Role of 20-hydroxyeicosatetraenoic acid in pulmonary hypertension and proliferation of pulmonary arterial smooth muscle cells

Abstract

Objective: To investigate the level of 20-Hydroxyeicosatetraenoic acid (20-HETE) in model of pulmonary hypertension (PH) and its effect on the proliferation of pulmonary arterial smooth muscle cells (PASMCs).

Methods

Twenty male Sprague-Dawley rats were randomly divided into two groups, including control group and PH group. PH was induced by intra-peritoneal injection of 20 mg/kg monocrotaline (MCT) twice in a week in 10 rats, and control rats were given equal amount of saline. Mean pulmonary arterial pressure (mPAP), right ventricular hypertrophy index (RVHI) and pulmonary vascular remodeling index (WA%, WT%) were assessed at the week 4. The levels of 20-HETE were analysed by liquid chromatography tandem-mass spectrometry (LC-MS/MS). EdU test was used to determine the proliferation of PASMCs. Intracellular levels of reactive oxygen species (ROS) were detected using DCFH-DA dye.

Results

(1) Prominent right ventricular hypertrophy and pulmonary vascular remodeling were verified in PH rats; (2) 20-HETE levels in lung tissue and serum were significantly lifted in PH rats; (3) Increased 20-HETE levels in cell culture supernatants were significantly noted in hypoxia condition; (4) Proliferation of PASMCs was induced by 20-HETE and hypoxia, and was inhibited by HET0016; (5) Production of ROS was elevated by 20-HETE and hypoxia, and was reduced by HET0016;

Conclusion

Vascular remodeling was demonstrated in PH rats. 20-HETE levels were significantly increased in PH rats and under hypoxia condition. PASMCs proliferation and ROS production were elevated by 20-HETE and could be inhibited by HET0016, a specific inhibitor of 20-HETE. Taken together, changes in the level of 20-HETE may be related to the excessive proliferation of PASMCs in PH rats.

Introduction

Pulmonary hypertension (PH) is well recognized as a complication of chronic hypoxic lung disease [1] with a progressive-increase in pulmonary circulation resistance [2], eventually leading to right heart failure, even death. Pathologically, PH were mainly manifested as abnormal proliferation of pulmonary arterial smooth muscle cells (PASMCs) and endothelial cells (ECs), increased contraction capacity of pulmonary arteries, endothelial dysfunction, pulmonary vascular remodeling and formation of the thrombus in situ [3]. As was described by Humbert et al. [4], the obstruction and hypoxic vasoconstriction led to an increased pulmonary vascular resistance and a decreased arterial compliance, resulting in PH. Clinically, the mortality of patients with PH was high. However, there is a huge limitation in the treatment of PH [5]. So it is critical to find out a new target for the treatment of PH.

Recent studies have shown that lipid metabolism is involved in the development of PH [6,7]. Free fatty acids in blood were nearly two-fold higher in PH patients compared with controls [8]. A number of studies have found that increased level of arachidonic acid (AA) metabolism could lead to an enhanced pulmonary artery contractions [9]. Recent years, a novel pathway for metabolism of AA by cytochrome P450 enzyme have been identified in which 20-Hydroxyeicosatetraenoic acid (20-HETE) plays an essential role in the regulation of vascular function (Fig. 1) [10].

20-HETE is one of products of AA metabolized by the cytochrome P450 pathway [11]. Recent researches have revealed that 20-HETE production could be induced by hypoxia [12,13]. It was shown that abnormal proliferation of smooth muscle cells and ECs was promoted by 20-HETE through RAS/MAPK pathway and PI3K/AKT pathway [14], leading to the vascular remodeling. Endothelial dysfunction could be induced by 20-HETE through nuclear factor-кB (NF-κB) pathway [15]. It was showed that the influx of intracellular calcium ions and enhanced sensitivity of blood vessels to calcium ions could be induced by 20-HETE, resulting in the vasoconstriction [16,17]. Vascular oxidative stress and inflammatory response could also be induced by 20-HETE through nicotinamide adenine dinucleotide phosphate (NADPH) [18] pathway and NF-κB pathway. Recent studies have found that 20-HETE may play roles in the developments of a series of cardiovascular diseases by binding to its specific receptor G protein-coupled receptor 75 (GPR75) [19]. However, the relationship between 20-HETE and PH remains unclear. Moreover, in the occurrence and development of PH, the change of level of 20-HETE was not yet clear. In this investigation, the aims were to quantify the level of 20-HETE in PH and the effect of 20-HETE on proliferation of PASMCs.