Cytochromes P450 and the Bioactivation of Arachidonic Acid
Environmental Cardiopulmonary Disease Group
The Zeldin group was the first to:
- Clone CYP2J2, a human P450 enzyme that is active in the metabolism of arachidonic acid to epoxyeicosatrienoic acids (EETs; Figure 1) and is abundant in heart (Wu et al., J. Biol. Chem., 1996)
- Show that EETs are endogenous constituents of cardiovascular tissues ( Wu et al., J. Biol. Chem., 1996; Wu et al., J. Biol. Chem., 1997)
- Localize CYP2J2 expression to cardiac myocytes and endothelial cells ( Wu et al., J. Biol. Chem., 1997; Node et al., Science, 1999)
P450-derived eicosanoid effects on cardiovascular system function
Subsequent studies demonstrated that EETs improved heart contractile function following ischemia ( Wu et al., J. Biol. Chem., 1997; Seubert et al., Circ. Res., 2004; Seubert et al., Circ. Res., 2006), inhibited cardiac L-type calcium channels (Chen et al., Mol. Pharmacol., 1999), and activated ATP-sensitive potassium channels (Lu et al., J. Physiol., 2006; Figure 2). and activated ATP-sensitive potassium channels (Lu et al., J. Physiol., 2006; Firgure 2). The group developed cardiomyocyte-specific CYP2J2 transgenic mice that have improved post-ischemic cardiac function and represent the first in vivo animal model for evaluating P450 functions in heart (Seubert et al., Circ. Res., 2004).
In collaboration with Harvard University’s James Liao, M.D., the group demonstrated that CYP2J2-derived eicosanoids decreased cytokine-induced endothelial adhesion molecule expression by a mechanism that involves inhibition of the transcription factor NF-kB (Node et al., Science, 1999). The group showed that exposure of endothelial cells to hypoxia-reoxygenation decreased CYP2J2 expression and that maintenance of CYP2J2 protein levels attenuated hypoxia-reoxygenation-induced cell death (Yang et al., Mol. Pharmacol., 2001).
More recently, Zeldin's group has developed several transgenic mice with endothelial-specific expression of human CYP2C8 or CYP2J2 to investigate the role of endothelial EETs on cardiovascular function. Transgenic expression of either CYP2J2 or CYPC8 in endothelial cells increased EET generation enhanced vasodilatory responses, and attenuated vasoconstriction and hypertension in mice (Lee et al., FASEB J, 2010). In addition, endothelial CYP2J2 or CYP2C8 expression was protective against lung inflammation induced by intraperitoneal injection of bacterial lipopolysaccharide. Endothelial cell adhesion molecule, inflammatory cytokine production, and cellular infiltration was reduced in lungs from these mice, which suggests an important role for CYP epoxygenases in regulating host responses to infection (Deng et al., FASEB J, 2011).
The group reported the detrimental effect of endothelial CYP2C8 expression on post-ischemic cardiac functional recovery. While increased EETs were cardioprotective following ischemia (Seubert et al., Circ. Res., 2004; Seubert et al., Circ. Res., 2006), endothelial CYP2C8 expression led to increased reactive oxygen species and dihydroxyoctadecanoic acid (DiHOME) generation, which had vasoconstrictive and cardiodepressive effects in the post-ischemic heart (Edin et al., FASEB J 2011). In collaboration with Craig Lee, Pharm.D, Ph.D. of the University of North Carolina and Nancy Brown, M.D. of Vanderbilt University, the group found that EPHX2 (soluble epoxide hydrolase) polymorphisms correlate with vasodilatory responses in humans. This finding provides a functional link between human EPHX2 polymorphism and regulation of vascular tone (Lee, et al., Hypertension. 2011).
Together, these studies demonstrate that CYP2J2, CYP2C8 and their eicosanoid metabolites play critical roles in cardiac myocytes and endothelial cells and may be involved in limiting vascular inflammation and regulating cardiac dysfunction following ischemia.
P450-derived eicosanoid effects on renal system function
Initial studies showed that P450-derived eicosanoids contribute substantially to integrated renal function (Figure 3). In 1999, the group cloned a new mouse P450 called CYP2J5 that is primarily expressed in the kidney, is active in the metabolism of arachidonic acid to EETs and localizes to proximal tubules and collecting ducts, sites where EETs affect renal fluid/electrolyte transport and mediate the actions of several hormones ( Ma et al., J. Biol. Chem., 1999; Mol. Pharmacol., 2004). Interestingly, maximal CYP2J5 expression occurs during a critical time in postnatal renal development when abnormalities in renal function appear in animals that develop hypertension.
Further evidence to support the hypothesis that CYP2J products contribute to the development or maintenance of hypertension comes from two subsequent studies: one showing upregulation of a CYP2J immunoreactive protein and increased EET biosynthesis in kidneys of spontaneously hypertensive rats (Yu et al., Mol. Pharmacol., 2000) and another showing the chromosomal mapping of the Cyp2j cluster to an area that co-segregates with the hypertensive phenotype in Dahl salt-sensitive rats (Ma et al., Genomics, 1998).
The group generated Cyp2j5 knockout mice to investigate the role of CYP2J5 products on renal homeostasis. Female Cyp2j5-/- mice have increased blood pressure, enhanced tubular transport rates, and increased response to endogenous vasoconstrictors. This data suggests an important role for renal CYP epoxygenases in blood pressure regulation (Athirakul et al., FASEB J, 2008).
P450-derived eicosanoid effects in brain
Zeldin's group identified several functionally relevant EPHX2 polymorphic variants (Przybyla-Zawislak et al., Mol. Pharmacol, 2003) and demonstrated that one of these variants associates with cardiovascular disease and stroke in humans (Fornage et al., Hum. Mol. Genetics, 2005; Lee et al., Hum. Mol. Genetics, 2006). In collaboration with Daowen Wang, Ph.D., the group found that carriers of the EPHX2 R287Q polymorphism (reduced hydrolase activity, increased EETs) were protected against incidence of ischemic stroke in Han Chinese (Zhang, et al., Pharmacogenet Genomics, 2008).
P450-derived eicosanoid effects on angiogenesis and cancer
In collaboration with Daowen Wang, Ph.D., our group has defined an important role for CYP epoxygenases and EETs in tumor growth and metastasis. CYP2J2 expression is abundant in human tumor tissues. Expression of CYP2J2 or treatment with synthetic EETs accelerates carcinoma cell proliferation, prevents apoptosis and enhances primary tumor grown in vivo (Jiang et al., Cancer Res, 2005). CYP2J2 is also upregulated in leukemia cells from peripheral blood and bone marrow in patients with malignant hematologic diseases (Chen, et al., J Pharmacol Exp Ther, 2011). CYP epoxygenase expression or EET treatment also promotes tumor metastasis independent of effects on primary tumor growth. CYP2J2 expression induces cancer cell migration, invasion, adhesion, and colony formation in soft agar in vitro. Importantly, CYP epoxygenase expression enhances pro-metastatic proteins such as CD44 and leads to lung metastasis in vivo. (Jiang et al., Cancer Res, 2007).
EETs also activate metalloproteinases which cleave and release heparin-bound EGF (HB-EGF). HB-EGF activates the EGF receptor to induce mitogenic signaling events and cancer cell proliferation (Cheng, et al., Acta Pharmacol Sin, 2010). Interestingly, CYP epoxygenase inhibition or antisense to CYP2J2 blocks these effects. Together, these studies suggest the possibility that selective CYP2J2 inhibition may represent a novel strategy for treatment of cancer. In collaboration with Daniel Mansuy, Ph.D., and Pierre Lafite, Ph.D., we generated a 3D homology model of CYP2J2 and characterized the unusual regioselectivity and active site topology of CYP2J2 using terfenadine derivatives (Lafite, et al., Bioorg Med Chem Lett, 2006). In addition a series of terfenadine and ebastine derivatives were found to be highly selective CYP2J2 inhibitors (Lafite, et al., Arch Biochem Biophys, 2007).
In collaboration with Daowen Wang, Ph.D., we tested these and similar compounds for in vitro and in vivo anti-tumor activity. Each of the compounds decrease tumor cell proliferation, decrease tumor invasion and increase induction of apoptosis in vitro. In mice inoculated with MDA-MB-235 tumor cells, these terfenadine derivatives decreased tumor volume and increased median survival from 58 to 80 days, while showing no toxic effects in the mice (Chen, et al., J Pharmacol Exp Ther, 2009). These studies provide proof-of-concept that terfenadine derivatives may represent novel chemotherapeutics for cancer in humans.
Previous reports revealed that CYP epoxygenases and EETs prevent tumor cell apoptosis while stimulating tumor cell growth, migration, and metastasis; however, the contribution of endothelial-derived EETs to tumor growth and metastasis was unknown. In collaboration with Dr. Dipak Panigrahy, we examined mice with increased endothelial expression of human P450 epoxygenases or with global Ephx2 disruption. These ‘high-EET’ mice have increased endothelial proliferation and migration in vitro, and increased primary tumor growth and metastasis in vivo. In contrast, mice with endothelial overexpression of human EPHX2 have decreased EETs and exhibit reciprocal phenotypes. Pharmacological treatment with synthetic EETs or EPHX2 inhibitors promoted tumor growth and metastasis, whereas treatment with the EET antagonist, 14,15-EEZE, reduced tumor growth and promoted survival (Panigrahy, et al., J. Clin. Invest., 2012). Our subsequent study with these mice showed that P450-derived EETs and sEH regulate angiogenesis in other disease models, including retinal vascularization, organ regeneration and wound healing (Panigrahy, et al., PNAS, 2013). Together, these findings have clinical relevance as EPHX2 inhibitors have been proposed as a novel therapeutics for various diseases. In addition, our data suggests the potential use of EET antagonists as novel anti-neoplastic agents.
Additional studies revealed novel roles for P450-derived eicosanoids in endothelial function. In collaboration with Dr. Lois Smith, we determined that EPHX2 and CYP2C enzymes regulate the aberrant retinal vascular development and detachment in oxygen-induced retinopathy, a model of retinal disease in premature infants (Shao, et al, Arterioscler. Thromb. Vasc. Biol., 2014). In collaboration with Dr. Leonard Zon, we showed that EETs enhance hematopoietic stem and progenitor cell (HSPC) engraftment in a zebrafish bone marrow transplantation model (Li, et al., Nature, 2015). Prior to these studies, our understanding of the role of CYPs, sEH and EETs in angiogenesis and related phenomena was limited to a handful of in vitro studies. CYP-derived EETs, acting through a common VEGF pathway involving PI3K activation, are now known to regulate angiogenesis in a wide variety of disease models in vivo.
The group cloned and characterized the human CYP2J2 gene, and identified several functionally relevant CYP2J2 polymorphic variants (King et al., Mol. Pharmacol., 2002). One of these variants associates with reduced CYP2J2 expression/activity and is present at higher frequency in patients with cardiovascular disease versus normals (Spiecker et al., Circulation, 2004).
In addition to the CYP2J work, Zeldin’s group has cloned and characterized CYP2C8, the major human liver P450 arachidonic acid epoxygenase ( Zeldin et al., Arch. Biochem. Biophys., 1995; Zeldin et al., Arch. Biochem. Biophys.,1996) and characterized several mouse CYP2C isoforms ( Luo et al., Arch. Biochem. Biophys., 1998; Tsao et al., Mol. Pharmacol., 2000; Tsao et al., J. Pharmacol. Exp. Therap., 2001; DeLozier et al., J. Pharmacol. Exp. Therap., 2004; Wang et al., Mol. Pharmacol., 2004).
The group has identified two new P450 subfamilies, the CYP2Ns and the CYP2Ps, which relate phylogenetically to the CYP2Js. CYP2Ns and the CYP2Ps are abundant in extrahepatic tissues and are active in the metabolism of arachidonic acid to EETs ( Oleksiak et al., J. Biol. Chem., 2000; Oleksiak et al., Arch. Biochem. Biophys., 2003).
The group was the first to document a role for soluble epoxide hydrolase (EPHX2, sEH) in the regio- and stereoselective metabolism of EETs to dihydroxyeicosatrienoic acids (DHETs) and to show that DHETs were endogenous constituents of liver and lung ( Zeldin et al., Arch. Biochem. Biophys., 1995; Zeldin et al., Arch. Biochem. Biophys., 1996; Zeldin et al., J. Clin. Invest., 1995). In collaboration with Deanna Kroetz, Ph.D. of the University of California, San Francisco, the group’s data showed that sEH regulates EET hydrolysis and blood pressure in spontaneously hypertensive rats (Yu et al., Circ. Res., 2000).
Zeldin's research has led to a better understanding of the role of cytochromes P450 in the metabolism of arachidonic acid to compounds which modulate fundamental biological processes in extrahepatic tissues, especially the heart, kidney and vasculature. The opportunities for translational research in this area include development of novel therapeutics for blood pressure control, vascular inflammation, atherosclerosis, ischemic heart disease, and stroke. The identification of individuals who are at increased risk for these disorders due to P450 or sEH genetic variation may also lead to new approaches to disease prevention.