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

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 development 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 convert 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. 3). application (24). However, more recent studies have strongly im- fl Immunohistochemical staining of LTA4H in AAA tissue plicated LTs as mediators of vascular in ammation, and in most fi showed the presence of LTA4H in the media layer (Fig. S1A), cases LTB4 was identi ed as the culprit. Consequently, a role for adventitia layer (Fig. S1B), and luminal thrombus (Fig. S1C), the LTB4 arm of the 5-LO pathway, including FLAP, LTA4H, and indicating that the branch of 5-LO pathway responsible for LTB4 BLT1, in cardiovascular diseases is currently in the focus of intense biosynthesis is also present. research efforts, both within academia and industry (16, 25). AAA is a vascular pathology characterized by weakening of the Biosynthesis of LTs. Homogenates of human aortic tissue from medial elastic lamina layer and infiltration of media and adven- AAA surgery were used to study the metabolism of arachidonic titia by immune cells, such as macrophages and T cells, which acid. Incubations of AAA wall tissue with arachidonic acid pro- release a variety of chemokines and other mediators that can duced significant amounts of cysLTs (2.62 ± 0.61 ng/μg of protein) enhance degradation of the extracellular matrix (10). The most when compared with LTB4 production (1.25 ± 0.51 ng/μgof known and studied mediators involved in aortic wall degeneration protein) (Fig. 4), as assessed by reverse-phase HPLC coupled to are MMPs, and several investigations have indicated a pivotal enzyme immunoassay (EIA). Similarly, more cysLTs (2.15 ± 0.97 role for MMPs in the pathological process leading to AAA ng/μg of protein) than LTB4 (1.02 ± 0.50 ng/μg of protein) were growth and rupture (26). Furthermore, it has been suggested that produced in the thrombus, although the differences did not reach the presence of intraluminal thrombus could affect the structural statistical significance (Fig. 4). and cellular composition of the underlying vessel wall (15) and In selected experiments we directly assessed the LTA4H and sustain the proteolytic activity by releasing MMPs (11). LTC4S activities by incubations of subcellular fractions with the Thus far, only a limited number of animal studies have unstable epoxide LTA4. To do that, we isolated (by ultracentri- addressed the potential role of LTs in AAA, yielding different fugation) cytosolic (rich in LTA4H) and membrane (rich in results. Funk and coworkers found a protective effect of 5-LO

Fig. 2. Coexpression of 5-LO, FLAP, and LTC4S protein with inﬂammatory cell markers in human AAA. Serial sections of human AAA (n = 3) were stained for 5- LO, FLAP, LTC4S, and CD68 (macrophage), CD66b (neutrophil), CD3 (T lymphocyte), and tryptase (mast cell) markers, revealing similar distribution in media (A) and adventitia (B) layers. (Magniﬁcation: 20×. Scale bars, 50 μm.)

2of5 | www.pnas.org/cgi/doi/10.1073/pnas.1015166107 Di Gennaro et al. Downloaded by guest on September 30, 2021 Fig. 3. Expression of 5-LO and LTC S in the luminal part of intraluminal Fig. 5. Synthesis of LTC4 and LTB4 in human AAA. The AAA wall and the 4 × thrombus of human AAA. Serial sections of the luminal side of human AAA thrombus (n = 8) were homogenized at 100,000 g for 1 h at 4 °C. The cy- (n = 3) were stained for 5-LO, LTC S, and CD68 (macrophage) and CD66b tosolic fraction (supernatant) and the membrane fraction (pellet) were in- 4 μ (neutrophil) markers, showing coexpression in that area. L, lumen. (Magni- cubated with 10 MofLTA4.LTC4 and LTB4 were measured by tandem HPLC/ ± < ﬁcation: 40×. Scale bar, 100 μm.) EIA, and data are presented as media SEM. *P 0.05.

− − gene deletion against AAA in atherosclerosis-prone, ApoE / markers of human neutrophils and T lymphocytes, respectively, mice (19). In a later study, the same authors failed to detect a role and these cells clustered together with macrophages. for 5-LO in the angiotensin II-induced AAA model in the ApoE- Going deeper into the AAA wall, through the adventitia layer, deficient mouse (21). On the other hand, other investigators found immunostaining showed a pattern similar to the one observed in fl that genetic deletion or pharmacological inhibition of the BLT1 the media, with presence of focal accumulations of in ammatory receptor afforded significant protection against AngII-induced cells, mostly macrophages and neutrophils, which are positive for AAA (20, 27). In line with the latter observations, Houard et al. 5-LO, FLAP, LTC4S, and LTA4H. However, in the adventitia, (14) recently proposed a role for LTB4, derived from neutrophils cells staining strongly for tryptase were also detected, indicating within the intraluminal thrombus, as a chemotactic factor in AAA. the presence of mast cells that are known to be involved in the In the present study we used a collection of fresh AAA speci- early and late phases of inflammatory reactions by release of mens obtained from patients undergoing elective surgery for re- chemokines, cytokines, and lipid mediators, such as cysLTs (29).

pair of infrarenal aneurysms, to assess the expression proﬁle and MEDICAL SCIENCES activity of enzymes in the 5-LO pathway. Surprisingly, we found AAA Wall Converts Arachidonic Acid Primarily into cysLTs. Next, we assessed whether our mRNA and morphological data correlated that cysLTs, rather than LTB4, may be involved in human AAA. with differences in enzyme activities. Thus, we incubated ho-

5-LO, FLAP, and LTC4S mRNA Levels Are Increased in Human AAA Wall. mogenized human AAA wall tissue with arachidonic acid and Analysis of 28 patients revealed a striking increase in the expres- analyzed the formation of LTs to probe the entire 5-LO pathway sion of 5-LO mRNA. In addition, we found significant increases in machinery. Tandem HPLC/EIA analysis revealed a significantly the mRNA levels of FLAP and LTC4S (Fig. 1), thus completing the increased ability of the wall to convert arachidonic acid into triplet of key proteins required for cysLTs biosynthesis. In contrast, cysLTs and lesser amounts of LTB4, in accord with the results LTA4H did not display significantly elevated levels of mRNA, demonstrating an important difference between human AAA and human atherosclerosis, in which LTA4H is up-regulated and has been shown to correlate with symptoms of plaque instability (17).

LT-Synthesizing Enzymes Are Present in Media and Adventitia Layers at Sites Rich in Inﬂammatory Cells. By immunostaining it was possible to detect the presence of proteins of the whole LT enzymatic pathway in several segments of the AAA wall. Thus, the media layer presented focal accumulations of 5-LO-, FLAP-, LTA4H-, and LTC4S-positive cells. The same areas also stained positively for CD68, indicating that macrophages could be the main source of LTs and conﬁrming previous results obtained in animal models (19, 28). Moreover, in agreement with previous studies demonstrating the presence of different types of immune cells in AAA (10, 13, 15), we could detect CD66b- and CD3-positive cells,

Fig. 6. Release of LTD4-mediated MMP-2 in human AAA and inhibition by montelukast. Minced vascular wall derived from AAA lesions (n =4)waspre- incubated for 30 min in the absence or presence of montelukast (1 μM), fol-

lowed by incubation with LTD4 (10 μM) for 24 h at 37 °C. The conditioned media

Fig. 4. Synthesis of cysLTs and LTB4 in human AAA. Homogenates of human were analyzed by gelatin gel zymography, and bands relative to proMMP-9, AAA wall and thrombus (n = 9) were incubated with arachidonic acid. CysLTs proMMP-2, and MMP-2 were analyzed by Image J software. (A) Representative

and LTB4 were measured by HPLC/EIA, and data are presented as mean ± SEM. gelatin gel; (B) densitometric analysis of gelatin gel zymography (n = 4). Data *P < 0.05. are expressed as mean ± SEM. *P < 0.05.

Di Gennaro et al. PNAS Early Edition | 3of5 Downloaded by guest on September 30, 2021 obtained by real-time PCR (Fig. 1). These data indicated geous, given the steadily increasing number and complex cross- a greater LTC4S activity as compared with LTA4H activity. To regulations reported for cysLT receptors (35–37). Finally, on the directly probe the LTC4S and LTA4H activities, we also carried basis of the results of the present study, an obvious approach would out incubations with the unstable epoxide intermediate LTA4, the be to use established antiasthma drugs, such as montelukast or immediate upstream substrate for both of these enzymes. Be- similar CysLT1 antagonists, for treatment of AAA, a possibility cause soluble GSTs could potentially interfere with the LTC4S that should be straightforward to test in a clinical trial. assay, we subjected the AAA wall homogenates to ultracentrifu- Materials and Methods gation and collected a cytosolic fraction containing LTA4H, as well as a membrane fraction enriched in membrane-bound Sample Collection. Patients (n = 28) who were about to undergo elective surgery for infrarenal AAA were selected for the study. Tissue was taken from LTC4S and devoid of soluble GSTs. Analysis of these incubates the anterior or lateral wall, because tissue sampling from the posterior wall clearly demonstrated a higher activity of LTC4S as compared with LTA H, yielding significantly more LTC than LTB (Fig. 5 and was considered to add to the risk of surgery. Control ascending aorta samples 4 4 4 for RNA studies were obtained from eight organ donors without clinical or Fig. S2). Although large amounts of LTs could be detected in fi macroscopic signs of aortic atherosclerosis at University Central Hospital, certain experiments (Fig. S2), the samples varied signi cantly Helsinki, Finland. The use of organ donor tissues was approved by The Na- with respect to enzyme activities. Comparison of data in Figs. 4 tional Authority for Medicolegal Affairs of Finland. and 5 also shows that a lot more LTs were formed in incubations AAA samples were immediately fixed in 4% formaldehyde for light mi- with LTA4. This is probably explained by the fact that we used croscopy or snap-frozen in liquid nitrogen for RNA isolation. All of the pro- homogenized tissues in our activity measurements, in which the cedures were approved by the local ethics committees, and all of the patients membrane-associated assembly of 5-LO and FLAP has been gave written informed consent. disrupted. In incubations with LTA4, this step is circumvented, Real-Time PCR. Total RNA from frozen samples was isolated with TRIzol (BRL- allowing direct assessment of downstream LTC4S and LTA4H. Life Technologies) and an RNEasy Mini Kit (Qiagen) after homogenization ’ Intraluminal Thrombus Is also a Potential Source of cysLTs. Because using FastPrep (MP Biomedicals) according to the manufacturer s instructions. the intraluminal thrombus has been suggested as an important The quality of RNA was analyzed with an Agilent 2100 bioanalyzer (Agilent Technologies) and quantity assessed by a NanoDrop. cDNA was synthesized source of chemotactic LTB4 (14), we also analyzed this component from total RNA by using SuperScript II reverse transcriptase (Invitrogen), of the aneurysm for presence of 5-LO pathway proteins and re- and 2 μl of cDNA were amplified by real-time PCR performed in a TaqMan lated enzyme activities. Immunohistochemical analysis of throm- 7300 instrument (Applied Biosystems). Normalization was made to cyclophilin A. bus revealed the presence of inflammatory cells only at the luminal The following primer/probe pairs were obtained by Assay-on-Demand (Applied side. Staining for cell markers CD68 and CD66b showed the Biosystems): 5-LO, FLAP, LTA4H, and LTC4S, with assay IDs Hs00386528_m1, presence of macrophages and neutrophils, but no T cells. These Hs00233463_m1, Hs00168505_m1, and Hs00168529_m1, respectively. The PCR amplification was related to a standard curve. cells also stained for 5-LO, LTC4S, and LTA4H, indicating that cells in the thrombus could potentially produce and release fi cysLTs, as well as the chemotactic factor LTB . In fact, further Immunohistochemistry. Paraf n sections were cleared in xylene, rehydrated in 4 graded ethanol (100–70%), and subjected to antigen retrieval by boiling in activity measurements, including incubations of thrombus with DIVA buffer (Biocare Medical) for 20 min. After cooling to room temperature both arachidonic acid and LTA , revealed a predominant pro- 4 in water, sections were incubated in 3% hydrogen peroxide (H2O2)for5min duction of cysLTs that in the latter case reached statistical sig- to inhibit endogenous peroxidase activity. The specimens were then rinsed nificance (Fig. 5). three times for 5 min each in PBS, blocked for 30 min in PBS with normal goat serum (1:5), and incubated overnight at 4 °C in a humidified chamber with Challenge with LTD4 Increases Release of MMP-2 from AAA Wall and without primary antibodies against human 5-LO (a gift from Olof Tissue. Production of cysLTs in the vascular wall may have several Rådmark, Karolinska Institutet, Stockholm, Sweden), human FLAP (a gift effects that potentially could promote inflammation and aneurysm from Jilly Evans, Amira Pharmaceuticals, San Diego, CA), human LTA4Hand formation. For instance, MMPs have been implicated in the LTC4S (prepared in house), human CD68 (Dako), human CD66b (Fitzgerald pathogenesis of aortic aneurysm (8, 9), and a recent study showed Industries), human CD3 (Santa Cruz Biotechnology), and Tryptase (Dako). The samples were then rinsed in PBS and incubated with biotinylated secondary that reduced formation of AAA in 5-LO knockout mice is associ- antibody (1:1,500) for 1 h at room temperature followed by avidin–biotin ated with a decrease in aortic MMP-2 activity (19). Along these amplification (ABC Elite) for 30 min, and developed with 3,3′-diamino- lines, we show that challenge of human AAA wall tissue with LTD4 benzidine (Sigma). Sections were counterstained with Mayer’s hematoxylin induces a significant increase of proMMP-2 and MMP-2 levels that for 1 min and mounted. Negative controls were obtained by substituting the potentially could contribute to matrix degradation in the vessel primary antibodies with PBS. wall. This effect seems to be mediated via CysLT1 receptors, because montelukast, a selective CysLT1 antagonist, could block the Reverse-Phase HPLC Coupled to EIA. Human AAA (n = 9) were collected im- increased release of MMP-2 (Fig. 6). mediately after surgery, dissected, weighed, homogenized in 0.1 M PBS (pH In addition to increased MMP activity, previous studies have 7.8) containing proteinase inhibitor mixture (Roche) and EDTA (1 mM), and shown that cysLTs and their receptors, in particular CysLT1, sonicated three times for 10 s on ice. Homogenates were incubated with 1 mM ATP, 2 mM Ca2+, and 40 μM arachidonic acid for 10 min at room temperature. are involved in de novo expression and secretion of several proin- fl fl The reaction was stopped by adding two volumes of cold methanol. Samples ammatory cytokines, such as IL-5, macrophage in ammatory pro- were centrifuged at 10,000 × g for 10 min at 4 °C, and the resulting super- α β α tein-1 and - ,aswellasTNF- from macrophages and mast cells natants were acidified to pH 3–4 and 5.6 for LTB4 or cysLTs, respectively. (30–32). These activities of cysLTs are believed to be involved in the Samples were purified by solid-phase extraction (Supelco LC18) and analyzed amplification of innate as well as adaptative immune responses. by reverse-phase HPLC. The column (Nova-Pak C18; Waters) was eluted with acetonitrile/methanol/water/acetic acid (30:35:35:0.01 by volume) at 0.8 mL/ Anti-LTs as Potential Drugs Against AAA. Assuming that production min, and absorbance was monitored at 270 nm or 280 nm for LTB4 and cysLTs, of cysLTs occurs during the course of AAA development, it respectively. Fractions corresponding to the retention times of LTB4, LTC4, should be possible to pharmacologically block its synthesis and LTD4, and LTE4 standards (Cayman Chemical) were collected, dried under ni- actions with already-existing anti-LT drugs. Inhibition of 5-LO trogen, and resuspended with EIA buffer. Levels of LTB4 or cysLTs were determined in duplicate assays with an LTB or cysLT EIA kit (Cayman Chemical) or FLAP would block synthesis of all LTs, as well as antiin- 4 fl by using dilutions within the linear portion of the standard curves. ammatory lipoxins. Selective inhibition of LTC4S, on the other In selected experiments (n = 8), AAA wall tissue was homogenized in PBS (pH hand, could block synthesis of cysLTs, while potentially sparing 7.4) 1:3/wt/vol and then centrifuged at 100,000 × g for 60 min at 4 °C to prepare lipoxin biosynthesis, as previously demonstrated for a selective cytosolic (supernatant) and membrane (pellet) fractions. The cytosolic fraction μ inhibitor of LTA4H (33, 34). This approach may also be advanta- was incubated with 10 MLTA4 (Cayman Chemical) and 0.2% BSA for 10 min

4of5 | www.pnas.org/cgi/doi/10.1073/pnas.1015166107 Di Gennaro et al. Downloaded by guest on September 30, 2021 at room temperature, and the pellet was resuspended in PBS (initial volume) mental media samples were diluted into 2× Tris-glycine SDS sample buffer and μ and incubated with 10 M LTA4, 5 mM glutathione, and 0.2% BSA for 10 min electrophoretically separated under nonreducing conditions. Proteins were in- at room temperature. The reaction was stopped with two volumes of cold cubated in renaturating buffer (Invitrogen) for 30 min at room temperature. The fi methanol. Samples were puri ed, extracted, and analyzed by HPLC as gels were incubated overnight at 37 °C in developing buffer (Invitrogen). After described above. 1 h staining with Coomassie brilliant blue and destaining for 2 d with 10% acetic acid and 40% methanol in water, gelatinase activity was evident by clear bands MMP Assays and Gel Zymography. The aneurysm walls from selected surgeries against a dark blue background. Quantification of the bands was performed (n = 4) were cut into pieces of ≈1mm3 and divided equally into three sep- using digital camera Fujifilm LAS-1000 and densitometry software ImageJ. arate samples. They were incubated in RPMI medium 1640 (Sigma) with LTD4 (10 μM) with or without pretreatment with montelukast (Cayman Chemical; 1 μM, 30 min at 37 °C) for 24 h at 37 °C (2 mL/g wet tissue), as previously ACKNOWLEDGMENTS. This work was supported by the Swedish Research described (12, 14). The conditioned media containing the released material Council (Grants 10350, 04342, 20854, and Linnéus Grant 70870302), Euro- pean Union integrated projects Eicosanox (005033), Atheroremo (201668), were collected and stored at −80 °C until use. and Fad (200647), the Vinnova consortium (2007-01999), The Strategic Car- Zymography supplies were purchased from Invitrogen. ProMMP-2, MMP-2, diovascular Program supported by Karolinska Institutet/Stockholm County proMMP-9, and active MMP-9 distributions were determined from conditioned Council, and the Italian Ministry of University and Research (“Internaziona- media. Gelatin substrate zymograms were prepared using precast 10% SDS- lizzazione del Sistema Universitario”). J.Z.H. was supported by a Distin- polyacrylamide gels containing 1 mg/mL of gelatin. Equal volumes of experi- guished Professor Award from Karolinska Institutet.

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