Introduction● Cux1 is a transcriptional

repressor gene and part of the network controlling G1-S phase transition. It represses the expression of the cyclin kinase inhibitors (CKI) p21 and p27.

● We have previously characterized the changes induced by this gene in kidney development, spermatogenesis, and liver development in Cux1 transgenic mice.

● In this study, we report the changes in the lung resulting from constitutive Cux1 expression in transgenic mice.Methods

● Transgenic mice constitutively expressing Cux1 were maintained and bred at Wheaton College.

● To ensure genotype accuracy, tail biopsies of mice were regularly sent to Transnetyx for genotyping.

● Mice were sacrificed by cervical dislocation following anesthetization with isofluorane as recommended by the Panel on Euthanasia of the American Veterinary Association.

● Lungs were harvested following euthanasia and fixed with formalin until histological analysis could be performed.

● H&E staining was performed on the eleven lung slides sent for blind review by UMKC School of Medicine.

● Each slide was photographed at different magnifications. At 1000x, macrophages were counted and averaged. At 100/400x, slides were graded on a scale from one to ten (one being least severe, ten being most severe) for the categories listed: inflammation, bronchitis, emphysema, vasculitis, and bronchiectasis.

● Trichrome staining was done to evaluate the presence of fibrosis.

Results● Histological staining (H&E) of lungs of all the mice showed extensive inflammation with few

patchy areas of emphysema. ● Severe bronchiectasis was also observed, particularly involving the bronchi of large or medium-

sized caliber. These bronchi were surrounded by large areas of inflammation. Bronchial basal membrane was thickened. The bronchial epithelium presented areas of pluristratification alternating with areas of necrosis. Peribronchial arteries had mild media thickening and large adventitia with scattered fibroblasts.

● Trichrome staining showed evidence of collagen in lungs of all the mice with most of its presence in the adventitia of the peribronchial arteries and of the bronchial basal membrane.

● Bronchiectasis, inflammation, and presence of collagen in particular, were more evident lungs of Cux1 mice.

● The average number of counted macrophages in the group of Cux1 transgenic mice was 54.6 versus 35.7 in the group of controls.

● The difference in lung tissue macrophage counts between Cux1 and wild type mice was significant with a p-value of 0.003.

Summary/Conclusion● Cux1 has been noted to be expressed in the

kidneys, liver, spleen, and testes.

● Transgenic mice constitutively expressing Cux1 developed significantly greater amounts of inflammation and bronchiectasis compared to wild type mice. In addition, extensive collagen was found in the adventitia of the pulmonary vessels and the airways.

● This suggests that over expression of Cux1 promotes lung pathology in addition to the multiorgan hyperplasia and fibrosis demonstrated in previous studies in other organs such as the kidneys.

● In conclusion, possible abnormal regulation of p21 and p27 via constitutive Cux1 expression led to multiple lung pathologies in transgenic mice.

● Further investigation is necessary to determine the specific mechanism by which Cux1 overexpression leads to the lung pathologies described and its relation to the organ pathologies demonstrated in previous studies.

References1. Ledford et al, Dev Biol 245: 157-71, 2002.2. Sharma et al, Dev Dyn 231: 828-38, 2004. 3. Kroll et al, Biol Repro 84, 455-65, 2011. 4. Alcalay et al, Am J Physiol Renal Physiol 295,

F1725-34, 2008. 5. Alcalay et al, Front Biosci 14, 4978-91, 2009.

Representative sections of lungs of Cux1 mice at 100x (A) and 400x (B) magnifications versus sections of WTFA (C and D). Bronchiectasis, inflammation, and collagen are more severe in the Cux1 mice. Staining: Trichrome.

A

C D

B

Pathological Changes in the Lungs of Cux1 Transgenic MiceCaroline Doo1, Sahar Safavi1, Betty Herndon1, Agostino Molteni1, Richard Baybutt2, Gregory Vanden Heuvel3, Ethan Harris2

1 UMKC School of Medicine, 2 Wheaton College, Wheaton, IL 3 Univ. Western Michigan, Lansing MI