BBAMCB-57681; No. of pages: 6; 4C: 2 Biochimica et Biophysica Acta xxx (2014) xxx–xxx

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Review

Transcellular biosynthesis of eicosanoid lipid mediators☆ Valérie Capra a, G. Enrico Rovati b, Paolo Mangano c, Carola Buccellati b, Robert C. Murphy d, Angelo Sala b,e,⁎ a

Department of Health Sciences, Università degli Studi di Milano, Milan, Italy Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milan, Italy c Department of Experimental Medicine, Università degli Studi di Messina, Messina, Italy d Department of Pharmacology, University of Colorado at Denver, Denver, USA e IBIM, CNR, Palermo, Italy b

a r t i c l e

i n f o

Article history: Received 30 April 2014 Received in revised form 1 September 2014 Accepted 2 September 2014 Available online xxxx Keywords: Transcellular biosynthesis Eicosanoids Arachidonic acid Lipoxygenases Cyclooxygenases

a b s t r a c t The synthesis of oxygenated eicosanoids is the result of the coordinated action of several enzymatic activities, from phospholipase A2 that releases the polyunsaturated fatty acids from membrane phospholipids, to primary oxidative enzymes, such as cyclooxygenases and lipoxygenases, to isomerases, synthases and hydrolases that carry out the final synthesis of the biologically active metabolites. Cells possessing the entire enzymatic machinery have been studied as sources of bioactive eicosanoids, but early on evidence proved that biosynthetic intermediates, albeit unstable, could move from one cell type to another. The biosynthesis of bioactive compounds could therefore be the result of a coordinated effort by multiple cell types that has been named transcellular biosynthesis of the eicosanoids. In several cases cells not capable of carrying out the complete biosynthetic process, due to the lack of key enzymes, have been shown to efficiently contribute to the final production of prostaglandins, leukotrienes and lipoxins. We will review in vitro studies, complex functional models, and in vivo evidences of the transcellular biosynthesis of eicosanoids and the biological relevance of the metabolites resulting from this unique biosynthetic pathway. This article is part of a Special Issue entitled “Oxygenated metabolism of PUFA: analysis and biological relevance”. © 2014 Published by Elsevier B.V.

1. Introduction Arachidonic acid (AA) is an abundant polyunsaturated fatty acid of the membrane phospholipids, where it is stored in the sn-2 position of phosphatidylinositol and/or phosphatidylcholine. AA regulates the physical properties of the membranes but when released as free acid by phospholipases (PLs) it has an essential role as a precursor for enzymatic and non-enzymatic biosynthetic pathways leading to the production of eicosanoids, a large family of potent local hormones acting at nanomolar concentration in autocrine/paracrine way [1]. PLs are sensitive to the increase in intracellular free calcium concentration as a result of physical stimuli and can be activated by growth factors, hormones, cytokines and eicosanoids acting through specific receptors [2]. Several families of eicosanoids are produced from AA, but the main biologically relevant are: prostanoids (prostaglandins, PGs, prostacyclin, PGI2 and thromboxane A2, TxA2), synthesized by the cyclooxygenase (COX) pathway (Fig. 1); leukotrienes (LTs, i.e. LTB4 and cysteinyl-LTs),

☆ This article is part of a Special Issue entitled “Oxygenated metabolism of PUFA: analysis and biological relevance”. ⁎ Corresponding author at: Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milan, Italy. Tel.: +39 025 318308. E-mail address: [email protected] (A. Sala).

derived from 5-lipoxygenase (5-LO) pathway (Fig. 1); and lipoxins (LXs), originating from the interaction of 5-LO, 12-LO and 15-LO (Fig. 2). The COX pathway was the first AA metabolic pathway elucidated. Two isoforms of COX have been identified: COX-1 is constitutively expressed in most cells and tissues, and is involved in vascular homeostasis, protection of the gastric mucosa and regulation of renal function; and the inducible isoform COX-2, which is expressed in response to inflammatory stimuli, even though it is constitutively present in some types of endothelial cells, brain and kidney [3]. The double catalytic activity of COX leads to the production of a highly unstable endoperoxide intermediate PGH2, which is further metabolized by specific synthase enzymes to produce PGs (PGE2, PGI2, PGF2〈, PGD2) and TxA2 (Fig. 1). Prostanoids exert various biological functions in several cells and tissues by interacting with specific G-protein-coupled receptors (GPCRs), integral membrane proteins that transmit signals inside the cells (for a comprehensive review, see [4]). Prostanoid roles in physiology and pathophysiology were initially derived from the observation of the effect of aspirin [5,6] or by exogenous addition of each prostanoid, and lately by the characterization of knock-out mice phenotypes [7]. They are mainly involved in inflammation, pain, fever, platelet and vascular homeostasis, immunity, and reproduction [8]. The 5-LO pathway leads to the production of another large family of potent lipid mediators called LTs (Fig. 1), first discovered by Samuelsson et al. in rabbit polymorphonuclear leukocytes (PMN) [9,10]. Furthermore,

http://dx.doi.org/10.1016/j.bbalip.2014.09.002 1388-1981/© 2014 Published by Elsevier B.V.

Please cite this article as: V. Capra, et al., Transcellular biosynthesis of eicosanoid lipid mediators, Biochim. Biophys. Acta (2014), http://dx.doi.org/ 10.1016/j.bbalip.2014.09.002

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Cysteinyl-LTs, which are known to increase vascular permeability and regulate smooth muscle tone, have a clear role in asthma and allergic rhinitis and have been implicated in other inflammatory conditions including cardiovascular, gastrointestinal, immune, and neurodegenerative diseases [22,23].

2. Initial evidence for transcellular metabolism: communication among platelets and ECs to produce PGI2

Fig. 1. Prostanoid and leukotriene biosynthetic pathways. Several proteins have been identified as possessing the ability to convert PGH2 into PGE2, PGD2 or PGF2α as well as to convert LTA4 into LTC4. FLAP (five-lipoxygenase activating protein) binds 5-LO on the nuclear membrane and contributes to handle AA to the 5-LO.

5-LO participates, in coordination with 12/15-LO, to the formation of LXs [11] (see Fig. 2 and the specific section below). 5-LO is expressed by cells of myeloid origin, particularly neutrophils, eosinophils, monocytes/macrophages, and mast cells [12–15], as well as in B lymphocytes [16]. To synthesize the highly unstable intermediate LTA4, 5-LO requires the intervention of 5-lipoxygenase activating protein (FLAP) that, in intact cells, associates to the membrane and presents AA to 5-LO [17–19]. LTA4 is subsequently metabolized by LTA4 hydrolase into LTB4 or conjugated with glutathione by LTC4 synthase to yield the cysteinylLTs, i.e. LTC4, LTD4 and LTE4 (Fig. 1). LTs interact with specific GPCRs ([20]) to display proinflammatory activities both in health and disease. LTB4 is a potent chemotactic for neutrophils and eosinophils and plays a number of roles in inflammation and immune regulation [21].

Since the discovery of prostanoids, lipoxins and leukotrienes as AA derivatives, investigations focused on the identification of cells possessing all the enzymes involved in the individual steps of their biosynthesis. However, these investigations were soon paralleled by the observation that many cells were found to contain only the enzymes responsible for the biochemical conversion of unstable intermediates (such as LTA4 or PGH2) into the final bioactive compounds. For example, 5-LO lacks in platelets, endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), but all these cells seemed to express secondary enzymes such as LTC4-synthases capable to convert LTA4 into cysteinyl-LTs (Fig. 2). Bunting et al. [24] were the first to postulate that an anti-aggregating compound (later named prostacyclin, PGI2) was produced by the arterial wall with COX pharmacologically inactivated with indomethacin following incubation with activated platelet-rich plasma, suggesting that the unstable intermediate PGH2 from platelets was converted by the arterial rings into the biologically active, anti-aggregating compound. This observation introduced the hypothesis that eicosanoid production could result from the cooperation of cells, one of which is activated to biosynthesize through a primary oxidative enzyme (COX or LO) an intermediate that is transferred to a neighboring cell to carry out the final synthesis of lipid mediators. The latter has no need of being activated and is impeded or lacks the ability to produce the intermediate but is able of converting it into biologically active metabolite(s) by the presence of secondary enzymes, such as prostaglandin isomerases, LTA4 hydrolase or LTC4 synthases (Table 1). We will see below that also the free AA could serve as a biosynthetic intermediate in this process (see Section 3). This mechanism of cell–cell cooperation is termed transcellular metabolism or transcellular biosynthesis and the seminal work by Bunting et al. indicates that this is a suitable event under normal conditions, where accumulation of platelets on the vessel should be

Fig. 2. Simplified schematic overview of the lipoxin biosynthetic pathways.

Please cite this article as: V. Capra, et al., Transcellular biosynthesis of eicosanoid lipid mediators, Biochim. Biophys. Acta (2014), http://dx.doi.org/ 10.1016/j.bbalip.2014.09.002

V. Capra et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx Table 1 Transcellular metabolism leading to the production of prostanoids.

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Table 2 Examples of transcellular metabolism leading to the production of LTs.

Cell–cell cooperation donor–acceptor)

Exported intermediate

End product

Ref.

Cell–cell cooperation (donor–acceptor)

Exported intermediate

End product

Ref.

Platelets–ECs Lymphocytes–ECs Neutrophils–platelets ECs–platelets

PGH2 PGH2 AA PGH2

PGI2 PGI2 TxA2 TxA2

[24] [30] [33] [31]

Neutrophils–erythrocytes Neutrophils–ECs Neutrophils–VSMCs Neutrophils–platelets Monocytes–platelets Platelets–Neutrophils

LTA4 LTA4 LTA4 LTA4 LTA4 AA

LTB4 LTC4 LTC4 LTC4 LTC4 LTC4

[37] [39–41] [45] [42,43] [49] [32]

Abbreviations: ECs, endothelial cells.

Abbreviations: ECs, endothelial cells; VSMCs, vascular smooth muscle cells.

prevented by active mechanisms that maintain a balance between platelet-derived TxA2 and EC-derived PGI2. Extending this observation, it follows that transcellular metabolism could represent an important contributor to the final profile of prostanoids, LTs and LXs produced in pathophysiologic circumstances, as for example it is well known that leukocytes, platelets, and endothelial cells interact at sites of vascular injury and inflammation and may have great impact on organ function [25,26]. Further studies that used co-cultures of purified cell populations confirmed the initial observation by Vane's laboratory [27–29]. In these studies human umbilical cord vascular endothelial cells (HUVECs) pharmacologically treated to inactivate COX, produced PGI2 following uptake of platelet-derived [27,29] or radiolabeled PGH2 [28]. More recently, a study examined the regulation of endothelial-derived PGI2 by peripheral blood lymphocytes [30]. In this study prostacyclin production by ECs was demonstrated to be, at least in part, dependent on PGH2 arising from lymphocytes and on the number of direct cell–cell interactions. 3. Cell–cell cooperation to synthesize TxA2 Platelet–EC cooperation originally observed in Vane's lab [24] was demonstrated to operate also in the opposite direction, when platelets were COX-inactivated [31]. This investigation demonstrates that platelets are able to bypass COX inhibition and synthesize TxA2 through their constitutive synthase enzyme by taking endothelial PGH2 following incubation with thrombin-activated ECs [31]. Nevertheless the effectiveness of low dose aspirin in suppressing the production of thromboxane suggests that transcellular TxA2 formation does not play a significant role. Furthermore, it was also evident that cells cooperate not only to bypass a temporary or permanent inhibition in their capacity to synthesize AA metabolites, but also to take advantage of unmetabolized precursors produced in the neighborhood. This is the case of neutrophils and platelets, which are able to uptake and make use of free AA produced in excess in a bidirectional way [32] (see also Section 4). It was initially observed that the amount of TxB2 found in supernatants of platelet/ PMN suspensions challenged with fMLP was 2–4 fold higher than that measured when supernatants from fMLP-activated PMNs were used to stimulate platelets [33]. 3H-AA-labeling of neutrophils was used to demonstrate that unlabeled platelets used neutrophil-derived unmetabolized AA to synthesize TxA2 [33] and that the extra TxA2 was generated only following formation of cell–cell contacts through the expression of the adhesive glycoprotein P-selectin, as reduction in mixed cell aggregates by means of an antibody against P-selectin significantly diminished production of TxA2 [32]. 4. Transcellular metabolism of LTA4 Transcellular metabolism as a potential mechanism to produce 5-LO metabolites is well documented in the literature. It has been demonstrated that over 50% of the LTA4 resulting from the activation of the 5-LO is released in the extracellular environment instead of being metabolized by the cell that synthesized it [34,35]. Neutrophil emerges as

a widespread donor for the release of LTA4 to neighboring acceptor cells lacking 5-LO such as platelets, VSMCs, ECs, etc. (Table 2). Also erythrocytes, for a long time considered inert regarding the biosynthesis of eicosanoids for they lack both COX and any of the LO, have the potential to significantly contribute to the synthesis of LTB4 when provided with LTA4 stabilized with albumin [36]. The same group later provided the first evidence for transcellular biosynthesis of LTB4 when they observed that following coincubation of erythrocytes with activated neutrophils the amount of LTB4 produced was greater than that observed upon stimulation of neutrophils alone [37]. Red blood cells may therefore assist neutrophils in generating LTB4 given that LTA4 hydrolase is subjected to suicide inactivation by covalent binding of LTA4 to a tyrosine residue [38], and unconverted LTA4 may therefore escape from neutrophils in significant amounts [37]. At the same time Feinmark et al. provided the first indications of cell–cell cooperation to synthesize LTC4 in studies in which neutrophils were coincubated with ECs, which were unable to actively produce LTs but able to produce a significant amount of LTC4 from exogenous LTA4, and more importantly, from neutrophil-derived LTA4 [39]. The transfer of LTA4 from neutrophils and the presence of a LTC4 synthase activity in ECs were convincingly demonstrated by labeling intracellular glutathione in ECs with [35S]cysteine and observing the production of [35S] LTC4 [39]. Additional reports further indicate significant production of LTs following coincubation of HUVECs and neutrophils [40,41]. Platelets, which were considered to produce mainly the eicosanoid TxA2 from COX, were found to be able to synthesize LTC4 from exogenous LTA4 [42,43] or following LTA4 transfer from activated neutrophils in co-cultures [43]. The production of LTC4 was dependent on the number of platelets coincubated and opened the hypothesis that platelets, interacting with neutrophils and vessels, could also be an important contributor to the production of vasoconstrictor LTs in the pathophysiological setting of the vascular wall, even if aspirin treatment has blocked platelet TxA2 synthesis [44]. This line of evidence, together with the demonstrated ability of endothelial cells and vascular smooth muscle cells (VSMCs) to synthesize LTC4 from LTA4 released from activated neutrophils in coincubation [45] provided additional support to possible role for LTC4 in ischemic and vascular inflammatory disorders. Maugeri et al. [32] demonstrated that platelets and neutrophils participate to the transcellular biosynthesis process in a bidirectional way, where LTB4 can be synthesized by neutrophils from AA donated by platelets. Interestingly, exogenous AA (such as that derived from activated platelets) appears nevertheless to be preferentially used for the additional transfer of LTA4 leading to the formation of LTC4 by transcellular metabolism [46]. Other types of cells that may contribute to transcellular biosynthesis of LTs have been identified by different research groups. In particular murine mast cells have been shown to efficiently convert LTA4 into LTC4 [47] while human B and T lymphocytes synthesized sizable amounts of LTA4 only when added with leukotriene A4 [48] and monocyte/platelet coincubations resulted in increased production of LTC4 as well as 12-HETE and 5(S),12(S)-dihydroxy-ETE [49] resulting from transfer of LTA4, AA and 5-HETE, respectively, from monocytes to platelets.

Please cite this article as: V. Capra, et al., Transcellular biosynthesis of eicosanoid lipid mediators, Biochim. Biophys. Acta (2014), http://dx.doi.org/ 10.1016/j.bbalip.2014.09.002

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Table 3 Examples of transcellular metabolism leading to the production of LXs or resolvins. Cell–cell cooperation (donor–acceptor)

Exported intermediate

End product

Ref.

AM–PMN Monocytes–PMN PMN–platelets Epithelial or Endothelial cells–PMN Endothelial cells–PMN

15(S)-HETE 15(S)-HETE LTA4 15(R)-HETE 18(R)-HEPE

LXA4/LXB4 LXA4/LXB4 LXA4/LXB4 15 epi-LXA4/15 epi-LXB4 RvE1/RvE2

[83] [82] [61–63] [64,65] [76]

Abbreviations: PMN, polymorphonuclear leucocytes; AM, alveolar macrophages.

5. Evidence for transcellular eicosanoid production in organ preparations and in vivo Indirect evidence that a transcellular biosynthetic event can indeed take place in humans was obtained in a wound model studying the production of TxA2 and PGI2 in blood before and after treatment with a TxA2 synthase inhibitor to inhibit formation of TxA2 from platelets [50]. The concentrations of TxB2, the stable hydrolysis product used to evaluate the formation of TxA2, dropped rapidly, whereas those of 6keto-PGF1α, the stable hydrolysis product of PGI2, and PGE2, significantly increased, most likely due to the uptake of platelet-derived PGH2 by ECs and subsequent transcellular biosynthesis of PGI2 and PGE2 [50]. Isolated organ preparations infused with cellular preparation leading to transcellular metabolism have been used to clarify the biological relevance of transcellular biosynthesis products. Seeger et al. using isolated rabbit lungs were able to show nearly quantitative conversion of exogenous LTA4 into LTC4 by the intact pulmonary vasculature [51] and provided evidence that cooperative leukotriene synthesis in pulmonary vasculature resulting from neutrophil–endothelial cell interactions was associated with significant changes in pulmonary microvascular permeability and oedema formation [52]. Similarly, using a model of neutrophil-perfused, isolated rabbit heart Sala et al. reported that transcellular biosynthesis of cysLTs caused coronary vessel vasoconstriction and oedema formation leading to myocardial dysfunction [53,54]. Interestingly, adhesion of neutrophils to the endothelial cell monolayer appeared to be a critical event for the transcellular biosynthesis to occur [25]. Transcellular biosynthesis of cysLTs causing alterations of vascular permeability and oedema formation was also observed in neutrophilperfused, isolated guinea-pig brain [55], further supporting the role played by transcellular metabolism products in the control of the permeability of microvessels. More recently, conclusive evidence that transcellular metabolism is indeed occurring in vivo has been obtained taking advantage of chimeric mice obtained rescuing lethally irradiated, 5-LO knockout mice with bone marrow cells obtained from either LTA4-hydrolase knockout mice [56] or LTC4-synthase knockout mice [57]. Depending on the genetic features of the rescuing bone marrow, no single cell in these animals would carry the complete enzymatic machinery (5-LO and LTA4hydrolase or 5-LO and LTC4-synthase) to carry out the synthesis of LTB4 or LTC4, respectively. Elicitation of an acute inflammatory reaction in these chimeric animals nevertheless resulted in sizeable formation of both leukotrienes families, clearly pointing out to the occurrence of transcellular cooperation between bone marrow derived cells and resident cells toward the synthesis of LTB4 and LTC4. Evaluation of neutrophil chemotaxis and plasma extravasation confirmed that the leukotrienes resulting from transcellular metabolism were biologically active and contributing to the inflammatory response in chimeric animals. 6. Transcellular biosynthesis of lipoxins and resolvins Lipoxins (LXs), lipoxygenase interaction products, are lipid mediators that act as a “stop signal” during inflammatory reactions [22,58], and represent a classical example of mandatory transcellular metabolites derived from cell–cell interactions. Chemically and functionally

different from LTs they are generated by the dual lipoxygenation of AA by either 5- and 12-LO or 15- and 5-LO yielding trihydroxytetraenecontaining eicosanoids, namely LXA4 (5S,6R,15S-trihydroxy-eicosa7E,9E,11Z,13E-tetraenoic acid) and LXB4 (5S,14R,15S-trihydroxyeicosa-6E,8Z,10E,12E-tetraenoic acid) [11,59]. Because of the requirement of a sequential action of two distinct LOs, it was early hypothesized that they were generated in vivo by transcellular biosynthesis through the interaction of two cell types, such as neutrophils, eosinophils, macrophages, endothelial cells, epithelial cells, parenchymal cells or platelets [60]. LXA4 and LXB4 can be generated, classically, through an initial lipoxygenation of AA by 15-LO, usually present in abundance in eosinophil, alveolar macrophage, monocytes and epithelial cells, generating (15S)-HPETE and its reduced form (15S)-HETE, followed by a second lipoxygenation through 5-LO to generate 5(6) epoxytetraene which, in turn, is rapidly converted by two distinct hydrolases in either LXA4 or LXB4. Alternatively, these LXs can be generated through the action of 12-LO [61], usually present in platelets, on LTA4 donated by neutrophils, giving rise to an intraluminal source of lipid mediators [62,63] in alternative to generation of LTC4 (see above) (Table 3). However, there is also a third transcellular biosynthetic route for the generation of a different set of LXs, more specifically 15-epi-LXs, involving COX-2 and 5-LO. While both isoforms of COX, upon acetylation by aspirin, permanently lose the ability to produce prostanoids, acetylation of COX-2 leaves the enzyme capable of oxidizing AA to give 15(R)HPETE and then 15(R)-HETE. These compounds can then be used as substrate by 5-LO to form 15epi-LXA4 or 15epi-LXB4, therefore named aspirin-trigged lipoxins (ATLs) [64,65] (Table 3). Of notice, inhibition of LTA4 hydrolase stimulates synthesis of ATLs and attenuates allergic airway inflammation and hyperresponsiveness in humans [61], and may be part of the cardioprotective action of low dose aspirin in vivo [66]. Despite several lines of evidence have persuasively demonstrated that LXA4 and 15-epi-LXA4 can limit neutrophil infiltration (see [67] for a complete review) and promote activation of both monocytes [68] and of a pro-resolving phenotype of NK cells [69], some functional in vitro studies with endogenous and recombinant ALX/FPR2, a GPCR with high sequence homology (70%) to the formyl peptide receptors (FPR) [67], have generated contradictory results in terms of LXs binding and signaling [20]. For example, while LXA4 and 15 epi-LXA4 have been demonstrated to bind ALX/FPR2 receptor with a Kd in the nanomolar range (0.7–1.7 nM) [70,71] and to promote β-arrestin translocation [72,73], other groups had failed to replicate these data [74,75]. However, because some of the above studies lacked a positive control for LXA4induced responses, these negative results are somewhat of difficult interpretation. Besides AA, also ω-3 essential polyunsaturated fatty acids (PUFAs), such as eicosapentanoic acid (EPA; 20:5, n-3) and docosahexaenoic acid (DHA; 22:6, n-3), are metabolized by lipoxygenases in human cells to produce specialized pro-resolving lipid mediators named resolvins [58]. As for AA, after acetylation by aspirin, COX-2 (or cytochrome P450) develops the ability to metabolize EPA to 18(R)hydroxyeicosapentaenoic acid (18(R)-HEPE), which is then released from endothelial cells and converted into resolvin E1 (RvE1) by PMN 5-LO [76]. Despite RvE1 also binding to the human BLT1 receptor [77], it is ChemR23 that was initially identified as a high affinity RvE1

Please cite this article as: V. Capra, et al., Transcellular biosynthesis of eicosanoid lipid mediators, Biochim. Biophys. Acta (2014), http://dx.doi.org/ 10.1016/j.bbalip.2014.09.002

V. Capra et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx

receptor [78] in cells transfected with an orphan GPCR related to chemokine receptors. RvE1 potently reduces neutrophil–endothelial cell interactions and transmigration as well as neutrophils inflammation, activating the resolution process of inflammation [79]. On the other hand, if the substrate of acetylated COX-2 or cytochrome P450 is DHA, 17(R)-HpDHA is formed which has been demonstrated to be converted by 5-LO to different oxygenated product named aspirin-triggered resolvins (AT-RvD1) [80,81]. Alternatively, DHA is converted by 15-LO to 17(S)-HpDHA which, upon a second lipoxygenation by either 15-LO or 5-LO, is converted into the D series of resolvins (RvD1-6) [80]. 7. Concluding remarks Results from a number of experiments using purified populations of cells, tissues, and organs, are consistent with a model in which transcellular biosynthesis accounts for the production of a portion of LTs present in the tissue because the amount and type of eicosanoid is different from that expected from the individual cells. Transcellular metabolism is an example of communication between cells in close vicinity or forming direct contacts, which can significantly alter the panel of active mediators biosynthesized in response to a specific stimulus adding flexibility to the biological response. Transcellular biosynthesis of eicosanoids can take place during the inflammatory response, contributing both the acute response and to its resolution. Transcellular metabolism can also contribute to the vascular homeostasis, modulating platelet adhesion to vessel walls through their contribution to the synthesis of PGI2 by ECs. It may also have pathological implications when for example leukocytes during an inflammatory response may switch their biosynthetic activity from LTB4 to LTC4 in cooperation with ECs. As such it may represent a potential pharmacological target, and indeed research program is targeted to identify inhibitors of secondary enzymes such as LTC4synthases. Acknowledgments This work was supported in part by the Fulbright Commission (AS). References [1] B. Samuelsson, Role of basic science in the development of new medicines: examples from the eicosanoid field, J. Biol. Chem. 287 (2012) 10070–10080. [2] J. Balsinde, M.V. Winstead, E.A. Dennis, Phospholipase A(2) regulation of arachidonic acid mobilization, FEBS Lett. 531 (2002) 2–6. [3] W.L. Smith, D.L. DeWitt, R.M. Garavito, Cyclooxygenases: structural, cellular, and molecular biology, Annu. Rev. Biochem. 69 (2000) 145–182. [4] D.F. Woodward, R.L. Jones, S. Narumiya, International Union of Basic and Clinical Pharmacology. LXXXIII: classification of prostanoid receptors, updating 15 years of progress, Pharmacol. Rev. 63 (2011) 471–538. [5] J.B. Smith, A.L. Willis, Aspirin selectively inhibits prostaglandin production in human platelets, Nat. New Biol. 231 (1971) 235–237. [6] J.R. Vane, Inhibition of prostaglandin synthesis as a mechanism of action for aspirinlike drugs, Nat. New Biol. 231 (1971) 232–235. [7] T. Kobayashi, S. Narumiya, Function of prostanoid receptors: studies on knockout mice, Prostaglandins Other Lipid Mediat. 68–69 (2002) 557–573. [8] S. Narumiya, Y. Sugimoto, F. Ushikubi, Prostanoid receptors: structures, properties, and functions, Physiol. Rev. 79 (1999) 1193–1226. [9] B. Samuelsson, Leukotrienes: mediators of immediate hypersensitivity reactions and inflammation, Science 220 (1983) 568–575. [10] B. Samuelsson, The discovery of the leukotrienes, Am. J. Respir. Crit. Care Med. 161 (2000) S2–S6. [11] C.N. Serhan, M. Hamberg, B. Samuelsson, Lipoxins: novel series of biologically active compounds formed from arachidonic acid in human leukocytes, Proc. Natl. Acad. Sci. U. S. A. 81 (1984) 5335–5339. [12] C.A. Rouzer, B. Samuelsson, On the nature of the 5-lipoxygenase reaction in human leukocytes: enzyme purification and requirement for multiple stimulatory factors, Proc. Natl. Acad. Sci. U. S. A. 82 (1985) 6040–6044. [13] A.R. Brash, J.J. Murray, J.A. Oates, The 5-lipoxygenase and 15-lipoxygenase of neutrophils and eosinophils, Prog. Clin. Biol. Res. 199 (1985) 143–152. [14] T. Nabe, M. Miura, T. Kamiki, S. Kohno, Arachidonate 5-lipoxygenase and cyclooxygenase metabolites from guinea pig eosinophils and macrophages, Jpn. J. Pharmacol. 83 (2000) 261–264.

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