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Title: The expression and function of the asthma susceptibility gene, a Disintegrin and Metalloprotease (ADAM) 33, in airways of normal and asthmatic subjects and in developing lungs
Author: Haitchi, Hans Michael
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2008
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Asthma affects 1 in 12 adults and 1 in 10 children in the UK. It is a complex disease involving genetic and environmental factors. ADAM33 is an asthma susceptibility gene whose polymorphic variation has been linked to asthma and bronchial hyperresponsiveness, as well as decline in lung function in asthma and COPD and reduced lung function in young children. ADAM33 mRNA is almost exclusively expressed in mesenchymal cells, such as fibroblasts, myofibroblasts and smooth muscle cells. These cells play an important role in modelling of the airways during lung development and in remodelling of the airway in established disease. As the function of ADAM33 and its role in asthma is not known, this thesis tested the hypotheses that: ADAM33 is differentially expressed in normal and asthmatic lungs; and ADAM33 is involved in embryonic/fetal lung development where the influence of an allergic maternal environment affects ADAM33 to contribute to the development of asthma. To test the hypothesis that ADAM33 is differentially expressed in asthma, bronchial biopsies and brushings from adult subjects were examined. Using RT-qPCR all previously described splice variants were detected in the biopsies. No disease specific difference for any of the mRNA splice variants could be detected. Using immunohistochemistry, it was shown, for the first time that ADAM33 is found mainly in the bronchial smooth muscle, consistent with its genetic association with BHR. Computer-aided image analysis of ADAM33 expression did not reveal a disease-specific difference, consistent with the mRNA data. No expression of ADAM33 could be demonstrated in bronchial brushings containing more than 95% epithelial cells. To test the hypothesis that ADAM33 is involved in embryonic lung development human embryonic lung (HEL) and mouse tissues were examined. Using RT-qPCR, the same splice variants were detected in HEL as in adult bronchial tissue, however Western blot analysis revealed an extra ADAM33 protein band in HELs compared to adult lungs suggesting a different role of ADAM33 in developing lung. Expression of ADAM33 increased in HELs from 7 to 9 weeks post conception and a similar increase occurred when HELs were cultured in a newly developed HEL explant culture system suggesting that this is a useful model for studying human lung development. ADAM33 could be successfully knocked down using siRNA in the mesenchymal progenitor cells grown in culture from HELs paving the way for knock-down in whole HEL tissue. When ADAM33 mRNA expression was studied during murine lung development it was detectable from as early as embryonic day 11 and two significant increments in expression could be seen. The first occurred during the pseudoglandular stage when spontaneous peristaltic contractions occur and the second one after birth when the lungs inflate at the beginning of air breathing, suggesting that mechanical forces might induce ADAM33. To test the hypothesis that ADAM33 and asthma are influenced by maternal allergy, a mouse model using bronchial hyperresponsiveness susceptible mice (A/J mice) was used. Maternal allergy was induced using ovalbumin and the offspring were studied for ADAM33 expression and lung function. Maternal allergy had a suppressive effect on ADAM33 mRNA directly after birth which was similar to the findings in HEL cultured for 18 days in the presence of IL-13. Maternal allergy also induced increased BHR to methacholine in 4 weeks old offspring. Although no direct causal relationship with ADAM33 was established, these findings suggest a potential gene-environment interaction. In conclusion this thesis provides novel data regarding the expression and localisation of ADAM33 in embryonic and adult lung. It also suggests a potential role for ADAM33 in lung development and highlights the effect that maternal allergy has on airway reactivity of offspring that carry the ADAM33 susceptibility gene, consistent with a role for ADAM33 in the early-life origins of asthma.
Supervisor: Davies, Donna ; Holgate, Stephen Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: RC Internal medicine