Endothelial cells can synthesize leukotriene B4 Kevin D. Nolan, M_D, Blair A. Keagy, MD, Fuad M. Ramadan, MD, George Johnson, Jr,, MD, and David C. Henke, MD, Chapel Hill, N.C. Leukotriene B4 (LTB,) from vascular endothelium may play a key role in the genesis of atherosclerotic lesions. However, the ability of this tissue to synthesize LTB~ is controversial. To resolve this issue arachidonic acid metabolism was characterized in cultures of confluent monolayers of a rabbit aortic endothelial cell line by use o f both high-pressure liquid chromatography and radioimmunoassay. Cells were grown to confluence in Dulbecco's modified Eagle's medium/Ham's F12 with 5% fetal bovine serum. Lipoxygenase activity was studied by placing the cells in Hank's balanced salt solution with 2 wmol/L indomethacin. After 30 minutes preincubation with indomethacin cells were exposed to either arachidonic acid (10 ~mol/L) or arachidonic acid labeled with radioactive carbon (14C) (1 ~tCi; SA 58 mCi/mmol) and then stimulated with 9.5 ~ m o l / L calcium ionophore A23187 for 55 minutes. Studies of the cyclooxygenase activity were performed without preincubating with indomethacin. Samples were prepared for high-pressure liquid chromatography by evaporation to dryness under a vacuum and resuspending in 2 ml o f 1 : I methanol/water. Tritium-labeled standards were added before loading the ~4C-labeled samples on the column. Radiolabeled arachidouic acid metabolites were separated by high-pressure liquid chromatography and detected by means o f a dual channel flowthrough radiodetector that monitors both ~4C and SH. Based on coelution with authentic standards three lipoxygenase metabolites of arachidonic acid have been identified: LTB~, 12- and 5-hydroxyeicosatetraneoic acid. Leukotriene B4 was further characterized by ultraviolet spectral analysis and inhibition studies with use of nordihydroguaiaretic acid. Quantitation was facilitated by commercially available radioimmunoassay kits. An average of 600 pg L T B J 106 cells was measured from separate experiments. Negative controls produced no LTB4. These data demonstrate that rabbit aortic endothelial cells when stimulated with calcium ionophore in the presence o f exogenous arachidouic acid synthesize LTB4, an inflammatory mediator that may play an important role in the pathogenesis of atheroslcerosis. (J VASC SURG 1990;12:298-304.)

The endothelial cell's ability to synthesize cyclooxygenase products of arachidonic acid (AA) metabolism has been established, ~ and these products have been implicated in a number of arterial processes including atherosclerosis.2 Lipoxygenase products of AA metabolism are involved in various aspects of inflammation and may also play a key role in the genesis of atherosclerotic lesions? For example, the intimal accumulation of smooth muscle cells in rabbit carotid arteries, an early stage of atherosclerosis, is inhibited by dexamethasone, which prevents formarion of both lipoxygenase and cyclooxygenase products, but is not inhibited by nonsteroidal antiinflammatory drugs such as indomethacin, which From the Department of Surgery, University of North Carolina at Chapel Hill. Presented at the Fourteenth Annual Meeting of the Southern Association for Vascular Surgery, Acapulco, Mexico, Jan. 2427, 1990. Reprint requests: George Johnson, Jr., MD, University of North Carolina Division of Vascular Surgery, 210 Burnett-Womack Building, CB 7210, Chapel Hill, NC 27599-7210. 24/6/21688 298

blocks only the cyclooxygenase pathway of AA metabolism. 4 These findings are further corroborated in cholesterol-fed rabbits, s where steroids such as dexa~ methasone, have been shown to inhibit plaque formation. Lipoxygenase metabolites of AA, therefore, may be key players in the genesis of atherosclerosis. Leukotriene B4 (LTB4), a product of the lipoxygenase pathway, mediates chemotaxis ofneutrophils, eosinophils, and monocytes,6 causes adhesion of neutrophils to the endothelium, and extravasation of white cells from the vascnlature. 7 Leukocytes, once present at a site of injury, produce other leukotrienes (C4, D4, and E4), which promote platelet aggregation and cause vascular constriction, s Endothelial-derived LTB4 may, therefore, be initiating vascular inflammarion; acting as a mediator in the genesis of atherosclerosis. Leukotriene B4 a 5-hpoxygenase metabolite of AA, however, has not clearly been shown to be produced by vascular endothelium.9-11 Controversy has existed with regard to endothelial cell synthesis of LTB, ever since it was first described by Piper et a1.12-

Volume 12 Number 3 September 1990

Endothelial cell metabolism of arachidonic acid 299

PGD2

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0 80

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Fig. 1. Reverse phase-high pressure liquid chromatography with an acetanitrile/water mobile phase (as described in text) provided good separation of these 3H-authentic standards. Recovery of radiolabeled standards from the column was approximately 82%. who demonstrated LTB4 production by this tissue using a bioassay) 2 Since then Jackson et al.~3 and Piper et al.X4have published reports of LTB4 synthesis from cultured endothelial cells using primarily immunochemical analysis. Our investigation has used reverse phase-high pressure liquid chromatography (RP-HPLC), spectrophotometry, and radioimmunoassay to characterize the lipoxygenase metabolites produced by rabbit aortic endothelial cells in culture. By use of these techniques we have demonstrated the -nzymatic production of LTB4 from AA by rabbit aortic endothelium. Under these in vitro conditions, rabbit aortic endothelium produces small amounts of LTB4 (541 pg/106 cells) compared with its ability to produce prostacyclin (PGI2; 8697 pg/106 cells). Whether this ratio of PGI2: LTB4 production can be altered in vitro or in vivo in association with disease states or therapeutic manipulation remains to be studied. MATERIAL AND METHODS

Reagents Calcium ionophore A23187 (Calbiochem, San Diego, Calif.); LTB4 standard (Caymen chemicals, Ann Arbor, Mich.); 3H LTB4 (SA 200 Ci/mmol), AA, 14C-AA (10 p~mol/L), (NEN Research Products, Boston, Mass.); Nordihydroguaiaretic acid (NDGA) (Sigma Chemical Co., St. Louis, Mo.); Indomethacin (Merck and Co., Inc., Rahway, N.J.).

All cell culture reagents were obtained through the Lineberger Cancer Research Center, Chapel Hill, N.C. Cell culture A rabbit aortic endothelial cell line (transformed with SV 40 viral oncogene by Dr, Fred C. Jensen, Scripps-Miles, Inc., La Jolla, Calif.), was obtained from the Lineberger Cancer Research and maintained in Dulbecco's modified Eagle's medium/Ham's F12, 1 : 1 mixture, and antibiotics (penicillin sodium: 100 IU/ml, streptomycin sulfate: 100 I~g/ml, and amphotericin B: 2 ~g/ml). The media were supplemented with 5% fetal calf serum for routine maintenance and passage of the cultures. One hour before the study of AA metabolism, the cells were washed three times and placed in Hank's balanced salt solution with calcium (185 mg/L) and magnesium (98 rag/L). Cells were maintained for the duration of the experiment in this environment. Experiments were conducted in 75 cmz tissue culture flasks, which were kept in a humidified 5% CO2 incubator at 37 ° C. Endothelial cell identity was estabfished and has been reported in earlier work from this laboratory. ~s Experimental protocol and product characterization Arachidonic acid metabolism in rabbit aortic endothelial cells was studied by use of sonicated and

300

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Journal o f VASCULAR SURGERY

et aI.

15 Rabbit Endothelium PGD2

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Fig. 2. Metabolites of SH-AAin media from sonicated rabbit aortic endothelial cell preparations. These cells were not exposed to A23187. Superimposed is the chromatograph obtained from authentic eicosanoid standards by use of identical HPLC technique. Comigration identifies the metabolites.

intact cells. Sonicated preparations were used to bypassthe regulatory mechanisms in the membrane that might mask the enzymatic activity in the cells. The goal was to present substrate, that is AA, directly to the 5-1ipoxygenase whose activity might otherwise be masked. In experiments designed to characterize AA metabolites in rabbit aortic endothelial cells, harvested cells were suspended in buffer solution (NaC1 0.16 mol/L, sucrose 0.79 mol/L, TES 0.2 mol/L, MgCI2 0.16 mol/L EGTA 0.004 mol/L, CaCI2 0.003 mol/L), sonicated on ice, and incubated for 5 minutes in tritiated (SH) AA. The incubation was stopped with 1 ml of cold methanol. Cellular debris was removed by centrifuging in an Epindorph at high settings for 5 minutes. Supematants were stored at - 8 0 ° C before analysis. Reverse phase-high pressure liquid chromatography consisted of an (A) acetanitrile and (B) water mobile phase. Experimental residues dissolved in 2000 mm s acetanitrile and water (1 : 1). The following program was used: initial conditions of 30% A/70% B run until 25 minutes, 32% A/68% B until 38 minutes, 55% A/45% B until 49 minutes, 60% A/40% B until 79 minutes and then to 100% A. Eighteen hundred mm s were injected onto the column (LC-18, 25 cm x 4.6 ram, particle size 5 p~ [Supelco, Bellefonte, Pa]), and radioactivity eluting off the column was compared to that of authentic eicosanoid standards for prostaglandin FI~ (PGFI~), thromboxane B2 (TXB2), prostaglandin F2~

(PGF2~), prostaglandin D2, and E2, LTB4, 15-, 12-, and 5-hydroxyeicosatetraneoic acid (HETE). Intact cell preparations were studied in two ways. First, AA metabolites produced after incubation with: ~C-AA were separated and identified by use of RPHPLC. In these experiments the cell monolayers were preincubated with 2 ~mol/L indomethacin for 30 minutes after which 14C-AA (lt~Ci; SA 58 mCi/mmol) was added and cells were stimulated with 9.5 I~mol/L A23187. The incubation was" stopped after 55 minutes by adding an equal volun~., of ethanol (4 ° C). The media were collected in po[ypropylene tubes and taken to dryness under a vacuum. The residue was dissolved in 2000 mm s methanol and water (1:1). Tritium-labeled authentic standards including PGFI~, TXB2, PGF2~, LTB4~~ 15-, 12-, and 5-HETE were then added to each sample. Samples were injected onto the H P L C column. Radiolabeled products were separated by this system with (A) methanol and (B) water with 10% methanol, p H adjusted to 5.05. Initial conditions were 52% A and 48% B. Program gradient was as follows:' initial conditions to 26 minutes then linear gradient to 61% A and 39% B. Seventy percent A and 30% B at 52 minutes, and finally to 100% A after 76 minutes. Flow rate was 1.01 ml/minute. Radioactivity eluted from the H P L C column was measured by a dual label flow-through radiodetector monitor-' ing both ~C and SH channels simultaneously. Leu-

Volume 12 Number 3 September 1990

Endothelial cell metabolism of arachidonic acid 301

20-

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kotriene B4 and HETEs were tentatively identified based on the coelution of peaks from sample and authentic leukotriene and hydroxylated eicosanoid standards. The second protocol with intact cells was designed specifically to look for LTB4 generation by rabbit aortic endothelial cells. In these experiments, 10 i~mol/L AA was substituted for its radiolabelled counterpart, but the experimental protocol was otherwise unchanged. The resultant residue was dissolved in acetanitrile and water (1: 1). These solvents were used for the mobile phase rather than methanol/water to optimize ultraviolet detection of the LTB4 fractions. Initial conditions were 55% A and 45% B. At 13 minutes the gradient was changed to 100% A as authentic LTB4 was found to elude at approximately 9 minutes. All fractions coeluding with authentic standard were collected, dried under a vacuum, and reconstituted in 500 man 3 of distilled &ionized water for radioimmunoassay. By use of this same protocol the effect of incubation time on LTB4 synthesis was assessed. Thirty-, 45-, 60-, and 75minute incubation times, after stimulation, were compared to LTB, levels from nonstimulated cells. The metabolism of AA to LTB4 by rabbit aortic endothelium was inhibited in a dose-dependent fashion by means of low-dose (10 ~mol/L) and a high-dose (100 ~mol/L) N D G A incubation before stimulation with A23187. 6-keto PGFI~ (6KPGFI~) production

was measured by radioimmunoassay and compared with LTB4 production to provide perspective. Leukotriene B4 production by rabbit aortic endothelial for characterization by ultraviolet spectraphotometric scanning was generated by combining the media from 9 x 10 6 cells. RESULTS Both RP-HPLC programs provide good separation of most of the prostaglandins, leukotrienes, and HETEs that might be generated by rabbit aortic endothelial cells. A representative chromatograph demonstrating the separation of 3H-labeled authentic eicosanoid standards achieved with the acetanitrile/water technique is shown in Fig. 1. The same system was used for sonicated cell preparations. The major metabolites produced by rabbit aortic endothelial cells sonicated and incubated with 3H-AA, cochromatographed with 6KPGF~, the stable decomposition product of PGI2 (Fig. 2). Products comigrating with TXB2 the stable decomposition product of thromboxane -42, PGF2~, 15- and 12-HETE were also produced. Chromatographic peaks representing LTB4 and 5-HETE were not detected. These profiles are consistent with profiles reported by others from cells in primary cultureF ° Media extracts from intact cell preparations incubated with ~*C-AA and stimulated with A23187 in the presence of indomethacin were analyzed by use of a methano! and

302

Journal o f VASCULAR SURGERY

Nolan et al.

HBSS NDGA Standard

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NDGA High NDGA Low

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0

1000

2000

3000

pg LTB4 / 100 ul Fraction

Fig. 4. 10 I~mol/L and 100 Ixmol/L NDGA preincubation with rabbit aortic endothelial cells before A23187 stimulation. Leukotriene B~ standard and both HBSS as well as NDGA are shown as positive and negative controls, respectively.

unstimulated controls

75

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60

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Fig. 5. Leukotriene B~ production as determined by radioimmunoassay in rabbit aortic endothelial cells incubated for differing lengths of time with A23187. Comparison is illustrated between groups and versus cells not exposed to A23187.

water H P L C system described previously. Authentic standards labeled with 3H were added directly m the 14C-containing extracts, and a dual channel flowthrough detector provided simultaneous chromatographs for 14C-labeled metabolites and 3H-labeled standards. Arachidonic acid metabolites were identiffed coeluting with LTB4, with 12- and 5-HETE (Fig. 3). When incubations were completed ha the pres-

ence of both high- and low-dose NDGA, LTB4 production was clearly inhibited in a dose-dependent fashion. Authentic LTB4 was included as a positive control, and both N D G A alone and Hank's balanced salt solution alone were run as negative controls (Fig. 4). When H P L C was used specifically for LTB4 fraction collection and fractions then subjected to radioimmunoassay, 55-minute incubations with

Volume 12 Number 3 September 1990

Endothelial cell metabolism of arachidonic acid 303

LTB4

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541

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Cells Produced

Fig. 6. Arachidonic acid metabolites from A23187 stimulated rabbit aortic endothelial cells in the presence of indomethacin and after addition of exogenous AA; as determined by radioimrnunoassay. A23187 produced 600 pg/106 cells. Stimulation with A23187 was clearly associated with higher LTB4 levels when compared to unstimulated controis, however, no statistically significant effects were appreciated when changes in the length of incubation times made during this investigation were reviewed (Fig. 5). Leukotriene B4 production is not as impressive when compared with 6KPGFa~ (Fig. 6). When we attempted to characterize the AA metabolite cochromatographing with LTB4 spectrophotometrically, H P L C fractions comigrating with LTB4 standard were collected and scanned over a wavelength range from 255 to 310 nm. They were then compared to authentic LTB4 processed in a similar fashion (Fig. 7). DISCUSSION Lipoxygenase products have been postulated to be involved in atherosclerosis. ~° Leukotriene B4, a 5lipoxygenase metabolite of AA, has many characteristics suggesting its possible role as an inflammatory mediator in the genesis of atherosclerotic lesions. Reports from Piper et al. ~2 and then from Jackson et al.~3 suggesting the presnce of a 5-1ipoxygenase system and the synthesis of LTB~ from vascular endothelium were challenged in subsequent papers from Johnson et al.9 and Simon et al. 1° The former studies were conducted in porcine and bovine pulmonary artery endothelium, respectively, whereas, the later used human umbilical vein and rabbit aortic endothelium. Study designs were variable, however,

LTB4 U.V.

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