Original Article
Lysophosphatidylcholine regulates human microvascular endothelial cell expression of chemokines

https://doi.org/10.1016/j.yjmcc.2003.08.004Get rights and content

Abstract

The role of lysophosphatidylcholine (LPC) in the induction of MCP-1, IL-8 and RANTES, which are chemotactic factors to monocytes, neutrophils and lymphocytes, respectively, by human vascular endothelial cells (EC), was examined. LPC induced the expression of MCP-1 and IL-8 in a concentration- and time-dependent manner in microvascular EC (MVEC) and in large vessel EC from aorta, pulmonary artery and umbilical vein. LPC also induced RANTES in MVEC but not in large vessel EC. Signaling pathways responsible for LPC induction of chemokines were examined in MVEC. LPC and TNFα, a cytokine secreted in sites of inflammation, additively stimulated RANTES expression. LPC did not augment TNFα induction of MCP-1 or IL-8. A platelet-activating factor receptor antagonist (BN52021) failed to block LPC induction of MVEC chemokines, but the Gi-protein inhibitor pertussis toxin partially blocked LPC induction of RANTES and IL-8. LPC activated multiple kinases in MVEC; it increased the phosphorylation of ERK1/2, AKT and p38 MAP kinase in a time-dependent manner. An inhibitor of the MAPK/ERK pathway, PD98059, blocked the phosphorylation of ERK1/2 and RANTES induction by LPC, but augmented IL-8 induction. LY294002, a specific inhibitor of phosphoinositide 3 kinase (PI3 kinase), blunted the phosphorylation of AKT and inhibited LPC induction of RANTES more strongly than IL-8. Inhibition of p38 MAP kinase pathway by SB202190 also blocked LPC-induced expression of IL-8 and RANTES. Our results suggest that LPC induction of chemokines in MVEC is distinct from that in large vessel EC, and required the activities of MAP kinases and PI3 kinase for the induction of RANTES and IL-8. We speculate that the presence of LPC, a bioactive lipid product of phospholipase A2 (PLA2) and a constituent of oxidized low-density lipoprotein, can differentially influence the chemotaxis of particular leukocyte subpopulations during inflammation.

Introduction

Vascular endothelium maintains the non-thrombogenic and selective permeability properties of blood vessels. Endothelium also plays a role in inflammation when it is activated by multiple adverse conditions, including infection, hypoxia, altered blood flow or vascular injury [1]. Activated endothelial cells (EC) express adhesion molecules and secrete chemokines, which facilitate the recruitment of leukocytes to the sites of inflammation [1], [2], [3]. If unregulated, EC chemokines can promote pathogenic inflammation [2], [3].

We have focused on the role of lysophosphatidylcholine (LPC), a bioactive product of phospholipase A2 (PLA2) activity [4] and a component of oxidized low-density lipoprotein (LDL) [5], [6], in the induction of chemokines in EC. LPC elicits multiple responses in EC. It has been shown to induce the elaboration of growth factors [7], [8], adhesion molecules [9], [10] and the chemokine MCP-1 [11], [12], [13], but the mechanisms underlying these effects are not fully understood. LPC has been reported to be chemotactic for human leukocytes [14], [15], [16], and PLA2 inhibitors can block chemotaxis regulated by PLA2 [16]. PLA2 has also been implicated in inflammation [17]. These findings suggest a role for LPC in inflammation.

In this study, we have examined tissue-specific induction of chemokines by LPC using human EC isolated from three large vessels (pulmonary artery, aorta and umbilical vein) and microvessels, each representing a distinct area of the vasculature with differing vessel sizes and hemodynamics [18]. Of eight chemokines examined using a multi-probe ribonuclease protection assay (RPA), LPC markedly regulated the expression of only RANTES, MCP-1 and IL-8, which are chemotactic factors to T cells, monocytes and neutrophils, respectively. We show that LPC differentially regulates the expression of chemokines in small vs. large vessel EC. We investigated the signaling mechanisms for LPC induction of chemokines in microvascular EC (MVEC) because of the preferential expression of RANTES. We report that LPC induction of chemokines in MVEC is Gi-protein dependent, and the activities of phosphoinositide 3 kinase (PI3 kinase) and p38 MAP kinase are required for optimal induction of RANTES and IL-8 by LPC.

Section snippets

Cell culture

EC were isolated by collagenase digestion from sterile segments of human non-atherosclerotic thoracic aorta (HAEC) and pulmonary artery (HPAEC) after obtaining institutional review board approved family consent [19]. Human umbilical vein EC (HUVEC) were isolated as described earlier [20], and human dermal and lung MVEC were obtained commercially (Clonetics, MD). All EC were subject to two or more passages on fibronectin (1 μg/cm2) coated dishes under the same culture conditions prior to

Differential induction of chemokines by LPC in small and large vessel EC

Three EC preparations from large vessels (aorta, pulmonary artery and umbilical vein) and one from tissue microvessels were examined for the expression of a panel of eight chemokines in response to 100 μmol/l LPC. LPC regulated the expression of only three chemokines, viz. RANTES, MCP-1 and IL-8, which attract different subsets of leukocytes. Autoradiographs from an RPA are shown in Fig. 2. LPC induced the expression of RANTES in MVEC, but not in large vessel EC (Fig. 2, panels A and D). IL-8

Discussion

This is the first report of LPC induction of the T-cell chemoattractant RANTES [27] in EC. In this report, we demonstrate that LPC can differentially regulate the expression of chemokines in small vs. large vessel EC. We also show that LPC induction of chemokines in MVEC involves differential mechanisms.

Acknowledgements

This work was supported by a beginning grant-in aid from the Ohio Valley Affiliate of the American Heart Association (No. 0060321B to G.M.), and NIH grants (2R01 NS 32151 to R.M.R. and HL 29582 to G.M.C.). The authors thank the Perinatal Clinical Research Center (NIH GCRC RR-00080) of the Cleveland MetroHealth Hospital and Dr. Paul DiCorleto of the Cleveland Clinic Foundation for HUVEC.

References (52)

  • K. Murao et al.

    TNF-alpha stimulation of MCP-1 expression is mediated by the Akt/PKB signal transduction pathway in vascular endothelial cells

    Biochem Biophys Res Commun

    (2000)
  • M. Kawaguchi et al.

    Activation of extracellular signal-regulated kinase (ERK)1/2, but not p38 and c-Jun N-terminal kinase, is involved in signaling of a novel cytokine, ML-1

    J Biol Chem

    (2002)
  • C.H. Macphee

    Lipoprotein-associated phospholipase A2: a potential new risk factor for coronary artery disease and a therapeutic target

    Curr Opin Pharmacol

    (2001)
  • J.T. Wong et al.

    Lysophosphatidylcholine stimulates the release of arachidonic acid in human endothelial cells

    J Biol Chem

    (1998)
  • M. Goebeler et al.

    The chemokine repertoire of human dermal microvascular endothelial cells and its regulation by inflammatory cytokines

    J Invest Dermatol

    (1997)
  • G. Krishnaswamy et al.

    Human endothelium as a source of multifunctional cytokines: molecular regulation and possible role in human disease

    J Interf Cytokine Res

    (1999)
  • A.D. Luster

    Chemokines—chemotactic cytokines that mediate inflammation

    New Engl J M

    (1998)
  • R. Ross

    Atherosclerosis—an inflammatory disease

    New Engl J M

    (1999)
  • E. Hurt-Camejo et al.

    Phospholipase A(2) in vascular disease

    Circ Res

    (2001)
  • G. Murugesan et al.

    Role of lysophosphatidylcholine in the inhibition of endothelial cell motility by oxidized low density lipoprotein

    J Clin Invest

    (1996)
  • N. Kume et al.

    Lysophosphatidylcholine transcriptionally induces growth factor gene expression in cultured human endothelial cells

    J Clin Invest

    (1994)
  • T. Kita et al.

    Induction of endothelial platelet-derived growth factor-B-chain and intercellular adhesion molecule-1 by lysophosphatidylcholine

    Ann NY Acad Sci

    (1997)
  • M.I. Cybulsky et al.

    Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis

    Science

    (1991)
  • T. Murohara et al.

    Lysophosphatidylcholine promotes P-selectin expression in platelets and endothelial cells. Possible involvement of protein kinase C activation and its inhibition by nitric oxide donors

    Circ Res

    (1996)
  • N. Kume et al.

    Lysophosphatidylcholine, a component of atherogenic lipoproteins, induces mononuclear leukocyte adhesion molecules in cultured human and rabbit arterial endothelial cells

    J Clin Invest

    (1992)
  • J.A. Berliner et al.

    Atherosclerosis: basic mechanisms. Oxidation, inflammation, and genetics

    Circulation

    (1995)
  • Cited by (116)

    View all citing articles on Scopus
    View full text