Implications And Mechanisms Of Epithelial Mesenchymal Transition (EMT) In Chronic Rhinosinusitis

Part I Implication of epithelial mesenchymal transition in mucous membrane of nasal cavity/sinus in patients with chronic rhinosinusitisObjectives:To detect the expression of epithelial mesenchymal transition (EMT) related markers in the mucous membrane of nasal cavity/sinus in patients with chronic rhinosinusitis (CRS) and to elucidate the implications of EMT in CRS patients.Methods:A total of39patients were recruited, including13CRS patients with NP (CRSwNP),13CRS patients without NP (CRSsNP) and13non-CRS patients. Gene expression levels of alpha smooth muscle actin (a-SMA), transforming growth factor beta1(TGF-(31), matrix metalloproteinase2(MMP-2), matrix metalloproteinase9(MMP-9), E-cadherin, fibronectin and vimentin were determined in mucous membrane samples of39patients by Real-time PCR. Protein levels of the EMT related markers were detected by Western blot and they were localized by immunofluorescence staining, respectively.Results:Compared with non-CRS patients, mRNA levels of a-SMA, TGF-β1, E-cadherin were elevated selectively in the CRSsNP group (P=0.032; P=0.048; P=0.001), whereas mRNA levels of a-SMA, vimentin were decreased in the CRSwNP Group (P=0.001; P=0.011). The protein levels of a-SMA, TGF-β1, E-cadherin, MMP-9increased in the CRSsNP group as compared with non-CRS controls (P<0.001; P<0.001; P=0.006; P=0.033). Co-expression of a-SMA and cytokeratin was confirmed in the epithelial layer of mucous membrane in patients with CRSsNP by immunofluorescence staining. There was a positive correlation between mRNA level of a-SMA and TGF-β1, fibronectin, vimentin (r=0.443, P=0.005; r=0.596, P=0.000; r=0.501, P<0.001), as well as protein levels of a-SMA and TGF-β1, MMP-9, E-cadherin, fibronectin (r=0.918, P=0.000; r=0.415, P=0.009; r=0.395, P=0.013; r=0.486, P=0.002). Conclusion:Elevated a-SMA expression was observed in mucous membrane of CRSsNP patients, which is positively correlated with elevated TGF-β1and fibronectin expression in such cases. Part II The relationships of EMT and CRS and the influence of EMT on the prognosis of CRS after endoscopic sinus surgeryObjectives:To investigate the relationship of EMT and CRS and to evaluate the influence of EMT on the prognosis of CRS after endoscopic sinus surgery (ESS).Methods:Of the39CRS patients in part I,26cases underwent further investigations. Clinical information including disease duration, sinus surgery history, smoking, co-existence of allergic rhinitis, asthma, aspirin sensitivity and gastroesophageal reflux disease were collected. Visual analogue scale (VAS), rhinosinusitis outcome measure-31(RSOM-31) symptom score, and Lund-Kennedy endoscopy score were assessed pre-and post-operation. Sinus CT scan was scored according to Lund-Mackay scoring system before surgery. The correlations of a-SMA protein level and all the clinical information, symptom scores, endoscopic scores, CT scores were analyzed pre-operation to determine the relationship of EMT and CRS. The correlation of a-SMA level and post-operative symptom scores, endoscopic scores were analyzed to evaluate the influence of EMT on the prognosis of CRS after ESS.Results:Except for smoking (r=-0.402, P=0.042), disease duration, sinus surgery history, co-existence of allergic rhinitis, asthma, aspirin sensitivity, gastroesophageal reflux disease couldn’t affect EMT. Serum total IgE showed the tendercy of negative correlation with protein level of a-SMA. The CT score of anterior ethmoid was negatively correlated with the protein level of a-SMA (r=-0.389,P=0.049). All the pre-operative symptoms were not related to EMT, while the hyposmia, hearing loss, fatigue, reduced productivity after surgery were negatively correlated with the protein level of α-SMA (r=-0.472, P=0.015; r=-0.413,P=0.036; r=-0.537, P=0.005; r=-0.420, P=0.033). Both pre-and post-operative endoscopic scores were negatively correlated with a-SMA protein level (r=-0.408,P=0.038; r=-0.640, P<0.001).Conclusion:CRS involves other sinuses but antierior ethmoid may tend to undergo EMT. EMT was not related any of the symptoms before surgery, but it was negatively correlated to the post-operative hyposmia, hearing loss, fatigue, reduced productivity, pre-and post-operative endoscopic signs. Part Ⅲ Establishment of Primary Epithelial Cellular Model of patients with CRSObjectives:To establish a primary epithelial cellular model of patients with CRS.Methods:Nasal polyps, middle turbinate or uncinate processes were obtained from CRS patients during the sinus surgery. Cells digested from the tissue samples were purified and cultured using two different methods:differential adhesion+trypsin digestion, magnetic activated cell sorting (MACS). The epithelial purity of cultured cells was tested by immunofluorescence staining and flow cytometry technology.Results:For the cells cultured using the meshod of differential adhesion+trypsin digestion, the purity of epithelial cells was85.6%determined by immunofluorescence staining,78.37%by flow cytometry technology. For the cells cultured using the meshod of MACS, the purity of epithelial cells was89.3%determined by immunofluorescence staining,92.12%by flow cytometry technology.Conclusion:We have established a primary epithelial cellular model of patients with CRS, which laid the foundation for further functional studies. Part Ⅳ EMT induced by TGF-β1on primary epithelial cellular model of patients with CRSObjectives:To investigate the role of EMT in mucous membrane of CRS patients.Methods:The study subject in this part was the primary epithelial cellular model of patients with CRS established in part Ⅲ. After induction of TGF-β1. primary epithelial cells were investigated by immunofluorescence staining for both a-SMA and cytokeratin and western blot for both a-SMA and E-cadherin. Scratch experiment and invision experiment were conducted to determine the transfer and invision ability of the cells induced by TGF-β1respectively.Results:The cells induced by TGF-β1underwent morphological changes and immunofluorescence staining showed combination of a-SMA and cytokeratin. The protein level of a-SMA in cells induced by TGF-β1increased compared with the primary epithelial cell, and the level of E-cadherin decreased. The cells induced by TGF-β1could move faster but showed no invasion ability.Conclusion:The primary epithelial cells of patients with CRS can transfer to mesenchymal cells under the induction of TGF-β1, which testified the implications of EMT in mucous membrane of patients with CRS.