Search

Sepsis is a complex disorder caused by a dysregulated host response to infection, with high levels of morbidity and mortality. Treatment aimed to modulate immune response and maintain vascular function is still one of the major clinical challenges. This study was designed to test the effect of the small molecule 1-Piperidine Propionic Acid (1-PPA) as molecular targeted agent to block protease-activated receptor 2 (PAR2), one of the major modulators of inflammatory response in LPS-induced experimental endotoxemia. In the THP-1 cell line, LPS-induced cytokine expression was inhibited by 1-PPA in a dose-dependent manner. In LPS-injected mice, treatment with 1-PPA was effective in reducing mortality and sepsis-related symptoms and improved cardiac function parameters. After 6 h from LPS injection, a significant decrease in IL-6, IL-1β, and IL-10 was observed in the lung tissue of 1-PPA-treated mice, compared to controls. In these mice, a significant decrease in vasoactive molecules, especially kininogen-1, was also observed, mainly in the liver. Histopathological analysis confirmed typical features of sepsis in different organs and these findings were markedly reduced in mice treated with 1-PPA. These data demonstrate the effectiveness of 1-PPA in protecting the whole organism from sepsis-induced damage.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is spreading worldwide, largely due to unhealthy lifestyles that contribute to the rise in diabetes, metabolic syndrome, and obesity. In this situation, the progression of injury to metabolic steatohepatitis can evolve to cirrhosis and, eventually, to hepatocellular carcinoma (HCC). It is well known that serine protease enzymes with different functions in cellular homeostasis act as signaling molecules that regulate liver inflammation by activating the protease-activated receptors (PARs) family members, expressed on the cellular plasma membrane. Among them, PAR2 plays a central role in the activation of signaling pathways in response to changes in the extracellular microenvironment. Experimental data have provided evidence that PAR2 is involved not only in inflammatory response but also in insulin resistance, lipid metabolism, and cancer. The major aims of this narrative review are addressed to assess PAR2 involvement in inflammation, metabolism, and liver disease progression and to explore possible therapeutic strategies, based on PAR2 inhibition, in order to prevent its biological effects in the context of MAFLD and cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Villano and Pontisso.)

Background and Purpose: Airway epithelial cells (AECs) regulate the activation of epithelial-mesenchymal trophic units (EMTUs) during airway remodelling through secretion of signalling mediators. However, the major trigger and the intrinsic pathogenesis of airway remodelling is still obscure., Experimental Approach: The differing expressed genes in airway epithelia related to airway remodelling were screened and verified by RNA-sequencing and signalling pathway analysis. Then, the effects of increased cathepsin K (CTSK) in airway epithelia on airway remodelling and EMTU activation were identified both in vitro and in vivo, and the molecular mechanism was elucidated in the EMTU model. The potential of CTSK as an an effective biomarker of airway remodelling was analysed in an asthma cohort of differing severity. Finally, an inhibitor of CTSK was administered for potential therapeutic intervention for airway remodelling in asthma., Key Results: The expression of CTSK in airway epithelia increased significantly along with the development of airway remodelling in a house dust mite (HDM)-stressed asthma model. Increased secretion of CTSK from airway epithelia induced the activation of EMTUs by activation of the PAR2-mediated pathway. Blockade of CTSK inhibited EMTU activation and alleviated airway remodelling as an effective intervention target of airway remodelling., Conclusion and Implications: Increased expression of CTSK in airway epithelia is involved in the development of airway remodelling in asthma through EMTU activation, mediated partly through the PAR2-mediated signalling pathway. CTSK is a potential biomarker for airway remodelling, and may also be a useful intervention target for airway remodelling in asthma patients., (© 2024 British Pharmacological Society.)

Protease-activated receptor 2 (PAR2) has garnered attention as a potential therapeutic target in breast cancer. PAR2 is implicated in the activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) via G protein and beta-arrestin pathways, contributing to the proliferation and metastasis of breast cancer cells. Despite the recognized role of PAR2 in breast cancer progression, clinically effective PAR2 antagonists remain elusive. To address this unmet clinical need, we synthesized and evaluated a series of novel compounds that target the orthosteric site of PAR2. Using in silico docking simulations, we identified compound 9a, an optimized derivative of compound 1a ((S)-N-(1-(benzylamino)-1-oxo-3-phenylpropan-2-yl)benzamide), which exhibited enhanced PAR2 antagonistic activity. Subsequent molecular dynamics simulations comparing 9a with the partial agonist 9d revealed that variations in ligand-induced conformational changes and interactions dictated whether the compound acted as an antagonist or agonist of PAR2. The results of this study suggest that further development of 9a could contribute to the advancement of PAR2 antagonists as potential therapeutic agents for breast cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Punicalagin is recorded to be a potent anti-inflammatory drug, while its effect on inflammation existing in ventilator-induced lung injury (VILI) requires further verification. Rats were pretreated with punicalagin, followed by VILI modeling. Lung histopathological examination was performed with hematoxylin-eosin staining accompanied by the lung injury score. The lung wet/dry (W/D) weight ratio and total bronchoalveolar lavage fluid (BALF) protein level were measured. After transfection with protease-activated receptor-2 (PAR2) overexpression plasmids, mouse alveolar epithelial MLE-12 cells were treated with punicalagin and then subjected to cyclic stretching. Punicalagin's cytotoxicity to MLE-12 cells were measured by MTT assay. The levels of inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6), PAR2, NLR family pyrin domain containing-3 (NLRP3), and apoptosis-associated speck-like protein containing a CARD (ASC) in the BALF, lung tissues or cells were analyzed by enzyme-linked immune-sorbent assay (ELISA), qRT-PCR or/and western blot. Punicalagin treatment attenuated VILI-induced lung histopathological changes and counteracted VILI-induced increases in the lung injury score, W/D weight ratio and total protein level in BALF. Also, punicalagin treatment counteracted in vivo VILI/cyclic stretching-induced increases in the levels of PAR2, inflammatory cytokines, NLRP3, and ASC. PAR2 overexpression potentiated the cyclic stretching-induced effects, while punicalagin treatment revoked this PAR2 overexpression-induced potentiation effect. In turn, PAR2 overexpression partly resisted the punicalagin treatment-induced counteractive effects on the cyclic stretching-induced effects. Punicalagin suppresses inflammation in VILI through PAR2 inhibition-induced inhibition of NLRP3 inflammasome activation., (© 2022 John Wiley & Sons A/S.)

Osimertinib, as the third-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs), is a first-line molecularly targeted drug for non-small cell lung cancer (NSCLC). However, the emergence of therapeutic resistance to osimertinib markedly impairs its efficiency and efficacy, leading to the failure of clinical applications. Novel molecular targets and drugs are urgently needed for reversing osimertinib resistance in NSCLC. Protease-activated receptor 2 (PAR2) that belongs to a subfamily of G protein-coupled receptors can stimulate the transactivation of EGFR to regulate multiple cellular signalling, actively participating in tumour progression. This study firstly discovered that PAR2 expression was notably enhanced when NSCLC cells became resistant to osimertinib. A PAR2 inhibitor facilitated osimertinib to attenuate EGFR transactivation, ERK phosphorylation, EMT and PD-L1 expression which were associated to osimertinib resistance. The combination of the PAR2 inhibitor and osimertinib also notably blocked cell viability, migration, 3D sphere formation and in vivo tumour growth whereas osimertinib itself lost such inhibitory effects in osimertinib-resistant NSCLC cells. Importantly, this reversal effect of PAR2 blockade was uncovered to depend on ERK-mediated EMT and PD-L1, since inhibition of β-arrestin or ERK, which could be modulated by PAR2, sensitized osimertinib to prevent EMT, PD-L1 expression and consequently overcame osimertinib resistance. Thus, this study demonstrated that PAR2 antagonism could limit ERK-mediated EMT and immune checkpoints, consequently attenuating EGFR transactivation and reactivate osimertinib. It suggested that PAR2 may be a novel drug target for osimertinib resistance, and PAR2 inhibition may be a promising strategy candidate for reversing EGFR-TKI resistance in NSCLC., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Oxidized low-density lipoprotein (ox-LDL)-induced endothelial dysfunction plays an important role in the initiation and development of cardiovascular diseases, especially atherosclerosis (AS). Protease-activated receptor 2 (PAR-2) is a receptor for inflammatory proteases. However, the biological function of PAR-2 in endothelial cells and the pathophysiological process of AS are still unknown. In the current study, we found that treatment with ox-LDL increased the gene and protein expressions of PAR-2 in EA.hy926 endothelial cells. Interestingly, we found that antagonism of PAR-2 with its specific antagonist AZ3451 could ameliorate ox-LDL-induced lactate dehydrogenase (LDH) release. Treatment with AZ3451 considerably improved the mitochondrial function by restoring the mitochondrial membrane potential and increasing the levels of intracellular adenosine triphosphate (ATP). Also, we found that AZ3451 attenuated ox-LDL-induced expression and production of pro-inflammatory cytokines such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-8 (IL-8). Treatment with AZ3451 also mitigated the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). Notably, our results demonstrated that the presence of AZ3451 alleviated ox-LDL-induced expression of the endothelial cell adhesion molecules vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1). Mechanistically, we found that AZ3451 attenuated ox-LDL-induced activation of nuclear factor-κB (NF-κB) by reducing the levels of intracellular NF-κB p65 and the luciferase activity of NF-κB promoter. Based on these findings, we conclude that PAR-2 might become a novel therapeutic target for the treatment of AS.

Previously we have shown that trypsin, a protein typically involved in digestion, is released from gills of both fresh and saltwater fishes into surrounding water under stress or injury. We have also shown that each species produces trypsin with different specific activities. In this report, using zebrafish as a model, we identified that trypsin induces an aversive response in zebrafish larvae and adult zebrafish. Since Protease-Activated Receptor 2 (PAR2) responds to trypsin, we tested whether the aversive response is dependent on the activation of PAR2 located on the zebrafish skin cells. Zebrafish larvae treated separately with neomycin and zinc sulfate also showed aversive response indicating neuromast, and olfactory cells are not involved in this aversion. Cultured keratinocytes from zebrafish showed a response to trypsin. Zebrafish larvae subjected to knockdown of par2a also exhibited reduced escape response. Similarly, par2a-deficient mutant larvae displayed no response to trypsin. Since it has been shown that stress activates PAR2 and sends signals to the brain as shown by the increased c-fos expression, we tested c-fos expression in adult zebrafish brains after trypsin treatment of adults and found enhanced c-fos expression by qRT-PCR. Taken together, our results show that the trypsin activates PAR2 on keratinocytes signaling the brain, and this pathway of trypsin-induced escape response will provide a unique communication mechanism in zebrafish. Furthermore, since PAR2 activation also occurs in pain/pruritus sensing, this model might be useful in elucidating components of signaling pathways in pain/pruritus., Competing Interests: The authors have declared that no competing interests exist.

The occurrence of allergic diseases induced by aeroallergens has increased in the past decades. Among inhalant allergens, mites remain the important causal agent of allergic diseases. Storage mites- Tyrophagus putrescentiae are found in stored products or domestic environments. Major allergen Tyr-p3 plays a significant role in triggering IgE-mediated hypersensitivity. However, its effects on pulmonary inflammation, internalization, and activation in human epithelium remain elusive. Protease-activated receptors (PARs) are activated upon cleavage by proteases. A549 cells were used as an epithelial model to examine the PAR activation by Tyr-p3 and therapeutic potential of PAR-2 antagonist (GB88) in allergic responses. Enzymatic properties and allergen localization of Tyr-p3 were performed. The release of inflammatory mediators, phosphorylation of mitogen-activated protein kinase (MAPK), and cell junction disruptions were evaluated after Tyr-p3 challenge. Enzymatic properties determined by substrate digestion and protease inhibitors indicated that Tyr-p3 processes a trypsin-like serine protease activity. The PAR-2 mRNA levels were significantly increased by nTyr-p3 but inhibited by protease inhibitors or GB88. Protease allergen of nTyr-p3 significantly increased the levels of pro-inflammatory cytokines (IL-6 and TNF-α), chemokine (IL-8), and IL-1β in epithelial cells. nTyr-p3 markedly increased phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and MAP kinase. When cells were pretreated with GB88 then added nTyr-p3, the phosphorylated ERK1/2 did not inhibit by GB88. GB88 increased ERK1/2 phosphorylation in human epithelium cells. GB88 is able to block PAR-2-mediated calcium signaling which inhibits the nTyr-p3-induced Ca 2+ release. Among the pharmacologic inhibitors, the most effective inhibitor of the nTyr-p3 in the induction of IL-8 or IL-1β levels was GB88 followed by SBTI, MAPK/ERK, ERK, and p38 inhibitors. Levels of inflammatory mediators, including GM-CSF, VEGF, COX-2, TSLP, and IL-33 were reduced by treatment of GB88 or SBTI. Further, GB88 treatment down-regulated the nTyr-p3-induced PAR-2 expression in allergic patients with asthma or rhinitis. Tight junction and adherens junction were disrupted in epithelial cells by nTyr-p3 exposure; however, this effect was avoided by GB88. Immunostaining with frozen sections of the mite body showed the presence of Tyr-p3 throughout the intestinal digestive system, especially in the hindgut around the excretion site. In conclusion, our findings suggest that Tyr-p3 from domestic mites leads to disruption of the airway epithelial barrier after inhalation. Proteolytic activity of Tyr-p3 causes the PAR-2 mRNA expression, thus leading to the release of numerous inflammatory mediators. Antagonism of PAR2 activity suggests GB88 as the therapeutic potential for anti-inflammation medicine, especially in allergy development triggered by protease allergens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wang, Yu, Tsai, Yu and Liao.)

Conditions that resemble osteoarthritis (OA) were produced by injection of sodium monoiodoacetate (MIA) into the knee joints of mice. Bone marrow derived mast cells (BMMCs) injected into the OA knee joints enhanced spontaneous pain. Since no spontaneous pain was observed when BMMCs were injected into the knee joints of control mice that had not been treated with MIA, BMMCs should be activated within the OA knee joints and release some pain-inducible factors. Protease activated receptor-2 (PAR2) antagonist (FSLLRY-NH2) almost abolished the pain-enhancing effects of BMMCs injected into the OA knee joints, suggesting that tryptase, a mast cell protease that is capable of activating PAR2, should be released from the injected BMMCs and enhance pain through activation of PAR2. When PAR2 agonist (SLIGKV-NH2) instead of BMMCs was injected into the OA knee joints, it was also enhanced pain. Apyrase, an ATP degrading enzyme, injected into the OA knee joints before BMMCs suppressed the pain enhanced by BMMCs. We showed that purinoceptors (P2X4 and P2X7) were expressed in BMMCs and that extracellular ATP stimulated the release of tryptase from BMMCs. These observations suggest that ATP may stimulate degranulation of BMMCs and thereby enhanced pain. BMMCs injected into the OA knee joints stimulated expression of IL-1β, IL-6, TNF-α, CCL2, and MMP9 genes in the infrapatellar fat pads, and PAR2 antagonist suppressed the stimulatory effects of BMMCs. Our study suggests that intermittent pain frequently observed in OA knee joints may be due, at least partly, to mast cells through activation of PAR2 and action of ATP, and that intraarticular injection of BMMCs into the OA knee joints may provide a useful experimental system for investigating molecular mechanisms by which pain is induced in OA knee joints., Competing Interests: The authors have declared that no competing interests exist.

Protease-activated receptor-2 (PAR2) has been extensively studied since its discovery in the mid-1990. Despite the advances in understanding PAR2 pharmacology, it has taken almost 25 years for the first inhibitor to reach clinical trials, and so far, no PAR2 antagonist has been approved for human use. Research has employed classical approaches to develop a wide array of PAR2 agonists and antagonists, consisting of peptides, peptoids and antibodies to name a few, with a surge in patent applications over this period. Recent breakthroughs in PAR2 structure determination has provided a unique insight into proposed PAR2 ligand binding sites. Publication of the first crystal structures of PAR2 resolved in complex with two novel non-peptide small molecule antagonists (AZ8838 and AZ3451) revealed two distinct binding pockets, originally presumed to be allosteric sites, with a PAR2 antibody (Fab3949) used to block tethered ligand engagement with the peptide-binding domain of the receptor. Further studies have proposed orthosteric site occupancy for AZ8838 as a competitive antagonist. One company has taken the first PAR2 antibody (MEDI0618) into phase I clinical trial (NCT04198558). While this first-in-human trial is at the early stages of the assessment of safety, other research into the structural characterisation of PAR2 is still ongoing in an attempt to identify new ways to target receptor activity. This review will focus on the development of novel PAR2 modulators developed to date, with an emphasis placed upon the advances made in the pharmacological targeting of PAR2 activity as a strategy to limit chronic inflammatory disease., (© 2020 The Author(s).)

Protease-activated receptor-2 (PAR2) has been implicated in multiple pathophysiologies but drug discovery is challenging due to low small molecule tractability and a complex activation mechanism. Here we report the pharmacological profiling of a potent new agonist, suggested by molecular modelling to bind in the putative orthosteric site, and two novel PAR2 antagonists with distinctly different mechanisms of inhibition. We identify coupling between different PAR2 binding sites. One antagonist is a competitive inhibitor that binds to the orthosteric site, while a second antagonist is a negative allosteric modulator that binds at a remote site. The allosteric modulator shows probe dependence, more effectively inhibiting peptide than protease activation of PAR2 signalling. Importantly, both antagonists are active in vivo, inhibiting PAR2 agonist-induced acute paw inflammation in rats and preventing activation of mast cells and neutrophils. These results highlight two distinct mechanisms of inhibition that potentially could be targeted for future development of drugs that modulate PAR2.

Atopic Dermatitis (AD) is characterized by skin barrier disruption and an aberrant immune response. Doxycycline is tetracycline antibiotics broadly used systemically to treat inflammatory dermatologic conditions. Several studies have shown doxycycline has anti-inflammatory and pro-healing properties, mainly by blocking tissue proteolytic activity. It is our hypothesis that daily application of a novel doxycycline topical formulation in AD subjects will reduce severity of the disease, by blocking cutaneous proteases activity and restoring skin barrier function and inflammation. To test this hypothesis, we performed a proof of concept, open-label clinical study. Subjects enrolled in the study (n = 15) applied NanoDOX ® Hydrogel 1% daily for 4 weeks on a chosen eczematous area. Investigational drug was well tolerated, and no local or systemic adverse events due to investigational drug were reported. Notably, a significant clinical improvement was observed based on a modified Eczema Area & Severity Index (EASI) score of the treated area from start of treatment to 14 and 28 days post-treatment (P < .001). A significant improvement of pruritus was also observed (P = .02). This proof of concept clinical trial is first to explore the impact of a non-systemic doxycycline treatment on AD patients. Our results provide evidence to investigate novel AD treatment strategies targeting cutaneous proteases activity., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Protease-activated receptor-2 (PAR2) is involved in inflammatory responses and pain, therefore representing a promising therapeutic target for the treatment of immune-mediated inflammatory diseases. However, as for other GPCRs, PAR2 can activate multiple signaling pathways and those involved in inflammatory responses remain poorly defined. Here, we describe a new selective and potent PAR2 inhibitor (I-287) that shows functional selectivity by acting as a negative allosteric regulator on Gα q and Gα 12/13 activity and their downstream effectors, while having no effect on G i/o signaling and βarrestin2 engagement. Such selective inhibition of only a subset of the pathways engaged by PAR2 was found to be sufficient to block inflammation in vivo. In addition to unraveling the PAR2 signaling pathways involved in the pro-inflammatory response, our study opens the path toward the development of new functionally selective drugs with reduced liabilities that could arise from blocking all the signaling activities controlled by the receptor.

There is strong evidence showing that the activation of peripheral proteinase-activated receptors type 2 (PAR-2) can initiate hyperalgesic and inflammatory responses in the joint. However, to date, there is no report of functional spinal PAR-2 receptors in arthritis models. The primary aim of this study was to evaluate the activity of PAR-2 receptors at the spinal cord by using a potent agonist (FLIGRL) in naïve animals, and an antagonist (GB83) in different models of joint pain. Saline or FLIGRL (10 nmol) were injected intrathecally in naïve animals and nociceptive behaviour was evaluated over a 24 h time period by von Frey hair algesiometry. Paw withdrawal threshold decreased from 3 to 24 h and this allodynic effect was blocked by GB83 (90 nmol; i.p.). Acute inflammatory joint pain was induced by injecting 0.5 % kaolin/carrageenan (50 μL each) into the right knee joint of male Wistar rats (24 h recovery). Chronic inflammatory joint pain was modelled by intraarticular injection of Freund's complete adjuvant (FCA; 50 μL; 7 days recovery) or chronic osteoarthritis pain by sodium monoiodoacetate (MIA; 3 mg; 14 days recovery). Animals were then treated with either intrathecal vehicle or 10 nmol of GB83 (10 μL); joint pain was evaluated throughout the subsequent 3 h period. The acute inflammatory pain induced by kaolin/carrageenan was not affected by treatment with GB83. Conversely, both chronic arthritis models demonstrated increased hind paw withdrawal threshold after spinal injection of the PAR-2 antagonist. Based on these results, spinal PAR-2 receptors are involved in joint nociceptive processing in chronic but not acute arthritic conditions., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Asthma is a chronic inflammatory and multifactorial respiratory tract disease. It affects over 18 million adults and 6 million children in the USA with Puerto Ricans showing the highest prevalence (12%-19%). This airways illness can be triggered by an environmental stimulus such as grass pollen, fungi spores, cockroaches allergens, dust mites metabolic compounds, and importantly, by environmental proteases such as trypsin and tryptase. Because of the pivotal role of proteases in the onset of asthma pathophysiology, we focused this study on the serine Protease Activated Receptor-2 (PAR-2), a G-protein-coupled receptor widely expressed in cells across the respiratory tract. Herein, we measured the activation of PAR-2 on primary pulmonary bronchial/tracheal epithelial cells, human small airway epithelial cells, lung bronchial smooth muscle cells (with and without asthma). We tested human-derived eosinophils from 61 Puerto Rican participants (33 asthmatic and 28 non-asthmatic). As surrogate of PAR-2 activation or inhibition we used intracellular calcium mobilization assay. We hypothesized that following exposure of the PAR-2 agonist (AC264613), the studied human primary cell types will increase the mobilization of intracellular calcium levels. In contrast, we expected a decrease of the intracellular calcium levels upon exposure to a PAR-2 antagonist (FSLLRY-NH2). The Puerto Rican-derived eosinophils were analyzed for the proinflammatory markers MAPK/PI3K using flow cytometry (n = 8). As expected, the PAR-2 agonist significantly increased the activation of PAR-2 on the bronchial/tracheal epithelial cells, bronchial smooth muscle cells and human small airway epithelial cells (P = .01). The PAR-2 antagonist significantly decreased the intracellular calcium levels of these lung primary down to undetectable levels (P = .01). Remarkably, the asthmatic-derived eosinophils showed a striking 300% increase of intracellular calcium mobilization suggesting a severe response to the PAR-2 agonist stimuli in asthmatics. In contrast, there were no significant changes between groups after adding the PAR-2 antagonist. Our outcomes revealed that PAR-2 antagonist effectively inhibited the studied primary cells, expecting to decrease the immune response of eosinophils. Most importantly, our results reveal a promising role for the PAR-2 antagonist in targeting bronchial/tracheal epithelial cells, human small airway epithelial cells and bronchial smooth muscle cells with the potential to oblige an asthma adjuvant therapy.

Objective: This study aims to investigate the role of protease-activated receptor (PAR) 2 and mast cell (MC) tryptase in LPS-induced lung inflammation and neutrophil recruitment in the lungs of C57BL/6 mice., Methods: C57BL/6 mice were pretreated with the PAR2 antagonist ENMD-1068, compound 48/80 or aprotinin prior to intranasal instillation of MC tryptase or LPS. Blood leukocytes, C-X-C motif chemokine ligand (CXCL) 1 production leukocytes recovered from bronchoalveolar lavage fluid (BALF), and histopathological analysis of the lung were evaluated 4 h later. Furthermore, we performed experiments to determine intracellular calcium signaling in RAW 264.7 cells stimulated with LPS in the presence or absence of a protease inhibitor cocktail or ENMD-1068 and evaluated PAR2 expression in the lungs of LPS-treated mice., Results: Pharmacological blockade of PAR2 or inhibition of proteases reduced neutrophils recovered in BALF and LPS-induced calcium signaling. PAR2 blockade impaired LPS-induced lung inflammation, PAR2 expression in the lung and CXCL1 release in BALF, and increased circulating blood neutrophils. Intranasal instillation of MC tryptase increased the number of neutrophils recovered in BALF, and MC depletion with compound 48/80 impaired LPS-induced neutrophil migration., Conclusion: Our study provides, for the first time, evidence of a pivotal role for MCs and MC tryptase in neutrophil migration, lung inflammation and macrophage activation triggered by LPS, by a mechanism dependent on PAR2 activation.

Animal studies have suggested that transient receptor potential ion channels and G-protein coupled receptors play important roles in itch transmission. TRPV3 gain-of-function mutations have been identified in patients with Olmsted syndrome, which is associated with severe pruritus. However, the mechanisms causing itch remain poorly understood. Here, we show that keratinocytes lacking TRPV3 impair the function of protease-activated receptor 2 (PAR2), resulting in reduced neuronal activation and scratching behavior in response to PAR2 agonists. Moreover, we show that TRPV3 and PAR2 were upregulated in skin biopsies from patients and mice with atopic dermatitis, whereas their inhibition attenuated scratching and inflammatory responses in mouse atopic dermatitis models. These results reveal a previously unrecognized link between TRPV3 and PAR2 in keratinocytes to convey itch information and suggest that a blockade of PAR2 or TRPV3 individually or both may serve as a potential approach for antipruritic therapy in atopic dermatitis., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Protease-activated receptors (PARs) are coagulation protease targets, and they increase expression of inflammatory cytokines and chemokines in various diseases. Of all PARs, previous reports have shown that PAR1 or PAR2 inhibition is protective against diabetic glomerular injury. However, how PAR1 and PAR2 cooperatively contribute to diabetic kidney disease (DKD) pathogenesis and whether dual blockade of PARs is more effective in DKD remain elusive. To address this issue, male type I diabetic Akita mice heterozygous for endothelial nitric oxide synthase were used as a model of DKD. Mice (4 mo old) were divided into four treatment groups and administered vehicle, PAR1 antagonist (E5555, 60 mg·kg -1 ·day -1 ), PAR2 antagonist (FSLLRY, 3 mg·kg -1 ·day -1 ), or E5555 + FSLLRY for 4 wk. The results showed that the urinary albumin creatinine ratio was significantly reduced when both PAR1 and PAR2 were blocked with E5555 + FSLLRY compared with the vehicle-treated group. Dual blockade of PAR1 and PAR2 by E5555 + FSLLRY additively ameliorated histological injury, including mesangial expansion, glomerular macrophage infiltration, and collagen type IV deposition. Marked reduction of inflammation- and fibrosis-related gene expression in the kidney was also observed. In vitro, PAR1 and PAR2 agonists additively increased mRNA expression of macrophage chemoattractant protein 1 or plasminogen activator inhibitor-1 in human endothelial cells. Changes induced by the PAR1 agonist were blocked by a NF-κB inhibitor, whereas those of the PAR2 agonist were blocked by MAPK and/or NF-κB inhibitors. These findings suggest that PAR1 and PAR2 additively contribute to DKD pathogenesis and that dual blockade of both could be a novel therapeutic option for treatment of patients with DKD.

Recently, protease-activated receptor 2 (PAR2) has been proved to be involved in the inflammatory response including osteoarthritis (OA). In the present study, we found that PAR2 antagonist could remarkably improve the pathological condition of OA rats in vivo. In addition, we also found that PAR2 antagonist could suppress the production of inflammatory factors (TNF-α and Cox-2), decrease the levels of MMP-1 and MMP-13, and restrain the levels of P62 proteins and aggravate the expression of LC3-II both in vivo and in vitro. Besides, in vitro, PAR2 antagonist could increase the proliferation and colony formation of chondrocytes induced with IL-1β. Moreover, PAR2 antagonist could decrease the expression of expressions of p-p38, p-IκBα and p-NF-κB in vitro. However, PAR2 agonist exhibited the opposite effects. Furthermore, SB203580, a p38 MAPK inhibitor, could remarkably promote the proliferation of chondrocytes induced with IL-1β, could alleviate the production of TNF-α and Cox-2, could down-regulate the protein expressions of MMP-1 and MMP-13, and could decrease the expression of P62 and increase the expressions of LC3-II of chondrocytes induced with IL-1β. Importantly, SB203580 could reverse the effects of PAR2 agonist on the functions of chondrocytes induced with IL-1β. Taken together, the present data suggest that down-regulation of PAR2 can ameliorate OA through inducing autophagy via regulation of MAPK/NF-κB signaling pathway in vivo and in vitro, and PAR2 can be considered as a potential candidate to treat OA., (© 2020 The Author(s).)

Aim: To evaluate the effect of FSLLRY-NH2, a protease-activated receptor 2 (PAR2) inhibitor, on neurocognitive impairment and hippocampal neuronal degeneration in the setting of asphyxial cardiac arrest (ACA)-induced global cerebral ischemia (GCI) in rats., Material and Methods: A total of 43 Sprague-Dawley male rats were used. Shams and rats resuscitated from 9 minutes of ACA were randomized to two separate experiments including time course and short-term neurological outcomes. FSLLRY-NH2 (50 microgram [μg] per rat) was administered intranasally at 1 hour postresuscitation. Neurological function and hippocampal neuronal degeneration were evaluated after ACA., Results: Significant neurological function decline and hippocampal neuron degeneration were observed in ACA animals as compared with the shams. Treatment with FSLLRY-NH2 significantly improved neurological outcome and reduced the number of degenerating hippocampal neurons after ACA., Conclusion: Targeting PAR2 may be a novel therapeutic approach in the management of neurological dysfunction after cardiac arrest-associated ischemic injury.

Background and Aim: Oxidative stress (OS) is accused in pathogenesis of many diseases, including liver cirrhosis by many mechanisms. One of them is the disturbance of long non coding maternally expressed 3 (MEG3)/protease activated receptor 2 (PAR2) downstream pathway. We aimed to investigate the role of this axis in cirrhotic neuropathy and whether an antioxidant compound such as N-acetylcysteine (NAC) could improve the peripheral nerve function through repression of MEG3/PAR2., Methods: Thirty Wistar rats were used and divided into 5 groups; naive, thiacetamide (TAA) (200 mg/kg 3 times/week. i.p. for 8 weeks) and TAA+NAC (50 or 100 or 200 mg/kg/day) groups. Von Frey (VF) test for mechanical nociceptive responses, hepatic& neural MEG3, NF-ҡB and neural PAR2 expression by PCR, histological studies for liver and sciatic nerve together with the dorsopedal skin thickness were done., Results: TAA induced significant decrease in liver function, negative VF test, an increase in the expression of hepatic& neural MEG3, NF-ҡB and neural PAR2. The histological studies showed cirrhotic changes with atrophy of the sciatic nerve and the dorsal skin. NAC improved the liver function together with reversal of the neural: functional, biochemical and histological changes in a dose dependent manner., Conclusions: NAC could improve the peripheral neuropathy in cirrhotic rat through suppression of MEG3/PAR2 expression., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Osteoarthritis (OA) is a highly prevalent joint disorder blamed for pain and disability in older individuals. It's commonly accepted that inflammation, apoptosis, autophagy and cellular senescence participate in the progress of OA. Protease activated receptor 2 (PAR2), a member of the G-protein coupled receptors, is involved in the regulation of various inflammation diseases. Previous studies have identified PAR2 as a potential therapeutic target for the treatment of OA. Here, we investigated the role of PAR2 antagonist AZ3451 in inflammation response, apoptosis, autophagy and cellular senescence during OA. We confirmed that PAR2 expression was significantly up-regulated in OA articular cartilage tissues as well as in interleukin 1β (IL-1β) stimulated chondrocytes. We demonstrated AZ3451 could prevent the IL-1β-induced inflammation response, cartilage degradation and premature senescence in chondrocytes. Further study showed that AZ3451 attenuated chondrocytes apoptosis by activating autophagy in vitro. The P38/MAPK, NF-κB and PI3K/AKT/mTOR pathways were involved in the protective effect of AZ3451. In vivo, we found that intra-articular injection of AZ3451 could ameliorate the surgery induced cartilage degradation in rat OA model. Our work provided a better understanding of the mechanism of PAR2 in OA, and indicated that PAR2 antagonist AZ3451 might serve as a promising strategy for OA treatment.

Systemic lupus erythematosus (SLE) is characterized by the production of autoantibodies, which causes multi-organ injury such as lupus nephritis. SLE is associated with hypercoagulability. Activated coagulation factors such as tissue factor and VIIa complex and factor Xa activate protease-activated receptor 2 (PAR2). PAR2 promotes cytokine production through mitogen-activated protein kinase or nuclear factor kappa B signaling, and previous reports demonstrated that inhibition of PAR2 alleviated kidney injuries such as diabetic kidney disease and renal fibrosis in animal models. However, the involvement of PAR2 in the pathogenesis of SLE remains unclear. We therefore administered a selective PAR2 peptide antagonist, FSLLRY-NH2, to SLE-prone 4-month-old MRL-Fas lpr mice for 4 weeks. Treatment with FSLLRY-NH2 caused the significant increases in the glomerular mesangial proliferation, glomerular deposition of both immunoglobulin G and complement factor C3d, and glomerular infiltration of Mac2-positive macrophages and CD3-positive T cells, compared with MRL-Fas lpr mice treated with saline. In addition, the treatment with the PAR2 antagonist increased renal expression levels of tumor necrosis factor-α (Tnfa) and monocyte chemoattractant protein 1 (Mcp1) mRNA. Collectively, these results suggest that inhibition of PAR2 may increase the severity of inflammation in lupus nephritis; namely, opposite to previous observations, PAR2 has anti-inflammatory properties. We propose that activation of PAR2 could serve as a potential therapeutic option for patients with SLE.

Demonstration of target binding is a key requirement for understanding the mode of action of new therapeutics. The cellular thermal shift assay (CETSA) has been introduced as a powerful label-free method to assess target engagement in physiological environments. Here, we present the application of live-cell CETSA to different classes of integral multipass transmembrane proteins using three case studies, the first showing a large and robust stabilization of the outer mitochondrial five-pass transmembrane protein TSPO, the second being a modest stabilization of SERCA2, and the last describing an atypical compound-driven stabilization of the GPCR PAR2. Our data demonstrated that using modified protocols with detergent extraction after the heating step, CETSA can reliably be applied to several membrane proteins of different complexity. By showing examples with distinct CETSA behaviors, we aim to provide the scientific community with an overview of different scenarios to expect during CETSA experiments, especially for challenging, membrane bound targets.

Itch treatment is a major challenge in the dermatologist's practice. We encounter patients suffering from pruritus on a regular basis, and often lack diverse treatment options to adequately respond to the patients' needs. In the last 20 years, novel pathways have been investigated that were beyond the scope of histamine. Although most did not result in a molecule available on the Canadian market, it is interesting and important as health care providers to stay up to date with new neuronal pathways involved in itch transmission and potential new therapeutic options. In this review, we will discuss pathways targeted in new topical treatments such as antagonist of proteinase-activated receptor-2, the endocannabinoid system, neurotrophins and tropomyosin-related kinase A receptor, the transient receptor potential-vanilloid or transient receptor potential-melastatine ion channels. New systemic therapies are now focusing on antagonizing the neurokinin receptor, modulating the opioidergic system, or targeting itch cytokines such as interleukin-31.

The aim of this study was to investigate the underlying mechanisms that transforming growth factor-β (TGF-β)-mediated epithelial-to-mesenchymal transition (EMT) in tumor cells contributes to 5-FU resistance. A series of experiments involving cell viability and caspase activity analyses, siRNA transfection, RNA isolation, and quantitative-PCR (qPCR) assay, cell migration analysis, Western blotting analysis of total protein and membrane protein were performed in this study. Mouse xenograft model was used to determine the effect of the PAR2 inhibitor in vivo. In this study, we found that protease-activated receptor 2 (PAR2) induction in 5-FU therapy is correlated with TGF-β-mediated EMT and apoptosis resistance. PAR2 and TGF-β were both activated in response to 5-FU treatment in vivo and in vitro, and whereas TGF-β inhibition sensitized CRC cells to 5-FU and suppressed cell migration, PAR2 activation eliminated the effect of TGF-β inhibition. Conversely, siRNA-mediated PAR2 depletion or PAR2 inhibition with a specific inhibitor produced a similar phenotype as TGF-β signal inhibition: 5-FU sensitization and cell migration suppression. Moreover, the results of xenograft experiments indicated that the PAR2 inhibitor can enhance cell killing by 5-FU in vivo and suppress EMT signaling. Our results reveal that the TGF-β effects require the coordinating action of PAR2, suggesting that PAR2 inhibition could be a new therapeutic strategy to combat 5-FU resistance in CRC.

The collagenase subfamily of matrix metalloproteinases (MMPs) have important roles in the remodeling of collagenous matrices. The proteinase-activated receptor (PAR) family has a unique mechanism of activation requiring proteolysis of an extracellular domain forming a neo-N terminus that acts as a tethered ligand, a process that has been associated with the development of arthritis. Canonical PAR2 activation typically occurs via a serine proteinase at Arg 36 -Ser 37 , but other proteinases can cleave PARs downstream of the tethered ligand and "disarm" the receptor. To identify additional cleavage sites within PAR2, we synthesized a 42-amino-acid peptide corresponding to the extracellular region. We observed that all three soluble MMP collagenases, MMP-1, MMP-8, and MMP-13, cleave PAR2 and discovered a novel cleavage site (Ser 37 -Leu 38 ). Metalloproteinases from resorbing bovine nasal cartilage and recombinant human collagenases could cleave a quenched fluorescent peptide mimicking the canonical PAR2 activation region, and kinetic constants were determined. In PAR2-overexpressing SW1353 chondrocytes, we demonstrated that the activator peptide SLIGKV-NH 2 induces rapid calcium flux, inflammatory gene expression (including MMP1 and MMP13 ), and the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 kinase. The corresponding MMP cleavage-derived peptide (LIGKVD-NH 2 ) exhibited no canonical activation; however, we observed phosphorylation of ERK, providing evidence of biased agonism. Importantly, we demonstrated that preincubation with active MMP-1 reduced downstream PAR2 activation by a canonical activator, matriptase, but not SLIGKV-NH 2 These results support a role for collagenases as proteinases capable of disarming PAR2, revealing a mechanism that suppresses PAR2-mediated inflammatory responses., (© 2019 Falconer et al.)

Glomerular crescent formation is a hallmark of rapidly progressive forms of glomerulonephritis. Thrombosis and macrophage infiltration are features of crescent formation in human and experimental kidney disease. Protease-activated receptor-2 (PAR-2) is a G-protein coupled receptor that links coagulation and inflammation. This study investigated whether pharmacological inhibition of PAR-2 can suppress glomerular crescent formation in rat nephrotoxic serum nephritis (NTN). Disease was induced in Wistar Kyoto rats by immunisation with sheep IgG followed by administration of sheep nephrotoxic serum. Rats (n = 8/group) received the PAR-2 antagonist (GB88, 10 mg/kg/p.o.), vehicle or no treatment starting 3 days before nephrotoxic serum injection and continuing until day 14. Vehicle and untreated rats developed thrombosis and macrophage infiltration in the glomerular tuft and Bowman's space in conjunction with prominent crescent formation. Activation of JNK signalling and proliferation in parietal epithelial cells was associated with crescent formation. GB88 treatment significantly reduced crescent formation with a substantial reduction in glomerular thrombosis, reduced macrophage infiltration in Bowman's space, and reduced activation of parietal epithelial cells. However, GB88 did not protect against the development of proteinuria, renal function impairment, inflammation or tubular cell damage in the NTN model. In conclusion, PAR-2 plays a specific role in glomerular crescent formation by promoting glomerular thrombosis, macrophage accumulation in Bowman's space and activation of parietal epithelial cells., (© 2019 John Wiley & Sons Australia, Ltd.)

Asthma, a chronic inflammatory disease affecting the airways is primarily caused due to immune system dysfunction. Different inhaled allergens such as house dust mites (HDM), fungi, cockroach allergens are the main contributors to allergic asthma. Protease activated receptor-2 (PAR-2) signaling plays an important role in allergic asthma through modulation of immune mediators in airway epithelial cells (AECs). Interleukin-17A (IL-17A) signals via subunits of IL-17 receptor (IL-17R), i.e. interleukin-17 receptor A (IL-17RA) and interleukin-17 receptor C (IL-17RC), and plays a necessary role in neutrophilic infiltration in response to infectious/allergenic stimuli, however it is not known if PAR-2 activation affects IL-17A/IL-17R signaling during acute exposure to house dust mite (HDM) allergens. Therefore, our study exposed mice to HDM allergens for five days and evaluated its effect on IL-17A/IL-17R signaling, chemokine/cytokines and neutrophilic inflammation in mice. Our study shows that HDM allergens upregulate IL-17A levels in the lung and IL-17RA/IL-17RC expression in AECs. PAR-2 activation by trypsin also upregulates neutrophilic influx and IL-17A/IL-17R signaling in the lung. Upregulated IL-17A/IL-17R signaling was associated with increased BAL neutrophils, pulmonary MPO activity and proinflammatory chemokines and cytokines (IL-23, IL-6, and MCP-1 in AECs/lung) in HDM exposed mice. Further, HDM-induced IL-17A, IL-17R and chemokines/cytokines were attenuated by PAR-2 antagonist, ENMD-1068. Furthermore, HDM-primed mice treated with IL-17A had greater neutrophilic inflammation and higher levels of inflammatory cytokines/chemokines than PBS-exposed mice treated with IL-17A. This proposes that acute exposure to HDM allergens activate AECs at a very early stage where PAR-2/IL-17R signaling serves a crucial role in neutrophilic inflammation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The study aimed to determine the effects of protease-activated receptor-2 (PAR-2) on glial scar formation after spinal cord injury (SCI) in Sprague-Dawley (SD) rats and the underlying mechanisms. Rivlin and Tator's acute extradural clip compression injury (CCI) model of severe SCI was established in this study. Animals were divided into four groups: 1) sham group (laminectomy only); 2) model group, treated with normal saline; 3) PAR-2 inhibitor group; 4) PAR-2 activator group. Enhanced GFAP and vimentin expression were the markers of glial scar formation. To determine whether JNK was involved in the effects of PAR-2 on GFAP and vimentin expression, we administered anisomycin (a JNK activator) in the presence of PAR-2 inhibitor and SP600125 (a JNK inhibitor) in the presence of PAR-2 activator. At 1, 7, 14 and 28 day after SCI, Basso, Beattie, and Bresnahan (BBB) locomotor score test was used to assess the locomotor functional recovery; immunofluorescence and western blot analysis were used to assess the expression level of GFAP, vimentin and p-JNK. Double immunofluorescence staining with GFAP and tubulin beta was used to assess the glial scar formation and the remaining neurons. Results suggested that PAR-2 is involved in glial scar formation and reduces neurons residues which can cause a further worsening in the functional outcomes after SCI via JNK signaling. Therefore, it may be effective to target PAR-2 in the treatment of SCI.

PAR2 has been proposed to contribute to lesion formation and intense itch in atopic dermatitis. Here, we tested the ability of a cell-penetrating pepducin, PZ-235, to mitigate the potentially deleterious effects of PAR2 in models of atopic dermatitis. PZ-235 significantly inhibited PAR2-mediated expression of inflammatory factors NF-κB, TSLP, TNF-α, and differentiation marker K10 by 94%-98% (P < 0.001) in human keratinocytes and suppressed IL-4 and IL-13 by 68%-83% (P < 0.05) in mast cells. In delayed pepducin treatment models of oxazolone- and DNFB-induced dermatitis, PZ-235 significantly attenuated skin thickening by 43%-100% (P < 0.01) and leukocyte crusting by 57% (P < 0.05), and it inhibited ex vivo chemotaxis of leukocytes toward PAR2 agonists. Daily PZ-235 treatment of filaggrin-deficient mice exposed to dust mite allergens for 8 weeks significantly suppressed total leukocyte and T-cell infiltration by 50%-68%; epidermal thickness by 60%-77%; and skin thickening, scaling, excoriation, and total lesion severity score by 46%-56%. PZ-235 significantly reduced itching caused by wasp venom peptide degranulation of mast cells in mice by 51% (P < 0.05), which was comparable to the protective effects conferred by PAR2 deficiency. Taken together, these results suggest that a PAR2 pepducin may confer broad therapeutic benefits as a disease-modifying treatment for atopic dermatitis and itch., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Microvesicles (MVs) represent a subgroup of extracellular vesicles (EVs) emerging from various cells by blebbing of their outer membrane. Therefore, they share features such as membrane composition and antigenicity with their parental cells. Released by many immune and tumor cells, MVs act as intercellular messengers, account for horizontal gene transfer and can activate the coagulation system. With the aim to investigate their relevance for tumor cell biology, we characterized MVs released by human tumor cell lines of various origins in the absence or presence of TNF-α. After stimulation, we used the combination of low and high-speed centrifugation to enrich MVs from cell culture supernatants. We analyzed the presentation of phosphatidylserine (PS) and tissue factor (TF) activity on the cell surface and investigated their potency to induce tumor cell migration. In all tumor cell lines, TNF-α stimulation enhanced the release of MVs. While the expression of PS was universally increased, an elevated activity of procoagulant TF could be detected on MVs from lung, pancreatic, and colon carcinoma, but not from breast and ovarian cancer cell lines. Functionally, TNF-α stimulation significantly increased the potency of MVs to induce tumor cell migration. In conclusion, inflammatory conditions promote the release of MVs with increased procoagulant activity from tumor cell lines in vitro. PS-containing and TF-expressing MVs may account for systemic activation of the coagulation system as seen in cancer patients and, since they induce tumor cell migration, they may serve as biomarkers for tumor progression., (© 2018 The Authors. Cell Biology International Published by John Wiley & Sons Ltd on behalf of International Federation for Cell Biology.)

We synthesized and investigated the effects of a novel peptide B, RRFSLLRY as a novel PAR-2 antagonist. Peptide B decreased calcium levels in HaCat cells stimulated with trypsin and PAR-2 activator (SLIGKV) and cytokeratin 14 and PCNA was decreased by peptide B. Peptides B also reduced the expression of inflammatory cytokines such as TNF-α and interleukin-6. Significant increase of oxazolone-induced transepidermal water loss (TEWL) was decreased by the addition of peptide B in hairless mice. These findings suggest that the anti-inflammatory effect of peptide B is due to inhibition of PAR-2 signaling. Skin safety of peptide B was demonstrated by performing MTT assay and human repeated insult patch test. Our findings indicate that peptide B might have potential as an anti-inflammatory agent without irritating skin for use in cosmetics and medical treatment of dermatologic disorders., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A new class of isoxazole-tethered diarylheptanoids having characteristic 1,3-syn-diol and 1,3-anti-diol chemophoric moieties, e.g. 4a-d and 5a-c respectively, have been designed and synthesized starting from d-glucose following a stereo-conserved general synthetic strategy. The isoxazole heterocycle was installed using our recently elaborated methodology deploying Magtrieve™ as a selective oxidizing agent. Two of these new analogs 4a and 5a exhibited significantly improved in vitro drug-like properties including solubility, metabolic stability, cell permeability and lack of nonspecific cytotoxicity when compared with curcumin-I. In a HEK293 cell-based intracellular calcium [Ca 2+ ] i release assay, 4a and 5a, when tested at 30 μM, inhibited the trypsin agonist induced protease-activated receptor-2 (PAR2) activity by 80% and 70% respectively. IC 50 of 4a (SB70) has been determined as 6 μM which is in the same range of current benchmarks for PAR2 antagonists., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Patients with obstructive sleep apnea (OSA) have a high prevalence of atrial fibrillation (AF). Rivaroxaban, a coagulation factor Xa inhibitor, has recently been reported to show pleiotropic effects. This study investigated the influence of rivaroxaban on cardiac remodeling caused by intermittent hypoxia (IH). Male C57BL/6J mice were exposed to IH (repeated cycles of 5% oxygen for 1.5 min followed by 21% oxygen for 5 min) for 28 days with/without rivaroxaban (12 mg/kg/day) or FSLLRY, a protease-activated receptor (PAR)-2 antagonist (10 μg/kg/day). IH caused endothelial cell degeneration in the small arteries of the right atrial myocardium and increased the level of %fibrosis and 4-hydroxy-2-nonenal protein adducts in the left ventricular myocardium. IH also increased the expression of PAR-2 as well as the phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 and nuclear factor-kappa B (NF-κB) were increased in human cardiac microvascular endothelial cells. However, rivaroxaban and FSLLRY significantly suppressed these changes. These findings demonstrate that rivaroxaban attenuates both atrial and ventricular remodeling induced by IH through the prevention of oxidative stress and fibrosis by suppressing the activation of ERK and NF-κB pathways via PAR-2. Treatment with rivaroxaban could potentially become a novel therapeutic strategy for cardiac remodeling in patients with OSA and AF., (Copyright © 2018 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Background and Purpose: Previously, we demonstrated that exogenous heat shock protein 27 (HSP27/gene, HSPB1) treatment of human endothelial progenitor cells (EPCs) increases the synthesis and secretion of VEGF, improves EPC-migration/re-endothelialization and decreases neo-intima formation, suggesting a role for HSPB1 in regulating EPC function. We hypothesized that HSPB1 also affects mature endothelial cells (ECs) to alter EC-mediated vasoreactivity in vivo. Our work focused on endothelial NOS (eNOS)/NO-dependent relaxation induced by ACh and the coagulation pathway-activated receptor, proteinase-activated receptor 2 (PAR2)., Experimental Approach: Aorta rings from male and female wild-type, HSPB1-null and HSPB1 overexpressing (HSPB1o/e) mice were contracted with phenylephrine, and NOS-dependent relaxation responses to ACh and PAR2 agonist, 2-furoyl-LIGRLO-NH 2 , were measured without and with L-NAME and ODQ, either alone or in combination to block NO synthesis/action. Tissues from female HSPB1-null mice were treated in vitro with recombinant HSP27 and then used for bioassay as above. Furthermore, oestrogen-specific effects were evaluated using a bioassay of aorta isolated from ovariectomized mice., Key Results: Relative to males, HSPB1-null female mice exhibited an increased L-NAME-resistant relaxation induced by activation of either PAR2 or muscarinic ACh receptors that was blocked in the concurrent presence of both L-NAME and ODQ. mRNAs (qPCR) for eNOS and ODQ-sensitive guanylyl-cyclase were increased in females versus males. Treatment of isolated aorta tissue with HSPB1 improved tissue responsiveness in the presence of L-NAME. Ovariectomy did not affect NO sensitivity, supporting an oestrogen-independent role for HSPB1., Conclusions and Implications: HSPB1 can regulate intact vascular endothelial function to affect NO-mediated vascular relaxation, especially in females., (© 2018 The British Pharmacological Society.)

Several classes of ligands for Protease-Activated Receptors (PARs) have shown impressive anti-inflammatory and cytoprotective activities, including PAR2 antagonists and the PAR1-targeting parmodulins. In order to support medicinal chemistry studies with hundreds of compounds and to perform detailed mode-of-action studies, it became important to develop a reliable PAR assay that is operational with endothelial cells, which mediate the cytoprotective effects of interest. We report a detailed protocol for an intracellular calcium mobilization assay with adherent endothelial cells in multiwell plates that was used to study a number of known and new PAR1 and PAR2 ligands, including an alkynylated version of the PAR1 antagonist RWJ-58259 that is suitable for the preparation of tagged or conjugate compounds. Using the cell line EA.hy926, it was necessary to perform media exchanges with automated liquid handling equipment in order to obtain optimal and reproducible antagonist concentration-response curves. The assay is also suitable for study of PAR2 ligands; a peptide antagonist reported by Fairlie was synthesized and found to inhibit PAR2 in a manner consistent with reports using epithelial cells. The assay was used to confirm that vorapaxar acts as an irreversible antagonist of PAR1 in endothelium, and parmodulin 2 (ML161) and the related parmodulin RR-90 were found to inhibit PAR1 reversibly, in a manner consistent with negative allosteric modulation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Protease-activated receptor 2 (PAR2) is a cell surface protein linked to G-protein dependent and independent intracellular signaling pathways that produce a wide range of physiological responses, including those related to metabolism, inflammation, pain, and cancer. Certain proteases, peptides, and nonpeptides are known to potently activate PAR2. However, no effective potent PAR2 antagonists have been reported yet despite their anticipated therapeutic potential. This study investigates antagonism of key PAR2-dependent signaling properties and functions by the imidazopyridazine compound I-191 (4-(8-( tert -butyl)-6-(4-fluorophenyl)imidazo[1,2- b ]pyridazine-2-carbonyl)-3,3-dimethylpiperazin-2-one) in cancer cells. At nanomolar concentrations, I-191 inhibited PAR2 binding of and activation by structurally distinct PAR2 agonists (trypsin, peptide, nonpeptide) in a concentration-dependent manner in cells of the human colon adenocarcinoma grade II cell line (HT29). I-191 potently attenuated multiple PAR2-mediated intracellular signaling pathways leading to Ca 2+ release, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, Ras homologue gene family, member A (RhoA) activation, and inhibition of forskolin-induced cAMP accumulation. The mechanism of action of I-191 was investigated using binding and calcium mobilization studies in HT29 cells where I-191 was shown to be noncompetitive and a negative allosteric modulator of the agonist 2f-LIGRL-NH 2 The compound alone did not activate these PAR2-mediated pathways, even at high micromolar concentrations, indicating no bias in these signaling properties. I-191 also potently inhibited PAR2-mediated downstream functional responses, including expression and secretion of inflammatory cytokines and cell apoptosis and migration, in human colon adenocarcinoma grade II cell line (HT29) and human breast adenocarcinoma cells (MDA-MB-231). These findings indicate that I-191 is a potent PAR2 antagonist that inhibits multiple PAR2-induced signaling pathways and functional responses. I-191 may be a valuable tool for characterizing PAR2 functions in cancer and in other cellular, physiological, and disease settings., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: Proteinase-Activated Receptor-2 (PAR 2 ) is a G protein-coupled receptor activated by serine proteinases. We have shown that PAR 2 activation in the airways is involved in the development of allergic inflammation and airway hyperresponsiveness (AHR) in acute murine models. We hypothesized that functional inhibition of PAR 2 prevents allergic inflammation, AHR and airway remodeling in chronic allergic airway inflammation models., Material and Methods: We developed and used a 12 week model of cockroach extract (CE)-mediated AHR, airway inflammation and remodeling in BALB/c mice., Results: Mice sensitized and challenged with CE for 12 weeks exhibit AHR, increased numbers of eosinophils in bronchoalveolar lavage (BAL) and increased collagen content in the lung tissue compared to saline controls. Administration of an anti-PAR 2 antibody, SAM-11, after the initial development of airway inflammation significantly inhibited all these parameters., Conclusions: Our data demonstrate that PAR 2 signaling plays a key role in CE-induced AHR and airway inflammation/remodeling in long term models of allergic airway inflammation. Targeting PAR 2 activation may be a successful therapeutic strategy for allergic asthma., (© 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Human proteinase-activated receptor 2 (PAR2), a transmembrane G-protein-coupled receptor (GPCR), is an attractive target for a novel anticancer therapy, as it plays a critical role in cell migration and invasion. Selective PAR2 inhibitors therefore have potential as anti-metastatic drugs. Knowing that the natural product teleocidin A2 is able to inhibit PAR2 in tumor cells, the goal of the present study was to elaborate structure-activity relationships and to identify potent PAR2 inhibitors with lower activity against the adverse target, protein kinase C (PKC). For this purpose, an efficient gram-scale total synthesis of indolactam V (i.e., the parent structure of all teleocidins) was developed, and a library of derivatives was prepared. Some compounds were indeed found to exhibit high potency as PAR2 inhibitors at low nanomolar concentrations with improved selectivity (relative to teleocidin A2). The pseudopeptidic fragment bridging the C3 and C4 positions of the indole core proved to be essential for target binding, whereas activity and target selectivity depends on the substituents at N1 or C7. This study revealed novel derivatives that show high efficacy in PAR2 antagonism combined with increased selectivity., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Hypertension is one of the most prevalent diseases worldwide and can cause harmful complications within the vascular system. Further research on vascular responsiveness to different ligands and diverse receptors in various arteries is required to understand the mechanisms underlying the development of these vascular complications. Here, we investigated the vasorelaxant effect of the protease-activated receptor 2 (PAR2) agonist 2-furoyl-LIGRLO-amide (2-Fly) and two commonest agents, namely endothelium-dependent dilator acetylcholine (ACh) and endothelium-independent dilator sodium nitroprusside (SNP), on the thoracic aorta isolated from aged spontaneously hypertensive rats (SHR) (age, 52±1 weeks). The effects of these agents were compared between aortas isolated from SHR and age-matched normotensive Wistar Kyoto (WKY) rats. Compared with the WKY group, in the SHR group, 2-Fly-induced relaxation was impaired, ACh-induced relaxation was slightly decreased at low concentrations, and SNP-induced relaxation was similar. In addition, 2-Fly-induced aortic relaxation was largely decreased by a PAR2 antagonist (FSLLRY), endothelial denudation, and a nitric oxide (NO) synthase inhibitor N G -nitro-L-arginine (L-NNA) but not by an Akt inhibitor. These results suggested that PAR2-induced relaxations of aortas of aged SHR was impaired, and this impaired aortic relaxation may be attributed to decreased NO bioavailability rather than altered NO sensitivity unrelated to the Akt activity.

Background and Purpose: The majority of the severe vascular complications in fibrosis are a consequence of a deregulated activity of mediators controlling vasomotor tone. One of the most important of these mediators is endothelin-1 (ET-1). Here, we have investigated the role of proteinase-activated receptor 2 (PAR2) in the vascular dysfunction in a model of fibrosis, using tight-skin (Tsk) mice., Experimental Approach: Aortas were collected from Tsk, transgenic over-expressing PAR2 (TgPAR2), PAR2 deficient (PAR2 -/- ) or the corresponding WT mice. Histological and immunohistochemistry analysis for α-smooth muscle actin, PAR2 and ET-1 receptors were performed on aorta sections. Vascular responses to phenylephrine, ET-1 and PAR2 activating peptide (PAR2-AP) were assessed on aortic rings., Key Results: In aortas from Tsk mice, responses to phenylephrine were reduced, contractions to ET-1 were increased and vasorelaxation to PAR2-AP was enhanced. These alterations matched changes observed in whole vessel architecture such as vascular fibre re-organization, increased collagen deposition and enhanced α-smooth muscle actin expression. Expression of both ET A receptors and PAR2 was enhanced in Tsk mice. Antagonism of PAR2 potentiated vascular effects of ET-1, whereas antagonism of ET A receptors increased vasorelaxation induced by PAR2-AP. In TgPAR2 mice, responses to ET-1 and ET-1 plasma levels were reduced. Conversely, PAR2 -/- mice showed enhanced ET-1 induced contraction in aortic rings and higher circulating ET-1 levels., Conclusions and Implications: Our data show that PAR2 counterbalanced enhanced contractions to ET-1 in aortas from Tsk mice. PAR2 could represent a possible target for novel drugs in the treatment of vascular complications in fibrosis., Linked Articles: This article is part of a themed section on Targeting Inflammation to Reduce Cardiovascular Disease Risk. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.22/issuetoc and http://onlinelibrary.wiley.com/doi/10.1111/bcp.v82.4/issuetoc., (© 2016 The British Pharmacological Society.)

Objectives: Proteases are key mediators of pain and altered enteric neuronal signalling, although the types and sources of these important intestinal mediators are unknown. We hypothesised that intestinal epithelium is a major source of trypsin-like activity in patients with IBS and this activity signals to primary afferent and enteric nerves and induces visceral hypersensitivity., Design: Trypsin-like activity was determined in tissues from patients with IBS and in supernatants of Caco-2 cells stimulated or not. These supernatants were also applied to cultures of primary afferents. mRNA isoforms of trypsin ( PRSS1 , 2 and 3 ) were detected by reverse transcription-PCR, and trypsin-3 protein expression was studied by western blot analysis and immunohistochemistry. Electrophysiological recordings and Ca 2+ imaging in response to trypsin-3 were performed in mouse primary afferent and in human submucosal neurons, respectively. Visceromotor response to colorectal distension was recorded in mice administered intracolonically with trypsin-3., Results: We showed that stimulated intestinal epithelial cells released trypsin-like activity specifically from the basolateral side. This activity was able to activate sensory neurons. In colons of patients with IBS, increased trypsin-like activity was associated with the epithelium. We identified that trypsin-3 was the only form of trypsin upregulated in stimulated intestinal epithelial cells and in tissues from patients with IBS. Trypsin-3 was able to signal to human submucosal enteric neurons and mouse sensory neurons, and to induce visceral hypersensitivity in vivo, all by a protease-activated receptor-2-dependent mechanism., Conclusions: In IBS, the intestinal epithelium produces and releases the active protease trypsin-3, which is able to signal to enteric neurons and to induce visceral hypersensitivity., Competing Interests: Competing interests: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Bortezomib (BTZ) is generally used as a chemotherapeutic agent for the treatment of multiple myeloma; however, one of the significant limiting complications of BTZ is painful peripheral neuropathy observed during BTZ therapy. There is a lack of drugs which can prevent and/or treat the painful symptoms induced by BTZ, as the underlying molecular mechanism leading to neuropathic pain remains largely unclear. In the present study, we examined engagement of proteinase-activated receptor 2 (PAR2) and transient receptor potential ankyrin 1 (TRPA1) in neuropathic pain induced by BTZ in rats. Our results demonstrated that systemic injection of BTZ increased mechanical pain and cold sensitivity as compared with control animals (P less than 0.05 vs control rats). Our data further showed that blocking respective PAR2 and TRPA1 attenuated mechanical pain and cold sensitivity observed in control rats and BTZ rats (P less than 0.05 vs vehicle control). Notably, the attenuating effect of blocking PAR2 and TRPA1 on mechanical pain and cold sensitivity was significantly less in BTZ rats than that in control rats. In addition, protein expression of PAR2 and TRPA1 was upregulated in the lumbar dorsal root ganglion of BTZ rats, and inhibition of PAR2 decreased the levels of TRPA1 and attenuated its downstream pathways (namely, PKCɛ and PKA). Overall, we revealed specific signaling pathways leading to neuropathic pain induced by chemotherapeutic BTZ and that blocking PAR2 and TRPA1 in sensory nerves is beneficial to improve neuropathic pain during BTZ intervention.

Factor (F) Xa reactive IgG isolated from patients with antiphospholipid syndrome (APS) display higher avidity binding to FXa with greater coagulant effects compared to systemic lupus erythematosus (SLE) non APS IgG. FXa signalling via activation of protease-activated receptors (PAR) leads to increased intracellular calcium (Ca 2+ ). Therefore, we measured alterations in Ca 2+ levels in human umbilical vein endothelial cells (HUVEC) following FXa-mediated PAR activation and investigated whether FXa reactive IgG from patients with APS or SLE/APS- alter these responses. We observed concentration-dependent induction of Ca 2+ release by FXa that was potentiated by APS-IgG and SLE/APS- IgG compared to healthy control subjects' IgG, and FXa alone. APS-IgG and SLE/APS- IgG increased FXa mediated NFκB signalling and this effect was fully-retained in the affinity purified anti-FXa IgG sub-fraction. Antagonism of PAR-1 and PAR-2 reduced FXa-induced Ca 2+ release. Treatment with a specific FXa inhibitor, hydroxychloroquine or fluvastatin significantly reduced FXa-induced and IgG-potentiated Ca 2+ release. In conclusion, PAR-1 and PAR-2 are involved in FXa-mediated intracellular Ca 2+ release in HUVEC and FXa reactive IgG from patients with APS and/or SLE potentiate this effect. Further work is required to explore the potential use of IgG FXa reactivity as a novel biomarker to stratify treatment with FXa inhibitors in these patients.

Cell migration and invasion are very characteristic features of cancer cells that promote metastasis, which is one of the most common causes of mortality among cancer patients. Emerging evidence has shown that coagulation factors can directly mediate cancer-associated complications either by enhancing thrombus formation or by initiating various signaling events leading to metastatic cancer progression. It is well established that, apart from its distinct role in blood coagulation, coagulation factor FVIIa enhances aggressive behaviors of breast cancer cells, but the underlying signaling mechanisms still remain elusive. To this end, we investigated FVIIa's role in the migration and invasiveness of the breast cancer cell line MDA-MB-231. Consistent with previous observations, we observed that FVIIa increased the migratory and invasive potential of these cells. We also provide molecular evidence that protease-activated receptor 2 activation followed by PI3K-AKT activation and GSK3β inactivation is involved in these processes and that β-catenin, a well known tumor-regulatory protein, contributes to this signaling pathway. The pivotal role of β-catenin was further indicated by the up-regulation of its downstream targets cyclin D1, c-Myc, COX-2, MMP-7, MMP-14, and Claudin-1. β-Catenin knockdown almost completely attenuated the FVIIa-induced enhancement of breast cancer migration and invasion. These findings provide a new perspective to counteract the invasive behavior of breast cancer, indicating that blocking PI3K-AKT pathway-dependent β-catenin accumulation may represent a potential therapeutic approach to control breast cancer., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Proteinase activated receptor 2 (PAR2), which is localized in the GI tract, the respiratory system, and the kidney tubules is a G protein-coupled receptor associated with inflammation, metabolism, and disease. The aim of this study was to explore the role of PAR2 in hydrogen peroxide (H 2 O 2 )-induced HepG2 cells by using FSLLRY-NH 2 a PAR2 antagonist. H 2 O 2 treatment resulted in induction of PAR2 in esophageal, gastric, and liver cells, with the most robust response being in HepG2 cells. Furthermore, this effect was dose-dependent in HepG2 cells. Treatment with H 2 O 2 at concentrations above 400 μM for 24 h also reduced HepG2 cell viability. H 2 O 2 treatment increased both the protein and mRNA levels of IL-1β, IL-8, and TNF-α, as well as those of SAPK/JNK. The increased levels of these pro-inflammatory genes and SAPK/JNK induced by H 2 O 2 were attenuated in a dose-dependent manner when cells were co-treated with H 2 O 2 and FSLLRY-NH2. In summary, the PAR2 antagonist peptide, FSLLRY-NH2, reduces the level of the pro-inflammatory genes IL-8, IL-1β, and TNF-α induced by H 2 O 2 , through the SAPK/JNK pathways in HepG2 cells. These data suggest that a PAR2 antagonist could be an anti-inflammatory agent in HepG2 cells.

Protease-activated receptors (PARs) are a family of G-protein-coupled receptors (GPCRs) that are irreversibly activated by proteolytic cleavage of the N terminus, which unmasks a tethered peptide ligand that binds and activates the transmembrane receptor domain, eliciting a cellular cascade in response to inflammatory signals and other stimuli. PARs are implicated in a wide range of diseases, such as cancer and inflammation. PARs have been the subject of major pharmaceutical research efforts but the discovery of small-molecule antagonists that effectively bind them has proved challenging. The only marketed drug targeting a PAR is vorapaxar, a selective antagonist of PAR1 used to prevent thrombosis. The structure of PAR1 in complex with vorapaxar has been reported previously. Despite sequence homology across the PAR isoforms, discovery of PAR2 antagonists has been less successful, although GB88 has been described as a weak antagonist. Here we report crystal structures of PAR2 in complex with two distinct antagonists and a blocking antibody. The antagonist AZ8838 binds in a fully occluded pocket near the extracellular surface. Functional and binding studies reveal that AZ8838 exhibits slow binding kinetics, which is an attractive feature for a PAR2 antagonist competing against a tethered ligand. Antagonist AZ3451 binds to a remote allosteric site outside the helical bundle. We propose that antagonist binding prevents structural rearrangements required for receptor activation and signalling. We also show that a blocking antibody antigen-binding fragment binds to the extracellular surface of PAR2, preventing access of the tethered ligand to the peptide-binding site. These structures provide a basis for the development of selective PAR2 antagonists for a range of therapeutic uses.