application): The hypothesis of this proposal is that alveolar epithelial stem cells that express telomerase resist hyperoxic damage, proliferate, and repopulate the damaged alveolar epithelium. This hypothesis is based on preliminary data showing that telomerase activity increases in developing alveolar epithelial cells (AEC) and in hyperoxic AEC during the proliferative repair phase following hyperoxic injury. To test the hypothesis there are four specific aims. 1) To determine the developmental function of telomerase expression in alveolar epithelial progenitor/stem cells in fetal rodent lung by FACS analysis of cell cycle status and immunohistochemical co-localization of telomerase and markers for proliferation and differentiation. 2) To determine the function of telomerase in adult rat AEC which survive hyperoxic injury by segregation of survivors from AEC that succumb to injury and undergo apoptosis, using FACS, TUNEL and telomeric repeat amplification protocol (TRAP) analyses. 3) To determine if telomerase expression is necessary and sufficient for the key epithelial stem cell functions of survival and repopulation following injury, by ectopic over-expression of the telomerase catalytic subunit, TERT, in adult rat AEC in vitro and in vivo (gain of function). 4) To determine if telomerase expression is necessary and sufficient for the key epithelial stem cell functions of survival and repopulation following injury, by analysis of AEC in which telomerase expression has been knocked down in vitro and in vivo (loss of function).
