations of bond lifetimes govern L-selectin-dependent cell adhesion below and above the shear optimum. These findings establish the first biological function for catch bonds as a mechanism for flow-enhanced cell adhesion.

Two E-selectin ligands, BST-2 and LGALS3BP, predict metastasis and poor survival of ER-negative breast cancer.

PubMed

Woodman, Natalie; Pinder, Sarah E; Tajadura, Virginia; Le Bourhis, Xuefen; Gillett, Cheryl; Delannoy, Philippe; Burchell, Joy M; Julien, Sylvain

2016-07-01

Distant metastases account for the majority of cancer-related deaths in breast cancer. The rate and site of metastasis differ between estrogen receptor (ER)-negative and ER-positive tumours, and metastatic fate can be very diverse even within the ER-negative group. Characterisation of new pro-metastatic markers may help to identify patients with higher risk and improve their care accordingly. Selectin ligands aberrantly expressed by cancer cells promote metastasis by enabling interaction between circulating tumour cells and endothelial cells in distant organs. These ligands consist in carbohydrate molecules, such as sialyl-Lewis x antigen (sLex), borne by glycoproteins or glycolipids on the cancer cell surface. We have previously demonstrated that the molecular scaffold presenting sLex to selectins (e.g. glycolipid vs. glycoproteins) was crucial for these interactions to occur. Moreover, we reported that detection of sLex alone in breast carcinomas was only of limited prognostic value. However, since sLex was found to be carried by several glycoproteins in cancer cells, we hypothesized that the combination of the carbohydrate with its carriers could be more relevant than each marker independently. In this study, we addressed this question by analysing sLex expression together with two glycoproteins (BST-2 and LGALS3BP), shown to interact with E-selectin in a carbohydrate-dependent manner, in a cohort of 249 invasive breast cancers. We found both glycoproteins to be associated with distant metastasis risk and poorer survival. Importantly, concomitant high expression of BST-2 with sLex defined a sub-group of patients with ER-negative tumours displaying higher risks of liver and brain metastasis and a 3-fold decreased survival rate.

Biomechanics of leukocyte rolling

PubMed Central

Sundd, Prithu; Pospieszalska, Maria K.; Cheung, Luthur Siu-Lun; Konstantopoulos, Konstantinos; Ley, Klaus

2011-01-01

Leukocyte rolling on endothelial cells and other P-selectin substrates is mediated by P-selectin binding to P-selectin glycoprotein ligand-1 expressed on the tips of leukocyte microvilli. Leukocyte rolling is a result of rapid, yet balanced formation and dissociation of selectin-ligand bonds in the presence of hydrodynamic shear forces. The hydrodynamic forces acting on the bonds may either increase (catch bonds) or decrease (slip-bonds) their lifetimes. The force-dependent ‘catch-slip’ bond kinetics are explained using the ‘two pathway model’ for bond dissociation. Both the ‘sliding-rebinding’ and the ‘allosteric’ mechanisms attribute ‘catch-slip’ bond behavior to the force-induced conformational changes in the lectin-EGF domain hinge of selectins. Below a threshold shear stress, selectins cannot mediate rolling. This ‘shear-threshold’ phenomenon is a consequence of shear-enhanced tethering and catch-bond enhanced rolling. Quantitative dynamic footprinting microscopy has revealed that leukocytes rolling at venular shear stresses (> 0.6 Pa) undergo cellular deformation (large footprint) and form long tethers. The hydrodynamic shear force and torque acting on the rolling cell are thought to be synergistically balanced by the forces acting on tethers and stressed microvilli, however, their relative contribution remains to be determined. Thus, improvement beyond the current understanding requires in silico models that can predict both cellular and microvillus deformation and experiments that allow measurement of forces acting on individual microvilli and tethers. PMID:21515934

Expression of E-selectin ligand-1 (CFR/ESL-1) on hepatic stellate cells: implications for leukocyte extravasation and liver metastasis.

PubMed

Antoine, Marianne; Tag, Carmen G; Gressner, Axel M; Hellerbrand, Claus; Kiefer, Paul

2009-02-01

Leukocytes and tumor cells use E-selectin binding ligands to attach to activated endothelial cells expressing E-selectin during inflammation or metastasis. The cysteine-rich fibroblast growth factor receptor (CFR) represents the main E-selectin ligand (ESL-1) on granulocytes and its expression is exclusively modified by alpha(1,3)-fucosyltransferases IV or VII (FucT4 and FucT7). Hepatic stellate cells (HSC) are pericytes of liver sinusoidal endothelial cells. The activation of HSC and transdifferentiation into a myofibroblastic phenotype is involved in the repair of liver tissue injury, liver regeneration and angiogenesis of liver metastases. In the present study, we demonstrated that HSC expressed CFR together with FucT7 and exhibited a functional E-selectin binding activity on their cell surface. Since HSC appear to be oxygen-sensing cells, the expression of E-selectin binding activity was analyzed in HSC under a hypoxic atmosphere. While the expression of the glycoprotein CFR was unaffected by hypoxia, the cell-associated E-selectin binding activity decreased. However, under the same conditions, mRNA expression of the modifying enzyme FucT7 increased. The loss of E-selectin binding activity, therefore, appears to be neither the result of a reduced expression of the modifying transferase nor the expression of the backbone glycoprotein. After the transient transfection of HSC with CFR cDNA, the E-selectin binding activity (ESL-1) was efficiently released into the supernatant. Therefore, we hypothesize that under hypoxia, ESL-1 is shed from activated HSC. Our findings provide a novel perspective on the function of HSC in liver metastasis and inflammatory liver diseases.

Targeting Selectins and Their Ligands in Cancer.

PubMed

Natoni, Alessandro; Macauley, Matthew S; O'Dwyer, Michael E

2016-01-01

Aberrant glycosylation is a hallmark of cancer cells with increased evidence pointing to a role in tumor progression. In particular, aberrant sialylation of glycoproteins and glycolipids has been linked to increased immune cell evasion, drug evasion, drug resistance, tumor invasiveness, and vascular dissemination, leading to metastases. Hypersialylation of cancer cells is largely the result of overexpression of sialyltransferases (STs). Differentially, humans express twenty different STs in a tissue-specific manner, each of which catalyzes the attachment of sialic acids via different glycosidic linkages (α2-3, α2-6, or α2-8) to the underlying glycan chain. One important mechanism whereby overexpression of STs contributes to an enhanced metastatic phenotype is via the generation of selectin ligands. Selectin ligand function requires the expression of sialyl-Lewis X and its structural isomer sialyl-Lewis A, which are synthesized by the combined action of alpha α1-3-fucosyltransferases, α2-3-sialyltransferases, β1-4-galactosyltranferases, and N-acetyl-β-glucosaminyltransferases. The α2-3-sialyltransferases ST3Gal4 and ST3Gal6 are critical to the generation of functional E- and P-selectin ligands and overexpression of these STs have been linked to increased risk of metastatic disease in solid tumors and poor outcome in multiple myeloma. Thus, targeting selectins and their ligands as well as the enzymes involved in their generation, in particular STs, could be beneficial to many cancer patients. Potential strategies include ST inhibition and the use of selectin antagonists, such as glycomimetic drugs and antibodies. Here, we review ongoing efforts to optimize the potency and selectivity of ST inhibitors, including the potential for targeted delivery approaches, as well as evaluate the potential utility of selectin inhibitors, which are now in early clinical development.

Targeting Selectins and Their Ligands in Cancer

PubMed Central

Natoni, Alessandro; Macauley, Matthew S.; O’Dwyer, Michael E.

2016-01-01

Aberrant glycosylation is a hallmark of cancer cells with increased evidence pointing to a role in tumor progression. In particular, aberrant sialylation of glycoproteins and glycolipids has been linked to increased immune cell evasion, drug evasion, drug resistance, tumor invasiveness, and vascular dissemination, leading to metastases. Hypersialylation of cancer cells is largely the result of overexpression of sialyltransferases (STs). Differentially, humans express twenty different STs in a tissue-specific manner, each of which catalyzes the attachment of sialic acids via different glycosidic linkages (α2-3, α2-6, or α2-8) to the underlying glycan chain. One important mechanism whereby overexpression of STs contributes to an enhanced metastatic phenotype is via the generation of selectin ligands. Selectin ligand function requires the expression of sialyl-Lewis X and its structural isomer sialyl-Lewis A, which are synthesized by the combined action of alpha α1-3-fucosyltransferases, α2-3-sialyltransferases, β1-4-galactosyltranferases, and N-acetyl-β-glucosaminyltransferases. The α2-3-sialyltransferases ST3Gal4 and ST3Gal6 are critical to the generation of functional E- and P-selectin ligands and overexpression of these STs have been linked to increased risk of metastatic disease in solid tumors and poor outcome in multiple myeloma. Thus, targeting selectins and their ligands as well as the enzymes involved in their generation, in particular STs, could be beneficial to many cancer patients. Potential strategies include ST inhibition and the use of selectin antagonists, such as glycomimetic drugs and antibodies. Here, we review ongoing efforts to optimize the potency and selectivity of ST inhibitors, including the potential for targeted delivery approaches, as well as evaluate the potential utility of selectin inhibitors, which are now in early clinical development. PMID:27148485

Cell–specific Variation in E-selectin Ligand Expression Among Human Peripheral Blood Mononuclear Cells: Implications for Immunosurveillance and Pathobiology

PubMed Central

Silva, Mariana; Fung, Ronald Kam Fai; Donnelly, Conor Brian; Videira, Paula Alexandra; Sackstein, Robert

2017-01-01

Both host defense and immunopathology are shaped by the ordered recruitment of circulating leukocytes to affected sites, a process initiated by binding of blood-borne cells to E-selectin displayed at target endothelial beds. Accordingly, knowledge of the expression and function of leukocyte E-selectin ligands is key to understanding the tempo and specificity of immunoreactivity. Here, we performed E-selectin adherence assays under hemodynamic flow conditions coupled with flow cytometry and western blot analysis to elucidate the function and structural biology of glycoprotein E-selectin ligands expressed on human peripheral blood mononuclear cells (PBMCs). Circulating monocytes uniformly express high levels of the canonical E-selectin binding determinant sLeX and display markedly greater adhesive interactions with E-selectin than do circulating lymphocytes, which exhibit variable E-selectin binding among CD4+ and CD8+ T-cells but no binding by B-cells. Monocytes prominently present sLeX decorations on an array of protein scaffolds including PSGL-1, CD43, and CD44 (rendering the E-selectin ligands CLA, CD43E, and HCELL, respectively), and B-cells altogether lack E-selectin ligands. Quantitative PCR gene expression studies of glycosyltransferases that regulate display of sLeX reveal high transcript levels among circulating monocytes and low levels among circulating B-cells, and, commensurately, cell surface α(1,3)-fucosylation reveals that acceptor sialyllactosaminyl glycans convertible into sLeX are abundantly expressed on human monocytes yet are relatively deficient on B-cells. Collectively, these findings unveil distinct cell-specific patterns of E-selectin ligand expression among human PBMCs, indicating that circulating monocytes are specialized to engage E-selectin and providing key insights into the molecular effectors mediating recruitment of these cells at inflammatory sites. PMID:28330896

Theoretical modeling of the catch-slip bond transition in biological adhesion

NASA Astrophysics Data System (ADS)

Gunnerson, Kim; Pereverzev, Yuriy; Prezhdo, Oleg

2006-05-01

The mechanism by which leukocytes leave the blood stream and enter inflamed tissue is called extravasation. This process is facilitated by the ability of selectin proteins, produced by the endothelial cells of blood vessels, to form transient bonds with the leukocytes. In the case of P-selectin, the protein bonds with P-selectin glycoprotein ligands (PSGL-1) produced by the leukocyte. Recent atomic force microscopy and flow chamber analyses of the binding of P-selectin to PSGL-1 provide evidence for an unusual biphasic catch-bond/slip-bond behavior in response to the strength of exerted force. This biphasic process is not well-understood. There are several theoretical models for describing this phenomenon. These models use different profiles for potential energy landscapes and how they change under forces. We are exploring these changes using molecular dynamics. We will present a simple theoretical model as well as share some of our early MD results for describing this phenomenon.

L-selectin mechanochemistry restricts neutrophil priming in vivo.

PubMed

Liu, Zhenghui; Yago, Tadayuki; Zhang, Nan; Panicker, Sumith R; Wang, Ying; Yao, Longbiao; Mehta-D'souza, Padmaja; Xia, Lijun; Zhu, Cheng; McEver, Rodger P

2017-05-12

Circulating neutrophils must avoid premature activation to prevent tissue injury. The leukocyte adhesion receptor L-selectin forms bonds with P-selectin glycoprotein ligand-1 (PSGL-1) on other leukocytes and with peripheral node addressin (PNAd) on high endothelial venules. Mechanical forces can strengthen (catch) or weaken (slip) bonds between biological molecules. How these mechanochemical processes influence function in vivo is unexplored. Here we show that mice expressing an L-selectin mutant (N138G) have altered catch bonds and prolonged bond lifetimes at low forces. Basal lymphocyte homing and neutrophil recruitment to inflamed sites are normal. However, circulating neutrophils form unstable aggregates and are unexpectedly primed to respond robustly to inflammatory mediators. Priming requires signals transduced through L-selectin N138G after it engages PSGL-1 or PNAd. Priming enhances bacterial clearance but increases inflammatory injury and enlarges venous thrombi. Thus, L-selectin mechanochemistry limits premature activation of neutrophils. Our results highlight the importance of probing how mechanochemistry functions in vivo.

L-selectin mechanochemistry restricts neutrophil priming in vivo

PubMed Central

Liu, Zhenghui; Yago, Tadayuki; Zhang, Nan; Panicker, Sumith R.; Wang, Ying; Yao, Longbiao; Mehta-D'souza, Padmaja; Xia, Lijun; Zhu, Cheng; McEver, Rodger P.

2017-01-01

Circulating neutrophils must avoid premature activation to prevent tissue injury. The leukocyte adhesion receptor L-selectin forms bonds with P-selectin glycoprotein ligand-1 (PSGL-1) on other leukocytes and with peripheral node addressin (PNAd) on high endothelial venules. Mechanical forces can strengthen (catch) or weaken (slip) bonds between biological molecules. How these mechanochemical processes influence function in vivo is unexplored. Here we show that mice expressing an L-selectin mutant (N138G) have altered catch bonds and prolonged bond lifetimes at low forces. Basal lymphocyte homing and neutrophil recruitment to inflamed sites are normal. However, circulating neutrophils form unstable aggregates and are unexpectedly primed to respond robustly to inflammatory mediators. Priming requires signals transduced through L-selectin N138G after it engages PSGL-1 or PNAd. Priming enhances bacterial clearance but increases inflammatory injury and enlarges venous thrombi. Thus, L-selectin mechanochemistry limits premature activation of neutrophils. Our results highlight the importance of probing how mechanochemistry functions in vivo. PMID:28497779

Separable requirements for cytoplasmic domain of PSGL-1 in leukocyte rolling and signaling under flow

PubMed Central

Miner, Jonathan J.; Xia, Lijun; Yago, Tadayuki; Kappelmayer, János; Liu, Zhenghui; Klopocki, Arkadiusz G.; Shao, Bojing; McDaniel, J. Michael; Setiadi, Hendra; Schmidtke, David W.

2008-01-01

In inflamed venules, leukocytes use P-selectin glycoprotein ligand-1 (PSGL-1) to roll on P-selectin and E-selectin and to activate integrin αLβ2 (lymphocyte function-associated antigen-1, LFA-1) to slow rolling on intercellular adhesion molecule-1 (ICAM-1). Studies in cell lines have suggested that PSGL-1 requires its cytoplasmic domain to localize in membrane domains, to support rolling on P-selectin, and to signal through spleen tyrosine kinase (Syk). We generated “ΔCD” mice that express PSGL-1 without the cytoplasmic domain. Unexpectedly, neutrophils from these mice localized PSGL-1 normally in microvilli, uropods, and lipid rafts. ΔCD neutrophils expressed less PSGL-1 on their surfaces because of inefficient export from the endoplasmic reticulum. Limited digestion of wild-type neutrophils with O-sialoglycoprotein endopeptidase was used to reduce the PSGL-1 density to that on ΔCD neutrophils. At matched PSGL-1 densities, both ΔCD and wild-type neutrophils rolled similarly on P-selectin. However, ΔCD neutrophils rolling on P-selectin did not trigger Syk-dependent activation of LFA-1 to slow rolling on ICAM-1. These data demonstrate that the PSGL-1 cytoplasmic domain is dispensable for leukocyte rolling on P-selectin but is essential to activate β2 integrins to slow rolling on ICAM-1. PMID:18550846

PSGL-1–mediated activation of EphB4 increases the proangiogenic potential of endothelial progenitor cells

PubMed Central

Foubert, Philippe; Silvestre, Jean-Sébastien; Souttou, Boussad; Barateau, Véronique; Martin, Coralie; Ebrahimian, Téni G.; Leré-Déan, Carole; Contreres, Jean Olivier; Sulpice, Eric; Levy, Bernard I.; Plouët, Jean; Tobelem, Gérard; Le Ricousse-Roussanne, Sophie

2007-01-01

Endothelial progenitor cell (EPC) transplantation has beneficial effects for therapeutic neovascularization; however, only a small proportion of injected cells home to the lesion and incorporate into the neocapillaries. Consequently, this type of cell therapy requires substantial improvement to be of clinical value. Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors and their ephrin ligands are key regulators of vascular development. We postulated that activation of the EphB4/ephrin-B2 system may enhance EPC proangiogenic potential. In this report, we demonstrate in a nude mouse model of hind limb ischemia that EphB4 activation with an ephrin-B2–Fc chimeric protein increases the angiogenic potential of human EPCs. This effect was abolished by EphB4 siRNA, confirming that it is mediated by EphB4. EphB4 activation enhanced P selectin glycoprotein ligand-1 (PSGL-1) expression and EPC adhesion. Inhibition of PSGL-1 by siRNA reversed the proangiogenic and adhesive effects of EphB4 activation. Moreover, neutralizing antibodies to E selectin and P selectin blocked ephrin-B2–Fc–stimulated EPC adhesion properties. Thus, activation of EphB4 enhances EPC proangiogenic capacity through induction of PSGL-1 expression and adhesion to E selectin and P selectin. Therefore, activation of EphB4 is an innovative and potentially valuable therapeutic strategy for improving the recruitment of EPCs to sites of neovascularization and thereby the efficiency of cell-based proangiogenic therapy. PMID:17510705

Biomechanics of P-selectin PSGL-1 bonds: Shear threshold and integrin-independent cell adhesion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiao, Zhihua; Goldsmith, Harry L.; MacIntosh, Fiona A.

2006-03-01

Platelet-leukocyte adhesion may contribute to thrombosis and inflammation. We examined the heterotypic interaction between unactivated neutrophils and either thrombin receptor activating peptide (TRAP) stimulated platelets or P-selectin bearing beads (Ps-beads) in suspension. Cone-plate viscometers were used to apply controlled shear rates from 14-3000/s. Platelet-neutrophil and bead-neutrophil adhesion analysis was performed using both flow cytometry and high-speed videomicroscopy. We observed that while blocking antibodies against either P-selectin or P-selectin glycoprotein ligand-1 (PSGL-1) alone inhibited platelet-neutrophil adhesion by ~60% at 140/s, these reagents completely blocked adhesion at 3000/s. Anti-Mac-1 alone did not alter platelet-neutrophil adhesion rates at any shear rate, though inmore » synergy with selectin antagonists it abrogated cell binding. Unstimulated neutrophils avidly bound Ps-beads and activated platelets in an integrin-independent manner, suggesting that purely selectin-dependent cell adhesion is possible. In support of this, antagonists against P-selectin or PSGL-1 dissociated previously formed platelet-neutrophil and Ps-bead neutrophil aggregates under shear in a variety of experimental systems, including in assays performed with whole blood. In studies where medium viscosity and shear rate were varied, a subtle shear threshold for P-selectin PSGL-1 binding was also noted at shear rates<100/s and at force loading rates of ~300pN/sec. Results are discussed in light of biophysical computations that characterize the collision between unequal size particles in linear shear flow. Overall, our studies reveal an integrin-independent regime for cell adhesion that may be physiologically relevant.« less

Sulfation-dependent recognition of high endothelial venules (HEV)- ligands by L-selectin and MECA 79, and adhesion-blocking monoclonal antibody

PubMed Central

1994-01-01

L-selectin is a lectin-like receptor that mediates the attachment of lymphocytes to high endothelial venules (HEV) of lymph nodes during the process of lymphocyte recirculation. Two sulfated, mucin-like glycoproteins known as Sgp50/GlyCAM-1 and Sgp90/CD34 have previously been identified as HEV-associated ligands for L-selectin. These proteins were originally detected with an L-selectin/Ig chimera called LEC-IgG. GlyCAM-1 and CD34 are also recognized by an antiperipheral node addressin (PNAd) mAb called MECA 79, which blocks L-selectin- dependent adhesion and selectively stains lymph node HEV. The present study compares the requirements for the binding of MECA 79 and LEC-IgG to HEV-ligands. Whereas desialylation of GlyCAM-1 and CD34 drastically reduced binding to LEC-IgG, this treatment enhanced the binding of GlyCAM-1 to MECA 79. In contrast, the binding of both MECA 79 and LEC- IgG to GlyCAM-1 and CD34 was greatly decreased when the sulfation of these ligands was reduced with chlorate, a metabolic inhibitor of sulfation. Because MECA 79 stains HEV-like vessels at various sites of inflammation, recognition by L-selectin of ligands outside of secondary lymphoid organs may depend on sulfation. In addition to their reactivity with GlyCAM-1 and CD34, both MECA 79 and LEC-IgG recognize an independent molecule of approximately 200 kD in a sulfate-dependent manner. Thus, this molecule, which we designate Sgp200, is an additional ligand for L-selectin. PMID:7525849

Novel Molecular Imaging Approach for Subclinical Detection of Iritis and Evaluation of Therapeutic Success

PubMed Central

Xie, Fang; Sun, Dawei; Schering, Alexander; Nakao, Shintaro; Zandi, Souska; Liu, Ping; Hafezi-Moghadam, Ali

2010-01-01

There is an urgent need for early diagnosis in medicine, whereupon effective treatments could prevent irreversible tissue damage. Acute anterior chamber inflammation is the most common form of uveitis and a major cause of vision loss. The proximity of the iris vasculature to the light-permeable cornea and its involvement in ocular inflammation make it an ideal target for noninvasive molecular imaging. To accomplish this, carboxylated fluorescent microspheres (MSs) were conjugated with recombinant P-selectin glycoprotein ligand-1 and systemically injected in endotoxin-induced uveitic animals. MS adhesion in the microcirculation of the anterior and posterior chamber was visualized by intravital microscopy and scanning laser ophthalmoscopy. In iritic animals, significantly higher numbers of recombinant P-selectin glycoprotein ligand-1-conjugated MSs adhered to the endothelium (P = 0.03) matching the increase in leukocyte adhesion. Conjugated MSs specifically interacted with firmly adhering leukocytes, allowing quantification of the endogenous immune response. Topical eye drop treatment with dexamethasone (P < 0.01) or cyclosporine A (P < 0.01) significantly lowered MS adhesion in iris vessels. Surprisingly, topical dexamethasone significantly reduced MS interaction in the fundus vessels (P < 0.01), while cyclosporine A did not. In vivo MS accumulation preceded clinical signs of anterior uveitis and leukocyte adhesion in iris vasculature. This work introduces noninvasive subclinical detection of endothelial injury in the iris vasculature, providing a unique opportunity for quantifying vascular injury and immune response in vivo. PMID:20581051

T Cells Prevent Hemorrhagic Transformation in Ischemic Stroke by P-Selectin Binding.

PubMed

Salas-Perdomo, Angélica; Miró-Mur, Francesc; Urra, Xabier; Justicia, Carles; Gallizioli, Mattia; Zhao, Yashu; Brait, Vanessa H; Laredo, Carlos; Tudela, Raúl; Hidalgo, Andrés; Chamorro, Ángel; Planas, Anna M

2018-06-14

Hemorrhagic transformation is a serious complication of ischemic stroke after recanalization therapies. This study aims to identify mechanisms underlying hemorrhagic transformation after cerebral ischemia/reperfusion. We used wild-type mice and Selplg -/- and Fut7 -/- mice defective in P-selectin binding and lymphopenic Rag2 -/- mice. We induced 30-minute or 45-minute ischemia by intraluminal occlusion of the middle cerebral artery and assessed hemorrhagic transformation at 48 hours with a hemorrhage grading score, histological means, brain hemoglobin content, or magnetic resonance imaging. We depleted platelets and adoptively transferred T cells of the different genotypes to lymphopenic mice. Interactions of T cells with platelets in blood were studied by flow cytometry and image stream technology. We show that platelet depletion increased the bleeding risk only after large infarcts. Lymphopenia predisposed to hemorrhagic transformation after severe stroke, and adoptive transfer of T cells prevented hemorrhagic transformation in lymphopenic mice. CD4 + memory T cells were the subset of T cells binding P-selectin and platelets through functional P-selectin glycoprotein ligand-1. Mice defective in P-selectin binding had a higher hemorrhagic score than wild-type mice. Adoptive transfer of T cells defective in P-selectin binding into lymphopenic mice did not prevent hemorrhagic transformation. The study identifies lymphopenia as a previously unrecognized risk factor for secondary hemorrhagic transformation in mice after severe ischemic stroke. T cells prevent hemorrhagic transformation by their capacity to bind platelets through P-selectin. The results highlight the role of T cells in bridging immunity and hemostasis in ischemic stroke. © 2018 American Heart Association, Inc.

Probing Dynamic Cell-Substrate Interactions using Photochemically Generated Surface-Immobilized Gradients: Application to Selectin-Mediated Leukocyte Rolling

PubMed Central

Herman, Christine T.; Potts, Gregory K.; Michael, Madeline C.; Tolan, Nicole V.

2014-01-01

Model substrates presenting biochemical cues immobilized in a controlled and well-defined manner are of great interest for their applications in biointerface studies that elucidate the molecular basis of cell receptor-ligand interactions. Herein, we describe a direct, photochemical method to generate one-component surface-immobilized biomolecular gradients that are applied to the study of selectin-mediated leukocyte rolling. The technique employs benzophenone-modified glass substrates, which upon controlled exposure to UV light (350 – 365 nm) in the presence of protein-containing solutions facilitate the generation of covalently immobilized protein gradients. Conditions were optimized to generate gradient substrates presenting P-selectin and PSGL-1 (P-selectin Glycoprotein Ligand-1) immobilized at site densities over a 5- to 10-fold range (from as low as ~200 molecules/μm2 to as high as 6000 molecules/μm2). The resulting substrates were quantitatively characterized via fluorescence analysis and radioimmunoassays before their use in the leukocyte rolling assays. HL-60 promyelocytes and Jurkat T lymphocytes were assessed for their ability to tether to and roll on substrates presenting immobilized P-selectin and PSGL-1 under conditions of physiologically relevant shear stress. The results of these flow assays reveal the combined effect of immobilized protein site density and applied wall shear stress on cell rolling behavior. Two-component substrates presenting P-selectin and ICAM-1 (intercellular adhesion molecule-1) were also generated to assess the interplay between these two proteins and their effect on cell rolling and adhesion. These proof-of-principle studies verify that the described gradient generation approach yields well-defined gradient substrates that present immobilized proteins over a large range of site densities that are applicable for investigation of cell-materials interactions, including multi-parameter leukocyte flow studies. Future applications of this enabling methodology may lead to new insights into the biophysical phenomena and molecular mechanism underlying complex biological processes such as leukocyte recruitment and the inflammatory response. PMID:21614364

Venous levels of shear support neutrophil-platelet adhesion and neutrophil aggregation in blood via P-selectin and beta2-integrin

NASA Technical Reports Server (NTRS)

Konstantopoulos, K.; Neelamegham, S.; Burns, A. R.; Hentzen, E.; Kansas, G. S.; Snapp, K. R.; Berg, E. L.; Hellums, J. D.; Smith, C. W.; McIntire, L. V.;

Venous levels of shear support neutrophil-platelet adhesion and neutrophil aggregation in blood via P-selectin and beta2-integrin.

PubMed

Konstantopoulos, K; Neelamegham, S; Burns, A R; Hentzen, E; Kansas, G S; Snapp, K R; Berg, E L; Hellums, J D; Smith, C W; McIntire, L V; Simon, S I

1998-09-01

After activation, platelets adhere to neutrophils via P-selectin and beta2-integrin. The molecular mechanisms and adhesion events in whole blood exposed to venous levels of hydrodynamic shear in the absence of exogenous activation remain unknown. Whole blood was sheared at approximately 100 s(-1). The kinetics of neutrophil-platelet adhesion and neutrophil aggregation were measured in real time by flow cytometry. P-selectin was upregulated to the platelet surface in response to shear and was the primary factor mediating neutrophil-platelet adhesion. The extent of neutrophil aggregation increased linearly with platelet adhesion to neutrophils. Blocking either P-selectin, its glycoprotein ligand PSGL-1, or both simultaneously by preincubation with a monoclonal antibody resulted in equivalent inhibition of neutrophil-platelet adhesion (approximately 30%) and neutrophil aggregation (approximately 70%). The residual amount of neutrophil adhesion was blocked with anti-CD11b/CD18. Treatment of blood with prostacyclin analogue ZK36374, which raises cAMP levels in platelets, blocked P-selectin upregulation and neutrophil aggregation to baseline. Complete abrogation of platelet-neutrophil adhesion required both ZK36374 and anti-CD18. Electron microscopic observations of fixed blood specimens revealed that platelets augmented neutrophil aggregation both by forming bridges between neutrophils and through contact-mediated activation. The results are consistent with a model in which venous levels of shear support platelet adherence to neutrophils via P-selectin binding PSGL-1. This interaction alone is sufficient to mediate neutrophil aggregation. Abrogation of platelet adhesion and aggregation requires blocking Mac-1 in addition to PSGL-1 or P-selectin. The described mechanisms are likely of key importance in the pathogenesis and progression of thrombotic disorders that are exacerbated by leukocyte-platelet aggregation.

Tyrosine kinase Btk regulates E-selectin-mediated integrin activation and neutrophil recruitment by controlling phospholipase C (PLC) gamma2 and PI3Kgamma pathways.

PubMed

Mueller, Helena; Stadtmann, Anika; Van Aken, Hugo; Hirsch, Emilio; Wang, Demin; Ley, Klaus; Zarbock, Alexander

2010-04-15

Selectins mediate leukocyte rolling, trigger beta(2)-integrin activation, and promote leukocyte recruitment into inflamed tissue. E-selectin binding to P-selectin glycoprotein ligand 1 (PSGL-1) leads to activation of an immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway, which in turn activates the spleen tyrosine kinase (Syk). However, the signaling pathway linking Syk to integrin activation after E-selectin engagement is unknown. To identify the pathway, we used different gene-deficient mice in autoperfused flow chamber, intravital microscopy, peritonitis, and biochemical studies. We report here that the signaling pathway downstream of Syk divides into a phospholipase C (PLC) gamma2- and phosphoinositide 3-kinase (PI3K) gamma-dependent pathway. The Tec family kinase Bruton tyrosine kinase (Btk) is required for activating both pathways, generating inositol-3,4,5-trisphosphate (IP(3)), and inducing E-selectin-mediated slow rolling. Inhibition of this signal-transduction pathway diminished Galpha(i)-independent leukocyte adhesion to and transmigration through endothelial cells in inflamed postcapillary venules of the cremaster. Galpha(i)-independent neutrophil recruitment into the inflamed peritoneal cavity was reduced in Btk(-/-) and Plcg2(-/-) mice. Our data demonstrate the functional importance of this newly identified signaling pathway mediated by E-selectin engagement.

G-CSF Induces Membrane Expression of a Myeloperoxidase Glycovariant that Operates as an E-selectin Ligand on Human Myeloid Cells

PubMed Central

Silvescu, Cristina I.; Sackstein, Robert

2014-01-01

The host defense response critically depends on the production of leukocytes by the marrow and the controlled delivery of these cells to relevant sites of inflammation/infection. The cytokine granulocyte-colony stimulating factor (G-CSF) is commonly used therapeutically to augment neutrophil recovery following chemo/radiation therapy for malignancy, thereby decreasing infection risk. Although best known as a potent inducer of myelopoiesis, we previously reported that G-CSF also promotes the delivery of leukocytes to sites of inflammation by stimulating expression of potent E-selectin ligands, including an uncharacterized ∼65-kDa glycoprotein. To identify this ligand, we performed integrated biochemical analysis and mass spectrometry studies of G-CSF–treated primary human myeloid cells. Our studies show that this novel E-selectin ligand is a glycoform of the heavy chain component of the enzyme myeloperoxidase (MPO), a well-known lysosomal peroxidase. This specialized MPO glycovariant, referred to as “MPO–E-selectin ligand” (MPO–EL), is expressed on circulating G-CSF–mobilized leukocytes and is naturally expressed on blood myeloid cells in patients with febrile leukocytosis. In vitro biochemical studies show that G-CSF programs MPO–EL expression on human blood leukocytes and marrow myeloid cells via induction of N-linked sialofucosylations on MPO, with concomitant cell surface display of the molecule. MPO–EL is catalytically active and mediates angiotoxicity on human endothelial cells that express E-selectin. These findings thus define a G-CSF effect on MPO chemical biology that endows unsuspected functional versatility upon this enzyme, unveiling new perspectives on the biology of G-CSF and MPO, and on the role of E-selectin receptor/ligand interactions in leukocyte migration and vascular pathology. PMID:25002508

PSGL-1 is highly expressed on Ly-6Chi monocytes and a major determinant for Ly-6Chi monocyte recruitment to sites of atherosclerosis in mice

PubMed Central

An, Guangyu; Wang, Huan; Tang, Rong; Yago, Tadayuki; McDaniel, J. Michael; McGee, Samuel; Huo, Yuqing; Xia, Lijun

2008-01-01

Background Ly-6Chi monocytes are key contributors to atherosclerosis in mice. However, how Ly-6Chi monocytes selectively accumulate in atherosclerotic lesions is largely unknown. Monocyte homing to sites of atherosclerosis is primarily initiated by rolling on P- and E-selectin expressed on endothelium. We hypothesize that P-selectin glycoprotein ligand-1 (PSGL-1), the common ligand of P- and E-selectin on leukocytes, contributes to the preferential homing of Ly-6Chi monocytes to atherosclerotic lesions. Methods and Results To test this hypothesis, we examined the expression and function of PSGL-1 on Ly-6Chi and Ly-6Clo monocytes from wild-type mice, ApoE-/- mice, and mice lacking both ApoE and PSGL-1 genes (ApoE-/-/PSGL-1-/-). We found that Ly-6Chi monocytes expressed a higher level of PSGL-1, and had enhanced binding to fluid-phase P- and E-selectin, compared to Ly-6Clo monocytes. Under in vitro flow conditions, more Ly-6Chi monocytes rolled on P-, E-, and L-selectin at slower velocities than Ly-6Clo cells. In an ex vivo perfused carotid artery model, Ly-6Chi monocytes interacted preferentially with atherosclerotic endothelium compared with Ly-6Clo monocytes in a PSGL-1-dependent manner. In vivo, ApoE-/- mice lacking PSGL-1 had impaired Ly-6Chi monocyte recruitment to atherosclerotic lesions. Moreover, ApoE-/-/PSGL-1-/- mice exhibited significantly reduced monocyte infiltration in wire injury-induced neointima and in atherosclerotic lesions. ApoE-/-/PSGL-1-/- mice also developed smaller neointima and atherosclerotic plaques. Conclusions These data indicate that PSGL-1 is a new marker for Ly-6Chi monocytes and a major determinant for Ly-6Chi cell recruitment to sites of atherosclerosis in mice. PMID:18519846

Conditional control of selectin ligand expression and global fucosylation events in mice with a targeted mutation at the FX locus.

PubMed

Smith, Peter L; Myers, Jay T; Rogers, Clare E; Zhou, Lan; Petryniak, Bronia; Becker, Daniel J; Homeister, Jonathon W; Lowe, John B

2002-08-19

Glycoprotein fucosylation enables fringe-dependent modulation of signal transduction by Notch transmembrane receptors, contributes to selectin-dependent leukocyte trafficking, and is faulty in leukocyte adhesion deficiency (LAD) type II, also known as congenital disorder of glycosylation (CDG)-IIc, a rare human disorder characterized by psychomotor defects, developmental abnormalities, and leukocyte adhesion defects. We report here that mice with an induced null mutation in the FX locus, which encodes an enzyme in the de novo pathway for GDP-fucose synthesis, exhibit a virtually complete deficiency of cellular fucosylation, and variable frequency of intrauterine demise determined by parental FX genotype. Live-born FX(-/-) mice exhibit postnatal failure to thrive that is suppressed with a fucose-supplemented diet. FX(-/-) adults suffer from an extreme neutrophilia, myeloproliferation, and absence of leukocyte selectin ligand expression reminiscent of LAD-II/CDG-IIc. Contingent restoration of leukocyte and endothelial selectin ligand expression, general cellular fucosylation, and normal postnatal physiology is achieved by modulating dietary fucose to supply a salvage pathway for GDP-fucose synthesis. Conditional control of fucosylation in FX(-/-) mice identifies cellular fucosylation events as essential concomitants to fertility, early growth and development, and leukocyte adhesion.

Global vascular expression of murine CD34, a sialomucin-like endothelial ligand for L-selectin.

PubMed

Baumhueter, S; Dybdal, N; Kyle, C; Lasky, L A

1994-10-15

Extravasation of leukocytes into organized lymphoid tissues and into sites of inflammation is critical to immune surveillance. Leukocyte migration to peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN) and Peyer's patches (PP) depends on L-selectin, which recognizes carbohydrate-bearing, sialomucin-like endothelial cell surface glycoproteins. Two of these ligands have been identified at the molecular level. One is the potentially soluble mucin, GlyCAM 1, which is almost exclusively produced by high endothelial venules (HEV) of PLN and MLN. The second HEV ligand for L-selectin is the membrane-bound sialomucin CD34. Historically, this molecule has been successfully used to purify human pluripotent bone marrow stem cells, and limited data suggest that human CD34 is present on the vascular endothelium of several organs. Here we describe a comprehensive analysis of the vascular expression of CD34 in murine tissues using a highly specific antimurine CD34 polyclonal antibody. CD34 was detected on vessels in all organs examined and was expressed during pancreatic and skin inflammatory episodes. A subset of HEV-like vessels in the inflamed pancreas of nonobese diabetic (NOD) mice are positive for both CD34 and GlyCAM 1, and bind to an L-selectin/immunoglobulin G (IgG) chimeric probe. Finally, we found that CD34 is present on vessels of deafferentiated PLN, despite the fact that these vessels are no longer able to interact with L-selectin or support lymphocyte binding in vitro or trafficking in vivo. Our data suggest that the regulation of posttranslational carbohydrate modifications of CD34 is critical in determining its capability to act as an L-selectin ligand. Based on its ubiquitous expression, we propose that an appropriately glycosylated form of vascular CD34 may act as a ligand for L-selectin-mediated leukocyte trafficking to both lymphoid and nonlymphoid sites.

Discovery of a potent nanoparticle P-selectin antagonist with anti-inflammatory effects in allergic airway disease

PubMed Central

John, Alison E.; Lukacs, Nicholas W.; Berlin, Aaron A.; Palecanda, Aiyappa; Bargatze, Robert F.; Stoolman, Lloyd M.; Nagy, Jon O.

2010-01-01

The severity of allergic asthma is dependent, in part, on the intensity of peribronchial inflammation. P-selectin is known to play a role in the development of allergen-induced peribronchial inflammation and airway hyperreactivity. Selective inhibitors of P-selectin-mediated leukocyte endothelial-cell interactions may therefore attenuate the inflammatory processes associated with allergic airway disease. Novel P-selectin inhibitors were created using a polyvalent polymer nanoparticle capable of displaying multiple synthetic, low molecular weight ligands. By assembling a particle that presents an array of groups, which as monomers interact with only low affinity, we created a construct that binds extremely efficiently to P-selectin. The ligands acted as mimetics of the key binding elements responsible for the high-avidity adhesion of P-selectin to the physiologic ligand, PSGL-1. The inhibitors were initially evaluated using an in vitro shear assay system in which interactions between circulating cells and P-selectin-coated capillary tubes were measured. The nanoparticles were shown to preferentially bind to selectins expressed on activated endothelial cells. We subsequently demonstrated that nanoparticles displaying P-selectin blocking arrays were functionally active in vivo, significantly reducing allergen-induced airway hyperreactivity and peribronchial eosinophilic inflammation in a murine model of asthma. PMID:14563683

Inhibition of endothelial receptor expression and of T-cell ligand activity by mycophenolate mofetil.

PubMed

Blaheta, R A; Leckel, K; Wittig, B; Zenker, D; Oppermann, E; Harder, S; Scholz, M; Weber, S; Schuldes, H; Encke, A; Markus, B H

1998-12-01

The novel immunosuppressive drug mycophenolate mofetil (CellCept, MMF) blocks DNA-synthesis by the inhibition of the enzyme inosine monophosphate dehydrogenase (IMDH). IMDH is also involved in the synthesis of adhesion receptors which are known to play an important role in the regulation of cell-cell contacts. Therefore, application of MMF might lead to a reduction of cellular infiltrates in the course of transplant rejection. To evaluate the therapeutic value of MMF, we investigated to what extent MMF blocks T-lymphocyte infiltration in vitro with regard to (a) adhesion to endothelial cells, (b) horizontal migration along these cells and (c) penetration through the endothelial cells. The results demonstrated a strong inhibition of both CD4+ and CD8+ T-cell adhesion and penetration by MMF. The ID50 value for CD4+ T-cell adhesion was calculated to be 0.03 microM and the ID50 value for CD4+ T-cell penetration 1.21 microM. MMF did not significantly influence the horizontal migration of T-lymphocytes along the human vascular endothelial cell (HUVEC) borders. FACS-analysis revealed a diminished E-selectin and P-selectin expression on endothelial cell membranes in the presence of MMF. Although MMF did not interfere with the synthesis of T-cell adhesion ligands, the binding activity of lymphocytic leucocyte function associated antigen 1 (LFA-1), very late antigen 4 (VLA-4) and PSGL-1 (P-selectin glycoprotein ligand 1) to immobilized intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P-selectin was impaired. Moreover, MMF prevented VLA-4 and PSGL-1 receptor accumulation on the membranes of T-cell pseudopodia. It can be concluded that MMF possesses potent infiltration blocking properties. MMF evoked down-regulation of specific endothelial membrane molecules and the loss of protein localization in the lymphocyte protrusions might be predominantly responsible for the observed blockade of cell adhesion and penetration.

Cell adhesion during bullet motion in capillaries.

PubMed

Takeishi, Naoki; Imai, Yohsuke; Ishida, Shunichi; Omori, Toshihiro; Kamm, Roger D; Ishikawa, Takuji

2016-08-01

A numerical analysis is presented of cell adhesion in capillaries whose diameter is comparable to or smaller than that of the cell. In contrast to a large number of previous efforts on leukocyte and tumor cell rolling, much is still unknown about cell motion in capillaries. The solid and fluid mechanics of a cell in flow was coupled with a slip bond model of ligand-receptor interactions. When the size of a capillary was reduced, the cell always transitioned to "bullet-like" motion, with a consequent decrease in the velocity of the cell. A state diagram was obtained for various values of capillary diameter and receptor density. We found that bullet motion enables firm adhesion of a cell to the capillary wall even for a weak ligand-receptor binding. We also quantified effects of various parameters, including the dissociation rate constant, the spring constant, and the reactive compliance on the characteristics of cell motion. Our results suggest that even under the interaction between P-selectin glycoprotein ligand-1 (PSGL-1) and P-selectin, which is mainly responsible for leukocyte rolling, a cell is able to show firm adhesion in a small capillary. These findings may help in understanding such phenomena as leukocyte plugging and cancer metastasis. Copyright © 2016 the American Physiological Society.

Dynamic biochemical tissue analysis detects functional L-selectin ligands on colon cancer tissues

PubMed Central

Carlson, Grady E.; Martin, Eric W.; Shirure, Venktesh S.; Malgor, Ramiro; Resto, Vicente A.; Goetz, Douglas J.; Burdick, Monica M.

2017-01-01

A growing body of evidence suggests that L-selectin ligands presented on circulating tumor cells facilitate metastasis by binding L-selectin presented on leukocytes. Commonly used methods for detecting L-selectin ligands on tissues, e.g., immunostaining, are performed under static, no-flow conditions. However, such analysis does not assay for functional L-selectin ligands, specifically those ligands that promote adhesion under shear flow conditions. Recently our lab developed a method, termed dynamic biochemical tissue analysis (DBTA), to detect functional selectin ligands in situ by probing tissues with L-selectin-coated microspheres under hemodynamic flow conditions. In this investigation, DBTA was used to probe human colon tissues for L-selectin ligand activity. The detection of L-selectin ligands using DBTA was highly specific. Furthermore, DBTA reproducibly detected functional L-selectin ligands on diseased, e.g., cancerous or inflamed, tissues but not on noncancerous tissues. In addition, DBTA revealed a heterogeneous distribution of functional L-selectin ligands on colon cancer tissues. Most notably, detection of L-selectin ligands by immunostaining using HECA-452 antibody only partially correlated with functional L-selectin ligands detected by DBTA. In summation, the results of this study demonstrate that DBTA detects functional selectin ligands to provide a unique characterization of pathological tissue. PMID:28282455

Dynamic biochemical tissue analysis detects functional L-selectin ligands on colon cancer tissues.

PubMed

Carlson, Grady E; Martin, Eric W; Shirure, Venktesh S; Malgor, Ramiro; Resto, Vicente A; Goetz, Douglas J; Burdick, Monica M

2017-01-01

A growing body of evidence suggests that L-selectin ligands presented on circulating tumor cells facilitate metastasis by binding L-selectin presented on leukocytes. Commonly used methods for detecting L-selectin ligands on tissues, e.g., immunostaining, are performed under static, no-flow conditions. However, such analysis does not assay for functional L-selectin ligands, specifically those ligands that promote adhesion under shear flow conditions. Recently our lab developed a method, termed dynamic biochemical tissue analysis (DBTA), to detect functional selectin ligands in situ by probing tissues with L-selectin-coated microspheres under hemodynamic flow conditions. In this investigation, DBTA was used to probe human colon tissues for L-selectin ligand activity. The detection of L-selectin ligands using DBTA was highly specific. Furthermore, DBTA reproducibly detected functional L-selectin ligands on diseased, e.g., cancerous or inflamed, tissues but not on noncancerous tissues. In addition, DBTA revealed a heterogeneous distribution of functional L-selectin ligands on colon cancer tissues. Most notably, detection of L-selectin ligands by immunostaining using HECA-452 antibody only partially correlated with functional L-selectin ligands detected by DBTA. In summation, the results of this study demonstrate that DBTA detects functional selectin ligands to provide a unique characterization of pathological tissue.

Human Synovial Lubricin Expresses Sialyl Lewis x Determinant and Has L-selectin Ligand Activity*

PubMed Central

Jin, Chunsheng; Ekwall, Anna-Karin Hultgård; Bylund, Johan; Björkman, Lena; Estrella, Ruby P.; Whitelock, John M.; Eisler, Thomas; Bokarewa, Maria; Karlsson, Niclas G.

2012-01-01

Lubricin (or proteoglycan 4 (PRG4)) is an abundant mucin-like glycoprotein in synovial fluid (SF) and a major component responsible for joint lubrication. In this study, it was shown that O-linked core 2 oligosaccharides (Galβ1–3(GlcNAcβ1–6)GalNAcα1-Thr/Ser) on lubricin isolated from rheumatoid arthritis SF contained both sulfate and fucose residues, and SF lubricin was capable of binding to recombinant L-selectin in a glycosylation-dependent manner. Using resting human polymorphonuclear granulocytes (PMN) from peripheral blood, confocal microscopy showed that lubricin coated circulating PMN and that it partly co-localized with L-selectin expressed by these cells. In agreement with this, activation-induced shedding of L-selectin also mediated decreased lubricin binding to PMN. It was also found that PMN recruited to inflamed synovial area and fluid in rheumatoid arthritis patients kept a coat of lubricin. These observations suggest that lubricin is able to bind to PMN via an L-selectin-dependent and -independent manner and may play a role in PMN-mediated inflammation. PMID:22930755

Changes in Blood Factors and Ultrasound Findings in Mild Cognitive Impairment and Dementia

PubMed Central

Cho, Kyoungjoo; Kim, Jihye; Kim, Gyung W.

2017-01-01

The present study aimed to assess the changes in blood factors and ultrasound measures of atherosclerosis burden patient with mild cognitive impairment (MCI) and dementia. Peripheral blood samples and ultrasonography findings were obtained for 53 enrolled participants. Flow cytometry was used to evaluate levels of activated platelets and platelet-leukocyte aggregates (PLAs). The number of platelets expressing p-selectin was correlated with intima media thickness (IMT) and plaque number in both the MCI and dementia groups. The number of platelets expressing p-selectin glycoprotein ligand (PSGL) was strongly correlated with IMT in patients with MCI, whereas the number of platelets expressing PGSL was correlated with plaque number rather than IMT in patients with dementia. PLAs was associated with both IMT and plaque number in patients with MCI but not in those with dementia. Our findings demonstrate that alterations in IMT and plaque number are associated with an increased risk of cognitive decline as well as conversion from MCI to dementia and that blood factor analysis may aid to detect the severity of cognitive decline. PMID:29311909

Thymic progenitor homing and lymphocyte homeostasis are linked via S1P-controlled expression of thymic P-selectin/CCL25.

PubMed

Gossens, Klaus; Naus, Silvia; Corbel, Stephane Y; Lin, Shujun; Rossi, Fabio M V; Kast, Jürgen; Ziltener, Hermann J

2009-04-13

Thymic T cell progenitor (TCP) importation is a periodic, gated event that is dependent on the expression of functional P-selectin ligands on TCPs. Occupancy of intrathymic TCP niches is believed to negatively regulate TCP importation, but the nature of this feedback mechanism is not yet resolved. We show that P-selectin and CCL25 are periodically expressed in the thymus and are essential parts of the thymic gate-keeping mechanism. Periodicity of thymic TCP receptivity and the size of the earliest intrathymic TCP pool were dependent on the presence of functional P-selectin ligand on TCPs. Furthermore, we show that the numbers of peripheral blood lymphocytes directly affected thymic P-selectin expression and TCP receptivity. We identified sphingosine-1-phosphate (S1P) as one feedback signal that could mediate influence of the peripheral lymphocyte pool on thymic TCP receptivity. Our findings suggest a model whereby thymic TCP importation is controlled by both early thymic niche occupancy and the peripheral lymphocyte pool via S1P.

Adhesion behavior of endothelial progenitor cells to endothelial cells in simple shear flow

NASA Astrophysics Data System (ADS)

Gong, Xiao-Bo; Li, Yu-Qing; Gao, Quan-Chao; Cheng, Bin-Bin; Shen, Bao-Rong; Yan, Zhi-Qiang; Jiang, Zong-Lai

2011-12-01

The adhesion of endothelial progenitor cells (EPCs) on endothelial cells (ECs) is one of the critical physiological processes for the regenesis of vascular vessels and the prevention of serious cardiovascular diseases. Here, the rolling and adhesion behavior of EPCs on ECs was studied numerically. A two-dimensional numerical model was developed based on the immersed boundary method for simulating the rolling and adhesion of cells in a channel flow. The binding force arising from the catch bond of a receptor and ligand pair was modeled with stochastic Monte Carlo method and Hookean spring model. The effect of tumor necrosis factor alpha (TNF- α) on the expression of the number of adhesion molecules in ECs was analyzed experimentally. A flow chamber system with CCD camera was set up to observe the top view of the rolling of EPCs on the substrate cultivated with ECs. Numerical results prove that the adhesion of EPC on ECs is closely related to membrane stiffness of the cell and shear rate of the flow. It also suggests that the adhesion force between EPC and EC by P-selectin glycoprotein ligand-1 only is not strong enough to bond the cell onto vessel walls unless contributions of other catch bond are considered. Experimental results demonstrate that TNF- α enhanced the expressions of VCAM, ICAM, P-selectin and E-selectin in ECs, which supports the numerical results that the rolling velocity of EPC on TNF- α treated EC substrate decreases obviously compared with its velocity on the untreated one. It is found that because the adhesion is affected by both the rolling velocity and the deformability of the cell, an optimal stiffness of EPC may exist at a given shear rate of flow for achieving maximum adhesion rates.

Base-modified UDP-sugars reduce cell surface levels of P-selectin glycoprotein 1 (PSGL-1) on IL-1β-stimulated human monocytes.

PubMed

Kanabar, Varsha; Tedaldi, Lauren; Jiang, Jingqian; Nie, Xiaodan; Panina, Irina; Descroix, Karine; Man, Francis; Pitchford, Simon C; Page, Clive P; Wagner, Gerd K

2016-10-01

P-selectin glycoprotein ligand-1 (PSGL-1, CD162) is a cell-surface glycoprotein that is expressed, either constitutively or inducibly, on all myeloid and lymphoid cell lineages. PSGL-1 is implicated in cell-cell interactions between platelets, leukocytes and endothelial cells, and a key mediator of inflammatory cell recruitment and transmigration into tissues. Here, we have investigated the effects of the β-1,4-galactosyltransferase inhibitor 5-(5-formylthien-2-yl) UDP-Gal (5-FT UDP-Gal, compound 1: ) and two close derivatives on the cell surface levels of PSGL-1 on human peripheral blood mononuclear cells (hPBMCs). PSGL-1 levels were studied both under basal conditions, and upon stimulation of hPBMCs with interleukin-1β (IL-1β). Between 1 and 24 hours after IL-1β stimulation, we observed initial PSGL-1 shedding, followed by an increase in PSGL-1 levels on the cell surface, with a maximal window between IL-1β-induced and basal levels after 72 h. All three inhibitors reduce PSGL-1 levels on IL-1β-stimulated cells in a concentration-dependent manner, but show no such effect in resting cells. Compound 1: also affects the cell surface levels of adhesion molecule CD11b in IL-1β-stimulated hPBMCs, but not of glycoproteins CD14 and CCR2. This activity profile may be linked to the inhibition of global Sialyl Lewis presentation on hPBMCs by compound 1: , which we have also observed. Although this mechanistic explanation remains hypothetical at present, our results show, for the first time, that small molecules can discriminate between IL-1β-induced and basal levels of cell surface PSGL-1. These findings open new avenues for intervention with PSGL-1 presentation on the cell surface of primed hPBMCs and may have implications for anti-inflammatory drug development. © The Author 2016. Published by Oxford University Press.

Base-modified UDP-sugars reduce cell surface levels of P-selectin glycoprotein 1 (PSGL-1) on IL-1β-stimulated human monocytes

PubMed Central

Kanabar, Varsha; Tedaldi, Lauren; Jiang, Jingqian; Nie, Xiaodan; Panina, Irina; Descroix, Karine; Man, Francis; Pitchford, Simon C; Page, Clive P; Wagner, Gerd K

2016-01-01

P-selectin glycoprotein ligand-1 (PSGL-1, CD162) is a cell-surface glycoprotein that is expressed, either constitutively or inducibly, on all myeloid and lymphoid cell lineages. PSGL-1 is implicated in cell–cell interactions between platelets, leukocytes and endothelial cells, and a key mediator of inflammatory cell recruitment and transmigration into tissues. Here, we have investigated the effects of the β-1,4-galactosyltransferase inhibitor 5-(5-formylthien-2-yl) UDP-Gal (5-FT UDP-Gal, compound 1) and two close derivatives on the cell surface levels of PSGL-1 on human peripheral blood mononuclear cells (hPBMCs). PSGL-1 levels were studied both under basal conditions, and upon stimulation of hPBMCs with interleukin-1β (IL-1β). Between 1 and 24 hours after IL-1β stimulation, we observed initial PSGL-1 shedding, followed by an increase in PSGL-1 levels on the cell surface, with a maximal window between IL-1β-induced and basal levels after 72 h. All three inhibitors reduce PSGL-1 levels on IL-1β-stimulated cells in a concentration-dependent manner, but show no such effect in resting cells. Compound 1 also affects the cell surface levels of adhesion molecule CD11b in IL-1β-stimulated hPBMCs, but not of glycoproteins CD14 and CCR2. This activity profile may be linked to the inhibition of global Sialyl Lewis presentation on hPBMCs by compound 1, which we have also observed. Although this mechanistic explanation remains hypothetical at present, our results show, for the first time, that small molecules can discriminate between IL-1β-induced and basal levels of cell surface PSGL-1. These findings open new avenues for intervention with PSGL-1 presentation on the cell surface of primed hPBMCs and may have implications for anti-inflammatory drug development. PMID:27233805

Endothelial progenitor cells bind and inhibit platelet function and thrombus formation.

PubMed

Abou-Saleh, Haissam; Yacoub, Daniel; Théorêt, Jean-François; Gillis, Marc-Antoine; Neagoe, Paul-Eduard; Labarthe, Benoit; Théroux, Pierre; Sirois, Martin G; Tabrizian, Maryam; Thorin, Eric; Merhi, Yahye

2009-12-01

Interactions of endothelial progenitor cells (EPCs) with vascular and blood cells contribute to vascular homeostasis. Although platelets promote the homing of EPCs to sites of vascular injury and their differentiation into endothelial cells, the functional consequences of such interactions on platelets remain unknown. Herein, we addressed the interactions between EPCs and platelets and their impact on platelet function and thrombus formation. Cultured on fibronectin in conditioned media, human peripheral blood mononuclear cells differentiated, within 10 days of culture, into EPCs, which uptake acetylated low-density lipoprotein, bind ulex-lectin, lack monocyte/leukocyte markers (CD14, P-selectin glycoprotein ligand-1, L-selectin), express progenitor/endothelial markers (CD34, vascular endothelial growth factor receptor-2, von Willebrand factor, and vascular endothelial cadherin), and proliferate in culture. These EPCs bound activated platelets via CD62P and inhibited its translocation, glycoprotein IIb/IIIa activation, aggregation, and adhesion to collagen, mainly via prostacyclin secretion. Indeed, this was associated with upregulation of cyclooxygenase-2 and inducible nitric oxide synthase. However, the effects on platelets in vitro were reversed by cyclooxygenase and cyclooxygenase-2 inhibition but not by nitric oxide or inducible nitric oxide synthase inhibition. Moreover, in a ferric chloride-induced murine arterial thrombosis model, injection of EPCs led to their incorporation into sites of injury and impaired thrombus formation, leading to an incomplete occlusion with 50% residual flow. Peripheral blood mononuclear cell-derived EPCs bind platelets via CD62P and inhibit platelet activation, aggregation, adhesion to collagen, and thrombus formation, predominantly via upregulation of cyclooxygenase-2 and secretion of prostacyclin. These findings add new insights into the biology of EPCs and define their potential roles in regulating platelet function and thrombosis.

Endothelial Progenitor Cells Bind and Inhibit Platelet Function and Thrombus Formation

PubMed Central

Abou-Saleh, Haissam; Yacoub, Daniel; Théorêt, Jean-François; Gillis, Marc-Antoine; Neagoe, Paul-Eduard; Labarthe, Benoit; Théroux, Pierre; Sirois, Martin G.; Tabrizian, Maryam; Thorin, Eric; Merhi, Yahye

2013-01-01

Background Interactions of endothelial progenitor cells (EPCs) with vascular and blood cells contribute to vascular homeostasis. Although platelets promote the homing of EPCs to sites of vascular injury and their differentiation into endothelial cells, the functional consequences of such interactions on platelets remain unknown. Herein, we addressed the interactions between EPCs and platelets and their impact on platelet function and thrombus formation. Methods and Results Cultured on fibronectin in conditioned media, human peripheral blood mononuclear cells differentiated, within 10 days of culture, into EPCs, which uptake acetylated low-density lipoprotein, bind ulex-lectin, lack monocyte/leukocyte markers (CD14, P-selectin glycoprotein ligand-1, L-selectin), express progenitor/endothelial markers (CD34, vascular endothelial growth factor receptor-2, von Willebrand factor, and vascular endothelial cadherin), and proliferate in culture. These EPCs bound activated platelets via CD62P and inhibited its translocation, glycoprotein IIb/IIIa activation, aggregation, and adhesion to collagen, mainly via prostacyclin secretion. Indeed, this was associated with upregulation of cyclooxygenase-2 and inducible nitric oxide synthase. However, the effects on platelets in vitro were reversed by cyclooxygenase and cyclooxygenase-2 inhibition but not by nitric oxide or inducible nitric oxide synthase inhibition. Moreover, in a ferric chloride–induced murine arterial thrombosis model, injection of EPCs led to their incorporation into sites of injury and impaired thrombus formation, leading to an incomplete occlusion with 50% residual flow. Conclusions Peripheral blood mononuclear cell– derived EPCs bind platelets via CD62P and inhibit platelet activation, aggregation, adhesion to collagen, and thrombus formation, predominantly via upregulation of cyclooxygenase-2 and secretion of prostacyclin. These findings add new insights into the biology of EPCs and define their potential roles in regulating platelet function and thrombosis. PMID:19917882

Rapid and label-free microfluidic neutrophil purification and phenotyping in diabetes mellitus

NASA Astrophysics Data System (ADS)

Hou, Han Wei; Petchakup, Chayakorn; Tay, Hui Min; Tam, Zhi Yang; Dalan, Rinkoo; Chew, Daniel Ek Kwang; Li, King Ho Holden; Boehm, Bernhard O.

2016-07-01

Advanced management of dysmetabolic syndromes such as diabetes will benefit from a timely mechanistic insight enabling personalized medicine approaches. Herein, we present a rapid microfluidic neutrophil sorting and functional phenotyping strategy for type 2 diabetes mellitus (T2DM) patients using small blood volumes (fingerprick ~100 μL). The developed inertial microfluidics technology enables single-step neutrophil isolation (>90% purity) without immuno-labeling and sorted neutrophils are used to characterize their rolling behavior on E-selectin, a critical step in leukocyte recruitment during inflammation. The integrated microfluidics testing methodology facilitates high throughput single-cell quantification of neutrophil rolling to detect subtle differences in speed distribution. Higher rolling speed was observed in T2DM patients (P < 0.01) which strongly correlated with neutrophil activation, rolling ligand P-selectin glycoprotein ligand 1 (PSGL-1) expression, as well as established cardiovascular risk factors (cholesterol, high-sensitive C-reactive protein (CRP) and HbA1c). Rolling phenotype can be modulated by common disease risk modifiers (metformin and pravastatin). Receiver operating characteristics (ROC) and principal component analysis (PCA) revealed neutrophil rolling as an important functional phenotype in T2DM diagnostics. These results suggest a new point-of-care testing methodology, and neutrophil rolling speed as a functional biomarker for rapid profiling of dysmetabolic subjects in clinical and patient-oriented settings.

A new approach to explore the binding space of polysaccharide-based ligands: selectin antagonists.

PubMed

Calosso, Mickael; Charpentier, Daniel; Vaillancourt, Marc; Bencheqroun, Mohammed; St-Pierre, Gabrielle; Wilkes, Brian C; Guindon, Yvan

2012-12-13

The discovery of molecules that interfere with the binding of a ligand to a receptor remains a topic of great interest in medicinal chemistry. Herein, we report that a monosaccharide unit of a polysaccharide ligand can be replaced advantageously by a conformationally locked acyclic molecular entity. A cyclic component of the selectin ligand Sialyl Lewis(x), GlcNAc, is replaced by an acyclic tether, tartaric esters, which link two saccharide units. The conformational bias of this acyclic tether originates from the minimization of intramolecular dipole-dipole interaction and the gauche effect. The evaluation of the binding of these derivatives to P-selectin was measured by surface plasmon resonance spectroscopy. The results obtained in our pilot study suggest that the discovery of tunable tethers could facilitate the exploration of the carbohydrate recognition domain of various receptors.

Production and characterization of monoclonal antibodies against conserved epitopes of P-selectin (CD62P).

PubMed

Massaguer, A; Engel, P; Pérez-del-Pulgar, S; Bosch, J; Pizcueta, P

2000-08-01

P-selectin (CD62P) is an adhesion molecule expressed on the activated endothelium and activated platelets that is involved in the initial attachment of leukocytes to inflamed vascular endothelium. Blocking monoclonal antibodies (mAbs) and P-selectin-deficient mice have shown that P-selectin is a potential target in anti-inflammatory therapy. Most mAbs against P-selectin do not bind to conserved epitopes, including the ligand-binding region, since P-selectin from mammalian species shares high amino acid sequence homology. The aim of this study was to generate a novel panel of anti-P-selectin mAbs against the conserved epitopes present in several animal species. To produce these mAbs, P-selectin-deficient mice were immunized with a pre-B-cell line transfected with human P-selectin cDNA. Twelve mouse mAbs that recognize human P-selectin were obtained. Individual mAbs that bound to human, rat, mouse, rabbit and pig activated platelets were characterized by flow-cytometry, immunohistochemistry, adhesion assays and immunoprecipitation. Four of these mAbs (P-sel.KO.2.3, P-sel.KO.2.4, P-sel.KO.2.7 and P-sel.KO.2.12) cross-reacted with human, rat and mouse P-selectin. Another three mAbs (P-sel.KO.2.2, P-sel.KO.2.11 and P-sel.KO.2.12) blocked the attachment of HL60 cells to P-selectin-transfected COS cells, demonstrating that these mAbs inhibit P-selectin-mediated adhesion. MAb cross-blocking experiments showed that these three mAbs bind to very close and overlapping epitopes. An ELISA assay using mAbs P-sel.KO.2.3 and P-sel.KO.2.12 was designed to measure soluble rat, mouse and human P-selectin. These anti-P-selectin mAbs are unique since they recognize common epitopes conserved during mammalian evolution and they may be useful for studying P-selectin function in inflammatory models in various species.

Tyrosine phosphorylation of P-selectin in intact platelets and in a disulphide-linked complex with immunoprecipitated pp60c-src.

PubMed Central

Modderman, P W; von dem Borne, A E; Sonnenberg, A

1994-01-01

P-selectin is a 140 kDa membrane glycoprotein found in secretory granules of platelets and endothelial cells where it is rapidly translocated to the plasma membrane upon cell activation. It then functions as a receptor for various types of leucocytes. Metabolic labelling of resting platelets with 32Pi showed that P-selectin is primarily phosphorylated on serine residues, although some tyrosine phosphorylation was observed as well. However, tyrosine phosphorylation of P-selectin was greatly stimulated by treatment with the permeating phosphatase inhibitor, pervanadate. When P-selectin immunoprecipitates were incubated with [gamma-32P]ATP (in vitro kinase assay), a fraction of P-selectin was phosphorylated on its tyrosine residues by a co-precipitated kinase. P-selectin phosphorylated in vitro co-migrated with 140 kDa surface-labelled 125I-P-selectin during SDS/PAGE under reducing conditions. Under non-reducing conditions, however, phosphorylated P-selectin was disulphide-linked to unknown protein(s) in a 205 kDa complex. In vitro kinase assays of the most abundant platelet tyrosine kinase, pp60c-src, demonstrated the presence of similar 140 and 205 kDa phosphorylated proteins in SDS/PAGE under reducing and non-reducing conditions respectively. Extraction and reprecipitation studies with proteins phosphorylated in vitro indicated that P-selectin and pp60c-src form a 205 kDa 1:1 disulphide-linked complex. In the complex, pp60c-src autophosphorylation is inhibited and P-selectin is phosphorylated on tyrosine residues. As protein disulphides in the cytoplasm of intact cells are extremely rare, our results suggest that P-selectin and pp60c-src, which co-localize in platelet dense granules, may be non-covalently associated and spontaneously form disulphide bridges during lysis. In addition, the observed tyrosine phosphorylation of P-selectin in intact platelets suggests that its function might be regulated by phosphorylation by pp60c-src. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7514867

Raised plasma soluble P-selectin in peripheral arterial occlusive disease enhances leukocyte adhesion.

PubMed

Woollard, K J; Kling, D; Kulkarni, S; Dart, A M; Jackson, S; Chin-Dusting, J

2006-01-06

Raised levels of soluble P-selectin (sP-selectin) have been reported in the plasma of patients with vascular diseases; however, the functional importance of this ligand remains unclear. In this study we have examined a potential role for plasma sP-selectin in regulating neutrophil adhesion in patients with peripheral arterial occlusive disease (PAOD). Patients with PAOD had significantly higher levels of sP-selectin (mean+/-SD: 73.3+/-13.0 versus 16.7+/-6.4 ng/mL) and enhanced whole blood leukocyte adhesion to platelets under shear. To examine whether the raised sP-selectin levels can directly influence leukocyte adhesion, isolated neutrophils were incubated with plasma from PAOD patients before and after immunodepletion of sP-selectin. Neutrophil adhesion to fibrinogen increased 2-fold following incubation with PAOD plasma, which was abrogated on sP-selectin immunodepletion. We subsequently demonstrated that recombinant sP-selectin dose-dependently (75 to 250 ng/mL) increased leukocyte adhesion to fibrinogen and platelet monolayers. This increase was PSGL-1 and Src kinase-dependent and correlated with an increase in sP-selectin-mediated Mac-1 activation. sP-selectin-stimulated neutrophil adhesion to platelet monolayers was inversely correlated with shear, such that at low shear (50 s(-1)) a 92.7%+/-15.7 increase in adhesion was observed decreasing to 38.5%+/-11.9 at 150 s(-1) and 10.1%+/-7.4 at 300 s(-1). These studies suggest a potentially important role for sP-selectin in modulating neutrophil adhesion in patients with PAOD, particularly at sites of low shear, where it raises the possibility that raised plasma sP-selectin levels may enhance leukocyte recruitment to vascular injury and promote disease progression.

Interplay between Rolling and Firm Adhesion Elucidated with a Cell-Free System Engineered with Two Distinct Receptor-Ligand Pairs

PubMed Central

Eniola, A. Omolola; Willcox, P. Jeanene; Hammer, Daniel A.

2003-01-01

The firm arrest of leukocytes to the endothelium during inflammation is known to be mediated by endothelial intercellular adhesion molecules (ICAMs) binding to activated integrins displayed on leukocyte surface. Selectin-ligand interactions, which mediate rolling, are believed to be important for facilitating firm adhesion, either by activating integrins or by facilitating the transition to firm adhesion by making it easier for integrins to bind. Although leukocytes employ two distinct adhesion molecules that mediate different states of adhesion, the fundamental biophysical mechanisms by which two pairs of adhesion molecules facilitate cell adhesion is not well understood. In this work, we attempt to understand the interaction between two molecular systems using a cell-free system in which polystyrene microspheres functionalized with the selectin ligand, sialyl LewisX (sLeX), and an antibody against ICAM-1, aICAM-1, are perfused over P-selectin/ICAM-1 coated surfaces in a parallel plate flow chamber. Separately, sLeX/P-selectin interactions support rolling and aICAM-1/ICAM-1 interactions mediate firm adhesion. Our results show that sLeX/aICAM-1 microspheres will firmly adhere to P-selectin/ICAM-1 coated surfaces, and that the extent of firm adhesion of microspheres is dependent on wall shear stress within the flow chamber, sLeX/aICAM-1 microsphere site density, and P-selectin/ICAM-1 surface density ratio. We show that P-selectin's interaction with sLeX mechanistically facilitates firm adhesion mediated by antibody binding to ICAM-1: the extent of firm adhesion for the same concentration of aICAM-1/ICAM-1 interaction is greater when sLeX/P-selectin interactions are present. aICAM-1/ICAM-1 interactions also stabilize rolling by increasing pause times and decreasing average rolling velocities. Although aICAM-1 is a surrogate for β2-integrin, the kinetics of association between aICAM-1 and ICAM-1 is within a factor of 1.5 of activated integrin binding ICAM-1, suggesting the findings from this model system may be insightful to the mechanism of leukocyte firm adhesion. In particular, these experimental results show how two molecule systems can interact to produce an effect not achievable by either system alone, a fundamental mechanism that may pervade leukocyte adhesion biology. PMID:14507735

Using affinity capillary electrophoresis and computational models for binding studies of heparinoids with p-selectin and other proteins.

PubMed

Mozafari, Mona; Balasupramaniam, Shantheya; Preu, Lutz; El Deeb, Sami; Reiter, Christian G; Wätzig, Hermann

2017-06-01

A fast and precise affinity capillary electrophoresis (ACE) method has been developed and applied for the investigation of the binding interactions between P-selectin and heparinoids as potential P-selectin inhibitors in the presence and absence of calcium ions. Furthermore, model proteins and vitronectin were used to appraise the binding behavior of P-selectin. The normalized mobility ratios (∆R/R f ), which provided information about the binding strength and the overall charge of the protein-ligand complex, were used to evaluate the binding affinities. It was found that P-selectin interacts more strongly with heparinoids in the presence of calcium ions. P-selectin was affected by heparinoids at the concentration of 3 mg/L. In addition, the results of the ACE experiments showed that among other investigated proteins, albumins and vitronectin exhibited strong interactions with heparinoids. Especially with P-selectin and vitronectin, the interaction may additionally induce conformational changes. Subsequently, computational models were applied to interpret the ACE experiments. Docking experiments explained that the binding of heparinoids on P-selectin is promoted by calcium ions. These docking models proved to be particularly well suited to investigate the interaction of charged compounds, and are therefore complementary to ACE experiments. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Differential regulation and impact of fucosyltransferase VII and core 2 β1,6-N-acetyl-glycosaminyltransferase for generation of E-selectin and P-selectin ligands in murine CD4+ T cells

PubMed Central

Schroeter, Micha F; Ratsch, Boris A; Lehmann, Jeanette; Baumgrass, Ria; Hamann, Alf; Syrbe, Uta

2012-01-01

Ligands for E-selectin and P-selectin (E-lig and P-lig) are induced on CD4+ T cells upon differentiation into effector T cells. Glycosyltransferases, especially α 1,3-fucosyltransferase VII (FucT-VII) and core 2 β1,6-N-acetyl-glycosaminyltransferase I (C2GlcNAcT-I), are critical for their synthesis. We here analysed the signals that control the expression of E-lig, P-lig and mRNA coding for FucT-VII and C2GlcNAcT-I. In line with previous reports, we found that P-lig expression correlates with the regulation of C2GlcNAcT-I, whereas E-lig expression can occur at low levels of C2GlcNAcT-I mRNA but requires high FucT-VII mRNA expression. Interestingly, the two enzymes are regulated by different signals. Activation-induced C2GlcNAcT-I up-regulation under permissive (T helper type 1) conditions was strongly reduced by cyclosporin A (CsA), suggesting the involvement of T-cell receptor-dependent, calcineurin/NFAT-dependent signals in combination with interleukin-12 (IL-12) -mediated signals in the regulation of C2GlcNAcT-I. In contrast, expression of FucT-VII mRNA was not significantly inhibited by CsA. Interleukin-4 inhibited the expression of FucT-VII but IL-2 and IL-7 were found to support induction of FucT-VII and E-lig. E-selectin, P-selectin and their ligands initially appeared to have rather overlapping functions. These findings however, unravel striking differences in the regulation of E-lig and P-lig expression, dictated by the dominance of FucT-VII and C2GlcNAcT-I, respectively, and their dependency on signals from either promiscuous or homeostatic cytokines (FucT-VII) or a strong T-cell receptor signal in combination with inflammatory cytokines in case of C2GlcNAcT-I. PMID:23039181

Staining of E-selectin ligands on paraffin-embedded sections of tumor tissue.

PubMed

Carrascal, Mylène A; Talina, Catarina; Borralho, Paula; Gonçalo Mineiro, A; Henriques, Ana Raquel; Pen, Cláudia; Martins, Manuela; Braga, Sofia; Sackstein, Robert; Videira, Paula A

2018-05-02

The E-selectin ligands expressed by cancer cells mediate adhesion of circulating cancer cells to endothelial cells, as well as within tissue microenvironments important for tumor progression and metastasis. The identification of E-selectin ligands within cancer tissue could yield new biomarkers for patient stratification and aid in identifying novel therapeutic targets. The determinants of selectin ligands consist of sialylated tetrasaccharides, the sialyl Lewis X and A (sLe X and sLe A ), displayed on protein or lipid scaffolds. Standardized procedures for immunohistochemistry make use of the antibodies against sLe X and/or sLe A . However, antibody binding does not define E-selectin binding activity. In this study, we developed an immunohistochemical staining technique, using E-selectin-human Ig Fc chimera (E-Ig) to characterize the expression and localization of E-selectin binding sites on paraffin-embedded sections of different cancer tissue. E-Ig successfully stained cancer cells with high specificity. The E-Ig staining show high reactivity scores in colon and lung adenocarcinoma and moderate reactivity in triple negative breast cancer. Compared with reactivity of antibody against sLe X/A , the E-Ig staining presented higher specificity to cancer tissue with better defined borders and less background. The E-Ig staining technique allows the qualitative and semi-quantitative analysis of E-selectin binding activity on cancer cells. The development of accurate techniques for detection of selectin ligands may contribute to better diagnostic and better understanding of the molecular basis of tumor progression and metastasis.

[Correlation of Plasma Co-stimulatory Molecules B7-H2 and B7-H3 with Platelet Auto-antibodies in Patients with Immune Thrombocytopenic Purpura].

PubMed

Zuo, Bin; Zhao, Yun-Xiao; Yang, Jian-Feng; He, Yang

2015-08-01

To investigate whether the plasma level of platelet auto- antibodies in ITP patients is related to that of co-stimulatory molecules sB7-H2 and sB7-H3. A total of 61 ITP patients and 25 healthy controls from the First Affiliated Hospital of Soochow University from June 2012 to August 2013 were enrolled in this study. The expression levels of platelet auto-antibodies against 5 glycoproteins (GPIX, GP Ib, GP IIIa, GPIIb and P-selectin) in plasma were detected by flow cytometric immuno-beads array, and the expression of soluable co-stimulatory molecules sB7-H2 and sB7-H3 was measured by ELISA. The plasma levels of 5 auto-antibodies against platelet membrance glycoproteins significantly increased in ITP patiens (P < 0.01). Compared with healthy controls, sB7-H2 levels increased (P < 0.05), while the sB7-H3 level did not significantly change (r = 0.13, P > 0.05). However, the correlation analysis showed that sB7-H3 negatively correlated with platelet P-selectin auto-antibody (r = -0.46, P < 0.05), and sB7-H2 and sB7-H3 significantly reduced in ITP patients with positive P-selectin auto-antibody (P < 0.01). In ITP patients, platelet counts negatively correlated with sB7-H2 (r = -0.3907, P < 0.01), but did not correlate with sB7-H3. Soluble costimulatory molecule sB7-H2 elevates in ITP patients, and the level of sB7-H3 is associated with auto-antibodies against P-selectin, suggesting that costimulatory molecules B7-H2 and B7-H3 may be involved in the pathogenesis of immune regulation abnormality in ITP.

Intravascular Inflammation Triggers Intracerebral Activated Microglia and Contributes to Secondary Brain Injury After Experimental Subarachnoid Hemorrhage (eSAH).

PubMed

Atangana, Etienne; Schneider, Ulf C; Blecharz, Kinga; Magrini, Salima; Wagner, Josephin; Nieminen-Kelhä, Melina; Kremenetskaia, Irina; Heppner, Frank L; Engelhardt, Britta; Vajkoczy, Peter

2017-04-01

Activation of innate immunity contributes to secondary brain injury after experimental subarachnoid hemorrhage (eSAH). Microglia accumulation and activation within the brain has recently been shown to induce neuronal cell death after eSAH. In isolated mouse brain capillaries after eSAH, we show a significantly increased gene expression for intercellular adhesion molecule-1 (ICAM-1) and P-selectin. Hence, we hypothesized that extracerebral intravascular inflammatory processes might initiate the previously reported microglia accumulation within the brain tissue. We therefore induced eSAH in knockout mice for ICAM-1 (ICAM-1 -/- ) and P-selectin glycoprotein ligand-1 (PSGL-1 -/- ) to find a significant decrease in neutrophil-endothelial interaction within the first 7 days after the bleeding in a chronic cranial window model. This inhibition of neutrophil recruitment to the endothelium results in significantly ameliorated microglia accumulation and neuronal cell death in knockout animals in comparison to controls. Our results suggest an outside-in activation of the CNS innate immune system at the vessel/brain interface following eSAH. Microglia cells, as part of the brain's innate immune system, are triggered by an inflammatory reaction in the microvasculature after eSAH, thus contributing to neuronal cell death. This finding offers a whole range of new research targets, as well as possible therapy options for patients suffering from eSAH.

Role of P-selectin in thromboembolic events in patients with cancer.

PubMed

Fernandes, Lúcio Flávio Barbour; Fregnani, José Humberto T G; Strunz, Célia Maria Cássaro; de Andrade Ramos Nogueira, Adriana; Longatto-Filho, Adhemar

2018-01-01

The objective of the present study was to evaluate the role of P-selectin in patients with cancer with suspected thromboembolic events (TEEs). Patients with cancer have a four times greater risk of developing TEEs. P-selectin is a glycoprotein that has the function of facilitating the interaction (adhesion) of leukocytes with the endothelium, or with platelets. There is a well-defined relationship between P-selectin and thrombosis; however, it is likely that the cut-off value of P-selectin for patients with cancer should be considered differently from that of the general population. In the present report, a prospective cross-sectional study was performed with patients of the Cancer Hospital of Barretos who were suspected of having TEEs. Among the 178 study participants, 167 (93.82%) were suspected of having deep vein thrombosis, while 59 of them (35.33%) were confirmed as such; and 11 (6.18%) were suspected of having pulmonary thromboembolism, while 3 of them were confirmed as such (27.69%). The mean results obtained were: P-selectin, 25.37 ng/ml; and D-dimer, 2,181.22 ng/ml. The P-selectin levels averaged 33.60 ng/ml with the confirmed TEE group compared with 20.40 ng/ml with the unconfirmed TEE group, with a standard deviation of 23.35 compared with 6.92 (P<0.001); and the level of D-dimer was 4,615.38 ng/ml compared with 977.52 ng/ml, with a standard deviation of 6,460.54 compared with 2,145.50 (P<0.001). Multiple logistic regression adjusted for distant metastases and the Eastern Cooperative Oncology Group (ECOG) score (2,3 and 4) were constructed. The cut-off value of P-selectin for patients with cancer was identified to be different from that reported in the literature for the general population, and the models using D-dimer and P-selectin therefore have been demonstrated to be a potentially useful tool to be used in a panel of tests to predict TEEs, either independently or in a prediction score.

Polymeric Selectin Ligands Mimicking Complex Carbohydrates: From Selectin Binders to Modifiers of Macrophage Migration.

PubMed

Moog, Kai E; Barz, Matthias; Bartneck, Matthias; Beceren-Braun, Figen; Mohr, Nicole; Wu, Zhuojun; Braun, Lydia; Dernedde, Jens; Liehn, Elisa A; Tacke, Frank; Lammers, Twan; Kunz, Horst; Zentel, Rudolf

2017-01-24

Novel polymeric cell adhesion inhibitors were developed in which the selectin tetrasaccharide sialyl-Lewis X (SLe X ) is multivalently presented on a biocompatible poly(2-hydroxypropyl)methacrylamide (PHPMA) backbone either alone (P1) or in combination with O-sulfated tyramine side chains (P2). For comparison, corresponding polymeric glycomimetics were prepared in which the crucial "single carbohydrate" substructures fucose, galactose, and sialic acid side chains were randomly linked to the PHPMA backbone (P3 or P4 (O-sulfated tyramine)). All polymers have an identical degree of polymerization, as they are derived from the same precursor polymer. Binding assays to selectins, to activated endothelial cells, and to macrophages show that polyHPMA with SLe X is an excellent binder to E-, L-, and P-selectins. However, mimetic P4 can also achieve close to comparable binding affinities in in vitro measurements and surprisingly, it also significantly inhibits the migration of macrophages; this provides new perspectives for the therapy of severe inflammatory diseases. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Increased E-selectin in hepatic ischemia-reperfusion injury mediates liver metastasis of pancreatic cancer

PubMed Central

YOSHIMOTO, KATSUHIRO; TAJIMA, HIDEHIRO; OHTA, TETSUO; OKAMOTO, KOICHI; SAKAI, SEISHO; KINOSHITA, JUN; FURUKAWA, HIROYUKI; MAKINO, ISAMU; HAYASHI, HIRONORI; NAKAMURA, KEISHI; OYAMA, KATSUNOBU; INOKUCHI, MASAFUMI; NAKAGAWARA, HISATOSHI; ITOH, HIROSHI; FUJITA, HIDETO; TAKAMURA, HIROYUKI; NINOMIYA, ITASU; KITAGAWA, HIROHISA; FUSHIDA, SACHIO; FUJIMURA, TAKASHI; WAKAYAMA, TOMOHIKO; ISEKI, SHOICHI; SHIMIZU, KOICHI

2012-01-01

Several recent studies have reported that selectins are produced during ischemia-reperfusion injury, and that selectin ligands play an important role in cell binding to the endothelium and in liver metastasis. Portal clamping during pancreaticoduodenectomy with vessel resection for pancreatic head cancer causes hepatic ischemia-reperfusion injury, which might promote liver metastasis. We investigated the liver colonization of pancreatic cancer cells under hepatic ischemia-reperfusion and examined the involvement of E-selectin and its ligands. A human pancreatic cancer cell line (Capan-1) was injected into the spleen of mice after hepatic ischemia-reperfusion (I/R group). In addition, to investigate the effect of an anti-E-selectin antibody on liver colonization in the IR group, mice received an intraperitoneal injection of the anti-E-selectin antibody following hepatic ischemia-reperfusion and tumor inoculation (IR+Ab group). Four weeks later, mice were sacrificed and the number of tumor nodules on the liver was compared to mice without hepatic ischemia-reperfusion (control group). The incidence of liver metastasis in the I/R group was significantly higher (16 of 20, 80%) than that in the control group (6 of 20, 30%) (P<0.01). Moreover, mice in the I/R group had significantly more tumor nodules compared to those in the control group (median, 9.9 vs. 2.7 nodules) (P<0.01). In the I/R+Ab group, only 2 of 5 (40%) mice developed liver metastases. RT-PCR and southern blotting of the liver extracts showed that the expression of IL-1 and E-selectin mRNA after hepatic ischemia-reperfusion was significantly higher than the basal levels. Hepatic ischemia-reperfusion increases liver metastases and E-selectin expression in pancreatic cancer. These results suggest that E-selectin produced due to hepatic ischemia-reperfusion is involved in liver metastasis. PMID:22766603

E-selectin: sialyl Lewis, a dependent adhesion of colon cancer cells, is inhibited differently by antibodies against E-selectin ligands.

PubMed

Srinivas, U; Påhlsson, P; Lundblad, A

1996-09-01

Recent studies have demonstrated that selectins, a new family of cell-adhesion molecules with similar domain structures, mediate the adhesion of peripheral blood cells to interleukin-1 (IL-1)-activated endothelium. In the present study the authors evaluated the role of E-selectin-Sialyl Lewis x (SLe(x))/ Sialyl Lewis a (SLe(a)) interaction in mediating in vitro adhesion of two colon cancer cell lines, HT-29 and COLO 201, to human umbilical cord endothelial cells (HUVEC). Colon cancer cell lines had a strong expression of blood group-related carbohydrate epitopes as evaluated by fluorescence-activated cell sorter (FACS) analysis. It was established that adhesion of HT-29 and COLO 201 cells to IL-1 stimulated HUVEC was calcium dependent and could be inhibited by a monoclonal antibody directed against E-selectin. Prior incubation of cells with two different antibodies directed against SLe(x) and antibodies directed against related Lewis epitopes, Le(x) and Le(a), had no significant effect on adhesion. Three antibodies directed against SLe(a) differed in their capacity to inhibit the adhesion of HT-29 and COLO 201 cells to HUVEC. Only one antibody directed against the SLe(a) structure was effective in inhibiting adhesion of both COLO 201 and HT-29 cells. The difference could not be attributed to titre, the type or number of glycoproteins, or to a difference in the amount of SLe(a) present on individual proteins, suggesting that presence and right presentation of SLe(a) epitope might be important for adhesion of colon cancer cells. Finally, in the in vitro system used, adhesion of HT-29 and COLO 201 cells to activated HUVEC is mediated predominantly by E-selectin/SLe(a) interaction. SLe(x) and related epitopes, Le(x) and Le(a), seem to have limited relevance for colon cancer cell recognition of E-selectin.

E-Selectin Ligands in the Human Mononuclear Phagocyte System: Implications for Infection, Inflammation, and Immunotherapy.

PubMed

Silva, Mariana; Videira, Paula A; Sackstein, Robert

2017-01-01

The mononuclear phagocyte system comprises a network of circulating monocytes and dendritic cells (DCs), and "histiocytes" (tissue-resident macrophages and DCs) that are derived in part from blood-borne monocytes and DCs. The capacity of circulating monocytes and DCs to function as the body's first-line defense against offending pathogens greatly depends on their ability to egress the bloodstream and infiltrate inflammatory sites. Extravasation involves a sequence of coordinated molecular events and is initiated by E-selectin-mediated deceleration of the circulating leukocytes onto microvascular endothelial cells of the target tissue. E-selectin is inducibly expressed by cytokines (tumor necrosis factor-α and IL-1β) on inflamed endothelium, and binds to sialofucosylated glycan determinants displayed on protein and lipid scaffolds of blood cells. Efficient extravasation of circulating monocytes and DCs to inflamed tissues is crucial in facilitating an effective immune response, but also fuels the immunopathology of several inflammatory disorders. Thus, insights into the structural and functional properties of the E-selectin ligands expressed by different monocyte and DC populations is key to understanding the biology of protective immunity and the pathobiology of several acute and chronic inflammatory diseases. This review will address the role of E-selectin in recruitment of human circulating monocytes and DCs to sites of tissue injury/inflammation, the structural biology of the E-selectin ligands expressed by these cells, and the molecular effectors that shape E-selectin ligand cell-specific display. In addition, therapeutic approaches targeting E-selectin receptor/ligand interactions, which can be used to boost host defense or, conversely, to dampen pathological inflammatory conditions, will also be discussed.

Aspirin inhibits surface glycoprotein IIb/IIIa, P-selectin, CD63, and CD107a receptor expression on human platelets.

PubMed

McKenzie, Marcus E; Malinin, Alex I; Bell, Christopher R; Dzhanashvili, Alex; Horowitz, Eric D; Oshrine, Benjamin R; Atar, Dan; Serebruany, Victor L

2003-04-01

Platelet inhibition after aspirin therapy reduces the risk for the development of acute coronary syndromes. However, the mechanism by which aspirin affect platelets other than by prostaglandin blockade is unclear. We sought to determine the in vitro effects of aspirin on the surface expression of nine platelet receptors using whole blood flow cytometry. Blood from 24 healthy volunteers was incubated for 30 min with 1.8 and 7.2 mg/l phosphate-buffered saline-diluted acetylsalicylic acid in the presence or absence of apyrase. Platelet serotonin release, and the surface expression of platelet receptors with or without apyrase were determined using the following monoclonal antibodies: anit-CD41 [glycoprotein (GP)IIb/IIIa], CD42b (GPIb), CD62p (P-selectin), CD51/CD61 (vitronectin receptor), CD31 [platelet/endothelial cellular adhesion molecule-1 (PECAM-1)], CD107a [lysosomal associated membrane protein (LAMP)-1], CD107b (LAMP-2), CD63 (LIMP or LAMP-3), and CD151 (PETA-3). Samples were then immediately fixed with 2% paraformaldehyde, and run on the flow cytometer within 48 h. Aspirin does not affect serotonin release from human platelets. Dose-dependent inhibition of GPIIb/IIIa, P-selectin, CD63, and CD107a receptor expression was observed in the aspirin-treated whole-blood samples. Apyrase potentiates the effects of aspirin, and independently inhibits PECAM-1. In addition to the known effect of irreversibly inhibiting platelet cyclooxygenase-1, thereby blocking thromboxane A(2) synthesis, it appears that aspirin exhibits direct effects on selective major platelet receptors.

Enforced hematopoietic cell E- and L-selectin ligand (HCELL) expression primes transendothelial migration of human mesenchymal stem cells.

PubMed

Thankamony, Sai P; Sackstein, Robert

2011-02-08

According to the multistep model of cell migration, chemokine receptor engagement (step 2) triggers conversion of rolling interactions (step 1) into firm adhesion (step 3), yielding transendothelial migration. We recently reported that glycosyltransferase-programmed stereosubstitution (GPS) of CD44 on human mesenchymal stem cells (hMSCs) creates the E-selectin ligand HCELL (hematopoietic cell E-selectin/L-selectin ligand) and, despite absence of CXCR4, systemically administered HCELL(+)hMSCs display robust osteotropism visualized by intravital microscopy. Here we performed studies to define the molecular effectors of this process. We observed that engagement of hMSC HCELL with E-selectin triggers VLA-4 adhesiveness, resulting in shear-resistant adhesion to ligand VCAM-1. This VLA-4 activation is mediated via a Rac1/Rap1 GTPase signaling pathway, resulting in transendothelial migration on stimulated human umbilical vein endothelial cells without chemokine input. These findings indicate that hMSCs coordinately integrate CD44 ligation and integrin activation, circumventing chemokine-mediated signaling, yielding a step 2-bypass pathway of the canonical multistep paradigm of cell migration.

Inhibition of selectin binding

DOEpatents

Nagy, Jon O.; Spevak, Wayne R.; Dasgupta, Falguni; Bertozzi, Caroline

2001-10-09

This invention provides compositions for inhibiting the binding between two cells, one expressing P- or L-selectin on the surface and the other expressing the corresponding ligand. A covalently crosslinked lipid composition is prepared having saccharides and acidic group on separate lipids. The composition is then interposed between the cells so as to inhibit binding. Inhibition can be achieved at an effective oligosaccharide concentration as low as 10.sup.6 fold below that of the free saccharide. Since selectins are involved in recruiting cells to sites of injury, these composition scan be used to palliate certain inflammatory and immunological conditions.

Inhibition of selectin binding

DOEpatents

Nagy, Jon O.; Spevak, Wayne R.; Dasgupta, Falguni; Bertozzi, Caroline

1999-01-01

This invention provides compositions for inhibiting the binding between two cells, one expressing P- or L-selectin on the surface and the other expressing the corresponding ligand. A covalently crosslinked lipid composition is prepared having saccharides and acidic group on separate lipids. The composition is then interposed between the cells so as to inhibit binding. Inhibition can be achieved at an effective oligosaccharide concentration as low as 10.sup.6 fold below that of the free saccharide. Since selectins are involved in recruiting cells to sites of injury, these composition scan be used to palliate certain inflammatory and immunological conditions.

Inhibition of selectin binding

DOEpatents

Nagy, Jon O.; Spevak, Wayne R.; Dasgupta, Falguni; Bertozzi, Carolyn

1999-10-05

This invention provides a system for inhibiting the binding between two cells, one expressing P- or L-selectin on the surface and the other expressing the corresponding ligand. A covalently crosslinked lipid composition is prepared having saccharides and acidic group on separate lipids. The composition is then interposed between the cells so as to inhibit binding. Inhibition can be achieved at an effective oligosaccharide concentration as low as 10.sup.6 fold below that of the free saccharide. Since selectins are involved in recruiting cells to sites of injury, this system can be used to palliate certain inflammatory and immunological conditions.

Dendritic polyglycerol sulfates as multivalent inhibitors of inflammation.

PubMed

Dernedde, Jens; Rausch, Alexandra; Weinhart, Marie; Enders, Sven; Tauber, Rudolf; Licha, Kai; Schirner, Michael; Zügel, Ulrich; von Bonin, Arne; Haag, Rainer

2010-11-16

Adhesive interactions of leukocytes and endothelial cells initiate leukocyte migration to inflamed tissue and are important for immune surveillance. Acute and chronic inflammatory diseases show a dysregulated immune response and result in a massive efflux of leukocytes that contributes to further tissue damage. Therefore, targeting leukocyte trafficking may provide a potent form of anti-inflammatory therapy. Leukocyte migration is initiated by interactions of the cell adhesion molecules E-, L-, and P-selectin and their corresponding carbohydrate ligands. Compounds that efficiently address these interactions are therefore of high therapeutic interest. Based on this rationale we investigated synthetic dendritic polyglycerol sulfates (dPGS) as macromolecular inhibitors that operate via a multivalent binding mechanism mimicking naturally occurring ligands. dPGS inhibited both leukocytic L-selectin and endothelial P-selectin with high efficacy. Size and degree of sulfation of the polymer core determined selectin binding affinity. Administration of dPGS in a contact dermatitis mouse model dampened leukocyte extravasation as effectively as glucocorticoids did and edema formation was significantly reduced. In addition, dPGS interacted with the complement factors C3 and C5 as was shown in vitro and reduced C5a levels in a mouse model of complement activation. Thus, dPGS represent an innovative class of a fully synthetic polymer therapeutics that may be used for the treatment of inflammatory diseases.

Dendritic polyglycerol sulfates as multivalent inhibitors of inflammation

PubMed Central

Dernedde, Jens; Rausch, Alexandra; Weinhart, Marie; Enders, Sven; Tauber, Rudolf; Licha, Kai; Schirner, Michael; Zügel, Ulrich; von Bonin, Arne; Haag, Rainer

2010-01-01

Adhesive interactions of leukocytes and endothelial cells initiate leukocyte migration to inflamed tissue and are important for immune surveillance. Acute and chronic inflammatory diseases show a dysregulated immune response and result in a massive efflux of leukocytes that contributes to further tissue damage. Therefore, targeting leukocyte trafficking may provide a potent form of anti-inflammatory therapy. Leukocyte migration is initiated by interactions of the cell adhesion molecules E-, L-, and P-selectin and their corresponding carbohydrate ligands. Compounds that efficiently address these interactions are therefore of high therapeutic interest. Based on this rationale we investigated synthetic dendritic polyglycerol sulfates (dPGS) as macromolecular inhibitors that operate via a multivalent binding mechanism mimicking naturally occurring ligands. dPGS inhibited both leukocytic L-selectin and endothelial P-selectin with high efficacy. Size and degree of sulfation of the polymer core determined selectin binding affinity. Administration of dPGS in a contact dermatitis mouse model dampened leukocyte extravasation as effectively as glucocorticoids did and edema formation was significantly reduced. In addition, dPGS interacted with the complement factors C3 and C5 as was shown in vitro and reduced C5a levels in a mouse model of complement activation. Thus, dPGS represent an innovative class of a fully synthetic polymer therapeutics that may be used for the treatment of inflammatory diseases. PMID:21041668

Functional contributions of N- and O-glycans to L-selectin ligands in murine and human lymphoid organs.

PubMed

Arata-Kawai, Hanayo; Singer, Mark S; Bistrup, Annette; Zante, Annemieke van; Wang, Yang-Qing; Ito, Yuki; Bao, Xingfeng; Hemmerich, Stefan; Fukuda, Minoru; Rosen, Steven D

2011-01-01

L-selectin initiates lymphocyte interactions with high endothelial venules (HEVs) of lymphoid organs through binding to ligands with specific glycosylation modifications. 6-Sulfo sLe(x), a sulfated carbohydrate determinant for L-selectin, is carried on core 2 and extended core 1 O-glycans of HEV-expressed glycoproteins. The MECA-79 monoclonal antibody recognizes sulfated extended core 1 O-glycans and partially blocks lymphocyte-HEV interactions in lymphoid organs. Recent evidence has identified the contribution of 6-sulfo sLe(x) carried on N-glycans to lymphocyte homing in mice. Here, we characterize CL40, a novel IgG monoclonal antibody. CL40 equaled or surpassed MECA-79 as a histochemical staining reagent for HEVs and HEV-like vessels in mouse and human. Using synthetic carbohydrates, we found that CL40 bound to 6-sulfo sLe(x) structures, on both core 2 and extended core 1 structures, with an absolute dependency on 6-O-sulfation. Using transfected CHO cells and gene-targeted mice, we observed that CL40 bound its epitope on both N-glycans and O-glycans. Consistent with its broader glycan-binding, CL40 was superior to MECA-79 in blocking lymphocyte-HEV interactions in both wild-type mice and mice deficient in forming O-glycans. This superiority was more marked in human, as CL40 completely blocked lymphocyte binding to tonsillar HEVs, whereas MECA-79 inhibited only 60%. These findings extend the evidence for the importance of N-glycans in lymphocyte homing in mouse and indicate that this dependency also applies to human lymphoid organs. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Derivation of Cinnamon Blocks Leukocyte Attachment by Interacting with Sialosides

PubMed Central

Lin, Wei-Ling; Guu, Shih-Yun; Tsai, Chan-Chuan; Prakash, Ekambaranellore; Viswaraman, Mohan; Chen, Hsing-Bao; Chang, Chuan-Fa

2015-01-01

Molecules derived from cinnamon have demonstrated diverse pharmacological activities against infectious pathogens, diabetes and inflammatory diseases. This study aims to evaluate the effect of the cinnamon-derived molecule IND02 on the adhesion of leukocytes to host cells. The anti-inflammatory ability of IND02, a pentameric procyanidin type A polyphenol polymer isolated from cinnamon alcohol extract, was examined. Pretreatment with IND02 significantly reduced the attachment of THP-1 cells or neutrophils to TNF-α-activated HUVECs or E-selectin/ICAM-1, respectively. IND02 also reduced the binding of E-, L- and P-selectins with sialosides. Furthermore, IND02 could agglutinate human red blood cells (RBC), and the agglutination could be disrupted by sialylated glycoprotein. Our findings demonstrate that IND02, a cinnamon-derived compound, can interact with sialosides and block the binding of selectins and leukocytes with sialic acids. PMID:26076445

Associations of combined polymorphisms of the platelet membrane glycoproteins Ia and IIIa and the platelet-endothelial cell adhesion molecule-1 and P-Selectin genes with IVF implantation failures.

PubMed

Vlachadis, Nikolaos; Tsamadias, Vasileios; Vrachnis, Nikolaos; Kaparos, Georgios; Vitoratos, Nikolaos; Kouskouni, Evaggelia; Economou, Emmanuel

2017-04-01

The aim of the study was to investigate the combined impact of the genetic heterogeneity of the glycoproteins Ia (GpIa) and IIIa (GpIIIa) and the platelet-endothelial cell adhesion molecule-1 (PECAM-1) and P-Selectin genes on IVF embryo transfer implantation failures (IVF-ET failures). Sixty nulligravida women with previous IVF-ET failures and 60 fertile controls were genotyped for the GpIa-C807T, GpIIIa-PlA1/PA2, PECAM-1-C373G (Leu125Val) and P-Selectin-A37674C (Thr715Pro) polymorphisms by pyrosequencing. Compared with wild-type combined homozygotes, carriers of combinations of risk alleles in two gene loci were at significantly increased risk for IVF-ET failure, whereas carriers of the combination of GpIa-807T, GpIIIa-PlA2 and PECAM-1-373G alleles had OR = 52.50 (95%CI: 4.05-680.95, p < .001). The area under the receiver-operating characteristic curve (AUC) based on the number of polymorphisms and the number of risk alleles per subject was 75.4% (95%CI: 66.7%-82.8%, p < .001) and 72.5% (95%CI: 63.6%-80.3%, p < .001), respectively. The OR per polymorphism and risk allele increase was 4.26 (95%CI: 2.15-8.41, p < .001) and 2.85 (95%CI: 1.71-4.76, p < .001), respectively. The above associations were more robust among younger women. The combined analysis of these polymorphisms revealed strong association of combined carriers with IVF-ET failures especially for younger women and provided a genetic risk score with good diagnostic accuracy in the prediction of IVF-ET failures.

Definition of molecular determinants of prostate cancer cell bone extravasation.

PubMed

Barthel, Steven R; Hays, Danielle L; Yazawa, Erika M; Opperman, Matthew; Walley, Kempland C; Nimrichter, Leonardo; Burdick, Monica M; Gillard, Bryan M; Moser, Michael T; Pantel, Klaus; Foster, Barbara A; Pienta, Kenneth J; Dimitroff, Charles J

2013-01-15

Advanced prostate cancer commonly metastasizes to bone, but transit of malignant cells across the bone marrow endothelium (BMEC) remains a poorly understood step in metastasis. Prostate cancer cells roll on E-selectin(+) BMEC through E-selectin ligand-binding interactions under shear flow, and prostate cancer cells exhibit firm adhesion to BMEC via β1, β4, and αVβ3 integrins in static assays. However, whether these discrete prostate cancer cell-BMEC adhesive contacts culminate in cooperative, step-wise transendothelial migration into bone is not known. Here, we describe how metastatic prostate cancer cells breach BMEC monolayers in a step-wise fashion under physiologic hemodynamic flow. Prostate cancer cells tethered and rolled on BMEC and then firmly adhered to and traversed BMEC via sequential dependence on E-selectin ligands and β1 and αVβ3 integrins. Expression analysis in human metastatic prostate cancer tissue revealed that β1 was markedly upregulated compared with expression of other β subunits. Prostate cancer cell breaching was regulated by Rac1 and Rap1 GTPases and, notably, did not require exogenous chemokines as β1, αVβ3, Rac1, and Rap1 were constitutively active. In homing studies, prostate cancer cell trafficking to murine femurs was dependent on E-selectin ligand, β1 integrin, and Rac1. Moreover, eliminating E-selectin ligand-synthesizing α1,3 fucosyltransferases in transgenic adenoma of mouse prostate mice dramatically reduced prostate cancer incidence. These results unify the requirement for E-selectin ligands, α1,3 fucosyltransferases, β1 and αVβ3 integrins, and Rac/Rap1 GTPases in mediating prostate cancer cell homing and entry into bone and offer new insight into the role of α1,3 fucosylation in prostate cancer development.

High-polyphenol chocolate reduces endothelial dysfunction and oxidative stress during acute transient hyperglycaemia in Type 2 diabetes: a pilot randomized controlled trial.

PubMed

Mellor, D D; Madden, L A; Smith, K A; Kilpatrick, E S; Atkin, S L

2013-04-01

To investigate the effects of high-polyphenol chocolate upon endothelial function and oxidative stress in Type 2 diabetes mellitus during acute transient hyperglycaemia induced following a 75-g oral glucose challenge. Ten subjects with Type 2 diabetes underwent a double-blinded randomized controlled crossover study. A 75-g oral glucose load was used to induce hyperglycaemia, which was administered to participants 60 min after they had ingested either low (control) or high-polyphenol chocolate. Participants undertook testing at weekly intervals, following an initial cocoa-free period. Endothelial function was assessed by both functional [reactive hyperaemia peripheral artery tonometry (EndoPAT-2000) and serum markers (including intercellular adhesion molecule 1, P-selectin and P-selectin glycoprotein ligand 1]. Urinary 15-F2t-isoprostane adjusted for creatinine was used as an oxidative stress marker. Measurements were made at baseline and 2 h post-ingestion of the glucose load. Prior consumption of high-polyphenol chocolate before a glucose load improved endothelial function (1.7 ± 0.1 vs. 2.3 ± 0.1%, P = 0.01), whereas prior consumption of control chocolate resulted in a significant increase in intercellular adhesion molecule 1 (321.1 ± 7.6 vs. 373.6 ± 10.5 ng/ml, P = 0.04) and 15-F2t-isoprostane (116.8 ± 5.7 vs. 207.1 ± 5.7 mg/mol, P = 0.02). Analysis of percentage changes from baseline comparing control and high-polyphenol chocolate showed a significant improvement for high-polyphenol chocolate in both measures of endothelial function (P < 0.05) and for urinary 15-F2t-isoprostane (P = 0.04). High-polyphenol chocolate protected against acute hyperglycaemia-induced endothelial dysfunction and oxidative stress in individuals with Type 2 diabetes mellitus. © 2012 The Authors. Diabetic Medicine © 2012 Diabetes UK.

An automatic braking system that stabilizes leukocyte rolling by an increase in selectin bond number with shear.

PubMed

Chen, S; Springer, T A

1999-01-11

Wall shear stress in postcapillary venules varies widely within and between tissues and in response to inflammation and exercise. However, the speed at which leukocytes roll in vivo has been shown to be almost constant within a wide range of wall shear stress, i.e., force on the cell. Similarly, rolling velocities on purified selectins and their ligands in vitro tend to plateau. This may be important to enable rolling leukocytes to be exposed uniformly to activating stimuli on endothelium, independent of local hemodynamic conditions. Wall shear stress increases the rate of dissociation of individual selectin-ligand tether bonds exponentially (, ) thereby destabilizing rolling. We find that this is compensated by a shear-dependent increase in the number of bonds per rolling step. We also find an increase in the number of microvillous tethers to the substrate. This explains (a) the lack of firm adhesion through selectins at low shear stress or high ligand density, and (b) the stability of rolling on selectins to wide variation in wall shear stress and ligand density, in contrast to rolling on antibodies (). Furthermore, our data successfully predict the threshold wall shear stress below which rolling does not occur. This is a special case of the more general regulation by shear of the number of bonds, in which the number of bonds falls below one.

E-selectin ligand-1 controls circulating prostate cancer cell rolling/adhesion and metastasis

PubMed Central

Yasmin-Karim, Sayeda; King, Michael R.; Messing, Edward M.; Lee, Yi-Fen

2014-01-01

Circulating prostate cancer (PCa) cells preferentially roll and adhere on bone marrow vascular endothelial cells, where abundant E-selectin and stromal cell-derived factor 1 (SDF-1) are expressed, subsequently initiating a cascade of activation events that eventually lead to the development of metastases. To elucidate the roles of circulating PCa cells' rolling and adhesion behaviors in cancer metastases, we applied a dynamic cylindrical flow-based microchannel device that is coated with E-selectin and SDF-1, mimicking capillary endothelium. Using this device we captured a small fraction of rolling PCa cells. These rolling cells display higher static adhesion ability, more aggressive cancer phenotypes and stem-like properties. Importantly, mice received rolling PCa cells, but not floating PCa cells, developed cancer metastases. Genes coding for E-selectin ligands and genes associated with cancer stem cells and metastasis were elevated in rolling PCa cells. Knock down of E-selectin ligand 1(ESL-1), significantly impaired PCa cells' rolling capacity and reduced cancer aggressiveness. Moreover, ESL-1 activates RAS and MAP kinase signal cascade, consequently inducing the downstream targets. In summary, circulating PCa cells' rolling capacity contributes to PCa metastasis, and that is in part controlled by ESL-1. PMID:25301730

Haploinsufficiency of E-selectin ligand-1 is Associated with Reduced Atherosclerotic Plaque Macrophage Content while Complete Deficiency Leads to Early Embryonic Lethality in Mice

PubMed Central

Luo, Wei; Wang, Hui; Guo, Chiao; Wang, Jintao; Kwak, Jeffrey; Bahrou, Kristina L; Eitzman, Daniel T.

2012-01-01

E-selectin-1 (ESL-1), also known as golgi complex-localized glycoprotein-1 (GLG1), homocysteine-rich fibroblast growth factor receptor (CGR-1), and latent transforming growth factor-β complex protein 1 (LTCP-1), is a multifunctional protein with widespread tissue distribution. To determine the functional consequences of ESL-1 deficiency, mice were generated carrying an ESL-1 gene trap. After backcrossing to C57BL6/J for 6 generations, mice heterozygous for the gene trap (ESL-1+/-) were intercrossed to produce ESL-1-/- mice, however ESL-1-/- mice were not viable, even at embryonic day E10.5. To determine the effect of heterozygous ESL-1 deficiency on atherosclerosis, apolipoprotein E deficient (ApoE-/-), ESL-1+/- mice were generated and fed western diet. Compared to ApoE-/-, ESL-1++ mice, atherosclerotic lesions from ApoE-/-, ESL-1+/- contained more collagen and fewer macrophages, suggesting increased plaque stability. In conclusion, heterozygous deficiency of ESL-1 is associated with features of increased atherosclerotic plaque stability while complete deficiency of ESL-1 leads to embryonic lethality. PMID:22939356

SAP Suppresses the Development of Experimental Autoimmune Encephalomyelitis in C57BL6 Mice

PubMed Central

Ji, Zhe; Ke, Zun-Ji; Geng, Jian-Guo

2012-01-01

Experimental autoimmune encephalomyelitis (EAE) is a CD4+ T cell-mediated disease of the CNS. Serum amyloid P component (SAP) is a highly conserved plasma protein named for its universal presence in amyloid deposits. Here we report SAP transgenic mice had unexpectedly attenuated EAE due to impaired encephalitogenic responses. Following induction with myelin oligodendroglial glycoprotein (MOG) peptide 35–55 in CFA, SAP transgenic mice showed reduced spinal cord inflammation with lower severity of EAE attacks as compared with control C57BL/6 mice. However in SAP-KO mice, the severity of EAE is enhanced. Adoptive transfer of Ag-restimulated T cells from wild-type to SAP transgenic mice or transfer of SAP transgenic Ag-restimulated T cells to control mice induced milder EAE. T cells from MOG-primed SAP transgenic mice showed weak proliferative responses. Furthermore, in SAP transgenic mice, there is little infiltration of CD45-positive cells in the spinal cord. In vitro, SAP suppressed the secretion of IL-2 stimulated by P-selectin, and blocked P-selectin binding to T cells. Moreover, SAP could change the affinity between α4-integrin and T cells. These data suggested that SAP could antagonize the development of the acute phase of inflammation accompanying EAE by modulating the function of P-selectin. PMID:21647172

Selectin catch-bonds mechanotransduce integrin activation and neutrophil arrest on inflamed endothelium under shear flow.

PubMed

Morikis, Vasilios A; Chase, Shannon; Wun, Ted; Chaikof, Elliot L; Magnani, John L; Simon, Scott I

2017-11-09

E-selectin extends from the plasma membrane of inflamed endothelium and serves to capture leukocytes from flowing blood via long-lived catch-bonds that support slow leukocyte rolling under shear stress. Its ligands are glycosylated with the tetrasaccharide sialyl Lewis x (sLe x ), which contributes to bond affinity and specificity. E-selectin-mediated rolling transmits signals into neutrophils that trigger activation of high-affinity β 2 -integrins necessary for transition to shear-resistant adhesion and transendothelial migration. Rivipansel is a glycomimetic drug that inhibits E-selectin-mediated vaso-occlusion induced by integrin-dependent sickle-red blood cell-leukocyte adhesion. How Rivipansel antagonizes ligand recognition by E-selectin and blocks outside-in signaling of integrin-mediated neutrophil arrest while maintaining rolling immune-surveillance is unknown. Here, we demonstrate that sLe x expressed on human L-selectin is preferentially bound by E-selectin and, on ligation, initiates secretion of MRP8/14 that binds TLR4 to elicit the extension of β 2 -integrin to an intermediate affinity state. Neutrophil rolling over E-selectin at precise shear stress transmits tension and catch-bond formation with L-selectin via sLe x , resulting in focal clusters that deliver a distinct signal to upshift β 2 -integrins to a high-affinity state. Rivipansel effectively blocked formation of selectin catch-bonds, revealing a novel mechanotransduction circuit that rapidly converts extended β 2 -integrins to high-affinity shear-resistant bond clusters with intracellular adhesion molecule 1 on inflamed endothelium.

Langerhans' cell expression of the selectin ligand, sialyl Lewis x.

PubMed Central

Ross, E L; Barker, J N; Allen, M H; Chu, A C; Groves, R W; MacDonald, D M

1994-01-01

Cellular adhesion molecules play a central role in leucocyte migration through peripheral blood and tissues. A crucial stage in these events in selectin-mediated adhesion involving E-selectin expressed on activated endothelium interacting with a range of carbohydrate ligands expressed by specific subpopulations of leucocytes. As such mechanisms may be relevant to bone marrow-derived dendritic epidermal Langerhans' cell (LC) migration, expression of these carbohydrate ligands was assessed immunocytochemically in whole skin biopsies and in epidermal cell suspensions obtained from adult humans. Double-labelling experiments revealed that sialyl Lewis x, recognized by the monoclonal antibody CSLEX1, was expressed on epidermal LC (n = 9). Furthermore, expression was enhanced at 24 hr following epicutaneous application of antigen and in the inflammatory disorder psoriasis (n = 10). E-selectin was concomitantly strongly expressed on dermal endothelium in psoriasis and allergic contact dermatitis. Intradermal injection of the T-cell-derived cytokine interferon-gamma (IFN-gamma) led to increased LC expression of sialyl Lewis x. In epidermal cell suspensions, in contrast to keratinocytes, CD1a+ cells expressed sialyl Lewis x, intensity of which was enhanced after 4 days in culture. CSLEX1 staining could be abolished and CD15 (non-sialated Lewis x) expression induced by saponification and treatment with neuraminidase. Expression of other selectin ligands was also examined. While the cutaneous lymphocyte antigen defined by the monoclonal antibody HECA-452 reacted with a small minority of LC, sialyl Lewis a and sulphatide were not expressed under any experimental conditions. These studies indicate that E-selectin-sialyl Lewis x interactions are potentially important in LC migration, both into and out of skin. Images Figure 2 Figure 3 Figure 5 Figure 6 PMID:7512530

HETEROTYPIC INTERACTIONS ENABLED BY POLARIZED NEUTROPHIL MICRODOMAINS MEDIATE THROMBO-INFLAMMATORY INJURY

PubMed Central

Hidalgo, Andrés; Chang, Jungshan; Jang, Jung-Eun; Peired, Anna J.; Chiang, Elaine Y.; Frenette, Paul S.

2009-01-01

Selectins and their ligands mediate leukocyte rolling allowing interactions with chemokines that lead to integrin activation and arrest. Here, we demonstrate that E-selectin is critical to induce a secondary wave of activating signals transduced specifically by E-selectin ligand-1, that induces polarized, activated αMβ2 integrin clusters at the leading edge of crawling neutrophils, allowing the capture of circulating erythrocytes or platelets. In a humanized model of sickle cell disease (SCD), the capture of erythrocytes by αMβ2 microdomains leads to acute lethal vascular occlusions. In a model of transfusion-related acute lung injury, polarized neutrophils capture circulating platelets, resulting in the generation of oxidative species that produces vascular damage and lung injury. Inactivation of E-selectin or αMβ2 prevented tissue injury in both inflammatory models, suggesting broad implications of this paradigm in thrombo-inflammatory diseases. These results indicate that endothelial selectins can influence neutrophil behavior beyond its canonical rolling step through delayed, organ-damaging, polarized activation. PMID:19305412

Equid Herpesvirus Type 1 Activates Platelets

PubMed Central

Stokol, Tracy; Yeo, Wee Ming; Burnett, Deborah; DeAngelis, Nicole; Huang, Teng; Osterrieder, Nikolaus; Catalfamo, James

2015-01-01

Equid herpesvirus type 1 (EHV-1) causes outbreaks of abortion and neurological disease in horses. One of the main causes of these clinical syndromes is thrombosis in placental and spinal cord vessels, however the mechanism for thrombus formation is unknown. Platelets form part of the thrombus and amplify and propagate thrombin generation. Here, we tested the hypothesis that EHV-1 activates platelets. We found that two EHV-1 strains, RacL11 and Ab4 at 0.5 or higher plaque forming unit/cell, activate platelets within 10 minutes, causing α-granule secretion (surface P-selectin expression) and platelet microvesiculation (increased small events double positive for CD41 and Annexin V). Microvesiculation was more pronounced with the RacL11 strain. Virus-induced P-selectin expression required plasma and 1.0 mM exogenous calcium. P-selectin expression was abolished and microvesiculation was significantly reduced in factor VII- or X-deficient human plasma. Both P-selectin expression and microvesiculation were re-established in factor VII-deficient human plasma with added purified human factor VIIa (1 nM). A glycoprotein C-deficient mutant of the Ab4 strain activated platelets as effectively as non-mutated Ab4. P-selectin expression was abolished and microvesiculation was significantly reduced by preincubation of virus with a goat polyclonal anti-rabbit tissue factor antibody. Infectious virus could be retrieved from washed EHV-1-exposed platelets, suggesting a direct platelet-virus interaction. Our results indicate that EHV-1 activates equine platelets and that α-granule secretion is a consequence of virus-associated tissue factor triggering factor X activation and thrombin generation. Microvesiculation was only partly tissue factor and thrombin-dependent, suggesting the virus causes microvesiculation through other mechanisms, potentially through direct binding. These findings suggest that EHV-1-induced platelet activation could contribute to the thrombosis that occurs in clinically infected horses and provides a new mechanism by which viruses activate hemostasis. PMID:25905776

Activation states of blood eosinophils in asthma

PubMed Central

Johansson, Mats W.

2014-01-01

Asthma is characterized by airway inflammation rich in eosinophils. Airway eosinophilia is associated with exacerbations and has been suggested to play a role in airway remodeling. Recruitment of eosinophils from the circulation requires that blood eosinophils become activated, leading to their arrest on the endothelium and extravasation. Circulating eosinophils can be envisioned as potentially being in different activation states, including non-activated, pre-activated or “primed”, or fully activated. In addition, the circulation can potentially be deficient of pre-activated or activated eosinophils, because such cells have marginated on activated endothelium or extravasated into the tissue. A number of eosinophil-surface proteins, including CD69, L-selectin, intercellular adhesion molecule-1 (ICAM-1, CD54), CD44, P-selectin glycoprotein ligand-1 (PSGL-1, CD162), cytokine receptors, Fc receptors, integrins including αM integrin (CD11b), and activated conformations of Fc receptors and integrins have been proposed to report cell activation. Variation in eosinophil activation states may be associated with asthma activity. Eosinophil-surface proteins proposed to be activation markers, with a particular focus on integrins, and evidence for associations between activation states of blood eosinophils and features of asthma are reviewed here. Partial activation of β1 and β2 integrins on blood eosinophils, reported by monoclonal antibodies (mAb) N29 and KIM-127, is associated with impaired pulmonary function and airway eosinophilia, respectively, in non-severe asthma. The association with lung function does not occur in severe asthma, presumably due to greater eosinophil extravasation, specifically of activated or pre-activated cells, in severe disease. PMID:24552191

L-selectin-carbohydrate interactions: relevant modifications of the Lewis x trisaccharide.

PubMed

Sanders, W J; Katsumoto, T R; Bertozzi, C R; Rosen, S D; Kiessling, L L

1996-11-26

Protein-carbohydrate interactions are known to mediate cell-cell recognition and adhesion events. Specifically, three carbohydrate binding proteins termed selectins (E-, P-, and L-selectin) have been shown to be essential for leukocyte rolling along the vascular endothelium, the first step in the recruitment of leukocytes from the blood into inflammatory sites or into secondary lymphoid organs. Although this phenomenon is well-established, little is known about the molecular-level interactions on which it depends. All three selectins recognize sulfated and sialylated derivatives of the Lewis x [Le(x):Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc] and Lewis a [Le(a): Gal beta 1-->3(Fuc alpha 1-->4)GlcNAc] trisaccharide cores with affinities in the millimolar range, and it is believed that variants of these structures are the carbohydrate determinants of selectin recognition. Recently it was shown that the mucin GlyCAM-1, a secreted physiological ligand for L-selectin, is capped with sulfated derivatives of sialyl Lewis x [sLe(x): Sia alpha 2-->3Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc] and that sulfation is required for the high-affinity interaction between GlyCAM-1 and L-selectin. To elucidate the important sites of sulfation on Le(x) with respect to L-selectin recognition, we have synthesized six sulfated Le(x) analogs and determined their abilities to block binding of a recombinant L-selectin-Ig chimera to immobilized GlyCAM-1. Our results suggest that 6-sulfo sLe(x) binds to L-selectin with higher affinity than does sLe(x) or 6'-sulfo sLe(x) and that sulfation of sLe(x) capping groups on GlyCAM-1 at the 6-position is important for L-selectin recognition.

Detection of site specific glycosylation in proteins using flow cytometry†

PubMed Central

Jayakumar, Deepak; Marathe, Dhananjay D.; Neelamegham, Sriram

2009-01-01

We tested the possibility that it is possible to express unique peptide probes on cell surfaces and detect site-specific glycosylation on these peptides using flow cytometry. Such development can enhance the application of flow cytometry to detect and quantify post-translational modifications in proteins. To this end, the N-terminal section of the human leukocyte glycoprotein PSGL-1 (P-selectin glycoprotein ligand-1) was modified to contain a poly-histidine tag followed by a proteolytic cleavage site. Amino acids preceding the cleavage site have a single O-linked glycosylation site. The recombinant protein called PSGL-1 (HT) was expressed on the surface of two mammalian cell lines, CHO and HL-60, using a lentiviral delivery approach. Results demonstrate that the N-terminal portion of PSGL-1 (HT) can be released from these cells by protease, and the resulting peptide can be readily captured and detected using cytometry-bead assays. Using this strategy, the peptide was immunoprecipitated onto beads bearing mAbs against either the poly-histidine sequence or the human PSGL-1. The carbohydrate epitope associated with the released peptide was detected using HECA-452 and CSLEX-1, monoclonal antibodies that recognize the sialyl Lewis-X epitope. Finally, the peptide released from cells could be separated and enriched using nickel chelate beads. Overall, such an approach that combines recombinant protein expression with flow cytometry, may be useful to quantify changes in site-specific glycosylation for basic science and clinical applications. PMID:19735085

Circulating Tumor Cells from Prostate Cancer Patients Interact with E-Selectin under Physiologic Blood Flow

PubMed Central

Gakhar, Gunjan; Navarro, Vicente N.; Jurish, Madelyn; Lee, Guang Yu.; Tagawa, Scott T.; Akhtar, Naveed H.; Seandel, Marco; Geng, Yue; Liu, He; Bander, Neil H.; Giannakakou, Paraskevi; Christos, Paul J.; King, Michael R.; Nanus, David M.

2013-01-01

Hematogenous metastasis accounts for the majority of cancer-related deaths, yet the mechanism remains unclear. Circulating tumor cells (CTCs) in blood may employ different pathways to cross blood endothelial barrier and establish a metastatic niche. Several studies provide evidence that prostate cancer (PCa) cell tethering and rolling on microvascular endothelium via E-selectin/E-selectin ligand interactions under shear flow theoretically promote extravasation and contribute to the development of metastases. However, it is unknown if CTCs from PCa patients interact with E-selectin expressed on endothelium, initiating a route for tumor metastases. Here we report that CTCs derived from PCa patients showed interactions with E-selectin and E-selectin expressing endothelial cells. To examine E-selectin-mediated interactions of PCa cell lines and CTCs derived from metastatic PCa patients, we used fluorescently-labeled anti-prostate specific membrane antigen (PSMA) monoclonal antibody J591-488 which is internalized following cell-surface binding. We employed a microscale flow device consisting of E-selectin-coated microtubes and human umbilical vein endothelial cells (HUVECs) on parallel-plate flow chamber simulating vascular endothelium. We observed that J591-488 did not significantly alter the rolling behavior in PCa cells at shear stresses below 3 dyn/cm2. CTCs obtained from 31 PCa patient samples showed that CTCs tether and stably interact with E-selectin and E-selectin expressing HUVECs at physiological shear stress. Interestingly, samples collected during disease progression demonstrated significantly more CTC/E-selectin interactions than samples during times of therapeutic response (p=0.016). Analysis of the expression of sialyl Lewis X (sLex) in patient samples showed that a small subset comprising 1.9-18.8% of CTCs possess high sLex expression. Furthermore, E-selectin-mediated interactions between prostate CTCs and HUVECs were diminished in the presence of anti-E-selectin neutralizing antibody. CTC-Endothelial interactions provide a novel insight into potential adhesive mechanisms of prostate CTCs as a means to initiate metastasis. PMID:24386459

Directing stem cell trafficking via GPS.

PubMed

Sackstein, Robert

2010-01-01

The success of stem-cell-based regenerative therapeutics critically hinges on delivering relevant stem/progenitor cells to sites of tissue injury. To achieve adequate parenchymal infiltration following intravascular administration, it is first necessary that circulating cells bind to target tissue endothelium with sufficient strength to overcome the prevailing forces of hemodynamic shear. The principal mediators of these shear-resistant binding interactions consist of a family of C-type lectins known as "selectins" that bind discrete sialofucosylated glycans on their respective ligands. One member of this family, E-selectin, is an endothelial molecule that is inducibly expressed on postcapillary venules at all sites of tissue injury, but is also constitutively expressed on the luminal surface of bone marrow and dermal microvascular endothelium. Most stem/progenitor cells express high levels of CD44, and, in particular, human hematopoietic stem cells express a specialized sialofucosylated glycoform of CD44 known as "hematopoietic cell E-/L-selectin ligand" (HCELL) that functions as a potent E-selectin ligand. This chapter describes a method called "glycosyltransferase-programmed stereosubstitution" (GPS) for custom-modifying CD44 glycans to create HCELL on the surface of living cells that natively lack HCELL. Ex vivo glycan engineering of HCELL via GPS licenses trafficking of infused cells to endothelial beds that express E-selectin, thereby enabling efficient vascular delivery of stem/progenitor cells to sites where they are needed. Copyright (c) 2010 Elsevier Inc. All rights reserved.

E-selectin mediates stem cell adhesion and formation of blood vessels in a murine model of infantile hemangioma.

PubMed

Smadja, David M; Mulliken, John B; Bischoff, Joyce

2012-12-01

Hemangioma stem cells (HemSCs) are multipotent cells isolated from infantile hemangioma (IH), which form hemangioma-like lesions when injected subcutaneously into immune-deficient mice. In this murine model, HemSCs are the primary target of corticosteroid, a mainstay therapy for problematic IH. The relationship between HemSCs and endothelial cells that reside in IH is not clearly understood. Adhesive interactions might be critical for the preferential accumulation of HemSCs and/or endothelial cells in the tumor. Therefore, we studied the interactions between HemSCs and endothelial cells (HemECs) isolated from IH surgical specimens. We found that HemECs isolated from proliferating phase IH, but not involuting phase, constitutively express E-selectin, a cell adhesion molecule not present in quiescent endothelial cells. E-selectin was further increased when HemECs were exposed to vascular endothelial growth factor-A or tumor necrosis factor-α. In vitro, HemSC migration and adhesion was enhanced by recombinant E-selectin but not P-selectin; both processes were neutralized by E-selectin-blocking antibodies. E-selectin-positive HemECs also stimulated migration and adhesion of HemSCs. In vivo, neutralizing antibodies to E-selectin strongly inhibited formation of blood vessels when HemSCs and HemECs were co-implanted in Matrigel. These data suggest that endothelial E-selectin could be a major ligand for HemSCs and thereby promote cellular interactions and vasculogenesis in IH. We propose that constitutively expressed E-selectin on endothelial cells in the proliferating phase is one mediator of the stem cell tropism in IH. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Comparison of platelet function and viscoelastic test results between healthy dogs and dogs with naturally occurring chronic kidney disease.

PubMed

Dudley, Alicia; Byron, Julie K; Burkhard, Mary Jo; Warry, Emma; Guillaumin, Julien

2017-05-01

OBJECTIVE To compare platelet function and viscoelastic test results between healthy dogs and dogs with chronic kidney disease (CKD) to assess whether dogs with CKD have platelet dysfunction and altered blood coagulation. ANIMALS 10 healthy control dogs and 11 dogs with naturally occurring CKD. PROCEDURES Blood and urine were collected once from each dog for a CBC, serum biochemical analysis, urinalysis, and determination of the urine protein-to-creatinine ratio, prothrombin time, activated partial thromboplastin time, plasma fibrinogen concentration, and antithrombin activity. Closure time was determined by use of a platelet function analyzer and a collagen-ADP platelet agonist. Thromboelastography (TEG) variables (reaction time, clotting time, α angle, maximum amplitude, and global clot strength [G value]) were determined by use of recalcified nonactivated TEG. Platelet expression of glycoprotein Ib (GPIb; receptor for von Willebrand factor), integrin αIIbβ3 (αIIbβ3; receptor for fibrinogen), and P-selectin (marker for platelet activation) was assessed by flow cytometry. RESULTS Compared with healthy control dogs, the median closure time was prolonged, the median maximum amplitude and G value were increased, and the median clotting time was decreased for dogs with CKD. Platelet expression of both αIIbβ3 and P-selectin was also significantly increased for dogs with CKD, compared with that for control dogs. Platelet expression of GPIb, αIIbβ3, and P-selectin was not correlated with closure time or any TEG variable. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that dogs with CKD frequently had evidence of platelet dysfunction and hypercoagulability that were not totally attributable to alterations in platelet surface expression of GPIb, αIIbβ3, and P-selectin.

Soluble P-selectin rescues mice from anthrax lethal toxin-induced mortality through PSGL-1 pathway-mediated correction of hemostasis.

PubMed

Sun, Der-Shan; Chang, Yao-Wen; Kau, Jyh-Hwa; Huang, Hsin-Hsien; Ho, Pei-Hsun; Tzeng, Yin-Jeh; Chang, Hsin-Hou

2017-10-03

As one of the virulence factors of Bacillus anthracis, lethal toxin (LT) induces various pathogenic responses including the suppression of the coagulation system. In this study, we observed that LT markedly increased the circulating soluble P-selectin (sP-sel) levels and microparticle (MP) count in wild-type but not P-selectin (P-sel, Selp -/- ) or P-sel ligand-1 (PSGL-1, Selplg -/- ) knockout mice. Because sP-sel induces a hypercoagulable state through PSGL-1 pathway to generate tissue factor-positive MPs, we hypothesized that the increase in plasma sP-sel levels can be a self-rescue response in hosts against the LT-mediated suppression of the coagulation system. In agreement with our hypothesis, our results indicated that compared with wild-type mice, Selp -/- and Selplg -/- mice were more sensitive to LT. In addition, the recombinant sP-sel treatment markedly ameliorated LT-mediated pathogenesis and reduced mortality. As a result, elicitation of circulating sP-sel is potentially a self-rescue response, which is beneficial to host recovery from an LT-induced hypocoagulation state. These results suggest that the administration of sP-sel is likely to be useful in the development of a new strategy to treat anthrax.

Circulating interleukin-6 and high-sensitivity C-reactive protein decrease after periodontal therapy in otherwise healthy subjects.

PubMed

Marcaccini, Andrea M; Meschiari, César A; Sorgi, Carlos A; Saraiva, Maria C P; de Souza, Ana M; Faccioli, Lúcia H; Tanus-Santos, José E; Novaes, Arthur B; Gerlach, Raquel F

2009-04-01

Periodontal disease has been associated with many chronic inflammatory systemic diseases, and a common chronic inflammation pathway has been suggested for these conditions. However, few studies have evaluated whether periodontal disease, in the absence of other known inflammatory conditions and smoking, affects circulating markers of chronic inflammation. This study compared chronic inflammation markers in control individuals and patients with periodontal disease and observed whether non-surgical periodontal therapy affected inflammatory disease markers after 3 months. Plasma and serum of 20 controls and 25 patients with periodontal disease were obtained prior to and 3 months after non-surgical periodontal therapy. All patients were non-smokers, they did not use any medication, and they had no history or detectable signs and symptoms of systemic diseases. Periodontal and systemic parameters included probing depth, bleeding on probing, clinical attachment level, hematologic parameters, as well as the following inflammatory markers: interleukin (IL)-6, high-sensitivity C-reactive protein (hs-CRP), CD40 ligand, monocyte chemoattractant protein (MCP)-1, soluble P-selectin (sP-selectin), soluble vascular adhesion molecule (sVCAM)-1, and soluble intercellular adhesion molecule (sICAM)-1. There were no differences in the hematologic parameters of the patients in the control and periodontal disease groups. Among the tested inflammatory markers, IL-6 concentrations were higher in the periodontal disease group at baseline compared to the controls (P = 0.006). Therapy was highly effective (P <0.001 for all the analyzed clinical parameters), and a decrease in circulating IL-6 and hs-CRP concentrations was observed 3 months after therapy (P = 0.001 and P = 0.006, respectively). Our results also suggest that the CD40 ligand marker may have been different in the control and periodontal disease groups prior to the therapy (P = 0.009). In apparently otherwise healthy patients, periodontal disease is associated with increased circulating concentrations of IL-6 and hs-CRP, which decreased 3 months after non-surgical periodontal therapy. With regard to the CD40 ligand, MCP-1, sP-selectin, sVCAM-1, and sICAM-1, no changes were seen in the periodontal disease group between baseline and 3 months after therapy.

Correlation and association between plasma platelet-, monocyte- and endothelial cell-derived microparticles in hypertensive patients with type 2 diabetes mellitus.

PubMed

Nomura, Shosaku; Inami, Norihito; Shouzu, Akira; Urase, Fumiaki; Maeda, Yasuhiro

2009-09-01

Elevated platelet-derived mircoparticles (MP) (PDMP), endothelial cell-derived MP (EDMP), and monocyte-derived MP (MDMP) concentrations are documented in almost all thrombotic diseases. However, the intricate interactions between PDMP, MDMP and EDMP in hypertensive patients with or without type 2 diabetes remains poorly understood. Therefore, to clarify the correlation and association of MPs, we measured and analysed the levels of MPs in 359 hypertensive patients. We compared the results of chemokines, cell adhesion molecules, platelet activation markers and microparticles in hypertensive patients with and without type 2 diabetes mellitus. The levels of all markers were significantly higher in the hypertensive patients with diabetes than in the non-diabetic patients. For hypertensive patients with diabetes, univariate analysis showed that age, body mass index, systolic blood pressure, high density lipoprotein cholesterol (HDL-CHO), creatinine (CRTN), soluble P-selectin (sP-selectin), soluble E-selectin (sE-selectin), soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble CD40 ligand (sCD40L), regulated on activation normally T-cell expressed and secreted (RANTES), monocyte chemotactic peptide-1 (MCP-1), MDMP and EDMP were significantly associated with PDMP. In addition, systolic blood pressure, HDL cholesterol, sP-selectin, sE-selectin, sVCAM-1, sCD40L, RANTES, MDMP and EDMP were significant factors in the multivariate model with PDMP. Furthermore, a correlation between plasma PDMP and MDMP or EDMP in hypertensive patients were observed both with and without diabetes. These results suggest that the existence of diabetes mellitus affects PDMP generation in hypertensive patients and that enhanced plasma levels of PDMP and an association between the plasma levels of PDMP, MDMP and EDMP may result in the development of atherothrombotic complications in hypertensive patients.

Minimal sulfated carbohydrates for recognition by L-selectin and the MECA-79 antibody.

PubMed

Bruehl, R E; Bertozzi, C R; Rosen, S D

2000-10-20

Sulfated forms of sialyl-Le(X) containing Gal-6-SO(4) or GlcNAc-6-SO(4) have been implicated as potential recognition determinants on high endothelial venule ligands for L-selectin. The optimal configuration of sulfate esters on the N-acetyllactosamine (Galbeta1-->4GlcNAc) core of sulfosialyl-Le(X), however, remains unsettled. Using a panel of sulfated lactose (Galbeta1-->4Glc) neoglycolipids as substrates in direct binding assays, we found that 6',6-disulfolactose was the preferred structure for L-selectin, although significant binding to 6'- and 6-sulfolactose was observed as well. Binding was EDTA-sensitive and blocked by L-selectin-specific monoclonal antibodies. Surprisingly, 6', 6-disulfolactose was poorly recognized by MECA-79, a carbohydrate- and sulfate-dependent monoclonal antibody that binds competitively to L-selectin ligands. Instead, MECA-79 bound preferentially to 6-sulfolactose. The difference in preferred substrates between L-selectin and MECA-79 may explain the variable activity of MECA-79 as an inhibitor of lymphocyte adhesion to high endothelial venules in lymphoid organs. Our results suggest that both Gal-6-SO(4) and GlcNAc-6-SO(4) may contribute to L-selectin recognition, either as components of sulfosialyl-Le(X) capping groups or in internal structures. By contrast, only GlcNAc-6-SO(4) appears to contribute to MECA-79 binding.

The role of dimension in multivalent binding events: structure-activity relationship of dendritic polyglycerol sulfate binding to L-selectin in correlation with size and surface charge density.

PubMed

Weinhart, Marie; Gröger, Dominic; Enders, Sven; Riese, Sebastian B; Dernedde, Jens; Kainthan, Rajesh K; Brooks, Donald E; Haag, Rainer

2011-08-11

L-, P-, and E-Selectin are cell adhesion molecules that play a crucial role in leukocyte recruitment from the blood stream to the afflicted tissue in an acute and chronic inflammatory setting. Since selectins mediate the initial contact of leukocytes to the vascular endothelium, they have evolved as a valuable therapeutic target in diseases related to inflammation by inhibition of the physiological selectin-ligand interactions. In a previous study, it was demonstrated that dPGS, a fully synthetic heparin analogue, works as an efficient inhibitor towards L- and P-selectin in vitro as well as in vivo. Herein, the focus is directed towards the effect of size and charge density of the polyanion. The efficiency of L-selectin inhibition via an SPR-based in vitro assay and a cell-based flow chamber assay is investigated with dPGS ranging from approximately 4 to 2000 kDa. SPR measurements show that the inhibitory potential of highly sulfated dPGS increases with size and charge density. Thereby, IC(50) values from the micromolar to the low picomolar range are determined. The same tendency could be observed in a cell-based flow chamber assay with three representative dPGS samples. This structure-affinity relationship of dPGS suggests that the strong inhibitory potential of dPGS is not only based on the strong electrostatic interaction with areas of cationic surface potential on L-selectin but is also due to a steric shielding of the carbohydrate binding site by large, flexible dPGS particles. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An Automatic Braking System That Stabilizes Leukocyte Rolling by an Increase in Selectin Bond Number with Shear

PubMed Central

Chen, Shuqi; Springer, Timothy A.

1999-01-01

Wall shear stress in postcapillary venules varies widely within and between tissues and in response to inflammation and exercise. However, the speed at which leukocytes roll in vivo has been shown to be almost constant within a wide range of wall shear stress, i.e., force on the cell. Similarly, rolling velocities on purified selectins and their ligands in vitro tend to plateau. This may be important to enable rolling leukocytes to be exposed uniformly to activating stimuli on endothelium, independent of local hemodynamic conditions. Wall shear stress increases the rate of dissociation of individual selectin–ligand tether bonds exponentially (1, 4) thereby destabilizing rolling. We find that this is compensated by a shear-dependent increase in the number of bonds per rolling step. We also find an increase in the number of microvillous tethers to the substrate. This explains (a) the lack of firm adhesion through selectins at low shear stress or high ligand density, and (b) the stability of rolling on selectins to wide variation in wall shear stress and ligand density, in contrast to rolling on antibodies (14). Furthermore, our data successfully predict the threshold wall shear stress below which rolling does not occur. This is a special case of the more general regulation by shear of the number of bonds, in which the number of bonds falls below one. PMID:9885254

E-Selectin Mediates Stem Cell Adhesion and Formation of Blood Vessels in a Murine Model of Infantile Hemangioma

PubMed Central

Smadja, David M.; Mulliken, John B.; Bischoff, Joyce

2013-01-01

Hemangioma stem cells (HemSCs) are multipotent cells isolated from infantile hemangioma (IH), which form hemangioma-like lesions when injected subcutaneously into immune-deficient mice. In this murine model, HemSCs are the primary target of corticosteroid, a mainstay therapy for problematic IH. The relationship between HemSCs and endothelial cells that reside in IH is not clearly understood. Adhesive interactions might be critical for the preferential accumulation of HemSCs and/or endothelial cells in the tumor. Therefore, we studied the interactions between HemSCs and endothelial cells (HemECs) isolated from IH surgical specimens. We found that HemECs isolated from proliferating phase IH, but not involuting phase, constitutively express E-selectin, a cell adhesion molecule not present in quiescent endothelial cells. E-selectin was further increased when HemECs were exposed to vascular endothelial growth factor–A or tumor necrosis factor–α. In vitro, HemSC migration and adhesion was enhanced by recombinant E-selectin but not P-selectin; both processes were neutralized by E-selectin–blocking antibodies. E-selectin–positive HemECs also stimulated migration and adhesion of HemSCs. In vivo, neutralizing antibodies to E-selectin strongly inhibited formation of blood vessels when HemSCs and HemECs were co-implanted in Matrigel. These data suggest that endothelial E-selectin could be a major ligand for HemSCs and thereby promote cellular interactions and vasculogenesis in IH. We propose that constitutively expressed E-selectin on endothelial cells in the proliferating phase is one mediator of the stem cell tropism in IH. PMID:23041613

Influence of haplotypes, gene expression and soluble levels of L-selectin on the risk of acute coronary syndrome.

PubMed

Sandoval-Pinto, Elena; Padilla-Gutiérrez, Jorge Ramón; Hernández-Bello, Jorge; Martínez-Fernández, Diana Emilia; Valdés-Alvarado, Emmanuel; Muñoz-Valle, José Francisco; Flores-Salinas, H E; Valle, Yeminia

2017-08-20

L-selectin gene (SELL) is a candidate gene for the development of acute coronary syndrome (ACS) that contributes to endothelial dysfunction. The -642C>T (rs2205849) and 725C>T (rs2229569) polymorphisms have been associated with changes in gene expression, ligand affinity and increased risk of cardiovascular disease. The aim of this study was to investigate the association between the haplotypes constructed with the -642C>T and 725C>T polymorphisms of the SELL gene, the expression levels of its mRNA and the serum levels of soluble L-selectin with ACS. We recruited 615 individuals of Mexican origin matched by age, including 342 patients with ACS and 273 individuals without personal history of ischemic cardiopathy as control group (CG). Genotyping was performed by PCR-RFLP. The qPCR technique was used to analyze the expression of mRNA using TaqMan® UPL probes. The levels of soluble L-selectin were measured with ELISA. The allele variants in both polymorphisms were over-represented in the CG compared to the ACS (OR range: 0.371-0.716, p<0.006). The CT and TT haplotypes had a protective effect against the development of ACS (OR=0.401, p<0.0001; OR=0.628, p<0.0001, respectively). SELL expression was 3.076 times higher in the ACS group compared to CG (p<0.001). The levels of soluble L-selectin were similar between ACS and CG. Both polymorphisms had no effect on mRNA expression and soluble protein levels. The polymorphisms -642C>T and 725C>T of the SELL gene are protective factors against the development of ACS. There is an increased gene expression of L-selectin in ACS compared to CG in the population of Western Mexico. Copyright © 2017 Elsevier B.V. All rights reserved.

E-selectin liposomal and nanotube-targeted delivery of doxorubicin to circulating tumor cells

PubMed Central

Mitchell, Michael J.; Chen, Christina S.; Ponmudi, Varun; Hughes, Andrew D.; King, Michael R.

2012-01-01

The presence of circulating tumor cells (CTCs) is believed to lead to the formation of secondary tumors via an adhesion cascade involving interaction between adhesion receptors of endothelial cells and ligands on CTCs. Many CTCs express sialylated carbohydrate ligands on their surfaces that adhere to selectin protein found on inflamed endothelial cells. We have investigated the feasibility of using immobilized selectin proteins as a targeting mechanism for CTCs under flow. Herein, targeted liposomal doxorubicin (L-DXR) was functionalized with recombinant human E-selectin (ES) and polyethylene glycol (PEG) to target and kill cancer cells under shear flow, both when immobilized along a microtube device or sheared in a cone-and-plate viscometer in a dilute suspension. Healthy circulating cells such as red blood cells were not targeted by this mechanism and were left to freely circulate, and minimal leukocyte death was observed. Halloysite nanotube (HNT)-coated microtube devices immobilized with nanoscale liposomes significantly enhanced the targeting, capture, and killing of cancer cells. This work demonstrates that E-selectin functionalized L-DXR, sheared in suspension or immobilized onto microtube devices, provides a novel approach to selectively target and deliver chemotherapeutics to CTCs in the bloodstream. PMID:22421423

IL-5 Induces Suspended Eosinophils to Undergo Unique Global Reorganization Associated with Priming

PubMed Central

Han, Shih-Tsung

2014-01-01

The experiments described herein define a unique program of polarization of suspended human eosinophils stimulated with IL-5 family cytokines. We found that eosinophil granules and the nucleus move in opposite directions to form, respectively, a granular compartment and the nucleopod, a specialized uropod occupied by the nucleus and covered with adhesion receptors, including P-selectin glycoprotein ligand-1, CD44, and activated αMβ2 integrin. Ligated IL-5 family receptors localize specifically at the tip of the nucleopod in proximity to downstream signaling partners Janus tyrosine kinase 2, signal transducer and activator of transcription-1 and -5, and extracellular signal–regulated kinase. Microscopy and effects of cytochalasin B and nocodazole indicate that remodeling of filamentous actin and reorientation of the microtubule network are required for eosinophil polarization and nucleopod formation. IL-5 induces persistent polarization and extracellular signal–regulated kinase redistribution that are associated with eosinophil priming, a robust response on subsequent stimulation with N-formyl-methionyl-leucyl-phenylalanine. Global reorganization of cytoskeleton, organelles, adhesion receptors, and signaling molecules likely facilitates vascular arrest, extravasation, migration, granule release, and survival of eosinophils entering inflamed tissues from the bloodstream. PMID:24156300

Microvascular Channel Device to Study Aggressiveness in Prostate Cancer Metastasis

DTIC Science & Technology

2014-08-01

contributes to PCa’s distant metastasis, which is mediated via an E- selectin ligand, ESL -1. Consequently, the interaction of E-selectin/ ESL -1 transduces...cancer cell, E-selectin, ESL -1. OVERALL PROJECT SUMMARY A. Major goals of the project: 3 Task 1: Correlation of cancers’ aggressiveness with...Determination of the aggressive/metastatic related gene I. 1. ESL -1 expression is high in rolling cells and tissue When PCa cells come in contact with

Standardizing a simpler, more sensitive and accurate tail bleeding assay in mice

PubMed Central

Liu, Yang; Jennings, Nicole L; Dart, Anthony M; Du, Xiao-Jun

2012-01-01

AIM: To optimize the experimental protocols for a simple, sensitive and accurate bleeding assay. METHODS: Bleeding assay was performed in mice by tail tip amputation, immersing the tail in saline at 37 °C, continuously monitoring bleeding patterns and measuring bleeding volume from changes in the body weight. Sensitivity and extent of variation of bleeding time and bleeding volume were compared in mice treated with the P2Y receptor inhibitor prasugrel at various doses or in mice deficient of FcRγ, a signaling protein of the glycoprotein VI receptor. RESULTS: We described details of the bleeding assay with the aim of standardizing this commonly used assay. The bleeding assay detailed here was simple to operate and permitted continuous monitoring of bleeding pattern and detection of re-bleeding. We also reported a simple and accurate way of quantifying bleeding volume from changes in the body weight, which correlated well with chemical assay of hemoglobin levels (r2 = 0.990, P < 0.0001). We determined by tail bleeding assay the dose-effect relation of the anti-platelet drug prasugrel from 0.015 to 5 mg/kg. Our results showed that the correlation of bleeding time and volume was unsatisfactory and that compared with the bleeding time, bleeding volume was more sensitive in detecting a partial inhibition of platelet’s haemostatic activity (P < 0.01). Similarly, in mice with genetic disruption of FcRγ as a signaling molecule of P-selectin glycoprotein ligand-1 leading to platelet dysfunction, both increased bleeding volume and repeated bleeding pattern defined the phenotype of the knockout mice better than that of a prolonged bleeding time. CONCLUSION: Determination of bleeding pattern and bleeding volume, in addition to bleeding time, improved the sensitivity and accuracy of this assay, particularly when platelet function is partially inhibited. PMID:24520531

Bifidobacteria isolated from infants and cultured on human milk oligosaccharides affect intestinal epithelial function

PubMed Central

Chichlowski, Maciej; De Lartigue, Guillaume; German, J. Bruce; Raybould, Helen E.; Mills, David A.

2012-01-01

Objectives Human milk oligosaccharides (HMO) are the third most abundant component of breast milk. Our laboratory has previously revealed gene clusters specifically linked to HMO metabolism in select bifidobacteria isolated from fecal samples of infants. Our objective was to test the hypothesis that growth of select bifidobacteria on HMO stimulates the intestinal epithelium. Methods Caco-2 and HT-29 cells were incubated with lactose (LAC) or HMO-grown Bifidobacterium longum subsp. infantis (B. infantis) or B. bifidum. Bacterial adhesion and translocation was measured by real-time quantitative PCR. Expression of pro- and anti-inflammatory cytokines and tight junction proteins was analyzed by real time reverse transcriptase. Distribution of tight junction proteins was measured using immunofluorescent microscopy. Results We showed that HMO-grown B. infantis had significantly higher rate of adhesion to HT-29 cells compared to B. bifidum. B. infantis also induced expression of a cell membrane glycoprotein, P-selectin glycoprotein ligand -1. Both B. infantis and B. bifidum grown on HMO caused less occludin relocalization and higher expression of anti-inflammatory cytokine, interleukin (IL)-10 compared to LAC-grown bacteria in Caco-2 cells. B. bifidum grown on HMO showed higher expression of junctional adhesion molecule and occludin in Caco-2 cell and HT-29 cells. There were no significant differences between LAC or HMO treatments in bacterial translocation. Conclusions This study provides evidence for the specific relationship between HMO-grown bifidobacteria and intestinal epithelial cells. To our knowledge, this is the first study describing HMO-induced changes in the bifidobacteria-intestinal cells interaction. PMID:22383026

A microcantilever device to assess the effect of force on the lifetime of selectin-carbohydrate bonds.

PubMed Central

Tees, D F; Waugh, R E; Hammer, D A

2001-01-01

A microcantilever technique was used to apply force to receptor-ligand molecules involved in leukocyte rolling on blood vessel walls. E-selectin was adsorbed onto 3-microm-diameter, 4-mm-long glass fibers, and the selectin ligand, sialyl Lewis(x), was coupled to latex microspheres. After binding, the microsphere and bound fiber were retracted using a computerized loading protocol that combines hydrodynamic and Hookean forces on the fiber to produce a range of force loading rates (force/time), r(f). From the distribution of forces at failure, the average force was determined and plotted as a function of ln r(f). The slope and intercept of the plot yield the unstressed reverse reaction rate, k(r)(o), and a parameter that describes the force dependence of reverse reaction rates, r(o). The ligand was titrated so adhesion occurred in approximately 30% of tests, implying that >80% of adhesive events involve single bonds. Monte Carlo simulations show that this level of multiple bonding has little effect on parameter estimation. The estimates are r(o) = 0.048 and 0.016 nm and k(r)(o) = 0.72 and 2.2 s(-1) for loading rates in the ranges 200-1000 and 1000-5000 pN s(-1), respectively. Levenberg-Marquardt fitting across all values of r(f) gives r(o) = 0.034 nm and k(r)(o) = 0.82 s(-1). The values of these parameters are in the range required for rolling, as suggested by adhesive dynamics simulations. PMID:11159435

Pathophysiological levels of soluble P-selectin mediate adhesion of leukocytes to the endothelium through Mac-1 activation.

PubMed

Woollard, Kevin J; Suhartoyo, Andreas; Harris, Emma E; Eisenhardt, Steffen U; Jackson, Shaun P; Peter, Karlheinz; Dart, Anthony M; Hickey, Michael J; Chin-Dusting, Jaye P F

2008-11-07

Plasma soluble P-selectin (sP-selectin) levels are increased in pathologies associated with atherosclerosis, including peripheral arterial occlusive disease (PAOD). However, the role of sP-selectin in regulating leukocyte-endothelial adhesion is unclear. The aim of this study was to assess the ability of exogenous and endogenous sP-selectin to induce leukocyte responses that promote their adhesion to various forms of endothelium. In flow chamber assays, sP-selectin dose-dependently increased neutrophil adhesion to resting human iliac artery endothelial cells. Similarly, sP-selectin induced neutrophil adhesion to the endothelial surface of murine aortae and human radial venous segments in ex vivo flow chamber experiments. Using intravital microscopy to examine postcapillary venules in the mouse cremaster muscle, in vivo administration of sP-selectin was also found to significantly increase leukocyte rolling and adhesion in unstimulated postcapillary venules. Using a Mac-1-specific antibody and P-selectin knockout mouse, it was demonstrated that this finding was dependent on a contribution of Mac-1 to leukocyte rolling and endothelial P-selectin expression. This was confirmed in an ex vivo perfusion model using viable mouse aorta and human radial vessels. In contrast, with tumor necrosis factor-alpha-activated endothelial cells and intact endothelium, where neutrophil adhesion was already elevated, sP-selectin failed to further increase adhesion. Plasma samples from PAOD patients containing pathologically elevated concentrations of sP-selectin also increased neutrophil adhesion to the endothelium in a sP-selectin-dependent manner, as demonstrated by immunodepletion of sP-selectin. These studies demonstrate that raised plasma sP-selectin may influence the early progression of vascular disease by promoting leukocyte adhesion to the endothelium in PAOD, through Mac-1-mediated rolling and dependent on endothelial P-selectin expression.

Echocardiographic Ischemic Memory Imaging Through Complement-Mediated Vascular Adhesion of Phosphatidylserine-Containing Microbubbles.

PubMed

Mott, Brian; Packwood, William; Xie, Aris; Belcik, J Todd; Taylor, Ronald P; Zhao, Yan; Davidson, Brian P; Lindner, Jonathan R

2016-08-01

This study hypothesized that microvascular retention of phosphatidylserine-containing microbubbles (MB-PS) would allow detection of recent but resolved myocardial ischemia with myocardial contrast echocardiographic (MCE) molecular imaging. Techniques for ischemic memory imaging which can detect and spatially assess resolved myocardial ischemia are being developed for rapid evaluation of patients with chest pain. MCE molecular imaging with MB-PS was performed 1.5 h, 3.0 h, and 6.0 h after brief (10 min) myocardial ischemia in mice; data were compared to selectin-targeted microbubbles. MCE molecular imaging with Sonazoid (GE Healthcare, Amersham, United Kingdom), a commercially produced phosphatidylserine (PS) - containing agent, was performed in separate mice at 1.5 h and 3.0 h after ischemia-reperfusion; and in dogs undergoing 135 min of ischemia and 60 min of reflow as well as in closed-chest nonischemic control dogs. The mechanism for MB-PS attachment was assessed by intravital microscopy of post-ischemic muscle and by flow cytometry analysis of cell-MB interactions. In mice undergoing ischemia-reperfusion without infarction, signal enhancement in the risk area for MB-PS and p-selectin glycoprotein ligand-1-targeted microbubbles was similar at reflow times of 1.5 h (23.3 ± 7.3 IU vs. 30.7 ± 4.1 IU), 3.0 h (42.2 ± 6.2 IU vs. 33.9 ± 7.4 IU), and 6.0 h (24.1 ± 4.3 IU vs. 25.5 ± 4.7 IU). For both agents, signal in the risk area was significantly (p < 0.05) higher than remote region at all reflow times. Sonazoid also produced strong risk area enhancement at 1.5 h (34.7 ± 5.0 IU) and 3.0 h (52.5 ± 4.5 IU) which was approximately 3-fold greater than in the control region, and which correlated spatially with the microsphere-derived risk area. In dogs, Sonazoid signal in the risk area was >5-fold higher than in closed-chest control myocardium (42.2 ± 8.1 IU vs. 7.9 ± 3.3 IU; p < 0.001). Mechanistic studies indicated that MB-PS attached directly to venular endothelium and adherent leukocytes which was dependent on serum complement components C1q and C3. Ischemic memory imaging with MCE is possible using MB-PS which may obviate the need for ligand-directed targeting. Copyright © 2016 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Multiple, targeted deficiencies in selectins reveal a predominant role for P-selectin in leukocyte recruitment

PubMed Central

Robinson, Stephen D.; Frenette, Paul S.; Rayburn, Helen; Cummiskey, Marge; Ullman-Culleré, Mollie; Wagner, Denisa D.; Hynes, Richard O.

1999-01-01

We extend our previous analyses of mice deficient in selectins by describing the generation and comparative phenotype of mice lacking one, two, or three selectins after sequential ablation of the murine genes encoding P-, E-, and L-selectins. All mice deficient in selectins are viable and fertile as homozygotes. However, mice missing both P- and E-selectins (PE−/−), and mice missing all three selectins (ELP−/−) develop mucocutaneous infections that eventually lead to death. Mice deficient in multiple selectins display varying degrees of leukocytosis, resulting in part from alterations in leukocyte rolling and recruitment. PE−/− mice, ELP−/− mice, and mice missing both P- and L-selectins (PL−/−) show drastic reductions in leukocyte rolling and in extravasation of neutrophils in thioglycollate-induced peritonitis. In a separate inflammatory model (ragweed-induced peritoneal eosinophilia), we demonstrate P-selectin to be both necessary and sufficient for the recruitment of eosinophils. The phenotype of mice missing both E- and L-selectins (EL−/−) is less severe than those seen in the other double knockouts. Comparisons among the double knockouts suggest that P-selectin normally cooperates with both E- and L-selectins. Our results indicate a preeminent role for P-selectin in regulating leukocyte behavior in mice. Data from the ELP−/− mice indicate, however, that all three selectins are important to leukocyte homeostasis and efficient neutrophil recruitment. PMID:10500197

Serum concentrations of soluble (s)L- and (s)P-selectins in women with ovarian cancer.

PubMed

Majchrzak-Baczmańska, Dominika B; Głowacka, Ewa; Wilczyński, Miłosz; Malinowski, Andrzej

2018-03-01

The aim of the study was to compare serum concentration of soluble L- and P-selectins in women with ovarian cancer (OC) and healthy controls, and to investigate sL- and sP-selectin levels with regard to clinical and pathological parameters. Correlation analysis was used to measure the following: sL- and sP-selectin concentration and Ca125; sP-selectin and platelet concentrations; and sL-selectin and serum leukocyte levels in women with OC. The study included 29 patients with OC and 23 healthy controls. Serum concentrations of sL- and sP-selectins were measured in all subjects. Routine diagnostic tests: CBC and USG (both groups) and Ca125 (study group) were performed. Significantly higher serum concentrations of sL- and sP-selectins were found in the study group as compared to controls. Lower levels of serum sL-selectin were observed in women with poorly-differentiated OC (G3) and advanced stages of the disease (FIGO III, IV), but the results were statistically insignificant. No statistically significant relationship was detected between sP-selectin serum concentration in women with OC and tumour differentiation, histological type, and stage of the disease. No significant correlation was found between sL- and sP-selectins and Ca125 levels. A weak correlation was found between serum concentration of sP-selectin in women with OC and platelet count. No statistically significant correlation was observed between sL-selectin concentration and serum leukocyte levels in women with OC. The analysis of sL- and sP-selectin concentrations may be a useful tool in the diagnosis of OC. The levels of sL-selectin decrease with disease progression.

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

PubMed Central

Gutsaeva, Diana R.; Parkerson, James B.; Yerigenahally, Shobha D.; Kurz, Jeffrey C.; Schaub, Robert G.; Ikuta, Tohru

2011-01-01

Adhesive 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. PMID:20926770

In situ trans ligands of CD22 identified by glycan-protein photocross-linking-enabled proteomics.

PubMed

Ramya, T N C; Weerapana, Eranthie; Liao, Lujian; Zeng, Ying; Tateno, Hiroaki; Liao, Liang; Yates, John R; Cravatt, Benjamin F; Paulson, James C

2010-06-01

CD22, a regulator of B-cell signaling, is a siglec that recognizes the sequence NeuAcalpha2-6Gal on glycoprotein glycans as ligands. CD22 interactions with glycoproteins on the same cell (in cis) and apposing cells (in trans) modulate its activity in B-cell receptor signaling. Although CD22 predominantly recognizes neighboring CD22 molecules as cis ligands on B-cells, little is known about the trans ligands on apposing cells. We conducted a proteomics scale study to identify candidate trans ligands of CD22 on B-cells by UV photocross-linking CD22-Fc chimera bound to B-cell glycoproteins engineered to carry sialic acids with a 9-aryl azide moiety. Using mass spectrometry-based quantitative proteomics to analyze the cross-linked products, 27 glycoproteins were identified as candidate trans ligands. Next, CD22 expressed on the surface of one cell was photocross-linked to glycoproteins on apposing B-cells followed by immunochemical analysis of the products with antibodies to the candidate ligands. Of the many candidate ligands, only the B-cell receptor IgM was found to be a major in situ trans ligand of CD22 that is selectively redistributed to the site of cell contact upon interaction with CD22 on the apposing cell.

P-selectin mediates neutrophil adhesion to endothelial cell borders.

PubMed

Burns, A R; Bowden, R A; Abe, Y; Walker, D C; Simon, S I; Entman, M L; Smith, C W

1999-03-01

During an acute inflammatory response, endothelial P-selectin (CD62P) can mediate the initial capture of neutrophils from the free flowing bloodstream. P-selectin is stored in secretory granules (Weibel-Palade bodies) and is rapidly expressed on the endothelial surface after stimulation with histamine or thrombin. Because neutrophil transmigration occurs preferentially at endothelial borders, we wished to determine whether P-selectin-dependent neutrophil capture (adhesion) occurs at endothelial cell borders. Under static or hydrodynamic flow (2 dyn/cm2) conditions, histamine (10(-4) M) or thrombin (0.2 U/mL) treatment induced preferential (> or = 75%) neutrophil adhesion to the cell borders of endothelial monolayers. Blocking antibody studies established that neutrophil adhesion was completely P-selectin dependent. P-selectin surface expression increased significantly after histamine treatment and P-selectin immunostaining was concentrated along endothelial borders. We conclude that preferential P-selectin expression along endothelial borders may be an important mechanism for targeting neutrophil migration at endothelial borders.

Inflammatory Mediators in Smoke Inhalation Injury

DTIC Science & Technology

2009-01-01

during inhalation injury significantly add to the acute lung injury physiology. Since vitamin E (alpha- tocopherol ) is an oxygen superoxide scav...with nebulized alpha- tocopherol [46]. Selectin Binding Sulfo Lewis C, a sulfated oligosaccharide, is a ligand of selectins with reported activity to...depletes vitamin E: kinetic studies using deu- terated tocopherols . Free Radic. Biol. Med., 2007, 42, 1421-1429. [45] Morita, N.; Shimoda, K.; Traber

Iron oxide nanoparticles stabilized with dendritic polyglycerols as selective MRI contrast agents

NASA Astrophysics Data System (ADS)

Nordmeyer, Daniel; Stumpf, Patrick; Gröger, Dominic; Hofmann, Andreas; Enders, Sven; Riese, Sebastian B.; Dernedde, Jens; Taupitz, Matthias; Rauch, Ursula; Haag, Rainer; Rühl, Eckart; Graf, Christina

2014-07-01

Monodisperse small iron oxide nanoparticles functionalized with dendritic polyglycerol (dPG) or dendritic polyglycerol sulfate (dPGS) are prepared. They are highly stable in aqueous solutions as well as physiological media. In particular, oleic acid capped iron oxide particles (core diameter = 11 +/- 1 nm) were modified by a ligand exchange process in a one pot synthesis with dPG and dPGS bearing phosphonate as anchor groups. Dynamic light scattering measurements performed in water and different biological media demonstrate that the hydrodynamic diameter of the particles is only slightly increased by the ligand exchange process resulting in a final diameter of less than 30 nm and that the particles are stable in these media. It is also revealed by magnetic resonance studies that their magnetic relaxivity is reduced by the surface modification but it is still sufficient for high contrast magnetic resonance imaging (MRI). Additionally, incubation of dPGS functionalized iron oxide nanoparticles with human umbilical vein endothelial cells showed a 50% survival at 85 nM (concentration of nanoparticles). Surface plasmon resonance (SPR) studies demonstrate that the dPGS functionalized iron oxide nanoparticles inhibit L-selectin ligand binding whereas the particles containing only dPG do not show this effect. Experiments in a flow chamber with human myelogenous leukemia cells confirmed L-selectin inhibition of the dPGS functionalized iron oxide nanoparticles and with that the L-selectin mediated leukocyte adhesion. These results indicate that dPGS functionalized iron oxide nanoparticles are a promising contrast agent for inflamed tissue probed by MRI.Monodisperse small iron oxide nanoparticles functionalized with dendritic polyglycerol (dPG) or dendritic polyglycerol sulfate (dPGS) are prepared. They are highly stable in aqueous solutions as well as physiological media. In particular, oleic acid capped iron oxide particles (core diameter = 11 +/- 1 nm) were modified by a ligand exchange process in a one pot synthesis with dPG and dPGS bearing phosphonate as anchor groups. Dynamic light scattering measurements performed in water and different biological media demonstrate that the hydrodynamic diameter of the particles is only slightly increased by the ligand exchange process resulting in a final diameter of less than 30 nm and that the particles are stable in these media. It is also revealed by magnetic resonance studies that their magnetic relaxivity is reduced by the surface modification but it is still sufficient for high contrast magnetic resonance imaging (MRI). Additionally, incubation of dPGS functionalized iron oxide nanoparticles with human umbilical vein endothelial cells showed a 50% survival at 85 nM (concentration of nanoparticles). Surface plasmon resonance (SPR) studies demonstrate that the dPGS functionalized iron oxide nanoparticles inhibit L-selectin ligand binding whereas the particles containing only dPG do not show this effect. Experiments in a flow chamber with human myelogenous leukemia cells confirmed L-selectin inhibition of the dPGS functionalized iron oxide nanoparticles and with that the L-selectin mediated leukocyte adhesion. These results indicate that dPGS functionalized iron oxide nanoparticles are a promising contrast agent for inflamed tissue probed by MRI. Electronic supplementary information (ESI) available: A detailed description of the synthesis of the ligands as well as the preparation and functionalization of the iron oxide nanoparticles including their physico-chemical characterization are presented. Further, details of the cell experiments and the SPR experiments are given. Two representative movies are provided showing leukocyte rolling on the ligand coated surface of the flow chamber. See DOI: 10.1039/c3nr04793h

Ultrasound-mediated vascular gene transfection by cavitation of endothelial-targeted cationic microbubbles.

PubMed

Xie, Aris; Belcik, Todd; Qi, Yue; Morgan, Terry K; Champaneri, Shivam A; Taylor, Sarah; Davidson, Brian P; Zhao, Yan; Klibanov, Alexander L; Kuliszewski, Michael A; Leong-Poi, Howard; Ammi, Azzdine; Lindner, Jonathan R

2012-12-01

Ultrasound-mediated gene delivery can be amplified by acoustic disruption of microbubble carriers that undergo cavitation. We hypothesized that endothelial targeting of microbubbles bearing cDNA is feasible and, through optimizing proximity to the vessel wall, increases the efficacy of gene transfection. Contrast ultrasound-mediated gene delivery is a promising approach for site-specific gene therapy, although there are concerns with the reproducibility of this technique and the safety when using high-power ultrasound. Cationic lipid-shelled decafluorobutane microbubbles bearing a targeting moiety were prepared and compared with nontargeted microbubbles. Microbubble targeting efficiency to endothelial adhesion molecules (P-selectin or intercellular adhesion molecule [ICAM]-1) was tested using in vitro flow chamber studies, intravital microscopy of tumor necrosis factor-alpha (TNF-α)-stimulated murine cremaster muscle, and targeted contrast ultrasound imaging of P-selectin in a model of murine limb ischemia. Ultrasound-mediated transfection of luciferase reporter plasmid charge coupled to microbubbles in the post-ischemic hindlimb muscle was assessed by in vivo optical imaging. Charge coupling of cDNA to the microbubble surface was not influenced by the presence of targeting ligand, and did not alter the cavitation properties of cationic microbubbles. In flow chamber studies, surface conjugation of cDNA did not affect attachment of targeted microbubbles at microvascular shear stresses (0.6 and 1.5 dyne/cm(2)). Attachment in vivo was also not affected by cDNA according to intravital microscopy observations of venular adhesion of ICAM-1-targeted microbubbles and by ultrasound molecular imaging of P-selectin-targeted microbubbles in the post-ischemic hindlimb in mice. Transfection at the site of high acoustic pressures (1.0 and 1.8 MPa) was similar for control and P-selectin-targeted microbubbles but was associated with vascular rupture and hemorrhage. At 0.6 MPa, there were no adverse bioeffects, and transfection was 5-fold greater with P-selectin-targeted microbubbles. We conclude that ultrasound-mediated transfection at safe acoustic pressures can be markedly augmented by endothelial juxtaposition. Copyright © 2012 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Cell surface glycan engineering of neural stem cells augments neurotropism and improves recovery in a murine model of multiple sclerosis

PubMed Central

Merzaban, Jasmeen S; Imitola, Jaime; Starossom, Sarah C; Zhu, Bing; Wang, Yue; Lee, Jack; Ali, Amal J; Olah, Marta; Abuelela, Ayman F; Khoury, Samia J; Sackstein, Robert

2015-01-01

Neural stem cell (NSC)-based therapies offer potential for neural repair in central nervous system (CNS) inflammatory and degenerative disorders. Typically, these conditions present with multifocal CNS lesions making it impractical to inject NSCs locally, thus mandating optimization of vascular delivery of the cells to involved sites. Here, we analyzed NSCs for expression of molecular effectors of cell migration and found that these cells are natively devoid of E-selectin ligands. Using glycosyltransferase-programmed stereosubstitution (GPS), we glycan engineered the cell surface of NSCs (“GPS-NSCs”) with resultant enforced expression of the potent E-selectin ligand HCELL (hematopoietic cell E-/L-selectin ligand) and of an E-selectin-binding glycoform of neural cell adhesion molecule (“NCAM-E”). Following intravenous (i.v.) injection, short-term homing studies demonstrated that, compared with buffer-treated (control) NSCs, GPS-NSCs showed greater neurotropism. Administration of GPS-NSC significantly attenuated the clinical course of experimental autoimmune encephalomyelitis (EAE), with markedly decreased inflammation and improved oligodendroglial and axonal integrity, but without evidence of long-term stem cell engraftment. Notably, this effect of NSC is not a universal property of adult stem cells, as administration of GPS-engineered mouse hematopoietic stem/progenitor cells did not improve EAE clinical course. These findings highlight the utility of cell surface glycan engineering to boost stem cell delivery in neuroinflammatory conditions and indicate that, despite the use of a neural tissue-specific progenitor cell population, neural repair in EAE results from endogenous repair and not from direct, NSC-derived cell replacement. PMID:26153105

Melanocortin 1 Receptor Deficiency Promotes Atherosclerosis in Apolipoprotein E-/- Mice.

PubMed

Rinne, Petteri; Kadiri, James J; Velasco-Delgado, Mauricio; Nuutinen, Salla; Viitala, Miro; Hollmén, Maija; Rami, Martina; Savontaus, Eriika; Steffens, Sabine

2018-02-01

The MC1-R (melanocortin 1 receptor) is expressed by monocytes and macrophages where it mediates anti-inflammatory actions. MC1-R also protects against macrophage foam cell formation primarily by promoting cholesterol efflux through the ABCA1 (ATP-binding cassette transporter subfamily A member 1) and ABCG1 (ATP-binding cassette transporter subfamily G member 1). In this study, we aimed to investigate whether global deficiency in MC1-R signaling affects the development of atherosclerosis. Apoe -/- (apolipoprotein E deficient) mice were crossed with recessive yellow (Mc1r e/e ) mice carrying dysfunctional MC1-R and fed a high-fat diet to induce atherosclerosis. Apoe -/- Mc1r e/e mice developed significantly larger atherosclerotic lesions in the aortic sinus and in the whole aorta compared with Apoe -/- controls. In terms of plaque composition, MC1-R deficiency was associated with less collagen and smooth muscle cells and increased necrotic core, indicative of more vulnerable lesions. These changes were accompanied by reduced Abca1 and Abcg1 expression in the aorta. Furthermore, Apoe -/- Mc1r e/e mice showed a defect in bile acid metabolism that aggravated high-fat diet-induced hypercholesterolemia and hepatic lipid accumulation. Flow cytometric analysis of leukocyte profile revealed that dysfunctional MC1-R enhanced arterial accumulation of classical Ly6C high monocytes and macrophages, effects that were evident in mice fed a normal chow diet but not under high-fat diet conditions. In support of enhanced arterial recruitment of Ly6C high monocytes, these cells had increased expression of L-selectin and P-selectin glycoprotein ligand 1. The present study highlights the importance of MC1-R in the development of atherosclerosis. Deficiency in MC1-R signaling exacerbates atherosclerosis by disturbing cholesterol handling and by increasing arterial monocyte accumulation. © 2017 The Authors.

Insertion of targeting domains into the envelope glycoprotein of Moloney murine leukemia virus (MoMLV)-based vectors modulates the route of mCAT-1-mediated viral entry.

PubMed

Viejo-Borbolla, A; Pizzato, M; Blair, E D; Schulz, T F

2005-03-01

Several groups have inserted targeting domains into the envelope glycoprotein (Env) of Moloney murine leukemia virus (MoMLV) in an attempt to produce targeted retroviral vectors for human gene therapy. While binding of these modified Envs to the target molecule expressed on the surface of human cells was observed, specific high-titer infection of human cells expressing the target molecule was not achieved. Here we investigate the initial steps in the entry process of targeted MoMLV vectors both in murine and human cells expressing the MoMLV receptor, the mouse cationic amino acid transporter-1 (mCAT-1). We show that insertion of a small ligand targeted to E-selectin and of a single chain antibody (scFv) targeted to folate-binding protein (FBP) into the N-terminus of MoMLV Env results in the reduction of the infectivity and the kinetics of entry of the MoMLV vectors. The use of soluble receptor-binding domain (sRBD), bafilomycin A1 (BafA1) and methyl-beta-cyclodextrin (MbetaC) increase the infectivity of the MoMLV vectors targeted to FBP (MoMLV-FBP) suggesting that the scFv targeted to FBP increases the threshold for fusion and might re-route entry of the targeted MoMLV-FBP vector towards an endocytic, non-productive pathway.

Synthesis and Functional Characterization of Novel Sialyl LewisX Mimic-Decorated Liposomes for E-selectin-Mediated Targeting to Inflamed Endothelial Cells.

PubMed

Chantarasrivong, Chanikarn; Ueki, Akiharu; Ohyama, Ryutaro; Unga, Johan; Nakamura, Shinya; Nakanishi, Isao; Higuchi, Yuriko; Kawakami, Shigeru; Ando, Hiromune; Imamura, Akihiro; Ishida, Hideharu; Yamashita, Fumiyoshi; Kiso, Makoto; Hashida, Mitsuru

2017-05-01

Sialyl LewisX (sLeX) is a natural ligand of E-selectin that is overexpressed by inflamed and tumor endothelium. Although sLeX is a potential ligand for drug targeting, synthesis of the tetrasaccharide is complicated with many reaction steps. In this study, structurally simplified novel sLeX analogues were designed and linked with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-2000 (DSPE-PEG) for E-selectin-mediated liposomal delivery. The sLeX structural simplification strategies include (1) replacement of the Gal-GlcNAc disaccharide unit with lactose to reduce many initial steps and (2) substitution of neuraminic acid with a negatively charged group, i.e., 3'-sulfo, 3'-carboxymethyl (3'-CM), or 3'-(1-carboxy)ethyl (3'-CE). While all the liposomes developed were similar in particle size and charge, the 3'-CE sLeX mimic liposome demonstrated the highest uptake in inflammatory cytokine-treated human umbilical vein endothelial cells (HUVECs), being even more potent than native sLeX-decorated liposomes. Inhibition studies using antiselectin antibodies revealed that their uptake was mediated primarily by overexpressed E-selectin on inflamed HUVECs. Molecular dynamics simulations were performed to gain mechanistic insight into the E-selectin binding differences among native and mimic sLeX. The terminally branched methyl group of the 3'-CE sLeX mimic oriented and faced the bulk hydrophilic solution during E-selectin binding. Since this state is entropically unfavorable, the 3'-CE sLeX mimic molecule might be pushed toward the binding pocket of E-selectin by a hydrophobic effect, leading to a higher probability of hydrogen-bond formation than native sLeX and the 3'-CM sLeX mimic. This corresponded with the fact that the 3'-CE sLeX mimic liposome exhibited much greater uptake than the 3'-CM sLeX mimic liposome.

New Insight on a Combination of Policosanol and 10-Dehydrogingerdione Phytochemicals as Inhibitors for Platelet Activation Biomarkers and Atherogenicity Risk in Dyslipidemic Rabbits: Role of CETP and PCSK9 Inhibition.

PubMed

Elseweidy, Mohamed Mahmoud; Amin, Rawia Sarhan; Atteia, Hebatallah Husseini; El-Zeiky, Reham Raafat; Al-Gabri, Naif A

2018-05-09

Platelet markers [soluble p selectin (sP-selectin) and soluble CD40 ligand (sCD40L)] are associated with platelet activation and cardiovascular risk. Both policosanol and 10-dehydrogingerdione are natural products with proven CETP inhibitory and antiatherogenic effects. Present work aimed mainly to investigate the levels of platelet activation biomarkers in the serum of dyslipidemic rabbits and the potential of these phytochemicals either alone or in a combination form to protect against atherogenicity. Additionally, this work clarified their effect on PCSK9, a key player in atherosclerosis progression. Daily administration of policosanol and/or 10-dehydrogingerdione at a dose level 10 mg/kg bw resulted in a CETP inhibitory activity, increasing HDL-C level. This protective effect was associated with improvement in lipid profile components and a reduction in PCSK9 level. Interestingly, this combination strengthened the CETP inhibitory activity of these phytochemicals, leading to a greater increase in serum HDL-C level than monotherapy. However, this combination did not enhance the reduction in PCSK9 level. Both drugs also decreased platelet activation and inflammation markers such as sCD40L, sP-selectin, and interferon-gamma (IFN-γ), and their combination showed a synergistic effect. Therefore, such phytochemicals may be regarded as promising agents in the protection against atherothrombosis risk.

Flow cytometric immunobead assay for quantitative detection of platelet autoantibodies in immune thrombocytopenia patients.

PubMed

Zhai, Juping; Ding, Mengyuan; Yang, Tianjie; Zuo, Bin; Weng, Zhen; Zhao, Yunxiao; He, Jun; Wu, Qingyu; Ruan, Changgeng; He, Yang

2017-10-23

Platelet autoantibody detection is critical for immune thrombocytopenia (ITP) diagnosis and prognosis. Therefore, we aimed to establish a quantitative flow cytometric immunobead assay (FCIA) for ITP platelet autoantibodies evaluation. Capture microbeads coupled with anti-GPIX, -GPIb, -GPIIb, -GPIIIa and P-selectin antibodies were used to bind the platelet-bound autoantibodies complex generated from plasma samples of 250 ITP patients, 163 non-ITP patients and 243 healthy controls, a fluorescein isothiocyanate (FITC)-conjugated secondary antibody was the detector reagent and mean fluorescence intensity (MFI) signals were recorded by flow cytometry. Intra- and inter-assay variations of the quantitative FCIA assay were assessed. Comparisons of the specificity, sensitivity and accuracy between quantitative and qualitative FCIA or monoclonal antibody immobilization of platelet antigen (MAIPA) assay were performed. Finally, treatment process was monitored by our quantitative FCIA in 8 newly diagnosed ITPs. The coefficient of variations (CV) of the quantitative FCIA assay were respectively 9.4, 3.8, 5.4, 5.1 and 5.8% for anti-GPIX, -GPIb, -GPIIIa, -GPIIb and -P-selectin autoantibodies. Elevated levels of autoantibodies against platelet glycoproteins GPIX, GPIb, GPIIIa, GPIIb and P-selectin were detected by our quantitative FCIA in ITP patients compared to non-ITP patients or healthy controls. The sensitivity, specificity and accuracy of our quantitative assay were respectively 73.13, 81.98 and 78.65% when combining all 5 autoantibodies, while the sensitivity, specificity and accuracy of MAIPA assay were respectively 41.46, 90.41 and 72.81%. A quantitative FCIA assay was established. Reduced levels of platelet autoantibodies could be confirmed by our quantitative FCIA in ITP patients after corticosteroid treatment. Our quantitative assay is not only good for ITP diagnosis but also for ITP treatment monitoring.

Increase in the adhesion molecule P-selectin in endothelium overlying atherosclerotic plaques. Coexpression with intercellular adhesion molecule-1.

PubMed Central

Johnson-Tidey, R. R.; McGregor, J. L.; Taylor, P. R.; Poston, R. N.

1994-01-01

P-selectin (GMP-140) is an adhesion molecule present within endothelial cells that is rapidly translocated to the cell membrane upon activation, where it mediates endothelial-leukocyte interactions. Immunohistochemical analysis of human atherosclerotic plaques has shown strong expression of P-selectin by the endothelium overlying active atherosclerotic plaques. P-selectin is not, however, detected in normal arterial endothelium or in endothelium overlying inactive fibrous plaques. Color image analysis was used to quantitate the degree of P-selectin expression in the endothelium and demonstrates a statistically significant increase in P-selectin expression by atherosclerotic endothelial cells. Double immunofluorescence shows that some of this P-selectin is expressed on the luminal surface of the endothelial cells. Previous work has demonstrated a significant up-regulation in the expression of the intercellular adhesion molecule-1 in atherosclerotic endothelium and a study on the expression of intercellular adhesion molecule-1 and P-selectin in atherosclerosis shows a highly positive correlation. These results suggest that the selective and cooperative expression of P-selectin and intercellular adhesion molecule-1 may be involved in the recruitment of monocytes into sites of atherosclerosis. Images Figure 1 Figure 3 Figure 4 Figure 5 PMID:7513951

Elevated Production of Nociceptive CC Chemokines and sE-Selectin in Patients With Low Back Pain and the Effects of Spinal Manipulation: A Nonrandomized Clinical Trial.

PubMed

Teodorczyk-Injeyan, Julita A; McGregor, Marion; Triano, John J; Injeyan, Stephen H

2018-01-01

The involvement of inflammatory components in the pathophysiology of low back pain (LBP) is poorly understood. It has been suggested that spinal manipulative therapy (SMT) may exert anti-inflammatory effects. The purpose of this study was to determine the involvement of inflammation-associated chemokines (CC series) in the pathogenesis of nonspecific LBP and to evaluate the effect of SMT on that process. Patients presenting with nonradicular, nonspecific LBP (minimum pain score 3 on 10-point visual analog scale) were recruited according to stringent inclusion criteria. They were evaluated for appropriateness to treat using a high velocity low amplitude manipulative thrust in the lumbar-lumbosacral region. Blood samples were obtained at baseline and following the administration of a series of 6 high velocity low amplitude manipulative thrusts on alternate days over the period of 2 weeks. The in vitro levels of CC chemokine ligands (CCL2, CCL3, and CCL4) production and plasma levels of an inflammatory biomarker, soluble E-selectin (sE-selectin), were determined at baseline and at the termination of treatments 2 weeks later. Compared with asymptomatic controls baseline production of all chemokines was significantly elevated in acute (P=0.004 to <0.0001), and that of CCL2 and CCL4 in chronic LBP patients (P<0.0001). Furthermore, CCL4 production was significantly higher (P<0.0001) in the acute versus chronic LBP group. sE-selectin levels were significantly higher (P=0.003) in chronic but not in acute LBP patients. Following SMT, patient-reported outcomes showed significant (P<0.0001) improvements in visual analog scale and Oswestry Disability Index scores. This was accompanied by a significant decline in CCL3 production (P<0.0001) in both groups of patients. Change scores for CCL4 production differed significantly (P<0.0001) only for the acute LBP cohort, and no effect on the production of CCL2 or plasma sE-selectin levels was noted in either group. The production of chemotactic cytokines is significantly and protractedly elevated in LBP patients. Changes in chemokine production levels, which might be related to SMT, differ in the acute and chronic LBP patient cohorts.

[Value of adhesion molecules for evaluating the efficiency of therapy for ulcerative colitis and Crohn's disease].

PubMed

Parfenov, A I; Boldyreva, O N; Ruchkina, I N; Knyazev, O V; Sagynbaeva, V E; Shcherbakov, P L; Khomeriki, S G; Lazebnik, L B; Konoplyannikov, A G

2014-01-01

To define the value of adhesion molecules (sVCAM-1 integrin, P-selectin, E-selectin, and L-selectin) for the prediction and evaluation of the efficiency of treatment in patients with ulcerative colitis (UC) and Crohn's disease. Twenty-six patients with UC and 14 patients with CD were examined. Of them, 16 patients took infliximab (INF) in a dose of 5 mg/kg of body weight according to the standard scheme; 14 patients received cultured mesenchymal stem stromal cells (MSSCs) in a quantity of 150 x 10(8) cells, and 10 had azathioprine (AZA) 2 mg/kg and glucocorticosteroids (GCS) 1 mg/kg of body weight. Enzyme immunoassay was used to determine the serum concentration of the adhesion molecules (L-selectin, E-selectin, P-selectin, and sVCAM-1 integrin) before and 2 months after treatment. The signs of bowel inflammatory disease activity and the elevated levels of adhesion molecules whose synthesis did not occur under normal conditions remained in the patients receiving GCS and AZA. INF treatment caused a decrease in P-selectin, E-selectin, and sVCAM-1 levels to 8.9 +/- 1.0, 5.5 +/- 1.7, and 9.5 +/- 4.4 ng/ml, respectively (p < 0.001). Incorporation of MSSCs was followed by a reduction of the concentrations of P-selectin and E-selectin to 6.9 +/- 1.1 and 5.7 +/- 1.3 ng/ml, respectively (p < 0.001). The level of integrin (cVCAM-1) fell to 12.2 +/- 2.2 ng/ml (p > 0.1); that of L-selectin did not drop after MSSC administration and INF induction therapy. P-selectin, E-selectin, L-selectin, and sVCAM-1 integrin are current inflammatory markers and may be used to evaluate the efficiency of standard and biological therapies for inflammatory bowel diseases and to predict disease course.

The profiles of soluble adhesion molecules in the "great obstetrical syndromes".

PubMed

Docheva, Nikolina; Romero, Roberto; Chaemsaithong, Piya; Tarca, Adi L; Bhatti, Gaurav; Pacora, Percy; Panaitescu, Bogdan; Chaiyasit, Noppadol; Chaiworapongsa, Tinnakorn; Maymon, Eli; Hassan, Sonia S; Erez, Offer

2018-02-01

The objective of this study was to determine the profiles of maternal plasma soluble adhesion molecules in patients with preeclampsia, small-for-gestational-age (SGA) fetuses, acute pyelonephritis, preterm labor with intact membranes (PTL), preterm prelabor rupture of the membranes (preterm PROM), and fetal death. A cross-sectional study was conducted to determine maternal plasma concentrations of sE-selectin, sL-selectin, and sP-selectin as well as sICAM-1, sVCAM-1, and sPECAM-1 in patients with (1) an uncomplicated pregnancy (control, n = 100); (2) preeclampsia (n = 94); (3) SGA fetuses (in women without preeclampsia/hypertension, n = 45); (4) acute pyelonephritis (n = 25); (5) PTL (n = 53); (6) preterm PROM (n = 24); and (7) fetal death (n = 34). Concentrations of soluble adhesion molecules and inflammatory cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-8) were determined with sensitive and specific enzyme-linked immunoassays. In comparison to women with a normal pregnancy, (1) women with preeclampsia had higher median concentrations of sE-selectin, sP-selectin, and sVCAM-1, and a lower concentration of sL-selectin (all p values < .001); (2) patients with SGA fetuses had higher median concentrations of sE-selectin, sP-selectin, and sVCAM-1 (all p values < .05); (3) patients with a fetal death had higher median concentrations of sE-selectin and sP-selectin (all p values < .05); (4) patients with acute pyelonephritis had higher median plasma concentrations of sE-selectin, sICAM-1, and sVCAM-1 (all p values < .001); (5) patients with preeclampsia and acute pyelonephritis, plasma concentrations of sVCAM-1, sE-selectin, and sP-selectin correlated with those of the proinflammatory cytokines TNF-α and interleukin (IL)-8 (all p values < .05); (6) patients with PTL had a higher median concentration of sP-selectin and a lower median concentration of VCAM-1 (all p values < .05); and (7) women with preterm PROM had lower median concentrations of sL-selectin and sVCAM-1 (all p values < .05). The results of this study show that endothelial cell activation/dysfunction reflected by the plasma concentration of sE-selectin is not specific to preeclampsia but is present in pregnancies complicated by SGA fetuses, acute pyelonephritis, and fetal death. Collectively, we report that each obstetrical syndrome appears to have a stereotypical profile of soluble adhesion molecules in the peripheral circulation.

Fulfilling Koch's postulates in glycoscience: HCELL, GPS and translational glycobiology.

PubMed

Sackstein, Robert

2016-06-01

Glycoscience-based research that is performed expressly to address medical necessity and improve patient outcomes is called "translational glycobiology". In the 19th century, Robert Koch proposed a set of postulates to rigorously establish causality in microbial pathogenesis, and these postulates can be reshaped to guide knowledge into how naturally-expressed glycoconjugates direct molecular processes critical to human well-being. Studies in the 1990s indicated that E-selectin, an endothelial lectin that binds sialofucosylated carbohydrate determinants, is constitutively expressed on marrow microvessels, and investigations in my laboratory indicated that human hematopoietic stem cells (HSCs) uniquely express high levels of a specialized glycoform of CD44 called "hematopoietic cell E-/L-selectin ligand" (HCELL) that functions as a highly potent E-selectin ligand. To assess the role of HCELL in directing HSC migration to marrow, a method called "glycosyltransferase-programmed stereosubstitution" (GPS) was developed to custom-modify CD44 glycans to enforce HCELL expression on viable cell surfaces. Human mesenchymal stem cells (MSCs) are devoid of E-selectin ligands, but GPS-based glycoengineering of CD44 on MSCs licenses homing of these cells to marrow in vivo, providing direct evidence that HCELL serves as a "bone marrow homing receptor". This review will discuss the molecular basis of cell migration in historical context, will describe the discovery of HCELL and its function as the bone marrow homing receptor, and will inform on how glycoengineering of CD44 serves as a model for adapting Koch's postulates to elucidate the key roles that glycoconjugates play in human biology and for realizing the immense impact of translational glycobiology in clinical medicine. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Cell surface glycan engineering of neural stem cells augments neurotropism and improves recovery in a murine model of multiple sclerosis.

PubMed

Merzaban, Jasmeen S; Imitola, Jaime; Starossom, Sarah C; Zhu, Bing; Wang, Yue; Lee, Jack; Ali, Amal J; Olah, Marta; Abuelela, Ayman F; Khoury, Samia J; Sackstein, Robert

2015-12-01

Neural stem cell (NSC)-based therapies offer potential for neural repair in central nervous system (CNS) inflammatory and degenerative disorders. Typically, these conditions present with multifocal CNS lesions making it impractical to inject NSCs locally, thus mandating optimization of vascular delivery of the cells to involved sites. Here, we analyzed NSCs for expression of molecular effectors of cell migration and found that these cells are natively devoid of E-selectin ligands. Using glycosyltransferase-programmed stereosubstitution (GPS), we glycan engineered the cell surface of NSCs ("GPS-NSCs") with resultant enforced expression of the potent E-selectin ligand HCELL (hematopoietic cell E-/L-selectin ligand) and of an E-selectin-binding glycoform of neural cell adhesion molecule ("NCAM-E"). Following intravenous (i.v.) injection, short-term homing studies demonstrated that, compared with buffer-treated (control) NSCs, GPS-NSCs showed greater neurotropism. Administration of GPS-NSC significantly attenuated the clinical course of experimental autoimmune encephalomyelitis (EAE), with markedly decreased inflammation and improved oligodendroglial and axonal integrity, but without evidence of long-term stem cell engraftment. Notably, this effect of NSC is not a universal property of adult stem cells, as administration of GPS-engineered mouse hematopoietic stem/progenitor cells did not improve EAE clinical course. These findings highlight the utility of cell surface glycan engineering to boost stem cell delivery in neuroinflammatory conditions and indicate that, despite the use of a neural tissue-specific progenitor cell population, neural repair in EAE results from endogenous repair and not from direct, NSC-derived cell replacement. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Effects of a detergent micelle environment on P-glycoprotein (ABCB1)-ligand interactions

PubMed Central

Shukla, Suneet; Abel, Biebele; Chufan, Eduardo E.; Ambudkar, Suresh V.

2017-01-01

P-glycoprotein (P-gp) is a multidrug transporter that uses energy from ATP hydrolysis to export many structurally dissimilar hydrophobic and amphipathic compounds, including anticancer drugs from cells. Several structural studies on purified P-gp have been reported, but only limited and sometimes conflicting information is available on ligand interactions with the isolated transporter in a dodecyl-maltoside detergent environment. In this report we compared the biochemical properties of P-gp in native membranes, detergent micelles, and when reconstituted in artificial membranes. We found that the modulators zosuquidar, tariquidar, and elacridar stimulated the ATPase activity of purified human or mouse P-gp in a detergent micelle environment. In contrast, these drugs inhibited ATPase activity in native membranes or in proteoliposomes, with IC50 values in the 10–40 nm range. Similarly, a 30–150-fold decrease in the apparent affinity for verapamil and cyclic peptide inhibitor QZ59-SSS was observed in detergent micelles compared with native or artificial membranes. Together, these findings demonstrate that the high-affinity site is inaccessible because of either a conformational change or binding of detergent at the binding site in a detergent micelle environment. The ligands bind to a low-affinity site, resulting in altered modulation of P-gp ATPase activity. We, therefore, recommend studying structural and functional aspects of ligand interactions with purified P-gp and other ATP-binding cassette transporters that transport amphipathic or hydrophobic substrates in a detergent-free native or artificial membrane environment. PMID:28283574

Glycosyltransferase-programmed stereosubstitution (GPS) to create HCELL: engineering a roadmap for cell migration.

PubMed

Sackstein, Robert

2009-07-01

During evolution of the vertebrate cardiovascular system, the vast endothelial surface area associated with branching vascular networks mandated the development of molecular processes to efficiently and specifically recruit circulating sentinel host defense cells and tissue repair cells at localized sites of inflammation/tissue injury. The forces engendered by high-velocity blood flow commensurately required the evolution of specialized cell surface molecules capable of mediating shear-resistant endothelial adhesive interactions, thus literally capturing relevant cells from the blood stream onto the target endothelial surface and permitting subsequent extravasation. The principal effectors of these shear-resistant binding interactions comprise a family of C-type lectins known as 'selectins' that bind discrete sialofucosylated glycans on their respective ligands. This review explains the 'intelligent design' of requisite reagents to convert native CD44 into the sialofucosylated glycoform known as hematopoietic cell E-/L-selectin ligand (HCELL), the most potent E-selectin counter-receptor expressed on human cells, and will describe how ex vivo glycan engineering of HCELL expression may open the 'avenues' for the efficient vascular delivery of cells for a variety of cell therapies.

A differential role for cell shape in neutrophil tethering and rolling on endothelial selectins under flow.

PubMed

Finger, E B; Bruehl, R E; Bainton, D F; Springer, T A

1996-12-01

We investigated the role of neutrophil microvilli in interactions with E-selectin and P-selectin in hydrodynamic shear flow by disruption with cytochalasin B, hypotonic swelling, and chilling. Cytochalasin B only marginally reduced microvilli numbers (from 30 +/- 6 to 16 +/- 6 per cell perimeter, p < 0.005) as shown by electron microscopy, completely disrupted tethering in shear flow to E-selectin and P-selectin, increased the strength of rolling adhesions on E-selectin and P-selectin, and increased cell deformability in shear flow with a likely increase in the area of cell:substrate contact. Hypoosmotic swelling markedly reduced microvilli number (to 6 +/- 5 per perimeter, p < 0.005), almost completely inhibited tethering on E- and P-selectin, and increased the strength of rolling adhesions on P-selectin but not on E-selectin. Chilling almost completely abolished microvilli (to 3 +/- 3 per perimeter, p < 0.005), but pseudopod-like structures were present, and had little effect on tethering in flow. Immunogold labeling of L-selectin, which is normally clustered on tips of microvilli, showed that in the absence of microvilli it remained in small clusters. Our studies show that alterations in cell morphology and viscoelasticity can have opposing effects on tethering and rolling, showing that they are independently regulatable. Furthermore, our results suggest that the association of molecules that mediate rolling with microvilli tips may be important not just to enhance presentation, but for other functions such as to promote resistance to extraction from the membrane or cooperative interactions among clustered receptors.

[Expression of EV71-VP1, PSGL-1 and SCARB2 in Tissues of Infants with Brain Stem Encephalitis].

PubMed

Li, Ming; Kong, Xiao-ping; Liu, Hong; Cheng, Ling-xi; Huang, Jing-lu; Quan, Li; Wu, Fang-yu; Hao, Bo; Liu, Chao; Luo, Bin

2015-04-01

To understand the correlation of enterovirus 71 (EV71), P-selectin glycoprotein ligand-1 (PSGL-1), and scavenger receptor B2 (SCARB2) and to explore the possible pathway and mechanism of EV71 infection by observing the expression of EV71, PSGL-1 and SCARB2 in tissues of infants with brain stem encephalitis. The organs and tissues of infants with EV71-VP1 positivity in their brain stems were chosen. Expression and distribution of EV71-VP1, PSGL-1, and SCARB2 were detected and compared by immunohistochemistry. Strong staining of EV71 -VP1 was observed in the neuron, glial cells, the inflammatory cells of perivascular cuffing, parietal cells of the gastric fundus gland while alveolar macrophages, intestinal gland epithelium cells, mucosa lymphoid nodule and lymphocyte of palatine tonsil showed moderate staining and weak staining were displayed in mesenteric lymph nodes and lymphocyte of spleen. PSGL-1 expression was detected in parietal cells of the gastric fundus gland, tonsillar crypt squamous epithelium, alveolar macrophages and leukocytes in each tissue. SCARB2 expression was observed in all the above tissues except the intestines and spleen. The distribution of EV71 correlates with SCARB2 expression. SCARB2 plays an important role in virus infection and replication. Stomach may be an important site for EV71 replication.

In vivo regulation of Bcl6 and T follicular helper cell development1

PubMed Central

Poholek, Amanda C.; Hansen, Kyle; Hernandez, Sairy G.; Eto, Danelle; Chandele, Anmol; Weinstein, Jason S.; Dong, Xuemei; Odegard, Jared M.; Kaech, Susan M.; Dent, Alexander L.; Crotty, Shane; Craft, Joe

2010-01-01

Follicular helper T (TFH) cells, defined by expression of the surface markers CXCR5 and PD-1 and synthesis of IL-21, require upregulation of the transcriptional repressor Bcl6 for their development and function in B cell maturation in germinal centers. We have explored the role of B cells, and the cytokines IL-6 and IL-21, in the in vivo regulation of Bcl6 expression and TFH cell development. We found that TFH cells are characterized by a Bcl6-dependent downregulation of P-selectin glycoprotein ligand-1 (PSGL1, a CCL19- and CCL21-binding protein), indicating that, like CXCR5 and PD-1 upregulation, modulation of PSGL1 expression is part of the TFH cell program of differentiation. B cells were neither required for initial upregulation of Bcl6 nor PSGL1 downregulation, suggesting these events preceded T-B cell interactions, although they were required for full development of the TFH cell phenotype, including CXCR5 and PD-1 upregulation, and IL-21 synthesis. Bcl6 upregulation and TFH cell differentiation were independent of IL-6 and IL-21, revealing that either cytokine is not absolutely required for development of Bcl6+ TFH cells in vivo. These data increase our understanding of Bcl6 regulation in TFH cells and their differentiation in vivo, and identifies a new surface marker that may be functionally relevant in this subset. PMID:20519643

Glutamine supplementation attenuates expressions of adhesion molecules and chemokine receptors on T cells in a murine model of acute colitis.

PubMed

Hou, Yu-Chen; Wu, Jin-Ming; Wang, Ming-Yang; Wu, Ming-Hsun; Chen, Kuen-Yuan; Yeh, Sung-Ling; Lin, Ming-Tsan

2014-01-01

Migration of T cells into the colon plays a major role in the pathogenesis in inflammatory bowel disease. This study investigated the effects of glutamine (Gln) supplementation on chemokine receptors and adhesion molecules expressed by T cells in mice with dextran sulfate sodium- (DSS-) induced colitis. C57BL/6 mice were fed either a standard diet or a Gln diet replacing 25% of the total nitrogen. After being fed the diets for 5 days, half of the mice from both groups were given 1.5% DSS in drinking water to induce colitis. Mice were killed after 5 days of DSS exposure. DSS colitis resulted in higher expression levels of P-selectin glycoprotein ligand- (PSGL-) 1, leukocyte function-associated antigen- (LFA-) 1, and C-C chemokine receptor type 9 (CCR9) by T helper (Th) and cytotoxic T (Tc) cells, and mRNA levels of endothelial adhesion molecules in colons were upregulated. Gln supplementation decreased expressions of PSGL-1, LFA-1, and CCR9 by Th cells. Colonic gene expressions of endothelial adhesion molecules were also lower in Gln-colitis mice. Histological finding showed that colon infiltrating Th cells were less in the DSS group with Gln administration. Gln supplementation may ameliorate the inflammation of colitis possibly via suppression of T cell migration.

mRNA-engineered mesenchymal stem cells for targeted delivery of interleukin-10 to sites of inflammation.

PubMed

Levy, Oren; Zhao, Weian; Mortensen, Luke J; Leblanc, Sarah; Tsang, Kyle; Fu, Moyu; Phillips, Joseph A; Sagar, Vinay; Anandakumaran, Priya; Ngai, Jessica; Cui, Cheryl H; Eimon, Peter; Angel, Matthew; Lin, Charles P; Yanik, Mehmet Fatih; Karp, Jeffrey M

2013-10-03

Mesenchymal stem cells (MSCs) are promising candidates for cell-based therapy to treat several diseases and are compelling to consider as vehicles for delivery of biological agents. However, MSCs appear to act through a seemingly limited "hit-and-run" mode to quickly exert their therapeutic impact, mediated by several mechanisms, including a potent immunomodulatory secretome. Furthermore, MSC immunomodulatory properties are highly variable and the secretome composition following infusion is uncertain. To determine whether a transiently controlled antiinflammatory MSC secretome could be achieved at target sites of inflammation, we harnessed mRNA transfection to generate MSCs that simultaneously express functional rolling machinery (P-selectin glycoprotein ligand-1 [PSGL-1] and Sialyl-Lewis(x) [SLeX]) to rapidly target inflamed tissues and that express the potent immunosuppressive cytokine interleukin-10 (IL-10), which is not inherently produced by MSCs. Indeed, triple-transfected PSGL-1/SLeX/IL-10 MSCs transiently increased levels of IL-10 in the inflamed ear and showed a superior antiinflammatory effect in vivo, significantly reducing local inflammation following systemic administration. This was dependent on rapid localization of MSCs to the inflamed site. Overall, this study demonstrates that despite the rapid clearance of MSCs in vivo, engineered MSCs can be harnessed via a "hit-and-run" action for the targeted delivery of potent immunomodulatory factors to treat distant sites of inflammation.

Structure and ligand-based design of P-glycoprotein inhibitors: a historical perspective.

PubMed

Palmeira, Andreia; Sousa, Emilia; Vasconcelos, M Helena; Pinto, Madalena; Fernandes, Miguel X

2012-01-01

Computer-assisted drug design (CADD) is a valuable approach for the discovery of new chemical entities in the field of cancer therapy. There is a pressing need to design and develop new, selective, and safe drugs for the treatment of multidrug resistance (MDR) cancer forms, specifically active against P-glycoprotein (P-gp). Recently, a crystallographic structure for mouse P-gp was obtained. However, for decades the design of new P-gp inhibitors employed mainly ligand-based approaches (SAR, QSAR, 3D-QSAR and pharmacophore studies), and structure-based studies used P-gp homology models. However, some of those results are still the pillars used as a starting point for the design of potential P-gp inhibitors. Here, pharmacophore mapping, (Q)SAR, 3D-QSAR and homology modeling, for the discovery of P-gp inhibitors are reviewed. The importance of these methods for understanding mechanisms of drug resistance at a molecular level, and design P-gp inhibitors drug candidates are discussed. The examples mentioned in the review could provide insights into the wide range of possibilities of using CADD methodologies for the discovery of efficient P-gp inhibitors.

The state diagram for cell adhesion under flow: leukocyte rolling and firm adhesion.

PubMed

Chang, K C; Tees, D F; Hammer, D A

2000-10-10

Leukocyte adhesion under flow in the microvasculature is mediated by binding between cell surface receptors and complementary ligands expressed on the surface of the endothelium. Leukocytes adhere to endothelium in a two-step mechanism: rolling (primarily mediated by selectins) followed by firm adhesion (primarily mediated by integrins). Using a computational method called "Adhesive Dynamics," we have simulated the adhesion of a cell to a surface in flow, and elucidated the relationship between receptor-ligand functional properties and the dynamics of adhesion. We express this relationship in a state diagram, a one-to-one map between the biophysical properties of adhesion molecules and various adhesive behaviors. Behaviors that are observed in simulations include firm adhesion, transient adhesion (rolling), and no adhesion. We varied the dissociative properties, association rate, bond elasticity, and shear rate and found that the unstressed dissociation rate, k(r)(o), and the bond interaction length, gamma, are the most important molecular properties controlling the dynamics of adhesion. Experimental k(r)(o) and gamma values from the literature for molecules that are known to mediate rolling adhesion fall within the rolling region of the state diagram. We explain why L-selectin-mediated rolling, which has faster k(r)(o) than other selectins, is accompanied by a smaller value for gamma. We also show how changes in association rate, shear rate, and bond elasticity alter the dynamics of adhesion. The state diagram (which must be mapped for each receptor-ligand system) presents a concise and comprehensive means of understanding the relationship between bond functional properties and the dynamics of adhesion mediated by receptor-ligand bonds.

Host defense against systemic infection with Streptococcus pneumoniae is impaired in E-, P-, and E-/P-selectin-deficient mice.

PubMed Central

Munoz, F M; Hawkins, E P; Bullard, D C; Beaudet, A L; Kaplan, S L

1997-01-01

Endothelial selectins mediate rolling of leukocytes on endothelium, a crucial step for leukocyte firm adhesion and emigration into sites of tissue injury and infection. To characterize the role of the endothelial selectins during bacterial sepsis in vivo, Streptococcus pneumoniae (1-10 x 10(6) colony-forming units) was inoculated intraperitoneally into wild-type mice and mice with E-, P-, or E-/P-selectin deficiencies. Mice were followed 10 d for morbidity, survival, clearance of bacteremia, and leukocyte migration to the peritoneal cavity and organs 48 h after infection. All selectin-deficient mice showed a more pronounced morbidity, a significantly higher mortality associated with persistent bacteremia, and a higher bacterial load when compared with wild-type mice. These differences were most remarkable in the E-selectin-deficient mice, which showed the highest rate of mortality and bacteremia (P

Polymorphisms and linkage analysis for ICAM-1 and the selectin gene cluster

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vora, D.K.; Rosenbloom, C.L.; Cottingham, R.W.

1994-06-01

Genetic polymorphisms in leukocyte and endothelial cell adhesion molecules may be important variables with regard to susceptibility to multifactorial disease processes that include an inflammatory component. For this reason, polymorphisms were sought for intercellular adhesion molecule-1 (ICAM-1; gene symbol ICAM1) and for the three genes in the selectin cluster, P-selectin, L-selectin, and E-selectin (gene symbols SELP, SELL, and SELE, respectively). Two amino acid polymorphisms were identified for ICAM-1; Gly or Arg at codon 241 and Lys or Glu at codon 469. Dinucleotide repeat polymorphisms were identified in the 3{prime}-untranslated region for ICAM-1 and in intron 9 for P-selectin. Restriction fragmentmore » length polymorphisms were found using cDNAs for each of the three selectin genes as probes; E-selectin with BglII, P-selectin with ScaI, and L-selectin with HincII. Linkage analysis was performed for the selectin gene cluster and for ICAM-1 using the CEPH families; ICAM-1 is very tightly linked to the LDL receptor on chromosome 19, and the selectin cluster is linked to markers at chromosome 1q23. 41 refs., 2 tabs.« less

Differential patterns of endothelial and leucocyte activation in ‘typhus-like’ illnesses in Laos and Thailand

PubMed Central

Paris, D H; Jenjaroen, K; Blacksell, S D; Phetsouvanh, R; Wuthiekanun, V; Newton, P N; Day, N P J; Turner, G D H

2008-01-01

Scrub typhus is responsible for a large proportion of undifferentiated fevers in south-east Asia. The cellular tropism and pathophysiology of the causative agent, Orientia tsutsugamushi, remain poorly understood. We measured endothelial and leucocyte activation by soluble cell adhesion molecule enzyme-linked immunosorbent assays in 242 Lao and Thai patients with scrub or murine typhus, leptospirosis, dengue, typhoid and uncomplicated falciparum malaria on admission to hospital. Soluble E-selectin (sE-selectin) levels were lowest in dengue, sL-selectin highest in scrub typhus with a high sE-selectin to sL-selectin ratio in leptospirosis patients. In scrub typhus patients elevated sL-selectin levels correlated with the duration of skin rash (P = 0·03) and the presence of eschar (P = 0·03), elevated white blood cell (WBC) count (P = 0·007), elevated lymphocyte (P = 0·007) and neutrophil counts (P = 0·015) and elevated levels of sE-selectin correlated with the duration of illness before admission (P = 0·03), the presence of lymphadenopathy (P = 0·033) and eschar (P = 0·03), elevated WBC (P = 0·005) and neutrophil counts (P = 0·0003). In comparison, soluble selectin levels in murine typhus patients correlated only with elevated WBC counts (P = 0·03 for sE-selectin and sL-selectin). Soluble intercellular adhesion molecule-1 and soluble vascular adhesion molecule-1 levels were not associated significantly with any clinical parameters in scrub or murine typhus patients. The data presented suggest mononuclear cell activation in scrub typhus. As adhesion molecules direct leucocyte migration and induce inflammatory and immune responses, this may represent O. tsutsugamushi tropism during early dissemination, or local immune activation within the eschar. PMID:18505434

Differential patterns of endothelial and leucocyte activation in 'typhus-like' illnesses in Laos and Thailand.

PubMed

Paris, D H; Jenjaroen, K; Blacksell, S D; Phetsouvanh, R; Wuthiekanun, V; Newton, P N; Day, N P J; Turner, G D H

2008-07-01

Scrub typhus is responsible for a large proportion of undifferentiated fevers in south-east Asia. The cellular tropism and pathophysiology of the causative agent, Orientia tsutsugamushi, remain poorly understood. We measured endothelial and leucocyte activation by soluble cell adhesion molecule enzyme-linked immunosorbent assays in 242 Lao and Thai patients with scrub or murine typhus, leptospirosis, dengue, typhoid and uncomplicated falciparum malaria on admission to hospital. Soluble E-selectin (sE-selectin) levels were lowest in dengue, sL-selectin highest in scrub typhus with a high sE-selectin to sL-selectin ratio in leptospirosis patients. In scrub typhus patients elevated sL-selectin levels correlated with the duration of skin rash (P = 0.03) and the presence of eschar (P = 0.03), elevated white blood cell (WBC) count (P = 0.007), elevated lymphocyte (P = 0.007) and neutrophil counts (P = 0.015) and elevated levels of sE-selectin correlated with the duration of illness before admission (P = 0.03), the presence of lymphadenopathy (P = 0.033) and eschar (P = 0.03), elevated WBC (P = 0.005) and neutrophil counts (P = 0.0003). In comparison, soluble selectin levels in murine typhus patients correlated only with elevated WBC counts (P = 0.03 for sE-selectin and sL-selectin). Soluble intercellular adhesion molecule-1 and soluble vascular adhesion molecule-1 levels were not associated significantly with any clinical parameters in scrub or murine typhus patients. The data presented suggest mononuclear cell activation in scrub typhus. As adhesion molecules direct leucocyte migration and induce inflammatory and immune responses, this may represent O. tsutsugamushi tropism during early dissemination, or local immune activation within the eschar.

Characterization of the ligand-binding site of the transferrin receptor in Trypanosoma brucei demonstrates a structural relationship with the N-terminal domain of the variant surface glycoprotein.

PubMed

Salmon, D; Hanocq-Quertier, J; Paturiaux-Hanocq, F; Pays, A; Tebabi, P; Nolan, D P; Michel, A; Pays, E

1997-12-15

The Trypanosoma brucei transferrin (Tf) receptor is a heterodimer encoded by ESAG7 and ESAG6, two genes contained in the different polycistronic transcription units of the variant surface glycoprotein (VSG) gene. The sequence of ESAG7/6 differs slightly between different units, so that receptors with different affinities for Tf are expressed alternatively following transcriptional switching of VSG expression sites during antigenic variation of the parasite. Based on the sequence homology between pESAG7/6 and the N-terminal domain of VSGs, it can be predicted that the four blocks containing the major sequence differences between pESAG7 and pESAG6 form surface-exposed loops and generate the ligand-binding site. The exchange of a few amino acids in this region between pESAG6s encoded by different VSG units greatly increased the affinity for bovine Tf. Similar changes in other regions were ineffective, while mutations predicted to alter the VSG-like structure abolished the binding. Chimeric proteins containing the N-terminal dimerization domain of VSG and the C-terminal half of either pESAG7 or pESAG6, which contains the ligand-binding domain, can form heterodimers that bind Tf. Taken together, these data provided evidence that the T.brucei Tf receptor is structurally related to the N-terminal domain of the VSG and that the ligand-binding site corresponds to the exposed surface loops of the protein.

Markers of endothelial dysfunction and leucocyte activation in Saudi and non-Saudi haplotypes of sickle cell disease.

PubMed

Al Najjar, Salwa; Adam, Soheir; Ahmed, Nessar; Qari, Mohamed

2017-01-01

Sickle cell disease (SCD) is an autosomal recessive inherited hemoglobinopathy, characterized by chronic hemolysis and recurrent vaso-occlusive crisis (VOC). This study investigates changes in leucocyte subsets and the relationship between cell adhesion molecule expression and disease manifestations in patients during steady state and acute VOC. We compared soluble E-selectin and P-selectin levels in 84 SCD patients, in steady state and during VOC to 84 healthy controls. Using immunophenotyping, we also compared lymphocyte subsets in these three groups. Further, we compared E-selectin and P-selectin levels in patients of Saudi ethnicity to non-Saudi patients, in all three groups. Lymphocyte subsets showed high percentages of total T lymphocytes, T helper and suppressor lymphocytes, B lymphocytes as well as NK cells in patients with SCD during steady state, while B lymphocytes and NK cells were significantly higher during acute VOC crisis. High levels of both soluble E-selectin (sE-selectin) and soluble P-selectin (sP-selectin) markers were demonstrated in the serum of patients with SCD during both steady state and acute VOC. Levels of selectins were significantly higher in acute VOC. The immunophenotypic expression of L-selectin, on leucocytes, was high in SCD both during steady state and during acute VOC in comparison to normal control subjects. There was no significant difference in all three study groups between Saudi and non-Saudi patients. These findings suggest that patients with SCD have increased expression of adhesion molecules: E-selectin and P-selectin, which play an important role in the pathogenesis of VOC. Despite the distinct phenotype of Saudi patients with SCD, there was no significant difference in levels of soluble E-selectin and soluble P-selectin between Saudi and non-Saudi patients in all three groups. While sickle cell disease is a well-recognized state of chronic inflammation, the role of specific adhesion molecules is steadily unraveling. Studies are underway to investigate the potential role of selectin antagonists, for prevention and reversal of acute vascular occlusions in SCD patients.

Protein mobilities and P-selectin storage in Weibel-Palade bodies.

PubMed

Kiskin, Nikolai I; Hellen, Nicola; Babich, Victor; Hewlett, Lindsay; Knipe, Laura; Hannah, Matthew J; Carter, Tom

2010-09-01

Using fluorescence recovery after photobleaching (FRAP) we measured the mobilities of EGFP-tagged soluble secretory proteins in the endoplasmic reticulum (ER) and in individual Weibel-Palade bodies (WPBs) at early (immature) and late (mature) stages in their biogenesis. Membrane proteins (P-selectin, CD63, Rab27a) were also studied in individual WPBs. In the ER, soluble secretory proteins were mobile; however, following insertion into immature WPBs larger molecules (VWF, Proregion, tPA) and P-selectin became immobilised, whereas small proteins (ssEGFP, eotaxin-3) became less mobile. WPB maturation led to further decreases in mobility of small proteins and CD63. Acute alkalinisation of mature WPBs selectively increased the mobilities of small soluble proteins without affecting larger molecules and the membrane proteins. Disruption of the Proregion-VWF paracrystalline core by prolonged incubation with NH(4)Cl rendered P-selectin mobile while VWF remained immobile. FRAP of P-selectin mutants revealed that immobilisation most probably involves steric entrapment of the P-selectin extracellular domain by the Proregion-VWF paracrystal. Significantly, immobilisation contributed to the enrichment of P-selectin in WPBs; a mutation of P-selectin preventing immobilisation led to a failure of enrichment. Together these data shed new light on the transitions that occur for soluble and membrane proteins following their entry and storage into post-Golgi-regulated secretory organelles.

P-selectin is a nanotherapeutic delivery target in the tumor microenvironment.

PubMed

Shamay, Yosi; Elkabets, Moshe; Li, Hongyan; Shah, Janki; Brook, Samuel; Wang, Feng; Adler, Keren; Baut, Emily; Scaltriti, Maurizio; Jena, Prakrit V; Gardner, Eric E; Poirier, John T; Rudin, Charles M; Baselga, José; Haimovitz-Friedman, Adriana; Heller, Daniel A

2016-06-29

Disseminated tumors are poorly accessible to nanoscale drug delivery systems because of the vascular barrier, which attenuates extravasation at the tumor site. We investigated P-selectin, a molecule expressed on activated vasculature that facilitates metastasis by arresting tumor cells at the endothelium, for its potential to target metastases by arresting nanomedicines at the tumor endothelium. We found that P-selectin is expressed on cancer cells in many human tumors. To develop a targeted drug delivery platform, we used a fucosylated polysaccharide with nanomolar affinity to P-selectin. The nanoparticles targeted the tumor microenvironment to localize chemotherapeutics and a targeted MEK (mitogen-activated protein kinase kinase) inhibitor at tumor sites in both primary and metastatic models, resulting in superior antitumor efficacy. In tumors devoid of P-selectin, we found that ionizing radiation guided the nanoparticles to the disease site by inducing P-selectin expression. Radiation concomitantly produced an abscopal-like phenomenon wherein P-selectin appeared in unirradiated tumor vasculature, suggesting a potential strategy to target disparate drug classes to almost any tumor. Copyright © 2016, American Association for the Advancement of Science.

Detection of early stage atherosclerotic plaques using PET and CT fusion imaging targeting P-selectin in low density lipoprotein receptor-deficient mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakamura, Ikuko, E-mail: nakamuri@riken.jp; Department of Cardiovascular Medicine, Saga University, Saga; Hasegawa, Koki

2013-03-29

Highlights: ► P-selectin regulates leukocyte recruitment as an early stage event of atherogenesis. ► We developed an antibody-based molecular imaging probe targeting P-selectin for PET. ► This is the first report on successful PET imaging for delineation of P-selectin. ► P-selectin is a candidate target for atherosclerotic plaque imaging by clinical PET. -- Abstract: Background: Sensitive detection and qualitative analysis of atherosclerotic plaques are in high demand in cardiovascular clinical settings. The leukocyte–endothelial interaction mediated by an adhesion molecule P-selectin participates in arterial wall inflammation and atherosclerosis. Methods and results: A {sup 64}Cu-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid conjugated anti-P-selectin monoclonal antibody ({sup 64}Cu-DOTA-anti-P-selectinmore » mAb) probe was prepared by conjugating an anti-P-selectin monoclonal antibody with DOTA followed by {sup 64}Cu labeling. Thirty-six hours prior to PET and CT fusion imaging, 3 MBq of {sup 64}Cu-DOTA-anti-P-selectin mAb was intravenously injected into low density lipoprotein receptor-deficient Ldlr-/- mice. After a 180 min PET scan, autoradiography and biodistribution of {sup 64}Cu-DOTA-anti-P-selectin monoclonal antibody was examined using excised aortas. In Ldlr-/- mice fed with a high cholesterol diet for promotion of atherosclerotic plaque development, PET and CT fusion imaging revealed selective and prominent accumulation of the probe in the aortic root. Autoradiography of aortas that demonstrated probe uptake into atherosclerotic plaques was confirmed by Oil red O staining for lipid droplets. In Ldlr-/- mice fed with a chow diet to develop mild atherosclerotic plaques, probe accumulation was barely detectable in the aortic root on PET and CT fusion imaging. Probe biodistribution in aortas was 6.6-fold higher in Ldlr-/- mice fed with a high cholesterol diet than in those fed with a normal chow diet. {sup 64}Cu-DOTA-anti-P-selectin mAb accumulated selectively in aortic atherosclerotic plaques and was detectable by PET and CT fusion imaging in Ldlr-/- mice. Conclusions: P-selectin is a candidate target molecule for early-phase detection by PET and CT fusion imaging of atherosclerotic plaques.« less

Multi-ligand nanoparticles for targeted drug delivery to the injured vascular wall

NASA Astrophysics Data System (ADS)

Kona, Soujanya

Pathological conditions like coronary artery disease, acute myocardial infarction, stroke, and peripheral artery diseases as well as cardiovascular interventions used in the treatment of coronary artery diseases such as angioplasty and stenting damage/injure the blood vessel wall, leading to inflamed or activated endothelial cells that have been implicated in events leading to thrombosis, inflammation, and restenosis. Oral administration of anti-coagulant and anti-inflammatory drugs causes systemic toxicity, bleeding, patient incompliance, and inadequate amounts of drugs at the injured area. Though drug-eluting stents have shown therapeutic benefits, complications such as in-stent restenosis and late thrombosis still remain and are a cause for concern. Rapid growth in the field of nanotechnology and nanoscience in recent years has paved the way for new targeted and controlled drug delivery strategies. In this perspective, the development of biodegradable nanoparticles for targeted intracellular drug delivery to the inflamed endothelial cells may offer an improved avenue for treatment of cardiovascular diseases. The major objective of this research was to develop "novel multi-ligand nanoparticles," as drug carriers that can efficiently target and deliver therapeutic agents to the injured/inflamed vascular cells under dynamic flow conditions. Our approach mimics the natural binding ability of platelets to injured/activated endothelial cells through glycoprotein Ib (GPIb) bound to P-selectin expressed on inflamed endothelial cells and to the subendothelium through GPIb binding to von Willebrand factor (vWF) deposited onto the injured vascular wall. Our design also exploits the natural cell membrane translocation ability of the internalizing cell peptide - trans-activating transcriptor (TAT) to enhance the nanoparticle uptake by the targeted cells. Our hypothesis is that these multi-ligand nanoparticles would show an increased accumulation at the injury site since GPIb specially binds to both P-selectin expressed on damaged endothelial cells and vWF deposited on injured subendothelium while the cell penetrating peptide -- TAT would facilitate enhanced uptake of these nanoparticles by the damaged vascular cells. To test this hypothesis, fluorescent drug loaded poly (D, L-lactic-co-glycolic acid) (PLGA)-polyethylene glycol (PEG) nanoparticles (PLGA-PEG NPs) were formulated using a standard double emulsion method. We further conjugated GPIb and TAT via carbodiimide and avidin-biotin chemistry to the PLGA-PEG nanoparticles. Characterization of these nanoparticles indicated the average size to be about 200nm. Endothelial cell uptake studies indicated an optimal nanoparticle incubation time of one hour and optimal dose of 400 mug/ml. Biocompatibility results showed these particles to be non-toxic to endothelial cells. Moreover, dexamethasone release profiles from the nanoparticles demonstrated their ability to provide a sustained drug release over four weeks. Static and dynamic uptake studies of control, GPIb-conjugated, and GPIb-TAT-conjugated PLGA-PEG nanoparticles on activated endothelial cells exhibited an increased adhesion and uptake of GPIb-TAT conjugated PLGA-PEG nanoparticles compared to control nanoparticles. A similar trend of significantly higher adhesion of GPIb-TAT conjugated PLGA-PEG nanoparticles to the injured vessel wall was also observed in preliminary ex-vivo studies using the rat carotid injury model. These results suggest that "our novel multi-ligand NPs" would provide a unique active targeting strategy. This system would rapidly target and deliver therapeutic agents to the injured vascular wall under flow conditions. It could also serve as an effective therapeutic delivery system to treat the complications associated with cardiovascular diseases.

Structure-activity relationships for xenobiotic transport substrates and inhibitory ligands of P-glycoprotein.

PubMed Central

Bain, L J; McLachlan, J B; LeBlanc, G A

1997-01-01

The multixenobiotic resistance phenotype is characterized by the reduced accumulation of xenobiotics by cells or organisms due to increased efflux of the compounds by P-glycoprotein (P-gp) or related transporters. An extensive xenobiotic database, consisting primarily of pesticides, was utilized in this study to identify molecular characteristics that render a xenobiotic susceptible to transport by or inhibition of P-gp. Transport substrates were differentiated by several molecular size/shape parameters, lipophilicity, and hydrogen bonding potential. Electrostatic features differentiated inhibitory ligands from compounds not catagorized as transport substrates and that did no interact with P-gp. A two-tiered system was developed using the derived structure-activity relationships to identify P-gp transport substrates and inhibitory ligands. Prediction accuracy of the approach was 82%. We then validated the system using six additional pesticides of which tow were predicted to be P-gp inhibitors and four were predicted to be noninteractors, based upon the structure-activity analyses. Experimental determinations using cells transfected with the human MDR1 gene demonstrated that five of the six pesticides were properly catagorized by the structure-activity analyses (83% accuracy). Finally, structure-activity analyses revealed that among P-gp inhibitors, relative inhibitory potency can be predicted based upon the surface area or volume of the compound. These results demonstrate that P-gp transport substrates and inhibitory ligands can be distinguished using molecular characteristics. Molecular characteristics of transport substrates suggest that P-gp may function in the elimination of hydroxylated metabolites of xenobiotics. Images Figure 1. A Figure 1. B Figure 1. C Figure 1. D Figure 1. E Figure 1. F Figure 1. G Figure 1. H Figure 2. Figure 2. Figure 2. Figure 2. Figure 2. Figure 2. Figure 3. A Figure 3. B PMID:9347896

In Silico Screening for Inhibitors of P-Glycoprotein That Target the Nucleotide Binding Domains

PubMed Central

Brewer, Frances K.; Follit, Courtney A.; Vogel, Pia D.

2014-01-01

Multidrug resistances and the failure of chemotherapies are often caused by the expression or overexpression of ATP-binding cassette transporter proteins such as the multidrug resistance protein, P-glycoprotein (P-gp). P-gp is expressed in the plasma membrane of many cell types and protects cells from accumulation of toxins. P-gp uses ATP hydrolysis to catalyze the transport of a broad range of mostly hydrophobic compounds across the plasma membrane and out of the cell. During cancer chemotherapy, the administration of therapeutics often selects for cells which overexpress P-gp, thereby creating populations of cancer cells resistant to a variety of chemically unrelated chemotherapeutics. The present study describes extremely high-throughput, massively parallel in silico ligand docking studies aimed at identifying reversible inhibitors of ATP hydrolysis that target the nucleotide-binding domains of P-gp. We used a structural model of human P-gp that we obtained from molecular dynamics experiments as the protein target for ligand docking. We employed a novel approach of subtractive docking experiments that identified ligands that bound predominantly to the nucleotide-binding domains but not the drug-binding domains of P-gp. Four compounds were found that inhibit ATP hydrolysis by P-gp. Using electron spin resonance spectroscopy, we showed that at least three of these compounds affected nucleotide binding to the transporter. These studies represent a successful proof of principle demonstrating the potential of targeted approaches for identifying specific inhibitors of P-gp. PMID:25270578

Computational predictive models for P-glycoprotein inhibition of in-house chalcone derivatives and drug-bank compounds.

PubMed

Ngo, Trieu-Du; Tran, Thanh-Dao; Le, Minh-Tri; Thai, Khac-Minh

2016-11-01

The human P-glycoprotein (P-gp) efflux pump is of great interest for medicinal chemists because of its important role in multidrug resistance (MDR). Because of the high polyspecificity as well as the unavailability of high-resolution X-ray crystal structures of this transmembrane protein, ligand-based, and structure-based approaches which were machine learning, homology modeling, and molecular docking were combined for this study. In ligand-based approach, individual two-dimensional quantitative structure-activity relationship models were developed using different machine learning algorithms and subsequently combined into the Ensemble model which showed good performance on both the diverse training set and the validation sets. The applicability domain and the prediction quality of the developed models were also judged using the state-of-the-art methods and tools. In our structure-based approach, the P-gp structure and its binding region were predicted for a docking study to determine possible interactions between the ligands and the receptor. Based on these in silico tools, hit compounds for reversing MDR were discovered from the in-house and DrugBank databases through virtual screening using prediction models and molecular docking in an attempt to restore cancer cell sensitivity to cytotoxic drugs.

P-selectin expressed by a human SELP transgene is atherogenic in apolipoprotein E-deficient mice

PubMed Central

Zhang, Nan; Liu, Zhenghui; Yao, Longbiao; Mehta-D’souza, Padmaja; McEver, Rodger P.

2016-01-01

Objective During inflammation, P-selectin expressed on activated endothelial cells and platelets mediates rolling adhesion of leukocytes. Atherosclerosis-prone mice crossed with P-selectin-deficient (Selp−/−) mice develop smaller lesions. Cytokines such as tumor necrosis factor-α increase Selp transcripts and augment atherosclerosis in mice. However, they decrease SELP transcripts in humans, challenging assumptions that human P-selectin is atherogenic. We used mice expressing a human SELP transgene to examine the atherogenic role of P-selectin. Approach and results We crossed apolipoprotein E-deficient (Apoe−/−) mice with Selp−/− mice and/or transgenic mice expressing the entire human SELP gene (TgSELP+/−). Aortas developed larger, macrophage-rich atheromas in Apoe−/−Selp−/−TgSELP+/− mice than in Apoe−/−Selp−/− mice after 8 or 16 weeks on a Western diet. Confocal microscopy of Apoe−/−Selp−/−TgSELP+/− aortas revealed staining for human P-selectin in endothelial cells overlying atheromas, but not in lesional macrophages. We also observed staining for human P-selectin in aortic endothelial cells of 3–4-week-old Apoe−/−Selp−/−TgSELP+/− weanlings before atheromas developed. Furthermore, human SELP transcripts were ~3-fold higher in aortas of Apoe−/−Selp+/−TgSELP+/− weanlings than in Selp+/−TgSELP+/− weanlings, whereas murine Selp and Sele transcripts were equivalent in weanlings of both genotypes. Human SELP transcripts in aortas of Apoe−/−Selp+/−TgSELP+/− mice remained nearly constant during 16 weeks on a Western diet, whereas murine Selp and Sele transcripts progressively increased. Bone marrow transplantation in Apoe−/−Selp−/− and Apoe−/−Selp−/−TgSELP+/− mice demonstrated that both platelets and endothelial cells must express human P-selectin to promote atherogenesis. Conclusions P-selectin expressed by human SELP is atherogenic in Apoe−/− mice, suggesting that P-selectin contributes to atherogenesis in humans. PMID:27102967

Machine learning-, rule- and pharmacophore-based classification on the inhibition of P-glycoprotein and NorA.

PubMed

Ngo, T-D; Tran, T-D; Le, M-T; Thai, K-M

2016-09-01

The efflux pumps P-glycoprotein (P-gp) in humans and NorA in Staphylococcus aureus are of great interest for medicinal chemists because of their important roles in multidrug resistance (MDR). The high polyspecificity as well as the unavailability of high-resolution X-ray crystal structures of these transmembrane proteins lead us to combining ligand-based approaches, which in the case of this study were machine learning, perceptual mapping and pharmacophore modelling. For P-gp inhibitory activity, individual models were developed using different machine learning algorithms and subsequently combined into an ensemble model which showed a good discrimination between inhibitors and noninhibitors (acctrain-diverse = 84%; accinternal-test = 92% and accexternal-test = 100%). For ligand promiscuity between P-gp and NorA, perceptual maps and pharmacophore models were generated for the detection of rules and features. Based on these in silico tools, hit compounds for reversing MDR were discovered from the in-house and DrugBank databases through virtual screening in an attempt to restore drug sensitivity in cancer cells and bacteria.

Spatiotemporal Targeting of a Dual-Ligand Nanoparticle to Cancer Metastasis

PubMed Central

Doolittle, Elizabeth; Peiris, Pubudu M.; Doron, Gilad; Goldberg, Amy; Tucci, Samantha; Rao, Swetha; Shah, Shruti; Sylvestre, Meilyn; Govender, Priya; Turan, Oguz; Lee, Zhenghong; Schiemann, William P.; Karathanasis, Efstathios

2015-01-01

Various targeting strategies and ligands have been employed to direct nanoparticles to tumors that upregulate specific cell-surface molecules. However, tumors display a dynamic, heterogeneous microenvironment, which undergoes spatiotemporal changes including the expression of targetable cell-surface biomarkers. Here, we investigated a dual-ligand nanoparticle to effectively target two receptors overexpressed in aggressive tumors. By using two different chemical specificities, the dual-ligand strategy considered the spatiotemporal alterations in the expression patterns of the receptors in cancer sites. As a case study, we used two mouse models of metastasis of triple-negative breast cancer using the MDA-MB-231 and 4T1 cells. The dual-ligand system utilized two peptides targeting P-selectin and αvβ3 integrin, which are functionally linked to different stages of the development of metastatic disease at a distal site. Using in vivo multimodal imaging and post mortem histological analyses, this study shows that the dual-ligand nanoparticle effectively targeted metastatic disease that was otherwise missed by single-ligand strategies. The dual-ligand nanoparticle was capable of capturing different metastatic sites within the same animal that overexpressed either receptor or both of them. Furthermore, the highly efficient targeting resulted in 22% of the injected dual-ligand nanoparticles being deposited in early-stage metastases within 2 h after injection. PMID:26203676

Spatiotemporal Targeting of a Dual-Ligand Nanoparticle to Cancer Metastasis.

PubMed

Doolittle, Elizabeth; Peiris, Pubudu M; Doron, Gilad; Goldberg, Amy; Tucci, Samantha; Rao, Swetha; Shah, Shruti; Sylvestre, Meilyn; Govender, Priya; Turan, Oguz; Lee, Zhenghong; Schiemann, William P; Karathanasis, Efstathios

2015-08-25

Various targeting strategies and ligands have been employed to direct nanoparticles to tumors that upregulate specific cell-surface molecules. However, tumors display a dynamic, heterogeneous microenvironment, which undergoes spatiotemporal changes including the expression of targetable cell-surface biomarkers. Here, we investigated a dual-ligand nanoparticle to effectively target two receptors overexpressed in aggressive tumors. By using two different chemical specificities, the dual-ligand strategy considered the spatiotemporal alterations in the expression patterns of the receptors in cancer sites. As a case study, we used two mouse models of metastasis of triple-negative breast cancer using the MDA-MB-231 and 4T1 cells. The dual-ligand system utilized two peptides targeting P-selectin and αvβ3 integrin, which are functionally linked to different stages of the development of metastatic disease at a distal site. Using in vivo multimodal imaging and post mortem histological analyses, this study shows that the dual-ligand nanoparticle effectively targeted metastatic disease that was otherwise missed by single-ligand strategies. The dual-ligand nanoparticle was capable of capturing different metastatic sites within the same animal that overexpressed either receptor or both of them. Furthermore, the highly efficient targeting resulted in 22% of the injected dual-ligand nanoparticles being deposited in early-stage metastases within 2 h after injection.

Levels of platelet-derived microparticles and soluble p-selectin in patients of acute myocardial infarction (case control study).

PubMed

Hameed, Aisha; Rubab, Zille; Abbas Rizvi, Syed Khizar; Hussain, Shabbir; Latif, Waqas; Mohsin, Shahida

2017-07-01

TTo measure levels of platelet-derived microparticles and soluble P-selectin in patients of acute myocardial infarction and their comparison with healthy controls. This case-control study was conducted in Department of Haematology, University of Health Sciences Lahore from April to September 2013, and comprised patients of acute myocardial infarction in group 1 and healthy controls in group 2. Platelet-derived microparticles and soluble P-selectin were measured by enzyme-linked immunosorbent assay. SPSS21 was used for data analysis. Of the 80 participants, 50(62.5%) were patients and 30(37.5%) were controls. The mean levels of platelet-derived microparticles and soluble P-selectin were significantly higher in group 1 compared to group 2 (45.70±10.30 vs 10.60±0.96, and 51.46±9.30 vs 9.16±1.04, respectively) (p<0.001). There was no significant difference in levels of platelet-derived microparticles and soluble P-selectin in three intervals after acute myocardial infarction (p>0.05). Although levels of platelet-derived microparticles and soluble P-selectin did not correlate to creatinekinase-myocardial band levels (p>0.05), but there was a trend of significant correlation with cardiac troponin T (p<0.05). Levels of platelet-derived microparticles and soluble P-selectin can be used as novel early diagnostic marker of acute myocardial infarction.

Pharmacological modulation of endothelial cell-associated adhesion molecule expression: implications for future treatment of dermatological diseases.

PubMed

Foster, C A; Dreyfuss, M; Mandak, B; Meingassner, J G; Naegeli, H U; Nussbaumer, A; Oberer, L; Scheel, G; Swoboda, E M

1994-11-01

Skin diseases with an inflammatory component, regardless of their etiology, are characterized at some point by the extravasation and subsequent infiltration of leukocytes into the dermal and/or epidermal compartments. This trafficking pattern is determined by a complex series of events whereby the leukocytes interact with cell adhesion molecules (CAM), particularly those induced on endothelial cells following activation with various inflammatory mediators. Vascular CAMs belonging to the selectin family (i.e., P-selectin and E-selectin) are thought to mediate early and reversible events involving leukocyte rolling and margination along the lumenal surface of microvascular cells (post-capillary venules). Certain members of the immunoglobulin supergene family (i.e., VCAM-1 and ICAM-1) regulate later and irreversible steps which lead to firm attachment and subsequent diapedesis of leukocytes. Accumulating evidence suggests that if one blocks the ligand-binding sites between leukocytes and endothelial cells, or inhibits vascular CAM expression, hematopoietic cell extravasation and progressive inflammatory events can be greatly diminished. To identify such inhibitors we developed a cell-based Elisa using the human microvascular cell line HMEC-1. As reported in the present paper, this approach yielded a naturally-occurring, low molecular weight compound which potently inhibits cytokine-induced adhesion molecule expression on cultured endothelial cells, without modulating "house-keeping" proteins.

Anti-IL-5 attenuates activation and surface density of β(2) -integrins on circulating eosinophils after segmental antigen challenge.

PubMed

Johansson, M W; Gunderson, K A; Kelly, E A B; Denlinger, L C; Jarjour, N N; Mosher, D F

2013-03-01

IL-5 activates α(M) β(2) integrin on blood eosinophils in vitro. Eosinophils in bronchoalveolar lavage (BAL) following segmental antigen challenge have activated β(2) -integrins. To identify roles for IL-5 in regulating human eosinophil integrins in vivo. Blood and BAL eosinophils were analysed by flow cytometry in ten subjects with allergic asthma who underwent a segmental antigen challenge protocol before and after anti-IL-5 administration. Blood eosinophil reactivity with monoclonal antibody (mAb) KIM-127, which recognizes partially activated β(2) -integrins, was decreased after anti-IL-5. Before anti-IL-5, surface densities of blood eosinophil β(2) , α(M) and α(L) integrin subunits increased modestly post challenge. After anti-IL-5, such increases did not occur. Before or after anti-IL-5, surface densities of β(2) , α(M) , α(L) and α(D) and reactivity with KIM-127 and mAb CBRM1/5, which recognizes high-activity α(M) β(2) , were similarly high on BAL eosinophils 48 h post-challenge. Density and activation state of β(1) -integrins on blood and BAL eosinophils were not impacted by anti-IL-5, even though anti-IL-5 ablated a modest post-challenge increase on blood or BAL eosinophils of P-selectin glycoprotein ligand-1 (PSGL-1), a receptor for P-selectin that causes activation of β(1) -integrins. Forward scatter of blood eosinophils post-challenge was less heterogeneous and on the average decreased after anti-IL-5; however, anti-IL-5 had no effect on the decreased forward scatter of eosinophils in post-challenge BAL compared with eosinophils in blood. Blood eosinophil KIM-127 reactivity at the time of challenge correlated with the percentage of eosinophils in BAL post-challenge. IL-5 supports a heterogeneous population of circulating eosinophils with partially activated β(2) -integrins and is responsible for up-regulation of β(2) -integrins and PSGL-1 on circulating eosinophils following segmental antigen challenge but has minimal effects on properties of eosinophils in BAL. Dampening of β(2) -integrin function of eosinophils in transit to inflamed airway may contribute to the decrease in lung inflammation caused by anti-IL-5. © 2012 Blackwell Publishing Ltd.

Anti-IL-5 attenuates activation and surface density of β2-integrins on circulating eosinophils after segmental antigen challenge

PubMed Central

Johansson, Mats W.; Gunderson, Kristin A.; Kelly, Elizabeth A. B.; Denlinger, Loren C.; Jarjour, Nizar N.; Mosher, Deane F.

2013-01-01

Background IL-5 activates αMβ2 integrin on blood eosinophils in vitro. Eosinophils in bronchoalveolar lavage (BAL) following segmental antigen challenge have activated β2-integrins. Objective To identify roles for IL-5 in regulating human eosinophil integrins in vivo. Methods Blood and BAL eosinophils were analyzed by flow cytometry in ten subjects with allergic asthma who underwent a segmental antigen challenge protocol before and after anti-IL-5 administration. Results Blood eosinophil reactivity with monoclonal antibody (mAb) KIM-127, which recognizes partially activated β2-integrins, was decreased after anti-IL-5. Before anti-IL-5, surface densities of blood eosinophil β2, αM, and αL integrin subunits increased modestly post-challenge. After anti-IL-5, such increases did not occur. Before or after anti-IL-5, surface densities of β2,αM, αL, and αD and reactivity with KIM-127 and mAb CBRM1/5, which recognizes high-activity αMβ2, were similarly high on BAL eosinophils 48 h post-challenge. Density and activation state of β1-integrins on blood and BAL eosinophils were not impacted by anti-IL-5, even though anti-IL-5 ablated a modest post-challenge increase on blood or BAL eosinophils of P-selectin glycoprotein ligand-1 (PSGL-1), a receptor for P-selectin that causes activation of β1-integrins. Forward scatter of blood eosinophils post-challenge was less heterogeneous and on the average decreased after anti-IL-5; however, anti-IL-5 had no effect on the decreased forward scatter of eosinophils in post-challenge BAL compared to eosinophils in blood. Blood eosinophil KIM-127 reactivity at the time of challenge correlated with the percentage of eosinophils in BAL post-challenge. Conclusion and Clinical Relevance IL-5 supports a heterogeneous population of circulating eosinophils with partially activated β2-integrins and is responsible for upregulation of β2-integrins and PSGL-1 on circulating eosinophils following segmental antigen challenge but has minimal effects on properties of eosinophils in BAL. Dampening of β2-integrin function of eosinophils in transit to inflamed airway may contribute to the decrease in lung inflammation caused by anti-IL-5. PMID:23414537

Quantitative Glycoproteomic Analysis Identifies Platelet-Induced Increase of Monocyte Adhesion via the Up-Regulation of Very Late Antigen 5.

PubMed

Huang, Jiqing; Kast, Juergen

2015-08-07

Physiological stimuli, such as thrombin, or pathological stimuli, such as lysophosphatidic acid (LPA), activate platelets circulating in blood. Once activated, platelets bind to monocytes via P-selectin-PSGL-1 interactions but also release the stored contents of their granules. These platelet releasates, in addition to direct platelet binding, activate monocytes and facilitate their recruitment to atherosclerotic sites. Consequently, understanding the changes platelet releasates induce in monocyte membrane proteins is critical. We studied the glyco-proteome changes of THP-1 monocytic cells affected by LPA- or thrombin-induced platelet releasates. We employed lectin affinity chromatography combined with filter aided sample preparation to achieve high glyco- and membrane protein and protein sequence coverage. Using stable isotope labeling by amino acids in cell culture, we quantified 1715 proteins, including 852 membrane and 500 glycoproteins, identifying the up-regulation of multiple proteins involved in monocyte extracellular matrix binding and transendothelial migration. Flow cytometry indicated expression changes of integrin α5, integrin β1, PECAM-1, and PSGL-1. The observed increase in monocyte adhesion to fibronectin was determined to be mediated by the up-regulation of very late antigen 5 via a P-selectin-PSGL-1 independent mechanism. This novel aspect could be validated on CD14+ human primary monocytes, highlighting the benefits of the improved enrichment method regarding high membrane protein coverage and reliable quantification.

Role of the endothelial surface layer in neutrophil recruitment.

PubMed

Marki, Alex; Esko, Jeffrey D; Pries, Axel R; Ley, Klaus

2015-10-01

Neutrophil recruitment in most tissues is limited to postcapillary venules, where E- and P-selectins are inducibly expressed by venular endothelial cells. These molecules support neutrophil rolling via binding of PSGL-1 and other ligands on neutrophils. Selectins extend ≤ 38 nm above the endothelial plasma membrane, and PSGL-1 extends to 50 nm above the neutrophil plasma membrane. However, endothelial cells are covered with an ESL composed of glycosaminoglycans that is ≥ 500 nm thick and has measurable resistance against compression. The neutrophil surface is also covered with a surface layer. These surface layers would be expected to completely shield adhesion molecules; thus, neutrophils should not be able to roll and adhere. However, in the cremaster muscle and in many other models investigated using intravital microscopy, neutrophils clearly roll, and their rolling is easily and quickly induced. This conundrum was thought to be resolved by the observation that the induction of selectins is accompanied by ESL shedding; however, ESL shedding only partially reduces the ESL thickness (to 200 nm) and thus is insufficient to expose adhesion molecules. In addition to its antiadhesive functions, the ESL also presents neutrophil arrest-inducing chemokines. ESL heparan sulfate can also bind L-selectin expressed by the neutrophils, which contributes to rolling and arrest. We conclude that ESL has both proadhesive and antiadhesive functions. However, most previous studies considered either only the proadhesive or only the antiadhesive effects of the ESL. An integrated model for the role of the ESL in neutrophil rolling, arrest, and transmigration is needed. © Society for Leukocyte Biology.

Role of the endothelial surface layer in neutrophil recruitment

PubMed Central

Marki, Alex; Esko, Jeffrey D.; Pries, Axel R.; Ley, Klaus

2015-01-01

Neutrophil recruitment in most tissues is limited to postcapillary venules, where E- and P-selectins are inducibly expressed by venular endothelial cells. These molecules support neutrophil rolling via binding of PSGL-1 and other ligands on neutrophils. Selectins extend ≤38 nm above the endothelial plasma membrane, and PSGL-1 extends to 50 nm above the neutrophil plasma membrane. However, endothelial cells are covered with an ESL composed of glycosaminoglycans that is ≥500 nm thick and has measurable resistance against compression. The neutrophil surface is also covered with a surface layer. These surface layers would be expected to completely shield adhesion molecules; thus, neutrophils should not be able to roll and adhere. However, in the cremaster muscle and in many other models investigated using intravital microscopy, neutrophils clearly roll, and their rolling is easily and quickly induced. This conundrum was thought to be resolved by the observation that the induction of selectins is accompanied by ESL shedding; however, ESL shedding only partially reduces the ESL thickness (to 200 nm) and thus is insufficient to expose adhesion molecules. In addition to its antiadhesive functions, the ESL also presents neutrophil arrest-inducing chemokines. ESL heparan sulfate can also bind L-selectin expressed by the neutrophils, which contributes to rolling and arrest. We conclude that ESL has both proadhesive and antiadhesive functions. However, most previous studies considered either only the proadhesive or only the antiadhesive effects of the ESL. An integrated model for the role of the ESL in neutrophil rolling, arrest, and transmigration is needed. PMID:25979432

Effect of long-term clopidogrel treatment on platelet function and inflammation in patients undergoing coronary arterial stenting.

PubMed

Antonino, Mark J; Mahla, Elisabeth; Bliden, Kevin P; Tantry, Udaya S; Gurbel, Paul A

2009-06-01

A clopidogrel loading dose administered during stenting attenuates inflammation marker release. However, less is known of the anti-inflammatory effect of clopidogrel maintenance therapy. Platelet reactivity to adenosine diphosphate and inflammation markers were measured in 110 consecutive patients (69 clopidogrel-naive patients and 41 patients receiving long-term clopidogrel therapy for >6 months) before nonemergent stenting by turbidimetric aggregometry and flow cytometry and multianalyte profiling, respectively. All patients were treated with aspirin. Prestenting adenosine diphosphate-induced platelet aggregation, P-selectin, and activated glycoprotein IIb/IIIa expression were lower in patients receiving long-term clopidogrel therapy compared with the clopidogrel-naive group (p <0.001), accompanied by lower levels of selected inflammation markers (p < or = 0.05). Additionally, there were strong correlations between platelet aggregation and flow cytometric measurements (p < or = 0.04) and between specific inflammation markers (p < or = 0.02). In conclusion, in addition to markedly lowering platelet reactivity to adenosine diphosphate, long-term clopidogrel therapy is associated with an anti-inflammatory effect.

Evaluation of the Value of d-Dimer, P-Selectin, and Platelet Count for Prediction of Portal Vein Thrombosis After Devascularization.

PubMed

Fei, Yang; Zong, Guang-Quan; Chen, Jian; Liu, Ren-Min

2016-07-01

To evaluate the value of d-dimer, P-selectin, and platelet count in patients with cirrhotic portal hypertension (PHT) for prediction of portal vein thrombosis (PVT) after devascularization. A total of 137 patients with cirrhotic PHT who undergone devascularization from January 2012 to April 2014 were retrospectively reviewed, all of them were divided into 2 groups (PVT group and non-PVT group) by Doppler ultrasonography (DU) examination. The level of d-dimer, P-selectin, and platelet count was tested during the perioperative period. In all, 38 (27.7%) patients were found to have PVT by DU examination postoperatively. In contrast to the non-PVT group, the level of d-dimer, P-selectin, and platelet count in the PVT group was much higher significantly at 1, 3, and 7 days after devascularization. (P < .05). However, in the 15 days after surgery, the difference in P-selectin between the 2 groups was not significant (P = .260). It was shown that the highest sensitivity of the 3 markers for PVT was d-dimer, the highest specificity belonged to P-selectin. The area under receiver-operating characteristic (ROC) curve of P-selectin was the biggest of the 3 markers. When the 3 markers were combined to be used to diagnose PVT, the sensitivity was increased to 0.907, with a slight drop of specificity to 0.693, the area under the ROC curve was 0.927. The level of d-dimer, P-selectin, and platelet count might be good candidate predictive markers for PVT in patients with cirrhotic PHT after devascularization. The combined test of the 3 markers can increase the value of prediction. © The Author(s) 2015.

Fetal wound healing using a genetically modified murine model: the contribution of P-selectin

USDA-ARS?s Scientific Manuscript database

During early gestation, fetal wounds heal with paucity of inflammation and absent scar formation. P-selectin is an adhesion molecule that is important for leukocyte recruitment to injury sites. We used a murine fetal wound healing model to study the specific contribution of P-selectin to scarless wo...

P-selectin- and heparanase-dependent antimetastatic activity of non-anticoagulant heparins.

PubMed

Hostettler, Nina; Naggi, Annamaria; Torri, Giangiacomo; Ishai-Michaeli, Riva; Casu, Benito; Vlodavsky, Israel; Borsig, Lubor

2007-11-01

Vascular cell adhesion molecules, P- and L-selectins, facilitate metastasis of cancer cells in mice by mediating interactions with platelets, endothelium, and leukocytes. Heparanase is an endoglycosidase that degrades heparan sulfate of extracellular matrix, thereby promoting tumor invasion and metastasis. Heparin is known to efficiently attenuate metastasis in different tumor models. Here we identified modified, nonanticoagulant species of heparin that specifically inhibit selectin-mediated cell-cell interactions, heparanase enzymatic activity, or both. We show that selective inhibition of selectin interactions or heparanase with specific heparin derivatives in mouse models of MC-38 colon carcinoma and B16-BL6 melanoma attenuates metastasis. Selectin-specific heparin derivatives attenuated metastasis of MC-38 carcinoma, but heparanase-specific derivatives had no effect, in accordance with the virtual absence of heparanase activity in these cells. Heparin derivatives had no further effect on metastasis in mice deficient in P- and L-selectin, indicating that selectins are the primary targets of heparin antimetastatic activity. Selectin-specific and heparanase-specific derivatives attenuated metastasis of B16-BL6 melanomas to a similar extent. When mice were injected with a derivative containing both heparanase and selectin inhibitory activity, no additional attenuation of metastasis could be observed. Thus, selectin-specific heparin derivatives efficiently attenuated metastasis of both tumor cell types whereas inhibition of heparanase led to reduction of metastasis only in tumor cells producing heparanase.

In silico screening for inhibitors of p-glycoprotein that target the nucleotide binding domains.

PubMed

Brewer, Frances K; Follit, Courtney A; Vogel, Pia D; Wise, John G

2014-12-01

Multidrug resistances and the failure of chemotherapies are often caused by the expression or overexpression of ATP-binding cassette transporter proteins such as the multidrug resistance protein, P-glycoprotein (P-gp). P-gp is expressed in the plasma membrane of many cell types and protects cells from accumulation of toxins. P-gp uses ATP hydrolysis to catalyze the transport of a broad range of mostly hydrophobic compounds across the plasma membrane and out of the cell. During cancer chemotherapy, the administration of therapeutics often selects for cells which overexpress P-gp, thereby creating populations of cancer cells resistant to a variety of chemically unrelated chemotherapeutics. The present study describes extremely high-throughput, massively parallel in silico ligand docking studies aimed at identifying reversible inhibitors of ATP hydrolysis that target the nucleotide-binding domains of P-gp. We used a structural model of human P-gp that we obtained from molecular dynamics experiments as the protein target for ligand docking. We employed a novel approach of subtractive docking experiments that identified ligands that bound predominantly to the nucleotide-binding domains but not the drug-binding domains of P-gp. Four compounds were found that inhibit ATP hydrolysis by P-gp. Using electron spin resonance spectroscopy, we showed that at least three of these compounds affected nucleotide binding to the transporter. These studies represent a successful proof of principle demonstrating the potential of targeted approaches for identifying specific inhibitors of P-gp. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Correlation between serum E-selectin levels and panoramic nailfold capillaroscopy in systemic sclerosis.

PubMed

Valim, V; Assis, L S S; Simões, M F J; Trevisani, V F M; Pucinelli, M L C; Andrade, L E C

2004-09-01

E-selectin is expressed by the activated endothelium and its plasma levels are increased in patients with systemic sclerosis. Eighteen patients fulfilling the American Rheumatism Association criteria for systemic sclerosis, 15 females and 3 males, 42-70 years old, 9 with diffuse and 9 with limited forms, were sequentially recruited for this study. Serum E-selectin levels were determined by commercially available ELISA and their association with nailfold capillaroscopic abnormalities was investigated. Nailfold capillaries were analyzed by 16X magnification wide-field capillaroscopy. Two parameters on capillaroscopy were used to correlate to serum E-selectin: deletion and ectasia. Data were analyzed statistically by the Student t-test and Spearman correlation. Two-tailed P values below 0.05 were considered significant. E-selectin range was 38 to 200 ng/ml (80 +/- 39.94). There was a correlation between serum E-selectin levels and the deletion capillaroscopic score (r = 0.50, P < 0.035). This correlation was even stronger within the first 48 months of diagnosis (r = 0.63, P < 0.048). On the other hand, no association was observed between selectin and ectasia. Patients with diffuse disease presented higher serum E-selectin levels than patients with limited disease, although the difference was not statistically significant (96.44 +/- 48.04 vs 63.56 +/- 21.77 ng/dl; P = 0.08). The present study is the first showing a correlation between soluble serum E-selectin levels and alterations in capillaroscopy. The stronger correlation of deletion score in capillaroscopy in early disease suggests that serum E-selectin levels might be a useful biochemical marker of disease activity in systemic sclerosis.

Dendritic cell immunization route determines CD8+ T cell trafficking to inflamed skin: role for tissue microenvironment and dendritic cells in establishment of T cell-homing subsets.

PubMed

Dudda, Jan C; Simon, Jan C; Martin, Stefan

2004-01-15

The effector/memory T cell pool branches in homing subsets selectively trafficking to organs such as gut or skin. Little is known about the critical factors in the generation of skin-homing CD8+ T cells, although they are crucial effectors in skin-restricted immune responses such as contact hypersensitivity and melanoma defense. In this study, we show that intracutaneous, but not i.v. injection of bone marrow-derived dendritic cells induced skin-homing CD8+ T cells with up-regulated E-selectin ligand expression and effector function in contact hypersensitivity. The skin-homing potential and E-selectin ligand expression remained stable in memory phase without further Ag contact. In contrast, i.p. injection induced T cells expressing the gut-homing integrin alpha(4)beta(7). Although differential expression of these adhesion molecules was strictly associated with the immunization route, the postulated skin-homing marker CCR4 was transiently up-regulated in all conditions. Interestingly, dendritic cells from different tissues effectively induced the corresponding homing markers on T cells in vitro. Our results suggest a crucial role for the tissue microenvironment and dendritic cells in the instruction of T cells for tissue-selective homing and demonstrate that Langerhans cells are specialized to target T cells to inflamed skin.

Non-natural amino acid peptide microarrays to discover Ebola virus glycoprotein ligands.

PubMed

Rabinowitz, Joshua A; Lainson, John C; Johnston, Stephen Albert; Diehnelt, Chris W

2018-02-06

We demonstrate a platform to screen a virus pseudotyped with Ebola virus glycoprotein (GP) against a library of peptides that contain non-natural amino acids to develop GP affinity ligands. This system could be used for rapid development of peptide-based antivirals for other emerging or neglected tropical infectious diseases.

Anti-citrullinated protein antibodies contribute to platelet activation in rheumatoid arthritis.

PubMed

Habets, Kim L L; Trouw, Leendert A; Levarht, E W Nivine; Korporaal, Suzanne J A; Habets, Petra A M; de Groot, Philip; Huizinga, Tom W J; Toes, René E M

2015-08-24

Although the role of platelets in rheumatoid arthritis (RA) is relatively unexplored, recent studies point towards a contribution of platelets in arthritis. We set out to determine platelet phenotype in RA and studied whether this could be influenced by the presence of anti-citrullinated protein antibodies (ACPA). Platelets from healthy controls were incubated in the presence of plasma of patients with RA or age- and sex-matched healthy controls and plasma from ACPA(neg) or ACPA(pos) patients or in the presence of plate-bound ACPA. Characteristics of platelets isolated from patients with RA were correlated to disease activity. Platelets isolated from healthy controls displayed markers of platelet activation in the presence of plasma derived from RA patients, as determined by P-selectin expression, formation of aggregates and secretion of soluble CD40 ligand (sCD40L). Furthermore, levels of P-selectin expression and sCD40L release correlated with high ACPA titres. In accordance with these findings, enhanced platelet activation was observed after incubation with ACPA(pos) plasma versus ACPA(neg) plasma. Pre-incubation of platelets with blocking antibodies directed against low-affinity immunoglobulin G receptor (FcγRIIa) completely inhibited the ACPA-mediated activation. In addition, expression of P-selectin measured as number of platelets correlated with Disease Activity Score in 44 joints, C-reactive protein level, ACPA status and ACPA level. We show for the first time that ACPA can mediate an FcγRIIa-dependent activation of platelets. As ACPA can be detected several years before RA disease onset and activated platelets contribute to vascular permeability, these data implicate a possible role for ACPA-mediated activation of platelets in arthritis onset.

Platelet-activated clotting time does not measure platelet reactivity during cardiac surgery.

PubMed

Shore-Lesserson, L; Ammar, T; DePerio, M; Vela-Cantos, F; Fisher, C; Sarier, K

1999-08-01

Platelet dysfunction is a major contributor to bleeding after cardiopulmonary bypass (CPB), yet it remains difficult to diagnose. A point-of-care monitor, the platelet-activated clotting time (PACT), measures accelerated shortening of the kaolin-activated clotting time by addition of platelet activating factor. The authors sought to evaluate the clinical utility of the PACT by conducting serial measurements of PACT during cardiac surgery and correlating postoperative measurements with blood loss. In 50 cardiac surgical patients, blood was sampled at 10 time points to measure PACT. Simultaneously, platelet reactivity was measured by the thrombin receptor agonist peptide-induced expression of P-selectin, using flow cytometry. These tests were temporally analyzed. PACT values, P-selectin expression, and other coagulation tests were analyzed for correlation with postoperative chest tube drainage. PACT and P-selectin expression were maximally reduced after protamine administration. Changes in PACT did not correlate with changes in P-selectin expression at any time interval. Total 8-h chest tube drainage did not correlate with any coagulation test at any time point except with P-selectin expression after protamine administration (r = -0.4; P = 0.03). The platelet dysfunction associated with CPB may be a result of depressed platelet reactivity, as shown by thrombin receptor activating peptide-induced P-selectin expression. Changes in PACT did not correlate with blood loss or with changes in P-selectin expression suggesting that PACT is not a specific measure of platelet reactivity.

Platelet morphology, soluble P selectin and platelet P-selectin in acute ischaemic stroke. The West Birmingham Stroke Project.

PubMed

Nadar, Sunil K; Lip, Gregory Y H; Blann, Andrew D

2004-12-01

The pathophysiology of ischaemic stroke involves the platelet. In this study, we hypothesised that abnormalities in platelet morphology, as well as soluble (sPsel) and total platelet P-selectin (pPsel) levels would be present in patients presenting with an acute ischaemic stroke, and that these changes would improve at > or = 3 months' follow-up. We studied 59 hypertensive patients (34 male; mean age 68 +/- 12 years) who presented with an acute ischaemic stroke (ictus < 24 hours), and compared them with 2 groups: (i) age-, sex- and ethnic- origin matched normotensive healthy controls; and (ii) uncomplicated 'high risk' hypertensive patients as 'risk factor control' subjects. Platelet morphology (volume and mass) was quantified, and sPsel (plasma marker of platelet activation) was measured (ELISA) in citrated plasma. The mass of P-selectin in each platelet (pPsel) was determined by lysing a fixed number of platelets and then determining the levels of P-selectin in the lysate. Results show that patients who presented with a stroke had significantly higher levels of sPsel and pPsel (both p < 0.001), compared to the normal controls and the hypertensive patients. Patients with an acute stroke had lower mean platelet mass (MPM) and mean platelet volume (MPV) as compared to the uncomplicated hypertensive patients, who had significantly higher mean MPM and MPV values, as compared to normal controls. On follow-up, the levels of both sPsel (p = 0.011), pPsel (< 0.001) and MPV (p = 0.03) were significantly lower. Mean MPM levels remained unchanged. We conclude that patients presenting with an acute ischaemic stroke have activated platelets, as evident by the increased levels of soluble and platelet P-selectin. Further study of platelet activation and the role of P-selectin is warranted.

Reduced recruitment of inflammatory cells in a contact hypersensitivity response in P-selectin-deficient mice

PubMed Central

1995-01-01

The inflammatory response at sites of contact hypersensitivity induced by oxazolone was examined in the ears of P-selectin-deficient and wild- type mice. Accumulation of CD4+ T lymphocytes, monocytes, and neutrophils was reduced significantly in the mutant mice, as well as mast cell degranulation. In contrast, there was no significant difference in vascular permeability or edema between the two genotypes. The results demonstrate a role for P-selectin in recruitment of CD4+ T lymphocytes and show that P-selectin plays a role in long-term inflammation as well as in acute responses. PMID:7539046

Minimizing antibody surface density on liposomes while sustaining cytokine-activated EC targeting.

PubMed

Almeda, Dariela; Wang, Biran; Auguste, Debra T

2015-02-01

Liposomes may be engineered to target inflamed endothelium by mimicking ligand-receptor interactions between leukocytes and cytokine-activated endothelial cells (ECs). The upregulation and assembly of vascular cell adhesion molecule-1 (VCAM1) and E-selectin on the cell membrane upon exposure to cytokines have shown potential for drug delivery vehicles to target sites of chronic endothelial inflammation, such as atherosclerosis and cancer. Herein, we characterized EC surfaces by measuring the E-selectin and VCAM1 surface densities and adhesion forces of aVCAM1 and aE-selectin to ECs. We quantified the antibody density, ratio, and diffusivity of liposomes to achieve significant binding and internalization. At 1 h, the 1:1 ratio of VCAM1:E-selectin antibodies was significantly higher than 1:0 and 0:1. Significant binding and uptake was achieved at aE-selectin densities as low as 400 molecules/μm(2). The highest levels of binding and uptake were achieved when using a 1:1 ratio of VCAM1:E-selectin antibodies at a density of 1000 molecules/μm(2); this density is 85% lower than previous reports. The binding and uptake of functionalized liposomes were reduced to levels comparable to IgG functionalized liposomes upon a 10-fold reduction in liposome membrane diffusivity. We conclude with a liposomal design that discriminates between healthy and inflamed endothelium while reducing antibody surface presentation. Copyright © 2014 Elsevier Ltd. All rights reserved.

Reduced background autofluorescence for cell imaging using nanodiamonds and lanthanide chelates.

PubMed

Cordina, Nicole M; Sayyadi, Nima; Parker, Lindsay M; Everest-Dass, Arun; Brown, Louise J; Packer, Nicolle H

2018-03-14

Bio-imaging is a key technique in tracking and monitoring important biological processes and fundamental biomolecular interactions, however the interference of background autofluorescence with targeted fluorophores is problematic for many bio-imaging applications. This study reports on two novel methods for reducing interference with cellular autofluorescence for bio-imaging. The first method uses fluorescent nanodiamonds (FNDs), containing nitrogen vacancy centers. FNDs emit at near-infrared wavelengths typically higher than most cellular autofluorescence; and when appropriately functionalized, can be used for background-free imaging of targeted biomolecules. The second method uses europium-chelating tags with long fluorescence lifetimes. These europium-chelating tags enhance background-free imaging due to the short fluorescent lifetimes of cellular autofluorescence. In this study, we used both methods to target E-selectin, a transmembrane glycoprotein that is activated by inflammation, to demonstrate background-free fluorescent staining in fixed endothelial cells. Our findings indicate that both FND and Europium based staining can improve fluorescent bio-imaging capabilities by reducing competition with cellular autofluorescence. 30 nm nanodiamonds coated with the E-selectin antibody was found to enable the most sensitive detective of E-selectin in inflamed cells, with a 40-fold increase in intensity detected.

NF-kB activity-dependent P-selectin involved in ox-LDL-induced foam cell formation in U937 cell

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yi, E-mail: wangyi2004a@126.com; Wang, Xiang; Sun, Minghui

Highlights: {yields} Ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. {yields} Ox-LDL induced expression of P-selectin through degradation of IkBa and augment of NF-kB activity and protein level during macrophage-derived foam cell formation. {yields} P-selectin and NF-kB may be identified as pivotal regulators of ox-LDL-induced foam cell formation. {yields} Therapy based on the inhibition of P-selectin and NF-kB may complement conventional treatments to prevent atherosclerosis. -- Abstract: Oxidized low-density lipoprotein (ox-LDL) plays a critical role in regulation of atherosclerosis. However, little is known about the role of Nuclear factor kBmore » (NF-kB) activity-dependent P-selectin in ox-LDL-induced foam cell formation during atherosclerosis. In this study, we first investigated ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. Treatment of U937 cells with ox-LDL increased lipid accumulation as well as intracellular cholesterol content. Next, a comparative analysis of gene expression profiling using cDNA microarray and Real-time-PCR indicated that ox-LDL exposure induced, in three treated groups, an extremely marked increase in the mRNA level of P-selectin. Protein levels of P-selectin and its upstream regulators IkBa and NF-kB showed that NF-kB pathway is involved in the ox-LDL-induced foam cell formation. Finally, overexpression of NF-kB significantly accelerated, whereas, inhibition of NF-kB with siRNA remarkably attenuated ox-LDL-induced macrophage-derived foam cell formation. It was concluded that the activity of NF-kB is augmented during macrophage-derived foam cell formation. Activation of NF-kB increased, whereas, inhibition of NF-kB decreased ox-LDL-induced P-selectin expression and lipid accumulation in macrophages, suggesting ox-LDL induced expression of P-selectin through degradation of IkBa and activation of NF-kB in the regulation of foam cell formation.« less

Loss of the endothelial glycocalyx is associated with increased E-selectin mediated adhesion of lung tumour cells to the brain microvascular endothelium.

PubMed

Rai, Srijana; Nejadhamzeeigilani, Zaynab; Gutowski, Nicholas J; Whatmore, Jacqueline L

2015-09-25

Arrest of metastasising lung cancer cells to the brain microvasculature maybe mediated by interactions between ligands on circulating tumour cells and endothelial E-selectin adhesion molecules; a process likely to be regulated by the endothelial glycocalyx. Using human cerebral microvascular endothelial cells and non-small cell lung cancer (NSCLC) cell lines, we describe how factors secreted by NSCLC cells i.e. cystatin C, cathepsin L, insulin-like growth factor-binding protein 7 (IGFBP7), vascular endothelial growth factor (VEGF) and tumour necrosis factor-alpha (TNF-α), damage the glycocalyx and enhance initial contacts between lung tumour and cerebral endothelial cells. Endothelial cells were treated with tumour secreted-proteins or lung tumour conditioned medium (CM). Surface levels of E-selectin were quantified by ELISA. Adhesion of A549 and SK-MES-1 cells was examined under flow conditions (1 dyne/cm(2)). Alterations in the endothelial glycocalyx were quantified by binding of fluorescein isothiocyanate-linked wheat germ agglutinin (WGA-FITC). A549 and SK-MES-1 CM and secreted-proteins significantly enhanced endothelial surface E-selectin levels after 30 min and 4 h and tumour cell adhesion after 30 min, 4 and 24 h. Both coincided with significant glycocalyx degradation; A549 and SK-MES-1 CM removing 55 ± 12 % and 58 ± 18.7 % of WGA-FITC binding, respectively. Inhibition of E-selectin binding by monoclonal anti-E-selectin antibody completely attenuated tumour cell adhesion. These data suggest that metastasising lung cancer cells facilitate their own adhesion to the brain endothelium by secreting factors that damage the endothelial glycocalyx, resulting in exposure of the previously shielded adhesion molecules and engagement of the E-selectin-mediated adhesion axis.

Alternatively activated macrophages upregulate mesothelial expression of P-selectin to enhance adhesion of ovarian cancer cells.

PubMed

Carroll, Molly J; Fogg, Kaitlin C; Patel, Harin A; Krause, Harris B; Mancha, Anne-Sophie; Patankar, Manish S; Weisman, Paul S; Barroilhet, Lisa; Kreeger, Pamela K

2018-05-08

Peritoneal metastasis of high-grade serous ovarian cancer (HGSOC) occurs when tumor cells suspended in ascites adhere to mesothelial cells. Despite the strong relationship between metastatic burden and prognosis in HGSOC, there are currently no therapies specifically targeting the metastatic process. We utilized a co-culture model and multivariate analysis to examine how interactions between tumor cells, mesothelial cells, and alternatively-activated macrophages (AAMs) influence the adhesion of tumor cells to mesothelial cells. We found that AAM-secreted MIP-1β activates CCR5/PI3K signaling in mesothelial cells, resulting in expression of P-selectin on the mesothelial cell surface. Tumor cells attached to this de novo P-selectin through CD24, resulting in increased tumor cell adhesion in static conditions and rolling under flow. C57/BL6 mice treated with MIP-1β exhibited increased P-selectin expression on mesothelial cells lining peritoneal tissues, which enhanced CaOV3 adhesion ex vivo and ID8 adhesion in vivo. Analysis of samples from HGSOC patients confirmed increased MIP-1β and P-selectin, suggesting that this novel multi-cellular mechanism could be targeted to slow or stop metastasis in HGSOC by repurposing anti- CCR5 and P-selectin therapies developed for other indications. Copyright ©2018, American Association for Cancer Research.

CTC-Endothelial Cell Interactions during Metastasis

DTIC Science & Technology

2013-04-01

endothelial cells via a variety of E-selectin ligands ( ESL ). These ESLs express a unique carbohydrate motif, sLex, which appears to be required for... ESL binding. The chemokine receptor CXCR4 has also been reported to supporting transendothelial migration of prostate cells through bone marrow

Monocyte-endothelial adhesion in chronic rheumatoid arthritis. In situ detection of selectin and integrin-dependent interactions.

PubMed Central

Grober, J S; Bowen, B L; Ebling, H; Athey, B; Thompson, C B; Fox, D A; Stoolman, L M

1993-01-01

Blood monocytes are the principal reservoir for tissue macrophages in rheumatoid synovitis. Receptor-mediated adhesive interactions between circulating cells and the synovial venules initiate recruitment. These interactions have been studied primarily in cultured endothelial cells. Thus the functional activities of specific adhesion receptors, such as the endothelial selectins and the leukocytic integrins, have not been evaluated directly in diseased tissues. We therefore examined monocyte-microvascular interactions in rheumatoid synovitis by modifying the Stamper-Woodruff frozen section binding assay initially developed to study lymphocyte homing. Specific binding of monocytes to venules lined by low or high endothelium occurred at concentrations as low as 5 x 10(5) cells/ml. mAbs specific for P-selectin (CD62, GMP-140/PADGEM) blocked adhesion by > 90% in all synovitis specimens examined. In contrast, P-selectin-mediated adhesion to the microvasculature was either lower or absent in frozen sections of normal foreskin and placenta. mAbs specific for E-selectin (ELAM-1) blocked 20-50% of monocyte attachment in several RA synovial specimens but had no effect in others. mAbs specific for LFA-1, Mo1/Mac 1, the integrin beta 2-chain, and L-selectin individually inhibited 30-40% of adhesion. An mAb specific for the integrin beta 1-chain inhibited the attachment of elutriated monocytes up to 20%. We conclude that P-selectin associated with the synovial microvasculature initiates shear-resistant adhesion of monocytes in the Stamper-Woodruff assay and stabilizes bonds formed by other selectins and the integrins. Thus the frozen section binding assay permits direct evaluation of leukocyte-microvascular adhesive interactions in inflamed tissues and suggests a prominent role for P-selectin in monocyte recruitment in vivo. Images PMID:7685772

Positron Emission Tomography and Autoradiography Imaging of P-selectin Activation Using 68Ga-Fucoidan in Photothrombotic Stroke in Rats.

PubMed

Israel, Ina; Fluri, Felix; Schadt, Fabian; Buck, Adreas; Samnick, Samuel

2018-03-19

P-selectin is activated early after stroke, followed by a rapid decline. This time course can be used to generate important information on stroke onset. The latter is crucial for therapeutic decision-making of wake-up strokes (i.e. thrombolysis or not). Here we evaluated the specific p-selectin inhibitor 68Ga-Fucoidan for assessing p-selectin activation after ischemic stroke using PET. 68Ga-Fucoidan was investigated in rats brain at 2-5 h (n=16), and additionally at 24-26 h (n=9) and 48 h (n=3) after induction of photothrombic stroke or in sham operated animals (n=6). Correlation of cerebral 68Ga-Fucoidan uptake with p-selectin activation was determined by exposing freshly cut brain cryosections to autoradiography and immunostaining using specific antibodies against p-selectin. PET scans showed an increased accumulation of 68Ga-Fucoidan in the histologically proven ischemic stroke, as compared to the corresponding contralateral hemisphere in all except one animal. The median ratio between the uptake in the ischemic lesion and the contralateral region was 1.95 (1.45-2.41) at 2-5 h, 1.38 (1.05-1.89) at 24-26 h, and 1.09 (0.81-1.38) at 48 h after stroke, compared to 1.22 (0.99-1.49) for sham operated animals. In the ex vivo autoradiography, 68Ga-Fucoidan accumulation co-localized with p-selectin as assessed by immunostaining. Control animals and those scanned at 24-26 h and 48 h after stroke exhibited no elevated 68Ga-Fucoidan uptake in either hemisphere. PET imaging using 68Ga-Fucoidan represents a valuable tool for assessing p-selectin activation in vivo and to discriminate ischemic stroke early after stroke onset. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

PPAR-α, a lipid-sensing transcription factor, regulates blood–brain barrier efflux transporter expression

PubMed Central

More, Vijay R; Campos, Christopher R; Evans, Rebecca A; Oliver, Keith D; Chan, Gary NY; Miller, David S

2016-01-01

Lipid sensor peroxisome proliferator-activated receptor alpha (PPAR-α) is the master regulator of lipid metabolism. Dietary release of endogenous free fatty acids, fibrates, and certain persistent environmental pollutants, e.g. perfluoroalkyl fire-fighting foam components, are peroxisome proliferator-activated receptor alpha ligands. Here, we define a role for peroxisome proliferator-activated receptor alpha in regulating the expression of three ATP-driven drug efflux transporters at the rat and mouse blood–brain barriers: P-glycoprotein (Abcb1), breast cancer resistance protein (Bcrp/Abcg2), and multidrug resistance-associated protein 2 (Mrp2/Abcc2). Exposing isolated rat brain capillaries to linoleic acid, clofibrate, or PKAs increased the transport activity and protein expression of the three ABC transporters. These effects were blocked by the PPAR-α antagonist, GW6471. Dosing rats with 20 mg/kg or 200 mg/kg of clofibrate decreased the brain accumulation of the P-glycoprotein substrate, verapamil, by 50% (in situ brain perfusion; effects blocked by GW6471) and increased P-glycoprotein expression and activity in capillaries ex vivo. Fasting C57Bl/6 wild-type mice for 24 h increased both serum lipids and brain capillary P-glycoprotein transport activity. Fasting did not alter P-glycoprotein activity in PPAR-α knockout mice. These results indicate that hyperlipidemia, lipid-lowering fibrates and exposure to certain fire-fighting foam components activate blood–brain barrier peroxisome proliferator-activated receptor alpha, increase drug efflux transporter expression and reduce drug delivery to the brain. PMID:27193034

Recombinant Mucin-Type Fusion Proteins with a Galα1,3Gal Substitution as Clostridium difficile Toxin A Inhibitors

PubMed Central

Jin, Chunsheng; Liu, Jining; Karlsson, Niclas G.; Holgersson, Jan

2016-01-01

The capability of a recombinant mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b), carrying Galα1,3Galβ1,4GlcNAc determinants to bind and inhibit Clostridium difficile toxin A (TcdA) was investigated. The fusion protein, produced by a glyco-engineered stable CHO-K1 cell line and designated C-PGC2, was purified by affinity and gel filtration chromatography from large-scale cultures. Liquid chromatography-mass spectrometry was used to characterize O-glycans released by reductive β-elimination, and new diagnostic ions to distinguish Galα1,3Gal- from Galα1,4Gal-terminated O-glycans were identified. The C-PGC2 cell line, which was 20-fold more sensitive to TcdA than the wild-type CHO-K1, is proposed as a novel cell-based model for TcdA cytotoxicity and neutralization assays. The C-PGC2-produced fusion protein could competitively inhibit TcdA binding to rabbit erythrocytes, making it a high-efficiency inhibitor of the hemagglutination property of TcdA. The fusion protein also exhibited a moderate capability for neutralization of TcdA cytotoxicity in both C-PGC2 and CHO-K1 cells, the former with and the latter without cell surface Galα1,3Galβ1,4GlcNAc sequences. Future studies in animal models of C. difficile infection will reveal its TcdA-inhibitory effect and therapeutic potential in C. difficile-associated diseases. PMID:27456831

The Tie2 receptor antagonist angiopoietin-2 in systemic lupus erythematosus: its correlation with various disease activity parameters.

PubMed

Salama, Maysa K; Taha, Fatma M; Safwat, Miriam; Darweesh, Hanan E A; Basel, Mohamed El

2012-01-01

Systemic lupus erythematosus is one of the autoimmune diseases characterized by multisystem involvement associated with autoantibody and immune complex vasculitis along with endothelial cell damage. to study the possible role of Angiopoietin- 2 (Ang-2) as a recently highlighted inflammatory and angiogenic mediator in the pathogenesis of SLE and its correlation with the state of another inflammatory marker, P-Selectin, as well as with various markers of the disease activity. The present study included 3 main groups: active SLE patients (group I), inactive SLE patients (group II) and healthy normal control subjects (group III). Groups I and II were subjected to disease activity assessment using the SLEDAI scoring system and measurement of plasma Ang-2 and P-Selectin by ELISA in addition to various laboratory investigations to assess disease activity as: Complete blood count, ESR, serum creatinine, C3, C4 and 24-h urinary proteins. The mean level of Plasma Ang-2 and P-selectin showed a high significant increase in active group compared to inactive SLE patients and control subjects (p < 0.001).There was a significant positive correlation between Ang-2, P-Selectin, and each of SLEDAI score and 24-h urinary proteins in all SLE patients as well as in the active group, and Ang-2 was a significant independent marker for proteinuria. A significant negative correlation was found between Ang-2, P-Selectin and each of C3, C4. Ang-2 and P-Selectin showed a high sensitivity and specificity in the patients with SLE. Our study suggests that Ang-2 may be a more useful marker than P-Selectin, C3 and C4 in the assessment of disease activity.

Carbohydrate moieties of myelin-associated glycoprotein, major glycoprotein of the peripheral nervous system myelin and other myelin glycoproteins potentially involved in cell adhesion.

PubMed

Badache, A; Burger, D; Villarroya, H; Robert, Y; Kuchler, S; Steck, A J; Zanetta, J P

1992-01-01

The myelin-associated glycoprotein (MAG) and the major glycoprotein of the peripheral nervous system myelin (P0) are two members of the family of cell adhesion molecules (CAMs). A role in cell adhesion of the carbohydrate moiety of these molecules has been attributed to the presence of N-glycans bearing the HNK-1 carbohydrate epitope. On the other hand, it has been suggested that these glycoproteins could be ligands of an endogenous mannose-binding lectin present in myelin, the cerebellar soluble lectin (CSL). In order to further document the heterogeneity of the glycans of these two CAMs, we have used several probes: an anti-carbohydrate antibody of the HNK-1 type, called Elec-39, the plant lectin concanavalin A (ConA), and the endogenous lectin CSL involved in myelin compaction. This study shows that CSL binds to a small proportion of the polypeptide chains of MAG found in adult CNS of rats and man and the polypeptide chains of P0 molecules from adult human and rat sciatic nerve. For MAG from adult rat brain, the binding of CSL is restricted to glycans of polypeptide chains which could be separated from the others according to their solubility properties. These MAG molecular entities react also with the Elec-39 antibody and with ConA. These results confirm that P0 and MAG are heterogeneous in their carbohydrate moieties.(ABSTRACT TRUNCATED AT 250 WORDS)

Leukocyte-inspired biodegradable particles that selectively and avidly adhere to inflamed endothelium in vitro and in vivo

NASA Astrophysics Data System (ADS)

Sakhalkar, Harshad S.; Dalal, Milind K.; Salem, Aliasger K.; Ansari, Ramin; Fu, Jie; Kiani, Mohammad F.; Kurjiaka, David T.; Hanes, Justin; Shakesheff, Kevin M.; Goetz, Douglas J.

2003-12-01

We exploited leukocyte-endothelial cell adhesion chemistry to generate biodegradable particles that exhibit highly selective accumulation on inflamed endothelium in vitro and in vivo. Leukocyte-endothelial cell adhesive particles exhibit up to 15-fold higher adhesion to inflamed endothelium, relative to noninflamed endothelium, under in vitro flow conditions similar to that present in blood vessels, a 6-fold higher adhesion to cytokine inflamed endothelium relative to non-cytokine-treated endothelium in vivo, and a 10-fold enhancement in adhesion to trauma-induced inflamed endothelium in vivo due to the addition of a targeting ligand. The leukocyte-inspired particles have adhesion efficiencies similar to that of leukocytes and were shown to target each of the major inducible endothelial cell adhesion molecules (E-selectin, P-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1) that are up-regulated at sites of pathological inflammation. The potential for targeted drug delivery to inflamed endothelium has significant implications for the improved treatment of an array of pathologies, including cardiovascular disease, arthritis, inflammatory bowel disease, and cancer.

CD40 Ligand Promotes Mac-1 Expression, Leukocyte Recruitment, and Neointima Formation after Vascular Injury

PubMed Central

Li, Guohong; Sanders, John M.; Bevard, Melissa H.; Sun, ZhiQi; Chumley, James W.; Galkina, Elena V.; Ley, Klaus; Sarembock, Ian J.

2008-01-01

High levels of circulating soluble CD40 ligand (sCD40L) are frequently found in patients with hypercholesterolemia, diabetes, ischemic stroke, or acute coronary syndromes, predicting an increased rate of atherosclerotic plaque rupture and restenosis after coronary/carotid interventions. Clinical restenosis is characterized in part by exaggerated neointima formation, but the underlying mechanism remains incompletely understood. This study investigated the role of elevated sCD40L in neointima formation in response to vascular injury in an atherogenic animal model and explored the molecular mechanisms involved. apoE−/− mice fed a Western diet developed severe hypercholesterolemia, significant hyperglycemia, and high levels of plasma sCD40L. Neointima formation after carotid denudation injury was exaggerated in the apoE−/− mice. In vivo, blocking CD40L with anti-CD40L monoclonal antibody attenuated the early accumulation of Ly-6G+ neutrophils and Gr-1+ monocytes (at 3 days) and the late accumulation of Mac-2+ macrophages (at 28 days) in the denudated arteries; it also reduced the exaggerated neointima formation at 28 days. In vitro, recombinant CD40L stimulated platelet P-selectin and neutrophil Mac-1 expression and platelet-neutrophil co-aggregation and adhesive interaction. These effects were abrogated by anti-CD40L or anti-Mac-1 monoclonal antibody. Moreover, recombinant CD40L stimulated neutrophil oxidative burst and release of matrix metalloproteinase-9 in vitro. We conclude that elevated sCD40L promotes platelet-leukocyte activation and recruitment and neointima formation after arterial injury, potentially through enhancement of platelet P-selectin and leukocyte Mac-1 expression and oxidative activity. PMID:18349125

Sulfated Hexasaccharides Attenuate Metastasis by Inhibition of P-selectin and Heparanase1

PubMed Central

Borsig, Lubor; Vlodavsky, Israel; Ishai-Michaeli, Rivka; Torri, Giangiacomo; Vismara, Elena

2011-01-01

Development of compounds that target both heparanase and selectins is emerging as a promising approach for cancer therapy. Selectins are vascular cell adhesion molecules that mediate tumor cell interactions with platelets, leukocytes, and the vascular endothelium. Heparanase is an endoglycosidase that degrades heparan sulfate in the tumor microenvironment, cell surfaces, and vessel wall. Acting together, these molecules facilitate tumor cell arrest, extravasation, and metastasis. Here, we report the preparation of novel semisynthetic sulfated tri mannose C-C-linked dimers (STMCs) endowed with heparanase and selectin inhibitory activity. The P-selectin specificity of the STMC was defined by the anomeric linkage of the C-C bond. This STMC hexasaccharide is an effective inhibitor of P-selectin in vivo. We show that selective inhibition of heparanase attenuates metastasis in B16-BL6 melanoma cells, expressing high levels of this endoglycosidase, but has no effect on the metastasis of MC-38 carcinoma cells that express little or no heparanase activity. P-selectin-specific STMC attenuated metastasis in both animal models, indicating that inhibition of tumor cell interaction with the vascular endothelium is critical for cancer dissemination. Thus, the small size, the stability of the C-C bond, and the chemically defined structure of the newly generated STMCs make them superior to heparin derivatives and signify STMCs as valuable candidates for further evaluation. PMID:21532885

Upregulation of β-1,4-galactosyltransferase I in rat spinal cord with experimental autoimmune encephalomyelitis.

PubMed

Zhao, Jianmei; Gao, Ying; Cheng, Chun; Yan, Meijuan; Wang, Jian

2013-03-01

Inflammatory infiltration has been recently emphasized in the demyelinating diseases of the central nervous system including multiple sclerosis. β-1,4-Galactosyltransferase I (β-1,4-GalT-I) is a major galactosyltransferase responsible for selectin-ligand biosynthesis, mediating rolling of the inflammatory lymphocytes. In the present study, Western blot showed that expression of β-1,4-GalT-I was low in normal or complete Freund's adjuvant (CFA) control rats' spinal cords, and it began to increase since early stage and peaked at E4 stage of experimental autoimmune encephalomyelitis (EAE) and restored approximately at normal level in the recovery stage. Immunohistochemisty revealed that upregulation of β-1,4-GalT-I was predominantly distributed in the white matter of spinal cord , while there was also some increased staining of β-1,4-GalT-I in the grey matter. Meanwhile, the expression of E-selectin, the substrate of β-1,4-GalT-I, was significantly increased, with a peak at E4 stage of EAE, and gradually decreased thereafter. Lectin blot showed that the protein bands with molecular weights of 65-25 kDa reacted a remarkable increase at the peak stage of EAE when compared with the normal and CFA control. Ricinus Communis Agglutinin-I (RCA-I) histochemistry revealed that RCA-Ι-positive signals were most intense in white matter of lumbosacral spinal cord at the peak stage of EAE (E4). Immunohistochemistry showed that β-1,4-GalT-I and CD62E, a marker for E-selectin stainings located in a considerable number of ED1 (+) macrophages in perivascular or in the white matter in EAE lesions, and a good co-localization of ED1 (+) cells with CD62E was observed. All these results suggest that β-1,4-GalT-I might serve as an inflammatory mediator regulating adhesion and migration of inflammatory cells in EAE, possibly through influencing the modification of galactosylated carbohydrate chains to modulate selectin-ligand biosynthesis and interaction with E-selectin.

Significant sE-Selectin levels reduction after 6 months of anti-TNF-α therapy in non-diabetic patients with moderate-to-severe psoriasis.

PubMed

Genre, Fernanda; Armesto, Susana; Corrales, Alfonso; López-Mejías, Raquel; Remuzgo-Martínez, Sara; Pina, Trinitario; Ubilla, Begoña; Mijares, Verónica; Martín-Varillas, José Luis; Rueda-Gotor, Javier; Portilla, Virginia; Dierssen-Sotos, Trinidad; González-López, Marcos Antonio; González-Vela, María Del Carmen; Blanco, Ricardo; Llorca, Javier; Hernández, José Luis; González-Gay, Miguel Ángel

2017-12-01

Psoriasis patients have high risk of atherosclerosis, characterized by endothelial dysfunction. We aimed to study the association of the endothelial activation biomarkers monocyte chemoattractant protein 1 (MCP-1), soluble (s) E-selectin and P-selectin with disease activity and severity in psoriasis patients treated with anti-TNF-α therapy. Also, to evaluate the relationship of metabolic syndrome features with these biomarkers and the effect of anti-TNF-α therapy on these molecules. Twenty-nine consecutive non-diabetic patients with moderate-to-severe psoriasis who underwent 6 months of anti-TNF-α-adalimumab therapy were studied. Metabolic and clinical evaluation was performed prior to anti-TNF-α treatment (time 0) and 6 months later. MCP-1, sE-selectin and sP-selectin serum levels were determined by ELISA. Dyslipidemic and obese patients showed higher MCP-1 levels at month 6 from the onset of anti-TNF-α therapy (p = .05 and .01, respectively). sE-selectin positively correlated with pro-inflammatory molecules such as asymmetric dimethylarginine, sP-selectin and resistin at baseline and month 6 (p < .05). sE-selectin levels significantly reduced after 6 months of therapy (p = .0006). Metabolic syndrome features are associated with endothelial activation in patients with moderate-to-severe psoriasis. Adalimumab therapy led to a reduction in sE-selectin levels, supporting the beneficial effect of anti-TNF-α therapy on mechanisms associated with the development of atherosclerosis in psoriasis.

N-acetylcysteine induces shedding of selectins from liver and intestine during orthotopic liver transplantation

PubMed Central

Taut, F J H; Schmidt, H; Zapletal, C M; Thies, J C; Grube, C; Motsch, J; Klar, E; Martin, E

2001-01-01

In orthotopic liver transplantation (OLT), N-acetylcysteine (NAC) reduces ischaemia/reperfusion (I/R) injury, improves liver synthesis function and prevents primary nonfunction of the graft. To further elucidate the mechanisms of these beneficial effects of NAC, we investigated influence of high-dose NAC therapy on the pattern of adhesion molecule release from liver and intestine during OLT. Nine patients receiving allograft OLT were treated with 150 mg NAC/kg during the first hour after reperfusion; 10 patients received the carrier only. One hour after reperfusion, samples of arterial, portal venous and hepatic venous plasma were taken and blood flow in the hepatic artery and the portal vein was measured. Absolute concentrations of sICAM-1, sVCAM-1, sP-selectin and sE-selectin were not markedly different. However, balance calculations showed release of selectins from NAC-treated livers as opposed to net uptake in controls (P ≤ 0·02 for sP-selectin). This shedding of selectins might be a contributing factor to the decrease in leucocyte adherence and improved haemodynamics found experimentally with NAC-treatment. PMID:11422213

Endothelial expression of selectins during endotoxin preconditioning.

PubMed

Bauer, P; Welbourne, T; Shigematsu, T; Russell, J; Granger, D N

2000-12-01

Although bacterial endotoxins [lipopolysaccharide (LPS)] can confer tissue resistance to subsequent inflammatory insults, the mechanisms that underlie this LPS-preconditioning (LPS-PC) response remain poorly defined. The dual-radiolabeled monoclonal antibody technique was used to examine whether LPS-PC alters the upregulation (protein) of E- and P-selectins after subsequent LPS challenge. In the gut of wild-type (C57BL/6J) mice, LPS-PC was associated with a reduction in E- (66%) and P-selectin (33%) expression. A similar reduction in E-selectin expression was observed in mutant mice that were genetically deficient in either the endothelial or inducible isoform of nitric oxide synthase or that overexpressed the human gene for Cu/Zn superoxide dismutase. Severe combined immunodeficient mice, genetically devoid of lymphocytes, did exhibit partial inhibition of the LPS-PC response. We conclude that 1) LPS-PC can be demonstrated for E- and P-selectins in some vascular beds (e.g., gut), 2) the mechanism(s) underlying this blunted selectin response does not include a major role for either nitric oxide and superoxide, and 3) circulating lymphocytes may contribute to the LPS-PC response.

Bullous pemphigoid and pemphigus vulgaris: correlated behaviour of serum VEGF, sE-selectin and TNF-alpha levels.

PubMed

Ameglio, F; D'Auria, L; Cordiali-Fei, P; Mussi, A; Valenzano, L; D'Agosto, G; Ferraro, C; Bonifati, C; Giacalone, B

1997-01-01

Recently, we reported that soluble E-selectin (sE-selectin), an isoform of the cell membrane E-selectin, an adhesion molecule synthesized only by endothelial cells, is significantly increased in sera of the patients with bullous pemphigoid (PB) or pemphigus vulgaris. A significant correlation was also found between the serum sE-selectin levels and the number of skin lesions, suggesting the possible use of this molecule to gauge disease intensity before therapy. One of the sE-selectin inducers is tumor nerosis factor-alpha (TNF-alpha), that is also able to enhance vascular endothelial growth factor (VEGF), a strong endothelium activator. On the basis of these observations, the present study was conducted to analyze the serum levels of VEGF, sE-selectin, and TNF-alpha in 8 patients with BP (age: 82, range 54-87, 7 males, 1 female) and in 6 patients affected affected with PV (age: 55, range 44-65; 5 males, 1 female) and to verify possible correlations between these variables and the disease activity, In addition, serum sE-selectin levels were measured over time and compared with the serum anti-epithelium antibodies titers. The sE-selectin, VEGF and TNF-alpha levels were measured in the samples by means of commercially available ELISA kit. The same samples were also employed to measure the anti-epithelium antibody titers. Serum VEGF, sE-selectin and TNF-alpha levels were significantly correlated each other (p at least < 0.01). All three variables were also significantly correlated with the number of lesions (p at least < 0.01). Serum VEGF levels were found increased (median = 178 pg/ml, range 37-595) as compared to 28 healthy controls (median = 135 pg/ml, range 18/269, p < 0.05). Also serum TNF-alpha levels were found increased (median = 5.5 pg/ml, range < 0.1-41.0) as compared to 28 healthy controls (median < 0.1 pg/ml, range < 0.1-5.3), p < 0.01). When the patients were observed over time, serum sE-selectin levels highly correlated with the disease intensity in both dermatoses, although with different regression curves. These data further underline the endothelium involvement in these bullous dermatoses and stress the possibility of employing sE-selectin as a non-specific follow-up marker of both BP and PV.

Calreticulin discriminates the proximal region at the N-glycosylation site of Glc1Man9GlcNAc2 ligand

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hirano, Makoto; Adachi, Yuka; Ito, Yukishige

Calreticulin (CRT) is well known as a lectin-like chaperone that recognizes Glc1Man9GlcNAc2 (G1M9)-glycoproteins in the endoplasmic reticulum (ER). However, whether CRT can directly interact with the aglycone moiety (protein portion) of the glycoprotein remains controversial. To improve our understanding of CRT interactions, structure-defined G1M9-derivatives with different aglycones (–OH, –Gly–NH{sub 2}, and –Gly–Glu–{sup t}Bu) were used as CRT ligands, and their interactions with recombinant CRT were analyzed using thermal shift analysis. The results showed that CRT binds strongly to a G1M9-ligand in the order –Gly–Glu–{sup t}Bu > –Gly–NH{sub 2} > –OH, which is the same as that of the reglucosylation of Man9GlcNAc2 (M9)-derivatives by themore » folding sensor enzyme UGGT (UDP-glucose: glycoprotein glucosyltransferase). Our results indicate that, similar to UGGT, CRT discriminates the proximal region at the N-glycosylation site, suggesting a similar mechanism mediating the recognition of aglycone moieties in the ER glycoprotein quality control system. - Highlights: • Glc1Man9GlcNAc2 (G1M9) ligands with different aglycones were chemically prepared. • Calreticulin (CRT) discriminates the aglycone of Glc1Man9GlcNAc2 (G1M9) ligand. • CRT binds with G1M9 ligands in a similar manner to folding sensor enzyme.« less

VCAM-1 blockade delays disease onset, reduces disease severity and inflammatory cells in an atopic dermatitis model.

PubMed

Chen, Lin; Lin, Shao-xia; Amin, Sanober; Overbergh, Lut; Maggiolino, Giacomo; Chan, Lawrence S

2010-01-01

We investigated the functions of critical adhesion molecules ICAM-1 and VCAM-1 in a keratin-14 IL-4-transgenic (Tg) mouse model of atopic dermatitis, the skin lesions of which are characterized by prominent inflammatory cell infiltration, significantly increased mRNAs and proteins of ICAM-1, VCAM-1, E-selectin, P-selectin, L-selectin, and PSGL-1, and significantly increased numbers of dermal vessels expressing these adhesion molecules. We tested the hypotheses that deletion or blockade of these molecules may impede the inflammation by examining the disease progresses in the Tg mice crossed with ICAM-1-knockout mice and Tg mice received anti-VCAM-1-neutralizing antibody. Although the findings of the ICAM-1-knockout Tg mice (Tg/ICAM-1(-/-)) developed skin lesions similar to wide-type ICAM-1 Tg mice (Tg/ICAM-1(+/+)) were surprising, a compensatory mechanism may account for it: the frequency of VCAM-1 ligand, CD49d, on CD3(+) T cells in the lesional skin significantly increased in the Tg/ICAM-1(-/-) mouse, compared with the Tg/ICAM-1(+/+) mice. In contrast, anti-VCAM-1-treated Tg/ICAM-1(-/-) or Tg/ICAM-1(+/+) mice had significantly delayed onset of skin inflammation compared with isotype antibody-treated groups. Moreover, anti-VCAM-1 significantly reduced the skin inflammation severity in Tg/ICAM-1(+/+) mice, accompanied with reduction of mast cell, eosinophil, and CD3(+) T cell infiltration. VCAM-1 is more critical in developing skin inflammation in this model.

Atorvastatin Reduces Plasma Levels of Chemokine (CXCL10) in Patients with Crohn's Disease

PubMed Central

Grip, Olof; Janciauskiene, Sabina

2009-01-01

Background In Crohn's disease high tissue expression and serum levels of chemokines and their receptors are known to correlate with disease activity. Because statins can reduce chemokine expression in patients with coronary diseases, we wanted to test whether this can be achieved in patients with Crohn's disease. Methodology/Principal Findings We investigated plasma levels of chemokines (CCL2, CCL4, CCL11, CCL13, CCL17, CCL22, CCL26, CXCL8, CXCL10) and endothelial cytokines (sP-selectin, sE-selectin, sICAM-3, thrombomodulin) in ten Crohn's disease patients before and after thirteen weeks' daily treatment with 80 mg atorvastatin. Of the 13 substances investigated, only CXCL10 was found to be significantly reduced (by 34%, p = 0.026) in all of the treated patients. Levels of CXCL10 correlated with C-reactive protein (r = 0.82, p<0.01). Conclusions/Significance CXCL10 is a ligand for the CXCR3 receptor, the activation of which results in the recruitment of T lymphocytes and the perpetuation of mucosal inflammation. Hence the reduction of plasma CXCL10 levels by atorvastatin may represent a candidate for an approach to the treatment of Crohns disease in the future. Trial Registration ClinicalTrials.gov NCT00454545 PMID:19421322

Cimetidine attenuates vinorelbine-induced phlebitis in mice by militating E-selectin expression.

PubMed

Wang, Zhuo; Ma, Lijuan; Wang, Xuebin; Cai, Heping; Huang, Jin; Liu, Jiyong; Hu, Jinhong; Su, Dingfeng

2014-08-01

We investigated E-selectin expression in mice and rabbits with vinorelbine-induced phlebitis and the effect of cimetidine. To find the relationship between E-selectin expression and vinorelbine-induced phlebitis. Mouse and rabbit model of vinorelbine-induced phlebitis was established by intravenous infusion of vinorelbine. Pathological observation, molecular-biological determination of E-selectin and protein function of it was evaluated. Grossly, we observed swelling, edema and cord-like vessel changes in mice receiving vinorelbine but only mild edema in mice pretreated with cimetidine. Pathological scoring yielded a total score of 37 for vinorelbine-treated mice and 17 for mice pretreated with cimetidine (P < 0.05). ELISA revealed that rabbits treated with vinorelbine had markedly higher serum contents of E-selectin than normal saline (NS) controls (vinorelbine 1.534 ± 0.449 vs. NS 0.746 ± 0.170 ng/mL, P < 0.05), which was markedly attenuated by cimetidine (cimetidine 0.717 ± 0.468 vs. vinorelbine 1.534 ± 0.449 ng/mL, P < 0.05). Rose Bengal staining assays showed that vinorelbine markedly increased the adhesion rate of neutrophils for endothelial cells (vinorelbine 38.70 ± 8.34% vs. controls 8.93 ± 4.85%, P < 0.01), which, however, was significantly suppressed by cimetidine (9.93 ± 5.91%, P < 0.01 vs. vinorelbine). In E-selectin knockout mice, we found no apparent difference in tail swelling in mice receiving vinorelbine or cimetidine and vinorelbine. In conclusion, cimetidine attenuates vinorelbine-induced phlebitis in mice probably by suppressing increased expression of E-selectin.

A congenital activating mutant of WASp causes altered plasma membrane topography and adhesion under flow in lymphocytes

PubMed Central

Burns, Siobhan O.; Killock, David J.; Moulding, Dale A.; Metelo, Joao; Nunes, Joao; Taylor, Ruth R.; Forge, Andrew; Thrasher, Adrian J.

2010-01-01

Leukocytes rely on dynamic actin-dependent changes in cell shape to pass through blood vessels, which is fundamental to immune surveillance. Wiskott-Aldrich Syndrome protein (WASp) is a hematopoietic cell–restricted cytoskeletal regulator important for modulating cell shape through Arp2/3-mediated actin polymerization. A recently identified WASpI294T mutation was shown to render WASp constitutively active in vivo, causing increased filamentous (F)–actin polymerization, high podosome turnover in macrophages, and myelodysplasia. The aim of this study was to determine the effect of WASpI294T expression in lymphocytes. Here, we report that lymphocytes isolated from a patient with WASpI294T, and in a cellular model of WASpI294T, displayed abnormal microvillar architecture, associated with an increase in total cellular F-actin. Microvillus function was additionally altered as lymphocytes bearing the WASpI294T mutation failed to roll normally on L-selectin ligand under flow. This was not because of defects in L-selectin expression, shedding, cytoskeletal anchorage, or membranal positioning; however, under static conditions of adhesion, WASpI294T-expressing lymphocytes exhibited altered dynamic interaction with L-selectin ligand, with a significantly reduced rate of adhesion turnover. Together, our results demonstrate that WASpI294T significantly affects lymphocyte membrane topography and L-selectin–dependent adhesion, which may be linked to defective hematopoiesis and leukocyte function in affected patients. PMID:20354175

Identifying Carbohydrate Ligands of a Norovirus P Particle using a Catch and Release Electrospray Ionization Mass Spectrometry Assay

NASA Astrophysics Data System (ADS)

Han, Ling; Kitova, Elena N.; Tan, Ming; Jiang, Xi; Klassen, John S.

2014-01-01

Noroviruses (NoVs), the major cause of epidemic acute gastroenteritis, recognize human histo-blood group antigens (HBGAs), which are present as free oligosaccharides in bodily fluid or glycolipids and glycoproteins on the surfaces of cells. The subviral P particle formed by the protruding (P) domain of the NoV capsid protein serves as a useful model for the study NoV-HBGA interactions. Here, we demonstrate the application of a catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay for screening carbohydrate libraries against the P particle to rapidly identify NoV ligands and potential inhibitors. Carbohydrate libraries of 50 and 146 compounds, which included 18 and 24 analogs of HBGA receptors, respectively, were screened against the P particle of VA387, a member of the predominant GII.4 NoVs. Deprotonated ions corresponding to the P particle bound to carbohydrates were isolated and subjected to collision-induced dissociation to release the ligands in their deprotonated forms. The released ligands were identified by ion mobility separation followed by mass analysis. All 13 and 16 HBGA ligands with intrinsic affinities >500 M-1 were identified in the 50 and the 146 compound libraries, respectively. Furthermore, screening revealed interactions with a series of oligosaccharides with structures found in the cell wall of mycobacteria and human milk. The affinities of these newly discovered ligands are comparable to those of the HBGA receptors, as estimated from the relative abundance of released ligand ions.

Investigating the feasibility of stem cell enrichment mediated by immobilized selectins.

PubMed

Charles, Nichola; Liesveld, Jane L; King, Michael R

2007-01-01

Hematopoietic stem cell therapy is used to treat both malignant and non-malignant diseases, and enrichment of the hematopoietic stem and progenitor cells (HSPCs) has the potential to reduce the likelihood of graft vs host disease or relapse, potentially fatal complications associated with the therapy. Current commercial HSPC isolation technologies rely solely on the CD34 surface marker, and while they have proven to be invaluable, they can be time-consuming with variable recoveries reported. We propose that selectin-mediated enrichment could prove to be a quick and effective method for recovering HSPCs from adult bone marrow (ABM) on the basis of differences in rolling velocities and independently of CD34 expression. Purified CD34+ ABM cells and the unselected CD34- ABM cells were perfused over immobilized P-, E-, and L-selectin-IgG at physiologic wall shear stresses, and rolling velocities and cell retention data were collected. CD34+ ABM cells generally exhibited lower rolling velocities and higher retention than the unselected CD34- ABM cells on all three selectins. For initial CD34+ ABM cell concentrations ranging from 1% to 5%, we predict an increase in purity ranging from 5.2% to 36.1%, depending on the selectin used. Additionally, selectin-mediated cell enrichment is not limited to subsets of cells with inherent differences in rolling velocities. CD34+ KG1a cells and CD34- HL60 cells exhibited nearly identical rolling velocities on immobilized P-selectin-IgG over the entire range of shear stresses studied. However, when anti-CD34 antibody was co-immobilized with the P-selectin-IgG, the rolling velocity of the CD34+ KG1a cells was significantly reduced, making selectin-mediated cell enrichment a feasible option. Optimal cell enrichment in immobilized selectin surfaces can be achieved within 10 min, much faster than most current commercially available systems.

P-selectin, endocan, and some adhesion molecules in obese children and adolescents with non-alcoholic fatty liver disease.

PubMed

Ustyol, Ala; Aycan Ustyol, Esra; Gurdol, Figen; Kokali, Funda; Bekpınar, Seldag

2017-05-01

There is increasing evidence for a direct relationship between the vascular system and non-alcoholic fatty liver disease (NAFLD). The aim of this study was to investigate endocan and adhesion molecules such as P-selectin derived from the endothelium and platelets in obese children and adolescents with NAFLD. One hundred obese patients and 40 lean controls were enrolled. The obese subjects were divided into two subgroups based on the presence or absence of fatty liver. Blood samples were assayed for endocan, P-selectin, platelet-derived growth factor (PDGF), intercellular cell adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1. Obese patients with NAFLD presented higher ALT and insulin levels, as well as more profound dyslipidemia when compared with their counterparts without NAFLD. Serum levels of high-sensitivity C-reactive protein, VCAM-1 and ICAM-1 were found increased in both obese groups, regardless of NAFLD. In obese subjects with NAFLD, decreased P-selectin levels (51.6 ± 4.14 ng/mL) were detected as compared with the obese (72.3 ± 4.23) and control (74.2 ± 6.97) subjects. Furthermore, circulating P-selectin levels were closely associated with endocan levels (r = 0.852, p < 0.001). Childhood obesity leads to vascular inflammation and therefore may cause a predisposition to atherosclerosis at an early age. The possible outcome of decreased P-selectin levels with NAFLD development must be further investigated.

Moderate consumption of red wine and human platelet responsiveness.

PubMed

Tozzi Ciancarelli, Maria Giuliana; Di Massimo, Caterina; De Amicis, Daniela; Ciancarelli, Irene; Carolei, Antonio

2011-08-01

Available studies showed an inverse association between red wine consumption and prevalence of vascular risk factors in coronary hearth disease and stroke. Effects were mainly associated to wine antioxidant and antiaggregant properties. Actually, in vitro studies indicate a favourable effect of wine and/or of its non-alcoholic components in decreasing platelet sensitivity and aggregability. In a 4-week supplementation in 15 healthy male volunteers, we evaluated whether moderate red wine consumption might improve antioxidant defence mechanisms and promote positive modulation of inflammatory cytokines and cell adhesion molecules in relation to platelet responsiveness. We did not find any change of ADP- and collagen-induced platelet aggregation ex vivo, any change of biomarkers of oxidative stress, and any change of plasma lipid profile and haemostatic parameters, with the only exception of decreased fibrinogen levels (P<0.05). We also found an increase of mean platelet volume (P<0.05) without any significant modification of CD40 Ligand and P-selectin levels. Increased expressions of intercellular adhesion molecule-1, soluble E-selectin and interleukin-6 (P<0.05) were also observed. According to our findings increased circulating levels of inflammatory and endothelial cell activation markers may indicate a low-grade systemic inflammation and vascular activation that could be responsible for the lack of inhibition or of decreased platelet responsiveness, possibly because the plasmatic increase of wine antioxidant compounds is insufficient to improve endothelial function and to counteract the influence of ethanol on endothelial activation. Copyright © 2011 Elsevier Ltd. All rights reserved.

Relationship Between Markers of Platelet Activation and Inflammation with Disease Activity in Wegener’s Granulomatosis

PubMed Central

TOMASSON, GUNNAR; LAVALLEY, MICHAEL; TANRIVERDI, KAHRAMAN; FINKIELMAN, JAVIER D.; DAVIS, JOHN C.; HOFFMAN, GARY S.; McCUNE, W. JOSEPH; St. CLAIR, E. WILLIAM; SPECKS, ULRICH; SPIERA, ROBERT; STONE, JOHN H.; FREEDMAN, JANE E.; MERKEL, PETER A.

2013-01-01

Objective There remains a need for biomarkers to guide therapy in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. Our objective was to determine whether measures of platelet activation or inflammation are associated with disease activity in Wegener’s granulomatosis (WG). Methods Study subjects were participants in a clinical trial. Soluble CD40 ligand (sCD40L), C-reactive protein, interleukin 6 (IL-6), IL-8, monocyte chemoattractant protein 1 (MCP-1), P-selectin, vascular endothelial growth factor, and proteinase 3 (PR3)-specific ANCA were measured by ELISA using plasma samples obtained at baseline (active disease), at remission, and prior to, during, and after first flares. Disease activity was assessed by the Birmingham Vasculitis Activity Score for WG (BVAS/WG). Association of biomarkers with disease activity was determined with conditional logistic and linear regression. Results Over a mean followup of 27 months, 180 subjects underwent 2044 visits; markers were measured in 563 samples. Longitudinally, all markers other than IL-6 were associated with disease activity. The strongest associations for active disease at baseline versus remission were observed for sCD40L (OR 4.72, 95% CI 2.47–9.03), P-selectin (OR 6.26, 95% CI 2.78–14.10), PR3-ANCA (OR 9.41, 4.03–21.99), and inversely for MCP-1 (OR 0.36, 95% CI 0.22–0.57). BVAS/WG increased by 0.80 (95% CI 0.44–1.16), 0.83 (95% CI 0.42–1.25), and 0.81 (95% CI 0.48–1.15) per unit-increase in PR3-ANCA, sCD40L, and P-selectin, respectively; and decreased by 1.54 (95% CI 0.96–2.12) per unit-increase in MCP-1. Conclusion Cytokines arising from within the circulation, including those of platelet activation, correlate with disease activity in WG. PMID:21411717

Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging.

PubMed

Chan, Joyce M S; Monaco, Claudia; Wylezinska-Arridge, Marzena; Tremoleda, Jordi L; Cole, Jennifer E; Goddard, Michael; Cheung, Maggie S H; Bhakoo, Kishore K; Gibbs, Richard G J

2018-05-01

Identification of patients with high-risk asymptomatic carotid plaques remains an elusive but essential step in stroke prevention. Inflammation is a key process in plaque destabilization and a prelude to clinical sequelae. There are currently no clinical imaging tools to assess the inflammatory activity within plaques. This study characterized inflammation in atherosclerosis using dual-targeted microparticles of iron oxide (DT-MPIO) as a magnetic resonance imaging (MRI) probe. DT-MPIO were used to detect and characterize inflammatory markers, vascular cell adhesion molecule 1 (VCAM-1). and P-selectin on (1) tumor necrosis factor-α-treated cells by immunocytochemistry and (2) aortic root plaques of apolipoprotein-E deficient mice by in vivo MRI. Furthermore, apolipoprotein E-deficient mice with focal carotid plaques of different phenotypes were developed by means of periarterial cuff placement to allow in vivo molecular MRI using these probes. The association between biomarkers and the magnetic resonance signal in different contrast groups was assessed longitudinally in these models. Immunocytochemistry confirmed specificity and efficacy of DT-MPIO to VCAM-1 and P-selectin. Using this in vivo molecular MRI strategy, we demonstrated (1) the DT-MPIO-induced magnetic resonance signal tracked with VCAM-1 (r = 0.69; P = .014), P-selectin (r = 0.65; P = .022), and macrophage content (r = 0.59; P = .045) within aortic root plaques and (2) high-risk inflamed plaques were distinguished from noninflamed plaques in the murine carotid artery within a practical clinical imaging time frame. These molecular MRI probes constitute a novel imaging tool for in vivo characterization of plaque vulnerability and inflammatory activity in atherosclerosis. Further development and translation into the clinical arena will facilitate more accurate risk stratification in carotid atherosclerotic disease in the future. Copyright © 2017 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.

Basigin (CD147), a multifunctional transmembrane glycoprotein with various binding partners

PubMed Central

Muramatsu, Takashi

2016-01-01

Basigin, also called CD147 or EMMPRIN, is a transmembrane glycoprotein that belongs to the immunoglobulin superfamily. Basigin has isoforms; the common form (basigin or basigin-2) has two immunoglobulin domains, and the extended form (basigin-1) has three. Basigin is the receptor for cyclophilins, S100A9 and platelet glycoprotein VI, whereas basigin-1 serves as the receptor for the rod-derived cone viability factor. Basigin tightly associates with monocarboxylate transporters and is essential for their cell surface translocation and activities. In the same membrane plane, basigin also associates with other proteins including GLUT1, CD44 and CD98. The carbohydrate portion of basigin is recognized by lectins, such as galectin-3 and E-selectin. These molecular recognitions form the basis for the role of basigin in the transport of nutrients, migration of inflammatory leukocytes and induction of matrix metalloproteinases. Basigin is important in vision, spermatogenesis and other physiological phenomena, and plays significant roles in the pathogenesis of numerous diseases, including cancer. Basigin is also the receptor for an invasive protein RH5, which is present in malaria parasites. PMID:26684586

Metabolic Flux Increases Glycoprotein Sialylation: Implications for Cell Adhesion and Cancer Metastasis*

PubMed Central

Almaraz, Ruben T.; Tian, Yuan; Bhattarcharya, Rahul; Tan, Elaine; Chen, Shih-Hsun; Dallas, Matthew R.; Chen, Li; Zhang, Zhen; Zhang, Hui; Konstantopoulos, Konstantinos; Yarema, Kevin J.

2012-01-01

This study reports a global glycoproteomic analysis of pancreatic cancer cells that describes how flux through the sialic acid biosynthetic pathway selectively modulates a subset of N-glycosylation sites found within cellular proteins. These results provide evidence that sialoglycoprotein patterns are not determined exclusively by the transcription of biosynthetic enzymes or the availability of N-glycan sequons; instead, bulk metabolic flux through the sialic acid pathway has a remarkable ability to increase the abundance of certain sialoglycoproteins while having a minimal impact on others. Specifically, of 82 glycoproteins identified through a mass spectrometry and bioinformatics approach, ∼31% showed no change in sialylation, ∼29% exhibited a modest increase, whereas ∼40% experienced an increase of greater than twofold. Increased sialylation of specific glycoproteins resulted in changes to the adhesive properties of SW1990 pancreatic cancer cells (e.g. increased CD44-mediated adhesion to selectins under physiological flow and enhanced integrin-mediated cell mobility on collagen and fibronectin). These results indicate that cancer cells can become more aggressively malignant by controlling the sialylation of proteins implicated in metastatic transformation via metabolic flux. PMID:22457533

Glycopeptide Analogues of PSGL-1 Inhibit P-Selectin In Vitro and In Vivo

PubMed Central

Krishnamurthy, Venkata R; Sardar, Mohammed Y. R.; Yu, Ying; Song, Xuezheng; Haller, Carolyn; Dai, Erbin; Wang, Xiacong; Hanjaya-Putra, Donny; Sun, Lijun; Morikis, Vasilios; Simon, Scott I.; Woods, Robert; Cummings, Richard D.; Chaikof, Elliot L.

2015-01-01

Blockade of P-selectin/PSGL-1 interactions holds significant potential for treatment of disorders of innate immunity, thrombosis, and cancer. Current inhibitors remain limited due to low binding affinity or by the recognized disadvantages inherent to chronic administration of antibody therapeutics. Here we report an efficient approach for generating glycosulfopeptide mimics of N-terminal PSGL-1 through development of a stereoselective route for multi-gram scale synthesis of the C2 O-glycan building block and replacement of hydrolytically labile tyrosine sulfates with isosteric sulfonate analogs. Library screening afforded a compound of exceptional stability, GSnP-6, that binds to human P-selectin with nanomolar affinity (Kd ~ 22 nM). Molecular dynamics simulation defines the origin of this affinity in terms of a number of critical structural contributions. GSnP-6 potently blocks P-selectin/PSGL-1 interactions in vitro and in vivo and represents a promising candidate for the treatment of diseases driven by acute and chronic inflammation. PMID:25824568

Widening and diversifying the proteome capture by combinatorial peptide ligand libraries via Alcian Blue dye binding.

PubMed

Candiano, Giovanni; Santucci, Laura; Petretto, Andrea; Lavarello, Chiara; Inglese, Elvira; Bruschi, Maurizio; Ghiggeri, Gian Marco; Boschetti, Egisto; Righetti, Pier Giorgio

2015-01-01

Combinatorial peptide ligand libraries (CPLLs) tend to bind complex molecules such as dyes due to their aromatic, heterocyclic, hydrophobic, and ionic nature that may affect the protein capture specificity. In this experimental work Alcian Blue 8GX, a positively charged phthalocyanine dye well-known to bind to glycoproteins and to glucosaminoglycans, was adsorbed on a chemically modified CPLL solid phase, and the behavior of the resulting conjugate was then investigated. The control and dye-adsorbed beads were used to harvest the human urinary proteome at physiological pH, this resulting in a grand total of 1151 gene products identified after the capture. Although the Alcian Blue-modified CPLL incremented the total protein capture by 115 species, it particularly enriched some families among the harvested proteins, such as glycoproteins and nucleotide-binding proteins. This study teaches that it is possible, via the two combined harvest mechanisms, to drive the CPLL capture toward the enrichment of specific protein categories.

Levels of Soluble Adhesion Molecules PECAM-1 and P-Selectin are Decreased in Children with Autism Spectrum Disorder

PubMed Central

Onore, Charity E.; Nordahl, Christine Wu; Young, Gregory S.; Van de Water, Judy A.; Rogers, Sally J.; Ashwood, Paul

2012-01-01

Background Although the etiopathology of Autism Spectrum Disorder (ASD) is not clear there is increasing evidence that dysfunction in the immune system affects many children with ASD. Findings of immune dysfunction in ASD include increases in inflammatory cytokines, chemokines and microglial activity in brain tissue and CSF, as well as abnormal peripheral immune cell function. Methods Adhesion molecules, such as platelet endothelial adhesion molecule-1 (PECAM-1), intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), P-Selectin, and L-Selectin, function to facilitate leukocyte transendothelial migration. We assessed concentrations of soluble adhesion molecules, sPECAM-1, sICAM-1, sVCAM-1, sP-Selectin, and sL-Selectin in the plasma of 49 participants with ASD, and 31 typically developing controls of the same age, all of whom were enrolled as part of the Autism Phenome Project (APP). Behavioral assessment, the levels of soluble adhesion molecules, head circumference and MRI measurements of brain volume were compared in the same subjects. Results Levels of sPECAM-1 and sP-Selectin were significantly reduced in the ASD group compared to typically developing controls (p < 0.02). Soluble PECAM-1 levels were negatively associated with repetitive behavior and abnormal brain growth in children with ASD (p=0.03). Conclusions As adhesion molecules modulate the permeability and signaling at the blood brain barrier as well as leukocyte infiltration into the CNS, current data suggests a role for these molecules in the complex pathophysiology of ASD. PMID:22717029

Platelet granule release is associated with reactive oxygen species generation during platelet storage: A direct link between platelet pro-inflammatory and oxidation states.

PubMed

Ghasemzadeh, Mehran; Hosseini, Ehteramolsadat

2017-08-01

Upon platelet stimulation with agonists, reactive oxygen species (ROS) generation enhances platelet activation and granule release. Whether ROS generation during platelet storage could be directly correlated with the expression of proinflammatory molecules and granule release has been investigated in this study. PRP-platelet concentrates were subjected to flowcytometry analysis to assess the expression of platelet activation marker, P-selectin and CD40L during storage. Intracellular ROS generation was also detected in platelet by flowcytometry using dihydrorhodamine (DHR) 123. Through the dual staining, ROS production was analyzed in either P-selectin positive or negative populations. ROS formation in platelet population was significantly increased by either TRAP (a potent agonist that induces granule release) or PMA (a classic inducer of ROS generation), while the effects of each agonists on P-selectin expression and ROS generation in platelets were comparable. Platelet storage was also associated with the increasing levels of ROS (day 0 vs. day 5; p<0.001) while this increasing pattern was directly correlated with the either expressed P-selectin or CD40L. In addition, in 5 day-stored platelets, samples with ROS levels above 40% showed significantly higher levels of P-selectin and CD40L expression. P-selectin negative population of platelet did not show significant amount of ROS. Our data demonstrated decreased levels of important platelet pro-inflammatory molecules in stored platelets with lower levels of intraplatelet ROS. However, whether quenching of ROS generation during platelet storage can attenuate adverse transfusion reactions raised by platelet pro-inflammatory status is required to be further studied. Copyright © 2017 Elsevier Ltd. All rights reserved.

Farnesoid X Receptor and Liver X Receptor Ligands Initiate Formation of Coated Platelets

PubMed Central

Unsworth, Amanda J.; Bye, Alexander P.; Tannetta, Dionne S.; Desborough, Michael J.R.; Kriek, Neline; Sage, Tanya; Allan, Harriet E.; Crescente, Marilena; Yaqoob, Parveen; Warner, Timothy D.; Jones, Chris I.

2017-01-01

Objectives— The liver X receptors (LXRs) and farnesoid X receptor (FXR) have been identified in human platelets. Ligands of these receptors have been shown to have nongenomic inhibitory effects on platelet activation by platelet agonists. This, however, seems contradictory with the platelet hyper-reactivity that is associated with several pathological conditions that are associated with increased circulating levels of molecules that are LXR and FXR ligands, such as hyperlipidemia, type 2 diabetes mellitus, and obesity. Approach and Results— We, therefore, investigated whether ligands for the LXR and FXR receptors were capable of priming platelets to the activated state without stimulation by platelet agonists. Treatment of platelets with ligands for LXR and FXR converted platelets to the procoagulant state, with increases in phosphatidylserine exposure, platelet swelling, reduced membrane integrity, depolarization of the mitochondrial membrane, and microparticle release observed. Additionally, platelets also displayed features associated with coated platelets such as P-selectin exposure, fibrinogen binding, fibrin generation that is supported by increased serine protease activity, and inhibition of integrin αIIbβ3. LXR and FXR ligand-induced formation of coated platelets was found to be dependent on both reactive oxygen species and intracellular calcium mobilization, and for FXR ligands, this process was found to be dependent on cyclophilin D. Conclusions— We conclude that treatment with LXR and FXR ligands initiates coated platelet formation, which is thought to support coagulation but results in desensitization to platelet stimuli through inhibition of αIIbβ3 consistent with their ability to inhibit platelet function and stable thrombus formation in vivo. PMID:28619996

Prostate Cancer Progression and Serum SIBLING (Small Integrin Binding N-Linked Glycoprotein)Levels

DTIC Science & Technology

2007-10-01

termed SIBLINGs (for small integrin binding ligand N-linked glycoproteins) whose members include bone sialoprotein (BSP), osteopontin (OPN), dentin...enzyme-linked immunosorbent assays (ELISAs) for quantitatively determining the levels of bone sialoprotein (BSP), osteopontin (OPN), dentin...synthesized as a chimeric protein, composed of three parts: dentin sialoprotein (DSP), dentin glycoprotein (DGP) and dentin phosphoprotein (DPP, also

Increased levels of markers of vascular inflammation in patients with coronary heart disease.

PubMed

Schumacher, A; Seljeflot, I; Sommervoll, L; Christensen, B; Otterstad, J E; Arnesen, H

2002-01-01

Elevated levels of soluble cell adhesion molecules (sCAMs), inflammatory cytokines and C-reactive protein (CRP) have been associated with atherosclerotic disease states. The aim of the present study was to evaluate whether circulating levels of vascular cell adhesion molecule-1 (sVCAM-1), intercellular adhesion molecule-1 (sICAM-1), E- and P-selectin were significantly elevated in patients with coronary heart disease (CHD) compared with healthy controls, and to study possible associations between these sCAMs, tumour necrosis factor alpha (TNFalpha). interleukin-6 (IL-6), CRP and major CHD risk factors. The study included 193 patients in various stages of CHD and 193 matched controls. To evaluate any possible influence of acute phase reaction, reinvestigation was performed after 6 months. After adjustment for major CHD risk factors, sVCAM-1, sICAM-1, P-selectin, IL-6 and CRP remained significantly elevated in the CHD patients (p for all <0.001). In multivariate analysis sVCAM-1 was predicted by age (p=0.015), sICAM-1 by smoking (p<0.001) and total cholesterol (p=0.026), E-selectin by body mass index (BMI) (p=0.004) and P-selectin by male gender (p=0.015). TNFalpha significantly predicted sICAM-1 and E-selectin levels, while IL-6 predicted CRP but none of the sCAMs measured. This might indicate that TNFalpha, but not IL-6, plays a major role in the regulation of sCAM levels in vivo.

Microvascular Channel Device to Study Aggressiveness in Prostate Cancer Metastasis

DTIC Science & Technology

2013-06-01

Aigner S, Ramos CL, Hafezi- Moghadam A, Lawrence MB, Friederichs J, Altevogt P, Ley K CD24 mediates rolling of breast carcinoma cells on P-selectin...1998 FASEB J 12:1241-1251 9. Friederichs J, Zeller Y, Hafezi- Moghadam A, Grone HJ, Ley K, Altevogt P The CD24/P-selectin binding pathway initiates

Role of ROS in Aβ42 Mediated Activation of Cerebral Endothelial Cells.

PubMed

Tsoy, Andrey; Umbayev, Bauyrzhan; Shalakhmetova, Tamara; Askarova, Sholpan

2014-01-01

There is substantial evidence that the deposition of aggregated amyloid-beta peptide (Aβ) in brain parenchyma and brain vessels is the main cause of neuronal dysfunction and death in Alzheimer's disease (AD). Aβ exhibits multiple cytotoxic effects on neurons and glial cells and causes dysfunction of the blood brain barrier (BBB). In AD brains, an increased deposition of Aβ in the cerebral vasculature has been found to be correlated with increased transmigration of blood-borne inflammatory cells and neurovascular inflammation. However, regulatory mediators of these processes remain to be elucidated. In this study, we examined the role of ROS in actin polymerization and expression of adhesion molecules (P-selectin) on the surface of the cerebral endothelial cells (CECs) that are activated by Aβ42. Mouse BEnd3 line (ATCC) was used in this research. BEnd3 cells respond to Aβ treatment similarly to human primary CECs and are a common model to investigate CECs' function. We used immortalized bEnd3 cells as the following: controls; cells incubated with Aβ42 for 10, 30, and 60 minutes; cells incubated with 30 mM of antioxidant N-acetylcysteine (NAC) for 1 hr; and, cells pre-treated with NAC followed by Aβ42 exposure. We measured DHE fluorescence to investigate intracellular ROS production. Immunofluorescent microscopy of anti-P-selectin and oregon green phalloidin was used to quantify the surface P-selectin expression and actin polymerization, and Western blot analysis was used to analyze total P-selectin expression. The results of this study have demonstrated a significant time-dependent ROS accumulation after 10 minutes, 30 minutes, and 60 minutes of Aβ42 treatment, while Aβ42 stimulated ROS production in CECs was attenuated by pre-treatment with the NAC antioxidant. We also found that Aβ42 increased P-selectin fluorescence at the surface of bEnd3 cells in a time dependent manner in parallel to ROS elevation. However, total expression levels of P-selectin were not changed following exposure to Aβ42. Pretreatment with NAC attenuated Aβ42 induced P-selectin localization, while NAC alone did not significantly affect P selectin localization. As a positive control, H 2 O 2 also increased P-selectin expression on the cell surface, which peaked after 30 minutes of H 2 O 2 treatment. Exposure of CECs with Aβ42 promoted actin polymerization, which peaked after 10 minutes of Aβ42 treatment, while no significant increase of F-actin intensity was observed when cells were pre-treated with NAC. H 2 O 2 was able to mimic Aβ42 induced oxidative stress, causing increased actin polymerization with similar timing. The results of our study have indicated that Aβ42 induced accumulation of P-selectin on the surface of bEnd3 cells and promoted actin polymerization, and all these events were correlated with ROS generation. The rapid post-translational cell signaling response mediated by ROS may well represent an important physiological trigger of the microvascular inflammatory responses in AD and requires further investigations.

How does measurement of platelet P-selectin compare with other methods of measuring platelet function as a means of determining the effectiveness of antiplatelet therapy?

PubMed

Fox, Susan C; May, Jane A; Dovlatova, Natalia; Glenn, Jackie R; Johnson, Andrew; White, Ann E; Radhakrishnan, Ashwin; Heptinstall, Stan

2018-02-20

Measurement of P-selectin on activated platelets as a means of measuring platelet function utilizing the technology described here has the advantage of not requiring immediate access to specialist equipment and expertise. Blood samples are activated, fixed, stored, and transported to a central laboratory for flow cytometric analysis. Here we have compared P-selectin with other more traditional approaches to measuring platelet function in blood and/or platelet-rich plasma (PRP) from patients with acute coronary syndromes on treatment for at least 1 month with either aspirin and clopidogrel or aspirin with prasugrel. The comparators were light transmission aggregometry (LTA), VerifyNow and Multiplate aggregometry (for determining the effects of aspirin) and LTA, VerifyNow and Multiplate together with the BioCytex VASP phosphorylation assay (for the P2Y 12 antagonists). The P-selectin Aspirin Test revealed substantial inhibition of platelet function in all but three of 96 patients receiving aspirin with clopidogrel and in none of 51 patients receiving aspirin and prasugrel. The results were very similar to those obtained using LTA. There was only one patient with high residual platelet aggregation and low P-selectin expression. The same patients identified as "non-responders" to aspirin also presented with the highest residual platelet activity as measured using the VerifyNow system, although not quite as well separated from the other values. With the Multiplate test only one of these patients clearly stood out from the others. The results obtained using the P-selectin P2Y 12 Test in 102 patients taking aspirin and clopidogrel were similar to the more traditional approaches in that a wide scatter of results was obtained. Generally, high values seen with the P-selectin P2Y 12 Test were also high with the LTA, VerifyNow, Multiplate, and BioCytex VASP P2Y 12 Tests. Similarly, low residual platelet function using the P2Y 12 test was seen irrespective of the testing procedure used. However, there were differences in some patients. Prasugrel was always more effective than clopidogrel in inhibiting platelet function with none of 56 patients (P-selectin and VerifyNow), only 2 of 56 patients (Multiplate) and only 3 of 56 patients (Biocytex VASP) demonstrating high on-treatment residual platelet reactivity (HRPR) defined using previously published cut-off values. The exception was LTA where there were 11 of 56 patients with HRPR. It remains to be seen which experimental approach provides the most useful information regarding outcomes after adjusting therapies in treated patients.

Thrombomodulin, von Willebrand factor and E-selectin as plasma markers of endothelial damage/dysfunction and activation in pregnancy induced hypertension.

PubMed

Nadar, Sunil K; Al Yemeni, Eman; Blann, Andrew D; Lip, Gregory Y H

2004-01-01

Endothelial disturbance (whether activation, dysfunction or damage) is a likely pathogenic mechanism in pre-eclampsia and pregnancy-induced hypertension (PIH). We set out to determine which of three plasma markers of endothelial disturbance, indicating endothelial activation (E-selectin) or damage/dysfunction (von Willebrand factor (vWf), soluble thrombomodulin), would provide the best discriminator of PIH compared to normotensive pregnancy. Cross-sectional study of 36 consecutive women with PIH (age 31+/-6 years) and 36 consecutive women with normotensive pregnancies (age 29+/-5 years) of similar parity. Plasma levels of vWf, E-selectin and thrombomodulin were measured using ELISA. As expected, women with PIH had significantly higher levels of plasma vWf (by 19%, p=0.003), E-selectin (by 40%, p<0.001) and thrombomodulin (by 61%, p=0.01) than normotensive women. However, on stepwise multiple regression analysis, only thrombomodulin was an independent significant predictor of the presence of PIH (p=0.023). We conclude that although vWf, E-selectin and thrombomodulin are all raised in PIH, only thrombomodulin was independently associated with PIH. This molecule could potentially be useful in monitoring and in providing clues in aetiology and pathophysiology, and may have implications for the clinical complications associated with PIH.

Apheresis platelet concentrates contain platelet-derived and endothelial cell-derived microparticles.

PubMed

Rank, A; Nieuwland, R; Liebhardt, S; Iberer, M; Grützner, S; Toth, B; Pihusch, R

2011-02-01

Microparticles (MP) are membrane vesicles with thrombogenic and immunomodulatory properties. We determined MP subgroups from resting platelets, activated platelets and endothelial cells in donors and apheresis platelet concentrates (PC). MP were double stained with annexin V and CD61 (platelet-derived MP; PMP), P-selectin or CD63 (MP from activated platelets) and CD144 plus E-selectin (endothelial cell-derived MP; EMP) and detected by flow cytometry in platelet donors (n=36) and apheresis PC (n=11; Trima™). PC contained MP, mainly from resting platelets [93% (90-95)], and minor fractions of PMP from activated platelets [P-selectin(+) or CD63(+); 4·8% (3·2-7·7) and 2·6% (2·0-4·0)]. Compared to donors, levels of annexin V+ MP, PMP, P-selectin(+) and CD63(+) MP were 1·7-, 2·3-, 8·6- and 3·1-fold higher in PC (all P<0·05). During storage (1-5 days), levels of annexin V+ MP and PMP did not increase, although small increases in the fraction of P-selectin(+) or CD63(+) MP occurred (both P<0·05). PC also contained EMP, which were 2·6- to 3·7-fold enriched in PC compared to donors (P<0·05). Transfusion of apheresis PC also results in transfusion of HLA-carrying PMP and EMP. This might counteract the aim of reducing transfused HLA load by leucodepletion. The increases in PMP exposing P-selectin or CD63 reflect mild platelet activation during storage. We conclude that in leucodepleted platelet apheresis using fluidized particle bed technology, MP are harvested mainly from the donor by apheresis. Improvement in apheresis technology might reduce MP load. © 2010 The Author(s). Vox Sanguinis © 2010 International Society of Blood Transfusion.

Analytical cell adhesion chromatography reveals impaired persistence of metastatic cell rolling adhesion to P-selectin

PubMed Central

Oh, Jaeho; Edwards, Erin E.; McClatchey, P. Mason; Thomas, Susan N.

2015-01-01

ABSTRACT Selectins facilitate the recruitment of circulating cells from the bloodstream by mediating rolling adhesion, which initiates the cell–cell signaling that directs extravasation into surrounding tissues. To measure the relative efficiency of cell adhesion in shear flow for in vitro drug screening, we designed and implemented a microfluidic-based analytical cell adhesion chromatography system. The juxtaposition of instantaneous rolling velocities with elution times revealed that human metastatic cancer cells, but not human leukocytes, had a reduced capacity to sustain rolling adhesion with P-selectin. We define a new parameter, termed adhesion persistence, which is conceptually similar to migration persistence in the context of chemotaxis, but instead describes the capacity of cells to resist the influence of shear flow and sustain rolling interactions with an adhesive substrate that might modulate the probability of extravasation. Among cell types assayed, adhesion persistence to P-selectin was specifically reduced in metastatic but not leukocyte-like cells in response to a low dose of heparin. In conclusion, we demonstrate this as an effective methodology to identify selectin adhesion antagonist doses that modulate homing cell adhesion and engraftment in a cell-subtype-selective manner. PMID:26349809

E-selectin ligand-1 (ESL-1) is a novel adiponectin binding protein on cell adhesion.

PubMed

Yamamoto, Hiroyasu; Kuroda, Nana; Uekita, Hiromi; Kochi, Ikoi; Matsumoto, Akane; Niinaga, Ryu; Funahashi, Tohru; Shimomura, Iichiro; Kihara, Shinji

2016-02-05

Adiponectin (APN) is an adipocyte-derived bioactive molecule with anti-diabetic and anti-atherogenic properties. Although anti-diabetic effects are mostly mediated by the adiponectin receptors AdipoR1 and AdipoR2, the anti-atherogenic mechanisms have not been fully elucidated. In this study, we identified E-selectin ligand (ESL)-1 as a novel APN-binding protein by mass spectrometry analysis of HepG2 cell-derived immunoprecipitant with an anti-APN antibody. Cell adhesion assays using fluorescence-labelled monocyte cell line THP-1 cells and human umbilical vein endothelial cells (HUVECs) revealed that APN-pre-treated THP-1 cells had reduced binding ability to HUVECs. This APN-mediated suppressive effect on monocyte binding to endothelial cells was partially abrogated by targeting ESL-1 with shRNA in THP-1 cells. In addition, serial mutagenesis analysis disclosed that five extracellular amino acids close to the N-terminus of ESL-1 were essential for binding with APN. Our results highlight the fact that interaction between APN and ESL-1 could provide a fundamental mechanism underlying the anti-atherogenic properties of APN. Copyright © 2016 Elsevier Inc. All rights reserved.

Glycosylation: a hallmark of cancer?

PubMed

Vajaria, Bhairavi N; Patel, Prabhudas S

2017-04-01

The hallmarks of cancer are characterized by functional capabilities that allow cancer cells to survive, proliferate and disseminate during the multistep tumorigenesis. Cancer being a cellular disease, changes in cellular glycoproteins play an important role in malignant transformation and cancer progression. The present review summarizes various studies that depicted correlation of glycosylation with tumor initiation, progression and metastasis, which are helpful in early diagnosis, disease monitoring and prognosis. The results are further strengthened by our reports, which depicted alterations in sialylation and fucosylation in different cancers. Alterations in glycosyltransferases are also involved in formation of various tumor antigens (e.g. Sialyl Lewis x) which serves as ligand for the cell adhesion molecule, selectin which is involved in adhesion of cancer cells to vascular endothelium and thus contributes to hematogenous metastasis. Increased glycosylation accompanied by alterations in glycosyltranferases, glycosidases, glycans and mucins (MUC)s are also involved in loss of E-cadherin, a key molecule implicated in metastatic dissemination of cells. The present review also summarizes the correlation of glycosylation with all the hallmarks of cancer. The enormous progress in the design of novel inhibitors of pathway intermediates of sialylation and fucosylation can prove wonders in combating the dreadful disease. The results provide the evidence that altered glycosylation is linked to tumor initiation, progression and metastasis. Hence, it can be considered as a new hallmark of cancer development and strategies to develop novel glycosylation targeted molecules should be strengthened.

Isolation and characterization of N-feruloyltyramine as the P-selectin expression suppressor from garlic (Allium sativum)

USDA-ARS?s Scientific Manuscript database

Because garlic (Allium sativum) is believed to have positive health effects on cardiovascular disease, the screening of isolated fractions from a garlic extract against cardiovascular disease related-processes should help identify active compounds. Both P-selectin expression suppressing activity ag...

Soluble Siglec-5 associates to PSGL-1 and displays anti-inflammatory activity

PubMed Central

Pepin, Marion; Mezouar, Soraya; Pegon, Julie; Muczynski, Vincent; Adam, Frédéric; Bianchini, Elsa P.; Bazaa, Amine; Proulle, Valerie; Rupin, Alain; Paysant, Jerome; Panicot-Dubois, Laurence; Christophe, Olivier D.; Dubois, Christophe; Lenting, Peter J.; Denis, Cécile V.

2016-01-01

Interactions between endothelial selectins and the leukocyte counter-receptor PSGL1 mediates leukocyte recruitment to inflammation sites. PSGL1 is highly sialylated, making it a potential ligand for Siglec-5, a leukocyte-receptor that recognizes sialic acid structures. Binding assays using soluble Siglec-5 variants (sSiglec-5/C4BP and sSiglec-5/Fc) revealed a dose- and calcium-dependent binding to PSGL1. Pre-treatment of PSGL1 with sialidase reduced Siglec-5 binding by 79 ± 4%. In confocal immune-fluorescence assays, we observed that 50% of Peripheral Blood Mononuclear Cells (PBMCs) simultaneously express PSGL1 and Siglec-5. Duolink-proximity ligation analysis demonstrated that PSGL1 and Siglec-5 are in close proximity (<40 nm) in 31 ± 4% of PBMCs. In vitro perfusion assays revealed that leukocyte-rolling over E- and P-selectin was inhibited by sSiglec-5/Fc or sSiglec-5/C4BP, while adhesion onto VCAM1 was unaffected. When applied to healthy mice (0.8 mg/kg), sSiglec-5/C4BP significantly reduced the number of rolling leukocytes under basal conditions (10.9 ± 3.7 versus 23.5 ± 9.3 leukocytes/field/min for sSiglec-5/C4BP-treated and control mice, respectively; p = 0.0093). Moreover, leukocyte recruitment was inhibited over a 5-h observation period in an in vivo model of TNFalpha-induced inflammation following injection sSiglec-5/C4BP (0.8 mg/kg). Our data identify PSGL1 as a ligand for Siglec-5, and soluble Siglec-5 variants appear efficient in blocking PSGL1-mediated leukocyte rolling and the inflammatory response in general. PMID:27892504

A Targeting Microbubble for Ultrasound Molecular Imaging

PubMed Central

Yeh, James Shue-Min; Sennoga, Charles A.; McConnell, Ellen; Eckersley, Robert; Tang, Meng-Xing; Nourshargh, Sussan; Seddon, John M.; Haskard, Dorian O.; Nihoyannopoulos, Petros

2015-01-01

Rationale Microbubbles conjugated with targeting ligands are used as contrast agents for ultrasound molecular imaging. However, they often contain immunogenic (strept)avidin, which impedes application in humans. Although targeting bubbles not employing the biotin-(strept)avidin conjugation chemistry have been explored, only a few reached the stage of ultrasound imaging in vivo, none were reported/evaluated to show all three of the following properties desired for clinical applications: (i) low degree of non-specific bubble retention in more than one non-reticuloendothelial tissue; (ii) effective for real-time imaging; and (iii) effective for acoustic quantification of molecular targets to a high degree of quantification. Furthermore, disclosures of the compositions and methodologies enabling reproduction of the bubbles are often withheld. Objective To develop and evaluate a targeting microbubble based on maleimide-thiol conjugation chemistry for ultrasound molecular imaging. Methods and Results Microbubbles with a previously unreported generic (non-targeting components) composition were grafted with anti-E-selectin F(ab’)2 using maleimide-thiol conjugation, to produce E-selectin targeting microbubbles. The resulting targeting bubbles showed high specificity to E-selectin in vitro and in vivo. Non-specific bubble retention was minimal in at least three non-reticuloendothelial tissues with inflammation (mouse heart, kidneys, cremaster). The bubbles were effective for real-time ultrasound imaging of E-selectin expression in the inflamed mouse heart and kidneys, using a clinical ultrasound scanner. The acoustic signal intensity of the targeted bubbles retained in the heart correlated strongly with the level of E-selectin expression (|r|≥0.8), demonstrating a high degree of non-invasive molecular quantification. Conclusions Targeting microbubbles for ultrasound molecular imaging, based on maleimide-thiol conjugation chemistry and the generic composition described, may possess properties (i)–(iii) desired for clinical applications. PMID:26161541

Borrelia burgdorferi upregulates the adhesion molecules E-selectin, P-selectin, ICAM-1 and VCAM-1 on mouse endothelioma cells in vitro.

PubMed

Böggemeyer, E; Stehle, T; Schaible, U E; Hahne, M; Vestweber, D; Simon, M M

1994-06-01

In order to obtain more information on processes leading to Borrelia burgdorferi-induced inflammation in the host, we have developed an in vitro model to study the upregulation of cell surface expression of adhesion molecules on endothelial cells by spirochetes. A mouse endothelioma cell line, derived from brain capillaries, bEnd3, was used as indicator population. bEnd3 cells were incubated with preparations of viable, inactivated or sonicated spirochetes and the expression of E-selectin, P-selectin, ICAM-1 and VCAM-1 was monitored by immunocytochemistry and quantified by cell surface ELISA. We show that all three spirochetal preparations are able to upregulate cell surface expression of E-selectin, P-selectin, ICAM-1 and VCAM-1 on bEnd 3 cells in a dose-dependent manner. The kinetics of cell surface expression of the individual adhesion molecules in the presence of Borrelia burgdorferi showed maxima at about 50 h of incubation or later; this was distinct from results obtained with sonicated-preparations of Escherichia coli bacteria or with enterobacterial LPS where peak expression was observed between 4 h and 16 h. The fact that Borrelia burgdorferi does not contain conventional LPS suggests that the mode of induction of adhesion molecules on endothelial cells is influenced by the phenotype of bacteria. At the peak of spirochete-induced cell surface expression of adhesion molecules (approximately 50 h), bEnd3 cells were found to bind cells of a VLA-4+ B lymphoma line (L1-2) much more efficiently than untreated control cells. The binding of L1-2 cells to presensitized bEnd3 cells was significantly inhibited (more than 75%) in the presence of monoclonal antibodies to both VLA-4 and its endothelial counterreceptor VCAM-1. These findings demonstrate that Borrelia burgdorferi organisms are able to induce functionally active adhesion molecules on endothelial cells in vitro and suggest that E-selectin, P-selectin, ICAM-1 and VCAM-1 play an important role in the pathogenesis of spirochetal infection.

Basigin (CD147), a multifunctional transmembrane glycoprotein with various binding partners.

PubMed

Muramatsu, Takashi

2016-05-01

Basigin, also called CD147 or EMMPRIN, is a transmembrane glycoprotein that belongs to the immunoglobulin superfamily. Basigin has isoforms; the common form (basigin or basigin-2) has two immunoglobulin domains, and the extended form (basigin-1) has three. Basigin is the receptor for cyclophilins, S100A9 and platelet glycoprotein VI, whereas basigin-1 serves as the receptor for the rod-derived cone viability factor. Basigin tightly associates with monocarboxylate transporters and is essential for their cell surface translocation and activities. In the same membrane plane, basigin also associates with other proteins including GLUT1, CD44 and CD98. The carbohydrate portion of basigin is recognized by lectins, such as galectin-3 and E-selectin. These molecular recognitions form the basis for the role of basigin in the transport of nutrients, migration of inflammatory leukocytes and induction of matrix metalloproteinases. Basigin is important in vision, spermatogenesis and other physiological phenomena, and plays significant roles in the pathogenesis of numerous diseases, including cancer. Basigin is also the receptor for an invasive protein RH5, which is present in malaria parasites. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society.

[Correlation analysis between biochemical and biophysical markers of endothelium damage in children with diabetes type 1].

PubMed

Głowińska-Olszewska, Barbara; Urban, Mirosława; Tołwińska, Joanna; Peczyńska, Jadwiga; Florys, Bozena

2005-01-01

Endothelial damage is one of the earliest stages in the atherosclerosis process. Adhesion molecules, secreted from dysfunctional endothelial cells are considered as early markers of atherosclerotic disease. Ultrasonographic evaluation of brachial arteries serves to detect biophysical changes in endothelial function, and evaluation of carotid arteries intima-media thickness allows to evaluate the earliest structural changes in the vessels. The aim of the study was to the evaluate levels of selected adhesion molecules (sICAM-1, sVCAM-1, sE-selectin, sP-selectin) and endothelial function with use of brachial artery dilatation study (flow mediated dilation--FMD, nitroglycerine mediated dilation--NTGMD) and IMT in carotid arteries in children and adolescents with diabetes type 1, as well as the correlation analysis between biochemical and biophysical markers of endothelial dysfunction. We studied 76 children and adolescents, with mean age--15.6+/-2.5 years, suffering from diabetes mean 7.8+/-2.8 years, mean HbA1c--8.4+/-1.5%. Control group consisted of 33 healthy children age and gender matched. Adhesion molecules levels were estimated with the use of immunoenzymatic methods (R&D Systems). Endothelial function was evaluated by study of brachial arteries dilation--FMD, NTGMD, with ultrasonographic evaluation (Hewlett Packard Sonos 4500) after Celermajer method, and IMT after Pignoli method. In the study group we found elevated levels of sICAM-1: 309.54+/-64 vs. 277.85+/-52 ng/ml in the control group (p<00.05) and elevated level of sE-selectin: 87.81+/-35 vs. 66.21+/-22 ng/ml (p<00.05). We found significantly impaired FMD in brachial arteries in the study group--7.51+/-4.52 vs. 12.61+/-4.65% (p<00.05) and significantly higher IMT value: 0.51+/-0.07 vs. 0.42+/-0.05 mm (p<00.001). Correlation analysis revealed a significant negative correlation between sE-selectin and FMD - r=-0.33 (p=0.004), and a positive correlation between E-selectin and IMT: r=0.32 (p=0.005). 1. In children and adolescents with diabetes type 1 we found elevated levels of adhesion molecules sICAM-1 and sE-selectin, what can confirm an endothelial dysfunction in these patients. 2. Significant negative correlation between sE-selectin level and FMD, and positive correlation between sE-selectin and IMT were found. 3. Biophysical proof of this damage is impaired brachial artery dilatation--FMD, and increased IMT values provide information about structural changes in the vessels.

Endothelial progenitor cells inhibit platelet function in a P-selectin-dependent manner.

PubMed

Abou-Saleh, Haissam; Hachem, Ahmed; Yacoub, Daniel; Gillis, Marc-Antoine; Merhi, Yahye

2015-05-07

The role of endothelial progenitor cells (EPCs) in vascular repair is related to their recruitment at the sites of injury and their interaction with different components of the circulatory system. We have previously shown that EPCs bind and inhibit platelet function and impair thrombus formation via prostacyclin secretion, but the role of EPC binding to platelet P-selectin in this process has not been fully characterized. In the present study, we assessed the impact of EPCs on thrombus formation and we addressed the implication of P-selectin in this process. EPCs were generated from human peripheral blood mononuclear cells cultured on fibronectin in conditioned media. The impact of EPCs on platelet aggregation and thrombus formation was investigated in P-selectin deficient (P-sel(-/-)) mice and their wild-type (WT) counterparts. EPCs significantly and dose-dependently impaired collagen-induced whole blood platelet aggregation in WT mice, whereas no effects were observed in P-sel(-/-) mice. Moreover, in a ferric chloride-induced arterial thrombosis model, infusion of EPCs significantly reduced thrombus formation in WT, but not in P-sel(-/-) mice. Furthermore, the relative mass of thrombi generated in EPC-treated P-sel(-/-) mice were significantly larger than those in EPC-treated WT mice, and the number of EPCs recruited within the thrombi and along the arterial wall was reduced in P-sel(-/-) mice as compared to WT mice. This study shows that EPCs impair platelet aggregation and reduce thrombus formation via a cellular mechanism involving binding to platelet P-selectin. These findings add new insights into the role of EPC-platelet interactions in the regulation of thrombotic events during vascular repair.

Crucial Role of the Aryl Hydrocarbon Receptor (AhR) in Indoxyl Sulfate-Induced Vascular Inflammation.

PubMed

Ito, Shunsuke; Osaka, Mizuko; Edamatsu, Takeo; Itoh, Yoshiharu; Yoshida, Masayuki

2016-08-01

The aryl hydrocarbon receptor (AhR), a ligand-inducible transcription factor mediating toxic effects of dioxins and uremic toxins, has recently emerged as a pathophysiological regulator of immune-inflammatory conditions. Indoxyl sulfate, a uremic toxin, is associated with cardiovascular disease in patients with chronic kidney disease and has been shown to be a ligand for AhR. The aim of this study was to investigate the potential role of AhR in indoxyl sulfate-induced leukocyte-endothelial interactions. Endothelial cell-specific AhR knockout (eAhR KO) mice were produced by crossing AhR floxed mice with Tie2 Cre mice. Indoxyl sulfate was administered for 2 weeks, followed by injection of TNF-α. Leukocyte recruitment to the femoral artery was assessed by intravital microscopy. Vascular endothelial cells were transfected with siRNA specific to AhR (siAhR) and treated with indoxyl sulfate, followed by stimulation with TNF-α. Indoxyl sulfate dramatically enhanced TNF-α-induced leukocyte recruitment to the vascular wall in control animals but not in eAhR KO mice. In endothelial cells, siAhR significantly reduced indoxyl sulfate-enhanced leukocyte adhesion as well as E-selectin expression, whereas the activation of JNK and nuclear factor-κB was not affected. A luciferase assay revealed that the region between －153 and －146 bps in the E-selectin promoter was responsible for indoxyl sulfate activity via AhR. Mutational analysis of this region revealed that activator protein-1 (AP-1) is responsible for indoxyl sulfate-triggered E-selectin expression via AhR. AhR mediates indoxyl sulfate-enhanced leukocyte-endothelial interactions through AP-1 transcriptional activity, which may constitute a new mechanism of vascular inflammation in patients with renal disease.

Fibrin, γ’-fibrinogen, and trans-clot pressure gradient control hemostatic clot growth during human blood flow over a collagen/tissue factor wound

PubMed Central

Muthard, Ryan W.; Welsh, John D.; Brass, Lawrence F.; Diamond, Scott L.

2015-01-01

SUMMARY Objective Biological and physical factors interact to modulate blood response in a wounded vessel, resulting in a hemostatic clot or an occlusive thrombus. Flow and pressure differential (ΔP) across the wound from the lumen to the extravascular compartment may impact hemostasis and the observed core/shell architecture. We examined physical and biological factors responsible for regulating thrombin mediated clot growth. Approach and Results Using factor XIIa-inhibited human whole blood perfused in a microfluidic device over collagen/tissue factor at controlled wall shear rate and ΔP, we found thrombin to be highly localized in the P-selectin+ core of hemostatic clots. Increasing ΔP from 9 to 29 mm-Hg (wall shear rate = 400 s−1) reduced P-selectin+ core size and total clot size due to enhanced extravasation of thrombin. Blockade of fibrin polymerization with 5 mM GPRP dysregulated hemostasis by enhancing both P-selectin+ core size and clot size at 400 s−1 (20 mm-Hg). For whole blood flow (no GPRP), the thickness of the P-selectin-negative shell was reduced under arterial conditions (2000 s−1, 20 mm-Hg). Consistent with the antithrombin-1 activity of fibrin implicated with GPRP, anti-γ’-fibrinogen antibody enhanced core-localized thrombin, core size, and overall clot size, especially at venous (100 s−1) but not arterial wall shear rates (2000 s−1). Pathological shear (15,000 s−1) and GPRP synergized to exacerbate clot growth. Conclusions Hemostatic clotting was dependent on core-localized thrombin that (1) triggered platelet P-selectin display and (2) was highly regulated by fibrin and the trans-clot ΔP. Also, γ’-fibrinogen had a role in venous but not arterial conditions. PMID:25614284

Meal-induced platelet activation in Type 2 diabetes mellitus: effects of treatment with repaglinide and glibenclamide.

PubMed

Yngen, M; Ostenson, C-G; Hjemdahl, P; Wallén, N H

2006-02-01

To compare the effects of treatment with repaglinide and glibenclamide on platelet function and endothelial markers in patients with Type 2 diabetes mellitus, before and after a standardized meal. Fifteen patients with Type 2 diabetes were investigated on three occasions: at baseline without oral hypoglycaemic drug treatment, and after 6 weeks' treatment with repaglinide or glibenclamide, respectively, in an open randomized cross-over study. Agonist-induced platelet P-selectin expression and platelet aggregation, urinary thromboxane, soluble P-selectin, von Willebrand factor (VWF), soluble E-selectin, intercellular adhesion molecule (ICAM-1) and C-reactive protein (CRP) were measured. In addition, pre-meal data were compared with non-diabetic control subjects (n = 15), matched for sex, age and BMI. Adenosine diphosphate (ADP)-induced platelet P-selectin expression increased post-meal in Type 2 diabetic patients both at baseline and after treatment with repaglinide and glibenclamide (P < 0.01 for all; repeated measures anova). Repaglinide treatment reduced fasting ADP-induced P-selectin expression compared with baseline (P = 0.01), but did not influence meal-induced platelet hyper-reactivity (P = 0.32). No significant anti-platelet effects of glibenclamide treatment were found. Plasma concentrations of VWF and ICAM-1 were elevated in patients with Type 2 diabetes compared with control subjects (P < 0.05 for both) and were reduced during treatment with repaglinide (P < 0.01 for both) but did not change during glibenclamide treatment. The post-meal state is associated with enhanced platelet reactivity in patients with Type 2 diabetes mellitus. Pre-meal treatment with repaglinide or glibenclamide does not inhibit postprandial platelet activation, but repaglinide treatment is associated with attenuated platelet and endothelial activity in the fasting state.

Annexin A8 controls leukocyte recruitment to activated endothelial cells via cell surface delivery of CD63

NASA Astrophysics Data System (ADS)

Poeter, Michaela; Brandherm, Ines; Rossaint, Jan; Rosso, Gonzalo; Shahin, Victor; Skryabin, Boris V.; Zarbock, Alexander; Gerke, Volker; Rescher, Ursula

2014-04-01

To enable leukocyte adhesion to activated endothelium, the leukocyte receptor P-selectin is released from Weibel-Palade bodies (WPB) to the endothelial cell surface where it is stabilized by CD63. Here we report that loss of annexin A8 (anxA8) in human umbilical vein endothelial cells (HUVEC) strongly decreases cell surface presentation of CD63 and P-selectin, with a concomitant reduction in leukocyte rolling and adhesion. We confirm the compromised leukocyte adhesiveness in inflammatory-activated endothelial venules of anxA8-deficient mice. We find that WPB of anxA8-deficient HUVEC contain less CD63, and that this is caused by improper transport of CD63 from late multivesicular endosomes to WPB, with CD63 being retained in intraluminal vesicles. Consequently, reduced CD63 cell surface levels are seen following WPB exocytosis, resulting in enhanced P-selectin re-internalization. Our data support a model in which anxA8 affects leukocyte recruitment to activated endothelial cells by supplying WPB with sufficient amounts of the P-selectin regulator CD63.

Immunologic changes in TNF-alpha, sE-selectin, sP-selectin, sICAM-1, and IL-8 in pediatric patients treated for psoriasis with the Goeckerman regimen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Borska, L.; Fiala, Z.; Krejsek, J.

2007-11-15

Psoriasis is a chronic inflammatory skin disease which is often manifested during childhood. The present study investigated changes in the serum levels of proinflammatory cytokines and soluble forms of adhesion molecules in children with psoriasis. The observed patient group of 26 children was treated with the Goeckerman regimen. This therapy combines dermal application of crude coal tar with ultraviolet radiation. The Psoriasis Area Severity Index decreased significantly after treatment by with the Goeckerman regimen (p < 0.001). Serum levels of the proinflammatory cytokine TNF-alpha and adhesion molecules sICAM-1, sP-selectin and sE-selectin decreased after the Goeckerman regimen. The TNF-alpha and sICAM-1more » decreased significantly (p < 0.05). Our findings support the complex role of these immune parameters in the immunopathogenesis of psoriasis in children. The serum level of IL-8 increased after the Goeckerman regimen. This fact indicates that the chemokine pathway of IL-8 activity could be modulated by this treatment, most likely by polycyclic aromatic hydrocarbons.« less

Novel aspects of sialoglycan recognition by the Siglec-like domains of streptococcal SRR glycoproteins.

PubMed

Bensing, Barbara A; Khedri, Zahra; Deng, Lingquan; Yu, Hai; Prakobphol, Akraporn; Fisher, Susan J; Chen, Xi; Iverson, Tina M; Varki, Ajit; Sullam, Paul M

2016-11-01

Serine-rich repeat glycoproteins are adhesins expressed by commensal and pathogenic Gram-positive bacteria. A subset of these adhesins, expressed by oral streptococci, binds sialylated glycans decorating human salivary mucin MG2/MUC7, and platelet glycoprotein GPIb. Specific sialoglycan targets were previously identified for the ligand-binding regions (BRs) of GspB and Hsa, two serine-rich repeat glycoproteins expressed by Streptococcus gordonii While GspB selectively binds sialyl-T antigen, Hsa displays broader specificity. Here we examine the binding properties of four additional BRs from Streptococcus sanguinis or Streptococcus mitis and characterize the molecular determinants of ligand selectivity and affinity. Each BR has two domains that are essential for sialoglycan binding by GspB. One domain is structurally similar to the glycan-binding module of mammalian Siglecs (sialic acid-binding immunoglobulin-like lectins), including an arginine residue that is critical for glycan recognition, and that resides within a novel, conserved YTRY motif. Despite low sequence similarity to GspB, one of the BRs selectively binds sialyl-T antigen. Although the other three BRs are highly similar to Hsa, each displayed a unique ligand repertoire, including differential recognition of sialyl Lewis antigens and sulfated glycans. These differences in glycan selectivity were closely associated with differential binding to salivary and platelet glycoproteins. Specificity of sialoglycan adherence is likely an evolving trait that may influence the propensity of streptococci expressing Siglec-like adhesins to cause infective endocarditis. Published by Oxford University Press 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

An oncological view on the blood-testis barrier.

PubMed

Bart, Joost; Groen, Harry J M; van der Graaf, Winette T A; Hollema, Harry; Hendrikse, N Harry; Vaalburg, Willem; Sleijfer, Dirk T; de Vries, Elisabeth G E

2002-06-01

The function of the blood-testis barrier is to protect germ cells from harmful influences; thus, it also impedes the delivery of chemotherapeutic drugs to the testis. The barrier has three components: first, a physicochemical barrier consisting of continuous capillaries, Sertoli cells in the tubular wall, connected together with narrow tight junctions, and a myoid-cell layer around the seminiferous tubule. Second, an efflux-pump barrier that contains P-glycoprotein in the luminal capillary endothelium and on the myoid-cell layer; and multidrug-resistance associated protein 1 located basolaterally on Sertoli cells. Third, an immunological barrier, consisting of Fas ligand on Sertoli cells. Inhibition of P-glycoprotein function offers the opportunity to increase the delivery of cytotoxic drugs to the testis. In the future, visualisation of function in the blood-testis barrier may also be helpful to identify groups of patients in whom testis conservation is safe or to select drugs that are less harmful to fertility.

Physical activity initiated by employer induces improvements in a novel set of biomarkers of inflammation: an 8-week follow-up study.

PubMed

Lunde, Lars-Kristian; Skare, Øivind; Aass, Hans C D; Mamen, Asgeir; Einarsdóttir, Elín; Ulvestad, Bente; Skogstad, Marit

2017-03-01

We investigated the level of pro- and anti-inflammatory biomarkers before and after 8 weeks of unsupervised physical activity (PA) initiated by employer. During autumn 2014, background data, blood samples and self-reported exercise level were collected from 76 men and 41 women in a Norwegian road maintenance company. Monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), leptin, adiponectin, p-selectin and CD40 ligand (CD40L) were analyzed. [Formula: see text] was measured in a subgroup of 50 subjects. With reference point of exercise ≤1 time/week, we found that participants who exercised 2-3 times/week had higher [Formula: see text] values (5.6 mL kg -1  min -1 ; 95% CI [1.3, 9.9]). MCP-1 was lower in those who exercised ≥ 4 times/week (-81.98 pg/ml [-142.9, -21.0]). IL-6 and p-selectin levels were lower in females who exercised ≥4 times/week (-1.04 pg/ml [-2.04, -0.03] and -13.75 ng/ml [-24.03, -3.48]). Leptin was lower in participants who exercised 2-3 times/week (-0.39 µg/ml ln [-0.68, -0.09]) and ≥4 times/week (-0.69 µg/ml ln [-1.10, -0.28]). During follow-up, [Formula: see text] increased (2.9 mL kg -1  min -1 [1.5, 4.3]), while p-selectin and CD40L decreased (-2.33 ng/ml [-3.78, -0.87] and 718.14 ng/ml [-1368, -68]). MCP-1 levels decreased among men (-32.70 pg/ml [-51.21, -14.19]). A joint analysis of all biomarkers (inversed adiponectin) showed that those who exercised ≥4 times/week at baseline had lower total levels of biomarkers and that total biomarker levels decreased during follow-up. Exercising several times a week was associated with less inflammation compared to exercising once a week or less. During the 8-week follow-up, total levels of biomarkers of inflammation improved.

Melanoma upregulates ICAM-1 expression on endothelial cells through engagement of tumor CD44 with endothelial E-selectin and activation of a PKCα–p38–SP-1 pathway

PubMed Central

Zhang, Pu; Goodrich, Chris; Fu, Changliang; Dong, Cheng

2014-01-01

Cancer metastasis involves multistep adhesive interactions between tumor cells (TCs) and endothelial cells (ECs), but the molecular mechanisms of intercellular communication in the tumor microenvironment remain elusive. Using static and flow coculture systems in conjunction with flow cytometry, we discovered that certain receptors on the ECs are upregulated on melanoma cell adhesion. Direct contact but not separate coculture between human umbilical endothelial cells (HUVECs) and a human melanoma cell line (Lu1205) increased intercellular adhesion molecule 1 (ICAM-1) and E-selectin expression on HUVECs by 3- and 1.5-fold, respectively, compared with HUVECs alone. The nonmetastatic cell line WM35 failed to promote ICAM-1 expression changes in HUVECs on contact. Enzyme-linked immunosorbent assay (ELISA) revealed that EC–TC contact has a synergistic effect on the expression of the cytokines interleukin (IL)-8, IL-6, and growth-related oncogene α (Gro-α). By using E-selectin cross-linking and beads coated with CD44 immunopurified from Lu1205 cells, we showed that CD44/selectin ligation was responsible for the ICAM-1 up-regulation on HUVECs. Protein kinase Cα (PKC-α) activation was found to be the downstream target of the CD44/selectin-initiated signaling, as ICAM-1 elevation was inhibited by siRNA targeting PKCα or a dominant negative form of PKCα (PKCα DN). Western blot analysis and electrophoretic mobility shift assays (EMSAs) showed that TC–EC contact mediated p38 phosphorylation and binding of the transcription factor SP-1 to its regulation site. In conclusion, CD44/selectin binding signals ICAM-1 up-regulation on the EC surface through a PKCα–p38–SP-1 pathway, which further enhances melanoma cell adhesion to ECs during metastasis.—Zhang, P., Goodrich, C., Fu, C., Dong, C. Melanoma upregulates ICAM-1 expression on ECs through engagement of tumor CD44 with endothelial E-selectin and activation of a PKCα–p38–SP-1 pathway. PMID:25138157

Surfactant Functionalization Induces Robust, Differential Adhesion of Tumor Cells and Blood Cells to Charged Nanotube-Coated Biomaterials Under Flow

PubMed Central

Mitchell, Michael J.; Castellanos, Carlos A.; King, Michael R.

2015-01-01

The metastatic spread of cancer cells from the primary tumor to distant sites leads to a poor prognosis in cancers originating from multiple organs. Increasing evidence has linked selectin-based adhesion between circulating tumor cells (CTCs) and endothelial cells of the microvasculature to metastatic dissemination, in a manner similar to leukocyte adhesion during inflammation. Functionalized biomaterial surfaces hold promise as a diagnostic tool to separate CTCs and potentially treat metastasis, utilizing antibody and selectin-mediated interactions for cell capture under flow. However, capture at high purity levels is challenged by the fact that CTCs and leukocytes both possess selectin ligands. Here, a straightforward technique to functionalize and alter the charge of naturally occurring halloysite nanotubes using surfactants is reported to induce robust, differential adhesion of tumor cells and blood cells to nanotube-coated surfaces under flow. Negatively charged sodium dodecanoate-functionalized nanotubes simultaneously enhanced tumor cell capture while negating leukocyte adhesion, both in the presence and absence of adhesion proteins, and can be utilized to isolate circulating tumor cells regardless of biomarker expression. Conversely, diminishing nanotube charge via functionalization with decyltrimethylammonium bromide both abolished tumor cell capture while promoting leukocyte adhesion. PMID:25934290

Flow cytometric analysis reveals the high levels of platelet activation parameters in circulation of multiple sclerosis patients.

PubMed

Morel, Agnieszka; Rywaniak, Joanna; Bijak, Michał; Miller, Elżbieta; Niwald, Marta; Saluk, Joanna

2017-06-01

The epidemiological studies confirm an increased risk of cardiovascular disease in multiple sclerosis, especially prothrombotic events directly associated with abnormal platelet activity. The aim of our study was to investigate the level of blood platelet activation in the circulation of patients with chronic phase of multiple sclerosis (SP MS) and their reactivity in response to typical platelets' physiological agonists. We examined 85 SP MS patients diagnosed according to the revised McDonald's criteria and 50 healthy volunteers as a control group. The platelet activation and reactivity were assessed using flow cytometry analysis of the following: P-selectin expression (CD62P), activation of GP IIb/IIIa complex (PAC-1 binding), and formation of platelet microparticles (PMPs) and platelet aggregates (PA) in agonist-stimulated (ADP, collagen) and unstimulated whole blood samples. Furthermore, we measured the level of soluble P-selectin (sP-selectin) in plasma using ELISA method, to evaluate the in vivo level of platelet activation, both in healthy and SP MS subjects. We found a statistically significant increase in P-selectin expression, GP IIb/IIIa activation, and formation of PMPs and PA, as well as in unstimulated and agonist-stimulated (ADP, collagen) platelets in whole blood samples from patients with SP MS in comparison to the control group. We also determined the higher sP-selectin level in plasma of SP MS subjects than in the control group. Based on the obtained results, we might conclude that during the course of SP MS platelets are chronically activated and display hyperreactivity to physiological agonists, such as ADP or collagen.

Serological responses in chimpanzees inoculated with human immunodeficiency virus glycoprotein (gp120) subunit vaccine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arthur, L.O.; Pyle, S.W.; Nara, P.L.

1987-12-01

The major envelope glycoprotein of a human immunodeficiency virus (HIV) has been purified and was utilized as a prototype vaccine in chimpanzees. The 120,000-dalton glycoprotein (gp120) was purified from membranes of human T-lymphotropic virus (HTLV)-IIIB-infected cells and the final preparation contained low levels to no detectable HTLV-IIIB core antigen (p24) and low levels of endotoxin. Chimpanzees inoculated with gp120 responded by developing antibodies that precipitated radiolabeled gp120 and neutralized in vitro infection of HTLV-IIIB. Antibodies to HTLV-IIIB p24 were not detected in the gp120-immunized chimpanzees. Peripheral blood leukocytes from the vaccinated animals were examined for T4/sup +/ and T8/sup +/more » cells, and no decrease in the T4/T8 ratio was found, indicating that immunization with a ligand (gp120) that binds to T4 has not detectable adverse effect on the population of T4/sup +/ cells. The only current animal model that can be reproducibly infected with HIV is the chimpanzee. Immunization of chimpanzees with HIV proteins will provide an experimental system for testing the effectiveness of prototype vaccines for preventing HIV infection in vivo.« less

Selectins and chemokines use shared and distinct signals to activate β2 integrins in neutrophils

PubMed Central

Yago, Tadayuki; Zhang, Nan; Zhao, Liang; Abrams, Charles S.

2018-01-01

Rolling neutrophils receive signals while engaging P- and E-selectin and chemokines on inflamed endothelium. Selectin signaling activates β2 integrins to slow rolling velocities. Chemokine signaling activates β2 integrins to cause arrest. Despite extensive study, key aspects of these signaling cascades remain unresolved. Using complementary in vitro and in vivo assays, we found that selectin and chemokine signals in neutrophils triggered Rap1a-dependent and phosphatidylinositol-4-phosphate 5-kinase γ (PIP5Kγ90)–dependent pathways that induce integrin-dependent slow rolling and arrest. Interruption of both pathways, but not either pathway alone, blocked talin-1 recruitment to and activation of integrins. An isoform of PIP5Kγ90 lacking the talin-binding domain (PIP5Kγ87) could not activate integrins. Chemokines, but not selectins, used phosphatidylinositol-4,5-bisphosphate 3-kinase γ (PI3Kγ) in cooperation with Rap1a to mediate integrin-dependent slow rolling (at low chemokine concentrations), as well as arrest (at high chemokine concentrations). High levels of chemokines activated β2 integrins without selectin signals. When chemokines were limiting, they synergized with selectins to activate β2 integrins. PMID:29592875

Selectins and chemokines use shared and distinct signals to activate β2 integrins in neutrophils.

PubMed

Yago, Tadayuki; Zhang, Nan; Zhao, Liang; Abrams, Charles S; McEver, Rodger P

2018-04-10

Rolling neutrophils receive signals while engaging P- and E-selectin and chemokines on inflamed endothelium. Selectin signaling activates β2 integrins to slow rolling velocities. Chemokine signaling activates β2 integrins to cause arrest. Despite extensive study, key aspects of these signaling cascades remain unresolved. Using complementary in vitro and in vivo assays, we found that selectin and chemokine signals in neutrophils triggered Rap1a-dependent and phosphatidylinositol-4-phosphate 5-kinase γ (PIP5Kγ90)-dependent pathways that induce integrin-dependent slow rolling and arrest. Interruption of both pathways, but not either pathway alone, blocked talin-1 recruitment to and activation of integrins. An isoform of PIP5Kγ90 lacking the talin-binding domain (PIP5Kγ87) could not activate integrins. Chemokines, but not selectins, used phosphatidylinositol-4,5-bisphosphate 3-kinase γ (PI3Kγ) in cooperation with Rap1a to mediate integrin-dependent slow rolling (at low chemokine concentrations), as well as arrest (at high chemokine concentrations). High levels of chemokines activated β2 integrins without selectin signals. When chemokines were limiting, they synergized with selectins to activate β2 integrins. © 2018 by The American Society of Hematology.

Inhibition of Na+/H+ exchanger enhances low pH-induced L-selectin shedding and β2-integrin surface expression in human neutrophils

PubMed Central

Kaba, Nubia K.; Schultz, Joanne; Law, Foon-Yee; Lefort, Craig T.; Martel-Gallegos, Guadalupe; Kim, Minsoo; Waugh, Richard E.; Arreola, Jorge; Knauf, Philip A.

2008-01-01

Ischemia-reperfusion injury is a common pathological occurrence causing tissue damage in heart attack and stroke. Entrapment of neutrophils in the vasculature during ischemic events has been implicated in this process. In this study, we examine the effects that lactacidosis and consequent reductions in intracellular pH (pHi) have on surface expression of adhesion molecules on neutrophils. When human neutrophils were exposed to pH 6 lactate, there was a marked decrease in surface L-selectin (CD62L) levels, and the decrease was significantly enhanced by inclusion of Na+/H+ exchanger (NHE) inhibitor 5-(N,N-hexamethylene)amiloride (HMA). Similar effects were observed when pHi was reduced while maintaining normal extracellular pH, by using an NH4Cl prepulse followed by washes and incubation in pH 7.4 buffer containing NHE inhibitors [HMA, cariporide, or 5-(N,N-dimethyl)amiloride (DMA)]. The amount of L-selectin shedding induced by different concentrations of NH4Cl in the prepulse correlated with the level of intracellular acidification with an apparent pK of 6.3. In contrast, β2-integrin (CD11b and CD18) was only slightly upregulated in the low-pHi condition and was enhanced by NHE inhibition to a much lesser extent. L-selectin shedding was prevented by treating human neutrophils with inhibitors of extracellular metalloproteases (RO-31-9790 and KD-IX-73-4) or with inhibitors of intracellular signaling via p38 MAP kinase (SB-203580 and SB-239063), implying a transmembrane effect of pHi. Taken together, these data suggest that the ability of NHE inhibitors such as HMA to reduce ischemia-reperfusion injury may be related to the nearly complete removal of L-selectin from the neutrophil surface. PMID:18829897

TREM-1 SNP rs2234246 regulates TREM-1 protein and mRNA levels and is associated with plasma levels of L-selectin

PubMed Central

Aldasoro Arguinano, Alex-Ander; Dadé, Sébastien; Stathopoulou, Maria; Derive, Marc; Coumba Ndiaye, Ndeye; Xie, Ting; Masson, Christine; Gibot, Sébastien

2017-01-01

High levels of TREM-1 are associated with cardiovascular and inflammatory diseases risks and the most recent studies have showed that TREM-1 deletion or blockade is associated with up to 60% reduction of the development of atherosclerosis. So far, it is unknown whether the levels of TREM-1 protein are genetically regulated. Moreover, TREM family receptors have been suggested to regulate the cellular adhesion process. The goal of this study was to investigate whether polymorphisms within TREM-1 are regulating the variants of serum TREM-1 levels and the expression levels of their mRNA. Furthermore, we aimed to point out associations between polymorphisms on TREM-1 and blood levels of selectins. Among the 10 SNPs studied, the minor allele T of rs2234246, was associated with increased sTREM-1 in the discovery population (p-value = 0.003), explaining 33% of its variance, and with increased levels of mRNA (p-value = 0.007). The same allele was associated with increased soluble L-selectin levels (p-value = 0.011). The higher levels of sTREM-1 and L-selectin were confirmed in the replication population (p-value = 0.0007 and p-value = 0.018 respectively). We demonstrated for the first time one SNP on TREM-1, affecting its expression levels. These novel results, support the hypothesis that TREM-1 affects monocytes extravasation and accumulation processes leading to atherogenesis and atherosclerotic plaque progression, possibly through increased inflammation and subsequent higher expression of sL-selectin. PMID:28771614

An Analytical Model for Determining Two-Dimensional Receptor-Ligand Kinetics

PubMed Central

Cheung, Luthur Siu-Lun; Konstantopoulos, Konstantinos

2011-01-01

Cell-cell adhesive interactions play a pivotal role in major pathophysiological vascular processes, such as inflammation, infection, thrombosis, and cancer metastasis, and are regulated by hemodynamic forces generated by blood flow. Cell adhesion is mediated by the binding of receptors to ligands, which are both anchored on two-dimensional (2-D) membranes of apposing cells. Biophysical assays have been developed to determine the unstressed (no-force) 2-D affinity but fail to disclose its dependence on force. Here we develop an analytical model to estimate the 2-D kinetics of diverse receptor-ligand pairs as a function of force, including antibody-antigen, vascular selectin-ligand, and bacterial adhesin-ligand interactions. The model can account for multiple bond interactions necessary to mediate adhesion and resist detachment amid high hemodynamic forces. Using this model, we provide a generalized biophysical interpretation of the counterintuitive force-induced stabilization of cell rolling observed by a select subset of receptor-ligand pairs with specific intrinsic kinetic properties. This study enables us to understand how single-molecule and multibond biophysics modulate the macroscopic cell behavior in diverse pathophysiological processes. PMID:21575567

E-selectin ligand 1 regulates bone remodeling by limiting bioactive TGF-β in the bone microenvironment.

PubMed

Yang, Tao; Grafe, Ingo; Bae, Yangjin; Chen, Shan; Chen, Yuqing; Bertin, Terry K; Jiang, Ming-Ming; Ambrose, Catherine G; Lee, Brendan

2013-04-30

TGF-β is abundantly produced in the skeletal system and plays a crucial role in skeletal homeostasis. E-selectin ligand-1 (ESL-1), a Golgi apparatus-localized protein, acts as a negative regulator of TGF-β bioavailability by attenuating maturation of pro-TGF-β during cartilage homeostasis. However, whether regulation of intracellular TGF-β maturation by ESL-1 is also crucial during bone homeostasis has not been well defined. Here, we show that Esl-1(-/-) mice exhibit a severe osteopenia with elevated bone resorption and decreased bone mineralization. In primary culture, Esl-1(-/-) osteoclast progenitors show no difference in osteoclastogenesis. However, Esl-1(-/-) osteoblasts show delayed differentiation and mineralization and stimulate osteoclastogenesis more potently in the osteoblast-osteoclast coculture, suggesting that ESL-1 primarily acts in osteoblasts to regulate bone homeostasis. In addition, Esl-1(-/-) calvaria exhibit an elevated mature TGF-β/pro-TGF-β ratio, with increased expression of TGF-β downstream targets (plasminogen activator inhibitor-1, parathyroid hormone-related peptide, connective tissue growth factor, and matrix metallopeptidase 13, etc.) and a key regulator of osteoclastogenesis (receptor activator of nuclear factor κB ligand). Moreover, in vivo treatment with 1D11, a pan-TGF-β antibody, significantly improved the low bone mass of Esl-1(-/-) mice, suggesting that elevated TGF-β signaling is the major cause of osteopenia in Esl-1(-/-) mice. In summary, our study identifies ESL-1 as an important regulator of bone remodeling and demonstrates that the modulation of TGF-β maturation is pivotal in the maintenance of a homeostatic bone microenvironment and for proper osteoblast-osteoclast coupling.

Modulation of P-selection and platelet aggregation in chronic periodontitis: A clinical study

PubMed Central

Perumal, Ramesh; Rajendran, Maheashwari; Krishnamurthy, Malathi; Ganji, Kiran Kumar; Pendor, Sunil Dattuji

2014-01-01

Background: The primary etiologic factor of periodontitis is the subgingival infection with a group of Gram negative pathogens. Transient bacteremia in periodontitis patients underlie chronic production and systemic increases of various proinflammatory mediators, including Interleukin (IL)-1α, IL-6, C-reactive protein and Tumor necrosis factor (TNF)-α. P- selectin is a member of selectin family of cell surface receptor which is located in the membrane of the secretory granules (alpha granules) of platelets and in the membrane of the Weibel-Palade bodies of the vascular endothelial cells. P selectin redistributes from the membrane of the granules to the plasma membrane when platelets and endothelial cells are activated and thus degranulated. Aim: To compare the level of platelet activation, soluble P Selectin level and morphological changes and aggregation of platelets in patients in periodontitis patients compared to healthy controls. Materials and Methods: 80 patients were included in the study with the age group of 35-60. The patients were divided into 2 groups, 40 subjects with generalized chronic periodontitis and 40 healthy subjects taken as control. Periodontal Examination using clinical parameters namely, Bleeding Index, Plaque Index, Probing Pocket Depth and Clinical Attachment Level were recorded. Collection of blood samples for estimation of serum soluble P- selectin level by ELISA method. Evaluation of Platelet morphology and grading the platelet aggregation. Results: P-selectin expression shows that the mean value for control group is 4.97 ± 16.56 ng/mL and study group 13.05 ± 29.94 ng/mL which was significantly higher than control group with P value 0.001. Platelet morphological changes shows small form – mean value for control group is 75.83% ± 14.24% while for study group is 39.08%. ± 21.59; Big form – mean value for control group 0.80% ± 0.35% while for study group 0.48% ± 1.3%and Spider form- mean value for control group 23.88% ± 14.13 while study group 59.32% ±. 23.42. The observation showed high statistical significance with P- value < 0.001 for small and spider form and no statistical significance for big form P = 0.075. Conclusion: Increased expression of P-selectin, spider form of platelets and pathological aggregation pattern which indicates that platelet activation may be associated with chronic periodontitis. The results of the study showed, higher number of spider forms and significant pathological aggregation pattern in periodontitis patients which indicates activation of platelets thus emphasized that periodontitis can be an contributing factor in the development of cardiovascular disease. PMID:25024540

Identification of the first small-molecule ligand of the neuronal receptor sortilin and structure determination of the receptor–ligand complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Andersen, Jacob Lauwring, E-mail: jla@mb.au.dk; Schrøder, Tenna Juul; Christensen, Søren

2014-02-01

The identification of the first small-molecule ligand of the neuronal receptor sortilin and structure determination of the receptor–ligand complex are reported. Sortilin is a type I membrane glycoprotein belonging to the vacuolar protein sorting 10 protein (Vps10p) family of sorting receptors and is most abundantly expressed in the central nervous system. Sortilin has emerged as a key player in the regulation of neuronal viability and has been implicated as a possible therapeutic target in a range of disorders. Here, the identification of AF40431, the first reported small-molecule ligand of sortilin, is reported. Crystals of the sortilin–AF40431 complex were obtained bymore » co-crystallization and the structure of the complex was solved to 2.7 Å resolution. AF40431 is bound in the neurotensin-binding site of sortilin, with the leucine moiety of AF40431 mimicking the binding mode of the C-terminal leucine of neurotensin and the 4-methylumbelliferone moiety of AF40431 forming π-stacking with a phenylalanine.« less

Platelet Glycoprotein Ib-IX and Malignancy

DTIC Science & Technology

2010-09-01

provide a unique microenvironment supporting the accumulation of more platelets and the elaboration of a fibrin - rich network produced by coagulation...process and can initiate the formation of a platelet - rich thrombus by tethering the platelet to a thrombogenic surface. Several ligands binding to GP Ib... Platelet Glycoprotein Ib-IX and Malignancy PRINCIPAL INVESTIGATOR: Jerry Ware, Ph.D

Indoxyl sulfate enhances IL-1β-induced E-selectin expression in endothelial cells in acute kidney injury by the ROS/MAPKs/NFκB/AP-1 pathway.

PubMed

Shen, Wen-Ching; Liang, Chan-Jung; Huang, Tao-Ming; Liu, Chen-Wei; Wang, Shu-Huei; Young, Guang-Huar; Tsai, Jaw-Shiun; Tseng, Ying-Chin; Peng, Yu-Sen; Wu, Vin-Cent; Chen, Yuh-Lien

2016-11-01

Uremic toxins are considered a risk factor for cardiovascular disorders in kidney diseases, but it is not known whether, under inflammatory conditions, they affect adhesion molecule expression on endothelial cells, which may play a critical role in acute kidney injury (AKI). In the present study, in cardiovascular surgery-related AKI patients, who are known to have high plasma levels of the uremic toxin indoxyl sulfate (IS), plasma levels of IL-1β were found to be positively correlated with plasma levels of the adhesion molecule E-selectin. In addition, high E-selectin and IL-1β expression were seen in the kidney of ischemia/reperfusion mice in vivo. We also examined the effects of IS on E-selectin expression by IL-1β-treated human umbilical vein endothelial cells (HUVECs) and the underlying mechanism. IS pretreatment of HUVECs significantly increased IL-1β-induced E-selectin expression, monocyte adhesion, and the phosphorylation of mitogen-activated protein kinases (ERK, p38, and JNK) and transcription factors (NF-κB and AP-1), and phosphorylation was decreased by pretreatment with inhibitors of ERK1/2 (PD98059), p38 MAPK (SB202190), and JNK (SP600125). Furthermore, IS increased IL-1β-induced reactive oxygen species (ROS) production and this effect was inhibited by pretreatment with N-acetylcysteine (a ROS scavenger) or apocynin (a NADPH oxidase inhibitor). Gel shift assays and ChIP-PCR demonstrated that IS enhanced E-selectin expression in IL-1-treated HUVECs by increasing NF-κB and AP-1 DNA-binding activities. Moreover, IS-enhanced E-selectin expression in IL-1β-treated HUVECs was inhibited by Bay11-7082, a NF-κB inhibitor. Thus, IS may play an important role in the development of cardiovascular disorders in kidney diseases during inflammation by increasing endothelial expression of E-selectin.

Usefulness of circulating E-selectin to early detection of the atherosclerotic process in the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil).

PubMed

de Almeida-Pititto, Bianca; Ribeiro-Filho, Fernando Flexa; Bittencourt, Marcio Sommer; Lotufo, Paulo A; Bensenor, Isabela; Ferreira, Sandra R G

2016-01-01

This cross-sectional analysis evaluated whether determination of E-selectin concentrations could identify deterioration of cardiometabolic risk profile or subclinical atherosclerosis in individuals at low-to-moderate risk included in The Brazilian Longitudinal Study of Adult Health-ELSA-Brasil. A sample of 984 individuals from ELSA-Brasil (35-54 years) without cardiovascular disease or diabetes was stratified according to E-selectin tertiles. Traditional risk factors, inflammatory markers and categories of coronary artery calcium (CAC) scores were evaluated across the tertiles by ANOVA or Chi-squared test. In linear regression models, associations of E-selectin levels with insulin resistance index, adjusted for age, sex and adiposity were tested. The mean age of the participants was 45.8 (SD 4.9) years and 55 % were women. Mean values of age, anthropometric data, biochemical variables and inflammatory status increased across E-selectin tertiles. Also, a gradual deterioration of the cardiometabolic profile was reflected by increments in frequencies (95 % CI) of BMI ≥ 25 kg/m(2) [53.7 % (48.5-58.8), 61.0 % (56.1-66.5) and 64.2 % (59.0-69.4), p = 0.019], hypertension [18.0 % (14.1-22.8), 19.8 % (15.4-24.6) and 24.8 % (20.4-29.9), p = 0.048], pre-diabetes [62.5 % (57.4-68.3), 63.1 % (58.4-69.6) and 73.8 % (68.8-78.3), p = 0.003] and hypertriglyceridemia [22.4 % (17.9-27.2), 27.3 % (22.5-32.8) and 33.4 % (28.3-38.5), p = 0.013]. Insulinemia and HOMA-IR were independently associated with E-selectin concentration. A greater proportion of individuals with CAC scores different from zero was found in the third tertile when compared with the first and second tertiles (16.1 versus 11 %, p = 0.04, respectively). Direct associations of E-selectin with traditional risk factors slightly above their normal ranges, components of the metabolic syndrome, insulin resistance and presence of CAC suggest that this biomarker may indicate an initial atherogenic process.

Increased plasma soluble adhesion molecules; ICAM-1, VCAM-1, and E-selectin levels in patients with slow coronary flow.

PubMed

Turhan, Hasan; Saydam, Gul Sevim; Erbay, Ali Riza; Ayaz, Selime; Yasar, Ayse Saatci; Aksoy, Yuksel; Basar, Nurcan; Yetkin, Ertan

2006-04-04

Inflammation has been reported to be a major contributing factor to many cardiovascular events. In the present study, we aimed to evaluate plasma soluble adhesion molecules; intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin as possible indicators of endothelial activation or inflammation in patients with slow coronary flow. Study population included 17 patients with angiographically proven normal coronary arteries and slow coronary flow in all three coronary vessels (group I, 11 male, 6 female, mean age=48+/-9 years), and 20 subjects with angiographically proven normal coronary arteries without associated slow coronary flow (group II, 11 male, 9 female, mean age=50+/-8 years). Coronary flow rates of all patients and control subjects were documented by Thrombolysis In Myocardial Infarction frame count (TIMI frame count). All patients in group I had TIMI frame counts greater than two standard deviation above those of control subjects (group II) and, therefore, were accepted as exhibiting slow coronary flow. Serum levels of ICAM-1, VCAM-1, and E-selectin were measured in all patients and control subjects using commercially available ELISA kits. Serum ICAM-1, VCAM-1, and E-selectin levels of patients with slow coronary flow were found to be significantly higher than those of control subjects with normal coronary flow (ICAM-1: 545+/-198 ng/ml vs. 242+/-113 ng/ml respectively, p<0.001, VCAM-1: 2040+/-634 ng/ml vs. 918+/-336 ng/ml respectively, p<0.001, E-selectin: 67+/-9 ng/ml vs. 52+/-8 ng/ml respectively, p<0.001). Average TIMI frame count was detected to be significantly correlated with plasma soluble ICAM-1 (r=0.550, p<0.001), VCAM-1 (r=0.569, p<0.001) and E-selectin (r=0.443, p=0.006). Increased levels of soluble adhesion molecules in patients with slow coronary flow may be an indicator of endothelial activation and inflammation and are likely to be in the causal pathway leading to slow coronary flow.

Glycosyltransferase Function in Core 2-Type Protein O Glycosylation▿

PubMed Central

Stone, Erica L.; Ismail, Mohd Nazri; Lee, Seung Ho; Luu, Ying; Ramirez, Kevin; Haslam, Stuart M.; Ho, Samuel B.; Dell, Anne; Fukuda, Minoru; Marth, Jamey D.

2009-01-01

Three glycosyltransferases have been identified in mammals that can initiate core 2 protein O glycosylation. Core 2 O-glycans are abundant among glycoproteins but, to date, few functions for these structures have been identified. To investigate the biological roles of core 2 O-glycans, we produced and characterized mice deficient in one or more of the three known glycosyltransferases that generate core 2 O-glycans (C2GnT1, C2GnT2, and C2GnT3). A role for C2GnT1 in selectin ligand formation has been described. We now report that C2GnT2 deficiency impaired the mucosal barrier and increased susceptibility to colitis. C2GnT2 deficiency also reduced immunoglobulin abundance and resulted in the loss of all core 4 O-glycan biosynthetic activity. In contrast, the absence of C2GnT3 altered behavior linked to reduced thyroxine levels in circulation. Remarkably, elimination of all three C2GnTs was permissive of viability and fertility. Core 2 O-glycan structures were reduced among tissues from individual C2GnT deficiencies and completely absent from triply deficient mice. C2GnT deficiency also induced alterations in I-branching, core 1 O-glycan formation, and O mannosylation. Although the absence of C2GnT and C4GnT activities is tolerable in vivo, core 2 O glycosylation exerts a significant influence on O-glycan biosynthesis and is important in multiple physiological processes. PMID:19349303

Triple selectin knockout (ELP-/-) mice fail to develop OVA-induced acute asthma phenotype

PubMed Central

2011-01-01

Objective The recruitment of leukocytes from circulation to sites of inflammation requires several families of adhesion molecules among which are selectins expressed on a variety of cells. In addition, they have also been shown to play key roles in the activation of cells in inflammation. Methods To explore the collective role of E-, L-, and P- selectins in OVA-induced Th2 mediated response in acute asthma pathophysiology, ELP-/- mice were used and compared with age-matched wildtype (WT). Results Asthma phenotype was assessed by measuring pulmonary function, inflammation and OVA-specific serum IgE, which were completely abrogated in ELP-/- mice. Adoptive transfer of sensitized L selectin+CD4+ T cells into naïve ELP-/- mice which post-OVA challenge, developed asthma, suggesting that L-selectin may be critically involved in the onset of Th2 response in asthma. Tissue resident ELP-deficient cells were otherwise functionally competent as proved by normal proliferative response. Conclusions: Comparative studies between ELP-/- and WT mice uncovered functional roles of these three integrins in inflammatory response in allergic asthma. All three selectins seem to impede inflammatory migration while only L-selectin also possibly regulates activation of specific T cell subsets in lung and airways. PMID:21835035

Markers of Vascular Perturbation Correlate with Airway Structural Change in Asthma

PubMed Central

Kruger, Stanley J.; Schiebler, Mark L.; Evans, Michael D.; Sorkness, Ronald L.; Denlinger, Loren C.; Busse, William W.; Jarjour, Nizar N.; Montgomery, Robert R.; Mosher, Deane F.; Fain, Sean B.

2013-01-01

Rationale: Air trapping and ventilation defects on imaging are characteristics of asthma. Airway wall thickening occurs in asthma and is associated with increased bronchial vascularity and vascular permeability. Vascular endothelial cell products have not been explored as a surrogate to mark structural airway changes in asthma. Objectives: Determine whether reporters of vascular endothelial cell perturbation correlate with airway imaging metrics in patients with asthma of varying severity. Methods: Plasma from Severe Asthma Research Program subjects was analyzed by ELISAs for soluble von Willebrand factor mature protein (VWF:Ag) and propeptide (VWFpp), P-selectin, and platelet factor 4. Additional subjects were analyzed over 48 hours after whole-lung antigen challenge. We calculated ventilation defect volume by hyperpolarized helium-3 magnetic resonance imaging and areas of low signal density by multidetector computed tomography (less than −856 Hounsfield units [HU] at functional residual capacity and −950 HU at total lung capacity [TLC]). Measurements and Main Results: VWFpp and VWFpp/Ag ratio correlated with and predicted greater percentage defect volume on hyperpolarized helium-3 magnetic resonance imaging. P-selectin correlated with and predicted greater area of low density on chest multidetector computed tomography less than −950 HU at TLC. Platelet factor 4 did not correlate. Following whole-lung antigen challenge, variation in VWFpp, VWFpp/Ag, and P-selectin among time-points was less than that among subjects, indicating stability and repeatability of the measurements. Conclusions: Plasma VWFpp and P-selectin may be useful as surrogates of functional and structural defects that are evident on imaging. The results raise important questions about why VWFpp and P-selectin are associated specifically with different imaging abnormalities. PMID:23855693

[Inflammatory cytokines and oxidative stress markers in the inhibition of osteoarthritis by curcumin].

PubMed

Liu, Jun; He, Xiaole; Zhen, Ping; Zhou, Shenghu; Li, Xusheng

2016-05-25

Objective: To observe the influence of matrix metalloproteinases-2 (MMP-2), monocyte chemoattractant protein-1 (MCP-1), CD47, L-selectin and advanced oxidation proteinproducts (AOPP) in osteoarthritis and the intervention of curcumin. Methods: A total of 20 male C57BL/6 mice (10.05-15.00 g) were randomly divided into control group, OA group, Cur25 group and Cur50 group (intraperitoneal injected 25 μmol/L or 50 μmol/L of curcumin everyday after modeling). After 4 weeks treatment, we observed the morphological changes of the gross specimen by immunohistochemical method, and observed the ultrastructure of cartilage tissue under electron microscope. The expression of MMP-2, MCP-1 and CD47 were detected by western blotting, and L-selectin and AOPP were detected by ELISA and spectrophotometer, respectively. Results: In the cartilage tissue morphology, the chondrocytes of OA group showed obvious change, while Cur25 and Cur50 groups maintained the good cartilage cell membrane intact. Compared with control group, the expressions of MMP-2, MCP-1, L-selectin and AOPP in OA group, Cur25 group and Cur50 group were increased (all P <0.05), while CD47 levels were decreased (all P <0.05). Compared with OA group, the expressions of MMP-2, MCP-1, L-selectin and AOPP in Cur25 group and Cur50 group were decreased (all P <0.05), while CD47 levels were increased (all P <0.05), and such changes were more significant in Cur50 group (all P <0.05). Conclusion: The MMP-2, MCP-1, CD47, L-selectin and AOPP are closely associated with the pathology course of OA. Curcumin has protection effect on cartilage, which can relieve joint cartilage degeneration, reduce cartilage inflammation and increase the metabolic activity of chondrocytes.

Surfactant functionalization induces robust, differential adhesion of tumor cells and blood cells to charged nanotube-coated biomaterials under flow.

PubMed

Mitchell, Michael J; Castellanos, Carlos A; King, Michael R

2015-07-01

The metastatic spread of cancer cells from the primary tumor to distant sites leads to a poor prognosis in cancers originating from multiple organs. Increasing evidence has linked selectin-based adhesion between circulating tumor cells (CTCs) and endothelial cells of the microvasculature to metastatic dissemination, in a manner similar to leukocyte adhesion during inflammation. Functionalized biomaterial surfaces hold promise as a diagnostic tool to separate CTCs and potentially treat metastasis, utilizing antibody and selectin-mediated interactions for cell capture under flow. However, capture at high purity levels is challenged by the fact that CTCs and leukocytes both possess selectin ligands. Here, a straightforward technique to functionalize and alter the charge of naturally occurring halloysite nanotubes using surfactants is reported to induce robust, differential adhesion of tumor cells and blood cells to nanotube-coated surfaces under flow. Negatively charged sodium dodecanoate-functionalized nanotubes simultaneously enhanced tumor cell capture while negating leukocyte adhesion, both in the presence and absence of adhesion proteins, and can be utilized to isolate circulating tumor cells regardless of biomarker expression. Conversely, diminishing nanotube charge via functionalization with decyltrimethylammonium bromide both abolished tumor cell capture while promoting leukocyte adhesion. Copyright © 2015 Elsevier Ltd. All rights reserved.

Platelet Glycoprotein lb-1X and Malignancy

DTIC Science & Technology

2010-09-01

supporting the accumulation of more platelets and the elaboration of a fibrin - rich network produced by coagulation factors. This paradigm has been...a platelet - rich thrombus by tethering the platelet to a thrombogenic surface. Several ligands binding to GP Ib-IX have been identified, including...08-1-0576 TITLE: Platelet Glycoprotein lb-1X and Malignancy PRINCIPAL INVESTIGATOR: Dr. Jerry Ware

Analysis of correlations between selected endothelial cell activation markers, disease activity, and nailfold capillaroscopy microvascular changes in systemic lupus erythematosus patients.

PubMed

Ciołkiewicz, Mariusz; Kuryliszyn-Moskal, Anna; Klimiuk, Piotr Adrian

2010-02-01

The aim of the study was to evaluate the correlation between selected serum endothelial cell activation markers such as vascular endothelial growth factor (VEGF), endothelin-1 (ET-1), soluble thrombomodulin (sTM), soluble E-selectin (sE-selectin), disease activity, and microvascular changes determined by nailfold capillaroscopy in patients with systemic lupus erythematosus (SLE). Serum levels of VEGF, ET-1, sTM, and sE-selectin were determined by an enzyme-linked immunosorbent assay in 80 SLE patients. The disease activity was measured with Systemic Lupus Erythematosus Disease Activity Index score. Nailfold capillaroscopy was performed in all patients. Positive correlation was found between VEGF and both ET-1 (r = 0.294, p < 0.01) and sE-selectin (r = 0.274, p < 0.05) serum levels as well as between sTM and ET-1 (r = 0.273, p < 0.05) serum concentrations. We noticed also positive correlation between VEGF (r = 0.224, p < 0.05) and ET-1 (r = 0.471, p < 0.001) serum levels and disease activity, and also between VEGF serum concentration and grade of morphological changes observed by nailfold capillaroscopy (r = 0.458, p < 0.001). There was also positive correlation between capillaroscopic score and disease activity (r = 0.339, p < 0.01). Our data suggest that correlation between VEGF and both ET-1 and E-selectin serum levels as well as between sTM and ET-1 serum concentrations may reflect their participation in the pathogenesis of SLE. VEGF seems to reflect changes in microcirculation in the course of SLE, visualised by nailfold capillaroscopy. The relationship between changes in nailfold capillaroscopy, endothelial cell activation markers, and the clinical activity of SLE points to an important role of microvascular abnormalities in the clinical manifestation of the disease.

An anti-von Willebrand factor aptamer reduces platelet adhesion among patients receiving aspirin and clopidogrel in an ex vivo shear-induced arterial thrombosis.

PubMed

Arzamendi, Dabit; Dandachli, Firas; Théorêt, Jean-François; Ducrocq, Gregory; Chan, Mark; Mourad, Walid; Gilbert, James C; Schaub, Robert G; Tanguay, Jean-François; Merhi, Yahye

2011-01-01

The von Willebrand factor (vWF) aptamer, ARC1779 that blocks the binding of vWF A1-domain to platelet glycoprotein 1b (GPIb) at high shear, may deliver a site-specific antithrombotic effect. We investigated the efficiency of ARC1779 on platelet function in patients with coronary artery disease (CAD) on double antiplatelet therapy. Blood from patients taking aspirin and clopidogrel and from normal volunteers was treated ex vivo with ARC1779 or abciximab, either prior to perfusion (pretherapy) or 10 minutes following the initiation of perfusion (posttherapy) on damaged arteries. Under pre- but not posttherapy, platelet adhesion was significantly reduced by ARC1779 at 83 and 250 nmol/L and by abciximab (100 nmol/L) versus placebo (4.8, 3.8, and 2.9 vs 7.3 platelets × 10(6)/cm(2), P < .05). In contrast to abciximab, ARC1779 did not significantly affect platelet aggregation, P-selectin expression, and platelet-leukocyte binding. These proof-of-concept data may constitute the framework for randomized clinical investigations of this novel antiplatelet therapy among patients with CAD.

The Galectin CvGal1 from the Eastern Oyster (Crassostrea virginica) Binds to Blood Group A Oligosaccharides on the Hemocyte Surface*

PubMed Central

Feng, Chiguang; Ghosh, Anita; Amin, Mohammed N.; Giomarelli, Barbara; Shridhar, Surekha; Banerjee, Aditi; Fernández-Robledo, José A.; Bianchet, Mario A.; Wang, Lai-Xi; Wilson, Iain B. H.; Vasta, Gerardo R.

2013-01-01

The galectin CvGal1 from the eastern oyster (Crassostrea virginica), which possesses four tandemly arrayed carbohydrate recognition domains, was previously shown to display stronger binding to galactosamine and N-acetylgalactosamine relative to d-galactose. CvGal1 expressed by phagocytic cells is “hijacked” by the parasite Perkinsus marinus to enter the host, where it proliferates and causes systemic infection and death. In this study, a detailed glycan array analysis revealed that CvGal1 preferentially recognizes type 2 blood group A oligosaccharides. Homology modeling of the protein and its oligosaccharide ligands supported this preference over type 1 blood group A and B oligosaccharides. The CvGal ligand models were further validated by binding, inhibition, and competitive binding studies of CvGal1 and ABH-specific monoclonal antibodies with intact and deglycosylated glycoproteins, hemocyte extracts, and intact hemocytes and by surface plasmon resonance analysis. A parallel glycomic study carried out on oyster hemocytes (Kurz, S., Jin, C., Hykollari, A., Gregorich, D., Giomarelli, B., Vasta, G. R., Wilson, I. B. H., and Paschinger, K. (2013) J. Biol. Chem. 288,) determined the structures of oligosaccharides recognized by CvGal1. Proteomic analysis of the hemocyte glycoproteins identified β-integrin and dominin as CvGal1 “self”-ligands. Despite strong CvGal1 binding to P. marinus trophozoites, no binding of ABH blood group antibodies was observed. Thus, parasite glycans structurally distinct from the blood group A oligosaccharides on the hemocyte surface may function as potentially effective ligands for CvGal1. We hypothesize that carbohydrate-based mimicry resulting from the host/parasite co-evolution facilitates CvGal1-mediated cross-linking to β-integrin, located on the hemocyte surface, leading to cell activation, phagocytosis, and host infection. PMID:23824193

Architecture effects on multivalent interactions by polypeptide-based multivalent ligands

NASA Astrophysics Data System (ADS)

Liu, Shuang

Multivalent interactions are characterized by the simultaneous binding between multiple ligands and multiple binding sites, either in solutions or at interfaces. In biological systems, most multivalent interactions occur between protein receptors and carbohydrate ligands through hydrogen-bonding and hydrophobic interactions. Compared with weak affinity binding between one ligand and one binding site, i.e. monovalent interaction, multivalent interactioins provide greater avidity and specificity, and therefore play unique roles in a broad range of biological activities. Moreover, the studies of multivalent interactions are also essential for producing effective inhibitors and effectors of biological processes that could have important therapeutic applications. Synthetic multivalent ligands have been designed to mimic the biological functions of natural multivalent interactions, and various types of scaffolds have been used to display multiple ligands, including small molecules, linear polymers, dendrimers, nanoparticle surfaces, monolayer surfaces and liposomes. Studies have shown that multivalent interactions can be highly affected by various architectural parameters of these multivalent ligands, including ligand identities, valencies, spacing, ligand densities, nature of linker arms, scaffold length and scaffold conformation. Most of these multivalent ligands are chemically synthesized and have limitations of controlling over sequence and conformation, which is a barrier for mimicking ordered and controlled natural biological systems. Therefore, multivalent ligands with precisely controlled architecture are required for improved structure-function relationship studies. Protein engineering methods with subsequent chemical coupling of ligands provide significant advantages of controlling over backbone conformation and functional group placement, and therefore have been used to synthesize recombinant protein-based materials with desired properties similar to natural protein materials, including structural as well as functional proteins. Therefore, polypeptide-based multivalent scaffolds are used to display ligands to assess the contribution of different architectural parameters to the multivalent binding events. In this work, a family of alanine-rich alpha-helical glycopolypeptides was designed and synthesized by a combination of protein engineering and chemical coupling, to display two types of saccharide ligands for two different multivalent binding systems. The valencies, chain length and spacing between adjacent ligands of these multivalent ligands were designed in order to study architecture effects on multivalent interactions. The polypeptides and their glycoconjugates were characterized via various methods, including SDS-PAGE, NMR, HPLC, amino acid analysis (AAA), MALDI, circular dichroism (CD) and GPC. In the first multivalent binding system, cholera toxin B pentamer (CT B5) was chosen to be the protein receptor due to its well-characterized structure, lack of significant steric interference of binding to multiple binding sites, and requirement of only simple monosaccharide as ligands. Galactopyranoside was incorporated into polypeptide scaffolds through amine-carboxylic acid coupling to the side chains of glutamic acid residues. The inhibition and binding to CT B5 of these glycopolypeptide ligands were evaluated by direct enzyme-linked assay (DELA). As a complement method, weak affinity chromatography (WAC) was also used to evaluate glycopolypeptides binding to a CT B5 immobilized column. The architecture effects on CT B 5 inhibition are discussed. In the second system, cell surface receptor L-selectin was targeted by polypeptide-based multivalent ligands containing disulfated galactopyranoside ligands, due to its important roles in various immunological activities. The effects of glycopolypeptide architectural variables L-selectin shedding were evaluated via ELISA-based assays. These polypeptide-based multivalent ligands are suggested to be useful for elucidating architecture effects on multivalent interactions, manipulating multivalent interactions and the subsequent cellular responses in different systems. These materials have great potential applications in therapeutics and could also provide guidelines for design of multivalent ligands for other protein receptors.

Failure of platelet parameters and biomarkers to correlate platelet function to severity and etiology of heart failure in patients enrolled in the EPCOT trial. With special reference to the Hemodyne hemostatic analyzer. Whole Blood Impedance Aggregometry for the Assessment of Platelet Function in Patients with Congestive Heart Failure.

PubMed

Serebruany, Victor L; McKenzie, Marcus E; Meister, Andrew F; Fuzaylov, Sergey Y; Gurbel, Paul A; Atar, Dan; Gattis, Wendy A; O'Connor, Christopher M

2002-01-01

Data from small studies have suggested the presence of platelet abnormalities in patients with congestive heart failure (CHF). We sought to characterize the diagnostic utility of different platelet parameters and platelet-endothelial biomarkers in a random outpatient CHF population investigated in the EPCOT ('Whole Blood Impedance Aggregometry for the Assessment of Platelet Function in Patients with Congestive Heart Failure') Trial. Blood samples were obtained for measurement of platelet contractile force (PCF), whole blood aggregation, shear-induced closure time, expression of glycoprotein (GP) IIb/IIIa, and P-selectin in 100 consecutive patients with CHF. Substantial interindividual variability of platelet characteristics exists in patients with CHF. There were no statistically significant differences when patients were grouped according to incidence of vascular events, emergency revascularization needs, survival, or etiology of heart failure. Aspirin use did not affect instrument readings either. PCF correlates very poorly with whole blood aggregometry (r(2) = 0.023), closure time (r(2) = 0.028), platelet GP IIb/IIIa (r(2) = 0.0028), and P-selectin (r(2) = 0.002) expression. Furthermore, there was no correlation with brain natriuretic peptide concentrations, a marker of severity and prognosis in heart failure reflecting the neurohumoral status. Patients with heart failure enrolled in the EPCOT Trial exhibited a marginal, sometimes oppositely directed change in platelet function, challenging the diagnostic utility of these platelet parameters and biomarkers to serve as useful tools for the identification of platelet abnormalities, for predicting clinical outcomes, or for monitoring antiplatelet strategies in this population. The usefulness of these measurements for assessing platelets in the different clinical settings remains to be explored. Taken together, opposite to our expectations, major clinical characteristics of heart failure did not correlate well with the platelet characteristics investigated in this study. Copyright 2002 S. Karger AG, Basel

Sialylated multivalent antigens engage CD22 in trans and inhibit B cell activation.

PubMed

Courtney, Adam H; Puffer, Erik B; Pontrello, Jason K; Yang, Zhi-Qiang; Kiessling, Laura L

2009-02-24

CD22 is an inhibitory coreceptor on the surface of B cells that attenuates B cell antigen receptor (BCR) signaling and, therefore, B cell activation. Elucidating the molecular mechanisms underlying the inhibitory activity of CD22 is complicated by the ubiquity of CD22 ligands. Although antigens can display CD22 ligands, the receptor is known to bind to sialylated glycoproteins on the cell surface. The propinquity of CD22 and cell-surface glycoprotein ligands has led to the conclusion that the inhibitory properties of the receptor are due to cis interactions. Here, we examine the functional consequences of trans interactions by employing sialylated multivalent antigens that can engage both CD22 and the BCR. Exposure of B cells to sialylated antigens results in the inhibition of key steps in BCR signaling. These results reveal that antigens bearing CD22 ligands are powerful suppressors of B cell activation. The ability of sialylated antigens to inhibit BCR signaling through trans CD22 interactions reveals a previously unrecognized role for the Siglec-family of receptors as modulators of immune signaling.

ARQ 092, an orally-available, selective AKT inhibitor, attenuates neutrophil-platelet interactions in sickle cell disease

PubMed Central

Kim, Kyungho; Li, Jing; Barazia, Andrew; Tseng, Alan; Youn, Seock-Won; Abbadessa, Giovanni; Yu, Yi; Schwartz, Brian; Andrews, Robert K.; Gordeuk, Victor R.; Cho, Jaehyung

2017-01-01

Previous studies identified the Ser/Thr protein kinase, AKT, as a therapeutic target in thrombo-inflammatory diseases. Here we report that specific inhibition of AKT with ARQ 092, an orally-available AKT inhibitor currently in phase Ib clinical trials as an anti-cancer drug, attenuates the adhesive function of neutrophils and platelets from sickle cell disease patients in vitro and cell-cell interactions in a mouse model of sickle cell disease. Studies using neutrophils and platelets isolated from sickle cell disease patients revealed that treatment with 50–500 nM ARQ 092 significantly blocks αMβ2 integrin function in neutrophils and reduces P-selectin exposure and glycoprotein Ib/IX/V-mediated agglutination in platelets. Treatment of isolated platelets and neutrophils with ARQ 092 inhibited heterotypic cell-cell aggregation under shear conditions. Intravital microscopic studies demonstrated that short-term oral administration of ARQ 092 or hydroxyurea, a major therapy for sickle cell disease, diminishes heterotypic cell-cell interactions in venules of sickle cell disease mice challenged with tumor necrosis factor-α. Co-administration of hydroxyurea and ARQ 092 further reduced the adhesive function of neutrophils in venules and neutrophil transmigration into alveoli, inhibited expression of E-selectin and intercellular adhesion molecule-1 in cremaster vessels, and improved survival in these mice. Ex vivo studies in sickle cell disease mice suggested that co-administration of hydroxyurea and ARQ 092 efficiently blocks neutrophil and platelet activation and that the beneficial effect of hydroxyurea results from nitric oxide production. Our results provide important evidence that ARQ 092 could be a novel drug for the prevention and treatment of acute vaso-occlusive complications in patients with sickle cell disease. PMID:27758820

E-selectin ligand 1 regulates bone remodeling by limiting bioactive TGF-β in the bone microenvironment

PubMed Central

Yang, Tao; Grafe, Ingo; Bae, Yangjin; Chen, Shan; Chen, Yuqing; Bertin, Terry K.; Jiang, Ming-Ming; Ambrose, Catherine G.; Lee, Brendan

2013-01-01

TGF-β is abundantly produced in the skeletal system and plays a crucial role in skeletal homeostasis. E-selectin ligand-1 (ESL-1), a Golgi apparatus-localized protein, acts as a negative regulator of TGF-β bioavailability by attenuating maturation of pro–TGF-β during cartilage homeostasis. However, whether regulation of intracellular TGF-β maturation by ESL-1 is also crucial during bone homeostasis has not been well defined. Here, we show that Esl-1−/− mice exhibit a severe osteopenia with elevated bone resorption and decreased bone mineralization. In primary culture, Esl-1−/− osteoclast progenitors show no difference in osteoclastogenesis. However, Esl-1−/− osteoblasts show delayed differentiation and mineralization and stimulate osteoclastogenesis more potently in the osteoblast–osteoclast coculture, suggesting that ESL-1 primarily acts in osteoblasts to regulate bone homeostasis. In addition, Esl-1−/− calvaria exhibit an elevated mature TGF-β/pro–TGF-β ratio, with increased expression of TGF-β downstream targets (plasminogen activator inhibitor-1, parathyroid hormone-related peptide, connective tissue growth factor, and matrix metallopeptidase 13, etc.) and a key regulator of osteoclastogenesis (receptor activator of nuclear factor κB ligand). Moreover, in vivo treatment with 1D11, a pan–TGF-β antibody, significantly improved the low bone mass of Esl-1−/− mice, suggesting that elevated TGF-β signaling is the major cause of osteopenia in Esl-1−/− mice. In summary, our study identifies ESL-1 as an important regulator of bone remodeling and demonstrates that the modulation of TGF-β maturation is pivotal in the maintenance of a homeostatic bone microenvironment and for proper osteoblast–osteoclast coupling. PMID:23589896

Temperature and pH Dual-Responsive Core-Brush Nanocomposite for Enrichment of Glycoproteins.

PubMed

Jiang, Lingdong; Messing, Maria E; Ye, Lei

2017-03-15

In this report, we present a novel modular approach to the immobilization of a high density of boronic acid ligands on thermoresponsive block copolymer brushes for effective enrichment of glycoproteins via their synergistic multiple covalent binding with the immobilized boronic acids. Specifically, a two-step, consecutive surface-initiated atom transfer radical polymerization (SI-ATRP) was employed to graft a flexible block copolymer brush, pNIPAm-b-pGMA, from an initiator-functionalized nanosilica surface, followed by postpolymerization modification of the pGMA moiety with sodium azide. Subsequently, an alkyne-tagged boronic acid (PCAPBA) was conjugated to the polymer brush via a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction, leading to a silica-supported polymeric hybrid material, Si@pNIPAm-b-pBA, with a potent glycol binding affinity. The obtained core-brush nanocomposite was systematically characterized with regard to particle size, morphology, organic content, brush density, and number of immobilized boronic acids. We also studied the characteristics of glycoprotein binding of the nanocomposite under different conditions. The nanocomposite showed high binding capacities for ovalbumin (OVA) (98.0 mg g -1 ) and horseradish peroxidase (HRP) (26.8 mg g -1 ) in a basic buffer (pH 9.0) at 20 °C. More importantly, by adjusting the pH and temperature, the binding capacities of the nanocomposite can be tuned, which is meaningful for the separation of biological molecules. In general, the synthetic approach developed for the fabrication of block copolymer brushes in the nanocomposite opened new opportunities for the design of more functional hybrid materials that will be useful in bioseparation and biomedical applications.

Platelet aggregation but not activation and degranulation during the acute post-ischemic reperfusion phase in livers with no underlying disease

PubMed Central

van Golen, Rowan F.; Stevens, Katarzyna M.; Colarusso, Pina; Jaeschke, Hartmut; Heger, Michal

2016-01-01

Background Platelets and P-selectin (CD62P) play an unequivocal role in the pathology of hepatic ischemia/reperfusion (I/R) injury. Inhibition or knock-out of P-selectin or immunodepletion of platelets results in amelioration of post-ischemic inflammation, reduced hepatocellular damage, and improved survival. However, P-selectin expression on platelets and endothelial cells, which concurs with platelet activation, has never been clearly demonstrated in I/R-subjected livers. Aims To determine whether platelets become activated and degranulate in the acute phase of liver I/R and whether the platelets interact with neutrophils. Methods Hepatic I/R was induced in male C57BL/6J mice (N = 12) using 37.5-min ischemia time. Platelets, endothelial cells, and neutrophils were fluorescently labeled by systemic administration of non-blocking antibodies. Cell kinetics were monitored by intravital spinning disk confocal microscopy during 90 min of reperfusion. Image analysis and quantification was performed with dedicated software. Results Platelets adhered to sinusoids more extensively in post-ischemic livers compared to livers not subjected to I/R and formed aggregates, which occurred directly after ischemia. Platelets and endothelial cells did not express P-selectin in post-ischemic livers. There was no interaction between platelets and neutrophils. Conclusions Platelets aggregate but do not become activated and do not degranulate in post-ischemic livers. There is no platelet-neutrophil interplay during the early reperfusion phase in a moderate model of hepatic I/R injury. The mechanisms underlying the biological effects of platelets and P-selectin in this setting warrant further investigation. Relevance for patients I/R in surgical liver patients may compromise outcome due to post-ischemic oxidative stress and sterile inflammation. Both processes are mediated in part by platelets. Understanding platelet function during I/R is key to developing effective interventions for I/R injury and improving clinical outcomes. PMID:26925465

Biocompatibility Assessment of a Long-Term Wearable Artificial Pump-Lung in Sheep

PubMed Central

Zhou, Kang; Niu, Shuqiong; Bianchi, Giacomo; Wei, Xufeng; Garimella, Narayana; Griffith, Bartley P; Wu, Zhongjun J

2013-01-01

The purpose of this study was to assess the biocompatibility of a newly developed long-term wearable artificial pump-lung (APL) in a clinically relevant ovine animal mode. The wearable APL device was implanted in five sheep through a left thoracotomy. The device was connected between the right atrium (RA) and pulmonary artery (PA) and evaluated for 30 days. Three sheep were used as the sham control. Platelet activation was assessed by measuring platelet surface P-selectin (CD62P) expression with flow cytometry and plasma soluble P-selectin with an enzyme-linked immunosorbent assay (ELISA). Thrombotic deposition on the device components and hollow fiber membranes (HFM) were analyzed with digital imaging and scanning electron microscopy (SEM). Surface P-selectin of the APL and sham groups changed significantly over the study period, but without significant differences between the two groups. Soluble P-selectin for the two groups peaked in the first 24 hours after the surgery. Soluble P-selectin of the APL group remained slightly elevated over the study period compared to the pre-surgical baseline value and was slightly higher compared to that of the sham group. Plasma free hemoglobin (PFH) remained in the normal ranges in all the animals. In spite of the surgery related alteration in laboratory tests and elevation of platelet activation status, the APL devices in all the animals functioned normally (oxygen transfer and blood pumping) during the 30 day study period. The device flow path and membrane surface were free of gross thrombus. Electron microscopy images showed only scattered thrombi on the fibers (membrane surface and weft). In summary, the APL exhibited excellent biocompatibility. Two forms of platelet activation, surgery related and device induced, in the animals implanted with the wearable APL were observed. The limited device-induced platelet activation did not cause gross thrombosis and impair the long-term device performance. PMID:23452221

Cytoskeletal regulation of CD44 membrane organization and interactions with E-selectin.

PubMed

Wang, Ying; Yago, Tadayuki; Zhang, Nan; Abdisalaam, Salim; Alexandrakis, George; Rodgers, William; McEver, Rodger P

2014-12-19

Interactions of CD44 on neutrophils with E-selectin on activated endothelial cells mediate rolling under flow, a prerequisite for neutrophil arrest and migration into perivascular tissues. How CD44 functions as a rolling ligand despite its weak affinity for E-selectin is unknown. We examined the nanometer scale organization of CD44 on intact cells. CD44 on leukocytes and transfected K562 cells was cross-linked within a 1.14-nm spacer. Depolymerizing actin with latrunculin B reduced cross-linking. Fluorescence resonance energy transfer (FRET) revealed tight co-clustering between CD44 fused to yellow fluorescent protein (YFP) and CD44 fused to cyan fluorescent protein on K562 cells. Latrunculin B reduced FRET-reported co-clustering. Number and brightness analysis confirmed actin-dependent CD44-YFP clusters on living cells. CD44 lacking binding sites for ankyrin and for ezrin/radixin/moesin (ERM) proteins on its cytoplasmic domain (ΔANKΔERM) did not cluster. Unexpectedly, CD44 lacking only the ankyrin-binding site (ΔANK) formed larger but looser clusters. Fluorescence recovery after photobleaching demonstrated increased CD44 mobility by latrunculin B treatment or by deleting the cytoplasmic domain. ΔANKΔERM mobility increased only modestly, suggesting that the cytoplasmic domain engages the cytoskeleton by an additional mechanism. Ex vivo differentiated CD44-deficient neutrophils expressing exogenous CD44 rolled on E-selectin and activated Src kinases after binding anti-CD44 antibody. In contrast, differentiated neutrophils expressing ΔANK had impaired rolling and kinase activation. These data demonstrate that spectrin and actin networks regulate CD44 clustering and suggest that ankyrin enhances CD44-mediated neutrophil rolling and signaling. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Cytoskeletal Regulation of CD44 Membrane Organization and Interactions with E-selectin*

PubMed Central

Wang, Ying; Yago, Tadayuki; Zhang, Nan; Abdisalaam, Salim; Alexandrakis, George; Rodgers, William; McEver, Rodger P.

2014-01-01

Interactions of CD44 on neutrophils with E-selectin on activated endothelial cells mediate rolling under flow, a prerequisite for neutrophil arrest and migration into perivascular tissues. How CD44 functions as a rolling ligand despite its weak affinity for E-selectin is unknown. We examined the nanometer scale organization of CD44 on intact cells. CD44 on leukocytes and transfected K562 cells was cross-linked within a 1.14-nm spacer. Depolymerizing actin with latrunculin B reduced cross-linking. Fluorescence resonance energy transfer (FRET) revealed tight co-clustering between CD44 fused to yellow fluorescent protein (YFP) and CD44 fused to cyan fluorescent protein on K562 cells. Latrunculin B reduced FRET-reported co-clustering. Number and brightness analysis confirmed actin-dependent CD44-YFP clusters on living cells. CD44 lacking binding sites for ankyrin and for ezrin/radixin/moesin (ERM) proteins on its cytoplasmic domain (ΔANKΔERM) did not cluster. Unexpectedly, CD44 lacking only the ankyrin-binding site (ΔANK) formed larger but looser clusters. Fluorescence recovery after photobleaching demonstrated increased CD44 mobility by latrunculin B treatment or by deleting the cytoplasmic domain. ΔANKΔERM mobility increased only modestly, suggesting that the cytoplasmic domain engages the cytoskeleton by an additional mechanism. Ex vivo differentiated CD44-deficient neutrophils expressing exogenous CD44 rolled on E-selectin and activated Src kinases after binding anti-CD44 antibody. In contrast, differentiated neutrophils expressing ΔANK had impaired rolling and kinase activation. These data demonstrate that spectrin and actin networks regulate CD44 clustering and suggest that ankyrin enhances CD44-mediated neutrophil rolling and signaling. PMID:25359776

Global analysis of glycoproteins identifies markers of endotoxin tolerant monocytes and GPR84 as a modulator of TNFα expression.

PubMed

Müller, Mario M; Lehmann, Roland; Klassert, Tilman E; Reifenstein, Stella; Conrad, Theresia; Moore, Christoph; Kuhn, Anna; Behnert, Andrea; Guthke, Reinhard; Driesch, Dominik; Slevogt, Hortense

2017-04-12

Exposure of human monocytes to lipopolysaccharide (LPS) induces a temporary insensitivity to subsequent LPS challenges, a cellular state called endotoxin tolerance. In this study, we investigated the LPS-induced global glycoprotein expression changes of tolerant human monocytes and THP-1 cells to identify markers and glycoprotein targets capable to modulate the immunosuppressive state. Using hydrazide chemistry and LC-MS/MS analysis, we analyzed glycoprotein expression changes during a 48 h LPS time course. The cellular snapshots at different time points identified 1491 glycoproteins expressed by monocytes and THP-1 cells. Label-free quantitative analysis revealed transient or long-lasting LPS-induced expression changes of secreted or membrane-anchored glycoproteins derived from intracellular membrane coated organelles or from the plasma membrane. Monocytes and THP-1 cells demonstrated marked differences in glycoproteins differentially expressed in the tolerant state. Among the shared differentially expressed glycoproteins G protein-coupled receptor 84 (GPR84) was identified as being capable of modulating pro-inflammatory TNFα mRNA expression in the tolerant cell state when activated with its ligand Decanoic acid.

Combined QSAR and molecule docking studies on predicting P-glycoprotein inhibitors

NASA Astrophysics Data System (ADS)

Tan, Wen; Mei, Hu; Chao, Li; Liu, Tengfei; Pan, Xianchao; Shu, Mao; Yang, Li

2013-12-01

P-glycoprotein (P-gp) is an ATP-binding cassette multidrug transporter. The over expression of P-gp leads to the development of multidrug resistance (MDR), which is a major obstacle to effective treatment of cancer. Thus, designing effective P-gp inhibitors has an extremely important role in the overcoming MDR. In this paper, both ligand-based quantitative structure-activity relationship (QSAR) and receptor-based molecular docking are used to predict P-gp inhibitors. The results show that each method achieves good prediction performance. According to the results of tenfold cross-validation, an optimal linear SVM model with only three descriptors is established on 857 training samples, of which the overall accuracy (Acc), sensitivity, specificity, and Matthews correlation coefficient are 0.840, 0.873, 0.813, and 0.683, respectively. The SVM model is further validated by 418 test samples with the overall Acc of 0.868. Based on a homology model of human P-gp established, Surflex-dock is also performed to give binding free energy-based evaluations with the overall accuracies of 0.823 for the test set. Furthermore, a consensus evaluation is also performed by using these two methods. Both QSAR and molecular docking studies indicate that molecular volume, hydrophobicity and aromaticity are three dominant factors influencing the inhibitory activities.

Platelet indexes in relation to target organ damage in high-risk hypertensive patients: a substudy of the Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT).

PubMed

Nadar, Sunil K; Blann, Andrew D; Kamath, Sridhar; Beevers, D Gareth; Lip, Gregory Y H

2004-07-21

We sought to investigate the relationship between target organ damage (TOD) in hypertension and a prothrombotic/hypercoagulable state, using a new technique of "platelet lysis" to quantify the amount of P-selectin per platelet (pP-sel), and to correlate it with other platelet markers (e.g., mass, volume and granularity, soluble P-selectin [sP-sel], and beta-thromboglobulin [beta-TG]). The increased risk of TOD in hypertension may be related to a prothrombotic/hypercoagulable state, with abnormalities in platelets, such as increased expression of P-selectin. We studied 199 patients (mean age 68 years, 75% men) with hypertension. Of these, 125 had TOD (e.g., stroke, previous myocardial infarction, angina, left ventricular hypertrophy). Values obtained were compared with those from 59 healthy normotensive control subjects (mean age 68 years, 58% men). Hypertensive patients had a higher mean platelet volume, mass, pP-sel, sP-sel, and beta-TG and lower platelet granularity (all p < 0.01), but a similar platelet count, as compared with controls. Within the hypertensive group, those with evidence of TOD had significantly larger platelets with greater mass but had lower granularity, sP-sel, and pP-sel levels than those without TOD, possibly reflecting increased aspirin use. On multivariate analysis, aspirin use was a determinant of pP-sel (p = 0.03) and sP-sel (p = 0.01), but the use of other drugs or other co-morbidity (e.g., diabetes, smoking) did not influence either P-selectin value. Patients with hypertension have evidence of changes in platelet physiology, as reflected by a higher level of pP-sel. Patients with TOD also had larger platelets, with greater mass, and the use of aspirin lowered pP-sel and sP-sel levels. These changes may have implications for the pathophysiology of cardiovascular and cerebrovascular disease in hypertension.

[Effect of acidic oligosaccharides on P-selectin of pulmonary hypertensive rats induced by monocrotaline].

PubMed

Feng, Z; Hu, Y; An, N N; Feng, W J; Hu, T; Mao, Y J

2018-02-12

Objective: To observe the effects of acidic oligosaccharides (AOS) on P-selectin levels in the serum and the pulmonary arteries of pulmonary hypertensive rats induced by monocrotaline. Methods: Sixty healthy adult male Sprague-Dawley rats were randomly divided into control group ( n =10), model group ( n =10), Alprostadil group ( n =10), low-dose AOS group (AOS-L, n =10), medium-dose AOS group (AOS-M, n =10) and high-dose AOS group (AOS-H, n =10). The rat model of pulmonary arterial hypertension was made by a single intraperitoneal injection of monocrotaline(60 mg/kg). Five weeks after injection, pulmonary arterial (PA) acceleration time (PAT) and ejection time (ET) were measured by color Doppler ultrasound. Then, the Alprostadil group was treated by Alprostadil 5 μg·kg(-1)·d(-1)intraperitoneally. Acidic oligosaccharides was administered by intraperitoneal injection to rats in the AOS-L group(5 kg(-1)·d(-1)), AOS-M group (10 mg·kg(-1)·d(-1))and AOS-H group (20 mg·kg(-1)·d(-1)). Control group and model group were given normal saline instead. At the end of experiments, the death rate was recorded and PAT/ET was measured. We calculated the right ventricular hypertrophy index (RVHI) of all rats sacrificed under anesthesia. Precision-cut lung slices were stained with HE for observation of the structure of middle and small arteries. The expression level of P-selectin in serum and pulmonary arterial tissues were detected by ELISA and Western blot respectively. Results: AOS significantly increased the level of PAT/ET ( P <0.01), and attenuated RVHI ( P <0.01). AOS significantly improved intima-media proliferation in small-to medium-sized pulmonary arteries, and attenuated perivascular inflammation. AOS and Alprostadil significantly down-regulated the protein expression of P-selectin in serum and pulmonary arteries ( P <0.01). Conclusion: In this rat model of monocrotaline-induced pulmonary hypertension, AOS decreased the expressions of P-selectin in serum and pulmonary arteries in a dose-dependent manner.

Continuous high throughput molecular adhesion based cell sorting using ridged microchannels

NASA Astrophysics Data System (ADS)

Tasadduq, Bushra; Wang, Gonghao; Alexeev, Alexander; Sarioglu, Ali Fatih; Sulchek, Todd

2016-11-01

Cell molecular interactions govern important physiological processes such as stem cell homing, inflammation and cancer metastasis. But due to a lack of effective separation technologies selective to these interactions it is challenging to specifically sort cells. Other label free separation techniques based on size, stiffness and shape do not provide enough specificity to cell type, and correlation to clinical condition. We propose a novel microfluidic device capable of high throughput molecule dependent separation of cells by flowing them through a microchannel decorated with molecule specific coated ridges. The unique aspect of this sorting design is the use of optimized gap size which is small enough to lightly squeeze the cells while flowing under the ridged part of the channel to increase the surface area for interaction between the ligand on cell surface and coated receptor molecule but large enough so that biomechanical markers, stiffness and viscoelasticity, do not dominate the cell separation mechanism. We are able to separate Jurkat cells based on its expression of PSGL-1ligand using ridged channel coated with P selectin at a flow rate of 0.045ml/min and achieve 2-fold and 5-fold enrichment of PSGL-1 positive and negative Jurkat cells respectively.

Haematopoietic ESL-1 enables stem cell proliferation in the bone marrow by limiting TGFβ availability.

PubMed

Leiva, Magdalena; Quintana, Juan A; Ligos, José M; Hidalgo, Andrés

2016-01-08

The life-long maintenance of haematopoietic stem and progenitor cells (HSPCs) critically relies on environmental signals produced by cells that constitute the haematopoietic niche. Here we report a cell-intrinsic mechanism whereby haematopoietic cells limit proliferation within the bone marrow, and show that this pathway is repressed by E-selectin ligand 1 (ESL-1). Mice deficient in ESL-1 display aberrant HSPC quiescence, expansion of the immature pool and reduction in niche size. Remarkably, the traits were transplantable and dominant when mutant and wild-type precursors coexisted in the same environment, but were independent of E-selectin, the vascular receptor for ESL-1. Instead, quiescence is generated by unrestrained production of the cytokine TGFβ by mutant HSPC, and in vivo or in vitro blockade of the cytokine completely restores the homeostatic properties of the haematopoietic niche. These findings reveal that haematopoietic cells, including the more primitive compartment, can actively shape their own environment.

Haematopoietic ESL-1 enables stem cell proliferation in the bone marrow by limiting TGFβ availability

PubMed Central

Leiva, Magdalena; Quintana, Juan A.; Ligos, José M.; Hidalgo, Andrés

2016-01-01

The life-long maintenance of haematopoietic stem and progenitor cells (HSPCs) critically relies on environmental signals produced by cells that constitute the haematopoietic niche. Here we report a cell-intrinsic mechanism whereby haematopoietic cells limit proliferation within the bone marrow, and show that this pathway is repressed by E-selectin ligand 1 (ESL-1). Mice deficient in ESL-1 display aberrant HSPC quiescence, expansion of the immature pool and reduction in niche size. Remarkably, the traits were transplantable and dominant when mutant and wild-type precursors coexisted in the same environment, but were independent of E-selectin, the vascular receptor for ESL-1. Instead, quiescence is generated by unrestrained production of the cytokine TGFβ by mutant HSPC, and in vivo or in vitro blockade of the cytokine completely restores the homeostatic properties of the haematopoietic niche. These findings reveal that haematopoietic cells, including the more primitive compartment, can actively shape their own environment. PMID:26742601

CD44 fucosylation on mesenchymal stem cell enhances homing and macrophage polarization in ischemic kidney injury.

PubMed

Chou, Kang-Ju; Lee, Po-Tsang; Chen, Chien-Liang; Hsu, Chih-Yang; Huang, Wei-Chieh; Huang, Chien-Wei; Fang, Hua-Chang

2017-01-01

The lack of homing ability possibly reduces the healing potential of bone-marrow-derived mesenchymal stem cells (MSCs). Therefore, transforming native CD44 on MSCs into a hematopoietic cell E-/L-selectin ligand (HCELL) that possesses potent E-selectin affinity might enhance the homing and regenerative abilities of MSCs. Through fucosyltransferase VI (FTVI) transfection, MSCs were fucosylated on N-glycans of CD44 to become HCELL positive, thus interacting with E-selectin on injured endothelial cells. HCELL expression facilitated MSC homing in kidneys within 24h after injury and reduced lung stasis. An in vitro adhesion assay revealed that transfection enhanced the association between MSCs and hypoxic endothelial cells. In mice treated with HCELL-positive MSCs, the injured kidneys exhibited clusters of homing MSCs, whereas MSCs were rarely observed in mouse kidneys treated with HCELL-negative MSCs. Most MSCs were initially localized at the renal capsule, and some MSCs later migrated inward between tubules. Most homing MSCs were in close contact with inflammatory cells without tubular transdifferentiation. Furthermore, HCELL-positive MSCs substantially alleviated renal injury, partly by enhancing the polarization of infiltrating macrophages. In conclusion, engineering the glycan of CD44 on MSCs through FTVI transfection might enhance renotropism and the regenerating ability of MSCs in ischemic kidney injury. Copyright © 2016 Elsevier Inc. All rights reserved.

Dynamic pattern of endothelial cell adhesion molecule expression in muscle and perineural vessels from patients with classic polyarteritis nodosa.

PubMed

Coll-Vinent, B; Cebrián, M; Cid, M C; Font, C; Esparza, J; Juan, M; Yagüe, J; Urbano-Márquez, A; Grau, J M

1998-03-01

To investigate endothelial cell adhesion molecule expression in vessels from patients with classic polyarteritis nodosa (PAN). Frozen sections of 21 muscle and 16 nerve samples from 30 patients with biopsy-proven PAN and 12 histologically normal muscle and 2 histologically normal nerve samples from 12 controls were studied immunohistochemically, using specific monoclonal antibodies (MAb) that recognize adhesion molecules. Adhesion molecules identified were intercellular adhesion molecule 1 (ICAM-1), ICAM-2, ICAM-3, vascular cell adhesion molecule 1 (VCAM-1), platelet endothelial cell adhesion molecule 1 (PECAM-1), E-selectin, P-selectin, L-selectin, lymphocyte function-associated antigen 1 (LFA-1), and very late activation antigen 4 (VLA-4). Neutrophils were identified with a MAb recognizing neutrophil elastase. Endothelial cells were identified with the lectin ulex europaeus. In early lesions, expression of PECAM-1, ICAM-1, ICAM-2, and P-selectin was similar to that in control samples, and VCAM-1 and E-selectin were induced in vascular endothelium. In advanced lesions, immunostaining for adhesion molecules diminished or disappeared in luminal endothelium, whereas these molecules were clearly expressed in microvessels within and surrounding inflamed vessels. Staining in endothelia from vessels in a healing stage tended to be negative. A high proportion of infiltrating leukocytes expressed LFA-1 and VLA-4, and only a minority expressed L-selectin. No relationship between the expression pattern of adhesion molecules and clinical features, disease duration, or previous corticosteroid treatment was observed. Endothelial adhesion molecule expression in PAN is a dynamic process that varies according to the histopathologic stage of the vascular lesions. The preferential expression of constitutive and inducible adhesion molecules in microvessels suggests that angiogenesis contributes to the persistence of inflammatory infiltration in PAN.

Direct Peritoneal Resuscitation Reduces Leukocyte Infiltration in the Kidney after Acute Brain Death.

PubMed

Weaver, Jessica Lee; Matheson, Paul J; Matheson, Amy; Graham, Victoria S; Downard, Cynthia; Garrison, Richard Neal; Smith, Jason W

2018-04-18

Brain death is associated with significant inflammation within the kidneys, which may contribute to reduced graft survival. Direct peritoneal resuscitation (DPR) has been shown to reduce systemic inflammation after brain death. To determine its effects, brain dead rats were resuscitated with normal saline (targeted intravenous fluid, TIVF) to maintain a mean arterial pressure of 80 mmHg and DPR animals also received 30cc of intraperitoneal peritoneal dialysis solution. Rats were euthanized at zero, two, four, and six hours after brain death. Pro-inflammatory cytokines were measured using ELISA. Levels of IL-1β, TNF-α, and IL-6 in the kidney were significantly increased as early as two hours after brain death and significantly decreased with DPR. Levels of leukocyte adhesion molecules ICAM and VCAM increased after brain death and were decreased with DPR (ICAM 2.33{plus minus}0.14 v 0.42{plus minus}0.04 p=0.002, VCAM 82.6{plus minus}5.8 v 37.3{plus minus}1.9 p=0.002 at four hours) as were E-selectin and P-selectin (E-selectin 25605 v 16144 p=0.005, P-selectin 82.5{plus minus}3.3 v 71.0{plus minus}2.3 p=0.009 at four hours). Use of DPR reduces inflammation and adhesion molecule expression in the kidneys, and is associated with reduced macrophages and neutrophils on immunohistochemistry. Using DPR in brain dead donors has the potential to reduce the immunologic activity of transplanted kidneys and could improve graft survival.

Circulating endothelial cells in acute ischaemic stroke.

PubMed

Nadar, Sunil K; Lip, Gregory Y H; Lee, Kaeng W; Blann, Andrew D

2005-10-01

Increased numbers of CD146-bearing circulating endothelial cells (CECs) in the peripheral blood probably represent the most direct evidence of endothelial cell damage. As acute ischaemic strokes are associated with endothelial abnormalities, we hypothesised that these CECs are raised in acute stroke, and that they would correlate with the other indices of endothelial perturbation, i.e. plasma von Willebrand factor (vWf) and soluble E-selectin. We studied 29 hypertensive patients (19 male; mean age 63 years) who presented with an acute stroke and compared them with 30 high risk hypertensive patients (21 male; mean age 62 years) and 30 normotensive controls (16 male; mean age 58 years). CECs were estimated by CD146 immunobead capture, vWf and soluble E-selectin by ELISA. Patients with an acute ischaemic stroke had significantly higher numbers of CECs/ml of blood (p<0.001) plasma vWf (p=0.008) soluble E-selectin (p=0.002) and higher systolic blood pressure (SBP) as compared to the other groups. The number of CECs significantly correlated with soluble E-selectin (r=0.432, p<0.001) and vWf (r=0.349, p=0.001) but not with SBP (r=0.198, p=0.069). However, in multivariate analysis, only disease group (i.e. health, hypertension or stroke) was associated with increased CECs. Acute ischaemic stroke is associated with increased numbers of CECs. The latter correlate well with established plasma markers of endothelial dysfunction or damage, thus unequivocally confirming severe vasculopathy in this condition. However, the greatest influence on CECs numbers was clinical group.

Amorphous titania modified with boric acid for selective capture of glycoproteins.

PubMed

Jin, Shanxia; Liu, Liping; Zhou, Ping

2018-05-22

Amorphous titania was modified with boric acid, and the resulting material was characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray powder diffraction and X-ray photoelectron spectrometry. The new material, in contrast to conventional boronate affinity materials containing boronic acid ligands, bears boric acid groups. It is shown to exhibit high specificity for glycoproteins, and this was applied to design a method for solid phase extraction of glycoproteins as shown for ribonuclease B, horse radish peroxidase and ovalbumin. Glycoproteins were captured under slightly alkaline environment and released in acidic solutions. The glycoproteins extracted were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The binding capacities for ribonuclease B, horse radish peroxidase and ovalbumin typically are 9.3, 26.0 and 53.0 mg ∙ g -1 , respectively. The method was successfully applied to the selective enrichment of ovalbumin from egg white. Graphical abstract Schematic presentation of the capture of glycoproteins by amorphous titania modified with boric acid.

A solid-phase glycosyltransferase assay for high-throughput screening in drug discovery research.

PubMed

Donovan, R S; Datti, A; Baek, M G; Wu, Q; Sas, I J; Korczak, B; Berger, E G; Roy, R; Dennis, J W

1999-10-01

Glycosyltransferases mediate changes in glycosylation patterns which, in turn, may affect the function of glycoproteins and/or glycolipids and, further downstream, processes of development, differentiation, transformation and cell-cell recognition. Such enzymes, therefore, represent valid targets for drug discovery. We have developed a solid-phase glycosyltransferase assay for use in a robotic high-throughput format. Carbohydrate acceptors coupled covalently to polyacrylamide are coated onto 96-well plastic plates. The glycosyltransferase reaction is performed with recombinant enzymes and radiolabeled sugar-nucleotide donor at 37 degrees C, followed by washing, addition of scintillation counting fluid, and measurement of radioactivity using a 96-well beta-counter. Glycopolymer construction and coating of the plastic plates, enzyme and substrate concentrations, and linearity with time were optimized using recombinant Core 2 beta1-6-N-acetylglucosaminyltransferase (Core 2 GlcNAc-T). This enzyme catalyzes a rate-limiting reaction for expression of polylactosamine and the selectin ligand sialyl-Lewis(x) in O-glycans. A glycopolymer acceptor for beta1-6-N-acetylglucosaminyltransferase V was also designed and shown to be effective in the solid-phase assay. In a high-throughput screen of a microbial extract library, the coefficient of variance for positive controls was 9.4%, and high concordance for hit validation was observed between the Core 2 GlcNAc-T solid-phase assay and a standard solution-phase assay. The solid-phase assay format, which can be adapted for a variety of glycosyltransferase enzymes, allowed a 5-6 fold increase in throughput compared to the corresponding solution-phase assay.

Enhanced tumor cell isolation by a biomimetic combination of E-selectin and anti-EpCAM: implications for the effective separation of circulating tumor cells (CTCs).

PubMed

Myung, Ja Hye; Launiere, Cari A; Eddington, David T; Hong, Seungpyo

2010-06-01

The selective detection of circulating tumor cells (CTCs) is of significant clinical importance for the clinical diagnosis and prognosis of cancer metastasis. However, largely because of the extremely low number of CTCs (as low as 1 in 10(9) hematologic cells) in the blood of patients, effective detection and separation of the rare cells remain a tremendous challenge. Cell rolling is known to play a key role in physiological processes such as the recruitment of leukocytes to sites of inflammation and selectin-mediated CTC metastasis. Furthermore, because CTCs typically express the epithelial-cell adhesion molecule (EpCAM) on the surface whereas normal hematologic cells do not, substrates with immobilized antibody against EpCAM may specifically interact with CTCs. In this article, we created biomimetic surfaces functionalized with P- and E-selectin and anti-EpCAM that induce different responses in HL-60 (used as a model of leukocytes in this study) and MCF-7 (a model of CTCs) cells. HL-60 and MCF-7 cells showed different degrees of interaction with P-/E-selectin and anti-EpCAM at a shear stress of 0.32 dyn/cm(2). HL-60 cells exhibited rolling on P-selectin-immobilized substrates at a velocity of 2.26 +/- 0.28 microm/s whereas MCF-7 cells had no interaction with the surface. Both cell lines, however, had interactions with E-selectin, and the rolling velocity of MCF-7 cells (4.24 +/- 0.31 microm/s) was faster than that of HL-60 cells (2.12 +/- 0.15 microm/s). However, only MCF-7 cells interacted with anti-EpCAM-coated surfaces, forming stationary binding under flow. More importantly, the combination of the rolling (E-selectin) and stationary binding (anti-EpCAM) resulted in substantially enhanced separation capacity and capture efficiency (more than 3-fold enhancement), as compared to a surface functionalized solely with anti-EpCAM that has been commonly used for CTC capture. Our results indicate that cell-specific detection and separation may be achieved through mimicking the biological processes of combined dynamic cell rolling and stationary binding, which will likely lead to a CTC detection device with significantly enhanced specificity and sensitivity without a complex fabrication process.

Ligand Promiscuity between the Efflux Pumps Human P-Glycoprotein and S. aureus NorA.

PubMed

Brincat, Jean Pierre; Broccatelli, Fabio; Sabatini, Stefano; Frosini, Maria; Neri, Annalisa; Kaatz, Glenn W; Cruciani, Gabriele; Carosati, Emanuele

2012-03-08

Thirty-two diverse compounds were evaluated for their ability to inhibit both Pgp-mediated efflux in mouse T-lymphoma L5178 MDR1 and NorA-mediated efflux in S. aureus SA-1199B. Only four compounds were strong inhibitors of both efflux pumps. Three compounds were found to inhibit Pgp exclusively and strongly, while seven compounds inhibited only NorA. These results demonstrate that Pgp and NorA inhibitors do not necessarily overlap, opening the way to safer therapeutic use of effective NorA inhibitors.

Double-chimera proteins to enhance recruitment of endothelial cells and their progenitor cells.

PubMed

Behjati, M; Kazemi, M; Hashemi, M; Zarkesh-Esfahanai, S H; Bahrami, E; Hashemi-Beni, B; Ahmadi, R

2013-08-20

Enhanced attraction of selective vascular reparative cells is of great importance in order to increase vascular patency after endovascular treatments. We aimed to evaluate efficient attachment of endothelial cells and their progenitors on surfaces coated with mixture of specific antibodies, L-selectin and VE-cadherin, with prohibited platelet attachment. The most efficient conditions for coating of L-selectin-Fc chimera and VE-cadherin-Fc chimera proteins were first determined by protein coating on ELISA plates. The whole processes were repeated on titanium substrates, which are commonly used to coat stents. Endothelial progenitor cells (EPCs) and human umbilical vein endothelial cells (HUVECs) were isolated and characterized by flow cytometry. Cell attachment, growth, proliferation, viability and surface cytotoxicity were evaluated using nuclear staining and MTT assay. Platelet and cell attachment were evaluated using scanning electron microscopy. Optimal concentration of each protein for surface coating was 50 ng/ml. The efficacy of protein coating was both heat and pH independent. Calcium ions had significant impact on simultaneous dual-protein coating (P<0.05). Coating stability data revealed more than one year stability for these coated proteins at 4°C. L-selectin and VE-cadherin (ratio of 50:50) coated surface showed highest EPC and HUVEC attachment, viability and proliferation compared to single protein coated and non-coated titanium surfaces (P<0.05). This double coated surface did not show any cytotoxic effect. Surfaces coated with L-selectin and VE-cadherin are friendly surface for EPC and endothelial cell attachment with less platelet attachment. These desirable factors make the L-selectin and VE-cadherin coated surfaces perfect candidate endovascular device. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Lipid raft-associated β-adducin is required for PSGL-1-mediated neutrophil rolling on P-selectin.

PubMed

Xu, Tingshuang; Liu, Wenai; Yang, Chen; Ba, Xueqing; Wang, Xiaoguang; Jiang, Yong; Zeng, Xianlu

2015-02-01

Lipid rafts, a liquid-ordered plasma membrane microdomain, are related to cell-surface receptor function. PSGL-1, a major surface receptor protein for leukocyte, also acts as a signaling receptor in leukocyte rolling. To investigate the role of lipid raft in PSGL-1 signaling in human neutrophils, we quantitatively analyzed lipid raft proteome of human promyelocytic leukemia cell line HL-60 cells and identified a lipid raft-associated protein β-adducin. PSGL-1 ligation induced dissociation of the raft-associated protein β-adducin from lipid rafts and actin, as well as phosphorylation of β-adducin, indicating a transient uncoupling of lipid rafts from the actin cytoskeleton. Knockdown of β-adducin greatly attenuated HL-60 cells rolling on P-selectin. We also showed that Src kinase is crucial for PSGL-1 ligation-induced β-adducin phosphorylation and relocation. Taken together, these results show that β-adducin is a pivotal lipid raft-associated protein in PSGL-1-mediated neutrophil rolling on P-selectin. © Society for Leukocyte Biology.

Highly sensitivity adhesion molecules detection in hereditary haemochromatosis patients reveals altered expression.

PubMed

Norris, S; White, M; Mankan, A K; Lawless, M W

2010-04-01

Several abnormalities in the immune status of patients with hereditary haemochromatosis (HH) have been reported, suggesting an imbalance in their immune function. This may include persistent production of, or exposure to, altered immune signalling contributing to the pathogenesis of this disorder. Adhesion molecules L-, E- and P-Selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) are some of the major regulators of the immune processes and altered levels of these proteins have been found in pathological states including cardiovascular diseases, arthritis and liver cancer. The aim of this study was to assess L-, E- and P-Selectin, ICAM-1 and VCAM-1 expression in patients with HH and correlate these results with HFE mutation status and iron indexes. A total of 139 subjects were diagnosed with HH (C282Y homozygotes = 87, C282Y/H63D = 26 heterozygotes, H63D homozygotes = 26), 27 healthy control subjects with no HFE mutation (N/N), 18 normal subjects heterozygous for the H63D mutation served as age-sex-matched controls. We observed a significant decrease in L-selectin (P = 0.0002) and increased E-selectin and ICAM-1 (P = 0.0006 and P = 0.0059) expression in HH patients compared with healthy controls. This study observes for the first time that an altered adhesion molecules profile occurs in patients with HH that is associated with specific HFE genetic component for iron overload, suggesting that differential expression of adhesion molecules may play a role in the pathogenesis of HH.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamamoto, Hiroyasu; Kuroda, Nana; Uekita, Hiromi

Background: Adiponectin (APN) is an adipocyte-derived bioactive molecule with anti-diabetic and anti-atherogenic properties. Although anti-diabetic effects are mostly mediated by the adiponectin receptors AdipoR1 and AdipoR2, the anti-atherogenic mechanisms have not been fully elucidated. Methods and Results: In this study, we identified E-selectin ligand (ESL)-1 as a novel APN-binding protein by mass spectrometry analysis of HepG2 cell-derived immunoprecipitant with an anti-APN antibody. Cell adhesion assays using fluorescence-labelled monocyte cell line THP-1 cells and human umbilical vein endothelial cells (HUVECs) revealed that APN-pre-treated THP-1 cells had reduced binding ability to HUVECs. This APN-mediated suppressive effect on monocyte binding to endothelial cells was partiallymore » abrogated by targeting ESL-1 with shRNA in THP-1 cells. In addition, serial mutagenesis analysis disclosed that five extracellular amino acids close to the N-terminus of ESL-1 were essential for binding with APN. Conclusion: Our results highlight the fact that interaction between APN and ESL-1 could provide a fundamental mechanism underlying the anti-atherogenic properties of APN. - Highlights: • E-selectin ligand (ESL)-1 was identified as an adiponectin (APN)-binding protein. • ESL-1 bound to APN at its N-terminal 6th-10th amino acids. • shESL-1 reduced the suppressive effect of APN on adhesion of THP-1 cells to HUVECs. • Interaction with ESL may be involved in the anti-atherogenic effects of APN.« less

Nanoparticle-mediated codelivery of myelin antigen and a tolerogenic small molecule suppresses experimental autoimmune encephalomyelitis

PubMed Central

Yeste, Ada; Nadeau, Meghan; Burns, Evan J.; Weiner, Howard L.; Quintana, Francisco J.

2012-01-01

The immune response is normally controlled by regulatory T cells (Tregs). However, Treg deficits are found in autoimmune diseases, and therefore the induction of functional Tregs is considered a potential therapeutic approach for autoimmune disorders. The activation of the ligand-activated transcription factor aryl hydrocarbon receptor by 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) or other ligands induces dendritic cells (DCs) that promote FoxP3+ Treg differentiation. Here we report the use of nanoparticles (NPs) to coadminister ITE and a T-cell epitope from myelin oligodendrocyte glycoprotein (MOG)35–55 to promote the generation of Tregs by DCs. NP-treated DCs displayed a tolerogenic phenotype and promoted the differentiation of Tregs in vitro. Moreover, NPs carrying ITE and MOG35–55 expanded the FoxP3+ Treg compartment and suppressed the development of experimental autoimmune encephalomyelitis, an experimental model of multiple sclerosis. Thus, NPs are potential new tools to induce functional Tregs in autoimmune disorders. PMID:22745170

Cardio-Metabolic Disease Risks and Their Associations with Circulating 25-Hydroxyvitamin D and Omega-3 Levels in South Asian and White Canadians.

PubMed

Xiao, Chao-Wu; Wood, Carla M; Swist, Eleonora; Nagasaka, Reiko; Sarafin, Kurtis; Gagnon, Claude; Fernandez, Lois; Faucher, Sylvie; Wu, Hong-Xing; Kenney, Laura; Ratnayake, Walisundera M N

2016-01-01

This study compared cardio-metabolic disease risk factors and their associations with serum vitamin D and omega-3 status in South Asian (SAC) and White Canadians (WC) living in Canada's capital region. Fasting blood samples were taken from 235 SAC and 279 WC aged 20 to 79 years in Ottawa, and 22 risk factors were measured. SAC men and women had significantly higher fasting glucose, insulin, homeostasis model assessment for insulin resistance (HOMA-IR), apolipoprotein B (ApoB), ratios of total (TC) to HDL cholesterol (HDLC) and ApoB to ApoA1, leptin, E-selectin, P-selectin, ICAM-1 and omega-3 (p < 0.05), but lower HDLC, ApoA1, vitamin D levels than WC (p < 0.05). SAC women had higher CRP and VEGF than WC women. Adequate (50-74.9 nmol/L) or optimal (≥ 75 nmol/L) levels of 25(OH)D were associated with lower BMI, glucose, insulin, HOMA-IR, TG, TC, low density lipoprotein cholesterol (LDLC), ApoB/ApoA1 ratio, CRP, leptin, and higher HDLC, ApoA1, omega-3 index, L-selectin levels in WC, but not in SAC. Intermediate (>4%-<8%) or high (≥ 8%) levels of omega-3 indices were related to lower E-selectin, P-selectin, ICAM-1 and higher HDLC, 25(OH)D levels in WC, but not in SAC. The BMIs of ≤ 25 kg/m2 were related to lower LDLC, ApoB, VEGF, creatinine and higher 25(OH)D in WC, but not in SAC. The associations of vitamin D, omega-3 status, BMI and risk factors were more profound in the WC than SAC. Compared to WC, vitamin D status and omega-3 index may not be good predictive risk factors for the prevalence of CVD and diabetes in SAC.

Cardio-Metabolic Disease Risks and Their Associations with Circulating 25-Hydroxyvitamin D and Omega-3 Levels in South Asian and White Canadians

PubMed Central

Xiao, Chao-Wu; Wood, Carla M.; Swist, Eleonora; Nagasaka, Reiko; Sarafin, Kurtis; Gagnon, Claude; Fernandez, Lois; Faucher, Sylvie; Wu, Hong-Xing; Kenney, Laura; Ratnayake, Walisundera M. N.

2016-01-01

Objectives This study compared cardio-metabolic disease risk factors and their associations with serum vitamin D and omega-3 status in South Asian (SAC) and White Canadians (WC) living in Canada’s capital region. Methods Fasting blood samples were taken from 235 SAC and 279 WC aged 20 to 79 years in Ottawa, and 22 risk factors were measured. Results SAC men and women had significantly higher fasting glucose, insulin, homeostasis model assessment for insulin resistance (HOMA-IR), apolipoprotein B (ApoB), ratios of total (TC) to HDL cholesterol (HDLC) and ApoB to ApoA1, leptin, E-selectin, P-selectin, ICAM-1 and omega-3 (p < 0.05), but lower HDLC, ApoA1, vitamin D levels than WC (p < 0.05). SAC women had higher CRP and VEGF than WC women. Adequate (50–74.9 nmol/L) or optimal (≥ 75 nmol/L) levels of 25(OH)D were associated with lower BMI, glucose, insulin, HOMA-IR, TG, TC, low density lipoprotein cholesterol (LDLC), ApoB/ApoA1 ratio, CRP, leptin, and higher HDLC, ApoA1, omega-3 index, L-selectin levels in WC, but not in SAC. Intermediate (>4%-<8%) or high (≥ 8%) levels of omega-3 indices were related to lower E-selectin, P-selectin, ICAM-1 and higher HDLC, 25(OH)D levels in WC, but not in SAC. The BMIs of ≤ 25 kg/m2 were related to lower LDLC, ApoB, VEGF, creatinine and higher 25(OH)D in WC, but not in SAC. Conclusions The associations of vitamin D, omega-3 status, BMI and risk factors were more profound in the WC than SAC. Compared to WC, vitamin D status and omega-3 index may not be good predictive risk factors for the prevalence of CVD and diabetes in SAC. PMID:26809065

Effects of aspirin on intra-platelet vascular endothelial growth factor, angiopoietin-1, and p-selectin levels in hypertensive patients.

PubMed

Nadar, Sunil; Blann, Andrew D; Lip, Gregory Y H

2006-09-01

Although aspirin is useful in reducing platelet activation and cardiovascular events, its effects on platelet levels of angiogenic factors, such as vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1), and markers of platelet activation in hypertension are unknown. The aim of this study was to study the effects of aspirin on the platelet morphology, plasma and platelet levels of VEGF (sVEGF and pVEGF respectively), Ang-1 (sAng-1 and pAng-1 respectively), and P-selectin (sPsel and pPsel respectively) in patients with well controlled hypertension. A total of 35 aspirin-naive, hypertensive patients (29 male and six female; mean age 64 years) were compared with 30 (23 male, seven female, mean age 59 years) normotensive control subjects. Blood was collected for plasma VEGF, P-selectin, and Ang-1 (enzyme-linked immunoassay), intra-platelet levels of VEGF, Ang-1, and P-selectin, and platelet volume and mass. Research indices in hypertensive patients were studied before and after 3 months treatment with aspirin 75 mg daily. Hypertensive patients had significantly higher plasma levels of VEGF (P=.04), Ang-1 (P<.001), as well as pVEGF (P=.008), pAng-1(P=.001), sPsel (P=.02), pPsel (P<.001), and mean platelet mass (P=.01) when compared with control subjects. After treatment with aspirin for 3 months, there were significant reductions in plasma VEGF (P=.01), pAng-1 (P=.04), sPsel (P=.001), and pPsel (P<.001) levels, but not levels of platelet VEGF and plasma Ang-1. Neither pVEGF nor pAng-1 correlated with blood pressure or with their respective plasma levels. The use of aspirin in high-risk hypertensive patients leads to a reduction in intra-platelet angiogenic growth factors and platelet activation. This may have implications for the use of aspirin in conditions (such as vascular disease) that have been associated with an increase in angiogenesis and platelet activation.

Impact of Blood Mixing and ABO Compatibility on Platelet-Leukocyte Aggregations and Platelet P-Selectin Expression: An in Vitro Study.

PubMed

Huang, Go-Shine; Hu, Mei-Hua; Lin, Tso-Chou; Tsai, Yi-Ting; Lin, Chih-Yuan; Ke, Hung-Yen; Zheng, Xu-Zhi; Lin, Yi-Chang; Tsai, Chien-Sung

2018-05-01

Effects of blood transfusions on platelet- and leukocyte-related inflammation are unclear. We simulated transfusion using in vitro blood mixing to evaluate platelet-leukocyte aggregations (PLA) and platelet P-selectin expression, and the mechanism of PLA. Donor packed red blood cells (pRBCs) were obtained from a blood bank. Recipient whole blood samples were obtained from patients undergoing cardiac surgery. Blood sample mixtures were divided into four groups: group M, cross-matched blood type mixing; group O, donor type O with other blood type mixing (A, B, or AB); group S, ABO type-specific uncross-matched blood mixing; and group I, ABO incompatibility mixing. Donor pRBCs were added to recipient blood to reach 1%, 5%, and 10% (vol/vol) concentrations. Blood sample mixtures were analyzed to determine the PLA; P-selectin expression; and leukocyte CD11a, CD11b, and CD18 subunits of integrin expression. Analysis of variance tests were used to analyze differences. PLA significantly increased only in groups O and I (P = 0.003 and P < 0.001). Subpopulations of leukocytes significantly increased in all groups. There were no significant differences among the four groups (P = 0.578) in PLA increase. Although there was no significant effect on P-selectin expression (P = 1.000) and leukocyte CD11a and CD18 expression (P = 0.999, P = 0.422) within and between the groups, there was an increase in CD11b expression (P = 0.018). Blood mixing can increase PLA, especially in platelet-neutrophil and platelet-monocyte aggregations, possibly through nonhemolytic reactions. The CD11b integrin with CD18 may play a role in the formation of PLA.

Nano- to microscale dynamics of P-selectin detachment from leukocyte interfaces. II. Tether flow terminated by P-selectin dissociation from PSGL-1.

PubMed

Heinrich, Volkmar; Leung, Andrew; Evans, Evan

2005-03-01

We have used a biomembrane force probe decorated with P-selectin to form point attachments with PSGL-1 receptors on a human neutrophil (PMN) in a calcium-containing medium and then to quantify the forces experienced by the attachment during retraction of the PMN at fixed speed. From first touch to final detachment, the typical force history exhibited the following sequence of events: i), an initial linear-elastic displacement of the PMN surface, ii), an abrupt crossover to viscoplastic flow that signaled membrane separation from the interior cytoskeleton and the beginning of a membrane tether, and iii), the final detachment from the probe tip most often by one precipitous step of P-selectin:PSGL-1 dissociation. Analyzing the initial elastic response and membrane unbinding from the cytoskeleton in our companion article I, we focus in this article on the regime of tether extrusion that nearly always occurred before release of the extracellular adhesion bond at pulling speeds > or =1 microm/s. The force during tether growth appeared to approach a plateau at long times. Examined over a large range of pulling speeds up to 150 microm/s, the plateau force exhibited a significant shear thinning as indicated by a weak power-law dependence on pulling speed, f(infinity) = 60 pN(nu(pull)/microm/s)(0.25). Using this shear-thinning response to describe the viscous element in a nonlinear Maxwell-like fluid model, we show that a weak serial-elastic component with a stiffness of approximately 0.07 pN/nm provides good agreement with the time course of the tether force approach to the plateau under constant pulling speed.

Role of the recombinant protein of the platelet receptor for type I collagen in the release of nitric oxide during platelet aggregation.

PubMed

Chiang, T M; Wang, Y B; Kang, E S

2000-12-01

Nitric oxide plays an important role in platelet function and platelets possess the endothelial isoform of nitric oxide synthase. Several reports have indicated that nitric oxide is released upon exposure of platelets to collagen. We have reported that a non-integrin platelet protein of 65 kDa is a receptor for type I collagen. By direct measurement of NO release from washed human platelets suspended in Tyrode buffer with a ISO-NO Mark II, World Precision Instruments, Sarasota, FL, USA, p30 sensor, type I collagen, but not ADP and epinephrine, induces the release of NO in a time-dependent manner. The production of NO is inhibited either by preincubation of type I collagen with the platelet type I collagen receptor recombinant protein or by preincubation of platelets with the antibody to the receptor protein, the anti-65 antibody. However, preincubation of platelets with anti-P-selectin and anti-glycoprotein IIb/IIIa did not affect the release of NO by platelets. These results suggest that the 65 kDa platelet receptor for type I collagen is specifically linked to the generation of NO, and that the 65 kDa platelet receptor for type I collagen plays an important new role in platelet function.

Tapered oral dexamethasone for the acute chest syndrome of sickle cell disease

PubMed Central

Quinn, Charles T.; Stuart, Marie J.; Kesler, Karen; Ataga, Kenneth I.; Wang, Winfred C.; Styles, Lori; Smith-Whitley, Kim; Wun, Ted; Raj, Ahsok; Hsu, Lewis L.; Krishnan, Suba; Kuypers, Frans A; Setty, B. N. Yamaja; Rhee, Seungshin; Key, Nigel S.; Buchanan, George R.

2011-01-01

Summary Tapered oral dexamethasone for acute chest syndrome (ACS) in sickle cell anaemia was studied using a novel ACS assessment tool and investigational biomarkers. Twelve participants were randomized (mean age 17.3 years) before early study termination. Dexamethasone decreased duration of hospitalization for ACS by 20.8 h compared to placebo (P=0.024). Rebound pain occurred in both groups (3 dexamethasone vs. 1 placebo). Overall, dexamethasone decreased the leucocyte activation biomarker, sL-selectin; however, participants with rebound pain had higher sL-selectin within 24 h of treatment (dexamethasone or placebo). This ACS assessment tool was feasibly applied, and sL-selectin is a promising biomarker of ACS therapy. PMID:21848879

Catalytic transitions in the human MDR1 P-glycoprotein drug binding sites.

PubMed

Wise, John G

2012-06-26

Multidrug resistance proteins that belong to the ATP-binding cassette family like the human P-glycoprotein (ABCB1 or Pgp) are responsible for many failed cancer and antiviral chemotherapies because these membrane transporters remove the chemotherapeutics from the targeted cells. Understanding the details of the catalytic mechanism of Pgp is therefore critical to the development of inhibitors that might overcome these resistances. In this work, targeted molecular dynamics techniques were used to elucidate catalytically relevant structures of Pgp. Crystal structures of homologues in four different conformations were used as intermediate targets in the dynamics simulations. Transitions from conformations that were wide open to the cytoplasm to transition state conformations that were wide open to the extracellular space were studied. Twenty-six nonredundant transitional protein structures were identified from these targeted molecular dynamics simulations using evolutionary structure analyses. Coupled movement of nucleotide binding domains (NBDs) and transmembrane domains (TMDs) that form the drug binding cavities were observed. Pronounced twisting of the NBDs as they approached each other as well as the quantification of a dramatic opening of the TMDs to the extracellular space as the ATP hydrolysis transition state was reached were observed. Docking interactions of 21 known transport ligands or inhibitors were analyzed with each of the 26 transitional structures. Many of the docking results obtained here were validated by previously published biochemical determinations. As the ATP hydrolysis transition state was approached, drug docking in the extracellular half of the transmembrane domains seemed to be destabilized as transport ligand exit gates opened to the extracellular space.

Gold(I) NHC Complexes: Antiproliferative Activity, Cellular Uptake, Inhibition of Mammalian and Bacterial Thioredoxin Reductases, and Gram-Positive Directed Antibacterial Effects.

PubMed

Schmidt, Claudia; Karge, Bianka; Misgeld, Rainer; Prokop, Aram; Franke, Raimo; Brönstrup, Mark; Ott, Ingo

2017-02-03

Gold complexes with N-heterocyclic carbene (NHC) ligands represent a promising class of metallodrugs for the treatment of cancer or infectious diseases. In this report, the synthesis and the biological evaluation of halogen-containing NHC-Au I -Cl complexes are described. The complexes 1 and 5 a-5 f displayed good cytotoxic activity against tumor cells, and cellular uptake studies suggested that an intact Au-NHC fragment is essential for the accumulation of high amounts of both the metal and the NHC ligand. However, the bioavailability was negatively affected by serum components of the cell culture media and was influenced by likely transformations of the complex. One example (5 d) efficiently induced apoptosis in vincristine- and daunorubicin-resistant P-glycoprotein overexpressing Nalm-6 leukemia cells. Cellular uptake studies with this compound showed that both the wild-type and resistant Nalm-6 cells accumulated comparable amounts of gold, indicating that the gold drug was not excreted by P-glycoprotein or other efflux transporters. The effective inhibition of mammalian and bacterial thioredoxin reductases (TrxR) was confirmed for all of the gold complexes. Antibacterial screening of the gold complexes showed a particularly high activity against Gram-positive strains, reflecting their high dependence on an intact Trx/TrxR system. This result is of particular interest as the inhibition of bacterial TrxR represents a relatively little explored mechanism of new anti-infectives. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Peracetylated 4-Fluoro-glucosamine Reduces the Content and Repertoire of N- and O-Glycans without Direct Incorporation*

PubMed Central

Barthel, Steven R.; Antonopoulos, Aristotelis; Cedeno-Laurent, Filiberto; Schaffer, Lana; Hernandez, Gilberto; Patil, Shilpa A.; North, Simon J.; Dell, Anne; Matta, Khushi L.; Neelamegham, Sriram; Haslam, Stuart M.; Dimitroff, Charles J.

2011-01-01

Prior studies have shown that treatment with the peracetylated 4-fluorinated analog of glucosamine (4-F-GlcNAc) elicits anti-skin inflammatory activity by ablating N-acetyllactosamine (LacNAc), sialyl Lewis X (sLeX), and related lectin ligands on effector leukocytes. Based on anti-sLeX antibody and lectin probing experiments on 4-F-GlcNAc-treated leukocytes, it was hypothesized that 4-F-GlcNAc inhibited sLeX formation by incorporating into LacNAc and blocking the addition of galactose or fucose at the carbon 4-position of 4-F-GlcNAc. To test this hypothesis, we determined whether 4-F-GlcNAc is directly incorporated into N- and O-glycans released from 4-F-GlcNAc-treated human sLeX (+) T cells and leukemic KG1a cells. At concentrations that abrogated galectin-1 (Gal-1) ligand and E-selectin ligand expression and related LacNAc and sLeX structures, MALDI-TOF and MALDI-TOF/TOF mass spectrometry analyses showed that 4-F-GlcNAc 1) reduced content and structural diversity of tri- and tetra-antennary N-glycans and of O-glycans, 2) increased biantennary N-glycans, and 3) reduced LacNAc and sLeX on N-glycans and on core 2 O-glycans. Moreover, MALDI-TOF MS did not reveal any m/z ratios relating to the presence of fluorine atoms, indicating that 4-F-GlcNAc did not incorporate into glycans. Further analysis showed that 4-F-GlcNAc treatment had minimal effect on expression of 1200 glycome-related genes and did not alter the activity of LacNAc-synthesizing enzymes. However, 4-F-GlcNAc dramatically reduced intracellular levels of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), a key precursor of LacNAc synthesis. These data show that Gal-1 and E-selectin ligand reduction by 4-F-GlcNAc is not caused by direct 4-F-GlcNAc glycan incorporation and consequent chain termination but rather by interference with UDP-GlcNAc synthesis. PMID:21493714

Human epidermal growth factor receptor bispecific ligand trap RB200: abrogation of collagen-induced arthritis in combination with tumour necrosis factor blockade

PubMed Central

2011-01-01

Introduction Rheumatoid arthritis (RA) is a chronic disease associated with inflammation and destruction of bone and cartilage. Although inhibition of TNFα is widely used to treat RA, a significant number of patients do not respond to TNFα blockade, and therefore there is a compelling need to continue to identify alternative therapeutic strategies for treating chronic inflammatory diseases such as RA. The anti-epidermal growth factor (anti-EGF) receptor antibody trastuzumab has revolutionised the treatment of patients with EGF receptor-positive breast cancer. Expression of EGF ligands and receptors (known as HER) has also been documented in RA. The highly unique compound RB200 is a bispecific ligand trap that is composed of full-length extracellular domains of HER1 and HER3 EGF receptors. Because of its pan-HER specificity, RB200 inhibits responses mediated by HER1, HER2 and HER3 in vitro and in vivo. The objective of this study was to assess the effect of RB200 combined with TNF blockade in a murine collagen-induced arthritis (CIA) model of RA. Methods Arthritic mice were treated with RB200 alone or in combination with the TNF receptor fusion protein etanercept. We performed immunohistochemistry to assess CD31 and in vivo fluorescent imaging using anti-E-selectin antibody labelled with fluorescent dye to elucidate the effect of RB200 on the vasculature in CIA. Results RB200 significantly abrogated CIA by reducing paw swelling and clinical scores. Importantly, low-dose RB200 combined with a suboptimal dose of etanercept led to complete abrogation of arthritis. Moreover, the combination of RB200 with etanercept abrogated the intensity of the E-selectin-targeted signal to the level seen in control animals not immunised to CIA. Conclusions The human pan-EGF receptor bispecific ligand trap RB200, when combined with low-dose etanercept, abrogates CIA, suggesting that inhibition of events downstream of EGF receptor activation, in combination with TNFα inhibitors, may hold promise as a future therapy for patients with RA. PMID:21982514

Interleukin‑12B is upregulated by decoy receptor 3 in rheumatoid synovial fibroblasts.

PubMed

Fukuda, Koji; Miura, Yasushi; Maeda, Toshihisa; Hayashi, Shinya; Kurosaka, Masahiro

2016-04-01

Decoy receptor 3 (DcR3) competitively binds to three ligands, Fas ligand, lymphotoxin‑related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells and tumor necrosis factor‑like ligand 1A (TL1A), to prevent their effects. Recent studies have suggested that DcR3 directly affects cells as a ligand. Using a microarray assay, our group newly identified interleukin (IL)‑12B, which encodes the p40 subunit common to IL‑12 and IL‑23, as one of the genes for which expression in fibroblast‑like synoviocytes from patients with rheumatoid arthritis (RA‑FLS) is induced by DcR3. The present study demonstrated that IL‑12B mRNA expression was upregulated by DcR3‑Fc in RA‑FLS in a dose‑dependent manner, but not in OA‑FLS. IL‑12B p40 protein in RA‑FLS was increased when stimulated with DcR3‑Fc. Pre‑treatment with anti‑TL1A antibody suppressed the upregulation of IL‑12B mRNA in RA‑FLS stimulated with DcR3‑Fc. DcR3 mRNA expression in RA‑FLS was induced by IL‑23, but not by IL‑12. These results indicated that DcR3 may increase IL‑12 or IL‑23 by inducing IL‑12B p40 expression via membrane‑bound TL1A on RA‑FLS and that IL‑23 reciprocally induces DcR3 expression in RA‑FLS. DcR3 and IL‑23 may interact in a feedback loop that aggravates local inflammation in patients with RA.

Decoy receptor 3 suppresses TLR2-mediated B cell activation by targeting NF-κB.

PubMed

Huang, Zi-Ming; Kang, Jhi-Kai; Chen, Chih-Yu; Tseng, Tz-Hau; Chang, Chien-Wen; Chang, Yung-Chi; Tai, Shyh-Kuan; Hsieh, Shie-Liang; Leu, Chuen-Miin

2012-06-15

Decoy receptor 3 (DcR3) is a soluble protein in the TNFR superfamily. Its known ligands include Fas ligand, homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes, TNF-like molecule 1A, and heparan sulfate proteoglycans. DcR3 has been reported to modulate the functions of T cells, dendritic cells, and macrophages; however, its role in regulating B cell activation is largely unknown. In this study, we found that the DcR3.Fc fusion protein bound to human and mouse B cells and suppressed the activation of B cells. DcR3.Fc attenuated Staphylococcus aureus, IgM-, Pam(3)CSK(4)-, and LPS-mediated B cell proliferation but did not affect cytokine-induced B cell growth. In the presence of these mitogens, DcR3.Fc did not induce B cell apoptosis, suggesting that DcR3 may inhibit the signal(s) important for B cell activation. Because the combination of Fas.Fc, LT-βR.Fc (homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes receptor), and DR3.Fc (TNF-like molecule 1A receptor) did not suppress B cell proliferation and because the biological effect of DcR3.Fc on B cells was not blocked by heparin, we hypothesize that a novel ligand(s) of DcR3 mediates its inhibitory activity on B cells. Moreover, we found that TLR2-stimulated NF-κB p65 activation and NF-κB-driven luciferase activity were attenuated by DcR3.Fc. The TLR2-induced cytokine production by B cells was consistently reduced by DcR3. These results imply that DcR3 may regulate B cell activation by suppressing the activation of NF-κB.

The effects of vitamin E-coated membrane dialyzer compared to simvastatin in patients on chronic hemodialysis.

PubMed

Kirmizis, Dimitrios; Papagianni, Aikaterini; Dogrammatzi, Fani; Belechri, Anna-Maria; Alexopoulos, Efstathios; Efstratiadis, Georgios; Memmos, Dimitrios

2012-01-01

We investigated the effects of the use of vitamin E-coated membrane (VEM) dialyzer in comparison to simvastatin on markers of chronic inflammation, oxidative stress, and endothelial cell apoptosis in ten patients on chronic hemodialysis (HD), aiming at distinguishing the different treatment effects and their time sequence on these pathogenetic routes. Ten HD patients were sequentially submitted to a 6-month treatment with the use of VEM and 10 mg of simvastatin daily, interrupted by a 3-month washout period. At baseline, at 3, and 6 months of each trial, serum C-reactive protein (CRP), apolipoprotein (Apo) A1 and B, lipoprotein-a [Lp(a)], high-sensitivity interleukin-6 (hsIL-6), monocyte chemoattractant protein-1 (MCP-1), soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble E-selectin (sE-selectin), soluble Fas (sFas), soluble Fas ligand (sFasL), and plasma oxidized low-density lipoproteins (oxLDL) levels were determined. VEM treatment resulted in a significant decrease in CRP, IL-6, sICAM-1 at 3 months, and oxLDL at 6 months, compared to baseline. Simvastatin resulted in a significant decrease in CRP, which correlated with decreases in both total (r = 0.87, p < 0.05) and low-density lipoprotein cholesterol, IL-6, sICAM-1, sVCAM-1, oxLDL, and sFas at 6 months, compared to baseline. Simvastatin effects on sVCAM-1 (mean difference = 652 ng/mL; 95% CI = 294 to 2686; p < 0.05) and sFas (mean difference = 1284 pg/mL; 95% CI = 510 to 1910; p < 0.05) differed significantly from the corresponding VEM effects. The 6-month use of VEM resulted in more direct and immediate anti-inflammatory effects compared with those caused by the 6-month treatment with simvastatin. Simvastatin caused a more intense decrease in the markers of inflammation, which was in part correlated with its lipid-lowering effects.

The cytoplasmic tail of L-selectin interacts with the adaptor-protein complex AP-1 subunit μ1A via a novel basic binding motif

PubMed Central

Tikhonova, Irina G.; Ivetic, Aleksandar; Schu, Peter

2017-01-01

L-selectin regulates leukocyte adhesion and rolling along the endothelium. Proteins binding to the cytoplasmic tail of L-selectin regulate L-selectin functions. We used L-selectin cytoplasmic tail peptide pulldown assays combined with high sensitivity liquid chromatography/mass spectrometry to identify novel L-selectin tail-binding proteins. Incubation of the L-selectin tail with cell extracts from phorbol 12-myristate 13-acetate-stimulated Raw 264.7 macrophages resulted in the binding of μ1A of the clathrin-coated vesicle AP-1 complex. Furthermore, full-length GST-μ1A and the GST-μ1A C-terminal domain, but not the GST-μ1A N-terminal domain, bind to L-selectin tail peptide, and the intracellular pool of L-selectin colocalizes with AP-1 at the trans-Golgi network. We identified a novel basic protein motif consisting of a cluster of three dibasic residues (356RR357, 359KK360, and 362KK363) in the membrane-proximal domain of the L-selectin tail as well as a doublet of aspartic acid residues (369DD370) in the membrane-distal end of the L-selectin tail involved in μ1A binding. Stimulation of Raw 264.7 macrophages with PMA augmented the amount of μ1A associated with anti-L-selectin immunoprecipitates. However, full-length GST-μ1A did not bind to the phospho-L-selectin tail or phospho-mimetic S364D L-selectin tail. Accordingly, we propose that phosphorylation of μ1A is required for interaction with the L-selectin tail and that L-selectin tail phosphorylation may regulate this interaction in vivo. Molecular docking of the L-selectin tail to μ1A was used to identify the μ1A surface domain binding the L-selectin tail and to explain how phosphorylation of the L-selectin tail abrogates μ1A interaction. Our findings indicate that L-selectin is transported constitutively by the AP-1 complex, leading to the formation of a trans-Golgi network reserve pool and that phosphorylation of the L-selectin tail blocks AP-1-dependent retrograde transport of L-selectin. PMID:28235798

The cytoplasmic tail of L-selectin interacts with the adaptor-protein complex AP-1 subunit μ1A via a novel basic binding motif.

PubMed

Dib, Karim; Tikhonova, Irina G; Ivetic, Aleksandar; Schu, Peter

2017-04-21

L-selectin regulates leukocyte adhesion and rolling along the endothelium. Proteins binding to the cytoplasmic tail of L-selectin regulate L-selectin functions. We used L-selectin cytoplasmic tail peptide pulldown assays combined with high sensitivity liquid chromatography/mass spectrometry to identify novel L-selectin tail-binding proteins. Incubation of the L-selectin tail with cell extracts from phorbol 12-myristate 13-acetate-stimulated Raw 264.7 macrophages resulted in the binding of μ1A of the clathrin-coated vesicle AP-1 complex. Furthermore, full-length GST-μ1A and the GST-μ1A C-terminal domain, but not the GST-μ1A N-terminal domain, bind to L-selectin tail peptide, and the intracellular pool of L-selectin colocalizes with AP-1 at the trans -Golgi network. We identified a novel basic protein motif consisting of a cluster of three dibasic residues ( 356 RR 357 , 359 KK 360 , and 362 KK 363 ) in the membrane-proximal domain of the L-selectin tail as well as a doublet of aspartic acid residues ( 369 DD 370 ) in the membrane-distal end of the L-selectin tail involved in μ1A binding. Stimulation of Raw 264.7 macrophages with PMA augmented the amount of μ1A associated with anti-L-selectin immunoprecipitates. However, full-length GST-μ1A did not bind to the phospho-L-selectin tail or phospho-mimetic S364D L-selectin tail. Accordingly, we propose that phosphorylation of μ1A is required for interaction with the L-selectin tail and that L-selectin tail phosphorylation may regulate this interaction in vivo Molecular docking of the L-selectin tail to μ1A was used to identify the μ1A surface domain binding the L-selectin tail and to explain how phosphorylation of the L-selectin tail abrogates μ1A interaction. Our findings indicate that L-selectin is transported constitutively by the AP-1 complex, leading to the formation of a trans -Golgi network reserve pool and that phosphorylation of the L-selectin tail blocks AP-1-dependent retrograde transport of L-selectin. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Annual FEV1 changes and numbers of circulating endothelial microparticles in patients with COPD: a prospective study

PubMed Central

Takahashi, Toru; Kobayashi, Seiichi; Fujino, Naoya; Suzuki, Takaya; Ota, Chiharu; Tando, Yukiko; Yamada, Mitsuhiro; Yanai, Masaru; Yamaya, Mutsuo; Kurosawa, Shin; Yamauchi, Masanori; Kubo, Hiroshi

2014-01-01

Objective Growing evidence suggests that endothelial injury is involved in the pathophysiology of chronic obstructive pulmonary disease (COPD). Circulating endothelial microparticles (EMPs) increase in patients with COPD because of the presence of endothelial injury. We examined the relationship between EMP number and changes in forced expiratory volume in 1 s (FEV1) in patients with COPD. Design Prospective study. Setting One hospital in Japan. Participants A total 48 outpatients with stable COPD coming to the hospital from September 2010 to September 2011. Primary and secondary outcomes measured Blood samples were collected and vascular endothelial (VE)-cadherin EMPs (CD144+ EMPs), E-selectin EMPs (CD62E+ EMPs) and platelet endothelial cell adhesion molecule EMPs (CD31+/CD41− EMPs) were measured using fluorescence-activated cell sorting. Annual FEV1 changes were evaluated using FEV1 data acquired a year before and a year after sample collection. Results The number of E-selectin and VE-cadherin EMPs showed significant negative correlations with annual FEV1 changes (rs=−0.65, p<0.001, rs=−0.43, p=0.003, respectively). Leucocyte counts tended to be correlated with annual FEV1 changes, but this correlation was not significant (rs=−0.28, p=0.057). There were significant differences in annual FEV1 changes between with and without history of frequent exacerbation (p=0.006), and among Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages (p=0.009). Multiple linear regression analysis revealed E-selectin EMP to be the only significant parameter associated with annual FEV1 changes, independent of VE-cadherin EMP, GOLD stages, leucocyte counts, and history of frequent exacerbation. Receiver operating characteristic curves showed the optimum E-selectin EMP cut-off level for prediction of rapid FEV1 decline (>66 mL/year) to be 153.0/µL (areas under curve 0.78 (95% CI 0.60 to 0.89); sensitivity, 67%; specificity, 81%). Conclusions The high E-selectin EMP levels in stable patients with COPD are predictive of rapid FEV1 decline. Trial registration number UMIN000005168. PMID:24604485

Natural Products based P-glycoprotein Activators for Improved β-amyloid Clearance in Alzheimer's Disease: An in silico Approach.

PubMed

Shinde, Pravin; Vidyasagar, Nikhil; Dhulap, Sivakami; Dhulap, Abhijeet; Hirwani, Raj

2015-01-01

Alzheimer's disease is an age related disorder and is defined to be progressive, irreversible neurodegenerative disease. The potential targets which are associated with the Alzheimer's disease are cholinesterases, N-methyl-D-aspartate receptor, Beta secretase 1, Pregnane X receptor (PXR) and P-glycoprotein (Pgp). P-glycoprotein is a member of the ATP binding cassette (ABC) transporter family, which is an important integral of the blood-brain, blood-cerebrospinal fluid and the blood-testis barrier. Reports from the literature provide evidences that the up-regulation of the efflux pump is liable for a decrease in β -amyloid intracellular accumulation and is an important hallmark in Alzheimer's disease (AD). Thus, targeting β-amyloid clearance by stimulating Pgp could be a useful strategy to prevent Alzheimer's advancement. Currently available drugs provide limited effectiveness and do not assure to cure Alzheimer's disease completely. On the other hand, the current research is now directed towards the development of synthetic or natural based therapeutics which can delay the onset or progression of Alzheimer's disease. Since ancient time medicinal plants such as Withania somnifera, Bacopa monieri, Nerium indicum have been used to prevent neurological disorders including Alzheimer's disease. Till today around 125 Indian medicinal plants have been screened on the basis of ethnopharmacology for their activity against neurological disorders. In this paper, we report bioactives from natural sources which show binding affinity towards the Pgp receptor using ligand based pharmacophore development, virtual screening, molecular docking and molecular dynamics simulation studies for the bioactives possessing acceptable ADME properties. These bioactives can thus be useful to treat Alzheimer's disease.

Thrombopoietin contributes to enhanced platelet activation in cigarette smokers.

PubMed

Lupia, Enrico; Bosco, Ornella; Goffi, Alberto; Poletto, Cesare; Locatelli, Stefania; Spatola, Tiziana; Cuccurullo, Alessandra; Montrucchio, Giuseppe

2010-05-01

Thrombopoietin (TPO) is a humoral growth factor that primes platelet activation in response to several agonists. We recently showed that TPO enhances platelet activation in unstable angina and sepsis. Aim of this study was to investigate the role of TPO in platelet function abnormalities described in cigarette smokers. In a case-control study we enrolled 20 healthy cigarette smokers and 20 nonsmokers, and measured TPO and C-reactive protein (CRP), as well as platelet-leukocyte binding and P-selectin expression. In vitro we evaluated the priming activity of smoker or control plasma on platelet activation, and the role of TPO in this effect. We then studied the effects of acute smoking and smoking cessation on TPO levels and platelet activation indices. Chronic cigarette smokers had higher circulating TPO levels than nonsmoking controls, as well as increased platelet-leukocyte binding, P-selectin expression, and CRP levels. Serum cotinine concentrations correlated with TPO concentrations, platelet-monocyte aggregates and P-selectin expression. In addition, TPO levels significantly correlated with ex vivo platelet-monocyte aggregation and P-selectin expression. In vitro, the plasma from cigarette smokers, but not from nonsmoking controls, primed platelet-monocyte binding, which was reduced when an inhibitor of TPO was used. We also found that acute smoking slightly increased TPO levels, but did not affect platelet-leukocyte binding, whereas smoking cessation induced a significant decrease in both circulating TPO and platelet-leukocyte aggregation. Elevated TPO contributes to enhance platelet activation and platelet-monocyte cross-talk in cigarette smokers. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.

Pathological Fingerprints, Systems Biology and Biomarkers of Blast Brain Injury

DTIC Science & Technology

2010-06-01

in triplicate. *=pɘ.05 vs. sham ( noise exposed rats) according unpaired t-test analysis. NS- Not significant. E-selectin and L-selectin are adhesion...an energy balance, food intake, wakefulness . We measured serum Resistin and Orexin A levels after blast exposure using 2 different methods...penetrating flesh wounds (Belanger et al., 2005; Lew, 2005). However, even mild and moderate brain injuries can produce significant deficits , and when repeated

CLA and CD62E expression in oral lichen planus lesions.

PubMed

Werneck, Juliana Tristão; Dias, Eliane Pedra; Gonçalves, Lucio Souza; Silva Junior, Arley

2016-03-01

There are few reports on the migration of CLA+ T cells through E-selectin in cutaneous lichen planus, with only one study on oral lichen planus (OLP). This study aimed to analyze CLA expression and assess whether there is a correlation with E-selectin (CD62E) in OLP lesions. Biopsies were performed on 11 patients including two areas: one without clinical and histopathological features of OLP [perilesional group (PLG)] and the other with clinical and histopathological features of OLP [OLP group (OLPG)]. The specimens obtained were divided into two: One was fixed in formalin for routine analysis (H&E), and the other was frozen for CD3, CD4, CD8, CLA, and CD62E immunofluorescence markers. More CD4+ (median 1409, range 860-2519), CD8+ (median 1568, range 654-3258), and CLA+ T cells (median 958, range 453-2198) and higher CD62E expression (median 37, range 27-85) were identified in OLPG (P = 0.003; P = 0.003; P = 0.004; P = 0.003, respectively) than those in PLG. The median prevalence analysis was also significantly higher for CLA+CD8+ T cells in OLPG (OLPG = 39.4%, range 18.4-64.2; PLG = 29.4%, range 12.1-47.1) (P = 0.026). None of the correlations between CD3+ or CLA+ T cells and CD62E in OLPG and in PLG were significant. The significant presence of CLA+ T cells and E-selectin expressions in the OLPG suggests their involvement in the etiopathogenesis of OLP; however, only a weak correlation between CLA+ T cells and E-selectin was observed. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Association of the 98T ELAM-1 polymorphism with increased bleeding after cardiac surgery.

PubMed

Welsby, Ian J; Podgoreanu, Mihai V; Phillips-Bute, Barbara; Morris, Richard; Mathew, Joseph P; Smith, Peter K; Newman, Mark F; Schwinn, Debra A; Stafford-Smith, Mark

2010-06-01

Hemorrhage continues to be a major problem after cardiac surgery despite the routine use of antifibrinolytic drugs, with striking inter-patient variability poorly explained by already known risk factors. The authors tested the hypothesis that genetic polymorphisms of inflammatory mediators and cellular adhesion molecules are associated with bleeding after cardiac surgery. Prospective, observational study. Single, tertiary referral university heart center. Adult patients undergoing aortocoronary surgery with cardiopulmonary bypass. Patients (n = 759) had 10 mL of blood drawn preoperatively and genomic DNA isolated then genotyped for 17 polymorphisms in 7 candidate genes: tumor necrosis factor, interleukins 1beta and 6, interleukin 1 receptor antagonist, intercellular adhesion molecule-1 (ICAM-1), P-selectin and endothelial leucocyte adhesion molecule-1 (E-selectin). Multivariate analyses were used to relate clinical and genetic factors to bleeding and transfusion. The 98G/T polymorphism of the E-selectin gene was independently associated with bleeding after cardiac surgery (p = 0.002), after adjusting for significant clinical predictors (patient size and baseline hemoglobin concentration). There was a gene dose effect according to the number of minor alleles in the genotype; carriers of the minor allele bled 17% (GT) and 54% (TT) more than wild type (GG) genotypes, respectively (p = 0.01). Carriers of the minor allele also had longer activated partial thromboplastin times (p = 0.0023) and increased fresh frozen plasma transfusion (p = 0.03) compared with wild type. The authors found a dose-related association between the 98T E-selectin polymorphism and bleeding after cardiac surgery, independent of and additive to standard clinical risk factors. Copyright 2010 Elsevier Inc. All rights reserved.

The Human Cytomegalovirus-Specific UL1 Gene Encodes a Late-Phase Glycoprotein Incorporated in the Virion Envelope

PubMed Central

Shikhagaie, Medya; Mercé-Maldonado, Eva; Isern, Elena; Muntasell, Aura; Albà, M. Mar; López-Botet, Miguel; Hengel, Hartmut

2012-01-01

We have investigated the previously uncharacterized human cytomegalovirus (HCMV) UL1 open reading frame (ORF), a member of the rapidly evolving HCMV RL11 family. UL1 is HCMV specific; the absence of UL1 in chimpanzee cytomegalovirus (CCMV) and sequence analysis studies suggest that UL1 may have originated by the duplication of an ancestor gene from the RL11-TRL cluster (TRL11, TRL12, and TRL13). Sequence similarity searches against human immunoglobulin (Ig)-containing proteins revealed that HCMV pUL1 shows significant similarity to the cellular carcinoembryonic antigen-related (CEA) protein family N-terminal Ig domain, which is responsible for CEA ligand recognition. Northern blot analysis revealed that UL1 is transcribed during the late phase of the viral replication cycle in both fibroblast-adapted and endotheliotropic strains of HCMV. We characterized the protein encoded by hemagglutinin (HA)-tagged UL1 in the AD169-derived HB5 background. UL1 is expressed as a 224-amino-acid type I transmembrane glycoprotein which becomes detectable at 48 h postinfection. In infected human fibroblasts, pUL1 colocalized at the cytoplasmic site of virion assembly and secondary envelopment together with TGN-46, a marker for the trans-Golgi network, and viral structural proteins, including the envelope glycoprotein gB and the tegument phosphoprotein pp28. Furthermore, analyses of highly purified AD169 UL1-HA epitope-tagged virions revealed that pUL1 is a novel constituent of the HCMV envelope. Importantly, the deletion of UL1 in HCMV TB40/E resulted in reduced growth in a cell type-specific manner, suggesting that pUL1 may be implicated in regulating HCMV cell tropism. PMID:22345456

Fluorescent carbohydrate probes for cell lectins

NASA Astrophysics Data System (ADS)

Galanina, Oxana; Feofanov, Alexei; Tuzikov, Alexander B.; Rapoport, Evgenia; Crocker, Paul R.; Grichine, Alexei; Egret-Charlier, Marguerite; Vigny, Paul; Le Pendu, Jacques; Bovin, Nicolai V.

2001-09-01

Fluorescein labeled carbohydrate (Glyc) probes were synthesized as analytical tools for the study of cellular lectins, i.e. SiaLe x-PAA-flu, Sia 2-PAA-flu, GlcNAc 2-PAA-flu, LacNAc-PAA-flu and a number of similar ones, with PAA a soluble polyacrylamide carrier. The binding of SiaLe x-PAA-flu was assessed using CHO cells transfected with E-selectin, and the binding of Sia 2-PAA-flu was assessed by COS cells transfected with siglec-9. In flow cytometry assays, the fluorescein probes demonstrated a specific binding to the lectin-transfected cells that was inhibited by unlabeled carbohydrate ligands. The intense binding of SiaLe x-PAA- 3H to the E-selectin transfected cells and the lack of binding to both native and permeabilized control cells lead to the conclusion that the polyacrylamide carrier itself and the spacer arm connecting the carbohydrate moiety with PAA did not contribute anymore to the binding. Tumors were obtained from nude mice by injection of CHO E-selectin or mock transfected cells. The fluorescent SiaLe x-PAA-flu probe could bind to the tumor sections from E-selectin positive CHO cells, but not from the control ones. Thus, these probes can be used to reveal specifically the carbohydrate binding sites on cells in culture as well as cells in tissue sections. The use of the confocal spectral imaging technique with Glyc-PAA-flu probes offered the unique possibility to detect lectins in different cells, even when the level of lectin expression was rather low. The confocal mode of spectrum recording provided an analysis of the probe localization with 3D submicron resolution. The spectral analysis (as a constituent part of the confocal spectral imaging technique) enabled interfering signals of the probe and intrinsic cellular fluorescence to be accurately separated, the distribution of the probe to be revealed and its local concentration to be measured.

[Behavior of soluble L-selectin in HIV infected children].

PubMed

Gaddi, E; Balbaryski, J; Cantisano, C; Barboni, G; Candi, M; Quiroz, H; Giraudi, V

2001-01-01

L-selectin is an adhesion molecule that is responsible for the initial attachment of leukocytes to endothelium. After leukocyte activation L-selectin is endoproteolytically released from the cell surface. In order to analyze the relationship between soluble L-selectin (sL-selectin) and parameters of immune activation and disease progression, 51 HIV infected children and 15 healthy controls were studied. Serum L-selectin concentrations were significantly higher in HIV infected children than in the control group. Levels of sL-selectin were higher in HIV infected patients with severe immunologic suppression than in those with moderate or no evidence of suppression. A positive correlation between sL-selectin levels and LTCD8 counts, sL-selectin and soluble intercellular adhesion molecule-1 (sICAM-1) and immunogobulin A (IgA) levels was detected. On the contrary sL-selectin concentration did not correlate with plasmatic viral load. The correlation with parameters of immune activation may implicate involvement of sL-selectin in the immunopathogenesis of HIV infection.

Neutrophil cell surface receptors and their intracellular signal transduction pathways☆

PubMed Central

Futosi, Krisztina; Fodor, Szabina; Mócsai, Attila

2013-01-01

Neutrophils play a critical role in the host defense against bacterial and fungal infections, but their inappropriate activation also contributes to tissue damage during autoimmune and inflammatory diseases. Neutrophils express a large number of cell surface receptors for the recognition of pathogen invasion and the inflammatory environment. Those include G-protein-coupled chemokine and chemoattractant receptors, Fc-receptors, adhesion receptors such as selectins/selectin ligands and integrins, various cytokine receptors, as well as innate immune receptors such as Toll-like receptors and C-type lectins. The various cell surface receptors trigger very diverse signal transduction pathways including activation of heterotrimeric and monomeric G-proteins, receptor-induced and store-operated Ca2 + signals, protein and lipid kinases, adapter proteins and cytoskeletal rearrangement. Here we provide an overview of the receptors involved in neutrophil activation and the intracellular signal transduction processes they trigger. This knowledge is crucial for understanding how neutrophils participate in antimicrobial host defense and inflammatory tissue damage and may also point to possible future targets of the pharmacological therapy of neutrophil-mediated autoimmune or inflammatory diseases. PMID:23994464

E-selectin S128R polymorphism and severe coronary artery disease in Arabs

PubMed Central

Abu-Amero, Khaled K; Al-Boudari, Olayan M; Mohamed, Gamal H; Dzimiri, Nduna

2006-01-01

Background The E-selectin p. S128R (g. A561C) polymorphism has been associated with the presence of angiographic coronary artery disease (CAD) in some populations, but no data is currently available on its association with CAD in Arabs. Methods In the present study, we determined the potential relevance of the E-selectin S128R polymorphism for severe CAD and its associated risk factors among Arabs. We genotyped Saudi Arabs for this polymorphism by PCR, followed by restriction enzyme digestion. Results The polymorphism was determined in 556 angiographically confirmed severe CAD patients and 237 control subjects with no CAD as established angiographically (CON). Frequencies of the S/S, S/R and R/R genotypes were found as 81.1%, 16.6% and 2.3% in CAD patients and 87.8%, 11.8%, and 0.4% in CON subjects, respectively. The frequency of the mutant 128R allele was higher among CAD patients compared to CON group (11% vs. 6%; odds ratio = 1.76; 95% CI 1.14 – 2.72; p = .007), thus indicating a significant association of the 128R allele with CAD among our population. However, the stepwise logistic regression for the 128R allele and different CAD risk factors showed no significant association. Conclusion Among the Saudi population, The E-selectin p. S128R (g. A561C) polymorphism was associated with angiographic CAD in Univariate analysis, but lost its association in multivariate analysis. PMID:16756647

Regular consumption of vitamin D-fortified yogurt drink (Doogh) improved endothelial biomarkers in subjects with type 2 diabetes: a randomized double-blind clinical trial

PubMed Central

2011-01-01

Background Endothelial dysfunction has been proposed as the underlying cause of diabetic angiopathy that eventually leads to cardiovascular disease, the major cause of death in diabetes. We recently demonstrated the ameliorating effect of regular vitamin D intake on the glycemic status of patients with type 2 diabetes (T2D). In this study, the effects of improvement of vitamin D status on glycemic status, lipid profile and endothelial biomarkers in T2D subjects were investigated. Methods Subjects with T2D were randomly allocated to one of the two groups to receive either plain yogurt drink (PYD; containing 170 mg calcium and no vitamin D/250 mL, n1 = 50) or vitamin D3-fortified yogurt drink (FYD; containing 170 mg calcium and 500 IU/250 mL, n2 = 50) twice a day for 12 weeks. Anthropometric measures, glycemic status, lipid profile, body fat mass (FM) and endothelial biomarkers including serum endothelin-1, E-selectin and matrix metalloproteinase (MMP)-9 were evaluated at the beginning and after the 12-week intervention period. Results The intervention resulted in a significant improvement in fasting glucose, the Quantitative Insulin Check Index (QUICKI), glycated hemoglobin (HbA1c), triacylglycerols, high-density lipoprotein cholesterol (HDL-C), endothelin-1, E-selectin and MMP-9 in FYD compared to PYD (P < 0.05, for all). Interestingly, difference in changes of endothelin-1, E-selectin and MMP-9 concentrations in FYD compared to PYD (-0.35 ± 0.63 versus -0.03 ± 0.55, P = 0.028; -3.8 ± 7.3 versus 0.95 ± 8.3, P = 0.003 and -2.3 ± 3.7 versus 0.44 ± 7.1 ng/mL, respectively, P < 0.05 for all), even after controlling for changes of QUICKI, FM and waist circumference, remained significant for endothelin-1 and MMP-9 (P = 0.009 and P = 0.005, respectively) but disappeared for E-selectin (P = 0.092). On the contrary, after controlling for serum 25(OH)D, the differences disappeared for endothelin-1(P = 0.066) and MMP-9 (P = 0.277) but still remained significant for E-selectin (P = 0.011). Conclusions Ameliorated vitamin D status was accompanied by improved glycemic status, lipid profile and endothelial biomarkers in T2D subjects. Our findings suggest both direct and indirect ameliorating effects of vitamin D on the endothelial biomarkers. Trial registration ClinicalTrials.gov: NCT01236846 PMID:22114787

A Lectin Purified from Blood Red Bracket Mushroom, Pycnoporus sanguineus (Agaricomycetidae), Mycelium Displayed Affinity Toward Bovine Transferrin.

PubMed

Albores, Silvana; Moros, Maria; Cerdeiras, Maria Pia; de la Fuente, Jesus Martinez; Grazu, Valeria; Fraguas, Laura Franco

2016-01-01

Fungal lectins constitute excellent ligands for development of affinity adsorbents useful in affinity chromatography. In this work, a lectin was purified from Pycnoporus sanguineus (PSL) mycelium using 3 procedures: by affinity chromatography, using magnetic galactosyl-nanoparticles or galactose coupled to Sepharose, and by ionic exchange chromatography (IEC). The highest lectin yield was achieved by IEC (55%); SDS-PAGE of PSL showed 2 bands with molecular mass of 68.7 and 55.2 kDa and IEC displayed 2 bands at pi 5.5 and 5.2. The lectin agglutinates rat erythrocytes, exhibiting broad specificity toward several monosaccharides, including galactose. The agglutination was also inhibited by the glycoproteins fetal calf fetuin, bovine lactoferrin, bovine transferrin, and horseradish peroxidase. The lectin was then used to synthesize an affinity adsorbent (PSL-Sepharose) and the interaction with glycoproteins was evaluated by analyzing their chromatographic behaviors. The strongest interaction with the PSL-derivative was observed with transferrin, although lower interactions were also displayed toward fetuin and lactoferrin. These results indicate that the purified PSL constitutes an interesting ligand for the design of affinity adsorbents to be used (i.e., in glycoprotein purification).

The inhibitory and combinative mechanism of HZ08 with P-glycoprotein expressed on the membrane of Caco-2 cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yanyan; Hu, Yahui; Feng, Yidong

2014-01-15

Recently, the research and development of agents to reverse the phenomenon of multidrug resistance has been an attractive goal as well as a key approach to elevating the clinical survival of cancer patients. Although three generations of P-glycoprotein modulators have been identified, poor clearance and metabolism render these agents too toxic to be used in clinical application. HZ08, which has been under investigation for several years, shows a dramatic reversal effect with low cytotoxicity. For the first time, we aimed to describe the interaction between HZ08 and P-glycoprotein in Caco-2 cell line in which P-glycoprotein is overexpressed naturally. Cytotoxicity andmore » multidrug resistance reversal assays, together with flow cytometry, fluorescence microscopy and siRNA interference as well as Caco-2 monolayer transport model were employed in this study to evaluate the interaction between HZ08 and P-glycoprotein. This study revealed that HZ08 was capable of reversing adriamycin resistance mediated by P-glycoprotein as a result of intracellular enhancement of adriamycin accumulation, which was found to be superior to verapamil. In addition, we confirmed that HZ08 suppressed the transport of Rhodamine123 in the Caco-2 monolayer model but had little effect on P-glycoprotein expression. The transport of HZ08 was diminished by P-glycoprotein inhibitors (verapamil and LY335979) and its accumulation was increased via siRNA targeting MDR1 in Caco-2 cells. Furthermore, considering the binding site of P-glycoprotein, verapamil performed as a competitive inhibitor with HZ08. In conclusion, as a P-glycoprotein substrate, HZ08 inhibited P-glycoprotein activity and may share the same binding site of verapamil to P-glycoprotein. - Highlights: • The cytotoxicity and reversing effect of HZ08 was measured in Caco-2 cell line. • HZ08 inhibited the transport of Rhodamine123 across Caco-2 cell monolayer. • The efflux ratio of HZ08 was dropped when combined with P-glycoprotein inhibitors. • The accumulation of HZ08 increased via gene interference targeting P-glycoprotein. • HZ08 competitively bound to P-glycoprotein under the presence of verapamil.« less

Tapered oral dexamethasone for the acute chest syndrome of sickle cell disease.

PubMed

Quinn, Charles T; Stuart, Marie J; Kesler, Karen; Ataga, Kenneth I; Wang, Winfred C; Styles, Lori; Smith-Whitley, Kim; Wun, Ted; Raj, Ashok; Hsu, Lewis L; Krishnan, Suba; Kuypers, Frans A; Setty, Yamaja; Rhee, Seungshin; Key, Nigel S; Buchanan, George R

2011-10-01

Tapered oral dexamethasone for acute chest syndrome (ACS) in sickle cell anaemia was studied using a novel ACS assessment tool and investigational biomarkers. Twelve participants were randomized (mean age 17·3 years) before early study termination. Dexamethasone decreased duration of hospitalization for ACS by 20·8 h compared to placebo (P = 0·024). Rebound pain occurred in both groups (3 dexamethasone versus 1 placebo). Overall, dexamethasone decreased the leucocyte activation biomarker, sL-selectin; however, participants with rebound pain had higher sL-selectin within 24 h of treatment (dexamethasone or placebo). This ACS assessment tool was feasibly applied, and sL-selectin is a promising biomarker of ACS therapy. © 2011 Blackwell Publishing Ltd.

Ultrasound-Mediated Vascular Gene Transfection by Cavitation of Endothelial-Targeted Cationic Microbubbles

PubMed Central

Xie, Aris; Belcik, Todd; Qi, Yue; Morgan, Terry K.; Champaneri, Shivam A.; Taylor, Sarah; Davidson, Brian P.; Zhao, Yan; Klibanov, Alexander L.; Kuliszewski, Michael A.; Leong-Poi, Howard; Ammi, Azzdine; Lindner, Jonathan R.

2013-01-01

OBJECTIVES Ultrasound-mediated gene delivery can be amplified by acoustic disruption of microbubble carriers that undergo cavitation. We hypothesized that endothelial targeting of microbubbles bearing cDNA is feasible and, through optimizing proximity to the vessel wall, increases the efficacy of gene transfection. BACKGROUND Contrast ultrasound-mediated gene delivery is a promising approach for site-specific gene therapy, although there are concerns with the reproducibility of this technique and the safety when using high-power ultrasound. METHODS Cationic lipid-shelled decafluorobutane microbubbles bearing a targeting moiety were prepared and compared with nontargeted microbubbles. Microbubble targeting efficiency to endothelial adhesion molecules (P-selectin or intercellular adhesion molecule [ICAM]-1) was tested using in vitro flow chamber studies, intravital microscopy of tumor necrosis factor-alpha (TNF-α)–stimulated murine cremaster muscle, and targeted contrast ultrasound imaging of P-selectin in a model of murine limb ischemia. Ultrasound-mediated transfection of luciferase reporter plasmid charge coupled to microbubbles in the post-ischemic hindlimb muscle was assessed by in vivo optical imaging. RESULTS Charge coupling of cDNA to the microbubble surface was not influenced by the presence of targeting ligand, and did not alter the cavitation properties of cationic microbubbles. In flow chamber studies, surface conjugation of cDNA did not affect attachment of targeted microbubbles at microvascular shear stresses (0.6 and 1.5 dyne/cm2). Attachment in vivo was also not affected by cDNA according to intravital microscopy observations of venular adhesion of ICAM-1–targeted microbubbles and by ultrasound molecular imaging of P-selectin–targeted microbubbles in the post-ischemic hindlimb in mice. Transfection at the site of high acoustic pressures (1.0 and 1.8 MPa) was similar for control and P-selectin–targeted microbubbles but was associated with vascular rupture and hemorrhage. At 0.6 MPa, there were no adverse bioeffects, and transfection was 5-fold greater with P-selectin–targeted microbubbles. CONCLUSIONS We conclude that ultrasound-mediated transfection at safe acoustic pressures can be markedly augmented by endothelial juxtaposition. PMID:23236976

In silico Analysis for Predicting Fatty Acids of Black Cumin Oil as Inhibitors of P-Glycoprotein.

PubMed

Ali, Babar; Jamal, Qazi Mohd Sajid; Mir, Showkat R; Shams, Saiba; Al-Wabel, Naser A; Kamal, Mohammad A

2015-10-01

Black cumin oil is obtained from the seeds of Nigella sativa L. which belongs to family Ranunculaceae. The seed oil has been reported to possess antitumor, antioxidant, antibacterial, anti-inflammatory, hypoglycemic, central nervous system depressant, antioxidant, and immunostimulatory activities. These bioactivities have been attributed to the fixed oil, volatile oil, or their components. Seed oil consisted of 15 saturated fatty acids (17%) and 17 unsaturated fatty acids (82.9%). Long chain fatty acids and medium chain fatty acids have been reported to increase oral bioavailability of peptides, antibiotics, and other important therapeutic agents. In earlier studies, permeation enhancement and bioenhancement of drugs has been done with black cumin oil. In order to recognize the mechanism of binding of fatty acids to P-glycoprotein (P-gp), linoleic acid, oleic acid, margaric acid, cis-11, 14-eicosadienoic acid, and stearic acid were selected for in silico studies, which were carried out using AutoDock 4.2, based on the Lamarckian genetic algorithm principle. Template search with BLAST and HHblits has been performed against the SWISS-MODEL template library. The target sequence was searched with BLAST against the primary amino acid sequence of P-gp from Rattus norvegicus. The amount of energy needed by linoleic acid, oleic acid, eicosadienoic acid, margaric acid, and stearic acid to bind with P-gp were found to be - 10.60, -10.48, -9.95, -11.92, and - 10.37 kcal/mol, respectively. The obtained data support that all the selected fatty acids have contributed to inhibit P-gp activity thereby enhances the bioavailability of drugs. This study plays a significant role in finding hot spots in P-gp and may offer the further scope of designing potent and specific inhibitors of P-gp. Generation of 3D structure of fatty acid compounds from Black cumin oil and 3D homology modeling of Rat P glycoprotein as a receptor.Rat P-gp structure quality shows 88.5% residues in favored region obtained by Ramchandran plot analysis.Docking analysis revealed that Some amino acids common for all compounds like Ser221, Pro222, Ile224, Gly225, Ser228, Ala229, Lys233, Tyr302, Tyr309, Ile337, Leu338 and Thr341 in the P-gp and ligands binding patterns.Eicosadeinoic acid has highest binding affinity with P-gp as the amount of energy needed to bind with P-gp was lowest (-11.92 kcal/mol). Abbreviations used: P-gp: P-glycoprotein.

Platelet activation in pregnancy-induced hypertension.

PubMed

Karalis, Ioannis; Nadar, Sunil K; Al Yemeni, Eman; Blann, Andrew D; Lip, Gregory Y H

2005-01-01

Although excess platelet activation, as indicated by increased plasma beta thromboglobulin (beta-TG), has been shown in pregnancy-induced hypertension (PIH), platelet adhesion, platelet morphology and a comparison of platelet and soluble (plasma) levels of the adhesion molecules P-selectin (pPsel and sPsel, respectively) have not been studied. We conducted a cross-sectional study of 35 consecutive women with PIH (age 31+/-6 years), 31 consecutive women with normotensive pregnancies (age 29+/-5 years) and 30 normotensive non pregnant women (age 30+/-5 years). Platelet adhesion was studied in vitro by binding to fibrinogen-coated microwells, platelet morphology [mass and volume by flow cytometry], whole-platelet P-selectin (pPsel) by ELISA of the lysate of 2 x 10(8) cells, and the plasma markers soluble P-selectin (sP-sel) and beta-TG, by ELISA. The women with PIH had significantly raised sPsel, pPsel and (as expected) beta-TG (all p<0.05), when compared to the normotensive pregnant women and controls. However, in PIH platelet adhesion was similar to that in the normotensive pregnancy, but still higher than the normal controls (p<0.001). There was no difference among the three groups with respect to platelet mass and volume. pPsel and platelet adhesion correlated with gestational age and with systolic and diastolic blood pressure (all p<0.05). Increased platelet activation and adhesion develop during normal pregnancy, with some indices being further altered in PIH.

Multiple Drug Transport Pathways through human P-Glycoprotein(†)

PubMed Central

McCormick, James W.; Vogel, Pia D.; Wise, John G.

2015-01-01

P-glycoprotein (P-gp) is a plasma membrane efflux pump that is commonly associated with therapy resistances in cancers and infectious diseases. P-gp can lower the intracellular concentrations of many drugs to subtherapeutic levels by translocating them out of the cell. Because of the broad range of substrates transported by P-gp, overexpression of P-gp causes multidrug resistance. We reported previously on dynamic transitions of P-gp as it moved through conformations based on crystal structures of homologous ABCB1 proteins using in silico targeted molecular dynamics techniques. We expanded these studies here by docking transport substrates to drug binding sites of P-gp in conformations open to the cytoplasm, followed by cycling the pump through conformations that opened to the extracellular space. We observed reproducible transport of two substrates, daunorubicin and verapamil, by an average of 11 to 12 Å through the plane of the membrane as P-gp progressed through a catalytic cycle. Methyl-pyrophosphate, a ligand that should not be transported by P-gp, did not show this movement through P-gp. Drug binding to either of two subsites on P-gp appeared to determine the initial pathway used for drug movement through the membrane. The specific side-chain interactions with drugs within each pathway seemed to be, at least in part, stochastic. The docking and transport properties of a P-gp inhibitor, tariquidar, were also studied. A mechanism of inhibition by tariquidar is presented that involves stabilization of an outward open conformation with tariquidar bound in intracellular loops or at the drug binding domain of P-gp. PMID:26125482

Multiple Drug Transport Pathways through Human P-Glycoprotein.

PubMed

McCormick, James W; Vogel, Pia D; Wise, John G

2015-07-21

P-Glycoprotein (P-gp) is a plasma membrane efflux pump that is commonly associated with therapy resistances in cancers and infectious diseases. P-gp can lower the intracellular concentrations of many drugs to subtherapeutic levels by translocating them out of the cell. Because of the broad range of substrates transported by P-gp, overexpression of P-gp causes multidrug resistance. We reported previously on dynamic transitions of P-gp as it moved through conformations based on crystal structures of homologous ABCB1 proteins using in silico targeted molecular dynamics techniques. We expanded these studies here by docking transport substrates to drug binding sites of P-gp in conformations open to the cytoplasm, followed by cycling the pump through conformations that opened to the extracellular space. We observed reproducible transport of two substrates, daunorubicin and verapamil, by an average of 11-12 Å through the plane of the membrane as P-gp progressed through a catalytic cycle. Methylpyrophosphate, a ligand that should not be transported by P-gp, did not show this movement through P-gp. Drug binding to either of two subsites on P-gp appeared to determine the initial pathway used for drug movement through the membrane. The specific side-chain interactions with drugs within each pathway seemed to be, at least in part, stochastic. The docking and transport properties of a P-gp inhibitor, tariquidar, were also studied. A mechanism of inhibition by tariquidar that involves stabilization of an outward open conformation with tariquidar bound in intracellular loops or at the drug binding domain of P-gp is presented.

The immunosuppressive drug mycophenolate mofetil impairs the adhesion capacity of gastrointestinal tumour cells

PubMed Central

LECKEL, K; BEECKEN, W-D; JONAS, D; OPPERMANN, E; COMAN, M C; BECK, K-F; CINATL, J; HAILER, N P; AUTH, M K H; BECHSTEIN, W O; SHIPKOVA, M; BLAHETA, R A

2003-01-01

Immunosuppression correlates with the development and recurrence of cancer. Mycophenolate mofetil (MMF) has been shown to reduce adhesion molecule expression and leucocyte recruitment into the donor organ. We have hypothesized that MMF might also prevent receptor-dependent tumour dissemination. Therefore, we have investigated the effects of MMF on tumour cell adhesion to human umbilical vein endothelial cells (HUVEC) and compared them with the effects on T cell–endothelial cell interactions. Influence of MMF on cellular adhesion to HUVEC was analysed using isolated CD4+ and CD8+ T cells, or WiDr colon adenocarcinoma cells as the model tumour. HUVEC receptors ICAM-1, VCAM-1, E-selectin and P-selectin were detected by flow cytometry, Western blot or Northern blot analysis. Binding activity of T cells or WiDr cells in the presence of MMF were measured using immobilized receptor globulin chimeras. MMF potently blocked both T cell and WiDr cell binding to endothelium by 80%. Surface expression of the endothelial cell receptors was reduced by MMF in a dose-dependent manner. E-selectin mRNA was concurrently reduced with a maximum effect at 1 µm. Interestingly, MMF acted differently on T cells and WiDr cells. Maximum efficacy of MMF was reached at 10 and 1 µm, respectively. Furthermore, MMF specifically suppressed T cell attachment to ICAM-1, VCAM-1 and P-selectin. In contrast, MMF prevented WiDr cell attachment to E-selectin. In conclusion, our data reveal distinct effects of MMF on both T cell adhesion and tumour cell adhesion to endothelial cells. This suggests that MMF not only interferes with the invasion of alloactivated T cells, but might also be of value in managing post-transplantation malignancy. PMID:14616783

The immunosuppressive drug mycophenolate mofetil impairs the adhesion capacity of gastrointestinal tumour cells.

PubMed

Leckel, K; Beecken, W-D; Jonas, D; Oppermann, E; Coman, M C; Beck, K-F; Cinatl, J; Hailer, N P; Auth, M K H; Bechstein, W O; Shipkova, M; Blaheta, R A

2003-11-01

Immunosuppression correlates with the development and recurrence of cancer. Mycophenolate mofetil (MMF) has been shown to reduce adhesion molecule expression and leucocyte recruitment into the donor organ. We have hypothesized that MMF might also prevent receptor-dependent tumour dissemination. Therefore, we have investigated the effects of MMF on tumour cell adhesion to human umbilical vein endothelial cells (HUVEC) and compared them with the effects on T cell-endothelial cell interactions. Influence of MMF on cellular adhesion to HUVEC was analysed using isolated CD4+ and CD8+ T cells, or WiDr colon adenocarcinoma cells as the model tumour. HUVEC receptors ICAM-1, VCAM-1, E-selectin and P-selectin were detected by flow cytometry, Western blot or Northern blot analysis. Binding activity of T cells or WiDr cells in the presence of MMF were measured using immobilized receptor globulin chimeras. MMF potently blocked both T cell and WiDr cell binding to endothelium by 80%. Surface expression of the endothelial cell receptors was reduced by MMF in a dose-dependent manner. E-selectin mRNA was concurrently reduced with a maximum effect at 1 microm. Interestingly, MMF acted differently on T cells and WiDr cells. Maximum efficacy of MMF was reached at 10 and 1 microm, respectively. Furthermore, MMF specifically suppressed T cell attachment to ICAM-1, VCAM-1 and P-selectin. In contrast, MMF prevented WiDr cell attachment to E-selectin. In conclusion, our data reveal distinct effects of MMF on both T cell adhesion and tumour cell adhesion to endothelial cells. This suggests that MMF not only interferes with the invasion of alloactivated T cells, but might also be of value in managing post-transplantation malignancy.

HMGB1 induces an inflammatory response in endothelial cells via the RAGE-dependent endoplasmic reticulum stress pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Luo, Ying; Li, Shu-Jun; Yang, Jian

Highlights: •Mechanisms of inflammatory response induced by HMGB1 are incompletely understood. •We found that endoplasmic reticulum stress mediate the inflammatory response induced by HMGB1. •RAGE-mediated ERS pathways are involved in those processes. •We reported a new mechanism for HMGB1 induced inflammatory response. -- Abstract: The high mobility group 1B protein (HMGB1) mediates chronic inflammatory responses in endothelial cells, which play a critical role in atherosclerosis. However, the underlying mechanism is unknown. The goal of our study was to identify the effects of HMGB1 on the RAGE-induced inflammatory response in endothelial cells and test the possible involvement of the endoplasmic reticulummore » stress pathway. Our results showed that incubation of endothelial cells with HMGB1 (0.01–1 μg/ml) for 24 h induced a dose-dependent activation of endoplasmic reticulum stress transducers, as assessed by PERK and IRE1 protein expression. Moreover, HMGB1 also promoted nuclear translocation of ATF6. HMGB1-mediated ICAM-1 and P-selectin production was dramatically suppressed by PERK siRNA or IRE1 siRNA. However, non-targeting siRNA had no such effects. HMGB1-induced increases in ICAM-1 and P-selectin expression were also inhibited by a specific eIF2α inhibitor (salubrinal) and a specific JNK inhibitor (SP600125). Importantly, a blocking antibody specifically targeted against RAGE (anti-RAGE antibody) decreased ICAM-1, P-selectin and endoplasmic reticulum stress molecule (PERK, eIF2α, IRE1 and JNK) protein expression levels. Collectively, these novel findings suggest that HMGB1 promotes an inflammatory response by inducing the expression of ICAM-1 and P-selectin via RAGE-mediated stimulation of the endoplasmic reticulum stress pathway.« less

The Role of VP1 Amino Acid Residue 145 of Enterovirus 71 in Viral Fitness and Pathogenesis in a Cynomolgus Monkey Model

PubMed Central

Kataoka, Chikako; Suzuki, Tadaki; Kotani, Osamu; Iwata-Yoshikawa, Naoko; Nagata, Noriyo; Ami, Yasushi; Wakita, Takaji; Nishimura, Yorihiro; Shimizu, Hiroyuki

2015-01-01

Enterovirus 71 (EV71), a major causative agent of hand, foot, and mouth disease, occasionally causes severe neurological symptoms. We identified P-selectin glycoprotein ligand-1 (PSGL-1) as an EV71 receptor and found that an amino acid residue 145 in the capsid protein VP1 (VP1-145) defined PSGL-1-binding (PB) and PSGL-1-nonbinding (non-PB) phenotypes of EV71. However, the role of PSGL-1-dependent EV71 replication in neuropathogenesis remains poorly understood. In this study, we investigated viral replication, genetic stability, and the pathogenicity of PB and non-PB strains of EV71 in a cynomolgus monkey model. Monkeys were intravenously inoculated with cDNA-derived PB and non-PB strains of EV71, EV71-02363-EG and EV71-02363-KE strains, respectively, with two amino acid differences at VP1-98 and VP1-145. Mild neurological symptoms, transient lymphocytopenia, and inflammatory cytokine responses, were found predominantly in the 02363-KE-inoculated monkeys. During the early stage of infection, viruses were frequently detected in clinical samples from 02363-KE-inoculated monkeys but rarely in samples from 02363-EG-inoculated monkeys. Histopathological analysis of central nervous system (CNS) tissues at 10 days postinfection revealed that 02363-KE induced neuropathogenesis more efficiently than that induced by 02363-EG. After inoculation with 02363-EG, almost all EV71 variants detected in clinical samples, CNS, and non-CNS tissues, possessed a G to E amino acid substitution at VP1-145, suggesting a strong in vivo selection of VP1-145E variants and CNS spread presumably in a PSGL-1-independent manner. EV71 variants with VP1-145G were identified only in peripheral blood mononuclear cells in two out of four 02363-EG-inoculated monkeys. Thus, VP1-145E variants are mainly responsible for the development of viremia and neuropathogenesis in a non-human primate model, further suggesting the in vivo involvement of amino acid polymorphism at VP1-145 in cell-specific viral replication, in vivo fitness, and pathogenesis in EV71-infected individuals. PMID:26181772

IL-5-stimulated eosinophils adherent to periostin undergo stereotypic morphological changes and ADAM8-dependent migration.

PubMed

Johansson, M W; Khanna, M; Bortnov, V; Annis, D S; Nguyen, C L; Mosher, D F

2017-10-01

IL-5 causes suspended eosinophils to polarize with filamentous (F)-actin and granules at one pole and the nucleus in a specialized uropod, the "nucleopod," which is capped with P-selectin glycoprotein ligand-1 (PSGL-1). IL-5 enhances eosinophil adhesion and migration on periostin, an extracellular matrix protein upregulated in asthma by type 2 immunity mediators. Determine how the polarized morphology evolves to foster migration of IL-5-stimulated eosinophils on a surface coated with periostin. Blood eosinophils adhering to adsorbed periostin were imaged at different time points by fluorescent microscopy, and migration of eosinophils on periostin was assayed. After 10 minutes in the presence of IL-5, adherent eosinophils were polarized with PSGL-1 at the nucleopod tip and F-actin distributed diffusely at the opposite end. After 30-60 minutes, the nucleopod had dissipated such that PSGL-1 was localized in a crescent or ring away from the cell periphery, and F-actin was found in podosome-like structures. The periostin layer, detected with monoclonal antibody Stiny-1, shown here to recognize the FAS1 4 module, was cleared in wide areas around adherent eosinophils. Clearance was attenuated by metalloproteinase inhibitors or antibodies to disintegrin metalloproteinase 8 (ADAM8), a major eosinophil metalloproteinase previously implicated in asthma pathogenesis. ADAM8 was not found in podosome-like structures, which are associated with proteolytic activity in other cell types. Instead, immunoblotting demonstrated proteoforms of ADAM8 that lack the cytoplasmic tail in the supernatant. Anti-ADAM8 inhibited migration of IL-5-stimulated eosinophils on periostin. Migrating IL-5-activated eosinophils on periostin exhibit loss of nucleopodal features and appearance of prominent podosomes along with clearance of the Stiny-1 periostin epitope. Migration and epitope clearance are both attenuated by inhibitors of ADAM8. We propose, therefore, that eosinophils remodel and migrate on periostin-rich extracellular matrix in the asthmatic airway in an ADAM8-dependent manner, making ADAM8 a possible therapeutic target. © 2017 John Wiley & Sons Ltd.

Phase 1 Study of the E-Selectin Inhibitor GMI 1070 in Patients with Sickle Cell Anemia

PubMed Central

Wun, Ted; Styles, Lori; DeCastro, Laura; Telen, Marilyn J.; Kuypers, Frans; Cheung, Anthony; Kramer, William; Flanner, Henry; Rhee, Seungshin; Magnani, John L.; Thackray, Helen

2014-01-01

Background Sickle cell anemia is an inherited disorder of hemoglobin that leads to a variety of acute and chronic complications. Abnormal cellular adhesion, mediated in part by selectins, has been implicated in the pathophysiology of the vaso-occlusion seen in sickle cell anemia, and selectin inhibition was able to restore blood flow in a mouse model of sickle cell disease. Methods We performed a Phase 1 study of the selectin inhibitor GMI 1070 in patients with sickle cell anemia. Fifteen patients who were clinically stable received GMI 1070 in two infusions. Results The drug was well tolerated without significant adverse events. There was a modest increase in total peripheral white blood cell count without clinical symptoms. Plasma concentrations were well-described by a two-compartment model with an elimination T1/2 of 7.7 hours and CLr of 19.6 mL/hour/kg. Computer-assisted intravital microscopy showed transient increases in red blood cell velocity in 3 of the 4 patients studied. Conclusions GMI 1070 was safe in stable patients with sickle cell anemia, and there was suggestion of increased blood flow in a subset of patients. At some time points between 4 and 48 hours after treatment with GMI 1070, there were significant decreases in biomarkers of endothelial activation (sE-selectin, sP-selectin, sICAM), leukocyte activation (MAC-1, LFA-1, PM aggregates) and the coagulation cascade (tissue factor, thrombin-antithrombin complexes). Development of GMI 1070 for the treatment of acute vaso-occlusive crisis is ongoing. Trial Registration ClinicalTrials.gov NCT00911495 PMID:24988449

Whole blood flow cytometry measurements of in vivo platelet activation in critically-Ill patients are influenced by variability in blood sampling techniques.

PubMed

Rondina, Matthew T; Grissom, Colin K; Men, Shaohua; Harris, Estelle S; Schwertz, Hansjorg; Zimmerman, Guy A; Weyrich, Andrew S

2012-06-01

Flow cytometry is often used to measure in vivo platelet activation in critically-ill patients. Variability in blood sampling techniques, which may confound these measurements, remains poorly characterized. Platelet activation was measured by flow cytometry performed on arterial and venous blood from 116 critically-ill patients. We determined how variability in vascular sampling site, processing times, and platelet counts influenced levels of platelet-monocyte aggregates (PMA), PAC-1 binding (for glycoprotein (GP) IIbIIIa), and P-selectin (P-SEL) expression. Levels of PMA, but not PAC-1 binding or P-SEL expression, were significantly affected by variability in vascular sampling site. Average PMA levels were approximately 60% higher in whole blood drawn from an arterial vessel compared to venous blood (16.2±1.8% vs. 10.7±1.2%, p<0.05). Levels of PMA in both arterial and venous blood increased significantly during ex vivo processing delays (1.7% increase for every 10 minute delay, p<0.05). In contrast, PAC-1 binding and P-SEL expression were unaffected by processing delays. Levels of PMA, but not PAC-1 binding or P-SEL expression, were correlated with platelet count quartiles (9.4±1.6% for the lowest quartile versus 15.4±1.6% for the highest quartile, p<0.05). In critically-ill patients, variability in vascular sampling site, processing times, and platelet counts influence levels of PMA, but not PAC-1 binding or P-SEL expression. These data demonstrate the need for rigorous adherence to blood sampling protocols, particularly when levels of PMA, which are most sensitive to variations in blood collection, are measured for detection of in vivo platelet activation. Copyright © 2011 Elsevier Ltd. All rights reserved.

Biomarkers of coronary endothelial health: correlation with invasive measures of collateral function, flow and resistance in chronically occluded coronary arteries and the effect of recanalization.

PubMed

Ladwiniec, Andrew; Ettelaie, Camille; Cunnington, Michael S; Rossington, Jennifer; Thackray, Simon; Alamgir, Farquad; Hoye, Angela

2016-06-01

In the presence of a chronically occluded coronary artery, the collateral circulation matures by a process of arteriogenesis; however, there is considerable variation between individuals in the functional capacity of that collateral network. This could be explained by differences in endothelial health and function. We aimed to examine the relationship between the functional extent of collateralization and levels of biomarkers that have been shown to relate to endothelial health. We measured four potential biomarkers of endothelial health in 34 patients with mature collateral networks who underwent a successful percutaneous coronary intervention (PCI) for a chronic total coronary occlusion (CTO) before PCI and 6-8 weeks after PCI, and examined the relationship of biomarker levels with physiological measures of collateralization. We did not find a significant change in the systemic levels of sICAM-1, sE-selectin, microparticles or tissue factor 6-8 weeks after PCI. We did find an association between estimated retrograde collateral flow before CTO recanalization and lower levels of sICAM-1 (r=0.39, P=0.026), sE-selectin (r=0.48, P=0.005) and microparticles (r=0.38, P=0.03). Recanalization of a CTO and resultant regression of a mature collateral circulation do not alter systemic levels of sICAM-1, sE-selectin, microparticles or tissue factor. The identified relationship of retrograde collateral flow with sICAM-1, sE-selectin and microparticles is likely to represent an association with an ability to develop collaterals rather than their presence and extent.

Targeted Entry via Somatostatin Receptors Using a Novel Modified Retrovirus Glycoprotein That Delivers Genes at Levels Comparable to Those of Wild-Type Viral Glycoproteins

PubMed Central

Li, Fang; Ryu, Byoung Y.; Krueger, Robin L.; Heldt, Scott A.

2012-01-01

Here we report a novel viral glycoprotein created by replacing a natural receptor-binding sequence of the ecotropic Moloney murine leukemia virus envelope glycoprotein with the peptide ligand somatostatin. This new chimeric glycoprotein, which has been named the Sst receptor binding site (Sst-RBS), gives targeted transduction based on three criteria: (i) a gain of the use of a new entry receptor not used by any known virus; (ii) targeted entry at levels comparable to gene delivery by wild-type ecotropic Moloney murine leukemia virus and vesicular stomatitis virus (VSV) G glycoproteins; and (iii) a loss of the use of the natural ecotropic virus receptor. Retroviral vectors coated with Sst-RBS gained the ability to bind and transduce human 293 cells expressing somatostatin receptors. Their infection was specific to target somatostatin receptors, since a synthetic somatostatin peptide inhibited infection in a dose-dependent manner and the ability to transduce mouse cells bearing the natural ecotropic receptor was effectively lost. Importantly, vectors coated with the Sst-RBS glycoprotein gave targeted entry of up to 1 × 106 transducing U/ml, a level comparable to that seen with infection of vectors coated with the parental wild-type ecotropic Moloney murine leukemia virus glycoprotein through the ecotropic receptor and approaching that of infection of VSV G-coated vectors through the VSV receptor. To our knowledge, this is the first example of a glycoprotein that gives targeted entry of retroviral vectors at levels comparable to the natural capacity of viral envelope glycoproteins. PMID:22013043

Podocalyxin Is a Glycoprotein Ligand of the Human Pluripotent Stem Cell-Specific Probe rBC2LCN

PubMed Central

Tateno, Hiroaki; Matsushima, Asako; Hiemori, Keiko; Onuma, Yasuko; Ito, Yuzuru; Hasehira, Kayo; Nishimura, Ken; Ohtaka, Manami; Takayasu, Satoko; Nakanishi, Mahito; Ikehara, Yuzuru; Nakanishi, Mio; Ohnuma, Kiyoshi; Chan, Techuan; Toyoda, Masashi; Akutsu, Hidenori; Umezawa, Akihiro; Asashima, Makoto

2013-01-01

In comprehensive glycome analysis with a high-density lectin microarray, we have previously shown that the recombinant N-terminal domain of the lectin BC2L-C from Burkholderia cenocepacia (rBC2LCN) binds exclusively to undifferentiated human induced pluripotent stem (iPS) cells and embryonic stem (ES) cells but not to differentiated somatic cells. Here we demonstrate that podocalyxin, a heavily glycosylated type 1 transmembrane protein, is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. When analyzed by DNA microarray, podocalyxin was found to be highly expressed in both iPS cells and ES cells. Western and lectin blotting revealed that rBC2LCN binds to podocalyxin with a high molecular weight of more than 240 kDa in undifferentiated iPS cells of six different origins and four ES cell lines, but no binding was observed in either differentiated mouse feeder cells or somatic cells. The specific binding of rBC2LCN to podocalyxin prepared from a large set of iPS cells (138 types) and ES cells (15 types) was also confirmed using a high-throughput antibody-overlay lectin microarray. Alkaline digestion greatly reduced the binding of rBC2LCN to podocalyxin, indicating that the major glycan ligands of rBC2LCN are presented on O-glycans. Furthermore, rBC2LCN was found to exhibit significant affinity to a branched O-glycan comprising an H type 3 structure (Ka, 2.5 × 104 M−1) prepared from human 201B7 iPS cells, indicating that H type 3 is a most probable potential pluripotency marker. We conclude that podocalyxin is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. PMID:23526252

Serum levels of endothelial and neural cell adhesion molecules in prostate cancer.

PubMed

Lynch, D F; Hassen, W; Clements, M A; Schellhammer, P F; Wright, G L

1997-08-01

Tumorigenesis and progression to metastatic disease are accompanied by changes in the expression of cell adhesion molecules (CAMs). Normally expressed CAMs, such as E-cadherin, are lost, while others, i.e., ICAM-1, VCAM-1, NCAM, and E-selectin, are altered and overexpressed in progressive disease and metastases. Abnormal levels of these latter CAMs have been observed in melanoma and carcinomas of the colon and breast, and NCAM is overexpressed in small-cell lung carcinoma (SCLC). The objective of this study was to determine if serum levels of ICAM-1, VCAM-1, NCAM, and E-selectin could differentiate patients with benign prostate hypertrophy (BPH) from those with prostate carcinoma (CaP) and identify prostate cancers with high potential for progression to metastatic disease. Serum levels of these CAMs were determined by ELISA in serum from normal males and females and from patients with BPH and CaP before and after treatment. Sera from patients with breast carcinoma, colon carcinoma, melanoma, and small-cell lung carcinoma were also evaluated, as soluble CAMs have been reported to be elevated in these cancer patients. ICAM-1 levels were elevated in sera from patients with breast carcinoma (P = 0.0004) and melanoma (P = 0.0001). VCAM-1 levels were elevated in sera from patients with colon carcinoma (P = 0.0001). NCAM levels were elevated in the sera of patients with SCLC (P = 0.0001). Normal levels of ICAM-1, E-selectin, and NCAM were found in both BPH and pretreatment CaP patients. Median NCAM levels in hormone-refractive CaP patients were significantly greater than in BPH (P = 0.0005) and CaP patients with pathologically determined organ-confined (P = 0.0014) or nonorgan-confined disease (P = 0.0385). VCAM-1 levels were significantly elevated in both BPH patients (P = 0.0002) and CaP patients (P = 0.0002) when compared with levels for normal age-matched donors. None of the CAMs were found to offer an advantage over prostatic-specific antigen (PSA) for monitoring CaP patients following definitive radiotherapy, radical prostatectomy, or hormonal therapy. The results of this study indicate that serum ICAM-1, VCAM-1, NCAM, and E-selectin are not clinically useful biomarkers for differentiating CaP from BPH, for predicting progression, for identifying metastatic potential, or for monitoring treatment.

Effects of plasma nitric oxide levels on platelet activation in single donor apheresis and random donor concentrates.

PubMed

Büyükkağnici, Demet Iren; Ilhan, Osman; Kavas, Güzin Ozelçi; Arslan, Onder; Arat, Mutlu; Dalva, Klara; Ayyildiz, Erol

2007-02-01

P-selectin is an useful marker to determine platelet activation and nitric oxide inhibits platelet activation, secretion, adhesion and aggregation. The aim of this study was to investigate the relationship between nitric oxide and P-selectin values in both single donor apheresis and random donor platelet concentrates. According to the results of this study, we found that the best platelet concentrate is freshly prepared single donor apheresis concentrate and it is important to prevent activation at the beginning of the donation. Nitric oxide, which is synthesized from platelets during the storage period, is not sufficient to prevent platelet activation.

Vitamin D receptor activation induces P-glycoprotein and increases brain efflux of quinidine: an intracerebral microdialysis study in conscious rats.

PubMed

Durk, Matthew R; Fan, Jianghong; Sun, Huadong; Yang, Yingbo; Pang, Henrianna; Pang, K Sandy; de Lannoy, Inés A M

2015-03-01

Since the vitamin D receptor (VDR) was found to up-regulate cerebral P-glycoprotein expression in vitro and in mice, we extend our findings to rats by assessing the effect of rat Vdr activation on brain efflux of quinidine, a P-gp substrate that is eliminated primarily by cytochrome P450 3a. We treated rats with vehicle or the active VDR ligand, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] (4.8 or 6.4 nmol/kg i.p. every 2nd day × 4) and examined P-gp expression and cerebral quinidine disposition via microdialysis in control and treatment studies conducted longitudinally in the same rat. The 6.4 nmol/kg 1,25(OH)2D3 dose increased cerebral P-gp expression 1.75-fold whereas hepatic Cyp3a remained unchanged. Although there was no change in systemic clearance elicited by 1,25(OH)2D3, brain extracellular fluid quinidine concentrations were lower in treated rats. We noted that insertion of indwelling catheters increased plasma protein binding of quinidine and serial sampling decreased the blood:plasma concentration ratio, factors that alter distribution ratios in microdialysis studies. After appropriate correction, KECF/P,uu and KECF/B,uu, or ratios of quinidine unbound concentrations in brain extracellular fluid to plasma or blood at steady-state, were more than halved. We demonstrate that VDR activation increases cerebral P-gp expression and delimits brain penetration of P-gp substrates.

Non-Carbohydrate Glycomimetics and Glycoprotein Surrogates as DC-SIGN Antagonists and Agonists

PubMed Central

Prost, Lynne R.; Grim, Joseph C.; Tonelli, Marco; Kiessling, Laura L.

2012-01-01

An understanding of the biological roles of lectins will be advanced by ligands that can inhibit or even recruit lectin function. To this end, glycomimetics, non-carbohydrate ligands that function analogously to endogenous carbohydrates, are being sought. The advantage of having such ligands is illustrated by the many roles of the protein DC-SIGN. DC-SIGN is a C-type lectin displayed on dendritic cells, where it binds to mannosides and fucosides to mediate interactions with other host cells or bacterial or viral pathogens. DC-SIGN engagement can modulate host immune responses (e.g., suppress autoimmunity) or benefit pathogens (e.g., promote HIV dissemination). DC-SIGN can bind to glycoconjugates, internalize glycosylated cargo for antigen processing, and transduce signals. DC-SIGN ligands can serve as inhibitors as well as probes of the lectin’s function, so they are especially valuable for elucidating and controlling DC-SIGN’s roles in immunity. We previously reported a small molecule that embodies key features of the carbohydrates that bind DC-SIGN. Here, we demonstrate that this non-carbohydrate ligand acts as a true glycomimetic. Using NMR HSQC experiments, we found that the compound mimics saccharide ligands: It occupies the same carbohydrate-binding site and interacts with the same side chain residues on DC-SIGN. The glycomimetic also is functional. It had been shown previously to antagonize DC-SIGN function but here we use it to generate DC-SIGN agonists. Specifically, appending this glycomimetic to a protein scaffold affords a conjugate that elicits key cellular signaling responses. Thus, the glycomimetic can give rise to functional glycoprotein surrogates that elicit lectin-mediated signaling. PMID:22747463

Increased Expression of P-Glycoprotein Is Associated With Chlorpyrifos Resistance in the German Cockroach (Blattodea: Blattellidae).

PubMed

Hou, Weiyuan; Jiang, Chu; Zhou, Xiaojie; Qian, Kun; Wang, Lei; Shen, Yanhui; Zhao, Yan

2016-12-01

A principal method for control of the German cockroach, Blattella germanica (L.), is the broad-spectrum organophosphorus insecticide, chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate); however, extensive and repeated application has resulted in the development of resistance to chlorpyrifos in this insect. Evidence suggests that ATP-binding cassette protein transporters, including P-glycoprotein, are involved in insecticide resistance. However, little is known of the role of P-glycoprotein in insecticide resistance in the German cockroach. Here, we developed a chlorpyrifos-resistant strain of German cockroach and investigated the relationship between P-glycoprotein and chlorpyrifos resistance using toxicity assays; inhibition studies with two P-glycoprotein inhibitors, verapamil and quinine; P-glycoprotein-ATPase activity assays; and western blotting analysis. After 23 generations of selection from susceptible strain cockroaches, we obtained animals with high resistance to chlorpyrifos. When P-glycoprotein-ATPase activity was inhibited by verapamil and quinine, we observed enhanced susceptibility to chlorpyrifos in both control and chlorpyrifos-resistant cockroaches. No significant alterations of P-glycoprotein expression or ATPase activity were observed in cockroaches acutely exposed to LD50 doses of chlorpyrifos for 24 h, while P-glycoprotein expression and ATPase activity were clearly elevated in the chlorpyrifos-resistant cockroach strain. Thus, we conclude that P-glycoprotein is associated with chlorpyrifos resistance in the German cockroach and that elevated levels of P-glycoprotein expression and ATPase activity may be an important mechanism of chlorpyrifos resistance in the German cockroach. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Amino Acids 270 to 510 of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein Are Required for Interaction with Receptor

PubMed Central

Babcock, Gregory J.; Esshaki, Diana J.; Thomas, William D.; Ambrosino, Donna M.

2004-01-01

A novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), has recently been identified as the causative agent of severe acute respiratory syndrome (SARS). SARS-CoV appears similar to other coronaviruses in both virion structure and genome organization. It is known for other coronaviruses that the spike (S) glycoprotein is required for both viral attachment to permissive cells and for fusion of the viral envelope with the host cell membrane. Here we describe the construction and expression of a soluble codon-optimized SARS-CoV S glycoprotein comprising the first 1,190 amino acids of the native S glycoprotein (S1190). The codon-optimized and native S glycoproteins exhibit similar molecular weight as determined by Western blot analysis, indicating that synthetic S glycoprotein is modified correctly in a mammalian expression system. S1190 binds to the surface of Vero E6 cells, a cell permissive to infection, as demonstrated by fluorescence-activated cell sorter analysis, suggesting that S1190 maintains the biologic activity present in native S glycoprotein. This interaction is blocked with serum obtained from recovering SARS patients, indicating that the binding is specific. In an effort to map the ligand-binding domain of the SARS-CoV S glycoprotein, carboxy- and amino-terminal truncations of the S1190 glycoprotein were constructed. Amino acids 270 to 510 were the minimal receptor-binding region of the SARS-CoV S glycoprotein as determined by flow cytometry. We speculate that amino acids 1 to 510 of the SARS-CoV S glycoprotein represent a unique domain containing the receptor-binding site (amino acids 270 to 510), analogous to the S1 subunit of other coronavirus S glycoproteins. PMID:15078936

Structural Insights into SraP-Mediated Staphylococcus aureus Adhesion to Host Cells

PubMed Central

Zhang, Juan; Wang, Lei; Bai, Xiao-Hui; Zhang, Shi-Jie; Ren, Yan-Min; Li, Na; Zhang, Yong-Hui; Zhang, Zhiyong; Gong, Qingguo; Mei, Yide; Xue, Ting; Zhang, Jing-Ren; Chen, Yuxing; Zhou, Cong-Zhao

2014-01-01

Staphylococcus aureus, a Gram-positive bacterium causes a number of devastating human diseases, such as infective endocarditis, osteomyelitis, septic arthritis and sepsis. S. aureus SraP, a surface-exposed serine-rich repeat glycoprotein (SRRP), is required for the pathogenesis of human infective endocarditis via its ligand-binding region (BR) adhering to human platelets. It remains unclear how SraP interacts with human host. Here we report the 2.05 Å crystal structure of the BR of SraP, revealing an extended rod-like architecture of four discrete modules. The N-terminal legume lectin-like module specifically binds to N-acetylneuraminic acid. The second module adopts a β-grasp fold similar to Ig-binding proteins, whereas the last two tandem repetitive modules resemble eukaryotic cadherins but differ in calcium coordination pattern. Under the conditions tested, small-angle X-ray scattering and molecular dynamic simulation indicated that the three C-terminal modules function as a relatively rigid stem to extend the N-terminal lectin module outwards. Structure-guided mutagenesis analyses, in addition to a recently identified trisaccharide ligand of SraP, enabled us to elucidate that SraP binding to sialylated receptors promotes S. aureus adhesion to and invasion into host epithelial cells. Our findings have thus provided novel structural and functional insights into the SraP-mediated host-pathogen interaction of S. aureus. PMID:24901708

Intercellular transfer of P-glycoprotein from the drug resistant human bladder cancer cell line BIU-87 does not require cell-to-cell contact.

PubMed

Zhou, Hui-liang; Zheng, Yong-jun; Cheng, Xiao-zhi; Lv, Yi-song; Gao, Rui; Mao, Hou-ping; Chen, Qin

2013-09-01

The efflux activity of transmembrane P-glycoprotein prevents various therapeutic drugs from reaching lethal concentrations in cancer cells, resulting in multidrug resistance. We investigated whether drug resistant bladder cancer cells could transfer functional P-glycoprotein to sensitive parental cells. Drug sensitive BIU-87 bladder cancer cells were co-cultured for 48 hours with BIU-87/ADM, a doxorubicin resistant derivative of the same cell line, in a Transwell® system that prevented cell-to-cell contact. The presence of P-glycoprotein in recipient cell membranes was established using fluorescein isothiocyanate, laser scanning confocal microscopy and Western blot. P-glycoprotein mRNA levels were compared between cell types. Rhodamine 123 efflux assay was done to confirm that P-glycoprotein was biologically active. The amount of P-glycoprotein protein in BIU-87 cells co-cultured with BIU-87/ADM was significantly higher than in BIU-87 cells (0.44 vs 0.25) and BIU-87/H33342 cells (0.44 vs 0.26, each p <0.001), indicating P-glycoprotein transfer. P-glycoprotein mRNA expression was significantly higher in BIU-87/ADM cells than in co-cultured BIU-87 cells (1.28 vs 0.30), BIU-87/H33342 (0.28) and BIU-87 cells (0.25, each p <0.001), ruling out a genetic mechanism. After 30 minutes of efflux, rhodamine 123 fluorescence intensity was significantly lower in BIU-87/ADM cells (5.55 vs 51.45, p = 0.004) and co-cultured BIU-87 cells than in BIU-87 cells (14.22 vs 51.45, p <0.001), indicating that P-glycoprotein was functional. Bladder cancer cells can acquire functional P-glycoprotein through a nongenetic mechanism that does not require direct cell contact. This mechanism is consistent with a microparticle mediated process. Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Neem leaf glycoprotein enhances carcinoembryonic antigen presentation of dendritic cells to T and B cells for induction of anti-tumor immunity by allowing generation of immune effector/memory response.

PubMed

Sarkar, Koustav; Goswami, Shyamal; Roy, Soumyabrata; Mallick, Atanu; Chakraborty, Krishnendu; Bose, Anamika; Baral, Rathindranath

2010-08-01

Vaccination with neem leaf glycoprotein matured carcinoembryonic antigen (CEA) pulsed dendritic cells (DCs) enhances antigen-specific humoral and cellular immunity against CEA and restricts the growth of CEA(+) murine tumors. NLGP helps better CEA uptake, processing and presentation to T/B cells. This vaccination (DCNLGPCEA) elicits mitogen induced and CEA specific T cell proliferation, IFN gamma secretion and induces specific cytotoxic reactions to CEA(+) colon tumor cells. In addition to T cell response, DCNLGPCEA vaccine generates anti-CEA antibody response, which is principally IgG2a in nature. This antibody participates in cytotoxicity of CEA(+) cells in antibody-dependent manner. This strong anti-CEA cellular and humoral immunity protects mice from tumor development and these mice remained tumor free following second tumor inoculation, indicating generation of effector memory response. Evaluation of underlying mechanism suggests vaccination generates strong CEA specific CTL and antibody response that can completely prevent the tumor growth following adoptive transfer. In support, significant upregulation of CD44 on the surface of lymphocytes from DCNLGPCEA immunized mice was noticed with a substantial reduction in L-selectin (CD62L). (c) 2010 Elsevier B.V. All rights reserved.

Stable knock-down of the sphingosine 1-phosphate receptor S1P1 influences multiple functions of human endothelial cells.

PubMed

Krump-Konvalinkova, Vera; Yasuda, Satoshi; Rubic, Tina; Makarova, Natalia; Mages, Jörg; Erl, Wolfgang; Vosseler, Claudia; Kirkpatrick, C James; Tigyi, Gabor; Siess, Wolfgang

2005-03-01

Sphingosine 1-phosphate (S1P) is a bioactive phospholipid acting both as a ligand for the G protein-coupled receptors S1P1-5 and as a second messenger. Because S1P1 knockout is lethal in the transgenic mouse, an alternative approach to study the function of S1P1 in endothelial cells is needed. All human endothelial cells analyzed expressed abundant S1P1 transcripts. We permanently silenced (by RNA interference) the expression of S1P1 in the human endothelial cell lines AS-M.5 and ISO-HAS.1. The S1P1 knock-down cells manifested a distinct morphology and showed neither actin ruffles in response to S1P nor an angiogenic reaction. In addition, these cells were more sensitive to oxidant stress-mediated injury. New S1P1-dependent gene targets were identified in human endothelial cells. S1P1 silencing decreased the expression of platelet-endothelial cell adhesion molecule-1 and VE-cadherin and abolished the induction of E-selectin after cell stimulation with lipopolysaccharide or tumor necrosis factor-alpha. Microarray analysis revealed downregulation of further endothelial specific transcripts after S1P1 silencing. Long-term silencing of S1P1 enabled us for the first time to demonstrate the involvement of S1P1 in key functions of endothelial cells and to identify new S1P1-dependent gene targets.

Influence of atrial fibrillation on plasma von willebrand factor, soluble E-selectin, and N-terminal pro B-type natriuretic peptide levels in systolic heart failure.

PubMed

Freestone, Bethan; Gustafsson, Finn; Chong, Aun Yeong; Corell, Pernille; Kistorp, Caroline; Hildebrandt, Per; Lip, Gregory Y H

2008-05-01

Endothelial dysfunction is present in patients with heart failure (HF) due to left ventricular systolic dysfunction, as well as in patients with atrial fibrillation (AF) who have normal cardiac function. It is unknown whether AF influences the degree of endothelial dysfunction in patients with systolic HF. We measured levels of plasma von Willebrand factor (vWF) and E-selectin (as indexes of endothelial damage/dysfunction and endothelial activation, respectively; both enzyme-linked immunosorbent assay) in patients with AF and HF (AF-HF), who were compared to patients with sinus rhythm and HF (SR-HF), as well as in age-matched, healthy, control subjects. We also assessed the relationship of vWF and E-selectin to plasma N-terminal pro B-type natriuretic peptide (NTpro-BNP), a marker for HF severity and prognosis. One hundred ninety patients (73% men; mean age, 69.0 +/- 10.1 years [+/- SD]) with systolic HF were studied, who were compared to 117 healthy control subjects: 52 subjects (27%) were in AF, while 138 subjects (73%) were in sinus rhythm. AF-HF patients were older than SR-HF patients (p = 0.046), but left ventricular ejection fraction and New York Heart Association class were similar. There were significant differences in NT-proBNP (p < 0.0001) and plasma vWF (p = 0.003) between patients and control subjects. On Tukey post hoc analysis, AF-HF patients had significantly increased NT-proBNP (p < 0.001) and vWF (p = 0.0183) but not E-selectin (p = 0.071) levels when compared to SR-HF patients. On multivariate analysis, the presence of AF was related to plasma vWF levels (p = 0.018). Plasma vWF was also significantly correlated with NT-proBNP levels (Spearman r = 0.139; p = 0.017). There is evidence of greater endothelial damage/dysfunction in AF-HF patients when compared to SR-HF patients. The clinical significance of this is unclear but may have prognostic value.

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

PubMed Central

Victor, Victor M.; Rovira-Llopis, Susana; Bañuls, Celia; Diaz-Morales, Noelia; Martinez de Marañon, Arantxa; Rios-Navarro, Cesar; Alvarez, Angeles; Gomez, Marcelino; Rocha, Milagros; Hernández-Mijares, Antonio

2016-01-01

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. PMID:27007571

Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation, platelet aggregation, and P-selectin expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nygaard, Gyrid; Department of Biomedicine, University of Bergen, Bergen; Herfindal, Lars

Highlights: • We investigated the impact of cyclic nucleotide analogues on platelet activation. • Different time dependence were found for inhibition of platelet activation. • Additive effect was found using PKA- and PKG-activating analogues. • Our results may explain some of the discrepancies reported for cNMP signalling. - Abstract: In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigatedmore » whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation.« less

Iris Damage Is Associated With Elevated Cytokine Levels in Aqueous Humor.

PubMed

Aketa, Naohiko; Yamaguchi, Takefumi; Suzuki, Terumasa; Higa, Kazunari; Yagi-Yaguchi, Yukari; Satake, Yoshiyuki; Tsubota, Kazuo; Shimazaki, Jun

2017-05-01

To evaluate the association between iris damage and cytokine levels in the aqueous humor (AqH). A total of 201 AqH samples from 201 consecutive patients (mean age 73.7 ± 10.6) were collected at the beginning of corneal transplantation or cataract surgery. Iris damage of each case was assessed from preoperative slit-lamp findings based on its severity. The subjects were classified into three groups: eyes without iris damage (126 eyes), eyes with mild iris damage (51 eyes), and eyes with severe iris damage (24 eyes). The levels of cytokines (IL-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17a, interferon gamma-induced protein [IP]-10, monocyte chemotactic protein [MCP]-1, IFN-α, IFN-γ, macrophage inflammatory protein [MIP]-1α, MIP-1β, P-selectin, E-selectin, soluble intercellular adhesion molecule [sICAM]-1, TNF-α, and granulocyte-macrophage colony-stimulating factor [GM-CSF]) in AqH were measured by multiplex beads immunoassay. The levels of aqueous protein, IL-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-17A, MCP-1, TNF-α, E-selectin, P-selectin, and sICAM-1 in eyes with mild and severe iris damage were higher than in those without iris damage (P < 0.033). Multivariate analyses of clinical factors revealed that iris damage was associated with the history of complicated glaucoma, and the number of previous intraocular surgeries. The levels of AqH IL-6, IL-8, IL-13, MIP-1α, TNF-α, and sICAM-1 were significantly elevated in eyes with mild and severe iris damage in phakic eyes, and the levels of AqH IL-8 and sICAM-1 were significantly elevated in eyes with severe iris damage in pseudophakic eyes, compared with the eyes without iris damage (P < 0.045). Iris damage was associated with the elevation in the levels of aqueous protein and cytokines.

Plasma and serum L-selectin and clinical and subclinical the Multi-Ethnic Study of Atherosclerosis (MESA)cardiovascular disease

PubMed Central

BERARDI, CECILIA; DECKER, PAUL A.; KIRSCH, PHILLIP S.; DE ANDRADE, MARIZA; TSAI, MICHAEL Y.; PANKOW, JAMES S.; SALE, MICHELE M.; SICOTTE, HUGUES; TANG, WEIHONG; HANSON, NAOMI; POLAK, JOSEPH F.; BIELINSKI, SUZETTE J.

2014-01-01

L-selectin has been suggested to play a role in atherosclerosis. Previous studies on cardiovascular disease (CVD) and serum or plasma L-selectin are inconsistent. The association of serum L-selectin (sL-selectin) with carotid intima-media thickness, coronary artery calcium, ankle-brachial index (subclinical CVD) and incident CVD was assessed within 2403 participants in the Multi-Ethnic Study of Atherosclerosis (MESA). Regression analysis and the Tobit model were used to study subclinical disease; Cox Proportional Hazards regression for incident CVD. Mean age was 63 ± 10, 47% were males; mean sL-selectin was significantly different across ethnicities. Within each race/ethnicity, sL-selectin was associated with age and sex; among Caucasians and African Americans, it was associated with smoking status and current alcohol use. sL-selectin levels did not predict subclinical or clinical CVD after correction for multiple comparisons. Conditional logistic regression models were used to study plasma L-selectin and CVD within 154 incident CVD cases, occurred in a median follow up of 8.5 years, and 306 age-, sex-, and ethnicity-matched controls. L-selectin levels in plasma were significantly lower than in serum and the overall concordance was low. Plasma levels were not associated with CVD. In conclusion, this large multi-ethnic population, soluble L-selectin levels did not predict clinical or subclinical CVD. PMID:24631064

Glaucocalyxin A Inhibits Platelet Activation and Thrombus Formation Preferentially via GPVI Signaling Pathway

PubMed Central

Li, Qiang; Ren, Lijie; Liu, Xiaohui; Chu, Chunjun; Ozaki, Yukio; Zhang, Jian; Zhu, Li

2013-01-01

Platelets play a pivotal role in atherothrombosis and the antiplatelet agents have been proved to be useful in preventing onset of acute clinical events including myocardial infarction and stroke. Increasing number of natural compounds has been identified to be potential antiplatelet agents. Here we report the antiplatelet effect of glaucocalyxin A (GLA), an ent-diterpenoid that we isolated and purified from the aerial parts of Rabdosia japonica (Burm. f.) var. glaucocalyx (Maxim.) Hara, and investigate the molecular mechanisms by which GLA inhibits platelet activation and thrombus formation. The effect of GLA on platelet activation was measured using platelets freshly isolated from peripheral blood of healthy donors. Results showed that pretreatment of human platelets with lower concentrations of GLA (0.01μg/ml, 0.1μg/ml) significantly inhibited platelet aggregation induced by collagen (P<0.001) and CRP (P<0.01), a synthetic GPVI ligand, but not by ADP and U46619. Accordingly, GLA inhibited collagen-stimulated tyrosine phosphorylation of Syk, LAT, and phospholipase Cγ2, the signaling events in collagen receptor GPⅥ pathway. GLA also inhibited platelet p-selectin secretion and integrin activation by convulxin, a GPVI selective ligand. Additionally, GLA was found to inhibit low-dose thrombin-induced platelet activation. Using a flow chamber device, GLA was found to attenuate platelet adhesion on collagen surfaces in high shear condition. In vivo studies showed that GLA administration increased the time for complete occlusion upon vascular injury in mice, but did not extend tail-bleeding time when mice were administered with relatively lower doses of GLA. Therefore, the present results provide the molecular basis for the inhibition effect of GLA on platelet activation and its in vivo effect on thrombus formation, suggesting that GLA could potentially be developed as an antiplatelet and antithrombotic agent. PMID:24386454

Surgical treatment of haemorrhoids according to Longo and Milligan Morgan: an evaluation of postoperative tissue response.

PubMed

Krska, Z; Kvasnièka, J; Faltýn, J; Schmidt, D; Sváb, J; Kormanová, K; Hubík, J

2003-11-01

To compare by prospective randomised trial the postoperative tissue reaction of stapled vs. conventional haemorrhoidectomy. Fifty patients with stage III haemorrhoids underwent surgery for haemorrhoids. Group 1 (n = 25) had the Milligan-Morgan procedure; Group 2 (n = 25) had a stapled haemorrhoidectomy. All patients underwent measurements of endothelial dysfunction markers including E-selectin, P-selectin and intercellular adhesion molecule (ICAM). Acute-phase proteins including C-reactive protein, orosomucoid and fibrinogen were also measured. Estimations were made prior to surgery, immediately afterward surgery and on the first and fifth postoperative days. Assessment of clinical outcome was made one month after the surgery. There was a postoperative increase of acute-phase reactants in both groups. The patterns of the cures of the monitored parameters appeared similar in both groups. Lower values were found in Group 1, but the difference was not statistically significant except the level of fibrinogen on day 5, which was significantly higher in Group 2. E-selectin, P-selectin and ICAM showed similar time curves. Statistical analysis found the differences to be significant only when individual days were compared and not for the types of surgery. Raised ICAM and P-selectin on the fifth postoperative day was found in both groups. In Group 1, pain assessment by patients remained in the lower part of the pain rating scale, while in Group 2 it did not start declining until one week after surgery and became normal in the third to fourth weeks. In Group 1, the duration of hospitalization and the duration of incapacity for work were 50% of the values in Group 2. Patients having stapled haemorrhoidectomy have less pain and experience more rapid recovery when compared to classical haemorroidectomy. This was mirrored by the acute-phase protein CRP and fibrinogen levels postoperatively. There was no significant difference in other acute-phase reactants monitored, nor was there any difference in parameters of endothelial dysfunction. The techniques differ in extent of pain and duration of hospital stay and incapacity for work.

Assessment of Molecular Acoustic Angiography for Combined Microvascular and Molecular Imaging in Preclinical Tumor Models

PubMed Central

Lindsey, Brooks D.; Shelton, Sarah E.; Foster, F. Stuart; Dayton, Paul A.

2017-01-01

Purpose To evaluate a new ultrasound molecular imaging approach in its ability to image a preclinical tumor model and to investigate the capacity to visualize and quantify co-registered microvascular and molecular imaging volumes. Procedures Molecular imaging using the new technique was compared with a conventional ultrasound molecular imaging technique (multi-pulse imaging) by varying the injected microbubble dose and scanning each animal using both techniques. Each of the 14 animals was randomly assigned one of three doses; bolus dose was varied, and the animals were imaged for three consecutive days so that each animal received every dose. A microvascular scan was also acquired for each animal by administering an infusion of non-targeted microbubbles. These scans were paired with co-registered molecular images (VEGFR2-targeted microbubbles), the vessels were segmented, and the spatial relationships between vessels and VEGFR2 targeting locations were analyzed. In 5 animals, an additional scan was performed in which the animal received a bolus of microbubbles targeted to E- and P-selectin. Vessel tortuosity as a function of distance from VEGF and selectin targeting was analyzed in these animals. Results Although resulting differences in image intensity due to varying microbubble dose were not significant between the two lowest doses, superharmonic imaging had significantly higher contrast-to-tissue ratio (CTR) than multi-pulse imaging (mean across all doses: 13.98 dB for molecular acoustic angiography vs. 0.53 dB for multi-pulse imaging; p = 4.9 × 10−10). Analysis of registered microvascular and molecular imaging volumes indicated that vessel tortuosity decreases with increasing distance from both VEGFR2 and selectin targeting sites. Conclusions Molecular acoustic angiography (superharmonic molecular imaging) exhibited a significant increase in CTR at all doses tested due to superior rejection of tissue artifact signals. Due to the high resolution of acoustic angiography molecular imaging, it is possible to analyze spatial relationships in aligned microvascular and molecular superharmonic imaging volumes. Future studies are required to separate the effects of biomarker expression and blood flow kinetics in comparing local tortuosity differences between different endothelial markers such as VEGFR2, E-selectin and P-selectin. PMID:27519522

Monocyte B7 and Sialyl Lewis X modulates the efficacy of IL-10 down-regulation of LPS-induced monocyte tissue factor in whole blood.

PubMed

Warnes, G; Biggerstaff, J P; Francis, J L

1998-07-01

Recent studies have investigated the use of anti-inflammatory cytokine, interleukin 10 (IL-10) to control the development of disseminated intravascular coagulation (DIC) in sepsis by down-regulation of monocyte tissue factor (MTF) induced by lipopolysaccharide (LPS) in the initial phase of the disease. In vitro and in vivo human studies have shown that a minimal (<1 h) delay in IL-10 treatment significantly reduces the cytokines ability to inhibit LPS-induced MTF expression and the end products of coagulation. In this whole blood in vitro study we investigated the role of lymphocyte and platelet interactions with monocytes to up-regulate MTF expression in the presence of IL-10 in the initial phase of exposure to LPS. Individual blockade of monocyte B7 or platelet P-selectin significantly (35%) reduced MTF expression (P<0.05). IL-10 showed a dose-dependent inhibition of LPS (0.1 microg/ml) induced MTF expression, with 56% inhibition at 1 ng/ml, maximizing at 5 ng/ml IL-10 (75%; P<0.05). Simultaneous exposure to LPS and IL-10 (1 ng/ml) or addition of IL-10 1 h after LPS, with individual B7 and P-selectin blockade significantly enhanced the inhibition of MTF expression by IL-10 (P<0.05). We conclude that the efficacy of IL-10 to control DIC could be enhanced by a simultaneous B7 and P-selectin blockade.

Amyloid-β peptide on sialyl-Lewis(X)-selectin-mediated membrane tether mechanics at the cerebral endothelial cell surface.

PubMed

Askarova, Sholpan; Sun, Zhe; Sun, Grace Y; Meininger, Gerald A; Lee, James C-M

2013-01-01

Increased deposition of amyloid-β peptide (Aβ) at the cerebral endothelial cell (CEC) surface has been implicated in enhancement of transmigration of monocytes across the brain blood barrier (BBB) in Alzheimer's disease (AD). In this study, quantitative immunofluorescence microscopy (QIM) and atomic force microscopy (AFM) with cantilevers biofunctionalized by sialyl-Lewis(x) (sLe(x)) were employed to investigate Aβ-altered mechanics of membrane tethers formed by bonding between sLe(x) and p-selectin at the CEC surface, the initial mechanical step governing the transmigration of monocytes. QIM results indicated the ability for Aβ to increase p-selectin expression at the cell surface and promote actin polymerization in both bEND3 cells (immortalized mouse CECs) and human primary CECs. AFM data also showed the ability for Aβ to increase cell stiffness and adhesion probability in bEND3 cells. On the contrary, Aβ lowered the overall force of membrane tether formation (Fmtf ), and produced a bimodal population of Fmtf , suggesting subcellular mechanical alterations in membrane tethering. The lower Fmtf population was similar to the results obtained from cells treated with an F-actin-disrupting drug, latrunculin A. Indeed, AFM results also showed that both Aβ and latrunculin A decreased membrane stiffness, suggesting a lower membrane-cytoskeleton adhesion, a factor resulting in lower Fmtf . In addition, these cerebral endothelial alterations induced by Aβ were abrogated by lovastatin, consistent with its anti-inflammatory effects. In sum, these results demonstrated the ability for Aβ to enhance p-selectin expression at the CEC surface and induce cytoskeleton reorganization, which in turn, resulted in changes in membrane-cytoskeleton adhesion and membrane tethering, mechanical factors important in transmigration of monocytes through the BBB.

Amyloid-β Peptide on Sialyl-LewisX-Selectin-Mediated Membrane Tether Mechanics at the Cerebral Endothelial Cell Surface

PubMed Central

Askarova, Sholpan; Sun, Zhe; Sun, Grace Y.; Meininger, Gerald A.; Lee, James C-M.

2013-01-01

Increased deposition of amyloid-β peptide (Aβ) at the cerebral endothelial cell (CEC) surface has been implicated in enhancement of transmigration of monocytes across the brain blood barrier (BBB) in Alzheimer's disease (AD). In this study, quantitative immunofluorescence microscopy (QIM) and atomic force microscopy (AFM) with cantilevers biofunctionalized by sialyl-Lewisx (sLex) were employed to investigate Aβ-altered mechanics of membrane tethers formed by bonding between sLex and p-selectin at the CEC surface, the initial mechanical step governing the transmigration of monocytes. QIM results indicated the ability for Aβ to increase p-selectin expression at the cell surface and promote actin polymerization in both bEND3 cells (immortalized mouse CECs) and human primary CECs. AFM data also showed the ability for Aβ to increase cell stiffness and adhesion probability in bEND3 cells. On the contrary, Aβ lowered the overall force of membrane tether formation (Fmtf), and produced a bimodal population of Fmtf, suggesting subcellular mechanical alterations in membrane tethering. The lower Fmtf population was similar to the results obtained from cells treated with an F-actin-disrupting drug, latrunculin A. Indeed, AFM results also showed that both Aβ and latrunculin A decreased membrane stiffness, suggesting a lower membrane-cytoskeleton adhesion, a factor resulting in lower Fmtf. In addition, these cerebral endothelial alterations induced by Aβ were abrogated by lovastatin, consistent with its anti-inflammatory effects. In sum, these results demonstrated the ability for Aβ to enhance p-selectin expression at the CEC surface and induce cytoskeleton reorganization, which in turn, resulted in changes in membrane-cytoskeleton adhesion and membrane tethering, mechanical factors important in transmigration of monocytes through the BBB. PMID:23593361

Numerical analysis of cell adhesion in capillary flow

NASA Astrophysics Data System (ADS)

Takeishi, Naoki; Imai, Yohsuke; Ishida, Shunichi; Omori, Toshihiro; Kamm, Roger; Ishikawa, Takuji

2016-11-01

Numerical simulation of cell adhesion was performed for capillaries whose diameter is comparable to or smaller than that of the cell. Despite a lot of works about leukocyte and tumor cell rolling, cell motion in capillaries has remained unclear. The solid and fluid mechanics of a cell in flow was coupled with a slip bond model of ligand-receptor interactions. When the size of a capillary was reduced, the cell always transitioned to "bullet-like" motion, with a consequent decrease in the velocity of the cell. A state diagram is obtained for various values of capillary diameter and receptor density. According to our numerical results, bullet motion enables firm adhesion of a cell to the capillary wall even for a weak ligand-receptor binding. We also quantified effects of various parameters, including the dissociation rate constant, the spring constant, and the reactive compliance on the characteristics of cell motion. Our results suggest that even under the interaction between PSGL-1 and P-selectin, which is mainly responsible for leukocyte rolling, a cell is able to show firm adhesion in a small capillary. These findings may help in understanding such phenomena as leukocyte plugging and cancer metastasis. This research was supported by JSPS KAKENHI Grant Numbers 25000008, 26107703, 14J03967. We also acknowledge support from the Tohoku University Division for International Advanced Research and Education Organization.

Acetaminophen Modulates P-Glycoprotein Functional Expression at the Blood-Brain Barrier by a Constitutive Androstane Receptor–Dependent Mechanism

PubMed Central

Thompson, Brandon J.; Sanchez-Covarrubias, Lucy; Zhang, Yifeng; Laracuente, Mei-Li; Vanderah, Todd W.; Ronaldson, Patrick T.; Davis, Thomas P.

2013-01-01

Effective pharmacologic treatment of pain with opioids requires that these drugs attain efficacious concentrations in the central nervous system (CNS). A primary determinant of CNS drug permeation is P-glycoprotein (P-gp), an endogenous blood-brain barrier (BBB) efflux transporter that is involved in brain-to-blood transport of opioid analgesics (i.e., morphine). Recently, the nuclear receptor constitutive androstane receptor (CAR) has been identified as a regulator of P-gp functional expression at the BBB. This is critical to pharmacotherapy of pain/inflammation, as patients are often administered acetaminophen (APAP), a CAR-activating ligand, in conjunction with an opioid. Our objective was to investigate, in vivo, the role of CAR in regulation of P-gp at the BBB. Following APAP treatment, P-gp protein expression was increased up to 1.4–1.6-fold in a concentration-dependent manner. Additionally, APAP increased P-gp transport of BODIPY-verapamil in freshly isolated rat brain capillaries. This APAP-induced increase in P-gp expression and activity was attenuated in the presence of CAR pathway inhibitor okadaic acid or transcriptional inhibitor actinomycin D, suggesting P-gp regulation is CAR-dependent. Furthermore, morphine brain accumulation was enhanced by P-gp inhibitors in APAP-treated animals, suggesting P-gp–mediated transport. A warm-water (50°C) tail-flick assay revealed a significant decrease in morphine analgesia in animals treated with morphine 3 or 6 hours after APAP treatment, as compared with animals treated concurrently. Taken together, our data imply that inclusion of APAP in a pain treatment regimen activates CAR at the BBB and increases P-gp functional expression, a clinically significant drug-drug interaction that modulates opioid analgesic efficacy. PMID:24019224

Celastraceae sesquiterpenes as a new class of modulators that bind specifically to human P-glycoprotein and reverse cellular multidrug resistance.

PubMed

Muñoz-Martínez, Francisco; Lu, Peihua; Cortés-Selva, Fernando; Pérez-Victoria, José María; Jiménez, Ignacio A; Ravelo, Angel G; Sharom, Frances J; Gamarro, Francisco; Castanys, Santiago

2004-10-01

Overexpression of ABCB1 (MDR1) P-glycoprotein, a multidrug efflux pump, is one mechanism by which tumor cells may develop multidrug resistance (MDR), preventing the successful chemotherapeutic treatment of cancer. Sesquiterpenes from Celastraceae family are natural compounds shown previously to reverse MDR in several human cancer cell lines and Leishmania strains. However, their molecular mechanism of reversion has not been characterized. In the present work, we have studied the ability of 28 dihydro-beta-agarofuran sesquiterpenes to reverse the P-glycoprotein-dependent MDR phenotype and elucidated their molecular mechanism of action. Cytotoxicity assays using human MDR1-transfected NIH-3T3 cells allowed us to select the most potent sesquiterpenes reversing the in vitro resistance to daunomycin and vinblastine. Flow cytometry experiments showed that the above active compounds specifically inhibited drug transport activity of P-glycoprotein in a saturable, concentration-dependent manner (K(i) down to 0.24 +/- 0.01 micromol/L) but not that of ABCC1 (multidrug resistance protein 1; MRP1), ABCC2 (MRP2), and ABCG2 (breast cancer resistance protein; BCRP) transporters. Moreover, sesquiterpenes inhibited at submicromolar concentrations the P-glycoprotein-mediated transport of [(3)H]colchicine and tetramethylrosamine in plasma membrane from CH(R)B30 cells and P-glycoprotein-enriched proteoliposomes, supporting that P-glycoprotein is their molecular target. Photoaffinity labeling in plasma membrane and fluorescence spectroscopy experiments with purified protein suggested that sesquiterpenes interact with transmembrane domains of P-glycoprotein. Finally, sesquiterpenes modulated P-glycoprotein ATPase-activity in a biphasic, concentration-dependent manner: they stimulated at very low concentrations but inhibited ATPase activity as noncompetitive inhibitors at higher concentrations. Sesquiterpenes from Celastraceae are promising P-glycoprotein modulators with potential applications in cancer chemotherapy because of their MDR reversal potency and specificity for P-glycoprotein.

Divergent effects of laughter and mental stress on arterial stiffness and central hemodynamics.

PubMed

Vlachopoulos, Charalambos; Xaplanteris, Panagiotis; Alexopoulos, Nikolaos; Aznaouridis, Konstantinos; Vasiliadou, Carmen; Baou, Katerina; Stefanadi, Elli; Stefanadis, Christodoulos

2009-05-01

To investigate the effect of laughter and mental stress on arterial stiffness and central hemodynamics. Arterial stiffness and wave reflections are independent predictors of cardiovascular risk. Chronic psychological stress is an independent risk factor for cardiovascular events, whereas acute stress deteriorates vascular function. Eighteen healthy individuals were studied on three occasions, according to a randomized, single-blind, crossover, sham procedure-controlled design. The effects of viewing a 30-minute segment of two films inducing laughter or stress were assessed. Carotid-femoral pulse wave velocity was used as an index of arterial stiffness; augmentation index was used as a measure of wave reflections. Laughter decreased pulse wave velocity (by 0.30 m/sec, p = .01), and augmentation index (by 2.72%, p = .05). Conversely, stress increased pulse wave velocity (by 0.29 m/sec, p = .05) and augmentation index (by 5.1%, p = .005). Laughter decreased cortisol levels by 1.67 microg/dl (p = .02), soluble P-selectin by 26 ng/ml (p = .02) and marginally von Willebrand factor (by 2.4%, p = .07) and increased total oxidative status (by 61 micromol/L, p < .001). Stress decreased interleukin-6 (by 0.11 pg/ml, p = .04) and increased total oxidative status (by 44 micromol/L, p = .007). Soluble CD40 ligand and fibrinogen remained unchanged. Positive (laughter) and negative (stress) behavioral interventions have divergent acute effects on arterial stiffness and wave reflections. These findings have important clinical implications extending the spectrum of lifestyle modifications that can ameliorate arterial function.

Low molecular weight fucoidan attenuates experimental abdominal aortic aneurysm through interfering the leukocyte-endothelial cells interaction.

PubMed

Zhou, Min; Ding, Yong; Cai, Liang; Wang, Yonggang; Lin, Changpo; Shi, Zhenyu

2018-05-01

Low molecular weight fucoidan (LMWF) is a sulfated polysaccharide extracted from Saccharina Japonica that presents high affinity for P-selectin and abolish selectin-dependent recruitment of leukocytes. We hypothesized that dietary intake of LMWF, as a competitive binding agent of P‑selectin, could limit the inflammatory infiltration and aneurysmal growth in an Angiotensin II‑induced abdominal aortic aneurysm (AAA) mouse model. The Gene Expression Omnibus database was used for gene expressions and gene set enrichment analysis. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that focal adhesion was involved in the development of AAA. However, dietary intake of LMWF could limit the enlargement of AAA, decreasing maximal aortic diameter and preserving elastin lamellae. Although LMWF did not decrease the circulatory monocytes count and lower the expression of P‑selectin in endothelium, it reduced macrophages infiltration in media and adventitia. Furthermore, matrix metalloproteinase expression was markedly downregulated, accompanied with reduced expression of inflammatory mediators, including interleukin 1β, tumor necrosis factor‑α and monocyte chemotactic protein‑1. The present study revealed a novel target for the treatment of AAA and the anti‑inflammatory effects of LMWF.

Targeted transport of nanocarriers into brain for theranosis with rabies virus glycoprotein-derived peptide.

PubMed

Fu, Chen; Xiang, Yonggang; Li, Xiaorong; Fu, Ailing

2018-06-01

For successful theranosis of brain diseases, limited access of therapeutic molecules across blood-brain barrier (BBB) needs be overcome in brain delivery. Currently, peptide derivatives of rabies virus glycoprotein (RVG) have been exploited as delivery ligands to transport nanocarriers across BBB and specifically into the brain. The targeting peptides usually conjugate to the nanocarrier surface, and the cargoes, including siRNA, miRNA, DNA, proteins and small molecular chemicals, are complexed or encapsulated in the nanocarriers. The peptide ligand of the RVG-modified nanocarriers introduces the conjugated targeted-delivery into the brain, and the cargoes are involved in disease theranosis. The peptide-modified nanocarriers have been applied to diagnose and treat various brain diseases, such as glioma, Alzheimer's disease, ischemic injury, protein misfolding diseases etc. Since the targeting delivery system has displayed good biocompatibility and desirable therapeutic effect, it will raise a potential application in treating brain diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Repurposing a Library of Human Cathepsin L Ligands: Identification of Macrocyclic Lactams as Potent Rhodesain and Trypanosoma brucei Inhibitors.

PubMed

Giroud, Maude; Dietzel, Uwe; Anselm, Lilli; Banner, David; Kuglstatter, Andreas; Benz, Jörg; Blanc, Jean-Baptiste; Gaufreteau, Delphine; Liu, Haixia; Lin, Xianfeng; Stich, August; Kuhn, Bernd; Schuler, Franz; Kaiser, Marcel; Brun, Reto; Schirmeister, Tanja; Kisker, Caroline; Diederich, François; Haap, Wolfgang

2018-04-26

Rhodesain (RD) is a parasitic, human cathepsin L (hCatL) like cysteine protease produced by Trypanosoma brucei ( T. b.) species and a potential drug target for the treatment of human African trypanosomiasis (HAT). A library of hCatL inhibitors was screened, and macrocyclic lactams were identified as potent RD inhibitors ( K i < 10 nM), preventing the cell-growth of Trypanosoma brucei rhodesiense (IC 50 < 400 nM). SARs addressing the S2 and S3 pockets of RD were established. Three cocrystal structures with RD revealed a noncovalent binding mode of this ligand class due to oxidation of the catalytic Cys25 to a sulfenic acid (Cys-SOH) during crystallization. The P-glycoprotein efflux ratio was measured and the in vivo brain penetration in rats determined. When tested in vivo in acute HAT model, the compounds permitted up to 16.25 (vs 13.0 for untreated controls) mean days of survival.

ACUTE CARDIOVASCULAR EFFECTS OF FIREFIGHTING AND ACTIVE COOLING DURING REHABILITATION

PubMed Central

Burgess, Jefferey L.; Duncan, Michael D.; Hu, Chengcheng; Littau, Sally R.; Caseman, Delayne; Kurzius-Spencer, Margaret; Davis-Gorman, Grace; McDonagh, Paul F.

2012-01-01

Objectives To determine the cardiovascular and hemostatic effects of fire suppression and post-exposure active cooling. Methods Forty-four firefighters were evaluated prior to and after a 12 minute live-fire drill. Next, 50 firefighters undergoing the same drill were randomized to post-fire forearm immersion in 10°C water or standard rehabilitation. Results In the first study, heart rate and core body temperature increased and serum C-reactive protein decreased but there were no significant changes in fibrinogen, sE-selectin or sL-selectin. The second study demonstrated an increase in blood coagulability, leukocyte count, factors VIII and X, cortisol and glucose, and a decrease in plasminogen and sP-selectin. Active cooling reduced mean core temperature, heart rate and leukocyte count. Conclusions Live-fire exposure increased core temperature, heart rate, coagulability and leukocyte count; all except coagulability were reduced by active cooling. PMID:23090161

Randomized comparison of intracoronary tirofiban versus urokinase as an adjunct to primary percutaneous coronary intervention in patients with acute ST-elevation myocardial infarction: results of the ICTUS-AMI trial.

PubMed

Zhu, Tian-qi; Zhang, Qi; Ding, Feng-hua; Qiu, Jian-ping; Jin, Hui-geng; Jiang, Li; Lu, Lin; Zhang, Rui-yan; Hu, Jian; Yang, Zhen-kun; Shen, Ying; Shen, Wei-feng

2013-08-01

No randomized trial has been performed to compare the efficacy of an intracoronary bolus of tirofiban versus urokinase during primary percutaneous coronary intervention (PCI). We investigated whether the effects of adjunctive therapy with an intracoronary bolus of urokinase was noninferior to the effects of an intracoronary bolus of tirofiban in patients with ST-elevation myocardial infarction (STEMI) undergoing PCI. A total of 490 patients with acute STEMI undergoing primary PCI were randomized to an intracoronary bolus of tirofiban (10 µg/kg; n = 247) or urokinase (250 kU/20 ml; n = 243). Serum levels of P-selectin, von Willebrand factor (vWF), CD40 ligand (CD40L), and serum amyloid A (SAA) in the coronary sinus were measured before and after intracoronary drug administration. The primary endpoint was the rate of complete ( ≥ 70%) ST-segment resolution (STR) at 90 minutes after intervention, and the noninferiority margin was set to 15%. In the intention-to-treat analysis, complete STR was achieved in 54.4% of patients treated with an intracoronary bolus of urokinase and in 60.6% of those treated with an intracoronary bolus of tirofiban (adjusted difference: -7.0%; 95% confidence interval: -15.7% to 1.8%). The corrected TIMI frame count of the infarct-related artery was lower, left ventricular ejection fraction was higher, and the 6-month major adverse cardiac event-free survival tended to be better in the intracoronary tirofiban group. An intracoronary bolus of tirofiban resulted in lower levels of P-selectin, vWF, CD40L, and SAA in the coronary sinus compared with an intracoronary bolus of urokinase after primary PCI (P < 0.05). An intracoronary bolus of urokinase as an adjunct to primary PCI for acute STEMI is not equally effective to an intracoronary bolus of tirofiban with respect to improvement in myocardial reperfusion assessed by STR. This may be caused by less reduction in coronary circulatory platelet activation and inflammation.

Chronic exposure to biomass fuel smoke and markers of endothelial inflammation

PubMed Central

Caravedo, Maria A; Herrera, Phabiola M; Mongilardi, Nicole; de Ferrari, Aldo; Davila-Roman, Victor G; Gilman, Robert H; Wise, Robert A; Miele, Catherine H; Miranda, J Jaime; Checkley, William

2016-01-01

Indoor smoke exposure may affect cardiovascular disease (CVD) risk via lung-mediated inflammation, oxidative stress, and endothelial inflammation. We sought to explore the association between indoor smoke exposure from burning biomass fuels and a selected group of markers for endothelial inflammation. We compared serum concentrations of amyloid A protein, E-selectin, soluble ICAM-1 and VCAM-1, von Willebrand factor (VWF), and high sensitivity C-reactive protein (hs-CRP) in 228 biomass exposed vs. 228 non-exposed participants living in Puno, Peru. Average age was 56 years (SD=13), average BMI was 26.5 kg/m2 (SD=4.4), 48% were male, 59.4% completed high school and 2% reported a physician diagnosis of CVD. In unadjusted analysis, serum levels of soluble ICAM-1 (330 vs. 302 ng/mL; p<0.001), soluble VCAM-1 (403 vs. 362 ng/mL; p<0.001), and E-selectin (54.2 vs. 52.7 ng/mL; p=0.05) were increased in biomass exposed vs. non-exposed participants, respectively; whereas serum levels of vWF (1148 vs. 1311 mU/mL; p<0.001) and hs-CRP (2.56 vs. 3.12 mg/L; p<0.001) were decreased, respectively. In adjusted analyses, chronic exposure to biomass fuels remained positively associated with serum levels of soluble ICAM-1 (p=0.03) and VCAM-1 (p=0.05) and E-selectin (p=0.05), and remained negatively associated with serum levels of vWF (p=0.02) and hs-CRP (p<0.001). Daily exposure to biomass fuel smoke was associated with important differences in specific biomarkers of endothelial inflammation and may help explain accelerated atherosclerosis among those who are chronically exposed. PMID:26476302

Clinical significance of circulating vascular cell adhesion molecule-1 to white matter disintegrity in Alzheimer's dementia.

PubMed

Huang, Chi-Wei; Tsai, Meng-Han; Chen, Nai-Ching; Chen, Wei-Hsi; Lu, Yan-Ting; Lui, Chun-Chung; Chang, Ya-Ting; Chang, Wen-Neng; Chang, Alice Y W; Chang, Chiung-Chih

2015-11-25

Endothelial dysfunction leads to worse cognitive performance in Alzheimer's dementia (AD). While both cerebrovascular risk factors and endothelial dysfunction lead to activation of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin, it is not known whether these biomarkers extend the diagnostic repertoire in reflecting intracerebral structural damage or cognitive performance. A total of 110 AD patients and 50 age-matched controls were enrolled. Plasma levels of VCAM-1, ICAM-1 and E-selectin were measured and correlated with the cognitive performance, white matter macro-structural changes, and major tract-specific fractional anisotropy quantification. The AD patients were further stratified by clinical dementia rating score (mild dementia, n=60; moderate-to-severe dementia, n=50). Compared with the controls, plasma levels of VCAM-1 (p< 0.001), ICAM-1 (p=0.028) and E-selectin (p=0.016) were significantly higher in the patients, but only VCAM-1 levels significantly reflected the severity of dementia (p< 0.001). In addition, only VCAM-1 levels showed an association with macro- and micro- white matter changes especially in the superior longitudinal fasciculus (p< 0.001), posterior thalamic radiation (p=0.002), stria terminalis (p=0.002) and corpus callosum (p=0.009), and were independent of, age and cortical volume. These tracts show significant association with MMSE, short term memory and visuospatial function. Meanwhile, while VCAM-1 level correlated significantly with short-term memory (p=0.026) and drawing (p=0.025) scores in the AD patients after adjusting for age and education, the significance disappeared after adjusting for global FA. Endothelial activation, especially VCAM-1, was of clinical significance in AD that reflects macro- and micro-structural changes and poor short term memory and visuospatial function.

Circulating endothelial progenitor cells in obese children and adolescents.

PubMed

Pires, António; Martins, Paula; Paiva, Artur; Pereira, Ana Margarida; Marques, Margarida; Castela, Eduardo; Sena, Cristina; Seiça, Raquel

2015-01-01

This study aimed to investigate the relationship between circulating endothelial progenitor cell count and endothelial activation in a pediatric population with obesity. Observational and transversal study, including 120 children and adolescents with primary obesity of both sexes, aged 6-17 years, who were recruited at this Cardiovascular Risk Clinic. The control group was made up of 41 children and adolescents with normal body mass index. The variables analyzed were: age, gender, body mass index, systolic and diastolic blood pressure, high-sensitivity C-reactive protein, lipid profile, leptin, adiponectin, homeostasis model assessment-insulin resistance, monocyte chemoattractant protein-1, E-selectin, asymmetric dimethylarginine and circulating progenitor endothelial cell count. Insulin resistance was correlated to asymmetric dimethylarginine (ρ=0.340; p=0.003), which was directly, but weakly correlated to E-selectin (ρ=0.252; p=0.046). High sensitivity C-reactive protein was not found to be correlated to markers of endothelial activation. Systolic blood pressure was directly correlated to body mass index (ρ=0.471; p<0.001) and the homeostasis model assessment-insulin resistance (ρ=0.230; p=0.012), and inversely correlated to adiponectin (ρ=-0.331; p<0.001) and high-density lipoprotein cholesterol (ρ=-0.319; p<0.001). Circulating endothelial progenitor cell count was directly, but weakly correlated, to body mass index (r=0.211; p=0.016), leptin (ρ=0.245; p=0.006), triglyceride levels (r=0.241; p=0.031), and E-selectin (ρ=0.297; p=0.004). Circulating endothelial progenitor cell count is elevated in obese children and adolescents with evidence of endothelial activation, suggesting that, during infancy, endothelial repairing mechanisms are present in the context of endothelial activation. Copyright © 2015 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

Comprehensive analog synthesis of (S)-valine thiazole peptidomimetic TTT-28 to understand enigmatic drug-binding sites of P-glycoprotein

NASA Astrophysics Data System (ADS)

Patel, Bhargav A.

P-glycoprotein (P-gp) is considered an important therapeutic target for reversal of multidrug resistance (MDR) in cancer. It recognizes a diverse range of chemically and mechanistically dissimilar drugs. It has been postulated that the efflux by P-gp plays a major role in failure of chemotherapy. Hence, researchers have been trying to obtain a potent inhibitor of P-gp with specificity to tumor sites. In this pursuit, we previously were able to obtain a novel (S)-valine thiazole-derived peptidomimetic compound 1 ( TTT-28), which showed potent reversal of MDR in vitro as well as in vivo compared to verapamil, a well-known MDR modulator. We have also found that compound 1 triggers ATPase stimulation when incubated with P-gp alike verapamil, which implies its mechanism of action as competitive in nature. In this study, we attempted to understand structural requirements of ligands binding to a perplexing drug-binding site of P-gp and affecting its ATPase function. Toward this goal, we prepared a novel set of 64 analogues by fine tuning lead compound 1. These synthesized analogues were tested using ATPase activity assay. During the course of the study, a potent stimulator (1) of ATPase activity was transformed into an ATPase inhibitory leads such as compounds 43 , 57 and 113. The ATPase inhibitory activity of these compounds is predominantly contributed by the presence of a cyclohexyl group in place of the 2-aminobenzophenone moiety of ATPase activity stimulatory lead compound 1. Molecular modeling studies suggested a need for specific interactions with the drug-binding site of P-gp to induce different conformational states of P-gp to produce either stimulation or inhibition of ATPase activity. Collectively, this comprehensive synthesis work will facilitate further research towards P-gp inhibitor development.

Relationship and significance between anti-β2-glycoproteinI antibodies and platelet activation state in patients with ulcerative colitis

PubMed Central

Gao, Yan-Hang; Gao, Pu-Jun; Wang, Chun-Guang; Wang, Xiao-Cong; Piao, Yun-Feng

2008-01-01

AIM: To study the relationship between anti-β2-glycoprotein I (aβ2GPI) antibodies and platelet activation state in patients with ulcerative colitis (UC) and its significance. METHODS: Peripheral blood samples were collected from 56 UC patients (34 males and 22 females, aged 43.5 years, range 21-66 years), including 36 at active stage and 20 at remission stage, and 25 sex-and age-matched controls. The level of aβ2GPI was measured by ELISA. The platelet activation markers, platelet activation complex-I (PAC-I) and P-selectin (CD62P) were detected by flow cytometry. RESULTS: The A value for IgG aβ2GPI in the active UC group was 0.61 ± 0.13, significantly higher than that in the remittent UC and control groups (0.50 ± 0.13 and 0.22 ± 0.14, P < 0.01). There was a significant difference between the two groups (P < 0.01). The A value for IgM aβ2GPI in the active and remittent UC groups was 0.43 ± 0.13 and 0.38 ± 0.12, significantly higher than that in the control group (0.20 ± 0.12, P < 0.01). However, there was no significant difference between the two groups (P > 0.05). The PAC-I positive rate for the active and remittent UC groups was 30.6% ± 7.6% and 19.6% ± 7.8% respectively, significantly higher than that for the control group (6.3% ± 1.7%, P < 0.01). There was a significant difference between the two groups (P < 0.01). The CD62P positive rate for the active and remittent UC groups was 45.0% ± 8.8% and 31.9% ± 7.8% respectively, significantly higher than that for the control group (9.2% ± 2.7%, P < 0.01). There was a significant difference between the two groups (P < 0.01). In the active UC group, the more severe the state of illness was, the higher the A value for IgG aβ2GPI was, and the positive rate for PAC-I and CD62P was positively correlated with the state of illness (Faβ2GPI = 3.679, P < 0.05; FPAC-I (%) = 5.346, P < 0.01; and FCD62P (%) = 5. 418, P < 0.01). Meanwhile, in the same state of illness, the A value for IgG aβ2GPI was positively correlated to the positive rates for PAC-I and CD62P. CONCLUSION: aβ2GPI level, platelet activation state and their relationship of them are closely correlated with the pathogenesis and development of UC. PMID:18205270

Relationship and significance between anti-beta2-glycoprotein I antibodies and platelet activation state in patients with ulcerative colitis.

PubMed

Gao, Yan-Hang; Gao, Pu-Jun; Wang, Chun-Guang; Wang, Xiao-Cong; Piao, Yun-Feng

2008-02-07

To study the relationship between anti-beta2-glycoprotein I (abeta2GPI) antibodies and platelet activation state in patients with ulcerative colitis (UC) and its significance. Peripheral blood samples were collected from 56 UC patients (34 males and 22 females, aged 43.5 years, range 21-66 years), including 36 at active stage and 20 at remission stage, and 25 sex-and age-matched controls. The level of abeta2GPI was measured by ELISA. The platelet activation markers, platelet activation complex-I (PAC-I) and P-selectin (CD62P) were detected by flow cytometry. The A value for IgG abeta2GPI in the active UC group was 0.61 +/- 0.13, significantly higher than that in the remittent UC and control groups (0.50 +/- 0.13 and 0.22 +/- 0.14, P < 0.01). There was a significant difference between the two groups (P < 0.01). The A value for IgM abeta2GPI in the active and remittent UC groups was 0.43 +/- 0.13 and 0.38 +/- 0.12, significantly higher than that in the control group (0.20 +/- 0.12, P < 0.01). However, there was no significant difference between the two groups (P > 0.05). The PAC-I positive rate for the active and remittent UC groups was 30.6% +/- 7.6% and 19.6% +/- 7.8% respectively, significantly higher than that for the control group (6.3% +/- 1.7%, P < 0.01). There was a significant difference between the two groups (P < 0.01). The CD62P positive rate for the active and remittent UC groups was 45.0% +/- 8.8% and 31.9% +/- 7.8% respectively, significantly higher than that for the control group (9.2% +/- 2.7%, P < 0.01). There was a significant difference between the two groups (P < 0.01). In the active UC group, the more severe the state of illness was, the higher the A value for IgG abeta2GPI was, and the positive rate for PAC-I and CD62P was positively correlated with the state of illness (Fabeta2GPI = 3.679, P < 0.05; FPAC-I (%) = 5.346, P < 0.01; and FCD62P (%) = 5. 418, P < 0.01). Meanwhile, in the same state of illness, the A value for IgG abeta2GPI was positively correlated to the positive rates for PAC-I and CD62P. abeta2GPI level, platelet activation state and their relationship of them are closely correlated with the pathogenesis and development of UC.

Influence of sialic acids on the galactose-recognizing receptor of rat peritoneal macrophages.

PubMed

Lee, H Y; Kelm, S; Michalski, J C; Schauer, R

1990-04-01

The interaction of the galactose-recognizing receptor from rat peritoneal macrophages with ligands containing terminal galactose residues, such as asialoorosomucoid, desialylated erythrocytes or lymphocytes, can be inhibited by free N-acetylneuraminic acid (Neu5Ac) and oligosaccharides or glycoproteins containing this sugar in terminal position. This effect of Neu5Ac on the receptor is specific. The other naturally occurring or most of synthetic neuraminic acid derivatives tested do not exhibit an equivalent inhibitory potency as Neu5Ac. Although free Neu5Ac inhibits 5-fold stronger (K50 = 0.2mM) than free galactose, clustering of Neu5Ac in oligosaccharides and glycoproteins does not lead to stronger inhibition, which is in contrast to galactose-containing ligands. A more branched (triantennary) sialooligosaccharide inhibits less than biantennary and unbranched sialooligosaccharides. This may be the reason, why complex sialic acid-containing ligands like native orosomucoid or blood cells are not bound and internalized by the macrophages. The dissociation of asialoorosomucoid from the receptor is slow under the influence of Neu5Ac and requires relatively high concentrations of this sugar, whereas the dissociation mediated by galactose is rapid and requires lower concentrations. An allosteric influence of Neu5Ac on the binding of galactose by the receptor is discussed.

Circulating microparticles and endogenous estrogen in newly menopausal women

PubMed Central

Jayachandran, M.; Litwiller, R. D.; Owen, W. G.; Miller, V. M.

2011-01-01

Background Estrogen modulates antithrombotic characteristics of the vascular endothelium and the interaction of blood elements with the vascular surface. A marker of these modulatory activities is formation of cell-specific microparticles. This study examined the relationship between blood-borne microparticles and endogenous estrogen at menopause. Methods Platelet activation and plasma microparticles were characterized from women being screened (n = 146) for the Kronos Early Estrogen Prevention Study. Women were grouped according to serum estrogen (< 20 pg/ml; low estrogen, n = 21 or > 40 pg/ml; high estrogen, n = 11). Results Age, body mass index, blood pressure and blood chemistries were the same in both groups. No woman was hypertensive, diabetic or a current smoker. Platelet counts, basal and activated expression of P-selectin on platelet membranes were the same, but activated expression of glycoprotein IIb/IIIa was greater in the high-estrogen group. Numbers of endothelium-, platelet-, monocyte- and granulocyte-derived microparticles were greater in the low-estrogen group. Of the total numbers of microparticles, those positive for phosphatidylserine and tissue factor were also greater in the low-estrogen group. Conclusion These results suggest that, with declines in endogenous estrogen at menopause, numbers of procoagulant microparticles increase and thus may provide a means to explore mechanisms for cardiovascular risk development in newly menopausal women. PMID:19051075

Bypassing P-Glycoprotein Drug Efflux Mechanisms: Possible Applications in Pharmacoresistant Schizophrenia Therapy

PubMed Central

Hoosain, Famida G.; Choonara, Yahya E.; Tomar, Lomas K.; Tyagi, Charu; du Toit, Lisa C.

2015-01-01

The efficient noninvasive treatment of neurodegenerative disorders is often constrained by reduced permeation of therapeutic agents into the central nervous system (CNS). A vast majority of bioactive agents do not readily permeate into the brain tissue due to the existence of the blood-brain barrier (BBB) and the associated P-glycoprotein efflux transporter. The overexpression of the MDR1 P-glycoprotein has been related to the occurrence of multidrug resistance in CNS diseases. Various research outputs have focused on overcoming the P-glycoprotein drug efflux transporter, which mainly involve its inhibition or bypassing mechanisms. Studies into neurodegenerative disorders have shown that the P-glycoprotein efflux transporter plays a vital role in the progression of schizophrenia, with a noted increase in P-glycoprotein function among schizophrenic patients, thereby reducing therapeutic outcomes. In this review, we address the hypothesis that methods employed in overcoming P-glycoprotein in cancer and other disease states at the level of the BBB and intestine may be applied to schizophrenia drug delivery system design to improve clinical efficiency of drug therapies. In addition, the current review explores polymers and drug delivery systems capable of P-gp inhibition and modulation. PMID:26491671

A comparison of cardiovascular risk indices in patients with polycystic ovary syndrome with and without coexisting nonalcoholic fatty liver disease.

PubMed

Dawson, Alison J; Sathyapalan, Thozhukat; Smithson, Jacqueline A J; Vince, Rebecca V; Coady, Anne-Marie; Ajjan, Ramzi; Kilpatrick, Eric S; Atkin, Stephen L

2014-06-01

Women with polycystic ovary syndrome (PCOS) have an adverse cardiovascular risk profile and an increased prevalence of nonalcoholic fatty liver disease (NAFLD), which is also associated with an adverse cardiovascular risk profile. To compare the cardiovascular risk profile of women with PCOS alone and women with PCOS and NAFLD. Twenty-five oligoanovulatory women with PCOS were screened for NAFLD (including liver biopsy if appropriate) and had their cardiovascular risk factors measured which included the inflammatory marker C-reactive protein (CRP), endothelial function {measured using endoPAT 2000 and serum markers [intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin and P-selectin]}, clot structure and function [maximum absorbance (MA) and lysis potential (LT)]. Twelve patients had confirmed PCOS without evidence of NAFLD, and 13 patients had confirmed PCOS with evidence of NAFLD. The PCOS and NAFLD group were heavier (BMI 43·9 ± 2·2 kg/m(2) ) compared with the PCOS alone group (BMI 37·6 ± 1·4 kg/m(2) P = 0·03). There was no difference in CRP (7·57 ± 0·95 vs 6·59 ± 1·87 mm P = 0·62) or endothelial function (RH-PAT 1·96 ± 0·1 vs 1·74 ± 0·16 P = 0·25), ICAM-1 (221 ± 48 vs 250 ± 60 ng/ml P = 0·19), VCAM-1 (2124 ± 78 vs 2314 ± 91 ng/ml P = 0·13), E-selectin (33·9 ± 3·3 vs 39·5 ± 15·5 ng/ml P = 0·31) and P-selectin (101·0 ± 6·6 vs 95·9 ± 10·2 ng/ml P = 0·69). There was no difference in clot formation or lysis. The patients with PCOS and NAFLD were heavier compared with patients with PCOS alone. Despite this, we were unable to demonstrate differences in inflammatory markers, endothelial function or clot structure and function, suggesting that severity of steatosis is not the most important determinant of cardiovascular risk in PCOS. © 2013 John Wiley & Sons Ltd.

Effects of propionyl-L-carnitine on ischemia-reperfusion injury in hamster cheek pouch microcirculation.

PubMed

Lapi, Dominga; Sabatino, Lina; Altobelli, Giovanna Giuseppina; Mondola, Paolo; Cimini, Vincenzo; Colantuoni, Antonio

2010-01-01

Propionyl-l-carnitine (pLc) exerts protective effects in different experimental models of ischemia-reperfusion (I/R). The aim of the present study was to assess the effects of intravenously and topically applied pLc on microvascular permeability increase induced by I/R in the hamster cheek pouch preparation. The hamster cheek pouch microcirculation was visualized by fluorescence microscopy. Microvascular permeability, leukocyte adhesion to venular walls, perfused capillary length, and capillary red blood cell velocity (V(RBC)) were evaluated by computer-assisted methods. E-selectin expression was assessed by in vitro analysis. Lipid peroxidation and reactive oxygen species (ROS) formation were determined by thiobarbituric acid-reactive substances (TBARS) and 2'-7'-dichlorofluorescein (DCF), respectively. In control animals, I/R caused a significant increase in permeability and in the leukocyte adhesion in venules. Capillary perfusion and V(RBC) decreased. TBARS levels and DCF fluorescence significantly increased compared with baseline. Intravenously infused pLc dose-dependently prevented leakage and leukocyte adhesion, preserved capillary perfusion, and induced vasodilation at the end of reperfusion, while ROS concentration decreased. Inhibition of nitric oxide synthase prior to pLc caused vasoconstriction and partially blunted the pLc-induced protective effects; inhibition of the endothelium-derived hyperpolarizing factor (EDHF) abolished pLc effects. Topical application of pLc on cheek pouch membrane produced the same effects as observed with intravenous administration. pLc decreased the E-selectin expression. pLc prevents microvascular changes induced by I/R injury. The reduction of permeability increase could be mainly due to EDHF release induce vasodilatation together with NO. The reduction of E-selectin expression prevents leukocyte adhesion and permeability increase.

Clinical significance of nailfold capillaroscopy in systemic lupus erythematosus: correlation with endothelial cell activation markers and disease activity.

PubMed

Kuryliszyn-Moskal, A; Ciolkiewicz, M; Klimiuk, P A; Sierakowski, S

2009-01-01

To evaluate whether nailfold capillaroscopy (NC) changes are associated with the main serum endothelial cell activation markers and the disease activity of systemic lupus erythematosus (SLE). Serum levels of vascular endothelial growth factor (VEGF), endothelin-1 (ET-1), soluble E-selectin (sE-selectin), and soluble thrombomodulin (sTM) were determined by an enzyme-linked immunosorbent assay (ELISA) in 80 SLE patients and 33 healthy controls. Nailfold capillary abnormalities were seen in 74 out of 80 (92.5%) SLE patients. A normal capillaroscopic pattern or mild changes were found in 33 (41.25%) and moderate/severe abnormalities in 47 (58.75%) of all SLE patients. In SLE patients a capillaroscopic score >1 was more frequently associated with the presence of internal organ involvement (p < 0.001) as well as with immunosuppressive therapy (p < 0.01). Significant differences were found in VEGF (p < 0.001), ET-1 (p < 0.001), sE-selectin (p < 0.01), and sTM (p < 0.001) serum concentrations between SLE patients with a capillaroscopic score > 1 and controls. SLE patients with severe/moderate capillaroscopic abnormalities showed significantly higher VEGF serum levels than patients with mild changes (p < 0.001). Moreover, there was a significant positive correlation between the severity of capillaroscopic changes and the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) (p < 0.005) as well as between capillaroscopic score and VEGF serum levels (p < 0.001). Our findings confirm the usefulness of NC as a non-invasive technique for the evaluation of microvascular involvement in SLE patients. A relationship between changes in NC, endothelial cell activation markers and clinical features of SLE suggest an important role for microvascular abnormalities in clinical manifestation of the disease.

Effects of Propionyl-L-Carnitine on Ischemia–Reperfusion Injury in Hamster Cheek Pouch Microcirculation

PubMed Central

Lapi, Dominga; Sabatino, Lina; Altobelli, Giovanna Giuseppina; Mondola, Paolo; Cimini, Vincenzo; Colantuoni, Antonio

2010-01-01

Background and purpose Propionyl-l-carnitine (pLc) exerts protective effects in different experimental models of ischemia–reperfusion (I/R). The aim of the present study was to assess the effects of intravenously and topically applied pLc on microvascular permeability increase induced by I/R in the hamster cheek pouch preparation. Methods The hamster cheek pouch microcirculation was visualized by fluorescence microscopy. Microvascular permeability, leukocyte adhesion to venular walls, perfused capillary length, and capillary red blood cell velocity (VRBC) were evaluated by computer-assisted methods. E-selectin expression was assessed by in vitro analysis. Lipid peroxidation and reactive oxygen species (ROS) formation were determined by thiobarbituric acid-reactive substances (TBARS) and 2′-7′-dichlorofluorescein (DCF), respectively. Results In control animals, I/R caused a significant increase in permeability and in the leukocyte adhesion in venules. Capillary perfusion and VRBC decreased. TBARS levels and DCF fluorescence significantly increased compared with baseline. Intravenously infused pLc dose-dependently prevented leakage and leukocyte adhesion, preserved capillary perfusion, and induced vasodilation at the end of reperfusion, while ROS concentration decreased. Inhibition of nitric oxide synthase prior to pLc caused vasoconstriction and partially blunted the pLc-induced protective effects; inhibition of the endothelium-derived hyperpolarizing factor (EDHF) abolished pLc effects. Topical application of pLc on cheek pouch membrane produced the same effects as observed with intravenous administration. pLc decreased the E-selectin expression. Conclusions pLc prevents microvascular changes induced by I/R injury. The reduction of permeability increase could be mainly due to EDHF release induce vasodilatation together with NO. The reduction of E-selectin expression prevents leukocyte adhesion and permeability increase. PMID:21423374

Comparison of In Vitro Assays in Selecting Radiotracers for In Vivo P-Glycoprotein PET Imaging

PubMed Central

Savolainen, Heli; Cantore, Mariangela; van de Steeg, Evita; Colabufo, Nicola A.; Elsinga, Philip H.; Windhorst, Albert D.

2017-01-01

Positron emission tomography (PET) imaging of P-glycoprotein (P-gp) in the blood-brain barrier can be important in neurological diseases where P-gp is affected, such as Alzheimer´s disease. Radiotracers used in the imaging studies are present at very small, nanomolar, concentration, whereas in vitro assays where these tracers are characterized, are usually performed at micromolar concentration, causing often discrepant in vivo and in vitro data. We had in vivo rodent PET data of [11C]verapamil, (R)-N-[18F]fluoroethylverapamil, (R)-O-[18F]fluoroethyl-norverapamil, [18F]MC225 and [18F]MC224 and we included also two new molecules [18F]MC198 and [18F]KE64 in this study. To improve the predictive value of in vitro assays, we labeled all the tracers with tritium and performed bidirectional substrate transport assay in MDCKII-MDR1 cells at three different concentrations (0.01, 1 and 50 µM) and also inhibition assay with P-gp inhibitors. As a comparison, we used non-radioactive molecules in transport assay in Caco-2 cells at a concentration of 10 µM and in calcein-AM inhibition assay in MDCKII-MDR1 cells. All the P-gp substrates were transported dose-dependently. At the highest concentration (50 µM), P-gp was saturated in a similar way as after treatment with P-gp inhibitors. Best in vivo correlation was obtained with the bidirectional transport assay at a concentration of 0.01 µM. One micromolar concentration in a transport assay or calcein-AM assay alone is not sufficient for correct in vivo prediction of substrate P-gp PET ligands. PMID:29036881

Drug resistance in epithelial ovarian cancer: P-glycoprotein and glutation S-transferase. Can they play an important role in detecting response to platinum-based chemotherapy as a first-line therapy.

PubMed

Simşek, T; Ozbilim, G; Gülkesen, H; Kaya, H; Sargin, F; Karaveli, S

2001-01-01

Drug resistance is important for the treatment of ovarian cancer. P-glycoprotein and glutation S-transferase as resistance markers play an important role in the effectivity of chemotherapeutical agents. The role of P-glycoprotein and glutation S-transferase in the treatment of epithelial ovarian cancer is not well understood. We investigated the relation between P-glycoprotein and glutation S-transferase level for response to platinum-based chemotherapy in epithelial ovarian cancer. We reviewed 30 cases diagnosed as epithelial ovarian cancer and treated with platinum-based chemotherapy in the Department of Obstetrics and Gynecology, Akdeniz University School of Medicine. The material was attained from initial parafin-embeded blocks stained for P-glycoprotein and glutation S-transferase. The cases that were diagnosed and treated before attending our clinic were not enrolled in the study. Mean age was 58.2 (25-70) and mean gravida 4.1 (0-10). Twenty-four patients (80%) were glutation S-transferase positive. Three cases (10%) out of 30 had positive reaction for P-glycoprotein. No difference was revealed regarding chemotherapy response rate among the cases showing glutation S-transferase positivity and P-glycoprotein negativity. Detection of glutation S-transferase and P-glycoprotein levels in epithelial ovarian cancer tissue is not important for response to platinum-based chemotherapy as a first line.

A sea lamprey glycoprotein hormone receptor similar with gnathostome thyrotropin hormone receptor.

PubMed

Freamat, Mihael; Sower, Stacia A

2008-10-01

The specificity of the vertebrate hypothalamic-pituitary-gonadal and hypothalamic-pituitary-thyroid axes is explained by the evolutionary refinement of the specificity of expression and selectivity of interaction between the glycoprotein hormones GpH (FSH, LH, and TSH) and their cognate receptors GpH-R (FSH-R, LH-R, and TSH-R). These two finely tuned signaling pathways evolved by gene duplication and functional divergence from an ancestral GpH/GpH-R pair. Comparative analysis of the protochordate and gnathostome endocrine systems suggests that this process took place prior or concomitantly with the emergence of the gnathostome lineage. Here, we report identification and characterization of a novel glycoprotein hormone receptor (lGpH-R II) in the Agnathan sea lamprey. This 781 residue protein was found approximately 43% identical with mammalian TSH-R and FSH-R representative sequences, and similarly with these two classes of mammalian receptors it is assembled from ten exons. A synthetic ligand containing the lamprey glycoprotein hormone beta-chain tethered upstream of a mammalian alpha-chain activated the lGpH-R II expressed in COS-7 cells but in a lesser extent than lGpH-R I. Molecular phylogenetic analysis of vertebrate GpH-R protein sequences suggests a closer relationship between lGpH-R II and gnathostome thyrotropin receptors. Overall, the presence and characteristics of the lamprey glycoprotein hormone receptors suggest existence of a primitive functionally overlapping glycoprotein hormone/glycoprotein hormone receptor system in this animal.

Prednisolone reduces experimental arthritis, and inflammatory tissue destruction in SCID mice infected with Borrelia burgdorferi.

PubMed

Hurtenbach, U; Böggemeyer, E; Stehle, T; Museteanu, C; Del Pozo, E; Simon, M M

1996-05-01

Glucocorticosteroids (GC) are widely used as anti-inflammatory agents. The effects of Prednisolone on the development of Borrelia (B.) burgdorferi-induced clinical arthritis and organ inflammation was studied in severe combined immunodeficiency (SCID) mice. The drug was administered orally at a dose of 3, 10 and 30 mg/kg, starting shortly before experimental infection of the mice. A dose dependent inhibition of arthritic joint swelling was observed. Full protection was obtained with 30 mg/kg until 21 days after infection, subsequently, mild joint swelling developed but progression and severity of the disease was considerably less than in the other treated as well as in the untreated mice. Inhibition of clinical arthritis coincided with reduction of inflammatory cell infiltration in the joints, liver and muscle. Prednisolone was ineffective when application was initiated after arthritis was fully developed, i.e., 22 days after infection. Since the activated endothelium plays a critical role in development of inflammatory lesions, the expression of the cellular adhesion molecules (CAMs) E-selectin, P-selectin, ICAM-1 and VCAM-1 was determined in vitro using the bEnd3 endothelial cell line. Stimulation with a sonicated B. burgdorferi preparation in the presence of the water-soluble compound Prednisolone-21-hemisuccinate considerably reduced expression of ICAM-1, and marginally also of E-selectin, whereas the level of P-selectin and VCAM-1 remained unaltered. Thus, downregulation of ICAM-1 might be a critical factor in Prednisolone-mediated inhibition of B. burgdorferi-induced inflammation; the flare up of the disease after the initial protection indicates that additional therapy, e.g. with antibiotics, is necessary.

Contribution of tumor endothelial cells to drug resistance: anti-angiogenic tyrosine kinase inhibitors act as p-glycoprotein antagonists.

PubMed

Bani, MariaRosa; Decio, Alessandra; Giavazzi, Raffaella; Ghilardi, Carmen

2017-05-01

Tumor endothelial cells (TEC) differ from the normal counterpart, in both gene expression and functionality. TEC may acquire drug resistance, a characteristic that is maintained in vitro. There is evidence that TEC are more resistant to chemotherapeutic drugs, substrates of ATP-binding cassette (ABC) transporters. TEC express p-glycoprotein (encoded by ABCB1), while no difference in other ABC transporters was revealed compared to normal endothelia. A class of tyrosine kinase inhibitors (TKI), used as angiostatic compounds, interferes with the ATPase activity of p-glycoprotein, thus impairing its functionality. The exposure of ovarian adenocarcinoma TEC to the TKIs sunitinib or sorafenib was found to abrogate resistance (proliferation and motility) to doxorubicin and paclitaxel in vitro, increasing intracellular drug accumulation. A similar effect has been reported by the p-glycoprotein inhibitor verapamil. No beneficial effect was observed in combination with cytotoxic drugs that are not p-glycoprotein substrates. The current paper reviews the mechanisms of TEC chemoresistance and shows the role of p-glycoprotein in mediating such resistance. Inhibition of p-glycoprotein by anti-angiogenic TKI might contribute to the beneficial effect of these small molecules, when combined with chemotherapy, in counteracting acquired drug resistance.

[Clinical study of hemostatic coagulation markers in prethrombosis state of pregnancy induced hypertension].

PubMed

Shi, Qing; Chen, Chen; Wang, Xue-Feng; Wang, Hong-Li

2004-11-01

To investigate the relationship between the hemostatic coagulation markers of prethrombosis state and pregnancy induced hypertension (PIH). Forty-five PIH patients and 20 control patients were studied. P-selectin, prothrombin fragments 1 + 2 (F1+2), D-dimers (D-D) and plasmin-antiplasmin complex (PAP) were measured by enzyme linked immunosorbent assay. Antithrombin activity (AT: A) was measured by chromogenic peptide substrate assay. (1) The P-selectin level of pre-delivery in moderate and severe PIH patients was (66 +/- 24) microg/L and (80 +/- 30) microg/L, it was (49 +/- 15) microg/L in the control group (both P < 0.05). The P-selectin level of post partum in severe PIH group and control group was (65 +/- 34) microg/L and (40 +/- 12) microg/L, with significant difference between them (P < 0.05). (2) The F1+2 level of pre-delivery in mild, moderate and severe PIH groups was respectively (2.2 +/- 0.2), (2.3 +/- 0.4) and (2.2 +/- 0.2) nmol/L, being all significantly higher than that in the control group, which was (1.2 +/- 0.3) nmol/L, but there was no obvious difference between three PIH groups. (3) The D-D level in mild, moderate and severe PIH groups was respectively (0.7 +/- 0.1), (0.7 +/- 0.3) and (0.8 +/- 0.2) mg/L, and it was (0.4 +/- 0.1) mg/L in the control group. The D-D level was increased when PIH became severe. (4) The PAP level in moderate and severe PIH groups was (0.8 +/- 0.4) mg/L and (0.8 +/- 0.4) mg/L, being significantly higher than that in control group (0.7 +/- 0.3) mg/L (both P < 0.05). (5) The AT: Aactivity was obviously decreased in PIH groups, being respectively (44 +/- 37)%, (64 +/- 25)% and (83 +/- 39)% in severe, moderate and mild PIH groups. There was obvious difference between severe and mild groups (P < 0.01). Elevated P-selectin levels and increased platelet activity were observed in PIH patients. F1+2 may be useful as a screening test for risk pregnancy. D-D can be used as an early monitor of DIC. AT: A reflects the severity of illness. These molecular markers may be used to predict the prethrombosis state in PIH.

Mitomycin C induces multidrug resistance in glaucoma surgery.

PubMed

Hueber, Arno; Esser, Johannes M; Kociok, Norbert; Welsandt, Gerhard; Lüke, Christoph; Roters, Sigrid; Esser, Peter J

2008-02-01

Despite the adjuvant use of mitomycin C during trabeculectomy, failures still occur. We investigated whether cultured human Tenon fibroblasts exposed to low-dose mitomycin C developed a multidrug resistance phenotype in vitro, and whether mitomycin C treatment during previous filtration surgery induces P-glycoprotein expression in vivo. Cultured human Tenon fibroblasts treated with low-dose 0.01 nM mitomycin C for 2 weeks were subsequently treated with 0.1 to 100 microM mitomycin C in the absence or presence of 4 microM verapamil, and allowed to recover for 24 hours. Low-dose mitomycin C-treated fibroblasts were analysed for P-glycoprotein expression using flow cytometry, immunoblotting, and RT-PCR for mdr-1 mRNA. In addition, fibroblasts were treated with low dose 0.1 nM 5-fluorouracil for 2 weeks and analysed for P-glycoprotein expression using flow cytometry. Expression of P-glycoprotein was analysed in surgically removed Tenon tissue (n = 30) using immunohistochemistry. Of the 30 patients, 20 had a previous trabeculectomy, of which nine had previous adjuvant therapy with mitomycin C during trabeculectomy. Partial resistance to mitomycin C after low-dose mitomycin C pre-treatment was significantly neutralised by the addition of verapamil. Low-dose mitomycin C up-regulated P-glycoprotein expression, but not mdr-1 mRNA expression. 5-Fluorouracil did not induce P-glycoprotein expression. P-glycoprotein expression was detected in all nine patients exposed to mitomycin C during previous trabeculectomies. Only six of 21 specimens from patients not previously exposed to mitomycin C showed faint P-glycoprotein expression. The induction of P-glycoprotein by mitomycin C could explain some failures that occur after repeated use of mitomycin C during trabeculectomy. The concomitant use of verapamil or the use of 5-fluorouracil alone could increase the success rate of repeat trabeculectomies.

VCAM-1 expression is upregulated by CD34+/CD133+-stem cells derived from septic patients

PubMed Central

Remmé, Christoph; Betzen, Christian; Tönshoff, Burkhard; Yard, Benito A.; Beck, Grietje; Rafat, Neysan

2018-01-01

CD34+/CD133+- cells are a bone marrow derived stem cell population, which presumably contain vascular progenitor cells and are associated with improved vascular repair. In this study, we investigated whether the adhesion molecules ICAM-1 (intercellular adhesion molecule-1), VCAM-1 (vascular adhesion molecule-1), E-selectin und L-selectin, which are involved in homing of vascular stem cells, are upregulated by CD34+/CD133+-stem cells from septic patients and would be associated with improved clinical outcome. Peripheral blood mononuclear cells from intensive care unit (ICU) patients with (n = 30) and without sepsis (n = 10), and healthy volunteers (n = 15) were isolated using Ficoll density gradient centrifugation. The expression of VCAM-1, ICAM-1, E-selectin and L-selectin was detected on CD34+/CD133+-stem cells by flow cytometry. The severity of disease was assessed by the Simplified Acute Physiology Score (SAPS) II. Serum concentrations of vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-2 were determined by Enzyme-linked immunosorbent assay. The expression of VCAM-1, ICAM-1, E-selectin and L-selectin by CD34+/CD133+-stem cells was significantly upregulated in septic patients, and correlated with sepsis severity. Furthermore, high expression of VCAM-1 by CD34+/CD133+-stem cells revealed a positive association with mortalitiy (p<0.05). Furthermore, significantly higher serum concentrations of VEGF and Ang-2 were found in septic patients, however none showed a strong association with survival. Our data suggest, that VCAM-1 upregulation on CD34+/CD133+-stem cells could play a crucial role in their homing in the course of sepsis. An increase in sepsis severity resulted in both and increase in CD34+/CD133+-stem cells and VCAM-1-expression by those cells, which might reflect an increase in need for vascular repair. PMID:29601599

Circulating sICAM-1 and sE-Selectin as biomarker of infection and prognosis in patients with systemic inflammatory response syndrome.

PubMed

de Pablo, Raúl; Monserrat, Jorge; Reyes, Eduardo; Díaz, David; Rodríguez-Zapata, Manuel; de la Hera, Antonio; Prieto, Alfredo; Álvarez-Mon, Melchor

2013-03-01

Vascular endothelium activation is a key pathogenic step in systemic inflammatory response syndrome (SIRS) that can be triggered by both microbial and sterile proinflammatory stimuli. The relevance of soluble adhesion molecules as clinical biomarkers to discriminate between infectious and non-infectious SIRS, and the individual patient prognosis, has not been established. We prospectively measured by sandwich ELISA, serum levels of soluble E-Selectin (sE-Selectin), soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular adhesion molecule-1 (sICAM-1) and soluble intercellular adhesion molecule-2 (sICAM-2) at ICU admission and at days 3, 7, 14 and 28 in patients with sepsis and at days 3 and 7 in patients with non-infectious SIRS. At ICU admission, sE-Selectin, sVCAM-1 and sICAM-1 in patients with infectious SIRS were significantly higher than those found in patients with non-infectious SIRS. ROC analysis revealed that the AUC for infection identification was best for sICAM-1 (0.900±0.041; 95% CI 0.819-0.981; p<0.0001). Moreover, multivariate analysis showed that 4 variables were significantly and independently associated with mortality at 28 days: male gender (OR 15.90; 95% CI, 2.54-99.32), MODS score (OR 5.60; 95% CI, 1.67-18.74), circulating sE-Selectin levels (OR 4.81; 95% CI, 1.34-17.19) and sVCAM-1 concentrations (OR 4.80; 95% CI, 1.34-17.14). Patients with SIRS secondary to infectious or non-infectious etiology show distinctive patterns of disturbance in serum soluble adhesion molecules. Serum ICAM-1 is a reliable biomarker for classifying patients with infectious SIRS from those with non-infectious SIRS. In addition, soluble E-Selectin is a prognostic biomarker with higher levels in patients with SIRS and fatal outcome. Copyright © 2012 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.

Phenylboronate chromatography selectively separates glycoproteins through the manipulation of electrostatic, charge transfer, and cis-diol interactions.

PubMed

Carvalho, Rimenys J; Woo, James; Aires-Barros, M Raquel; Cramer, Steven M; Azevedo, Ana M

2014-10-01

Phenylboronate chromatography (PBC) has been applied for several years, however details regarding the mechanisms of interactions between the ligand and biomolecules are still scarce. The goal of this work is to investigate the various chemical interactions between proteins and their ligands, using a protein library containing both glycosylated and nonglycosylated proteins. Differences in the adsorption of these proteins over a pH range from 4 to 9 were related to two main properties: charge and presence of glycans. Acidic or neutral proteins were strongly adsorbed below pH 8 although the uncharged trigonal form of phenylboronate (PB) is less susceptible to forming electrostatic and cis-diol interactions with proteins. The glycosylated proteins were only adsorbed above pH 8 when the electrostatic repulsion between the boronate anion and the protein surface was mitigated (at 200 mM NaCl). All basic proteins were highly adsorbed above pH 8 with PB also acting as a cation-exchanger with binding occurring through electrostatic interactions. Batch adsorption performed at acidic conditions in the presence of Lewis base showed that charge-transfer interactions are critical for protein retention. This study demonstrates the multimodal interaction of PBC, which can be a selective tool for separation of different classes of proteins. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The role of Nox1 and Nox2 in GPVI-dependent platelet activation and thrombus formation☆

PubMed Central

Walsh, T.G.; Berndt, M.C.; Carrim, N.; Cowman, J.; Kenny, D.; Metharom, P.

2014-01-01

Background Activation of the platelet-specific collagen receptor, glycoprotein (GP) VI, induces intracellular reactive oxygen species (ROS) production; however the relevance of ROS to GPVI-mediated platelet responses remains unclear. Objective The objective of this study was to explore the role of the ROS-producing NADPH oxidase (Nox)1 and 2 complexes in GPVI-dependent platelet activation and collagen-induced thrombus formation. Methods and results ROS production was measured by quantitating changes in the oxidation-sensitive dye, H2DCF-DA, following platelet activation with the GPVI-specific agonist, collagen related peptide (CRP). Using a pharmacological inhibitor specific for Nox1, 2-acetylphenothiazine (ML171), and Nox2 deficient mice, we show that Nox1 is the key Nox homolog regulating GPVI-dependent ROS production. Nox1, but not Nox2, was essential for CRP-dependent thromboxane (Tx)A2 production, which was mediated in part through p38 MAPK signaling; while neither Nox1 nor Nox2 was significantly involved in regulating CRP-induced platelet aggregation/integrin αIIbβ3 activation, platelet spreading, or dense granule and α-granule release (ATP release and P-selectin surface expression, respectively). Ex-vivo perfusion analysis of mouse whole blood revealed that both Nox1 and Nox2 were involved in collagen-mediated thrombus formation at arterial shear. Conclusion Together these results demonstrate a novel role for Nox1 in regulating GPVI-induced ROS production, which is essential for optimal p38 activation and subsequent TxA2 production, providing an explanation for reduced thrombus formation following Nox1 inhibition. PMID:24494191

Endogenous pro-thrombotic biomarkers from the arm and leg may not have the same value.

PubMed

Lattimer, Christopher R; Kalodiki, Evi; Geroulakos, George; Hoppensteadt, Debra; Fareed, Jawed

2016-05-01

Assessments of endogenous pro-thrombotic biomarkers are performed invariably on arm blood. However, the commonest site for thrombosis is in the leg. A leg blood sample may reflect local pro-thrombotic processes more accurately than systemic arm blood. The aim was to determine whether pro-thrombotic biomarkers from standard venous arm samples differed significantly from leg samples. Concurrent blood samples were taken from an ankle/lower calf varicose vein and an ante-cubital vein in 24 patients awaiting laser treatment as well as age approximated and sex matched healthy controls without venous disease. The following assays were performed: thrombin-antithrombin (ng/ml), antithrombin (%) activity, microparticles (nM), fibrinogen (mg/dl), prothrombin fragment 1.2 (F1.2) (pM) and P-selectin (ng/ml). Expressed as median (inter-quartile range). Significant arm/leg differences were observed in thrombin-antithrombin, antithrombin, prothrombin fragment 1.2 and P-selectin. The legs of patients had significantly reduced antithrombin activity and P-selectin concentrations compared to their arms (leg: 101 (90-108) versus arm: 112 (99-126), P = 0.001 and leg: 42 (26-52) versus 45 (27-52), P = 0.044, respectively). Control leg samples had significantly increased thrombin-antithrombin and P-selectin compared to control arm samples (leg: 2.1 (0.9-3.2) versus arm: 0.8 (0.5-1.7), P = 0.015 and leg: 36 (24-50) versus arm: 30 (23-41), P = 0.007, respectively). However, the control legs had significantly reduced F1.2 (leg: 265 (230-333) versus arm: 299 (236-361), P = 0.028). No significant arm/leg differences were detected in the microparticle or fibrinogen levels. These findings indicate that venous arm blood is significantly different from venous leg blood in four out of six biomarkers studied. Recognition of local venous leg sampling as a site for investigation may unravel why the leg has a greater predisposition to thrombosis and lead the way towards an arm/leg differential test. © The Author(s) 2015.

Dexamethasone enhances interaction of endogenous annexin 1 with L-selectin and triggers shedding of L-selectin in the monocytic cell line U-937.

PubMed

de Coupade, Catherine; Solito, Egle; Levine, Jon D

2003-09-01

(1) L-selectin, constitutively expressed by leukocytes, is involved in the initial binding of leukocytes to activated endothelium. Anti-inflammatory drugs like glucocorticoids can induce shedding of L-selectin, but the mechanism is still unknown. Annexin 1, a protein whose synthesis and externalization/secretion are induced during the inflammatory response, has been proposed as a mediator of the anti-inflammatory actions of glucocorticoids. (2) The monocytic cell line U-937 strongly expresses Annexin 1 after 24 h of phorbol 12-myristate 13-acetate (PMA, 1 nm) treatment and externalizes/releases the protein after additional 16 h of dexamethasone (1 microm) treatment. (3) This study investigated the possible regulation of cell surface L-selectin shedding by endogenous Annexin 1, and its role in glucocorticoid-induced L-selectin shedding in the U-937 cell line. (4) PMA- and dexamethasone treatment-induced L-selectin shedding was potentially mediated by Annexin 1, since neutralizing antibodies against Annexin 1 reduced dexamethasone- and Annexin 1-induced shedding. (5) Immunoprecipitation and binding assays provided support for the suggestion that this effect could be mediated by an interaction between externalized Annexin 1 and L-selectin. Such interaction involved the N-terminal domain of Annexin 1 and was calcium-dependent. Confocal microscopy studies demonstrated increased colocalization of Annexin 1 and L-selectin on the cell surface. (6) Overall, our study provides new insights into the potential role of endogenous ANXA1 as a mediator of dexamethasone-induced L-selectin shedding, which may contribute to the anti-inflammatory activity of glucocorticoids.

Glycoprotein hormone receptors: determinants in leucine-rich repeats responsible for ligand specificity

PubMed Central

Smits, Guillaume; Campillo, Mercedes; Govaerts, Cédric; Janssens, Véronique; Richter, Christine; Vassart, Gilbert; Pardo, Leonardo; Costagliola, Sabine

2003-01-01

Glycoprotein hormone receptors [thyrotropin (TSHr), luteinizing hormone/chorionic gonadotropin (LH/CGr), follicle stimulating hormone (FSHr)] are rhodopsin-like G protein-coupled receptors with a large extracellular N-terminal portion responsible for hormone recognition and binding. In structural models, this ectodomain is composed of two cysteine clusters flanking nine leucine-rich repeats (LRRs). The LRRs form a succession of β-strands and α-helices organized into a horseshoe-shaped structure. It has been proposed that glycoprotein hormones interact with residues of the β-strands making the concave surface of the horseshoe. Gain-of-function homology scanning of the β-strands of glycoprotein hormone receptors allowed identification of the critical residues responsible for the specificity towards human chorionic gonadotropin (hCG). Substitution of eight or two residues of the LH/CGr into the TSHr or FSHr, respectively, resulted in constructs displaying almost the same affinity and sensitivity for hCG as wild-type LH/CGr. Molecular dynamics simulations and additional site-directed mutagenesis provided a structural rationale for the evolution of binding specificity in this duplicated gene family. PMID:12773385

Off-target effect of the Epac agonist 8-pCPT-2'-O-Me-cAMP on P2Y12 receptors in blood platelets.

PubMed

Herfindal, Lars; Nygaard, Gyrid; Kopperud, Reidun; Krakstad, Camilla; Døskeland, Stein Ove; Selheim, Frode

2013-08-09

The primary target of the cAMP analogue 8-pCPT-2'-O-Me-cAMP is exchange protein directly activated by cAMP (Epac). Here we tested potential off-target effects of the Epac activator on blood platelet activation signalling. We found that the Epac analogue 8-pCPT-2'-O-Me-cAMP inhibits agonist-induced-GPCR-stimulated, but not collagen-stimulated, P-selectin surface expression on Epac1 deficient platelets. In human platelets, 8-pCPT-2'-O-Me-cAMP inhibited P-selectin expression elicited by the PKC activator PMA. This effect was abolished in the presence of the extracellular ADP scavenger system CP/CPK. In silico modelling of 8-pCPT-2'O-Me-cAMP binding into the purinergic platelet receptor P2Y12 revealed that the analogue docks similar to the P2Y12 antagonist 2MeSAMP. The 8-pCPT-2'-O-Me-cAMP analogue per se, did not provoke Rap 1 (Rap 1-GTP) activation or phosphorylation on the vasodilator-stimulated phosphoprotein (VASP) at Ser-157. In addition, the protein kinase A (PKA) antagonists Rp-cAMPS and Rp-8-Br-cAMPS failed to block the inhibitory effect of 8-pCPT-2'-O-Me-cAMP on thrombin- and TRAP-induced Rap 1 activation, thus suggesting that PKA is not involved. We conclude that the 8-pCPT-2'-O-Me-cAMP analogue is able to inhibit agonist-induced-GPCR-stimulated P-selectin independent from Epac1; the off-target effect of the analogue appears to be mediated by antagonistic P2Y12 receptor binding. This has implications when using cAMP analogues on specialised system involving such receptors. We found, however that the Epac agonist 8-Br-2'-O-Me-cAMP did not affect platelet activation at similar concentrations. Copyright © 2013 Elsevier Inc. All rights reserved.

Impact of soy supplementation on sex steroids and vascular inflammation markers in postmenopausal women using tibolone: role of equol production capability.

PubMed

Törmälä, R; Appt, S; Clarkson, T B; Mueck, A O; Seeger, H; Mikkola, T S; Ylikorkala, O

2008-10-01

Tibolone is often taken concurrently with soy. Tibolone, soy and equol-producing capacity each affect vascular health, whereas their concomitant effects are unknown. We studied the effects of soy on sex steroids and vascular inflammation markers in long-term tibolone users. Postmenopausal women (n = 110) on tibolone were screened with a soy challenge to find 20 equol producers and 20 non-producers. All women were treated for 8 weeks in a cross-over trial with soy (52 g of soy protein containing 112 mg of isoflavones) or placebo. Serum estrone, 17beta-estradiol, testosterone, androstenedione, dehydroepiandrosterone sulfate (DHEAS), sex hormone binding globulin (SHBG), C-reactive protein (CRP), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and platelet-selectin (P-selectin) were assessed. Soy decreased (7.1%) the estrone level, significantly (12.5%) only in equol producers (from 80.2 +/- 10.8 to 70.3 +/- 7.0 pmol/l; p = 0.04). Testosterone was reduced (15.5%; from 586 +/- 62.6 to 495 +/- 50.1 pmol/l, p = 0.02) during soy treatment, and more markedly in equol producers than non-producers (22.1% vs. 10.0%). No changes appeared in SHBG, CRP or ICAM-1, but VCAM-1 increased (9.2%) and P-selectin decreased (10.3%) during soy treatment. Soy modified the concentrations of estrone, testosterone and some endothelial markers. Equol production enforced these effects. Soy supplementation may be clinically significant in tibolone users.

Acidosis downregulates platelet haemostatic functions and promotes neutrophil proinflammatory responses mediated by platelets.

PubMed

Etulain, Julia; Negrotto, Soledad; Carestia, Agostina; Pozner, Roberto Gabriel; Romaniuk, María Albertina; D'Atri, Lina Paola; Klement, Giannoula Lakka; Schattner, Mirta

2012-01-01

Acidosis is one of the hallmarks of tissue injury such as trauma, infection, inflammation, and tumour growth. Although platelets participate in the pathophysiology of all these processes, the impact of acidosis on platelet biology has not been studied outside of the quality control of laboratory aggregation assays or platelet transfusion optimization. Herein, we evaluate the effect of physiologically relevant changes in extracellular acidosis on the biological function of platelets, placing particular emphasis on haemostatic and secretory functions. Platelet haemostatic responses such as adhesion, spreading, activation of αIIbβ3 integrin, ATP release, aggregation, thromboxane B2 generation, clot retraction and procoagulant activity including phosphatidilserine exposure and microparticle formation, showed a statistically significant inhibition of thrombin-induced changes at pH of 7.0 and 6.5 compared to the physiological pH (7.4). The release of alpha granule content was differentially regulated by acidosis. At low pH, thrombin or collagen-induced secretion of vascular endothelial growth factor and endostatin were dramatically reduced. The release of von Willebrand factor and stromal derived factor-1α followed a similar, albeit less dramatic pattern. In contrast, the induction of CD40L was not changed by low pH, and P-selectin exposure was significantly increased. While the generation of mixed platelet-leukocyte aggregates and the increased chemotaxis of neutrophils mediated by platelets were further augmented under acidic conditions in a P-selectin dependent manner, the increased neutrophil survival was independent of P-selectin expression. In conclusion, our results indicate that extracellular acidosis downregulates most of the haemostatic platelet functions, and promotes those involved in amplifying the neutrophil-mediated inflammatory response.

Thrombogenesis with continuous blood flow in the inferior vena cava. A novel mouse model.

PubMed

Diaz, José A; Hawley, Angela E; Alvarado, Christine M; Berguer, Alexandra M; Baker, Nichole K; Wrobleski, Shirley K; Wakefield, Thomas W; Lucchesi, Benedict R; Myers, Daniel D

2010-08-01

Several rodent models have been used to study deep venous thrombosis (DVT). However, a model that generates consistent venous thrombi in the presence of continuous blood flow, to evaluate therapeutic agents for DVT, is not available. Mice used in the present study were wild-type C57BL/6 (WT), plasminogen activator inhibitor-1 (PAI-1) knock out (KO) and Delta Cytoplasmic Tail (DCT). An electrolytic inferior vena cava (IVC) model (EIM) was used. A 25G stainless-steel needle, attached to a silver coated copper wire electrode (anode), was inserted into the exposed caudal IVC. Another electrode (cathode) was placed subcutaneously. A current of 250 muAmps over 15 minutes was applied. Ultrasound imaging was used to demonstrate the presence of IVC blood flow. Analyses included measurement of plasma soluble P-selectin (sP-Sel), thrombus weight (TW), vein wall morphometrics, P-selectin and Von Willebrand factor (vWF) staining, transmission electron microscopy (TEM), scanning electron microscopy (SEM); and the effect of enoxaparin on TW was evaluated. A current of 250 muAmps over 15 minutes consistently promoted thrombus formation in the IVC. Plasma sP-Sel was decreased in PAI-1 KO and increased in DCT vs. WT (WT/PAI-1: p=0.003, WT/DCT: p=0.0002). Endothelial activation was demonstrated by SEM, TEM, P-selectin and vWF immunohistochemistry and confirmed by inflammatory cell counts. Ultrasound imaging demonstrated thrombus formation in the presence of blood flow. Enoxaparin significantly reduced the thrombus size by 61% in this model. This EIM closely mimics clinical venous disease and can be used to study endothelial cell activation, leukocyte migration, thrombogenesis and therapeutic applications in the presence of blood flow.

Olive Oil-Based Lipid Emulsions Do Not Influence Platelet Receptor Expression in Comparison to Medium and Long Chain Triglycerides In vitro.

PubMed

Stoetzer, Carsten; Nickel, Katja; Weißig, Annette; Großheim, Marieke; Scheinichen, Dirk; Doll, Thorben; Jüttner, Björn

2016-11-01

Lipid emulsions influence platelet aggregation and receptor expression. However, the effect on platelet function is not fully explained. Therefore, the aim of this study was to examine the influence of the lipids Lipofundin ® , Lipidem ® and ClinOleic ® on surface expressions of P-selectin, GPIb and GPIIb/IIIa on platelets in vitro. Whole blood was incubated in two different concentrations (0.06 and 0.6 mg/ml) of LCT/MCT, n-3/LCT/MCT and LCT-MUFA for 30 min, followed by activation with TRAP-6 or ADP for flow-cytometric assay. Rates of P-selectin, GPIb and GPIIb/IIIa expression were analyzed. There was a significant increase in GPIIb/IIIa- and P-selectin-expression after incubation with LCT/MCT and n-3/LCT/MCT at the concentration of 0.6 mg/ml, without and after stimulation with TRAP-6 and ADP. GPIb was significantly decreased. Accordingly, LCT-MUFA had no effect on receptor expression of platelets in vitro. We demonstrated that LCT-MUFA did not activate receptor expression of platelets whereas LCT/MCT significantly increased platelet aggregation in vitro. This finding should be noted for parenteral nutrition of intensive care patients and, in the future, might provide further insight into the pathogenic pathways of acute thromboembolic events. However, prospectively designed clinical studies are needed to support our results.

Impact of adiposity on cellular adhesion: The Multi-Ethnic Study of atherosclerosis (MESA).

PubMed

Christoph, Mary J; Allison, Matthew A; Pankow, James S; Decker, Paul A; Kirsch, Phillip S; Tsai, Michael Y; Sale, Michele M; de Andrade, Mariza; Sicotte, Hugues; Tang, Weihong; Hanson, Naomi Q; Berardi, Cecilia; Wassel, Christina L; Larson, Nicholas B; Bielinski, Suzette J

2016-01-01

At the cellular level, how excess adiposity promotes atherogenesis is not fully understood. One pathway involves secretion of adipokines that stimulate endothelial dysfunction through increased expression of adhesion molecules. However, the relationship of adiposity to adhesion molecules that promote atherosclerosis is largely unknown. Linear regression models were used to assess the sex-specific associations of soluble cellular adhesion molecules (sP- and sL-selectin, sICAM-1, sVCAM-1, and sHGF) and adiposity in 5,974 adults examined as part of the Multi-Ethnic Study of Atherosclerosis (MESA). Adiposity measures included body mass index (BMI), waist-to-hip-ratio (WHR), and computed tomography measures of subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT). The mean age was 64 years and 52% were female. In multivariable models adjusting for traditional cardiovascular risk factors, sHGF was positively associated with BMI, WHR, and VAT in both males and females, and sP-selectin with WHR and VAT in males. sVCAM-1 was inversely associated with VAT in females only. Our results showed the relation of adiposity to soluble cellular adhesion proteins was similar across adiposity measures and for both sexes. However, the relationship between adiposity and sVCAM-1 and P-selectin may be modified by sex and the measure used to assess adiposity. © 2015 The Obesity Society.

P-Selectin Targeted Dexamethasone-Loaded Lipid Nanoemulsions: A Novel Therapy to Reduce Vascular Inflammation

PubMed Central

Simion, Viorel; Constantinescu, Cristina Ana; Stan, Daniela; Deleanu, Mariana; Tucureanu, Monica Madalina; Butoi, Elena; Manduteanu, Ileana; Simionescu, Maya

2016-01-01

Inflammation is a common process associated with numerous vascular pathologies. We hypothesized that targeting the inflamed endothelium by coupling a peptide with high affinity for P-selectin to the surface of dexamethasone-loaded lipid nanoemulsions will highly increase their specific binding to activated endothelial cells (EC) and reduce the cell activation. We developed and characterized dexamethasone-loaded lipid nanoemulsions directed towards P-selectin (PLN-Dex) and monitored their anti-inflammatory effects in vitro using cultured EC (EA.hy926 cells) and in vivo using a mouse model of acute inflammation [lipopolysaccharides (LPS) intravenously administered in C57BL/6 mice]. We found that PLN-Dex bound specifically to the surface of activated EC are efficiently internalized by EC and reduced the expression of proinflammatory genes, thus preventing the monocyte adhesion and transmigration to/through activated EC. Given intravenously in mice with acute inflammation, PLN-Dex accumulated at a significant high level in the lungs (compared to nontargeted nanoemulsions) and significantly reduced mRNA expression level of key proinflammatory cytokines such as IL-1β, IL-6, and MCP-1. In conclusion, the newly developed nanoformulation, PLN-Dex, is functional in vitro and in vivo, reducing selectively the endothelium activation and the consequent monocyte infiltration and diminishing significantly the lungs' inflammation, in a mouse model of acute inflammation. PMID:27703301

VEGF controls lung Th2 inflammation via the miR-1-Mpl (myeloproliferative leukemia virus oncogene)-P-selectin axis.

PubMed

Takyar, Seyedtaghi; Vasavada, Hema; Zhang, Jian-ge; Ahangari, Farida; Niu, Naiqian; Liu, Qing; Lee, Chun Geun; Cohn, Lauren; Elias, Jack A

2013-09-23

Asthma, the prototypic Th2-mediated inflammatory disorder of the lung, is an emergent disease worldwide. Vascular endothelial growth factor (VEGF) is a critical regulator of pulmonary Th2 inflammation, but the underlying mechanism and the roles of microRNAs (miRNAs) in this process have not been defined. Here we show that lung-specific overexpression of VEGF decreases miR-1 expression in the lung, most prominently in the endothelium, and a similar down-regulation occurs in lung endothelium in Th2 inflammation models. Intranasal delivery of miR-1 inhibited inflammatory responses to ovalbumin, house dust mite, and IL-13 overexpression. Blocking VEGF inhibited Th2-mediated lung inflammation, and this was restored by antagonizing miR-1. Using mRNA arrays, Argonaute pull-down assays, luciferase expression assays, and mutational analysis, we identified Mpl as a direct target of miR-1 and showed that VEGF controls the expression of endothelial Mpl during Th2 inflammation via the regulation of miR-1. In vivo knockdown of Mpl inhibited Th2 inflammation and indirectly inhibited the expression of P-selectin in lung endothelium. These experiments define a novel VEGF-miR-1-Mpl-P-selectin effector pathway in lung Th2 inflammation and herald the utility of miR-1 and Mpl as potential therapeutic targets for asthma.

VEGF controls lung Th2 inflammation via the miR-1–Mpl (myeloproliferative leukemia virus oncogene)–P-selectin axis

PubMed Central

Vasavada, Hema; Zhang, Jian-ge; Ahangari, Farida; Niu, Naiqian; Liu, Qing; Lee, Chun Geun; Cohn, Lauren

2013-01-01

Asthma, the prototypic Th2-mediated inflammatory disorder of the lung, is an emergent disease worldwide. Vascular endothelial growth factor (VEGF) is a critical regulator of pulmonary Th2 inflammation, but the underlying mechanism and the roles of microRNAs (miRNAs) in this process have not been defined. Here we show that lung-specific overexpression of VEGF decreases miR-1 expression in the lung, most prominently in the endothelium, and a similar down-regulation occurs in lung endothelium in Th2 inflammation models. Intranasal delivery of miR-1 inhibited inflammatory responses to ovalbumin, house dust mite, and IL-13 overexpression. Blocking VEGF inhibited Th2-mediated lung inflammation, and this was restored by antagonizing miR-1. Using mRNA arrays, Argonaute pull-down assays, luciferase expression assays, and mutational analysis, we identified Mpl as a direct target of miR-1 and showed that VEGF controls the expression of endothelial Mpl during Th2 inflammation via the regulation of miR-1. In vivo knockdown of Mpl inhibited Th2 inflammation and indirectly inhibited the expression of P-selectin in lung endothelium. These experiments define a novel VEGF–miR-1–Mpl–P-selectin effector pathway in lung Th2 inflammation and herald the utility of miR-1 and Mpl as potential therapeutic targets for asthma. PMID:24043765

Association between MDR1 gene of gastrointestinal tumors, the expression of P-glycoprotein and resistance to chemotherapeutic drugs.

PubMed

Su, Jian-Li; Wang, Cheng-Hong; Kang, Hong-Gang; Zhang, Jing; Wang, Bao-Zhong; Liu, Mei-Rong; Zhao, Jun; Liu, Lin

2017-09-01

The aim of the present study was to examine and discuss the association between multidrug resistance 1 gene ( MDR1 ) of gastrointestinal tumors, the expression of P-glycoprotein and resistance to chemotherapeutic drugs. In this study, 126 cases of patients with gastrointestinal tumors admitted to hospital from February 2013 to February 2015 were selected. The expression levels of MDR1 gene were obsreved in the control population and patients before and after treatment by fluoresecent quantitative PCR. The protein expression level of P-glycoprotein was determined using western blotting and enzyme-linked immunosorbent assay. In addition, drug resistance was assessed by ATP-TCA chemosensitivity experiments. The results showed that before treatment, the expression of mRNA in MDR1 of tissues of gastrointestinal tract of the 126 cases was 108-fold larger than that of the gastrointestinal tract of the controls (p<0.05), P-glycoprotein was 87-fold larger than the expression level of the controls (p<0.05). The sensitivity of 126 tumor tissues to different chemotherapeutic drugs was determined, and the results showed that most of the tumor tissues were sensitive to chemotherapeutic drugs, and the sensitivity rate reached 96.4%. Following chemotherapy, the expression of mRNA in MDR1 of tumor tissues and the expression of P-glycoprotein decreased (p<0.05). In conclusion, the MDR1 gene and P-glycoprotein have a positive correlation with the occurrence of gastrointestinal tumors, and a negative correlation between the MDR1 gene and P-glycoprotein with resistance of chemotherapeutic drugs. Therefore, the MDR1 gene and P-glycoprotein can be used as references in the identification and diagnosis of gastrointestinal tumors.

Strategies to overcome or circumvent P-glycoprotein mediated multidrug resistance.

PubMed

Yuan, Hongyu; Li, Xun; Wu, Jifeng; Li, Jinpei; Qu, Xianjun; Xu, Wenfang; Tang, Wei

2008-01-01

Cancer patients who receive chemotherapy often experience intrinsic or acquired resistance to a broad spectrum of chemotherapeutic agents. The phenomenon, termed multidrug resistance (MDR), is often associated with the over-expression of P-glycoprotein, a transmembrane protein pump, which can enhance efflux of a various chemicals structurally unrelated at the expense of ATP depletion, resulting in decrease of the intracellular cytotoxic drug accumulation. The MDR has been a big threaten to the human health and the war fight for it continues. Although several other mechanisms for MDR are elucidated in recent years, considerable efforts attempting to inverse MDR are involved in exploring P-glycoprotein modulators and suppressing P-glycoprotein expression. In this review, we will report on the recent advances in various strategies for overcoming or circumventing MDR mediated by P-glycoprotein.

Platelet reactivity and thrombogenicity in postmenopausal women.

PubMed

Singla, Anand; Bliden, Kevin P; Jeong, Young-Hoon; Abadilla, Katrina; Antonino, Mark J; Muse, William C; Mathew, Denny P; Bailon, Oscar; Tantry, Udaya S; Gurbel, Paul A

2013-01-01

Age-adjusted incidence of cardiovascular disease, including myocardial infarction, is significantly lower in premenopausal women than in men, which is thought to be caused by the cardioprotective effects of estrogen. However, there is a consistent increase in the incidence of coronary artery disease in postmenopausal women in comparison with premenopausal women. The protective benefit of hormone therapy has not been observed in postmenopausal women. It is unknown whether measures of platelet reactivity and clot strength contribute to the disproportionate incidence of cardiovascular disease between premenopausal and postmenopausal women. Fifty healthy volunteers, including 25 premenopausal women and 25 postmenopausal women, aged between 40 and 65 years were enrolled. Total estradiol and follicle-stimulating hormone levels were measured for confirmation of menopausal state and comparison testing. Platelet reactivity was assessed using light transmission aggregometry and P-selectin, and glycoprotein IIb/IIIa receptor expression was assessed using flow cytometry. Thrombelastography was used to measure clot strength, clotting time, and fibrinogen activity. Serum cholesterol, C-reactive protein, complete blood count, and comprehensive metabolic panel were also measured. Platelet reactivity did not differ among menopausal states or hormone levels. Clotting time was increased in postmenopausal women (6.6 ± 2.0 vs. 7.8 ± 1.2 min, P = 0.013) and significantly correlated with estradiol levels (r = 0.68, P < 0.001). A significantly higher low-density lipoprotein cholesterol level was observed in postmenopausal women (P = 0.05). Mean C-reactive protein levels were numerically higher in the postmenopausal group. The thrombotic risk profile between premenopausal and postmenopausal women is similar. However, improved management of cholesterol may be of clinical benefit. Large-scale studies are required to validate these findings.

Relationship between Platelet PPARs, cAMP Levels, and P-Selectin Expression: Antiplatelet Activity of Natural Products.

PubMed

Fuentes, Eduardo; Palomo, Iván

2013-01-01

Platelets are no longer considered simply as cells participating in thrombosis. In atherosclerosis, platelets are regulators of multiple processes, with the recruitment of inflammatory cells towards the lesion sites, inflammatory mediators release, and regulation of endothelial function. The antiplatelet therapy has been used for a long time in an effort to prevent and treat cardiovascular diseases. However, limited efficacy in some patients, drug resistance, and side effects are limitations of current antiplatelet therapy. In this context, a large number of natural products (polyphenols, terpenoids, alkaloids, and fatty acids) have been reported with antiplatelet activity. In this sense, the present paper describes mechanisms of antiplatelet action of natural products on platelet P-selectin expression through cAMP levels and its role as peroxisome proliferator-activated receptors agonists.

Herpes simplex virus glycoprotein D relocates nectin-1 from intercellular contacts

PubMed Central

Bhargava, Arjun K.; Rothlauf, Paul W.; Krummenacher, Claude

2016-01-01

Herpes simplex virus (HSV) uses the cell adhesion molecule nectin-1 as a receptor to enter neurons and epithelial cells. The viral glycoprotein D (gD) is used as a non-canonical ligand for nectin-1. The gD binding site on nectin-1 overlaps with a functional adhesive site involved in nectin-nectin homophilic trans-interaction. Consequently, when nectin-1 is engaged with a cellular ligand at cell junctions, the gD binding site is occupied. Here we report that HSV gD is able to disrupt intercellular homophilic trans-interaction of nectin-1 and induce a rapid redistribution of nectin-1 from cell junctions. This movement does not require the receptor’s interaction with the actin-binding adaptor afadin. Interaction of nectin-1 with afadin is also dispensable for virion surfing along nectin-1-rich filopodia. Cells seeded on gD-coated surfaces also fail to accumulate nectin-1 at cell contact. These data indicate that HSV gD affects nectin-1 locally through direct interaction and more globally through signaling. PMID:27723487

Partial purification of the mu opioid receptor irreversibly labeled with (/sup 3/H)b-funaltrexamine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu-Chen, L.Y.; Phillips, C.A.; Tam, S.W.

1986-03-01

The mu opioid receptor in bovine striatal membranes was specifically and irreversibly labeled by incubation with 5 nM (/sup 3/H)..beta..-funaltrexamine (approx.-FNA) at 37/sup 0/C for 90 min in the presence of 100 mM NaCl. The specific and irreversible binding of (/sup 3/H)..beta..-FNA as defined by that blocked by 1 /sup +/M naloxone was about 60% of total irreversible binding. The specific irreversible binding was saturable, stereospecific, time-, temperature, and tissue-dependent. Mu opioid ligands were much more potent than delta or kappa ligands in inhibiting the specific irreversible labeling. SDS polyacrylamide gel electrophoresis of solubilized membranes in the presence of 2-mercaptoethanolmore » yielded a major radiolabeled broad band of MW 68-97K daltons, characteristic of a glycoprotein band. This band was not observed in membranes labeled in the presence of excess unlabeled naloxone. The glycoprotein nature of the (/sup 3/H)..beta..-FNA-labeled opioid receptor was confirmed by its binding to a wheat germ agglutinin-Sepharose column and its elution with N-acetylglucosamine.« less

Herpes simplex virus glycoprotein D relocates nectin-1 from intercellular contacts.

PubMed

Bhargava, Arjun K; Rothlauf, Paul W; Krummenacher, Claude

2016-12-01

Herpes simplex virus (HSV) uses the cell adhesion molecule nectin-1 as a receptor to enter neurons and epithelial cells. The viral glycoprotein D (gD) is used as a non-canonical ligand for nectin-1. The gD binding site on nectin-1 overlaps with a functional adhesive site involved in nectin-nectin homophilic trans-interaction. Consequently, when nectin-1 is engaged with a cellular ligand at cell junctions, the gD binding site is occupied. Here we report that HSV gD is able to disrupt intercellular homophilic trans-interaction of nectin-1 and induce a rapid redistribution of nectin-1 from cell junctions. This movement does not require the receptor's interaction with the actin-binding adaptor afadin. Interaction of nectin-1 with afadin is also dispensable for virion surfing along nectin-1-rich filopodia. Cells seeded on gD-coated surfaces also fail to accumulate nectin-1 at cell contact. These data indicate that HSV gD affects nectin-1 locally through direct interaction and more globally through signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

Application of Physiologically Based Pharmacokinetic Modeling in Understanding Bosutinib Drug-Drug Interactions: Importance of Intestinal P-Glycoprotein.

PubMed

Yamazaki, Shinji; Loi, Cho-Ming; Kimoto, Emi; Costales, Chester; Varma, Manthena V

2018-05-08

Bosutinib is an orally available Src/Abl tyrosine kinase inhibitor indicated for the treatment of patients with Ph+ chronic myelogenous leukemia at a clinically recommended dose of 500 mg once daily. Clinical results indicated that increases in bosutinib oral exposures were supra-proportional at the lower doses (50 to 200 mg) and approximately dose-proportional at the higher doses (200 to 600 mg). Bosutinib is a substrate of CYP3A4 and P-glycoprotein and exhibits pH-dependent solubility with moderate intestinal permeability. These findings led us to investigate the factors influencing the underlying pharmacokinetic mechanisms of bosutinib with physiologically-based pharmacokinetic (PBPK) models. Our primary objectives were to: 1) refine the previously developed bosutinib PBPK model based on the latest oral bioavailability data and 2) verify the refined PBPK model with P-glycoprotein kinetics based on the bosutinib drug-drug interaction (DDI) results with ketoconazole and rifampin. Additionally, the verified PBPK model was applied to predict bosutinib DDIs with dual CYP3A/P-glycoprotein inhibitors. The results indicated that 1) the refined PBPK model adequately described the observed plasma concentration-time profiles of bosutinib and 2) the verified PBPK model reasonably predicted the effects of ketoconazole and rifampin on bosutinib exposures by accounting for intestinal P-gp inhibition/induction. These results suggested that bosutinib DDI mechanism could involve not only CYP3A4-mediated metabolism but also P-glycoprotein-mediated efflux on absorption. In summary, P-glycoprotein kinetics could constitute a critical element in the PBPK models to understand the pharmacokinetic mechanism of dual CYP3A/P-glycoprotein substrates such as bosutinib exhibiting nonlinear pharmacokinetics due largely to a saturation of intestinal P-glycoprotein-mediated efflux. The American Society for Pharmacology and Experimental Therapeutics.

Determining P-glycoprotein-drug interactions: evaluation of reconstituted P-glycoprotein in a liposomal system and LLC-MDR1 polarized cell monolayers

PubMed Central

Melchior, Donald L.; Sharom, Frances J.; Evers, Raymond; Wright, George E.; Chu, Joseph W.K.; Wright, Stephen E.; Chu, Xiaoyan; Yabut, Jocelyn

2012-01-01

Introduction P-Glycoprotein (ABCB1, MDR1) is a multidrug efflux pump that is a member of the ATP-binding cassette (ABC) superfamily. Many drugs in common clinical use are either substrates or inhibitors of this transporter. Quantitative details of P-glycoprotein inhibition by pharmaceutical agents are essential for assessment of their pharmacokinetic behavior and prevention of negative patient reactions. Cell-based systems have been widely used for determination of drug interactions with P-glycoprotein, but they suffer from several disadvantages, and results are often widely variable between laboratories. We aimed to demonstrate that a novel liposomal system employing contemporary biochemical methodologies could measure the ability of clinically used drugs to inhibit the P-glycoprotein pump. To accomplish this we compared results with those of cell-based approaches. Methods Purified transport-competent hamster Abcb1a P-glycoprotein was reconstituted into a unilamellar liposomal system, Fluorosome-trans-pgp, whose aqueous interior contains fluorescent drug sensors. This provides a well-defined system for measuring P-glycoprotein transport inhibition by test drugs in real time using rapid fluorescence-based technology. Results Inhibition of ATP-driven transport by Fluorosome-trans-pgp employed a panel of 46 representative drugs. Resulting IC50 values correlated well (r2 = 0.80) with Kd values for drug binding to purified P-glycoprotein. They also showed a similar trend to transport inhibition data obtained using LLC-MDR1 cell monolayers. Fluorosome-trans-pgp IC50 values were in agreement with published results of digoxin drug-drug interaction studies in humans. Discussion This novel approach using a liposomal system and fluorescence-based technology is shown to be suitable to study whether marketed drugs and drug candidates are P-glycoprotein inhibitors. The assay is rapid, allowing a 7-point IC50 determination in <6 minutes, and requires minimal quantities of test drug. The method is amenable to robotics and offers a cost advantage relative to conventional cell-based assays. The well-defined nature of this assay also obviates many of the inherent complications and ambiguities of cell-based systems. PMID:22394995

Determining P-glycoprotein-drug interactions: evaluation of reconstituted P-glycoprotein in a liposomal system and LLC-MDR1 polarized cell monolayers.

PubMed

Melchior, Donald L; Sharom, Frances J; Evers, Raymond; Wright, George E; Chu, Joseph W K; Wright, Stephen E; Chu, Xiaoyan; Yabut, Jocelyn

2012-03-01

P-Glycoprotein (ABCB1, MDR1) is a multidrug efflux pump that is a member of the ATP-binding cassette (ABC) superfamily. Many drugs in common clinical use are either substrates or inhibitors of this transporter. Quantitative details of P-glycoprotein inhibition by pharmaceutical agents are essential for assessment of their pharmacokinetic behavior and prevention of negative patient reactions. Cell-based systems have been widely used for determination of drug interactions with P-glycoprotein, but they suffer from several disadvantages, and results are often widely variable between laboratories. We aimed to demonstrate that a novel liposomal system employing contemporary biochemical methodologies could measure the ability of clinically used drugs to inhibit the P-glycoprotein pump. To accomplish this we compared results with those of cell-based approaches. Purified transport-competent hamster Abcb1a P-glycoprotein was reconstituted into a unilamellar liposomal system, Fluorosome-trans-pgp, whose aqueous interior contains fluorescent drug sensors. This provides a well-defined system for measuring P-glycoprotein transport inhibition by test drugs in real time using rapid fluorescence-based technology. Inhibition of ATP-driven transport by Fluorosome-trans-pgp employed a panel of 46 representative drugs. Resulting IC50 values correlated well (r2=0.80) with Kd values for drug binding to purified P-glycoprotein. They also showed a similar trend to transport inhibition data obtained using LLC-MDR1 cell monolayers. Fluorosome-trans-pgp IC50 values were in agreement with published results of digoxin drug-drug interaction studies in humans. This novel approach using a liposomal system and fluorescence-based technology is shown to be suitable to study whether marketed drugs and drug candidates are P-glycoprotein inhibitors. The assay is rapid, allowing a 7-point IC50 determination in <6 min, and requires minimal quantities of test drug. The method is amenable to robotics and offers a cost advantage relative to conventional cell-based assays. The well-defined nature of this assay also obviates many of the inherent complications and ambiguities of cell-based systems. Copyright © 2012 Elsevier Inc. All rights reserved.

Participation of the 39-kDa glycoprotein (gp39) of the vitelline envelope of Bufo arenarum eggs in sperm-egg interaction.

PubMed

Barrera, Daniel; Llanos, Ricardo J; Miceli, Dora C

2012-05-01

The acquisition of egg fertilizability in Bufo arenarum takes place during the oviductal transit and during this process the extracellular coelomic envelope (CE) of the eggs is converted into the vitelline envelope (VE). It has been stated that one of the necessary events leading to a fertilizable state is the proteolytic cleavage of CE glycoproteins in the oviductal pars recta by oviductin, a serine protease. Consequently, there is a marked increase in the relative quantity of glycoproteins with 39 (gp39) and 42 kDa (gp42) in the VE. In the present study, sperm-VE binding assays using heat-solubilized biotin-conjugated VE glycoproteins revealed that both gp39 and gp42 have sperm binding capacity. According to this result, our study was focused on gp39, a glycoprotein that we have previously reported as a homologue of mammalian ZPC. For this purpose, rabbit polyclonal antibodies against gp39 were generated at our laboratory. The specificity of the antibodies was confirmed with western blot of VE glycoproteins separated on SDS-PAGE. Immunohistochemical and immunoelectron studies showed gp39 distributed throughout the width of the VE. In addition, immunofluorescence assays probed that gp39 bound to the sperm head. Finally, as an approach to elucidate the possible involvement of gp39 in fertilization, inhibition assays showed that pretreatment of eggs with antibodies against gp39 generated a significant decrease in the fertilization rate. Therefore, our findings suggest that gp39, which is modified by oviductal action, participates as a VE glycoprotein ligand for sperm in Bufo arenarum fertilization.

Correlation of leukocyte adhesiveness, adhesion molecule expression and leukocyte-induced contraction following balloon angioplasty

PubMed Central

Kennedy, Simon; McPhaden, Allan R; Wadsworth, Roger M; Wainwright, Cherry L

2000-01-01

The aim of this study was to examine the changes in leukocyte adhesion and leukocyte-induced contraction in balloon-injured rabbit subclavian artery and to correlate these changes with vessel morphology and expression of adhesion molecules on the injured arteries.Rabbits were anaesthetized and their left subclavian arteries were injured by balloon inflation and withdrawal followed by sacrifice at 2, 24, 48 h or 8 days after injury. The left and right subclavian arteries were removed and leukocytes were isolated from autologous rabbit blood. Leukocyte-induced contraction was measured in 5-HT precontracted artery rings and leukocyte adhesion was measured using 51Cr-labelled leukocytes. Immunocytochemistry using paraffin-embedded tissue was employed to detect changes in the expression of adhesion molecules on injured arteries.Autologous leukocytes caused a contraction of rabbit subclavian artery rings, which was prevented by L-NAME (10−3 M). Balloon-induced injury abolished the contractile response to leukocytes, which correlated with loss of carbachol-induced relaxationBalloon injury markedly enhanced the adhesiveness of the subclavian artery for leukocytes, most notably at 24 and 48 h after injury (1.7 and 1.8 fold respectively). Increased leukocyte adhesion at these two time points correlated with an upregulation of E-selectin, P-selectin and VCAM-1 expression on the remaining endothelium of the injured artery.Vessel morphology revealed that balloon inflation had induced an infiltration of inflammatory cells into the vessel wall, the greatest increase being seen at 24 h after injury.It is concluded that an increase in the expression of E-selectin, P-selectin and VCAM-1 following balloon-induced injury leads to enhanced leukocyte adhesion and migration into the injured vessel. PMID:10781003

Changes in Serum Markers of Inflammation and Endothelial Activation in HIV-Infected Antiretroviral Naive Patients Starting A Treatment with Abacavir-Lamivudine or Tenofovir-Emtricitabine Plus Efavirenz.

PubMed

Calza, Leonardo; Magistrelli, Eleonora; Danese, Ilaria; Colangeli, Vincenzo; Borderi, Marco; Bon, Isabella; Re, Maria Carla; Mancini, Rita; Conti, Matteo; Motta, Roberto; Viale, Pierluigi

2016-01-01

The association between abacavir use and increased risk of myocardial infarction has been heavily debated, but cohort studies and randomized trials have provided conflicting results. Aim of our study is to compare the effect of abacavir and tenofovir on the inflammation and endothelial activation markers. We performed an observational study of HIV-infected naïve patients starting tenofovir/emtricitabine (group A) or abacavir/lamivudine (group B) plus efavirenz. In the present analysis, we measured serum levels of high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), soluble vascular adhesion molecule-1 (VCAM-1), soluble intercellular adhesion molecule-1 (ICAM-1), E-selectin and P-selectin at baseline and during a 48-week follow-up. As a whole, 118 patients (93 males; mean age ± SD of 42.8 ± 10.1 years) were enrolled: 61 in group A and 57 in group B. In group A at weeks 24 and 48 the mean concentrations of IL-6, TNF-α, ICAM-1, VCAM-1, E-selectin and Pselectin decreased significantly in comparison with respective baseline values. In group B at week 24 a significant increase in mean values of these markers was reported in comparison with group A, but after 48 weeks they significantly decreased in group B too and no significant differences between groups A and B were found. In our study, naïve patients starting tenofovir/emtricitabine or abacavir/lamivudine plus efavirenz showed after 48 weeks a significant and comparable decrease in serum concentrations of IL-6, TNF-α, ICAM-1, VCAM-1, Eselectin and P-selectin, while the mean level of hs-CRP did not change significantly in any group.

Combined aspirin and cilostazol treatment is associated with reduced platelet aggregation and prevention of exercise-induced platelet activation.

PubMed

Cleanthis, M; Bhattacharya, V; Smout, J; Ashour, H; Stansby, G

2009-05-01

Cilostazol has proven efficacy in increasing walking distance in claudicants, but it has not been demonstrated to be more effective than placebo in secondary cardiovascular prevention. The direct effect of exercise on platelet function remains less well defined. We have investigated the effect of combination treatment with aspirin and cilostazol on platelet activity in claudicants subjected to repeated treadmill exercise. Nineteen claudicants completed a double-blind, randomised, controlled, cross-over trial. Each subject received a 2-week course of aspirin (75mg) and placebo and aspirin and cilostazol (100mg twice daily). Following each 2-week treatment period, patients participated in a standardised treadmill test (3.2kmh(-1), 10 degrees incline) walking to maximal claudication distance. The exercise was repeated thrice in total, and blood was sampled before and after exercise. Platelet activation was measured using free platelet counting aggregation, flow cytometry for surface markers of platelet activation and soluble P-selectin assay. Compared to aspirin and placebo, combination treatment with aspirin and cilostazol was associated with reduced arachidonic-acid-induced platelet aggregation (p<0.01, Wilcoxon signed-rank test). Aspirin and placebo treatment were associated with elevated P-selectin expression, platelet-monocyte aggregation and reduced CD42b expression (p<0.05, Wilcoxon signed-rank test) post-exercise. No difference was seen in spontaneous platelet aggregation whilst soluble P-selectin was reduced post-exercise with combination treatment with aspirin and cilostazol (p<0.05, Wilcoxon signed-rank test). Combination treatment with aspirin and cilostazol results in suppression of platelet activation and reduces the effect of exercise on platelets. The benefit seen may be a result of cilostazol enhancing the inhibitory effect of aspirin on the cyclo-oxygenase pathway.

L-selectin Is Essential for Delivery of Activated CD8+ T Cells to Virus-Infected Organs for Protective Immunity

PubMed Central

Mohammed, Rebar N.; Watson, H. Angharad; Vigar, Miriam; Ohme, Julia; Thomson, Amanda; Humphreys, Ian R.; Ager, Ann

2016-01-01

Summary Cytotoxic CD8+ T lymphocytes play a critical role in the host response to infection by viruses. The ability to secrete cytotoxic chemicals and cytokines is considered pivotal for eliminating virus. Of equal importance is how effector CD8+ T cells home to virus-infected tissues. L-selectin has not been considered important for effector T cell homing, because levels are low on activated T cells. We report here that, although L-selectin expression is downregulated following T cell priming in lymph nodes, L-selectin is re-expressed on activated CD8+ T cells entering the bloodstream, and recruitment of activated CD8+ T cells from the bloodstream into virus-infected tissues is L-selectin dependent. Furthermore, L-selectin on effector CD8+ T cells confers protective immunity to two evolutionally distinct viruses, vaccinia and influenza, which infect mucosal and visceral organs, respectively. These results connect homing and a function of virus-specific CD8+ T cells to a single molecule, L-selectin. PMID:26804910

Synthetic E-selectin prevents postoperative vascular restenosis by inhibiting nuclear factor κB in rats

PubMed Central

Liu, Jiangang; Liu, Zhongjie; Hu, Xiaohui; Zhang, Yuan; Zhang, Shiming

2018-01-01

During the development of postoperative vascular restenosis, the aberrant proliferation of vascular smooth muscle cells (VSMCs) is a critical event resulting in intimal hyperplasia. Inflammatory responses involving the activation of nuclear factor (NF)-κB are among the major molecular processes underlying restenosis. The present study aimed to investigate the roles of NF-κB in VSMC proliferation and restenosis following vascular anastomosis, as well as to evaluate the potential of synthetic E-selectin to downregulate NF-κB and thus inhibit vascular hyperplasia. A total of 72 adult male Sprague-Dawley rats were randomly assigned to three groups: Control, operation and treatment groups. Rats in the operation and treatment groups received longitudinal incisions in the right carotid arteries, which were closed using interrupted sutures. Following vascular anastomosis, synthetic E-selectin (10 mg/kg), or an equal volume of saline, was immediately injected into the right femoral vein of rats in the treatment and operation groups, respectively. Following surgery, the mRNA and protein expression levels of NF-κB at the site of anastomosis, the levels of tumor necrosis factor-α and interleukin-6 in the serum, NF-κB binding activity, and the presence of proliferating cell nuclear antigen (PCNA)-positive cells were evaluated by western blotting, reverse transcription-quantitative polymerase chain reaction, ELISA, electrophoretic mobility shift assay and immunofluorescence staining. The present results demonstrated that following treatment with synthetic E-selectin, the levels of NF-κB and the inflammatory response, as well as the presence of PCNA-positive cells, were significantly reduced (P<0.01). In conclusion, the results of the present study suggested that synthetic E-selectin may exert anti-inflammatory and anti-restenotic effects following vascular anastomosis in vivo. PMID:29393453

Comparison of the effects of Crataegus oxyacantha extract, aerobic exercise and their combination on the serum levels of ICAM-1 and E-Selectin in patients with stable angina pectoris.

PubMed

Jalaly, Leila; Sharifi, Gholamreza; Faramarzi, Mohammad; Nematollahi, Alireza; Rafieian-kopaei, Mahmoud; Amiri, Masoud; Moattar, Fariborz

2015-12-19

Adhesion molecules play an important role in the development and progression of coronary atherosclerosis. The aim of this study was comparing the effect of Cratagol herbal tablet, aerobic exercise and their combination on the serum levels of Intercellular adhesion molecule (ICAM)-1 and E-Selectin in patients with stable angina pectoris. Eighty stable angina pectoris patients aged between 45 and 65 years, were randomly divided into four groups including three experimental groups and one control group: aerobic exercise (E), Crataegus oxyacantha extract (S), aerobic exercise and Crataegus oxyacantha extract (S+E), and control (C). Blood sampling was taken 24 h before and after 12 weeks of aerobic exercise and Crataegus oxyacantha extract consumption. The results of serum levels of ICAM-1 and E-selectin were compared. Intergroup comparison of the data revealed a significant reduction (P <0.01) in serum levels of ICAM-1 and E-selectin in experimental groups. Analysis of data showed that the serum levels of ICAM-1 had significant difference when group S+E was compared with groups S and C, but not group E (P = 0.021, P = 0.000 and P = 0.068, respectively). Also the difference between the levels of E-selectin was significant comparing S+E and S but not E with group C (P = 0.021, P = 0.000 and P = 0.052, respectively). Twelve weeks effects of aerobic exercise and Crataegus oxyacantha extract consuming is an effective complementary strategy to significantly lower the risk of atherosclerosis and heart problems.

Fixation of Oligosaccharides to a Surface May Increase the Susceptibility to Human Parainfluenza Virus 1, 2, or 3 Hemagglutinin-Neuraminidase▿†

PubMed Central

Tappert, Mary M.; Smith, David F.; Air, Gillian M.

2011-01-01

The hemagglutinin-neuraminidase (HN) protein of human parainfluenza viruses (hPIVs) both binds (H) and cleaves (N) oligosaccharides that contain N-acetylneuraminic acid (Neu5Ac). H is thought to correspond to receptor binding and N to receptor-destroying activity. At present, N′s role in infection remains unclear: does it destroy only receptors, or are there other targets? We previously demonstrated that hPIV1 and 3 HNs bind to oligosaccharides containing the motif Neu5Acα2-3Galβ1-4GlcNAc (M. Amonsen, D. F. Smith, R. D. Cummings, and G. M. Air, J. Virol. 81:8341–8345, 2007). In the present study, we tested the binding specificity of hPIV2 on the Consortium for Functional Glycomics' glycan array and found that hPIV2 binds to oligosaccharides containing the same motif. We determined the specificities of N on red blood cells, soluble small-molecule and glycoprotein substrates, and the glycan array and compared them to the specificities of H. hPIV2 and -3, but not hPIV1, cleaved their ligands on red blood cells. hPIV1, -2, and -3 cleaved their NeuAcα2-3 ligands on the glycan array; hPIV2 and -3 also cleaved NeuAcα2-6 ligands bound by influenza A virus. While all three HNs exhibited similar affinities for all cleavable soluble substrates, their activities were 5- to 10-fold higher on small molecules than on glycoproteins. In addition, some soluble glycoproteins were not cleaved, despite containing oligosaccharides that were cleaved on the glycan array. We conclude that the susceptibility of an oligosaccharide substrate to N increases when the substrate is fixed to a surface. These findings suggest that HN may undergo a conformational change that activates N upon receptor binding at a cell surface. PMID:21917945

Interfacial Cavity Filling To Optimize CD4-Mimetic Miniprotein Interactions with HIV-1 Surface Glycoprotein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morellato-Castillo, Laurence; Acharya, Priyamvada; Combes, Olivier

Ligand affinities can be optimized by interfacial cavity filling. A hollow (Phe43 cavity) between HIV-1 surface glycoprotein (gp120) and cluster of differentiation 4 (CD4) receptor extends beyond residue phenylalanine 43 of CD4 and cannot be fully accessed by natural amino acids. To increase HIV-1 gp120 affinity for a family of CD4-mimetic miniproteins (miniCD4s), we targeted the gp120 Phe43 cavity with 11 non-natural phenylalanine derivatives, introduced into a miniCD4 named M48 (1). The best derivative, named M48U12 (13), bound HIV-1 YU2 gp120 with 8 pM affinity and showed potent HIV-1 neutralization. It contained a methylcyclohexyl derivative of 4-aminophenylalanine, and its cocrystalmore » structure with gp120 revealed the cyclohexane ring buried within the gp120 hydrophobic core but able to assume multiple orientations in the binding pocket, and the aniline nitrogen potentially providing a focus for further improvement. Altogether, the results provide a framework for filling the interfacial Phe43 cavity to enhance miniCD4 affinity.« less

pH protective Y1 receptor ligand functionalized antiphagocytosis BPLP-WPU micelles for enhanced tumor imaging and therapy with prolonged survival time.

PubMed

Jiang, Zhenqi; Tian, Yuchen; Shan, Dingying; Wang, Yinjie; Gerhard, Ethan; Xia, Jianbi; Huang, Rong; He, Yan; Li, Aiguo; Tang, Jianchao; Ruan, Huimin; Li, Yong; Li, Juan; Yang, Jian; Wu, Aiguo

2018-07-01

Nanoparticle-based tumor therapies are extensively studied; however, few are capable of improving patient survival time due to premature drug leakage, off target effects, and poor tissue penetration. Previously, we successfully synthesized a novel family of Y 1 receptor (Y 1 R) ligand modified, photoluminescent BPLP nanobubbles and nanoparticles for targeted breast cancer ultrasound imaging; however, increased accumulation could also be observed in the liver, kidney, and spleen, suggesting significant interaction of the particles with macrophages in vivo. Herein, for the first time, we imparted antiphagocytosis capability to Y 1 R ligand functionalized BPLP-WPU polymeric micelles through the incorporation of a CD47 human glycoprotein based self-peptide. Application of self-peptide modified, DOX loaded micelles in vivo resulted in a 100% survival rate and complete tumor necrosis over 100 days of treatment. In vivo imaging of SPION loaded, self-peptide modified micelles revealed effective targeting to the tumor site while analysis of iron content demonstrated reduced particle accumulation in the liver and kidney, demonstrating reduced macrophage interaction, as well as a 2-fold increase of particles in the tumor. As these results demonstrate, Y 1 R ligand, self-peptide modified BPLP-WPU micelles are capable of target specific cancer treatment and imaging, making them ideal candidates to improve survival rate and tumor reduction clinically. Copyright © 2018 Elsevier Ltd. All rights reserved.

Comparison of L-selectin blood level and gene polymorphism in tuberculosis patients with healthy individuals.

PubMed

Eini, Peyman; Shirvani, Maria; Hajilooi, Mehrdad; Esna-Ashari, Farzaneh

2018-02-12

The inflammatory response to Mycobacterium tuberculosis bacilli influences tuberculosis (TB) progression. In this study, we aimed to identify the Phe206Leu polymorphism and serum L-selectin level in TB patients, compared to healthy individuals. Ninety patients with a diagnosis of TB and 90 healthy controls were selected in this study. The serum L-selectin level was determined, using ELISA. L-selectin polymorphism was also evaluated using PCR. For data analysis, SPSS was used at a significance level of 0.05. According to the findings, the mean±SD age of the participants was 57.5 ± 18.4 and 56.5 ± 17.5 years in the TB and healthy groups, respectively. The TB group showed a significantly higher serum L-selectin level (1721.1 ± 330.9) versus the healthy controls (1624 ± 279). The L-selectin Phe allele frequencies were higher than the Leu allele frequencies in the main population, whereas the patients and controls were not significantly different. Eight (0.04%) subjects had Leu/Leu genotypes, 84 (46.6%) carried Phe/Leu genotypes, and 88 (48.8%) had Phe/Phe genotypes. Our results showed that the groups were not significantly different regarding L-selectin genotypes. TB patients had a significantly higher serum L-selectin level, compared to the controls. Based on the findings, the incidence of TB and L-selectin polymorphism in the Phe206Leu gene had no significant association. © 2018 Wiley Periodicals, Inc.

Barley as a green factory for the production of functional Flt3 ligand.

PubMed

Erlendsson, Lýdur S; Muench, Marcus O; Hellman, Ulf; Hrafnkelsdóttir, Soffía M; Jonsson, Anders; Balmer, Yves; Mäntylä, Einar; Orvar, Björn L

2010-02-01

Biologically active recombinant human Flt3 ligand was expressed and isolated from transgenic barley seeds. Its expression is controlled by a tissue specific promoter that confines accumulation of the recombinant protein to the endosperm tissue of the seed. The recombinant Flt3 ligand variant expressed in the seeds contains an HQ-tag for affinity purification on immobilized metal ion affinity chromatography (IMAC) resin. The tagged protein was purified from seed extracts to near homogeneity using sequential chromatography on IMAC affinity resin and cation exchange resin. We also show that the recombinant Flt3 ligand protein undergoes posttranslational modifications: it is a glycoprotein containing alpha-1,3-fucose and alpha-1,2-xylose. The HQ-tagged Flt3 ligand variant exhibits comparable biological activity to commercial Flt3 ligand. This is the first report showing expression and accumulation of recombinant human growth factor in barley seeds with a yield of active protein similar to a bacterial expression system. The present results demonstrate that plant molecular farming is a viable approach for the bioproduction of human-derived growth factors.

Theoretical and Computational Studies of Peptides and Receptors of the Insulin Family

PubMed Central

Vashisth, Harish

2015-01-01

Synergistic interactions among peptides and receptors of the insulin family are required for glucose homeostasis, normal cellular growth and development, proliferation, differentiation and other metabolic processes. The peptides of the insulin family are disulfide-linked single or dual-chain proteins, while receptors are ligand-activated transmembrane glycoproteins of the receptor tyrosine kinase (RTK) superfamily. Binding of ligands to the extracellular domains of receptors is known to initiate signaling via activation of intracellular kinase domains. While the structure of insulin has been known since 1969, recent decades have seen remarkable progress on the structural biology of apo and liganded receptor fragments. Here, we review how this useful structural information (on ligands and receptors) has enabled large-scale atomically-resolved simulations to elucidate the conformational dynamics of these biomolecules. Particularly, applications of molecular dynamics (MD) and Monte Carlo (MC) simulation methods are discussed in various contexts, including studies of isolated ligands, apo-receptors, ligand/receptor complexes and intracellular kinase domains. The review concludes with a brief overview and future outlook for modeling and computational studies in this family of proteins. PMID:25680077

L1 adhesion molecule on mouse leukocytes: regulation and involvement in endothelial cell binding.

PubMed

Hubbe, M; Kowitz, A; Schirrmacher, V; Schachner, M; Altevogt, P

1993-11-01

L1 is a cell surface glycoprotein of the immunoglobulin superfamily which was initially shown to mediate adhesion between neural cells. Recently we have reported that L1 is expressed by bone marrow cells and the majority of mature lymphocytes (Kowitz et al., Eur. J. Immunol. 1992. 22: 1199-1205). To analyze the function of L1 on leukocytes we studied its regulation following cell activation. In vitro activation of B lymphocytes with lipopolysaccharide or T lymphocytes with phorbol 12-myristate 13-acetate/Ca2+ ionophore, concanavalin A or anti-CD3 monoclonal antibody as well as in vivo activation of V beta 8+ T cells with staphylococcal enterotoxin B (SEB) revealed a down-regulation of L1 within 48 h. A rapid loss of L1 expression was seen when mouse neutrophils were activated with PMA alone. This rapid loss paralleled the shedding of L-selectin. We also studied a possible role of L1 in the binding of leukocytes to endothelial cells. ESb-MP lymphoma cells with a high expression of L1 (L1hi) could bind to bend3 endothelioma cells without prior activation with inflammatory cytokines. The interaction was inhibited by anti-L1 antibodies. In contrast, ESb-MP cells with low L1 expression (L1lo) were only marginally bound. Latex beads coated with affinity-isolated L1 antigen were also able to bind to the endothelioma cells in a specific fashion. The binding of ESb-MP lymphoma cells required Ca2+ and Mg2+ ions and was sensitive to cold temperature. Since the endothelioma cells did not express L1 the binding mechanism studied here is distinct from the established L1-L1 homotypic interaction. It is possible that the novel L1-mediated adhesion pathway involves an unidentified ligand and could play a role in leukocyte migration.

Microglial Lectins in Health and Neurological Diseases

PubMed Central

Siew, Jian Jing; Chern, Yijuang

2018-01-01

Microglia are the innate sentinels of the central nervous system (CNS) and are responsible for the homeostasis and immune defense of the CNS. Under the influence of the local environment and cell-cell interaction, microglia exhibit a multidimensional and context-dependent phenotypes that can be cytotoxic and neuroprotective. Recent studies suggest that microglia express multitudinous types of lectins, including galectins, Siglecs, mannose-binding lectins (MBLs) and other glycan binding proteins. Because most studies that examine lectins focus on the peripheral system, the functions of lectins have not been critically investigated in the CNS. In addition, the types of brain cells that contribute to the altered levels of lectins present in diseases are often unclear. In this review, we will discuss how galectins, Siglecs, selectins and MBLs contribute to the dynamic functions of microglia. The interacting ligands of these lectins are complex glycoconjugates, which consist of glycoproteins and glycolipids that are expressed on microglia or surrounding cells. The current understanding of the heterogeneity and functions of glycans in the brain is limited. Galectins are a group of pleotropic proteins that recognize both β-galactoside-containing glycans and non- β-galactoside-containing proteins. The function and regulation of galectins have been implicated in immunomodulation, neuroinflammation, apoptosis, phagocytosis and oxidative bursts. Most Siglecs are expressed at a low level on the plasma membrane and bind to sialic acid residues for immunosurveillance and cell-cell communication. Siglecs are classified based on their inhibitory and activatory downstream signaling properties. Inhibitory Siglecs negatively regulate microglia activation upon recognizing the intact sialic acid patterns and vice versa. MBLs are expressed upon infection in cytoplasm and can be secreted in order to recognize molecules containing terminal mannose as an innate immune defense machinery. Most importantly, multiple studies have reported dysregulation of lectins in neurological disorders. Here, we reviewed recent studies on microglial lectins and their functions in CNS health and disease, and suggest that these lectin families are novel, potent therapeutic targets for neurological diseases. PMID:29867350

Fragment-based virtual screening approach and molecular dynamics simulation studies for identification of BACE1 inhibitor leads.

PubMed

Manoharan, Prabu; Ghoshal, Nanda

2018-05-01

Traditional structure-based virtual screening method to identify drug-like small molecules for BACE1 is so far unsuccessful. Location of BACE1, poor Blood Brain Barrier permeability and P-glycoprotein (Pgp) susceptibility of the inhibitors make it even more difficult. Fragment-based drug design method is suitable for efficient optimization of initial hit molecules for target like BACE1. We have developed a fragment-based virtual screening approach to identify/optimize the fragment molecules as a starting point. This method combines the shape, electrostatic, and pharmacophoric features of known fragment molecules, bound to protein conjugate crystal structure, and aims to identify both chemically and energetically feasible small fragment ligands that bind to BACE1 active site. The two top-ranked fragment hits were subjected for a 53 ns MD simulation. Principle component analysis and free energy landscape analysis reveal that the new ligands show the characteristic features of established BACE1 inhibitors. The potent method employed in this study may serve for the development of potential lead molecules for BACE1-directed Alzheimer's disease therapeutics.

Evaluation of serum leaking enzymes and investigation into new biomarkers for exercise-induced muscle damage.

PubMed

Kanda, Kazue; Sugama, Kaoru; Sakuma, Jun; Kawakami, Yasuo; Suzuki, Katsuhiko

2014-01-01

This investigation determined whether existing muscle damage markers and organ damage markers respond to an acute eccentric exercise protocol and are associated with affected muscle symptoms. Nine healthy-young men completed one-leg calf-raise exercise with their right leg on a force plate. They performed 10 sets of 40 repetitions of exercise at 0.5 Hz with a load corresponding to half of their body weight, with 3 min rest between sets. The tenderness of medial gastrocnemius, lateral gastrocnemius and soleus, and the ankle active range of motion (ROM) were assessed before, immediately after, 24 h and 48 h, 72 h, 96 h and 168 h after exercise. Blood and urine were collected pre-exercise and 2 h, 4 h, 24 h, 48 h, 72 h and 96 h post-exercise. Serum was analyzed for creatine kinase (CK), aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and aldolase (ALD) activities. We also determined heart-type fatty acid-binding protein (H-FABP), intestinal-type fatty acid-binding protein (I-FABP) and liver-type fatty acid-binding protein (L-FABP), neutrophil gelatinase-associated lipocalin (NGAL), interleukin (IL)-17A, IL-23, nerve growth factor (NGF), soluble-Endothelial (sE)-selectin, s-Leukocyte (L)-selectin, s-Platelets (P)-selectin, and 8-isoprostane in plasma and urine. The tenderness of proximal and middle gastrocnemius increased significantly 72 h (p < 0.05, p < 0.01) after exercise. Ankle active ROM in dorsal flexion decreased significantly 48 h (p < 0.05) and 72 h (p < 0.01) after exercise. CK and ALD activities significantly increased at 72 h (p < 0.05) and remained elevated at 96 h (p < 0.01) postexercise compared to pre-exercise values. Also, ALD which showed relatively lower interindividual variability was significantly correlated with tenderness of middle gastrocnemius at 72 h. LDH activity significantly increased 96 h postexercise (p < 0.01), whereas the increase in AST and ALT activities 96 h post-exercise was not significantly different from pre-exercise values. There were no significant changes in FABPs, NGAL, IL-17A, IL-23, NGF, selectins and 8-isoprostanes in plasma and urine. In conclusion, calf-raise exercise induced severe local muscle damage symptoms which were accompanied by increases in both serum CK and ALD activities, but we could not detect any changes in examined markers of organ damage, inflammation and oxidative stress. Further research is needed to determine other more sensitive biomarkers and the underlying mechanisms of exercise-induced muscle damage.

GlyCAM1 negatively regulates monocyte entry into the optic nerve head and contributes to radiation-based protection in glaucoma.

PubMed

Williams, Pete A; Braine, Catherine E; Foxworth, Nicole E; Cochran, Kelly E; John, Simon W M

2017-04-26

We previously reported a profound long-term neuroprotection subsequent to a single radiation-therapy in the DBA/2J mouse model of glaucoma. This neuroprotection prevents entry of monocyte-like immune cells into the optic nerve head during glaucoma. Gene expression studies in radiation-treated mice implicated Glycam1 in this protection. Glycam1 encodes a proteoglycan ligand for L-selectin and is an excellent candidate to modulate immune cell entry into the eye. Here, we experimentally test the hypothesis that radiation-induced over-expression of Glycam1 is a key component of the neuroprotection. We generated a null allele of Glycam1 on a DBA/2J background. Gene and protein expression of Glycam1, monocyte entry into the optic nerve head, retinal ganglion cell death, and axon loss in the optic nerve were assessed. Radiation therapy potently inhibits monocyte entry into the optic nerve head and prevents retinal ganglion cell death and axon loss. DBA/2J mice carrying a null allele of Glycam1 show increased monocyte entry and increased retinal ganglion cell death and axon loss following radiation therapy, but the majority of optic nerves were still protected by radiation therapy. Although GlyCAM1 is an L-selectin ligand, its roles in immunity are not yet fully defined. The current study demonstrates a partial role for GlyCAM1 in radiation-mediated protection. Furthermore, our results clearly show that GlyCAM1 levels modulate immune cell entry from the vasculature into neural tissues. As Glycam1 deficiency has a more profound effect on cell entry than on neurodegeneration, further experiments are needed to precisely define the role of monocyte entry in DBA/2J glaucoma. Nevertheless, GlyCAM1's function as a negative regulator of extravasation may lead to novel therapeutic strategies for an array of common conditions involving inflammation.

Differential effects of low-carbohydrate and low-fat diets on inflammation and endothelial function in diabetes.

PubMed

Davis, Nichola J; Crandall, Jill P; Gajavelli, Srikanth; Berman, Joan W; Tomuta, Nora; Wylie-Rosett, Judith; Katz, Stuart D

2011-01-01

To characterize acute (postprandial) and chronic (after a 6-month period of weight loss) effects of a low-carbohydrate vs. a low-fat diet on subclinical markers of cardiovascular disease (CVD) in adults with type 2 diabetes. At baseline and 6 months, measures of C-reactive protein (CRP), interleukin-6 (IL-6), soluble intercellular adhesion molecule (sICAM) and soluble E-selectin were obtained from archived samples (n = 51) of participants randomized in a clinical trial comparing a low-carbohydrate and a low-fat diet. In a subset of participants (n = 27), postprandial measures of these markers were obtained 3 h after a low-carbohydrate or low-fat liquid meal. Endothelial function was also measured by reactive hyperemic peripheral arterial tonometry during the meal test. Paired t tests and unpaired t tests compared within- and between-group changes. There were no significant differences observed in postprandial measures of inflammation or endothelial function. After 6 months, CRP (mean ± S.E.) decreased in the low-fat arm from 4.0 ± 0.77 to 3.0 ± 0.77 (P = .01). In the low-carbohydrate arm, sICAM decreased from 234 ± 22 to 199 ± 23 (P = .001), and soluble E-selectin decreased from 93 ± 10 to 82 ± 10 (P = .05.) A significant correlation between change in high-density lipoprotein and change in soluble E-selectin (r = -0.33, P = .04) and with the change in ICAM (r = -0.43, P = .01) was observed. Low-carbohydrate and low-fat diets both have beneficial effects on CVD markers. There may be different mechanisms through which weight loss with these diets potentially reduces CVD risk. Copyright © 2011 Elsevier Inc. All rights reserved.

Relationship between Platelet PPARs, cAMP Levels, and P-Selectin Expression: Antiplatelet Activity of Natural Products

PubMed Central

Fuentes, Eduardo; Palomo, Iván

2013-01-01

Platelets are no longer considered simply as cells participating in thrombosis. In atherosclerosis, platelets are regulators of multiple processes, with the recruitment of inflammatory cells towards the lesion sites, inflammatory mediators release, and regulation of endothelial function. The antiplatelet therapy has been used for a long time in an effort to prevent and treat cardiovascular diseases. However, limited efficacy in some patients, drug resistance, and side effects are limitations of current antiplatelet therapy. In this context, a large number of natural products (polyphenols, terpenoids, alkaloids, and fatty acids) have been reported with antiplatelet activity. In this sense, the present paper describes mechanisms of antiplatelet action of natural products on platelet P-selectin expression through cAMP levels and its role as peroxisome proliferator-activated receptors agonists. PMID:24324520

Mechanisms of the priming effect of low doses of lipopoly-saccharides on leukocyte-dependent platelet aggregation in whole blood.

PubMed

Montrucchio, Giuseppe; Bosco, Ornella; Del Sorbo, Lorenzo; Fascio Pecetto, Paolo; Lupia, Enrico; Goffi, Alberto; Omedè, Paola; Emanuelli, Giorgio; Camussi, Giovanni

2003-11-01

Several studies focused on the ability of bacterial lipopolysac-charides (LPS) in triggering platelet and/or leukocyte activation. The aim of this study was to investigate the molecular mechanisms involved in the aggregation of platelets and in their interaction with leukocytes in whole blood after stimulation with low doses of LPS. LPS did not directly induce platelet aggregation in whole blood, but they primed the aggregation of platelets induced by epinephrine, adenosine diphosphate and arachidonic acid. As shown by cytofluorimetry, platelets neither bind FITC-LPS, nor express the LPS-receptors CD14 and toll-like receptor 4 (TLR4). On the contrary, LPS primed monocytes and to a lesser extent polymorphonuclear neutrophils to adhere to platelets. Both platelet-leukocyte interaction and platelet aggregation in whole blood were inhibited by blockade of CD14 and TLR4. Moreover, the interaction between platelets and leukocytes was inhibited by P-selectin, and by blockade of PAF and reactive oxygen species, suggesting a role of P-selectin and of leukocyte-derived mediators. In conclusion, these results elucidate the mechanisms leading to platelet activation and interaction with leukocytes triggered by LPS. They suggest that the activation of platelets by LPS is mainly dependent on leukocytes and especially monocytes as a result of CD14 and TLR4 engagement. Moreover, we found that leukocyte-platelet interaction was triggered by the synthesis of PAF and the generation of oxygen radicals that induced upregulation of surface expression of P-selectin.

Circulating soluble adhesion molecules in patients with giant cell arteritis. Correlation between soluble intercellular adhesion molecule-1 (sICAM-1) concentrations and disease activity

PubMed Central

Coll-Vinent, B.; Vilardell, C.; Font, C.; Oristrell, J.; Hernandez-Rodrigu..., J.; Yague, J.; Urbano-Marquez, A.; Grau, J.; Cid, M.

1999-01-01

OBJECTIVE—To evaluate whether changes in concentrations of circulating adhesion molecules are related to disease activity in patients with giant cell arteritis (GCA).
METHODS—A sandwich ELISA was used to measure soluble intercellular adhesion molecule-1 (sICAM-1), sICAM-3, vascular cell adhesion molecule-1 (sVCAM-1), E-selectin (sE-selectin), and L-selectin (sL-selectin) in serum and plasma samples from patients with GCA. A cross sectional study was performed on 64 GCA patients at different activity stages and on 35 age and sex matched healthy donors. Thirteen of these patients were evaluated at the time of diagnosis and serially during follow up.
RESULTS—At the time of diagnosis, sICAM-1 concentrations were significantly higher in active GCA patients than in controls (mean (SD) 360.55 (129.78) ng/ml versus 243.25 (47.43) ng/ml, p<0.001). In contrast, sICAM-3, sVCAM-1, sE-selectin, and sL-selectin values did not differ from those obtained in normal donors. With corticosteroid administration, a decrease in sICAM-1 concentrations was observed, reaching normal values when clinical remission was achieved (263.18 (92.7) ng/ml globally, 293.59 (108.39) ng/ml in the group of patients in recent remission, and 236.83 (70.02) ng/ml in those in long term remission). In the 13 patients followed up longitudinally, sICAM-1 values also normalised with clinical remission (225.87 (64.25) ng/ml in patients in recent remission, and 256.29 (75.15) ng/ml in those in long term remission).
CONCLUSIONS—Circulating sICAM-1 concentrations clearly correlate with clinically apparent disease activity in GCA patients. Differences with results previously found in patients with other vasculitides may indicate that different pathogenic mechanisms contribute to vascular inflammation in different disorders.

 Keywords: adhesion molecules; giant cell arteritis; inflammation PMID:10364919

Arenavirus Stable Signal Peptide Is the Keystone Subunit for Glycoprotein Complex Organization

PubMed Central

Bederka, Lydia H.; Bonhomme, Cyrille J.; Ling, Emily L.

2014-01-01

ABSTRACT The rodent arenavirus glycoprotein complex encodes a stable signal peptide (SSP) that is an essential structural component of mature virions. The SSP, GP1, and GP2 subunits of the trimeric glycoprotein complex noncovalently interact to stud the surface of virions and initiate arenavirus infectivity. Nascent glycoprotein production undergoes two proteolytic cleavage events: first within the endoplasmic reticulum (ER) to cleave SSP from the remaining precursor GP1/2 (glycoprotein complex [GPC]) glycoprotein and second within the Golgi stacks by the cellular SKI-1/S1P for GP1/2 processing to yield GP1 and GP2 subunits. Cleaved SSP is not degraded but retained as an essential glycoprotein subunit. Here, we defined functions of the 58-amino-acid lymphocytic choriomeningitis virus (LCMV) SSP in regard to glycoprotein complex processing and maturation. Using molecular biology techniques, confocal microscopy, and flow cytometry, we detected SSP at the plasma membrane of transfected cells. Further, we identified a sorting signal (FLLL) near the carboxyl terminus of SSP that is required for glycoprotein maturation and trafficking. In the absence of SSP, the glycoprotein accumulated within the ER and was unable to undergo processing by SKI-1/S1P. Mutation of this highly conserved FLLL motif showed impaired glycoprotein processing and secretory pathway trafficking, as well as defective surface expression and pH-dependent membrane fusion. Immunoprecipitation of SSP confirmed an interaction between the signal peptide and the GP2 subunit; however, mutations within this FLLL motif disrupted the association of the GP1 subunit with the remaining glycoprotein complex. PMID:25352624

The Biological Function of the Prion Protein: A Cell Surface Scaffold of Signaling Modules.

PubMed

Linden, Rafael

2017-01-01

The prion glycoprotein (PrP C ) is mostly located at the cell surface, tethered to the plasma membrane through a glycosyl-phosphatydil inositol (GPI) anchor. Misfolding of PrP C is associated with the transmissible spongiform encephalopathies (TSEs), whereas its normal conformer serves as a receptor for oligomers of the β-amyloid peptide, which play a major role in the pathogenesis of Alzheimer's Disease (AD). PrP C is highly expressed in both the nervous and immune systems, as well as in other organs, but its functions are controversial. Extensive experimental work disclosed multiple physiological roles of PrP C at the molecular, cellular and systemic levels, affecting the homeostasis of copper, neuroprotection, stem cell renewal and memory mechanisms, among others. Often each such process has been heralded as the bona fide function of PrP C , despite restricted attention paid to a selected phenotypic trait, associated with either modulation of gene expression or to the engagement of PrP C with a single ligand. In contrast, the GPI-anchored prion protein was shown to bind several extracellular and transmembrane ligands, which are required to endow that protein with the ability to play various roles in transmembrane signal transduction. In addition, differing sets of those ligands are available in cell type- and context-dependent scenarios. To account for such properties, we proposed that PrP C serves as a dynamic platform for the assembly of signaling modules at the cell surface, with widespread consequences for both physiology and behavior. The current review advances the hypothesis that the biological function of the prion protein is that of a cell surface scaffold protein, based on the striking similarities of its functional properties with those of scaffold proteins involved in the organization of intracellular signal transduction pathways. Those properties are: the ability to recruit spatially restricted sets of binding molecules involved in specific signaling; mediation of the crosstalk of signaling pathways; reciprocal allosteric regulation with binding partners; compartmentalized responses; dependence of signaling properties upon posttranslational modification; and stoichiometric requirements and/or oligomerization-dependent impact on signaling. The scaffold concept may contribute to novel approaches to the development of effective treatments to hitherto incurable neurodegenerative diseases, through informed modulation of prion protein-ligand interactions.

Higher platelet reactivity and platelet-monocyte complex formation in Gram-positive sepsis compared to Gram-negative sepsis.

PubMed

Tunjungputri, Rahajeng N; van de Heijden, Wouter; Urbanus, Rolf T; de Groot, Philip G; van der Ven, Andre; de Mast, Quirijn

2017-09-01

Platelets may play a role in the high risk for vascular complications in Gram-positive sepsis. We compared the platelet reactivity of 15 patients with Gram-positive sepsis, 17 with Gram-negative sepsis and 20 healthy controls using a whole blood flow cytometry-based assay. Patients with Gram-positive sepsis had the highest median fluorescence intensity (MFI) of the platelet membrane expression of P-selectin upon stimulation with high dose adenosine diphosphate (ADP; P = 0.002 vs. Gram-negative and P = 0.005 vs. control groups) and cross-linked collagen-related peptide (CRP-XL; P = 0.02 vs. Gram-negative and P = 0.0001 vs. control groups). The Gram-positive group also demonstrated significantly higher ADP-induced fibrinogen binding (P = 0.001), as wll as platelet-monocyte complex formation (P = 0.02), compared to the Gram-negative group and had the highest plasma levels of platelet factor 4, β-thromboglobulin and soluble P-selectin. In contrast, thrombin-antithrombin complex and C-reactive protein levels were comparable in both patient groups. In conclusion, common Gram-positive pathogens induce platelet hyperreactivity, which may contribute to a higher risk for vascular complications.

Structure based classification for bile salt export pump (BSEP) inhibitors using comparative structural modeling of human BSEP

NASA Astrophysics Data System (ADS)

Jain, Sankalp; Grandits, Melanie; Richter, Lars; Ecker, Gerhard F.

2017-06-01

The bile salt export pump (BSEP) actively transports conjugated monovalent bile acids from the hepatocytes into the bile. This facilitates the formation of micelles and promotes digestion and absorption of dietary fat. Inhibition of BSEP leads to decreased bile flow and accumulation of cytotoxic bile salts in the liver. A number of compounds have been identified to interact with BSEP, which results in drug-induced cholestasis or liver injury. Therefore, in silico approaches for flagging compounds as potential BSEP inhibitors would be of high value in the early stage of the drug discovery pipeline. Up to now, due to the lack of a high-resolution X-ray structure of BSEP, in silico based identification of BSEP inhibitors focused on ligand-based approaches. In this study, we provide a homology model for BSEP, developed using the corrected mouse P-glycoprotein structure (PDB ID: 4M1M). Subsequently, the model was used for docking-based classification of a set of 1212 compounds (405 BSEP inhibitors, 807 non-inhibitors). Using the scoring function ChemScore, a prediction accuracy of 81% on the training set and 73% on two external test sets could be obtained. In addition, the applicability domain of the models was assessed based on Euclidean distance. Further, analysis of the protein-ligand interaction fingerprints revealed certain functional group-amino acid residue interactions that could play a key role for ligand binding. Though ligand-based models, due to their high speed and accuracy, remain the method of choice for classification of BSEP inhibitors, structure-assisted docking models demonstrate reasonably good prediction accuracies while additionally providing information about putative protein-ligand interactions.

Developmental Stage-Specific Manifestations of Absent TPO/c-MPL Signalling in Newborn Mice.

PubMed

Lorenz, Viola; Ramsey, Haley; Liu, Zhi-Jian; Italiano, Joseph; Hoffmeister, Karin; Bihorel, Sihem; Mager, Donald; Hu, Zhongbo; Slayton, William B; Kile, Benjamin T; Sola-Visner, Martha; Ferrer-Marin, Francisca

2017-12-01

Congenital amegakaryocytic thrombocytopaenia (CAMT) is a disorder caused by c-MPL mutations that impair thrombopoietin (TPO) signalling, resulting in a near absence of megakaryocytes (MKs). While this phenotype is consistent in adults, neonates with CAMT can present with severe thrombocytopaenia despite normal MK numbers. To investigate this, we characterized MKs and platelets in newborn c-MPL –/– mice. Liver MKs in c-MPL –/– neonates were reduced in number and size compared with wild-type (WT) age-matched MKs, and exhibited ultrastructural abnormalities not found in adult c-MPL –/– MKs. Platelet counts were lower in c-MPL –/– compared with WT mice at birth and did not increase over the first 2 weeks of life. In vivo biotinylation revealed a significant reduction in the platelet half-life of c-MPL –/– newborn mice (P2) compared with age-matched WT pups, which was not associated with ultrastructural abnormalities. Genetic deletion of the pro-apoptotic Bak did not rescue the severely reduced platelet half-life of c-MPL –/– newborn mice, suggesting that it was due to factors other than platelets entering apoptosis early. Indeed, adult GFP+ (green fluorescent protein transgenic) platelets transfused into thrombocytopenic c-MPL –/– P2 pups also had a shortened lifespan, indicating the importance of cell-extrinsic factors. In addition, neonatal platelets from WT and c-MPL –/– mice exhibited reduced P-selectin surface expression following stimulation compared with adult platelets of either genotype, and platelets from c-MPL –/– neonates exhibited reduced glycoprotein IIb/IIIa (GPIIb/IIIa) activation in response to thrombin compared with age-matched WT platelets. Taken together, our findings indicate that c-MPL deficiency is associated with abnormal maturation of neonatal MKs and developmental stage-specific defects in platelet function.

Effects of intensive glucose control on platelet reactivity in patients with acute coronary syndromes. Results of the CHIPS Study ("Control de Hiperglucemia y Actividad Plaquetaria en Pacientes con Sindrome Coronario Agudo").

PubMed

Vivas, David; García-Rubira, Juan C; Bernardo, Esther; Angiolillo, Dominick J; Martín, Patricia; Calle-Pascual, Alfonso; Núñez-Gil, Iván; Macaya, Carlos; Fernández-Ortiz, Antonio

2011-05-01

Hyperglycaemia has been associated with increased platelet reactivity and impaired prognosis in patients with acute coronary syndrome (ACS). Whether platelet reactivity can be reduced by lowering glucose in this setting is unknown. The aim of this study was to assess the functional impact of intensive glucose control with insulin on platelet reactivity in patients admitted with ACS and hyperglycaemia. This is a prospective, randomised trial evaluating the effects of either intensive glucose control (target glucose 80-120 mg/dl) or conventional control (target glucose 180 mg/dl or less) with insulin on platelet reactivity in patients with ACS and hyperglycaemia. The primary endpoint was platelet aggregation following stimuli with 20 μM ADP at 24 h and at hospital discharge. Aggregation following collagen, epinephrine and thrombin receptor-activated peptide, as well as P2Y₁₂ reactivity index and surface expression of glycoprotein IIb/IIIa and P-selectin were also measured. Of the 115 patients who underwent random assignment, 59 were assigned to intensive and 56 to conventional glucose control. Baseline platelet functions and inhospital management were similar in both groups. Maximal aggregation after ADP stimulation at hospital discharge was lower in the intensive group (47.9 ± 13.2% vs 59.1 ± 17.3%; p=0.002), whereas no differences were found at 24 h. Similarly all other parameters of platelet reactivity measured at hospital discharge were significantly reduced in the intensive glucose control group. In this randomised trial, early intensive glucose control with insulin in patients with ACS presenting with hyperglycaemia was found to decrease platelet reactivity. Clinical Trial Registration Number http://www.controlledtrials.com/ISRCTN35708451/ISRCTN35708451.

Serum CXCL4 increase in primary Sjögren's syndrome characterizes patients with microvascular involvement and reduced salivary gland infiltration and lymph node involvement.

PubMed

Vettori, Serena; Irace, Rosaria; Riccardi, Antonella; Iacono, Daniela; Pellecchia, Luciana; Vicedomini, Lucia; Valentini, Gabriele

2016-10-01

CXCL4 is an antiangiogenic and immunomodulatory chemokine. We aimed to investigate serum levels of CXCL4 in primary Sjögren's syndrome (pSS), looking for associations with disease features. Thirty-nine consecutive pSS patients underwent clinical-serological assessment and nailfold videocapillaroscopy (NVC). Thirty-six patients and 30 controls affected by osteoarthritis were also investigated for serum levels of CXCL4 and soluble E-selectin (sE-selectin). CXCL4 was higher in pSS patients than in controls (1.79 [0.2-11.18] vs 1.023 ng/ml [0.02-14.45], p < 0.05), particularly in those without anti-La/SSB antibodies (2.89 [1.01-11.18] vs 1.69 ng/ml [0.2-2.72], p < 0.05), while it was lower in pSS patients with a focus score ≥1 at lip biopsy (1.44 [0.86-2.1] vs 2.24 ng/ml [1.64-3.25], p < 0.05) and clinically evident lymphadenopathy (1.53 [0.38-1.7] vs 2.08 ng/ml [1.45-3.03], p < 0.05). CXCL4 correlated with disease duration (r = 0.35, p < 0.05) and sE-selectin (r = 0.45, p < 0.01). Patients with Raynaud's phenomenon (RP) had more frequently abnormal CXCL4 levels than patients without RP (11/15 vs 3/21, p < 0.001), enlarged capillaries (14/16 vs 7/23, p < 0.001) and capillary loss at NVC (14/16 vs 6/23, p < 0.001). The hitherto unknown association of increased serum CXCL4 with features of microvascular impairment in pSS, along with the negative association with features of lymphocytic response (i.e., the absence of subset disease-specific autoantibodies, a low focus score, and the absence of lymphadenopathy) suggest clarifying the possible implication of this chemokine in pSS pathogenesis in larger studies.

Interaction of pyridostigmine bromide and N,N-diethyl-m-toluamide alone and in combination with P-glycoprotein expressed in Escherichia coli leaky mutant.

PubMed

El-Masry, Eman M; Abou-Donia, Mohamed B

2006-05-01

P-glycoprotein (P-gp), the most extensively studied ATP-binding transporter, functions as a biological barrier by extruding toxic substances and xenobiotics out of the cell. This study was carried out to determine the effect of N,N-diethyl-m-toluamide (DEET) and pyridostigmine bromide (PB), alone and in combination, on P-gp expression using Escherichia coli leaky mutant transformed with Mdr1 gene (pT5-7/mdr1), which codes for P-gp or lactose permease (pT5-7/lacY) as negative control. Also, daunomycin (a known P-gp sustrate) was used as a positive control and reserpine (a known P-gp inhibitor) served as a negative control. An in vitro cell-resistant assay was used to monitor the potential of test compounds to interact with P-gp. Following exposure of the cells to pyridostigmine bromide or daunomycin, P-gp conferred significant resistance against both compounds, while reserpine and DEET significantly inhibited the glycoprotein. Cells were grown in the presence of noncytotoxic concentrations of daunomycin, pyridostigmine bromide, reserpine, or DEET, and membrane fractions were examined by Western immunoblotting for expression of P-gp. Daunomycin induced P-gp expression quantitatively more than pyridostigmine bromide, while reserpine and DEET significantly inhibited P-gp expression in cells harboring mdr1. Photoaffinity labeling experiment performed with the P-gp ligand [125I]iodoarylazidoprazosin demonstrated that compounds that induced or inhibited P-gp transport activity also bound to P-gp. DEET was also found to be a potent inhibitor of P-gp-mediated ATPase activity, whereas pyridostigmine bromide increased P-gp ATPase activity. Cells expressing P-gp or lac permease were exposed to pyridostigmine bromide and DEET, alone and in combination. Noncytotoxic concentrations of DEET significantly inhibited P-gp-mediated resistance against pyridostigmine bromide, resulting in a reduction of the number of effective drug interactions with biological targets. An explanation of these results might be that DEET is a third-generation inhibitor of P-gp; it has high potency and specificity for P-gp, it inhibits hydrolysis of ATP, it exerts no appreciable impact on cytochrome P-450 3A4, and it prevents transport of xenobiotics, such as pyridostigmine bromide, out of the cell. This conclusion explains, at least in part, the increased toxicity and bioavailability of pyridostigmine bromide following combined administration with DEET. This study improves our understanding of the basis of chemical interactions with DEET by defining the ability of drugs to interact with P-gp either as inhibitors or substrates, which may in turn lead to altered efficacy or toxicity.

Down-regulation of histamine-induced endothelial cell activation as potential anti-atherosclerotic activity of peptides from Spirulina maxima.

PubMed

Vo, Thanh-Sang; Kim, Se-Kwon

2013-10-09

Histamine, a potent inflammatory mediator, has been known to cause the pathogenesis of atherosclerosis. In this sense, two bioactive peptides P1 (LDAVNR; 686Da) and P2 (MMLDF; 655Da) purified from gastric enzymatic hydrolysate of Spirulina maxima were examined for their protective effects against early atherosclerotic responses induced by histamine in EA.hy926 endothelial cells. Interestingly, both P1 and P2 exhibited inhibitory activities on the production and expression of IL-6 and MCP-1. Furthermore, P1 and P2 inhibited the production of adhesion molecules including P-selectin and E-selectin, and thus reducing in vitro cell adhesion of monocyte onto endothelial cells. In addition, the production of intracellular reactive oxygen species was observed to reduce in the presence of P1 or P2. Notably, the inhibitory activities of P1 and P2 were found due to down-regulating Egr-1 expression via histamine receptor and PKCδ-dependent MAPKs activation pathway. These results suggest that peptides P1 and P2 from S. maxima are effective to suppress histamine-induced endothelial cell activation that may contribute to the prevention of early atherosclerosis. Copyright © 2013 Elsevier B.V. All rights reserved.

Role of mannose-6-phosphate receptors in herpes simplex virus entry into cells and cell-to-cell transmission.

PubMed Central

Brunetti, C R; Burke, R L; Hoflack, B; Ludwig, T; Dingwell, K S; Johnson, D C

1995-01-01

Herpes simplex virus (HSV) glycoprotein D (gD) is essential for virus entry into cells, is modified with mannose-6-phosphate (M-6-P), and binds to both the 275-kDa M-6-P receptor (MPR) and the 46-kDa MPR (C. R. Brunetti, R. L. Burke, S. Kornfeld, W. Gregory, K. S. Dingwell, F. Masiarz, and D. C. Johnson, J. Biol. Chem. 269:17067-17074, 1994). Since MPRs are found on the surfaces of mammalian cells, we tested the hypothesis that MPRs could serve as receptors for HSV during virus entry into cells. A soluble form of the 275-kDa MPR, derived from fetal bovine serum, inhibited HSV plaques on monkey Vero cells, as did polyclonal rabbit anti-MPR antibodies. In addition, the number and size of HSV plaques were reduced when cells were treated with bovine serum albumin conjugated with pentamannose-phosphate (PM-PO4-BSA), a bulky ligand which can serve as a high-affinity ligand for MPRs. These data imply that HSV can use MPRs to enter cells; however, other molecules must also serve as receptors for HSV because a reasonable fraction of virus could enter cells treated with even the highest concentrations of these inhibitors. Consistent with the possibility that there are other receptors, HSV produced the same number of plaques on MPR-deficient mouse fibroblasts as were produced on normal mouse fibroblasts, but there was no inhibition with PM-PO4-BSA with either of these embryonic mouse cells. Together, these results demonstrate that HSV does not rely solely on MPRs to enter cells, although MPRs apparently play some role in virus entry into some cell types and, perhaps, act as one of a number of cell surface molecules that can facilitate entry. We also found that HSV produced small plaques on human fibroblasts derived from patients with pseudo-Hurler's polydystrophy, cells in which glycoproteins are not modified with M-6-P residues and yet production of infectious HSV particles was not altered in the pseudo-Hurler cells. In addition, HSV plaque size was reduced by PM-PO4-BSA; therefore, it appears that M-6-P residues and MPRs are required for efficient transmission of HSV between cells, a process which differs in some respects from entry of exogenous virus particles. PMID:7745699

Blocking the Adhesion Cascade at the Premetastatic Niche for Prevention of Breast Cancer Metastasis

PubMed Central

Kang, Shin-Ae; Hasan, Nafis; Mann, Aman P; Zheng, Wei; Zhao, Lichao; Morris, Lynsie; Zhu, Weizhu; Zhao, Yan D; Suh, K Stephen; Dooley, William C; Volk, David; Gorenstein, David G; Cristofanilli, Massimo; Rui, Hallgeir; Tanaka, Takemi

2015-01-01

Shear-resistant adhesion and extravasation of disseminated cancer cells at the target organ is a crucial step in hematogenous metastasis. We found that the vascular adhesion molecule E-selectin preferentially promoted the shear-resistant adhesion and transendothelial migration of the estrogen receptor (ER)–/CD44+ hormone-independent breast cancer cells, but not of the ER+/CD44-/low hormone-dependent breast cancer cells. Coincidentally, CD44+ breast cancer cells were abundant in metastatic lung and brain lesions in ER– breast cancer, suggesting that E-selectin supports hematogenous metastasis of ER–/CD44+ breast cancer. In an attempt to prevent hematogenous metastasis through the inhibition of a shear-resistant adhesion of CD44+ cancer cells to E-selectin-expressing blood vessels on the premetastatic niche, an E-selectin targeted aptamer (ESTA) was developed. We demonstrated that a single intravenous injection of ESTA reduced metastases to a baseline level in both syngeneic and xenogeneic forced breast cancer metastasis models without relocating the site of metastasis. The effect of ESTA was absent in E-selectin knockout mice, suggesting that E-selectin is a molecular target of ESTA. Our data highlight the potential application of an E-selectin antagonist for the prevention of hematogenous metastasis of ER–/CD44+ breast cancer. PMID:25815697

Blocking the adhesion cascade at the premetastatic niche for prevention of breast cancer metastasis.

PubMed

Kang, Shin-Ae; Hasan, Nafis; Mann, Aman P; Zheng, Wei; Zhao, Lichao; Morris, Lynsie; Zhu, Weizhu; Zhao, Yan D; Suh, K Stephen; Dooley, William C; Volk, David; Gorenstein, David G; Cristofanilli, Massimo; Rui, Hallgeir; Tanaka, Takemi

2015-06-01

Shear-resistant adhesion and extravasation of disseminated cancer cells at the target organ is a crucial step in hematogenous metastasis. We found that the vascular adhesion molecule E-selectin preferentially promoted the shear-resistant adhesion and transendothelial migration of the estrogen receptor (ER)(-)/CD44(+) hormone-independent breast cancer cells, but not of the ER(+)/CD44(-/low) hormone-dependent breast cancer cells. Coincidentally, CD44(+) breast cancer cells were abundant in metastatic lung and brain lesions in ER(-) breast cancer, suggesting that E-selectin supports hematogenous metastasis of ER(-)/CD44(+) breast cancer. In an attempt to prevent hematogenous metastasis through the inhibition of a shear-resistant adhesion of CD44(+) cancer cells to E-selectin-expressing blood vessels on the premetastatic niche, an E-selectin targeted aptamer (ESTA) was developed. We demonstrated that a single intravenous injection of ESTA reduced metastases to a baseline level in both syngeneic and xenogeneic forced breast cancer metastasis models without relocating the site of metastasis. The effect of ESTA was absent in E-selectin knockout mice, suggesting that E-selectin is a molecular target of ESTA. Our data highlight the potential application of an E-selectin antagonist for the prevention of hematogenous metastasis of ER(-)/CD44(+) breast cancer.

Carbohydrate-dependent binding of langerin to SodC, a cell wall glycoprotein of Mycobacterium leprae.

PubMed

Kim, Hee Jin; Brennan, Patrick J; Heaslip, Darragh; Udey, Mark C; Modlin, Robert L; Belisle, John T

2015-02-01

Langerhans cells participate in the immune response in leprosy by their ability to activate T cells that recognize the pathogen, Mycobacterium leprae, in a langerin-dependent manner. We hypothesized that langerin, the distinguishing C-type lectin of Langerhans cells, would recognize the highly mannosylated structures in pathogenic Mycobacterium spp. The coding region for the extracellular and neck domain of human langerin was cloned and expressed to produce a recombinant active trimeric form of human langerin (r-langerin). Binding assays performed in microtiter plates, by two-dimensional (2D) Western blotting, and by surface plasmon resonance demonstrated that r-langerin possessed carbohydrate-dependent affinity to glycoproteins in the cell wall of M. leprae. This lectin, however, yielded less binding to mannose-capped lipoarabinomannan (ManLAM) and even lower levels of binding to phosphatidylinositol mannosides. However, the superoxide dismutase C (SodC) protein of the M. leprae cell wall was identified as a langerin-reactive ligand. Tandem mass spectrometry verified the glycosylation of a recombinant form of M. leprae SodC (rSodC) produced in Mycobacterium smegmatis. Analysis of r-langerin affinity by surface plasmon resonance revealed a carbohydrate-dependent affinity of rSodC (equilibrium dissociation constant [KD] = 0.862 μM) that was 20-fold greater than for M. leprae ManLAM (KD = 18.69 μM). These data strongly suggest that a subset of the presumptively mannosylated M. leprae glycoproteins act as ligands for langerin and may facilitate the interaction of M. leprae with Langerhans cells. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Carbohydrate-Dependent Binding of Langerin to SodC, a Cell Wall Glycoprotein of Mycobacterium leprae

PubMed Central

Kim, Hee Jin; Brennan, Patrick J.; Heaslip, Darragh; Udey, Mark C.; Modlin, Robert L.

2014-01-01

Langerhans cells participate in the immune response in leprosy by their ability to activate T cells that recognize the pathogen, Mycobacterium leprae, in a langerin-dependent manner. We hypothesized that langerin, the distinguishing C-type lectin of Langerhans cells, would recognize the highly mannosylated structures in pathogenic Mycobacterium spp. The coding region for the extracellular and neck domain of human langerin was cloned and expressed to produce a recombinant active trimeric form of human langerin (r-langerin). Binding assays performed in microtiter plates, by two-dimensional (2D) Western blotting, and by surface plasmon resonance demonstrated that r-langerin possessed carbohydrate-dependent affinity to glycoproteins in the cell wall of M. leprae. This lectin, however, yielded less binding to mannose-capped lipoarabinomannan (ManLAM) and even lower levels of binding to phosphatidylinositol mannosides. However, the superoxide dismutase C (SodC) protein of the M. leprae cell wall was identified as a langerin-reactive ligand. Tandem mass spectrometry verified the glycosylation of a recombinant form of M. leprae SodC (rSodC) produced in Mycobacterium smegmatis. Analysis of r-langerin affinity by surface plasmon resonance revealed a carbohydrate-dependent affinity of rSodC (equilibrium dissociation constant [KD] = 0.862 μM) that was 20-fold greater than for M. leprae ManLAM (KD = 18.69 μM). These data strongly suggest that a subset of the presumptively mannosylated M. leprae glycoproteins act as ligands for langerin and may facilitate the interaction of M. leprae with Langerhans cells. PMID:25422308

Emodin plays an interventional role in epileptic rats via multidrug resistance gene 1 (MDR1).

PubMed

Yang, Tao; Kong, Bin; Kuang, Yongqin; Cheng, Lin; Gu, Jianwen; Zhang, Junhai; Shu, Haifeng; Yu, Sixun; Yang, Xiaokun; Cheng, Jingming; Huang, Haidong

2015-01-01

To observe the interventional effects of emodin in epileptic rats and elucidate its possible mechanism of action. Thirty-six female Wistar rats were randomly divided into normal control group, model group (intraperitoneal injection of kainic acid) and emodin group (intraperitoneal injection of kainic acid+emodin intervention). The rat epilepsy model was confirmed by behavioral tests and electroencephalography. The protein levels of P-glycoprotein and N-methyl-D-aspartate (NMDA) receptor in cerebral vascular tissue were analyzed by western blotting, and mRNA levels of multidrug resistance gene 1 (MDR1) and cyclooxygenase-2 (COX-2) were analyzed by real-time PCR. COX-2 and P-glycoprotein levels in the brains were detected by immunohistochemical assay. The seizures were relieved in emodin group. Laser scanning confocal microscopy showed P-glycoprotein fluorescence increased significantly after seizures, indicating that epilepsy can induce overexpression of P-glycoprotein. Compared with control group, protein levels of P-glycoprotein and NMDA receptor in cerebral vascular tissue were significantly higher in model group, and mRNA levels of MDR1 and COX-2 were also significantly increased. Compared with model group, P-glycoprotein and NMDA receptor levels in cerebral vascular tissue were significantly decreased in emodin group (P<0.05), and the levels of MDR1 and COX-2 were down-regulated (P<0.05). In the rat brain, seizures could significantly increase COX-2 and P-glycoprotein levels, while emodin intervention was able to significantly reduce the levels of both. These findings suggest that epileptic seizures are tightly associated with up-regulated MDR1 gene, and emodin shows good antagonistic effects on epileptic rats, possibly through inhibition of MDR1 gene and its associated genes.

Phage display vectors for in vivo recombination of immunoglobulin heavy and light chain genes to make large combinatorial libraries.

PubMed

Tsurushita, N; Fu, H; Warren, C

1996-06-12

New phage display vectors for in vivo recombination of immunoglobulin (Ig) heavy (VH) and light (VL) chain variable genes, to make single-chain Fv fragments (scFv), were constructed. The VH and VL genes of monoclonal antibody (mAb) EP-5C7, which binds to both human E- and P-selectin, were cloned into a pUC19-derived plasmid vector, pCW93, and a pACYC184-derived phagemid vector, pCW99, respectively. Upon induction of Cre recombinase (phage P1 recombinase), the VH and VL genes were efficiently recombined into the same plasmid via the two loxP sites (phage P1 recombination sites), one located downstream from a VH gene in pCW93 and another upstream from a VL gene in pCW99. In the resulting phagemid, the loxP sequence also encodes a polypeptide linker connecting the VH and VL domains to form a scFv of EP-5C7. Whether expressed on the phage surface or as a soluble form, the EP-5C7 scFv showed specific binding to human E- and P-selectin. This phagemid vector system provides a way to recombine VH and VL gene libraries efficiently in vivo to make extremely large Ig combinatorial libraries.

Sulfur mustard and respiratory diseases.

PubMed

Tang, Feng Ru; Loke, Weng Keong

2012-09-01

Victims exposed to sulfur mustard (HD) in World War I and Iran-Iraq war, and those suffered occupational or accidental exposure have endured discomfort in the respiratory system at early stages after exposure, and marked general physical deterioration at late stages due to pulmonary fibrosis, bronchiolitis obliterans or lung cancer. At molecule levels, significant changes of cytokines and chemokines in bronchoalveolar lavage and serum, and of selectins (in particular sE-selectin) and soluble Fas ligand in the serum have been reported in recent studies of patients exposed to HD in Iran-Iraq war, suggesting that these molecules may be associated with the pathophysiological development of pulmonary diseases. Experimental studies in rodents have revealed that reactive oxygen and nitrogen species, their product peroxynitrite (ONOO(-)), nitric oxide synthase, glutathione, poly (adenosine diphosphate-ribose) polymerase, activating protein-1 signaling pathway are promising drug targets for preventing HD-induced toxicity, whereas N-acetyl cysteine, tocopherols, melatonin, aprotinin and many other molecules have been proved to be effective in prevention of HD-induced damage to the respiratory system in different animal models. In this paper, we will systemically review clinical and pathophysiological changes of respiratory system in victims exposed to HD in the last century, update clinicians and researchers on the mechanism of HD-induced acute and chronic lung damages, and on the relevant drug targets for future development of antidotes for HD. Further research directions will also be proposed.

HG-829 Is a Potent Noncompetitive Inhibitor of the ATP-Binding Cassette Multidrug Resistance Transporter ABCB1

PubMed Central

Caceres, Gisela; Robey, Robert W.; Sokol, Lubomir; McGraw, Kathy L.; Clark, Justine; Lawrence, Nicholas J.; Sebti, Said M.; Wiese, Michael; List, Alan F.

2015-01-01

Transmembrane drug export mediated by the ATP-binding cassette (ABC) transporter P-glycoprotein contributes to clinical resistance to antineoplastics. In this study, we identified the substituted quinoline HG-829 as a novel, noncompetitive, and potent P-glycoprotein inhibitor that overcomes in vitro and in vivo drug resistance. We found that nontoxic concentrations of HG-829 restored sensitivity to P-glycoprotein oncolytic substrates. In ABCB1-overexpressing cell lines, HG-829 significantly enhanced cytotoxicity to daunorubicin, paclitaxel, vinblastine, vincristine, and etoposide. Coadministration of HG-829 fully restored in vivo antitumor activity of daunorubicin in mice without added toxicity. Functional assays showed that HG-829 is not a Pgp substrate or competitive inhibitor of Pgp-mediated drug efflux but rather acts as a noncompetitive modulator of P-glycoprotein transport function. Taken together, our findings indicate that HG-829 is a potent, long-acting, and noncompetitive modulator of P-glycoprotein export function that may offer therapeutic promise for multidrugresistant malignancies. PMID:22761337

Dark chocolate effect on platelet activity, C-reactive protein and lipid profile: a pilot study.

PubMed

Hamed, Miruais S; Gambert, Steven; Bliden, Kevin P; Bailon, Oscar; Singla, Anand; Anand, Singla; Antonino, Mark J; Hamed, Fatema; Tantry, Udaya S; Gurbel, Paul A

2008-12-01

Dark chocolate (DC) is one of the richest sources of flavonoids. Since DC has been demonstrated to have beneficial effects on the cardiovascular system, our study examined its effect on platelet reactivity, inflammation, and lipid levels in healthy subjects. In 28 healthy volunteers, we analyzed the effect of one week of DC (providing 700 mg of flavonoids/day). The primary outcome was to determine the effects of DC consumption on platelet activity measured by flow cytometry (adenosine diphosphate [ADP]- and arachidonic acid [AA]-induced total and activated glycoprotein (GP) IIb/IIIa as well as P-selectin expression). In addition to this, we measured the effect of DC on high-sensitivity C-reactive protein (hsCRP), high-density lipid cholesterol (HDL) and low-density lipid cholesterol (LDL) levels. Following seven days of regular DC ingestion, LDL fell by 6% (120 +/- 38 vs 112 +/- 37 mg/dL, P < 0.018) and HDL rose by 9% (66 +/- 23 vs 72 +/- 26 mg/dL, P < 0.0019). ADP- and AA-induced activated GPIIb/IIIa expression was reduced by DC [27.3 +/- 27.8 vs 17.4 +/- 20.5 mean fluorescence intensity (MFI), P < 0.006; and 9.2 +/- 6.5 vs. 6.1 +/- 2.2 MFI, P < 0.005, respectively]. DC reduced hsCRP levels in women (1.8 +/- 2.1 vs. 1.4 +/- 1.7 mg/dL, P < 0.04). One week of DC ingestion improved lipid profiles and decreased platelet reactivity within the total group while reducing inflammation only in women. Regular dark chocolate ingestion may have cardioprotective properties. Further long-term research is warranted to evaluate the effect of flavonoids on cardiovascular health and to determine whether DC's beneficial effects are related to flavonoids or some yet unknown component. This research is based on a larger study which was presented at the American Heart Association Scientific Sessions 2007.

The Effect of Regular Intake of Dry-Cured Ham Rich in Bioactive Peptides on Inflammation, Platelet and Monocyte Activation Markers in Humans

PubMed Central

Martínez-Sánchez, Sara María; Minguela, Alfredo; Prieto-Merino, David; Zafrilla-Rentero, María Pilar; Abellán-Alemán, José; Montoro-García, Silvia

2017-01-01

Background and aims: Dietary studies have shown that active biopeptides provide protective health benefits, although the mediating pathways are somewhat uncertain. To throw light on this situation, we studied the effects of consuming Spanish dry-cured ham on platelet function, monocyte activation markers and the inflammatory status of healthy humans with pre-hypertension. Methods: Thirty-eight healthy volunteers with systolic blood pressure of >125 mmHg were enrolled in a two-arm crossover randomized controlled trial. Participants received 80 g/day dry-cured pork ham of >11 months proteolysis or 100 g/day cooked ham (control product) for 4 weeks followed by a 2-week washout before “crossing over” to the other treatment for 4 more weeks. Soluble markers and cytokines were analyzed by ELISA. Platelet function was assessed by measuring P-selectin expression and PAC-1 binding after ADP (adenosine diphosphate) stimulation using whole blood flow cytometry. Monocyte markers of the pathological status (adhesion, inflammatory and scavenging receptors) were also measured by flow cytometry in the three monocyte subsets after the interventional period. Results: The mean differences between dry-cured ham and cooked ham followed by a time period adjustment for plasmatic P-selectin and interleukin 6 proteins slightly failed (p = 0.062 and p = 0.049, respectively), notably increased for MCP-1 levels (p = 0.023) while VCAM-1 was not affected. Platelet function also decreased after ADP stimulation. The expression of adhesion and scavenging markers (ICAM1R, CXCR4 and TLR4) in the three subsets of monocytes was significantly higher (all p < 0.05). Conclusions: The regular consumption of biopeptides contained in the dry-cured ham but absent in cooked ham impaired platelet and monocyte activation and the levels of plasmatic P-selectin, MCP-1 and interleukin 6 in healthy subjects. This study strongly suggests the existence of a mechanism that links dietary biopeptides and beneficial health effects. PMID:28333093

The effect of soy protein beverages on serum cell adhesion molecule concentrations in prehypertensive/stage 1 hypertensive individuals.

PubMed

Dettmer, Michelle; Alekel, D Lee; Lasrado, Joanne A; Messina, Mark; Carriquiry, Alicia; Heiberger, Kevin; Stewart, Jeanne W; Franke, Warren

2012-04-01

Prehypertensive and hypertensive individuals are at increased risk of atherosclerotic cardiovascular disease (CVD), in part because hypertension contributes to endothelial dysfunction and increased cell adhesion molecule expression. Soy protein and isoflavones may favorably alter CVD risk factors, and hence the aim of this study was to determine whether intake of cow's milk compared with soy beverage prepared from whole soy bean (WSB) or soy protein isolate (SPI) would lower soluble cell adhesion molecule concentrations as a means of decreasing CVD risk. We enrolled healthy prehypertensive/stage 1 hypertensive men (n = 60; 18-63 years) and premenopausal women (n = 8; 20-48 years). Participants were randomized to 1 of 3 groups for 8 weeks: cow's milk (600 mL/d), SPI beverage (840 mL/d; 30.1 mg total isoflavones/d), or WSB beverage (840 mL/d; 91.4 mg total isoflavones/d). We measured soluble vascular cell adhesion molecule-1 (VCAM-1), intercellular cell adhesion molecule-1 (ICAM-1), and endothelial-leukocyte adhesion molecule-1 (E-selectin) concentrations at baseline and week 8. Soluble CAM concentrations were not altered by treatment and did not differ between prehypertensive and hypertensive participants. However, analysis of variance indicated a treatment × gender interaction (gender effect) for ICAM-1 (p = 0.0037) but not for E-selectin (p = 0.067) or VCAM-1 (p = 0.16). Men had higher concentrations of ICAM-1 and E-selectin, respectively, at baseline (p = 0.0071, p = 0.049) and week 8 (p = 0.0054, p = 0.038) than women did. Neither intake of cow's milk nor soy beverage for 8 weeks altered soluble CAM concentrations in prehypertensive/stage 1 hypertensive individuals, suggesting that neither type of beverage diminished atherosclerotic CVD risk in mildly hypertensive individuals by way of improving circulating CAM concentrations.

The effect of Slit homolog 2 (SLIT2) on populations of preantral follicles in cultured cortical tissue from pig ovaries

USDA-ARS?s Scientific Manuscript database

SLIT guidance ligands are secreted glycoproteins involved in organogenesis. SLIT proteins and their receptor have been linked to ovarian development in fetal sheep and luteal function in adult humans. In pigs, SLIT proteins have been associated with age at puberty (GWAS) and as a major node in genom...

The effect of P-glycoprotein on methadone hydrochloride flux in equine intestinal mucosa.

PubMed

Linardi, R L; Stokes, A M; Andrews, F M

2013-02-01

Methadone is an effective analgesic opioid that may have a place for the treatment of pain in horses. However, its absorption seems to be impaired by the presence of a transmembrane protein, P-glycoprotein, present in different tissues including the small intestine in other species. This study aims to determine the effect of the P-glycoprotein on methadone flux in the equine intestinal mucosa, as an indicator of in vivo drug absorption. Jejunum tissues from five horses were placed into the Ussing chambers and exposed to methadone solution in the presence or absence of Rhodamine 123 or verapamil. Electrical measurements demonstrated tissue viability for 120 min, and the flux of methadone across the jejunal membrane (mucosal to submucosal direction) was calculated based on the relative drug concentration measured by ELISA. The flux of methadone was significantly higher only in the presence of verapamil. P-glycoprotein was immunolocalized in the apical membrane of the jejunal epithelial cells (enterocytes), mainly located in the tip of the villi compared to cells of the crypts. P-glycoprotein is present in the equine jejunum and may possibly mediate the intestinal transport of methadone. This study suggests that P-glycoprotein may play a role in the poor intestinal absorption of methadone in vivo. © 2012 Blackwell Publishing Ltd.

Activation of PKC isoform beta(I) at the blood-brain barrier rapidly decreases P-glycoprotein activity and enhances drug delivery to the brain.

PubMed

Rigor, Robert R; Hawkins, Brian T; Miller, David S

2010-07-01

P-glycoprotein is an ATP (adenosine triphosphate)-driven drug efflux transporter that is highly expressed at the blood-brain barrier (BBB) and is a major obstacle to the pharmacotherapy of central nervous system diseases, including brain tumors, neuro-AIDS, and epilepsy. Previous studies have shown that P-glycoprotein transport activity in rat brain capillaries is rapidly reduced by the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha) acting through protein kinase C (PKC)-dependent signaling. In this study, we used isolated rat brain capillaries to show that the TNF-alpha-induced reduction of P-glycoprotein activity was prevented by a PKCbeta(I/II) inhibitor, LY333531, and mimicked by a PKCbeta(I/II) activator, 12-deoxyphorbol-13-phenylacetate-20-acetate (dPPA). Western blotting of brain capillary extracts with phospho-specific antibodies showed that dPPA activated PKCbeta(I), but not PKCbeta(II). Moreover, in intact rats, intracarotid infusion of dPPA potently increased brain accumulation of the P-glycoprotein substrate, [(3)H]-verapamil without compromising tight junction integrity. Thus, PKCbeta(I) activation selectively reduced P-glycoprotein activity both in vitro and in vivo. Targeting PKCbeta(I) at the BBB may prove to be an effective strategy for enhancing the delivery of small molecule therapeutics to the brain.

Applications of transgenics in studies of bone sialoprotein.

PubMed

Zhang, Jin; Tu, Qisheng; Chen, Jake

2009-07-01

Bone sialoprotein (BSP) is a major non-collagenous protein in mineralizing connective tissues such as dentin, cementum and calcified cartilage tissues. As a member of the Small Integrin-Binding Ligand, N-linked Glycoprotein (SIBLING) gene family of glycoproteins, BSP is involved in regulating hydroxyapatite crystal formation in bones and teeth, and has long been used as a marker gene for osteogenic differentiation. In the most recent decade, new discoveries in BSP gene expression and regulation, bone remodeling, bone metastasis, and bone tissue engineering have been achieved with the help of transgenic mice. In this review, we discuss these new discoveries obtained from the literatures and from our own laboratory, which were derived from the use of transgenic mouse mutants related to BSP gene or its promoter activity.

Markers of mechanical asphyxia: immunohistochemical study on autoptic lung tissues.

PubMed

Cecchi, R; Sestili, C; Prosperini, G; Cecchetto, G; Vicini, E; Viel, G; Muciaccia, B

2014-01-01

Forensic pathologists are often asked to provide evidence of asphyxia death in the trial and a histological marker of asphyxiation would be of great help. Data from the literature indicate that the reaction of lung tissue cells to asphyxia may be of more interest for forensic purposes than migrating cells. The lungs of 62 medico-legal autopsy cases, 34 acute mechanical asphyxia (AMA), and 28 control cases (CC), were immunostained with anti-P-selectin, anti-E-selectin, anti-SP-A, and anti-HIF1-α antibodies, in order to verify if some of them may be used as markers of asphyxia death. Results show that P- and E-selectins expression in lung vessels, being activated by several types of trigger stimuli not specific to hypoxia, cannot be used as indicator of asphyxia. Intra-alveolar granular deposits of SP-A seem to be related to an intense hypoxic stimulus, and when massively present, they can suggest, together with other elements, a severe hypoxia as the mechanism of death. HIF1-α was expressed in small-, medium-, and large-caliber lung vessels of the vast majority of mechanical asphyxia deaths and CO intoxications, with the number and intensity of positive-stained vessels increasing with the duration of the hypoxia. Although further confirmation studies are required, these preliminary data indicate an interesting potential utility of HIF1-α as a screening test for asphyxia deaths.

The effect of adhesion molecule blockade on pulmonary reperfusion injury.

PubMed

Levine, Adrian J; Parkes, Karen; Rooney, Stephen J; Bonser, Robert S

2002-04-01

Selectins are the molecules involved in the initial adhesion of the activated neutrophil on pulmonary endothelium. We investigated the efficacy of selectin blockade in a selective (monoclonal antibody RMP-1) and nonselective (Fucoidin) manner in pulmonary reperfusion injury. Groups of six rat lungs were flushed with University of Wisconsin solution then stored at 4 degrees C for 4 hours. They then underwent sanguinous reperfusion for 30 minutes during which functional measures (gas exchange, pulmonary artery pressure, and airway pressure) of lung performance were made. After reperfusion we estimated their capillary filtration coefficient (Kfc units g/cm water/minute/g wet lung tissue) using a gravimetric technique. Four groups were studied: group I had no reperfusion, group II had 30 minutes of reperfusion, group III had infusion of 20 mg/kg Fucoidin before reperfusion, and group IV had infusion of 20 microg/mL RMP-1 before reperfusion. Reperfusion injury was found between groups I and II by an increase in capillary filtration coefficient (1.048 +/- 0.316 to 3.063 +/- 0.466, p < 0.01). Groups III and IV had a significantly lower Kfc than group II (0.967 +/- 0.134 and 1.205 +/- 0.164, respectively, p < 0.01). There was no significant functional difference between groups II, III, and IV. Reperfusion-induced hyperpermeability was ameliorated by selective (RMP-1) and nonselective (Fucoidin) selectin blockade.

Effect of aliskiren and valsartan combination versus aliskiren monotherapy on hemostatic biomarkers in hypertensive diabetics: Aliskiren and Valsartan Impact in Diabetics pilot trial.

PubMed

Serebruany, Victor L; Pokov, Alex N; Aradi, Daniel; Can, Mehmet; DiNicolantonio, James; Kipshidze, Nodar; Atar, Dan

2014-01-01

Valsartan is known to inhibit platelet activity in both in vitro and ex vivo clinical setting, whereas aliskiren in vitro modulates antithrombin-III in plasma. The authors tested how aliskiren and valsartan combination versus aliskiren monotherapy will affect hemostatic biomarkers in mild-to-moderate hypertensive diabetics in the frame of the Aliskiren and Valsartan Impact in Diabetics (AVID) trial. A total of 52 patients with type 2 diabetes and mild-to-moderate hypertension were equally randomized to aliskiren (150-300 mg/d) and valsartan (160 mg/d) versus aliskiren (150-300 mg/d) alone for 4 weeks. A total of 25 biomarkers were serially measured, of which 16 are related to platelet function, 6 to coagulation, and 3 to fibrinolysis. Aliskiren monotherapy has no significant impact on any of the assessed biomarkers. In contrast, valsartan on top of aliskiren provided significant inhibition of ADP-induced platelet aggregation (P=0.032), decreased shear-induced activation measured with PFA-100 analyzer (P=0.041), and diminished expression of GP IIb/IIIa activity (P=0.027) measured by PAC-1 antibody, GP Ib (CD42b, P=0.033), vitronectin receptor (CD51/61, P=0.046), P-selectin (CD62p, P=0.026), lysosome-associated membrane protein (CD107a, P=0.042), and CD40-ligand (CD154, P=0.048). In AVID trial, valsartan in combination with aliskiren mildly but significantly inhibited platelets, confirming previous observations. In contrast, aliskiren monotherapy does not enhance antithrombin activity, suggesting that previous data probably represent a laboratory artifact. Importantly, these randomized data were generated on top of low-dose daily aspirin, supporting extra benefit for combination use of angiotensin receptor blockers and renin inhibitors in high-risk diabetic population.

Neuronal apoptotic signaling pathways probed and intervened by synthetically and modularly modified (SMM) chemokines.

PubMed

Choi, Won-Tak; Kaul, Marcus; Kumar, Santosh; Wang, Jun; Kumar, I M Krishna; Dong, Chang-Zhi; An, Jing; Lipton, Stuart A; Huang, Ziwei

2007-03-09

As the main coreceptors for human immunodeficiency virus type 1 (HIV-1) entry, CXCR4 and CCR5 play important roles in HIV-associated dementia (HAD). HIV-1 glycoprotein gp120 contributes to HAD by causing neuronal damage and death, either directly by triggering apoptotic pathways or indirectly by stimulating glial cells to release neurotoxins. Here, to understand the mechanism of CXCR4 or CCR5 signaling in neuronal apoptosis associated with HAD, we have applied synthetically and modularly modified (SMM)-chemokine analogs derived from natural stromal cell-derived factor-1alpha or viral macrophage inflammatory protein-II as chemical probes of the mechanism(s) whereby these SMM-chemokines prevent or promote neuronal apoptosis. We show that inherently neurotoxic natural ligands of CXCR4, such as stromal cell-derived factor-1alpha or viral macrophage inflammatory protein-II, can be modified to protect neurons from apoptosis induced by CXCR4-preferring gp120(IIIB), and that the inhibition of CCR5 by antagonist SMM-chemokines, unlike neuroprotective CCR5 natural ligands, leads to neurotoxicity by activating a p38 mitogen-activated protein kinase (MAPK)-dependent pathway. Furthermore, we discover distinct signaling pathways activated by different chemokine ligands that are either natural agonists or synthetic antagonists, thus demonstrating a chemical biology strategy of using chemically engineered inhibitors of chemokine receptors to study the signaling mechanism of neuronal apoptosis and survival.

Effect of bisphenol A on drug efflux in BeWo, a human trophoblast-like cell line.

PubMed

Jin, H; Audus, K L

2005-04-01

Bisphenol A (BPA) is a monomer of polycarbonate plastics that has estrogenic activities and has been shown to be a substrate for multidrug resistant efflux mechanisms, specifically, P-glycoprotein. Since the natural hormone estrogen reverses multidrug resistance in some cell types, we hypothesized that BPA might have a similar activity in trophoblasts. We have used BeWo cells as an in vitro model for human trophoblasts and calcein AM as a substrate for drug efflux mechanism to characterize BPA interactions with placental P-glycoprotein. We found that chronic exposure of BeWo cells to BPA did not alter intracellular calcein accumulation in a fashion that would be reflective of changes in P-glycoprotein expression. Immunoblots affirmed that BPA had small effects on P-glycoprotein expression. However, BeWo cells acutely exposed to BPA pretreatment were observed to have a significantly decreased calcein accumulation. Addition of cyclosporin A, a P-glycoprotein inhibitor and substrate, completely reversed BPA's effects on calcein accumulation and resulted in a net increase, relative to controls, in calcein accumulation by the BeWo cells. BPA was found not to stimulate P-gp ATPase or alter intracellular esterases mediating calcein release from calcein AM. Therefore, our results suggested that BPA stimulated drug efflux by BeWo cells probably by direct effects on P-glycoprotein.

Microvascular Channel Device to Study Aggressiveness in Prostate Cancer Metastasis

DTIC Science & Technology

2012-06-01

metastatic inefficiency. Breast Cancer Res 2000 2:400-407 8. Aigner S, Ramos CL, Hafezi- Moghadam A, Lawrence MB, Friederichs J, Altevogt P, Ley K...CD24 mediates rolling of breast carcinoma cells on P-selectin. 1998 FASEB J 12:1241-1251 9. Friederichs J, Zeller Y, Hafezi- Moghadam A, Grone HJ, Ley

The Signaling Networks of the Herpesvirus Entry Mediator (TNFRSF14) in Immune Regulation

PubMed Central

Steinberg, Marcos; Cheung, Timothy C.; Ware, Carl F.

2012-01-01

Summary The tumor necrosis factor (TNF) receptor superfamily member herpesvirus entry mediator (HVEM) (TNFRSF14) regulates T-cell immune responses by activating both inflammatory and inhibitory signaling pathways. HVEM acts as both a receptor for the canonical TNF-related ligands, LIGHT [lymphotoxin-like, exhibits inducible expression, and competes with herpes simplex virus glycoprotein D for HVEM, a receptor expressed on T lymphocytes] and lymphotoxin-α, and as a ligand for the immunoglobulin superfamily proteins BTLA (B and T lymphocyte attenuator) and CD160, a feature distinguishing HVEM from other immune regulatory molecules. The ability of HVEM to interact with multiple ligands in distinct configurations creates a functionally diverse set of intrinsic and bidirectional signaling pathways that control both inflammatory and inhibitory responses. The HVEM system is integrated into the larger LTβR and TNFR network through extensive shared ligand and receptor usage. Experimental mouse models and human diseases indicate that dysregulation of HVEM network may contribute to autoimmune pathogenesis, making it an attractive target for drug intervention. PMID:22017438

Tc-99m galactosyl-neoglycoalbumin: in vitro characterization of receptor-mediated binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vera, D.R.; Krohn, K.A.; Stadalnik, R.C.

1984-07-01

Hepatic binding protein (HBP) is a membrane receptor that binds and transports plasma glycoproteins from hepatic blood to hepatocellular lysosomes. A characterization is made of the in vitro binding of Tc-99m galactosyl-neoglycoalbumin (Tc-NGA), a synthetic HBP ligand, to liver membrane. Structural modifications of NGA resulted in the alteration of the equilibrium constant, KA, and the forward-binding rate constant, kb. Binding was second-order; the relative amount of membrane-bound NGA depended on the initial concentrations of ligand and membrane. Membrane displacement studies, using carrier ligands in contrast to previously bound Tc-NGA or I-NGA, correlated with the binding characteristics of a native HBPmore » ligand, asialo-orosomucoid. Computer simulation was used to study the detectability of the changes in HBP concentration at different values of kb. The simulations indicated that radiopharmacokinetic sensitivity to alterations in (HBP) should be possible using a neoglycoalbumin preparation with a carbohydrate density within the range of 15 to 25 galactose units per albumin molecule.« less

Indomethacin induced gastropathy in CD18, intercellular adhesion molecule 1, or P-selectin deficient mice

PubMed Central

Morise, Z; Granger, D; Fuseler, J; Anderson, D; Grisham, M

1999-01-01

BACKGROUND—Neutrophil-endothelial cell interactions are thought to play a critical role in the pathophysiology of non-steroidal anti-inflammatory drug (NSAID) induced gastropathy.
AIMS—To optimise a mouse model of NSAID induced gastropathy and to evaluate the importance of adhesion molecules using adhesion molecule deficient mice.
METHODS—Gastropathy was induced in C57BL/6 mice or their adhesion molecule deficient counterparts via oral administration of indomethacin (20 mg/kg). Lesion scores, mucosal permeability, and histopathology were used to assess gastric mucosal injury.
RESULTS—Intragastric administration of indomethacin induced linear haemorrhagic mucosal lesions, primarily in the corpus of the stomach that were first observed at six hours. These lesions continued to develop over the next six hours with maximal lesion scores and mucosal permeabilities at 12 hours. When indomethacin was administered to mice deficient in CD18, intercellular adhesion molecule 1 (ICAM-1), or P-selectin, there were significant decreases in lesion scores compared with their C57BL/6 controls. In addition, mucosal permeabilities were found to be significantly lower in CD18 or ICAM-1 deficient mice observed at 12 hours.
CONCLUSION—Certain leucocyte and endothelial cell adhesion molecules are important determinants for full expression of indomethacin induced gastropathy. It is proposed that this modification of the mouse model may be useful for the investigation of other pathophysiological mechanisms of NSAID induced gastropathy.


Keywords: indomethacin; gastropathy; cyclooxygenase; intercellular adhesion molecule; VCAM; vascular cell adhesion molecule; P-selectin PMID:10486359

Prospective study of hemostatic alterations in children with acute lymphoblastic leukemia.

PubMed

Giordano, Paola; Molinari, Angelo Claudio; Del Vecchio, Giovanni Carlo; Saracco, Paola; Russo, Giovanna; Altomare, Maria; Perutelli, Paolo; Crescenzio, Nicoletta; Santoro, Nicola; Marchetti, Marina; De Mattia, Domenico; Falanga, Anna

2010-05-01

In a group of newly diagnosed acute lymphocytic leukemia (ALL) children we evaluated a number of hemostatic and inflammatory markers at diagnosis and at different time points during chemotherapy for the remission induction to identify alterations in the plasma levels of prothrombotic markers before and during the course of chemotherapy. The following plasma markers were evaluated: thrombin-antithrombin complex (TAT), D-Dimer, plasminogen activator inhibitor 1 (PAI-1), antithrombin, fibrinogen, von Willebrand factor (VWF) antigen and high molecular weight VWF (HMW-VWF) multimers, P-selectin, tumor necrosis factor alpha (TNF-alpha), and interleukin 6 (IL-6). Plasma samples were collected at the following time points: at T0 (baseline) and T1 (+24 days of therapy), T2 (+36 days therapy), and T3 (+64 days therapy). The results show that, at diagnosis, ALL children presented with laboratory signs of increased thrombin generation and fibrin formation (i.e. high TAT and D-dimer levels), fibrinolysis inhibition (i.e. high PAI-1 level), endothelial activation (i.e., high HMW-VWF and soluble P-selectin levels) and inflammation (i.e. high TNF-alpha and IL-6 levels). After starting induction therapy, the thrombin generation markers and inflammatory cytokines significantly decreased. To the opposite, PAI-1 and P-selectin significantly increased, suggesting an insult by chemotherapy on the vascular endothelium. These effects were more evident during steroid administration. Symptomatic venous thromboembolism (VTE) episodes developed in two cases during induction therapy, which did not allow the evaluation of the predictive value for VTE of laboratory markers. (c) 2010 Wiley-Liss, Inc.

Elevated thrombopoietin in plasma of burned patients without and with sepsis enhances platelet activation.

PubMed

Lupia, E; Bosco, O; Mariano, F; Dondi, A E; Goffi, A; Spatola, T; Cuccurullo, A; Tizzani, P; Brondino, G; Stella, M; Montrucchio, G

2009-06-01

Thrombopoietin (TPO) is a humoral growth factor that does not induce platelet aggregation per se, but enhances platelet activation in response to several agonists. Circulating levels of TPO are increased in patients with sepsis and are mainly related to sepsis severity. To investigate the potential contribution of elevated TPO levels in platelet activation during burn injury complicated or not by sepsis. We studied 22 burned patients, 10 without and 12 with sepsis, and 10 healthy subjects. We measured plasma levels of TPO, as well as leukocyte-platelet binding and P-selectin expression. The priming activity of plasma from burned patients or healthy subjects on platelet aggregation and leukocyte-platelet binding, and the role of TPO in these effects were also studied in vitro. Burned patients without and with sepsis showed higher circulating TPO levels and increased monocyte-platelet binding compared with healthy subjects. Moreover, TPO levels, monocyte-platelet binding and P-selectin expression were significantly higher in burned patients with sepsis than in burned patients without sepsis. In vitro, plasma from burned patients without and with sepsis, but not from healthy subjects, primed platelet aggregation, monocyte-platelet binding and platelet P-selectin expression. The effect of plasma from burned patients with sepsis was significantly higher than that of plasma from burned patients without sepsis. An inhibitor of TPO prevented the priming effect of plasma from burned patients. Increased TPO levels may enhance platelet activation during burn injury and sepsis, potentially participating in the pathogenesis of multi-organ failure in these diseases.

Assessment of canine autologous platelet-rich plasma produced with a commercial centrifugation and platelet recovery kit.

PubMed

Frye, Chris W; Enders, Andrew; Brooks, Marjory B; Struble, Angela M; Wakshlag, Joseph J

2016-01-01

To characterize the cellular composition (platelets, erythrocytes, and leukocytes) and confirm reproducibility of platelet enrichment, as well as determine the platelet activation status in the final product of a commercial platelet-rich plasma kit using canine blood. Venous blood from 20 sedated client-owned dogs was used to prepare platelet-rich plasma (PRP) from a commercial kit. Complete blood counts were performed to determine erythrocyte, leukocyte, and platelet numbers in both whole blood (WB) and resultant PRP. The WB and PRP samples from jugular (fast collection) and cephalic (slow collection) venipuncture were also compared. P-selectin externalization was measured in WB and PRP samples from 15 of 20 dogs. This commercial kit produced an average percent recovery in platelets of 64.7 ± 17.4; erythrocytes of 3.7 ± 0.8, and leukocytes of 31.6 ± 10.0. Neutrophil, monocyte, and lymphocyte percent recovery was 19.6 ± 7.2, 44.89 ± 19.8, and 57.5 ± 10.6, respectively. The recovery of platelets from jugular venipuncture (59.7 ± 13.6%) was lower than from cephalic recovery (68.8 ± 19.1%). The mean percent P-Selectin externalization for WB, PRP, and PRP with thrombin was 25.5 ± 30.9, 4.5 ± 6.4, and 90.6 ± 4.4 respectively. Cellular reproducibility of this kit was confirmed and platelets were concentrated within autologous serum. Additionally, measurements of P-selectin externalization showed that platelets are inactive in PRP unless stimulated to degranulate.

Fucosylated chondroitin sulfates from the body wall of the sea cucumber Holothuria forskali: conformation, selectin binding, and biological activity.

PubMed

Panagos, Charalampos G; Thomson, Derek S; Moss, Claire; Hughes, Adam D; Kelly, Maeve S; Liu, Yan; Chai, Wengang; Venkatasamy, Radhakrishnan; Spina, Domenico; Page, Clive P; Hogwood, John; Woods, Robert J; Mulloy, Barbara; Bavington, Charlie D; Uhrín, Dušan

2014-10-10

Fucosylated chondroitin sulfate (fCS) extracted from the sea cucumber Holothuria forskali is composed of the following repeating trisaccharide unit: → 3)GalNAcβ4,6S(1 → 4) [FucαX(1 → 3)]GlcAβ(1 →, where X stands for different sulfation patterns of fucose (X = 3,4S (46%), 2,4S (39%), and 4S (15%)). As revealed by NMR and molecular dynamics simulations, the fCS repeating unit adopts a conformation similar to that of the Le(x) blood group determinant, bringing several sulfate groups into close proximity and creating large negative patches distributed along the helical skeleton of the CS backbone. This may explain the high affinity of fCS oligosaccharides for L- and P-selectins as determined by microarray binding of fCS oligosaccharides prepared by Cu(2+)-catalyzed Fenton-type and photochemical depolymerization. No binding to E-selectin was observed. fCS poly- and oligosaccharides display low cytotoxicity in vitro, inhibit human neutrophil elastase activity, and inhibit the migration of neutrophils through an endothelial cell layer in vitro. Although the polysaccharide showed some anti-coagulant activity, small oligosaccharide fCS fragments had much reduced anticoagulant properties, with activity mainly via heparin cofactor II. The fCS polysaccharides showed prekallikrein activation comparable with dextran sulfate, whereas the fCS oligosaccharides caused almost no effect. The H. forskali fCS oligosaccharides were also tested in a mouse peritoneal inflammation model, where they caused a reduction in neutrophil infiltration. Overall, the data presented support the action of fCS as an inhibitor of selectin interactions, which play vital roles in inflammation and metastasis progression. Future studies of fCS-selectin interaction using fCS fragments or their mimetics may open new avenues for therapeutic intervention. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The adhesion of Pseudomonas aeruginosa to high molecular weight human tear film species corresponds to glycoproteins reactive with Sambucus nigra lectin.

PubMed

Aristoteli, Lina Panayiota; Willcox, Mark D P

2006-11-01

Pseudomonas aeruginosa is a pathogen gaining prevalence in contact lens-related corneal ulcers. Tear outflow protects the ocular surface, where high molecular weight tear glycoproteins bind bacteria for removal from the eye. The purpose of the present study was to identify glycoproteins in human tears involved in the adhesion of ocular P. aeruginosa isolates. Basal human tears were applied to a bacterial adhesion assay involving electrophoretic separation of tear components, transfer to nitrocellulose and incubation with biotin-labelled bacteria. Glycoproteins were further characterised using lectin profiling. The results showed large-dimension agarose gels were imperative for the detection of at least four glycoproteins with a migration >200 kDa, including species not previously identified. P. aeruginosa 6294 preferentially bound to a well-defined glycoprotein near the origin of the gel that, unlike other glycoproteins >200 kDa, reacted with Sambucus nigra lectin (sialic acid alpha2-6) but not WGA lectin (N-acetylglucosamine, sialic acid alpha2-3). Adhesion did not involve free biotin label or hydrophobic interactions. Also, the pre-incubation of separated tear glycoproteins with S. nigra lectin increased subsequent adhesion of 6294 to this tear glycoprotein. The less virulent Paer1 strain showed diffuse adhesion in the S. nigra-reactive region at the gel origin. In conclusion, an overlay adhesion assay was developed that identified slow-migrating sialylated glycoprotein species in human tears preferentially bound by P. aeruginosa ocular strains, and S. nigra lectin seemed to enhance the interaction. The study provides a basis for direct investigation of bacterial adhesion to glycoproteins with an apparent migration >200 kDa in tear fluid.

Enhanced platelet/endothelial activation in depressed patients with acute coronary syndromes: evidence from recent clinical trials.

PubMed

Serebruany, Victor L; Glassman, Alexander H; Malinin, Alex I; Sane, David C; Finkel, Mitchell S; Krishnan, Ranga R; Atar, Dan; Lekht, Vladimir; O'Connor, Christopher M

2003-09-01

Platelets play a key role in the progression of acute coronary syndromes (ACS). Clinical depression alone is also associated with enhanced platelet activation. The purpose of this study was to compare concentrations of established biomarkers of enhanced platelet/endothelial activation in clinically depressed versus non-depressed patients enrolled in recent clinical trials for ACS. Two hundred and eighty-one baseline plasma samples from patients with acute myocardial infarction (ASSENT-2; n = 41), with ACS (PRONTO; n = 126) and with clinical depression plus previous acute coronary syndrome within 6 months (SADHART; n = 64), and from normal healthy controls (n = 50) were analyzed. Blood was drawn before applying any therapeutic strategies including interventions, thrombolytics, infusions, and selective serotonin re-uptake inhibitors. Platelet factor 4, beta-thromboglobulin, platelet/endothelial cell adhesion molecule-1, P-selectin, thromboxane, prostacyclin, vascular cell adhesion molecule-1, and E-selectin were measured by enzyme-linked immunosorbent assay by a single core laboratory. Patients with ACS exhibited a higher degree of platelet activation than controls independently of the presence of depression. Plasma levels of P-selectin, thromboxane, prostacyclin, and vascular cell adhesion molecule-1 were the highest in the acute myocardial infarction group when compared with ACS despite the presence or absence of clinical depression. Surprisingly, patients with ACS and depression exhibited the highest levels of platelet factor 4, beta-thromboglobulin, and platelet/endothelial cell adhesion molecule-1 when compared with myocardial infarction or angina patients without clinical depression. E-selectin plasma level was constantly elevated compared with controls but did not differ among the groups dependent on the incidence of depression. The depressed plus ACS group had higher plasma levels of all biomarkers compared with the non-depressed patients. Retrospective analysis of the data from several clinical trials reveals that clinical depression is associated with enhanced activation of platelet/endothelial biomarkers even above the level expected in ACS. These findings may contribute to the unfavorable outcome associated with clinical depression in patients with ACS.

[Regulative effects of hydrogen-rich medium on monocytic adhesion and vascular endothelial permeability].

PubMed

Wang, Wei-na; Xie, Ke-liang; Chen, Hong-guang; Han, Huan-zhi; Wang, Guo-lin; Yu, Yong-hao

2013-11-19

To explore the regulative effects of hydrogen-rich medium on lipopolysaccharide (LPS)-induced monocytes adhesion to human umbilical vein endothelial cells (HUVEC) and vascular endothelial permeability in vitro. Endothelial cells were seeded in 6-well plates and randomly divided into 4 groups (n = 42 each):control (A), hydrogen-rich medium (B), LPS (C) and LPS+hydrogen-rich medium (D). Cells were cultured in plain culture medium in groups A and C or in hydrogen-saturated culture medium in groups B and D.LPS 1 µg/ml was added into groups C and D.When forming a monolayer, monocytes were added into each group after 6, 12 and 24 h respectively. After a 90-minute co-culturing, adhesion status was detected by Wright-Giemsa stain.Supernatants were collected to detect the concentrations of vascular cell adhesion molecule-1 (VCAM-1) and E-selectin by enzyme-linked immunosorbent assay (ELISA). The expression of VE-cadherin was measured by Western blot. Cells were stained with immunofluorescence to show the distribution of VE-cadherin after a 24-hour incubation. Compared with group A, the adhesion of monocytes to endothelial cells increased (P < 0.05) in group C, the levels of E-selectin and VCAM-1 became elevated (P < 0.05) while the expression of VE-cadherin decreased significantly (P < 0.05). Compared with group C, adhesion decreased in group D (P < 0.05), the levels of E-selectin and VCAM-1 decreased (P < 0.05) while there was an increased expression of VE-cadherin (P < 0.05). Three timepoints showed the same tendency. The results of 24 h fluorescence indicated that, compared with group A, VE-cadherin was incomplete in cell-cell connections in group C.However it was complete and well-distributed in group D versus group C. Hydrogen-rich medium may reduce the LPS-induced release of adhesion molecules, lessen monocytic adhesion to HUVEC and regulate the expression of VE-cadherin to protect vascular permeability.

Plasma E-selectin levels can play a role in the development of diabetic retinopathy.

PubMed

Kasza, Márta; Meleg, J; Vardai, J; Nagy, B; Szalai, E; Damjanovich, J; Csutak, A; Ujhelyi, B; Nagy, V

2017-01-01

Diabetic retinopathy is one of the leading causes of blindness. There are several risk factors, such as the duration of diabetes or glycemic control of the patient; however, several biochemical factors also alter the process. Our aim was to investigate the role of soluble E-selectin in the formation of diabetic retinopathy. Fifty-seven patients (37 female and 20 male, aged 61.71 ± 12.31 years) and 14 healthy control subjects (ten female and four male, aged 63.06 ± 10.46 years) were enrolled in the study. We measured the soluble E-selectin level in the plasma of patients by ELISA. All patients underwent careful ophthalmological examination, including ophthalmoscopy and color fundus photography, while diabetic retinopathy grading was performed in line with the 2012 classification of the American Academy of Ophthalmology (AAO). The soluble E-selectin level was significantly higher in patients with diabetes compared to controls (32.95 ng/ml vs. 26.55 ng/ml, p = 0.03). Dividing patients into groups by the presence of retinopathy, the E-selectin level was also significantly higher in the retinopathy group (p < 0.05). When we examined diabetic patients by the severity of retinopathy (groups A, B, and C, by the guidelines of the AAO), however, we did not find any significant difference in soluble E-selectin levels, although it tended to be higher in group B. An elevated E-selectin level can play a role in the development of diabetic retinopathy, but it does not seem to alter disease severity. However, glycemic control and the reduction of cardiovascular risk factors may also alter the level of E-selectin that might play a role in the prevention of diabetic retinopathy.

Multi-ethnic analysis reveals soluble L-selectin may be post-transcriptionally regulated by 3′UTR polymorphism: the Multi-Ethnic Study of Atherosclerosis (MESA)

PubMed Central

Berardi, Cecilia; Larson, Nicholas B.; Decker, Paul A.; Wassel, Christina L.; Kirsch, Phillip S.; Pankow, James S.; Sale, Michele M.; de Andrade, Mariza; Sicotte, Hugues; Tang, Weihong; Hanson, Naomi Q.; Tsai, Michael Y.; da Chen, Yii-Der I; Bielinski, Suzette J.

2015-01-01

L-selectin is constitutively expressed on leukocytes and mediates their interaction with endothelial cells during inflammation. Previous studies on the association of soluble L-selectin (sL-selectin) with cardiovascular disease (CVD) are inconsistent. Genetic variants associated with sL-selectin levels may be a better surrogate of levels over a lifetime. We explored the association of genetic variants and sL-selectin levels in a race/ethnicity stratified random sample of 2,403 participants in the Multi-Ethnic Study of Atherosclerosis (MESA). Through a genome-wide analysis with additive linear regression models, we found that rs12938 on the SELL gene accounted for a significant portion of the protein level variance across all four races/ethnicities. To evaluate potential additional associations, elastic net models were used for variants located in the SELL/SELP/SELE genetic region and an additional two SNPs, rs3917768 and rs4987361, were associated with sL-selectin levels in African Americans. These variants accounted for a portion of protein variance that ranged from 4% in Hispanic to 14% in African Americans. To investigate the relationship of these variants with CVD, 6,317 subjects were used. No significant association was found between any of the identified SNPs and carotid intima-media thickness or presence of carotid plaque using linear and logistic regression, respectively. Similarly no significant results were found for coronary artery calcium or coronary heart disease events. In conclusion, we found that variants within the SELL gene are associated with sL-selectin levels. Despite accounting for a significant portion of the protein level variance, none of the variants was associated with clinical or subclinical CVD. PMID:25576479

The G glycoprotein of respiratory syncytial virus depresses respiratory rates through the CX3C motif and substance P.

PubMed

Tripp, Ralph A; Dakhama, Azzeddine; Jones, Les P; Barskey, Albert; Gelfand, Erwin W; Anderson, Larry J

2003-06-01

Respiratory syncytial virus (RSV) infection in the neonate can alter respiratory rates, i.e., lead to episodes of apnea. We show that RSV G glycoprotein reduces respiratory rates associated with the induction of substance P (SP) and G glycoprotein-CX3CR1 interaction, an effect that is inhibited by treatment with anti-G glycoprotein, anti-SP, or anti-CX3CR1 monoclonal antibodies. These data suggest new approaches for treating some aspects of RSV disease.

The G Glycoprotein of Respiratory Syncytial Virus Depresses Respiratory Rates through the CX3C Motif and Substance P

PubMed Central

Tripp, Ralph A.; Dakhama, Azzeddine; Jones, Les P.; Barskey, Albert; Gelfand, Erwin W.; Anderson, Larry J.

2003-01-01

Respiratory syncytial virus (RSV) infection in the neonate can alter respiratory rates, i.e., lead to episodes of apnea. We show that RSV G glycoprotein reduces respiratory rates associated with the induction of substance P (SP) and G glycoprotein-CX3CR1 interaction, an effect that is inhibited by treatment with anti-G glycoprotein, anti-SP, or anti-CX3CR1 monoclonal antibodies. These data suggest new approaches for treating some aspects of RSV disease. PMID:12743318

P-glycoprotein substrate transport assessed by comparing cellular and vesicular ATPase activity.

PubMed

Nervi, Pierluigi; Li-Blatter, Xiaochun; Aänismaa, Päivi; Seelig, Anna

2010-03-01

We compared the P-glycoprotein ATPase activity in inside-out plasma membrane vesicles and living NIH-MDR1-G185 cells with the aim to detect substrate transport. To this purpose we used six substrates which differ significantly in their passive influx through the plasma membrane. In cells, the cytosolic membrane leaflet harboring the substrate binding site of P-glycoprotein has to be approached by passive diffusion through the lipid membrane, whereas in inside-out plasma membrane vesicles, it is accessible directly from the aqueous phase. Compounds exhibiting fast passive influx compared to active efflux by P-glycoprotein induced similar ATPase activity profiles in cells and inside-out plasma membrane vesicles, because their concentrations in the cytosolic leaflets were similar. Compounds exhibiting similar influx as efflux induced in contrast different ATPase activity profiles in cells and inside-out vesicles. Their concentration was significantly lower in the cytosolic leaflet of cells than in the cytosolic leaflet of inside-out membrane vesicles, indicating that P-glycoprotein could cope with passive influx. P-glycoprotein thus transported all compounds at a rate proportional to ATP hydrolysis (i.e. all compounds were substrates). However, it prevented substrate entry into the cytosol only if passive influx of substrates across the lipid bilayer was in a similar range as active efflux. Copyright 2009 Elsevier B.V. All rights reserved.