Elsevier

Atherosclerosis

Volume 196, Issue 1, January 2008, Pages 201-209
Atherosclerosis

Structure–function properties of the apoE-dependent COX-2 pathway in vascular smooth muscle cells

https://doi.org/10.1016/j.atherosclerosis.2007.03.038Get rights and content

Abstract

Apolipoprotein (apoE) E is a multifunctional protein that plays a critical role in atherogenesis, in part by regulating the intimal proliferation of vascular smooth muscle cells. Recently, a novel cyclooxygenase (COX)-2 pathway was shown to contribute to the anti-proliferative action of human apoE3 in vascular smooth muscle cells (VSMC). Here, we provide insight into the structure–function properties by which apoE mediates these effects. ApoE3 is most effective in promoting COX-2 expression as a lipid-free protein and is less active after lipidation. Alterations in the stability of the helix bundle N-terminal domain of apoE that contains the binding site for the low density lipoprotein (LDL) receptor and heparin do not affect the up-regulation of the COX-2 pathway. In addition, the apoE2, 3, and 4 isoforms are all capable of up-regulating the COX-2 pathway. Finally, the effect of apoE on COX-2 was found to be independent of expression on the VSMC surface of the LDL receptor and heparan sulfate proteoglycans (HSPG). In summary, our data indicates that apoE, particularly in the lipid-free state, can up-regulate COX-2 in murine vascular smooth muscle cells apparently independently of binding to the LDLR, LRP or HSPG.

Introduction

Apolipoprotein E (apoE) is a component of high density lipoproteins (HDL) and triglyceride-rich proteins (VLDL, chylomicrons) and, as such, is an important regulator of lipid homeostasis. The apoE molecule is composed of 22 kDa (residues 1–191) and 10 kDa (residues 216–299) domains connected by a hinge region. Each of these domains has distinct functions [1]. The 22 kDa N-terminal domain is important for receptor binding while the 10 kDa C-terminal domain is involved predominantly in lipid binding [2], [3]. The lipid transport functions of apoE depend on its interaction with the LDL receptor (LDLR) family and heparan sulfate proteoglycans (HSPG) [4], [5]. These apoE receptors are involved in lipoprotein metabolism, cholesterol homeostasis, brain development, vascular integrity, and protection against the development of atherosclerosis [6], [7], [8].

Independent of its ability to regulate lipoprotein metabolism, apoE affects the vascular system by modulating inflammation [9], suppressing oxidative stress [10], and inhibiting the proliferation of vascular smooth muscle cells [11], [12], [13], [14], [15]. The role of apoE in the proliferation of vascular smooth muscle cells (VSMC) in particular has important implications for atherosclerosis, vascular remodeling and restenosis.

The cyclooxygenase (COX) enzymes convert arachidonic acid to prostaglandin H2 that eventually leads to the biosynthesis of eicosanoids; these include prostaglandins (PGE2, PGF, PGD2), prostacyclin (PGI2) and thromboxane (TXA2). COX exist as two isoforms: COX-1 is constitutively expressed whereas COX-2 is up-regulated by various pro-inflammatory mediators, such as lipopolysaccharide, growth factors, cytokines and apolipoproteins [16]. The major eicosanoids produced by the activation of COX-2 in endothelial cells are prostacyclin (PGI2) and prostaglandin E2 (PGE2) [17]. The PGI2 signaling cascade is dependent upon activation of its prostacyclin receptor (IP), which is generally associated with a G stimulatory protein coupled receptor [18]. The physiological consequences of eicosanoid metabolite production by the COX-2 enzyme are tissue- and cell-specific. In vascular systems, COX-2-dependent production of prostacyclin can affect platelet coagulation [19], [20], [21] and leukocyte adhesion [22], [23]. COX-2 activation in endothelial cells and vascular smooth muscle cells plays a vital role in the development and stability of atherosclerotic plaque. COX-2 is up-regulated in atherosclerotic lesions either by inflammatory cytokines or platelet activation [24], [25].

In vitro studies have implicated several molecular pathways by which apoE may directly influence the proliferation of VSMC. Ishigami et al. showed that apoE inhibits platelet derived growth factor (PDGF)-induced VSMC proliferation by mitogen-activated protein kinase (MAPK) mediated suppression of the G1 cell cycle gene, cyclin D[14], [15]. The initial signal transduction mechanism describing apoE's anti-proliferative activity in VSMC involves binding of the C-terminal domain of apoE to perlecan HSPG through PDGF stimulation to induce iNOS which suppresses MAP kinase activity [13], [14]. This suppression subsequently arrests cells in the G0 phase of the cell cycle (21). We have identified another signaling pathway by which apoE binds to an unknown receptor to up-regulate COX-2 and prostacyclin production [11]. Prostacyclin activates the Gs protein-coupled (IP) receptor which eventually leads to inhibition of the CREBP and pocket protein-E2F of cyclin A, thereby, preventing cell progression to the S-phase [12]. In this study, we sought to further characterize important structure–functional properties of apoE regarding its ability to mediate up-regulation of the COX-2 pathway.

Section snippets

VSMC culture

Primary murine aortic smooth muscle cells were isolated from wild-type C57BL/6 (Jackson Laboratory, Bar Harbor, Maine) or LDLR/apobec double knockout mice as described [26] and used between passage three and five. The cells were maintained in 40:40 ratio of Dulbecco's modified Eagle's/Ham F12 medium containing 10% heat-inactivated fetal bovine serum (HI-FBS) and 25 mM HEPES, penicillin, streptomycin, and glutamine. The purity of the VSMC population was determined by the expression of α-smooth

The ability to up-regulate COX-2 is confined to the N-terminal 22 kDa domain of apoE3

Previous experimental data indicated that anti-mitogenic effect of apoE3 was contained within the 22 kDa N-terminal domain but not the C-terminal 10 kDa fragment of apoE [11]. Hence, to confirm that the anti-mitogenic effect of apoE3-22 kDa domain was up-stream of its ability to inhibit cell cycle progression, COX-2 mRNA and prostaglandin production were analyzed after stimulation of primary murine VSMC with apoE3 variants. As shown in Fig. 1A, the 22 kDa domain of human apoE3 exhibited similar

Discussion

In this study, some structure–function features of apoE were analyzed to determine their influence on COX-2 signaling pathway in VSMC. The results demonstrate that the receptor binding domain of the apoE molecule (spanning residues 140–150) is important, but proper orientation of the α-helix spanning residues 140–150 in the N-terminal domain is not necessary for appropriate receptor interactions. Furthermore, lipid-free apoE is more effective than lipid-bound apoE in the induction of COX-2 and

Acknowledgement

We thank Dr. Ellen Pure for assistance and advice. This work was supported by NIH grants HL56083 (SLK), HL07443 (KA), and P01-HL62250 (DJR).

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