11,12-Epoxyeicosatrienoic acid stimulates heme-oxygenase-1 in endothelial cells

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Abstract

As epoxyeicosatrienoic acids (EETs), particularly 11,12-EET, and the heme oxygenase/carbon monoxide (HO/CO) system share overlapping biological activities, we examined a possible link between 11,12-EET and HO activity in endothelial cells. Confocal microscopy analysis of immunostaining of HO-1 and HO-2 in cultured endothelial cells treated with 11,12-EET (1 μM) showed an increase in florescence of HO-1 protein in the various cellular compartments, but not of HO-2. Incubation of endothelial cells with 11,12-EET (1 μM) for 24 h increased the level of HO-1 protein by about three-fold. Similarly, incubation of endothelial cells with 8,9-EET and sodium nitroprussiate, a known inducer of HO-1, increased HO-1 protein without any effect on HO-2. Upregulation of HO-1 by 11,12-EET, as well as 8,9-EET, was associated with an increase in HO activity, which was inhibited by stannous mesoporphirin (10 μM). Incubation of rat aortas with 11,12-EET (1 μM for 60 min) increased HO activity.

These findings identify a novel effect of EETs on endothelial HO-1 and indicate that the signaling pathway of EETs in endothelial cells is possibly via an increase in HO-1 expression and activity.

Introduction

11,12-Epoxyeicosatrienoic acid (11,12-EET), derived through metabolism of arachidonic acid (AA) by cytochrome P-450 (CYP450) epoxygenase enzymes, has various biological activities: it dilates arterial vessels, causes inhibition of platelet aggregation, stimulates angiogenesis [1], inhibits both apical and basolateral Na(+)–K(+)–2Cl(−) cotransport in renal epithelial cells [2], is an important modulator of cardiac electrical excitability [3], induces polymorphonuclear leukocyte (PMN) aggregation [4], has antimigratory effects on smooth muscle cells [5], inhibits apoptosis [6], possesses fibrinolytic properties [7], is part of the endogenous antipyretic system [8]. EETs act as an endothelium-derived hyperpolarizing factor (EDHF) in some vascular beds [9], thus contributing to endothelium-dependent vasodilatation independently from nitric oxide.

Carbon monoxide (CO), a naturally produced gas by the action of heme-oxygenase (HO) on heme, causes endothelium-independent vasodilatation mediated both through cGMP and a direct effect on big-conductance calcium-activated K channels. The HO/CO system also modulates the proliferative response of vascular smooth muscle cells [10], has anti-atherosclerotic [11], and anti-hypertensive effects [12].

The major objective of this study was to determine the interactions between EETs and the HO/CO system in endothelial cells. Our results demonstrate that 11,12-EET is a potent inducer of HO-1 and, thus, CO production may mediate its effects in arterial vessels.

Section snippets

Chemicals

Metalloporphyrins were obtained from Porphyrin Products (Logan, UT, USA). 11,12-EET and 8,9-EET were provided by Dr. J.R. Falck. All other chemicals were obtained from Sigma Chemical (St. Louis, MO, USA). Stannous mesoporphyrin (SnMP) was dissolved in a solution of 50 mM NaCO3.

Cell culture conditions

Human dermal microvessel endothelial cells were grown in MCDB131 medium (GIBCO-BRL, City, ST, CO) supplemented with 10% FBS, 10 ng/mL epithelial growth factor (Sigma Chemicals, Saint Louis, MO, USA), and 1 μg/mL

Confocal microscopy analysis of HO-1 and HO-2 in endothelial cells

Fig. 1 shows the effect of 11,12-EET on HO-1 and HO-2 proteins. In EET-treated cells, membrane localization and cytoplasmic immunostaining of HO-1 (green signal) was increased (n = 4; p < 0.05) (Panel C compared to Panel A). HO-2 protein did not change with 11,12-EET incubation (Panels B and D). Staining intensity was computed as integrated optical density, measured in four different experiments, and digitally fixed images were analyzed as described before [16]. The integrated optical density of

Discussion

The results of this study showed for the first time that 11,12-EET causes induction of HO-1 in endothelial cells in culture and activation of HO in the aorta. This novel observation was substantiated by immunoflorescent microscopy, Western blot analysis, and enzymatic activity.

EETs are produced from AA in the endothelium. They are potent vasodilators of the arterial circulation, particularly of the coronary vessels. EETs also increase intracellular Ca2+ concentration in endothelial cells [17],

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    This work was supported by NIH grants HL55601 and HL34300 (NGA) and GM31278 (JRF) a grant from the Italian Ministry for University and Scientific and Technological Research (MURST).

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