Angiotensin-TGF-β1 Crosstalk in Human Idiopathic Pulmonary Fibrosis:Autocrine Mechanisms in Myofibroblasts and Macrophages

Bruce   D.   Uhal; Jong      Kyong Kim; Xiaopeng      Li; Maria      Molina-Molina

Abstract

Angiotensin II (ANGII) has been identified as a proapoptotic and profibrotic factor in experimental lung fibrosis models, and patients with the ID/DD polymorphism of ANG converting enzyme (ACE), which confers higher levels of ACE, are predisposed to lung fibrosis (Hum. Pathol. 32:521-528, 2001). Previous work from this laboratory has shown that human lung myofibroblasts isolated from patients with Idiopathic Pulmonary Fibrosis (IPF) synthesize the ANGII precursor angiotensinogen (AGT) constitutively. In attempts to understand the mechanisms and consequences of constitutive AGT synthesis by myofibroblasts, we studied myofibroblast-rich primary cultures of lung fibroblasts from patients with IPF (HIPF isolates), primary fibroblasts from normal human lung (NLFs), the IMR90 and WI38 human lung fibroblasts cell lines, and paraffin sections of lung biopsies from patients with IPF. Compared to the normal NLF isolates, HIPF primary fibroblast isolates constitutively synthesized more AGT and TGF-β1 mRNA, and released more AGT protein, ANGII and active TGF-β1 protein into serum-free conditioned media (both p < 0.01). Incubation of HIPF fibrotic isolates with the ANGII receptor antagonist saralasin reduced both TGF-β1 mRNA and active protein, suggesting that the constitutive expression of AGT drives the higher expression of TGF-β1 by the HIPF cells. Consistent with this premise, treatment of either the primary NLFs or the WI38 cell line with 10-7M ANGII increased both TGF-β1 mRNA and soluble active TGF-β1 protein. Moreover, induction of the myofibroblast transition in the IMR90 cell line with 2ng/ml TGF-??1 increased steady state AGT mRNA levels by realtime PCR (8-fold, p < 0.01) and induced expression of an AGT promoterluciferase reporter construct by over 10-fold (p < 0.001). Antisense oligonucleotides against TGF-β1 mRNA or TGF-β neutralizing antibodies, when applied to the fibrotic HIPF cells in serum-free medium, significantly reduced AGT expression. In lung sections from IPF patient biopsies, immunoreactive AGT/ANGI proteins were detected in myofibroblasts, epithelial cells and presumptive alveolar macrophages. Together, these data support the existence of an angiotensin/TGF- β1 “autocrine loop” in human lung myofibroblasts and also suggest ANG peptide expression by epithelia and macrophages in the IPF lung. These findings may explain the ability of ACE inhibitors and ANG receptor antagonists to block experimental lung fibrosis in animals, and support the need for evaluation of these agents for potential treatment of human IPF. This manuscript discusses the data described above and their implications regarding IPF pathogenesis.

Keywords: angiotensinogen, human lung fibroblasts, MCP-1, Reactive oxygen species, ACE inhibitors

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