The long-term goal of the P.I. is to define, at the cellular and molecular levels, regulation and mechanisms of type II pneumocyte proliferation and differentiation. The response of adult rat type II cells to partial lung resection has been chosen as the primary model in this proposal. The investigator has recently developed classical and flow cytometric protocols which permit isolation and analysis of type II cells from pneumonectomized rats, including the identification and isolation of a subgroup of hypertrophic cells. Preliminary data indicate that this subgroup may represent those epithelial cells. The objectives of this proposal will be: 1) to test this hypothesis through a combination of biochemical and flow cytometric techniques including cell cycle analyses; b) to conduct flow cytometric and biochemical characterization of specific metabolic alterations in type II pneumonectomized rats as a bioassay to identify paracrine and/or autocrine growth factors involved in the regulation of lung growth and repair following injury. Type II pneumocytes isolated from normal, sham and injured animals will be studied both in primary culture and as fresh isolates. Flow cytometric techniques of cytokinetic analysis will be adapted for the study of type II cells, and will be employed to correlate specific biochemical changes in these cells with cell cycle position and traverse. In addition, growth regulatory factors identified by bioassay will be characterized by both flow cytometric and biochemical techniques. IF hypertrophic type II cells are indeed in the process of proliferating and differentiating, this isolated subgroup will provide an excellent new model for the study of type II cell hyperplasia and differentiation following pneumonectomy. The flow cytometric techniques refined in these studies will clarify type II cell kinetics in this model and will provide a foundation for studies of cytokinetics following other forms of lung injury as well. These cytometric methods will also serve as a powerful tool for the long-term study of the functions of regulatory factors related to lung growth and repair following injury. The information gained from these studies will be vital to the future development of improved surgical and/or therapeutic strategies for the management of lung disease.
