The effect of levothyroxine on expression of inflammation-related genes in healthy subjects: a controlled randomized crossover study.

An excess of thyroid hormone leads to a prothrombotic state; however, the underlying pathophysiological mechanisms remain unknown. As evidence points towards an extensive "cross-talk" between the inflammatory and coagulation cascade, inflammation has been claimed as a possible mechanism through which different risk factors trigger venous thrombus formation. We aimed to study changes in expression of inflammation-related genes of the leukocyte RNA expression profile in healthy subjects in response to supraphysiological doses of levothyroxine. In a randomized single-blinded crossover design, 12 healthy volunteers (aged 18-40 years) received levothyroxine and no medication, both for 14 days with a wash-out period of at least 28 days between the periods. Blood was sampled at baseline and day 14 of each study period. MRNA was isolated from whole blood and used for multiplex ligation-dependent probe amplification to study the expression of inflammation-related genes. Compared to the control situation no significant changes were found in the expression of proinflammatory cytokines and mediators after the intake of levothyroxine. The results of this study show that high thyroid hormone levels do not lead to an altered inflammatory profile. This provides evidence against a major role of the inflammatory system as mediator in the effect of thyroid hormone on the coagulation system. The mechanisms by which thyroid hormone may influence coagulation proteins remain to be elucidated.

Alternatively spliced tissue factor induces angiogenesis through integrin ligation.

The initiator of coagulation, full-length tissue factor (flTF), in complex with factor VIIa, influences angiogenesis through PAR-2. Recently, an alternatively spliced variant of TF (asTF) was discovered, in which part of the TF extracellular domain, the transmembrane, and cytoplasmic domains are replaced by a unique C terminus. Subcutaneous tumors produced by asTF-secreting cells revealed increased angiogenesis, but it remained unclear if and how angiogenesis is regulated by asTF. Here, we show that asTF enhances angiogenesis in matrigel plugs in mice, whereas a soluble form of flTF only modestly enhances angiogenesis. asTF dose-dependently upregulates angiogenesis ex vivo independent of either PAR-2 or VIIa. Rather, asTF was found to ligate integrins, resulting in downstream signaling. asTF-alphaVbeta3 integrin interaction induces endothelial cell migration, whereas asTF-dependent formation of capillaries in vitro is dependent on alpha6beta1 integrin. Finally, asTF-dependent aortic sprouting is sensitive to beta1 and beta3 integrin blockade and a TF-antibody that disrupts asTF-integrin interaction. We conclude that asTF, unlike flTF, does not affect angiogenesis via PAR-dependent pathways but relies on integrin ligation. These findings indicate that asTF may serve as a target to prevent pathological angiogenesis.

Gross deletions/duplications in PROS1 are relatively common in point mutation-negative hereditary protein S deficiency.

Hereditary protein S (PS) deficiency is an autosomal disorder caused by mutations in the PS gene (PROS1). Conventional PCR-based mutation detection identifies PROS1 point mutations in approximately 50% of the cases. To verify if gross copy number variations (CNVs) are often present in point mutation-negative hereditary PS deficiency we used multiplex ligation-dependent probe amplification (MLPA) as a detection tool in samples from individuals with a high probability of having true PS deficiency. To this end, DNA samples from nine PS deficient probands with family members (seven type I and two type III) and nine isolated probands (three type I and six type III), in whom PROS1 mutations were not found by DNA sequencing, were evaluated. An independent quantitative PCR (qPCR) was performed to confirm the findings of the MLPA assay. Family members were also tested when DNA was available. Gross abnormalities of PROS1 were found in six out of eighteen probands. In three probands complete deletion of the gene was detected. Two probands had a partial deletion involving different parts of the gene (one from exon 4 through 9 and another from exon 9 through 11). One family showed a duplication of part of PROS1. qPCR analysis was in accordance with these results. In conclusion, this study substantiates that gross gene abnormalities in PROS1 are relatively common in hereditary PS deficient patients and that MLPA is a useful tool for direct screening of CNVs in PROS1 point mutation-negative individuals.

Gene expression profile comparison of Barrett's esophagus epithelial cell cultures and biopsies.

Barrett's esophagus (BE) is a metaplastic process in which the normal squamous epithelium of the distal esophagus is replaced by columnar lined epithelium. The aim was to gain more insight in the process of metaplasia and to identify which genes are specifically expressed by the epithelial cells and the surrounding tissues in BE. Hereto, the gene expression profile of a BE epithelial primary cell culture was compared to that of a BE biopsy. To specifically obtain the epithelial cell layer, epithelial cells from biopsies of BE were cultured using a Barrett specific culturing medium. Serial analysis of gene expression was applied to obtain a transcription library of the primary epithelial cell culture. The transcriptome was analyzed and compared to a previously described transcriptome of a BE biopsy. Validation of results by reverse transcriptase-polymerase chain reaction was performed using tissues of 16 BE patients and 16 primary cell cultures. Over 43,000 tags were sequenced. Genes specifically expressed by the Barrett epithelial cells were for instance Lipocalin 2 and Cyclin D1, whereas annexin A10, trefoil factor (TFF)1 and TFF2 were specifically expressed in the BE biopsies. The comparison of the gene expression profiles of BE primary cultured epithelial cells with BE biopsy defines a subset of genes that are specifically expressed by the epithelial cells and another subset that most likely is expressed by the underlying stromal tissues in the BE biopsy specimens.

Targeting coagulation factor receptors - protease-activated receptors in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease with a 5-year mortality rate of > 50% and unknown etiology. Treatment options remain limited and, currently, only two drugs are available, i.e. nintedanib and pirfenidone. However, both of these antifibrotic agents only slow down the progression of the disease, and do not remarkably prolong the survival of IPF patients. Hence, the discovery of new therapeutic targets for IPF is crucial. Studies exploring the mechanisms that are involved in IPF have identified several possible targets for therapeutic interventions. Among these, blood coagulation factor receptors, i.e. protease-activated receptors (PARs), are key candidates, as these receptors mediate the cellular effects of coagulation factors and play central roles in influencing inflammatory and fibrotic responses. In this review, we will focus on the controversial role of the coagulation cascade in the pathogenesis of IPF. In the light of novel data, we will attempt to reconciliate the apparently conflicting data and discuss the possibility of pharmacologic targeting of PARs for the treatment of fibroproliferative diseases.

Association between protein C levels and mortality in patients with advanced prostate, lung and pancreatic cancer.

INTRODUCTION: Procoagulant factors promote cancer progression and metastasis. Protein C is involved in hemostasis, inflammation and signal transduction, and has a protective effect on the endothelial barrier. In mice, administration of activated protein C reduced experimental metastasis. We assessed the association between protein C and mortality in patients with three types of cancer. METHODS: The study population consisted of patients with advanced prostate, non-small cell lung or pancreatic cancer, who participated in the INPACT trial (NCT00312013). The trial evaluated the addition of nadroparin to chemotherapy in patients with advanced malignancy. Patients were divided into tertiles based on protein C at baseline. The association between protein C levels and mortality was evaluated with Cox proportional hazard models. RESULTS: We analysed 477 patients (protein C tertiles: <97, 97-121 and ≥121%). Mean age was 65±9years; 390 (82%) were male; 191 patients (40%) had prostate cancer, 161 (34%) had lung cancer, and 125 (26%) pancreatic cancer. During a median follow-up of 10.4months, 291 patients (61%) died. Median protein C level was 107% (IQR 92-129). In the lowest tertile, 75 patients per 100 patient-years died, as compared to 60 and 54 in the middle and high tertile, respectively. Lower levels of protein C were associated with increased mortality (in tertiles: HR for trend 1.18, 95%CI 1.02-1.36, adjusted for age, sex and nadroparin use; as a continuous variable: HR 1.004, 95%CI 1.00-1.008, p=0.07). CONCLUSION: Protein C seems inversely associated with mortality in patients with advanced prostate, lung and pancreatic cancer. Further research should validate protein C as a biomarker for mortality, and explore the effects of protein C on progression of cancer.

Inhalation of activated protein C inhibits endotoxin-induced pulmonary inflammation in mice independent of neutrophil recruitment.

BACKGROUND AND PURPOSE: Intravenous administration of recombinant human activated protein C (rhAPC) is known to reduce lipopolysaccharide (LPS)-induced pulmonary inflammation by attenuating neutrophil chemotaxis towards the alveolar compartment. Ideally, one would administer rhAPC in pulmonary inflammation at the site of infection to minimize the risk of systemic bleeding complications. In this study, we therefore assessed the effect of inhaled rhAPC in a murine model of acute lung injury. EXPERIMENTAL APPROACH: Mice were exposed to LPS (0.5 mg kg(-1): intranasally) to induce acute lung injury. 30 minutes before and 3 hours after LPS exposure mice were subjected to vehicle or rhAPC inhalation (25 or 100 microg per mouse in each nebulization). In order to establish whether rhAPC inhalation affects neutrophil recruitment, neutrophil migration was determined in vitro using a trans-well migration assay. KEY RESULTS: rhAPC inhalation dose-dependently decreased LPS-induced coagulation and inflammation markers in bronchoalveolar lavage fluid (BALF), reduced protein leakage into the alveolar space and improved lung function. In contrast, rhAPC did not prevent LPS-induced neutrophil recruitment into the alveolar space. Neutrophil migration in vitro towards FCS or interleukin (IL)-8 was significantly inhibited by pretreatment with rhAPC (0.01-10 microg ml(-1)], whereas rhAPC (10 microg ml(-1)) added to the chemoattractant (modelling for topical rhAPC administration) did not affect neutrophil migration towards FCS or IL-8. CONCLUSIONS AND IMPLICATIONS: rhAPC inhalation significantly diminished LPS-induced pulmonary inflammation. The benefit of inhaled rhAPC appeared not to involve attenuation of neutrophil recruitment, in contrast to its effects after intravenous administration.

PO-28 - Protein C levels are associated with mortality in patients with advanced cancer.

INTRODUCTION: In cancer, tumor progression and metastasis are promoted by prohemostatic activity. Protein C (PC) is involved in hemostasis, inflammation and signal transduction, and has a protective effect on the endothelial barrier. In mice, administration of activated PC reduced experimental metastasis. It is unclear whether PC level is associated with mortality in patients with cancer. AIM: To assess the relation between PC level and survival in patients with advanced cancer. MATERIALS AND METHODS: A multicenter, randomized, open-label study was performed in 11 countries between May 2006 and August 2008 (INPACT study, van Doormaal et al, JCO 2011). Patients (n=503) with hormone-refractory prostate cancer, non-small cell lung cancer stage IIIB and locally advanced pancreatic cancer were randomized to receive nadroparin or placebo for 6 to 46 weeks following a specific schedule. Patients were followed till death or the end of the study in May 2009. PC activity levels were measured at baseline and categorized in tertiles. The association between PC level and mortality was evaluated with Cox proportional hazard models, adjustments were made by multivariate Cox proportional hazard models. RESULTS: PC activity could be measured in 479 (95%) patients (tertiles: <97, 97-120 and >120%). Two patients with missing information on type of cancer were excluded. Mean age was 65±10 years; 87 (18%) were female; and 161 patients had lung cancer, 125 pancreatic cancer and 191 prostate cancer. During median follow-up of 10.5 months, 291 (61%) patients died. Median PC activity was 107% (IQR 92-129). There was a clear inverse relation between PC activity and mortality (p for trend=0.036). In the lowest tertile, mortality was 66%, in the middle and high tertile 61% and 56%, respectively. Compared to the highest tertile, the lowest tertile was associated with a HR on mortality of 1.36 (95% CI 1.02-1.80). Adjustment for age, gender and nadroparin use did not affect this association. The association appeared to be strongest in the patients with lung cancer, HR 0.818 (p=0.11) as compared to the patients with prostate cancer, HR 0.972 (p=0.83) and pancreatic cancer, HR 0.950 (p=0.68). CONCLUSIONS: Lower PC activity is associated with increased mortality in patients with advanced cancer. However, validation of our findings in a larger cohort is necessary. When the association of PC and mortality has been proven to be consistent, we would suggest a trial on suppletion of PC in cancer patients.

Role of coagulation FVIII in septic peritonitis assessed in hemophilic mice.

BACKGROUND: Inhibition of blood coagulation appears to be an important therapeutic strategy to improve the outcome in sepsis. However, the beneficial effect of anticoagulant treatment in sepsis is solely based on experimental data using inhibitors of the extrinsic coagulant pathway. The role of the intrinsic pathway of coagulation in the pathogenesis of sepsis has not been explored yet. OBJECTIVE: In the current study, we contribute to determine the role of factor (F)VIII, the key player of the intrinsic coagulant pathway, on host defense against peritonitis. METHOD: Hemizygous FVIII-deficient mice and their wild-type littermates were challenged with 1 x 10(4) bacteria in a septic peritonitis model. RESULTS: The intraperitoneal injection of Escherichia coli led to growth and dissemination of bacteria and provoked an inflammatory response as evident from elevated cytokine levels, increased cell influx into tissues, liver necrosis, and endothelialitis resulting in mortality. The FVIII-deficient genotype slightly reduced bacterial outgrowth but had no effect on markers of inflammation and/or survival. In addition, FVIII-deficient mice showed profound activation of coagulation, thereby improving the hemophilic phenotype of FVIII-deficient mice. CONCLUSION: FVIII deficiency slightly modifies host defense in septic peritonitis in mice, but does not influence the final outcome of peritonitis. Therefore, we question the importance of the intrinsic coagulant pathway during sepsis.

Tissue factor haploinsufficiency during endotoxin induced coagulation and inflammation in mice.

Intervention studies blocking tissue factor (TF) driven coagulation show beneficial effects on survival in endotoxemia models by reducing cytokine production. It is unknown, however, if moderately reduced TF levels influence endotoxemia. The objective was to investigate whether TF haploinsufficiency reduces endotoxin-induced cytokine production in murine cells or in mice. We analyzed the intrinsic capacity of heterozygous TF deficient (TF+/-) leukocytes to produce cytokines. In addition, we determined the consequences of TF haploinsufficiency on endotoxin-induced inflammation during murine endotoxemia. Endotoxin induced the production of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and keratinocyte-derived chemokine (KC) in both whole blood and macrophages. Heterozygous TF deficiency reduced endotoxin induced IL-6 and KC levels about two-fold, while TNF-alpha levels were indistinguishable between TF+/- and wild-type cells. In vivo, endotoxin induced a biphasic coagulant response and significant increases in cytokine levels. Surprisingly, both the inflammatory and the coagulant responses were indistinguishable between wild-type and TF+/- mice. At baseline tissues of TF+/- mice showed a 50% reduction in TF activity compared to wild type. Upon endotoxin administration, TF activity increased and the difference between TF+/- and wild-type mice disappeared after 4 h. After 12 h the baseline difference in TF activity was re-established. TF deficiency reduces cytokine production in vitro, but an enhanced induction of TF during murine endotoxemia eliminates this effect in vivo.

Regulation of the p21Ras-MAP kinase pathway by factor VIIa.

BACKGROUND: In recent years it has become clear that factor (F)VIIa is not a passive mediator involved in the linear transduction of the coagulation cascade, but actively engages target cells to induce signal transduction and that this signal transduction fulfills critical functions in angiogenesis, arteriosclerosis and inflammatory processes. OBJECTIVES: The details of coagulation factor-dependent signal transduction are among the least understood in biology and thus we set out to establish the molecular events responsible for MAP kinase activation induced by the interaction of FVIIa with its cellular binding partner tissue factor (TF). METHODS: Two different TF-expressing cell types, BHKTF and HaCaT cells, were assayed for p21Ras activation using a pull-down assay that is specific for activated Ras. This activation was visualized by means of Western blotting. In addition, the upstream pathways leading to FVIIa-induced Ras activation were characterized using phosphospecific antibodies and specific inhibitors. RESULTS: We observed that in both BHKTF and HaCaT cells FVIIa-induced MAP kinase activation correlates with p21Ras activation, and that this p21Ras activation is essential for FVIIa-induced MAP kinase activation. In BHKTF cells, early p21Ras activation was mediated by the activation of protein kinase C (PKC), whereas late p21Ras activation employed alternative mechanisms. In HaCaT cells, stimulation of the Src kinase family mediated FVIIa-dependent p21Ras activation. Finally, in both cell types, Raf activity was mandatory for MAP kinase activation. CONCLUSIONS: p21Ras activation is instrumental in FVIIa signal transduction and the FVIIa-dependent activation of p21Ras involves either PKC or Src-dependent mechanisms, depending on the cell type investigated.

The pleiotropic effects of tissue factor: a possible role for factor VIIa-induced intracellular signalling?

Tissue factor, a 47 kDa membrane glycoprotein, lies at the basis of the extrinsic pathway of the coagulation cascade. Interaction of TF with factor VIIa results in the formation of fibrin from fibrinogen, thereby inducing the formation of a blood clot. In addition to this well-established role in blood coagulation, TF is associated with various other physiological processes such as sepsis, inflammation, angiogenesis, metastasis and atherosclerosis. The molecular basis of the latter events is slowly emerging. It has become clear that TF-FVIIa interaction elicits a variety of intracellular signalling events that may be implicated in these actions. These events include the sequential activation of Src-like kinases, MAP kinases, small GTPases and calcium signalling. How this progress in the understanding of TF associated signal transduction may generate answers as to the mechanism through which TF exerts it pleiotropic effects will be focus of this review.

Some association properties of bovine SH-kappa-casein.

(1) It is shown that kappa-casein association is characterized by a critical micelle concentration which decreases as the ionic strength is increased. (2) The kappa-casein polymer molecular weight was calculated from the weight-average apparent molecular weight by taking into consideration the monomer concentration and the excluded volume. The degree of polymerization is 30 and does not depend on ionic strength between 0.1 and 1 M. (3) The non-electrical contribution to the standard free energy of association is -38 kJ/mol monomer. The electrical part is small: 1-2 kJ/mol monomer depending on the ionic strength and kappa-casein genetic variant. (4) The limitation of size and the size itself of the kappa-casein polymer can be explained by the theory of self-assembly of virus particles by Caspar and Klug (D.L.D. Caspar and A. Klug, Cold Spring Harb. Symp. Quant. Biol. 27 (1962)1). (5) Extending this theory to casein micelle assembly, it is predicted that micelles are distributed preferentially over a restricted number of sizes.

Determination of the allelic and haplotype frequencies of three polymorphisms in the promoter region of the human protein C gene.

The frequencies of three polymorphisms in the promoter region of the human protein C gene (C/T at -1654, A/G at -1641 and A/T at -1476) have been determined. All three polymorphisms show allelic frequencies of about 0.60 and 0.40. The determination of the frequencies of the haplotypes of these polymorphisms showed that three of them (CGT, TAA and CAA) account for 88% of the observed haplotypes.

Protease-activated receptor-2 induces migration of pancreatic cancer cells in an extracellular ATP-dependent manner.

BACKGROUND: Protease-activated receptor 2 (PAR-2) is a G protein-coupled receptor suggested to play an important role in the proliferation and migration of tumor cells of epithelial origin. However, the role of PAR-2 in the setting of pancreatic cancer remains largely unexplored. OBJECTIVES: To understand the importance of PAR-2 in pancreatic cancer cell migration. METHODS AND RESULTS: The present study shows that PAR-2 does not affect pancreatic cancer cell proliferation but significantly induces the migration of pancreatic cancer cells in scratch assays. Interestingly, PAR-2 does not affect migration in a trans-well setting. This apparent discrepancy depends on extracellular ATP release in the scratch assays and the addition of exogenous (ATP)-induced PAR-2-dependent migration in trans-well assays, whereas a specific P2Y11 receptor antagonist prevents PAR-2-driven migration in scratch assays. In the scratch assays, inhibitors of Src, Rac, protein kinase C, mitogen-activated protein kinase kinase, p38, and epidermal growth factor (EGF) receptor blocked PAR-2-driven migration, whereas they did not affect fetal calf serum-driven wound closure. CONCLUSION: Taken together, PAR-2 activation drives pancreatic cancer cell migration via an EGF-Src-Rac-p38/mitogen-activated protein kinase kinase/EGF1/2 signaling pathway, which is facilitated by extracellular ATP. Targeting the PAR-2/ATP-driven signaling pathway may therefore limit cell migration, which could inhibit pancreatic cancer metastasis.