Sebastian Vogel

Platelets in tissue repair: control of apoptosis and interactions with regenerative cells

Besides mediating primary hemostasis and thrombosis, platelets play a critical role in tissue repair and regeneration. They regulate fundamental mechanisms involved in the healing process including cellular migration, proliferation, and angiogenesis. Control of apoptosis/cell survival and interaction with progenitor cells, which are clinically relevant but poorly understood aspects of platelets in tissue repair, will be highlighted in this review. Gaining deeper insight into the less well-characterized molecular mechanisms is necessary to develop new therapeutic platelet-based options.

Platelet-derived HMGB1 is a critical mediator of thrombosis

Thrombosis and inflammation are intricately linked in several major clinical disorders, including disseminated intravascular coagulation and acute ischemic events. The damage-associated molecular pattern molecule high-mobility group box 1 (HMGB1) is upregulated by activated platelets in multiple inflammatory diseases; however, the contribution of platelet-derived HMGB1 in thrombosis remains unexplored. Here, we generated transgenic mice with platelet-specific ablation of HMGB1 and determined that platelet-derived HMGB1 is a critical mediator of thrombosis. Mice lacking HMGB1 in platelets exhibited increased bleeding times as well as reduced thrombus formation, platelet aggregation, inflammation, and organ damage during experimental trauma/hemorrhagic shock. Platelets were the major source of HMGB1 within thrombi. In trauma patients, HMGB1 expression on the surface of circulating platelets was markedly upregulated. Moreover, evaluation of isolated platelets revealed that HMGB1 is critical for regulating platelet activation, granule secretion, adhesion, and spreading. These effects were mediated via TLR4- and MyD88-dependent recruitment of platelet guanylyl cyclase (GC) toward the plasma membrane, followed by MyD88/GC complex formation and activation of the cGMP-dependent protein kinase I (cGKI). Thus, we establish platelet-derived HMGB1 as an important mediator of thrombosis and identify a HMGB1-driven link between MyD88 and GC/cGKI in platelets. Additionally, these findings suggest a potential therapeutic target for patients sustaining trauma and other inflammatory disorders associated with abnormal coagulation.

Macrophage Migration Inhibitory Factor Limits Activation-Induced Apoptosis of Platelets via CXCR7-Dependent Akt Signaling

Rationale: Macrophage migration inhibitory factor (MIF) is released on platelet activation. Circulating MIF could potentially regulate platelets and thereby platelet-mediated inflammatory and regenerative mechanisms. However, the effect of MIF on platelets is unknown. Objective: The present study evaluated MIF in regulating platelet survival and thrombotic potential. Methods and Results: MIF interacted with CXCR4-CXCR7 on platelets, defining CXCR7 as a hitherto unrecognized receptor for MIF on platelets. MIF internalized CXCR4, but unlike CXCL12 (SDF-1&agr;), it did not phosphorylate Erk1/2 after CXCR4 ligation because of the lack of CD74 and failed in subsequent CXCR7 externalization. MIF did not alter the activation status of platelets. However, MIF rescued platelets from activation and BH3 mimetic ABT-737–induced apoptosis in vitro via CXCR7 and enhanced circulating platelet survival when administered in vivo. The antiapoptotic effect of MIF was absent in Cxcr7−/− murine embryonic cells but pronounced in CXCR7-transfected Madin–Darby canine kidney cells. This prosurvival effect was attributed to the MIF–CXCR7–initiated PI3K-Akt pathway. MIF induced CXCR7-Akt–dependent phosphorylation of BCL-2 antagonist of cell death (BAD) both in vitro and in vivo. Consequentially, MIF failed to rescue Akt−/− platelets from thrombin-induced apoptosis when challenged ex vivo, also in prolonging platelet survival and in inducing BAD phosphorylation among Akt−/− mice in vivo. MIF reduced thrombus formation under arterial flow conditions in vitro and retarded thrombotic occlusion after FeCl3-induced arterial injury in vivo, an effect mediated through CXCR7. Conclusion: MIF interaction with CXCR7 modulates platelet survival and thrombotic potential both in vitro and in vivo and thus could regulate thrombosis and inflammation.

SDF-1α induces differential trafficking of CXCR4-CXCR7 involving cyclophilin A, CXCR7 ubiquitination and promotes platelet survival

Platelet‐derived SDF‐1α (CXCL12) mediates inflammatory and regenerative mechanisms. The present study characterizes the effect of SDF‐1α ligation in platelets. SDF‐1α (0–100 μM) dose and time dependently caused internalization of its receptor CXCR4 (28.9±1.6 vs. 16.1±1.9 in SDF‐1α‐treated platelets), coupled to the surface externalization of CXCR7 (65.5±8 vs. 162.8±27.6 following SDF‐1α treatment), both in vitro and in vivo. This was inhibited in the presence of AMD3100 (100 μM), CXCR4 blocking and vesicular transport inhibitors (brefeldin A, 10 μM; rapamycin, 100 nM). SDF‐1α/CXCR‐4‐mediated CXCR7 translocation was significantly reduced by inhibitors of ERK1/2‐(U0126‐10 μM) and cyclophilinA (CyPA)‐(NIM811‐10 μM) by 28 and 46%, respectively. Further, SDF‐1α‐induced downstream phosphorylation of Erk1/2 led to CyPA‐dependent ubiquitination of CXCR7, which is essential for its surface translocation. CyPA‐PPIase‐activity inhibitor NIM‐811, Erk1/2, and E1‐ligase inhibitor‐(PYR‐41‐25 μM) significantly abolished SDF‐1α‐driven CXCR7 ubiquitination and subsequent surface translocation. SDF‐1α induced CXCR7 ubiquitination, and its surface exposure was observed in wild‐type murine platelets, but not in CyPA‐deficient platelets. SDF‐1α/CXCR4‐CyPA‐dependent CXCR7 translocation and its subsequent ligation attenuated activation‐induced apoptosis both in vitro and when administered in vivo. This antiapoptotic effect of SDF‐1α was abrogated by blocking CXCR7, also significantly affected in Cypa–/– platelets. Thus, we decipher a novel mechanism, whereby SDF‐1α regulates relative receptor availability in circulating platelets and exerts its prosurvival benefits.—Chatterjee, M., Seizer, P., Borst, O., Schönberger, T., Mack, A., Geisler, T., Langer, H. F., May, A. E., Vogel, S., Lang, F., Gawaz, M. SDF‐1α induces differential trafficking of CXCR4‐CXCR7 involving cyclophilin A, CXCR7 ubiquitination and promotes platelet survival. FASEB J. 28, 2864–2878 (2014). www.fasebj.org

Pivotal Role of Serum- and Glucocorticoid-Inducible Kinase 1 in Vascular Inflammation and Atherogenesis

Objective— Atherosclerosis, an inflammatory disease of arterial vessel walls, requires migration and matrix metalloproteinase (MMP)-9–dependent invasion of monocytes/macrophages into the vascular wall. MMP-9 expression is stimulated by transcription factor nuclear factor-&kgr;B, which is regulated by inhibitor &kgr;B (I&kgr;B) and thus I&kgr;B kinase. Regulators of nuclear factor-&kgr;B include serum- and glucocorticoid-inducible kinase 1 (SGK1). The present study explored involvement of SGK1 in vascular inflammation and atherogenesis. Approach and Results— Gene-targeted apolipoprotein E (ApoE)–deficient mice without (apoe −/− sgk1 +/+) or with (apoe −/− sgk1 −/−) additional SGK1 knockout received 16-week cholesterol-rich diet. According to immunohistochemistry atherosclerotic lesions in aorta and carotid artery, vascular CD45+ leukocyte infiltration, Mac-3+ macrophage infiltration, vascular smooth muscle cell content, MMP-2, and MMP-9 positive areas in atherosclerotic tissue were significantly less in apoe −/− sgk1 −/−mice than in apoe −/− sgk1 +/+mice. As determined by Boyden chamber, thioglycollate-induced peritonitis and air pouch model, migration of SGK1-deficient CD11b+F4/80+ macrophages was significantly diminished in vitro and in vivo. Zymographic MMP-2 and MMP-9 production, MMP-9 activity and invasion through matrigel in vitro were significantly less in sgk1 −/− than in sgk1 +/+macrophages and in control plasmid–transfected or inactive K127NSGK1-transfected than in constitutively active S422DSGK1-transfected THP-1 cells. Confocal microscopy revealed reduced macrophage number and macrophage MMP-9 content in plaques of apoe −/− sgk1 −/− mice. In THP-1 cells, MMP-inhibitor GM6001 (25 &mgr;mol/L) abrogated S422DSGK1-induced MMP-9 production and invasion. According to reverse transcription polymerase chain reaction, MMP-9 transcript levels were significantly reduced in sgk1 −/−macrophages and strongly upregulated in S422DSGK1-transfected THP-1 cells compared with control plasmid–transfected or K127NSGK1-transfected THP-1 cells. According to immunoblotting and confocal microscopy, phosphorylation of I&kgr;B kinase and inhibitor &kgr;B and nuclear translocation of p50 were significantly lower in sgk1 −/−macrophages than in sgk1 +/+macrophages and significantly higher in S422DSGK1-transfected THP-1 cells than in control plasmid–transfected or K127NSGK1-transfected THP-1 cells. Treatment of S422DSGK1-transfected THP-1 cells with I&kgr;B kinase-inhibitor BMS-345541 (10 &mgr;mol/L) abolished S422DSGK1-induced increase of MMP-9 transcription and gelatinase activity. Conclusions— SGK1 plays a pivotal role in vascular inflammation during atherogenesis. SGK1 participates in the regulation of monocyte/macrophage migration and MMP-9 transcription via regulation of nuclear factor-&kgr;B.

Activated Platelets Interfere with Recruitment of Mesenchymal Stem Cells to Apoptotic Cardiac Cells via High Mobility Group Box 1/Toll-like Receptor 4-mediated Down-regulation of Hepatocyte Growth Factor Receptor MET

Background: Mesenchymal stem cells (MSC) contribute to cardiac repair after myocardial injury. Underlying molecular mechanisms remain unexplored. Results: Activated platelets inhibit recruitment of MSC to apoptotic cardiac myocytes and fibroblasts via HMGB1/TLR-4-mediated down-regulation of HGF receptor MET. Conclusion: We identify a novel mechanism by which platelets impair MSC migration to damaged cardiac cells. Significance: The cross-talk between platelets and MSC might be critical for myocardial repair. Recruitment of mesenchymal stem cells (MSC) following cardiac injury, such as myocardial infarction, plays a critical role in tissue repair and may contribute to myocardial recovery. However, the mechanisms that regulate migration of MSC to the site of tissue damage remain elusive. Here, we demonstrate in vitro that activated platelets substantially inhibit recruitment of MSC toward apoptotic cardiac myocytes and fibroblasts. The alarmin high mobility group box 1 (HMGB1) was released by platelets upon activation and mediated inhibition of the cell death-dependent migratory response through Toll-like receptor (TLR)-4 expressed on the MSC. Migration of MSC to apoptotic cardiac myocytes and fibroblasts was driven by hepatocyte growth factor (HGF), and platelet activation was followed by HMGB1/TLR-4-dependent down-regulation of HGF receptor MET on MSC, thereby impairing HGF-driven MSC recruitment. We identify a novel mechanism by which platelets, upon activation, interfere with MSC recruitment to apoptotic cardiac cells, a process that may be of particular relevance for myocardial repair and regeneration.

Dynamic adhesion of eryptotic erythrocytes to immobilized platelets via platelet phosphatidylserine receptors

Glucose depletion of erythrocytes triggers suicidal erythrocyte death or eryptosis, which leads to cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptotic erythrocytes adhere to endothelial cells by a mechanism involving phosphatidylserine at the erythrocyte surface and CXCL16 as well as CD36 at the endothelial cell membrane. Nothing has hitherto been known about an interaction between eryptotic erythrocytes and platelets, the decisive cells in primary hemostasis and major players in thrombotic vascular occlusion. The present study thus explored whether and how glucose-depleted erythrocytes adhere to platelets. To this end, adhesion of phosphatidylserine-exposing erythrocytes to platelets under flow conditions was examined in a flow chamber model at arterial shear rates. Platelets were immobilized on collagen and further stimulated with adenosine diphosphate (ADP, 10 μM) or thrombin (0.1 U/ml). As a result, a 48-h glucose depletion triggered phosphatidylserine translocation to the erythrocyte surface and augmented the adhesion of erythrocytes to immobilized platelets, an effect significantly increased upon platelet stimulation. Adherence of erythrocytes to platelets was blunted by coating of erythrocytic phosphatidylserine with annexin V or by neutralization of platelet phosphatidylserine receptors CXCL16 and CD36 with respective antibodies. In conclusion, glucose-depleted erythrocytes adhere to platelets. The adhesive properties of platelets are augmented by platelet activation. Erythrocyte adhesion to immobilized platelets requires phosphatidylserine at the erythrocyte surface and CXCL16 as well as CD36 expression on platelets. Thus platelet-mediated erythrocyte adhesion may foster thromboocclusive complications in diseases with stimulated phosphatidylserine exposure of erythrocytes.

Preserved bioactivity and tunable release of a SDF1-GPVI bi-specific protein using photo-crosslinked PEGda hydrogels

Chemokine-induced stem cell recruitment is a promising strategy for post myocardial infarction treatment. Injection of stromal cell-derived factor 1 (SDF1) has been shown to attract bone marrow-derived progenitor cells (BMPCs) from the blood that have the potential to differentiate into cardiovascular cells, which support angiogenesis, enabling the improvement of myocardial function. SDF1-GPVI bi-specific protein contains a glycoprotein VI (GPVI)-domain that serves as an anchor for collagen type I (Col I) and III, which are exposed in the wall of injured vasculature. In this study, we generated a cytocompatible hydrogel via photo-crosslinking of poly(ethylene glycol) diacrylate that serves as a reservoir for SDF1-GPVI. Controlled and sustained release of SDF1-GPVI was demonstrated over a period of 7 days. Release features were modifiable depending on the degree of the crosslinking density. Functionality of the GPVI-domain was investigated using a GPVI-binding ELISA to Col I. Activity of the SDF1-domain was tested for its CXCR4 binding potential. Preserved functionality of SDF1-GPVI bi-specific protein after photo-crosslinking and controllable release was successfully demonstrated in vitro supporting the implementation of this drug delivery system as a powerful tool for therapeutic protein delivery in the treatment of cardiovascular ischemic disease.

Gremlin-1 inhibits macrophage migration inhibitory factor-dependent monocyte function and survival

BACKGROUND Monocyte migration and their differentiation into macrophages critically regulate vascular inflammation and atherogenesis and are governed by macrophage migration inhibitory factor (MIF). Gremlin-1 binds to MIF. Current experimental evidences present Gremlin-1 as a potential physiological agent that might counter-regulate the inflammatory attributes of MIF. METHODS AND RESULTS We found that Gremlin-1 inhibited MIF-dependent monocyte migration and adhesion to activated endothelial cells in flow chamber perfusion assay in vitro and to the injured carotid artery of WT and ApoE-/- mice in vivo as deciphered by intravital microscopy. Intravenous administration of Gremlin-1, but not of control protein, significantly reduced leukocyte recruitment towards the inflamed carotid artery of ApoE-/- mice. Besides, leukocytes from MIF-/- when administered into ApoE-/- mice showed lesser adhesion as compared to wild type. In the presence of Gremlin-1 however, adhesion of wild type, but not of MIF-/- leukocytes, to the carotid artery was significantly inhibited as compared to control. Gremlin-1 also inhibited the MIF-induced differentiation of monocytes into macrophages. Gremlin-1 substantially inhibited the anti-apoptotic impact of MIF on monocytes against BH3 mimetic ABT-737-induced apoptosis as verified by Annexin V-binding, caspase 3 activity, and mitochondrial depolarization. CONCLUSIONS Therefore Gremlin-1 can modulate MIF dependent monocyte adhesion, migration, differentiation and survival.

Impact of counterbalance between macrophage migration inhibitory factor and its inhibitor Gremlin-1 in patients with coronary artery disease.

OBJECTIVE Monocyte infiltration is a critical step in the pathophysiology of plaque instability in coronary artery disease (CAD). Macrophage migration inhibitory factor (MIF) is involved in atherosclerotic plaque progression and instability leading to intracoronary thrombosis. Gremlin-1 (Grem1) has been recently identified as endogenous inhibitor of MIF. To date there are no data on the clinical impact of this interaction in cardiovascular patients. METHODS AND RESULTS Plasma levels of MIF and Grem1 were determined by enzyme-linked immunoassay in patients with acute coronary syndromes (ACS, n = 120; stable CAD, n = 166 and healthy control subjects, n = 25). MIF levels were significantly increased in ACS compared to stable CAD and healthy control (ACS: median 2.85; IQR 3.52 ng/ml; versus SAP: median 1.22; IQR 2.99 ng/ml; versus healthy control: median 0.10; IQR 0.09 ng/ml, p < 0.001). Grem1 levels were significantly higher in ACS and stable CAD patients compared to healthy control (ACS: median 211.00; IQR 130.47 ng/ml; SAP: median 220.20; IQR 120.93 ng/ml, versus healthy control: median 90.57; IQR 97.68 ng/ml, p < 0.001). Grem1/MIF ratio was independently associated with ACS, whereas the single parameters were not associated with the presence of ACS. Furthermore, Grem1/MIF ratio was associated with angiographic signs of intracoronary thrombi and severity of thrombus burden. CONCLUSION These novel findings suggest a potential role of Grem1/MIF ratio to indicate acuity of CAD and the grade of plaque stability. Prospective angiographic cohort studies involving plaque imaging techniques are warranted to further characterize the prognostic role of this novel risk marker in CAD patients.