Formation of 15-hydroxyeicosatetraenoic acid (15-HETE) as the predominant eicosanoid in aortas from Watanabe Heritable Hyperlipidemic and cholesterol-fed rabbits

31

Atherosclerosis, 75 (1989) 31-38 Elsevier Scientific Publishers Ireland, Ltd.

ATH 04239

Formation of Shydroxyeicosatetraenoic acid (SHETE) as the predominant eicosanoid in aortas from Watanabe Heritable Hyperlipidemic and cholesterol-fed rabbits Theodore C. Simon, Amar N. Makheja and J. Martyn Bailey Biochemistry Department,

George Washington University School of Medicine, Washington, DC 20037 (U.S.A.) (Received 15 February, 1988) (Revised, received 3 August, 1988) (Accepted 8 August, 1988)

Atherosclerotic plaque formation is accompanied by hyperproliferative events which have many features of an inflammatory response. A high-performance liquid chromatography procedure was developed to analyze the inflammatory prostaglandins, leukotrienes and hydroxyeicosatetraenoic acids (HETEs) produced by aortic segments. Normal rabbit aortas incubated with tritiated arachidonic acid synthesized 12-HETE as the principal lipoxygenase metabolite, and prostacyclin as the major cyclooxygenase product. In contrast, atherosclerotic aortas from both cholesterol-fed and Watanabe Heritable Hyperlipidemic rabbits showed major increases in synthesis of lipoxygenase-derived SHETE, which became the predominant eicosanoid in the aortas of both types of rabbit. No leukotrienes or other 5-lipoxygenase products were detected to the detection limit of 0.5 pmol/cm aorta. 15HETE, which is chemotactic for smooth muscle cells, mitogenic for endothelial cells, and an inhibitor of prostacyclin synthesis may thus play a role in atherogenesis.

Key words: Atherosclerosis;

WHHL rabbit; Arachidonic acid; Lipoxygenase;

Introduction

Arachidonic cyclooxygenase

acid can be converted by the and lipoxygenase enzymes to a

Correspondence to: Dr. J.M. Bailey, Biochemistry Department, George Washington University School of Medicine, 2300 I St., N.W., Washington, DC 20037, U.S.A. 0021-9150/89/$03.50

15-HETE;

Eicosanoid

variety of biologically active molecules [l]. The cyclooxygenase-derived products include thromboxane, prostacyclin, and the prostaglandins, and are involved in many aspects of arterial homeostasis [2] and atherosclerosis [3]. The lipoxygenasederived products include hydroperoxy fatty acids (HPETEs), which can be reduced to the corresponding hydroxy fatty acids (HETEs), or enzymatically converted to the leukotrienes, for which a variety of biological functions have been

0 1989 Elsevier Scientific Publishers Ireland, Ltd.

32 described [4]. Lipoxygenase products are involved in various aspects of inflammation [4], and a role for both inflammatory processes [5,6] and lipoxygenase products [7] in atherosclerosis has been postulated. It is possible that lipoxygenase products may be involved in atherogenesis. Cuff-induced intimal accumulation of smooth muscle cells in rabbit carotid arteries was inhibited by dexamethasone, which prevents formation of both lipoxygenase and cyclooxygenase products, but not by inwhich inhibits only the cycloodomethacin, xygenase [8]. We have previously shown that antiinflammatory steroids can completely inhibit plaque formation in cholesterol-fed rabbits [9]. While certain non-steroidal anti-inflammatory drugs such as phenylbutazone partially duplicated this effect, others such as aspirin were ineffective. Mouse peritoneal macrophages incubated with acetylated LDL were found to synthesize 250% more 12-HETE than control macrophages [lo], although most of the 12-HETE released by these cells was taken up and esterified into cellular lipids over a 30-min period [ll]. 12-HETE [12] and 15-HETE [13] have been found to be chemotactic for cultured rat smooth muscle cells. In addition to these possible roles for lipoxygenase products in atherogenesis, the sulfidopeptide leukotrienes may be involved in the increased tendency toward vasospasm of arterial regions with plaques [14]. Leukotrienes can constrict normal arteries [2], and have been found to enhance histamine-induced vasospasm [15]. Formation of sulfidopeptide leukotrienes has been found in normal arterial tissue from a variety of species [2], including human arterial ring incubations [16] as well as in an individual with arterial spasm [17], although investigations in the rabbit have not been published. Since acetylated LDL was found to induce production of eicosanoids, including leukotriene C, (LTC,) in mouse peritoneal macrophages in vitro [18], and lipid-laden macrophages have been identified in atherosclerotic plaques [19], leukotrienes might be expected to be increased in atherosclerotic arteries. The occurrence of increased 15-HETE formation in incubations of cholesterol-fed rabbit aortas with arachidonic acid has been reported by Henriksson et al. [20]. The formation of cycloo-

xygenase and other lipoxygenase products including 12-HETE, 5-HETE, and the leukotrienes was not examined, nor was the enzymatic source of the 15-HETE identified. Cyclooxygenase-derived 15HETE and ll-HETE formation were observed in fetal calf aorta [21] and human umbilical arteries [22], and it has been shown that in cultured vascular smooth muscle cells ll-HETE and 15-HETE are formed exclusively by the cyclooxygenase pathway [23]. Lipoxygenase-derived 12-HETE formation was also found in the fetal calf aorta. Prostacyclin has been reported to be formed in both normal and hypercholesterolemic rabbit aortas [24]. Due to these possible roles of lipoxygenase metabolites in atherosclerosis, we have developed a high-performance liquid chromatography (HPLC) procedure to determine the eicosanoid synthesizing capability of arteries in two types of commonly used models of atherosclerotic plaque formation, the cholesterol-fed rabbit and the Watanabe Heritable Hyperlipidemic (WHHL) rabbit, in which atherosclerosis occurs spontaneously. Materials

and methods

Cholesterol was obtained from Sigma Chemical Co., radioisotopes from DuPont Corp., and prostaglandin standards from Cayman Chemical Co. HETE standards were prepared by singlet oxygen oxidation of arachidonic acid [25], and leukotriene standards were a generous gift from Dr. J. Rokach of Merck Frosst. Rabbits Rabbits used in this study were of three types: (1) male New Zealand White rabbits maintained on commercial rabbit chow, (2) male New Zealand White rabbits fed ad libitum with chow supplemented with 1% cholesterol for 19 weeks as previously described [26], or (3) male WHHL rabbits, which have a defective LDL receptor [27] and develop atherosclerosis spontaneously. All rabbits were between 4 and 12 months of age. Aortic incubations Each animal was killed with a l-ml injection of T61 euthanasia solution, and the aorta was ex-

33 cised, photographed for plaque quantitation, and divided as follows. Seven l-cm segments were taken one 0.5 cm apart for the eicosanoid assay, while the alternating 0.5-cm pieces were preserved for histological studies. Each l-cm piece was divided into 6 equal-sized segments which were randomly assigned to 6 assay tubes containing 2.0 ml of Dulbecco’s PBS, 5 mM glucose, pH 7.4. The aortic segments were incubated for 10 min at 37 o C with 2 ~1 ethanol carrier, or 300 PM aspirin, or 50 PM nordihydroguaiaretic acid (NDGA). 5 PM tritiated arachidonic acid (4 PCi in 4 ~1 of ethanol) was added, followed by 3 I_LMionophore A23187 in 4 ~1 ethanol after 2 min. After 20 min incubation, the reaction was terminated by adding 100 ~1 glacial acetic acid and an HPLC standards mixture. The tubes were then stored at - 80 o C until extraction. The standards added were 40 pg 6-keto-PGF,,, 20 pg PGD,, 20 pg PGE,, 20 pg PGF,,, 2 pg PGB,, a mixture of HETEs containing 0.552 pg of 15-, 12-, ll-, 9-, 8, and 5-HETE, and 10 pg BHT. Selected incubations had 200 ng each of LTC,, LTD,, LTE,, and 100 ng of LTB, added as internal standards to confirm that no radioactive peaks were occurring in the leukotriene region of the chromatogram.

tor. The HPLC column was a DuPont Zorbax Cl8 5-pm analytical column, 25 cm X 4.6 mm. The , following 4 solvents were used at a flow of 1 ml/mm, A: methanol, B: acetonitrile, C: water, and D: water with 0.2% trifluoroacetic acid and 0.1% triethylamine. The solvents were mixed by a linear gradient from the initial conditions of 20% B, 75% C, and 5% D to 42% B, 53% C, and 5% D from 0 to 5 min, and then changed by linear gradient from 23 to 28 min to 17% A, 50% B, 8% C, and 25% D, until a step change to 100% B at 72 min. Absorbance at 190 nm was followed from 16 to 35 mm to detect prostaglandins, at 280 nm from 35 to 52 mm to detect leukotrienes, and at 235 nm from 52 to 100 min to detect HETEs. Radioactivity was followed using a 3-ml mixing cell in the Berthold detector, with scintillation cocktail (3a70B, Research Products International) flowing at 3 ml/mm. Radioactive peaks comigrating with UV peaks from the added standards were quantitated by integration, and corrected for yield based on recovery of the added internal standards, which varied between 50 and 100%. Serum cholesterol levels were determined at The George Washington University Lipid Research Clinic using an automated enzymatic assay v91.

Extraction of samples for HPLC analysis The extraction and HPLC protocol are modifications of the method of Eskra et al. [28]. Samples were prepared for chromatography by passing the supematant of the aortic incubation through a Cl8 extraction column (Fisher) which had been washed with 10 ml methanol, 10 ml water, and 10 ml 0.1% disodium EDTA at pH = 6.5. Two 2-ml water rinses of the aortic pieces were done, passing the washes through the column, and discarding all three eluents. The aortic segments were rinsed with 5 separate l-ml methanol rinses. Each wash was passed through the column and filtered through a 0.45pm teflon, syringe tip filter. The combined methanol washes were dried to a volume of 0.75 ml under vacuum, and stored at - 80” C until analyzed by HPLC. HPLC assay The HPLC equipment used was a Waters 600 pump, 490 UV detector, U6K port, and 820 data system, and a Berthold 504B radioactivity moni-

Results An HPLC assay method was developed which can separate and quantitate all of the major arachidonic acid-derived prostaglandins, leukotrienes, and HETEs formed in an aortic incubation. Separation of eicosanoid standards by reverse phase HPLC is shown in Fig. 1. We found that aortic segments from normal rabbits converted [3H]arachidonic acid predominantly to 6keto-PGF,,, the stable decomposition product of prostacyclin, and also 1ZHETE as well as small amounts of aspirin-sensitive 15-HETE (Fig. 2). Aortic segments from hypercholesterolemic animals also formed 6-keto-PGF,, and 12-HETE, together with major amounts of a new aspirin-insensitive metabolite identified as 15-HETE (Fig. 3). No significant amounts of other eicosanoids were detected in either type of aorta, including 5-HETE, leukotrienes, or other 5-lipoxygenase products.

34

nh4E(minutes) Fig. 1. Chromatographic profile of eicosanoid standards separated by HPLC. Authentic standards of the major eicosanoids are separated by reverse phase HPLC as described in the Materials and Methods section. Standards are detected by UV absorbance at the indicated wavelengths. Recovery of standards added to each aortic incubation varied between 50 and 90%.

In the 4 normal aortas tested, 1ZHETE formed in the presence of aspirin was found to vary between 5 and 17 pmol/cm aorta (Table 1). Eicosanoid formation was corrected for yield based on added internal standards, and normalized to length of aorta, since in the atherosclerotic aorta, considerable changes in weight and protein content occur due to plaque formation. The amounts of 12-HETE formed in the 4 cholesterol-fed rabbit aortas and 3 WHHL rabbit aortas were similar to or lower than those found in the normal aortas, except that in 2 cholesterol-fed rabbit aortas, no 1Zlipoxygenase activity was detected. The observed variability in 1Zlipoxygenase activity is apparently due to differences in individual rabbits as well as variation in duplicate incubations, which was found to be less than 50%. 15-HETE production in normal rabbit aortas was below detection limits in all cases except for one aorta, in contrast to the hyperlipidemic animals where all the aortas displayed significant 15-lipoxygenase activity. The levels of 15-HETE formation in the hyperlipidemic animals were variable, ranging from 3 to 14 pmol/cm aorta, except for one animal, which produced 434 pmol/cm aorta. In this animal, the high 15-lipoxygenase activity was found in both the control and aspirin incubations, and was reduced to background levels by incubation with the lipoxygenase inhibitor, NDGA. No unusual plaque morphology

or hypercholesterolemia was observed which could explain the high lipoxygenase activity. 15-HETE formation did not appear to directly correlate either to serum cholesterol levels or percent plaque (Table 1). Identification of 15-HETE and 12-HETE synthesized in aortic incubations was confirmed by analyzing duplicate samples on both reverse and straight phase HPLC. Similar amounts of the 2 radioactive products corn&rated with authentic standards of 12-HETE and 15-HETE in both chromatography systems. The standards were produced by singlet oxygen oxidation of arachidonic acid as described in the Materials and Methods

II I

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I III

I

___/J:(

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9070

90

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TIME (minutes)

Fig. 2. Eicosanoid formation in normal rabbit aorta. The 3 panels show products formed from radioactive arachidonic acid by normal rabbit aorta, incubated with ethanol carrier, and in the presence of 300 pM aspirin and 50 pM NDGA. Arrows indicate the migration times of standards added to each incubation and detected by UV absorbance as shown in Fig. 1. In the control incubation, radioactive peaks are observed which comigrate with 6-keto-PGF,,, 15-HETE, 12 HETE and LTD,,/LTE,. In the presence of aspirin, the peaks corresponding to 6-keto-PGF,,, LTDJLTE,, and 15HETE peaks are decreased. In the presence of NDGA, a lipoxygenase inhibitor, the 12-HETE peak also is inhibited. The peak comigrating with LTD,/LTE, was seen in approximately half the incubations, and remains unidentified. Since it was inhibited by aspirin, it is not a lipoxygenase product. Formation of aspirin-sensitive 15-HETE in arterial tissue has been previously reported [21].

35 animals among a group of the highly inbred WHHL rabbits maintained on anti-inflammatory steroids. Alternatively therefore, the increase in 15-lipoxygenase activity could be an episodic event, with periods of high and low activity occurring at discrete times during the disease process. Plaque progression in humans is known to be episodic in nature 1301. Experiments to distinguish these possibilities are currently in progress. Contrary to the expectation that atherosclerotic aortas would produce a variety of leukotrienes or other lipoxygenase products, 15-HETE was the only additional product detected on these aortas. Significantly, no 5-HETE formation was observed

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TIME (minutes)

Fig. 3. Eicosanoid formation in hypercholesterolemic rabbit aorta. The 3 panels show products formed from radioactive arachidonic acid by hypercholesterolemic rabbit aorta, incubated with ethanol carrier, and in the presence of 300 pM aspirin and 50 pM NDGA. Note that the predominant eicosanoid is 15-HETE, which is aspirin-insensitive but inhibitable by NDGA.

section and their identity confirmed by GC/MS analysis of the hydrogenated trimethylsilyl ether, methyl ester derivatives as previously described 1231. Discussion The increase we found in the 15-lipoxygenase activity compared to the 1Zlipoxygenase activity indicates that the hypercholesterolemic condition selectively induces the 15-lipoxygenase. The absolute differences in lipoxygenase activity found in both types of hypercholesterolemic animals may result from the individual variation in the course of the atherosclerotic progression. However, the identification of a high-responding animal in which a 40-fold increase in 15-lipoxygenase activity, relative to the other atherosclerotic animals, is significant. It seems unlikely that this increase could be due to an inherent genetic difference in this animal, and no overt differences in plaque morphology or hypercholesterolemia were observed. In addition, we have since observed other hyperresponding

TABLE 1 LIPGXYGENASE PRODUCT FORMATION, PLAQUE FORMATION, AND SERUM CHOLESTEROL LEVELS IN NORMAL AND ATHEROSCLEROTIC RABBITS Rabbit aortas were sectioned and incubated with [‘H]arachidonic acid as described in the Materials and Methods section. The radioactive metabolites were separated by reverse-phase HPLC and quantitated by integrating peak area. The values for lipoxygenase-derived 15-HETE and 12-HETE were obtained from incubations in the presence of 300 pM aspirin, with background values of incubations in the presence of 50 pM NDGA subtracted. HETE formation is normalized to length of aorta since aortic weight and protein content varies with plaque formation. Percent plaque coverage was obtained from photographs of the aorta overlaid by a grid to quantify the affected areas, and serum cholesterol levels were measured by an enzymatic assay and autoanalyzer [29]. 15-HETE

12-HETE

(pmol/cm aorta) New Zealand White rabbits 1 0 6 2 2 11 3 0 17 4 0 5 Cholesterol-fed rabbits 1 11 0 2 14 3 3 3 0 4 434 21

Percent plaque

Serum cholesterol (mg/dl)

0 0 0 0

58 72 63 85

29 100 90 100

1510 1895 2424 2742

Watanabe Heritable Hyperlipidemic rabbits 1 11 20 47 2 13 I 11 3 9 6 54

438 777 568

36 in the atherosclerotic aorta incubations, indicating that the 5-lipoxygenase pathway, from which the inflammatory leukotrienes are produced, is not active in this tissue. The lower detection limit with this system is one half pmol of product formed per cm of aorta, based on the specific activity of the [3H]arachidonic acid and the smallest clearly identifiable peak. Other tissues of the rabbits were tested for eicosanoid production, and both 5HETE and LTC, were detected in homogenized spleen using the same incubation and HPLC procedures. The cellular sources of the increased tissue eicosanoids remain to be identified. 12-HETE could arise from contaminating platelets, but this is unlikely since no corresponding amounts of the other major platelet arachidonic acid metabolites, thromboxane B, and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), were observed. The increase in the 15-lipoxygenase may be related to infiltration of inflammatory cells, including macrophages, although Saito et al. [38] found no significant increase in HETEs from circulating macrophages from cholesterol-fed rabbits. Furthermore we observed no significant formation of other macrophage products such as PGE,, 5HETE, or LTB,. However, other cells such as eosinophils [39], which are known to produce 15HETE, may be involved. Alternatively, the 15lipoxygenase could be induced in the cells of the artery wall. Both smooth muscle cells [40] and endothelial cells [41] have been reported to possess a 15-lipoxygenase. Production of 15-HPETE or 15-HETE in vivo might have a variety of biological effects. Both 12-HETE [12] and 15-HETE [13] have been shown to be chemoattractants for smooth muscle cells, and 15-HETE has also been reported to be a mitogen for endothelial cells [31], and thus might play a role in the cellular events of atherogenesis. 15-HPETE has been shown to inhibit prostacyclin production from rabbit vessel wall microsomes [32], and 15-HETE to inhibit the cyclooxygenase of human umbilical arteries [33]. In addition to a role in atherogenesis, 15-lipoxygenase products may have a role in vessel tone. 15-HPETE was found to enhance the anti-aggregatory action of prostacyclin and to prevent platelet aggregation induced by arachidonic acid

[34]. 15-HPETE has also been found to induce relaxation of rat aortic rings contracted with PGF,, [35], canine cerebral and coronary artery strips contracted with PGF,, [36], and also rat aortic rings in which contraction was induced with a stable thromboxane analog [37]. It should be noted from the comparative data in Table 1 that the increase in 15-lipoxygenase activity observed in hypercholesterolemic animals did not correlate directly with the extent of atherosclerosis or the degree of hypercholesterolemia. It could thus represent a tissue response of a defensive nature to the hypercholesterolemic insult, rather than being an intrinsic product of the atherosclerotic lesion. Further experiments to determine the anatomical location of the 15-lipoxygenase and its possible biological function in atherogenesis are in progress. Acknowledgements We wish to thank Dr. Richard Muesing of The George Washington University Lipid Research Clinic for performing the serum cholesterol determinations. The authors gratefully acknowledge Dr. Michael S. Brown and Dr. Joseph L. Goldstein for providing breeding stock to form the WHHL rabbit colony. References Needleman, P., Turk, J., Jakschik, B.A., Morrison, A.R., and Lefkowith, J.B., Arachidonic acid metabolism, Ann. Rev. B&hem., 55 (1986) 69. Simmet, T. and Peskar, B.A., Eicosanoids and the coronary circulation, Rev. B&hem. Pharm., 104 (1986) 1. Willis, A.L., Smith, D.L. and Vigo, C., Suppression of principal atherosclerotic mechanisms by prostacyclins and other eicosanoids, Prog. Lipid Res., 25 (1986) 645. Davies, P., Bailey, P.J., Goldenberg, M.M. and FordHutchinson, A.W., The role of arachidonic acid oxygenation in pain and inflammation, Ann. Rev. Immunol., 2 (1984) 335. Lopes-Virella, M.F. and Virella, G., Immunological and microbiological factors in the pathogenesis of atherosclerosis, Clin. Immunol. Immunopath., 37 (1985) 377. Schwartz, C.J., Valente, A.J., Sprague, E.A., Kelley, J.L., Suenram, C.A., Graves, D.T., Rozek, M.M., Edwards, E.H. and Delgado, R., Monocyte-macrophage participation in atherogenesis: inflammatory components of pathogenesis, Sem. Thromb. Hemostasis, 12 (1986) 79. Heam, J.A., The pathogenesis of atherosclerosis, Lancet, 315 (1986) 643.

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38 Saeki, Y., Vascular effects of 15-hydroperoxyeicosatetraenoic acid and 15-hydroxyeicosatetraenoic acid on canine arteries, Jap. J. Pharm., 37 (1985) 325. 37 D’Alarao, M., Corey, E.J., Cunard, D., Ramwell, P., Uotila, P., Vargas, R. and Wroblewska, B., The vasodilation induced by hydroperoxy metabolites of arachidonic acid in the rat mesenteric and pulmonary circulation, Br. J. Pharmacol., 91 (1987) 627. 38 S&to, H., Salmon, J.A. and Moncada, S., Influence of cholesterol feeding on the production of eicosanoids, tissue plasminogen activator and superoxide anion (0; ) by rabbit blood monocytes, Atherosclerosis, 61 (1986) 141.

39 Turk, J., Maas, R.L., Brash, A.R., Roberts, L.J. and Oates, J.A., Arachidonic acid 15-lipoxygenase products from human eosinophils, J. Biol. Chem., 257 (1982) 7068. 40 Nakao, J., Koshihara, Y., Ito, H., Murota, S. and Chang, W., Enhancement of endogenous production of 12-L-hydroxy-5$X,11,13-eicosatetraenoic acid in aortic smooth muscle cells by platelet-derived growth factor, Life Sci., 37 (1985) 1435. 41 Hopkins, N.K., Oglesby, T.D., Bundy, G.L. and Gorman, R.R., Biosynthesis and metabolism of 15-hydroperoxy5,8,11,13-eicosatetraenoic acid by human umbilical vein endothelial cells, J. Biol. Chem., 259 (1984) 14048.