Unraveling the Role of 12- and 20- HETE in Cardiac Pathophysiology: G-Protein-Coupled Receptors, Pharmacological Inhibitors, and Transgenic Approaches.

ABSTRACT: Arachidonic acid-derived lipid mediators play crucial roles in the development and progression of cardiovascular diseases. Eicosanoid metabolites generated by lipoxygenases and cytochrome P450 enzymes produce several classes of molecules, including the epoxyeicosatrienoic acid (EET) and hydroxyeicosatetraenoic acids (HETE) family of bioactive lipids. In general, the cardioprotective effects of EETs have been documented across a number of cardiac diseases. In contrast, members of the HETE family have been shown to contribute to the pathogenesis of ischemic cardiac disease, maladaptive cardiac hypertrophy, and heart failure. The net effect of 12(S)- and 20-HETE depends upon the relative amounts generated, ratio of HETEs:EETs produced, timing of synthesis, as well as cellular and subcellular mechanisms activated by each respective metabolite. HETEs are synthesized by and affect multiple cell types within the myocardium. Moreover, cytochrome P450-derived and lipoxygenase- derived metabolites have been shown to directly influence cardiac myocyte growth and the regulation of cardiac fibroblasts. The mechanistic data uncovered thus far have employed the use of enzyme inhibitors, HETE antagonists, and the genetic manipulation of lipid-producing enzymes and their respective receptors, all of which influence a complex network of outcomes that complicate data interpretation. This review will summarize and integrate recent findings on the role of 12(S)-/20-HETE in cardiac diseases.

An ALOX12-12-HETE-GPR31 signaling axis is a key mediator of hepatic ischemia-reperfusion injury.

Hepatic ischemia-reperfusion (IR) injury is a common clinical issue lacking effective therapy and validated pharmacological targets. Here, using integrative 'omics' analysis, we identified an arachidonate 12-lipoxygenase (ALOX12)-12-hydroxyeicosatetraenoic acid (12-HETE)-G-protein-coupled receptor 31 (GPR31) signaling axis as a key determinant of the hepatic IR process. We found that ALOX12 was markedly upregulated in hepatocytes during ischemia to promote 12-HETE accumulation and that 12-HETE then directly binds to GPR31, triggering an inflammatory response that exacerbates liver damage. Notably, blocking 12-HETE production inhibits IR-induced liver dysfunction, inflammation and cell death in mice and pigs. Furthermore, we established a nonhuman primate hepatic IR model that closely recapitulates clinical liver dysfunction following liver resection. Most strikingly, blocking 12-HETE accumulation effectively attenuated all pathologies of hepatic IR in this model. Collectively, this study has revealed previously uncharacterized metabolic reprogramming involving an ALOX12-12-HETE-GPR31 axis that functionally determines hepatic IR procession. We have also provided proof of concept that blocking 12-HETE production is a promising strategy for preventing and treating IR-induced liver damage.

Pigment epithelium-derived factor inhibits retinal microvascular dysfunction induced by 12/15-lipoxygenase-derived eicosanoids.

We recently demonstrated that 12/15-lipoxygenase (LOX) derived metabolites, hydroxyeicosatetraenoic acids (HETEs), contribute to diabetic retinopathy (DR) via NADPH oxidase (NOX) and disruption of the balance in retinal levels of the vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). Here, we test whether PEDF ameliorates retinal vascular injury induced by HETEs and the underlying mechanisms. Furthermore, we pursue the causal relationship between LOX-NOX system and regulation of PEDF expression during DR. For these purposes, we used an experimental eye model in which normal mice were injected intravitreally with 12-HETE with/without PEDF. Thereafter, fluorescein angiography (FA) was used to evaluate the vascular leakage, followed by optical coherence tomography (OCT) to assess the presence of angiogenesis. FA and OCT reported an increased vascular leakage and pre-retinal neovascularization, respectively, in response to 12-HETE that were not observed in the PEDF-treated group. Moreover, PEDF significantly attenuated the increased levels of vascular cell and intercellular adhesion molecules, VCAM-1 and ICAM-1, elicited by 12-HETE injection. Accordingly, the direct relationship between HETEs and PEDF has been explored through in-vitro studies using Müller cells (rMCs) and human retinal endothelial cells (HRECs). The results showed that 12- and 15-HETEs triggered the secretion of TNF-α and IL-6, as well as activation of NFκB in rMCs and significantly increased permeability and reduced zonula occludens protein-1 (ZO-1) immunoreactivity in HRECs. All these effects were prevented in PEDF-treated cells. Furthermore, interest in PEDF regulation during DR has been expanded to include NOX system. Retinal PEDF was significantly restored in diabetic mice treated with NOX inhibitor, apocynin, or lacking NOX2 up to 80% of the control level. Collectively, our findings suggest that interfering with LOX-NOX signaling opens up a new direction for treating DR by restoring endogenous PEDF that carries out multilevel vascular protective functions.

Protection by 20-5,14-HEDGE against surgically induced ischemia reperfusion lung injury in rats.

BACKGROUND: We previously reported that the cytochrome P450 product 20-hydroxyeicosatetraenoic acid has prosurvival effects in pulmonary artery endothelial cells and ex vivo pulmonary arteries. We tested the potential of a 20-hydroxyeicosatetraenoic acid analog N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (20-5,14-HEDGE) to protect against lung ischemic reperfusion injury in rats. Furthermore, we examined activation of innate immune system components, high mobility group box 1 (HMGB1) and toll-like receptor 4 (TLR4), in this model as well as the effect of 20-5,14-HEDGE on this signaling pathway. METHODS: Sprague-Dawley rats treated with 20-5,14-HEDGE or vehicle were subjected to surgically induced, unilateral lung ischemia for 60 minutes followed by reperfusion for 2 hours in vivo. Injury was assessed histologically by hematoxylin and eosin, and with identification of myeloperoxidase immunohistochemically. The HMGB1 and TLR4 proteins were identified by Western blot. Caspase 3 activity or 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole, incorporation were used to measure apoptosis and cell survival. RESULTS: The ischemia reperfusion injury evoked atelectasis and hemorrhage, an influx of polymorphonuclear cells, and increased TLR4 and HMGB1 expression. Caspase 3 activity was increased, and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide incorporation was decreased. The 20-5,14-HEDGE protected against each of these endpoints, including infiltration of polymorphonuclear cells, with no changes in caspase 3 activity in other organs. CONCLUSIONS: Lung ischemia reperfusion produces apoptosis and activation of the innate immune system including HMGB1 and TLR4 within 2 hours of reperfusion. Treatment with 20-5,14-HEDGE decreases activation of this response system, and salvages lung tissue.

Discovery of potent and selective inhibitors of human platelet-type 12- lipoxygenase.

We report the discovery of novel small molecule inhibitors of platelet-type 12-human lipoxygenase, which display nanomolar activity against the purified enzyme, using a quantitative high-throughput screen (qHTS) on a library of 153607 compounds. These compounds also exhibit excellent specificity, >50-fold selectivity vs the paralogues, 5-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity vs ovine cyclooxygenase-1 and human cyclooxygenase-2. Kinetic experiments indicate this chemotype is a noncompetitive inhibitor that does not reduce the active site iron. Moreover, chiral HPLC separation of two of the racemic lead molecules revealed a strong preference for the (-)-enantiomers (IC(50) of 0.43 ± 0.04 and 0.38 ± 0.05 µM) compared to the (+)-enantiomers (IC(50) of >25 µM for both), indicating a fine degree of selectivity in the active site due to chiral geometry. In addition, these compounds demonstrate efficacy in cellular models, which underscores their relevance to disease modification.

In vitro 12(S)-HETE and leukotriene metabolism inhibitory activity of sesquiterpenes of Warburgia ugandensis.

Twelve drimane and coloratane sesquiterpenes, isolated from the stem bark of Ethiopian Warburgia ugandensis, were evaluated for inhibition of 12( S)-HETE using human platelets and of leukotriene B (4) formation using activated human neutrophile granulocytes. Among the compounds examined for inhibition of 12-LOX, ugandensidial and muzigadial displayed potent inhibitory activity with IC (50) values of 3.3 and 20.2 microM, respectively, in comparison to the positive control baicalein with an IC (50) value of 20.0 microM. In the 5-LOX assay muzigadial and ugandensidial also exhibited a dose dependent and potent inhibitory effect on LTB (4) biosynthesis with an IC (50) value of 10.2 and 12.7 microM, respectively, compared to the well known 5-LOX inhibitor zileuton with an IC (50) value of 10.4 microM. In contrast, none of the other sesquiterpenoids showed significant inhibition of either 12( S)-HETE or LTB (4) metabolism.

In vitro 12(S)-HETE inhibitory activities of naphthoquinones isolated from the root bark of Euclea racemosa ssp. schimperi.

Platelet 12-lipoxygenase is believed to play a role in cancer and other pathological conditions, such as psoriasis, atherosclerosis and arthritis. The inhibition of 12-LOX is a potential therapeutic approach in the treatment of tumor metastasis. The extracts of Euclea racemosa Murr. ssp. schimperi (A. DC.) F. White (Ebenaceae) obtained by maceration and naphthoquinones isolated from the dichloromethane extract have been investigated for their 12(S)-HETE inhibitory activity using human platelets. At 100 microg/ml, the dichloromethane extract inhibited the formation of 12(S)-HETE by 88.7% and compounds 7-methyljuglone (2), isodiospyrin (3), neodiospyrin (4) and mamegakinone (5), isolated from this extract, exhibited significant activities with IC(50) values ranging from 4 to 58 microg/ml (22.2-155.7 microM). Of these the most abundant compound, 7-methyljuglone displayed a potent inhibitory activity with an IC(50) value of 4.18 microg/ml (22.2 microM), which was comparable to the positive control baicalein with an IC(50) value of 5 microg/ml (18.5 microM). In contrast, 4(S)-shinanolone (1), the reduced form of compound 2, did not show any significant inhibitory activity even at a concentration of 60 microg/ml.

10-alpha-Aminoacyl-9(10H)-anthracenones: inhibition of 12(S)-HETE biosynthesis and HaCaT cell growth.

1,8-Dihydroxy-9(10H)-anthracenones with a 10-alpha-aminoacyl group were synthesized using either a mixed-anhydride coupling method or Boc-protected oxazolidinediones. The novel anthracenones were evaluated as inhibitors of the biosynthesis of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) in epidermal homogenate of mice and for inhibition of the growth of HaCaT keratinocytes. These cells were also tested for their susceptibility for the action of the most potent members of this series on plasma membrane integrity, in order to confirm that inhibition of cell growth is not a result of membrane damage induced by prooxidants released from anthracenones. Hydroxyl-radical generation as measure of the prooxidant potential of the compounds was determined by deoxyribose degradation. The most potent analogues of this series were equally potent as anthralin against 12(S)-HETE biosynthesis and keratinocyte proliferation, while oxygen-radical generation and the resulting damage to cell membrane was strongly reduced as compared to the antipsoriatic drug.

Platelet-derived 12-hydroxyeicosatetraenoic acid plays an important role in mediating canine coronary thrombosis by regulating platelet glycoprotein IIb/IIIa activation.

BACKGROUND: In the thrombotic process of acute coronary syndromes, the pathophysiological role of thromboxane A2 via cyclooxygenase is well established; however, the role of 12-HETE via 12-lipoxygenase is little known. Therefore, we used OPC-29030, a novel specific inhibitor of 12-HETE synthesis, to test whether platelet-derived 12-HETE is involved in mediating cyclic flow variations (CFVs) and platelet aggregation in stenosed and endothelium-injured canine coronary arteries. METHODS AND RESULTS: After developing CFVs, dogs received a vehicle or OPC-29030 intravenously. Plasma and intraplatelet 12-HETE levels increased after CFVs. OPC-29030 but not vehicle reduced CFVs, which was associated with decreases in plasma and intraplatelet 12-HETE levels. Cessation of OPC-29030 restored CFVs in association with increases in plasma and intraplatelet 12-HETE levels. ADP and U46619 induced ex vivo platelet 12-HETE production and aggregation. After OPC-29030 administration, the ADP- and U46619-induced increases in ex vivo platelet 12-HETE production and aggregation were inhibited significantly. Platelet aggregation was linearly correlated with platelet 12-HETE production. OPC-29030 suppressed activation of human platelet glycoprotein IIb/IIIa. CONCLUSIONS: OPC-29030 reduced intraplatelet 12-HETE levels, resulting in the inhibition of coronary thrombosis in vivo in dogs. OPC-29030 inhibited human platelet glycoprotein IIb/IIIa activation in vitro. Thus, platelet-derived 12-HETE may play an important role in mediating thrombotic process.