This proposal is a renewal application. The long-term goal of the applicant is to elucidate the control of alveolar epithelial injury/repair and its anomalies that lead to lung fibrosis. In the last award period, the P.I. has demonstrated that angiotensin II (ANGII) is a potent inducer of apoptosis in lung alveolar epithelial cells (AECs), and that de novo synthesis of ANGII by AECs is required for apoptosis in response to Fas ligand, TNF-alpha, bleomycin (BLEO) or amiodarone (AMIO). In vitro and whole animal experiments showed that ANGII synthesis occurs de novo in AECs, i.e. by expression of the ANG precursor angiotensinogen (ANGEN), and that apoptosis of human and rat AECs requires binding of ANGII to receptor subtype AT1. Accordingly, in this application we propose to begin defining the regulation of ANGEN gene expression by AECs, to elucidate AT1 receptor signaling mechanisms and to identify the AEC-specific aspartyl proteases involved in the conversion of ANGEN to ANGI.
Aim 1 will define the molecular mechanisms that control ANGEN gene expression in AECs in response to AMIO, BLEO, and TNF-alpha; regulation of ANGEN expression by AECs will be studied with a human ANGEN promoter-luciferase reporter.
Aim 2 will profile ANGII-induced mRNA and protein expression in AECs and identify signaling mechanisms in AT1-induced apoptosis; genechips and proteomics will define subsets of ANG-responsive genes and AT1-dependent signaling mechanisms in purified AECs.
Aim 3 will determine the role of aspartyl proteases and in particular, Cathepsin D (CatD), in AEC apoptosis and lung fibrosis using gene knockdown methods and aspartyl protease inhibitors.
Aim 4 will determine if specific gene deletion of angiotensin rece`ptor subtype AT1a or ANGEN prevents bleomycin-induced apoptosis of AECs in vivo and blocks lung fibrosis, using AT1a and ANGEN-null mice. Together, the proposed experiments will clarify our understanding of the local Angiotensin System in the alveolar epithelium and its regulation in the context of lung fibrogenesis. This information may ultimately be useful in the design of new therapeutic strategies for the treatment of both fibrotic and acute lung diseases.
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