Matrix metalloproteinase-7 induces E-cadherin cleavage in acid-exposed primary human pharyngeal epithelial cells via the ROS/ERK/c-Jun pathway.

Laryngopharyngeal reflux disease (LPRD) is caused by pharyngeal mucosal damage due to the reflux of gastric contents, including acid, pepsin, and bile juice. Our previous study has demonstrated that LPRD is associated with the cleavage of E-cadherin, which is facilitated by the acid-activated matrix metalloproteinase-7 (MMP-7); however, the mechanism by which the acid activates MMP-7 remains unclear. The purpose of this study was to investigate the mechanism by which MMP-7 is activated in the pharyngeal epithelial cells that are exposed to acid. The levels of reactive oxygen species (ROS) were measured in the epithelial cells exposed to acid. To investigate the signaling mechanism of ROS in the expression of MMP-7, the mechanism of action of the mitogen-activated protein kinase was examined. The expression of various signaling factors was determined, according to the presence or absence of each inhibitor in the acid-exposed pharyngeal epithelial cells. To identify changes in the cleavage of E-cadherin, the integrity of the mucosal membrane was assessed using a transepithelial permeability test. We found that acid exposure increased the levels of ROS, phosphorylated-extracellular signal-regulated kinase (p-ERK) 1/2, and phosphorylated-c-Jun (p-c-Jun) in pharyngeal epithelial cells. The ROS inhibitor reduced the expression of p-ERK and MMP-7, while the ERK inhibitor reduced the expression of p-c-Jun and MMP-7. Moreover, the c-Jun inhibitor reduced the expression of MMP-7 and blocked the degradation of E-cadherin. In addition, decrease in the levels of immunostained E-cadherin and increase in transepithelial permeability after acid exposure were collectively alleviated by the inhibitors of ROS, ERK, and c-Jun. The degradation of E-cadherin that occurs after human mucosal cells are exposed to acid appears to be caused by an increase in the expression of MMP-7 via the ROS/ERK/c-Jun pathway, which is thought to be an important mechanism associated with the development of LPRD. KEY MESSAGES: • ROS is triggered when reflux occurs. • ROS regulates the transcription factor c-Jun via the ERK pathway. • The increase in MMP-7 that induces LPRD is induced via the ROS/ERK/c-Jun pathway. • This study revealed for the first time the expression mechanism of MMP-7, which is one of the causes of LPRD.

Usefulness of matrix metalloproteinase-7 in saliva as a diagnostic biomarker for laryngopharyngeal reflux disease.

Several diagnostic methods are currently being used to diagnose LPRD (laryngopharyngeal reflux disease), but have the disadvantage of being invasive, subjective, or expensive. Our purpose in this study was to investigate the correlation between pepsin and MMP-7 (Matrix Metalloproteinase-7) in pharyngeal secretions of subjects according to RSI (Reflux Symptom Index) score to find out the diagnostic value of MMP-7. We recruited 173 subjects aged between 19 and 85 years who completed the RSI scale. All samples were taken after waking up, and the amount of the pepsin and MMP-7 in saliva were measured by means of an enzyme activity assay. There was a significant increase of pepsin and MMP-7 activity in the study group with an RSI score of 13 or higher. The sensitivity and specificity of MMP-7 for predicting the possibility of an RSI of 13 or more was higher than that of pepsin. When MMP-7 and pepsin were combined, this sensitivity and specificity increased. An enzyme assay of MMP-7 in saliva may be a noninvasive and easy technique for diagnosing LPRD.

Application of M1 macrophage as a live vector in delivering nanoparticles for in vivo photothermal treatment.

Introduction: To enhance photothermal treatment (PTT) efficiency, a delivery method that uses cell vector for nanoparticles (NPs) delivery has drawn attention and studied widely in recent years. Objectives: In this study, we demonstrated the feasibility of M1 activated macrophage as a live vector for delivering NPs and investigated the effect of NPs loaded M1 stimulated by Lipopolysaccharide on PTT efficiency in vivo. Methods: M1 was used as a live vector for delivering NPs and further to investigate the effect of NPs loaded M1 on PTT efficiency. Non-activated macrophage (MФ) was stimulated by lipopolysaccharide (LPS) into M1 and assessed for tumor cell phagocytic capacity towards NPs. Results: We found M1 exhibited a 20-fold higher uptake capacity of NPs per cell volume and 2.9-fold more active infiltration into the tumor site, compared with non-activated macrophage MФ. We injected M1 cells peritumorally and observed that these cells penetrated into the tumor mass within 12 h. Then, we conducted PTT using irradiation of a near-infrared laser for 1 min at 1 W/cm2. As a result, we confirmed that using M1 as an active live vector led to a more rapid reduction in tumor size within 1 day indicating that the efficacy of PTT with NPs-loaded M1 is higher than that with NPs-loaded MФ. Conclusion: Our study demonstrated the potential role of M1 as a live vector for enhancing the feasibility of PTT in cancer treatment.

Non-surgical animal model of gastroesophageal reflux disease by overeating induced in mice.

Previous animal models of gastroesophageal reflux disease (GERD) were not physiological and required a variety of surgical procedures. Therefore, the animal model developed by conditions that are similar to the pathogenesis of GERD is necessary. The aim is to establish a non-surgical animal model with GERD caused by overeating induced in mice. To induce mice to overeat, we designed dietary control protocols including repetitive fasting and feeding. The esophageal tissues were evaluated with GERD markers to prove the establishment of a GERD animal model. Mice fasted every other day (group 2) showed more pronounced overeating feature and demonstrated evident changes similar to the macroscopic and microscopic findings of GERD, the expressions of inducible nitric oxide synthase and substance P were stronger. The higher frequency of fasting and overeating could cause GERD effectively. The dietary control can make mice overeat, which elicits the change of lower esophageal mucosa similar to GERD. Thus, the overeating-induced mouse may be used as a GERD mouse model.

Actin-Associated Gene Expression is Associated with Early Regional Metastasis of Tongue Cancer.

OBJECTIVES: We aimed to analyze gene expression profile of tongue cancer associated with early lymph node metastasis using the cancer genome atlas (TCGA) data. STUDY DESIGN: Basic research. METHODS: A total of 515 patients with matched RNAseq data of primary tumor and clinical data from TCGA data were extracted. To compare gene expression profile between early T-stage tongue cancer with cervical lymph node metastasis and late T-stage tongue cancer without cervical metastasis, genomic data of following two groups was assessed; 1) group 1: T1/2 and N2/3 (n = 41), 2) group 2: T4 and N0 (n = 65). Using R and limma package in bioconductor program, differentially expressed genes (DEGs) were extracted. Gene ontology and pathway enrichment analysis were performed using the DAVID online tool. FFPE tissue of 285 patients were evaluated for the validation of relevant genes by imunofluorescence (IF) and immunohistochemical (IHC) stain. RESULTS: A total of 225 DEGs were found, and 50 genes were highly significant with absolute fold change over eight. Gene ontology and pathway enrichment analysis revealed that most of the upregulated genes were associated with actin cytoskeleton and included following genes: ANKRD23, NO3, PDLIM3, MUSTN1, TNNT3, MYBPC1, MB, MYH3, TTN, ACTA1, and ACTC1. When comparing tongue cancer with cN0pN0 vs. pN0pN+ using the total tongue cancer cohort of TCGA, ACTA1 was the only parameter which was associated with hidden lymph node metastasis in T1/2 (P = .019). Perineural invasion was significantly associated with high expression of ACTA1 (P < .001). IF and IHC analysis revealed that actin was overexpressed, while E-cadherin and N-cadherin were not significantly different. CONCLUSIONS: Actin associated genes, especially overexpression of ACTA1 may be associated with early regional metastasis of tongue cancer. LEVEL OF EVIDENCE: 3 Laryngoscope, 131:813-819, 2021.

Enhancement of aberrantly modified integrin­mediated cell motility in multicellular tumor spheroids.

Multicellular tumor spheroids (MTSs) of malignant cells can display cell­cell and cell­matrix interactions, different from monolayer cultures. The objective of the present study was to examine difference in intercellular and cell­matrix interaction between monolayered cultures and spheroid cultures. Expression levels of cell adhesion molecules (CAMs) and epithelial­mesenchymal transition (EMT) signaling molecules in monolayered cells and MTS cells were compared. The motility of single cells dispersed from each culture was evaluated using a live­cell imaging device. The effect of an E­cadherin neutralizing antibody, DECMA, was also compared between the two cultures. Among various CAMs, only E­cadherin was increased in MTSs. The motility of single cells dispersed from MTSs was higher than that from monolayered cells. Compared with monolayered cells, the molecular weight (MW) of ß1 integrin was decreased during MTS formation, particularly during the early stage. This notable reduction was maintained when DECMA was used to treat MTSs. Additionally, the expression levels of the EMT signaling molecules Snail and ILK increased more in MTSs than in monolayered cells. The blocking of E­cadherin elicited increased expression levels of EMT molecules and cellular motility only in MTSs. In conclusion, the alteration of E­cadherin expression and presence of low­MW ß1 integrin in MTS may enhance cell motility via the upregulation of EMT signaling molecules that may be intensified by blocking E­cadherin.

Role of Matrix Metalloproteinases 7 in the Pathogenesis of Laryngopharyngeal Reflux: Decreased E-cadherin in Acid exposed Primary Human Pharyngeal Epithelial Cells.

Cleavage of E-cadherin and the resultant weakness in the cell-cell links in the laryngeal epithelium lining is induced by exposure to acidic contents of the refluxate. Herein, we aimed to evaluate the role of matrix metalloproteinases (MMPs) in inducing E-cadherin level changes following acid exposure to the human pharyngeal mucosal cells. E-cadherin levels were inversely correlated with the duration of acid exposure. Treatment with actinonin, a broad MMP inhibitor, inhibited this change. Immunocytochemical staining and transepithelial permeability test revealed that the cell surface staining of E-cadherin decreased and transepithelial permeability increased after acid exposure, which was significantly inhibited by the MMP inhibitor. Among the various MMPs analyzed, the mRNA for MMP-7 in the cellular component was upregulated, and the secretion and enzymatic activity of MMP-7 in the culture media increased with the acid treatment. Consequently, MMP-7 plays a significant role in the degradation of E-cadherin after exposure to a relatively weak acidic condition that would be similar to the physiologic condition that occurs in Laryngopharyngeal reflux disease patients.

Decreased expression of CCL17 in the disrupted nasal polyp epithelium and its regulation by IL-4 and IL-5.

BACKGROUND: In airway epithelium, thymus and activation-regulated chemokine (CCL17) and macrophage-derived chemokine (CCL22) are induced by defective epithelial barriers such as E-cadherin and attract the effector cells of Th2 immunity. However, the association between the epithelial barrier and CCL17 expression has not been studied in chronic rhinosinusitis with nasal polyp (CRSwNP). Thus, we aimed to evaluate the expression of CCL17 and its regulation by Th cytokines in nasal polyp (NP) epithelial cells. METHODS: The expression and distribution of CCL17, CCL22, E-cadherin and/or epidermal growth factor receptor (EGFR) were measured using real-time PCR, western blot, and immunohistochemistry and compared between normal ethmoid sinus epithelium and NP epithelium. In addition, the expression level of CCL17 was determined in cultured epithelial cells treated with IL-4, IL-5, IL-13, TNF-α, and IFN-γ. RESULTS: The expression of CCL17 was decreased in the NP epithelium compared to the epithelium of normal ethmoid sinus, whereas the expression of CCL22 was not decreased. E-cadherin was differentially distributed between the epithelium of normal ethmoid sinus and NP epithelium. EGFR was also decreased in NPs. Interestingly, the stimulation of cultured epithelial cells with Th2 cytokines, IL-4 and IL-5, resulted in an upregulation of CCL17 expression only in NP epithelial cells whereas the expression of CCL17 was increased in both normal epithelial cells and NP epithelial cells by Th1 cytokines. CONCLUSION: Our results suggest that the decreased expression of CCL17 in defective NP epithelium may be closely connected to NP pathogenesis and can be differentially regulated by cytokines in the NP epithelium of patients with CRSwNP.

Effect of matrix metalloproteinase inhibitor on disrupted E-cadherin after acid exposure in the human nasal epithelium.

OBJECTIVE: Laryngopharyngeal reflux disease (LPRD) is one of potential factors in recalcitrant chronic rhinosinusitis with or without polyps. An increase in junctional permeability in the nasal mucosa in LPRD may be due to disrupted protein bridge formation with cell-to-cell adhesion molecules such as E-cadherin. Despite the relationship between nasal mucosal inflammation and LPRD, the clear mechanism by which acid reflux affects the nasal epithelium remains unclear. METHODS: The expression levels and distribution patterns of E-cadherin in primary culture of nasal epithelial cells after acid exposure with or without dexamethasone and matrix metalloproteinase (MMP) inhibitor were determined using Western blot and immunocytochemistry. The functional roles of MMP inhibitor in maintaining junctional permeability in the nasal epithelium were elucidated by transepithelial permeability test. RESULTS: By acid exposure to nasal epithelial cells, mature E-cadherin was decreased and cleaved E-cadherin was increased. This was thought to be caused by cleavage of mature E-cadherin between cells and was confirmed by the increment of E-cadherin inside a cell in immunocytochemical evaluation. Whereas disruption of E-cadherin was not recovered by steroid medication with various treatments of dexamethasone, disrupted E-cadherin was restored to normal by inhibition of MMPs with actinonin, a broad MMP inhibitor. This recovery was functionally demonstrated by transepithelial permeability test. CONCLUSION: Our results suggest that altered expression of E-cadherin in the nasal epithelium by acid exposure may be a possible mechanism for nasal tissue injury in chronic nasal inflammation with LPRD, and that MMP inhibition is a potential treatment. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:E1-E7, 2018.

In vivo photothermal treatment with real-time monitoring by optical fiber-needle array.

Photothermal treatment (PTT) using gold nanoshells (gold-NSs) is accepted as a method for treating cancer. However, owing to restrictions in therapeutic depth and skin damage caused by excessive light exposure, its application has been limited to lesions close to the epidermis. Here, we demonstrate an in vivo PTT method that uses gold-NSs with a flexible optical fiber-needle array (OFNA), which is an array of multiple needles in which multimode optical fibers are inserted, one in each, for light delivery. The light for PTT was directly administrated to subcutaneous tissues through the OFNA, causing negligible thermal damage to the skin. Enhancement of light energy delivery assisted by the OFNA in a target area was confirmed by investigation using artificial tissues. The ability of OFNA to treat cancer without causing cutaneous thermal damage was also verified by hematoxylin and eosin (H&E) staining and optical coherence tomography in cancer models in mice. In addition, the OFNA allowed for observation of the target site through an imaging fiber bundle. By imaging the activation of the injected gold-NSs, we were able to obtain information on the PTT process in real-time.

Decreased expression of E-cadherin and ZO-1 in the nasal mucosa of patients with allergic rhinitis: Altered regulation of E-cadherin by IL-4, IL-5, and TNF-alpha.

BACKGROUND: Allergic rhinitis is a chronic nasal inflammatory disease mediated by an immunoglobulin E mediated process to environmental allergens. Although atopy is a potent predisposing risk factor for allergic rhinitis, local tissue susceptibilities are inevitable for disease expression. The nasal epithelium maintains tissue homeostasis by providing a physical barrier controlled by epithelial junctional proteins. However, the expression of epithelial junctional proteins has not been studied in patients with allergic rhinitis. We sought to elucidate the expression and the regulation of epithelial junctional proteins in the nasal epithelium of patients with allergic rhinitis. METHODS: The expression of E-cadherin and zonula occludens (ZO) 1 in epithelium of turbinate was measured by using real-time polymerase chain reaction, Western blot, and immunohistochemical assays, and was compared between control subjects and patients with allergic rhinitis. In addition, the expression levels of E-cadherin and ZO-1 were determined in cultured epithelial cell treated with interleukin (IL) 4, IL-5, tumor necrosis factor (TNF) alpha, and interferon gamma. RESULTS: The expression and the immunoreactivity of E-cadherin and ZO-1 were decreased in the nasal epithelium of patients with allergic rhinitis. Interestingly, the stimulation of cultured epithelial cells with IL-4, IL-5, and TNF-alpha resulted in downregulation of E-cadherin expression only in cultured epithelial cells of patients with allergic rhinitis, whereas E-cadherin expression in cultured epithelial cells of controls was not affected by stimulation with the same panel of cytokines. CONCLUSION: Decreased expression of epithelial junctional proteins was found in patients with allergic rhinitis. The disruption of epithelial integrity by IL-4, IL-5, and TNF-alpha in vitro indicated a possible role for these cytokines in the pathogenesis of patients with allergic rhinitis.