Peptides of the renin angiotensin system and their potential roles in idiopathic pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible disease of the lung, characterised by excessive extracellular matrix (ECM) deposition and loss of pulmonary function.

Fibroblasts are the main cell type responsible for ECM deposition and peptides from the renin-angiotensin system (RAS) have been shown to modulate fibroblast function. Angiotensin (ANG)-II, has been shown to exert profibrotic effects on human cardiac fibroblasts, such as increased collagen deposition. ANGII is known to bind to two receptors (AT1R and AT2R), with profibrotic effects attributed to AT1R.

ANGII is cleaved to form ANG(1-7). This peptide has been shown to exert antifibrotic effects on fibroblasts, such as decreased cell proliferation, via binding to the Mas receptor. However, most investigations have been performed in rat fibroblasts. Another related peptide called ANG(1-9), has also been shown to exert antifibrotic effects on fibroblasts, via binding to AT2R, again this work has only been performed in rat fibroblasts.

The main aims of this thesis were to investigate the effects of ANGII and related peptides on signalling pathways in both IPF derived and healthy human lung fibroblasts (HLFs) and to explore the effects of ANG peptides on HLF function. A second aim was to characterise Mas receptor expression on HLFs.

The effects of ANGII, ANG(1-7) and six related peptides on intracellular Ca2+ release were explored and the ability of ANGII to phosphorylate ERK was also investigated. Fibroblast functions in response to both ANGII and ANG(1-7) were also examined, including proliferation, collagen deposition, matrix metalloproteinase (MMP) and cytokine secretion. Receptor involvement was investigated with telmisartan (AT1R antagonist), PD-123319 (AT2R antagonist) and A-779 (Mas receptor antagonist). Mas receptor expression on HLFs was also investigated with immunocytochemistry and radioligand binding studies.

ANGI ANGII and ANGIII caused intracellular Ca2+ release via AT1R. ANGII also induced ERK phosphorylation, although this did not translate to fibroblast function. No effects of ANGII were observed on cell proliferation, collagen deposition, MMP secretion or IL-6 production.

In the Ca2+ assay, ANG(1-7) and ANG(1-9) partially inhibited the responses to both ANGII and ANGIII. These effects did not appear to be modulated by AT2R or the Mas receptor, indicating a novel binding site for these two peptides. The effects of ANG(1-7) did not appear to correlate with functional activity, with no reduction in cell proliferation or collagen deposition observed with this peptide. Immunocytochemistry showed clear nuclear expression of the Mas receptor in HLFs, with very low cell surface expression, providing an explanation for the lack of functional response.

Together these results indicate that although many ANG peptides can affect intracellular signalling pathways, peptides from the RAS do not affect functional responses of HLFs.

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