Syeda T. Towhid

Dynamic adhesion of eryptotic erythrocytes to endothelial cells via CXCL16/SR-PSOX

Suicidal death of erythrocytes, or eryptosis, is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine exposure at the cell surface. Eryptosis is triggered by increase of cytosolic Ca2+ activity, which may result from treatment with the Ca2+ ionophore ionomycin or from energy depletion by removal of glucose. The present study tested the hypothesis that phosphatidylserine exposure at the erythrocyte surface fosters adherence to endothelial cells of the vascular wall under flow conditions at arterial shear rates and that binding of eryptotic cells to endothelial cells is mediated by the transmembrane CXC chemokine ligand 16 (CXCL16). To this end, human erythrocytes were exposed to energy depletion (for 48 h) or treated with the Ca2+ ionophore ionomycin (1 μM for 30 min). Phosphatidylserine exposure was quantified utilizing annexin-V binding, cell volume was estimated from forward scatter in FACS analysis, and erythrocyte adhesion to human vascular endothelial cells (HUVEC) was determined in a flow chamber model. As a result, both, ionomycin and glucose depletion, triggered eryptosis and enhanced the percentage of erythrocytes adhering to HUVEC under flow conditions at arterial shear rates. The adhesion was significantly blunted in the presence of erythrocyte phosphatidylserine-coating annexin-V (5 μl/ml), of a neutralizing antibody against endothelial CXCL16 (4 μg/ml), and following silencing of endothelial CXCL16 with small interfering RNA. The present observations demonstrate that eryptotic erythrocytes adhere to endothelial cells of the vascular wall in part by interaction of phosphatidylserine exposed at the erythrocyte surface with endothelial CXCL16.

Sphingomyelinase-induced adhesion of eryptotic erythrocytes to endothelial cells

Eryptosis, the suicidal erythrocyte death, leads to cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptotic erythrocytes adhere to the vascular wall by binding of phosphatidylserine to the CXC chemokine ligand 16 (CXCL16). Stimulators of eryptosis include increased cytosolic Ca(2+) activity, energy depletion, and activation of ceramide-producing sphingomyelinase. The present study explored whether sphingomyelinase triggers erythrocyte adhesion to endothelial cells. To this end, human erythrocytes were exposed for 6 h to bacterial sphingomyelinase (1-10 mU/ml) and phosphatidylserine exposure was estimated from fluorescent annexin-V-binding, cell volume from forward scatter in FACS-analysis, erythrocyte adhesion to human umbilical vein endothelial cells (HUVEC) from trapping of labeled erythrocytes in a flow chamber under flow conditions at arterial shear rates, and CXCL16 protein abundance utilizing Western blotting and FACS analysis of fluorescent antibody binding. As a result, sphingomyelinase (≥1 mU/ml) triggered cell shrinkage, phosphatidylserine exposure and erythrocyte adhesion to HUVEC, effects blunted by Ca(2+) removal. Adhesion was significantly blunted by phosphatidylserine-coating annexin-V (5 μl/ml), following addition of neutralizing antibodies against endothelial CXCL16 (4 μg/ml) and following silencing of the CXCL16 gene with small interfering RNA. Pretreatment of HUVEC with sphingomyelinase upregulated CXCL16 protein abundance. Six hours pretreatment of HUVEC with sphingomyelinase (10 mU/ml) or C6-ceramide (50 μM) augmented erythrocyte adhesion following a 30-min treatment with Ca(2+) ionophore ionomycin (1 μM) or following energy depletion by 48-h glucose removal. Thus exposure to sphingomyelinase or C6-ceramide triggers eryptosis followed by phosphatidylserine- and CXCL16-sensitive adhesion of eryptotic erythrocytes to HUVEC.

Sensitization of erythrocytes to suicidal erythrocyte death following water deprivation.

Background/Aims: Klotho deficiency results in excessive formation of 1,25(OH)2D3, accelerated ageing and early death. Moreover, klotho deficiency enhances eryptosis, the suicidal erythrocyte death characterized by phosphatidylserine exposure at the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i), glucose depletion, hyperosmotic shock and oxidative stress. Klotho expression is decreased and 1,25(OH)2D3-formation enhanced by dehydration. The present study thus explored whether dehydration influences eryptosis. Methods: Blood was drawn from hydrated or 36h dehydrated mice. Plasma osmolarity was determined by vapour pressure method, plasma 1,25(OH)2D3 and aldosterone concentrations using ELISA, and plasma Ca2+-concentration utilizing photometry. Erythrocytes were exposed to Ca2+-ionophore ionomycin (1 µM, 30 min), energy depletion (12 h glucose removal), hyperosmotic shock (500 mM sucrose added, 2 h) and oxidative stress (100 µM tert-butyl-hydroperoxide, 30 min) and phosphatidylserine exposure at the erythrocyte surface estimated from annexin V binding. Results: Dehydration increased plasma osmolarity and plasma 1,25(OH)2D3 and aldosterone concentrations. Dehydration did not significantly modify phosphatidylserine-exposure of freshly drawn erythrocytes but significantly enhanced the increase of phosphatidylserine-exposure under control conditions and following treatment with ionomycin, glucose-deprivation, hyperosmolarity or tert-butyl-hydroperoxide. Conclusions: Dehydration sensitizes the erythrocytes to spontaneous eryptosis and to the triggering of eryptosis by excessive Ca2+-entry, energy depletion, hyperosmotic shock and oxidative stress.

The Inflammatory Chemokine CXC Motif Ligand 16 Triggers Platelet Activation and Adhesion Via CXC Motif Receptor 6–Dependent Phosphatidylinositide 3-Kinase/Akt Signaling

Rationale: The recently discovered chemokine CXC motif ligand 16 (CXCL16) is highly expressed in atherosclerotic lesions and is a potential pathogenic mediator in coronary artery disease. Objective: The aim of this study was to test the role of CXCL16 on platelet activation and vascular adhesion, as well as the underlying mechanism and signaling pathway. Methods and Results: Reverse-transcriptase polymerase chain reaction, Western blotting, confocal microscopy, and flow cytometry revealed that CXCL16-specific receptor, CXC motif receptor 6, is highly expressed in platelets. According to flow cytometry and confocal microscopy, stimulation of platelets with CXCL16 induced platelet degranulation, integrin &agr;IIb&bgr;3 activation, and shape change. CXCL16 increased Akt phosphorylation (Thr308/Ser473), an effect abrogated by phosphatidylinositide 3-kinase inhibitors wortmannin (100 nmol/L) and LY294002 (25 µmol/L). The phosphatidylinositide 3-kinase inhibitors and Akt inhibitor SH-6 (20 µmol/L) further diminished CXCL16-induced platelet activation. CXCL16-mediated platelet degranulation, integrin &agr;IIb&bgr;3 activation, and Akt phosphorylation were blunted in platelets lacking CXCL16-specific receptor CXC motif receptor 6. CXCL16-induced platelet activation was abrogated in Akt1- or Akt2-deficient platelets. CXCL16 enhanced platelet adhesion to endothelium in vitro after high arterial shear stress (2000−s) and to injured vascular wall in vivo after carotid ligation. CXCL16-induced stimulation of platelet adhesion again was prevented by phosphatidylinositide 3-kinase and Akt inhibitors. Apyrase and antagonists of platelet purinergic receptors P2Y1 (MRS2179, 100 µmol/L) and especially P2Y12 (Cangrelor, 10 µmol/L) blunted CXCL16-triggered platelet activation as well as CXCL16-induced platelet adhesion under high arterial shear stress in vitro and after carotid ligation in vivo. Conclusions: The inflammatory chemokine CXCL16 triggers platelet activation and adhesion via CXC motif receptor 6–dependent phosphatidylinositide 3-kinase/Akt signaling and paracrine activation, suggesting a decisive role for CXCL16 in linking vascular inflammation and thrombo-occlusive diseases.Rationale: The recently discovered chemokine CXCL16 is highly expressed in atherosclerotic lesions and a potential pathogenic mediator in coronary artery disease. Objective: To test the role of CXCL16 on platelet activation and vascular adhesion as well as the underlying mechanism and signaling pathway. Methods and Results: RT-PCR, western blotting, confocal microscopy and flow cytometry revealed that CXCL16-specific receptor CXCR6 is highly expressed on platelets. According to flow cytometry and confocal microscopy stimulation of platelets with CXCL16 induced platelet degranulation, integrin α IIb β 3 activation and shape change. CXCL16 increased Akt phosphorylation (Thr 308 /Ser 473 ), an effect abrogated by phosphatidylinositide 3-kinase (PI3K) inhibitors wortmannin (100nM) and LY294002 (25 µM). The PI3K inhibitors and Akt inhibitor SH-6 (20 µM) further diminished CXCL16-induced platelet activation. CXCL16-mediated platelet degranulation, integrin α IIb β 3 activation and Akt phosphorylation were blunted in platelets lacking CXCL16-specific receptor CXCR6. CXCL16-induced platelet activation was abrogated in Akt1- or Akt2-deficient platelets. CXCL16 enhanced platelet adhesion to endothelium in vitro following high arterial shear stress (2000 -s ) and to injured vascular wall in vivo following carotis ligation. CXCL16-induced stimulation of platelet adhesion was again prevented by PI3K and Akt inhibitors. Apyrase and antagonists of platelet purinergic receptors P 2 Y 1 (MRS2179, 100µM) and especially P 2 Y 12 (Cangrelor, 10µM) blunted CXCL16-triggered platelet activation as well as CXCL16-induced platelet adhesion under high arterial shear stress in vitro and after carotis ligation in vivo. Conclusions: The inflammatory chemokine CXCL16 triggers platelet activation and adhesion via CXCR6-dependent PI3K/Akt signaling and paracrine activation suggesting a decisive role for CXCL16 in linking vascular inflammation and thrombo-occlusive diseases.

The serum- and glucocorticoid-inducible kinase 1 (SGK1) influences platelet calcium signaling and function by regulation of Orai1 expression in megakaryocytes

Platelets are activated on increase of cytosolic Ca2+ activity ([Ca2+](i)), accomplished by store-operated Ca2+ entry (SOCE) involving the pore-forming ion channel subunit Orai1. Here, we show, for the first time, that the serum- and glucocorticoid-inducible kinase 1 (SGK1) is expressed in platelets and megakaryocytes. SOCE and agonist-induced [Ca2+](i) increase are significantly blunted in platelets from SGK1 knockout mice (sgk1(-/-)). Similarly, Ca2+ -dependent degranulation, integrin α(IIb)β3 activation, phosphatidylserine exposure, aggregation, and in vitro thrombus formation were significantly impaired in sgk1(-/-) platelets, whereas tail bleeding time was not significantly enhanced. Platelet and megakaryocyte Orai1 transcript levels and membrane protein abundance were significantly reduced in sgk1(-/-) mice. In human megakaryoblastic cells (MEG-01), transfection with constitutively active (S422D)SGK1 but not with inactive (K127N)SGK1 significantly enhanced Orai1 expression and SOCE, while effects reversed by the SGK1 inhibitor GSK650394 (1μM). Transfection of MEG-01 cells with (S422D)SGK1 significantly increased phosphorylation of IκB kinase α/β and IκBα resulting in nuclear translocation of NF-κB subunit p65. Treatment of (S422D)SGK1-transfected MEG-01 cells with the IκB kinase inhibitor BMS-345541 (10μM) abolished SGK1-induced increase of Orai1 expression and SOCE. The present observations unravel SGK1 as novel regulator of platelet function, effective at least in part by NF-κB-dependent transcriptional up-regulation of Orai1 in megakaryocytes and increasing platelet SOCE.

The Inflammatory Chemokine CXCL16 Triggers Platelet Activation and Adhesion via CXCR6-Dependent PI3K/Akt Signaling

Rationale: The recently discovered chemokine CXC motif ligand 16 (CXCL16) is highly expressed in atherosclerotic lesions and is a potential pathogenic mediator in coronary artery disease. Objective: The aim of this study was to test the role of CXCL16 on platelet activation and vascular adhesion, as well as the underlying mechanism and signaling pathway. Methods and Results: Reverse-transcriptase polymerase chain reaction, Western blotting, confocal microscopy, and flow cytometry revealed that CXCL16-specific receptor, CXC motif receptor 6, is highly expressed in platelets. According to flow cytometry and confocal microscopy, stimulation of platelets with CXCL16 induced platelet degranulation, integrin &agr;IIb&bgr;3 activation, and shape change. CXCL16 increased Akt phosphorylation (Thr308/Ser473), an effect abrogated by phosphatidylinositide 3-kinase inhibitors wortmannin (100 nmol/L) and LY294002 (25 µmol/L). The phosphatidylinositide 3-kinase inhibitors and Akt inhibitor SH-6 (20 µmol/L) further diminished CXCL16-induced platelet activation. CXCL16-mediated platelet degranulation, integrin &agr;IIb&bgr;3 activation, and Akt phosphorylation were blunted in platelets lacking CXCL16-specific receptor CXC motif receptor 6. CXCL16-induced platelet activation was abrogated in Akt1- or Akt2-deficient platelets. CXCL16 enhanced platelet adhesion to endothelium in vitro after high arterial shear stress (2000−s) and to injured vascular wall in vivo after carotid ligation. CXCL16-induced stimulation of platelet adhesion again was prevented by phosphatidylinositide 3-kinase and Akt inhibitors. Apyrase and antagonists of platelet purinergic receptors P2Y1 (MRS2179, 100 µmol/L) and especially P2Y12 (Cangrelor, 10 µmol/L) blunted CXCL16-triggered platelet activation as well as CXCL16-induced platelet adhesion under high arterial shear stress in vitro and after carotid ligation in vivo. Conclusions: The inflammatory chemokine CXCL16 triggers platelet activation and adhesion via CXC motif receptor 6–dependent phosphatidylinositide 3-kinase/Akt signaling and paracrine activation, suggesting a decisive role for CXCL16 in linking vascular inflammation and thrombo-occlusive diseases.Rationale: The recently discovered chemokine CXCL16 is highly expressed in atherosclerotic lesions and a potential pathogenic mediator in coronary artery disease. Objective: To test the role of CXCL16 on platelet activation and vascular adhesion as well as the underlying mechanism and signaling pathway. Methods and Results: RT-PCR, western blotting, confocal microscopy and flow cytometry revealed that CXCL16-specific receptor CXCR6 is highly expressed on platelets. According to flow cytometry and confocal microscopy stimulation of platelets with CXCL16 induced platelet degranulation, integrin α IIb β 3 activation and shape change. CXCL16 increased Akt phosphorylation (Thr 308 /Ser 473 ), an effect abrogated by phosphatidylinositide 3-kinase (PI3K) inhibitors wortmannin (100nM) and LY294002 (25 µM). The PI3K inhibitors and Akt inhibitor SH-6 (20 µM) further diminished CXCL16-induced platelet activation. CXCL16-mediated platelet degranulation, integrin α IIb β 3 activation and Akt phosphorylation were blunted in platelets lacking CXCL16-specific receptor CXCR6. CXCL16-induced platelet activation was abrogated in Akt1- or Akt2-deficient platelets. CXCL16 enhanced platelet adhesion to endothelium in vitro following high arterial shear stress (2000 -s ) and to injured vascular wall in vivo following carotis ligation. CXCL16-induced stimulation of platelet adhesion was again prevented by PI3K and Akt inhibitors. Apyrase and antagonists of platelet purinergic receptors P 2 Y 1 (MRS2179, 100µM) and especially P 2 Y 12 (Cangrelor, 10µM) blunted CXCL16-triggered platelet activation as well as CXCL16-induced platelet adhesion under high arterial shear stress in vitro and after carotis ligation in vivo. Conclusions: The inflammatory chemokine CXCL16 triggers platelet activation and adhesion via CXCR6-dependent PI3K/Akt signaling and paracrine activation suggesting a decisive role for CXCL16 in linking vascular inflammation and thrombo-occlusive diseases.

Stimulation of suicidal death of erythrocytes by rifampicin

The antibiotic rifampicin is widely used in the treatment of tuberculosis. Side effects of rifampicin include hemolytic anemia. Loss of circulating erythrocytes resembling hemolytic anemia could result from stimulation of eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure at the cell surface. Stimulators of eryptosis include increase of cytosolic Ca(2+) activity ([Ca(2+)](i)) and formation of ceramide. The present study explored, whether and, if so, how rifampicin triggers eryptosis. To this end, [Ca(2+)](i) was estimated from Fluo3 fluorescence, cell volume from forward scatter in flow cytometry, PS exposure from annexin binding, ceramide formation from binding of fluorescent antibodies and hemolysis from hemoglobin release. As a result, a 48 h exposure to rifampicin (≥ 24 μg/ml) significantly increased Fluo3 fluorescence, ceramide abundance and annexin binding, and significantly decreased forward scatter. Rifampicin triggered slight, but significant hemolysis. Removal of extracellular Ca(2+) significantly blunted, but did not fully abolish rifampicin induced annexin binding. In conclusion, exposure of human erythrocytes to rifampicin is followed by suicidal erythrocyte death or eryptosis, an effect at least partially due to increase of cytosolic Ca(2+) concentration and stimulation of ceramide formation.

Down-Regulation of Na+/K+ ATPase Activity by Human Parvovirus B19 Capsid Protein VP1

Background/Aims: Human parvovirus B19 (B19V) may cause inflammatory cardiomyopathy (iCMP) which is accompanied by endothelial dysfunction. The B19V capsid protein VP1 contains a lysophosphatidylcholine producing phospholipase A2 (PLA) sequence. Lysophosphatidylcholine has in turn been shown to inhibit Na+/K+ ATPase. The present study explored whether VP1 modifies Na+/K+ ATPase activity. Methods: Xenopus oocytes were injected with cRNA encoding VP1 isolated from a patient suffering from fatal B19V-iCMP or cRNA encoding PLA2-negative VP1 mutant (H153A) and K+ induced pump current (Ipump) as well as ouabain-inhibited current (Iouabain) both reflecting Na+/K+-ATPase activity were determined by dual electrode voltage clamp. Results: Injection of cRNA encoding VP1, but not of VP1(H153A) or water, was followed by a significant decrease of both, Ipump and Iouabain in Xenopus oocytes. The effect was not modified by inhibition of transcription with actinomycin (10 µM for 36 hours) but was abrogated in the presence of PLA2 specific blocker 4-bromophenacylbromide (50 µM) and was mimicked by lysophosphatidylcholine (0.5 - 1 µg/ml). According to whole cell patch clamp, lysophosphatidylcholine (1 µg /ml) similarly decreased Ipump in human microvascular endothelial cells (HMEC). Conclusion: The B19V capsid protein VP1 is a powerful inhibitor of host cell Na+/K+ ATPase, an effect at least partially due to phospholipase A2 (PLA2) dependent formation of lysophosphatidylcholine.

Inhibition of Colonic Tumor Growth by the Selective SGK Inhibitor EMD638683

Background: The serum and glucocorticoid inducible kinase SGK1, which was originally cloned from mammary tumor cells, is highly expressed in some but not all tumors. SGK1 confers survival to several tumor cells. Along those lines, the number of colonic tumors following chemical carcinogenesis was decreased in SGK1 knockout mice. Recently, a highly selective SGK inhibitor (EMD638683) has been developed. The present study explored whether EMD638683 affects survival of colon carcinoma cells in vitro and impacts on development of colonic tumors in vivo. Methods: Colon carcinoma (Caco-2) cells were exposed to EMD638683 with or without exposure to radiation (3 Gray) and cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, mitochondrial potential from JC-9 fluorescence, caspase 3 activity from CaspGlow Fluorescein staining, DNA degradation from propidium iodide staining as well as late apoptosis from annexin-V FITC and propidium iodide double staining. In vivo tumor growth was determined in wild type mice subjected to chemical carcinogenesis (intraperitoneal injection of 20 mg/kg 1,2-dimethylhydrazine followed by three cycles of 30 g/L synthetic dextran sulfate sodium in drinking water for 7 days). Results: EMD638683 treatment significantly augmented the radiation-induced decrease of forward scatter, increase of phosphatidylserine exposure, decrease of mitochondrial potential, increase of caspase 3 activity, increase of DNA fragmentation and increase of late apoptosis. The in vivo development of tumors following chemical carcinogenesis was significantly blunted by treatment with EMD638683. Conclusions: EMD638683 promotes radiation-induced suicidal death of colon tumor cells in vitro and decreases the number of colonic tumors following chemical carcinogenesis in vivo.

SGK1 Sensitivity of Platelet Migration

Recent observations pointed to the ability of platelets to migrate and thus to invade the inflamed vascular wall. Platelet migration could be stimulated by stromal cell-derived factor-1 (SDF-1), an effect dependent on phosphatidylinositide-3-kinase (PI3K) and paralleled by activation and phosphorylation of Wiskott-Aldrich syndrome protein (WASP). Migration is inhibited by vinculin, which is similarly regulated by phosphorylation. PI3K-sensitive kinases include the serum- and glucocorticoid-inducible kinase 1 (SGK1). The present study explored whether SGK1 modifies WASP and vinculin phosphorylation in murine platelets and participates in the regulation of platelet migration. Platelets were isolated from gene-targeted mice lacking SGK1 (sgk1-/-) and from their wild type littermates (sgk1+/+). Platelet migration stimulated with SDF-1 was significantly less pronounced in sgk1-/-platelets than in sgk1+/+ platelets. Moreover, SDF-1 significantly induced WASP phosphorylation, an effect again reduced in platelets lacking SGK1. Phosphorylation of vinculin was significantly enhanced in sgk1-/-platelets and was significantly reduced following treatment of platelets with Ca2+ chelator BAPTA. Immunohistochemical analysis of in vivo experiments in intestinal vessels after vascular inflammation revealed that transmigration of platelets into inflamed vessel walls was significantly less pronounced in sgk1-/-than in sgk1+/+ mice. In conclusion, SGK1 is a powerful regulator of platelet migration.