Pirfenidone Inhibits Hypoxic Pulmonary Hypertension through the NADPH/ROS/p38 Pathway in Adventitial Fibroblasts in the Pulmonary Artery.

Song Zhang,Zongxiu Yin,Weidong Qin,Yao Zhang,Yanbiao Chu,Xiaoli Ma,Enxiu Liu

doi:10.1155/2020/2604967

Abstract

Hypoxic pulmonary hypertension (HPH) is a devastating disease characterized by progressive vasoconstriction and vascular remodeling. Pirfenidone (PFD) inhibits the progression of HPH, though the molecular mechanisms remain unknown. This study is aimed at determining the role and mechanism of PFD in HPH in human pulmonary artery adventitial fibroblasts (HPAAFs), which were cultured under normal or hypoxic conditions. NOX4 and Rac1 were inhibited or overexpressed by shRNA or pcDNA3.1, respectively. Proliferation of HPAAFs was quantified by colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assays to assess cellular metabolic activity, cell counts, and ethynyldeoxyuridine (EdU) assays to detect DNA synthesis. Migration of HPAAFs was assessed by a wound healing assay. The expression levels of smooth muscle alpha-actin (a-SMA) and procollagen I (COL1A1) were assessed by RT-PCR and western blot analysis. PFD suppressed hypoxia-induced proliferation and migration of HPAAFs. Compared with the hypoxic control group, PFD reduced the expression of a-SMA and procollagen I (COL1A1). PFD reduced hypoxia-induced phosphorylation of p38 through the NOX4/reactive oxygen species (ROS) signaling pathway. Moreover, Rac1 also decreased hypoxia-induced phosphorylation of p38, without any cross-interaction with NOX4. These findings demonstrate that PFD is a novel therapeutic agent to prevent cell proliferation, migration, and fibrosis, which might be useful in inhibiting vascular remodeling in patients with HPH.

Highlights

Hypoxic pulmonary hypertension (HPH) is a complicated multifactorial syndrome characterized by pulmonary vascular remodeling, and its treatment is largely palliative [1, 2]
human pulmonary artery adventitial fibroblasts (HPAAFs) were treated with pirfenidone (PFD) at 0.1mg/ml or 0.2mg/ml before hypoxia exposure to assess cellular proliferation by use of the MTT assay, cell counting, and EdU assay
The results demonstrated that compared with the control, hypoxia induced HPAAF proliferation, whereas PFD significantly attenuated cell proliferation (Figure 1(a), P < 0:001; Figure 1(b), P < 0:001; Figure 1(c), P = 0:001)

Summary

Introduction

Hypoxic pulmonary hypertension (HPH) is a complicated multifactorial syndrome characterized by pulmonary vascular remodeling, and its treatment is largely palliative [1, 2]. The extensive remodeling of the pulmonary artery (PA) occurs in all three layers of the vascular wall, including the intimal endothelial cells, medial smooth muscle cells, and adventitial fibroblasts. The adventitia plays a key regulatory function in the release, retrieval, integration, and storage of nutrients in the vascular wall [2, 4]. The proliferation and differentiation of pulmonary artery adventitial fibroblasts (PAAFs) are thought to be a critical mechanism for initiating the development of HPH [5]. In response to chronic hypoxia, PAAFs first become activated and differentiate into myofibroblasts,

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