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 TF⅐VIIa-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 TF⅐VIIa 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 TF⅐PAR2 complex and TF⅐VIIa 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 TF⅐VIIa signaling can be specifically inhibited with minimal effects on coagulation.allosteric disulfide ͉ protein disulfide ͉ isomerase ͉ S-nitrosylation ͉ G protein-coupled receptor

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 beta1 integrins is regulated by TF extracellular ligand binding and independent of PAR2 signaling or proteolytic activity of VIIa. In contrast, alpha3beta1 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.

These data identify exenatide as a potentially effective compound to reduce infarct size in adjunction to reperfusion therapy in patients with acute MI.

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 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.

Objective: Elective repair of abdominal aortic aneurysms (AAA) is associated with significant morbidity and mortality. Large amounts of AAA tissue are necessary to assess heterogeneity among AAA and to correct for potential confounders such as known risk factors. The Aneurysm-express study aims to identify different types of AAA using inflammatory markers in the aneurysm wall that predict postoperative cardiovascular adverse events and mortality, therefore allowing individual risk assessment. Methods: The Aneurysm-express is an ongoing prospective cohort study including AAA patients undergoing open repair. At baseline, blood is drawn, relevant clinical data are collected and the standard diagnostic modalities are performed. During surgery a specimen of the ventral AAA wall is collected and processed to study protein expressions and histology. Interim Results: The study commenced in 2003 in 2 medical centers and currently holds information and material of >300 AAA patients, making it the largest reported aneurysm biobank. Patients are followed for 3 years after surgery for occurring cardiovascular events. The current mean follow-up is 2.1 ± 1.3 years with an event rate of 27%. Conclusion: The large amount of structurally stored tissue and blood combined with clinical characteristics and follow-up provide an excellent soil for indepth pathophysiological analyses, with assessment of AAA heterogeneity in combination with postoperative clinical outcome.

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 Chlamydialike 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.

Streptococcus pneumoniae is the most prevalent pathogen causing community-acquired pneumonia. CD44 is a transmembrane adhesion molecule, expressed by a wide variety of cell types, that has several functions in innate and adaptive immune responses. In this study, we tested the hypothesis that CD44 is involved in the host response during pneumococcal pneumonia. On intranasal infection with a lethal dose of S. pneumoniae CD44-knockout (KO) mice showed a prolonged survival when compared with wild-type mice, which was accompanied by a diminished pulmonary bacterial growth and reduced dissemination to distant body sites. Whereas, proinflammatory cytokine responses and lung pathology were not affected, CD44 deficiency resulted in increased early neutrophil influx into the lung. In separate experiments, we confirmed a detrimental role of CD44 in host defense against pneumococci during sublethal pneumonia, as demonstrated by an improved capacity of CD44 KO mice to clear a low infectious dose. In addition, CD44 appeared important for the resolution of lung inflammation during sublethal pneumonia, as shown by histopathology of lung tissue slides. In conclusion, we show here that CD44 facilitates bacterial outgrowth and dissemination during pneumococcal pneumonia, which in lethal infection results in a prolonged survival of CD44 KO mice. Moreover, during sublethal pneumonia CD44 contributes to the resolution of the inflammatory response.

Recent studies have suggested that Chlamydia pneumoniae infection is a risk factor for abdominal aortic aneurysm. This study explores the presence of Chlamydia pneumoniae DNA in buffy-coat samples of control subjects and of patients with abdominal aortic aneurysm. The seroepidemiological association between abdominal aortic aneurysm and Chlamydia pneumoniae was also investigated. Buffy-coat samples and serum specimens were obtained from 88 patients and 88 control subjects. Detection of Chlamydia pneumoniae DNA in buffy-coat samples and measurement of IgG antibodies to Chlamydia pneumoniae in serum specimens were performed by polymerase chain reaction and microimmunofluorescence, respectively. Chlamydia pneumoniae DNA was detected in buffy-coat samples of 18 (20%) patients and 8 (9%) control subjects (adjusted odds ratio 2.9, 95% confidence interval 1-8.5). IgG antibodies to Chlamydia pneumoniae were detected in 85 (97%) patients and 71 (81%) control subjects (adjusted odds ratio 7.2, 95% confidence interval 1.7-31). The results show an association between abdominal aortic aneurysm and either the presence of Chlamydia pneumoniae DNA in buffy-coat samples or IgG antibodies to Chlamydia pneumoniae. These findings support the hypothesis that previous infection with Chlamydia pneumoniae might be a risk factor for abdominal aortic aneurysm.