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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 1, 2002 as doi:10.1096/fj.02-0092fje. |
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* Department of Comparative Medicine, The University of Tennessee, Knoxville, Tennessee, USA;
Center for Environmental Biotechnology,
Institute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania, USA; and
Department of Internal Medicine (Division of Pulmonary and Critical Care Medicine), University of Michigan, Ann Arbor, Michigan, USA
2Correspondence: The University of Tennessee, Dept. of Comparative Medicine, A205, College of Veterinary Medicine, 2407 River Dr., Knoxville, TN 37996-4543, USA. E-mail: ptithof{at}utk.edu
SPECIFIC AIMS
The aim of this study was to examine the role of the phospholipase A2/arachidonic acid cascade in apoptosis of human coronary artery endothelial cells caused by specific polycyclic aromatic hydrocarbons (PAHs) present in high concentrations in cigarette smoke. Identification of specific components of cigarette smoke that alter pathways known to be important in heart disease will likely provide means for discovering effective preventative and therapeutic strategies in smokers.
PRINCIPAL FINDINGS
1. PAHs induce release of 3H-arachidonic acid or 3H-linoleic acid from human coronary artery endothelial cells
Release of 3H-arachidonic acid from prelabeled cells was used as a measure of PLA2 activity in response to three different PAHs present in high concentrations in cigarette smoke: 1-methylanthracene (1-MA; 1500 ng/cigarette), benzo(a)pyrene (B(a)P; 25 ng/cigarette), and phenanthrene (PA; 362 ng/cigarette). When endothelial cells were incubated for 1 h with various concentrations of 1-methylanthracene, benzo(a)pyrene, or phenanthrene, all three compounds caused concentration-dependent release of 3H-arachidonic acid. 1-Methylanthracene and phenanthrene also induced release of 3H-linoleic acid, suggesting that these compounds activate an arachidonyl-nonselective PLA2. In contrast, benzo(a)pyrene did not cause release of 3H-linoleic acid, suggesting that this compound activates an arachidonyl-selective enzyme. Significant release of 3H-arachidonic acid was observed within 5 min from cells exposed to phenanthrene (30 µM) and within 10 min from cells exposed to 1-methylanthracene (30 µM) or benzo(a)pyrene (30 µM).
2. Specific PLA2 inhibitors attenuate PAH-induced release of 3H-arachidonic acid from human coronary artery endothelial cells
PAH-induced release of 3H-arachidonic acid was evaluated in the presence and absence of inhibitors of selective isoforms of PLA2. Methyl-arachidonyl fluorophosphonate (MAFP), an inhibitor of group IV and group VI enzymes, inhibited 3H-arachidonic acid released in response to all three PAHs. Bromoenol lactone (BEL), a selective inhibitor of group VI calcium-independent PLA2 (iPLA2), attenuated the response to 1-methylanthracene and phenanthrene, but not benzo(a)pyrene. MJ33, an inhibitor of the acidic, calcium-independent PLA2, aiPLA2 inhibited 3H-arachidonic acid release in response to phenanthrene but not 1-methylanthracene or benzo(a)pyrene (Fig. 1
). 4-Bromophenacyl bromide, an inhibitor of small molecular weight, calcium-dependent PLA2s, did not alter the response to any PAH. Reverse transcriptase-polymerase chain reaction analysis of PLA2 isoforms indicates that human coronary artery endothelial cells contain mRNA for at least five different PLA2 isozymes: groups IV
, IVß, IV
, group VI, and the acidic, calcium-independent PLA2. Western analysis confirmed the presence of four of these enzymes: group IV ß, IV, group VI, and the lysosomal, acidic, calcium-independent PLA2.
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3. PAHs induce apoptosis of human coronary artery endothelial cells
Apoptosis was evaluated by three techniques: Western analysis for the cleavage product of poly(ADP) ribose polymerase (PARP), ELISA for histone fragmentation, and terminal deoxyribonucleotide nick-end labeling (TUNEL). There was no evidence of PARP cleavage in vehicle-treated cells; however, significant amounts of the cleavage fragments were observed in cells after treatment for 6 h with 30 µM 1-methylanthracene, benzo(a)pyrene, phenanthrene, or 10 µM arachidonic acid or linoleic acid. To evaluate the time course of apoptosis, TUNEL assays were performed. No apoptotic cells were observed at 60 min, but a significant number of TUNEL-positive cells were seen at 2 h after treatment with the PAHs or fatty acids. For a more quantitative measure of PAH-induced apoptosis, histone fragmentation was evaluated by ELISA. These studies were performed in the presence and absence of MAFP, BEL, and MJ33. As can be seen in Fig. 2
, the same inhibitor profile observed for 3H-arachidonic acid release was seen with the apoptotic response. The response to 1-methylanthracene was attenuated by MAFP and BEL, but not MJ33. In contrast, the response to phenanthrene was inhibited by all three inhibitors, but benzo(a)pyrene-induced apoptosis was inhibited only by MAFP.
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CONCLUSIONS
It is well known from epidemiologic studies that the PLA2/arachidonic acid cascade is important in the mechanism whereby cigarette smoking causes heart disease; however, little work has focused on identifying specific components of cigarette smoke responsible for this effect. This study is the first to identify specific PAHs in high concentrations in cigarette smoke that stimulate PLA2-mediated release of membrane fatty acids. These results suggest that three distinct isoforms of PLA2 are activated by three different PAHs. The data indicate that 1-methylanthracene, a three-ring compound, activates the group VI iPLA2 whereas benzo(a)pyrene, a five-ring compound, activates a group IV enzyme. In contrast, phenanthrene, with a four-membered ring, activates both group VI and the acidic iPLA2. Moreover, this study is the first to link exposure of endothelial cells to cigarette smoke components with PLA2 activation, fatty acid release, and apoptosis, events known to be important in the etiology of atherosclerosis.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0092fje; to cite this article, use FASEB J. (July 1, 2002) 10.1096/fj.02-0092fje 
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