Crosstalk of platelets with macrophages and fibroblasts aggravates inflammation, aortic wall stiffening, and osteopontin release in abdominal aortic aneurysm.

AIMS: Abdominal aortic aneurysm (AAA) is a highly lethal disease with progressive dilatation of the abdominal aorta accompanied by degradation and remodelling of the vessel wall due to chronic inflammation. Platelets play an important role in cardiovascular diseases, but their role in AAA is poorly understood. METHODS AND RESULTS: The present study revealed that platelets play a crucial role in promoting AAA through modulation of inflammation and degradation of the extracellular matrix (ECM). They are responsible for the up-regulation of SPP1 (osteopontin, OPN) gene expression in macrophages and aortic tissue, which triggers inflammation and remodelling and also platelet adhesion and migration into the abdominal aortic wall and the intraluminal thrombus (ILT). Further, enhanced platelet activation and pro-coagulant activity result in elevated gene expression of various cytokines, Mmp9 and Col1a1 in macrophages and Il-6 and Mmp9 in fibroblasts. Enhanced platelet activation and pro-coagulant activity were also detected in AAA patients. Further, we detected platelets and OPN in the vessel wall and in the ILT of patients who underwent open repair of AAA. Platelet depletion in experimental murine AAA reduced inflammation and ECM remodelling, with reduced elastin fragmentation and aortic diameter expansion. Of note, OPN co-localized with platelets, suggesting a potential role of OPN for the recruitment of platelets into the ILT and the aortic wall. CONCLUSION: In conclusion, our data strongly support the potential relevance of anti-platelet therapy to reduce AAA progression and rupture in AAA patients.

Homophilic Interaction Between Transmembrane-JAM-A and Soluble JAM-A Regulates Thrombo-Inflammation: Implications for Coronary Artery Disease.

Genetic predisposition through F11R-single-nucleotide variation (SNV) influences circulatory soluble junctional adhesion molecule-A (sJAM-A) levels in coronary artery disease (CAD) patients. Homozygous carriers of the minor alleles (F11R-SNVs rs2774276, rs790056) show enhanced levels of thrombo-inflammatory sJAM-A. Both F11R-SNVs and sJAM-A are associated with worse prognosis for recurrent myocardial infarction in CAD patients. Platelet surface-associated JAM-A correlate with platelet activation markers in CAD patients. Activated platelets shed transmembrane-JAM-A, generating proinflammatory sJAM-A and JAM-A-bearing microparticles. Platelet transmembrane-JAM-A and sJAM-A as homophilic interaction partners exaggerate thrombotic and thrombo-inflammatory platelet monocyte interactions. Therapeutic strategies interfering with this homophilic interface may regulate thrombotic and thrombo-inflammatory platelet response in cardiovascular pathologies where circulatory sJAM-A levels are elevated.

Platelet ACKR3/CXCR7 favors antiplatelet lipids over an atherothrombotic lipidome and regulates thromboinflammation.

Platelet ACKR3/CXCR7 surface expression is enhanced and influences prognosis in coronary artery disease (CAD) patients, who exhibit a distinct atherothrombotic platelet lipidome. Current investigation validates the potential of ACKR3/CXCR7 in regulating thromboinflammatory response through its impact on the platelet lipidome. CAD patients with enhanced platelet ACKR3/CXCR7 expression exhibited reduced aggregation. Pharmacological CXCR7 agonist (VUF11207) significantly reduced prothrombotic platelet response in blood from acute coronary syndrome patients ex vivo. CXCR7 agonist administration reduced thrombotic functions and thromboinflammatory plateletleukocyte interactions post-myocardial infarction and arterial injury in vivo. ACKR3/CXCR7 ligation did not affect surface availability of surface receptors, coagulation profile, bleeding time, plasma-dependent thrombin generation (thrombinoscopy), or clot formation (thromboelastography) but counteracted activation-induced phosphatidylserine exposure and procoagulant platelet-assisted thrombin generation. Targeted (micro-UHPLC-ESI-QTrap-MS/MS) and untargeted (UHPLCESI-QTOF-MS/MS) lipidomics analysis revealed that ACKR3/CXCR7 ligation favored generation of antithrombotic lipids (dihomo-γ-linolenic acid [DGLA], 12-hydroxyeicosatrienoic acid [12-HETrE]) over cyclooxygenase-1 (COX-1) or 12-lipoxygenase (12-LOX) metabolized prothrombotic and phospholipase-derived atherogenic lipids in healthy subjects and CAD patients, contrary to antiplatelet therapy. Through 12-HETrE, ACKR3/CXCR7 ligation coordinated with Gαs-coupled prostacyclin receptor to trigger cyclic adenosine monophosphate/protein kinase A-mediated platelet inhibition. ACKR3/CXCR7 ligation reduced generation of lipid agonists and lipid signaling intermediates, which affected calcium mobilization, intracellular signaling, and consequently platelet interaction with physiological matrices and thromboinflammatory secretome. This emphasized its functional dichotomy from prothrombotic CXCR4. Moreover, CXCR7 agonist regulated heparin-induced thrombocytopenia-sera/immunoglobulin G-triggered platelet and neutrophil activation, heparin-induced platelet aggregation, generation of thromboinflammatory lipids, platelet-neutrophil aggregate formation, and thromboinflammatory secretion ex vivo. Therefore, ACKR3/CXCR7 may offer a novel therapeutic strategy in acute/chronic thromboinflammation exaggerated cardiovascular pathologies and CAD.

Evaluation of paraneoplastic antigens reveals TRIM21 autoantibodies as biomarker for early detection of ovarian cancer in combination with autoantibodies to NY-ESO-1 and TP53.

BACKGROUND: The majority of ovarian cancer cases are diagnosed at an advanced stage with poor prognosis. This study evaluates autoantibodies against tumor antigens to identify candidate biomarkers for early detection of ovarian cancer in women at increased risk. OBJECTIVE: To assess the immunoreactivity of paraneoplastic antigens and tumor associated antigens with high-grade serous ovarian cancer (HGSOC) samples. METHODS: Five paraneoplastic antigens along with three tumor-associated antigens were evaluated with HGSOC patient serum samples. Validation screening was performed with n= 164 serum samples consisting of: 50 late stage HGSOC, 14 early stage HGSOC, 50 benign ovarian cyst, and 50 healthy control samples on ELISA and western blot. The four markers TRIM21, NY-ESO-1, TP53, and PAX8 were evaluated on a second validation serum set, n= 150. RESULTS: TRIM21 achieved the highest sensitivity in the first validation screening of 33% with 100% specificity. Combining TRIM21 with NY-ESO-1, TP53, and PAX8 provided 67% sensitivity with 94% specificity, and 56% sensitivity at 98% specificity. These four markers resulted in 46% sensitivity with 98% specificity in the second validation cohort; TRIM21 achieved the highest individual sensitivity of 36%. CONCLUSIONS: Autoantibodies to TRIM21, NY-ESO-1, and TP53 may complement CA125 in screening of women at genetic risk for ovarian cancer.

Targeting PP2A inhibits the growth of triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) does not respond to widely used targeted/endocrine therapies because of the absence of progesterone and estrogen receptors and HER2 amplification. It has been shown that the majority of TNBC cells are highly sensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, but the development of TRAIL resistance limits its efficacy. We previously found that protein phosphatase 2A (PP2A) plays an important role in TRAIL resistance. In this study, we evaluated the effects of PP2A inhibition on cell death in TRAIL-resistant TNBC cells. We found that the PP2A inhibitor LB-100 effectively inhibits the growth of a panel of TNBC cell lines including lines that are intrinsically resistant to TRAIL. Using two TRAIL-resistant cell lines generated from TRAIL-sensitive parental cells (MDA231 and SUM159), we found that both TRAIL-sensitive and -resistant cell lines are equally sensitive to LB-100. We also found that LB-100 sensitizes TNBC cells to clinically used chemotherapeutical agents, including paclitaxel and cisplatin. Importantly, we found that LB-100 effectively inhibits the growth of MDA468 tumors in mice in vivo without apparent toxicity. Collectively, these data suggest that pharmacological inhibition of PP2A activity could be a novel therapeutic strategy for treating patients with TNBC in a clinical setting.

GP78 Cooperates with Dual-Specificity Phosphatase 1 To Stimulate Epidermal Growth Factor Receptor-Mediated Extracellular Signal-Regulated Kinase Signaling.

GP78 is an autocrine motility factor (AMF) receptor (AMFR) with E3 ubiquitin ligase activity that plays a significant role in tumor cell proliferation, motility, and metastasis. Aberrant extracellular signal-regulated kinase (ERK) activation via receptor tyrosine kinases promotes tumor proliferation and invasion. The activation of GP78 leads to ERK activation, but its underlying mechanism is not fully understood. Here, we show that GP78 is required for epidermal growth factor receptor (EGFR)-mediated ERK activation. On one hand, GP78 interacts with and promotes the ubiquitination and subsequent degradation of dual-specificity phosphatase 1 (DUSP1), an endogenous negative regulator of mitogen-activated protein kinases (MAPKs), resulting in ERK activation. On the other hand, GP78 maintains the activation status of EGFR, as evidenced by the fact that EGF fails to induce EGFR phosphorylation in GP78-deficient cells. By the regulation of both EGFR and ERK activation, GP78 promotes cell proliferation, motility, and invasion. Therefore, this study identifies a previously unknown signaling pathway by which GP78 stimulates ERK activation via DUSP1 degradation to mediate EGFR-dependent cancer cell proliferation and invasion.

Utility of paraneoplastic antigens as biomarkers for surveillance and prediction of recurrence in ovarian cancer.

BACKGROUND: Ovarian cancer is frequently diagnosed at an advanced stage and 70% of patients experience recurrence months to years from initial diagnosis. The expression of paraneoplastic antigens can result in the occurrence of onconeural autoantibodies in ovarian cancer that may be associated with neurological disorders that are clinically manifested in patients before diagnosis of ovarian cancer. These paraneoplastic antigens can serve as excellent biomarkers not only for early detection but also for monitoring ovarian cancer recurrence. OBJECTIVE: To assess the immunoreactivity of our previous 3 biomarkers along with 3 paraneoplastic antigens, HARS, Ro52 and CDR2 for the evaluation of their sensitivity in predicting recurrence before the clinical relapse of the ovarian cancer. METHODS: Western blot immunoassays were performed to assess the immunoreactivity of 6 antigens with 21 recurrent ovarian cancer patients. RESULTS: The results indicated that antibodies to HARS, Ro52, CDR2 and 5H6 antigens predicted ovarian cancer recurrence 5.03 months before the clinical or symptomatic relapse in 21 ovarian cancer patients with a sensitivity of 90.5% when CA125 levels were below the standard cutoff (35 U/ml). CONCLUSION: Our study suggests that appearance of onconeural antibodies prior to the rise in CA125 during post treatment surveillance can be a useful diagnostic to predict ovarian cancer recurrence.

Extracellular Cyclophilin A Augments Platelet-Dependent Thrombosis and Thromboinflammation.

Cyclophilin A (CyPA) is involved in the pathophysiology of several inflammatory and cardiovascular diseases. To our knowledge, there is no specific inhibitor targeting extracellular CyPA without affecting other extracellular cyclophilins or intracellular CyPA functions. In this study, we developed an antibody-based inhibitor of extracellular CyPA and analysed its effects in vitro and in vivo. To generate a specific antibody, mice and rats were immunized with a peptide containing the extracellular matrix metalloproteinase inducer binding site and various antibody clones were selected and purified. At first, antibodies were tested for their binding capacity to recombinant CyPA and their functional activity. The clone 8H7-mAb was chosen for further experiments. 8H7-mAb reduced the CyPA-induced migration of inflammatory cells in vitro and in vivo. Furthermore, 8H7-mAb revealed strong antithrombotic effects by inhibiting CyPA-dependent activation of platelets and thrombus formation in vitro and in vivo. Surprisingly, 8H7-mAb did not influence in vivo tail bleeding time or in vitro whole blood coagulation parameters. Our study provides first evidence that antibody-based inhibition of extracellular CyPA inhibits thrombosis and thromboinflammation without affecting blood homeostasis. Thus, 8H7-mAb may be a promising compound for thrombi modulation in inflammatory diseases to prevent organ dysfunction.

Paraneoplastic antigens as biomarkers for early diagnosis of ovarian cancer.

Paraneoplastic syndromes are a group of rare disorders that can be triggered by an abnormal immune response to proteins from tumors of the lung, ovary, lymphatics, or breast. Paraneoplastic clinical syndromes affect < 1% of patients with cancer; however, the frequency of subclinical levels of paraneoplastic autoantibodies in asymptomatic patients with cancer is unknown. Numerous studies have reported that ovarian cancer patients show signs of paraneoplastic neurological syndromes (PNSs) before or after their cancers are diagnosed. PNSs arise from a tumor-elicited immune response against onconeural antigens that are shared by tissues of nervous system, muscle, and tumor cells. Studies on the serum IgGs obtained from ovarian cancer patients have indicated the presence of onconeural antibodies in the absence of any PNS symptoms. The occurrence of PNSs is low in ovarian cancer patients and it can be accompanied by onconeural antibodies. The diagnosis of PNSs is accompanied by a suspicion of a malignant tumor such that neurologists typically refer such patients for a tumor diagnostic workup. There will be tremendous utility if subclinical levels (without paraneoplastic neurological symptoms or myositis) of these autoantibodies to paraneoplastic antigens can be exploited to screen asymptomatic high-risk patients for ovarian cancer, and used as biomarkers in immunoassays for the early detection or recurrence of ovarian cancer. Ovarian cancer overall survival is likely to be improved with early detection. Therefore, a panel of onconeural antigens that can detect paraneoplastic autoantibodies in patient sera should provide diagnostic utility for an earlier therapeutic intervention. Here we review the usefulness of PNS and other paraneoplastic syndromes and their association with paraneoplastic antigens to exploit these autoantibody biomarkers to form diagnostic multi-analyte panels for early detection of ovarian cancer.

Platelets as a Novel Source of Pro-Inflammatory Chemokine CXCL14.

OBJECTIVE: Platelets are a major source of chemokines. Here, we demonstrate for the first time that platelets express significant amounts of CXCL14 and disclose powerful effects of platelet-derived CXCL14 on monocyte and endothelial migration. METHODS: The expression of CXCL14 in platelets and in the supernatant of activated platelets was analysed by immunoblotting, ELISA, and flow cytometry. The effect of platelet-derived CXCL14 on monocyte migration was evaluated using a modified Boyden chamber. The effect of CXCL14 on monocyte phagocytosis was tested by using fluorochrome-labelled E.coli particles. The effect of platelet-derived CXCL14 on endothelial migration was explored by the use of an endothelial scratch assay. RESULTS: Hitherto unrecognized expression of CXCL14 in human and murine platelets was uncovered by immunoblotting. Activation with platelet agonists such as adenosine-di-phosphate (ADP), collagen-related peptide (CRP), or thrombin-receptor activating peptide (TRAP), increased CXCL14 surface expression (flow cytometry) and release into the supernatant (immunoblotting, ELISA). Since CXCL14 is known to be chemotactic for CD14+ monocytes, we investigated the chemotactic potential of platelet-derived CXCL14 on human monocytes. Activated platelet supernatant induced monocyte migration, which was counteracted upon neutralization of platelet-derived CXCL14 as compared to IgG control. Blocking of the chemokine receptor CXCR4, but not CXCR7, reduced the number of migratory monocytes towards recombinant CXCL14, suggesting the involvement of CXCR4 in the CXCL14-directed monocyte chemotaxis. Recombinant CXCL14 enhanced the phagocytic uptake of E.coli particles by monocytes. In scratch assays with cultured endothelial cells (HUVECs), platelet-derived CXCL14 counteracted the pro-angiogenic effects of VEGF, supporting its previously recognized angiostatic potential. CONCLUSIONS: Platelets are a relevant source of CXCL14. Platelet-derived CXCL14 at the site of vascular lesions might play an important role in vascular repair/regeneration.

Influence of γ-Secretase Inhibitor 24-Diamino-5-Phenylthiazole DAPT on Platelet Activation.

BACKGROUND/AIMS: DAPT (24-diamino-5-phenylthiazole) inhibits γ-secretase, which cleaves the signaling molecule CD44, a negative regulator of platelet activation and apoptosis. CD44 is a co-receptor for macrophage migration inhibitory factor (MIF) an anti-apoptotic pro-inflammatory cytokine expressed and released from blood platelets. Whether DAPT influences platelet function, remained, however, elusive. Activators of platelets include collagen related peptide (CRP). The present study thus explored whether DAPT modifies the stimulating effect of CRP on platelet function. METHODS: Platelets isolated from wild-type mice were exposed for 30 minutes to DAPT (10 µM). Flow cytometry was employed to estimate Orai1 abundance with specific antibodies, cytosolic Ca(2+)-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from α(IIb)ß3 integrin abundance, generation of reactive oxygen species (ROS) from DCFDA fluorescence, mitochondrial transmembrane potential from TMRE fluorescence, phospholipid scrambling of the cell membrane from annexin-V-binding, relative platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. RESULTS: Exposure of platelets to 2-5 µg/ml CRP was followed by significant increase of Orai1 abundance, [Ca2+]i, and P-selectin abundance, as well as by α(IIb)ß3 integrin activation, ROS generation, mitochondrial depolarization, enhanced annexin-V-binding, decreased cell volume, and aggregation. All CRP induced effects were significantly blunted in the presence of DAPT. CONCLUSIONS: The γ-secretase inhibitor DAPT counteracts agonist induced platelet activation, apoptosis and aggregation.

Endomyocardial expression of SDF-1 predicts mortality in patients with suspected myocarditis.

BACKGROUND: Risk stratification in patients with suspected myocarditis is pivotal for optimizing therapy. Stromal cell-derived factor 1 (SDF-1) is an inflammatory chemokine expressed in the inflamed and failing myocardium. Therefore, we aimed to investigate whether endomyocardial expression of SDF-1 identifies high-risk patients with suspected myocarditis. METHODS AND RESULTS: We prospectively enrolled 174 patients with non-ischemic HF who underwent endomyocardial biopsy for suspected myocarditis. Biopsies were analyzed using established histopathological and immunohistological criteria together with SDF-1 staining. SDF-1 was significantly enhanced in patients with inflammatory cardiomyopathy (65.4 % positive biopsies) as compared to patients with non-inflammatory cardiomyopathy (19.1 %, p < 0.001). SDF-1 expression levels correlated significantly with the degree of myocardial fibrosis (correlation coefficient r = 0.196; p = 0.010) since patients with severe myocardial fibrosis displayed high myocardial SDF-1 expression. During a mean follow-up of 27.5 months, 20 patients (11.5 %) died. The 4-year mortality rate was 26.0 % among the 92 SDF-1-positive patients vs. 9.5 % among the 82 SDF-1-negative patients (p = 0.001). On multivariable analysis which considered clinical (NYHA functional class, left ventricular ejection fraction), laboratory (brain natriuretic peptide, troponin I) and biopsy staining, SDF-1 was the strongest independent predictor of mortality (hazard ratio 6.1; 95 % confidence interval 1.4-27.5; p = 0.018). Subgroup analysis revealed SDF-1 as a predictor of mortality in both patients with inflammatory and non-inflammatory cardiomyopathy. CONCLUSIONS: Endomyocardial expression of SDF-1 is enhanced in inflammatory cardiomyopathy, positively correlates with myocardial fibrosis and identifies high-risk patients with suspected myocarditis.

The Novel Extracellular Cyclophilin A (CyPA) - Inhibitor MM284 Reduces Myocardial Inflammation and Remodeling in a Mouse Model of Troponin I -Induced Myocarditis.

Cyclophilins are a group of highly conserved cytosolic enzymes that have a peptidylprolyl cis/trans isomerase activity. Cyclophilin A (CyPA) can be secreted in the extracellular space by inflammatory cells and upon cell death. The presence of CyPA in patients with non-ischemic cardiomyopathy is associated with poor clinical prognosis. Here, we investigated the inhibition of extracellular CyPA in a mouse model of troponin I-induced autoimmune myocarditis using the strictly extracellular CyPA-inhibitor MM284. Since A/J mice develop severe inflammation and fibrosis after immunization with murine cardiac troponin I (mcTn I), we used this model to analyze the effects of an extracellular CyPA inhibition. As extracellular CyPA-inhibitor we used the recently described CsA-derivate MM284. In vitro studies confirmed that MM284 inhibits CyPA-induced monocytic migration and adhesion. A/J mice immunized with mcTnI were treated with MM284 or vehicle every second day. After 28 days, we found a considerable reduction of myocardial injury and fibrosis. Further analysis revealed a reduced myocardial presence of T-cells and macrophages compared to control treated animals. Whereas MMP-9 expression was reduced significantly by MM284, we observed no significant reduction of inflammatory cytokines such as IL-6 or TNFα. Extracellular CyPA plays an important role in autoimmune myocarditis for myocardial damage and fibrosis. Our data suggest a new pharmacological approach for the treatment of myocardial inflammation and reduction of cardiac fibrosis by inhibition of extracellular CyPA.

Extracellular cyclophilin A activates platelets via EMMPRIN (CD147) and PI3K/Akt signaling, which promotes platelet adhesion and thrombus formation in vitro and in vivo.

OBJECTIVE: Cyclophilin A (CyPA) is secreted under inflammatory conditions by various cell types. Whereas the important role of intracellular CyPA for platelet function has been reported, the effect of extracellular CyPA on platelet function has not been investigated yet. APPROACH AND RESULTS: Inhibition of extracellular CyPA through a novel specific inhibitor MM284 reduced thrombus after ferric chloride-induced injury in vivo. In vitro extracellular CyPA enhanced thrombus formation even in CyPA(-/-) platelets. Treatment of isolated platelets with recombinant CyPA resulted in platelet degranulation in a time- and dose-dependent manner. Inhibition of the platelet surface receptor extracellular matrix metalloproteinase inducer (cluster of differentiation 147) by an anticluster of differentiation 147 monoclonal antibody significantly reduced CyPA-dependent platelet degranulation. Pretreatment of platelets with CyPA enhanced their recruitment to mouse carotid arteries after arterial injury, which could be inhibited by an anticluster of differentiation 147 monoclonal antibody (intravital microscopy). The role of extracellular CyPA in adhesion could be confirmed by infusing CyPA(-/-) platelets in CyPA(+/+) mice and by infusing CyPA(+/+) platelets in CyPA(-/-) mice. Stimulation of platelets with CyPA induced phosphorylation of Akt, which could in turn be inhibited in the presence of phosphoinositid-3-kinase inhibitors. Akt-1(-/-) platelets revealed a markedly decreased degranulation on CyPA stimulation. Finally, ADP-induced platelet aggregation was attenuated by MM284, as well as by inhibiting paracrine-secreted CyPA without directly affecting Ca(2+)-signaling. CONCLUSIONS: Extracellular CyPA activates platelets via cluster of differentiation 147-mediated phosphoinositid-3-kinase/Akt-signaling, leading to enhanced adhesion and thrombus formation independently of intracellular CyPA. Targeting extracellular CyPA via a specific inhibitor may be a promising strategy for platelet inhibition without affecting critical functions of intracellular CyPA.

Experimentally induced psoriatic lesions associate with rapid but transient decrease in interleukin-33 immunostaining in epidermis.

A slight epidermal damage can induce the Köbner reaction in psoriasis, and the "alarmin", interleukin-33 (IL-33), may be involved in this process. Therefore, the uninvolved psoriatic skin was tape-stripped, and skin biopsies were collected at 0 day, 2 h and 3 days or at 0 day, 1 day and 7 days for immunohistochemistry. Eight patients out of 18 with the positive Köbner reaction showed a decrease in epidermal thickness and revealed transient reduction in epidermal nuclear immunostaining of IL-33 in 2-h, 1-day, 3-day biopsies compared to the 10 Köbner-negative patients. In keratinocyte cultures, the full-length 32-kDa IL-33 was detected after damaging the cells with freeze-thawing. Interestingly, a very low concentration of rh-IL-33 (0.001­0.01 ng/ml) significantly stimulated (3)H-thymidine uptake by human LAD2 mast cells, but not by psoriatic peripheral blood mononuclear cells. The results show that epidermal IL-33 associates with positive Köbner response, and only a small amount of the IL-33 apparently released may induce proliferation in dermal mast cells.

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-1α), 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.

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.

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.

Serum prognostic biomarkers in head and neck cancer patients.

OBJECTIVES/HYPOTHESIS: A reliable estimate of survival is important as it may impact treatment choice. The objective of this study is to identify serum autoantibody biomarkers that can be used to improve prognostication for patients affected with head and neck squamous cell carcinoma (HNSCC). STUDY DESIGN: Prospective cohort study. METHODS: A panel of 130 serum biomarkers, previously selected for cancer detection using microarray-based serological profiling and specialized bioinformatics, were evaluated for their potential as prognostic biomarkers in a cohort of 119 HNSCC patients followed for up to 12.7 years. A biomarker was considered positive if its reactivity to the particular patient's serum was greater than one standard deviation above the mean reactivity to sera from the other 118 patients, using a leave-one-out cross-validation model. Survival curves were estimated according to the Kaplan-Meier method, and statistically significant differences in survival were examined using the log rank test. Independent prognostic biomarkers were identified following analysis using multivariate Cox proportional hazards models. RESULTS: Poor overall survival was associated with African Americans (hazard ratio [HR] for death = 2.61; 95% confidence interval [CI]: 1.58-4.33; P = .000), advanced stage (HR = 2.79; 95% CI: 1.40-5.57; P = .004), and recurrent disease (HR = 6.66; 95% CI: 2.54-17.44; P = .000). On multivariable Cox analysis adjusted for covariates (race and stage), six of the 130 markers evaluated were found to be independent prognosticators of overall survival. CONCLUSIONS: The results shown here are promising and demonstrate the potential use of serum biomarkers for prognostication in HNSCC patients. Further clinical trials to include larger samples of patients across multiple centers may be warranted.

Acid sphingomyelinase regulates platelet cell membrane scrambling, secretion, and thrombus formation.

OBJECTIVE: Platelet activation is essential for primary hemostasis and acute thrombotic vascular occlusions. On activation, platelets release their prothrombotic granules and expose phosphatidylserine, thus fostering thrombin generation and thrombus formation. In other cell types, both degranulation and phosphatidylserine exposure are modified by sphingomyelinase-dependent formation of ceramide. The present study thus explored whether acid sphingomyelinase participates in the regulation of platelet secretion, phosphatidylserine exposure, and thrombus formation. APPROACH AND RESULTS: Collagen-related peptide-induced or thrombin-induced ATP release and P-selectin exposure were significantly blunted in platelets from Asm-deficient mice (Smpd1(-/-)) when compared with platelets from wild-type mice (Smpd1(+/+)). Moreover, phosphatidylserine exposure and thrombin generation were significantly less pronounced in Smpd1(-/-) platelets than in Smpd1(+/+) platelets. In contrast, platelet integrin αIIbß3 activation and aggregation, as well as activation-dependent Ca(2+) flux, were not significantly different between Smpd1(-/-) and Smpd1(+/+) platelets. In vitro thrombus formation at shear rates of 1700 s(-1) and in vivo thrombus formation after FeCl3 injury were significantly blunted in Smpd1(-/-) mice while bleeding time was unaffected. Asm-deficient platelets showed significantly reduced activation-dependent ceramide formation, whereas exogenous ceramide rescued diminished platelet secretion and thrombus formation caused by Asm deficiency. Treatment of Smpd1(+/+) platelets with bacterial sphingomyelinase (0.01 U/mL) increased, whereas treatment with functional acid sphingomyelinase-inhibitors, amitriptyline or fluoxetine (5 µmol/L), blunted activation-dependent platelet degranulation, phosphatidylserine exposure, and thrombus formation. Impaired degranulation and thrombus formation of Smpd1(-/-) platelets were again overcome by exogenous bacterial sphingomyelinase. CONCLUSIONS: Acid sphingomyelinase is a completely novel element in the regulation of platelet plasma membrane properties, secretion, and thrombus formation.

Gremlin-1 is an inhibitor of macrophage migration inhibitory factor and attenuates atherosclerotic plaque growth in ApoE-/- Mice.

Monocyte infiltration and macrophage formation are pivotal steps in atherosclerosis and plaque vulnerability. Gremlin-1/Drm is crucial in embryo-/organogenesis and has been shown to be expressed in the adult organism at sites of arterial injury and to inhibit monocyte migration. The purpose of the present study was to evaluate and characterize the role of Gremlin-1 in atherosclerosis. Here we report that Gremlin-1 is highly expressed primarily by monocytes/macrophages in aortic atherosclerotic lesions of ApoE(-/-) mice and is secreted from activated monocytes and during macrophage development in vitro. Gremlin-1 reduces macrophage formation by inhibiting macrophage migration inhibitory factor (MIF), a cytokine critically involved in atherosclerotic plaque progression and vulnerability. Gremlin-1 binds with high affinity to MIF (KD = 54 nm), as evidenced by surface plasmon resonance analysis and co-immunoprecipitation, and reduces MIF-induced release of TNF-α from macrophages. Treatment of ApoE(-/-) mice with a dimeric recombinant fusion protein, mGremlin1-Fc, but not with equimolar control Fc or inactivated mGremlin1-Fc, reduced TNF-α expression, the content of monocytes/macrophages of atherosclerotic lesions, and attenuated atheroprogression. The present data disclose that Gremlin-1 is an endogenous antagonist of MIF and define a role for Gremlin-1/MIF interaction in atherosclerosis.