Molecular Mechanisms Underlying Loss of Vascular and Epithelial Integrity in Irritable Bowel Syndrome.

BACKGROUND AND AIMS: The pathophysiology of irritable bowel syndrome (IBS) is multifactorial and includes epithelial barrier dysfunction, a key element at the interface between the gut lumen and the deeper intestinal layers. Beneath the epithelial barrier there is the vascular one representing the last barrier to avoid luminal antigen dissemination The aims of this study were to correlate morpho-functional aspects of epithelial and vascular barriers with symptom perception in IBS. METHODS: Seventy-eight healthy subjects (controls) and 223 patients with IBS were enrolled in the study and phenotyped according to validated questionnaires. Sugar test was used to evaluate in vivo permeability. Immunohistochemistry, western blot, and electron microscopy were used to characterize the vascular barrier. Vascular permeability was evaluated by assessing the mucosal expression of plasmalemma vesicle-associated protein-1 and vascular endothelial cadherin. Caco-2 or human umbilical vein endothelial cell monolayers were incubated with soluble mediators released by mucosal biopsies to highlight the mechanisms involved in permeability alteration. Correlation analyses have been performed among experimental and clinical data. RESULTS: The intestinal epithelial barrier was compromised in patients with IBS throughout the gastrointestinal tract. IBS-soluble mediators increased Caco-2 permeability via a downregulation of tight junction gene expression. Blood vessel density and vascular permeability were increased in the IBS colonic mucosa. IBS mucosal mediators increased permeability in human umbilical vein endothelial cell monolayers through the activation of protease-activated receptor-2 and histone deacetylase 11, resulting in vascular endothelial cadherin downregulation. Permeability changes correlated with intestinal and behavioral symptoms and health-related quality of life of patients with IBS. CONCLUSIONS: Epithelial and vascular barriers are compromised in patients with IBS and contribute to clinical manifestations.

The ecotin-like peptidase inhibitor of Trypanosoma cruzi prevents TMPRSS2-PAR2-TLR4 crosstalk downmodulating infection and inflammation.

Trypanosoma cruzi is the causative agent of Chagas disease, a chronic pathology that affects the heart and/or digestive system. This parasite invades and multiplies in virtually all nucleated cells, using a variety of host cell receptors for infection. T. cruzi has a gene that encodes an ecotin-like inhibitor of serine peptidases, ISP2. We generated ISP2-null mutants (Δisp2) in T. cruzi Dm28c using CRISPR/Cas9. Epimastigotes of Δisp2 grew normally in vitro but were more susceptible to lysis by human serum compared to parental and ISP2 add-back lines. Tissue culture trypomastigotes of Δisp2 were more infective to human muscle cells in vitro, which was reverted by the serine peptidase inhibitors aprotinin and camostat, suggesting that host cell epitheliasin/TMPRSS2 is the target of ISP2. Pretreatment of host cells with an antagonist to the protease-activated receptor 2 (PAR2) or an inhibitor of Toll-like receptor 4 (TLR4) selectively counteracted the increased cell invasion by Δisp2, but did not affect invasion by parental and add-back lines. The same was observed following targeted gene silencing of PAR2, TLR4 or TMPRSS2 in host cells by siRNA. Furthermore, Δisp2 caused increased tissue edema in a BALB/c mouse footpad infection model after 3 h differently to that observed following infection with parental and add-back lines. We propose that ISP2 contributes to protect T. cruzi from the anti-microbial effects of human serum and to prevent triggering of PAR2 and TLR4 in host cells, resulting in the modulation of host cell invasion and contributing to decrease inflammation during acute infection.

Filamin A regulates platelet shape change and contractile force generation via phosphorylation of the myosin light chain.

Platelets are critical mediators of hemostasis and thrombosis. Platelets circulate as discs in their resting form but change shape rapidly upon activation by vascular damage and/or soluble agonists such as thrombin. Platelet shape change is driven by a dynamic remodeling of the actin cytoskeleton. Actin filaments interact with the protein myosin, which is phosphorylated on the myosin light chain (MLC) upon platelet activation. Actin-myosin interactions trigger contraction of the actin cytoskeleton, which drives platelet spreading and contractile force generation. Filamin A (FLNA) is an actin-crosslinking protein that stabilizes the attachment between subcortical actin filaments and the cell membrane. In addition, FLNA binds multiple proteins and serves as a critical intracellular signaling scaffold. Here, we used platelets from mice with a megakaryocyte/platelet-specific deletion of FLNA to investigate the role of FLNA in regulating platelet shape change. Relative to controls, FLNA-null platelets exhibited defects in stress fiber formation, contractile force generation, and MLC phosphorylation in response to thrombin stimulation. Blockade of Rho kinase (ROCK) and protein kinase C (PKC) with the inhibitors Y27632 and bisindolylmaleimide (BIM), respectively, also attenuated MLC phosphorylation; our data further indicate that ROCK and PKC promote MLC phosphorylation through independent pathways. Notably, the activity of both ROCK and PKC was diminished in the FLNA-deficient platelets. We conclude that FLNA regulates thrombin-induced MLC phosphorylation and platelet contraction, in a ROCK- and PKC-dependent manner.

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.

Ubiquitin-driven G protein-coupled receptor inflammatory signaling at the endosome.

G protein-coupled receptors (GPCRs) are ubiquitously expressed cell surface receptors that mediate numerous physiological responses and are highly druggable. Upon activation, GPCRs rapidly couple to heterotrimeric G proteins and are then phosphorylated and internalized from the cell surface. Recent studies indicate that GPCRs not only localize at the plasma membrane but also exist in intracellular compartments where they are competent to signal. Intracellular signaling by GPCRs is best described to occur at endosomes. Several studies have elegantly documented endosomal GPCR-G protein and GPCR-ß-arrestin signaling. Besides phosphorylation, GPCRs are also posttranslationally modified with ubiquitin. GPCR ubiquitination has been studied mainly in the context of receptor endosomal-lysosomal trafficking. However, new studies indicate that ubiquitination of endogenous GPCRs expressed in endothelial cells initiates the assembly of an intracellular p38 mitogen-activated kinase signaling complex that promotes inflammatory responses from endosomes. In this mini-review, we discuss emerging discoveries that provide critical insights into the function of ubiquitination in regulating GPCR inflammatory signaling at endosomes.

MicroRNA-26a alleviates tubulointerstitial fibrosis in diabetic kidney disease by targeting PAR4.

Our previous study found that miR-26a alleviates aldosterone-induced tubulointerstitial fibrosis (TIF). However, the effect of miR-26a on TIF in diabetic kidney disease (DKD) remains unclear. This study clarifies the role and possible mechanism of exogenous miR-26a in controlling the progression of TIF in DKD models. Firstly, we showed that miR-26a was markedly decreased in type 2 diabetic db/db mice and mouse tubular epithelial cells (mTECs) treated with high glucose (HG, 30 mM) using RT-qPCR. We then used adeno-associated virus carrying miR-26a and adenovirus miR-26a to enhance the expression of miR-26a in vivo and in vitro. Overexpressing miR-26a alleviated the TIF in db/db mice and the extracellular matrix (ECM) deposition in HG-stimulated mTECs. These protective effects were caused by reducing expression of protease-activated receptor 4 (PAR4), which involved in multiple pro-fibrotic pathways. The rescue of PAR4 expression reversed the anti-fibrosis activity of miR-26a. We conclude that miR-26a alleviates TIF in DKD models by directly targeting PAR4, which may provide a novel molecular strategy for DKD therapy.

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.

KLK10 promotes the progression of KRAS mutant colorectal cancer via PAR1-PDK1-AKT signaling pathway.

Kirsten rat sarcoma virus (KRAS) gene mutation is common in colorectal cancer (CRC) and is often predictive of treatment failure and poor prognosis. To understand the mechanism, we compared the transcriptome of CRC patients with wild-type and mutant KRAS and found that KRAS mutation is associated with the overexpression of a secreted serine protease, kallikrein-related peptidase 10 (KLK10). Moreover, using in vitro and in vivo models, we found that KLK10 overexpression favors the rapid growth and liver metastasis of KRAS mutant CRC and can also impair the efficacy of KRAS inhibitors, leading to drug resistance and poor survival. Further functional assays revealed that the oncogenic role of KLK10 is mediated by protease-activated receptor 1 (PAR1). KLK10 cleaves and activates PAR1, which further activates 3-phosphoinositide-dependent kinase 1 (PDK1)-AKT oncogenic pathway. Notably, suppressing PAR1-PDK1-AKT cascade via KLK10 knockdown can effectively inhibit CRC progression and improve the sensitivity to KRAS inhibitor, providing a promising therapeutic strategy. Taken together, our study showed that KLK10 promotes the progression of KRAS mutant CRC via activating PAR1-PDK1-AKT signaling pathway. These findings expanded our knowledge of CRC development, especially in the setting of KRAS mutation, and also provided novel targets for clinical intervention.

CLIC4 Regulates Endothelial Barrier Control by Mediating PAR1 Signaling via RhoA.

BACKGROUND: Endothelial CLICs (chloride intracellular channel proteins) CLIC1 and CLIC4 are required for the GPCRs (G-protein-coupled receptors) S1PR1 (sphingosine-1-phosphate receptor 1) and S1PR3 to activate the small GTPases Rac1 (Ras-related C3 botulinum toxin substrate 1) and RhoA (Ras homolog family member A). To determine whether CLIC1 and CLIC4 function in additional endothelial GPCR pathways, we evaluated CLIC function in thrombin signaling via the thrombin-regulated PAR1 (protease-activated receptor 1) and downstream effector RhoA. METHODS: We assessed the ability of CLIC1 and CLIC4 to relocalize to cell membranes in response to thrombin in human umbilical vein endothelial cells (HUVEC). We examined CLIC1 and CLIC4 function in HUVEC by knocking down expression of each CLIC protein and compared thrombin-mediated RhoA or Rac1 activation, ERM (ezrin/radixin/moesin) phosphorylation, and endothelial barrier modulation in control and CLIC knockdown HUVEC. We generated a conditional murine allele of Clic4 and examined PAR1-mediated lung microvascular permeability and retinal angiogenesis in mice with endothelial-specific loss of Clic4. RESULTS: Thrombin promoted relocalization of CLIC4, but not CLIC1, to HUVEC membranes. Knockdown of CLIC4 in HUVEC reduced thrombin-mediated RhoA activation, ERM phosphorylation, and endothelial barrier disruption. Knockdown of CLIC1 did not reduce thrombin-mediated RhoA activity but prolonged the RhoA and endothelial barrier response to thrombin. Endothelial-specific deletion of Clic4 in mice reduced lung edema and microvascular permeability induced by PAR1 activating peptide. CONCLUSIONS: CLIC4 is a critical effector of endothelial PAR1 signaling and is required to regulate RhoA-mediated endothelial barrier disruption in cultured endothelial cells and murine lung endothelium. CLIC1 was not critical for thrombin-mediated barrier disruption but contributed to the barrier recovery phase after thrombin treatment.

Discovery of Protease-activated receptor 2 antagonists derived from phenylalanine for the treatment of breast cancer.

Protease-activated receptor 2 (PAR2) has garnered attention as a potential therapeutic target in breast cancer. PAR2 is implicated in the activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) via G protein and beta-arrestin pathways, contributing to the proliferation and metastasis of breast cancer cells. Despite the recognized role of PAR2 in breast cancer progression, clinically effective PAR2 antagonists remain elusive. To address this unmet clinical need, we synthesized and evaluated a series of novel compounds that target the orthosteric site of PAR2. Using in silico docking simulations, we identified compound 9a, an optimized derivative of compound 1a ((S)-N-(1-(benzylamino)-1-oxo-3-phenylpropan-2-yl)benzamide), which exhibited enhanced PAR2 antagonistic activity. Subsequent molecular dynamics simulations comparing 9a with the partial agonist 9d revealed that variations in ligand-induced conformational changes and interactions dictated whether the compound acted as an antagonist or agonist of PAR2. The results of this study suggest that further development of 9a could contribute to the advancement of PAR2 antagonists as potential therapeutic agents for breast cancer.

Single-Cell Analysis Reveals Distinct Immune and Smooth Muscle Cell Populations that Contribute to Chronic Thromboembolic Pulmonary Hypertension.

Rationale: Chronic thromboembolic pulmonary hypertension (CTEPH) is a sequela of acute pulmonary embolism (PE) in which the PE remodels into a chronic scar in the pulmonary arteries. This results in vascular obstruction, pulmonary microvasculopathy, and pulmonary hypertension. Objectives: Our current understanding of CTEPH pathobiology is primarily derived from cell-based studies limited by the use of specific cell markers or phenotypic modulation in cell culture. Therefore, our main objective was to identify the multiple cell types that constitute CTEPH thrombusy and to study their dysfunction. Methods: Here we used single-cell RNA sequencing of tissue removed at the time of pulmonary endarterectomy surgery from five patients to identify the multiple cell types. Using in vitro assays, we analyzed differences in phenotype between CTEPH thrombus and healthy pulmonary vascular cells. We studied potential therapeutic targets in cells isolated from CTEPH thrombus. Measurements and Main Results: Single-cell RNA sequencing identified multiple cell types, including macrophages, T cells, and smooth muscle cells (SMCs), that constitute CTEPH thrombus. Notably, multiple macrophage subclusters were identified but broadly split into two categories, with the larger group characterized by an upregulation of inflammatory signaling predicted to promote pulmonary vascular remodeling. CD4+ and CD8+ T cells were identified and likely contribute to chronic inflammation in CTEPH. SMCs were a heterogeneous population, with a cluster of myofibroblasts that express markers of fibrosis and are predicted to arise from other SMC clusters based on pseudotime analysis. Additionally, cultured endothelial, smooth muscle, and myofibroblast cells isolated from CTEPH fibrothrombotic material have distinct phenotypes from control cells with regard to angiogenic potential and rates of proliferation and apoptosis. Last, our analysis identified PAR1 (protease-activated receptor 1) as a potential therapeutic target that links thrombosis to chronic PE in CTEPH, with PAR1 inhibition decreasing SMC and myofibroblast proliferation and migration. Conclusions: These findings suggest a model for CTEPH similar to atherosclerosis, with chronic inflammation promoted by macrophages and T cells driving vascular remodeling through SMC modulation, and suggest new approaches for pharmacologically targeting this disease.

Beyond Anticoagulation: A Comprehensive Review of Non-Vitamin K Oral Anticoagulants (NOACs) in Inflammation and Protease-Activated Receptor Signaling.

Non-vitamin K oral anticoagulants (NOACs) have revolutionized anticoagulant therapy, offering improved safety and efficacy over traditional agents like warfarin. This review comprehensively examines the dual roles of NOACs-apixaban, rivaroxaban, edoxaban, and dabigatran-not only as anticoagulants, but also as modulators of inflammation via protease-activated receptor (PAR) signaling. We highlight the unique pharmacotherapeutic properties of each NOAC, supported by key clinical trials demonstrating their effectiveness in preventing thromboembolic events. Beyond their established anticoagulant roles, emerging research suggests that NOACs influence inflammation through PAR signaling pathways, implicating factors such as factor Xa (FXa) and thrombin in the modulation of inflammatory responses. This review synthesizes current evidence on the anti-inflammatory potential of NOACs, exploring their impact on inflammatory markers and conditions like atherosclerosis and diabetes. By delineating the mechanisms by which NOACs mediate anti-inflammatory effects, this work aims to expand their therapeutic utility, offering new perspectives for managing inflammatory diseases. Our findings underscore the broader clinical implications of NOACs, advocating for their consideration in therapeutic strategies aimed at addressing inflammation-related pathologies. This comprehensive synthesis not only enhances understanding of NOACs' multifaceted roles, but also paves the way for future research and clinical applications in inflammation and cardiovascular health.

Endothelial Protein C Receptor and 3K3A-Activated Protein C Protect Mice from Allergic Contact Dermatitis in a Contact Hypersensitivity Model.

Endothelial protein C receptor (EPCR) is a receptor for the natural anti-coagulant activated protein C (aPC). It mediates the anti-inflammatory and barrier-protective functions of aPC through the cleavage of protease-activated receptor (PAR)1/2. Allergic contact dermatitis is a common skin disease characterized by inflammation and defective skin barrier. This study investigated the effect of EPCR and 3K3A-aPC on allergic contact dermatitis using a contact hypersensitivity (CHS) model. CHS was induced using 1-Fluoro-2,4-dinitrobenzene in EPCR-deficient (KO) and matched wild-type mice and mice treated with 3K3A-aPC, a mutant form of aPC with diminished anti-coagulant activity. Changes in clinical and histological features, cytokines, and immune cells were examined. EPCRKO mice displayed more severe CHS, with increased immune cell infiltration in the skin and higher levels of inflammatory cytokines and IgE than wild-type mice. EPCR, aPC, and PAR1/2 were expressed by the skin epidermis, with EPCR presenting almost exclusively in the basal layer. EPCRKO increased the epidermal expression of aPC and PAR1, whereas in CHS, their expression was reduced compared to wild-type mice. 3K3A-aPC reduced CHS severity in wild-type and EPCRKO mice by suppressing immune cell infiltration/activation and inflammatory cytokines. In summary, EPCRKO exacerbated CHS, whereas 3K3A-aPC could reduce the severity of CHS in both EPCRKO and wild-type mice.

Characterization of Biomarkers of Thrombo-Inflammation in Patients with First-Diagnosed Atrial Fibrillation.

Patients with first-diagnosed atrial fibrillation (FDAF) exhibit major adverse cardiovascular events (MACEs) during follow-up. Preclinical models have demonstrated that thrombo-inflammation mediates adverse cardiac remodeling and atherothrombotic events. We have hypothesized that thrombin activity (FIIa) links coagulation with inflammation and cardiac fibrosis/dysfunction. Surrogate markers of the thrombo-inflammatory response in plasma have not been characterized in FDAF. In this prospective longitudinal study, patients presenting with FDAF (n = 80), and 20 matched controls, were included. FIIa generation and activity in plasma were increased in the patients with early AF compared to the patients with chronic cardiovascular disease without AF (controls; p < 0.0001). This increase was accompanied by elevated biomarkers (ELISA) of platelet and endothelial activation in plasma. Pro-inflammatory peripheral immune cells (TNF-α+ or IL-6+) that expressed FIIa-activated protease-activated receptor 1 (PAR1) (flow cytometry) circulated more frequently in patients with FDAF compared to the controls (p < 0.0001). FIIa activity correlated with cardiac fibrosis (collagen turnover) and cardiac dysfunction (NT-pro ANP/NT-pro BNP) surrogate markers. FIIa activity in plasma was higher in patients with FDAF who experienced MACE. Signaling via FIIa might be a presumed link between the coagulation system (tissue factor-FXa/FIIa-PAR1 axis), inflammation, and pro-fibrotic pathways (thrombo-inflammation) in FDAF.

Endothelial PAR2 activation evokes resistance artery relaxation.

Protease-activated receptor-1 & -2 (PAR1 and PAR2) are expressed widely in cardiovascular tissues including endothelial and smooth muscle cells. PAR1 and PAR2 may regulate blood pressure via changes in vascular contraction or relaxation mediated by endothelial Ca2+ signaling, but the mechanisms are incompletely understood. By using single-cell Ca2+ imaging across hundreds of endothelial cells in intact blood vessels, we explored PAR-mediated regulation of blood vessel function using PAR1 and PAR2 activators. We show that PAR2 activation evoked multicellular Ca2+ waves that propagated across the endothelium. The PAR2-evoked Ca2+ waves were temporally distinct from those generated by muscarinic receptor activation. PAR2 activated distinct clusters of endothelial cells, and these cells were different from those activated by muscarinic receptor stimulation. These results indicate that distinct cell clusters facilitate spatial segregation of endothelial signal processing. We also demonstrate that PAR2 is a phospholipase C-coupled receptor that evokes Ca2+ release from the IP3 -sensitive store in endothelial cells. A physiological consequence of this PAR2 signaling system is endothelium-dependent relaxation. Conversely, PAR1 activation did not trigger endothelial cell Ca2+ signaling nor relax or contract mesenteric arteries. Neither did PAR1 activators alter the response to PAR2 or muscarinic receptor activation. Collectively, these results suggest that endothelial PAR2 but not PAR1 evokes mesenteric artery relaxation by evoking IP3 -mediated Ca2+ release from the internal store. Sensing mediated by PAR2 receptors is distributed to spatially separated clusters of endothelial cells.

Effect of chimeric antigen receptor T cells against protease-activated receptor 1 for treating pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a devastating malignancy with a 5-year survival rate of 6% following a diagnosis, and novel therapeutic modalities are needed. Protease-activated receptor 1 (PAR1) is abundantly overexpressed by both tumor cells and multiple stroma cell subsets in the tumor microenvironment (TME), thereby offering a suitable immunotherapy target. METHODS: A chimeric antigen receptor (CAR) strategy was applied to target PAR1 using a human anti-PAR1 scFv antibody fused to the transmembrane region with two co-stimulatory intracellular signaling domains of cluster of differentiation 28 (CD28) and CD137 (4-1BB), added to CD3ζ in tandem. RESULTS: The engineered PAR1CAR-T cells eliminated PAR1 overexpression and transforming growth factor (TGF)-ß-mediated PAR1-upregulated cancer cells by approximately 80% in vitro. The adoptive transfer of PAR1CAR-T cells was persistently enhanced and induced the specific regression of established MIA PaCa-2 cancer cells by > 80% in xenograft models. Accordingly, proinflammatory cytokines/chemokines increased in CAR-T-cell-treated mouse sera, whereas Ki67 expression in tumors decreased. Furthermore, the targeted elimination of PAR1-expressing tumors reduced matrix metalloproteinase 1 (MMP1) levels, suggesting that the blocking of the PAR1/MMP1 pathway constitutes a new therapeutic option for PDAC treatment. CONCLUSIONS: Third-generation PAR1CAR-T cells have antitumor activity in the TME, providing innovative CAR-T-cell immunotherapy against PDAC.

The roles of proteases in prostate cancer.

Since the proposition of the pro-invasive activity of proteolytic enzymes over 70 years ago, several roles for proteases in cancer progression have been established. About half of the 473 active human proteases are expressed in the prostate and many of the most well-characterized members of this enzyme family are regulated by androgens, hormones essential for development of prostate cancer. Most notably, several kallikrein-related peptidases, including KLK3 (prostate-specific antigen, PSA), the most well-known prostate cancer marker, and type II transmembrane serine proteases, such as TMPRSS2 and matriptase, have been extensively studied and found to promote prostate cancer progression. Recent findings also suggest a critical role for proteases in the development of advanced and aggressive castration-resistant prostate cancer (CRPC). Perhaps the most intriguing evidence for this role comes from studies showing that the protease-activated transmembrane proteins, Notch and CDCP1, are associated with the development of CRPC. Here, we review the roles of proteases in prostate cancer, with a special focus on their regulation by androgens.

Elevated serum PAR-1 levels as an emerging biomarker of inflammation to predict the dengue infection severity.

The present study was designed to check the serum levels of protease-activated receptor (PAR-1) in patients during different phases of dengue severity. Moreover, a correlation between serum PAR-1 levels and hematological parameters, inflammatory cytokine levels, and liver functional changes was also determined. Based on the World Health Organization criteria, the study population was divided into: nonsevere dengue fever (DF; n = 30), severe dengue hemorrhagic fever (DHF; n = 19), and severe dengue shock syndrome (DSS; n = 11). The platelet count (PLT) and hematocrit (HCT) were analyzed using an automated hematology analyzer and liver function enzymes aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphate (ALP), bilirubin were checked by auto-analyzer using diagnostic kits. Moreover, the levels of inflammatory mediators C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-17 (IL-17), and PAR-1 were determined using respective ELISA kits. The HCT levels were elevated and platelet count decreased significantly during dengue complications (DHF and DSS) compared to the DF patients, while the levels of liver functional biomarkers AST, ALT, ALP, and bilirubin remained elevated in DHF and DSS groups than in the corresponding DF group. Similarly, the inflammatory cytokine levels of CRP, TNF-α, IL-6, and IL-17 in DHF and DSS subjects were markedly increased when observed against DF subjects. Notably, the PAR-1 levels were significantly elevated in DHF and DSS groups than in the DF group and positively correlated with changes in HCT levels, inflammatory biomarkers, and liver enzymes. Our findings conclude that PAR-1 levels persistently increased with the severity of the dengue infection and are strongly associated with various clinical manifestations. Thus, PAR-1 levels can be used as a diagnostic marker for assessing dengue severity.

Protease-Activated Receptor 2 Controls Vascular Smooth Muscle Cell Proliferation in Cyclic AMP-Dependent Protein Kinase/Mitogen-Activated Protein Kinase Kinase 1/2-Dependent Manner.

INTRODUCTION: Cardiovascular disorders are characterized by vascular smooth muscle (VSM) transition from a contractile to proliferative state. Protease-activated receptor 2 (PAR2) involvement in this phenotypic conversion remains unclear. We hypothesized that PAR2 controls VSM cell proliferation in phenotype-dependent manner and through specific protein kinases. METHODS: Rat clonal low (PLo; P3-P6) and high passage (PHi; P10-P15) VSM cells were established as respective models of quiescent and proliferative cells, based on reduced PKG-1 and VASP. Western blotting determined expression of cytoskeletal/contractile proteins, PAR2, and select protein kinases. DNA synthesis and cell proliferation were measured 24-72 h following PAR2 agonism (SLIGRL; 100 nM-10 µm) with/without PKA (PKI; 10 µm), MEK1/2 (PD98059; 10 µm), and PI3K (LY294002; 1 µm) blockade. RESULTS: PKG-1, VASP, SM22α, calponin, cofilin, and PAR2 were reduced in PHi versus PLo cells. Following PAR2 agonism, DNA synthesis and cell proliferation increased in PLo cells but decreased in PHi cells. Western analyses showed reduced PKA, MEK1/2, and PI3K in PHi versus PLo cells, and kinase blockade revealed PAR2 controls VSM cell proliferation through PKA/MEK1/2. DISCUSSION: Findings highlight PAR2 and PAR2-driven PKA/MEK1/2 in control of VSM cell growth and provide evidence for continued investigation of PAR2 in VSM pathology.

Protease allergens as initiators-regulators of allergic inflammation.

Tremendous progress in the last few years has been made to explain how seemingly harmless environmental proteins from different origins can induce potent Th2-biased inflammatory responses. Convergent findings have shown the key roles of allergens displaying proteolytic activity in the initiation and progression of the allergic response. Through their propensity to activate IgE-independent inflammatory pathways, certain allergenic proteases are now considered as initiators for sensitization to themselves and to non-protease allergens. The protease allergens degrade junctional proteins of keratinocytes or airway epithelium to facilitate allergen delivery across the epithelial barrier and their subsequent uptake by antigen-presenting cells. Epithelial injuries mediated by these proteases together with their sensing by protease-activated receptors (PARs) elicit potent inflammatory responses resulting in the release of pro-Th2 cytokines (IL-6, IL-25, IL-1ß, TSLP) and danger-associated molecular patterns (DAMPs; IL-33, ATP, uric acid). Recently, protease allergens were shown to cleave the protease sensor domain of IL-33 to produce a super-active form of the alarmin. At the same time, proteolytic cleavage of fibrinogen can trigger TLR4 signaling, and cleavage of various cell surface receptors further shape the Th2 polarization. Remarkably, the sensing of protease allergens by nociceptive neurons can represent a primary step in the development of the allergic response. The goal of this review is to highlight the multiple innate immune mechanisms triggered by protease allergens that converge to initiate the allergic response.