Effect Of House Dust Mites On Human Bronchial Epithelial Permeability And Airway Smooth Muscle Cells Migration

Objective:Asthma is a complex chronic respiratory disease lead by genetic and environmental factors. A number of clinical studies have shown that although most of correctly diagnosed patients after standard treatment have an effective symptom controlled, but it can not reverse or stop the natural history of asthma, and ultimately leads to irreversible airway structural changes. The airway epithelium paly an important role in asthma. Airway epithelial injury, desquamation and subepithelial fibrosis are characteristic features of asthmatic airway. There is immune imbalance in asthma, variety cytokines involved in the onset and development of asthma. Some studies have shown that vascular endothelial growth factor (VEGF) plays an important role in the pathophysiology of asthma. NHBE is one of major source of VEGF. At different treatment, VEGF expression from NHBE is different. It has been found that the number of myofibroblasts located subjacent to sub-epithelial basement membrane is increased in asthmatic airway, and significantly correlated with the thickness of the lamina reticularis. The main feature of the sub-epithelial myofibroblasts is represented by the expression of α-smooth muscle actin (a-SMA),which makes them with contractility and an increased collagen synthetic activity. The origin of the sub-epithelial myofibroblasts remains unclear. There are many triggering factors of asthma, studies have shown that house dust mite is the most common allergens in natural, house dust mites exposure in patients with asthma were significantly higher than other groups. To explore the effect of HDM on NHBE permeability and VEGF expression and if airway smooth muscle cells have an ability to migrate towards sub-epithelial basement membrane under the stimulation of the injured airway epithelial cells, we established the cell model stimulited by HDM. And we established a model of airway smooth muscle cells co-cultured with injured airway epithelial cells to investigate the possibility that injured airway epithelial cells induce migration of airway smooth muscle cells, and study its mechanism and medicinal targets involved in this process.Methods:1. Primary culture of human bronchial epithelium.2. Primary culture of airway smooth muscle cells by explant culture.3. Investigation the effect of HDM on VEGF expression from NHBE and NHBE permeability. We estimated HDM induced VEGF release in NHBE by using an ELISA and VEGF mRNA expression by using an RT-PCR reaction. The influence of HDM on NHBE barrier function was estimated by measuring electrical resistance with an electric cell substrate impedance-sensing system.4. Investigation the effects of injured bronchial epithelial cells on the migration of airway smooth muscle cells.Using co-culture instrument and fluorescence microscope to examine the effects of injured bronchial epithelial cells on the migration of airway smooth muscle cells. Measurement of cytokine levels in injured bronchial epithelial cells by ELISA.5. SPSS 13.0 analysis statistical software was used for date analysis. Data was expressed as mean±SD, One-way analysis of variance (one-way ANOVA) was used to compare the overall mean when the variance was Homogeneity, and LSD method was used for Multiple comparisons among the groups; when the variance was not Homogeneity, Welkch method was used to compare the overall mean, Tamhane’s was used for Multiple comparisons among the groups. Significance was accepted when p< 0.05.Result:1. We developed a model of primary NHBE.2. We developed a model of primary ASMC.3. Our results demonstrate that HDM induces VEGF release in NHBE in a time-dependent and concentration-dependent manner. HDM decreased electrical resistance across NHBE monolayers. Neutralizing antibodies to VEGF significantly inhibited the decrease in NHBE electrical resistance caused by HDM.4. ASMC migration was significantly increased by NHBE treated with HDM (P< 0.05). The neutralizing Ab to VEGF significantly attenuated the chemotactic effect of HDM-treated epithelial cells on the co-cultured ASMC.Conclusions:1. We have succeeded in establishment of a model of co-culture with primary airway smooth muscle cells and primary airway epithelial cells treated with HDM.2. HDM increases permeability of bronchial epithelial monolayer via VEGF induction.3. With HDM stimulation, injured NHBE can induce co-cultured ASMC migration toward the injured NHBE. HDM can induce NHBE to produce VEGF leading to the migration of ASMC. The ASMC migration induced by injured epithelial cells might participate in the resource of myofibroblasts in the lamina reticularis of asthmatic airway, contributing to airway remodeling in relation to airway hyperresponsiveness in asthma.