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

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-beta1 mRNA, and released more AGT protein, ANGII and active TGF-beta1 protein into serum-free conditioned media (both p<0.01). Incubation of HIPF fibrotic isolates with the ANGII receptor antagonist saralasin reduced both TGF-beta1 mRNA and active protein, suggesting that the constitutive expression of AGT drives the higher expression of TGF-beta1 by the HIPF cells. Consistent with this premise, treatment of either the primary NLFs or the WI38 cell line with 10(-7) M ANGII increased both TGF-beta1 mRNA and soluble active TGF-beta1 protein. Moreover, induction of the myofibroblast transition in the IMR90 cell line with 2 ng/ml TGF-beta1 increased steady state AGT mRNA levels by realtime PCR (8-fold, p<0.01) and induced expression of an AGT promoter-luciferase reporter construct by over 10-fold (p<0.001). Antisense oligonucleotides against TGF-beta1 mRNA or TGF-beta 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-beta1 "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.

Mas Deficiency in FVB/N Mice Produces Marked Changes in Lipid and Glycemic Metabolism

“…Signs of adipocyte hypertrophy were not observed in histological studies, suggesting that the increase in fat mass occurred by increased proliferation of adipocytes (hyperplasia). This observation was corroborated by the increase in TGF-␤ expression, an important cytokine involved in tissue proliferation (19). On the other hand, the absence of alterations in food intake indicates that the changes in fat mass were not a consequence of metabolic changes induced by increased appetite.…”

The matricellular protein CCN1 enhances TGF‐β1/SMAD3‐dependent profibrotic signaling in fibroblasts and contributes to fibrogenic responses to lung injury

“…There have been very few studies that have investigated the regulatory mechanisms of constitutively active IPF fibroblasts (53,(55)(56)(57). The observation that disease-associated fibroblasts from lungs of patients with IPF express higher levels of CCN1 and that this is mechanistically linked to heightened expression of profibrotic genes has potential therapeutic implications.…”

NOX Enzymes and Pulmonary Disease

“…A p47 phox -requiring NOX isoform is required for the development of fibrosis in a murine lung-injury model that is inflammation dependent, and the observed protection in p47 phoxÀ=À mice is associated with enhanced neutrophilic inflammation and matrix metalloproteinase (MMP)-9 activity (70). Significant crosstalk occurs between the reninangiotensin-aldosterone system and TGF-b1 in organ fibrosis (130,147), and this effect is, at least in part, mediated by induction=activation of NOX1, NOX2, NOX4, or a combination of these (4,12,112,120,139,143).…”