Protease-activated receptor-1 (PAR1) promotes epithelial-endothelial transition through Twist1 in hepatocellular carcinoma.

BACKGROUND: Tumor cells transfer into endothelial cells by epithelial-endothelial transition (EET), which is characterized by vasculagenic mimicry (VM) in morphology. VM can change tumor microcirculation, progression, and metastasis. However, the molecular mechanisms of endothelial-like transition remain unclear. EET is a subtype of epithelial-mesenchymal transition (EMT). Twist1, a transcriptional regulatory factor of EMT, is an important factor that induces EET in hepatocellular carcinoma(HCC), but the upstream signal of Twist1 is unclear. METHODS: Expression plasmids, Ca mobilization, and three-dimensional cultures were evaluated. Western blot assay, reporter gene assay, and immunofluorescence staining were conducted. A murine xenograft model was established. Analyses of immunohistochemistry, patient samples, and complementary DNA (cDNA) microarrays were also performed. RESULTS: This study demonstrated that protease-activated receptor-1 (PAR1) can increase the expression of endothelial markers and enhance VM formation by upregulating Twist1 both in vitro and in vivo through thrombin binding. Thrombin not only activates PAR1 but also promotes PAR1 internalization in a time-dependent manner. Clinical pathological analysis further confirms that PAR1 expression is directly correlated with the endothelial marker expression, VM formation, and metastasis and indicates poor survival rate of patients with tumors. CONCLUSION: PAR1 promotes EET through Twist1 in HCC.

Oral Antiplatelet Therapy for Secondary Prevention of Acute Coronary Syndrome.

Patients surviving an acute coronary syndrome (ACS) remain at increased risk of ischemic events long term. This paper reviews current evidence and guidelines for oral antiplatelet therapy for secondary prevention following ACS, with respect to decreased risk of ischemic events versus bleeding risk according to individual patient characteristics and risk factors. Specifically, data are reviewed from clinical studies of clopidogrel, prasugrel, ticagrelor and vorapaxar, as well as the results of systematic reviews and meta-analyses looking at the benefits and risks of oral antiplatelet therapy, and the relative merits of shorter versus longer duration of dual antiplatelet therapy, in different patient groups.

Thrombin induces protease-activated receptor 1 signaling and activation of human atrial fibroblasts and dabigatran prevents these effects.

BACKGROUND: Data with animal cells and models suggest that thrombin activates cardiac fibroblasts (Fib) to myofibroblasts (myoFib) via protease-activated receptor 1 (PAR1) cleavage, and in this way promotes adverse atrial remodeling and, thereby, atrial fibrillation (AF). OBJECTIVE: Here, we explored the effects of thrombin on human atrial Fib and whether they are antagonized by the clinically available direct thrombin inhibitor, dabigatran. METHODS: Fib isolated from atrial appendages of patients without AF undergoing elective cardiac surgery were evaluated for PAR expression and treated with thrombin with or without dabigatran. PAR1 cleavage, downstream signaling and myoFib markers were investigated by immunofluorescence and Western blot. Collagen synthesis, activity of matrix metalloprotease (MMP)-2 and proliferation were assessed by Picro-Sirius red staining, gelatinolytic zymography and BrdU incorporation, respectively. Fib function was studied as capability to contract a collagen gel and stimulate the chemotaxis of peripheral blood monocytes from healthy volunteers. RESULTS: Primary human atrial Fib expressed PAR1, while levels of the other PARs were very low. Thrombin triggered PAR1 cleavage and phosphorylation of ERK1/2, p38 and Akt, elicited a switch to myoFib enriched for αSMA, fibronectin and type I collagen, and induced paracrine/autocrine transforming growth factor beta-1, cyclooxygenase-2, endothelin-1 and chemokine (C-C motif) ligand 2 (CCL2); conversely, MMP-2 activity decreased. Thrombin-primed cells displayed enhanced proliferation, formed discrete collagen-containing cellular nodules, and stimulated the contraction of a collagen gel. Furthermore, their conditioned medium caused monocytes to migrate. All these effects were prevented by dabigatran. CONCLUSION: These results with human cells complete the knowledge about thrombin actions on cardiac Fib and strengthen the translational potential of the emerging paradigm that pharmacological blockade of thrombin may counteract molecular and cellular events underlying AF.

TGF-ß induced PAR-1 expression promotes tumor progression and osteoclast differentiation in giant cell tumor of bone.

Although protease activated receptor-1 (PAR-1) has been confirmed as an oncogene in many cancers, the role of PAR-1 in giant cell tumor (GCT) of bone has been rarely reported. The mechanism of PAR-1 in tumor-induced osteoclastogenesis still remains unclear. In the present study, we detected that PAR-1 was significantly upregulated in GCT of bone compared to normal tissues, while TGF-ß was also overexpressed in GCT tissues and could promote the expression of PAR-1 in a dose and time dependent manner. Using the luciferase reporter assay, we found that two downstreams of TGF-ß, Smad3 and Smad4, could activate the promoter of PAR-1, which might explain the mechanism of TGF-ß induced PAR-1 expression. Meanwhile, PAR-1 was also overexpressed in microvesicles from stromal cells of GCT (GCTSCs), and might be transported from GCTSCs to monocytes through microvesicles. In addition, knockout of PAR-1 by TALENs in GCTSCs inhibited tumor growth, angiogenesis and osteoclastogenesis in GCT in vitro. Using the chick CAM models, we further showed that inhibition of PAR-1 suppressed tumor growth and giant cell formation in vivo. Using microarray assay, we detected a number of genes involved in osteoclastogenesis as the possible downstreams of PAR-1, which may partly explain the mechanism of PAR-1 in GCT. In brief, for the first time, these results reveal an upstream regulatory role of TGF-ß in PAR-1 expression, and PAR-1 expression promotes tumor growth, angiogenesis and osteoclast differentiation in GCT of bone. Hence, PAR-1 represents a novel potential therapeutic target for GCT of bone.

Reduction of intracerebral hemorrhage by rivaroxaban after tPA thrombolysis is associated with downregulation of PAR-1 and PAR-2.

This study aimed to assess the risk of intracerebral hemorrhage (ICH) after tissue-type plasminogen activator (tPA) treatment in rivaroxaban compared with warfarin-pretreated male Wistar rat brain after ischemia in relation to activation profiles of protease-activated receptor-1, -2, -3, and -4 (PAR-1, -2, -3, and -4). After pretreatment with warfarin (0.2 mg/kg/day), low-dose rivaroxaban (60 mg/kg/day), high-dose rivaroxaban (120 mg/kg/day), or vehicle for 14 days, transient middle cerebral artery occlusion was induced for 90 min, followed by reperfusion with tPA (10 mg/kg/10 ml). Infarct volume, hemorrhagic volume, immunoglobulin G leakage, and blood parameters were examined. Twenty-four hours after reperfusion, immunohistochemistry for PARs was performed in brain sections. ICH volume was increased in the warfarin-pretreated group compared with the rivaroxaban-treated group. PAR-1, -2, -3, and -4 were widely expressed in the normal brain, and their levels were increased in the ischemic brain, especially in the peri-ischemic lesion. Warfarin pretreatment enhanced the expression of PAR-1 and PAR-2 in the peri-ischemic lesion, whereas rivaroxaban pretreatment did not. The present study shows a lower risk of brain hemorrhage in rivaroxaban-pretreated compared with warfarin-pretreated rats following tPA administration to the ischemic brain. It is suggested that the relative downregulation of PAR-1 and PAR-2 by rivaroxaban compared with warfarin pretreatment might be partly involved in the mechanism of reduced hemorrhagic complications in patients receiving rivaroxaban in clinical trials. © 2016 Wiley Periodicals, Inc.

Protease-activated receptor-1 inhibits proliferation but enhances leukemia stem cell activity in acute myeloid leukemia.

Eradication of leukemia stem cells (LSCs) is the ultimate goal of treating acute myeloid leukemia (AML). We recently showed that the combined loss of Runx1/Cbfb inhibited the development of MLL-AF9-induced AML. However, c-Kit+/Gr-1- cells remained viable in Runx1/Cbfb-deleted cells, indicating that suppressing RUNX activity may not eradicate the most immature LSCs. In this study, we found upregulation of several hemostasis-related genes, including the thrombin-activatable receptor PAR-1 (protease-activated receptor-1), in Runx1/Cbfb-deleted MLL-AF9 cells. Similar to the effect of Runx1/Cbfb deletion, PAR-1 overexpression induced CDKN1A/p21 expression and attenuated proliferation in MLL-AF9 cells. To our surprise, PAR-1 deficiency also prevented leukemia development induced by a small number of MLL-AF9 leukemia stem cells (LSCs) in vivo. PAR-1 deficiency also reduced leukemogenicity of AML1-ETO-induced leukemia. Re-expression of PAR-1 in PAR-1-deficient cells combined with a limiting-dilution transplantation assay demonstrated the cell-dose-dependent role of PAR-1 in MLL-AF9 leukemia: PAR-1 inhibited rapid leukemic proliferation when there were a large number of LSCs, while a small number of LSCs required PAR-1 for their efficient growth. Mechanistically, PAR-1 increased the adherence properties of MLL-AF9 cells and promoted their engraftment to bone marrow. Taken together, these data revealed a multifaceted role for PAR-1 in leukemogenesis, and highlight this receptor as a potential target to eradicate primitive LSCs in AML.

Upregulation of Thrombin/Matrix Metalloproteinase-1/Protease-Activated Receptor-1 Chain in Proliferative Diabetic Retinopathy.

PURPOSE: Selective proteolytic activation of protease-activated receptor-1 (PAR1) by thrombin and matrix metalloproteinase-1 (MMP-1) plays a central role in enhancing angiogenesis. We investigated the expression levels of thrombin, MMP-1, and PAR1 and correlated these levels with vascular endothelial growth factor (VEGF) in proliferative diabetic retinopathy (PDR). In addition, we examined the expression of PAR1 and thrombin in the retinas of diabetic rats and PAR1 in human retinal microvascular endothelial cells (HRMEC) following exposure to high-glucose, the proinflammatory cytokines interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and the hypoxia mimetic agent cobalt chloride (CoCl2). METHODS: Vitreous samples from 32 PDR and 23 nondiabetic patients, epiretinal membranes from 10 patients with PDR, retinas of rats, and HRMEC were studied by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and Western blot analysis. An assay for in vitro cell migration angiogenesis was performed in HRMEC. RESULTS: In epiretinal membranes, PAR1 was expressed in vascular endothelial cells, CD45-expressing leukocytes, and myofibroblasts. ELISA and Western blot assays revealed significant increases in the expression levels of thrombin, MMP-1, and VEGF in vitreous samples from PDR patients compared to nondiabetic controls. Significant positive correlations were found between the levels of VEGF and the levels of thrombin (r = 0.41; p = 0.006) and MMP-1 (r = 0.66; p < 0.0001). Significant increases of cleaved PAR1 (approximately 50 kDa) and the proteolytically active thrombin (approximately 50 kDa) were detected in rat retinas after induction of diabetes. The proinflammatory cytokines IL-1ß and TNF-α, but not high-glucose and CoCl2, induced upregulation of cleaved PAR1 (approximately 30 kDa) in HRMEC. In addition, thrombin and MMP-1 induced VEGF in HRMEC and vorapaxar, a PAR1 inhibitor, inhibited thrombin-induced migration in HRMEC. CONCLUSIONS: Interactions among thrombin, MMP-1, PAR1, and VEGF might facilitate angiogenesis in PDR.

Down-regulation of PAR1 activity with a pHLIP-based allosteric antagonist induces cancer cell death.

Even though abnormal expression of G protein-coupled receptors (GPCRs) and of their ligands is observed in many cancer cells of various origins, only a few anti-cancer compounds directly act on their signalling. One promising approach to modulate their activity consists of targeting the receptor cytoplasmic surfaces interacting with the associated G-proteins using peptides mimicking the intracellular loops of the receptor. Thus, to be fully effective, the peptide mimics must be selectively targeted to the tumour while sparing healthy tissues, translocated across the cell membrane and stay anchored to the cytoplasmic leaflet of the plasma membrane. In the present study, we introduce a novel way to selectively target and inhibit the activity of a GPCR in cancer cells under acidic conditions, such as those found in solid tumours. We find that the conjugation of a peptide fragment derived from the third intracellular loop (i3) of the protease-activated receptor 1 (PAR1) to a peptide that can selectively target tumours solely based on their acidity [pH(Low) Insertion Peptide (pHLIP)], produces a construct capable of effectively down-regulating PAR1 activity in a concentration- and pH-dependent manner and of inducing a potent cytotoxic effect in a panel of cancer cells that is proportional to the relative level of receptor expression at the cell surface. This strategy not only allows for a more selective targeting and specific intracellular delivery than current approaches, but also offers new possibilities for developing novel anti-cancer drugs targeting GPCRs.

Activation of platelet protease-activated receptor-1 induces epithelial-mesenchymal transition and chemotaxis of colon cancer cell line SW620.

The aim of the present study was to examine the role of protease-activated receptor-1 (PAR1)-stimulated platelet activation in the epithelial-mesenchymal transition (EMT) and migration of colon cancer cells, and to identify the underlying mechanisms. TFLLR-NH2, a PAR1 agonist, was used to activate platelets and the platelet supernatants were used to treat the SW620 colon cancer cell line. Expression of E-cadherin and vimentin on SW620 cells was detected by immunofluorescence and western blotting, and the level of the transforming growth factor ß1 (TGF-ß1) was measured using ELISA following the activation of platelets by TFLLR-NH2. miR-200b expression was detected using quantitative PCR in SW620 cells. In order to investigate the chemotactic ability of the SW620 cells, the expression of CXC chemokine receptor type 4 (CXCR4) was measured by flow cytometry. Transwell migration assays were performed following exposure of the cells to the supernatant of PAR1-activated platelets. SW620 cells cultured in the supernatant of TFLLR-NH2-activated platelets upregulated E-cadherin expression and downregulated the vimentin expression. In the in vitro platelet culture system, a TFLLR-NH2 dose-dependent increase of secreted TGF-ß1 was detected in the supernatant. The activation of PAR1 on the platelets led to the inhibition of miR-200b expression in the SW620 cells that were cultured in platelet-conditioned media. The number of SW620 cells that penetrated through the Transwell membrane increased with the dose of TFLLR-NH2 used to treat the platelets. The percentage of CXCR4-positive SW620 cells was significantly higher when they were exposed to the supernatant of platelets cultured for 24 h with PAR1 agonist than when cultured in non-conditioned media (40.89 ± 6.74 vs. 3.47 ± 1.40%, P < 0.01). Platelet activation with a PAR1 agonist triggered TGF-ß secretion, which induced EMT of SW620 human colon cancer cells via the downregulation of miR-200b expression, and activated platelets had a chemotactic effect on colon cancer cells mediated by the upregulation of CXCR4 on the cell surface.

A brief exposure to tryptase or thrombin potentiates fibrocyte differentiation in the presence of serum or serum amyloid p.

A key question in both wound healing and fibrosis is the trigger for the initial formation of scar tissue. To help form scar tissue, circulating monocytes enter the tissue and differentiate into fibroblast-like cells called fibrocytes, but fibrocyte differentiation is strongly inhibited by the plasma protein serum amyloid P (SAP), and healthy tissues contain very few fibrocytes. In wounds and fibrotic lesions, mast cells degranulate to release tryptase, and thrombin mediates blood clotting in early wounds. Tryptase and thrombin are upregulated in wound healing and fibrotic lesions, and inhibition of these proteases attenuates fibrosis. We report that tryptase and thrombin potentiate human fibrocyte differentiation at biologically relevant concentrations and exposure times, even in the presence of concentrations of serum and SAP that normally completely inhibit fibrocyte differentiation. Fibrocyte potentiation by thrombin and tryptase is mediated by protease-activated receptors 1 and 2, respectively. Together, these results suggest that tryptase and thrombin may be an initial trigger to override SAP inhibition of fibrocyte differentiation to initiate scar tissue formation.

Neuroprotective effect of ginsenoside-Rg1 on cerebral ischemia/reperfusion injury in rats by downregulating protease-activated receptor-1 expression.

AIMS: Ginsenoside-Rg1 (G-Rg1), a saponin that is a primary component of ginseng, is very useful and important in traditional Chinese medicine for stroke. The objective of this study was to explore the mechanisms underlying the neuroprotective effect of G-Rg1 on focal cerebral ischemia/reperfusion. MAIN METHODS: Focal cerebral ischemia was induced by middle cerebral artery occlusion. Neurological examinations were performed by using Longa's 5-point scale. The brain infarct volume was determined by the 2,3,5-triphenyltetrazolium chloride staining. The permeability of the blood-brain barrier (BBB) was evaluated by Evans blue dye. Western blot and quantitative RT-PCR were used to assess protease-activated receptor-1 (PAR-1) expression. KEY FINDINGS: After G-Rg1 treatment, there was a significant decrease in the neurobehavioral function score compared with normal saline (NS) treatment after ischemia/reperfusion (P<0.05). G-Rg1 significantly reduced the infarct volume compared with NS treatment after ischemia/reperfusion (P<0.001). The permeability of the BBB was significantly decreased in the G-Rg1 group compared with the NS group (P<0.05 or P<0.01). Western blot and quantitative real time RT-PCR indicated that G-Rg1 administration down-regulated the expression of PAR-1 in the ischemic hemisphere compared with NS administration (P<0.01 and P<0.05, respectively). The level of PAR-1 expression strongly correlated with BBB permeability in both the G-Rg1- and NS-treated rats (r=0.856 and r=0.908, respectively, P<0.01). SIGNIFICANCE: G-Rg1 may ameliorate the neurological injury, the brain infarct volume and the BBB permeability induced by focal cerebral ischemia in rats and its neuroprotective mechanism is related to the down-regulation of PAR-1 expression.

Thrombin conducts epithelial­mesenchymal transition via protease­activated receptor­1 in human gastric cancer.

Epithelial-mesenchymal transition (EMT) is thought to be a key step for cancer metastasis. Using an immunohistochemical approach with gastric carcinoma tissue, we found the expression of protease-activated receptor-1 (PAR1), along with a metalloproteinase known to activate PAR1, were associated with poorer prognosis, compared with expression-negative tumors, and activated PAR1 promotes gastric cancer cell invasion and proliferation in vivo. In this study we observed EMT induction by the PAR1 agonist α-thrombin, in human gastric cell lines stably expressing PAR1. We investigated α-thrombin-induced changes in the cell forms of pcDNA3.1-MKN45 (MKN45/Mock), pcDNA3.1­PAR1 transfected MKN45 (MKN45/PAR1), and MKN74. Expression levels of epithelial and mesenchymal markers as well as the distribution of transcriptional factors of E-cadherin in the cytoplasm and nucleus were also noted in these cell lines. We observed α-thrombin-induced morphological changes in MKN45/PAR1 and MKN74 cells. Western blotting and immunohistochemistry of these cells indicated a fall in the expression level of E-cadherin and an increase in fibronectin expression after 48 h. PAR1 activation also induced significant increases in nuclear levels of the Snail which is a repressor of E-cadherin gene expression. We found EMT in gastric cancer cell lines that underwent α-thrombin-induced PAR1 activation.

Advanced glycation end products potentiate citrated plasma-evoked oxidative and inflammatory reactions in endothelial cells by up-regulating protease-activated receptor-1 expression.

Advanced glycation end products (AGEs) and receptor RAGE interaction contribute to endothelial cell damage in diabetes. Several thrombogenic abnormalities are also involved in diabetic vascular complications. However, the pathological role of thrombin and protease-activated receptor-1 (PAR-1) system in AGE-induced endothelial cell (EC) damage remains unclear. In this study, we investigated the effects of rivaroxaban, an inhibitor of factor Xa on 3% citrated human plasma-evoked reactive oxygen species (ROS) generation and RAGE, monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1) gene expression in AGE-exposed ECs. We further examined whether FR171113, an inhibitor of PAR-1 blocked the plasma-induced EC damage and if AGEs increased PAR-1 expression in ECs. Human citrated plasma stimulated ROS generation and RAGE, MCP-1 and ICAM-1 expression in ECs, all of which were potentiated by the treatment with AGEs. Rivaroxaban or FR171113 significantly inhibited these derangements in plasma- or plasma plus AGE-exposed ECs. Moreover, AGEs significantly increased the PAR-1 levels in ECs. The present study suggests that citrated plasma could induce oxidative and inflammatory reactions in ECs via the activation of thrombin-PAR-1 system and that AGEs could potentiate the plasma-evoked EC damages via up-regulation of PAR-1. Blockade of the crosstalk between AGE-RAGE axis and coagulation system by rivaroxaban might be a novel therapeutic target for thromboembolic disorders in diabetes.

Thrombin promotes proliferation of human lung fibroblasts via protease activated receptor-1-dependent and NF-κB-independent pathways.

Acute and chronic respiratory diseases are associated with abnormal coagulation regulation and fibrolysis. However, the detailed mechanism by which coagulation regulation and fibrolysis affect the occurrence and development of lung diseases remain to be elucidated. Protease activated receptor-1 (PAR-1), a major high-affinity thrombin receptor, and nuclear factor kappa B (NF-κB), a transcription factor, are involved in cell survival, differentiation, and proliferation. We have investigated the potential mechanism of thrombin-induced fibroblast proliferation and roles of PAR-1 and NF-κB signalling in this process. The effect of thrombin on proliferation of human pulmonary fibroblasts (HPF) was assessed by 5-bromo-2-deoxyuridine (BrdU) incorporation assay. The expression of PAR1 and NF-κB subunit p65 protein was detected by Western blot. Nuclear translocation of p65 was examined by laser scanning confocal microscopy. We show that thrombin significantly increased proliferation of HPF as determined by induction of BrdU-positive incorporation ratio. Induced PAR1 protein expression was also seen in HPF cells treated with thrombin. However, thrombin had no significant effect on expression and translocation of NF-κB p65 in HPF cells. The results indicate that, by increasing protein expression and interacting with PAR1, thrombin promotes HPF proliferation. NF-κB signalling appears to play no role in this process.

Inhibition of airway epithelial-to-mesenchymal transition and fibrosis by kaempferol in endotoxin-induced epithelial cells and ovalbumin-sensitized mice.

Chronic airway remodeling is characterized by structural changes within the airway wall, including smooth muscle hypertrophy, submucosal fibrosis and epithelial shedding. Epithelial-to-mesenchymal transition (EMT) is a fundamental mechanism of organ fibrosis, which can be induced by TGF-ß. In the in vitro study, we investigated whether 1-20 µM kaempferol inhibited lipopolysaccharide (LPS)-induced bronchial EMT in BEAS-2B cells. The in vivo study explored demoting effects of 10-20 mg/kg kaempferol on airway fibrosis in BALB/c mice sensitized with ovalbumin (OVA). LPS induced airway epithelial TGF-ß1 signaling that promoted EMT with concurrent loss of E-cadherin and induction of α-smooth muscle actin (α-SMA). Nontoxic kaempferol significantly inhibited TGF-ß-induced EMT process through reversing E-cadherin expression and retarding the induction of N-cadherin and α-SMA. Consistently, OVA inhalation resulted in a striking loss of epithelial morphology by displaying myofibroblast appearance, which led to bronchial fibrosis with submucosal accumulation of collagen fibers. Oral administration of kaempferol suppressed collagen deposition, epithelial excrescency and goblet hyperplasia observed in the lung of OVA-challenged mice. The specific inhibition of TGF-ß entailed epithelial protease-activated receptor-1 (PAR-1) as with 20 µM kaempferol. The epithelial PAR-1 inhibition by SCH-79797 restored E-cadherin induction and deterred α-SMA induction, indicating that epithelial PAR-1 localization was responsible for resulting in airway EMT. These results demonstrate that dietary kaempferol alleviated fibrotic airway remodeling via bronchial EMT by modulating PAR1 activation. Therefore, kaempferol may be a potential therapeutic agent targeting asthmatic airway constriction.

miR-20b regulates expression of proteinase-activated receptor-1 (PAR-1) thrombin receptor in melanoma cells.

The proteinase-activated receptor 1 (PAR-1) plays a central role in melanoma progression and its expression level is believed to correlate with the degree of cancer invasiveness. Here, we show that PAR-1 is post-transcriptionally regulated by miR-20b microRNA in human melanoma cells. PAR-1 was found to be expressed in metastatic melanoma cells but was barely detectable in primary melanoma. By transducing primary melanoma cells with a lentivirus containing a 3'-UTR construct of PAR-1 mRNA, we could show that endogenous melanoma microRNAs interacted with PAR-1 3'-UTR and silenced a fused luciferase reporter. Transfection of an inhibitor against miR-20b into primary melanoma cells reversed this process. Finally, transfection of miR-20b mimic into metastatic melanoma cells caused downregulation of the luciferase reporter. We conclude that miR-20b regulates expression of melanoma PAR-1 receptor, which may explain the differential expression of PAR-1 observed in human melanoma.

Expression of tissue factor signaling pathway elements correlates with the production of vascular endothelial growth factor and interleukin-8 in human astrocytoma patients.

The expression levels of tissue factor (TF), the clotting initiator protein, have been correlated with angiogenesis and the histological grade of malignancy in glioma patients. The pro-tumor function of TF is linked to a family of G protein-coupled receptors known as protease-activated receptors (PARs), which may be activated by blood coagulation proteases. Activation of PARs elicits a number of responses, including the expression of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8). In the present study, we analyzed the expression of TF signaling pathway elements (TF, PAR1 and PAR2) and evaluated their correlation with the expression of downstream products (VEGF and IL-8) in human astrocytoma patients. Quantitative PCR (qPCR) showed a significant increase in TF expression in grade IV (glioblastoma) tumors, which was inversely correlated with the expression of the tumor-suppressor PTEN. Immunohistochemistry and qPCR analyses demonstrated a highly significant elevation in the expression of PAR1, but not PAR2, in tumor samples from high-grade astrocytoma patients. The elevated VEGF expression levels detected in the high-grade astrocytoma samples were positively correlated with TF, PAR1 and PAR2 expression. In addition, IL-8 was significantly increased in glioblastoma patients and positively correlated with TF and PAR2 expression. Further in vitro assays employing the human glioma cell lines U87-MG and HOG demonstrated that a synthetic peptide PAR2 agonist stimulated VEGF and IL-8 production. Our findings suggest a role for TF signaling pathway elements in astrocytoma progression, particularly in glioblastoma. Therefore, TF/PAR signaling elements may be suitable targets for the development of new therapies for the treatment of aggressive glioma.

Protein C anticoagulant system--anti-inflammatory effects.

Activated protein C (APC) plays active roles in preventing progression of a number of disease processes. These include thrombosis due to its direct anticoagulant activity which is likely augmented by its cytoprotective activity, thereby limiting exposure of procoagulant cellular membrane surfaces on cells. Beyond that, the pathway signals the cells to prevent apoptosis, to dampen inflammation, to increase endothelial barrier function, and to selectively downregulate some genes implicated in disease progression. Most of these functions are manifested to APC binding to endothelial protein C receptor (EPCR) allowing PAR1 activation, but activation of other PARS is also implicated in some cases. In addition to EPCR orchestrating these changes, CD11b is also capable of supporting APC signaling. Selective control of these pathways offers potential in new therapeutic approaches to disease.

PAR-1 and thrombin: the ties that bind the microenvironment to melanoma metastasis.

Progression of melanoma is dependent on cross-talk between tumor cells and the adjacent microenvironment. The thrombin receptor, protease-activated receptor-1 (PAR-1), plays a key role in exerting this function during melanoma progression. PAR-1 and its activating factors, which are expressed on tumor cells and the surrounding stroma, induce not only coagulation but also cell signaling, which promotes the metastatic phenotype. Several adhesion molecules, cytokines, growth factors, and proteases have recently been identified as downstream targets of PAR-1 and have been shown to modulate interactions between tumor cells and the microenvironment in the process of melanoma growth and metastasis. Inhibiting such interactions by targeting PAR-1 could potentially be a useful therapeutic modality for melanoma patients.

Systemic administration of argatroban inhibits protease-activated receptor-1 expression in perihematomal tissue in rats with intracerebral hemorrhage.

The present study investigated the role of thrombin in the expression of protease-activated receptor-1 (PAR-1), and the effect of argatroban (Arg) a direct thrombin inhibitor, on PAR-1 expression in perihematomal tissue with intracerebral hemorrhage (ICH). For these experiments 90 rats were divided into 5 groups: sham, ICH, argatroban-treated ICH (ICH+Arg), thrombin (TM) and argatroban-treated thrombin (TM+Arg). The ICH model or thrombin injection models were established by injecting autologous blood or thrombin, respectively. Rats in TM+Arg and ICH+Arg groups were administered argatroban (0.9mg/kg) after models were established for 3h and 12h, intraperitoneally. All rats were killed to harvest brains after models were established for 24h. The levels of PAR-1 protein and PAR-1 mRNA expression were detected by Western blot and RT-PCR, respectively. Brain water content was also measured. Our results showed that the levels of PAR-1 protein or PAR-1 mRNA in ICH and TM groups were up-regulated compared to that observed for the sham group; while the levels observed in ICH+Arg group and TM+Arg group were significantly lower than that observed for the ICH group and TM group (P<0.01 or P<0.05). The intraperitoneal administration argatroban also significantly reduced edema in ICH or TM group (P<0.05). Our observations suggested that the production of thrombin following ICH play a key role in the up-regulation of PAR-1 and anti-PAR-1 by systemic administration of argatroban, and may be a potential strategy for ICH therapy.