Inhibitors of Cytochrome P450 4A Suppress Angiogenic Responses

Abstract

Arachidonic acid is acutely released from membrane phospholipids in response to a variety of stimuli, including vascular endothelial growth factor (VEGF),¹ fibroblast growth factor (FGF)-2,² and epidermal growth factor (EGF).^3–5 Arachidonic acid can be metabolized in blood vessels by enzymes of the cytochrome P450 4A (CYP4A) family to 20-hydroxyeicosatetraenoic acid (20-HETE) and by CYP 2C and 2W to epoxyeicosatrienoic acid (EETs). Both of these factors have been implicated in the regulation of angiogenesis. CYP4A products, in particular 20-HETE, have been reported to serve as second messengers for the vasoactive and mitogenic responses of a number of compounds in blood vessel cells and other cells, including angiotensin II (ang II) norepinephrine, endothelin, vasopressin, 5-hydroxy-tryptamine, and EGF. Blockade of the formation of 20-HETE attenuated the growth response to serum, norepinephrine, and EGF.^6–9

Recently, Amaral and colleagues¹⁰ reported that CYP4A, and by extension 20-HETE, plays a critical role in the angiogenesis induced by electrical stimulation of skeletal muscle. This angiogenic response is ang II- and VEGF-dependent. Jiang and colleagues¹¹ have shown that overexpression of CYP4A1 in smooth muscle promotes endothelial sprouting in renal arterial microvessels. Norepinephrine, ang II, and many growth factors stimulate cytosolic phospholipase A₂ (cPLA₂) and the release of arachidonic acid.^1,2,12,13 This promotes the formation of CYP metabolites of arachidonic acid (EET and/or 20-HETE) in the microvasculature. In addition, activation of the VEGF receptor 2 (VEGF-R2) stimulates phospholipase C and the downstream Raf-MAP kinase pathway.¹⁴ Indeed, Muthalif and colleagues¹⁵ reported that activation of MAPK by norepinephrine, ang II, and EGF is dependent on the formation of 20-HETE, which is generated after stimulation of cPLA2 by calcium/calmodulin-dependent protein kinase II. Activation of the Ras/MAPK pathway by 20-HETE amplifies cPLA2 activity and additional release of arachidonic acid by a positive feedback mechanism. Muthalif and colleagues¹⁵ proposed that this mechanism may play a central role in the regulation of other cellular signaling molecules involved in cell proliferation and growth. Both Ras and MAPK activation are associated with angiogenesis.^16,17 Thus, there may be an association between the actions of angiogenic growth factors metabolites of arachidonic acid derived from CYP4A activity.

VEGF, FGF-2, and EGF are all prominent agonists of members of the membrane-bound tyrosine kinase receptor family. These growth factors play a pivotal role in the regulation of angiogenesis and in the pathology of disorders associated with angiogenesis including the growth of solid tumors. Thus, the present studies were designed to test the hypothesis that the metabolism of arachidonic acid by CYP4A enzymes plays a central role in tyrosine kinase-dependent growth factors involved in angiogenesis. To address this hypothesis, we studied whether inhibition of CYP4A activity using two chemically dissimilar inhibitors, HET0016 and dibromododecenyl methylsulfonimide (DDMS), decreased the angiogenic response in the cornea of rats in vivo and the mitogenic response in human umbilical vein endothelial cells (HUVECs) in vitro to VEGF, a prototypical angiogenic growth factor. In addition, we extended the in vivo studies using HET0016 to include FGF-2 and EGF. We also studied whether HET0016 would affect cancer-induced angiogenesis in the cornea of rats in vivo.

Acknowledgments

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