Antioxidant Activity Mediates Pirfenidone Antifibrotic Effects in Human Pulmonary Vascular Smooth Muscle Cells Exposed to Sera of Idiopathic Pulmonary Fibrosis Patients

Hindawi Oxidative Medicine and Cellular Longevity Volume 2018, Article ID 2639081, 8 pages https://doi.org/10.1155/2018/2639081 Research Article Antioxidant Activity Mediates Pirfenidone Antifibrotic Effects in Human Pulmonary Vascular Smooth Muscle Cells Exposed to Sera of Idiopathic Pulmonary Fibrosis Patients Alessandro Giuseppe Fois,1 Anna Maria Posadino,2 Roberta Giordo,3 Annalisa Cossu,2 Abdelali Agouni ,4 Nasser Moustafa Rizk ,5 Pietro Pirina ,1 Ciriaco Carru ,2 Angelo Zinellu ,2 and Gianfranco Pintus 2,3,5 1 Department of Clinical and Experimental Medicine, University of Sassari, Viale San Pietro 43, 07100 Sassari, Italy Department of Biomedical Sciences, University of Sassari, Viale San Pietro 43, 07100 Sassari, Italy 3 Biomedical Research Center, Qatar University, 2713 Doha, Qatar 4 Pharmaceutical Science Section, College of Pharmacy, Qatar University, 2713 Doha, Qatar 5 Department of Biomedical Sciences, College of Health Sciences, Qatar University, 2713 Doha, Qatar 2 Correspondence should be addressed to Gianfranco Pintus; Received 5 June 2018; Accepted 6 September 2018; Published 21 October 2018 Guest Editor: Carlo Tocchetti Copyright © 2018 Alessandro Giuseppe Fois et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by an exacerbated fibrotic response. Although molecular and cellular determinants involved in the onset and progression of this devastating disease are largely unknown, an aberrant remodeling of the pulmonary vasculature appears to have implications in IPF pathogenesis. Here, we demonstrated for the first time that an increase of reactive oxygen species (ROS) generation induced by sera from IPF patients drives both collagen type I deposition and proliferation of primary human pulmonary artery smooth muscle cells (HPASMCs). IPF sera-induced cellular effects were significantly blunted in cells exposed to the NADPH oxidase inhibitor diphenyleneiodonium (DPI) proving the causative role of ROS and suggesting their potential cellular source. Contrary to IPF naive patients, sera from Pirfenidonetreated IPF patients failed to significantly induce both ROS generation and collagen synthesis in HPASMCs, mechanistically implicating antioxidant properties as the basis for the in vivo effect of this drug. 1. Introduction Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease characterized by an abnormal fibrotic response involving several areas of the lung tissue [1]. An aberrant tissue structure, encompassing exacerbated collagen secretion and deposition, progressively replaces the healthy tissue architecture, dramatically compromising the lung functions and ultimately leading to death [2]. The molecular and cellular determinants that trigger and maintain these processes are largely unknown. However, it seems that repetitive microinjuries directed towards the alveolar epithelium may play a major role [2]. Indeed, the abovementioned process leads to the release of different growth factors and fibrotic mediators such as fibroblast growth factor (FGF), plateletderived growth factor (PDGF), and transforming growth factor-beta 1 (TGF-β1), which activate myofibroblast recruitment, proliferation, and accumulation of extracellular matrix in alveolar regions [2]. Although the fibroblast appears to be the most well established, other types of cells have been reported to be implicated in the IPF-associated fibrotic process [2]. In addition to a destroyed parenchyma [2], an aberrant microvascular and macrovascular remodeling of the pulmonary vasculature appears to be strongly implicated in IPF pathogenesis [3]. In this context, vascular smooth muscle cells (VSMCs) play a pivotal role in maintaining organ and tissue physiological remodeling. Indeed, under 2 Oxidative Medicine and Cellular Longevity Table 1: Patient demographics and clinical characteristics. Subjects characteristics PT0, n = 11 PT1, n = 11 HD, n = 11 P value Age, years, mean (SD) 71.27 (5.51) 71.27 (5.51) 67.0 (9.4) P = 0 21∗ Male, n (%) 8 (72.72) 8 (72.72) 9 (81.81) P = 0 62# Former smokers, n (%) 9 (81.8) 9 (81.8) 8 (72.72) P = 0 62# 2343.6 (777.88) 81.04 (26.95) 92.81 (4.43) 54.17 (18.11) 2385.45 (801.51) 77.90 (24.49) 91.66 (6.66) 56.1 (22.38) FVC, ml, mean (SD) FVC, % predicted, mean (SD) FEV1/FVC ratio, %, mean (SD) DLCO, % predicted, mean (SD) 0.6328§ 0.1813§ 0.7161§ 0.5770§ PT0, Pirfenidone T0, which refers to untreated patients just diagnosticated with IPF; PT1, Pirfenidone T1, which refers to IPF patients treated with Pirfenidone for 24 weeks; HD, healthy donors, which refers to healthy blood donors; FVC, forced vital capacity; FEV1, forced expiratory volume1; DLCO, carbon monoxide diffusing lung capacity. § P values were determined by paired t-test (PT0 versus PT1), ∗ unpaired t-test (HD versus PT0/PT1), and # Chi-square (HD versus PT0/PT1). physiological conditions, the contractile phenotype of VSMCs is actively involved in the control of organ microcirculation, architecture, and function [4]. However, when a vascular injury occurs in response to proinflammatory factors, VSMCs undergo a “phenotypic switch” that confers in them the ability to proliferate, migrate, and synthesize extracellular matrix, ultimately leading to a dramatic pathological restructuring of the involved tissue [4]. Despite the massive vascular remodeling associated to IPF [3], and the potential implication of VSMCs in this process [5, 6], their role in the onset and progression of IPF-associated fibrotic phenomena remains to be elucidated. For example, whether VSMCs are involved in IPF-associated vascular remodeling in terms of increased proliferation and collagen deposition has never been investigated. After decades of having no effective medical treatment for IPF, two recent antifibrotic agents have been introduced for the management of this pathology: Nintedanib, a potent kinase inhibitor blocking the effects of growth factors implicated in the pathogenesis of IPF (platelet-derived growth factor, vascular endothelial growth factor, and fibroblast growth factor) [7], and Pirfenidone, whose mechanisms of action are still unclear [8]. However, with Pirfenidone, some papers suggest that this molecule possesses antioxidant properties, which might account for its reported antifibrotic effect as evidenced in experimental models of lung fibrosis [9, 10]. Oxidative stress has been previously linked to IPF at both the systemic and tissue levels [11–14]. In particular, NOX-4, a ROS-generating enzyme member of the NADPH family has been reportedly implicated in IPF-associated vascular remodeling [15]. We hypothesized that prooxidant circulating factors may trigger VSMCs’ phenotypic switching and induce cell proliferation and collagen I synthesis and that the (...truncated)