PAR1 signaling on tumor cells limits tumor growth by maintaining a mesenchymal phenotype in pancreatic cancer.

Protease activated receptor-1 (PAR1) expression is associated with disease progression and overall survival in a variety of cancers. However, the importance of tumor cell PAR1 in pancreatic ductal adenocarcinomas (PDAC) remains unexplored. Utilizing orthotopic models with wild type and PAR1-targeted PDAC cells, we show that tumor cell PAR1 negatively affects PDAC growth, yet promotes metastasis. Mechanistically, we show that tumor cell-specific PAR1 expression correlates with mesenchymal signatures in PDAC and that PAR1 is linked to the maintenance of a partial mesenchymal cell state. Indeed, loss of PAR1 expression results in well-differentiated pancreatic tumors in vivo, with enhanced epithelial characteristics both in vitro and in vivo. Taken together, we have identified a novel growth inhibitory role of PAR1 in PDAC, which is linked to the induction, and maintenance of a mesenchymal-like phenotype. The recognition that PAR1 actively limits pancreatic cancer cell growth suggest that the contributions of PAR1 to tumor growth differ between cancers of epithelial origin and that its targeting should be applied with care.

Dabigatran potentiates gemcitabine-induced growth inhibition of pancreatic cancer in mice.

Pancreatic cancer is one of the most lethal solid malignancies with little treatment options. We have recently shown that expression of protease activated receptor (PAR)-1 in the tumor microenvironment drives progression and induces chemoresistance of pancreatic cancer. As thrombin is the prototypical PAR-1 agonist, here we addressed the effect of the direct thrombin inhibitor dabigatran on pancreatic cancer growth and drug resistance in an orthotropic pancreatic cancer model. We show that dabigatran treatment did not affect primary tumor growth whereas it significantly increased tumor dissemination throughout the peritoneal cavity. Increased dissemination was accompanied by intratumoral bleeding and increased numbers of aberrant and/or collapsed blood vessels in the primary tumors. In combination with gemcitabine, dabigatran treatment limited primary tumor growth, did not induce bleeding complications and prevented tumor cell dissemination. Dabigatran was however not as efficient as genetic ablation of PAR-1 in our previous study suggesting that thrombin is not the main PAR-1 agonist in the setting of pancreatic cancer. Overall, we show that dabigatran potentiates gemcitabine-induced growth inhibition of pancreatic cancer but does not affect primary tumor growth when used as a monotherapy.

Pharmacological Targeting of Protease-Activated Receptor 2 Affords Protection from Bleomycin-Induced Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease that remains refractory to therapy. Despite increasing evidence that protease-activated receptor 2 (PAR-2) contributes to fibrosis, its importance in pulmonary fibrosis is under debate. We addressed whether PAR-2 deficiency persistently reduces bleomycin-induced pulmonary fibrosis or merely delays disease progression and whether pharmacological PAR-2 inhibition limits experimental pulmonary fibrosis. Bleomycin was instilled intranasally into wild-type or PAR-2-deficient mice in the presence/absence of a specific PAR-2 antagonist (P2pal-18S). Pulmonary fibrosis was consistently reduced in PAR-2-deficient mice throughout the fibrotic phase, as evident from reduced Ashcroft scores (29%) and hydroxyproline levels (26%) at d 28. Moreover, P2pal-18S inhibited PAR-2-induced profibrotic responses in both murine and primary human pulmonary fibroblasts (p < 0.05). Once daily treatment with P2pal-18S reduced the severity and extent of fibrotic lesions in lungs of bleomycin-treated wild-type mice but did not further reduce fibrosis in PAR-2-deficient mice. Importantly, P2pal-18S treatment starting even 7 d after the onset of fibrosis limits pulmonary fibrosis as effectively as when treatment was started together with bleomycin instillation. Overall, PAR-2 contributes to the progression of pulmonary fibrosis, and targeting PAR-2 may be a promising therapeutic strategy for treating pulmonary fibrosis.

Protease-activated receptor (PAR)-2 is required for PAR-1 signalling in pulmonary fibrosis.

Idiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease of unknown aetiology. Compelling evidence suggests that both protease-activated receptor (PAR)-1 and PAR-2 participate in the development of pulmonary fibrosis. Previous studies have shown that bleomycin-induced lung fibrosis is diminished in both PAR-1 and PAR-2 deficient mice. We thus have been suggested that combined inactivation of PAR-1 and PAR-2 would be more effective in blocking pulmonary fibrosis. Human and murine fibroblasts were stimulated with PAR-1 and PAR-2 agonists in the absence or presence of specific PAR-1 or PAR-2 antagonists after which fibrotic markers like collagen and smooth muscle actin were analysed by Western blot. Pulmonary fibrosis was induced by intranasal instillation of bleomycin into wild-type and PAR-2 deficient mice with or without a specific PAR-1 antagonist (P1pal-12). Fibrosis was assessed by hydroxyproline quantification and (immuno)histochemical analysis. We show that specific PAR-1 and/or PAR-2 activating proteases induce fibroblast migration, differentiation and extracellular matrix production. Interestingly, however, combined activation of PAR-1 and PAR-2 did not show any additive effects on these pro-fibrotic responses. Strikingly, PAR-2 deficiency as well as pharmacological PAR-1 inhibition reduced bleomycin-induced pulmonary fibrosis to a similar extent. PAR-1 inhibition in PAR-2 deficient mice did not further diminish bleomycin-induced pulmonary fibrosis. Finally, we show that the PAR-1-dependent pro-fibrotic responses are inhibited by the PAR-2 specific antagonist. Targeting PAR-1 and PAR-2 simultaneously is not superior to targeting either receptor alone in bleomycin-induced pulmonary fibrosis. We postulate that the pro-fibrotic effects of PAR-1 require the presence of PAR-2.

Targeting protease activated receptor-1 with P1pal-12 limits bleomycin-induced pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis is the most devastating fibrotic diffuse parenchymal lung disease which remains refractory to pharmacological therapies. Therefore, novel treatments are urgently required. Protease-activated receptor (PAR)-1 is a G-protein-coupled receptor that mediates critical signalling pathways in pathology and physiology. Bleomycin-induced lung fibrosis has been shown to be diminished in PAR-1-deficient mice. The purpose of this study is to investigate whether pharmacological PAR-1 inhibition is a potential therapeutic option to combat pulmonary fibrosis. METHODS: Pulmonary fibrosis was induced by intranasal instillation of bleomycin into wild-type mice with or without a specific PAR-1 antagonist (ie, P1pal-12, a pepducin that blocks the PAR-1/G-protein interaction). Fibrosis was assessed by hydroxyproline analysis, immunohistochemistry, quantitative PCR and western blot for fibrotic markers expression. RESULTS: We first show that P1pal-12 effectively inhibits PAR-1-induced profibrotic responses in fibroblasts. Next, we show that once daily treatment with 0.5, 2.5 or 10 mg/kg P1pal-12 reduced the severity and extent of fibrotic lesions in a dose-dependent manner. These findings correlated with significant decreases in fibronectin, collagen and α smooth muscle actin expression at the mRNA and protein level in treated mice. To further establish the potential clinical applicability of PAR-1 inhibition, we analysed fibrosis in mice treated with P1pal-12 1 or 7 days after bleomycin instillation. Interestingly, when administered 7 days after the induction of fibrosis, P1pal-12 was as effective in limiting the development of pulmonary fibrosis as when administration was started before bleomycin instillation. CONCLUSIONS: Overall, targeting PAR-1 may be a promising treatment for pulmonary fibrosis.

CCAAT/enhancer-binding protein δ facilitates bacterial dissemination during pneumococcal pneumonia in a platelet-activating factor receptor-dependent manner.

CCAAT/enhancer-binding protein δ (C/EBPδ) recently emerged as an essential player in the inflammatory response to bacterial infections. C/EBPδ levels increase rapidly after a proinflammatory stimulus, and increasing C/EBPδ levels seem to be indispensable for amplification of the inflammatory response. Here we aimed to elucidate the role of C/EBPδ in host defense in community-acquired pneumococcal pneumonia. We show that C/EBPδ(-/-) mice are relatively resistant to pneumococcal pneumonia, as indicated by delayed and reduced mortality, diminished outgrowth of pneumococci in lungs, and reduced dissemination of the infection. Moreover, expression of platelet-activating factor receptor (PAFR), which is known to potentiate bacterial translocation of gram-positive bacteria, was significantly reduced during infection in C/EBPδ(-/-) mice compared with WT controls. Importantly, cell stimulation experiments revealed that C/EBPδ potentiates PAFR expression induced by lipoteichoic acid and pneumococci. Thus, C/EBPδ exaggerates bacterial dissemination during Streptococcus pneumoniae-induced pulmonary infection, suggesting an important role for PAFR-dependent bacterial translocation.

Betulinic acid delivered in liposomes reduces growth of human lung and colon cancers in mice without causing systemic toxicity.

Betulinic acid (BetA) is a plant-derived pentacyclic triterpenoid with potent anticancer capacity that targets the mitochondrial pathway of apoptosis. BetA has a broad efficacy in vitro against prevalent cancer types, including lung, colorectal, prostate, cervix and breast cancer, melanomas, neuroblastomas, and leukemias. The cytotoxic effects of the compound against healthy cells are minimal, rendering BetA a promising potential anticancer drug. However, because of the weak hydrosolubility of BetA, it has been difficult to study its efficacy in vivo and a pharmaceutical formulation is not yet available. We report the development of a liposome formulation of BetA and show its successful application in mice. Large liposomes, assembled without cholesterol to reduce their rigidity, efficiently incorporated BetA. Nude mice xenografted with human colon and lung cancer tumors were treated intravenously with the BetA-containing liposomes. Tumor growth was reduced to more than 50% compared with the control treatment, leading to an enhanced survival of the mice. Oral administration of the liposomal formulation of BetA also slowed tumor growth. Any signs of systemic toxicity caused by BetA treatment were absent. Thus, liposomes are an efficient formulation vehicle for BetA, enabling its preclinical development as a nontoxic compound for the treatment of cancers.

Protease-activated receptor-2 induces myofibroblast differentiation and tissue factor up-regulation during bleomycin-induced lung injury: potential role in pulmonary fibrosis.

Idiopathic pulmonary fibrosis constitutes the most devastating form of fibrotic lung disorders and remains refractory to current therapies. The coagulation cascade is frequently activated during pulmonary fibrosis, but this observation has so far resisted a mechanistic explanation. Recent data suggest that protease-activated receptor (PAR)-2, a receptor activated by (among others) coagulation factor (F)Xa, plays a key role in fibrotic disease; consequently, we assessed the role of PAR-2 in the development of pulmonary fibrosis in this study. We show that PAR-2 is up-regulated in the lungs of patients with idiopathic pulmonary fibrosis and that bronchoalveolar lavage fluid from these patients displays increased procoagulant activity that triggers fibroblast survival. Using a bleomycin model of pulmonary fibrosis, we show that bleomycin induces PAR-2 expression, as well as both myofibroblast differentiation and collagen synthesis. In PAR-2-/- mice, both the extent and severity of fibrotic lesions are reduced, whereas myofibroblast differentiation is diminished and collagen expression is decreased. Moreover, fibrin deposition in the lungs of fibrotic PAR-2-/- mice is reduced compared with wild-type mice due to differential tissue factor expression in response to bleomycin. Taken together, these results suggest an important role for PAR-2 in the development of pulmonary fibrosis, and the inhibition of the PAR-2-coagulation axis may provide a novel therapeutic approach to treat this devastating disease.

The cholinergic anti-inflammatory pathway regulates the host response during septic peritonitis.

BACKGROUND: The nervous system, through the vagus nerve, can down-regulate inflammation in vivo by decreasing the release of tumor necrosis factor- alpha by endotoxin-stimulated macrophages. This anti-inflammatory effect is mediated by an interaction between acetylcholine, the principal neurotransmitter of the vagus nerve, and cholinergic nicotinic acetylcholine receptors on macrophages. METHODS: We determined the role of this "cholinergic anti-inflammatory pathway" during septic peritonitis induced in mice by intraperitoneal injection of live Escherichia coli. Septic peritonitis was preceded by inhibition of the cholinergic anti-inflammatory pathway by unilateral cervical vagotomy, by stimulation of this pathway by pretreatment of mice with nicotine, or by a combination of both interventions. RESULTS: Initial cytokine release during septic peritonitis was enhanced after previous vagotomy and was decreased after nicotine pretreatment, independently of the integrity of the vagus nerve. Further study established that vagotomy before septic peritonitis resulted in an enhanced influx of neutrophils and a marked increase in proinflammatory cytokine levels and liver damage. Conversely, nicotine pretreatment strongly decreased cell influx, proinflammatory cytokine levels, and liver damage, whereas bacterial clearance and survival were impaired. DISCUSSION: These data provide the first evidence, to our knowledge, of an important role of the vagus nerve in regulating the innate immune response to a severe bacterial infection.

Therapeutic effects of troglitazone in experimental chronic pancreatitis in mice.

Peroxisome proliferator-activated receptor (PPAR)-gamma controls growth, differentiation, and inflammation. PPAR-gamma agonists exert anti-inflammatory effects in vitro and inhibit the activation of pancreas stellate cells, implicated in the formation and progression of fibrosis. We determined the influence of troglitazone, a ligand for PPAR-gamma, on pancreatic damage and fibrosis in experimental chronic pancreatitis. Mice received six hourly intraperitoneal injections with 50 microg/kg of cerulein or saline, three times a week for 6 weeks. One week after the last injection all mice were sacrificed. Untreated mice were compared with mice treated with troglitazone either during weeks 1 to 6 or weeks 4 to 6. All mice that received cerulein injections displayed histopathological signs of chronic pancreatitis at week 7. Troglitazone treatment improved all markers for severity of pancreatitis. Moreover, early and postponed troglitazone treatments were equally effective in diminishing intrapancreatic fibrosis as quantified by Sirius red staining, hydroxyproline content, and laminin staining as well as the increased number of pancreatic stellate cells and pancreas levels of transforming growth factor-beta. Thus, troglitazone attenuated pancreatic damage and inflammation in experimental chronic pancreatitis and remained beneficial in a therapeutic setting when given after initial damage had been established.