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Increased Expression of Leukotriene C4 Synthase and Predominant Formation of Cysteinyl-Leukotrienes in Human Abdominal Aortic Aneurysm

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Increased Expression of Leukotriene C4 Synthase and Predominant Formation of Cysteinyl-Leukotrienes in Human Abdominal Aortic Aneurysm Increased expression of leukotriene C4 synthase and predominant formation of cysteinyl-leukotrienes in human abdominal aortic aneurysm Antonio Di Gennaroa, Dick Wågsäterb, Mikko I. Mäyränpääc,d, Anders Gabrielsenb, Jesper Swedenborge, Anders Hamstenb, Bengt Samuelssona,1, Per Erikssonb, and Jesper Z. Haeggströma,1 aDivision of Chemistry II, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 171 77 Stockholm, Sweden; bAtherosclerosis Research Unit, Center for Molecular Medicine, Department of Medicine, Karolinska Institutet, 171 76 Stockholm, Sweden; cWihuri Research Institute, 00140, Helsinki, Finland; dDepartment of Pathology and HUSLAB Division of Pathology, University of Helsinki, 00014, Helsinki, Finland; and eDepartment of Molecular Medicine and Surgery, Vascular Surgery Unit, Karolinska Institutet, Center for Molecular Medicine, Karolinska University Hospital, 171 76 Stockholm, Sweden Contributed by Bengt Samuelsson, October 19, 2010 (sent for review September 10, 2010) Leukotrienes (LTs) are arachidonic acid-derived lipid mediators pointed to the intraluminal thrombus as a site of proteolytic ac- involved in the pathogenesis and progression of diverse inflamma- tivity (11, 12) and source of chemotactic factors (13, 14), poten- tory disorders. The cysteinyl-leukotrienes LTC4, LTD4, and LTE4 are tially influencing the structural and cellular composition of the important mediators of asthma, and LTB4 has recently been impli- AAA wall (15). LTs have been the subject of recent investigations cated in atherosclerosis. Here we report that mRNA levels for the indicating an important role for those lipid mediators in cardio- three key enzymes/proteins in the biosynthesis of cysteinyl-leuko- vascular diseases. Thus, genetic, morphological, biochemical, trienes, 5-lipoxygenase (5-LO), 5-LO-activating protein (FLAP), and pharmacological, and functional evidence collected from human fi LTC4 synthase (LTC4S), are signi cantly increased in the wall of hu- and animal studies have implicated LTs, in particular LTB4, in the man abdominal aortic aneurysms (AAAs). In contrast, mRNA levels development and progression of atherosclerosis (16, 17) and as of LTA4 hydrolase, the enzyme responsible for the biosynthesis of risk factors in human myocardial infarction (18). Less is known LTB4, are not increased. Immunohistochemical staining of AAA wall regarding LTs and AAA, and studies using animal models have revealed focal expression of 5-LO, FLAP, and LTC4S proteins in the fl yielded con icting results about the involvement of the 5-LO MEDICAL SCIENCES media and adventitia, localized in areas rich in inflammatory cells, pathway in aneurysmal disease (19–21). In the present study including macrophages, neutrophils, and mast cells. Human AAA we examined the expression of 5-LO cascade-related enzymes wall tissue converts arachidonic acid and the unstable epoxide LTA4 in human AAA. Biochemical analysis revealed an overexpression fi into signi cant amounts of cysteinyl-leukotrienes and to a lesser of 5-LO, FLAP, and LTC4S, but not of LTA4H, in the AAA wall. extent LTB4. Furthermore, challenge of AAA wall tissue with exog- Using immunohistochemistry, we also found that these three enous LTD4 increases the release of matrix metalloproteinase enzymes, required for cysLT biosynthesis, are expressed in the (MMP) 2 and 9, and selective inhibition of the CysLT1 receptor by media and adventitia layers of the AAA wall and colocalize with montelukast blocks this effect. The increased expression of LTC4S, the presence of inflammatory cells, in particular macrophages and together with the predominant formation of cysteinyl-leukotrienes mast cells. Moreover, this enzymatic machinery is catalytically and effects on MMPs production, suggests a mechanism by which active and capable of converting arachidonic acid into LTs. Fi- LTs may promote matrix degradation in the AAA wall and identify nally we show that LTD4 induces metalloproteinases in a CysLT the components of the cysteinyl-leukotriene pathway as potential receptor-dependent manner, suggesting a mechanism by which targets for prevention and treatment of AAA. these mediators may promote arterial wall degradation and de- velopment of AAA. cardiovascular disease | eicosanoid | inflammation Results eukotrienes (LTs) are powerful lipid mediators released by Expression of 5-LO, FLAP, and LTC4S mRNA in Human AAA. In human Linflammatory cells, such as macrophages, neutrophils, eosi- AAA wall we found increased transcript levels of 5-LO, FLAP, nophils, and mast cells (1, 2). In cellular biosynthesis of LTs, and LTC4S compared with control aortas, corresponding to 2.5- arachidonic acid is oxygenated by 5-lipoxygenase (5-LO) into the fold (2.46 ± 0.26, P = 0.002), 1.5-fold (1.51 ± 0.09, P = 0.0012), ± P unstable epoxide LTA4 with the aid of the accessory 5-LO acti- and 1.5-fold (1.81 0.21, = 0.0068) increases, respectively (Fig. vating protein (FLAP). Here, the pathway branches. The LTA 1). In contrast, the levels of LTA4H mRNA in AAA wall were not 4 fi hydrolase (LTA4H) can metabolize LTA4 to LTB4, which acts as signi cantly different when compared with control (Fig. 1). a potent chemoattractant. Alternatively, LTA4 can be conjugated with glutathione by LTC synthase (LTC S) to form cysteinyl- Expression of 5-LO, FLAP, and LTC4S Protein in Human AAA. To check 4 4 the presence of leukotriene cascade-related enzymes at the leukotrienes LTC4, LTD4, and LTE4 (cysLTs), which promote airway smooth-muscle constriction and increased vascular per- protein level, we performed immunostaining in serial sections of AAA wall. The tunica media expressed focal accumulations of 5- meability. LTB4 and cysLTs exert biological effects through two pairs of G protein-coupled receptors denoted BLT1/BLT2 and LO, FLAP, and LTC4S immunostaining that colocalize with the CysLT1/CysLT2, respectively (3–6). LTs and their receptors exert diverse functions in immune and inflammatory responses and Author contributions: A.D.G., D.W., A.G., J.S., A.H., B.S., P.E., and J.Z.H. designed research; have been associated with many diseases, in particular asthma (7). A.D.G. and D.W. performed research; M.I.M. and J.S. contributed new reagents/analytic Abdominal aortic aneurysm (AAA) is a cardiovascular disease tools; A.D.G., D.W., and J.Z.H. analyzed data; and A.D.G., P.E., and J.Z.H. wrote the paper. fl associated with aging, whereby chronic in ammation in the aortic The authors declare no conflict of interest. wall and proteinase-mediated degradation of structural matrix Freely available online through the PNAS open access option. proteins are believed to contribute to its development and rup- 1To whom correspondence may be addressed. E-mail: [email protected] or jesper. ture (8, 9). Inflammatory cells such as macrophages, neutrophils, [email protected]. and mast cells have been shown to be sources of proteolytic This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. enzymes in the AAA wall (10). Moreover, several studies have 1073/pnas.1015166107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1015166107 PNAS Early Edition | 1of5 Downloaded by guest on September 30, 2021 LTC4S) fractions from AAA wall and incubated them with LTA4 (10 μM). HPLC quantification showed a 2-fold higher formation of LTC4 (2.14 pmol/μg protein; Fig. S2A) compared with LTB4 (1.10 pmol/μg protein; Fig. S2B) in the wall samples. Moreover, the tandem EIA analysis of HPLC fractions showed a stronger LTC4S activity as compared with LTA4H activity (Fig. 5) to con- vert the same substrate both in the wall (LTC4 vs. LTB4:21.45± 7.37 vs. 10.25 ± 0.84 ng/μg protein) and in thrombus (LTC4 vs. LTB4:13.22± 3.75 vs. 3.34 ± 0.71 ng/μg protein), confirming the data obtained from incubations with arachidonic acid. Fig. 1. Expression of 5-LO, FLAP, LTA4H, and LTC4S mRNA in human AAA wall (n = 28) was determined by real-time quantitative RT-PCR and compared with human control aortas (CTRL; n = 8). Data are presented as mean ± SEM. LTD4-Induced Release of Matrix Metalloproteinases. It has been shown **P < 0.01; ***P < 0.001. that cysLTs can induce the release of matrix metalloproteinases (MMPs) from macrophages (19, 23), such as MMP-9 and MMP-2, and the presence of these proteinases has been associated with presence of CD68- and CD66b-positive cells, reflecting the pres- degradation of connective tissue (9). Therefore, we examined the A ence of macrophages and neutrophils, respectively (Fig. 2 ). In potential role of LTD4 asastimulusforthereleaseofMMPsfrom the same area it was also possible to see the presence of T lym- AAA tissue. phocytes, as shown by CD3-positive staining (Fig. 2A). Gelatin gel zymography showed an increased level of MMP-2 In the adventitia, once again, there were also plenty of 5-LO- and its proform when AAA wall was incubated with LTD4 (10 μM) and LTC4S-positive cells that colocalize with CD68 (macro- as compared with the control group (Fig. 6 A and B). Moreover, phages) and CD66b (neutrophils) (Fig. 2B). Moreover, in the pretreatment of AAA tissue with the selective CysLT1 antagonist adventitia, it was also possible to detect the presence of mast cells montelukast (1 μM) reduced the MMP signals to control levels in the same area, as determined by tryptase staining (Fig. 2B). (Fig. 6 A and B). We also carried out immunohistochemistry of AAA thrombus. The abluminal side did not show the presence of cells, confirming Discussion previous observations that this part of thrombus is mainly com- LTs are paracrine lipid mediators that have potent proinflam- posed of densely packed fibrin (22). In contrast, the luminal side matory biological activities. One class of LTs, the cysLTs, are well of the thrombus showed the presence of focal accumulations of recognized as important signaling molecules in human asthma, inflammatory cells, in particular macrophages and neutrophils, and leukotriene-modifying drugs have been produced for this that colocalized with cells positive for 5-LO and LTC4S (Fig.
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