Emphysema mediated by lung overexpression of ADAM10

Abstract

Cigarette smoking is the major risk factor for emphysema, a disorder of the lung parenchyma characterized by destruction of the alveolar walls. Current concepts of the pathogenesis of emphysema hold that the destruction of the lung parenchyma results, in part, from a local imbalance of proteases and antiproteases. Based on the knowledge that human alveolar macrophages express ADAM 10, a protease capable of destroying basement membrane collagen but not previously implicated in emphysema, we used adenovirus-mediated lung expression of ADAM 10 in a mouse model to assess whether an increased burden of ADAM 10 was capable of inducing emphysema. To assess this, the human ADAM 10 cDNA under control of a constitutive promoter was inserted into an adenovirus gene transfer vector (AdhADAMlO), and the vector (10(11) particle units) administered to the respiratory tract of wild type C57BI/6 mice. Lung levels of ADAM 10 mRNA and protein were upregulated following AdhADAMlO administration. After 8 weeks, quantitative morphometry of the lung parenchyma demonstrated that AdhADAMlO administration induced emphysema (mean linear intercept of 60.6 + 1.3 microm compared with 55.6 + 0.8 in mice treated with a control vector, p < 0.003). These results suggest a role of ADAM 10 in the pathogenesis of emphysema, adding to the list of proteases expressed in the lung that are capable of contributing to the development of lung destruction.

Figure 1

Expression of human ADAM10 mRNA and protein mediated by Ad‐gene transfer in the A549 cells. (A) TaqMan RT‐PCR relative quantification of Ad‐mediated ADAM10 gene expression. Total RNA was extracted from A549 cells 24 hours after infection with 2×10¹⁰ pu of AdhADAMlO, or AdNull, and subjected to quantification by TaqMan RT‐PCR. The data are shown relative to the endogenous expression of ADAM10 in naive A549 cells. (B) Expression of ADAM10 protein in A549 cells 48 hours postinfection with 2 × 10¹⁰ pu of AdhADAMlO or AdNull (control). Cell lysates of naive A549 cells were used as an additional control. ADAM10 was detected by Western analysis of the cell lysates using an anti‐ADAM10 polyclonal antibody. Two forms of ADAM10 are observed in naive, AdNull infected, or AdhADAMlO infected A549 cells (approximately 98 and 64 kDa), representing the precursor and mature form, respectively.

Figure 2

Time course of expression of human ADAM10 mRNA and protein in whole mouse lung following intratracheal administration of AdhADAMlO vector or the AdNull control vector (5 × 10¹¹ pu each). Total RNA was extracted from whole mouse lungs to serve as template for RT‐PCR using human ADAM10‐specific primers, at days 1, 3, 5, and 7 postinfection. Whole cell lysates were also prepared at days 1, 3, 5, and 7 postinfection for Western analysis using an anti‐ADAMlO polyclonal antibody. (A) Time course of human ADAM10 mRNA expression monitored by RT‐PCR. Naive and Ad Null‐infected were used as negative controls. Lane 1 = naive; lane 2 = AdNull, day 1; lane 3 = AdNull, day 3; lanes 4–7 = AdhADAMlO, days 1, 3, 5, and 7, respectively. Expression of human ADAM10 mRNA (2541 bp band) was detected at days 1 through 7 postinfection, peaking at days 1 and 3. Murine b‐actin primers were used as a control for template amount. (B) Time course of ADAM10 protein expression detected by Western analysis. Lane 1 = naive; lane 2 = AdNull; lanes 3–6 = ADhADAMlO, days 1, 3, 5, and 7 respectively. Since the antibody used is not specific for human ADAM10 but cross‐reacts with the murine protein, an approximately 64 kDa band corresponding to endogenous mouse ADAM10 can be seen in Ad Null‐transduced cells. At days 5 and 7 postinfection, a more intense approximately 64 kDa band can be seen in the AdhADAMlO‐transduced cells; the approximately 98 kDa band observed in the AdhADAMlO‐infected A549, as shown, can also be observed at those same time points, indicating the presence of the precursor protein.

Figure 3

Lung histology of mice 8 weeks following intratracheal administration of AdhADAM! 0 or AdNull. PBS (50 μl), AdNull or AdhADAM10 (10¹¹ pu, each in 50 μl) were delivered by intratracheal administration to 8 week old C57BI/6 mice. At week 8 posttreatment, the mice were sacrificed and the lungs inflated with 4% buffered paraformaldehyde to 25 cm water pressure, for 4 hours. The lungs were then removed and immersed in 4% paraformaldehyde, replaced by 70% ethanol, and stored overnight at 4°C. The fixed lungs were embedded in paraffin and 4μm‐thick sections were prepared and stained with hematoxoxylin and eosin. Shown are representative histological lung sections. (A) Naive (PBS); (B) AdNull‐treated; (C) AdhADAM10‐treated animals (low power); and (D) AdhADAM 10 (high power). (A)‐(C), bar = 500 μηη; (D), bar = 200 μηη.

Figure 4

Quantitative morphometric analysis of emphysema in the lungs of mice receiving intratracheal AdhADAM 10. Shown is the quantitative morphometric (Lm = mean linear intercept) analysis of alveolar septae of the lungs of five Ad Null‐treated and five AdhADAM 10‐treated mice. The lungs were prepared as described in Figure 3 .