Extravascular sources of lung angiotensin peptide synthesis in idiopathic pulmonary fibrosis

Previous work from this laboratory demonstrated de novo synthesis of angiotensin (ANG) peptides by apoptotic pulmonary alveolar epithelial cells (AEC) and by lung myofibroblasts in vitro and in bleomycin-treated rats. To determine whether these same cell types also synthesize ANG peptides de novo within the fibrotic human lung in situ, we subjected paraffin sections of normal and fibrotic (idiopathic pulmonary fibrosis, IPF) human lung to immunohistochemistry (IHC) and in situ hybridization to detect ANG peptides and angiotensinogen (AGT) mRNA. These were analyzed both alone and in combination with cell-specific markers of AEC [monoclonal antibody (MAb) MNF-116] and myofibroblasts [alpha-smooth muscle actin (alpha-SMA) MAb] and an in situ DNA end labeling (ISEL) method to detect apoptosis. In normal human lung, IHC detected AGT protein in smooth muscle underlying normal bronchi and vessels, but not elsewhere. Real-time RT-PCR and Western blotting revealed that AGT mRNA and protein were 21-fold and 3.6-fold more abundant, respectively, in IPF lung biopsies relative to biopsies of normal human lung (both P < 0.05). In IPF lung, both AGT protein and mRNA were detected in AEC that double-labeled with MAb MNF-116 and with ISEL, suggesting AGT expression by apoptotic epithelia in situ. AGT protein and mRNA also colocalized to myofibroblast foci detected by alpha-SMA MAb, but AGT mRNA was not detected in smooth muscle. These data are consistent with earlier data from isolated human lung cells in vitro and bleomycin-induced rat lung fibrosis models, and they suggest that apoptotic AEC and myofibroblasts constitute key sources of locally derived ANG peptides in the IPF lung.