Elsevier

Atherosclerosis

Volume 273, June 2018, Pages 75-83
Atherosclerosis

Myeloperoxidase modification of high-density lipoprotein suppresses human endothelial cell proliferation and migration via inhibition of ERK1/2 and Akt activation

https://doi.org/10.1016/j.atherosclerosis.2018.04.006Get rights and content

Highlights

  • Native HDL stimulates endothelial ERK and Akt phosphorylation.

  • MPO-modified HDL attenuates endothelial proliferation and migration.

  • MPO-mediated oxidation of HDL inhibits endothelial proliferation and migration by inhibiting Akt and ERK phosphorylation.

Abstract

Background and aims

Preclinical studies show high-density lipoproteins (HDL) have a protective and reparative effect on the endothelium. HDL is, however, susceptible to oxidation, which affects function. Myeloperoxidase (MPO)-induced modification of HDL results in loss of anti-apoptotic and anti-inflammatory functions, however, its effect on endothelial proliferation and migration has not been characterized.

Methods

HUVECs were co-incubated with MPO-oxidised- or native-HDL (nHDL) in proliferation and migration assays. Signalling proteins were assessed in Western blots.

Results

nHDL caused dose-dependent increases of endothelial proliferation and migration. Consistent with an increase in cellular proliferation, HDL also stimulated proliferative cellular nuclear antigen (PCNA) expression and ERK phosphorylation in a concentration-dependent manner, which did not occur with MPO-oxidised HDL. HDL increased Akt phosphorylation, a driver of cellular migration. Contrastingly, MPO-oxidised HDL was unable to increase Akt phosphorylation and extensively-oxidised HDL inhibited Akt phosphorylation.

Conclusions

HDL promotes endothelial proliferation and migration, mediated in part via activation of ERK and Akt signalling. MPO-induced oxidative modification of HDL attenuates the endothelial-protective effects of HDL. These findings suggest that in an oxidative milieu, present in ageing and disease, HDL is likely to become ineffective. This has implications for HDL-raising therapies and emphasizes the need for strategies that prevent oxidation-related HDL dysfunction.

Introduction

It is well established that high-density lipoprotein (HDL) cholesterol levels are inversely associated with coronary artery disease (CAD). This has been demonstrated in epidemiological studies [1,2]. HDL is highly heterogeneous and composed of cholesterol-carrying lipoprotein particles built upon a predominantly apolipoprotein AI (apoA-I) backbone. Myeloperoxidase (MPO) binds to HDL in vivo, selectively targeting the apoA-I protein for oxidative modification by utilising hydrogen peroxide (H2O2) and other substrates, resulting in a loss of cholesterol efflux and lecithin cholesterol acyl transferase (LCAT) activating activities, thus generating a “dysfunctional HDL” particle [3,4]. Plasma HDL levels are not always proportional to the degree of atheroprotection and this likely reflects loss of function [5] and supports the concept of dysfunctional HDL. Pathophysiologically relevant levels of MPO-catalysed oxidation also cause a loss of non-cholesterol efflux-related activities of HDL, including anti-apoptotic and anti-inflammatory functions [6,7]. A large body of evidence has demonstrated that MPO and its reactive oxidant by-products play a role in the promotion of pathological events at all stages of atherosclerosis [8].

Endothelial cells (EC) play critical reparative and athero-protective roles in the vasculature. Endothelial damage/dysfunction is considered the critical initiator of large vessel diseases such as atherosclerosis [9]. An intact EC monolayer modulates local haemostasis and thrombolysis and provides a non-permeable barrier protecting vascular smooth muscle cells of the vessel wall from circulating growth-promoting factors [10]. Vascular EC proliferation and migration are vital in many physiological and pathological processes, such as angiogenesis and healing of the injured endothelium [11]. HDL has been shown to protect EC from apoptosis and promote proliferation and migration [10]. This is reported to be mediated by interaction with SR-BI and downstream activation of eNOS and nitric oxide production, key promoters of proliferation and migration [12]. The rHDL-mediated induction of extracellular-signal regulated kinase (ERK1/2), an important transcription factor that promotes cellular proliferation, and phosphatidylinositol 3-kinase (PI3-K) and its downstream effector protein kinase B (Akt), which plays a key active role in the promotion of cell migration and survival, is facilitated via interaction with SR-B1 [13]. HDL has been shown to increase both ERK and Akt phosphorylation in ECs [11,14], however, little is known about MPO-modified HDL stimulation of these signalling pathways nor its effect on EC proliferation and/or migration.

The aim of this study was to determine whether MPO-induced oxidation of HDL affects the promotion of EC proliferation and migration by HDL, as well as the associated ERK and Akt pathways. We find that MPO-mediated oxidised HDL attenuates native HDL (nHDL)-induced EC proliferation and migration and supresses the activation of both ERK and Akt signalling pathways. These findings highlight the importance of identifying strategies that prevent oxidation-induced dysfunction of HDL.

Section snippets

Preparation of HDL and MPO modification of HDL: oxidation reaction

HDL (1.063 < d < 1.21) was isolated by sequential ultracentrifugation from pooled human plasma [15]. HDL was further delipidated and apoA-I was isolated by anion-exchange chromatography [16] and verified by standard SDS-PAGE gel electrophoresis. Reconstituted HDL (rHDL) was prepared as detailed previously [17] (ratio of 100:1, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine:apoA-I; mol:mol). All HDL used in these studies were native HDL except when stated otherwise. MPO was purified from

MPO chlorinates tyrosine residues in lipid-free apoA-I

A range of molar ratios of oxidant H2O2 to apoA-I (mol/mol, 0–5) were tested and the Cl-Tyr content in apoA-I (representing protein modification) was determined. It was found that there were increasing amounts of Cl-Tyr modifications with higher molar ratios of oxidant H2O2 to apoA-I, in the presence of MPO (Supplemental Fig. 1).

Effect of HDL or MPO-oxidised HDL on HUVECs proliferation

Treatment of HUVECs with HDL caused a concentration-dependent increase in cellular proliferation, reaching significance between 20 and 80 μg/ml, when compared to the

Discussion

Pre-clinical and in vitro studies have shown that HDL exhibits endothelial-protective effects. Despite this, HDL-raising clinical trials have failed to show benefit on cardiovascular outcome. HDL functionality, rather than HDL cholesterol, is increasingly being recognised as a more robust predictor of cardiovascular risk [25]. In this study, we show that whilst nHDL increases EC proliferation and migration, MPO-oxidised HDL significantly attenuates these functions. nHDL induces proliferative

Conflict of interest

The authors declared they do not have anything to disclose regarding conflict of interest with respect to this manuscript.

Financial support

Funding was provided by departmental funds at Cleveland Clinic Foundation.

Author contributions

XC conducted the experiments, analysed and interpreted the results and wrote the manuscript. MD provided experimental support and was a major contributor in writing the manuscript. CB was a major contributor to intellectual input, writing and editing the manuscript. SN provided intellectual and editorial input. All authors read and approved the final manuscript.

Acknowledgements

The authors thank Ruliang Li (Cleveland Clinic Foundation) for his technical support.

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    1

    Authors contributed equally to this work.

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