488. Adenovirus-Mediated Delivery of ADAM10, a Novel Candidate Gene for COPD, Results in Emphysematous Changes in the Mouse Lung

Abstract

Cigarette smoking is associated with a risk for emphysema, a disorder of the lung parenchyma characterized by destruction of the alveolar walls. Based on the knowledge that cigarette smoke activates alveolar macrophages (AM) on the alveolar epithelial surface, and that AM are a potential source of proteases that can damage the alveolar walls, we have initiated a search to identify human AM proteases that play a role in the pathogenesis of emphysema. The strategy is to combine Affymetrix gene analysis of human AM gene expression (to identify the proteases upregulated in association with smoking) with an adenovirus (Ad)-mediated gene transfer of the candidate protease to the lungs of experimental animals (to prove that the candidate protease can induce emphysema). Alveolar macrophages were recovered from the lower lung respiratory tract by bronchoalveolar lavage of phenotypically normal smokers (19 ± 3 pack yr smoker history, n = 6) and normal nonsmokers (n = 5). Affymetrix HuGeneFl Chip analysis of mRNA expression demonstrated >2-fold upregulation of 35 genes in association with smoking. Among these was ADAM10, a 64 kDa (catalytically active form) single chain member of the disintegrin and metalloprotease domain gene family that functions as a type IV collagenase. Expression of ADAM10 was upregulated 2.5-fold in smoker AM compared to non-smokers (p<0.02) an observation confirmed by TaqMan real time PCR analysis (p<0.008). To verify that ADAM10 has a potential role in the pathogenesis of emphysema, an Ad vector (AdhADAM10) was used to deliver the human ADAM10 to mouse lungs and, after 8 wk, the lungs evaluated histologically for the response to ADAM10 overexpression. The AdhADAM10 vector was characterized in vitro in various cell lines to demonstrate it directed the expression of the 64 kDa form of ADAM10 product as assessed by Western analysis. To evaluate the effect of overexpression of ADAM10 in the lung, AdhADAM10 (1011 particle units) was administered intratracheally to the lungs of C57Bl/6J mice, and the lungs were evaluated 8 wk after gene transfer to quantify emphysematous changes using quantitative morphometry. Visual evaluation of histological sections of lungs of AdhADAM10 treated-animals showed enlargement of the airspaces indicative of emphysema. Quantitative morphometry of the lung parenchyma demonstrated that AdhADAM10 treatment resulted in a mean linear intercept (Lm) of 51.2 ± 1.3 mm (mean ± SE), compared to 47.3 ± 0.7 in mice treated with a control vector expressing no transgene (AdNull, 1011 particle units; p < 0.003). These results indicate a role of ADAM10 in the pathogenesis of emphysema, and the ADAM10 gene as a potential candidate gene for susceptibility to smoking-induced emphysema. This study represents an example of the paradigm of combining microarray analysis of human cells with experimental animal gene transfer to demonstrate the relevance of candidate genes that play a role in the pathogenesis of human disease.