The novel thromboxane prostanoid receptor mediates CTGF production to drive human nasal fibroblast self-migration through NF-κB and PKCδ-CREB signaling pathways.

Chronic rhinosinusitis without nasal polyp (CRSsNP) is characterized by tissue repair/remodeling and the subepithelial stroma region in whose nasal mucosa has been reported by us to have thromboxane A2 (TXA2) prostanoid (TP) receptor and overexpress connective tissue growth factor (CTGF). Therefore, this study aimed to investigate the relationship between TP receptor activation and CTGF production/function in human CRSsNP nasal mucosa stromal fibroblasts. We found that TP agonists including U46619 and IBOP ([1S-[1α,2α(Z),3ß(1E,3 S*),4α]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid) could promote CTGF protein/messenger RNA expression and secretion. The pharmacological intervention and TP activation assay with U46619 identified the possible participation of PKCµ, PKCδ, nuclear factor-κB (NF-κB), and cyclic AMP response element-binding protein (CREB) phosphorylation/activation in the CTGF induction. Moreover, a phorbol ester-phorbol-12-myristate 13-acetate (PMA) exhibited a similar cellular signaling and CTGF production profile to that elicited by TP activation. However, further small interfering RNA interference analysis revealed that only NF-κB and PKCδ-CREB pathways were necessarily required for TP-mediated CTGF production, which could not be completely supported by those findings from PMA. Finally, in a functional assay, although CTGF did not affect fibroblast proliferation, TP-mediated CTGF could drive novel self-migration in fibroblasts both in the scratch/wound healing and transwell apparatus assays. Meanwhile, the overall staining for stress fibers and formation of the lamellipodia and filopodia-like structures was concomitantly increased in the treated migrating cells. Collectively, we provided here that novel TP mediates CTGF production and self-migration in human nasal fibroblasts through NF-κB and PKCδ-CREB signaling pathways. More importantly, we also demonstrated that thromboxane, TP receptor, CTGF, and stromal fibroblasts may act in concert in the tissue remodeling/repair process during CRSsNP development and progression.

Novel involvement of PLD-PKCδ-CREB axis in regulating FGF-2-mediated pentraxin 3 production in human nasal fibroblast cells.

A higher expression level of mitogenic fibroblast growth factor-2 (FGF-2) has been reported in human nasal mucus of both chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP). Meanwhile, we have shown that long pentraxin 3 (PTX3), an essential component of humoral innate immunity that is produced at sites of infection and inflammation, was overproduced in human nasal mucosae and secretions of CRSsNP. Therefore, this study was aimed to investigate how FGF-2 regulates PTX3 expression in human CRSsNP nasal mucosa-derived fibroblast cells (hNMDFs). The FGF-2 treatment caused ptx3 mRNA expression and PTX3 protein induction and secretion. In parallel, a differential expression of FGF receptor (FGFR)-1 to FGFR-4 was observed in hNMDFs and human nasal tissues. While conventionally known PI3K/Akt/mTOR and AP-1 pathways following FGFR activation were shown to be involved, the protein kinase Cδ (PKCδ) and cAMP response element-binding protein (CREB) were also found to be as critical signaling molecules in FGF-2-induced PTX3 induction. The PKCδ and CREB activation could be detected in total cells and in the cell nucleus. Accordingly, a novel CREB binding sequence was detected in the human ptx3 promoter region and could interact with activated CREB in cells challenged with FGF-2. Surprisingly, the phospholipase D (PLD), but not phosphoinositide- and phosphatidylcholine-phospholipase C, was necessarily required for PKCδ and CREB activation. Therefore, we demonstrated here for the first time that FGF-2 mediates PTX3 production not only through PI-3K/Akt/mTOR and AP-1 activation, but also through a novel FGFR-PLD-PKCδ-CREB cellular signaling pathway.

TGFß mediates collagen production in human CRSsNP nasal mucosa-derived fibroblasts through Smad2/3-dependent pathway and CTGF induction and secretion.

Chronic rhinosinusitis without nasal polyp (CRSsNP) is characterized by tissue remodeling and fibrosis. Transforming growth factor-ß (TGF-ß) is considered a master switch in the induction of the profibrotic program which can induce fibroblasts to synthesize and contract extracellular matrix (ECM) proteins. A previous study has shown TGF-ß1 signaling and collagen overproduction in the CRSsNP, but the responsible cells and mechanism of action remain unclear. Therefore, this study was aimed to investigate the relationship between TGF-ß1 stimulation and collagen expression and to explore the role of connective tissue growth factor (CTGF) during the remodeling process using human CRSsNP nasal mucosa tissues and mucosa-derived fibroblasts as main materials. We found that TGF-ß1 and its isoforms could promote collagen protein expression. Concomitantly, TGF-ß1 caused CTGF expression and secretion. An addition of exogenous CTGF to fibroblasts also caused collagen expression. In accordance with these observations, TGF-ß1, CTGF, and collagen were highly expressed in the subepithelial stroma region of CRSsNP nasal mucosa, as determined by immunohistochemistry. The TGF-ß1-mediated collagen expression could be blocked by actinomycin D and SIS3, suggesting that the induction was through transcriptional regulation and Smad2/3-dependent pathway. Finally, we demonstrated that CTGF small interfering RNA knockdown led to a substantial decrease in TGF-ß1-mediated collagen expression. Collectively, our results provide first and further evidence that TGF-ß1 mediates collagen expression-production through a canonical Smad2/3-dependent pathway and CTGF induction and secretion in human nasal fibroblasts. Moreover, TGF-ß1, CTGF, and collagen are highly expressed in human CRSsNP nasal mucosa specimens, suggesting their roles in tissue remodeling during CRSsNP progression.

Peptidoglycan induces bradykinin receptor 1 expression through Toll-like receptor 2 and NF-κB signaling pathway in human nasal mucosa-derived fibroblasts of chronic rhinosinusitis patients.

Numerous studies have demonstrated that Gram-positive microbiomes play an important role in the pathogenesis and in the way of treatment of chronic rhinosinusitis (CRS). Kinins are inflammatory mediators and one of their receptors, namely bradykinin receptor 1 (BKR1 or B1R), is believed to be induced and involved in inflammation in pathophysiological conditions. In the present study, we investigated the effect of peptidoglycan (PGN), a major cell wall component of G(+) bacteria, on BKR expression and its signaling pathway in nasal fibroblasts from CRS without nasal polyp (CRSsNP). The PGN induced increases in B1R mRNA and protein production. The induction was abolished by the NF-κB and protein kinase A inhibitor. In parallel, the PGN treatment directly activated IκB/NF-κB signaling and CREB phosphorylation. Interestingly, a further analysis suggested no involvement of cAMP/PKA/CREB pathway in this induction. The B1R expression and IκB/NF-κB signaling pathway could be attenuated by Toll-like receptor-2 (TLR2) blocking/neutralizing Ab. In a functional assay, the addition of B1R selective agonist (Des-Arg10 -kallidin) to the fibroblasts after PGN stimulation led to an increase in CXCL8 release and p38 MAPK and ERK1/2 phosphorylation, which could be inhibited by the B1R antagonist. Taken together, our results revealed for the first time that PGN can increase B1R expression in human nasal mucosa-derived fibroblasts through TLR2 activation and NF-κB signaling pathway. This induction functionally leads to MAPKs activation and CXCL8 release upon B1R stimulation. Our results also suggest that a major component of G(+) bacteria can participate in B1R upregulation in nasal mucosa during CRSsNP progression.

CXCL1 regulation in human pulmonary epithelial cells by tumor necrosis factor.

BACKGROUND/AIMS: The chemokine CXCL1 has been reported to be expressed in lung airway epithelium and non-small cell lung cancer biopsy specimens. In this study, we investigated the effects of TNF-α, an abundant cytokine detected in inflammation and various cancers, on CXCL1 release by human A549 lung carcinoma epithelial cells. METHODS: CXCL1 expression was determined by ELISA and RT-PCR. TNF-α signaling was examined by western blotting. Monocyte migration was assayed by a Transwell migration system. RESULTS: TNF-α stimulated CXCL1 release and mRNA expression, and this release was inhibited by inhibitors of JNK, p38 MAPK, PI-3K/Akt and AP-1 transcription factor. TNF-α treatment was followed by JNK, p38 MAPK and PI3K/Akt activation. However, only the JNK inhibitor could reduce the CXCL1 mRNA level, suggesting that JNK is required mainly for CXCL1 mRNA synthesis, whereas p38 MAPK and PI-3K/Akt might be responsible for CXCL1 secretion. Dexamethasone (dex) and TGF-ß reduced CXCL1 secretion, with dex upregulating the expression of MAP kinase phosphatase-1 and TGF-ß causing smad2/3 activation and nuclear translocation. A functional analysis showed that the released CXCL1 enhanced monocyte migration and could be abolished by a CXCL1 neutralizing antibody and CXCR antagonist. CONCLUSION: We demonstrate that TNF-α induces CXCL1 expression through the JNK, p38 MAPK and PI-3K/Akt signaling pathways in human pulmonary epithelial cells.

The cyclooxygenase-2 upregulation mediates production of PGE2 autacoid to positively regulate interleukin-6 secretion in chronic rhinosinusitis with nasal polyps and polyp-derived fibroblasts.

Chronic rhinosinusitis (CRS) can be traditionally classified as CRSwNP [with nasal polyps (NPs)] and CRSsNP (without NPs) based on the clinical phenotypes but recently suggested to be classified by the endotypes. We have identified overexpression of the cyclooxygenase-2 (COX-2) gene in NP tissues of Taiwanese CRSwNP patients. Therefore, in this study, we sought to investigate its protein expression/location/distribution in NP specimens and explore its roles in nasal polyposis. The COX-2 protein and mRNA expression was found higher in NPs than that in the control and CRSsNP patients' nasal tissues, mainly located at the epithelium and subepithelial stroma. Consistently, the CRS-related peptidoglycan (PGN) and bradykinin provoked COX-2 mRNA and protein upregulation in the human NP-derived fibroblasts and caused PGE2, thromboxane A2 (TXA2), and interleukin (IL-6) secretion in culture medium. Further analysis revealed that the PI3K/Akt activation and COX-2 induction were necessarily required for PGN-induced IL-6 production/secretion and the induced PGE2, but not TXA2, was speculated to affect IL-6 protein trafficking and production. Finally, the IL-6 increase observed in vitro could also be detected in NP tissues. Collectively, we demonstrated here that COX-2 protein and IL-6 are overexpressed in human NP tissues. In response to PGN challenge, the PI3K/Akt activation and COX-2-mediated PGE2 autacoid correlates with extracellular IL-6 protein trafficking/production in NP-derived fibroblasts, which can additionally contribute to the production of Th17-related cytokines such as IL-17 and TNF-α. This study also suggests COX-2 as a special biomarker for CRSwNP endotyping and may highlight the importance of COX-2 inhibitors in treating CRSwNP.

A naturally occurring carotenoid, lutein, reduces PDGF and H2O2 signaling and compromised migration in cultured vascular smooth muscle cells.

BACKGROUND: Platelet-derived growth factor (PDGF) is a potent stimulator of growth and motility of vascular smooth muscle cells (VSMCs). Abnormalities of PDGF/PDGF receptor (PDGFR) are thought to contribute to vascular diseases and malignancy. We previously showed that a carotenoid, lycopene, can directly bind to PDGF and affect its related functions in VSMCs. In this study we examined the effect of the other naturally occurring carotenoid, lutein, on PDGF signaling and migration in VSMCs. METHODS: Western blotting was performed to examine PDGF and H2O2 signaling. Flowcytometry was used to determine PDGF binding to VSMCs. Fluorescence microscopy was performed to examine intracellular ROS production. Modified Boyden chamber system (Transwell apparatus) was used for migration assay. RESULTS: Lutein reduced PDGF signaling, including phosphorylation of PDGFR-ß and its downstream protein kinases/enzymes such as phospholipase C-γ, Akt, and mitogen-activated protein kinases (MAPKs). Although lutein possesses a similar structure to lycopene, it was striking that lutein inhibited PDGF signaling through a different way from lycopene in VSMCs. Unlike lycopene, lutein not only interacted with (bound to) PDGF but also interfered with cellular components. This was evidenced that preincubation of PDGF with lutein and treatment of VSMCs with lutein followed by removing of lutein compromised PDGF-induced signaling. Lutein reduced PDGF-induced intracellular reactive oxygen species (ROS) production and attenuated ROS- (H2O2-) induced ERK1/2 and p38 MAPK activation. A further analysis indicated lutein could inhibit a higher concentration of H2O2-induced PDGFR signaling, which is known to act through an oxidative inhibition of protein tyrosine phosphatase. Finally, we showed that lutein functionally inhibited PDGF-induced VSMC migration, whereas its stereo-isomer zeaxanthin did not, revealing a special action of lutein on VSMCs. CONCLUSIONS: Our study reveals a differential action mechanism of lutein from other reported caroteinoids and suggests a possible beneficial effect of lutein but not zeaxanthin on prevention of vascular diseases.

A Lemierre syndrome variant caused by Klebsiella pneumoniae.

Lemierre syndrome is an extremely rare disease characterized by oropharyngeal infection, septicemia, internal jugular vein thrombosis, and skip lesions. The most common causative pathogen is Fusobacterium necrophorum. We reported a 45-year-old woman who presented with left neck painful swelling and septicemia. Magnetic resonance imaging of the head and neck demonstrated venous thrombosis extending from the left internal jugular vein to the sigmoid sinus. During admission we discovered that the patient had uncontrolled diabetes mellitus. We also found a metastatic lesion through chest radiography. Klebsiella pneumoniae was cultivated from both blood samples and pus from deep neck spaces. Surgical drainage, early and adequate antibiotic treatment, anticoagulation, and strict control of blood glucose led to the patient's complete recovery. Because Lemierre syndrome is a forgotten disease in the era of antibiotics, awareness of the signs and symptoms of this disease is important because of its associated high mortality rate. This case illustrated that the presence of K pneumoniae can lead to Lemierre syndrome.

Transcriptomic Analysis of Genes Associated with Oxidative Stress in Chronic Rhinosinusitis Patients with Nasal Polyps: Identifying Novel Genes Involved in Nasal Polyposis.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a complicated inflammatory disease, and the underlying mechanism remains unclear. While some reactive oxygen/nitrogen species-related gene products are reported to participate in CRSwNP, a systemic and full analysis of oxidative-stress-associated genes in CRSwNP has not been extensively studied. Therefore, this study sought to catalog the gene-expression patterns related to oxidative stress and antioxidant defense in control and CRSwNP patients. In total, 25 control and 25 CRSwNP patients were recruited. The distribution and expression of 4-hydroxynonenal and 3-nitrotyrosine as markers of oxidative stress-which is represented by lipid peroxidation and the protein nitration of tyrosine residues in CRSwNP nasal polyps (NPs)-were more apparently increased than those found in the control nasal mucosae, as determined by immunohistochemistry (IHC). The expression of 84 oxidative-stress-related genes in nasal mucosae and NP tissues was analyzed via real-time PCR, which showed that 19 genes and 4 genes were significantly up- and downregulated, respectively; among them, inducible nitric oxide synthase (iNOS) and heme oxygenase 1 (HO-1) were notably upregulated, whereas lactoperoxidase (LPO), myeloperoxidase (MPO), and superoxide dismutase 3 (SOD3) were highly downregulated. Changes in the mRNA and protein levels of these redox proteins were confirmed with a customized, real-time PCR array and RT-PCR analysis, as well as Western blotting and IHC assays. A receiver operating characteristic curve analysis further suggested that LPO, MPO, SOD3, HO-1, and iNOS are possible endotype predictors of CRSwNP development. Collectively, we present an oxidative-stress-related gene profile of CRSwNP NP tissues, providing evidence that the systemic changes in oxidative stress and the antioxidative defense system, including novel iNOS, heme peroxidases, and other genes, are closely linked to CRSwNP pathology, development, and progression.

Naringenin induces endoplasmic reticulum stress-mediated cell apoptosis and autophagy in human oral squamous cell carcinoma cells.

Human oral squamous cell carcinoma (OSCC) has been one of the most common oral cancers owing to high percentage of betel nuts chewers, smokers, and alcohol consumption. With current treatment strategies in OSCC, more than half patients relapse and develop distant metastases with poor prognosis. To overcome the incident, OSCC poses a challenge in current therapies and treatments. Naringenin, a natural flavonoid, has been noted for antitumor effects on various types of cancers; however, the effects of naringenin on OSCC remain bias. In this study, naringenin demonstrated the potential multifunction in human OSCC cells not only leading to cell apoptosis, but also alternating the general function of autophagy, serving as pro-survival mechanism by inducing the endoplasmic reticulum (ER) stress signaling through intracellular reactive oxygen species (ROS) production. In the process of programmed cell death, naringenin induced apoptotic signaling through caspase-cascade, mitochondrial dysfunction, and ER stress by aberrance of Ca2+ release. In contrast, under the presence of naringenin, the pro-survival has been altered into pro-death to activate the caspases-mediated apoptosis achieving cell death. The cross-function of apoptosis and autophagy has demonstrated the effect of naringenin-induced intracellular ROS activity in OSCC cells. Therefore, this study found that the effect of naringenin induces intracellular ROS to trigger programmed cell death and ER stress through the mechanisms of apoptosis and autophagy in human oral squamous carcinoma. PRACTICAL APPLICATIONS: This study revealed that naringenin debilitated the OSCC cell viability via the intracellular ROS production, ER stress, and autophagy, leading to cell apoptosis. Based on these studies and findings, naringenin provided an antitumor effect as a novel natural compound to improve the current therapies in OSCC.

Endonasal Septoplasty Using a Septal Cartilaginous Batten Graft for Managing Caudal Septal Deviation.

Caudal nasal septal deviation is an important condition altering nasal obstruction and cosmetic appearance and many surgical techniques have been published on how to correct caudal septal deviation, as successful management of caudal septal deviation is challenging. The goal of our study was to explore the effect of endonasal septoplasty using a septal cartilaginous batten graft for managing caudal septal deviation. We tested 26 participants with caudal septal deviation who received endonasal septoplasty using a septal cartilaginous batten graft from 1 April 2019 to 29 June 2022, and followed up for at least 6 months. Nasal Obstruction Symptom Evaluation (NOSE) Scale and visual analog scale (VAS) were recorded at baseline, 1 month, and 6 months after surgery. Valid samples were analyzed by repeated measures ANOVA and paired sample t-test. Average participant age was 36.15 ± 11.02 years old. The preoperative, 1-month postoperative, and 6-month postoperative NOSE scale decreased significantly (75.38 ± 15.62, 13.85 ± 7.79, and 14.04 ± 9.90; p < 0.001), while preoperative, 1-month postoperative, and 6-month postoperative VAS (convex/concave side) also improved (7.50 ± 0.81/3.38 ± 0.94, 2.27 ± 0.53/1.54 ± 0.58, and 2.31 ± 0.55/1.58 ± 0.58; p < 0.001). Our results showed that endonasal septoplasty using a septal cartilaginous batten graft had good surgical outcomes without an open scar or severe complications.

Liver X Receptor Expression and Pentraxin 3 Production in Chronic Rhinosinusitis and Sinonasal Mucosal Fibroblast Cells.

The long pentraxin 3 (PTX3) is a prototypic molecule for recognizing pathogens. Liver X receptors (LXRs), belonging to nuclear receptors (NRs) for cholesterol metabolism through heterodimerizing with other NRs, were recently reported to participate in inflammation. However, their roles in chronic rhinosinusitis without nasal polyps (CRSsNP) are unclear. Therefore, this study was sought to explore roles of LXRs in chronic rhinosinusitis (CRS) sinonasal tissues and derived fibroblasts. Immunohistochemistry indicated that LXRα and ß expression and lipid/fat deposition were differentially expressed in the control and CRSsNP nasal mucosa. GW7647 (a peroxisome proliferator activated receptor α (PPARα) agonist) and GW3965 (a dual agonist for LXRα and ß) significantly caused PTX3 induction in the fibroblast cells. GW3965 induced PTX3 mRNA and protein expression, and the induction substantially led to PTX3 secretion. Meanwhile, an endogenous agonist-cholesterol had a similar enhancing effect on the induction of PTX3 protein. LXR siRNA knockdown to lower LXRα or ß expression significantly compromised PTX3 induction. Interestingly, GW3965 also induced phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) activation and its inhibition reduced PTX3 expression. Collectively, we demonstrated here for the first time that CRSsNP nasal mucosa differentially expresses LXRα and ß and deposits lipids/fats that may contain cholesterol metabolites to activate LXRs. Activation of LXRs leads to PTX3 production in sinonasal mucosa-derived fibroblasts. Our previous study showed PTX3 overexpression in the nasal cavity of CRSsNP, whereas this study highlights that cholesterol metabolites and LXR activation regulate PTX3 production and may contribute to antimicrobial activity and tissue repair during CRSsNP progression.

CPAP Treatment Improves Pure Tone Audiometry Threshold in Sensorineural Hearing Loss Patients with Sleep-Disordered Breathing.

This article investigates the effects of continuous positive airway pressure (CPAP) on hearing impairment in sensorineural hearing loss (SNHL) patients with sleep-disordered breathing (SDB). This retrospective and observational study took place from September 2016 to February 2021, accumulating 77 subjects with SNHL and SDB (60.7 ± 11.1 years). Of which, 28 received CPAP treatment (63.0 ± 8.5 years). In our methodology, hearing thresholds at low, medium, high, and average frequencies are assessed by pure-tone audiometry at baseline (BL), three (3 m), six (6 m), and 12 (12 m) months. Our results show that the BL of at least three frequencies in all subjects is positively associated with old age, males, smoking, alcohol, coronary artery disease, hypertension, and apnea-hypopnea index [AHI] (all p < 0.05). Moreover, low, medium, and average frequencies are negatively correlated at CPAP-6 m (-5.60 ± 2.33, -5.82 ± 2.56, and -5.10 ± 2.26 dB; all p < 0.05) and CPAP-12 m (-7.97 ± 2.74, -8.15 ± 2.35, and -6.67 ± 2.37 dB; all p < 0.01) against corresponding measures of CPAP-BL. High, medium, and average frequencies positively correlated with age (p < 0.001 for high and average frequencies and <0.01 for medium frequencies). We conclude that in SNHL patients with SDB, hearing thresholds at low and medium frequencies improves under CPAP use after six months, which persists at least to the end of one year.

Expression of long pentraxin 3 in human nasal mucosa fibroblasts, tissues, and secretions of chronic rhinosinusitis without nasal polyps.

Numerous studies have shown that microbiomes play an important role in the pathogenesis of chronic rhinosinusitis (CRS). In addition to a known short pentraxin, C-reactive protein, long pentraxin 3 (PTX3) belongs to pentraxin family which detects conserved microbial pentraxin motifs and mobilizes early defense against foreign invaders, but its participation in CRS remains unclear. In the present study, through an intensive screening, peptidoglycan (PGN) was selected as a main material to investigate the action mechanism of a cell wall component on CRS without nasal polyps (CRSsNP) nasal mucosa-derived fibroblasts and the PTX3 expression in human nasal mucosa tissue and discharge. The PGN not only enhanced PTX3 mRNA and protein production in cells but also caused marked PTX3 secretion into extracellular space. The pharmacological interventions indicated that the PTX3 induction was mediated mainly through toll-like receptor 2 (TLR2), phosphoinositide-phospholipase C (PI-PLC), protein kinase C (PKC), NF-κB, and cAMP response element binding protein (CREB), which was further confirmed by the observations that a direct PKC activator (phorbol ester) had a similar inductory effect on PTX3 expression/production and the siRNA interference knockdown of PKCµ/δ, NF-κB, and CREB compromised PTX3 production. Meanwhile, PTX3 was found to be overexpressed/produced in nasal mucosa and discharge/secretion of the CRSsNP patients. Collectively, we first demonstrated here that PGN enhances PTX3 expression and release in nasal fibroblasts through TLR2, PI-PLC, PKCµ/δ, NF-κB, and CREB signaling pathways. The PTX3 is overexpressed in nasal mucosa and discharge/secretion of CRSsNP patients, revealing its possible importance in CRSsNP development and progression. KEY MESSAGES: Long pentraxin 3 (PTX3) is highly expressed in nasal mucosa and discharge/secretion of patients of chronic rhinosinusitis without nasal polyps (CRSsNP). The bacteria cell wall component-peptidoglycan (PGN) causes PTX3 expression in CRSsNP nasal mucosa-derived fibroblasts, contributing to the PTX3 increase in tissues. PGN induces PTX3 expression through a previously known IκB/NF-κB and a novel PKCµ/δ and CREB signaling pathway. The PTX3 may be used as a biomarker for CRS.

Thrombin induces cyclooxygenase-2 expression and prostaglandin E2 release via PAR1 activation and ERK1/2- and p38 MAPK-dependent pathway in murine macrophages.

Thrombin levels increase at sites of vascular injury and during acute coronary syndromes. It is also increased several fold by sepsis with a reciprocal decrease in the anti-thrombin III levels. In this study we investigate the effects of thrombin on the induction of cyclooxygenase-2 (COX-2) and prostaglandin (PG) production in macrophages. Thrombin-induced COX-2 protein and mRNA expression in RAW264.7 and primary cultured peritoneal macrophages. A serine proteinase, trypsin, also exerted a similar effect. The inducing effect by thrombin in macrophages was not affected by a lipopolysaccharide (LPS)-binding antibiotic, polymyxin B, excluding the possibility of LPS contamination. The increase of COX-2 expression by thrombin was functionally linked to release of PGE(2) and PGI(2) but not thromboxane A(2) into macrophage culture medium. Thrombin-induced COX-2 expression and PGE(2) production were significantly attenuated by PD98059 and SB202190 but not by SP600125, suggesting that ERK1/2 and p38 MAPK activation were involved in this process. This was supported by the observation that thrombin could directly activate ERK1/2 and p38 MAPK in macrophages. A further analysis indicated that the proteinase-activated receptor 1 (PAR1)-activating agonist induced effects similar to those induced by thrombin in macrophages and the PAR1 antagonist-SCH79797 could attenuate thrombin-induced COX-2 expression and PGE(2) release. Taken together, we provided evidence demonstrating that thrombin can induce COX-2 mRNA and protein expression and PGE(2) production in macrophages through PAR1 activation and ERK1/2 and p38 MAPK-dependent pathway. The results presented here may explain, at least in part, the possible contribution of thrombin and macrophages in these pathological conditions.

Thromboxane A2 Regulates CXCL1 and CXCL8 Chemokine Expression in the Nasal Mucosa-Derived Fibroblasts of Chronic Rhinosinusitis Patients.

BACKGROUND: Chronic rhinosinusitis without nasal polyps (CRSsNP) is a common chronic disease and the etiology remains unclear. Thromboxane A2 (TXA2) participates in platelet aggregation and tissue inflammation. In this study, the CXCL1/8 chemokine and TXA2-TP receptor expression in the CRSsNP mucosa was investigated. EXPERIMENTAL APPROACH: Immunohistochemistry, chemokine release assay by ELISA, RT-PCR, Real-time PCR, Western blotting, pharmacological and siRNA knockdown analysis were applied in the CRSsNP tissue specimen and cultured nasal mucosa-derived fibroblasts. RESULTS: The immunohistochemistry results indicated that CXCL1 and CXCL8 were highly expressed in the CRSsNP mucosa compared with the controls; however, the TP receptors were expressed in both mucosa. Therefore, U46619 and IBOP, a TXA2 analog and TP agonist, were used to explore the role of TP activation in CXCL1/8 expression; both of these induced CXCL1/8 mRNA and protein expression in CRSsNP mucosa-derived fibroblasts. U46619 phosphorylated PI-3K, cyclic AMP (cAMP)/PKA, PKC, and cAMP response element (CREB). Activation of cAMP/PKA, PKC, and CREB was the major pathway for cxcl1/8 gene transcription. Pharmacological and siRNA knockdown analyses revealed that activation of cAMP/PKA and PKCµ/PKD pathways were required for CREB phosphorylation and PKA/C crosstalked with the PI-3K pathway. CONCLUSION AND IMPLICATIONS: Our study provides the first evidence for abundant TP receptor and CXCL1/8 expression in human CRSsNP mucosa and for TXA2 stimulation inducing CXCL1/8 expression in nasal fibroblasts primarily through TP receptor, cAMP/PKA, PKCµ/PKD, and CREB-related pathways.

Involvement of B2 receptor in bradykinin-induced proliferation and proinflammatory effects in human nasal mucosa-derived fibroblasts isolated from chronic rhinosinusitis patients.

Chronic rhinosinusitis (CRS) is a chronic inflammatory disease of the sinonasal mucosa either accompanied by polyp formation (CRSwNP) or without polyps (CRSsNP). CRSsNP accounts for the majority of CRS cases and is characterized by fibrosis and neutrophilic inflammation. However, the pathogenesis of CRS, especially CRSsNP, remains unclear. Immunohistochemistry of CRSsNP specimens in the present study showed that the submucosa, perivascular areas, and the mucous glands were abundant in fibroblasts. Therefore, we investigated the effects bradykinin (BK), an autacoid known to participate in inflammation, on human CRSsNP nasal mucosa-derived fibroblasts (NMDFs). BK increased CXCL1 and -8 secretion and mRNA expression with EC50 ranging from 0.15~0.35 µM. Moreover, BK enhanced cell proliferation and upregulated the expressions of proinflammatory molecules, including cell adhesion molecules (CAMs) and cyclooxygenase (COX)-1 and -2. These functionally caused an increase in monocyte adhesion to fibroblast monolayer. Using pharmacological intervention and BKR siRNA knockdown, we demonstrated that the BK-induced CXCL chemokine release, cell proliferation and COX and CAM expressions were mainly through the B2 receptor (B2R). Accordingly, the B2R was preferentially expressed in the NMDFs than B1R. The B2R was highly expressed in the CRSsNP than the control specimens, while the B1R and kininogen (KNG)/BK expression slightly increased in the CRSsNP mucosa. Collectively, we report here for the first time that fibroblasts, KNG/BK, and BKRs are overexpressed in CRSsNP mucosa and BK upregulates chemokine expression, proliferation, and proinflammatory molecule expression in NMDFs via B2R activation, which lead to a functional increase in monocyte-fibroblast interaction. Our findings reveal a critical role of fibroblast, KNG/BK, and BKRs in the development of CRSsNP.

Biofilm formations in nasopharyngeal tissues of patients with nasopharyngeal osteoradionecrosis.

OBJECTIVE: Determine the presence of nasopharynx biofilms in patients with nasopharyngeal cancer (NPC) and osteoradionecrosis (ORN) and patients with NPC but no ORN. STUDY DESIGN: Cross-sectional study. SETTING: Tertiary referral medical center. SUBJECTS AND METHODS: We enrolled 27 patients with NPC from our outpatient clinic during January 2010 to June 2012. These patients were diagnosed with NPC between 1980 and 2012, and all had received radiotherapy. Of these 27 patients, 15 presented with ORN, and 12 did not. The nasopharynx biopsied specimens were processed and analyzed within 2 hours of collection with the FilmTracer LIVE_DEAD Biofilm Viability Kit (Molecular Probes, Invitrogen, Carlsbad, California). A blinded investigator determined the formation of biofilms by fluorescence microscopy. Bacterial cultures were collected. RESULTS: Eleven of 15 (73%) ORN patients had biofilm formations in nasopharynx biopsy specimens. Five of these samples (45%) yielded positive cultures, and 4 of these cultures indicated the presence of methicillin-resistant Staphylococcus aureus (MRSA). Only 1 of 12 NPC patients without ORN had nasopharynx biofilm formation, and all culture results were negative. CONCLUSION: Biofilm formations were common in nasopharynx samples of NPC patients with ORN but rare in samples of NPC patients without ORN. The presence of biofilms, especially MRSA, may have a role in the disease progression of ORN or may contribute to the chronicity and resistance to antibiotic treatment.

Can pillar suturing promote efficacy of adenotonsillectomy for pediatric OSAS? A prospective randomized controlled trial.

OBJECTIVES/HYPOTHESIS: Pediatric obstructive sleep apnea syndrome (OSAS) is a common disorder with serious clinical implications if left untreated. The recommended initial treatment for pediatric patients with obstructive sleep apnea syndrome (OSAS) is adenotonsillectomy. However, recent reports have shown variable surgical results with adenotonsillectomy in the treatment of pediatric OSAS. STUDY DESIGN: Prospective, controlled study. METHODS: From April 2007 to August 2010, 24 participants were assigned alternatively to either adenotonsillectomy with pillar suturing (intervention group) or adenotonsillectomy alone (control group). RESULT: The average improvement in apnea hypopnea index (AHI) was 42.6% in the control group and 79.9% in the intervention group (P=0.037). The success rate was 50% in the control group and 91.6% in the intervention group (P=0.034). Six patients (50%) in the intervention group achieved complete resolution of pediatric OSAS, as defined by an AHI <1/hour, compared to 2 patients (16.7%) in the control group (P=0.097). CONCLUSION: Adenotonsillectomy with pillar suturing showed significant improvement in treating pediatric patients with OSAS compared to adenotonsillectomy alone. The procedure is simple and safe. LEVEL OF EVIDENCE: 4.

Systematic analysis of cephalometry in obstructive sleep apnea in Asian children.

OBJECTIVES/HYPOTHESIS: This study was designed to systematically analyze the relationship between a cephalometric analysis and the apnea-hypopnea index in a group of Asian children with obstructive sleep apnea. STUDY DESIGN: Retrospective study. METHODS: Data were collected from 56 children with obstructive sleep apnea who were between 3 and 13 years old. Each child underwent attended overnight polysomnography and cephalometry. We measured nine angles, 10 lines, and two ratios as well as adenoid size on each cephalometric film. Data included five segments of the upper airway: nasal cavity (segment 1), nasopharyngeal space (segment 2), retropalatal space (segment 3), retroglossal space and hyoid (segment 4), and oral cavity-related space (segment 5). RESULTS: Four cephalometric anthropomorphic findings (Gn-Go-H, MP-H, MPH/GnGo, Ad/Na) were related to the apnea-hypopnea index. Three of the four parameters belonged to segment 4, indicating the importance of hyoid position in pediatric obstructive sleep apnea. CONCLUSIONS: This study showed that segment 4 was the most important segment affecting the apnea-hypopnea index. Most of the cephalometric parameters in segment 4 did not show a difference from the results of Caucasian groups, except that mandibular length and position appeared to have more positive findings in the Caucasian results. In segment 2, the apnea-hypopnea index was less affected by the skull base-related parameters in our data. The reason why the other segments appeared to play a lesser role in pediatric obstructive sleep apnea might due to the limitations of two-dimensional imaging. Further cephalometric studies with anterior-posterior view and on the differences between Caucasian and Asian children are mandatory.