Par-4 Expression Research Articles

Tissue factor cytoplasmic domain exacerbates post-infarct left ventricular remodeling via orchestrating cardiac inflammation and angiogenesis

Abstract Introduction The coagulation protein tissue factor (TF) regulates inflammation and angiogenesis via its cytoplasmic domain in infection, cancer and diabetes. While TF is highly abundant in the heart and implicated in cardiac injuries and dysfunction, the contribution of its cytoplasmic domain in cardiac pathology remains unclear. Purpose We aimed to investigate the contribution of the cytoplasmic domain of TF to post-infarct myocardial injury and adverse left ventricular (LV) remodeling. Methods and results Myocardial infarction was induced by permanent occlusion of the left anterior descending coronary artery. Male mice with C57BL/Jax background were used for the study. Compared with wild-type mice, mice lacking the TF cytoplasmic domain (TFΔCT) had a higher survival rate (90.5% versus 70%, p=0.0298) during a 28-day follow-up after myocardial infarction. Among surviving mice, TFΔCT mice had better cardiac function and less LV remodeling (ESV: 114.5±13.1mL for WT, 67.06±10.8mL for TFΔCT, p<0.001; EDV: 146.6±12.4mL for WT, 99.97±11.71mL for TFΔCT, p<0.001) than wild-type mice. Bone marrow chimerism indicated that deletion of the TF cytoplasmic domain in either bone marrow-derived cells or cardiac resident cells could alleviate post-infarct cardiac dysfunction. Speckle-tracking strain analysis revealed that the overall improvement of post-infarct cardiac performance in TFΔCT mice was attributed to reduced myocardial deformation in the peri-infarct region (strain-%: 11.14±0.97 for WT, 15.34±1.10 for TFΔCT, p=0.007; strain rate-/s: 3.89±0.26 for WT, 5.18±0.21 for TFΔCT, p=0.0005). Histological analysis demonstrated that TFΔCT hearts had in the infarct area greater proliferation of endothelial cells and myofibroblasts accompanied with better scar formation. Compared with wild-type hearts, infarcted TFΔCT hearts showed less infiltration of proinflammatory cells with concomitant lower expression of protease-activated receptor-1 (PAR1)-Rac1 axis. Furthermore, infarcted TFΔCT hearts presented markedly higher peri-infarct vessel density associated with enhanced endothelial cell proliferation and higher expression of PAR2 and PAR2-associated pro-angiogenic pathway factors. Conclusions Our findings demonstrate that the TF cytoplasmic domain exacerbates post-infarct cardiac injury and adverse LV remodeling via differential regulation of inflammation and angiogenesis. Targeted inhibition of the TF cytoplasmic domain-mediated intracellular signaling may ameliorate post-infarct LV remodeling without perturbing coagulation. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): National University Health System of Singapore

Expression/Activation of PAR-1 in Airway Epithelial Cells of COPD Patients: Ex Vivo/In Vitro Study.

The role of PAR-1 expression and activation was described in epithelial cells from the central and distal airways of COPD patients using an ex vivo/in vitro model. PAR-1 immunoreactivity was studied in epithelial cells from surgical specimens of the central and distal airways of COPD patients and healthy control (HC). Furthermore, PAR-1 expression and activation were measured in both the human bronchial epithelial cell line (16HBE) and normal human bronchial epithelial cells (NHBEs) exposed to cigarette smoke extract (CSE) (10%) or thrombin. Finally, cell proliferation, apoptosis, and IL-8 release were detected in stimulated NHBEs. We identified higher levels of PAR-1 expression/activation in epithelial cells from the central airways of COPD patients than in HC. Active PAR-1 increased in epithelial cells from central and distal airways of COPD, with higher levels in COPD smokers (correlated with pack-years) than in COPD ex-smokers. 16HBE and NHBEs exposed to CSE or thrombin showed increased levels of active PAR-1 (localized in the cytoplasm) than baseline conditions, while NHBEs treated with thrombin or CSE showed increased levels of IL-8 proteins, with an additional effect when used in combination. Smoking habits generate the upregulation of PAR-1 expression/activation in airway epithelial cells, and promoting IL-8 release might affect the recruitment of infiltrating cells in the airways of COPD patients.

Protease-Activated Receptor-1 Antagonist Protects Against Lung Ischemia/Reperfusion Injury.

Protease-activated receptor (PAR)-1 is a thrombin-activated receptor that plays an essential role in ischemia/reperfusion (IR)-induced acute inflammation. PAR-1 antagonists have been shown to alleviate injuries in various IR models. However, the effect of PAR-1 antagonists on IR-induced acute lung injury (ALI) has not yet been elucidated. This study aimed to investigate whether PAR-1 inhibition could attenuate lung IR injury. Lung IR was induced in an isolated perfused rat lung model. Male rats were treated with the specific PAR-1 antagonist SCH530348 (vorapaxar) or vehicle, followed by ischemia for 40 min and reperfusion for 60 min. To examine the role of PAR-1 and the mechanism of SCH530348 in lung IR injury, western blotting and immunohistochemical analysis of lung tissue were performed. In vitro, mouse lung epithelial cells (MLE-12) were treated with SCH530348 or vehicle and subjected to hypoxia-reoxygenation (HR). We found that SCH530348 decreased lung edema and neutrophil infiltration, attenuated thrombin production, reduced inflammatory factors, including cytokine-induced neutrophil chemoattractant-1, interleukin-6 and tumor necrosis factor-α, mitigated lung cell apoptosis, and downregulated the phosphoinositide 3-kinase (PI3K), nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways in IR-injured lungs. In addition, SCH530348 prevented HR-induced NF-κB activation and inflammatory chemokine production in MLE12 cells. Our results demonstrate that SCH530348 exerts protective effects by blocking PAR-1 expression and modulating the downstream PI3K, NF-κB and MAPK pathways. These findings indicate that the PAR-1 antagonist protects against IR-induced ALI and is a potential therapeutic candidate for lung protection following IR injury.

A medicinal plant Cudrania tricuspidata ameliorates allergic inflammation in mouse

Aim: This study aims at investigating the effect of ethanol extract of Cudrania tricuspidata twigs on in vitro and in vivo allergic inflammation. Methodology: The ethanol extract of twigs from Cudrania tricuspidata (CTE) was prepared. The effects of CTE on the lipopolysaccharide (LPS)-induced production of nitric oxide (NO) in Raw 264.7 cells were examined. 2,4-Dinitrochlorobenzene (DNCB)-induced allergic inflammation model of mouse was constructed for in vivo study. The anti-oxidative, anti-inflammatory and anti-allergic effects of CTE were examined through DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Results: The LPS-induced NO level was significantly reduced by Cudrania tricuspidata application in Raw 264.7 cells. LPS-induced inducible nitric oxide synthase (iNOS) expression was also reduced by CTE, showing CTE’s anti-inflammatory activity. Moreover, CTE reduced DNCB-induced IgE level in the mouse serum. The suppressive effect on the allergic inflammation stimulated by DNCB in the mouse model was investigated in terms of IL-4, IFN-γ, TNF-α level and PAR-2 expression. Interpretation: The ethanol extract of Cudrania tricuspidata twigs can be developed as a potent therapeutics for allergic inflammation.

Antagonism of Protease Activated Receptor-2 by GB88 Reduces Inflammation Triggered by Protease Allergen Tyr-p3

The occurrence of allergic diseases induced by aeroallergens has increased in the past decades. Among inhalant allergens, mites remain the important causal agent of allergic diseases. Storage mites- Tyrophagus putrescentiae are found in stored products or domestic environments. Major allergen Tyr-p3 plays a significant role in triggering IgE-mediated hypersensitivity. However, its effects on pulmonary inflammation, internalization, and activation in human epithelium remain elusive. Protease-activated receptors (PARs) are activated upon cleavage by proteases. A549 cells were used as an epithelial model to examine the PAR activation by Tyr-p3 and therapeutic potential of PAR-2 antagonist (GB88) in allergic responses. Enzymatic properties and allergen localization of Tyr-p3 were performed. The release of inflammatory mediators, phosphorylation of mitogen-activated protein kinase (MAPK), and cell junction disruptions were evaluated after Tyr-p3 challenge. Enzymatic properties determined by substrate digestion and protease inhibitors indicated that Tyr-p3 processes a trypsin-like serine protease activity. The PAR-2 mRNA levels were significantly increased by nTyr-p3 but inhibited by protease inhibitors or GB88. Protease allergen of nTyr-p3 significantly increased the levels of pro-inflammatory cytokines (IL-6 and TNF-α), chemokine (IL-8), and IL-1β in epithelial cells. nTyr-p3 markedly increased phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and MAP kinase. When cells were pretreated with GB88 then added nTyr-p3, the phosphorylated ERK1/2 did not inhibit by GB88. GB88 increased ERK1/2 phosphorylation in human epithelium cells. GB88 is able to block PAR-2-mediated calcium signaling which inhibits the nTyr-p3-induced Ca2+ release. Among the pharmacologic inhibitors, the most effective inhibitor of the nTyr-p3 in the induction of IL-8 or IL-1β levels was GB88 followed by SBTI, MAPK/ERK, ERK, and p38 inhibitors. Levels of inflammatory mediators, including GM-CSF, VEGF, COX-2, TSLP, and IL-33 were reduced by treatment of GB88 or SBTI. Further, GB88 treatment down-regulated the nTyr-p3-induced PAR-2 expression in allergic patients with asthma or rhinitis. Tight junction and adherens junction were disrupted in epithelial cells by nTyr-p3 exposure; however, this effect was avoided by GB88. Immunostaining with frozen sections of the mite body showed the presence of Tyr-p3 throughout the intestinal digestive system, especially in the hindgut around the excretion site. In conclusion, our findings suggest that Tyr-p3 from domestic mites leads to disruption of the airway epithelial barrier after inhalation. Proteolytic activity of Tyr-p3 causes the PAR-2 mRNA expression, thus leading to the release of numerous inflammatory mediators. Antagonism of PAR2 activity suggests GB88 as the therapeutic potential for anti-inflammation medicine, especially in allergy development triggered by protease allergens.

Abstract P245: Activation Of Protease-activated Receptors 1 Leads To Structural Changes In Immortalized Cultured Human Podocytes.

Previous studies have suggested that activation of the protease-activated receptors (PAR) exacerbates the development of diabetic nephropathy. PARs are transmembrane proteins activated by extracellular serine proteases, such as thrombin and trypsin, which are frequently elevated in disease states. We hypothesize that serine protease activation of the PAR1 signaling cascade upregulates calcium channel activity and promotes excessive intracellular Ca 2+ ([Ca 2+ ] i ), leading to podocyte foot retraction, apoptosis, and glomerular filtration barrier (GFB) damage. Immunofluorescent labeling revealed the co-localization of PAR1 and podocyte marker nephrin in freshly isolated human glomeruli. To explore the functional role of PAR1 mediated signaling, we used an immortalized cultured human podocyte (hPod) cell line. Live confocal imaging revealed rapid elevation of [Ca 2+ ] i in response to a PAR1 specific agonist (TFLLR-NH 2 ). Moreover, preincubation with a PAR1 antagonist (RWJ 56100) completely blocked this effect (1616±48 vs. 206±135 a.u., for TFLLR vs. TFLLR+RJW, n<100 cells, p<0.05). Furthermore, we tested potential downstream signaling proteins in hPod cells after PAR1 activation using Western blot analysis. Application of TFLLR-NH 2 led to a decrease in PAR1 expression due to internalization and lysosomal degradation, accompanied by an increase in TRPC6 expression and time-dependent changes in p-ERK1/2 and PLC-γ1 cascades. We further used scanning ion-conductance microscopy (SICM) to detect morphological changes in podocytes, which measures real-time surface topography in hPod cells. This imaging revealed normal protrusion of lamellipodium under control conditions. In contrast, PAR1 activation led to retraction of the lamellipodium, and a significant decrease in surface area (+51±46 vs. -15±8 μm 2 , 30 min after application of vehicle or TFLLR, respectively; n=5, p<0.05). Our studies suggested that PAR1 is involved in GPCR-induced calcium influx in podocytes. Furthermore, these data demonstrate that PAR1-mediated signaling is involved in podocyte structural alterations and may increase [Ca 2+ ] i , potentially leading to loss of GFB integrity and podocyte apoptosis.

Overexpression of Flii during Murine Embryonic Development Increases Symmetrical Division of Epidermal Progenitor Cells.

Epidermal progenitor cells divide symmetrically and asymmetrically to form stratified epidermis and hair follicles during late embryonic development. Flightless I (Flii), an actin remodelling protein, is implicated in Wnt/β-cat and integrin signalling pathways that govern cell division. This study investigated the effect of altering Flii on the divisional orientation of epidermal progenitor cells (EpSCs) in the basal layer during late murine embryonic development and early adolescence. The effect of altering Flii expression on asymmetric vs. symmetric division was assessed in vitro in adult human primary keratinocytes and in vivo at late embryonic development stages (E16, E17 and E19) as well as adolescence (P21 day-old) in mice with altered Flii expression (Flii knockdown: Flii+/−, wild type: WT, transgenic Flii overexpressing: FliiTg/Tg) using Western blot and immunohistochemistry. Flii+/− embryonic skin showed increased asymmetrical cell division of EpSCs with an increase in epidermal stratification and elevated talin, activated-Itgb1 and Par3 expression. FliiTg/Tg led to increased symmetrical cell division of EpSCs with increased cell proliferation rate, an elevated epidermal SOX9, Flap1 and β-cat expression, a thinner epidermis, but increased hair follicle number and depth. Flii promotes symmetric division of epidermal progenitor cells during murine embryonic development.

Rivaroxaban attenuates cardiac hypertrophy by inhibiting protease-activated receptor-2 signaling in renin-overexpressing hypertensive mice.

Rivaroxaban (Riv), a direct factor Xa (FXa) inhibitor, exerts anti-inflammatory effects in addition to anticoagulation. However, its role in cardiovascular remodeling is largely unknown. We tested the hypothesis that Riv attenuates the progression of cardiac hypertrophy and fibrosis induced by continuous activation of the renin-angiotensin system (RAS) in renin-overexpressing hypertensive transgenic (Ren-Tg) mice. We treated 12-week-old male Ren-Tg and wild-type (WT) mice with a diet containing Riv (12 mg/kg/day) or a regular diet for 4 weeks. After this, FXa in plasma significantly increased in Ren-Tg mice compared with WT mice, and Riv inhibited this increase. Left ventricular wall thickness (LVWT) and the area of cardiac fibrosis evaluated by Masson's trichrome staining were greater in Ren-Tg mice than in WT mice, and Riv decreased them. Cardiac expression levels of the protease-activated receptor (PAR)-2, tumor necrosis factor-α, transforming growth factor (TGF)-β1, and collagen type 3 α1 (COL3A1) genes were all greater in Ren-Tg mice than in WT mice, and Riv attenuated these increases. To investigate the possible involvement of PAR-2, we treated Ren-Tg mice with a continuous subcutaneous infusion of 10 μg/kg/day of the PAR-2 antagonist FSLLRY for 4 weeks. FSLLRY significantly decreased LVWT and cardiac expression of PAR-2, TGF-β1, and COL3A1. In isolated cardiac fibroblasts (CFs), Riv or FSLLRY pretreatment inhibited the FXa-induced increase in the phosphorylation of extracellular signal-regulated kinases. In addition, Riv or FSLLRY inhibited FXa-stimulated wound closure in CFs. Riv exerts a protective effect against cardiac hypertrophy and fibrosis development induced by continuous activation of the RAS, partly by inhibiting PAR-2.

Effect of Yiqi Huoxue Granules on Platelet Activation Induced by Thrombin.

Objective To study the effects of Yiqi Huoxue (YQHX) granules on platelet activation and aggregation induced by thrombin. Methods The effect of YQHX on platelet aggregation rate was detected by platelet aggregation instrument; the effect of YQHX on thrombosis time was observed by the mouse mesentery thrombosis model. DAMI cells were induced to transform into platelet-like granules using PMA, and the effects of SCH (PAR-1 inhibitor) on thrombin-induced changes in platelet intracellular calcium concentration, PAR-1 protein expression, and phosphorylation of MAPK were examined. Results Compared with the control group, the platelet aggregation rate, PAR-1 protein expression, phosphorylation of ERK1/2, and p38 protein in the YQHX group decreased (P < 0.05), and there was no significant difference between the YQHX + SCH group and YQHX group (P > 0.05). Conclusion YQHX suppresses the platelet activation induced by thrombin by inhibiting PAR-1 expression.

The Effect of a Novel Serine Protease Inhibitor on Inflammation and Intestinal Permeability in a Murine Colitis Transfer Model.

Background: A protease/antiprotease disbalance is observed in inflammatory bowel diseases (IBD). We therefore studied the effect of the novel serine protease inhibitor UAMC-00050 on intestinal inflammation and permeability in a chronic colitis T cell transfer mouse model to get further insight into the regulation of T cell-mediated immunopathology. Methods: Colitis was induced in severe combined immunodeficient (SCID) mice, by the adoptive transfer of CD4+CD25−CD62L+ T cells. Animals were treated intraperitoneally (i.p.) 2x/day with vehicle or UAMC-00050 (5 mg/kg) from week 2 onwards. Colonic inflammation was assessed by clinical parameters, colonoscopy, macroscopy, microscopy, myeloperoxidase activity and cytokine expression levels. At week 4, 4 kDa FITC-dextran intestinal permeability was evaluated and T helper transcription factors, protease-activated receptors and junctional proteins were quantified by RT-qPCR. Results: Adoptive transfer of CD4+CD25−CD62L+ T cells resulted in colonic inflammation and an altered intestinal permeability. The serine protease inhibitor UAMC-00050 ameliorated both the inflammatory parameters and the intestinal barrier function. Furthermore, a decrease in colonic mRNA expression of Tbet and PAR4 was observed in colitis mice after UAMC-00050 treatment. Conclusion: The beneficial effect of UAMC-00050 on inflammation was apparent via a reduction of Tbet, IFN-γ, TNF-α, IL-1β and IL-6. Based on these results, we hypothesize a pivotal effect of serine protease inhibition on the Th1 inflammatory profile potentially mediated via PAR4.

Loss of polarity protein Par3 is mediated by transcription factor Sp1 in breast cancer

Loss of polarity protein Par3 promotes breast cancer tumorigenesis and metastasis. The underlying molecular mechanisms of Par3 down-regulation and related prognostic significance in breast cancer remain unclear. Here, we discovered that Par3 down-regulation was associated with shorter relapse-free survival in Luminal A subtype of breast cancer. Par3 knockdown promoted breast cancer cells migration and invasion. Importantly, we identified that transcription factor Sp1 bound to PARD3 promoter region and induced Par3 expression. Breast cancer patients with low Sp1 showed significantly worse RFS and low expression level of Par3. Par3 over-expression partially reversed Sp1 knockdown induced migration and invasion. Together, decreased Sp1 level mediates Par3 down-regulation, which correlated with poor prognosis of ER + breast cancer patients, via reduced binding with PARD3 promoter.

Loss of polarity protein Par3, via transcription factor Snail, promotes bladder cancer metastasis.

Bladder cancer (BLCA) remains the leading cause of cancer‐related mortality among genitourinary malignancies worldwide. BLCA metastasis represents the primary reason for its poor prognosis. In this study, we report that decreased expression of partitioning defective 3 (Par3), a polarity protein (encoded by PARD3), is associated with tumor aggressive phenotypes and poor prognosis in BLCA patients. Consistently, ablation of Par3 promotes the metastasis and invasion of BLCA cells in vitro and in vivo. Further studies reveal that zinc finger protein Snail represses the expression of Par3 by binding to E2‐box (CAGGTG) of PARD3 promoter‐proximal. Inhibition of GSK‐3β promotes the expression and nuclear localization of Snail and then reduces the expression of Par3, resulting in the metastasis and invasion of BLCA cells. Moreover, we detected the interaction between Par3 (936‐1356 aa) and ZO‐1 (1372‐1748 aa), which is involved in the maintenance of tight junction. Together, our results demonstrate that the GSK‐3β/Snail/Par3/ZO‐1 axis regulates BLCA metastasis, and Snail is a major regulator for Par3 protein expression in BLCA.

Expression and functional characterization of protease-activated receptor 2 (PAR2) and PAR1 on human skin mast cells

Abstract Mast cells play an important role in the pathophysiology of asthma and allergic disease by releasing proinflammatory mediators and cytokines. Activation of protease-activated receptors (PARs) may also play a role in inflammatory response. We investigated the expression of PAR1, 2, 3 and 4 as well as the function of PAR1 and 2 in cultured human skin-derived mast cells (SMCs). Using a combination of flow cytometric analysis, reverse transcriptase-polymerase chain reaction and Western blotting, we demonstrate that human SMCs express PAR1, 2, 3 and 4. Confocal microscopy indicates PAR2 partially colocalize with tryptase within SMCs. Exposure to PAR2 agonist, trypsin and activating peptide (AP) 2-Furoyl-LIGRLO-NH2 induce Ca2+ mobilization in SMCs. The PAR1 APs, SFLLRN-NH2 and TFLLR-NH2 cause degranulation and induce GM-CSF and IL-8 synthesis. Similarly, the PAR2 APs, SLIGKV-NH2, 2-Furoyl-LIGRLO-NH2 also cause degranulation and induce GM-CSF and IL-8 synthesis in SMCs. Both PAR1 APs and PAR2 APs induce rapid phosphorylation of ERK and pertussis toxin (PTX) robustly inhibits phosphorylation of ERK by both PAR1 APs and PAR2 APs. These studies indicate that human SMCs can be activated by both PAR1 and PAR2 which may contribute to MC-mediated inflammatory process.

PAR1 regulation of CXCL1 expression and neutrophil recruitment to the lung in mice infected with influenza A virus

BackgroundProtease‐activated receptor 1 (PAR1) is expressed in various immune cells and in the lung. We showed that PAR1 plays a role in Coxsackievirus B3 infection by enhancing toll‐like receptor 3‐dependent interferon‐β expression in cardiac fibroblasts. ObjectivesWe investigated the role of PAR1 in a mouse model of influenza A virus (IAV) infection. MethodsWe used mice with either a global deficiency of PAR1, cell type‐specific deficiencies of PAR1, or mutation of PAR1 at the R41 or R46 cleavage sites. ResultsPAR1‐deficient mice had increased CXCL1 expression in the lung, increased neutrophil recruitment, increased protein levels in the bronchoalveolar lavage fluid, and increased mortality after IAV infection compared with control mice infected with IAV. Results from mice with cell type‐specific deletion of PAR1 indicated that PAR1 expression by hematopoietic cells suppressed CXCL1 expression, whereas PAR1 expression by endothelial cells enhanced CXCL1 expression in response to IAV infection. PAR1 activation also enhanced polyinosinic:polycytodylic acid induction of interleukin‐8 in a human endothelial cell line. Mutation of the R46 cleavage site of PAR1 was associated with increased CXCL1 expression in the lung in response to IAV infection, which suggested that R46 signaling suppresses CXCL1 expression. ConclusionsThese results indicate that PAR1 expression by different cell types and activation by different proteases modulates the immune response during IAV infection.

Mast Cells and Proteins Related to Myofibroblast Differentiation (PAR-2, IL-6, and TGFβ1) in Salivary Cancers: A Preliminary Study.

Salivary gland neoplasms represent an important group of cancers in the head and neck and myoepithelial cells play a key role on the development these tumors. This study evaluated the distribution of mast cells and related proteins (PAR-2, TGFβ1, IL-6) to the myofibroblastic differentiation in malignant tumors of salivary glands with and without myoepithelial differentiation. Immunohistochemical assessement for tryptase mast cells, SMA, PAR-2, TGFβ1, IL-6 was performed in 10 cases of polymorphous low-grade adenocarcinoma, 14 cases of mucoepidermoid carcinoma (MEC) and 10 cases of adenoid cystic carcinoma. When the density of mast cells were compared between tumors, their density was significantly higher in MEC (P=0.08). Tumors with high expression of PAR-2 (79.4%) exhibited a high density of mast cells. Myofibroblasts were more frequent in malignant tumors with low expression (<50%) of cell masts. Individual analysis of the tumors showed no significant difference between the expression of PAR-2, IL-6, TGFβ1, and myofibroblasts. When the density of mast cells, myofibroblasts and the expression of PAR-2 protein, IL-6, and TGFβ1 were compared, it was no statistically significant difference between tumors with and without myoepithelial differentiation. The results of present study suggest a possible participation of mast cells and especially of PAR-2 in the development and progression of malignant salivary cancers, regardless of myoepithelial content.

Astragalin Inhibits Cigarette Smoke-Induced Pulmonary Thrombosis and Alveolar Inflammation and Disrupts PAR Activation and Oxidative Stress-Responsive MAPK-Signaling.

Epidemiological evidence shows that smoking causes a thrombophilic milieu that may play a role in the pathophysiology of chronic obstructive pulmonary disease (COPD) as well as pulmonary thromboembolism. The increased nicotine level induces a prothrombotic status and abnormal blood coagulation in smokers. Since several anticoagulants increase bleeding risk, alternative therapies need to be identified to protect against thrombosis without affecting hemostasis. Astragalin is a flavonoid present in persimmon leaves and green tea seeds and exhibits diverse activities of antioxidant and anti-inflammation. The current study investigated that astragalin attenuated smoking-induced pulmonary thrombosis and alveolar inflammation. In addition, it was explored that molecular links between thrombosis and inflammation entailed protease-activated receptor (PAR) activation and oxidative stress-responsive mitogen-activated protein kinase (MAPK)-signaling. BALB/c mice were orally administrated with 10–20 mg/kg astragalin and exposed to cigarette smoke for 8 weeks. For the in vitro study, 10 U/mL thrombin was added to alveolar epithelial A549 cells in the presence of 1–20 µM astragalin. The cigarette smoking-induced the expression of PAR-1 and PAR-2 in lung tissues, which was attenuated by the administration of ≥10 mg/kg astragalin. The oral supplementation of ≥10 mg/kg astragalin to cigarette smoke-challenged mice attenuated the protein induction of urokinase plasminogen activator, plasminogen activator inhibitor-1and tissue factor, and instead enhanced the induction of tissue plasminogen activator in lung tissues. The astragalin treatment alleviated cigarette smoke-induced lung emphysema and pulmonary thrombosis. Astragalin caused lymphocytosis and neutrophilia in bronchoalveolar lavage fluid due to cigarette smoke but curtailed infiltration of neutrophils and macrophages in airways. Furthermore, this compound retarded thrombin-induced activation of PAR proteins and expression of inflammatory mediators in alveolar cells. Treating astragalin interrupted PAR proteins-activated reactive oxygen species production and MAPK signaling leading to alveolar inflammation. Accordingly, astragalin may interrupt the smoking-induced oxidative stress–MAPK signaling–inflammation axis via disconnection between alveolar PAR activation and pulmonary thromboembolism.

Par3/integrin β1 regulates embryo adhesion via changing endometrial luminal epithelium polarity†.

The objective is to investigate the pathophysiological significance of Par3 and integrin β1 with regard to the functionality of the endometrial luminal epithelium (LE). Design: laboratory study; setting: university research laboratory. Analysis involved endometrial aspirates and endometrial adenocarcinoma cells (HEC-1A) and endometrial carcinoma cells (RL95-2). We first examined the expression and localization of Par3 and integrin β1 in HEC-1A cells and RL95-2 cells. Then we knocked down Par3 and integrin β1 in HEC-1A cells and RL95-2 cells, respectively, and found that Par3/integrin β1 affected embryo adhesion by regulating the intercellular tight junctions' (TJs') structure and thus the polarity of the endometrial LE. These findings were also confirmed in the endometrium specimens from human and mice. The main outcome measures were the expression and localization of Par3 and integrin β1 in the endometrial epithelial cell lines and endometrium specimens and the regulations of Par3 and integrin β1 on TJs, polarity, and embryo adhesion. Following the knockdown of Par3 in HEC-1A cells, there was a reduction in the complexity of the TJs and cell polarity, and the adhered blastocysts number was significantly increased. However, the reduction of integrin β1 in RL95-2 cells resulted in effects that directly opposed those following the knockdown of Par3 in HEC-1A cells. Estrogen and progesterone reduced the expression of Par3 and promoted the expression of integrin β1 in HEC-1A cells. Par3/integrin β1 regulates embryo adhesion by regulating intercellular TJs' structure and polarity of endometrial LE under the action of ovarian hormones.

Effects of Mast Cells Induced by NSAIDs Impair Intestinal Epithelial Barrier Function In Vivo and In Vitro

To explore the correlation between altered expression of mast cells and PAR-2 and impaired mucosal barrier in NSAIDs enteropathy through animal and cell experiments, and to elucidate the role of mast cells and PAR-2 in the pathogenesis of NSAIDs enteropathy and the regulatory mechanism of the tight junction of intestinal epithelium. Animal experiments: the NSAIDs-related small intestine injury model was established by intragastric administration of diclofenac sodium, and mast cells were detected by toluidine blue staining. Cell experiments: Intestinal epithelial cell line (IEC-6) was applied with diclofenac sodium and its activity was detected by CCK-8.IEC-6 and RBL-2H3 were co-cultured to evaluate the permeability of intestinal epithelial cells by detecting the concentration of potassium ion and LDH. The expressions of tight junction proteins (zo-1, claudin-1, occludin), cytoskeletal components (actin, tubulin, keratin) and par-2 were analyzed by Western Blot. In animal experiments, the number of mast cells was significantly increased after 24 h of action of diclofenac sodium. In cell experiments, the survival rate of IEC-6 cells decreased significantly when the concentration of diclofenac sodium is more than 50 μg/mL; after 24 h of co-culture, the potassium and LDH concentration in the co-culture group were significantly higher, and the expression of ZO-1, claudin-1, occludin, tubulin, and keratin was decreased. Mast cells activate PAR-2 in intestinal epithelial cells, downregulate the related proteins of cell tight junctions and cytoskeletal proteins, and increase the permeability of intestinal epithelial cells.

Evidence from comparative genomic analyses indicating that Lactobacillus-mediated irritable bowel syndrome alleviation is mediated by conjugated linoleic acid synthesis.

Irritable bowel syndrome (IBS) is a chronic intestinal disorder accompanied by low-grade inflammation, visceral hypersensitivity, and gut microbiota dysbiosis. Several studies have indicated that Lactobacillus supplementation can help to alleviate IBS symptoms and that these effects are strain-specific. Therefore, this study aimed to investigate the key physiological characteristics and functional genes contributing to the IBS-alleviating effects of Lactobacillus. An IBS model was established by subjecting C57BL/6 mice to Citrobacter rodentium ingestion and water avoidance stress. Lactobacillus strains with different physiological characteristics were administered to mice intragastrically for 4 weeks (5 × 109 CFU/0.2 mL per mouse per day). Indicators of colonic inflammation, visceral hypersensitivity, and gut microbiota were also evaluated. Finally, differences in functional genes between Lactobacillus strains were analyzed by a comparative genomic analysis, and the relationships between the physiological characteristics, functional genes, and IBS-alleviating effects of the strains were quantified using correlation analysis. Among the eight tested Lactobacillus strains, only Lactobacillus plantarum CCFM8610 significantly inhibited the expression of IL-1β, IL-6, PAR-2, and mast cell tryptase. L. plantarum CCFM8610 also significantly increased the intestinal barrier function, inhibited visceral hypersensitivity symptoms, and modulated the gut microbiota diversity and composition. The correlation analysis of factors associated with the IBS-alleviating effects of Lactobacillus revealed the ability to synthesize conjugated linoleic acid as the most strongly associated physiological characteristic and COG1028-related genes as the most strongly associated functional genes. In conclusion, these findings can facilitate the rapid screening of Lactobacillus strains with IBS-alleviating effects and lay a foundation for studies of the related mechanisms.

Tissue factor cytoplasmic domain exacerbates post-infarct left ventricular remodeling via orchestrating cardiac inflammation and angiogenesis.

The coagulation protein tissue factor (TF) regulates inflammation and angiogenesis via its cytoplasmic domain in infection, cancer and diabetes. While TF is highly abundant in the heart and is implicated in cardiac pathology, the contribution of its cytoplasmic domain to post-infarct myocardial injury and adverse left ventricular (LV) remodeling remains unknown.Methods: Myocardial infarction was induced in wild-type mice or mice lacking the TF cytoplasmic domain (TF∆CT) by occlusion of the left anterior descending coronary artery. Heart function was monitored with echocardiography. Heart tissue was collected at different time-points for histological, molecular and flow cytometry analysis.Results: Compared with wild-type mice, TF∆CT had a higher survival rate during a 28-day follow-up after myocardial infarction. Among surviving mice, TF∆CT mice had better cardiac function and less LV remodeling than wild-type mice. The overall improvement of post-infarct cardiac performance in TF∆CT mice, as revealed by speckle-tracking strain analysis, was attributed to reduced myocardial deformation in the peri-infarct region. Histological analysis demonstrated that TF∆CT hearts had in the infarct area greater proliferation of myofibroblasts and better scar formation. Compared with wild-type hearts, infarcted TF∆CT hearts showed less infiltration of proinflammatory cells with concomitant lower expression of protease-activated receptor-1 (PAR1) - Rac1 axis. In particular, infarcted TF∆CT hearts displayed markedly lower ratios of inflammatory M1 macrophages and reparative M2 macrophages (M1/M2). In vitro experiment with primary macrophages demonstrated that deletion of the TF cytoplasmic domain inhibited macrophage polarization toward the M1 phenotype. Furthermore, infarcted TF∆CT hearts presented markedly higher peri-infarct vessel density associated with enhanced endothelial cell proliferation and higher expression of PAR2 and PAR2-associated pro-angiogenic pathway factors. Finally, the overall cardioprotective effects observed in TF∆CT mice could be abolished by subcutaneously infusing a cocktail of PAR1-activating peptide and PAR2-inhibiting peptide via osmotic minipumps.Conclusions: Our findings demonstrate that the TF cytoplasmic domain exacerbates post-infarct cardiac injury and adverse LV remodeling via differential regulation of inflammation and angiogenesis. Targeted inhibition of the TF cytoplasmic domain-mediated intracellular signaling may ameliorate post-infarct LV remodeling without perturbing coagulation.