Par-4 Expression Research Articles

Pharmacological investigation of new niclosamide-based isatin hybrids as antiproliferative, antioxidant, and apoptosis inducers

A group of Niclosamide-linked isatin hybrids (Xo, X1, and X2) was created and examined using IR, 1HNMR, 13C NMR, and mass spectrometry. These hybrids' cytotoxicity, antioxidant, cell cycle analysis, and apoptosis-inducing capabilities were identified. Using the SRB assay, their cytotoxicity against the human HCT-116, MCF-7, and HEPG-2 cancer cell lines, as well as VERO (African Green Monkey Kidney), was evaluated. Compound X1 was the most effective compound. In HCT-116 cells, compound X1 produced cell cycle arrest in the G1 phase, promoted cell death, and induced apoptosis through mitochondrial membrane potential breakdown in comparison to niclosamide and the control. Niclosamide and compound X1 reduced reactive oxygen species generation and modulated the gene expression of BAX, Bcl-2, Bcl-xL, and PAR-4 in comparison to the control. Docking modeling indicated their probable binding modalities with the XIAP BIR2 domain, which selectively binds caspase-3/7, and highlighted their structural drivers of activity for further optimization investigations. Computational in silico modeling of the new hybrids revealed that they presented acceptable physicochemical values as well as drug-like characteristics, which may introduce them as drug-like candidates. The study proved that compound X1 might be a novel candidate for the development of anticancer agents as it presents antiproliferative activity mediated by apoptosis.

Recombinant hirudin and PAR-1 regulate macrophage polarisation status in diffuse large B-cell lymphoma

BackgroundDiffuse large B-cell lymphoma (DLBCL) is a malignant tumour. Although some standard therapies have been established to improve the cure rate, they remain ineffective for specific individuals. Therefore, it is meaningful to find more novel therapeutic approaches. Macrophage polarisation is extensively involved in the process of tumour development. Recombinant hirudin (rH) affects macrophages and has been researched frequently in clinical trials lately. Our article validated the regulatory role of rH in macrophage polarisation and the mechanism of PAR-1 by collecting clinical samples and subsequently establishing a cellular model to provide a scientifically supported perspective for discovering new therapeutic approaches.MethodWe assessed the expression of macrophage polarisation markers, cytokines and PAR-1 in clinical samples. We established a cell model by co-culture with THP-1 and OCI-Ly10 cell. We determined the degree of cell polarisation and expression of validation cytokines by flow cytometry, ELISA, and RT-qPCR to confirm the success of the cell model. Subsequently, different doses of rH were added to discover the function of rH on cell polarisation. We confirmed the mechanism of PAR-1 in macrophage polarisation by transfecting si-PAR-1 and pcDNA3.1-PAR-1.ResultsWe found higher expression of M2 macrophage markers (CD163 + CMAF+) and PAR-1 in 32 DLBCL samples. After inducing monocyte differentiation into M0 macrophages and co-culturing with OCI-Ly10 lymphoma cells, we found a trend of these expressions in the cell model consistent with the clinical samples. Subsequently, we discovered that rH promotes the polarisation of M1 macrophages but inhibits the polarisation of M2 macrophages. We also found that PAR-1 regulates macrophage polarisation, inhibiting cell proliferation, migration, invasion and angiogenic capacity.ConclusionrH inhibits macrophage polarisation towards the M2 type and PAR-1 regulates polarisation, proliferation, migration, invasion, and angiogenesis of DLBCL-associated macrophages.

Elucidating PAR1 as a therapeutic target for delayed traumatic brain injury: Unveiling the PPAR-γ/Nrf2/HO-1/GPX4 axis to suppress ferroptosis and alleviate NLRP3 inflammasome activation in rats

Repetitive traumatic brain injury (RTBI) is acknowledged as a silent overlooked public health crisis, with an incomplete understanding of its pathomechanistic signaling pathways. Mounting evidence suggests the involvement of thrombin and its receptor, the protease-activated receptor (PAR)1, in the development of secondary injury in TBI; however, the consequences of PAR1 modulation and its impact on ferroptosis-redox signaling, and NLRP3 inflammasome activation in RTBI, remain unclear. Further, the utilitarian function of PAR1 as a therapeutic target in RTBI has not been elucidated. To study this crosstalk, RTBI was induced in Wistar rats by daily weight drops on the right frontal region for five days. Three groups were included: normal control, untreated RTBI, and RTBI+SCH79797 (a PAR1 inhibitor administered post-trauma at 25 μg/kg/day). The concomitant treatment of PAR1 antagonism improved altered behavior function, cortical histoarchitecture, and neuronal cell survival. Moreover, the receptor blockade downregulated mRNA expression of PAR1 but upregulatedthat of the neuroprotective receptor PPAR-γ. The anti-inflammatory impact of SCH79797 was signified by the low immune expression/levels of NF-κB p65,TNF-α, IL-1β, and IL-18. Consequently, the PAR1 blocker hindered the formation of inflammasome components NLRP3, ASC, and activated caspase-1. Ultimately, SCH79797 treatment abated ferroptosis-dependent iron redox signaling through the activation of the antioxidant Nrf2/HO-1 axis and its subsequent antioxidant machinery (GPX4, SOD) to limit lipid peroxidation, iron accumulation, and transferrin serum increment. Collectively, SCH79797 offered putative preventive mechanisms against secondary RTBI consequences in rats by impeding ferroptosis and NLRP3 inflammasome through activating the PPAR-γ/Nrf2 antioxidant cue.

Downregulation of adipose LPL by PAR2 contributes to the development of hypertriglyceridemia.

Lipoprotein lipase (LPL) hydrolyzes circulating triglycerides (TGs), releasing fatty acids (FA) and promoting lipid storage in white adipose tissue (WAT). However, the mechanisms regulating adipose LPL and its relationship with the development of hypertriglyceridemia are largely unknown. WAT from obese humans exhibited high PAR2 expression, which was inversely correlated with the LPL gene. Decreased LPL expression was also inversely correlated with elevated plasma TG levels, suggesting that adipose PAR2 might regulate hypertriglyceridemia by downregulating LPL. In mice, aging and high palmitic acid diet (PD) increased PAR2 expression in WAT, which was associated with a high level of macrophage migration inhibitory factor (MIF). MIF downregulated LPL expression and activity in adipocytes by binding with CXCR2/4 receptors and inhibiting Akt phosphorylation. In a MIF overexpression model, high-circulating MIF levels suppressed adipose LPL, and this suppression was associated with increased plasma TGs but not FA. Following PD feeding, adipose LPL expression and activity were significantly reduced, and this reduction was reversed in Par2-/- mice. Recombinant MIF infusion restored high plasma MIF levels in Par2-/- mice, and the levels decreased LPL and attenuated adipocyte lipid storage, leading to hypertriglyceridemia. These data collectively suggest that downregulation of adipose LPL by PAR2/MIF may contribute to the development of hypertriglyceridemia.

Evaluation the expression of Sp1 and Par3 in urinary bladder carcinoma

Evaluation the expression of Sp1 and Par3 in urinary bladder carcinoma

Oncocytic type has distinct immunohistochemical and recurrence-free survival than other histologic types of the intraductal papillary neoplasm of the bile duct

Although intraductal oncocytic papillary neoplasm (IOPN) was considered distinct from the intraductal papillary neoplasm of the pancreas, the oncocytic histologic type remained as a subtype of intraductal papillary neoplasms of the bile duct (IPNBs) with gastric, intestinal, and pancreatobiliary types based on the fifth edition of the WHO classification. To test the characteristics of the oncocytic type of IPNBs, the histopathologic, immunohistochemical (Hep Par-1 and CD117), and clinical characteristics of 13 oncocytic type were compared with 114 others (15 gastric, 39 pancreatobiliary, and 60 intestinal) IPNB types. The oncocytic type, which occupied about 9% of IPNBs, was more frequent in females (p < 0.05) and larger (mean, 5.3 vs. 3.6 cm; p < 0.002) than other IPNB types. Immunohistochemically, the oncocytic type had more frequent combined Hep Par-1 and CD117 expression than other IPNB types (all p < 0.05). The recurrence-free survival rate for patients with the oncocytic type (5-year survival, 100%) was significantly higher (p = 0.015) than for those with other histologic types (59.9%). The oncocytic type had distinct histopathologic, immunohistochemical, and survival outcomes from other IPNBs. Therefore, it can be separated from other IPNB types and classified as one independent entity, similar to IOPN of the pancreas.

Direct neuronal protection by the protease-activated receptor PAR4 antagonist ML354 after experimental stroke in mice.

Thrombo-inflammation is a key feature of stroke pathophysiology and provides multiple candidate drug targets. Thrombin exerts coagulation-independent actions via protease-activated receptors (PAR), of which PAR1 has been implicated in stroke-associated neuroinflammation. The role of PAR4 in this context is less clear. This study examined if the selective PAR4 antagonist ML354 provides neuroprotection in experimental stroke and explored the underlying mechanisms. Mouse primary cortical neurons were exposed to oxygen-glucose deprivation (OGD) and simulated reperfusion ± ML354. For comparison, functional Ca2+-imaging was performed upon acute stimulation with a PAR4 activating peptide or glutamate. Male mice underwent sham operation or transient middle cerebral artery occlusion (tMCAO), with ML354 or vehicle treatment beginning at recanalization. A subset of mice received a platelet-depleting antibody. Stroke size and functional outcomes were assessed. Abundance of target genes, proteins, and cell markers was determined in cultured cells and tissues by qPCR, immunoblotting, and immunofluorescence. Stroke up-regulated PAR4 expression in cortical neurons in vitro and in vivo. OGD augments spontaneous and PAR4-mediated neuronal activity; ML354 suppresses OGD-induced neuronal excitotoxicity and apoptosis. ML354 applied in vivo after tMCAO reduced infarct size, apoptotic markers, macrophage accumulation, and interleukin-1β expression. Platelet depletion did not affect infarct size in mice with tMCAO ± ML354. Selective PAR4 inhibition during reperfusion improves infarct size and neurological function after experimental stroke by blunting neuronal excitability, apoptosis, and local inflammation. PAR4 antagonists may provide additional neuroprotective benefits in patients with acute stroke beyond their canonical antiplatelet action.

Controlling autoimmune diabetes onset by targeting Protease-Activated Receptor 2

BackgroundType 1 diabetes (T1D) is a challenging autoimmune disease, characterized by an immune system assault on insulin-producing β-cells. As insulin facilitates glucose absorption into cells and tissues, β-cell deficiency leads to elevated blood glucose levels on one hand and target-tissues starvation on the other. Despite efforts to halt β-cell destruction and stimulate recovery, success has been limited. Our recent investigations identified Protease-Activated Receptor 2 (Par2) as a promising target in the battle against autoimmunity. We discovered that Par2 activation’s effects depend on its initial activation site: exacerbating the disease within the immune system but fostering regeneration in affected tissue. MethodsWe utilized tissue-specific Par2 knockout mice strains with targeted Par2 mutations in β-cells, lymphocytes, and the eye retina (as a control) in the NOD autoimmune diabetes model, examining T1D onset and β-cell survival. ResultsWe discovered that Par2 expression within the immune system accelerates autoimmune processes, while its presence in β-cells offers protection against β-cell destruction and T1D onset. This suggests a dual-strategy treatment for T1D: inhibiting Par2 in the immune system while activating it in β-cells, offering a promising strategy for T1D. ConclusionsThis study highlights Par2's potential as a drug target for autoimmune diseases, particularly T1D. Our results pave the way for precision medicine approaches in treating autoimmune conditions through targeted Par2 modulation.

Ex Vivo Antiplatelet Effects of Oral Anticoagulants.

The impact of non-vitamin K antagonist oral anticoagulants (NOACs) on platelet function is still unclear. We conducted a comprehensive ex vivo study aimed at assessing the effect of the four currently marketed NOACs on platelet function. We incubated blood samples from healthy donors with concentrations of NOACs (50, 150 and 250 ng/mL), in the range of those achieved in the plasma of patients during therapy. We evaluated generation of thrombin; light transmittance platelet aggregation (LTA) in response to adenosine diphosphate (ADP), thrombin receptor-activating peptide (TRAP), human γ-thrombin (THR) and tissue factor (TF); generation of thromboxane (TX)B2; and expression of protease-activated receptor (PAR)-1 and P-selectin on the platelet surface. All NOACs concentration-dependently reduced thrombin generation compared with control. THR-induced LTA was suppressed by the addition of dabigatran at any concentration, while TF-induced LTA was reduced by factor-Xa inhibitors. ADP- and TRAP-induced LTA was not modified by NOACs. TXB2 generation was reduced by all NOACs, particularly at the highest concentrations. We found a concentration-dependent increase in PAR-1 expression after incubation with dabigatran, mainly at the highest concentrations, but not with FXa inhibitors; P-selectin expression was not changed by any drugs. Treatment with the NOACs is associated with measurable ex vivo changes in platelet function, arguing for antiplatelet effects beyond the well-known anticoagulant activities of these drugs. There are differences, however, among the NOACs, especially between dabigatran and the FXa inhibitors.

Abstract 5456: Identifying the role of tissue factor in osteosarcoma metastasis

Abstract Patients with osteosarcoma who develop metastasis face very poor prognoses. Even when treated with the most effective medical and surgical interventions available, about forty percent of osteosarcoma patients will eventually succumb to lung metastasis. Despite the relevance of metastasis to clinical outcomes, we understand little about the mechanisms that drive these tumor cells into the lung and facilitate their metastatic growth. Consequently, there are no approved antimetastatic agents for osteosarcoma and outcomes have not improved in more than 40 years. Tissue factor is a tightly regulated physiologic initiator of coagulation with functions in inflammation, cytokinesis, angiogenesis, apoptosis, and growth factor secretion. These functions are divided between two separate pathways: initiation of the coagulation cascade via activation of factors VII and X, and propagation of cell signaling pathways via PAR activation. It is aberrantly expressed in many cancers and has been associated with metastatic behavior and inferior outcomes by affecting metastatic vascularity, growth, and migration. Osteosarcoma has been shown to also aberrantly express tissue factor, with descriptive studies suggesting similar effects on metastatic behavior. We hypothesized that tissue factor expressed on the surface of disseminated osteosarcoma cells activates procoagulant pathways and PAR signaling, and that both facilitate pulmonary metastasis. To test this, we first established and quantified expression of tissue factor across six osteosarcoma cell lines using Western blot, flow cytometry, and confocal microscopy in both monoculture and in coculture with human bronchial epithelial cells. PAR1 and PAR2 were also discovered to be expressed in osteosarcoma cells. RNA sequencing performed suggests that both tissue factor and PAR1 expression in osteosarcoma increase over the metastatic period, as does FVII expression by alveolar macrophages; this implies that tissue factor activation during metastasis is dynamic and dependent on host interactions. Ongoing work includes functional assays of osteosarcoma tissue factor and PARs activity as well as preclinical testing of tissue factor inhibition in a murine xenograft model of osteosarcoma. To define how tissue factor propagates metastasis, we are separately inhibiting its coagulant and cell signaling pathways after murine tail vein injection using apixaban or pathway-specific antibodies. We will also use an inducible F3 knockout to alter tumor expression of tissue factor. If we find that manipulation of tissue factor expression changes metastatic behavior across cell lines, this will suggest that tissue factor plays a crucial, mechanistic role in osteosarcoma metastasis. Varying effectiveness of coagulation or signaling pathway inhibition on metastasis will implicate tissue factor’s mechanism of metastatic propagation. Citation Format: Charles C. Treinen, Amy Gross, Maren Cam, Bryce Kerlin, Ryan Roberts. Identifying the role of tissue factor in osteosarcoma metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5456.

Linagliptin's impact on lymphatic barrier and lymphangiogenesis in oral cancer with high glucose.

Uncertainties remain regarding the effect of elevated glucose levels on lymphatic metastasis of cancer cells. Our study elucidated the mechanisms linking high glucose to lymphangiogenesis and lymphatic barrier-related factors and investigated the protective role of linagliptin against lymphatic barrier dysfunction. A CAL-27-LEC co-culture system was established. Sodium fluorescein permeability assay observed lymphatic endothelial cell permeability. Western blotting and RT-qPCR detected protein and mRNA expression under different conditions, respectively. CCK-8, scratch wound healing, and transwell assays revealed cell migration and proliferation. Tube formation experiment tested capacity for endothelial tube formation. Immunohistochemical staining analyzed tissue sections from 43 oral cancer individuals with/without diabetes. In high-glucose co-culture system, we observed increased lymphatic barrier permeability and decreased expression of ZO-1 and occludin, two tight-junction proteins; conversely, the expression of PAR2, a high permeability-related protein, was increased. Following linagliptin treatment, the expression levels of VEGF-C, VEGFR-3, and PAR2 decreased, while those of ZO-1 and occludin increased. Considerably higher levels of LYVE-1 expression in individuals with diabetes than in those without diabetes. By ameliorating the high glucose-induced disruption of the lymphatic endothelial barrier, linagliptin may reduce lymphangiogenesis and exhibit an inhibitory effect on lymphatic metastasis in oral cancer patients with diabetes.

Association of single nucleotide polymorphisms in the F2RL1 gene with clinical and inflammatory characteristics of patients with asthma

BackgroundProteinase-activated receptor 2 (PAR-2) is a G-protein coupled receptor associated with many inflammatory diseases, including asthma. We have shown an association between PAR-2 expression in peripheral blood monocytes and asthma severity as well as blood PAR-2 mRNA level and lung function. Since F2RL1 (the gene encoding PAR-2) polymorphisms affect PAR-2 expression, we hypothesize they may affect asthma severity.MethodsWe recruited 76 subjects with asthma of varying severity and collected clinical (FEV1 [% predicted], FEV1/FVC, IgE) and immunological (PAR-2 mRNA, blood eosinophils) disease parameters. We also genotyped these individuals for 3 F2RL1 SNPs (-45C/T, -149C/G, c.621C/T).ResultsWe found that the F2RL1 SNP “C” allele of -45C/T (rs1529505) was associated with PAR-2 mRNA and blood eosinophils. F2RL1 SNP c.621C/T (rs631465) was associated with PAR-2 mRNA. The F2RL1 SNP -149C/G (rs2242991) had no association with any of the parameters studied. This study identified one F2RL1 SNP rs1529505 is associated with parameters of asthma, but not asthma severity.ConclusionLarger studies are needed to further elucidate the role of PAR-2 in the pathophysiology of asthma and the influence of genetic variation.

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.

Electroacupuncture protects the intestinal mucosal barrier in diarrhea-predominant Irritable Bowel Syndrome rats by regulating the MCs/Tryptase/PAR-2/MLCK pathway.

The pathogenesis of diarrhea-predominant irritable bowel syndrome (IBS-D) is related to damage to the intestinal mucosal barrier function. Based on the Mast cell (MC)/Tryptase/Protease-activated receptor-2 (PAR-2)/Myosin light chain kinase (MLCK) pathway, this study explored the effect of electroacupuncture (EA) on IBS-D rats and its possible mechanism of protecting the intestinal mucosal barrier. The IBS-D rat model was established by mother-offspring separation, acetic acid enema, and chronic restraint stress. The efficacy of EA on IBS-D rats was evaluated by observing the rate of loose stool (LSP) and the minimum volume threshold of abdominal withdrawal reflex (AWR) in rats. Mast cells and the ultrastructure of intestinal mucosa were observed by H&E staining, toluidine blue staining, and transmission electron microscopy. The expression levels of Tryptase, PAR-2, MLCK, zonula occludens-1 (ZO-1), and Occludin in rats were detected by ELISA, qRT-PCR, and western blot. After 7 days of intervention, compared to the IBS-D group, the loose stool rates of rats in IBS-D + EA group and IBS-D + ketotifen group were decreased (P < 0.01), the minimum volume thresholds of AWR were improved (P < 0.01), the inflammation of colon tissue decreased, the number of MCs were decreased (P < 0.01), the expression of Tryptase, PAR-2, and MLCK were lowered (P < 0.01, P < 0.05), and the expression of ZO-1 and Occludin were enhanced (P < 0.01, P < 0.05). Compared to the EA group, there was no significant difference in each index between the ketotifen groups (P > 0.05). EA has a good therapeutic effect on IBS-D rats. Regulating the MCs/Tryptase/PAR-2/MLCK pathway may be a mechanism to protect the intestinal mucosal barrier.

Use of Immunohistochemical Markers in Differentiation of Hepatocellular Carcinoma from Intrahepatic Cholangiocarcinoma and Metastatic Adenocarcinomatous Deposits in the Liver from Colon: A Cross-sectional Study

Introduction: Hepatocellular Carcinoma (HCC) is a major worldwide health problem due to its high incidence and mortality rates. The liver often becomes a site for metastasis from various primary locations, benefiting from its abundant blood supply. Distinguishing liver metastatic tumours from HCC can pose a diagnostic challenge, significantly impacting prognosis and treatment decisions. Aim: To differentiate between HCC, Intrahepatic Cholangiocarcinoma (ICC), and metastatic colonic adenocarcinoma in the liver using Hep par-1, Cytokeratin (CK) 7, CK19, and CK20 as immunohistochemical markers. The manual Tissue Microarray (TMA) technique was employed for present study. Materials and Methods: The present cross-sectional study was conducted in Department of Pathology at Government Stanley Medical College, Chennai, Tamil Nadu, India, spanning a three-year duration from July 2012 to June 2015. A total of 60 cases diagnosed histologically with HCC, ICC, and metastatic colonic adenocarcinoma in the liver were included. The manual TMA technique was used to create recipient blocks, and immunohistochemistry was performed to assess the expression of Hep par-1, CK7, CK19 and CK20. The sensitivity, specificity, Positive Predictive Value (PPV), and Negative Predictive Values (NPV) of these markers in HCC, ICC, and metastatic colonic adenocarcinoma in the liver were analysed and tabulated using statistical software Statistical Package for Social Sciences (SPSS) version 16.0. Results: The study included a total of 60 cases, with 40 (66.7%) males and 20 (33.3%) females, ranging in age from 27 to 73 years with a mean age of 51.3 years. Among the cases, there were 30 (50%) cases of HCC, 14 (23%) cases of ICC, and 16 (27%) cases of metastatic colonic adenocarcinoma in the liver. The sensitivity, specificity, and PPV of Hep par-1 in distinguishing HCC from ICC and metastatic deposits were 80%, 100%, and 100%, respectively. The NPV of Hep par-1 in distinguishing HCC from ICC and metastatic deposits was 70% and 72%, respectively. The sensitivity, specificity, PPV, and NPV of CK7 in distinguishing ICC from HCC were 3.3%, 50%, 6.3%, and 34.1%, respectively. The sensitivity, specificity, PPV, and NPV of CK19 in distinguishing HCC from ICC and metastatic deposits were 0%, 50%, 0%, and 33.3%. The sensitivity, specificity, PPV, and NPV of CK20 in distinguishing HCC from ICC and metastatic deposits were 0%, 53.3%, 0%, and 34.8%, respectively. Conclusion: In conclusion, it was found that a panel of markers including Hep par-1, CK7, CK19 and CK20 can differentiate between HCC, ICC, and metastatic colonic adenocarcinoma in the liver. This differentiation is crucial for determining the appropriate treatment for patients by understanding the exact behaviour of the tumour.

Argatroban promotes recovery of spinal cord injury by inhibiting the PAR1/JAK2/STAT3 signaling pathway.

Argatroban is a synthetic thrombin inhibitor approved by U.S. Food and Drug Administration for the treatment of thrombosis. However, whether it plays a role in the repair of spinal cord injury is unknown. In this study, we established a rat model of T10 moderate spinal cord injury using an NYU Impactor Moder III and performed intraperitoneal injection of argatroban for 3 consecutive days. Our results showed that argatroban effectively promoted neurological function recovery after spinal cord injury and decreased thrombin expression and activity in the local injured spinal cord. RNA sequencing transcriptomic analysis revealed that the differentially expressed genes in the argatroban-treated group were enriched in the JAK2/STAT3 pathway, which is involved in astrogliosis and glial scar formation. Western blotting and immunofluorescence results showed that argatroban downregulated the expression of the thrombin receptor PAR1 in the injured spinal cord and the JAK2/STAT3 signal pathway. Argatroban also inhibited the activation and proliferation of astrocytes and reduced glial scar formation in the spinal cord. Taken together, these findings suggest that argatroban may inhibit astrogliosis by inhibiting the thrombin-mediated PAR1/JAK2/STAT3 signal pathway, thereby promoting the recovery of neurological function after spinal cord injury.

Expression and clinical implications of PARs in the stenotic tissue of ureteropelvic junction obstruction.

To explore the expression and clinical implications of protease activated receptors (PARs) in the pathogenesis of children with ureteropelvic junction obstruction (UPJO). Immunohistochemistry was employed to investigate the distribution of PARs in both normal human ureteropelvic junction (UPJ) and cases of UPJO. Furthermore, PAR gene expression levels were assessed using real-time PCR (RT-PCR), and the patients in the UPJO group were stratified according to the Onen grading system. Subsequently, the clinical implications of PARs in UPJO were explored through RT-PCR analysis. Immunofluorescence showed robust PAR2 expression in the control group compared with the UPJO group. The results of RT-PCR analysis revealed a significant decrease in the relative mRNA expression of PAR2 in the UPJO group compared to the control group. Notably, the relative RNA expression of PAR1 was significantly lower in the Onen-4 group compared to the control group. Furthermore, the relative mRNA expression of PAR2 exhibited a statistically significant difference among the Onen-3 group, Onen-4 group, and control group. PARs are widely distributed throughout the SIP syncytium of the UPJ and play a role in maintaining smooth muscle cells (SMCs) membrane potential by interacting with interstitial cells of Cajal (ICCs), as well as platelet-derived growth factor receptor alpha-positive cells (PDGFR α+ cells). The decreased expression of PAR1 suggests a higher preoperative Onen grade in UPJO patients. Furthermore, the downregulation of PAR2 effects at the UPJ may be involved in the loss of inhibitory neuromuscular transmission, disrupting the rhythmic peristalsis of the UPJ.

Coagulation Factor Xa Has No Effects on the Expression of PAR1, PAR2, and PAR4 and No Proinflammatory Effects on HL-1 Cells.

Atrial fibrillation (AF), characterised by irregular high-frequency contractions of the atria of the heart, is of increasing clinical importance. The reasons are the increasing prevalence and thromboembolic complications caused by AF. So-called atrial remodelling is characterised, among other things, by atrial dilatation and fibrotic remodelling. As a result, AF is self-sustaining and forms a procoagulant state. But hypercoagulation not only appears to be the consequence of AF. Coagulation factors can exert influence on cells via protease-activated receptors (PAR) and thereby the procoagulation state could contribute to the development and maintenance of AF. In this work, the influence of FXa on Heart Like-1 (HL-1) cells, which are murine adult atrial cardiomyocytes (immortalized), was investigated. PAR1, PAR2, and PAR4 expression was detected. After incubations with FXa (5-50 nM; 4-24 h) or PAR1- and PAR2-agonists (20 µM; 4-24 h), no changes occurred in PAR expression or in the inflammatory signalling cascade. There were no time- or concentration-dependent changes in the phosphorylation of the MAP kinases ERK1/2 or the p65 subunit of NF-κB. In addition, there was no change in the mRNA expression of the cell adhesion molecules (ICAM-1, VCAM-1, fibronectin). Thus, FXa has no direct PAR-dependent effects on HL-1 cells. Future studies should investigate the influence of FXa on human cardiomyocytes or on other cardiac cell types like fibroblasts.

Clinically uninvolved but not healthy-The skin of patients with atopic dermatitis is primed for itch and inflammation.

Atopic dermatitis (AD) is a highly prevalent inflammatory skin disorder characterized by episodic exacerbations and remissions. Why the clinically healthy skin of AD patients becomes rapidly inflamed and very pruritic is poorly understood. To investigate cowhage- and histamine-induced itch and skin expression levels of their target receptors in lesional and non-lesional skin of AD, compared to the skin of patients with psoriasis, chronic spontaneous urticaria (CSU) and healthy subjects. Patients with AD, psoriasis and chronic spontaneous urticaria (CSU) as well as healthy control subjects (HC) (n = 20 each) were assessed for differences in itch parameters, neurogenic flare reaction and local blood flow responses to skin provocations with cowhage and histamine. Skin biopsies from 10 AD, 10 psoriasis,11 CSU and 12 HC were obtained to assess expression of protease-activated receptors 2 and 4 (PAR-2, PAR-4), histamine H1 and H4 receptors (H1R, H4R), and mast cells. Provocation of non-lesional skin of AD patients with cowhage resulted in prolonged itch (p = 0.020), which was not observed in psoriasis and CSU. Significantly prolonged and more intense cowhage- and histamine-induced itch (for duration, peak and overall intensity) was also observed in lesional AD skin. Diminished neurogenic flare reaction and blood flow after histamine provocation were shown in AD and psoriasis patients. Non-lesional AD skin along with lesional AD and psoriasis skin showed an increased expression of PAR-2 and PAR-4, H1R and H4R. Mast cell number was higher in lesional AD and psoriasis skin (p = 0.006 and p = 0.006, respectively). The non-lesional skin of AD patients markedly differs from healthy skin in cowhage-induced itch responses and the expression of receptors for proteases and histamine. Proactive therapeutic interventions that downregulate these receptors may prevent episodic exacerbation in AD.

Delineation of the healthy rabbit tongue by immunohistochemistry – A technical note

In the oral cavity the tongue is an important muscular organ that supports the swallowing of food and liquids. It is responsible for the sense of taste, based on the many different taste buds it contains. Research in the field of tongue diseases demands for suitable preclinical models. The healthy rabbit tongue may therefore serve as baseline and reference for the pathological situation. With this consideration, we covered the fixation and histological stainings as well as the immunohistochemical labelling of the healthy rabbit tongue. In this technical note, initial choice of the fixative is discussed, with a comparison of formalin fixation and subsequent paraffin embedding versus cryopreservation. Moreover, we delineate the effect of an antigen retrieval step for formalin fixation by several examples. Finally, we provide ECM markers collagen I, collagen III, fibronectin, α-SMA and elastin staining as well as ki67 for proliferative status and PAR-2 protein expression as a marker for inflammatory status and nociception in tongue sections, mainly from the tongue body. Technically, we found superiority of paraffin sections for collagen I, collagen III, fibronectin, ki67 and α-SMA labelling, for selected detections systems. As for ECM components, the lamina propria was very rich in collagen and fibronectin, while the muscular body of the tongue showed only collagen and fibronectin positive areas between the muscle fibers. Moreover, α-SMA was clearly expressed in the walls of arteries and veins. The inflammatory marker PAR-2 on the other hand was prominently expressed in the salivary glands and to some extent in the walls of the vessels. Particular PAR-2 expression was found in the excretory ducts of the tongue. This technical note has the aim to provide baseline images that can be used to compare the pathological state of the diseased rabbit tongue as well as for inter-species comparison, such as mouse or rat tongue. Finally, it can be used for the comparison with the human situation.