Deletion of Microsomal Prostaglandin E Synthase-1 Augments Prostacyclin and Retards Atherogenesis

Deletion of microsomal prostaglandin E synthase-1 augments prostacyclin and retards atherogenesis

Miao Wang*, Alicia M. Zukas†, Yiqun Hui*, Emanuela Ricciotti*, Ellen Pure´ †, and Garret A. FitzGerald*‡

*Institute for Translational Medicine and Therapeutics, School of Medicine, University of Pennsylvania, 3620 Hamilton Walk, Philadelphia, PA 19104; and †Wistar Institute, 34th and Spruce Streets, Philadelphia, PA 19104

Communicated by Bengt Samuelsson, Karolinska Institutet, Stockholm, Sweden, August 3, 2006 (received for review May 3, 2006)

Prostaglandin (PG) E2 is formed from PGH2 by a series of PGE four E prostanoid receptors (EPs), which mediate contrasting synthase (PGES) enzymes. Microsomal PGES-1؊/؊ (mPGES-1؊/؊) biologies. Two of them, EP2 and EP4, are linked to Gs-mediated mice were crossed into low-density lipoprotein receptor knock- activation of adenylate cyclase, whereas the two others, EP1 and out (LDLR؊/؊) mice to generate mPGES-1؊/؊ LDLR؊/؊s. Urinary EP3, are linked to Gq and͞or Gi (18). Deletion of EP2, just like 11␣-hydroxy-9, 15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid the IP, results in salt-sensitive hypertension (9, 19), whereas EP4 (PGE-M) was depressed by mPGES-1 deletion. Vascular mPGES-1 mediates antiinflammatory effects, at least in vitro (20, 21). EP1 ؊/؊ was augmented during atherogenesis in LDLR s. Deletion of and EP3 mediate PGE2-induced vasoconstriction (22, 23), and ؊/؊ mPGES-1 reduced plaque burden in fat-fed LDLR s but did not PGE2 can either activate platelets by EP3 or, at higher concen- alter blood pressure. mPGES-1؊/؊ LDLR؊/؊ plaques were enriched trations, inhibit platelet aggregation by the IP (24). A further with ﬁbrillar collagens relative to LDLR؊/؊, which also contained layer of complexity has been added by the observation that small and intermediate-sized collagens. Macrophage foam cells COX-2, EP4, and metalloproteinase (MMP) 9 are all up- were depleted in mPGES-1؊/؊ LDLR؊/؊ lesions, whereas the total regulated in human atherosclerotic plaque ex vivo (25), and that areas rich in vascular smooth muscle cell (VSMC) and matrix were COX-2-dependent extracellular matrix-dependent activation of unaltered. mPGES-1 deletion augmented expression of both pros- MMP9 is mediated by the EP4 in vitro (26). tacyclin (PGI2) and thromboxane (Tx) synthases in endothelial cells, The product of COX-catalyzed metabolism of arachidonic ؊ ؊ and VSMCs expressing PGI synthase were enriched in mPGES-1 / acid, PGH2, is metabolized further by specific synthases and LDLR؊/؊ lesions. Stimulation of mPGES-1؊/؊ VSMC and macro- isomerases to generate PGs (27). Microsomal (m)-PGE synthase phages with bacterial LPS increased PGI2 and thromboxane A2 to (PGES)-1 (28, 29) is a member of the MAPEG (membrane- varied extents. Urinary PGE-M was depressed, whereas urinary associated proteins involved in eicosanoid and glutathione me- 2,3-dinor 6-keto PGF1␣, but not 2,3-dinor-TxB2, was increased in tabolism) superfamily and has previously been suggested as a ؊/؊ ؊/؊ mPGES-1 LDLR s. mPGES-1-derived PGE2 accelerates athero- potential drug target (28). Two other PGES have also been ,genesis in LDLR؊/؊ mice. Disruption of this enzyme retards athero- identified, mPGES-2 (30) and cytosolic PGES-1 (31). Recently genesis, without an attendant impact on blood pressure. This may mPGES-1 deletion in mice was found to modulate experimen- reﬂect, in part, rediversion of accumulated PGH2 to augment tally evoked pain and inflammation to a degree indistinguishable formation of PGI2. Inhibitors of mPGES-1 may be less likely than from treatment with traditional COX-nonspecific NSAIDs (32). those selective for cyclooxygenase 2 to result in cardiovascular This raises the possibility that specific inhibitors of mPGES-1 complications because of a divergent impact on the biosynthesis might retain the efficacy of NSAIDs, including those specific for inhibition of COX-2. However, given the contrasting effects of of PGI2. PGE2 on cardiovascular function, it is unclear whether such a atherosclerosis ͉ cyclooxygenase ͉ macrophage ͉ strategy would bypass or diminish the risk evident in trials of vascular smooth muscle cell NSAIDs specific for inhibition of COX-2. Recently, we have reported that mPGES-1 deletion, in contrast to deletion, dis-

ruption, or inhibition of COX-2, does not result in hypertension MEDICAL SCIENCES ecent placebo-controlled trials have revealed that nonste- or a predisposition to thrombosis in normolipidemic mice (13). Rroidal antiinflammatory drugs (NSAIDs) selective for inhi- Here, we consider the impact of mPGES-1 deletion in hyper- bition of cyclooxygenase 2 (COX-2) confer a small but absolute lipidemic mice. Studies with COX-2 inhibitors have described risk of myocardial infarction and stroke (1–5). Mechanistically, contrasting effects on atherogenesis in mice, perhaps reflecting this is attributable to suppression of COX-2-derived prostacyclin variation among the drugs and doses used and both the timing (PGI2; ref. 6), which acts as a general restraint on endogenous and duration of the intervention (33). Here, we report that stimuli, including platelet COX-1-derived thromboxane (Tx) A2, to platelet activation, vascular proliferation and remodeling, hypertension, atherogenesis, and cardiac function (7–13). Pre- Author contributions: G.A.F. designed research; M.W., A.M.Z., Y.H., and E.R. performed cise estimates of the incidence of this risk are not available. research; E.P. and G.A.F. analyzed data; and M.W., E.P., and G.A.F. wrote the paper. However, the relative risk is small and likely to be conditioned Conflict of interest statement: G.A.F. receives financial support for investigator-initiated by such factors as drug exposure, underlying cardiovascular risk research from Bayer, Merck, and Boehringer Ingelheim, all of which manufacture drugs that target COXs. G.A.F. is a member of the Steering Committee of the Multinational of the patients exposed and concomitant therapies (6, 13). Etoricoxib and Diclofenac Arthritis Long-Term (MEDAL) Study Program. G.A.F. also serves However, the consumption of celecoxib, rofecoxib, and valde- as a consultant for Bayer, Merck, GlaxoSmithKline, Genome Institute of the Novartis coxib by millions has raised concern that many patients may have Foundation, Boehringer Ingelheim, and NicOx. suffered cardiovascular adverse events from these drugs. Freely available online through the PNAS open access option. Inhibitors of COX-2 afford relief from pain and inflammation Abbreviations: PG, prostaglandin; PGES, PGE synthase; mPGES, microsomal PGES; PGE-M, ␣ principally by suppressing prostaglandin (PG) E2 and PGI2. 11 -hydroxy-9, 15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid; LDLR, low-density li- Deletion and͞or antagonism of receptors for both of these PGs poprotein receptor; VSMC, vascular smooth muscle cell; PGI2, prostacyclin; PGIS, PGI2 synthase; Tx, thromboxane; TxS, Tx synthase; PGI-M, 2,3-dinor 6-keto PGF1␣; Tx-M, 2,3- modulate the response to both painful and inflammatory stimuli dinor-TxB2; NSAID, nonsteroidal antiinflammatory drug; COX, cyclooxygenase; EP, E pro- (14–17). Although deletion of the PGI2 receptor, the I prosta- stanoid receptor; HFD, high-fat diet; TxA2, thromboxane A2; IP, I prostanoid receptor; TxS, noid receptor (IP), has revealed the important role that this PG TxA2 synthase. plays in protection of cardiac and vascular function, the role of ‡To whom correspondence should be addressed. E-mail: [email protected]. PGE2 is more complex. The effects of this PG are transduced by © 2006 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0606586103 PNAS ͉ September 26, 2006 ͉ vol. 103 ͉ no. 39 ͉ 14507–14512 Downloaded by guest on September 27, 2021 females [116.7 Ϯ 5.2 mmHg in LDLRϪ/Ϫ vs. 113.3 Ϯ 3.6 mmHg in mPGES-1Ϫ/Ϫ LDLRϪ/Ϫ (1 mmHg ϭ 133 Pa)] or males (112.8 Ϯ 3.3 mmHg in LDLRϪ/Ϫ vs. 113.4 Ϯ 2.7 mmHg in mPGES-1Ϫ/Ϫ LDLRϪ/Ϫ), as measured after 6 mo on the HFD (P Ͼ 0.8, n ϭ 10–15 per group).

Lesional Morphology Consequent to mPGES-1 Deletion in LDLR؊/؊ Mice. Macrophages, VSMCs, and collagen were immunostained in plaques of comparable size obtained at the aortic roots (Fig. 3). The VSMC content was variable in both groups, with no consistent difference observed between the groups in VSMC abundance (Fig. 3 A Left and B) or the percentage of lesion matrix rich (i.e., collagen) area. However, the birefringence of sirius red staining under polarized light indicates that the collagen that accumulates in the lesions of mPGES-1Ϫ/Ϫ LDLRϪ/Ϫ mice is in the form of mid-sized to large fibers (yellow to white reflection) typical of type I collagen compared with small fibers (red reflection) evident in the LDLRϪ/Ϫ (Fig. 3A Right). Strikingly, macrophages, as stained by CD11b (Fig. 3A Center), were depleted in lesions from the mPGES-1Ϫ/Ϫ LDLRϪ/Ϫs compared with littermate controls. Indeed, macrophage foam cells, as stained by CD11c antibody, occupied Ϸ60% Ϫ Ϫ Fig. 1. mPGES-1 is induced during atherogenesis. (A) mPGES-1 expression of the total plaque area in LDLR / s, contrasting Ϸ40% in the was examined by real-time PCR in aorta from mice fed chow diet and after mPGES-1Ϫ/Ϫ LDLRϪ/Ϫs (Fig. 3C). 6-mo HFD. Data shown are the mean Ϯ SEM from duplicate determinations Both PGIS and thromboxane A2 (TxA2) synthase (TxS) were with n ϭ 3 per group. *, P Ͻ 0.05. (B) mPGES-1 expression in an atherosclerotic detectable in LDLRϪ/Ϫs (Fig. 5, which is published as supporting Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ lesion. Lesions from LDLR and mPGES-1 LDLR mice were stained with information on the PNAS web site). TxS was particularly evident blocking peptide-preabsorbed mPGES-1 antibody (Left) and͞or the antibody alone (Center and Right). (Scale bar, 100 ␮m.) in macrophage foam cells and PGIS in VSMC. Lesions obtained from mPGES-1Ϫ/Ϫ LDLRϪ/Ϫs had less TxS, reflecting macrophage depletion; they also exhibited less necrosis of the lesional mPGES-1 is up-regulated in the vasculature during atherogen- core. Although structural integrity of the endothelium was esis in mice lacking the receptor for low-density lipoprotein retained over the disrupted sections of vasculature, and the (LDLR). Deletion of mPGES-1 results in marked depletion of endothelium was morphologically indistinguishable between the lesional macrophages and macrophage-derived foam cells, in- two groups, both PGIS and TxS were much more abundantly expressed in endothelium in the mPGES-1Ϫ/Ϫ LDLRϪ/Ϫs, sug- creased expression of vascular smooth muscle cell (VSMC) PGI2 gesting a functional distinction. Endothelial localization of the synthase (PGIS), depression of endogenous PGE2, and an two synthases was supported by apparent colocalization with increase in systemic PGI2, but not Tx, biosynthesis. These results raise the possibility that drugs that inhibit specifically mPGES-1 lesional CD31 (platelet endothelial cell adhesion molecule, PECAM). PGIS was relatively more abundant in mPGES-1Ϫ/Ϫ may confer cardiovascular benefit, at least in part by rediversion Ϫ/Ϫ of the COX product, PGH , to vascular PGIS. LDLR lesions, probably due to macrophage depletion and 2 the relative abundance of medial VSMCs, which are rich in the Results enzyme. Strikingly, VSMCs were radially orientated in mPGES- 1Ϫ/Ϫ LDLRϪ/Ϫ lesions, perhaps reflecting the relative deficiency, mPGES-1 Deletion Retards Atherogenesis. Neither a gender imbal- compared with LDLRϪ/Ϫ, of migratory cues from necrotic or ance nor a genotype-associated developmental morbidity was foam cell regions. observed when generating the double-knockout mice (mPGES- 1Ϫ/Ϫ LDLRϪ/Ϫ). Expression of aortic mPGES-1 transcripts Differential Impact of mPGES-1 Deletion on Prostanoid Generation in increased Ϸ2-fold on high-fat diet (HFD; Fig. 1A). Correspond- Macrophages and VSMCs. mPGES-1 was the primary source of ingly, immunostaining of the aortic root revealed mPGES-1 PGE formation under basal conditions and after LPS stimula- protein expressed in macrophages and macrophage-derived 2 Ϫ/Ϫ tion of either macrophages or VSMCs (Fig. 6, which is published foam cells, particularly in the shoulder region of mPGES-1 as supporting information on the PNAS web site). VSMCs were plaques (Fig. 1B). ␣ Ϫ/Ϫ Ϫ/Ϫ a more abundant source of 6-keto PGF1 (the hydrolysis product Starting at 8 wk of age, the mPGES-1 LDLR and of PGI ) than macrophages under basal conditions. Also, the Ϫ/Ϫ 2 control mice (LDLR ) of both genders were fed a HFD for 3 relative abundance of prostanoid formation in macrophages or 6 mo. Total plasma cholesterol, triglycerides, and body weight (PGE2 Ͼ TxB2 Ͼ 6-keto PGF1␣) differed from that in VSMC were not different between mPGES-1Ϫ/Ϫ LDLRϪ/Ϫs and con- (6-keto PGF1␣ Ͼ PGE2Ͼ TxB2). trols in either gender at any time point. All mice achieved a Deletion of mPGES-1 depressed PGE2 and augmented LPS- significant increase in plasma cholesterol during high-fat dietary stimulated 6-keto PGF1␣ and TxB2 in both cell types, although feeding (Table 1, which is published as supporting information the principal redirection product in VSMC was PGI2, whereas on the PNAS web site). TxA2 predominated in the macrophages. Indeed, under basal Deletion of mPGES-1 delayed atherogenesis significantly in conditions, the only evidence of substrate shift detectable was a Ϫ/Ϫ both genders in LDLR s (Fig. 2A). The lesion burden de- significant increase in 6-keto PGF1␣ production in VSMC. creased in females by a mean 41% and 35% after 3 and 6 mo on

HFD and in males by 15% and 50%, respectively. Consistent with mPGES-1 Deletion Decreases Systemic Biosynthesis of PGE2 and Aug- the en face data, crosssectional analysis of aortic root samples ments PGI2. Systemic production of PGE2, TxA2, and PGI2 was revealed a significant decrease (34%) in the total lesion area in determined by measuring their major urinary metabolites, 11␣- the aortic root from female mice fed HFD for 6 mo (Fig. 2B). hydroxy-9, 15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid mPGES-1 deletion did not affect blood pressure in either (PGE-M), 2,3-dinor-TxB2 (Tx-M), and 2,3-dinor 6-keto PGF1␣

14508 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0606586103 Wang et al. Downloaded by guest on September 27, 2021 Fig. 2. mPGES-1 deletion retards atherogenesis in LDLRϪ/Ϫ mice. (A) The extent of atherosclerosis, represented by the ratio of lesion area to total aortic area, was quantiﬁed by en face analysis of aortae from mice treated with a HFD for 3 or 6 mo (Upper). Representative en face graphs are shown (Lower), with each vertically matched to the corresponding data set in Upper.(B) Box-and-whiskers graph revealing the crosssectional analysis of aortic root samples from female mice after 6 mo on HFD, measuring total lesion area across the aortic root as detailed in Materials and Methods (Upper). A representative crosssection from each group is shown (Lower), vertically aligned with the corresponding data in Upper. (Scale bar, 500 ␮m.) *, P Ͻ 0.05; **, P Ͻ 0.01; n ϭ 10–15 per group.

(PGI-M), respectively. mPGES-1 deletion significantly decreased tion on the PNAS web site). Urinary Tx-M and PGI-M both PGE-M in the mPGES-1Ϫ/Ϫ LDLRϪ/Ϫs in each gender before increased during atherogenesis in LDLRϪ/Ϫs (Fig. 4). Urinary HFD treatment (Fig. 7, which is published as supporting informa- PGI-M increased further in the mPGES-1Ϫ/Ϫ LDLRϪ/Ϫs, which was evident at all time points in the study, whereas excretion of Tx-M was unaltered. The significant depression of urinary PGE-M in mPGES-1-deleted mice was sustained on the HFD at 3 (12.33 Ϯ 1.84 vs. 6.08 Ϯ 1.05 ng͞mg creatinine, P Ͻ 0.01 n ϭ 14 and 16, respectively) and 6 mo (8.74 Ϯ 0.99 vs. 5.16 Ϯ 0.89 ng͞mg MEDICAL SCIENCES creatinine, P Ͻ 0.05 n ϭ 11 per group).

Discussion Development of drugs that inhibit specifically mPGES-1 as alternatives to NSAIDs is configured on several assumptions. These include the premises that (i) mPGES-1 is a major source of the PGE2 formed in vivo, and that other PGES enzymes do not substitute for it when it is inhibited; (ii) an mPGES-1 inhibitor retains substantially the clinical efficacy of NSAIDs, despite the recognition that PGs other than PGE2, particularly PGI2, may contribute to pain and inflammation; (iii) the cardiovascular hazard attributable to specific inhibitors of COX-2 results from inhibition of PGI2 rather than PGE2; and (iv) the gastrointestinal consequences of mPGES-1 inhibition do not Fig. 3. Effects of mPGES-1 deletion on lesional morphology in LDLRϪ/Ϫ mice. exceed those of NSAIDs, particularly those selective for COX-2. Smooth muscle cells and macrophages were stained by antibodies against The present study was designed to test some of these assump- ␣-smooth muscle actin (␣-SMA; A Left) and CD11b (A Center), respectively. tions. We have previously reported that deletion of mPGES-1 Macrophages were depleted in the mPGES-1Ϫ/Ϫ LDLRϪ/Ϫs. Collagen content was depresses substantially endogenous PGE2 biosynthesis, as re- stained by sirius red (A Right). Note the ﬁbrillar collagen content of the mPGES- flected by urinary PGE-M, in normolipidemic mice (13). Here, 1Ϫ/Ϫ LDLRϪ/Ϫ neointima. All stainings in A were performed on one lesion of each genotype. Another set of ␣-SMA staining is shown in B. [Scale bar, 200 ␮m(A) and it is apparent that this observation extends to hyperlipidemic 100 ␮m(B).] Quantitative analysis (C) of macrophage-foam cells reveals a signif- mice, and this inhibition is sustained as they develop atheroscle- icant decrease in the ratio of foam cell area to lesion area. *, P ϭ 0.02. rosis on a HFD. Thus, other PGES do not appear to substitute

Wang et al. PNAS ͉ September 26, 2006 ͉ vol. 103 ͉ no. 39 ͉ 14509 Downloaded by guest on September 27, 2021 stained sections. Specifically, whereas lesions in LDLRϪ/Ϫs have significant levels of smaller fibers, the mPGES-1Ϫ/Ϫ LDLRϪ/Ϫ lesions were enriched in fibrillar collagens (presumably type I, III, and or V), the predominant collagens produced by VSMC. These changes and the depletion of lesional macrophages are two of three morphological hallmarks of plaque stabilization (33), evident when mPGES-1 deletion occurs in hyperlipidemic mice. The third, enhanced formation of a fibrous cap, was not observed. However, there was no suggestion of plaque destabi- lization (25) in the mPGES-1Ϫ/Ϫ LDLRϪ/Ϫs. Expression of both PGIS and TxS is increased in endothelial cells, and the increase in the ratio of VSMCs relative to foam cells is associated with an increase in the proportion of neointimal cells expressing PGIS consequent to deletion of mPGES-1. Increased endothelial expression of both PGIS and TxS suggests removal of an mPGES-1-derived regulatory restraint on these enzymes in the mPGES-1Ϫ/Ϫ LDLRϪ/Ϫs. Trebino et al. (34) have previously reported that synthesis of both TxA2 and PGI2 is increased in LPS-stimulated macrophages obtained from mPGES-1Ϫ/Ϫ mice ex vivo, which they attributed to rediversion of the accumulated PGH2 substrate to PGIS and TxS. Here, we replicate their observation in macrophages. Synthesis of both PGI2 and TxA2 is increased under LPS- stimulated conditions in mPGES-1-depleted macrophages. However, whereas this is also true of LPS-stimulated VSMCs, PGI2 is the more abundant product, and only PGI2 formation is increased in mPGES-1-depleted VSMC under basal conditions. Thus, the predominant product formed consequent to PGH2 rediversion due to mPGES-1 deletion or inhibition is likely to vary by cell type and pathophysiological condition (Fig. 8, which is published as supporting information on the PNAS web site). Analysis of urinary metabolites represents a time-integrated noninvasive approach to the study of PG biosynthesis in vivo Fig. 4. Biosynthesis of PGI2 and TxA2 during atherogenesis. Systemic pro- (35). Previously, we have shown that mPGES-1 deletion aug- duction of PGI2 and TxA2 was examined by measuring their urinary metabo- ments PGI , but not TxA , metabolite excretion in normolipi- lites, PGI-M and Tx-M. Both increased during atherogenesis in the LDLRϪ/Ϫ 2 2 demic mice in which PGE2 biosynthesis is suppressed substan- mice and substrate redirection to PGI2 (A), but not TxA2 (B), is observed in Ϫ/Ϫ Ϫ/Ϫ response to mPGES-1 deletion. This is sustained during the study period in tially. Here, we show that in the mPGES-1 LDLR s, hyperlipidemic mice. Data shown are from females. *, P Ͻ 0.05; **, P Ͻ 0.01; systemic biosynthesis of PGI2, but not TxA2, is augmented, and ***, P Ͻ 0.001; n ϭ 11–16 per group. that this increase is sustained during atherogenesis. This may reflect the increased vascular expression of PGIS, together with depletion of lesional macrophages, a major source of TxA2 (36). for the suppression of PGE2 formation consequent to mPGES-1 Despite the marked gender difference in biosynthesis of PGE2, deletion. apparent both in normolipidemic and here in hyperlipidemic We have previously reported that mPGES-1 deletion does not mice, the qualitative shift to an increase in PGI2 biosynthesis is result in an elevation of blood pressure or enhance the response apparent in both males and females. It is possible that initial to thrombogenic stimuli in normolipidemic mice in which genetic manipulation of mPGES-1 when atherosclerosis is advanced and manipulation or inhibition of COX-2 adversely influences both lesions are rich in macrophages might augment Tx biosynthesis. variables (13). Here again, we fail to observe a rise in blood However, inducible deletion of the enzyme or timed intervention pressure, this time when mPGES-1Ϫ/Ϫ mice are crossed into with a specific inhibitor would be necessary to address this those lacking the LDLR. Indeed, deficiency in mPGES-1 does hypothesis. not adversely affect blood pressure during prolonged HFD Previous studies have indicated that PGI2 is an important feeding. restraint on initiation and early development of atherogenesis in The impact of COX-2 deficiency on atherogenesis is complex. both male (10) and female (11) hyperlipidemic mice. This Investigators have reported that the development of plaque appears to reflect, at least in part, the induction of antioxidant burden is accelerated, retarded, or uninfluenced by COX-2 enzymes, such as hemeoxygenase-1 (11, 37), which serve to inhibitors. These contrasting effects may reflect the contrasting counter free radical induced vascular injury consequent to biologies of the products of COX-2 formed by cells that pre- platelet (10, 11) and neutrophil (10) activation. Furthermore, dominate variably at different stages in disease evolution (33). PGIS is uniquely susceptible to free radical-based inactivation Here, we observe that atherogenesis results in increased vascular (38), such as might pertain during atherogenesis. Although Ϫ/Ϫ expression of mPGES-1 in LDLR mice. Deletion of biosynthesis of PGI2 is augmented in patients with severe mPGES-1 retards atherogenesis strikingly on an LDLRϪ/Ϫ atherosclerosis (39), this reflects accelerated platelet and neu- background. trophil–vessel wall interactions and stimulated production in Lesion morphology is markedly altered in mPGES-1Ϫ/Ϫ response to physical and chemical stimuli. The capacity of Ϫ/Ϫ LDLR s. Macrophages, themselves rich in COX-2, TxS, and atherosclerotic vascular tissue to produce PGI2, by contrast, is mPGES-1, are markedly depleted. The VSMC content of the reduced compared with healthy vasculature (39). Biosynthesis of lesions, by contrast, is apparently unaltered. Interestingly, we PGI2 is augmented by mPGES-1 deletion by Ϸ50% in both observed changes in the type of collagen that accumulates in the normolipidemic and hyperlipidemic mice. Although the func- atherosclerotic lesions, based on birefringence analysis of sirius- tional relevance of this observation is unknown, haploinsuffi-

14510 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0606586103 Wang et al. Downloaded by guest on September 27, 2021 ciency of the IP results in a detectable cardiovascular phenotype Real-Time PCR Analysis of Gene Expression in Mouse Aorta. TaqMan (13), and increments in PGI2 biosynthesis of this order are often Gene Expression Assays (Applied Biosystems, Foster City, CA) observed in settings of platelet activation where it may serve a for mPGES1 (Mm00452105࿝m1) were performed on an ABI homeostatic purpose (39). Prism 7900 Sequence Detection System (Applied Biosystems). These experiments indicate that mPGES-1-derived PGE2 accel- Results were normalized with 18s rRNA (Hs99999901࿝s1). erates atherogenesis in both male and female LDLRϪ/Ϫ mice. Experiments in mPGES-1Ϫ/Ϫ mice raise the possibility that selec- Macrophage and VSMC Preparation. Mice were injected i.p. with 3 tive inhibitors of this enzyme not only may reduce the likelihood of ml of 3% thioglycollate media and killed after 4 days. Peritoneal the hypertension and predisposition to thrombosis associated with lavages were collected, filtered, centrifuged at 1,000 ϫ g at 4°C COX-2 inhibitors but also may retain their clinical efficacy and even for 10 min, and resuspended in phenol-free RPMI medium 1640 confer cardiovascular benefit during sustained dosing. The impact (Invitrogen Carlsbad, CA) supplemented with 10% FBS. Cells on atherogenesis may reflect depletion of lesional macrophages and were plated onto six-well plates, incubated at 37°C for 4 h, and foam cells, alterations in the nature of plaque collagen, and a then changed into serum-free media overnight. VSMCs were relative increase in VSMC, rich in PGIS, facilitating substrate isolated from mouse aorta as described (11) and incubated in rediversion to augment systemic biosynthesis of PGI2. Although serum-free media overnight. Cells were treated the next day with these results are encouraging, it remains to be determined whether fresh serum-free media with LPS (Sigma-Aldrich L-2654 from specific inhibitors of mPGES-1 will retain the properties conferred Escherichia coli 026:B6, 1 ␮g͞ml in PBS) or PBS (control) and by mPGES-1 deletion. then incubated for 24 h. Cell culture media were used for prostanoid determination. Materials and Methods Mice. mPGES-1Ϫ/Ϫ mice on DBA͞1lacJ genetic background (32) Histological Examination of Lesion Morphology. Fixed and peroxi- were crossed with LDLRϪ/Ϫ mice fully backcrossed onto a dase-quenched sections (8 ␮m) of OCT compound-embedded C57BL͞6 background (The Jackson Laboratory, Bar Harbor, tissue were blocked with 20 ␮g͞ml goat IgG (Jackson Immuno- ME), and the resulting mice (mPGES-1ϩ/Ϫ LDLRϩ/Ϫ) were Research, West Grove, PA), followed by incubation with pri- intercrossed to generate mPGES-1Ϫ/Ϫ LDLRϪ/Ϫ mice and their mary antibodies: rabbit antilaminin, FITC-conjugated mouse littermate controls (mPGES-1ϩ/ϩ LDLRϪ/Ϫ). Mice used in these anti-␣-smooth muscle actin clone 1A4 (Sigma-Aldrich), rabbit studies were on a Ϸ75% C57BL͞6͞25% DBA͞1lacJ genetic anti-mPGES-1, rabbit anti-PGIS, rabbit anti-TxS (Cayman background. All animals were housed according to guidelines of Chemicals, Ann Arbor, MI), biotinylated hamster anti-CD11c, the Institutional Animal Care and Usage Committee (IACUC) rat anti-CD11b, or rat anti-platelet endothelial cell adhesion of the University of Pennsylvania, and all experiments were molecule (anti-PECAM) (BD Biosciences, San Jose, CA). Sec- approved by the IACUC. Genotyping by PCR was derived from tions were then incubated with either biotinylated mouse anti- The Jackson Laboratory protocol (for ldlr) and from a previous rabbit, mouse anti-rat, or mouse anti-FITC (Jackson Immu- publication for mpges-1 (32). Mice of both genders were started noResearch), followed by Vectastain ABC avidin-biotin on a HFD (0.2% cholesterol͞21% saturated fat; formula TD amplification (Vector Laboratories, Burlingame, CA), and de- 88137, Harlan Teklad, Indianapolis, IN) at 8 wk of age; sub- veloped with diaminobenzidine (Dako, Carpinteria, CA). All groups were killed after 3 and 6 mo on the diet. sections were counterstained with Gill’s Formulation no. 1 hematoxylin (Fisher Scientific), and isotype controls were run in Blood Pressure Measurement. Systolic blood pressure was mea- parallel with negligible staining observed in all cases. Eosin was sured in conscious mice by using a computerized noninvasive obtained from Fisher Scientific, and Sirius Direct Red 80 was tail-cuff system (Visitech Systems, Apex, NC), as described (13). obtained from Sigma-Aldrich. Crosssectional analysis for mac- Blood pressure was recorded once each day between 15:00 and rophage foam cells (CD11c) was performed every 96 ␮m over 18:00 for 4 consecutive days, and the average blood pressure was 300 ␮m of the aortic root of five mice for each group. used.

Analysis of Prostanoids and Their Metabolites. Urine was collected MEDICAL SCIENCES Preparation of Mouse Aortae and Quantitation of Atherosclerosis. for 24 h at baseline and again after 3 and 6 mo of HFD feeding. Mice were killed, and the aortic tree was perfused with ice-cold Systemic production of PGE2, TxA2, and PGI2 was determined by PBS. The entire aorta (from the aortic root to the iliac bifur- mass spectrometric quantitation of their major urinary metabolites: cation) was dissected out, fixed in buffered formalin (Fisher 7-hydroxy-5,11-diketotetranorprostane-1,16-dioic acid (PGE-M), Scientific, Hampton, NH), cleaned of adventitial fat, opened Tx-M, and PGI-M, respectively. Briefly, 100 ␮l of urine was spiked longitudinally, and stained with Sudan IV (Sigma-Aldrich, St. with the corresponding stable isotope labeled internal standard: 18 Louis, MO). The extent of atherosclerosis (Phase 3 Imaging d6-PGE-M, O2-2,3-dinor TxB2 and d3-2,3-dinor-6-keto PGF1␣, Systems, Glen Mills, PA) was determined by using the en face allowed to react with mythoxylamine, purified with solid-phase method (40) and crosssectional analysis of lesion burden on extraction by using Strata-X cartridges (Phenomenex, Torrance, aortic roots sections. Total lesion areas over 300 ␮m of the aortic CA), and subjected to HPLC and tandem mass spectrometry root of nine mPGES-1Ϫ/Ϫ LDLRϪ/Ϫ mice and 11 LDLRϪ/Ϫ mice (Quantum Ultra, ThermoFinnigan, San Jose, CA). The following were measured on 8-␮m (every 96 ␮m) acetone-fixed serial mass transitions were monitored: m͞z 3853336 (PGE-M), m͞z sections by using average lesion areas derived from three 3913342 (d6-PGE-M), m͞z 3703155 (2,3-dinor TxB2), m͞z 18 sections. 3743155 ( O2-2,3-dinor TxB2), m͞z 3703232 (2,3-dinor-6-keto PGF1␣), and m͞z 3733235 (d3-2,3-dinor-6-keto PGF1␣). Quanti- Lipid Analyses. Blood was drawn by cardiac puncture from the fication of the endogenous metabolites was used the ratio of the killed mice, and EDTA (final concentration, 10 mM) was added peak areas of the analytes and their respective internal standards. immediately. Blood samples were obtained from animals at Data were corrected for urinary creatinine (Oxford Biomedical baseline after they fasted overnight by retroorbital bleeding with Research, Oxford, MI). EDTA-coated Microvette 200 (Sarstedt, Newton, NC). Plasma total cholesterol and triglyceride levels were measured enzymat- Statistical Analysis. Data are expressed as mean Ϯ SEM. Com- ically on a Cobas Fara II autoanalyzer (Roche Diagnostic parisons of multiple groups were performed by ANOVA and a Systems, Nutley, NJ) by using Wako Chemicals (Richmond, VA) Dunn’s post-ANOVA multiple comparison test when the reagents. ANOVA was significant. When only two mean values were

Wang et al. PNAS ͉ September 26, 2006 ͉ vol. 103 ͉ no. 39 ͉ 14511 Downloaded by guest on September 27, 2021 compared, the two-tailed Mann–Whitney t test was used. Dif- Ferrari, and John Lawson. mPGES-1Ϫ/Ϫ mice were generously ferences were considered statistically significant at P Ͻ 0.05. supplied by Dr. L. Audoly (Pfizer, New York, NY). This work was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health (Grants HL 70128, HL 62250, Technical support was kindly provided by Helen Zou and Ping Liu. and 2-P01-HL-06225). G.A.F. holds the Elmer Bobst Chair of We gratefully acknowledge the contributions of Karine Egan, Julien Pharmacology.

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