Tanja Schönberger

Adherent Platelets Recruit and Induce Differentiation of Murine Embryonic Endothelial Progenitor Cells to Mature Endothelial Cells In Vitro

The homing and differentiation mechanisms of endothelial progenitor cells (EPCs) at sites of vascular lesions are unclear. To investigate whether platelets play a role in the recruitment and differentiation of EPCs, we made use of a robust mouse embryonic EPC (eEPC) line that reliably differentiates to a mature endothelial phenotype. We found that platelets stimulate chemotaxis and migration of these murine eEPCs. Further, the substantial adhesion of murine eEPCs on immobilized platelets that occurs under dynamic flow conditions is inhibited by neutralizing anti–P-selectin glycoprotein ligand-1 and anti–VLA-4 (β1-integrin) monoclonal antibodies but not by anti-CD11b (aM-integrin; macrophage antigen-1). Coincubation of murine eEPCs with platelets for 5 days induced differentiation of EPCs to mature endothelial cells as verified by positive von Willebrand factor immunofluorescence and detection of Weibel Palade bodies through electron microscopy. We conclude that platelets may play a critical part in the capture and subsequent differentiation of murine eEPCs at sites of vascular lesions, revealing a possible new role of platelets in neoendothelization after vascular injury.

Platelets induce differentiation of human CD34+ progenitor cells into foam cells and endothelial cells

Recruitment of human CD34+ progenitor cells toward vascular lesions and differentiation into vascular cells has been regarded as a critical initial step in atherosclerosis. Previously we found that adherent platelets represent potential mediators of progenitor cell homing besides their role in thrombus formation. On the other hand, foam cell formation represents a key process in atherosclerotic plaque formation. To investigate whether platelets are involved in progenitor cell recruitment and differentiation into endothelial cells and foam cells, we examined the interactions of platelets and CD34+ progenitor cells. Cocultivation experiments showed that human platelets recruit CD34+ progenitor cells via the specific adhesion receptors P‐selectin/PSGL‐1 and β1‐ and β2‐integrins. Furthermore, platelets were found to induce differentiation of CD34+ progenitor cells into mature foam cells and endothelial cells. Platelet‐induced foam cell generation could be prevented partially by HMG coenzyme A reductase inhibitors via reduction of matrix metallo‐proteinase‐9 (MMP‐9) secretion. Finally, agonists of peroxisome proliferator‐activated receptor‐α and ‐γ attenuated platelet‐induced foam cell generation and production of MMP‐9. The present study describes a potentially important mechanism of platelet‐induced foam cell formation and generation of endothelium in atherogenesis and atheroprogression. The understanding and modulation of these mechanisms may offer new treatment strategies for patients at high risk for atherosclerotic diseases.—Daub, K., Langer, H., Seizer, P., Stellos, K., May, A. E., Goyal, P., Bigalke, B., Schönberger, T., Geisler, T., Siegel‐Axel, D., Oostendorp, R. A. J., Lindemann, S., Gawaz, M. Platelets induce differentiation of human CD34+ progenitor cells into foam cells and endothelial cells. FASEB J. 20, E1935–E1944 (2006)

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.

Platelets Recruit Human Dendritic Cells Via Mac-1/JAM-C Interaction and Modulate Dendritic Cell Function In Vitro

Objective—Thrombotic events and immunoinflammatory processes take place next to each other during vascular remodeling in atherosclerotic lesions. In this study we investigated the interaction of platelets with dendritic cells (DCs). Methods and Results—The rolling of DCs on platelets was mediated by PSGL-1. Firm adhesion of DCs was mediated through integrin &agr;M&bgr;2 (Mac-1). In vivo, adhesion of DCs to injured carotid arteries in mice was mediated by platelets. Pretreatment with soluble GPVI, which inhibits platelet adhesion to collagen, substantially reduced recruitment of DCs to the injured vessel wall. In addition, preincubation of DCs with sJAM-C significantly reduced their adhesion to platelets. Coincubation of DCs with platelets induced maturation of DCs, as shown by enhanced expression of CD83. In the presence of platelets, DC-induced lymphocyte proliferation was significantly enhanced. Moreover, coincubation of DCs with platelets resulted in platelet phagocytosis by DCs, as verified by different cell phagocytosis assays. Finally, platelet/DC interaction resulted in apoptosis of DCs mediated by a JAM-C–dependent mechanism. Conclusions—Recruitment of DCs by platelets, which is mediated via CD11b/CD18 (Mac-1) and platelet JAM-C, leads to DC activation and platelet phagocytosis. This process may be of importance for progression of atherosclerotic lesions.

EMMPRIN and its ligand cyclophilin A regulate MT1-MMP, MMP-9 and M-CSF during foam cell formation

UNLABELLED Upon coincubation with platelets, CD34(+) progenitor cells have the potential to differentiate into foam cells, and thereby may promote the progression of atherosclerosis. The exact mechanism of MMP-regulation during the cellular differentiation process to foam cells is still unclear. Thus, we investigated the role of EMMPRIN (CD147) and its ligand cyclophilin A (CyPA) during foam cell formation originating from both monocytes/macrophages and CD34(+) progenitor cells. METHODS AND RESULTS Differentiation of CD34(+) progenitor to foam cells was analyzed in a coculture model of progenitor cells and platelets. While CD34(+) cells did not express EMMPRIN or MT1-MMP, mature foam cells strongly expressed EMMPRIN, which was associated with MT1-MMP expression as well as MMP-9. Gene silencing of EMMPRIN by siRNA during the cell differentiation process hindered not only the upregulation of MMPs (MT1-MMP, MMP-9), but also the secretion of the cytokine M-CSF. During the differentiation process CyPA was substantially released into the supernatant. The presence of the CyPA inhibitor NIM811 significantly reduced MMP-9 secretion during the differentiation process. Similar results were obtained using the classical pathway of foam cell formation by coincubating human macrophages with AcLDL. Additionally, the presence of soluble EMMPRIN ligands (CyPA, recombinant EMMPRIN) further enhanced MMP-9 secretion by mature foam cells. Consistently, CyPA and EMMPRIN were found in atherosclerotic plaques of ApoE-deficient mice by immunohistochemistry. CONCLUSION EMMPRIN is upregulated during the differentiation process from CD34(+) progenitor cells to foam cells, whereas its ligand, CyPA, is released. The CyPA/EMMPRIN activation pathway may play a relevant role in promoting the vulnerability of atherosclerotic plaques.

Disrupting the EMMPRIN (CD147)–Cyclophilin A Interaction Reduces Infarct Size and Preserves Systolic Function After Myocardial Ischemia and Reperfusion

Objective—Inflammation and proteolysis crucially contribute to myocardial ischemia and reperfusion injury. The extracellular matrix metalloproteinase inducer EMMPRIN (CD147) and its ligand cyclophilin A (CyPA) may be involved in both processes. The aim of the study was to characterize the role of the CD147 and CyPA interplay in myocardial ischemia/reperfusion (I/R) injury. Methods and Results—Immunohistochemistry showed enhanced expression of CD147 and CyPA in myocardial sections from human autopsies of patients who had died from acute myocardial infarction and from mice at 24 hours after I/R. At 24 hours and 7 days after I/R, the infarct size was reduced in CD147+/− mice vs CD147+/+ mice (C57Bl/6), in mice (C57Bl/6) treated with monoclonal antibody anti-CD147 vs control monoclonal antibody, and in CyPA−/− mice vs CyPA+/+ mice (129S6/SvEv), all of which are associated with reduced monocyte and neutrophil recruitment at 24 hours and with a preserved systolic function at 7 days. The combination of CyPA−/− mice with anti-CD147 treatment did not yield further protection compared with either inhibition strategy alone. In vitro, treatment with CyPA induced monocyte chemotaxis in a CD147- and phosphatidylinositol 3-kinase–dependent manner and induced monocyte rolling and adhesion to endothelium (human umbilical vein endothelial cells) under flow in a CD147-dependent manner. Conclusion—CD147 and its ligand CyPA are inflammatory mediators after myocardial ischemia and reperfusion and represent potential targets to prevent myocardial I/R injury.

Impact of glycoprotein VI and platelet adhesion on atherosclerosis—A possible role of fibronectin

Glycoprotein VI (GPVI) mediates binding of platelets to subendothelial collagen during acute arterial thrombosis. GPVI interactions with the activated atherosclerotic vascular endothelium during early atherosclerosis, however, are not well understood. In ApoE-/- mice, platelet adhesion to atherosclerotic arteries was increased, as measured by intravital microscopy. This platelet adhesion was significantly inhibited by IV injection of GPVI-Fc (1 mg/kg body weight). Atherosclerosis in ApoE-/- mice was attenuated both after 7 and 10 weeks of treatment with the anti-GPVI antibody JAQ1 (2 mg/kg body weight i.p. twice weekly). Binding of GPVI-Fc (1 mg/kg IV) occurred to deeper layers, but also to the luminal site of plaques in atherosclerotic rabbits, but not to the vessel wall of healthy littermates. Gene transfer of GPVI-Fc to the carotid vascular wall significantly attenuated athero-progression and endothelial dysfunction in atherosclerotic rabbits in vivo. Specific binding of the soluble GPVI receptor (GPVI-Fc) to fibronectin was found in vitro to coated ELISA plates. Platelet adhesion to fibronectin was significantly inhibited both by GPVI-Fc and by the anti-GPVI antibody 5C4 ex vivo in flow chamber experiments. GPVI plays a role in platelet adhesion to atherosclerotic endothelium in the absence of plaque rupture. Inhibition of GPVI both via GPVI-Fc and anti-GPVI-antibodies results in protection against atherosclerosis in both cholesterol-fed rabbits and ApoE-/- mice. This novel mechanism of GPVI-mediated platelet adhesion-possibly via fibronectin-could relevantly contribute to platelet-triggered atheroprogression.

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.

Engagement of αIIbβ3 (GPIIb/IIIa) with ανβ3 Integrin Mediates Interaction of Melanoma Cells with Platelets A CONNECTION TO HEMATOGENOUS METASTASIS

A mutual relationship exists between metastasizing tumor cells and components of the coagulation cascade. The exact mechanisms as to how platelets influence blood-borne metastasis, however, remain poorly understood. Here, we used murine B16 melanoma cells to observe functional aspects of how platelets contribute to the process of hematogenous metastasis. We found that platelets interfere with a distinct step of the metastasis cascade, as they promote adhesion of melanoma cells to the endothelium in vitro under shear conditions. Constitutively active platelet receptor GPIIb/IIIa (integrin αIIbβ3) expressed on Chinese hamster ovary cells promoted melanoma cell adhesion in the presence of fibrinogen, whereas blocking antibodies to aνβ3 integrin on melanoma cells or to GPIIb/IIIa significantly reduced melanoma cell adhesion to platelets. Furthermore, using intravital microscopy, we observed functional platelet-melanoma cell interactions, as platelet depletion resulted in significantly reduced melanoma cell adhesion to the injured vascular wall in vivo. Using a mouse model of hematogenous metastasis to the lung, we observed decreased metastasis of B16 melanoma cells to the lung by treatment with a mAb blocking the aν subunit of aνβ3 integrin. This effect was significantly reduced when platelets were depleted in vivo. Thus, the engagement of GPIIb/IIIa with aνβ3 integrin interaction mediates tumor cell-platelet interactions and highlights how this interaction is involved in hematogenous tumor metastasis.