Biphasic influence of pravastatin on human cardiac microvascular endothelial cell functions under pathological and physiological conditions

https://doi.org/10.1016/j.bbrc.2019.02.090Get rights and content

Highlights

  • Pravastatin has biphasic effects on H2O2-induced HMVEC-C dysfunction.

  • Pravastatin at high doses inhibits HMVEC-C functions under normal condition.

  • Pravastatin at low doses alleviates H2O2-induced oxidative stress and damage.

  • Pravastatin at high doses acts on HMVEC-C via inhibiting prenylation.

Abstract

HMG-CoA reductase inhibitor statins are used to treat patients with hypercholesterolemia. The pleiotropic effects of statins have been recently extended to the regulation of angiogenesis. However, the observations on the effects of statins on endothelial cells seem to be contradictory. In this work, we systematically analysed the effects of pravastatin at concentrations covering 10,000-fold range on the functions of human cardiac microvascular endothelial cells (HMVEC-C) under H2O2-induced oxidative stress and normal physiological conditions. We observed the biphasic effects of pravastatin in protecting HMVEC-C dysfunctions induced by H2O2: pravastatin at low concentrations significantly enhanced vascular network formation, growth, migration and survival under H2O2-induced oxidative stress condition whereas this effect disappeared at higher concentrations. Interestingly, pravastatin at low concentrations did not affect HMVEC-C functions but at high concentrations significantly inhibited HMVEC-C vascular network formation, growth, migration and survival in a dose-dependent manner. We further demonstrated the different molecular mechanisms of the action of pravastatin at low and high concentrations on HMVEC-C: pravastatin at low concentrations alleviates H2O2-induced oxidative stress and damage and at high concentrations inhibits prenylation. Our work provides better understanding on the multiple differential effects and the underlying mechanisms of pravastatin on HMVEC-C, which may be of relevance to the influence of statins in cardiovascular system.

Introduction

HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase is a key enzyme of mevalonate pathway, the metabolic pathway that catalyzes the synthesis of a precursor of cholesterol as well as non-sterol isoprenoids, such as mevalonate [1]. Statins is a leading therapeutic class of lipid lowering drugs that are potent inhibitors of HMG-CoA reductase effectively subverting cholesterol metabolism through blocking the substrate accessibility to the enzyme [2]. These drugs, such lovastatin, simvastatin, atorvastatin, pravastatin and pitavastatin, have been commonly used to lower serum cholesterol as a means of reducing the risk for cardiovascular disease [3]. Statin therapy has contributed to the substantial decrease in coronary heart disease morbidity and mortality, and demonstrated benefits in both primary- and secondary-prevention of cardiovascular disease [4].

Increasing substantial evidence suggests that the beneficial effects of statins in cardiovascular disease may not only be attributed to their cholesterol lowering effects but also to their pleiotropic effects on endothelial cells [[5], [6], [7]]. However, the effects of statins on endothelial cells seem to be contradictory and the underlying mechanisms of their action vary in different types of endothelial cells. Simvastatin has been shown to promote cardiac microvascular endothelial cells functions by phosphorylation of p70 S6K and FoxO3a [8]. On the other hand, atorvastatin attenuates endothelial cell tube formation by regulating urokinase receptor-related signalling pathway [9]. While some studies report the proangiogenic activities of statins [10,11], others demonstrate the pro-apoptotic effect of statins on human vascular endothelial cells [12].

Pravastatin is approved for use in people with a high cholesterol level or heart disease. Its effects on human cardiac microvascular endothelial cells (HMVEC-C) are largely unknown. In this work, we systematically investigated the effects of pravastatin on the biological activities of HMVEC-C under both pathological (eg, H2O2-induced oxidative stress) and normal physiological conditions, and analysed the underlying mechanisms. Our work is the first to demonstrate the biphasic effects of pravastatin on HMVEC-C functions via multiple molecular mechanisms.

Section snippets

Primary cells and reagents

Human Cardiac Microvascular Endothelial Cells (HMVEC-C, Lonza, US) was used in our study. Cells were cultured in EGMTM -2 MV Microvascular Endothelial Cell Growth Medium-2 Bullet Kit (Lonza, US) at 37 °C cell culture incubator. HMVEC-C used for the experiments were from passages 3–10. Pravastatin, hydrogen peroxide (H2O2), farnesol and mevalonate were purchased from Sigma, US. Geranylgeraniol was purchased from ICN Biomedicals, The Netherlands.

Measurement of proliferation and apoptosis

HMVEC-C (20000 per well in 96-well plate) were

The differential effects of pravastatin on HMVEC-C capillary network formation under oxidative stress and normal physiological conditions

The formation of a vascular capillary network is a main biological function of endothelial cells. To investigate the effects of pravastatin on HMVEC-C functions, we firstly performed in vitro angiogenesis assay using HMVEC-C in the presence of pravastatin at concentrations ranging from 0.01 to 100 μM under oxidative stress condition induced by H2O2. Endothelial cells can rapidly align and form tubular structures when cultured on Complete Matrigel matrix which are rich in extracellular matrix

Discussion

In this work, we are the first to demonstrate that pravastatin, a cholesterol-lowering drug, has varying effects on HMVEC-C functions under physiological and pathological conditions via multiple mechanisms. We observed a biphasic effect of pravastatin on the angiogenic activity of HMVEC-C under H2O2-induced oxidative stress condition, being protective to reverse H2O2-induced angiogenic inhibition at low, nanomolar concentrations and this protective effect disappeared at higher concentrations.

Conflicts of interest

All authors declare no conflict of interest.

Acknowledgement

This work was supported by research grant provided by Jingzhou Science and Technology Bureau (No. 2016–87).

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    These authors contributed to this work equally and are co-first authors.

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