Serum from COVID-19 patients promotes endothelial cell dysfunction through protease-activated receptor 2.

BACKGROUND: Endothelial dysfunction plays a central role in the pathophysiology of COVID-19 and is closely linked to the severity and mortality of the disease. The inflammatory response to SARS-CoV-2 infection can alter the capacity of the endothelium to regulate vascular tone, immune responses, and the balance between anti-thrombotic and pro-thrombotic properties. However, the specific endothelial pathways altered during COVID-19 still need to be fully understood. OBJECTIVE: In this study, we sought to identify molecular changes in endothelial cells induced by circulating factors characteristic of COVID-19. METHODS AND RESULTS: To this aim, we cultured endothelial cells with sera from patients with COVID-19 or non-COVID-19 pneumonia. Through transcriptomic analysis, we were able to identify a distinctive endothelial phenotype that is induced by sera from COVID-19 patients. We confirmed and expanded this observation in vitro by showing that COVID-19 serum alters functional properties of endothelial cells leading to increased apoptosis, loss of barrier integrity, and hypercoagulability. Furthermore, we demonstrated that these endothelial dysfunctions are mediated by protease-activated receptor 2 (PAR-2), as predicted by transcriptome network analysis validated by in vitro functional assays. CONCLUSION: Our findings provide the rationale for further studies to evaluate whether targeting PAR-2 may be a clinically effective strategy to counteract endothelial dysfunction in COVID-19.

Regulation of tissue factor activity by interaction with the first PDZ domain of MAGI1.

BACKGROUND: Tissue factor (TF) activity is stringently regulated through processes termed encryption. Post-translational modification of TF and its interactions with various protein and lipid moieties allows for a multi-step de-encryption of TF and procoagulant activation. Membrane-associated guanylate kinase-with inverted configuration (MAGI) proteins are known to regulate the localisation and activity of a number of proteins including cell-surface receptors. METHODS: The interaction of TF with MAGI1 protein was examined as a means of regulating TF activity. MDA-MB-231 cell line was used which express TF and MAGI1, and respond well to protease activated receptor (PAR)2 activation. Proximity ligation assay (PLA), co-immunoprecipitation and pull-down experiments were used to examine the interaction of TF with MAGI1-3 proteins and to investigate the influence of PAR2 activation. Furthermore, by cloning and expressing the PDZ domains from MAGI1, the TF-binding domain was identified. The ability of the recombinant PDZ domains to act as competitors for MAGI1, allowing the induction of TF procoagulant and signalling activity was then examined. RESULTS: PLA and fluorescence microscopic analysis indicated that TF predominantly associates with MAGI1 and less with MAGI2 and MAGI3 proteins. The interaction of TF with MAGI1 was also demonstrated by both co-immunoprecipitation of TF with MAGI1, and co-immunoprecipitation of MAGI1 with TF. Moreover, activation of PAR2 resulted in reduction in the association of these two proteins. Pull-down assays using TF-cytoplasmic domain peptides indicated that the phosphorylation of Ser253 within TF prevents its association with MAGI1. Additionally, the five HA-tagged PDZ domains of MAGI1 were overexpressed separately, and the putative TF-binding domain was identified as PDZ1 domain. Expression of this PDZ domain in cells significantly augmented the TF activity measured both as thrombin-generation and also TF-mediated proliferative signalling. CONCLUSIONS: Our data indicate a stabilising interaction between TF and the PDZ-1 domain of MAGI1 and demonstrate that the activation of PAR2 disrupts this interaction. The release of TF from MAGI1 appears to be an initial step in TF de-encryption, associated with increased TF-mediated procoagulant and signalling activities. This mechanism is also likely to lead to further interactions and modifications leading to further enhancement of procoagulant activity, or the release of TF.

Genetic priming of sensory neurons in mice that overexpress PAR2 enhances allergen responsiveness.

Pruritus is a common symptom of inflammatory skin conditions, including atopic dermatitis (AD). Although primary sensory neurons that transmit pruritic signals are well-cataloged, little is known about the neuronal alterations that occur as a result of skin disruption in AD. To address this question, we examined the molecular and behavioral consequences of challenging Grhl3PAR2/+ mice, which overexpress PAR2 in suprabasal keratinocytes, with serial topical application of the environmental allergen house dust mite (HDM). We monitored behavior and used RNA sequencing, qPCR, and in situ hybridization to evaluate gene expression in trigeminal ganglia (TG), before and after HDM. We found that neither Grhl3PAR2/+ nor wild-type (WT) mice exhibited spontaneous scratching, and pruritogen-induced acute scratching did not differ. In contrast, HDM exacerbated scratching in Grhl3PAR2/+ mice. Despite the absence of scratching in untreated Grhl3PAR2/+ mice, several TG genes in these mice were up-regulated compared to WT. HDM treatment of the Grhl3PAR2/+ mice enhanced up-regulation of this set of genes and induced additional genes, many within the subset of TG neurons that express TRPV1. The same set of genes was up-regulated in HDM-treated Grhl3PAR2/+ mice that did not scratch, but at lesser magnitude. Finally, we recorded comparable transcriptional changes in IL31Tg mice, demonstrating that a common genetic program is induced in two AD models. Taken together, we conclude that transcriptional changes that occur in primary sensory neurons in dermatitis-susceptible animals underlie a genetic priming that not only sensitizes the animal to chronic allergens but also contributes to pruritus in atopic skin disease.

Targeting PAR2-mediated inflammation in osteoarthritis: a comprehensive in vitro evaluation of oleocanthal's potential as a functional food intervention for chondrocyte protection and anti-inflammatory effects.

BACKGROUND: Osteoarthritis (OA) is a prevalent degenerative joint disease characterized by chronic inflammation and progressive cartilage degradation, ultimately leading to joint dysfunction and disability. Oleocanthal (OC), a bioactive phenolic compound derived from extra virgin olive oil, has garnered significant attention due to its potent anti-inflammatory properties, which are comparable to those of non-steroidal anti-inflammatory drugs (NSAIDs). This study pioneers the investigation into the effects of OC on the Protease-Activated Receptor-2 (PAR-2) mediated inflammatory pathway in OA, aiming to validate its efficacy as a functional food-based therapeutic intervention. METHODS: To simulate cartilage tissue in vitro, human bone marrow-derived mesenchymal stem cells (BMSCs) were differentiated into chondrocytes. An inflammatory OA-like environment was induced in these chondrocytes using lipopolysaccharide (LPS) to mimic the pathological conditions of OA. The therapeutic effects of OC were evaluated by treating these inflamed chondrocytes with various concentrations of OC. The study focused on assessing key inflammatory markers, catabolic enzymes, and mitochondrial function to elucidate the protective mechanisms of OC. Mitochondrial function, specifically mitochondrial membrane potential (ΔΨm), was assessed using Rhodamine 123 staining, a fluorescent dye that selectively accumulates in active mitochondria. The integrity of ΔΨm serves as an indicator of mitochondrial and bioenergetic function. Additionally, Western blotting was employed to analyze protein expression levels, while real-time polymerase chain reaction (RT-PCR) was used to quantify gene expression of inflammatory cytokines and catabolic enzymes. Flow cytometry was utilized to measure cell viability and apoptosis, providing a comprehensive evaluation of OC's therapeutic effects on chondrocytes. RESULTS: The results demonstrated that OC significantly downregulated PAR-2 expression in a dose-dependent manner, leading to a substantial reduction in pro-inflammatory cytokines, including TNF-α, IL-1ß, and MCP-1. Furthermore, OC attenuated the expression of catabolic markers such as SOX4 and ADAMTS5, which are critically involved in cartilage matrix degradation. Importantly, OC was found to preserve mitochondrial membrane potential (ΔΨm) in chondrocytes subjected to inflammatory stress, as evidenced by Rhodamine 123 staining, indicating a protective effect on cellular bioenergetics. Additionally, OC modulated the Receptor Activator of Nuclear Factor Kappa-Β Ligand (RANKL)/Receptor Activator of Nuclear Factor Kappa-Β (RANK) pathway, suggesting a broader therapeutic action against the multifactorial pathogenesis of OA. CONCLUSIONS: This study is the first to elucidate the modulatory effects of OC on the PAR-2 mediated inflammatory pathway in OA, revealing its potential as a multifaceted therapeutic agent that not only mitigates inflammation but also protects cartilage integrity. The preservation of mitochondrial function and modulation of the RANKL/RANK pathway further underscores OC's comprehensive therapeutic potential in counteracting the complex pathogenesis of OA. These findings position OC as a promising candidate for integration into nutritional interventions aimed at managing OA. However, further research is warranted to fully explore OC's therapeutic potential across different stages of OA and its long-term effects in musculoskeletal disorders.

Comment on Lai et al. Dipeptidyl Peptidase 4 Stimulation Induces Adipogenesis-Related Gene Expression of Adipose Stromal Cells. Int. J. Mol. Sci. 2023, 24, 16101.

Adiponectin is a circulating hormone secreted by adipose tissue that exerts, unlike other adipokines such as leptin, anti-inflammatory, anti-atherosclerotic and other protective effects on health. Adiponectin receptor agonists are being tested in clinical trials and are expected to show benefits in many diseases. In a recent article, LW Chen's group used monocyte chemoattractant protein-1 (MCP-1/CCL2) to improve plasma levels of adiponectin, suggesting the involvement of dipeptidyl peptidase 4 (DPP4/CD26) in the mechanism. Here, we discuss the significance of the role of DPP4, favoring the increase in DPP4-positive interstitial progenitor cells, a finding that fits with the greater stemness and persistence of other DPP4/CD26-positive cells.

α-Arrestin ARRDC3 tumor suppressor function is linked to GPCR-induced TAZ activation and breast cancer metastasis.

The α-arrestin domain containing protein 3 (ARRDC3) is a tumor suppressor in triple-negative breast carcinoma (TNBC), a highly metastatic subtype of breast cancer that lacks targeted therapies. Thus, understanding the mechanisms and targets of ARRDC3 in TNBC is important. ARRDC3 regulates trafficking of protease-activated receptor 1 (PAR1, also known as F2R), a G-protein-coupled receptor (GPCR) implicated in breast cancer metastasis. Loss of ARRDC3 causes overexpression of PAR1 and aberrant signaling. Moreover, dysregulation of GPCR-induced Hippo signaling is associated with breast cancer progression. However, the mechanisms responsible for Hippo dysregulation remain unknown. Here, we report that the Hippo pathway transcriptional co-activator TAZ (also known as WWTR1) is the major effector of GPCR signaling and is required for TNBC migration and invasion. Additionally, ARRDC3 suppresses PAR1-induced Hippo signaling via sequestration of TAZ, which occurs independently of ARRDC3-regulated PAR1 trafficking. The ARRDC3 C-terminal PPXY motifs and TAZ WW domain are crucial for this interaction and are required for suppression of TNBC migration and lung metastasis in vivo. These studies are the first to demonstrate a role for ARRDC3 in regulating GPCR-induced TAZ activity in TNBC and reveal multi-faceted tumor suppressor functions of ARRDC3. This article has an associated First Person interview with the first author of the paper.

Reconstructing the epidermal proteolytic cascades in health and disease.

The epidermis is the outer stratified epithelium of the skin, forming the physical barrier that is indispensable for homeostasis. Epidermal proteolysis, mainly but not exclusively executed by kallikrein-related peptidases (KLKs), is tightly regulated to ensure maintenance of physiological skin renewal and an intact skin barrier. Perturbation of epidermal proteolytic networks is implicated in a wide array of rare and common skin pathologies of diverse genetic backgrounds. Recent studies of monogenic human skin diseases and newly developed animal models have revealed new mechanisms of regulation of proteolytic pathways in epidermal physiology and in disease states. These new data have challenged some accepted views, for example the role of matriptase in epidermal desquamation, which turned out to be restricted to mouse skin. The significance of PAR2 signaling in skin inflammation should also be reconsidered in the face of recent findings. Cumulatively, recent studies necessitate a sophisticated redefinition of the proteolytic and signaling pathways that operate in human skin. We elaborate how epidermal proteolysis is finely regulated at multiple levels, and in a spatial manner that has not been taken into consideration so far, in which specific proteases are confined to distinct epidermal sublayers. Of interest, transglutaminases have emerged as regulators of epidermal proteolysis and desquamation by spatially fixing endogenous protease inhibitors, constituting regulatory factors that were not recognized before. Furthermore, new evidence suggests a link between proteolysis and lipid metabolism. By synthesis of established notions and recent discoveries, we provide an up-to-date critical evaluation and synthesis of current knowledge and the extended complexity of proteolysis regulation and signaling pathways in skin. © 2022 The Pathological Society of Great Britain and Ireland.

Recent Advances in Pathology: the 2022 Annual Review Issue of The Journal of Pathology.

The 2022 Annual Review Issue of The Journal of Pathology, Recent Advances in Pathology, contains 15 invited reviews on research areas of growing importance in pathology. This year, the articles include those that focus on digital pathology, employing modern imaging techniques and software to enable improved diagnostic and research applications to study human diseases. This subject area includes the ability to identify specific genetic alterations through the morphological changes they induce, as well as integrating digital and computational pathology with 'omics technologies. Other reviews in this issue include an updated evaluation of mutational patterns (mutation signatures) in cancer, the applications of lineage tracing in human tissues, and single cell sequencing technologies to uncover tumour evolution and tumour heterogeneity. The tissue microenvironment is covered in reviews specifically dealing with proteolytic control of epidermal differentiation, cancer-associated fibroblasts, field cancerisation, and host factors that determine tumour immunity. All of the reviews contained in this issue are the work of invited experts selected to discuss the considerable recent progress in their respective fields and are freely available online (https://onlinelibrary.wiley.com/journal/10969896). © 2022 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Effect of a protease-activated receptor-2 antagonist (GB88) on inflammation-related loss of alveolar bone in periodontal disease.

BACKGROUND AND OBJECTIVE: Protease-activated receptor-2 (PAR2 ), a pro-inflammatory G-protein coupled receptor, has been associated with pathogenesis of periodontitis and the resulting bone loss caused by oral pathogens, including the keystone pathogen Porphyromonas gingivalis (P. gingivalis). We hypothesised that administration of a PAR2 antagonist, GB88, might prevent inflammation and subsequent alveolar bone resorption in a mouse model of periodontal disease. METHODS: Periodontitis was induced in mice by oral inoculations with P. gingivalis for a total of eight times over 24 days. The infected mice were treated with either GB88 or vehicle for the duration of the trial. Following euthanasia on day 56, serum was collected and used for the detection of mast cell tryptase. The right maxillae were defleshed and stained with methylene blue to measure the exposed cementum in molar teeth. The left maxillae were prepared for cryosections followed by staining for tartrate-resistant acid phosphatase to identify osteoclasts or with toluidine blue to identify mast cells. Reverse transcription quantitative PCR (RT-qPCR) was used to quantify the expression of inflammatory cytokines in the gingival tissue. Supernatants of T-lymphocyte cultures isolated from the regional lymph nodes were assayed using a cytometric bead array to measure the Th1/Th2/Th17 cytokine levels. RESULTS: Measurement of the exposed cementum showed that GB88 reduced P. gingivalis-induced alveolar bone loss by up to 69%. GB88 also prevented the increase in osteoclast numbers observed in the infected mice. Serum tryptase levels were significantly elevated in both the infected groups, and not altered by treatment. RT-qPCR showed that GB88 prevented the upregulation of Il1b, Il6, Ifng and Cd11b. In T-lymphocyte supernatants, only IFNγ and IL-17A levels were increased in response to infection, but this was prevented by GB88 treatment. CONCLUSIONS: GB88 significantly reduced osteoclastic alveolar bone loss in mice infected with P. gingivalis, seemingly by preventing the upregulation of several inflammatory cytokines. PAR2 antagonism may be an effective treatment strategy for periodontal disease.

Comparison of pancreatic enzyme abnormalities and protease-activated receptor-2-positive eosinophils in the duodenum of patients with functional dyspepsia-irritable bowel syndrome overlap with functional dyspepsia alone in Asian populations.

BACKGROUND AND AIM: Some patients with functional gastrointestinal disorders exhibit pancreatic dysfunctions and pancreatic enzyme abnormalities. Thus, we aimed to clarify whether significant differences in clinical characteristics, prevalence of pancreatic enzyme abnormalities, duodenal inflammation, and protease-activated receptor 2 (PAR2) expression levels related to hypersensitivity exist between functional dyspepsia (FD) alone and FD-irritable bowel syndrome (IBS) overlap group. METHODS: Ninety-three patients based on the Rome IV criteria, FD alone (n = 44) and FD overlapped with IBS (n = 49) group were enrolled. The patients scored their own clinical symptoms after consuming high-fat meals. Serum trypsin, PLA2, lipase, p-amylase, and elastase-1 levels were measured. PAR2, eotaxin-3, and TRPV4 mRNA levels in duodenum were determined using real-time polymerase chain reaction methods. PRG2- and PAR2 in the duodenum were evaluated using immunostaining. RESULTS: FD score and global GSRS in patients with FD-IBS overlap were significantly higher than FD alone. Although the prevalence of pancreatic enzyme abnormalities in patients with FD alone was significantly (P < 0.01) higher than that in FD-IBS overlap, the ratio of aggravation of clinical symptoms following high-fat intake in patients with FD-IBS overlap was significantly higher (P = 0.007) than that in patients with FD alone. PAR2- and PRG2-double positive cells were localized in the degranulated eosinophils in the duodenum of patients with FD-IBS overlap. The number of PAR2- and PRG2-double positive cells in FD-IBS overlap was significantly (P < 0.01) higher than FD alone. CONCLUSIONS: Pancreatic enzyme abnormalities and PAR2 expression on degranulated eosinophils infiltrations in the duodenum may be associated with the pathophysiology of patients with FD-IBS overlap in Asian populations.

Mast cells instruct keratinocytes to produce thymic stromal lymphopoietin: Relevance of the tryptase/protease-activated receptor 2 axis.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) promotes TH2 inflammation and is deeply intertwined with inflammatory dermatoses like atopic dermatitis. The mechanisms regulating TSLP are poorly defined. OBJECTIVE: We investigated whether and by what mechanisms mast cells (MCs) foster TSLP responses in the cutaneous environment. METHODS: Ex vivo and in vivo skin MC degranulation was induced by compound 48/80 in wild-type protease-activated receptor 2 (PAR-2)- and MC-deficient mice in the presence or absence of neutralizing antibodies, antagonists, or exogenous mouse MC protease 6 (mMCP6). Primary human keratinocytes and murine skin explants were stimulated with lysates/supernatants of human skin MCs, purified tryptase, or MC lysate diminished of tryptase. Chymase and histamine were also used. TSLP was quantified by ELISA, real-time quantitative PCR, and immunofluorescence staining. RESULTS: Mas-related G protein-coupled receptor X2 (Mrgprb2) activation elicited TSLP in intact skin, mainly in the epidermis. Responses were strictly MC dependent and relied on PAR-2. Complementarily, TSLP was elicited by tryptase in murine skin explants. Exogenous mMCP6 could fully restore responsiveness in MC-deficient murine skin explants. Conversely, PAR-2 knockout mice were unresponsive to mMCP6 while displaying increased responsiveness to other inflammatory pathways, such as IL-1α. Indeed, IL-1α acted in concert with tryptase. In primary human keratinocytes, MC-elicited TSLP generation was likewise abolished by tryptase inhibition or elimination. Chymase and histamine did not affect TSLP production, but histamine triggered IL-6, IL-8, and stem cell factor. CONCLUSION: MCs communicate with kerationocytes more broadly than hitherto suspected. The tryptase/PAR-2 axis is a crucial component of this cross talk, underlying MC-dependent stimulation of TSLP in neighboring kerationocytes. Interference specifically with MC tryptase may offer a treatment option for disorders initiated or perpetuated by aberrant TSLP, such as atopic dermatitis.

PAR2 activation in the dura causes acute behavioral responses and priming to glyceryl trinitrate in a mouse migraine model.

BACKGROUND: Migraine is a severely debilitating disorder that affects millions of people worldwide. Studies have indicated that activation of protease-activated receptor-2 (PAR2) in the dura mater causes headache responses in preclinical models. It is also well known that vasodilators such as nitric oxide (NO) donors can trigger migraine attacks in migraine patients but not controls. In the current study we examined whether activation of PAR2 in the dura causes priming to the NO donor glyceryl trinitrate (GTN). METHODS: A preclinical behavioral model of migraine was used where stimuli (PAR2 agonists: 2at-LIGRL-NH2 (2AT) or neutrophil elastase (NE); and IL-6) were applied to the mouse dura through an injection made at the intersection of the lamdoidal and sagittal sutures on the skull. Following dural injection, periorbital von Frey thresholds and facial grimace responses were measured until their return to baseline. GTN was then given by intraperitoneal injection and periorbital hypersensitivity and facial grimace responses observed until they returned to baseline. RESULTS: We found that application of the selective PAR2 agonist 2at-LIGRL-NH2 (2AT) onto the dura causes headache-related behavioral responses in WT but not PAR2-/- mice with no differences between sexes. Additionally, dural PAR2 activation with 2AT caused priming to GTN (1 mg/kg) at 14 days after primary dural stimulation. PAR2-/- mice showed no priming to GTN. We also tested behavioral responses to the endogenous protease neutrophil elastase, which can cleave and activate PAR2. Dural neutrophil elastase caused both acute responses and priming to GTN in WT but not PAR2-/- mice. Finally, we show that dural IL-6 causes acute responses and priming to GTN that is identical in WT and PAR2-/- mice, indicating that IL-6 does not act through PAR2 in this model. CONCLUSIONS: These results indicate that PAR2 activation in the meninges can cause acute headache behavioral responses and priming to an NO donor, and support further exploration of PAR2 as a novel therapeutic target for migraine.

PAR2 Overexpression is Involved in the Occurrence of Hyperoxygen-Induced Bronchopulmonary Dysplasia in Rats.

BACKGROUND: Bronchopulmonary dysplasia is a chronic lung disease commonly seen in preterm infants. It is characterized by delayed development of the alveoli and lung fibrosis. Protease-activated receptor 2 (PAR2) is an inflammatory driver that plays a proinflammatory role mainly through the P38 MAPK/NF-κB signaling pathway. METHODS: Newborn rat pups were kept under air or oxygen at >60% concentration. Lung tissues were collected at postnatal days (P) 1, 4, 7, and 10 to observe pathological changes and take measurements. RESULTS: In the hyperoxic group, P4 and P7 rats showed delayed alveolar development, septal thickening, and disturbances in alveolar structure.PAR2, P38 MAPK, NF-κB, and IL-18 expression at P4, P7, and P10 was significantly higher than in the air group. CONCLUSION: PAR2 is involved in lung injury induced by persistent hyperoxia. Activated PAR2 promotes IL-18 overexpression through the P38 MAPK/NF-κB signaling pathway, which may be an important mechanism of PAR2-mediated lung injury in bronchopulmonary dysplasia.

Esophageal mast cells may be associated with the perception of symptoms in patients with eosinophilic esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is a type 2 helper T-cell (Th2)-mediated allergic disease that involves mast cells. This study aimed to clarify the relationship between perception of symptoms and mast cell levels in patients with EoE. METHODS: We enrolled patients with asymptomatic esophageal eosinophilia (aEE) and those with symptomatic EoE. Immunofluorescence staining was performed on esophageal biopsy specimens to quantify mast cell-related molecules, such as tryptase, proteinase-activated receptor (PAR)-2, and vasoactive intestinal peptide receptor (VPAC)-1. RESULTS: We evaluated 28 and 58 patients with aEE and EoE, respectively. There were no significant differences in clinical and endoscopic features and peak eosinophil counts between both groups. Mast cell tryptase-positive areas were significantly higher in EoE than in aEE (4.9 [3.5-6.2] vs. 2.0 [1.2-3.4] %, p < 0.01). The number of PAR-2-positive cells was significantly higher in EoE than in aEE (14 [8.8-20.0] vs. 4 [2.8-8.0] cells/high-power field [HPF], p < 0.01). The number of VPAC-1-positive cells was significantly higher in the EoE group than in the aEE group (13 [8.8-16.0] vs. 6 [3.0-9.3] cells/HPF, p < 0.01). A positive correlation was observed between the numbers of PAR-2-positive cells and VPAC-1-positive cells (r = 0.851, p < 0.01). Moreover, mast cell tryptase-positive areas positively correlated with the number of PAR-2- and VPAC-1-positive cells (r = 0.352, p < 0.01; r = 0.355, p < 0.01, respectively). CONCLUSIONS: Esophageal mast cells and their receptors, PAR-2 and VPAC-1, may contribute to the perception of symptoms in patients with EoE.

Prenatal stress induces changes in PAR2- and M3-dependent regulation of colon primitive cells.

Prenatal stress is associated with a high risk of developing adult intestinal pathologies, such as irritable bowel syndrome, chronic inflammation, and cancer. Although epithelial stem cells and progenitors have been implicated in intestinal pathophysiology, how prenatal stress could impact their functions is still unknown. We have investigated the proliferative and differentiation capacities of primitive cells using epithelial crypts isolated from colons of adult male and female mice whose mothers have been stressed during late gestation. Our results show that stem cell/progenitor proliferation and differentiation in vitro are negatively impacted by prenatal stress in male progeny. This is promoted by a reinforcement of the negative proliferative/differentiation control by the protease-activated receptor 2 (PAR2) and the muscarinic receptor 3 (M3), two G protein-coupled receptors present in the crypt. Conversely, prenatal stress does not change in vitro proliferation of colon primitive cells in female progeny. Importantly, this maintenance is associated with a functional switch in the M3 negative control of colonoid growth, becoming proliferative after prenatal stress. In addition, the proliferative role of PAR2 specific to females is maintained under prenatal stress, even though PAR2-targeted stress signals Dusp6 and activated GSK3ß are increased, reaching the levels of males. An epithelial serine protease could play a critical role in the activation of the survival kinase GSK3ß in colonoids from prenatally stressed female progeny. Altogether, our results show that following prenatal stress, colon primitive cells cope with stress through sexually dimorphic mechanisms that could pave the way to dysregulated crypt regeneration and intestinal pathologies.NEW & NOTEWORTHY Primitive cells isolated from mouse colon following prenatal stress and exposed to additional stress conditions such as in vitro culture, present sexually dimorphic mechanisms based on PAR2- and M3-dependent regulation of proliferation and differentiation. Whereas prenatal stress reinforces the physiological negative control exerted by PAR2 and M3 in crypts from males, in females, it induces a switch in M3- and PAR2-dependent regulation leading to a resistant and proliferative phenotype of progenitor.

Protease-activated receptor 2 enhances innate and inflammatory mechanisms induced by lipopolysaccharide in macrophages from C57BL/6 mice.

OBJECTIVE: This study was conducted to investigate the effects of the synthetic PAR2 agonist peptide (PAR2-AP) SLIGRL-NH2 on LPS-induced inflammatory mechanisms in peritoneal macrophages. METHODS: Peritoneal macrophages obtained from C57BL/6 mice were incubated with PAR2-AP and/or LPS, and the phagocytosis of zymosan fluorescein isothiocyanate (FITC) particles; nitric oxide (NO), reactive oxygen species (ROS), and cytokine production; and inducible NO synthase (iNOS) expression in macrophages co-cultured with PAR-2-AP/LPS were evaluated. RESULTS: Co-incubation of macrophages with PAR2AP (30 µM)/LPS (100 ng/mL) enhanced LPS-induced phagocytosis; production of NO, ROS, and the pro-inflammatory cytokines interleukin (IL)-1ß, tumour necrosis factor (TNF)-α, IL-6, and C-C motif chemokine ligand (CCL)2; and iNOS expression and impaired the release of the anti-inflammatory cytokine IL-10 after 4 h of co-stimulation. In addition, PAR2AP increased the LPS-induced translocation of the p65 subunit of the pro-inflammatory transcription factor nuclear factor kappa B (NF-κB) and reduced the expression of inhibitor of NF-κB. CONCLUSION: This study provides evidence of a role for PAR2 in macrophage response triggered by LPS enhancing the phagocytic activity and NO, ROS, and cytokine production, resulting in the initial and adequate macrophage response required for their innate response mechanisms.

Biochanin-A has antidiabetic, antihyperlipidemic, antioxidant, and protective effects on diabetic nephropathy via suppression of TGF-ß1 and PAR-2 genes expression in kidney tissues of STZ-induced diabetic rats.

One of the major complications of diabetes is diabetic nephropathy, and often many patients suffer from diabetic nephropathy. That is why it is important to find the mechanisms that cause nephropathy and its treatment. This study was designed to examine the antidiabetic effects of biochanin A (BCA) and evaluate its effects on oxidative stress markers and the expression of transforming growth factor-ß1 (TGF-ß1) and protease-activated receptors-2 (PAR-2) genes in the kidney of type 1 diabetic rats. After induction of diabetes using streptozotocin (STZ), 55 mg/kg bw dose, rats were randomly divided into four groups with six rats in each group as follows: normal group: normal control receiving normal saline and a single dose of citrate buffer daily; diabetic control group: diabetic control receiving 0.5% dimethyl sulfoxide daily; diabetic+BCA (10 mg/kg) group: diabetic rats receiving biochanin A at a dose of 10 mg/kg bw daily; diabetic+BCA (15 mg/kg) group: diabetic rats receiving biochanin A at a dose of 15 mg/kg bw daily. TGF-ß1 and PAR-2 gene expression was assessed by real-time. Spectrophotometric methods were used to measure biochemical factors: fast blood glucose (FBG), urea, creatinine, albumin, lipids profiles malondialdehyde (MDA), and superoxide dismutase (SOD). The course of treatment in this study was 42 days. The results showed that in the diabetic control group, FBG, serum urea, creatinine, expression of TGF-ß1 and PAR-2 genes, and the levels of MDA in kidney tissue significantly increased and SOD activity in kidney tissue and serum albumin significantly decreased compared to the normal group (p < 0.001). The results showed that administration of biochanin A (10 and 15 mg/kg) after 42 days significantly reduced the expression of TGF-ß1 and PAR-2 genes and FBG, urea, creatinine in serum compared to the diabetic control group (p < 0.001), also significantly increased serum albumin compared to the diabetic control group (p < 0.001). The level of MDA and SOD activity in the tissues of diabetic rats that used biochanin A (10 and 15 mg/kg) was significantly reduced and increased, respectively, compared to the diabetic control group (p < 0.001). Also, the result showed that in the diabetic control group lipids profiles significantly is disturbed compared to the normal group (p < 0.001), the results also showed that biochanin A (10 and 15 mg/kg) administration could significantly improved the lipids profile compared to the control diabetic group (p < 0.001). It is noteworthy that it was found that the beneficial effects of the biochanin A were dose dependent. In conclusion, administration of biochanin A for 42 days has beneficial effect and improves diabetes and nephropathy in diabetic rats. So probably biochanin A can be used as an adjunct therapy in the treatment of diabetes.

Knockdown of PAR2 alleviates cancer-induced bone pain by inhibiting the activation of astrocytes and the ERK pathway.

OBJECTIVE: Cancer-induced bone pain (CIBP) is a kind of pain with complex pathophysiology. Proteinase-activated receptor 2 (PAR-2) is involved in CIBP. This study explored the effects of PAR-2 on CIBP rats. METHODS: CIBP rat model was established by injecting Walker 256 rat breast cancer cells into the left tibia of female Sprague-Dawley rats and verified by tibial morphology observation, HE staining, and mechanical hyperalgesia assay. CIBP rats were injected with PAR-2 inhibitor, ERK activator, and CREB inhibitor through the spinal cord sheath on the 13th day after operation. CIBP behaviors were measured by mechanical hyperalgesia assay. On the 14th day after operation, L4-5 spinal cord tissues were obtained. PAR-2 expression, co-expression of PAR-2 and astrocyte marker GFAP, GFAP mRNA and protein levels and the ERK pathway-related protein levels were detected by Western blot, immunofluorescence double staining, RT-qPCR, and Western blot. RESULTS: CIBP rats had obvious mechanical hyperalgesia and thermal hyperalgesia from the 7th day after modeling; mechanical hyperalgesia threshold and thermal threshold were decreased; PAR-2 was increased in spinal cord tissues and was co-expressed with GFAP. PAR-2 silencing alleviated rat CIBP by inhibiting astrocyte activation. p-ERK/t-ERK and p-CREB/t-CREB levels in CIBP spinal cord were elevated, the ERK/CREB pathway was activated, while the ERK/CREB pathway was inhibited by PAR-2 silencing. The alleviating effect of PAR-2 inhibitor on hyperalgesia behaviors in CIBP rats were weakened by ERK activator, while were partially restored by CREB inhibitor. CONCLUSIONS: PAR-2 knockdown inhibited the ERK/CREB pathway activation and astrocyte activation, thus alleviating CIBP in rats.

GB83, an Agonist of PAR2 with a Unique Mechanism of Action Distinct from Trypsin and PAR2-AP.

Protease-activated receptor 2 (PAR2) is a G-protein-coupled receptor (GPCR) activated by proteolytic cleavage of its N-terminal domain. Once activated, PAR2 is rapidly desensitized and internalized by phosphorylation and ß-arrestin recruitment. Due to its irreversible activation mechanism, some agonists that rapidly desensitized PAR2 have been misconceived as antagonists, and this has impeded a better understanding of the pathophysiological role of PAR2. In the present study, we found that GB83, initially identified as a PAR2 antagonist, is a bona fide agonist of PAR2 that induces unique cellular signaling, distinct from trypsin and PAR2-activating peptide (AP). Activation of PAR2 by GB83 markedly elicited an increase in intracellular calcium levels and phosphorylation of MAPKs, but in a delayed and sustained manner compared to the rapid and transient signals induced by trypsin and PAR2-AP. Interestingly, unlike PAR2-AP, GB83 and trypsin induced sustained receptor endocytosis and PAR2 colocalization with ß-arrestin. Moreover, the recovery of the localization and function of PAR2 was significantly delayed after stimulation by GB83, which may be the reason why GB83 is recognized as an antagonist of PAR2. Our results revealed that GB83 is a bona fide agonist of PAR2 that uniquely modulates PAR2-mediated cellular signaling and is a useful pharmacological tool for studying the pathophysiological role of PAR2.

Impact of High-Molecular-Weight Hyaluronic Acid on Gene Expression in Rabbit Achilles Tenocytes In Vitro.

(1) Background: Surgical tendon repair often leads to adhesion formation, leading to joint stiffness and a reduced range of motion. Tubular implants set around sutured tendons might help to reduce peritendinous adhesions. The lubricant hyaluronic acid (HA) is a viable option for optimizing such tubes with the goal of further enhancing the anti-adhesive effect. As the implant degrades over time and diffusion is presumed, the impact of HA on tendon cells is important to know. (2) Methods: A culture medium of rabbit Achilles tenocytes was supplemented with high-molecular-weight (HMW) HA and the growth curves of the cells were assessed. Additionally, after 3, 7 and 14 days, the gene expression of several markers was analyzed for matrix assembly, tendon differentiation, fibrosis, proliferation, matrix remodeling, pro-inflammation and resolution. (3) Results: The addition of HA decreased matrix marker genes, downregulated the fibrosis marker α-SMA for a short time and slightly increased the matrix-remodeling gene MMP-2. Of the pro-inflammatory marker genes, only IL-6 was significantly upregulated. IL-6 has to be kept in check, although IL-6 is also needed for a proper initial inflammation and efficient resolution. (4) Conclusions: The observed effects in vitro support the intended anti-adhesion effect and therefore, the use of HMW HA is promising as a biodegradable implant for tendon repair.