The arachidonic acid epoxygenase is a component of the signaling mechanisms responsible for VEGF-stimulated angiogenesis

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Abstract

Cultured lung endothelial cells (LEC) respond to VEGF or arachidonic acid with increases in cell proliferation, the formation of tube-like structures, and the activation of Akt and ERK1/2 mediated growth pathways. LECs express a VEGF inducible Cyp2c44 epoxygenase and its 11,12- and 14,15-EET metabolites increase cell proliferation, tubulogenic activity, and the phosphorylation states of the ERK1/2 and Akt kinases. Ketoconazole, an epoxygenase inhibitor, blocks the cellular responses to VEGF. LECs expressing a Cyp2c44 epoxygenase small interference RNA show reductions in Cyp2c44 mRNA levels, and in their VEGF-stimulated proliferative and tubulogenic capacities; effects that are associated with decreases in VEGF-induced phosphorylation of the ERK1/2 and Akt kinases. We conclude that the Cyp2c44 arachidonic acid epoxygenase is a component of the signaling pathways associated with VEGF-stimulated angiogenesis, and suggest a role for EETs in the growth factor-induced changes in the activation states of the ERK1/2 and Akt kinase pathways.

Introduction

Angiogenesis and de novo vascularization are key components of physiological responses to inflammation and wound healing, and play also important roles during organ recovery from injury [1], [2], [3], [4]. Due its recognized roles in tumor vascularization [5], [6], [7], [8], angiogenesis has become an attractive candidate for the development of anti-cancer therapies aimed to reduce tumor access to blood derived oxygen, nutrients and hormones [5], [6]. Consequently, the identification of novel angiogenic factors, and the analysis of their mechanisms of action has become an active area of research, since it holds a promise for more effective and potentially less invasive approaches to cancer treatment. Current efforts to develop anti-angiogenic approaches for cancer treatment are primarily directed towards disrupting the interactions between VEGF and its receptors [6], [7], [8], [9], [10], [11], [12], [13]. Angiogenesis is a complex process that requires the coordinated contributions of endothelial cell proliferation and migration, leading to the formation of nascent capillary structures and, ultimately, functional vessels. These processes, initiated upon VEGF binding to its receptors involve among other things, the activation of kinase cascades such as those of the PI3K/Akt and MAKP/ERK1/2 signaling pathways [7], [8]. In addition to VEGF, several products of the oxidative metabolism of arachidonic acid (AA)1 have been shown to have pro-angiogenic properties, including prostanoids, [14], [15], cis–trans conjugated hydroxyeicosatetraenoic acids [16], as well as products of the cytochrome P450 (P450) AA epoxygenase and ω-hydroxylase pathways [17], [18], [19], [20], [21], [22], [23], [24], [25].

The P450 epoxygenase metabolizes endogenous pools of AA to 5,6-, 8,9-, 11,12-, and 14,15-EET [26], [27], [28], [29], in reactions that are predominantly catalyzed by members of the CYP2C gene subfamily [26], [29]. Among CYP2C isoforms, human CYP2C8 and CYP2C9, and mouse Cyp2c44 are known to be expressed in endothelial cells and to catalyze EET formation [25], [30], [31], [32]. The EETs are enzymatically hydrated to 5,6-, 8,9-, 11,12-, or 14,15-dihydroxyeicosatrienoic acid (DHET) by cytosolic epoxide hydrolase [26]. A role for EETs in cell proliferation was first identified in 1990 when it was demonstrated that exogenously added 14,15-EET induced the proliferation of cultured rat glomerular mesangial cells [33]. Subsequently, 14,15-EET was shown to mediate the proliferative responses to EGF and HB-EGF, and to promote activation of the PI3K/Akt and MAPK/ERK1/2 pathways [34], [35]. Since those early studies, 11,12- and 14,15-EET have been characterized a potent mitogens in cells obtained from kidney, brain, and endothelium [17], [22], [23], [24], [25], and 20-hydroxyeicosatetraenoic acid has been identified as pro-angiogenic [17], [18], [19], [20]. Moreover, 5,6-, 8,9-, and 11,12-EET were shown to stimulate endothelial cell proliferation [25], to activate endothelial cell PI3K, MAPK pathways [25], [36], and to be potent in vitro and in vivo angiogenic factors [25].

Recent studies identified PPARα and its ligands as effective inhibitors of tumor angiogenesis and growth [37], [38], [39], [40], [41]. Thus, PPARα ligands such Wyeth 14,643 and Fenofibrate inhibit tumor angiogenesis and growth by interfering with the proliferative activity of the host endothelial cells in a PPARα-dependent fashion [40], [41]. A role for the murine Cyp2c epoxygenases in the anti-angiogenic, anti-tumor effects of Wyeth 14,643 was indicated by the demonstration that the ligand down-regulates endothelial cell Cyp2c44 expression and reduces the concentrations of plasma circulating pro-angiogenic EETs [40]. On the other hand, except for a postulated role for prostanoids and a VEGF receptor in EET-mediated mitogenesis [24], the mechanism of action and the role of the Cyp2c epoxygenase metabolites in VEGF-mediated angiogenesis remains uncertain. As part of ongoing efforts to understand the relevance of the AA epoxygenase to the angiogenic activities associated with tumor vascularization and growth, we have utilized cultured mouse pulmonary endothelial cells for studies of the role of the Cyp2c44 AA epoxygenase in VEGF signaling. Here, we provide evidence that the Cyp2c44 arachidonic acid epoxygenase and 11,12-EET are components of the metabolic responses elicited during VEGF signaling and angiogenic activity. Inasmuch as VEGF is a current target of efforts to develop novel anti-tumor strategies, these studies identify the Cyp2c epoxygenases as potentially useful targets for the development of new anti-angiogenic approaches for cancer treatment.

Section snippets

Cell culture and siRNA transfections

Pulmonary micro vascular endothelial cells (LEC) were isolated from the lungs of transgenic mice expressing a temperature sensitive large tumor (T) antigen (Tag) under the control of a mouse histocompatibility H-2 Kb interferon responsive promoter [42], [43], [44]. Cells were propagated at 33° in EBM2 media (EBM2, Cambrex Bio Science Inc.) containing interferon (10 units/ml), 5% fetal calf serum and a mixture of hydrocortisone, hFGF-B, VEGF, IGF-1, hEGF, and ascorbic acid (the concentrations of

Results and discussion

The isolation of endothelial cells from mice and the establishment of primary cultures of these cells in numbers and purity sufficient for biochemical analysis, is a difficult process that has hindered studies of the roles played by lipid mediators in growth factor signaling [25], [40]. To obviate some of these complications, we utilized as subrogates cultures of immortalized mouse lung endothelial cells or LEC. These cells express phenotypes similar to those of freshly isolated primary

Acknowledgments

This work was supported by National Institutes of Health Grants DK38226 and GM 37922 (to JHC), and CA094849 and 074359 (to AP), and by the Vanderbilt University Mass Spectrometry Center, supported in part by Cancer Center Grant CA-68485.

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