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A5825 - Cell Type Specific Activation of Heat Shock Proteins and Inhibition of Anti-Apoptotic Proteins May Explain the Beneficial Long Term Effect of Bronchial Thermoplasty
Author Block: L. Fang1, M. Tamm2, M. Roth1, D. Stolz3; 1Pneumology / Biomedicine, University Hospital of Basel, Basel, Switzerland, 2Univ Hosp Basel, 4031 Basel, Switzerland, 3University hospital of basel, Clinic of Pneumology and Respiratory Cell Research, 4031 Basel, Switzerland.
RATIONALE: Bronchial thermoplasty (BT) is the only therapy that improves life quality of patients with uncontrolled severe asthma. BT locally heats (65oC) airway tissue by radiofrequency, which results in reduced sub-epithelial mesenchymal cell numbers and re-epithelization. However, the beneficial lasting effect of BT is not sufficiently explained by the ‘burning out smooth muscle’ hypothesis. This study assessed cell type specific effects of experimental BT on primary human airway epithelial and sub-epithelial mesenchymal cells
METHODS: Human airway epithelial and mesenchymal cells were isolated and expanded from endoscopic bronchial tissue biopsies from patients with severe asthma (n=15). Cells were characterized by specific immunocytochemistry: (i) E-cadherin and cytokeratin for epithelial cells, and (ii) fibronectin and α-smooth muscle actin (α-SMA) for mesenchymal cells. BT was mimicked in vitro by submerging sub-confluent cells into a water bath pre-heated to 60-65°C for 10 seconds, followed by cooling the cells to 37oC immediately. Cells at 37oC served as control. Cells were kept at 37oC for up to 48 hours, and cell culture medium, protein and mRNA were collected prior to treatment, as well as 24 and 48 hours after treatment. Cell proliferation was determined by double blind manual cell counting. Heat stress response was evaluated by western-blotting for heat shock protein (HSP) 40, HSP60, HSP70, and HSP90, as well as by quantitative immunofluorescence microscopy. Expression of apoptosis regulating proteins (IXAP, cleaved caspase-3, annexin-V) was examined by western-blotting and immune-fluorescence microscopy.
RESULTS: In epithelial cells, experimental BT significantly increased proliferation, while mesenchymal cell proliferation was stopped. Western-blot analysis demonstrated significant increased expression of HSP40, HSP60, HSP70 and HSP90 after 24h and 48h. In contrast, no HSP was significantly up-regulated in mesenchymal cells. Immunofluorescence microscopy confirmed the cell type specific effect. The apoptotic analysis indicated that heat stress had less effect in epithelial cells, while apoptosis was significantly up-regulated in mesenchymal cells.
CONCLUSIONS: Cell type specific heat response in airway epithelial cells versus mesenchymal cells may explain the mechanisms by which BT achieves its beneficial long-term effects in patients with severe asthma. Beside other functions, increased expression of HSP will help epithelial cells to survive heat stress by BT and support re-epithelization by increased proliferation. The mesenchymal cell specific down-regulation of anti-apoptotic protein after heat stress may result in reduced numbers of airway smooth muscle cells and fibroblasts. Further in vivo studies are required to confirm if this effect of heat stress occur after BT in asthmatic airways.