Katherine N. Theken

Cytochrome P450 epoxygenases, soluble epoxide hydrolase, and the regulation of cardiovascular inflammation.

The cytochrome P450 (CYP) epoxygenase enzymes CYP2J and CYP2C catalyze the epoxidation of arachidonic acid to epoxyeicosatrienoic acids (EETs), which are rapidly hydrolyzed to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). It is well-established that CYP epoxygenase-derived EETs possess potent vasodilatory effects; however, the cellular effects of EETs and their regulation of various inflammatory processes have become increasingly appreciated in recent years, suggesting that the role of this pathway in the cardiovascular system extends beyond the maintenance of vascular tone. In particular, CYP epoxygenase-derived EETs inhibit endothelial activation and leukocyte adhesion via attenuation of nuclear factor-kappaB activation, inhibit hemostasis, protect against myocardial ischemia-reperfusion injury, and promote endothelial cell survival via modulation of multiple cell signaling pathways. Thus, the CYP epoxygenase pathway is an emerging target for pharmacological manipulation to enhance the cardiovascular protective effects of EETs. This review will focus on the role of the CYP epoxygenase pathway in the regulation of cardiovascular inflammation and (1) describe the functional impact of CYP epoxygenase-derived EET biosynthesis and sEH-mediated EET hydrolysis on key inflammatory process in the cardiovascular system, (2) discuss the potential relevance of this pathway to pathogenesis and treatment of cardiovascular disease, and (3) identify areas for future research.

Endothelial CYP epoxygenase overexpression and soluble epoxide hydrolase disruption attenuate acute vascular inflammatory responses in mice

Cytochrome P‐450 (CYP)‐derived epoxyei‐cosatrienoic acids (EETs) possess potent anti‐inflammatory effects in vitro. However, the effect of increased CYP‐mediated EET biosynthesis and decreased soluble epoxide hydrolase (sEH, Ephx2)‐mediated EET hydrolysis on vascular inflammation in vivo has not been rigorously investigated. Consequently, we characterized acute vascular inflammatory responses to endotoxin in transgenic mice with endothelial expression of the human CYP2J2 and CYP2C8 epoxygenases and mice with targeted disruption of Ephx2. Compared to wild‐type controls, CYP2J2 transgenic, CYP2C8 transgenic, and Ephx2−/− mice each exhibited a significant attenuation of endotoxin‐induced activation of nuclear factor (NF)‐κB signaling, cellular adhesion molecule, chemokine and cytokine expression, and neutrophil infiltration in lung in vivo. Furthermore, attenuation of endotoxin‐induced NF‐κB activation and cellular adhesion molecule and chemokine expression was observed in primary pulmonary endothelial cells isolated from CYP2J2 and CYP2C8 transgenic mice. This attenuationwas inhibited bya putative EET receptor antagonist and CYP epoxygenase inhibitor, directly implicating CYP epoxygenase‐derived EETs with the observed anti‐inflammatory phenotype. Collectively, these data demonstrate that potentiation of the CYP epoxygenase pathway by either increased endothelial EET biosynthesis or globally decreased EET hydrolysis attenuates NF‐κB‐dependent vascular inflammatory responses in vivo and may serve as a viable anti‐inflammatory therapeutic strategy.—Deng, Y., Edin, M. L., Theken, K N., Schuck, R N., Flake, G. P., Kannon, M. A., DeGraff, L. M., Lih, F. B., Foley, J., Bradbury, J. A., Graves, J. P., Tomer, K. B., Falck, J. R., Zeldin, D. C., Lee, C. R. Endothelial CYP epoxygenase overexpression and soluble epoxide hydrolase disruption attenuate acute vascular inflammatory responses in mice. FASEB J. 25, 703–713 (2011). www.fasebj.org

Activation of the Acute Inflammatory Response Alters Cytochrome P450 Expression and Eicosanoid Metabolism

Cytochrome P450 (P450)-mediated metabolism of arachidonic acid regulates inflammation in hepatic and extrahepatic tissue. CYP2C/CYP2J-derived epoxyeicosatrienoic and dihydroxyeicosatrienoic acids (EET+DHET) elicit anti-inflammatory effects, whereas CYP4A/CYP4F-derived 20-hydroxyeicosatetraenoic acid (20-HETE) is proinflammatory. Because the impact of inflammation on P450-mediated formation of endogenous eicosanoids is unclear, we evaluated P450 mRNA levels and P450 epoxygenase (EET+DHET) and ω-hydroxylase (20-HETE) metabolic activity in liver, kidney, lung, and heart in mice 3, 6, 24, and 48 h after intraperitoneal lipopolysaccharide (LPS) (1 mg/kg) or saline administration. Hepatic Cyp2c29, Cyp2c44, and Cyp2j5 mRNA levels and EET+DHET formation were significantly lower 24 and 48 h after LPS administration. Hepatic Cyp4a12a, Cyp4a12b, and Cyp4f13 mRNA levels and 20-HETE formation were also significantly lower at 24 h, but recovered to baseline at 48 h, resulting in a significantly higher 20-HETE/EET+DHET formation rate ratio compared with that for saline-treated mice. Renal P450 mRNA levels and P450-mediated eicosanoid metabolism were similarly suppressed 24 h after LPS treatment. Pulmonary EET+DHET formation was lower at all time points after LPS administration, whereas 20-HETE formation was suppressed in a time-dependent manner, with the lowest formation rate observed at 24 h. No differences in EET+DHET or 20-HETE formation were observed in heart. Collectively, these data demonstrate that acute activation of the innate immune response alters P450 expression and eicosanoid metabolism in mice in an isoform-, tissue-, and time-dependent manner. Further study is necessary to determine whether therapeutic restoration of the functional balance between the P450 epoxygenase and ω-hydroxylase pathways is an effective anti-inflammatory strategy.

Evaluation of cytochrome P450-derived eicosanoids in humans with stable atherosclerotic cardiovascular disease

OBJECTIVE Preclinical and genetic epidemiologic studies suggest that modulating cytochrome P450 (CYP)-mediated arachidonic acid metabolism may have therapeutic utility in the management of coronary artery disease (CAD). However, predictors of inter-individual variation in CYP-derived eicosanoid metabolites in CAD patients have not been evaluated to date. Therefore, the primary objective was to identify clinical factors that influence CYP epoxygenase, soluble epoxide hydrolase (sEH), and CYP ω-hydroxylase metabolism in patients with established CAD. METHODS Plasma levels of epoxyeicosatrienoic acids (EETs), dihydroxyeicosatrienoic acids (DHETs), and 20-hydroxyeicosatetraenoic acid (20-HETE) were quantified by HPLC-MS/MS in a population of patients with stable, angiographically confirmed CAD (N=82) and healthy volunteers from the local community (N=36). Predictors of CYP epoxygenase, sEH, and CYP ω-hydroxylase metabolic function were evaluated by regression. RESULTS Obesity was significantly associated with low plasma EET levels and 14,15-EET:14,15-DHET ratios. Age, diabetes, and cigarette smoking also were significantly associated with CYP epoxygenase and sEH metabolic activity, while only renin-angiotensin system inhibitor use was associated with CYP ω-hydroxylase metabolic activity. Compared to healthy volunteers, both obese and non-obese CAD patients had significantly higher plasma EETs (P<0.01) and epoxide:diol ratios (P<0.01), whereas no difference in 20-HETE levels was observed (P=NS). CONCLUSIONS Collectively, these findings suggest that CYP-mediated eicosanoid metabolism is dysregulated in certain subsets of CAD patients, and demonstrate that biomarkers of CYP epoxygenase and sEH, but not CYP ω-hydroxylase, metabolism are altered in stable CAD patients relative to healthy individuals. Future studies are necessary to determine the therapeutic utility of modulating these pathways in patients with CAD.

Genetic variation in the cytochrome P450 epoxygenase pathway and cardiovascular disease risk

The cytochrome (CYP) P450 epoxygenase pathway catalyzes the epoxidation of arachidonic acids to epoxyeicosatrienoic acids, which are subsequently hydrolyzed to less active dihydroxyeicosatrienoic acids by soluble epoxide hydrolase. Numerous preclinical studies have demonstrated that CYP-derived epoxyeicosatrienoic acids possess potent vasodilatory and anti-inflammatory properties in the cardiovascular system. In humans, functionally relevant polymorphisms, which may significantly modulate epoxyeicosatrienoic acid levels in vivo, have been identified in the genes encoding CYP2J2, CYP2C8, CYP2C9 and soluble epoxide hydrolase. Initial epidemiologic studies have demonstrated that genetic variation in the CYP epoxygenase pathway significantly modifies cardiovascular disease risk at the population level in humans, providing support for the hypothesis that modulation of this pathway may represent a novel approach to the prevention and treatment of cardiovascular disease. Future studies in humans validating these relationships and characterizing the underlying mechanisms will be necessary to fully understand the functional role of the CYP epoxygenase pathway in cardiovascular disease.

Enalapril reverses high-fat diet-induced alterations in cytochrome P450-mediated eicosanoid metabolism.

Metabolism of arachidonic acid by cytochrome P450 (CYP) to biologically active eicosanoids has been recognized increasingly as an integral mediator in the pathogenesis of cardiovascular and metabolic disease. CYP epoxygenase-derived epoxyeicosatrienoic and dihydroxyeicosatrienoic acids (EET + DHET) and CYP ω-hydroxylase-derived 20-hydroxyeicosatetraenoic acid (20-HETE) exhibit divergent effects in the regulation of vascular tone and inflammation; thus, alterations in the functional balance between these parallel pathways in liver and kidney may contribute to the pathogenesis and progression of metabolic syndrome. However, the impact of metabolic dysfunction on CYP-mediated formation of endogenous eicosanoids has not been well characterized. Therefore, we evaluated CYP epoxygenase (EET + DHET) and ω-hydroxylase (20-HETE) metabolic activity in liver and kidney in apoE(-/-) and wild-type mice fed a high-fat diet, which promoted weight gain and increased plasma insulin levels significantly. Hepatic CYP epoxygenase metabolic activity was significantly suppressed, whereas renal CYP ω-hydroxylase metabolic activity was induced significantly in high-fat diet-fed mice regardless of genotype, resulting in a significantly higher 20-HETE/EET + DHET formation rate ratio in both tissues. Treatment with enalapril, but not metformin or losartan, reversed the suppression of hepatic CYP epoxygenase metabolic activity and induction of renal CYP ω-hydroxylase metabolic activity, thereby restoring the functional balance between the pathways. Collectively, these findings suggest that the kinin-kallikrein system and angiotensin II type 2 receptor are key regulators of hepatic and renal CYP-mediated eicosanoid metabolism in the presence of metabolic syndrome. Future studies delineating the underlying mechanisms and evaluating the therapeutic potential of modulating CYP-derived EETs and 20-HETE in metabolic diseases are warranted.

PharmGKB summary: ibuprofen pathways.

Ibuprofen is a traditional nonsteroidal anti-inflammatory drug (NSAID) widely used for its analgesic, anti-inflammatory, and antipyretic properties [1,2]. At low over-the-counter doses (800–1200 mg/day), ibuprofen is indicated to relieve minor pain and inflammation, including headache, muscular aches, toothache, fever, backache, and dysmenorrhea. At prescription doses (1800–2400 mg/day), it is used for the long-term treatment of rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, and other chronic conditions [2]. Ibuprofen has also been used off-label to promote closure of patent ductus arteriosus (PDA) in preterm neonates [3]. It is commonly used in pediatric patients for the treatment of acute pain and fever (5–10 mg/kg every 6–8 h) due to its relative safety compared with aspirin and its high efficacy compared with acetaminophen [2]. Prescription doses of ibuprofen (adult: 200–800 mg every 6–8 h; pediatric: 5–10 mg/kg every 6–8 h) have greater antipyretic and analgesic effects in both children and adults compared with commonly used doses of acetaminophen (adult: 500–1000 mg every 6–8 h; pediatric: 10–15 mg/kg every 4–6 h) [4].

Dual modulation of cyclooxygenase and CYP epoxygenase metabolism and acute vascular inflammation in mice

Cyclooxygenase (COX)-derived prostaglandins and cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids are important regulators of inflammation; however, functional interactions between these pathways in the regulation of vascular inflammation in vivo have not been studied. We investigated the relative and additive effects of endothelial CYP2J2 overexpression (Tie2-CYP2J2-Tr), global sEH disruption (Ephx2(-/-)), and pharmacologic COX inhibition with indomethacin on the acute vascular inflammatory response to endotoxin in mice. Compared to vehicle-treated wild-type C57BL/6 controls, induction of myeloperoxidase (MPO) activity in lung and liver was similarly attenuated in Tie2-CYP2J2-Tr mice, Ephx2(-/-) mice and wild-type mice treated with moderate dose indomethacin. Dual modulation of both pathways, however, did not produce an additive anti-inflammatory effect. These findings demonstrate that both COX and CYP epoxygenase-mediated eicosanoid metabolism are important regulators of the acute vascular inflammatory response in vivo, and suggest that the anti-inflammatory effects of modulating each pathway may be mediated, at least in part, by overlapping mechanisms.

Cyclooxygenase Inhibition: Pain, Inflammation, and the Cardiovascular System

Inhibitors of the cyclooxygenases (COXs), the nonsteroidal antiinflammatory drugs (NSAIDs), relieve inflammatory pain, but are associated with gastrointestinal and cardiovascular complications. Given the widespread use of NSAIDs, there has been a longstanding interest in optimizing their risk–benefit ratio, for example by reducing their gastrointestinal risk. More recently, the focus has shifted toward the cardiovascular complications of NSAIDs and very large prospective studies have been performed to compare cardiovascular risk across distinct NSAIDs. Surprisingly, much less attention has been paid to the efficacy side of the risk–benefit ratio. There is marked variability in the degree of pain relief by NSAIDs due to the complex interplay of molecular mechanisms contributing to the pain sensation, variability in the disposition of NSAIDs, and imprecision in the quantification of human pain. Here we discuss how NSAIDs relieve pain, how molecular mechanisms relate to clinical efficacy, and how this may inform our interpretation of clinical trials.

Weight-adjusted aspirin for cardiovascular prevention

Understanding the sources of variability in patients’ responses to medicines has the potential to improve efficacy and safety through personalisation of treatment. Individual factors—ie, a patient’s risk of cardiovascular events—are already considered when deciding who is prescribed low-dose aspirin for cardio vascular prevention. However, aspirin inhibits just one of several pathways of platelet activation, so it is not surprising that many patients still exper ience cardiovascular events. Factors that potentially contribute to these treatment failures, sometimes named as aspirin resistance, have been studied ex ten sively, and several plausible mechanisms have been suggested, including non-adherence, accelerated platelet turnover in patients with diabetes, drug interactions with non-steroidal anti-inflammatory drugs, reduced bioavailability due to enteric coating, and bodyweight. Considering these factors when tailoring dose, dosing frequency, and perhaps even time of dosing to individual patients might have substantial public health effects. In The Lancet, Peter Rothwell and colleagues report their investigation of the effects of the interaction between bodyweight and aspirin dose on the risk of vascular events, bleeding, and cancer by combining individual patient data from randomised controlled trials. Their analysis included nine trials of aspirin in primary prevention of cardiovascular events and major bleeding (including roughly 103 000 patients) and four of aspirin in secondary prevention of stroke (including roughly 13 000 patients). The authors found that lowdose aspirin (≤100 mg/day) prevented cardiovascular events only in individuals with low bodyweight (<70 kg), whereas higher doses of aspirin (≥300 mg/day) were effective only in individuals weighing 70 kg or more. A similar interaction was observed for colorectal cancer and bleeding, albeit with an apparently higher weight cutoff (≥80 kg for colorectal cancer and ≥90 kg for major bleeds). Notably, the analysis implies that weightadjusted aspirin dosing might substantially improve the effectiveness of aspirin. Low-dose aspirin reduced cardiovascular events by 23% (hazard ratio [HR] 0·77, 95% CI 0·68–0·87; p<0·0001) in individuals weighing less than 70 kg, compared with a reduction of only 12% (0·88, 0·81–0·95; p=0·0008) in the overall primary prevention population when weight was not considered. Also, the authors suggest that sex differences in bodyweight—eg, more men than women weighed more than 70 kg— might explain aspirin’s long recognised lack of efficacy in preventing stroke in men. The difference in stroke risk between men and women was no longer detectable when bodyweight was accounted for. Weight can affect drug distribution and clearance, and is known to modulate platelet inhibition by clopidogrel and prasugrel. Moreover, weight-adjusted dosing is common for intravenous platelet inhibitors (glycoprotein IIb/IIIa antagonists and cangrelor). With regard to aspirin, extensive clearance by esterases in the intestine, plasma, blood cells, and the liver results in a systemic bioavailability of just 50%. It seems plausible that individuals with a larger body mass would have a greater quantity of these esterases than would smaller individuals, resulting in reduced aspirin bioavailability. This hypothesis is supported by the observation that the interaction between weight and risk of cardiovascular events was accentuated with enteric-coated aspirin, which has about 40% lower systemic bioavailability than does non-coated aspirin. The purpose of using low doses of aspirin is to limit systemic aspirin exposure and the consequent depression of homoeostatic cyclo-oxygenase (COX) products in vasculature and the stomach. Aspirin irreversibly acetylates COX-1 in platelets that are unable to synthesise new enzyme. Thus, low doses of aspirin can inhibit platelet function as long as more platelets are inhibited than are newly released from megakaryocytes. This inhibition can occur presystemically before inactivation by liver esterases. Prostacyclin, the vascular COX product intended to be spared by use of low doses of aspirin, is a potent platelet inhibitor and a central molecule in the important negative feedback mechanism that limits the growth of thrombi at sites of vascular injury. The apparent lack of efficacy of intermediate-dose and high-dose aspirin in individuals weighing less than 70 kg raises the question of whether these treatment failures might reflect inhibition of vascular prostacyclin because of the increased systemic bioavailability of aspirin in this subgroup. How might weight-adjusted dosing of aspirin affect bleeding risk? In the trials included in the analysis, 80% of men and nearly 50% of women weighed 70 kg or more. Published Online July 12, 2018 http://dx.doi.org/10.1016/ S0140-6736(18)31307-2