The Pulmonary Mesenchymal Tissue Layer Is Defective in an in Vitro Recombinant Model of Nitrofen-Induced Lung Hypoplasia Rhiannon B. van Loenhout, Irene.

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The Pulmonary Mesenchymal Tissue Layer Is Defective in an in Vitro Recombinant Model of Nitrofen-Induced Lung Hypoplasia Rhiannon B. van Loenhout, Irene Tseu, Emily K. Fox, Zhen Huang, Dick Tibboel, Martin Post, Richard Keijzer The American Journal of Pathology Volume 180, Issue 1, Pages 48-60 (January 2012) DOI: 10.1016/j.ajpath.2011.09.032 Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

Figure 1 Schematic representation of the formation of the homotypic and heterotypic recombinants. Black represents control cells (C), and gray indicates nitrofen-treated cells (N). The arcs denote fibroblasts (F), and the circles represent epithelial cells (E). Model 1 (FC)(EC) (control) and model 2 (FN)(EN) (nitrofen) are homotypic recombinants, whereas model 3 (FC)(EN) and model 4 (FN)(EC) are heterotypic recombinants. The American Journal of Pathology 2012 180, 48-60DOI: (10.1016/j.ajpath.2011.09.032) Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

Figure 2 Organogenesis in homotypic and heterotypic recombinants. Alveolar-like structures developed in all four types of recombinants spontaneously (A–D). IF staining of the recombinants for cytokeratin (epithelial cells; red) and vimentin (fibroblasts; green) that were counterstained with DAPI (nuclei; blue) (E–H) revealed tissue layer-specific morphological differences. In FN containing recombinants (F, H) the fibroblast layer appeared thickened and epithelial structures were less organized compared with FC-containing recombinants (E, G). Scale bar = 50 μm. The American Journal of Pathology 2012 180, 48-60DOI: (10.1016/j.ajpath.2011.09.032) Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

Figure 3 Quantitative analysis of epithelial cells versus fibroblasts surface area in recombinants. To quantify the observed differences in Figure 2E–H, the surface area of epithelial cells and fibroblasts was measured using Volocity4 software (Quorum Technologies Inc). The epithelial-to-fibroblast (E/F) ratio (A) and fraction (B) of fibroblasts in the recombinants were determined. The E/F ratio was significantly decreased whereas the number of fibroblasts increased in (FN)(EN) recombinants compared with FC-containing recombinants. Both the E/F ratio and number of fibroblasts trended to either decrease or increase in (FN)(EC) recombinants, respectively. *P <0.025. The bars represent SEM. The American Journal of Pathology 2012 180, 48-60DOI: (10.1016/j.ajpath.2011.09.032) Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

Figure 4 Nitrofen-treated fibroblasts exhibit reduced apoptosis in recombinants. Immunofluorescent TUNEL (green) analysis on recombinants counterstained with vimentin (fibroblasts; red) and DAPI (nuclei; blue) revealed less TUNEL-positive fibroblasts of both FN containing recombinants in comparison to FC containing recombinants (B, D versus A, C). Cleaved caspase-3 (brown) immunoreactivity was less in both FN containing recombinants in comparison to FC-containing recombinants (F, H versus E, G) as well. Scale bar = 50 μm. The American Journal of Pathology 2012 180, 48-60DOI: (10.1016/j.ajpath.2011.09.032) Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

Figure 5 Arrest in proliferation induced by nitrofen-treated fibroblasts in recombinants. Immunohistochemistry for ki67 (brown) demonstrated less proliferation in FN-containing recombinants (B, D) compared with FC-containing recombinants (A, C). Cyclin D3 (brown) demonstrated no significant differences between all four types of recombinants (E–H). EdU-uptake (green) by recombinants counterstained with vimentin (fibroblasts; red) and DAPI (nuclei; blue) revealed a decrease in cells in the S-phase in (FN)(EN) recombinants (J) compared with (FC)(EC) recombinants (I). PH3 (brown) staining indicated less cells undergoing mitosis in FN-containing recombinants (L, N) compared with FC-containing recombinants (K, M). Scale bar = 50 μm. The American Journal of Pathology 2012 180, 48-60DOI: (10.1016/j.ajpath.2011.09.032) Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

Figure 6 Decrease in mitosis in affected recombinants. To quantify the mitotic index of pH3 positive cells in Figure 5, they were counted in 20 slides per type of recombinant. The number of pH3-positive cells was significantly decreased in (FN)(EN) and (FN)(EC) recombinants compared with both (FC)(EC) and (FC)(EC) recombinants. *P <0.0001. The American Journal of Pathology 2012 180, 48-60DOI: (10.1016/j.ajpath.2011.09.032) Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

Figure 7 Increased expression of Cdk inhibitors in nitrofen-treated fibroblasts. Western blot analysis demonstrated that cultured fibroblasts from nitrofen-treated embryonic lungs have increased amounts of p27Kip1 and P57Kip2 proteins. The American Journal of Pathology 2012 180, 48-60DOI: (10.1016/j.ajpath.2011.09.032) Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

Figure 8 Nitrofen exposure does not alter cell differentiation in recombinants. IF for αSMA (smooth muscle cells; green) and cytokeratin (epithelial cells; orange) in recombinants counterstained with DAPI (nuclei; blue) revealed a similar pattern of myofibroblast staining in all four types of recombinants (A–D). Pro-SFTPC (type II epithelial cells; red) counterstained with DAPI (nuclei, blue) demonstrated also a similar pattern of staining in all four types of recombinants (E–H). Scale bar = 50 μm. The American Journal of Pathology 2012 180, 48-60DOI: (10.1016/j.ajpath.2011.09.032) Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

Figure 9 A schematic representation of our hypothesis of the pathogenesis in lung hypoplasia: the lung fibroblasts are the malfunctioning layer. Both a defect in mesenchymal apoptosis and generate the characteristic images seen in hypoplastic CDH lungs such as thickening of the fibroblast (mesenchymal) tissue layer and reduced alveolar spaces. The American Journal of Pathology 2012 180, 48-60DOI: (10.1016/j.ajpath.2011.09.032) Copyright © 2012 American Society for Investigative Pathology Terms and Conditions