4,5-EET
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The epoxyeicosatrienoic acids or EETs are signaling molecules formed within various types of cells by the metabolism of
arachidonic acid Arachidonic acid (AA, sometimes ARA) is a polyunsaturated omega-6 fatty acid 20:4(ω-6), or 20:4(5,8,11,14). It is structurally related to the saturated arachidic acid found in cupuaçu butter. Its name derives from the New Latin word ''arachi ...
by a specific subset of
Cytochrome P450 Cytochromes P450 (CYPs) are a Protein superfamily, superfamily of enzymes containing heme as a cofactor (biochemistry), cofactor that functions as monooxygenases. In mammals, these proteins oxidize steroids, fatty acids, and xenobiotics, and are ...
enzymes termed cytochrome P450 epoxygenases. These nonclassic eicosanoids are generally short-lived, being rapidly converted from epoxides to less active or inactive dihydroxy-eicosatrienoic acids (diHETrEs) by a widely distributed cellular enzyme,
Soluble epoxide hydrolase Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that in humans is encoded by the EPHX2 gene. sEH is a member of the epoxide hydrolase family. This enzyme, found in both the cytosol and peroxisomes, binds to specific epoxides and converts ...
(sEH), also termed Epoxide hydrolase 2. The EETs consequently function as transiently acting, short-range hormones; that is, they work locally to regulate the function of the cells that produce them (i.e. they are autocrine agents) or of nearby cells (i.e. they are paracrine agents). The EETs have been most studied in animal models where they show the ability to lower blood pressure possibly by a) stimulating arterial
vasorelaxation Vasodilation is the widening of blood vessels. It results from relaxation of smooth muscle cells within the vessel walls, in particular in the large veins, large arteries, and smaller arterioles. The process is the opposite of vasoconstriction, ...
and b) inhibiting the kidney's retention of salts and water to decrease intravascular blood volume. In these models, EETs prevent arterial occlusive diseases such as heart attacks and brain strokes not only by their anti-hypertension action but possibly also by their anti-inflammatory effects on blood vessels, their inhibition of platelet activation and thereby blood clotting, and/or their promotion of pro-fibrinolytic removal of blood clots. With respect to their effects on the heart, the EETs are often termed cardio-protective. Beyond these cardiovascular actions that may prevent various cardiovascular diseases, studies have implicated the EETs in the pathological growth of certain types of cancer and in the physiological and possibly pathological perception of neuropathic pain. While studies to date imply that the EETs, EET-forming epoxygenases, and EET-inactivating sEH can be manipulated to control a wide range of human diseases, clinical studies have yet to prove this. Determination of the role of the EETS in human diseases is made particularly difficult because of the large number of EET-forming epoxygenases, large number of epoxygenase substrates other than arachidonic acid, and the large number of activities, some of which may be pathological or injurious, that the EETs possess.


Structure

EETS are epoxide eicosatrienoic acid metabolites of
arachidonic acid Arachidonic acid (AA, sometimes ARA) is a polyunsaturated omega-6 fatty acid 20:4(ω-6), or 20:4(5,8,11,14). It is structurally related to the saturated arachidic acid found in cupuaçu butter. Its name derives from the New Latin word ''arachi ...
(a straight chain Eicosatetraenoic acid, omega-6 fatty acid). Arachidonic acid has 4 cis double bonds (see
Cis–trans isomerism ''Cis''–''trans'' isomerism, also known as geometric isomerism or configurational isomerism, is a term used in chemistry that concerns the spatial arrangement of atoms within molecules. The prefixes "''cis''" and "''trans''" are from Latin: " ...
which are abbreviated with the notation ''Z'' in the IUPAC
Chemical nomenclature A chemical nomenclature is a set of rules to generate systematic names for chemical compounds. The nomenclature used most frequently worldwide is the one created and developed by the International Union of Pure and Applied Chemistry (IUPAC). The ...
used here. These double bonds are located between carbons 5–6, 8–9, 11–12, and 14–15; arachidonic acid is therefore 5''Z'',8''Z'',11''Z'',14''Z''-eicosatetraenoic acid. Cytochrome P450 epoxygenases attack these double bonds to form their respective eicosatrienoic acid epoxide regioisomers (see
Structural isomer In chemistry, a structural isomer (or constitutional isomer in the IUPAC nomenclature) of a chemical compound, compound is another compound whose molecule has the same number of atoms of each element, but with logically distinct chemical bond, b ...
, section on position isomerism (regioisomerism)) viz., 5,6-EET (i.e. 5,6-epoxy-8''Z'',11''Z'',14''Z''-eicosatrienoic acid), 8,9-EET (i.e. 8,9-epoxy-5''Z'',11''Z'',14''Z''-eicosatrienoic acid), 11,12-EET (i.e. 11,12-epoxy-5''Z'',8''Z'',14''Z''-eicosatrienoic acid), or, as drawn in the attached figure, 14,15-EET (i.e. 14,15-epoxy-5''Z'',8''Z'',11''Z''-eicosatrienoic acid). The enzymes generally form both ''R''/''S''
enantiomers In chemistry, an enantiomer ( /ɪˈnænti.əmər, ɛ-, -oʊ-/ ''ih-NAN-tee-ə-mər''; from Ancient Greek ἐνάντιος ''(enántios)'' 'opposite', and μέρος ''(méros)'' 'part') – also called optical isomer, antipode, or optical anti ...
at each former double bond position; for example, cytochrome P450 epoxidases metabolize arachidonic acid to a mixture of 14''R'',15''S''-EET and 14''S'',15''R''-EET.


Production

The
Cytochrome P450 Cytochromes P450 (CYPs) are a Protein superfamily, superfamily of enzymes containing heme as a cofactor (biochemistry), cofactor that functions as monooxygenases. In mammals, these proteins oxidize steroids, fatty acids, and xenobiotics, and are ...
(CYP) superfamily of enzymes is distributed broadly throughout bacteria,
archaea Archaea ( ; singular archaeon ) is a domain of single-celled organisms. These microorganisms lack cell nuclei and are therefore prokaryotes. Archaea were initially classified as bacteria, receiving the name archaebacteria (in the Archaebac ...
, fungi, plants, animals, and even viruses. The superfamily comprises more than 11,000 genes categorized into 1,000 families. Humans have 57 putatively active CYP genes and 58 CYP pseudogenes; only a relatively few of the active CYP genes code for EET-forming epoxygenases, i.e. protein enzymes with the capacity to attach atomic oxygen (see Allotropes of oxygen#Atomic oxygen) to the carbon-carbon double bonds of unsaturated long chain fatty acids such as arachidonic acid. The CYP epoxygenases fall into several subfamilies including CYP1A, CYP2B, CYP2C, CYP2E, CYP2J, and within the CYP3A sub family,
CYP3A4 Cytochrome P450 3A4 (abbreviated CYP3A4) () is an important enzyme in the body, mainly found in the liver and in the intestine. It oxidizes small foreign organic molecules (xenobiotics), such as toxins or drugs, so that they can be removed from t ...
; in humans,
CYP2C8 Cytochrome P4502C8 (abbreviated CYP2C8), a member of the cytochrome P450 mixed-function oxidase system, is involved in the metabolism of xenobiotics in the body. Cytochrome P4502C8 also possesses epoxygenase activity, i.e. it metabolizes long-cha ...
, CYP2C9, CYP2C19, CYP2J2, and possibly CYP2S1 isoforms are the main producers of EETs although CYP2C9, CYP2C18,
CYP3A4 Cytochrome P450 3A4 (abbreviated CYP3A4) () is an important enzyme in the body, mainly found in the liver and in the intestine. It oxidizes small foreign organic molecules (xenobiotics), such as toxins or drugs, so that they can be removed from t ...
,
CYP4A11 Cytochrome P450 4A11 is a protein that in humans is codified by the ''CYP4A11'' gene. Function This gene encodes a member of the cytochrome P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases which catalyze many reacti ...
,
CYP4F8 Cytochrome P450 4F8 is a protein that in humans is encoded by the ''CYP4F8'' gene. Function This gene, CYP4F8, encodes a member of the cytochrome P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases which catalyze many r ...
, and
CYP4F12 Cytochrome P450 4F12 is a protein that in humans is encoded by the ''CYP4F12'' gene. This gene encodes a member of the cytochrome P450 superfamily of enzymes and is part of a cluster of cytochrome P450 genes on chromosome 19. The cytochrome P450 ...
are capable of producing the EETs and may do so in certain tissues. The CYP epoxygenases can epoxidize any of the double bounds in arachidonic acid but most of them are relatively selective in that they make appreciable amounts of only one or two EETs with 11,12-EET and 14,15-EET accounting for 67–80% of the product made by the cited CYP epoxidases as well as the main EETs made by mammalian tissues. CYP2C9, CYP2J9, and possibly the more recently characterized CYP2S1 appear to be the main produces of the EETs in humans with CYP2C9 being the main EET producer in vascular endothelial cells and CYP2J9 being highly expressed (although less catalytically active than CYP2C) in heart muscle, kidneys, pancreas, lung, and brain. CYP2S1 is expressed in macrophages, liver, lung, intestine, and spleen and is abundant in human and mouse atherosclerosis (i.e. Atheroma) plaques as well as inflamed tonsils. ETEs are commonly produced by the stimulation of specific cell types. The stimulation causes arachidonic acid to be released from the ''sn-2'' position of cellular
phospholipid Phospholipids, are a class of lipids whose molecule has a hydrophilic "head" containing a phosphate group and two hydrophobic "tails" derived from fatty acids, joined by an alcohol residue (usually a glycerol molecule). Marine phospholipids typ ...
s through the action of
Phospholipase A2 The enzyme phospholipase A2 (EC 3.1.1.4, PLA2, systematic name phosphatidylcholine 2-acylhydrolase) catalyse the cleavage of fatty acids in position 2 of phospholipids, hydrolyzing the bond between the second fatty acid “tail” and the glyce ...
-type enzymes and subsequent attack of the released arachidonic acid by a CYP epoxidase. In a typical example of this mechanism, bradykinin or
acetylcholine Acetylcholine (ACh) is an organic chemical that functions in the brain and body of many types of animals (including humans) as a neurotransmitter. Its name is derived from its chemical structure: it is an ester of acetic acid and choline. Part ...
acting through their respective
Bradykinin receptor B2 Bradykinin receptor B2 is a G-protein coupled receptor for bradykinin, encoded by the BDKRB2 gene in humans. Mechanism The B2 receptor is a G protein-coupled receptor, probably coupled to Gq and Gi. Gq stimulates phospholipase C to increase i ...
and muscarinic acetylcholine receptor M1 or muscarinic acetylcholine receptor M3 stimulate vascular endothelial cells to make and release EETs. The CYP epoxygenases, similar to essentially all CYP450 enzymes, are involved in the metabolism of diverse xenobiotics and natural compounds. Since many of these same compounds also induce increases in the levels of the epoxygenases, CYP oxygenase levels and consequently EET levels in humans vary widely and are highly dependent on their recent consumption history.


Metabolism of EETs

In cells, the EETs are rapidly metabolized by a cytosolic
soluble epoxide hydrolase Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that in humans is encoded by the EPHX2 gene. sEH is a member of the epoxide hydrolase family. This enzyme, found in both the cytosol and peroxisomes, binds to specific epoxides and converts ...
(sEH) which adds water (H2O) across the epoxide to form their corresponding Vicinal (chemistry)
diol A diol is a chemical compound containing two hydroxyl groups ( groups). An aliphatic diol is also called a glycol. This pairing of functional groups is pervasive, and many subcategories have been identified. The most common industrial diol is e ...
dihydroxyeicosatrienoic acids (diHETrEs or DHETs), i.e. sEH converts 14,15-ETE to 14,15-dihydroxy-eicosatrienoic acid (14,15-diHETrE), 11,12-ETE to 11,12-diHETrE, 8,9-ETE to 8,9-diHETrE, and 5,6-ETE to 5,6-diHETrE. The product diHETrEs, like their epoxy precursors, are
enantiomer In chemistry, an enantiomer ( /ɪˈnænti.əmər, ɛ-, -oʊ-/ ''ih-NAN-tee-ə-mər''; from Ancient Greek ἐνάντιος ''(enántios)'' 'opposite', and μέρος ''(méros)'' 'part') – also called optical isomer, antipode, or optical ant ...
mixtures; for instance, sEH converts 14,15-ETE to a mixture of 14(''S''),15(''R'')-diHETrE and 14(''R''),15(''S'')-diHETrE. However, 5,6-EET is a relatively poor substrate for sEH and in cells is more rapidly metabolized by cyclooxygenase-2 to form 5,6-epoxy-prostaglandin F1α. Since the diHETrE products are as a rule generally far less active than their epoxide precursors, the sEH pathway of EET metabolism is regarded as a critical EET-inactivating pathway. In some instances, however, the diHETrEs have been found to possess appreciable activity as indicated in the Biological activities section below. Membrane-bound Microsomal epoxide hydrolase (mEH or Epoxide hydrolase 1
C 3.2.2.9. C, or c, is the third letter in the Latin alphabet, used in the modern English alphabet, the alphabets of other western European languages and others worldwide. Its name in English is ''cee'' (pronounced ), plural ''cees''. History "C ...
can metabolize EETs to their dihydroxy products but is regarded as not contributing significantly to EET inactivation in vivo except perhaps in brain tissue where mEH activity levels far outstrip those of sEH. Furthermore, two other human sEH, epoxide hydrolases 3 and 4 (see epoxide hydrolase), have been defined but their role in attacking EETs (and other epoxides) in vivo has not yet been determined. Besides these four epoxide hydrolase pathways, EETs may be acylated into phospholipids in an Acylation-like reaction. This pathway may serve to limit the action of EETs or store them for future release. EETs are also inactivated by being further metabolized though three other pathways: Beta oxidation,
Omega oxidation Omega oxidation (ω-oxidation) is a process of fatty acid metabolism in some species of animals. It is an alternative pathway to beta oxidation that, instead of involving the β carbon, involves the oxidation of the ω carbon (the carbon most dist ...
, and elongation by enzymes involved in Fatty acid synthesis. These alternate to sEH pathways of EET metabolism ensure that blockade of sEH with drugs can increase EET levels only moderately in vivo.


Biological effects

Generally, EETs cause: * Calcium release from intracellular stores *Increased sodium-hydrogen antiporter activity *Increased cell proliferation *Decreased cyclooxygenase activity Other effects are specific to certain cells or locations; EETs: *Are cardioprotective after ischemic heart attack and reperfusion. *Act in the corpus cavernosum to maintain penile erection. *Specific epoxidation of EET sites produces endogenous
PPARα Peroxisome proliferator-activated receptor alpha (PPAR-α), also known as NR1C1 (nuclear receptor subfamily 1, group C, member 1), is a nuclear receptor protein functioning as a transcription factor that in humans is encoded by the ''PPARA'' gene ...
agonists. *Decrease release of
somatostatin Somatostatin, also known as growth hormone-inhibiting hormone (GHIH) or by several other names, is a peptide hormone that regulates the endocrine system and affects neurotransmission and cell proliferation via interaction with G protein-couple ...
,
insulin Insulin (, from Latin ''insula'', 'island') is a peptide hormone produced by beta cells of the pancreatic islets encoded in humans by the ''INS'' gene. It is considered to be the main anabolic hormone of the body. It regulates the metabolism o ...
and
glucagon Glucagon is a peptide hormone, produced by alpha cells of the pancreas. It raises concentration of glucose and fatty acids in the bloodstream, and is considered to be the main catabolic hormone of the body. It is also used as a Glucagon (medicati ...
from endocrine cells. *Stimulate blood vessel formation (
angiogenesis Angiogenesis is the physiological process through which new blood vessels form from pre-existing vessels, formed in the earlier stage of vasculogenesis. Angiogenesis continues the growth of the vasculature by processes of sprouting and splitting ...
. *Cause Vasodilation in the systemic arterial circulation. *Cause Vasoconstriction of the Liver sinusoidal and pulmonary venous systems. *Increased risk of
tumor adhesion Cell adhesion is the process by which cells interact and attach to neighbouring cells through specialised molecules of the cell surface. This process can occur either through direct contact between cell surfaces such as cell junctions or indire ...
on endothelial cells *Decrease platelet aggregation responses *Increase axon growth in neurons. Diol metabolites of the EETs, i.e. the diHETrEs (also termed DHETs), have relatively little or no activity compared to the EETs in most systems. However: *The
chemotaxis Chemotaxis (from '' chemo-'' + ''taxis'') is the movement of an organism or entity in response to a chemical stimulus. Somatic cells, bacteria, and other single-cell or multicellular organisms direct their movements according to certain chemica ...
response of human monocytes to monocyte chemotactic protein 1) in vivo and in vitro appears to depend on the generation of EETs and conversion of these EETs to diHETrEs. *Certain diHETrEs dilate human coronary arteries with efficacies approaching those of the EETs. *11,12-diHETrE but not 11,12-EET appears to support the maturation of the myelocyte cell line (i.e. support Myelopoiesis) in mice and to promote certain types of
angiogenesis Angiogenesis is the physiological process through which new blood vessels form from pre-existing vessels, formed in the earlier stage of vasculogenesis. Angiogenesis continues the growth of the vasculature by processes of sprouting and splitting ...
in mice and Zebrafish. *In opposition of the anti-inflammatory actions of EETs, diHETrEs may have some pro-inflammatory actions.


Clinical significance


Regulation of blood pressure

With respect to the regulation of blood pressure as well as the kidneys' regulation of salt and water absorption (which contributes to blood pressure regulation), EETS are counterpoises to another CYP-derived arachidonic acid metabolite,
20-Hydroxyeicosatetraenoic acid 20-Hydroxyeicosatetraenoic acid, also known as 20-HETE or 20-hydroxy-5''Z'',8''Z'',11''Z'',14''Z''-eicosatetraenoic acid, is an eicosanoid metabolite of arachidonic acid that has a wide range of effects on the vascular system including the regula ...
(20-HETE). In humans, the major CYPs making 20-HETE are
CYP4A11 Cytochrome P450 4A11 is a protein that in humans is codified by the ''CYP4A11'' gene. Function This gene encodes a member of the cytochrome P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases which catalyze many reacti ...
,
CYP4F2 Leukotriene-B(4) omega-hydroxylase 1 is an enzyme protein involved in the metabolism of various endogenous substrate (chemistry), substrates (mainly the fatty acids) and xenobiotics (including pharmaceutical drugs). The most notable substrate of t ...
, and
CYP4F3 Leukotriene-B(4) omega-hydroxylase 2 is an enzyme that in humans is encoded by the ''CYP4F3'' gene. CYP4F3 encodes two distinct enzymes, CYP4F3A and CYP4F3B, which originate from the alternative splicing of a single pre-mRNA precursor molecule; ...
. In animal models, 20-HETE raises blood pressure by contracting arteries and stimulating the kidney to reabsorb salt and water to increase the intravascular volume (see
20-Hydroxyeicosatetraenoic acid 20-Hydroxyeicosatetraenoic acid, also known as 20-HETE or 20-hydroxy-5''Z'',8''Z'',11''Z'',14''Z''-eicosatetraenoic acid, is an eicosanoid metabolite of arachidonic acid that has a wide range of effects on the vascular system including the regula ...
). EETs have the opposite effects. They are one type of
Endothelium-Derived Hyperpolarizing Factor In blood vessels Endothelium-Derived Hyperpolarizing Factor or EDHF is proposed to be a substance and/or electrical signal that is generated or synthesized in and released from the endothelium; its action is to hyperpolarize vascular smooth muscle ...
, i.e. a substance and/or electrical signal synthesized or generated in and released from the vascular endothelium that hyperpolarize nearby vascular smooth muscle cells. This causes these cells to relax and thereby lowers blood pressure. In animal (primarily rodent) models, EETs dilate smaller sized resistance arteries involved in causing hypertension as well as cardiac and renal arteries. They cause smooth muscle hyperpolarization by opening vascular smooth muscle
large-conductance calcium-activated potassium channels BK channels (big potassium), are large conductance calcium-activated potassium channels, also known as Maxi-K, slo1, or Kca1.1. BK channels are voltage-gated potassium channels that conduct large amounts of potassium ions (K+) across the ...
, opening certain vascular smooth muscle
transient receptor potential channels Transient receptor potential channels (TRP channels) are a group of ion channels located mostly on the plasma membrane of numerous animal cell types. Most of these are grouped into two broad groups: Group 1 includes TRPC ( "C" for canonical), T ...
, or facilitating the movement of excitatory signals through gap junctions between endothelium and smooth muscles or between smooth muscles. The actual mechanism(s) involved in these EET-induced effects have not been fully elucidated although some studies implicate EET binding to an unidentified Cell surface receptor and/or Gs protein-linked G protein–coupled receptor to initiate the signal pathway(s) leading to the cited channel and gap junction changes. With respect to the kidney, studies in rodents find that 20-HETE increases sodium and water reabsorption while the EETs, which are made in the proximal tubules and cortical collecting ducts, reduce sodium ion and water transport at both sites by inhibiting kidney
Sodium–hydrogen antiporter The sodium–hydrogen antiporter or sodium–proton exchanger (Na+/H+ exchanger) is a membrane protein that transports Na+ into the cell, and H+ out of the cell ( antiport). Function They are found in the membranes of many cells, and especially ...
(i.e. Na+/H+ exchanger) and/or
Epithelial sodium channel The epithelial sodium channel (ENaC), (also known as amiloride-sensitive sodium channel) is a membrane-bound ion channel that is selectively permeable to sodium ions (). It is assembled as a heterotrimer composed of three homologous subunits α ...
s. Mice lacking either of the EET-producing Cyp2c44 or Cyp4ac44 genes (by
gene knockout A gene knockout (abbreviation: KO) is a genetic technique in which one of an organism's genes is made inoperative ("knocked out" of the organism). However, KO can also refer to the gene that is knocked out or the organism that carries the gene kno ...
) develop hypertension when fed high sodium or high potassium diets. These and large number of other studies included in the cited references implicate the EETs in the control of at least certain forms of hypertension in rodents. In humans, vascular endothelium production of EETs involves mainly CYP2C9 and numerous indirect studies have implicated CYP epoxygenase, possibly CYP2C9, in producing a product which causes vasodilation. These studies find that selective (but not entirely specific) CYP epoxygenase-inhibiting drugs reduce human vasodilation responses elicited by the vasodilators bradykinin,
acetylcholine Acetylcholine (ACh) is an organic chemical that functions in the brain and body of many types of animals (including humans) as a neurotransmitter. Its name is derived from its chemical structure: it is an ester of acetic acid and choline. Part ...
, and methacholine; this suggests that these vasodilators operate by stimulation the production of EETs. Human studies also find that Caucasian but not African American subjects who have the Lys55Arg
single nucleotide polymorphism In genetics, a single-nucleotide polymorphism (SNP ; plural SNPs ) is a germline substitution of a single nucleotide at a specific position in the genome. Although certain definitions require the substitution to be present in a sufficiently larg ...
variant in the polyunsaturated fatty epoxide-inactivating enzyme, sEH, express hyperactive sEH and show reduced vasodilation responses to bradykinin. Other studies find that women with
pregnancy-induced hypertension Gestational hypertension or pregnancy-induced hypertension (PIH) is the development of new hypertension in a pregnant woman after 20 weeks' gestation without the presence of protein in the urine or other signs of pre-eclampsia. Gestational hyperte ...
and subjects with
Renovascular hypertension Renovascular hypertension is a condition in which high blood pressure is caused by the kidneys' hormonal response to narrowing of the arteries supplying the kidneys. When functioning properly this hormonal axis regulates blood pressure. Due to lo ...
exhibit low plasma ETE levels. Finally, 11,12-EET has been shown to relax the internal mammary artery in women, indicating that at least this EET has direct vasodilating actions in humans. On the other hand, several studies in humans with
single nucleotide polymorphism In genetics, a single-nucleotide polymorphism (SNP ; plural SNPs ) is a germline substitution of a single nucleotide at a specific position in the genome. Although certain definitions require the substitution to be present in a sufficiently larg ...
in CYP epxoygenase genes have given negative or confusing results. The most common variant of CYP2J2, rs890293, similarly contradictive or negative results are reported in studies on the rs11572082 (Arg1391Lys) variant of CYP2C8 and the rs1799853 (Arg144Cys) and rs1057910 (Ile359Leu) variants of CYP2C9, all of which code for an epoxygenase with reduced arachidonic acid-metabolizing and EET-forming activities. While many of the cited studies suggest that one or more of the EETs released by vascular endothelial cells are responsible for the actions of the vasodilators and that deficiencies in EET production or excessive EET inactivation by sEH underlie certain types of hypertension in humans, they are not conclusive. They do not exclude a possibility that other polyunsaturated fatty acid epoxides such as those derived from eicosatetraenoic, docosatetraenoic, or linoleic acids made by CYP2C9 or other CYP epoxygenases (see epoxygenase) contribute in small or large part to vasodilation responses and by this action promote blood flow to tissues and function in lowering high blood pressures. Furthermore, the genetic studies conducted to date on SNP variants do not give strong support for an antihypertensive role for the EETs or EET-forming epoxygenases in humans. Recently developed drugs which are metabolically stable analogs of the EETs and thereby mimic the EETs actions or, alternatively of drugs which inhibit sEH and thereby increase EET levels are in the
Pre-clinical development In drug development, preclinical development, also termed preclinical studies or nonclinical studies, is a stage of research that begins before clinical trials (testing in humans) and during which important feasibility, iterative testing and drug ...
stage for treating human hypertension. Testing for their usefulness in treating human hypertension is made difficult because of: 1) the large number of CYP epoxygenases along with their differing tissue distributions and sensitivities to drug inhibitors; 2) the diversity of EETs made by the CYP epoxygenases, some of which differ in activities; 3) the diversity of fatty acid substrates metabolized by the CYP epoxygenases some of which are converted to epoxides (e.g. the epoxide metabolites of linoleic, docosahexaenoic, eicosapentaenoic acids) with have different activities than the EETs or may even be overtly toxic to humans (see Coronaric acid); 4) the sEH-derived dihydroxy metabolites of the EETs some of which have potent vasodilating effects in the certain vascular networks in rodents and therefore potentially in humans; and 5) the non-specificity and side effects of the latter drugs. As indicated on the ClinicalTrials.gov web site, a National Institutes of Health-sponsored clinical trial entitled "Evaluation of Soluble Epoxide Hydrolase (s-EH) Inhibitor in Patients With Mild to Moderate Hypertension and Impaired Glucose Tolerance" has not been completed or reported on although started in 2009.


Heart disease

As indicated elsewhere on this page, EETs inhibit inflammation, inhibit blood clot formation, inhibit platelet activation, dilate blood vessels including the coronary arteries, reduce certain types of
hypertension Hypertension (HTN or HT), also known as high blood pressure (HBP), is a long-term medical condition in which the blood pressure in the arteries is persistently elevated. High blood pressure usually does not cause symptoms. Long-term high bl ...
, stimulate the survival of vascular endothelial and cardiac muscle cells by inhibiting
apoptosis Apoptosis (from grc, ἀπόπτωσις, apóptōsis, 'falling off') is a form of programmed cell death that occurs in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. These changes incl ...
, promote blood vessel growth (i.e.
angiogenesis Angiogenesis is the physiological process through which new blood vessels form from pre-existing vessels, formed in the earlier stage of vasculogenesis. Angiogenesis continues the growth of the vasculature by processes of sprouting and splitting ...
), and stimulate smooth muscle cell migration; these activities may protect the heart. Indeed, studies on in vivo animal and in vitro animal and human cell model systems indicate that the ETEs reduce infarct (i.e. injured tissue) size, reduce cardiac arrhythmias, and improve the strength of left ventricle contraction immediately after blockade of coronary artery blood flow in animal models of ischemia- reperfusion injury; EETs also reduce the size of heart enlargement that occurs long after these experiment-induced injuries. Humans with established
coronary artery disease Coronary artery disease (CAD), also called coronary heart disease (CHD), ischemic heart disease (IHD), myocardial ischemia, or simply heart disease, involves the reduction of blood flow to the heart muscle due to build-up of atherosclerotic pla ...
have higher levels of plasma EETs and higher ratios of 14,15-EET to 14,15-diHETrE (14,15-diHETrE is the less active or inactive metabolite 14,15-EET). This suggests that the EETs serve a protective role in this setting and that these plasma changes were a result of a reduction in cardiac sEH activity. Furthermore,
coronary artery disease Coronary artery disease (CAD), also called coronary heart disease (CHD), ischemic heart disease (IHD), myocardial ischemia, or simply heart disease, involves the reduction of blood flow to the heart muscle due to build-up of atherosclerotic pla ...
patients who had lower levels of EETs/14,15-di-ETE ratios exhibited evidence of a poorer prognosis based on the presence of poor prognostic indicators, cigarette smoking, obesity, old age, and elevation in inflammation markers.


Strokes and seizures

Indirect studies in animal models suggest that EETs have protective effects in
stroke A stroke is a medical condition in which poor blood flow to the brain causes cell death. There are two main types of stroke: ischemic, due to lack of blood flow, and hemorrhagic, due to bleeding. Both cause parts of the brain to stop functionin ...
s (i.e. cerbrovasular accidents). Thus, sEH inhibitors and sEH-
Gene knockout A gene knockout (abbreviation: KO) is a genetic technique in which one of an organism's genes is made inoperative ("knocked out" of the organism). However, KO can also refer to the gene that is knocked out or the organism that carries the gene kno ...
have been shown to reduce the damage to brain that occurs in several different models of ischemic stroke; this protective effect appears due to a reduction in systemic blood pressure and maintenance of blood flow to ischemic areas of the brain by arteriole dilation as a presumed consequence of inhibiting the degradation of EETs (and/or other fatty acid epoxides). sEH-gene knockout mice were also protected from that brain damage that followed induced-
subarachnoid hemorrhage Subarachnoid hemorrhage (SAH) is bleeding into the subarachnoid space—the area between the arachnoid membrane and the pia mater surrounding the brain. Symptoms may include a severe headache of rapid onset, vomiting, decreased level of consci ...
; this protective effect appeared due to a reduction in cerebral edema which was also presumable due to the prolongation of EET half-lives. 14,15-EET levels have been shown to be elevated in the cerebrospinal fluid of humans suffering subarachnoid hemorrhage. sEH inhibitors and gene knockout also reduce the number and severity of Epileptic seizures in several animal models; this effect is presumed due to the actions of EETs (and other epoxide fatty acids) in reducing cerebral blood flow changes, and reducing neuron production of
Neuroactive steroid Neurosteroids, also known as neuroactive steroids, are endogenous or exogenous steroids that rapidly alter neuronal excitability through interaction with ligand-gated ion channels and other cell surface receptors. The term ''neurosteroid'' was coin ...
s, reducing neuroinflammation,


Portal hypertension

Portal hypertension or hypertension in the venous hepatic portal system of blood flow is defined as an increase in portal pressure above normal values of 10 Millimeter of mercury. It is a serious, sometimes life-threatening complication of various diseases such as liver cirrhosis, liver fibrosis, massive Fatty liver, portal vein thrombosis, liver
schistosomiasis Schistosomiasis, also known as snail fever, bilharzia, and Katayama fever, is a disease caused by parasitic flatworms called schistosomes. The urinary tract or the intestines may be infected. Symptoms include abdominal pain, diarrhea, bloody s ...
, massive liver involvement in
miliary tuberculosis To disseminate (from lat. ''disseminare'' "scattering seeds"), in the field of communication, is to broadcast a message to the public without direct feedback from the audience. Meaning Dissemination takes on the theory of the traditional view ...
or sarcoidosis, and obstruction of the venous circuit at any level between liver and right heart (see
Portal hypertension Portal hypertension is abnormally increased portal venous pressure – blood pressure in the portal vein and its branches, that drain from most of the intestine to the liver. Portal hypertension is defined as a hepatic venous pressure gradient gr ...
). Vascular contraction in the portal system is mediated by several agents:
nitric oxide Nitric oxide (nitrogen oxide or nitrogen monoxide) is a colorless gas with the formula . It is one of the principal oxides of nitrogen. Nitric oxide is a free radical: it has an unpaired electron, which is sometimes denoted by a dot in its che ...
, carbon monoxide, prostacyclin I2, and
Endothelium-derived hyperpolarizing factor In blood vessels Endothelium-Derived Hyperpolarizing Factor or EDHF is proposed to be a substance and/or electrical signal that is generated or synthesized in and released from the endothelium; its action is to hyperpolarize vascular smooth muscle ...
s (EDHFs). EDHFs include endothelin,
angiotensin II Angiotensin is a peptide hormone that causes vasoconstriction and an increase in blood pressure. It is part of the renin–angiotensin system, which regulates blood pressure. Angiotensin also stimulates the release of aldosterone from the adre ...
, thromboxane A2, certain leukotrienes, and the EETs. In portal hypertension, portal vein endothelium appears to be dysfunctional in that it overproduces EDHFs. The EETs, particularly 11,12-EET, have a quite different effect on the Liver sinusoidal veins than on arteries of the
systemic circulation The blood circulatory system is a system of organs that includes the heart, blood vessels, and blood which is circulated throughout the entire body of a human or other vertebrate. It includes the cardiovascular system, or vascular system, tha ...
: they constrict the sinusoids. Levels of EETs in the plasma and liver of patients with cirrhosis and portal hypertension are reportedly elevated compared to normal subjects. These and other findings have led to the proposal that portal endothelium-derived EETs, perhaps acting in cooperation with another EDHF, endothelin, contribute to portal hypertension.


Cancer

The forced over-expression of CYP2J2 in or the addition of an EET to cultured human Tca-8113 oral squamous cancer cells, lung cancer A549 cells and NCL-H446 cells,
HepG2 Hep G2 (or HepG2) is a human liver cancer cell line. Hep G2 is an immortal cell line which was derived in 1975 from the liver tissue of a 15-year-old Caucasian male from Argentina with a well-differentiated hepatocellular carcinoma. T ...
liver cancer cells, LS-174 colon cancer cells,
SiHa Siha is one of the seven administrative districts of Kilimanjaro Region in Tanzania. The district covers approximately . It is bordered to the west by Meru District in Arusha Region and to the northeast by Rombo District and the southeast H ...
uterine cervix cancer cells, U251 glioblastoma cancer cells, ScaBER urinary bladder cancer cells, and
K562 K562 cells were the first human immortalised myelogenous leukemia cell line to be established. K562 cells are of the erythroleukemia type, and the cell line is derived from a 53-year-old female chronic myelogenous leukemia patient in blast crisis. ...
erythroleukemia Acute erythrocyte leukemia is a rare form of acute myeloid leukemia (less than 5% of AML cases) where the myeloproliferation is of erythrocytic precursors. It is defined as type "M6" under the FAB classification. Signs and symptoms The most com ...
and
HL-60 The HL-60 cell line is a human leukemia cell line that has been used for laboratory research on blood cell formation and physiology. HL-60 proliferates continuously in suspension culture in nutrient and antibiotic chemicals. The doubling time is ...
promyelocyte leukemic blood cancer cells caused an increase in their survival and proliferation. Putative inhibitors of CYP2J2 inhibit the growth in culture of several human cancer cell lines that express relatively high levels of CYP2J2 viz., Tca-8113 cells, HeLa uterine cervix cell lines, A549 cells, MDA-MB-435 breast cells, and
HepG2 Hep G2 (or HepG2) is a human liver cancer cell line. Hep G2 is an immortal cell line which was derived in 1975 from the liver tissue of a 15-year-old Caucasian male from Argentina with a well-differentiated hepatocellular carcinoma. T ...
cells but they had no significant inhibitory effects on two cell lines that expressed little or no CYP2J2. A putative inhibitor of CYPJ2 also inhibited the growth of human
K562 K562 cells were the first human immortalised myelogenous leukemia cell line to be established. K562 cells are of the erythroleukemia type, and the cell line is derived from a 53-year-old female chronic myelogenous leukemia patient in blast crisis. ...
erythroleukemia in a
mice A mouse ( : mice) is a small rodent. Characteristically, mice are known to have a pointed snout, small rounded ears, a body-length scaly tail, and a high breeding rate. The best known mouse species is the common house mouse (''Mus musculus' ...
model as well as the growth of mouse el4 lymphoma cells in mice that were forced to overexpress CYP2J2 cells in their vascular epithelium. Forced expression of CYP2J2 also enhanced, while forced inhibition of its expression (using Small interfering RNA) reduced, the survival, growth, and metastasis of MDA-MB-231 human breast carcinoma cells in the mouse model and likewise enhanced or reduced, respectively, the survival and growth of these cells in culture. Further studies found that the expression of CYP2J2 was in increased in the malignant cells, relative to the nearby normal cells, in the following specimens taken from humans suffering
squamous-cell carcinoma Squamous-cell carcinomas (SCCs), also known as epidermoid carcinomas, comprise a number of different types of cancer that begin in squamous cells. These cells form on the surface of the skin, on the lining of hollow organs in the body, and on the ...
and
adenocarcinoma Adenocarcinoma (; plural adenocarcinomas or adenocarcinomata ) (AC) is a type of cancerous tumor that can occur in several parts of the body. It is defined as neoplasia of epithelial tissue that has glandular origin, glandular characteristics, or ...
types of
esophageal cancer Esophageal cancer is cancer arising from the esophagus—the food pipe that runs between the throat and the stomach. Symptoms often include difficulty in swallowing and weight loss. Other symptoms may include pain when swallowing, a hoarse voice ...
and lung cancer,
small cell lung carcinoma Small-cell carcinoma is a type of highly malignant cancer that most commonly arises within the lung, although it can occasionally arise in other body sites, such as the cervix, prostate, and gastrointestinal tract. Compared to non-small cell c ...
, breast cancer,
stomach cancer Stomach cancer, also known as gastric cancer, is a cancer that develops from the lining of the stomach. Most cases of stomach cancers are gastric carcinomas, which can be divided into a number of subtypes, including gastric adenocarcinomas. Lymph ...
,
liver cancer Liver cancer (also known as hepatic cancer, primary hepatic cancer, or primary hepatic malignancy) is cancer that starts in the liver. Liver cancer can be primary (starts in liver) or secondary (meaning cancer which has spread from elsewhere to th ...
, and
colon adenocarcinoma Colorectal cancer (CRC), also known as bowel cancer, colon cancer, or rectal cancer, is the development of cancer from the colon or rectum (parts of the large intestine). Signs and symptoms may include blood in the stool, a change in bowel mo ...
; this CYP was also highly expressed in the malignant cells of patients with acute leukemia, chronic leukemia, and lymphoma. As a group, patients with these cancers exhibited increased levels of EETs in their urine and blood samples. Studies of the CYP epoxygenases have not been restricted to the CYP2J subfamily. Reduction in the expression of CYP3A4 or CYP2C using small interfering RNA inhibits the growth of cultured MCF7,
T47D T-47D is a human breast cancer cell line commonly used in biomedical research involving the hormonal expression of cancer cells. Characteristics The cells were derived from the pleural effusion of a ductal carcinoma found in the mammary gland ...
, and MDA-MB-231 human breast cancer cells; in these studies 14,15-EET stimulated the proliferation of cultured MCF7 cells, reduction in the expression of CYP3A4 by small interference RNA methods, inhibited these cells from proliferating, and 14,15-ETE reversed the effect of CYP3A4 interference; in other studies, the forced overexpression of CYP3A4 stimulated the growth of human liver cancer (
hepatoma Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer in adults and is currently the most common cause of death in people with cirrhosis. HCC is the third leading cause of cancer-related deaths worldwide. It occurs in t ...
) cell line, Hep3 . In human breast cancer, not only CYP2J2 but also CYP2C8 and CYP2C9 levels appear elevated while sEH levels appear reduced in malignant compared to nearby normal tissues; associated with this finding, the levels of 14,15-EET as well as the levels of 14,15-EET plus 14,15-dihydroxy-EET were significantly elevated in the cancerous compared to noncancerous cells and the levels of CYP2C8 and CYP2C9 proteins correlated positively and sEH levels correlated negatively with the tumor cells rate of proliferation as accessed by their
Ki67 Antigen KI-67, also known as Ki-67, Ki-67 or MKI67 (Marker Of Proliferation Ki-67), is a protein that in humans is encoded by the ''MKI67'' gene (antigen identified by monoclonal antibody Ki-67). Function Antigen KI-67 is a nuclear protein tha ...
levels while CYP2J2 levels correlated positively with poorer prognosis as predicted tumor histological grade and tumor size. The cited findings suggest that various CYP epoxygenases along with the epoxide metabolites which they make promote the growth and spread of diverse types of cancer in animals and humans. Their effects may reflect the ability of the epoxide metabolites to stimulate the proliferation and survival of the target cancer cells but perhaps also to stimulate these cells to trigger new capillary formation (see angiogenesis#Tumor angiogenesis), invade new tissues, and metastasize. A series of drugs derived from Terfenadine have been shown to inhibit CYP2J2 and to suppress the proliferation and cause the
apoptosis Apoptosis (from grc, ἀπόπτωσις, apóptōsis, 'falling off') is a form of programmed cell death that occurs in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. These changes incl ...
of various types of human cancer cell lines in culture as well as in animal models. However, clinical studies targeting CYP epoxygenases and EETs and to successfully suppress cancer in humans have not been reported. Pro-angiogenic and tumor promoting effects of EETs have been attributed to downstream cyclooxygenase (COX)-derived metabolites. Dual sEH/COX inhibitors or sEH inhibitors supplemented with an enhanced omega-3 fatty acid diet and a depleted omega-6 fatty acid diet have been shown to induce significant anti-angiogenic effects and blunt tumor growth.


Inflammation

In vitro and animal model studies indicate that the EETs possess anti-inflammatory activity that is directed toward reducing, resolving, and limiting the damage caused by inflammation. Most of these studies have focused on circulating leukocytes, blood vessel endothelium, and the occlusion of blood vessels due to pathological blood clotting. EETs a) inhibit vascular endothelial cells from expressing Cell adhesion molecules such as VCAM-1, ICAM-1, and E-selectin thereby limiting circulating leukocytes from adhering to blood vessel endothelium and migrating across this endothelium into tissues; 2) inhibit the expression and activity of cyclooxygenase-2 in blood
monocytes Monocytes are a type of leukocyte or white blood cell. They are the largest type of leukocyte in blood and can differentiate into macrophages and conventional dendritic cells. As a part of the vertebrate innate immune system monocytes also infl ...
thereby reducing their production of pro-inflammatory metabolites of arachidonic acid such as
prostaglandin E2 Prostaglandin E2 (PGE2), also known as dinoprostone, is a naturally occurring prostaglandin with oxytocic properties that is used as a medication. Dinoprostone is used in labor induction, bleeding after delivery, termination of pregnancy, and ...
; 3) inhibit platelet aggregation thereby reducing thrombus (i.e. blood clot) formation; 4) promote fibrinolysis thereby dissolving blood clots; and 5) inhibit vascular smooth muscle cell proliferation thereby reducing blood vessel hypertrophy and narrowing.


Diabetes, non-alcoholic fatty liver disease, and kidney disease

EETs, pharmacological inhibition of sEH, and/or inhibition of sEH expression enhance insulin actions on animal tissues in vitro and have protective effects in ameliorating insulin resistance as well as many of the neurological and kidney complications of diabetes in various animal models of diabetes; the studies suggest that the EETs have beneficial effects in
Type I diabetes Type 1 diabetes (T1D), formerly known as juvenile diabetes, is an autoimmune disease that originates when cells that make insulin (beta cells) are destroyed by the immune system. Insulin is a hormone required for the cells to use blood sugar for ...
as well as Type II diabetes. Treatment of EET analog is beneficial for hepatic insulin signaling in mouse model of insulin resistance. These interventions also gave beneficial results in animal models of
non-alcoholic fatty liver disease Non-alcoholic fatty liver disease (NAFLD), also known as metabolic (dysfunction) associated fatty liver disease (MAFLD), is excessive fat build-up in the liver without another clear cause such as alcohol use. There are two types; non-alcoholic ...
and certain types inflammation-related kidney diseases including chronic kidney disease, renal ischemia-reperfusion injury, and polycystic kidney disease. The protective role of EETs in these animal model diseases may reflect, at least in part, their anti-inflammatory actions.


Pain

EETs have been shown to have anti-hyperalgesic and pain-relieving activity in several animal models of pain including
Nociception Nociception (also nocioception, from Latin ''nocere'' 'to harm or hurt') is the sensory nervous system's process of encoding noxious stimuli. It deals with a series of events and processes required for an organism to receive a painful stimulus, co ...
resulting from tissue injury, inflammation, and Peripheral neuropathy (also see Neuropathic pain) including pain secondary to experimentally induced Diabetes in mice. The epoxides of omega-3 fatty acids appear far stronger and more involved in the relief of pain than the EETs (see
epoxydocosapentaenoic acid Epoxide docosapentaenoic acids (epoxydocosapentaenoic acids, EDPs, or EpDPEs) are metabolites of the 22-carbon straight-chain omega-3 fatty acid, docosahexaenoic acid (DHA). Cell types that express certain cytochrome P450 (CYP) epoxygenases metab ...
).


References

{{Transient receptor potential channel modulators Metabolic pathways Cytochrome P450 Eicosanoids Epoxides Cell biology Immunology Inflammations Blood pressure Human physiology Animal physiology