Areti Tzanetakis1,2, Lina Antounians1,2, Alyssa Belfiore1,2, Qi Ma1,2, Mark Stasiewicz1,2, Ornella Pellerito1,2, Augusto Zani3,4. 1. Developmental and Stem Cell Biology Program, PGCRL, The Hospital for Sick Children, Toronto, ON, Canada. 2. Division of General and Thoracic Surgery, Department of Surgery, The Hospital for Sick Children, 555 University Avenue, Toronto, ON, M5G 1X8, Canada. 3. Developmental and Stem Cell Biology Program, PGCRL, The Hospital for Sick Children, Toronto, ON, Canada. augusto.zani@sickkids.ca. 4. Division of General and Thoracic Surgery, Department of Surgery, The Hospital for Sick Children, 555 University Avenue, Toronto, ON, M5G 1X8, Canada. augusto.zani@sickkids.ca.
Abstract
PURPOSE: Pulmonary hypoplasia secondary to congenital diaphragmatic hernia (CDH) is characterized by impaired epithelial homeostasis. Recently, amniotic fluid stem cells (AFSCs) have been shown to promote growth in hypoplastic lungs of rat fetuses with CDH. Herein, we investigated whether CDH hypoplastic lungs mount an endoplasmic reticulum (ER) stress response and whether AFSCs could re-establish pulmonary epithelial homeostasis. METHODS: Primary epithelial cells were isolated from fetal rat lungs at E14.5 from control and nitrofen-exposed dams at E9.5. Nitrofen-exposed epithelial cells were grown in medium alone or co-cultured with AFSCs. Epithelial cell cultures were compared for apoptosis (TUNEL), cytotoxicity (LIVE/DEAD assay), proliferation (5'EdU), and ER stress (CHOP, Bcl-2) using one-way ANOVA (Dunn's post-test). RESULTS: Compared to control, nitrofen-exposed epithelial cells had increased cytotoxicity and apoptosis, reduced proliferation, and activated ER stress. AFSCs restored apoptosis, proliferation, and ER stress back to control levels, and significantly reduced cytotoxicity. CONCLUSIONS: This study shows for the first time that ER stress-induced apoptosis is activated in the pulmonary epithelium of hypoplastic lungs from fetuses with CDH. AFSC treatment restores epithelial cellular homeostasis by attenuating the ER stress response and apoptosis, by increasing proliferation and migration ability, and by reducing cytotoxicity.
PURPOSE:Pulmonary hypoplasia secondary to congenital diaphragmatic hernia (CDH) is characterized by impaired epithelial homeostasis. Recently, amniotic fluid stem cells (AFSCs) have been shown to promote growth in hypoplastic lungs of rat fetuses with CDH. Herein, we investigated whether CDH hypoplastic lungs mount an endoplasmic reticulum (ER) stress response and whether AFSCs could re-establish pulmonary epithelial homeostasis. METHODS: Primary epithelial cells were isolated from fetal rat lungs at E14.5 from control and nitrofen-exposed dams at E9.5. Nitrofen-exposed epithelial cells were grown in medium alone or co-cultured with AFSCs. Epithelial cell cultures were compared for apoptosis (TUNEL), cytotoxicity (LIVE/DEAD assay), proliferation (5'EdU), and ER stress (CHOP, Bcl-2) using one-way ANOVA (Dunn's post-test). RESULTS: Compared to control, nitrofen-exposed epithelial cells had increased cytotoxicity and apoptosis, reduced proliferation, and activated ER stress. AFSCs restored apoptosis, proliferation, and ER stress back to control levels, and significantly reduced cytotoxicity. CONCLUSIONS: This study shows for the first time that ER stress-induced apoptosis is activated in the pulmonary epithelium of hypoplastic lungs from fetuses with CDH. AFSC treatment restores epithelial cellular homeostasis by attenuating the ER stress response and apoptosis, by increasing proliferation and migration ability, and by reducing cytotoxicity.
Entities:
Keywords:
CDH; Cellular homeostasis; ER stress; Nitrofen; Regenerative medicine
Authors: Susan Buckley; Wei Shi; Gianni Carraro; Sargis Sedrakyan; Stefano Da Sacco; Barbara A Driscoll; Laura Perin; Roger E De Filippo; David Warburton Journal: Am J Respir Cell Mol Biol Date: 2011-06-23 Impact factor: 6.914