Activation Of Protease-activated Receptor Research Articles

1339-P: Plasma Heparin Cofactor II Activity Is Inversely Associated with the Development NAFLD in Patients with Diabetes

Aims and Introduction: The prevalence and incidence of nonalcoholic fatty liver disease (NAFLD) have been increasing worldwide. Thrombin exerts various pathophysiological functions via activation of protease-activated receptors (PARs) , and activation of PARs promotes the development of NAFLD. Since heparin cofactor II (HCII) specifically inhibits thrombin action by binding to dermatan sulfate proteoglycans in various tissue matrices, we hypothesized that plasma HCII activity is associated with the development of NAFLD. Materials and Methods: Plasma HCII activity and clinical markers for assessment of hepatic fibrosis including Fibrosis 4 (FIB-4) index, NAFLD fibrosis score (NFS) , and aspartate aminotransferase to platelet ratio index (APRI) were determined in 322 Japanese patients with diabetes mellitus (183 males and 139 females; mean age, 66.0±11.3 years) . The relationships between plasma HCII activities and the clinical markers were statistically evaluated. Results: The mean plasma HCII activity in all participants was 94.0 ± 17.7%. Multivariate regression analysis with adjustments for confounding factors including age, sex, and serum albumin showed that plasma HCII activity independently contributed to reduction of the FIB-4 index (p < 0.0001) , NFS (p < 0.0001) , and APRI (p < 0.05) . Conclusions: Plasma HCII activity was independently and inversely associated with the clinical markers of hepatic fibrosis. The results suggest that HCII can serve as a novel therapeutic target for the development of NAFLD in patients with diabetes. Disclosure T.Hara: None. I.Endo: None. M.Matsuhisa: Research Support; Nissui , Sysmex Corp., Speaker’s Bureau; Astellas Pharma Inc., Eli Lilly and Company, Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk, Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited. M.Abe: None. K.Aihara: None. H.Yamagami: None. S.Masuda: None. Y.Mitsui: None. K.Kurahashi: None. S.Yoshida: None. T.Otoda: None. T.Yuasa: None. A.Kuroda: None.

Melanocore uptake by keratinocytes occurs through phagocytosis and involves protease-activated receptor-2 internalization.

In the skin epidermis, melanin is produced and stored within melanosomes in melanocytes, and then transferred to keratinocytes. Different models have been proposed to explain the melanin transfer mechanism, which differ essentially in how melanin is transferred—either in a membrane‐bound melanosome or as a melanosome core, that is, melanocore. Here, we investigated the endocytic route followed by melanocores and melanosomes during internalization by keratinocytes, by comparing the uptake of melanocores isolated from the supernatant of melanocyte cultures, with melanosomes isolated from melanocytes. We show that inhibition of actin dynamics impairs the uptake of both melanocores and melanosomes. Moreover, depletion of critical proteins involved in actin‐dependent uptake mechanisms, namely Rac1, CtBP1/BARS, Cdc42 or RhoA, together with inhibition of Rac1‐dependent signaling pathways or macropinocytosis suggest that melanocores are internalized by phagocytosis, whereas melanosomes are internalized by macropinocytosis. Interestingly, we found that Rac1, Cdc42 and RhoA are differently activated by melanocore or melanosome stimulation, supporting the existence of two distinct routes of melanin internalization. Furthermore, we show that melanocore uptake induces protease‐activated receptor‐2 (PAR‐2) internalization by keratinocytes to a higher extent than melanosomes. Because skin pigmentation was shown to be regulated by PAR‐2 activation, our results further support the melanocore‐based mechanism of melanin transfer and further refine this model, which can now be described as coupled melanocore exo/phagocytosis.

Protease Activated Receptor‐2 (PAR‐2) Antagonist potentially ameliorates the SARS‐CoV‐2 virus‐induced Inflammatory Storm

The severe acute respiratory syndrome‐ 2 (SARS‐CoV‐2) caused the coronavirus disease COVID‐19 responsible for the current pandemic presenting a high mortality rate. The development of vaccines provided a transient relief to the social, economic, and health care systems. However, the virus is constantly mutating to survive, generating new pathogenic variants that jeopardize the ongoing efforts and up‐to‐date accomplishments against it. The TMPRSS2 is a crucial player for SARS‐CoV‐2 cell entry. This transmembrane serine protease intervenes with the viral Spike/Angiotensin‐Converting Enzyme 2 (ACE2) host receptor axis, enabling to initiate the SARS‐CoV‐2 entry to target cells. The subsequent viral replication causes pathophysiological changes including a major complication of the COVID‐19 known as the inflammatory storm (IS). This is characterized by an enhanced release of inflammatory modulators leading to a hyper‐inflammation. TMPRSS2 is expressed in the epithelial cells of several tissues, including the epithelium of the respiratory tract, that is the focus of our study. However, this catalytic enzyme shows poor anchoring in the cell membrane and is consequently released into the extracellular space with the potential to activate or cleave neighboring cells dependent on serine proteases. An example of this cellular scenario is represented by the Protease Activated Receptor‐2 (PAR‐2), associated with activation of inflammatory pathways upon cleavage of the N‐terminus by a serine protease. We hypothesized: 1) that TMPRSS2 co‐localizes with PAR‐2 at the cell membrane of target epithelial cells; 2) TMPRSS2 activates PAR‐2 and associated proinflammatory signaling pathways; 3) the SARS‐CoV‐2 chaotic inflammation together with PAR‐2 activation will worsen the resulting IS. Based on our lab prior work on PAR‐2 and its antagonist, we anticipate that by inhibiting PAR‐2 activation, we will observe a decrease in the COVID‐19 associated IS. In this study, we used the MCF‐7 cell line to standardize the protocols, since this cell line expresses both proteins. They are more resilient to adverse conditions in the lab (due to the pandemicconsequences at research labs) and will allow us to establish a proof of concept to be compare with the human primary airway epithelial cells that we will subsequently use. Using MCF‐7 as a model, we established the presence of PAR‐2 and TMPRSS2 via western blot. In addition, by using immuno‐cyto‐fluorescence we confirmed that these two proteins colocalize at the cell membrane. Activation of intracellular calcium‐dependent pathways, detected by intracellular calcium fluorogenic assay, showed that after treating cells with trypsin (Gold Standard for PAR‐2 activation), TMPRSS2, PAR‐2 agonist, and PAR‐2 antagonist, trypsin caused a significant (p < 0.05) increase compared to TMPRSS2 either alone, with the PAR‐2 agonist or the PAR‐2 antagonist with a decreasing of 50 % trypsin activity. Importantly, BrdU assay results showed that PAR‐2 antagonist decreased the proliferation of MCF‐7 cells when compared to trypsin (p < 0.005). Currently, we are working to test our hypothesis with human primary cells from different locations of the respiratory tract and compare the outcomes with the MCF‐7 model. Furthermore, our study will contribute to having a better understanding of PAR‐2 role in SARS‐ CoV‐2 complications.

Application of Scanning Ion Conductance Microscopy in the Studies of Podocytes Morphological Changes

High‐resolution imaging of glomerular podocytes morphology is becoming an essential and helpful tool for studying their role in renal disease progression. Electron microscopy (EM) is commonly used to analyze the effacement of the podocytes’ cell foot processes and the glomerular filtration barrier integrity loss. However, EM does not allow studying living samples and requires multiple complex procedures for sample preparation. Here, we propose and demonstrate the successful application of Scanning Ion Conductance Microscopy (SICM) for studying morphological changes of cultured podocytes and freshly isolated glomeruli.SICM provides a high‐resolution (comparable to EM) non‐optical imaging of cell topography using hopping probe microscopy principles and new dynamic reconstruction software. This technique can be used for analysis of cell membrane morphology and different functional characteristics, such as cell volume, membrane potentials, single ion‐channel currents, and other parameters. The major advantage of the current method is that it can be applied to live samples with complex morphology, such as intact glomeruli, under physiologically relevant liquid conditions.We have established and demonstrated a successful application of the SICM method in the live human glomerulus and provided a proof of principle for future dynamic analysis of membrane morphology and varied functional parameters in the living glomerulus. As a proof of principle, we applied this method for estimation the pathological structural changes in podocyte foot processes in type 2 diabetic nephropathy (T2DN) rats. Using topographical quantification of the length and irregular shape of the podocyte foot processes, we showed an absence of interdigitated foot processes on the selected region of the glomerular slit diaphragm in isolated T2DN glomeruli.In addition to the application of SICM with freshly isolated glomeruli, we used this approach to visualize and quantify foot process dynamics in cultured human podocytes during pharmacological applications. Thus, we showed that destroying the F‐actin with cytochalasin D completely stopped the migration of the podocyte filopodium through the remodeling of the actin cytoskeleton. Here, we further applied SICM to detect morphological changes in human podocytes after activation of Protease‐activated receptor‐1 (PAR1) by acute application of agonist TFLLR‐NH2. A series of experiments revealed that PAR1 activation led to retraction of the lamellipodium and a significant decrease in cell surface area (+51±46 vs. ‐15±8 µm2, 30 min after application of vehicle or TFLLR, respectively; n=5, p&lt;0.05).In summary, SICM has numerous advantages and has excellent potential for precise quantitative analyses of the morphological changes in the glomerulus and cultured podocytes. Moreover, it opens the possibility of performing patch‐clamp recordings on the specific parts of the podocyte cell body and advances our understanding of the function and mechanism of the glomerulus filtration barrier.

Metformin down‐regulates TGF beta signal transduction and production of PAR2 N‐terminus cleaving protease activity in an NR4a1 dependent manner in a PC3 prostate cancer cell line

OBJECTIVEOur aim is to understand the mechanisms of metformin’s antitumor actionsHYPOTHESISMetformin is known to have antitumour effects in cancers like head &amp; neck cancer, that may involve its regulation of TGF‐β signalling, but the metformin receptor mechanisms for this action are not known. We recently discovered that the action of metformin to preserve hypoglycaemia‐impaired endothelial cell function regulated by proteinase‐activated receptor‐2 (PAR2) requires its interaction with the orphan nuclear receptor, NR4A1 (PubMed ID 34452975). PAR2 also plays a role in regulating prostate cancer PC3 cell function whereby autocrine signaling occurs in PC3 cells which secrete a PAR2‐cleaving/activating matrix metalloproteinase, MMP2. We hypothesized that metformin can act in PC3 prostate cancer‐derived cells via NR4A1 to change TGF‐β signalling and to alter the production of PAR‐cleaving proteinases.METHODSThe ability of TGF‐β to upregulate connective tissue growth factor (CTGF) mRNA in PC3 cells was measured for metformin treated cells (&lt;/=100 μM) that were transfected with NR4A1, C‐terminally tagged with red‐fluorescent protein (RFP): either wild‐type (WT) or mutant (PPGG) murine NR4A1 that does not interact with metformin. TGF‐β upregulated CTGF mRNA in PC3 cells was monitored after metformin treatment for 20 hours. In similarly transfected WT and mutant PPGG NR4A1‐expressing PC3 cells, treated or not with metformin (&lt;/=100 μM), the cleavage status of dually tagged transfected N‐terminal RFP/C‐terminal GFP‐PAR2 was monitored: (confocal imaging: yellow = intact receptor; green = cleaved receptor). Concurrently, the abundance of MMP activity of PC3 cell supernatants was measured by a fluorogenic MMP substrate, Mca‐RPKPVE‐Nval‐WRK(Dnp)‐NH2 (ES002).RESULTSThe expected TGF‐β upregulated CTGF mRNA in PC3 cells was prevented by metformin treatment, but only for cells transfected with the WT and not the mutant PPGG NR4A1. Metformin acted at concentrations (&lt;/=100 μM) far lower than those that affect mitochondrial complex I (&gt; mM). We also found that in WT, but not PPGG mutant‐transfected PC3 cells, metformin (&lt;/=100 μM) down‐regulated MMP PAR2‐cleaving proteinase activity.CONCLUSIONSOur results show that low concentrations of metformin (&lt;/=100 μM), in the presence of wild‐type but not PPGG mutant NR4A1 which does not enable metformin interactions at the NR4A1 alternative ligand binding site, can both block TGF‐ β signaling and reduce cell‐secreted MMP activity that in turn can drive tissue invasion and metastasis via PAR2 activation. Thus, NR4A1 may play a role in the antitumour actions of metformin.

Abstract 474: Macrophage Par2 Contributes To Influenza A Virus Pathology And Is A Potential Therapeutic Target To Treat Influenza A Virus Infection.

Proteinase-activated receptor 2 (PAR2) was shown to enhance proinflammatory toll-like receptor 3 (TLR3) responses, while dampening TLR3-dependent antiviral response in epithelial cells. Moreover, Par2 -/- mice exhibited improved survival after influenza A virus (IAV) infection. The objective of the study is to identify PAR2 expressing cells contributing to IAV pathology, and test if PAR2 inhibition is a potential new target to treat IAV. Infection with H1N1 IAV (PR8) was analyzed in global ( Par2 -/- ), myeloid ( Par2 fl/fl ;LysM Cre+ ), neutrophil ( Par2 fl/fl ;MRP8 Cre+ ) and lung epithelial cell (EpC) Par2 deficient ( Par2 fl/fl ;SPC Cre+ ) mice and respective controls ( Par2 +/+ and Par2 fl/fl ). The effect of PAR2 activation on poly I:C activation of TLR3 was analyzed in bone marrow-derived macrophages (BMDM) and murine macrophage-like J774A.1 cells. Lastly, we analyzed PAR2 inhibition in wild-type (WT) mice using mouse-anti PAR2 antibody (SAM11) and small molecule PAR2 inhibitor ENMD-1068. Par2 -/- and Par2 fl/fl ;LysM Cre+ mice exhibited increased survival, reduced proinflammatory mediators and cellular infiltration in bronchoalveolar lavage fluid (BALF) 3 days post infection (dpi) compared to control mice. Reduced IAV pathology resulted in significantly better lung function (PenH, airway resistance) in Par2 -/- mice compared to Par2 +/+ mice 7 dpi. Neither Par2 fl/fl ;MRP8 Cre+ or Par2 fl/fl ;SPC Cre+ mice showed any survival benefit compared to Par2 fl/fl . In vitro studies showed that Par2 -/- BMDM produced less IL6 and IL12p40 than Par2 +/+ BMDM after poly I:C stimulation. J774A.1 cells stimulated with PAR2 agonist and poly I:C showed an increase in NFκB activation compared to poly I:C alone. Prophylactic PAR2 inhibition with SAM11 before and daily after IAV infection, decreased BALF inflammation and lung virus load compared to control IgG treated mice 3dpi. Therapeutic PAR2 inhibition with ENMD-1068 daily from 3 dpi resulted in improved lung function (PenH) compared to vehicle treated mice 7 dpi. Global or macrophage cell but not neutrophil or lung EpC Par2 deficiency was associated with reduced IAV-induced lung pathology and mortality. Additionally, PAR2 inhibition experiments showed PAR2 may be a therapeutic target to reduce IAV pathology.

Platelet activation by SARS-CoV-2 implicates the release of active tissue factor by infected cells

AbstractPlatelets are hyperactivated in coronavirus disease 2019 (COVID-19). However, the mechanisms promoting platelet activation by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not well understood. This may be due to inherent challenges in discriminating the contribution of viral vs host components produced by infected cells. This is particularly true for enveloped viruses and extracellular vesicles (EVs), as they are concomitantly released during infection and share biophysical properties. To study this, we evaluated whether SARS-CoV-2 itself or components derived from SARS-CoV-2-infected human lung epithelial cells could activate isolated platelets from healthy donors. Activation was measured by the surface expression of P-selectin and the activated conformation of integrin αIIbβ3, degranulation, aggregation under flow conditions, and the release of EVs. We find that neither SARS-CoV-2 nor purified spike activates platelets. In contrast, tissue factor (TF) produced by infected cells was highly potent at activating platelets. This required trace amounts of plasma containing the coagulation factors FX, FII, and FVII. Robust platelet activation involved thrombin and the activation of protease-activated receptor (PAR)-1 and -4 expressed by platelets. Virions and EVs were identified by electron microscopy. Through size-exclusion chromatography, TF activity was found to be associated with a virus or EVs, which were indistinguishable. Increased TF messenger RNA (mRNA) expression and activity were also found in lungs in a murine model of COVID-19 and plasma of severe COVID-19 patients, respectively. In summary, TF activity from SARS-CoV-2–infected cells activates thrombin, which signals to PARs on platelets. Blockade of molecules in this pathway may interfere with platelet activation and the coagulation characteristic of COVID-19.

Immunomodulatory role of thrombin in cancer progression.

Coagulation proteases and the generation of thrombin are increased in tumors. In addition, chemotherapeutic agents commonly used to treat malignant cancers can exacerbate cancer-associated thromboses. Thrombin can modify tumor cell behavior directly through the activation of protease-activated receptors (PAR) or indirectly by generating fibrin matrices. In addition to its role in generating fibrin to promote hemostasis, thrombin acts directly on multiple effector cells of the immune system impacting both acute and chronic inflammatory processes. Thrombin-mediated release of interleukin-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 leads to the accumulation of multiple tumor-infiltrating immunosuppressive cell populations including myeloid derived suppresser cells, M2-like macrophages, and T regulatory cells. Ablation of PAR-1 from the tumor microenvironment, but not the tumor, has been shown to dramatically reduce tumor growth and metastasis in multiple tumor models. Thrombin-activated platelets release immunosuppressive cytokines including transforming growth factor-β that can inhibit natural killer cell activity, helping tumor cells to evade host immunosurveillance. Taken together, there is strong evidence that thrombin influences cancer progression via multiple mechanisms, including the tumor immune response, with thrombin emerging as a target for novel therapeutic strategies for cancer.

The Yin-Yang roles of protease-activated receptors in inflammatory signalling and diseases.

Inflammatory diseases have become increasingly prevalent throughout the world. Coronavirus disease 2019 (COVID-19), which has recently become pandemic, also exhibits hyperinflammation and cytokine release syndrome. To address inflammation-related diseases, numerous molecular targets have been explored in preclinical studies and clinical trials. Among them, the protease-activated receptors (PARs) that belong to G protein-coupled receptors are one of the most popular classes of drug targets, participating in inflammatory signalling and diseases. PARs activation can trigger downstream intracellular signalling to modulate a variety of inflammatory responses in multiple systems, including nervous, respiratory, digestive, circulatory, urinary and immune systems. Importantly, there are the Yin-Yang effects, comprising anti- and pro-inflammatory roles, of PARs activation in different types of inflammations, and these are context-dependent. Alternatively, it was recently revealed that PARs not only modulate inflammatory-related tumour progression, but also participate in inflammatory cytokine release related to COVID-19 via direct interaction with severe acute respiratory syndrome coronavirus 2 protein, suggesting that PARs also participate in other diseases via inflammatory responses. In this review, we renew and discuss the findings of PARs as molecular targets for treating inflammatory diseases, highlighting the novel roles of PARs and facilitating a better understanding of their designated values in the specific inflammatory environment.

Thrombin induces a temporal biphasic vascular response through the differential phosphorylation of endothelial nitric oxide synthase via protease-activated receptor-1 and protein kinase C

Endothelial nitric oxide synthase (eNOS) is a critical regulatory enzyme that controls vascular tone via the production of nitric oxide. Although thrombin also modulates vascular tone predominantly via the activation of protease-activated receptors (PARs), the time course and mechanisms involved in how thrombin controls eNOS enzymatic activity are unknown. eNOS enzymatic activity is enhanced by the phosphorylation of eNOS-Ser1177 and reduced by the phosphorylation of eNOS-Thr495. In this study, we hypothesized that thrombin regulates vascular tone through the differential phosphorylation of eNOS. Using rat descending aorta, we show that thrombin modulates vascular tone in an eNOS-dependent manner via activated PAR-1. We also show that thrombin causes a temporal biphasic response. Protein kinase C (PKC) is associated with second phase of thrombin-induced response. Western blot analysis demonstrated thrombin phosphorylated eNOS-Ser1177 and eNOS-Thr495 in human umbilical vein endothelial cells. A PKC inhibitor suppressed the thrombin-induced phosphorylation of eNOS-Thr495, but not that of eNOS-Ser1177. Our results suggest that thrombin induces a temporal biphasic vascular response through the differential phosphorylation of eNOS via activated PAR-1. Thrombin causes transient vasorelaxation by the phosphorylation of eNOS-Ser1177, and subsequent attenuation of vasorelaxation by the phosphorylation of eNOS-Thr495 via PKC, leading to the modulation of vascular tone.

Proteinase-activated receptor-2 antagonist C391 inhibits Alternaria-induced airway epithelial signalling and asthma indicators in acute exposure mouse models.

Despite the availability of a variety of treatment options, many asthma patients have poorly controlled disease with frequent exacerbations. Proteinase-activated receptor-2 (PAR2) has been identified in preclinical animal models as important to asthma initiation and progression following allergen exposure. Proteinase activation of PAR2 raises intracellular Ca2+ , inducing MAPK and β-arrestin signalling in the airway, leading to inflammatory and protective effects. We have developed C391, a potent PAR2 antagonist effective in blocking peptidomimetic- and trypsin-induced PAR2 signalling in vitro as well as reducing inflammatory PAR2-associated pain in vivo. We hypothesized that PAR2 antagonism by C391 would attenuate allergen-induced acutely expressed asthma indicators in murine models. We evaluated the ability of C391 to alter Alternaria alternata-induced PAR2 signalling pathways in vitro using a human airway epithelial cell line that naturally expresses PAR2 (16HBE14o-) and a transfected embryonic cell line (HEK 293). We next evaluated the ability for C391 to reduce A.alternata-induced acutely expressed asthma indicators in vivo in two murine strains. C391 blocked A.alternata-induced, PAR2-dependent Ca2+ and MAPK signalling in 16HBE14o- cells, as well as β-arrestin recruitment in HEK 293 cells. C391 effectively attenuated A.alternata-induced inflammation, mucus production, mucus cell hyperplasia and airway hyperresponsiveness in acute allergen-challenged murine models. To our best knowledge, this is the first demonstration of pharmacological intervention of PAR2 to reduce allergen-induced asthma indicators in vivo. These data support further development of PAR2 antagonists as potential first-in-class allergic asthma drugs.

Giardia duodenalis cysteine proteases cleave proteinase-activated receptor-2 to regulate intestinal goblet cell mucin gene expression

Giardia duodenalis cysteine proteases have been identified as key virulence factors and have been implicated in alterations to intestinal goblet cell activity and mucus production during Giardia infection. The present findings demonstrate a novel mechanism by which Giardia cysteine proteases modulate goblet cell activity via cleavage and activation of protease-activated receptor 2. Giardia duodenalis (assemblage A) increased MUC2 mucin gene expression in human colonic epithelial cells in a manner dependent upon both protease-activated receptor 2 activation and Giardia cysteine protease activity. Protease-activated receptor 2 cleavage within the N-terminal activation domain by Giardia proteases was confirmed using a nano-luciferase tagged recombinant protease-activated receptor 2. In keeping with these observations, the synthetic protease-activated receptor 2-activating peptide 2fLIGRLO-amide increased Muc2 gene expression in a time-dependent manner. Calcium chelation and inhibition of the ERK1/2 mitogen activated protein kinase pathway inhibited Muc2 upregulation during Giardia infection, consistent with canonical protease-activated receptor 2 signaling pathways. Giardia cysteine proteases cleaved both recombinant protease-activated receptor 1 and protease-activated receptor 2 within their extracellular activation domains with isolate-dependent efficiency that correlated with the production of cysteine protease activity. Protease-activated receptors represent a novel target for Giardia cysteine proteases, and these findings demonstrate that protease-activated receptor 2 can regulate mucin gene expression in intestinal goblet cells.

Activated protein C suppresses neuropathic pain through activation of proteinase-activated receptor 1 (PAR1)

Activated protein C suppresses neuropathic pain through activation of proteinase-activated receptor 1 (PAR1)

Targeting Proteinase Activated Receptor-4 Reduces Mechanonociception During the Acute Inflammatory Phase but not the Chronic Neuropathic Phase of Osteoarthritis in Rats

Serine proteases are elevated in arthritic joints where they can cleave protease activated receptors (PARs) to modulate pain and inflammation. Activation of protease-activated receptor 4 (PAR4) has been implicated in inflammatory joint pain. Whether PAR4 is involved in osteoarthritis (OA) pain has not yet been explored. The aim of this study was to compare the role of PAR4 in modulating early versus late stage OA pain using two models of OA viz. monoiodoacetate (MIA) and medial meniscal transection (MMT). G-ratio calculation and electron microscopy analysis revealed saphenous nerve demyelination and structural damage during late stage but not early OA in both models. Using immunohistochemistry, neuronal expression of PAR4 was higher in early versus late OA. Systemic administration of the PAR4 antagonist pepducin P4pal10 reduced both secondary allodynia (von Frey hair algesiometry) and joint nociceptor firing (single unit recordings) in MMT and MIA animals compared to vehicle-treated animals in early OA. The PAR4 antagonist was ineffective at altering pain or joint afferent firing in post-inflammatory OA. During the acute phase of the models, joint inflammation as determined by laser speckle contrast analysis and intravital microscopy could be partially blocked by pepducin P4pal10. Compared to late-stage disease, inflammatory cytokines were elevated in early MIA and MMT rats. These findings suggest that PAR4 may be a viable target to treat the pain of early onset OA or during episodic inflammatory flares.

Protease-activated receptor 2 activation induces behavioural changes associated with depression-like behaviour through microglial-independent modulation of inflammatory cytokines

RationaleMajor depressive disorder (MDD) is a leading cause of disability worldwide but currently prescribed treatments do not adequately ameliorate the disorder in a significant portion of patients. Hence, a better appreciation of its aetiology may lead to the development of novel therapies.ObjectivesIn the present study, we have built on our previous findings indicating a role for protease-activated receptor-2 (PAR2) in sickness behaviour to determine whether the PAR2 activator, AC264613, induces behavioural changes similar to those observed in depression-like behaviour.MethodsAC264613-induced behavioural changes were examined using the open field test (OFT), sucrose preference test (SPT), elevated plus maze (EPM), and novel object recognition test (NOR). Whole-cell patch clamping was used to investigate the effects of PAR2 activation in the lateral habenula with peripheral and central cytokine levels determined using ELISA and quantitative PCR.ResultsUsing a blood–brain barrier (BBB) permeable PAR2 activator, we reveal that AC-264613 (AC) injection leads to reduced locomotor activity and sucrose preference in mice but is without effect in anxiety and memory-related tasks. In addition, we show that AC injection leads to elevated blood sera IL-6 levels and altered cytokine mRNA expression within the brain. However, neither microglia nor peripheral lymphocytes are the source of these altered cytokine profiles.ConclusionsThese data reveal that PAR2 activation results in behavioural changes often associated with depression-like behaviour and an inflammatory profile that resembles that seen in patients with MDD and therefore PAR2 may be a target for novel antidepressant therapies.

APC/PAR1 confers endothelial anti-apoptotic activity via a discrete, β-arrestin-2–mediated SphK1-S1PR1-Akt signaling axis

Endothelial dysfunction is associated with vascular disease and results in disruption of endothelial barrier function and increased sensitivity to apoptosis. Currently, there are limited treatments for improving endothelial dysfunction. Activated protein C (aPC), a promising therapeutic, signals via protease-activated receptor-1 (PAR1) and mediates several cytoprotective responses, including endothelial barrier stabilization and anti-apoptotic responses. We showed that aPC-activated PAR1 signals preferentially via β-arrestin-2 (β-arr2) and dishevelled-2 (Dvl2) scaffolds rather than G proteins to promote Rac1 activation and barrier protection. However, the signaling pathways utilized by aPC/PAR1 to mediate anti-apoptotic activities are not known. aPC/PAR1 cytoprotective responses also require coreceptors; however, it is not clear how coreceptors impact different aPC/PAR1 signaling pathways to drive distinct cytoprotective responses. Here, we define a β-arr2-mediated sphingosine kinase-1 (SphK1)-sphingosine-1-phosphate receptor-1 (S1PR1)-Akt signaling axis that confers aPC/PAR1-mediated protection against cell death. Using human cultured endothelial cells, we found that endogenous PAR1 and S1PR1 coexist in caveolin-1 (Cav1)-rich microdomains and that S1PR1 coassociation with Cav1 is increased by aPC activation of PAR1. Our study further shows that aPC stimulates β-arr2-dependent SphK1 activation independent of Dvl2 and is required for transactivation of S1PR1-Akt signaling and protection against cell death. While aPC/PAR1-induced, extracellular signal-regulated kinase 1/2 (ERK1/2) activation is also dependent on β-arr2, neither SphK1 nor S1PR1 are integrated into the ERK1/2 pathway. Finally, aPC activation of PAR1-β-arr2-mediated protection against apoptosis is dependent on Cav1, the principal structural protein of endothelial caveolae. These studies reveal that different aPC/PAR1 cytoprotective responses are mediated by discrete, β-arr2-driven signaling pathways in caveolae.

Myeloid Protease-Activated Receptor-2 Contributes to Influenza A Virus Pathology in Mice.

BackgroundInnate immune responses to influenza A virus (IAV) infection are initiated in part by toll-like receptor 3 (TLR3). TLR3-dependent signaling induces an antiviral immune response and an NFκB-dependent inflammatory response. Protease-activated receptor 2 (PAR2) inhibits the antiviral response and enhances the inflammatory response. PAR2 deficiency protected mice during IAV infection. However, the PAR2 expressing cell-types contributing to IAV pathology in mice and the mechanism by which PAR2 contributes to IAV infection is unknown.MethodsIAV infection was analyzed in global (Par2-/- ), myeloid (Par2 fl/fl;LysMCre+) and lung epithelial cell (EpC) Par2 deficient (Par2fl/fl ;SPCCre+) mice and their respective controls (Par2 +/+ and Par2 fl/fl). In addition, the effect of PAR2 activation on polyinosinic-polycytidylic acid (poly I:C) activation of TLR3 was analyzed in bone marrow-derived macrophages (BMDM). Lastly, we determined the effect of PAR2 inhibition in wild-type (WT) mice.ResultsAfter IAV infection, Par2-/- and mice with myeloid Par2 deficiency exhibited increased survival compared to infected controls. The improved survival was associated with reduced proinflammatory mediators and reduced cellular infiltration in bronchoalveolar lavage fluid (BALF) of Par2-/- and Par2 fl/fl;LysMCre+ 3 days post infection (dpi) compared to infected control mice. Interestingly, Par2 fl/fl;SPCCre+ mice showed no survival benefit compared to Par2fl/fl . In vitro studies showed that Par2-/- BMDM produced less IL6 and IL12p40 than Par2 +/+ BMDM after poly I:C stimulation. In addition, activation of PAR2 on Par2 +/+ BMDM increased poly I:C induction of IL6 and IL12p40 compared to poly I:C stimulation alone. Importantly, PAR2 inhibition prior to IAV infection protect WT mice.ConclusionGlobal Par2 or myeloid cell but not lung EpC Par2 deficiency was associated with reduced BALF inflammatory markers and reduced IAV-induced mortality. Our study suggests that PAR2 may be a therapeutic target to reduce IAV pathology.

The activation fragment of PAR2 is elevated in serum from patients with rheumatoid arthritis and reduced in response to anti-IL6R treatment

Osteoarthritis (OA) and rheumatoid arthritis (RA) are serious and painful diseases. Protease-activated receptor 2 (PAR2) is involved in the pathology of both OA and RA including roles in synovial hyperplasia, cartilage destruction, osteophyogenesis and pain. PAR2 is activated via cleavage of its N-terminus by serine proteases. In this study a competitive ELISA assay was developed targeting the 36-amino acid peptide that is cleaved and released after PAR2 activation (PRO-PAR2). Technical assay parameters including antibody specificity, intra- and inter-assay variation (CV%), linearity, accuracy, analyte stability and interference were evaluated. PRO-PAR2 release was confirmed after in vitro cleavage of PAR2 recombinant protein and treatment of human synovial explants with matriptase. Serum levels of 22 healthy individuals, 23 OA patients and 15 RA patients as well as a subset of RA patients treated with tocilizumab were evaluated. The PRO-PAR2 antibody was specific for the neo-epitope and intra-inter assay CV% were 6.4% and 5.8% respectively. In vitro cleavage and matriptase treated explants showed increased PRO-PAR2 levels compared to controls. In serum, PRO-PAR2 levels were increased in RA patients and decreased in RA patients treated with tocilizumab. In conclusion, PRO-PAR2 may be a potential biomarker for monitoring RA disease and pharmacodynamics of treatment.

PAR-1 Antagonism to Promote Gut Mucosa Healing in Crohn's Disease Patients: A New Avenue for CVT120165.

A new paradigm has been added for the treatment of inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. In addition to resolving symptoms and inflammatory cell activation, the objective of tissue repair and mucosal healing is also now considered a primary goal. In the search of mediators that would be responsible for delayed mucosal healing, protease-activated receptor-1 (PAR-1) has emerged as a most interesting target. Indeed, in Crohn's disease, the endogenous PAR-1 agonist thrombin is drastically activated. Activation of PAR-1 is known to be associated with epithelial dysfunctions that hamper mucosal homeostasis. This review gathers the scientific evidences of a potential role for PAR-1 in mucosal damage and mucosal dysfunctions associated with chronic intestinal inflammation. The potential clinical benefits of PAR-1 antagonism to promote mucosal repair in CD patients are discussed. Targeted local delivery of a PAR-1 antagonist molecule such as CVT120165, a formulated version of the FDA-approved PAR-1 antagonist vorapaxar, at the mucosa of Crohn's disease patients could be proposed as a new indication for IBD that could be rapidly tested in clinical trials.

Differential Expression and Distinct Roles of Proteinase-Activated Receptor 2 in Microglia and Neurons in Neonatal Mouse Brain After Hypoxia-Ischemic Injury.

Regulation of microglial activation and neuroinflammation are critical factors in the pathogenesis of ischemic brain injury. Interest in protease-activated receptor 2 (PAR2) as a pharmaceutical target for various diseases is creasing. However, it is unclear the expression and functions of PAR2 in hypoxia-ischemic (HI) brain injury. Mice with HI and cells with oxygen-glucose deprivation and reoxygenation (OGD/R) were studied. Immunoblot and qRT-PCR were used to study the differential gene expression in cultured microglia and neurons. Immunofluorescent staining was used to study the expression pattern of PAR2 in the HI brain and phagocytotic activity of microglia after OGD/R. In neonatal mice brain after HI, we found PAR2 expression was abundant in neurons, but barely in microglia from the contralateral side of cortex and hippocampus. Conversely, PAR2 expression was barely in neurons while significantly increased in activated microglia from the ipsilateral side of cortex and hippocampus. The activations of PAR2 were increased in both microglia and neuron in a cell model of OGD/R. PAR2 activation mediated the cross-talk between microglia and neurons including the following: microglial PAR2 mediated inflammatory responses that induced neuronal damage; neuronal PAR2 regulated chemokines that recruited activated microglia to damage area; microglia PAR2 controlled the phagocytosis of degenerating neurons. These data suggested differential expression and distinct roles of PAR2 in microglia and neurons after HI injury; thereby, interventions targeting PAR2 may provide insights into the inflammatory-related diseases.