Proceedings of The Physiological Society

Physiology 2016 (Dublin, Ireland) (2016) Proc Physiol Soc 37, PCA068

Poster Communications

Pulmonary Epithelial Cells are a Source of Gremlin1 in the Lung

S. Devlin1, M. Pickering1, K. Rochfort1, P. McLoughlin1

1. University College Dublin, Dublin, Ireland.

Hypoxic pulmonary hypertension (HPH) is a common complication of chronic lung diseases. Current treatments are only partially effective. Gremlin1 contributes to pulmonary vascular homeostasis by antagonizing Bone Morphogenetic Protein signaling. Pulmonary vascular remodeling in response to hypoxia in the lung is accompanied by lung-specific changes in gremlin1 expression, suggesting gremlin 1 antagonism may be a novel therapeutic target. Ubiquitous gremlin1 halpodeficiency in mice attenuates the structural changes seen in the lung in response to hypoxia [1]. This work aimed to identify cellular sources of gremlin1 in the lung and quantify gremlin1 expression in Type II alveolar epithelial cells (ATIIs) using surfactant protein C (SPC) as a cell marker. Male mice (C57BL6/J,8 weeks old) were exposed to normoxic and hypoxic conditions (FIO2=0.10) for 2 days or 3 weeks. After exposure, mice were sedated using 2% isoflurane, deeply anaesthetised via IP injection of sodium pentobarbitone (60mg/Kg) and euthanised by exsanguination. Lungs were fixed in paraformaldehyde and embedded in paraffin wax. Immunohistochemical (IHC) staining and double immunofluorescence (IF) were employed to explore in vivo gremlin localisation. ‘Threshold' and ‘watershed segmentation' features in EBImage (Bioconductor) were used to identify ATIIs [2]. This automated method of identification was tested by comparing the number of ATIIs identified to the number identified by three independent observers. Fluorescence intensity was used as an index of gremlin1 expression, and quantified using the R programming language in EBImage. IHC staining of lung sections localised gremlin1 expression to small airway epithelial cells and endothelial cells of large vessels. Staining in alveolar walls suggested gremlin1 expression in alveolar epithelial cells and macrophages. Double IF staining for gremlin1 and SPC confirmed gremlin1 expression in ATIIs under normoxic and hypoxic conditions. The ATIIs identified by EBImage were also identified by three independent observers. EBImage reliably detected ATIIs when SPC expression was uniform throughout the cell. Differences in the number of ATIIs detected between EBImage and independent observers were attributed to cells where SPC staining was punctate. Such staining represented ~20% of ATIIs detected by independent observers. Gremlin1 fluorescence intensity within ATIIs was significantly greater than that in all other gremlin1 expressing cells combined in alveolar tissue (n=9; P<0.001; T-test). We report in vivo confirmation that ATIIs and small airway epithelium express gremlin1. EBImage software successfully identified ATIIs in which SPC distribution was uniform, but not when punctate. Gremlin1 quantification suggests ATIIs are a major cellular source of gremlin1 in alveolar tissue. Future work will investigate the functional role of ATII-derived gremlin1 in HPH.

Where applicable, experiments conform with Society ethical requirements