Protease-activated Receptor 1 Levels Research Articles

Exogenous autoinducer-2 alleviates intestinal damage in necrotizing enterocolitis via PAR2/MMP3 signaling pathway

BackgroundImbalanced intestinal microbiota and damage to the intestinal barrier contribute to the development of necrotizing enterocolitis (NEC). Autoinducer-2 (AI-2) plays a crucial role in repairing intestinal damage and reducing inflammation. ObjectiveThis study aimed to investigate the impact of AI-2 on the expression of intestinal zonula occludens-1 (ZO-1) and occludin proteins in NEC. We evaluated its effects in vivo using NEC mice and in vitro using lipopolysaccharide (LPS)-stimulated intestinal cells. MethodsPathological changes in the intestines of neonatal mice were assessed using histological staining and scoring. Cell proliferation was measured using the cell counting kit-8 (CCK-8) assay to determine the optimal conditions for LPS and AI-2 interventions. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the mRNA levels of matrix metalloproteinase-3 (MMP3), protease activated receptor-2 (PAR2), interleukin-1β (IL-1β), and IL-6. Protein levels of MMP3, PAR2, ZO-1, and occludin were evaluated using western blot, immunohistochemistry, or immunofluorescence. ResultsAI-2 alleviated NEC-induced intestinal damage (P < 0.05) and enhanced the proliferation of damaged IEC-6 cells (P < 0.05). AI-2 intervention reduced the mRNA and protein expressions of MMP3 and PAR2 in intestinal tissue and cells (P < 0.05). Additionally, it increased the protein levels of ZO-1 and occludin (P < 0.05), while reducing IL-1β and IL-6 mRNA expression (P < 0.05). ConclusionAI-2 intervention enhances the expression of tight junction proteins (ZO-1 and occludin), mitigates intestinal damage in NEC neonatal mice and IEC-6 cells, potentially by modulating PAR2 and MMP3 signaling. AI-2 holds promise as a protective intervention for NEC. AI-2 plays a crucial role in repairing intestinal damage and reducing inflammation.

The effect of GnRH-a on the angiogenesis of endometriosis

PurposeNeoangiogenesis is necessary for adhesion and invasiveness of endometriotic lesions in women affected by endometriosis. Vascular endothelial growth factor (VEGF) is one of the main components of angiogenesis and is part of the major pathway tissue factor (TF)-protease activated receptor-2 (PAR-2)-VEGF that leads to neoangiogenesis. Specificity protein 1 (SP1) is a transcriptional factor that has recently been studied for its crucial role in angiogenesis via a specific pathway. We hypothesize that by blocking angiogenetic pathways we can suppress endometriotic lesions. Gonadotrophin-releasing hormone-agonists (GnRH-a) are routinely used, especially preoperatively, in endometriosis. It would be of great interest to clarify which angiogenetic pathways are affected and, thereby, pave the way for further research into antiangiogenetic effects on endometriosis.MethodsWe used quantitative real-time polymerase chain reaction (qRT-PCR) to study mRNA expression levels of TF, PAR-2, VEGF, and SP1 in endometriotic tissues of women who underwent surgery for endometriosis and received GnRH-a (leuprolide acetate) preoperatively.ResultsVEGF, TF, and PAR-2 expression is significantly lower in patients who received treatment (p < 0,001) compared to those who did not, whereas SP1 expression is not altered (p = 0.779).ConclusionsGnRH-a administration does affect some pathways of angiogenesis in endometriotic lesions, but not all of them. Therefore, supplementary treatments that affect the SP1 pathway of angiogenesis should be developed to enhance the antiangiogenetic effect of GnRH-a in patients with endometriosis.Trial registrationClinicaltrial.gov ID: NCT06106932.

Effect of moxibustion on colonic low-grade inflammatory response in rats with diarrhea-predominant irritable bowel syndrome based on mast cell degranulation.

To observe the effects of moxibustion on colonic mast cell degranulation and inflammatory factor expression in rats with diarrhea-predominant irritable bowel syndrome (IBS-D), and explore the potential mechanism of moxibustion in treating IBS-D. Forty-five rat pups born from 5 healthy SPF-grade pregnant SD rats, with 8 rats were randomly selected as the normal group. The remaining 37 rats were intervened with maternal separation, acetic acid enema, and chronic restraint stress to establish the IBS-D model. The successfully modeled 32 rats were then randomly assigned to a model group, a ketotifen group, a moxibustion group, and a moxibustion-medication group, with 8 rats in each group. The rats in the ketotifen group were intervened with intragastric administration of ketotifen solution (10 mL/kg); the rats in the moxibustion group were intervened with suspended moxibustion on bilateral "Tianshu" (ST 25) and "Shangjuxu" (ST 37); the rats in the moxibustion-medication group were intervened with suspended moxibustion combined with intragastric administration of ketotifen solution. All interventions were administered once daily for 7 consecutive days. The diarrhea rate and minimum volume threshold of abdominal withdrawal reflex (AWR) were calculated before and after modeling, as well as after intervention. After intervention, colonic tissue morphology was observed using HE staining; colonic mucosal ultrastructure was examined by scanning electron microscopy; colonic mast cell ultrastructure was observed using transmission electron microscopy; mast cell degranulation was assessed by toluidine blue staining; serum and colonic levels of histamine, interleukin (IL)-1β, IL-6, IL-1α, trypsin-like enzyme, and protease-activated receptor 2 (PAR-2) were measured by ELISA; the Western blot and real-time quantitative PCR were employed to evaluate the protein and mRNA expression of colonic IL-1β, IL-6, IL-1α, trypsin-like enzyme, and PAR-2; the immunofluorescence was used to detect the positive expression of histamine, IL-1β, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 in the colonic tissue. Compared to the normal group, the rats in the model group exhibited extensive infiltration of inflammatory cells in colonic tissue, severe damage to the colonic mucosa, disordered arrangement of villi, reduced electron density, and a significant decrease in granule quantity within mast cells. The diarrhea rate and mast cell degranulation rate were increased (P<0.01), AWR minimum volume threshold was decreased (P<0.01); the serum and colonic levels of histamine, IL-1β, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 were elevated (P<0.01); the positive expression of histamine, as well as protein, mRNA and positive expression of IL-1β, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 in the colon were all elevated (P<0.01). Compared to the model group, the rats in the ketotifen group, the moxibustion group, and the moxibustion-medication group exhibited significantly reduced infiltration of inflammatory cells in colonic tissue, relatively intact colonic mucosa, orderly arranged villi, increased electron density, and an augmented number of mast cell granules; the diarrhea rate and mast cell degranulation rate were decreased (P<0.01), and AWR minimum volume threshold was increased (P<0.01); the serum and colonic levels of histamine, IL-1β, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 were reduced (P<0.01); the positive expression of histamine, as well as protein, mRNA and positive expression of IL-1β, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 in the colon were all decreased (P<0.01). Compared to the ketotifen group, the moxibustion group showed decreased serum levels of histamine, IL-6, and trypsin-like enzyme (P<0.01, P<0.05), as well as reduced colonic levels of IL-1β and IL-6 (P<0.01, P<0.05); the protein expression of colonic IL-1β, IL-1α, and PAR-2 was reduced (P<0.05), and the positive expression of colonic IL-1β and trypsin-like enzyme was reduced (P<0.01, P<0.05). Compared to both the ketotifen group and the moxibustion group, the moxibustion-medication group exhibited decreased diarrhea rate and mast cell degranulation rate (P<0.01), an increased AWR minimum volume threshold (P<0.01), reduced serum and colonic levels of histamine, IL-1β, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 (P<0.01), decreased protein expression of colonic IL-1β, trypsin-like enzyme, and PAR-2 (P<0.01, P<0.05), reduced mRNA and positive expression of colonic IL-1β, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 (P<0.01, P<0.05), and decreased positive expression of colonic histamine (P<0.01). Moxibustion on "Tianshu" (ST 25) and "Shangjuxu" (ST 37) might inhibit low-grade inflammatory reactions in the colon of IBS-D model rats. The mechanism may be related to the inhibition of histamine and trypsin-like enzyme secreted by mast cell, thereby reducing the expression of related inflammatory factors.

Jiawei guomin decoction regulates the degranulation of mast cells in atopic dermatitis mice via the HIS/PAR-2 pathway

Ethnopharmacological relevanceGuomin decoction (GMD) is a traditional Chinese medicine commonly used in clinical practice. It has traditionally been used to treat all allergic diseases. Currently, Jiawei Guomin Decoction (JWGMD) is used to treat sensitive skin after initial therapy. Although it has a significant clinical therapeutic effect, the exact role of mast cell degranulation in treating atopic dermatitis (AD) is still unclear. Aim of the studyGMD and JWGMD can both treat allergic diseases, while JWGMD focuses on skin allergies. This study aims to explore the potential effect of JWGMD on the degranulation of mast cells in an AD mouse model induced by 2,4-dinitrofluorobenzene (DNFB) and investigate the effectiveness of JWGMD in alleviating disease progression to further provide specific therapeutic targets for treating AD. Materials and methodsThe scratching times and skin lesions of model mice induced by DNFB were observed, and skin tissues were collected for subsequent measurement. Histopathological changes in the back skin of mice were observed by haematoxylin eosin (H&E) staining, Toluidine blue staining was used to detect the degranulation of mouse skin mast cells, and the relationship between the expression of histamine (HIS), mast cell tryptase (MCT) and mast cell degranulation was analysed by enzyme-linked immunosorbent assay (ELISA). The expression of protease-activated receptor-2 (PAR-2), histamine 1 receptor (H1R), H2R, H4R and MCT proteins in AD mice was detected by Western blot (WB). Immunofluorescence assay (IFA) further confirmed the localization of PAR-2, H1R, H2R, H4R, and MCT proteins in the skin. Quantitative real-time PCR (qPCR) was used to determine PAR-2, H1R, H2R and H4R mRNA levels in skin lesions to further clarify the mechanism by which JWGMD amplifies mast cell degranulation in AD. In addition, a reliable ultrahigh-performance liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry (UPLC-QE-MS) nontargeted metabolomics analysis was performed to analyse the differences in metabolite abundance between GMD and JWGMD, and these results were used to identify the active components in JWGMD that may have antipruritic and anti-inflammatory properties and inhibit mast cell degranulation. ResultsAfter intermittent stimulation with DNFB, the skin lesions showed extensive desquamation, dryness, scabbing, skin thickening, and slight bleeding. Both treatments alleviated this phenomenon and reduced the number of scratches, with JWGMD being the most effective. JWGMD can significantly reduce inflammatory cell infiltration, oedema, and some capillary neogenesis in mice and reduce the degranulation of mast cells. The ELISA results showed that JWGMD can increase the levels of MCT and HIS proteins. The WB and IFA results demonstrated that JWGMD reduced the expression levels of PAR-2, H1R, H4R, and MCT proteins in skin lesions, with protein localization mainly in the epidermal layer, while H2R protein levels were increased and mainly localized in the dermis. In addition, JWGMD downregulates the mRNA expression of PAR-2, H1R, H2R, and H4R. Interestingly, through UPLC-QE-MS nontargeted metabolomic analysis, we detected the anti-inflammatory and antiallergy active substances in JWGMD, such as methyl eugenol, dictamnine and sinapine. ConclusionsJWGMD may alleviate itching through methyl syringol, dictamnine, sinapine and other substances, and its mechanism may be related to inhibiting the HIS/PAR-2 pathway in AD model mice and further regulating the self-amplification of mast cell degranulation. JWGMD is a potential drug for treating AD. Therefore, it deserves continuous attention and research.

Abstract 4961: Novel biased PAR2 inhibitors with best-in-class properties reduce resistance to both chemotherapy and immunotherapy in oncology models

Abstract A large-scale meta-analysis has recently identified Protease-activated receptor 2 (PAR2) gene expression to be significantly associated with resistance to immune checkpoint blockade (ICB) in cancer patients and preclinical models. PAR2 and its ligands (proteases) are indeed upregulated in different cancer types and are expressed by various cells in the tumor microenvironment. In cancer cells, the PAR2 receptor controls cell migration, proliferation, survival, and expression of inflammatory cytokines. In immune cells, it influences the infiltration and phenotype of macrophages and T cells. Therefore, PAR2 represents a promising therapeutic target in oncology and immuno-oncology. A novel series of potent and selective PAR2 inhibitors has been developed at Domain Therapeutics. In vitro experiments demonstrated unique properties of our PAR2 small molecule antagonists when compared to those of competitors. The antagonists inhibit pathogenic signaling pathways (i.e. Gz, G13, Gq, G14 and G15 protein activation as well as intracellular calcium) but not βarrestin2 recruitment, potentially reducing the risks of drug resistance. Furthermore, they maintain high potency and insurmountability in conditions that mimic the tumor microenvironment (high concentration of proteases and acidic pH). Finally, their pharmacokinetic properties are compatible with a once-a-day oral administration and demonstrate no signs of in vivo toxicity except at high doses (&amp;gt;500 mg/kg). Proof-of-concept experiments showed that PAR2 antagonists prevented PAR2-mediated resistance to Gefitinib in vitro and increased the potency of anti-PD1 therapy in vivo in pre-clinical syngeneic mouse models. Immunohistochemistry analyses from cancer patient biopsies confirmed that high expression levels of PAR2 in cancer and stromal cells within the tumor microenvironment significantly associates with the patient overall survival. In conclusion, new potent and selective negative allosteric modulators of PAR2 have been developed, they demonstrate strong potency by alleviating the resistance to both chemo- and immunotherapy in cancer models. These findings confirm the high value of PAR2 as a therapeutic target and demonstrates the relevance of small molecule inhibitors targeting this receptor to treat cancer. Citation Format: Thibaut Brugat, Francesco Bergami, Baptiste Rugeri, Aurélie Janvier, Edith Steinberg, Luc Baron, Mandy Recolet, Xavier Wirth, Meriem Semache, Antoine Mousson, Camille Dietsch, Quentin Ruet, Orphée Blanchard, Célia Jacoberger-Foissac, Isabelle Cousineau, Maleck Kadiri, Anne-Laure Blayo, Christel Franchet, Stanislas Mayer, John Stagg, Nathalie Lenne, Stephan Schann. Novel biased PAR2 inhibitors with best-in-class properties reduce resistance to both chemotherapy and immunotherapy in oncology models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4961.

Punicalagin suppresses inflammation in ventilator-induced lung injury through protease-activated receptor-2 inhibition-induced inhibition of NLR family pyrin domain containing-3 inflammasome activation.

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.

Protease activated receptor 2 signaling promotes self-renewal and metastasis in colorectal cancer through β-catenin and periostin.

The maintenance and expansion of cancer stem-like cells (CSCs) is necessary for metastasis. Although protease-activated receptor 2 (PAR2) is strongly associated with colorectal cancer (CRC) progression, it is unclear how it regulates distal metastasis, and no studies have shown the involvement of CSCs. In this study, we demonstrated that high PAR2 protein expression was correlated with metastatic CRC and poor prognosis in patients with stage III-IV CRC. CSCs from cell lines and patients showed higher levels of PAR2 than that of corresponding non-CSCs, and PAR2 inhibition reduced the CSC properties of the cell lines. Mechanistically, PAR2 inhibition switched the division mode of CSCs from symmetrical to asymmetrical via the ERK/GSK-3β/β-catenin pathway. We also identified periostin as a direct transcriptional target of β-catenin that mediates CSC self-renewal via PAR2 signaling. In a mouse xenograft model, PAR2 knockdown significantly attenuated liver metastasis. Finally, PAR2 expression was positively correlated with β-catenin and periostin in the primary sites of CRC with distant metastasis. Overall, our results indicate that PAR2 activation enhances CSC self-renewal and promotes metastasis through β-catenin and its target gene, periostin, in CRC.

Recombinant Activated Protein C (rhAPC) Affects Lipopolysaccharide-Induced Mechanical Compliance Changes and Beat Frequency of mESC-Derived Cardiomyocyte Monolayers.

ABSTRACTBackground:Septic cardiomyopathy increases mortality by 70% to 90% and results in mechanical dysfunction of cells.Methods:Here, we created a LPS-induced in-vitro sepsis model with mouse embryonic stem cell-derived cardiomyocytes (mESC-CM) using the CellDrum technology which simultaneously measures mechanical compliance and beat frequency of mESCs. Visualization of reactive oxygen species (ROS), actin stress fibers, and mRNA quantification of endothelial protein C receptor (EPCR) and protease-activated receptor 1 (PAR1) before/after LPS incubation were used for method validation. Since activated protein C (APC) has cardioprotective effects, samples were treated with human recombinant APC (rhAPC) with/-out LPS predamage to demonstrate the application in therapeutic studies.Results:Twelve hours LPS treatment (5 μg/mL) increased ROS and decreased actin stress fiber density and significantly downregulated EPCR and PAR1 compared to control samples (0.26, 0.39-fold respectively). rhAPC application (5 μg/mL, 12 h) decreased ROS and recovered actin density, EPCR, and PAR1 levels were significantly upregulated compared to LPS predamaged samples (4.79, 3.49-fold respectively). The beat frequencies were significantly decreased after 6- (86%) and 12 h (73%) of LPS application. Mechanical compliance of monolayers significantly increased in a time-dependent manner, up to eight times upon 12-h LPS incubation compared to controls. rhAPC incubation increased the beat frequency by 127% (6h-LPS) and 123% (12h-LPS) and decreased mechanical compliance by 68% (12h-LPS) compared to LPS predamaged samples.Conclusion:LPS-induced contraction dysfunction and the reversal effects of rhAPC were successfully assessed by the mechanical properties of mESC-CMs. The CellDrum technology proved a decent tool to simulate sepsis in-vitro.

PAR2 promotes high-fat diet-induced hepatic steatosis by inhibiting AMPK-mediated autophagy

Protease-activated receptor 2 (PAR2) is a member of G protein-coupled receptors. There are two types of PAR2 signaling pathways: Canonical G-protein signaling and β-arrestin signaling. Although PAR2 signaling has been reported to aggravate hepatic steatosis, the exact mechanism is still unclear, and the role of PAR2 in autophagy remains unknown. In this study, we investigated the regulatory role of PAR2 in autophagy during high-fat diet (HFD)-induced hepatic steatosis in mice. Increased protein levels of PAR2 and β-arrestin-2 and their interactions were detected after four months of HFD. To further investigate the role of PAR2, male and female wild-type (WT) and PAR2-knockout (PAR2 KO) mice were fed HFD. PAR2 deficiency protected HFD-induced hepatic steatosis in male mice, but not in female mice. Interestingly, PAR2-deficient liver showed increased AMP-activated protein kinase (AMPK) activation with decreased interaction between Ca2+/calmodulin-dependent protein kinase kinase β (CAMKKβ) and β-arrestin-2. In addition, PAR2 deficiency up-regulated autophagy in the liver. To elucidate whether PAR2 plays a role in the regulation of autophagy and lipid accumulation in vitro, PAR2 was overexpressed in HepG2 cells. Overexpression of PAR2 decreased AMPK activation with increased interaction of CAMKKβ with β-arrestin-2 and significantly inhibited autophagic responses in HepG2 cells. Inhibition of autophagy by PAR2 overexpression further exacerbated palmitate-induced lipid accumulation in HepG2 cells. Collectively, these findings suggest that the increase in the PAR2-β-arrestin-2-CAMKKβ complex by HFD inhibits AMPK-mediated autophagy, leading to the alleviation of hepatic steatosis.

A novel PAR2 chemiluminescence immunoassay as a biomarker for arthritic diseases

Purpose: Arthritis is a heterogeneous group of joint diseases, with the most common to be osteoarthritis (OA) and rheumatoid arthritis (RA). Increasing evidence has shown that protease-activated receptor 2 (PAR2) is involved in many pathophysiological characteristics of these diseases, like inflammatory responses, synovial hyperplasia, cartilage destruction, osteophyogenesis and pain sensitization. PAR2 is a G-protein transmembrane coupled receptor expressed in different parts of the joint, like the synovial lining, bone and cartilage. It is activated via cleavage of its N-terminus by serine proteases (e.g., tryptase), unveiling an N-terminus tethered ligand which binds to the receptor and leads to its activation. The aim of this study was to develop a novel blood-based biomarker measuring PAR2 activation fragment levels and investigate the association of PAR2 fragments with disease. Methods: A monoclonal antibody against the neo-epitope generated when PAR2 is cleaved and then activated, was produced. More specifically, an enzyme-linked chemiluminescence immunoassay was developed targeting the C-terminal sequence of the fragment that is released after cleavage of the N-terminal of the total protein. For optimal assay performance different buffers, peptide and antibody concentrations, incubations times and temperatures were tested. Briefly, the assay protocol was described as follows: coater peptide was added to streptavidin pre-coated plates and was incubated at 20oC for 30min. Next, after washing the plates, standards, controls, samples and HRP-labeled antibody are added and incubated at 4oC overnight. Chemiluminescence substrate was added on the plates, and signal was detected on the reader after 3-minute incubation with the substrate. Assay parameters including antibody specificity, intra- and inter-assay variation (CV%) and linearity were evaluated. The selectivity of the antibody towards the epitope sequence was examined by testing reactivity with an elongated and a truncated peptide. specificity of the assay was investigated by in vitro cleavage of the human full-length recombinant PAR2 protein by matriptase was performed. Biological validation of the assay was investigated in serum of 22 young healthy human donors and compared with that of 23 OA patients and 15 RA patients. Results: The specificity of the antibody was confirmed by showing absence of reactivity in the assay with the elongated and truncated peptides (Figure 1). Inter-intra assay variation was determined less than 15% (6.39% and 5.82% accordingly) and the linearity of the assay was acceptable for both serum and plasma. Cleavage of recombinant PAR2 with matriptase showed increased PAR2 pro-fragment levels compared to non-cleaved control. In serum, PAR2 was significantly higher in RA patients but not OA, compared to healthy controls (Figure 2). Conclusions: The PAR2 assay showed high sensitivity and specificity and good technical performance. PAR2 levels were increased in patients with RA compared to healthy donors, while only a numeric but non-statistical difference was observed for OA patients. These data indicate that PAR2 may be a potential biomarker in rheumatic diseases.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Effects of norepinephrine on colonic tight junction protein expression during heat stress.

Stress induced by changes in the internal or external environment in humans and animals leads to intestinal epithelial damage, in a manner that is associated with impaired intestinal barrier function. However, the role of the stress hormone norepinephrine (NE) in impairments in barrier function remains poorly understood. In the present study, a rat heat-exposed model was used to observe changes in the tight junction proteins Occludin and zonula occludens-1 (ZO-1), in addition to those in protease-activated receptor 2 (PAR-2) and transient receptor potential ankyrin 1 channel (TRPA1) in colon. The levels of plasma NE were detected using an ELISA kit. Different concentrations of NE were used to culture the human colon cell line Caco-2 for 6 and 24 h to investigate the cell viability using Cell Counting Kit-8 assay, whilst the expression levels of Occludin, ZO-1, PAR-2 and TRPA1 were examined using western blotting and immunofluorescence in Caco-2 cells and immunohistrochemistry in rat colon tissues. Although there was no clear histological damage to the rat colonic mucosa, there were decreased expression levels of tight junction proteins Occludin and ZO-1 after heat exposure. In addition, PAR-2 expression was increased by heat exposure. It was found that TRPA1 expression was concentrated to the luminal surface of the colon in the heat exposed group compared with that in the control group. After the administration of increasing concentrations of NE for 6 h, treatment did not affect cell viability. Furthermore, after application of NE for 24 h, cell viability gradually increased as the NE concentration was elevated from 10 to 100 µM. However, no significant increase in viability was observed when the cells were treated with 120 and 160 µM NE. Occludin expression was decreased when 10 µM NE was applied for 6 or 24 h. By contrast, 60 µM NE significantly downregulated Occludin expression in the 6 h group, but caused an insignificant decrease in the 24 h group. It was found that ZO-1 expression was upregulated after treatment with 10 µM NE for 6 h, whilst downregulation was observed after treatment with 10 µM NE for 24 h. PAR-2 protein expression was increased after application of NE for both 6 and 24 h, but not after treatment with 60 µM NE. In addition, TRPA1 expression was not affected by the treatment of NE, but increased positive staining was observed on the luminal side of the mucosa, which appeared to be concentrated in the cells of the luminal side in the rat colon after heat exposure. Collectively, the present results suggested that expression of tight junction proteins Occludin and ZO-1, in addition to that of PAR-2, can be regulated by NE, which may contribute to impairments in barrier function observed during heat stress.

Blockage of protease-activated receptor 2 exacerbates inflammation in high-fat environment partly through autophagy inhibition.

Protease-activated receptor 2 (PAR2) regulates inflammatory responses and lipid metabolism. However, its precise role in colitis remains unclear. In this study, we aimed to investigate the function of PAR2 in high-fat diet-fed mice with colitis and its potential role in autophagy. PAR2+/+ and PAR2-/- mice were fed a high-fat diet (HFD) for 7 days before colitis induction with dextran sodium sulfate. Deletion of PAR2 and an HFD significantly exacerbated colitis, as shown by increased mortality, body weight loss, diarrhea or bloody stools, colon length shortening, and mucosal damage. Proinflammatory cytokine levels were elevated in HFD-fed PAR2-/- mice and in cells treated with the PAR2 antagonist GB83, palmitic acid (PA), and a cytokine cocktail (CC). Damaging effects of PAR2 blockage were associated with autophagy regulation by reducing the levels of YAP1, SIRT1, PGC-1α, Atg5, and LC3A/B-I/II. In addition, mitochondrial dysfunction was demonstrated only in cells treated with GB83, PA, and CC. Reduced cell viability and greater induction of apoptosis, as shown by increased levels of cleaved caspase-9, cleaved caspase-3, and cleaved poly(ADP-ribose) polymerase (PARP), were observed in cells treated with GB83, PA, and CC but not in those treated with only PA and CC. Collectively, protective effects of PAR2 were elucidated during inflammation accompanied by a high-fat environment by promoting autophagy and inhibiting apoptosis, suggesting PAR2 as a therapeutic target for inflammatory bowel disease co-occurring with metabolic syndrome.NEW & NOTEWORTHY Deletion of PAR2 with high-fat diet feeding exacerbates colitis in a murine colitis model. Proinflammatory effects of PAR2 blockage in a high-fat environment were associated with an altered balance between autophagy and apoptosis. Increased colonic levels of PAR2 represent as a therapeutic strategy for IBD co-occurring with metabolic syndrome.

Vorapaxar-modified polysulfone membrane with high hemocompatibility inhibits thrombosis

Hemodialysis therapy is intended for patients suffering from renal insufficiency, pancreatitis, and other serious diseases. Platelets are an important active ingredient in the thrombosis induced by hemodialysis membranes. So far, there are few studies of hemodialysis membranes focusing on the effects of protease-activated receptor 1 (PAR1) activation on the platelet membrane. Among various antithrombotic agents, vorapaxar is a novel PAR1 inhibitor with high efficacy. In this study, we constructed a vorapaxar-modified polysulfone (VMPSf) membrane using immersion-precipitation phase transformation methods and characterized the microstructure in terms of hydrophilicity and mechanical properties. The water contact angle of the VMPSf membrane was 22.45% lower than that of the PSf membrane. A focused determination of platelet morphology was obtained using scanning electron microscopy. Meanwhile, we evaluated the effects of a VMPSf membrane on platelet adhesion. We observed that the VMPSf membrane could reduce the number of adhered platelets without altering their spherical or elliptical shape. The PAR1 levels in VMPSf membranes were 7.4 MFI lower than those in PSf membranes, suggesting that this modified membrane can effectively inhibit platelet activation. Activated partial thromboplastin time (APTT, 5.3 s extension) and thrombin time (TT, 2.1 s extension) reflect good anticoagulant properties. Recalcification time (80.6 s extension) and fibrinogen adsorption (9.9 μg/cm2 reduction) were related to antithrombotic properties. To determine the biosafety of VMPSf membranes, we investigated antianaphylactic and anti-inflammatory properties in vitro and acute toxicity in vivo, it was obvious that C3a and C5a had decreased to 9.6 and 0.8 ng/mL, respectively. The results indicated that the VMPSf membrane has potential for clinical application.

Brain Protease Activated Receptor 1 Pathway: A Therapeutic Target in the Superoxide Dismutase 1 (SOD1) Mouse Model of Amyotrophic Lateral Sclerosis.

Glia cells are involved in upper motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Protease activated receptor 1 (PAR1) pathway is related to brain pathologies. Brain PAR1 is located on peri-synaptic astrocytes, adjacent to pyramidal motor neurons, suggesting possible involvement in ALS. Brain thrombin activity in superoxide dismutase 1 (SOD1) mice was measured using a fluorometric assay, and PAR1 levels by western blot. PAR1 was localized using immunohistochemistry staining. Treatment targeted PAR1 pathway on three levels; thrombin inhibitor TLCK (N-Tosyl-Lys-chloromethylketone), PAR1 antagonist SCH-79797 and the Ras intracellular inhibitor FTS (S-trans-trans-farnesylthiosalicylic acid). Mice were weighed and assessed for motor function and survival. SOD1 brain thrombin activity was increased (p < 0.001) particularly in the posterior frontal lobe (p = 0.027) and hindbrain (p < 0.01). PAR1 levels were decreased (p < 0.001, brain, spinal cord, p < 0.05). PAR1 and glial fibrillary acidic protein (GFAP) staining decreased in the cerebellum and cortex. SOD1 mice lost weight (≥17 weeks, p = 0.047), and showed shorter rotarod time (≥14 weeks, p < 0.01). FTS 40mg/kg significantly improved rotarod scores (p < 0.001). Survival improved with all treatments (p < 0.01 for all treatments). PAR1 antagonism was the most efficient, with a median survival improvement of 10 days (p < 0.0001). Our results support PAR1 pathway involvement in ALS.

Protease-activated receptor 2 exacerbates cisplatin-induced nephrotoxicity.

Acute kidney injury (AKI) is associated with hypercoagulability. Tissue factor/factor VIIa complex and factor Xa in the coagulation cascade activate protease-activated receptor 2 (PAR2). Previously, we have shown that PAR2-mediated inflammation aggravates kidney injury in models of diabetic kidney disease and adenine-induced renal fibrosis. However, the role of PAR2 in AKI remains unclear. To clarify the role of PAR2, we administered cisplatin, one of the most common causal factors of AKI, to wild-type and PAR2-deficient mice. The expression levels of tissue factor and PAR2 were significantly increased in the kidneys of mice that were administered cisplatin. A lack of PAR2 corrected the levels of plasma blood urea nitrogen and creatinine as well as ameliorated the acute tubular injury score in the kidney. A lack of PAR2 corrected the infiltration of neutrophils and the gene expression levels of proinflammatory cytokines/chemokines in these mouse kidneys. Similarly, apoptotic markers, such as cleaved caspase-3-positive area and Bax/Bcl2 ratio, were attenuated via PAR2 deletion. Thus, elevated PAR2 exacerbates cisplatin nephrotoxicity, and targeting PAR2 is a novel therapeutic option that aids in the treatment of patients with cisplatin-induced AKI.

Endothelial cells in the innate response to allergens and initiation of atopic asthma.

Protease-activated receptor 2 (PAR-2), an airway epithelial pattern recognition receptor (PRR), participates in the genesis of house dust mite-induced (HDM-induced) asthma. Here, we hypothesized that lung endothelial cells and proangiogenic hematopoietic progenitor cells (PACs) that express high levels of PAR-2 contribute to the initiation of atopic asthma. HDM extract (HDME) protease allergens were found deep in the airway mucosa and breaching the endothelial barrier. Lung endothelial cells and PACs released the Th2-promoting cytokines IL-1α and GM-CSF in response to HDME, and the endothelium had PAC-derived VEGF-C-dependent blood vessel sprouting. Blockade of the angiogenic response by inhibition of VEGF-C signaling lessened the development of inflammation and airway remodeling in the HDM model. Reconstitution of the bone marrow in WT mice with PAR-2-deficient bone marrow also reduced airway inflammation and remodeling. Adoptive transfer of PACs that had been exposed to HDME induced angiogenesis and Th2 inflammation with remodeling similar to that induced by allergen challenge. Our findings identify that lung endothelium and PACs in the airway sense allergen and elicit an angiogenic response that is central to the innate nonimmune origins of Th2 inflammation.

Knockdown of EPCR inhibits the proliferation and migration of human gastric cancer cells via the ERK1/2 pathway in a PAR-1-dependent manner.

Endothelial proteinC receptor (EPCR) has been implicated in the carcinogenesis of diverse tumor types. This tumor-promoting effect of EPCR is associated with the upregulation of activated proteinC and the activation of protease-activated receptor1(PAR-1). However, the exact role of EPCR in gastric cancer(GC) and the mechanisms underlying the regulation of EPCR remain elusive. In the present study, we investigated the effects of EPCR on human GC cells, as well as the underlying mechanisms. An siRNA inference system was used to knock down the expression of EPCR in GC cells, and CCK-8, colony formation and Transwell assays were performed to determine the effects of EPCR knockdown on the proliferation and migration of the tumor cells. Additionally, cell cycle distribution and apoptosis were assessed by flow cytometry, and activated PAR-1 levels were determined by cell ELISA. The results indicated that the proliferation, clonogenicity and migration were significantly reduced and that the cell cycle was arrested in the Gap1phase by EPCR knockdown in SGC7901 and AGS cells. Meanwhile, apoptosis was promoted by EPCR knockdown in the two cell lines. The activation of PAR-1 on the cell surface of SGC7901 and AGS cells was significantly reduced after the knockdown of EPCR. By contrast, blockade of PAR-1 reduced the proliferation and migration of gastric cells in vitro. Additionally, after the knockdown of EPCR or treatment with PAR-1 antibody, the expression of pERK1/2 was significantly downregulated in the SGC7901 and AGS cells, while the expression levels of p-AKT(S473) and p-AKT(T308) were unchanged. The findings of the present study demonstrated that EPCR exerts pro-carcinogenic effects in GC cells in a PAR-1-dependent manner via the ERK1/2-MAPK pathway. Thus, EPCR may be a potential molecular diagnostic or therapeutic target for GC.

Electroacupuncture Relieves Visceral Hypersensitivity by Inactivating Protease-Activated Receptor 2 in a Rat Model of Postinfectious Irritable Bowel Syndrome.

Background The role of protease-activated receptor 2 (PAR2) in the analgesic effect of electroacupuncture (EA) on visceral hypersensitivity (VH) in postinfectious irritable bowel syndrome (PI-IBS) has yet to be elucidated. Aim In this study, we investigated the molecular mechanisms underlying the analgesic effect of EA in a rat model of PI-IBS. Methods Visceral hypersensitivity was evaluated by the abdominal withdrawal reflex test before and after administration of the PAR2 agonist, PAR2-AP, and/or EA. The protein expression and mRNA levels of PAR2, CGRP, SP, and TPSP in colon tissues were measured by immunofluorescence, western blot, and RT-PCR. Results We found that EA could alleviate VH and significantly decrease protein and mRNA levels of PAR2, TPSP, CGRP, and SP in PI-IBS rats. The analgesic effect of EA on VH was slightly reduced in the presence of PAR2-AP. Conclusions These results suggest that EA alleviates VH symptoms through downregulation of the levels of the TPSP/PAR2/SP/CGRP signaling axis in colon tissues in PI-IBS rats. Together, our data suggests that PAR2 plays a critical role in the analgesic effect of EA on VH in PI-IBS.

A Linear Temporal Increase in Thrombin Activity and Loss of Its Receptor in Mouse Brain following Ischemic Stroke.

Brain thrombin activity is increased following acute ischemic stroke and may play a pathogenic role through the protease-activated receptor 1 (PAR1). In order to better assess these factors, we obtained a novel detailed temporal and spatial profile of thrombin activity in a mouse model of permanent middle cerebral artery occlusion (pMCAo). Thrombin activity was measured by fluorescence spectroscopy on coronal slices taken from the ipsilateral and contralateral hemispheres 2, 5, and 24 h following pMCAo (n = 5, 6, 5 mice, respectively). Its spatial distribution was determined by punch samples taken from the ischemic core and penumbra and further confirmed using an enzyme histochemistry technique (n = 4). Levels of PAR1 were determined using western blot. Two hours following pMCAo, thrombin activity in the stroke core was already significantly higher than the contralateral area (11 ± 5 vs. 2 ± 1 mU/ml). At 5 and 24 h, thrombin activity continued to rise linearly (r = 0.998, p = 0.001) and to expand in the ischemic hemisphere beyond the ischemic core reaching deleterious levels of 271 ± 117 and 123 ± 14 mU/ml (mean ± SEM) in the basal ganglia and ischemic cortex, respectively. The peak elevation of thrombin activity in the ischemic core that was confirmed by fluorescence histochemistry was in good correlation with the infarcts areas. PAR1 levels in the ischemic core decreased as stroke progressed and thrombin activity increased. In conclusion, there is a time- and space-related increase in brain thrombin activity in acute ischemic stroke that is closely related to the progression of brain damage. These results may be useful in the development of therapeutic strategies for ischemic stroke that involve the thrombin-PAR1 pathway in order to prevent secondary thrombin related brain damage.

Inverse PAR1 Activity of Hematopoietic Stem Cells and BM Stromal Cells Mediates G-CSF-Induced Mobilization By Regulation of Nitric Oxide Generation

Hematopoietic stem and progenitor cell (HSPC) egress from the bone marrow (BM) to the circulation is tightly regulated and is accelerated during stress conditions, a process utilized for BM harvest. Recently, we demonstrated that mouse long term repopulating hematopoietic stem cell (LT-HSC) BM retention and their rapid release to the blood circulation are governed by a switch in nitric oxide (NO) generation via distinct coagulation-related protease activated receptor 1 (PAR1) cascades (Gur-Cohen S. et al., NM, 2016).Herein we report that surface PAR1 expression can be exploited and serve as a positive predictive marker for the efficiency of human CD34+ HSPC mobilization among healthy donors in clinical G-CSF-induced mobilization for matched allogeneic transplantations. We found that PAR1 expression on circulating leukocytes before G-CSF administration was positively correlated with higher yields of mobilized leukocytes after stimulation (P<0.001). In addition, the percentage of PAR1-expressing CD34+ HSPCs in the blood and their absolute numbers before initiation of mobilization positively correlated with peripheral blood leukocyte counts (P<0.001) and yield of mobilized CD34+ HSPCs (P=0.002). Importantly, tracing the consequences of the follow-up BM transplantation, we found accelerated neutrophil recovery in patients transplanted with mobilized cells expressing higher surface PAR1 levels at baseline (P=0.074), shortening the time for neutrophil recovery (neutrophil count>0.5 x 109/L) from an average of 14.6 days to an average of 11 days post-transplant. In addition, a trend of accelerated platelet production was documented to be related with higher PAR1 expression by circulating leukocytes prior to G-CSF stimulation. Consequently, poor mobilizer donors were characterized by extremely low surface PAR1 expression on circulating CD34+ cells prior to G-CSF stimulations. Herein we present a case report of a thrombophilic donor carrying the MTHFR mutation, expressing exceedingly low PAR1 levels at baseline, with the outcome of inadequate numbers of mobilized CD34+ HSPC in the blood following G-CSF treatments.To further gain insight into the role played by PAR1 signaling in the regulation of G-CSF-induced HSPC mobilization, we used mice as a functional preclinical small animal model. We found that antagonizing PAR1 signaling attenuated both steady state release and G-CSF-induced HSPC mobilization. Furthermore, co-administration of G-CSF with PAR1 antagonist attenuated secretion of BM stromal CXCL12 and abrogated upregulation of surface CXCR4 and PAR1 expression by BM HSPCs, all leading to significantly reduced HSPC migration, differentiation and mobilization. In support, PAR1-/- mice failed to efficiently mobilize HSPCs in response to G-CSF compared to wild type counterparts. Enforced HSPC recruitment by G-CSF treatments dramatically accelerated PAR1-dependent NO production by eNOS, known to promote TACE-mediated EPCR shedding and rapid LT-HSC mobilization. Concomitantly, circulating steady state and G-CSF-mobilized stem cells lack surface EPCR expression. Intriguingly, while EPCR expression by primitive BM stem cells was transiently reduced after G-CSF treatments, antagonizing PAR1 signaling along with G-CSF stimuli blocked NO generation and synchronically expanded BM EPCR+ LT-HSC and their supportive stromal progenitor cells (MSPCs), as confirmed by increased repopulation in transplanted mice. Finally, we report an inverse PAR1 expression and regulation by BM HSPC and stromal MSPCs in mediating G-CSF-induced mobilization. G-CSF induced elevation of PAR1 expression on BM HSPCs, providing the driving force for their enhanced NO mediated migration, proliferation, differentiation and recruitment to the circulation. Contrary, the levels of PAR1 expression were reduced on MSPCs in response to G-CSF treatment, and following NO generation by eNOS activity and CXCL12 secretion, resulted in reduced mesenchymal differentiation leading to accumulated numbers of immature mesenchymal (CFU-F) and osteoblast (CFU-OB) progenitor cells.Taken together, our study identifies and highlights inverse PAR1 signaling and NO generation as essential regulator of G-CSF induced HSPC mobilization and MSPC development opening new avenues to advance therapeutics for enhancing clinical G-CSF induced stem cell mobilization and transplantation protocols. DisclosuresRuf:Iconic Therapeutics: Consultancy.