20-HETE in neovascularization

Prostaglandins & other Lipid Mediators 98 (2012) 63–68

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Prostaglandins and Other Lipid Mediators

Review

20-HETE in neovascularization Li Chen a,b,1 , Rachel Ackerman a,1 , Austin M. Guo a,b,∗ a b

Department of Pharmacology, New York Medical College, Valhalla, NY 10595, United States Department of Pharmacology, School of Medicine, Wuhan University, Wuhan, 430071, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 11 October 2011 Received in revised form 9 December 2011 Accepted 19 December 2011 Available online 28 December 2011 Keywords: Neovascularization Arachidonic acid metabolism 20-HETE Endothelial cells Endothelial progenitor cells Signaling

a b s t r a c t Cytochrome P450 4A/F (CYP4A/F) converts arachidonic acid (AA) to 20-HETE by ␻-hydroxylation. The contribution of 20-HETE to the regulation of myogenic response, blood pressure, and mitogenic actions has been well summarized. This review focuses on the emerging role of 20-HETE in physiological and pathological vascularization. 20-HETE has been shown to regulate vascular smooth muscle cells (VSMC) and endothelial cells (EC) by affecting their proliferation, migration, survival, and tube formation. Furthermore, the proliferation, migration, secretion of proangiogenic molecules (such as HIF-1␣, VEGF, SDF-1␣), and tube formation of endothelial progenitor cells (EPC) are stimulated by 20-HETE. These effects are mediated through c-Src- and EGFR-mediated downstream signaling pathways, including MAPK and PI3K/Akt pathways, eNOS uncoupling, and NOX/ROS system activation. Therefore, the CYP4A/F-20-HETE system may be a therapeutic target for the treatment of abnormal angiogenic diseases. © 2011 Elsevier Inc. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The CYP4A/F-20-HETE system: synthesis and distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20-HETE and vascular cell functions associated with angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20-HETE and endothelial progenitor cell functions associated with angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20-HETE and vascular cell signaling relevant to angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20-HETE and physiological and pathological angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The CYP4A/F-20-HETE system as a therapeutic target . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Neovascularization (including vasculogenesis and angiogenesis) is an important process that has been studied extensively over the last several decades. Vasculogenesis, the de novo formation of blood vessels, begins with islands of precursor angioblast cells that differentiate into endothelial cells (EC) which mature into a secondary vessel network [1]. On the other hand, angiogenesis is the sprouting of existing vascular structures into new capillary growth and occurs both as developmental and

∗ Corresponding author at: Department of Pharmacology, New York Medical College, Valhalla, NY 10595, United States. Tel.: +1 914 594 4625; fax: +1 914 594 4273. E-mail addresses: Li [email protected] (L. Chen), Rachel [email protected] (R. Ackerman), Austin [email protected] (A.M. Guo). 1 Authors equally contributed to the article. 1098-8823/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.prostaglandins.2011.12.005

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patho-physiological responses (e.g. cancer, atherosclerosis, ischemia, infectious diseases, diabetes, and retinopathy) to local hypoxia [2]. The angiogenic process involves a cascade of events that are initiated with the production, release, and binding of angiogenic factors to EC receptors, which leads to EC proliferation and directional migration toward gradients of pro-angiogenic factors. Consequently, there is lumen formation, branch development, anastomosis of the tip of one tube with another to form a loop (loop formation), and vessel stabilization to form mature blood vessels [3–7]. Angiogenesis requires many different cytokines and growth factors interacting with the endothelium and its microenvironment, such as hypoxia inducible factor-1␣ (HIF-1␣), vascular endothelial growth factor (VEGF), stromal-derived factor-1 (SDF-1), placental growth factor (PIGF), and platelet-derived growth factor-BB (PDGFBB) [8–10]. VEGF, the prominent angiogenic factor, promotes the proliferation, survival, migration, and tube formation of EC [11,12]. Furthermore, VEGF also mobilizes and recruits endothelial

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progenitor cells (EPC) from their bone marrow niche into blood circulation. EPC express C-X-C chemokine receptor-4 (CXCR4) that allows their migration to sites of neovascularization in response to its ligand, SDF-1. SDF-1 is typically released by target tissues, including ischemic tissues, primary tumors, and pre-metastatic sites of tumor [13–15]. Once EPC are recruited, they can promote neovascularization by secreting VEGF and other cytokines [16]. Thus, a positive feedback exists between VEGF secretion and EPC recruitment. Mounting evidence in recent years has implicated that the CYP4A/F-20-HETE system plays an important role in regulation of the neovascularization process. 20-hydroxyeicosatetraenoic acid (20-HETE), a key bioactive lipid mediator, arises from arachidonic acid (AA) via metabolism by cytochrome P450 4A/F (CYP4A/F). 20HETE is an important mitogen upstream of many growth factors [17]. In this review, we will focus on the role of the CYP4A/4F20-HETE system in the regulation of neovascularization processes and the potential mechanisms involved, which may facilitate the development of therapeutic approaches to modulate vascular growth. 2. The CYP4A/F-20-HETE system: synthesis and distribution Arachidonic acid is released from the cellular membrane by phospholipase A2 (PLA2) activation [18]. Free AA can then be metabolized via the cyclooxygenases, lipoxygenases, or CYP450 oxygenases to bioactive molecules such as prostaglandins, thromboxanes, leukotrienes, and CYP450-derived metabolites [19]. Specifically, the CYP450 family of enzymes catalyzes either epoxygenation (forms epoxy bonds across ␻-5,6, ␻-8,9, ␻-11,12, or ␻-14,15 via CYP2C and 2J) or hydroxylation (adds a hydroxyl group at the ␻, ␻-1, or ␻-2 carbons via CYP4A and 4F) of AA yielding epoxyeicosatrienoic acids (EETs) or hydroxyeicosatetraenoic acids (HETEs). 20-HETE is the major metabolite of the CYP ␻hydroxylases [18,20,21]. Although both CYP4A11 and 4F2 are highly expressed in human kidney and liver [22,23], CYP4A11catalyzed 20-HETE formation is quantitatively less important than the corresponding CYP4F2 component in human liver and kidney, while CYP4A22 has been identified as a highly homologous isoform of CYP4A11 [23–25]. The main sites of 20-HETE synthesis and action are kidneys, liver, vasculature, and lungs [26,27]. 20-HETE has also been shown to inhibit large conductance calcium-activated potassium channels in VSMC [21,28]. This action of 20-HETE, in turn, sensitizes the vasculature to myogenic and hormonal stimuli, which promotes vasoconstriction. Besides the human isoforms, different isoforms of 20-HETE synthases are also present in animals. Similar to the human isoforms, expression of these enzymes is organ- and gender-specific in mice [29]. Cyp4a12a has been identified as the major 20-HETE synthase that is prominent in males, and Cyp4a14 has been demonstrated to be the predominant 20-HETE synthase in females [29–31]. However, Cyp4a10 was found in the kidneys of all mice [29]. The main isoforms in rats [32] were CYP4A1 [32–36] and CYP4A2 [37–40], which also vary by gender. CYP4A protein has been found in the brain, prostate, intestine, and lungs of rat and rabbits [32,41,42]. 20HETE synthase expression and function can be altered by the states of diseases [28] and in response to drugs. For example, Cyp4a10 and Cyp4a14 were shown to be increased in the liver and ob/ob in db/db diabetic animals [43,44]. 3. 20-HETE and vascular cell functions associated with angiogenesis The production of 20-HETE has long been shown in EC from systemic circulations, pulmonary small arteries, as well as VSMC [28,32]. Recent studies have expanded our knowledge of the actions

of 20-HETE on vascular cell functions associated with angiogenesis. EC proliferation is one of the early steps in angiogenesis. 20-HETE was shown to induce the proliferation of human EC in vitro via stimulating superoxide formation and the production of both VEGF and HIF-1␣, both essential regulators of angiogenic responses in EC [45,46]. In addition, 20-HETE increases EC migration, another important step in the angiogenic cascade [25,45–48]. Furthermore, 20-HETE induces neovascularization in the rat cornea in vivo [49]. Thus, 20-HETE activates both release of angiogenic factors and the growth responses of vascular cells both in vitro and in vivo. Ishizuka et al. have also shown that both CYP4A overexpression in human umbilical vein endothelial cell (HUVEC) [48] and addition of exogenous 20-HETE to non-transfected EC induced their activation [48]. Activation of EC results in the production of cytokines [50], such as VEGF. In turn, elevated levels of VEGF increase EC proliferation. Similar to EC proliferation, the regulation of vascular cell survival and apoptosis is another cellular mechanism for neonatal vascular remodeling [51]. 20-HETE and its stable analog, 20hydroxy-eicosa-5(Z),14(Z)-dienoic acid (WIT003), were found to have a protective effect on bovine pulmonary artery ECs (BPAEC) and pulmonary arterial smooth muscle cells (PASMC) by promoting survival and preventing apoptosis by acting on, at least in part, the intrinsic apoptotic pathway [47,52]. By preserving the integrity of the endothelium, 20-HETE increases the chance of EC exposure to pro-angiogenic molecules and plays a role in the angiotensin II-induced neo-intimal formation [53]. The growth of new blood vessels depends on the formation of both capillary-like tubes of EC and the subsequent infiltration of VSMC [54]. The directional migration of EC toward gradients of proangiogenic factors is a prerequisite of lumen formation. 20-HETE treatment not only induced EC migration [46], but also promoted VSMC migration via PDGF [55], which is another crucial step in angiogenic responses. 20-HETE treatment induced cytoskeletal changes in EC, promoting a spindle shape [46], which would be easier for forming sprouts out of existing vessels. Therefore, 20-HETE plays a critical role in angiogenic responses via regulating the proliferation, migration, tube formation, and survival of both EC and VSMC.

4. 20-HETE and endothelial progenitor cell functions associated with angiogenesis Accumulating evidence suggests that the regulatory influence of 20-HETE on angiogenesis involves actions on the EPC function. Recent studies in stem cell biology suggest that bone-marrowderived EPC also play a pivotal role in postnatal vasculogenesis, the de novo formation of new blood vessels essential for organ and tissue growth, wound healing, and tumor neovascularization [56–65]. EPC activate the “angiogenesis switch”, a crucial step in the transition of an avascular, dormant area to a vascularized, rapidly growing tissue [66–69]. Under normal physiological conditions, EPC are in a quiescent state within the bone marrow niche, with a low frequency of EPC in circulating blood. Conversely, when the endothelium is perturbed, as occurs in tumor neovascularization, wounds, or ischemia, bone marrow EPC are mobilized and their numbers in the blood greatly increase. The most prominent factors promoting mobilization and differentiation of EPC include VEGF, angiopoietin-1, PIGF [70–73], and SDF-1␣. EPC and other CXCR4+ bone marrow derived cells can be recruited to ischemic tissues, primary tumor sites, and premetastatic tissues partially through increases in HIF and its target genes SDF-1␣ and VEGF [56,58,74–77]. After incorporation into the vasculature, EPC gradually differentiate toward EC and form neo-vessels.

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Recent data from our group showed that human umbilical cord-derived EPC express CYP4A11, CYP4A22, and CYP4F2, and synthesize 20-HETE [25]. 20-HETE promotes EPC proliferation and migration in an autocrine and paracrine manner. Furthermore, 20HETE enhanced secretion of pro-angiogenic molecules by EPC, such as HIF-1␣, VEGF, SDF-1␣ [25]. VEGF and SDF-1␣ are two of the most important regulators of EPC involvement in the angiogenic process, which could recruit bone marrow-derived stem cells to the sites of injury and promote angiogenesis [78–80]. Co-culturing EPC with EC dramatically increased tube formation. Moreover, treatment with selective inhibitors of 20-HETE synthesis (HET0016 and DDMS) or the antagonist of 20-HETE, abrogated EPC proliferation and migration toward VEGF and SDF-1␣ and, hence, EPC-induced EC tube formation, which is consistent with 20-HETE being an important mediator of EPC-induced EC differentiation [25]. These data suggest that the CYP4A/F-20-HETE system plays an important role in regulation of the EPC functions associated with angiogenic responses.

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molecules that can act as oxidizing agents or be converted into radicals such as hydrogen peroxide; it is a primary function of NOX to generate ROS [99]. Accumulating evidence has suggested that ROS are involved in VEGF induction and signaling [100,101]. Our previous data suggested that 20-HETE does increase HIF1␣ levels secondary to VEGF-induced superoxide formation by a NOX-dependent mechanism [45]. However, 20-HETE-induced VEGF expression is not NOX-dependent. In addition, NOX and ROS have been shown to be induced by VEGF and 20-HETE which leads to protection from apoptosis [47,99,102]. Furthermore, 20-HETE-mediated ROS is an important pathway to enhanced survival or proliferation of PAEC, BPAEC, and ischemia reoxygenation injury in ex vivo pulmonary arteries, through activation of pro-survival PI3k/Akt pathways, NADPH oxidase activation, and NADPH oxidase-derived superoxide in a manner that is dependent upon the NADPH oxidase and PI3K/Akt activation [47,94,102]. Fig. 1 has summarized the potential signaling mechanism by which 20-HETE regulates and contributes to angiogenesis via altering vascular cell and EPC functions.

5. 20-HETE and vascular cell signaling relevant to angiogenesis 6. 20-HETE and physiological and pathological angiogenesis Two candidate pathways involved in cellular proliferation and survival are the MAPK and PI-3 kinase pathways [81,82]. Inhibition of 20-HETE synthesis reduced ERK1/2 activation in various cell types [83–86], but the mechanism by which 20-HETE stimulated the ERK1/2 protein complex remains unclear. Epidermal growth factor receptors (EGFRs) are known to play an important role in mediating growth and cell proliferation through the activation of the Raf/MEK/ERK pathway in polycystic kidney disease and various epithelial cancers [87–90]. A non-receptor tyrosine kinase, c-Src, can be recruited to EGFR and promote phosphorylation of EGFR [91,92]. Recent studies showed that both EGFR and c-Src were involved in 20-HETE-mediated activation of downstream pathways. 20-HETE-mediated activation of Raf/MEK/ERK and PI3K/Akt was strictly dependent on EGFR and c-Src signaling in renal epithelial cells [17] and the downstream signaling cascades. Growing evidence shows that activation of a multitude of distinct signaling pathways dependent on EGFR and c-Src signaling are mediated by 20-HETE, leading to various biological responses. The activation of Ras/MAPK by 20-HETE amplifies cytosolic PLA2 activity and releases AA through a positive feedback mechanism, thereby, promoting cell proliferation and growth [93]. In addition, 20-HETE-induced PI3K/Akt phosphorylation acted as an upstream signal of eNOS uncoupling, leading to superoxide formation [94]. Furthermore, nuclear factor-␬B (NF-␬B) has also been reported to be a downstream target of the MAPK/ERK pathway [95,96]. In human EC, U0126, the specific inhibitor of MAPK/ERK pathway, suppressed NF-␬B activation in 20-HETE-stimulated EC, indicating that the MAPK/ERK pathway is upstream of the NF-␬B pathway and is important for NF-␬B activation [48]. These findings are consistent with reports showing that PI3K/Akt phosphorylation acted upstream of 20-HETE-endothelial nitric oxide synthase (eNOS) uncoupling [94]. Previous studies demonstrated that 20-HETE disrupted the balance between nitric oxide (NO) and superoxide (reduced scavenging of superoxide secondary to a reduction in NO production) produced by EC through interference with eNOS activity [45,94,97,98], and the increased superoxide levels brought about by 20-HETE may be due to the eNOS uncoupling via disruption of decreased levels of heat shock protein 90 (HSP90)-eNOS association [97]. Another signaling pathway involved in the actions of 20-HETE that affects endothelium function is the NADPH oxidase-reactive oxygen species (NOX-ROS) system. ROS are oxygen-derived small

Expression of 20-HETE synthase by arterioles, VSMC and EC [27,28,103–105], is consistent with the idea that 20-HETE plays a role in myogenic activation of small arterioles of the cerebral and renal circulations [55,106–108]. It is worth noting that 20HETE has dual biological effects. It is a potent vasoconstrictor in renal and cerebral microcirculation [28], but it plays a role as a vasodilator by promoting NO release in the pulmonary vascular bed [47,52,105]. In addition, 20-HETE formation has been shown to increase in skeletal muscle after chronic electrical stimulation and plays an important role in mediating skeletal muscle angiogenesis [109]. Although studies on the location and role of 20-HETE in physiological angiogenesis are rather scarce, growing evidence suggests that 20-HETE promotes pathological angiogenesis in various disease states such as cancer, atherosclerosis, ischemia, and diabetes. Neovascularization processes are a prerequisite for tumor progression and metastasis. The hypoxic environment within a tumor leads to the secretion of molecules like HIF-1␣ and VEGF that result in the promotion of angiogenesis [2,3]. 20-HETE synthases have been reported to be highly expressed in tumor tissues (including prostate cancer, hepatoma, renal carcinoma, lung carcinoma) and cell lines (human non-small cell lung adenocarcinoma (NSCLC) cell line A549, hepatocellular liver carcinoma cell line HepG2, HepaRG, renal cell adenocarcinoma lines 786-O and 769-P) [110–114]. Since 20-HETE is an important mitogen upstream of many growth factors [28,46,115], it stands to reason that 20-HETE may promote angiogenesis in the early hypoxic areas of tumor growth. Guo et al. reported that using inhibitors of 20-HETE synthesis HET0016 in 9L gliosarcoma decreases tumor vessel density in vivo [84]. Recently, Yu et al. reported CYP4F2 derived 20-HETE promotes angiogenesis and metastatic potential of NSCLC cells through the upregulation of VEGF and MMP-9 expression [114]. These findings are consistent with 20-HETE which may regulate tumor angiogenesis. However, further studies are urgently needed to elucidate whether 20-HETE is a potent mediator of tumor angiogenesis by studying additional tumor types that actively generate 20-HETE. 20-HETE can also promote angiogenesis in ischemia related disease conditions. When pulmonary arteries are exposed ex vivo to hypoxic conditions, 20-HETE was found to reduce caspase 3 activity [47] implying it has anti-apoptotic activity. By protecting the endothelium in a hypoxia-reperfusion model, 20-HETE keeps the vessel intact and allows for the secretion of growth molecules

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Fig. 1. Scheme of the signaling components involved in 20-HETE-induced angiogenesis. 20-HETE-mediated activation of c-Src and EGFR lies upstream of MAPK and PI3K/Akt signaling pathway phosphorylation, followed by activation of eNOS uncoupling. Uncoupling of eNOS releases superoxide leading to increased VEGF production in a NADPH oxidase-independent manner, later followed by HIF-1␣ translocation in NADPH oxidase-dependent, and NF-␬B activation (promote adhesion molecule secretions), all of which promotes vascular cell activation (survival, proliferation, migration, tube formation) and angiogenesis. On the other side, vascular-derived 20-HETE may also stimulate EPC proliferation, migration, cytokines secretion (such as SDF-1, VEGF) and homing to sites of angiogenesis, resulting in enhanced neovascularization.

that could lead to compensatory angiogenesis. In a rat cornea pocket angiogenesis experiment, WIT003, a stable 20-HETE analog, induced angiogenesis [49]. The stimulatory action of 20-HETE on angiogenesis in vivo is often due to downstream mediators such as NOX-derived ROS, which promotes angiogenesis through upregulation of VEGF or angiopoietin [101,116–118]. In the same study, Chen et al. reduced VEGF-, FGF-2-, and EGF-mediated cornea neovascularization using the 20-HETE inhibitor, HET0016 [49]. Furthermore, Stec et al. have shown that 20-HETE can stimulate VSMC migration, which is an important component of restenosis and atherosclerosis [55]. 7. The CYP4A/F-20-HETE system as a therapeutic target Abnormal neovascularization commonly occurs in cancer development, ischemia repair, atherosclerosis, diabetes, and retinopathy, and has a major patho-physiological impact that involves changes in tissue capillarization with significant consequences on morbidity and mortality [119]. Since 20-HETE plays an important role in postnatal neovascularization, the CYP4A/F20-HETE system merits consideration as a therapeutic target to increase or decrease angiogenesis in clinical settings featuring abnormal angiogenesis. Inhibitors of 20-HETE synthesis, such as DDMS (Nmethylsulfonyl-12,12-dibromododec-11-enamide) and HET0016 (N-hydroxy-N’-(4-n-butyl-2-methylphenyl) formamidine), as well as its competitive antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE), have been commonly used. These pharmacological compounds have been reported to block the angiogenic response of a variety of growth factors that induce neovascularization, including VEGF, FGF, and EGF in vivo as well as the proliferation of U251 human glioma and 9L rat gliosarcoma cells, 786-O and 769-P human renal cell carcinoma cells both in vitro and their tumors in vivo [45,83,84,112]. Furthermore, reduction of 20-HETE

in human NSCLC reduced the size of primary tumors and decreased the numbers of metastases in a therapeutic application. [112]. Traditional cancer chemotherapy or anti-angiogenic therapy (i.e. anti-VEGF antibody – Avastin) often leads to multi-drug resistance, tumor metastasis, or unwanted side effects. 20-HETE is an important upstream signal of many growth factors, suggesting that knocking down 20-HETE synthesis may potentially improve the outcome in these cancer types through blocking the multiple steps of tumor development, including tumor-growth, angiogenesis, and metastasis. However, the role of the CYP4A/F-20-HETE system in regulation of cancer is still emerging. Future studies are needed to further reveal how CYP4 therapy might be beneficial compared to traditional cancer therapies. In a model of kidney ischemia-reperfusion, 20-HETE analogs had a protective effect while HET0016 exacerbated the damage [120]. Our recent studies also found that 20-HETE can promote EPC migration toward SDF-1, which may hint at a potential effect in ischemic tissue repair and wound healing [25]. Additionally, it has been reported that HET0016 was partially effective in attenuating the decreases in retinal perfusion found in the initial weeks of STZ-induced diabetes [121]. However, there remains areas in 20-HETE regulation of angiogenesis that are not well understood, such as the levels of 20-HETE in both local tissue microenvironments and the general circulation, the mechanism by which the CYP4A/F20-HETE system regulates EPC homing, and the signaling triggered by 20-HETE and subsequent cross-talk. More research is required to further determine the function and the mechanisms involved with this important eicosanoid. Finally, there are limitations in our present research, such as the low specificity and selectivity of available pharmacological inhibitors, and potential compensatory responses between different 20-HETE synthase isoforms, and the difficulty in developing transgene or knockout animal models. Further development of the pharmacological competitive inhibitors of 20-HETE

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