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The G protein–coupled protease-activated receptors (PAR) are key signaling components for proteases in vascular biology and tumor progression. To address the contributions of PAR1 and PAR2 to breast cancer development, we established cohorts of mouse mammary tumor virus–polyoma middle T (PyMT) PAR1−/− and PAR2−/− mice, considering that the PyMT model recapitulates aspects of human disease. Appearance of palpable tumors, tumor expansion, and metastasis was indistinguishable between wild-type and PAR1−/− mice. PAR1−/− breast cancer cells were no longer responsive to thrombin in vitro, excluding compensatory up-regulation of alternative thrombin receptors and indicating that thrombin-PAR1 signaling is dispensable in breast tumor microenvironments. In contrast, palpable tumors and multifocal disease developed slower in PAR2−/− mice, and as a consequence of delayed tumor onset, metastasis was reduced. Analysis of early tumors showed persistence of adenomas with delayed appearance of vascularized adenocarcinomas in PAR2−/− mice. Furthermore, CXCL1 production by early PAR2−/− tumors was reduced. These results are consistent with previous xenograft data that implicated breast cancer PAR2 signaling in the induction of proangiogenic growth factors and chemokines. This study establishes that protease signaling contributes to mammary tumor development and that PAR2, rather than the thrombin receptor PAR1, plays a crucial role in the angiogenic switch. [Cancer Res 2008;68(17):7219–27]

Coagulation activation by tissue factor (TF) is implicated in cancer progression, cancer-associated thrombosis and metastasis. The role of direct TF signaling pathways in cancer, however, remains incompletely understood. Here we address how TF contributes to primary tumor growth by using a unique pair of isotype-matched antibodies that inhibit either coagulation (monoclonal antibody [Mab]-5G9) or direct signaling (Mab-10H10). We demonstrate that the inhibitory antibody of direct TF-VIIa signaling not only blocks TF-VIIa mediated activation of PAR2, but also disrupts the interaction of TF with integrins. In epithelial and TF-expressing endothelial cells, association of TF with β1 integrins is regulated by TF extracellular ligand binding and independent of PAR2 signaling or proteolytic activity of VIIa. In contrast, α3β1 integrin association of TF is constitutive in breast cancer cells and blocked by Mab-10H10 but not by Mab-5G9. Mab-5G9 has antitumor activity in vivo, but we show here that Mab-10H10 is at least as effective in suppressing human xenograft tumors in 2 different models. Breast tumor growth was also attenuated by blocking PAR2 signaling. These results show that tumor cell TF-PAR2 signaling is crucial for tumor growth and suggest that anti-TF strategies can be applied in cancer therapy with minor impairment of TF-dependent hemostatic pathways.

Cell-surface tissue factor (TF) binds the serine protease factor VIIa to activate coagulation or, alternatively, to trigger signaling through the G protein-coupled, protease-activated receptor 2 (PAR2) relevant to inflammation and angiogenesis. Here we demonstrate that TF·VIIa-mediated coagulation and cell signaling involve distinct cellular pools of TF. The surface-accessible, extracellular Cys 186 –Cys 209 disulfide bond of TF is critical for coagulation, and protein disulfide isomerase (PDI) disables coagulation by targeting this disulfide. A TF mutant (TF C209A) with an unpaired Cys 186 retains TF·VIIa signaling activity, and it has reduced affinity for VIIa, a characteristic of signaling TF on cells with constitutive TF expression. We further show that PDI suppresses TF coagulant activity in a nitric oxide-dependent pathway, linking the regulation of TF thrombogenicity to oxidative stress in the vasculature. Furthermore, a unique monoclonal antibody recognizes only the noncoagulant, cryptic conformation of TF. This antibody inhibits formation of the TF·PAR2 complex and TF·VIIa signaling, but it does not prevent coagulation activation. These experiments delineate an upstream regulatory mechanism that controls TF function, and they provide initial evidence that TF·VIIa signaling can be specifically inhibited with minimal effects on coagulation.

Objectives This study sought to examine whether exenatide is capable of reducing myocardial infarct size. Background Exenatide is a glucagon-like peptide (GLP)-1 analogue with insulinotropic and insulinomimetic properties. Because insulin and GLP-1 have been described as reducing apoptosis, exenatide might confer cardioprotection after acute myocardial infarction (MI). Methods Pigs were randomized to exenatide or phosphate-buffered saline (PBS) treatment after 75 min of coronary artery ligation and subsequent reperfusion. Infarct size was assessed with Evans Blue (Sigma-Aldrich, St. Louis, Missouri) and triphenyltetrazolium chloride. Cardiac function was measured with epicardial ultrasound and conductance catheter-based pressure-volume loops. Western blotting, histology, and activity assays were performed to determine markers of apoptosis/survival and oxidative stress. Results Exenatide reduced myocardial infarct size (32.7 +/- 6.4% vs. 53.6 +/- 3.9%; p = 0.031) and prevented deterioration of systolic and diastolic cardiac function (systolic wall thickening: 47.3 +/- 6.3% vs. 8.1 +/- 1.9%, p

Background: Tissue factor (TF) and factor (F) VIIa are the primary initiators of the coagulation cascade, but also promote non-hemostatic events, such as angiogenesis and tumor growth, via activation of protease activated receptor-2 (PAR2). Our previous findings indicated that the TF:FVIIa complex activates signal transducer and activator of transcription (STAT) signaling, leading to cell survival in TF-transfected baby hamster kidney (BHK) cells. Methods: Using BHK(TF), keratinocytes (HaCaT) and human umbilical vein endothelial cells (HUVEC), FVIIa-induced phosphorylation and activation of the transcription factor cyclic AMP-responsive binding protein (CREB) were tested and compared to that elicited by thrombin and FXa. In addition, the effect of these factors on cell survival and expression of apoptosis-associated proteins was monitored. Results:: Factor VIIa led to a TF-dependent, but TF cytoplasmic domain-independent phosphorylation and activation of CREB in BHK(TF), HaCaT and HUVEC. CREB activation was sensitive to blockade of the extracellular-signal regulated kinase 1/2 pathway and PAR2. Surprisingly, FVIIa decreased cell survival in HaCaT cells but not other cell types and upregulated the pro-apoptotic proteins Bak and Puma in a CREB-dependent manner. Factor Xa, but not FIIa, induced phosphorylation of CREB, but did not have an effect on apoptosis. Conclusion: TF:FVIIa induces CREB phosphorylation and activation in several cell types, but TF:FVIIa induces pro-apoptotic proteins and apoptosis only in selected cell types.

Inter- and intralaboratory inconsistencies in detection rates of Chlamydia pneumoniae in vascular specimens have been demonstrated. In this study, 66 vascular tissue specimens from 66 patients with vascular disease were tested by three PCR assays: a 16S PCR-based reverse line blot (RLB) assay, a single-step PCR, and a nested PCR. Also, we explored the impacts of different DNA polymerase enzymes on the results based on gel electrophoresis and hybridization. The PCR results by gel electrophoresis in the single-step PCR depended on which DNA polymerase was used. All samples were negative with AmpliTaq Gold DNA polymerase, and 54.5% (36 of 66) were positive with the conventional Taq DNA polymerase. All samples were negative after hybridization with a C. pneumoniae -specific probe. In the nested PCR, all specimens were negative by gel electrophoresis and after hybridization. The RLB assay failed to detect C. pneumoniae in any specimen; however, 20 specimens were Chlamydia sp. positive. The sequence analysis of six of these samples demonstrated Chlamydia -like organisms. RLB detected Chlamydia sp. DNA in water and in the elution buffer after passage of the Qiagen columns (11 of 40). This study identified factors that may influence the detection of C. pneumoniae DNA in vascular tissues and consequently bias the perception of a link between C. pneumoniae and vascular diseases. The following are strongly recommended: to use DNA polymerases that have to be activated, to decontaminate with dUTP-uracil-DNA glycosylase, to hybridize with specific probes, to include sufficient controls, and to use molecular grade water.

Tissue factor (TF) forms a catalytic complex with coagulation factor VIIa and thus triggers coagulation as well as cell signaling through protease activated receptor (PAR) 2. The molecular switch between these two functions remains elusive. We demonstrate in growth arrested keratinocytes that TF’s coagulant function remains unchanged when TF expression levels and TF-VIIa signaling are concordantly downregulated. Thus, coagulation and signaling are mediated by two distinct and differentially regulated pools of TF. TF-VIIa signaling, but not coagulation, is blocked by bacitracin, an inhibitor of protein disulfide isomerase, and TF co-immunoprecipitates protein disulfide isomerases. To test the role of isomerization, we expressed single thiol containing mutants of the cross-stranded, surface exposed TF Cys186-Cys209 disulfide bond. Retaining Cys186, but not Cys209, preserves signaling properties with high affinity for VIIa, but eliminates the TF-dependent coagulation. Cys186-containing TF is surface expressed predominantly in a dimeric form, indicating a trapped isomerization intermediate. Signaling by wild-type, but not the Cys186 dimer, is inhibited by blocking free thiols indicating that thiol-isomerization plays an essential role in regulating TF signaling. Furthermore, we identified a monoclonal antibody that blocks TF-VIIa signaling of wild-type, but not Cys186 dimer TF, suggesting interference with the dynamics of TF isomerization. This human TF specific antibody does not interfere with coagulation, but inhibits the growth of human xenograft tumors in mice. Thus, TF-VIIa signaling, not coagulation, is the primary pathway for TF-driven tumorigenesis. These experiments resolve the molecular mechanism that switches TF-VIIa from coagulation to signaling and show feasibility to block signaling TF for the therapeutic intervention in cancer.