Proc. Nati. Acad. Sci. USA Vol. 85, pp. 4511-4515, June 1988 Medical Sciences Increased prostacyclin and thromboxane A2 biosynthesis in atherosclerosis (arachidonic acid) JAWAHAR L. MEHTA*t, DANIEL LAWSON*, PAULETTE MEHTA*, AND TOM SALDEENt *University of Florida, College of Medicine and the Veterans Administration Medical Center, Gainesville, FL 32610; and tUniversity of Uppsala, 751 21 Uppsala, Sweden Communicated by George K. Davis, March 2, 1988 ABSTRACT It has been proposed that atherosclerotic PGI2 metabolite released in the urine of patients with severe arteries produce less prostacyclin (PGI2) than nonatheroscle- atherosclerosis, and they demonstrated increased PGI2 bio- rotic arteries do, thereby predisposing arteries to vasospasm synthesis in these patients relative to normal subjects. and thrombosis in vivo. We reexamined this concept by Since many previous studies employed bioassay or only measuring spontaneous as well as arachidonate-induced PGI2 radioimmunoassay (RIA) for measurement of PGI2, the biosynthesis in aortic segments from nonatherosclerotic and present studies were designed to reexamine the concept of cholesterol-fed atherosclerotic New Zealand White rabbits. altered PGI2 generation in atherosclerosis. We examined the Thromboxane A2 (TXA2) generation was also measured. For- uptake and incorporation of labeled arachidonic acid into the mation of PGI2, as well as TXA2, as measured by radio- phospholipids ofthe vessel wall and subsequent arachidonate immunoassay (RIA) of their metabolites, was increased in release and conversion to major metabolites of and atherosclerotic aortic segments relative to nonatherosclerotic PGI2 segments (P S 0.05) at 0, 5, 10, 15, and 30 min of incubation TXA2 in both normal and atherosclerotic vessels. with arachidonate. Pretreatment of arterial segments with indomethacin inhibited PGI2 as well as TXA2 formation, MATERIALS AND METHODS whereas pretreatment with the selective TXA2 inhibitor OKY- 046 inhibited only TXA2 release, thus confirming the identity Induction of Atherosclerosis. New Zealand White male of icosanoids. To confirm the RIA data, aortic segments were rabbits weighing 2.26-2.70 kg were fed an atherogenic diet of incubated with ['4C]arachidonate prior to stimulation with 1% cholesterol and 5% (wt/wt) lard (Bio-Serve, Frenchtown, unlabeled arachidonate. The uptake of arachidonate was NJ) for 2 weeks, which caused an increase of serum choles- similar, but the release of incorporated ['4C]arachidonate was terol to 981-1368 mg/dl (mean = 1191 mg/dl). Thereafter, significantly (P - 0.05) greater in atherosclerotic segments the rabbits were anesthetized (acepromazine at 0.5 mg/kg, than in nonatherosclerotic ones. Conversions of released Rompun at 3 mg/kg, and ketamine at 50 mg/kg), and their ["4C]arachidonate to 6-keto["4C]prostaglandin Fl and right groins were explored to isolate the right femoral artery. ["'C]thromboxane B2 were similar in the two types of aortic After arteriotomy, a no. 4F Fogarty catheter was inserted and segments. Thus, synthesis of PGI2 as well as TXA2 is increased advanced into the abdominal aorta to the level of the in atherosclerosis, and this alteration in arachidonate metab- diaphragm. The balloon was then inflated and retrieved, olism is related to increased release of arachidonate. resulting in endothelial stripping of the aorta and right iliac artery. The right femoral artery was then ligated, and the Decreased prostacyclin (PG12) biosynthesis and release by incision was closed. The atherogenic diet was thereafter atherosclerotic compared to normal vessels was described by continued for 3 months prior to surgical exposure of the Dembinska-Keic et al. in 1977 (1). These initial observations abdominal aorta. As controls, a group of rabbits underwent in rabbit vessels were subsequently confirmed in the post- aortic stripping, but no atherogenic diet was given either mortem examination of human atherosclerotic vessels by before or after stripping. D'Angelo et al. (2) and Sinzinger et al. (3). Larrue et al. (4) Preparation ofTissues for PGI2 and TXA2 Generation. After reported diminished PGI2 generation by cultured smooth 3-months of atherogenic diet, animals were sacrificed with muscle cells from atherosclerotic rabbit aortae. intravenous pentabarbital infusion and the abdominal aorta Thromboxane A2 (TXA2) production is also increased in was rapidly harvested. The vessel was collected in ice-cold experimental (5) and human atherosclerosis (6-8). Accord- Hanks' balanced salt solution (HBSS) without Ca2+ and ingly, it has been proposed that a relative excess ofTXA2 and Mg2+, pH 7.4, and carefully cleaned of all loose connective deficiency of PG12 may predispose atherosclerotic vessels to tissue and fat. The aorta was then perfused repeatedly with vasospasm and platelet aggregation in vivo (9, 10). HBSS to remove blood clots, remaining platelets, and leu- Although the plasma concentrations of 6-ketoprosta- kocytes until the perfusate contained no leukocytes or glandin Fla (6-keto-PGFia), the stable hydrolysis product of platelets visible by light microscopy. The tissues were then PGI2, are extremely low in normal plasma (-4 pg/ml) (11), it cut into 2-mm ring segments, weighed, and immediately has been reported that these concentrations are increased in placed into 2.0 ml of HBSS containing Ca2+ and Mg2 . patients with atherosclerosis (8, 12). Hirsh et al. (8) reported Approximately 30 mg of tissue (wet weight) was incubated mean plasma concentrations in patients with coronary ath- with 10 ,M arachidonate (Sigma) alone or with the selective erosclerosis of 140 pgfml. In another study (12), mean plasma TXA2 synthetase inhibitor OKY-046 (10 ,M) (Ono Pharma- 6-keto-PGF1a concentrations in similar patients were re- ceutical, Osaka, Japan) or the cyclooxygenase inhibitor ported to be 94 pg/ml (vs. 54 pg/ml in control normal indomethacin (10 (Merck Sharpe & Dohme) at 37°C for subjects). Recently, Fitzgerald et al. (13) measured a major AM) Abbreviations: 6-keto-PGFIa, 6-ketoprostaglandin Fla; PGI2, pros- The publication costs of this article were defrayed in part by page charge tacyclin; TXA2 or TXB2, thromboxane A2 or B2. payment. This article must therefore be hereby marked "advertisement" *To whom reprint requests should be addressed at: University of in accordance with 18 U.S.C. §1734 solely to indicate this fact. Florida, Box J-277, JHMHC, Gainesville, FL 32610. Downloaded by guest on September 29, 2021 4511 4512 Medical Sciences: Mehta et al. Proc. Natl. Acad. Sci. USA 85 (1988) 30 min. The supernate was collected at 0, 5, 15, and 30 min surrounding each 6-keto-PGFia and TXB2 spot was then for measurement of PGI2 and TXA2 metabolites (14). scraped from the plate and resuspended in 5.0 ml of Atom- Incorporation of ['4C]Arachidonate. To confirm the results light liquid scintillation counting fluid (New England Nu- of the PGI2 and TXA2 released into the supernates of aortic clear) and mixed well on a mechanical shaker for 1 hr, and its rings treated with arachidonate, 150-mg samples of aortic radioactivity was measured. rings were incubated with 1.0 puCi of [14C]arachidonate (390 RIAs for PGI2 and TXA2. 6-Keto-PGFia and TXB2, the mCi/mmol, New England Nuclear; 1 Ci = 37 GBq) for 5 min stable hydrolysis products ofPGI2 and TXA2, were measured at 250C. The rings were then washed twice in 2.0 ml of HBSS by RIA as described previously (14, 15). Lyophilized stan- to remove unincorporated radioactivity. Supernate radioac- dards, tracers, and antisera were obtained from New England tivity was quantified in a liquid scintillation counter (Tracor Nuclear. Cross-reactivity and anti-TXB2 antiserum with Analytic, Elk Grove Village, IL) to measure unincorporated other prostaglandins was less than 2%. Cross-reactivity of [14C]arachidonate. Aortic rings were then resuspended in 2.0 anti-6-keto-PGFia antiserum with other prostaglandins was ml of HBSS. also less than 2%. All measurements were performed in Conversion of [14C]Arachidonate to PGI2 and TXA2. To duplicate and results are expressed as ng/ml of supernate. stimulate the conversion of membrane-bound [14C]arachi- Statistical Analysis. All determinations made in duplicate donate into cyclooxygenase metabolites, 100 uM unlabeled were averaged. Data are expressed as mean + SD. Student's arachidonate was added to the rings, which were incubated t test (paired and unpaired data) was used for statistical at 37TC for 30 min in a shaking water bath. An aliquot of analysis. A P value of 0.05 was considered significant. supernate was then assayed for radioactivity to determine the release of [14C]arachidonate and its metabolites. The aortic rings were then isolated and washed and their radioactivities RESULTS were measured. PGI2 and TXA2 Release. As shown in Fig. 1, PGI2 (mea- To determine the conversion of [14C]arachidonate into sured as 6-keto-PGFia by RIA) released from atherosclerotic the 6-keto-PGFia and thromboxane B2 (TXB2), aortic ring rabbit aortic rings was greater (P - 0.05) than that released supernates, after stimulation with arachidonate, were ex- by normal rabbit aortic rings. This increase was seen at 0, 5, tracted and analyzed by thin-layer chromatography (TLC) as release continued for follows: To each supernate, unlabeled TXB2 and 6-keto- 10, 15, and 30 min of incubation. PGI2 PGFia standards (5 ,ug each) were added as carriers, the pH 15 min and then stabilized. was adjusted with 0.5 M formic acid to 3.5, and the supernate TXA2 (measured as TXB2 by RIA in aortic ring supernates) was extracted twice with 3.0 ml ofethyl acetate. The resulting was also greater (P - 0.05) in atherosclerotic rabbit rings. organic phase contained 75% of the standards. It was evap- Similar to PGI2 release, increased TXA2 release was ob- orated to dryness under a stream of nitrogen and the residue served at all time points. However, in contrast to PGI2 was dissolved in ethyl acetate and spotted onto TLC plates release, TXA2 release continued for the entire 30 min of (Fisher) along with 6-keto-PGFia and TXB2 standards (5 ,ug incubation.