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Airway Epithelial Repair: Cell Subtypes or Proliferation/Differentiation Dependent?
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A1275 - Airway Epithelial Repair: Cell Subtypes or Proliferation/Differentiation Dependent?
Author Block: G. K. Singhera, T. Guo, K. Yamasaki, D. D. Sin, D. R. Dorscheid; Centre for Heart Lung Innovation, University of British Columbia, Vancouver, BC, Canada.
RATIONALE: The airway epithelium is the first line of defense against environmental exposures. The injury-repair mechanisms depend primarily on migration, proliferation and the differentiation potential of airway epithelial cells (AEC) in particular non-ciliated AEC (basal cells) as a subset are thought to be the pluripotent stem cells that affect repair. Aging and the specific micro-environment that AEC are exposed to impact their reparative potential. Cigarette smoke (CS) can cause basal cell hyperplasia and affect the cilia number and length, as well as the expression of repair markers such as EGFR. The goal of this project is to characterize the AEC cellular profile from individuals with COPD and healthy controls using bronchial brushings and bronchial alveolar lavage (BAL).
HYPOTHESIS: The proportion of basal cells within the airway epithelium and their proliferation potential will be altered in COPD. Exposure to a smoking micro environment will reduce airway epithelium differentiation and repair potential.
METHODS: BAL and bronchial brushing samples were collected to either produce cytospins or to culture AEC, respectively. May-Grunwald Giemsa stained cytospins were analyzed to facilitate differential cell count by two blinded independent observers. Epithelial cell subtypes as a fraction were calculated as a percentage of ciliated and non-ciliated cells within a total of 300 AEC counted per slide. Unstained cytospins and first passage epithelial cells grown on chamber slides were used for immunofluorescence detection of the proliferation marker Ki67 and the basal cell specific marker CK5. Percentage of Ki67 and CK5 positive cells was determined after counting 300 total airway epithelial cells.
RESULTS: Cytospins made from BAL samples from control and COPD patients demonstrated significantly less non-ciliated AEC and more ciliated AEC in the COPD group relative to the Control group. Expression of basal cell specific marker (CK5) and the proliferation marker (Ki67) were both reduced in COPD samples compared to Control. These differences from Control were demonstrated for both BAL-cytospins and first passage cultured AEC.
CONCLUSION: Changes in the proportion of non-ciliated epithelial cells subset may alter the repair and differentiation potential for the airway epithelium. This could impact airway reparative and regenerative potential and the altered AEC subtypes may contribute to and affect regeneration of intact epithelial layer and barrier function. New therapeutic targets will need to be identified to address this.
Author Block: G. K. Singhera, T. Guo, K. Yamasaki, D. D. Sin, D. R. Dorscheid; Centre for Heart Lung Innovation, University of British Columbia, Vancouver, BC, Canada.
RATIONALE: The airway epithelium is the first line of defense against environmental exposures. The injury-repair mechanisms depend primarily on migration, proliferation and the differentiation potential of airway epithelial cells (AEC) in particular non-ciliated AEC (basal cells) as a subset are thought to be the pluripotent stem cells that affect repair. Aging and the specific micro-environment that AEC are exposed to impact their reparative potential. Cigarette smoke (CS) can cause basal cell hyperplasia and affect the cilia number and length, as well as the expression of repair markers such as EGFR. The goal of this project is to characterize the AEC cellular profile from individuals with COPD and healthy controls using bronchial brushings and bronchial alveolar lavage (BAL).
HYPOTHESIS: The proportion of basal cells within the airway epithelium and their proliferation potential will be altered in COPD. Exposure to a smoking micro environment will reduce airway epithelium differentiation and repair potential.
METHODS: BAL and bronchial brushing samples were collected to either produce cytospins or to culture AEC, respectively. May-Grunwald Giemsa stained cytospins were analyzed to facilitate differential cell count by two blinded independent observers. Epithelial cell subtypes as a fraction were calculated as a percentage of ciliated and non-ciliated cells within a total of 300 AEC counted per slide. Unstained cytospins and first passage epithelial cells grown on chamber slides were used for immunofluorescence detection of the proliferation marker Ki67 and the basal cell specific marker CK5. Percentage of Ki67 and CK5 positive cells was determined after counting 300 total airway epithelial cells.
RESULTS: Cytospins made from BAL samples from control and COPD patients demonstrated significantly less non-ciliated AEC and more ciliated AEC in the COPD group relative to the Control group. Expression of basal cell specific marker (CK5) and the proliferation marker (Ki67) were both reduced in COPD samples compared to Control. These differences from Control were demonstrated for both BAL-cytospins and first passage cultured AEC.
CONCLUSION: Changes in the proportion of non-ciliated epithelial cells subset may alter the repair and differentiation potential for the airway epithelium. This could impact airway reparative and regenerative potential and the altered AEC subtypes may contribute to and affect regeneration of intact epithelial layer and barrier function. New therapeutic targets will need to be identified to address this.
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