Elsevier

Biochemical Pharmacology

Volume 85, Issue 12, 15 June 2013, Pages 1753-1760
Biochemical Pharmacology

Statins protect human endothelial cells from TNF-induced inflammation via ERK5 activation

https://doi.org/10.1016/j.bcp.2013.04.009Get rights and content

Abstract

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) exert pleiotropic effects on the cardiovascular system, in part through a decrease in reactive oxygen species (ROS) formation and reduction of vascular inflammation. To elucidate the molecular mechanisms involved in these effects, we investigated the effect of statins on TNF-α-induced ROS production, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) expression in human aortic endothelial cells (HAECs). Exposure of HAECs to TNF-α caused production of ROS via Rac-1 membrane translocation and activation. The Rac-1 activation and ROS liberation mediated TNF-stimulated NF-κB activation and the subsequent VCAM-1 and ICAM-1 expression. Extracellular-signal-regulated kinase 5 (ERK5) plays a central role in inhibiting endothelial inflammation. Immune complex kinase assay of protein extracts from HAECs treated with atorvastatin revealed increased ERK5 activity in a time- and dose-dependent manner. In addition, pretreatment with atorvastatin inhibited TNF-α-induced ROS production and VCAM-1 and ICAM-1 expression. Chemical or genetic inhibition of ERK5 ablated the statins inhibition of Rac-1 activation, ROS formation, NF-κB, VCAM-1 and ICAM-1 expression induced by TNF-α. Taken together, statins, via ERK5 activation, suppress TNF-stimulated Rac-1 activation, ROS generation, NF-κB activation and VCAM-1 and ICAM-1 expression in human ECs, which provides a novel explanation for the pleiotropic effects of statins that benefit the cardiovascular system.

Graphical abstract

The proposed mechanism by which statins inhibit the pro-inflammatory effects of TNF.

  1. Download : Download full-size image

Introduction

Inflammation plays a critical role in cardiovascular disease, and the inflammatory cascade is particularly important in the atherosclerotic process. The inflammatory mediator tumor necrosis factor (TNF, also known as TNF-α) has been implicated in the pathogenesis of a number of cardiovascular diseases, including atherosclerosis, myocardial infarction, heart failure, myocarditis and cardiac allograft rejection [1]. In response to TNF, vascular endothelial cells promote inflammation changes, which increase leukocyte adhesion, transendothelial migration and vascular leak and promote thrombosis, by displaying, in a distinct temporal, spatial and anatomical pattern [2], [3], [4], different combinations of adhesion molecules for leukocytes, including vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin [5], [6]. In addition, TNF is vital for the ICAM-1-dependent recruitment of mononuclear cells and microvascular damage [7]. The central role of TNF in inflammation has also been demonstrated by the ability of TNF blocker to treat a range of cardiovascular disorders and inflammatory conditions, including acute myocardial infarction (AMI), heart failure, rheumatoid arthritis, diabetes and hyperlipidaemia [1], [8]. Despite the important pathological role of TNF in cardiovascular diseases, the exact mechanisms underlying TNF-induced vascular inflammation and dysfunction remain unresolved and there is no current scientific consensus.

Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (statins) are potent inhibitors of cholesterol biosynthesis widely used to reduce serum cholesterol levels in hyperlipidemic patients [9]. In addition to lowering lipids, these drugs exhibit potent anti-inflammatory effects mediated by inhibition of macrophage function. Statins are the most effective agents available today for the reduction of vascular inflammation. However, the mechanisms by which statins may exert beneficial anti-inflammatory effects independent of lipid-lowering have not been completely identified.

Extracellular-signal-regulated kinase 5 (ERK5) is the newest member of the mitogen-activated protein kinase (MAPK) family. Similar to other MAPK family members, ERK5 plays a significant role in cell growth and differentiation. Nevertheless, emerging evidence suggests ERK5's unique functional characteristics [10]. Recent studies have revealed distinctive features of the ERK5 pathway: ERK5 is a key factor to inhibit endothelial inflammation [10] and has a key role in cardiovascular development [11]. Intriguingly, ERK5 is strongly activated by steady laminar flow (s-flow) that generates a frictional dragging force on the endothelium surface (called fluid shear stress), which is known to possess anti-inflammatory and antiatherosclerotic effects and to protect endothelial cells (ECs) from becoming dysfunctional [12], [13]. However, whether pharmaceutical stimulated ERK5 activation has a similar anti-inflammatory vasoprotective effect is not clear.

The accumulation of data implicating ERK5 as a key factor to inhibit endothelial inflammation prompted us to analyze the effect of statins on ERK5 activation and cross-talk between ERK5 and TNF inflammatory cascade in human endothelial cells. We found that TNF significantly increases NF-κB activity and VCAM-1 and ICAM-1 expression via Rac-1 activation and ROS generation. We provide evidence that statins can directly activate ERK5 and potently block TNF-induced Rac-1/ROS/NF-κB/VCAM-1 inflammation pathway. Therefore, the ERK5 activation and subsequent inhibition of TNF inflammation pathway in endothelial cells mediate statins-elicited anti-inflammatory vasoprotective effect.

Section snippets

Materials

Antibodies used in the present study and their commercial sources were as follows: anti-Rac-1, anti-IκB and anti-ERK5 (EMD Millipore Corporation, Billerica, MA, USA); anti-PMCA4 (plasma membrane calcium ATPase 4) (Sigma–Aldrich, St. Louis, MO, USA); anti-VCAM-1, ICAM-1 (OriGene Technologies, Rockville, MD, USA). Rac-1 inhibitor was purchased from Calbiochem (San Diego, CA, USA); PEG-catalase were purchased from Sigma; BAY 11-7085 and XMD 8-92 were from Santa Cruz Biotechnology (Santa Cruz, CA,

Statins inhibits TNF-induced Rac-1 activation and ROS formation in cultured ECs

To investigate whether TNF-α induces the liberation of ROS in HAECs, intracellular ROS levels were determined by DCF fluorescence. Stimulation of the HAECs with TNF-α (100 U/ml) led to a time-dependent increase of intracellular ROS, which was maximally 3.2-fold over control at 1 h (Fig. 1A). Recent research focused on the molecular and biochemical characterization of TNF-α-induced ROS production in endothelial cells. Several authors proved the role of the small GTPase Rac-1 in this context. Rac-1

Discussion

In the present study, we reveal the newest member of the MAPK family ERK5 as a critical mediator of the endothelial vasoprotective phenotype conferred by statins. Our data demonstrate that statins activate ERK5 in cultured human ECs, and importantly, that this activation is necessary for the statins-mediated decrease in TNF-activated expression of adhesion molecules such as VCAM-1 and ICAM-1, important inflammatory factors in ECs. Additionally, we have shown that the inhibition of Rac-1

Disclosures

None.

Acknowledgment

This work was supported by American Heart Association award – AHA Award #10POST3530033 to Y. Wu.

References (66)

  • G. Rimbach et al.

    Macrophages stimulated with IFN-gamma activate NF-kappa B and induce MCP-1 gene expression in primary human endothelial cells

    Mol Cell Biol Res Commun

    (2000)
  • G.L. Schieven et al.

    Reactive oxygen intermediates activate NF-kappa B in a tyrosine kinase-dependent mechanism and in combination with vanadate activate the p56lck and p59fyn tyrosine kinases in human lymphocytes

    Blood

    (1993)
  • G. Pearson et al.

    ERK5 and ERK2 cooperate to regulate NF-kappaB and cell transformation

    J Biol Chem

    (2001)
  • Z.W. Yang et al.

    Mechanisms of hydrogen peroxide-induced contraction of rat aorta

    Eur J Pharmacol

    (1998)
  • J. Abe et al.

    c-Src is required for oxidative stress-mediated activation of big mitogen-activated protein kinase 1

    J Biol Chem

    (1997)
  • B.E. Xu et al.

    WNK1 activates ERK5 by an MEKK2/3-dependent mechanism

    J Biol Chem

    (2004)
  • T.H. Chao et al.

    MEKK3 directly regulates MEK5 activity as part of the big mitogen-activated protein kinase 1 (BMK1) signaling pathway

    J Biol Chem

    (1999)
  • W. Sun et al.

    MEKK2 associates with the adapter protein Lad/RIBP and regulates the MEK5-BMK1/ERK5 pathway

    J Biol Chem

    (2001)
  • G. Zhou et al.

    Components of a new human protein kinase signal transduction pathway

    J Biol Chem

    (1995)
  • C. Garcia-Hoz et al.

    G alpha(q) acts as an adaptor protein in protein kinase C zeta (PKCzeta)-mediated ERK5 activation by G protein-coupled receptors (GPCR)

    J Biol Chem

    (2010)
  • F.A. Inesta-Vaquera et al.

    Alternative ERK5 regulation by phosphorylation during the cell cycle

    Cell Signal

    (2010)
  • J.R. Bradley

    TNF-mediated inflammatory disease

    J Pathol

    (2008)
  • D.V. Messadi et al.

    Induction of an activation antigen on postcapillary venular endothelium in human skin organ culture

    J Immunol

    (1987)
  • J.R. Bradley et al.

    Prolonged cytokine exposure causes a dynamic redistribution of endothelial cell adhesion molecules to intercellular junctions

    Lab Invest

    (1996)
  • J.S. Pober et al.

    Two distinct monokines, interleukin 1 and tumor necrosis factor, each independently induce biosynthesis and transient expression of the same antigen on the surface of cultured human vascular endothelial cells

    J Immunol

    (1986)
  • J.M. Munro et al.

    Tumor necrosis factor and interferon-gamma induce distinct patterns of endothelial activation and associated leukocyte accumulation in skin of Papio anubis

    Am J Pathol

    (1989)
  • W. Rudin et al.

    Resistance to cerebral malaria in tumor necrosis factor-alpha/beta-deficient mice is associated with a reduction of intercellular adhesion molecule-1 up-regulation and T helper type 1 response

    Am J Pathol

    (1997)
  • H. Zhang et al.

    Role of TNF-alpha in vascular dysfunction

    Clin Sci (Lond)

    (2009)
  • D.J. Maron et al.

    Current perspectives on statins

    Circulation

    (2000)
  • N.T. Le et al.

    Reactive oxygen species, SUMOylation, and endothelial inflammation

    Int J Inflam

    (2012)
  • S. Nishimoto et al.

    MAPK signalling: ERK5 versus ERK1/2

    EMBO Rep

    (2006)
  • M.A. Gimbrone et al.

    Endothelial dysfunction, hemodynamic forces, and atherogenesis

    Ann N Y Acad Sci

    (2000)
  • P.F. Davies

    Hemodynamic shear stress and the endothelium in cardiovascular pathophysiology

    Nat Clin Pract Cardiovasc Med

    (2009)
  • Cited by (55)

    • Cell adhesion molecule-mediated therapeutic strategies in atherosclerosis: From a biological basis and molecular mechanism to drug delivery nanosystems

      2021, Biochemical Pharmacology
      Citation Excerpt :

      Hence, blocking the common inflammatory pathways is considered an effective therapeutics for the prevention and treatment of AS. Currently, various approaches are available in the AS field including statins, antiplatelet drugs (aspirin), and specific inflammatory inhibitors (monoclonal antibodies and antagonists), possessing anti-inflammatory properties that contributed to their anti-atherosclerotic effects [7–9]. Among them, inflammatory inhibitors hold great promise for the treatment of AS [10].

    • Repurposing of statins via inhalation to treat lung inflammatory conditions

      2018, Advanced Drug Delivery Reviews
      Citation Excerpt :

      GTPase family members Rac and RhoA, initiate downstream signalling that involves numerous intermediates (Fig. 3), most notably via the MAPK/NF-κB signalling axis to initiate synthesis of pro-inflammatory cytokines TNF-α, IL-6, IL-8 and pro-inflammatory intermediates intracellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 to name a few [29, 32, 53, 61, 63–66]. Importantly, cells treated with a variety of hydrophilic and lipophilic statins treatment show decreased Rac/RhoA-activity and therefore decreased downstream cytokine synthesis and secretion [32, 61, 65]. Additional studies have then further confirmed the role of prenylated GTPases play in regulating cytokine synthesis as chemical inhibition of prenylation reported similar results [18, 29, 30, 62, 67].

    View all citing articles on Scopus
    View full text