Nicotinic acid and DP1 blockade: studies in mouse models of atherosclerosis.

The use of nicotinic acid to treat dyslipidemia is limited by induction of a "flushing" response, mediated in part by the interaction of prostaglandin D(2) (PGD(2)) with its G-protein coupled receptor, DP1 (Ptgdr). The impact of DP1 blockade (genetic or pharmacologic) was assessed in experimental murine models of atherosclerosis. In Ptgdr(-/-)ApoE(-/-) mice versus ApoE(-/-) mice, both fed a high-fat diet, aortic cholesterol content was modestly higher (1.3- to 1.5-fold, P < 0.05) in Ptgdr(-/-)ApoE(-/-) mice at 16 and 24 weeks of age, but not at 32 weeks. In multiple ApoE(-/-) mouse studies, a DP1-specific antagonist, L-655, generally had a neutral to beneficial effect on aortic lipids in the presence or absence of nicotinic acid treatment. In a separate study, a modest increase in some atherosclerotic measures was observed with L-655 treatment in Ldlr(-/-) mice fed a high-fat diet for 8 weeks; however, this effect was not sustained for 16 or 24 weeks. In the same study, treatment with nicotinic acid alone generally decreased plasma and/or aortic lipids, and addition of L-655 did not negate those beneficial effects. These studies demonstrate that inhibition of DP1, with or without nicotinic acid treatment, does not lead to consistent or sustained effects on plaque burden in mouse atherosclerotic models.

Both strains of mice, particularly on a Western diet, were prone to develop skin lesions, which were exacerbated by fi ghting among cage mates; however, skin lesion development may also be facility-dependent. We excluded animals with large or unhealed lesions ( ‫ف‬ 5% of apoE knockouts and ‫ف‬ 15% of Ldlr knockouts), because the presence of these skin lesions is associated with decreased plaque volume (K. Cheng, unpublished observations), presumably because of the immune responses generated. Mice with small skin lesions that had healed may have been included; however, an attempt was made to closely monitor the mice across the duration of the experiments to minimize this potential variable.
All animals were kept in a controlled environment, with a 12 h light/dark cycle, with constant temperature and humidity, and had access to food and water ad libitum . The studies were conducted in conformity with the Public Health Service Policy on Humane Care and Use of Laboratory Animals. All procedures described here were approved by the Merck Research Laboratories (Rahway) Institutional Animal Care and Use Committee.

Determination of L-655 dose
Vasoconstriction/vasodilatation studies were designed to determine the dose of L-655 that would effectively block the DP1 receptor, without having measureable off-target effects at the TP receptor. Male and female ApoE Ϫ / Ϫ and Ldlr Ϫ / Ϫ mice were tested separately ( n = 5-6 per gender per group). Ldlr Ϫ / Ϫ mice were weaned at 4 weeks of age, and at 8 weeks of age were placed on the high-fat Western diet (HFWD) TD88137 (21% fat and 0.15% cholesterol; Harlan Teklad, Madison, Wisconsin) for at least 4 weeks prior to the beginning of the studies. ApoE Ϫ / Ϫ mice were weaned at 4 weeks of age and placed on a HFWD immediately after weaning and for at least 12 weeks prior to the beginning of the studies. These feeding conditions mimicked those selected for the long-term atherosclerosis studies (see below), with the limitation that mice had to be at least 12 weeks old to perform vasoconstriction/vasodilatation studies. U46619, a full TP agonist (Cayman Chemical; Ann Arbor, MI), was supplied by the manufacturer as a 10 mg/ml stock in methyl acetate. The compound was dried under nitrogen and resuspended in 100% DMSO to 8 mg/ml, and then diluted with water to 0.2 mg/ml to provide a fi nal dose of 2 mg/kg. Solid SQ-29,548, a TP antagonist (Cayman Chemical) was resuspended in 0.1 M sodium carbonate to a concentration of 2 mg/ml and then diluted in PBS to 0.3 mg/ml to provide a fi nal dose of 3 mg/kg. PGD 2 (Sigma-Aldrich; St. Louis, MO) was prepared as a stock solution at 40 mg/ml in 100% ethanol, and diluted to 0.2 mg/ml in saline to provide a fi nal dose of 2 mg/kg.
One week prior to performing the vasodilatation/vasoconstriction studies, mice were switched to a milled HFWD, which contained an admixture of L-655 at 0 (chow-alone control), 0.003%, 0.01%, or 0.03% (w/w). Mice had access to the diets ad libitum .
The fi rst series of studies was performed to test for the lack of TP antagonism in mice fed L-655 for 1 week. To demonstrate that these strains of mice were indeed responsive to TP antagonism, mice that had not been exposed to L-655 were used to monitor the effects of the TP antagonist SQ-29,548 on U46619induced vasoconstriction. Studies were carried out in the morning, immediately following the end of the feeding cycle (starting at 8:00 AM and not extending past 11:00 AM ), when plasma levels of L-655 were expected to be the highest.
To study the effects of L-655 on vasoconstriction, mice were anesthetized with sodium pentobarbital (60 mg/kg, intraperitoneal injection) 10 min prior to the start of the vasoconstriction studies. Cutaneous blood fl ow in the mouse ear was measured by laser Doppler perfusion imaging, employing a PeriScan PIMII that pharmacological blockade of DP1 should not in itself cause signifi cant adverse effects. However, there are no published reports of preclinical models that have measured potential effects of DP1 receptor antagonists on atherosclerosis endpoints, including plaque burden, in the presence or absence of nicotinic acid. The effects of nicotinic acid alone on plasma lipids have been explored in several animal models, including the mouse, rat, guinea pig, rabbit, dog, mini pig, and Rhesus monkey. However, for those species in which HDL-C metabolism has been examined, nicotinic acid generally lacks the effects seen in humans, with the possible exception of the mouse model, in which the human cholesteryl ester transfer protein (CETP) transgene is expressed ( 11 ). Recent studies in both rabbits and mini pigs suggest that nicotinic acid has benefi ts in atherosclerosis over and above its effects on serum lipids ( 12 ).
This report describes genetic and pharmacological approaches to antagonize DP1 function and to measure the impact on mouse models of atherosclerosis. In addition, we utilize well-established ApoE Ϫ / Ϫ and Ldlr Ϫ / Ϫ mouse models of atherosclerosis to study the effects of nicotinic acid treatment, with and without the presence of a specifi c DP1 antagonist. The DP1 antagonist L-655 (see supplementary Fig. I) was chosen because of its selectivity for murine DP1 over murine thromboxane A 2 (TxA 2 ) receptor (TP). TP has an often-used pro-atherogenic role in mouse models (13)(14)(15)(16); therefore, laropiprant was not chosen, because it displays signifi cant affi nity for the murine TP.
We assessed a battery of atherosclerotic endpoints in these mouse studies, including aortic cholesterol levels, en face staining of the aorta, and cross-sectional histological analyses that examine plaque composition.

In vitro characterization of L-655
The affi nity of L-655 for DP1, DP2, and TxA 2 receptors was determined using membrane-based radioligand binding assays employing recombinant murine receptors ( 17 ). The antagonist potency of L-655 was determined in a functional assay employing cells expressing recombinant mouse DP1 (whole-cell cAMP-based assay) ( 18 ). Antagonist activity at platelet TP was monitored in human platelet assays that measure the inhibition of U46619 (TxA 2 mimetic)-induced platelet aggregation in platelet-rich plasma ( 19 ).

ApoE
Ϫ / Ϫ mice (B6.129P2-ApoE tm1Unc /J) used in these studies were purchased from The Jackson Laboratory (Bar Harbor, ME) or bred in-house using the descendants of a breeding pair originally purchased from The Jackson Laboratory. Ptgdr double-defi cient mice were bred in-house from the abovementioned stock of ApoE Ϫ / Ϫ mice and Ptgdr tm1Dgen mice (defi cient in the prostaglandin 2 DP1 receptor) obtained from Deltagen, San Carlos, CA ( 8 ). Ldlr Ϫ / Ϫ mice used in these studies were bred from strain B6.129-Ldlr tm1Her /J, stock no. 002207, from the Jackson Laboratory. Both the ApoE and the Ldlr knockout mice were backcrossed into C57BL/6 at least 10 generations before being used in the study. the anti-atherogenic properties of nicotinic acid in ApoE Ϫ / Ϫ mice, male and female ApoE Ϫ / Ϫ mice were weaned at 4 weeks of age, and immediately fed a pelleted HFWD. At 15 weeks of age, mice were switched to a milled HFWD for 1 week. At 16 weeks of age, animals were assigned to different treatment groups, depending on study design. All studies consisted of a baseline group (terminated at 16 weeks of age); a vehicle group, which received a HFWD for 8 weeks; a nicotinic acid group, which received a HFWD supplemented with 3% nicotinic acid for 8 weeks; and one or more groups receiving either L-655 alone (0.003%, 0.01%, or 0.3% in a HFWD) or a combination of L-655 plus 3% nicotinic acid. All groups received the admixed diets for 8 weeks. Most studies used male mice; one included both males and females. Ten to 36 animals were analyzed per gender and treatment group.
To study whether pharmacological antagonism of DP1 had any effect on the development of atherosclerosis or on the antiatherogenic properties of nicotinic acid in Ldlr Ϫ / Ϫ mice, a large, three-part study was carried out ( Fig. 1A ). In all parts, male and female Ldlr Ϫ / Ϫ mice were kept on regular mouse chow until 8 weeks of age; n = 41-56 animals per gender per group were analyzed. The same dietary admixture of drugs was used as described for the ApoE Ϫ / Ϫ mice, except that all treatments with L-655 were at 0.01%. The three parts were performed concurrently so that the vehicle groups of the fi rst part could be used for all three parts. In Part 1, the time course of L-655 effects was examined. A baseline group, terminated at 8 weeks of age, was used to establish baseline values; vehicle and L-655 groups were treated for 8, 16, and 24 weeks, starting at 8 weeks of age. Lesion development as a function of time (histology) in the vehicle-treated group is shown in Fig. 1B . Parts 2 and 3 were focused on assessment of the effects of nicotinic acid treatment ± L-655 on animals on a HFWD for 16 (early therapeutic) and 24 weeks (late therapeutic), respectively. In Part 2, vehicle, L-655, nicotinic acid, and an L-655/nicotinic acid combination were administered to respective groups for 8 weeks, starting at 16 weeks of age and ending at 24 weeks. In Part 3, the same four treatment groups were also treated for 8 weeks, but this time beginning at age 24 weeks and ending at 32 weeks. Of the animals in each group, 7 to 12 were analyzed for aortic en face and aortic root histology; the remaining animals were analyzed for aortic lipids. Plasma from all animals was saved for measurement of cholesterol and TG.

Aortic and plasma lipid analysis
After the in vivo treatment phase, mice were euthanized with carbon dioxide, and blood was immediately collected from the vena cava into syringes rinsed with 0.5 M EDTA. Plasma was prepared via centrifugation at 850 g for 15 min at 4°C and stored at Ϫ 80°C until analysis. Levels of total cholesterol (TC) and TG were determined on an automated Roche P Module Clinical Chemistry Analyzer (Roche Diagnostics; Indianapolis, IN).
Aortic lipid content was determined as described in a published report ( 20 ). Briefl y, after mice were euthanized and blood collected, the vasculature was perfused through the left ventricle with cold PBS supplemented with 5 mM EDTA. A section of aorta from the aortic root to the right renal artery was used for biochemical determination of cholesterol after being cleaned of adventitious fat. TC and free cholesterol (FC) were determined by an enzymatic fl uorometric assay, and cholesteryl ester (CE) was calculated as the difference between TC and FC.

Aorta en face analysis
En face analysis was performed according to previously published procedures ( 21 ). Briefl y, after euthanasia and blood collection, mice were perfused with PBS through the left ventricle for 3 to 5 min, and aortas (from the point where the aorta exits the top of the heart to laser Doppler Perfusion Imager system (Perimed, Inc.; North Royalton, OH). The settings of the system were: repeated mode, image size 12 × 12, start method auto interval, 40 images, images every 30 s, medium resolution, intensity 7-9V, range 0-3V. All measurements were taken from the ventral side of the right ear. Mice were placed in left lateral recumbency, and the right ear was taped to a fl at surface, exposing the ventral side for measurements. After a 3 min baseline reading, U46619 (2 mg/kg) or vehicle (5% DMSO in water) was administered by subcutaneous injection, and cutaneous blood perfusion in the ear was measured for another 17 min. Perfusion values for the baseline in each animal were averaged, and subsequent values after injection of vehicle or U46619 were expressed as the percentage change over this initial value. Negative percentage changes were indicative of reduced blood perfusion (i.e., vasoconstriction). All procedures were performed under dim lights. In the control group of animals not exposed to L-655, SQ-29,548 (3 mg/kg) was administered by intraperitoneal injection after a 3 min baseline reading. Five minutes later, animals were challenged with a subcutaneous dose of U46619 at 2 mg/kg. Vasoconstriction was then monitored for another 17 min.
The second series of studies was designed to determine the minimal dose of L-655 that would inhibit PGD 2 -induced vasodilatation in male and female ApoE Ϫ / Ϫ or Ldlr Ϫ / Ϫ mice. Mice were fed the HFWD as described above. One week prior to performing the vasodilatation studies, mice were switched to a milled HFWD, which contained an admixture of L-655 at 0 (chow-alone control), 0.003%, 0.1%, or 0.03% (w/w). Mice had access to the diets ad libitum . After feeding on medicated diets for 1 week, the vasodilatatory response to 2 mg/kg PGD 2 was tested in the mice using the same laser doppler perfusion technique described above, with the following modifi cations: on the day of the study, mice were placed in clean cages in the morning (between 7:30 and 8:00 AM), and switched to a HFWD lacking L-655 to prevent any further exposure to the compound-containing diet. Vasodilatation was studied in the afternoon (after 3:00 PM), after the medicated diet had been removed for at least 7 h. Studying vasodilatation in the afternoon, many hours after the last possible dose of compound could be ingested, provided the most-stringent test of in vivo DP1 antagonism, because this time point probably refl ected the minimal plasma concentration of L-655 that would occur when mice were not actively feeding. After a 3 min baseline reading of cutaneous blood perfusion, PGD 2 (2 mg/kg) or vehicle (0.5% ethanol in saline) was administered by intraperitoneal injection, and measurement of cutaneous perfusion continued for another 17 min. Perfusion values for the baseline in each animal were averaged, and subsequent values after injection of vehicle or PGD 2 were expressed as percent change over this initial value. Positive-percentage changes were indicative of increased blood perfusion (i.e., vasodilatation).

Treatment groups
Genetic studies. To

Characterization of L-655, a selective DP1 antagonist
The affi nity of L-655 for murine DP2, murine and human DP1, and TP indicated that L-655 binds more tightly to DP1 the iliac bifurcation) were collected, cleaned of adventitious fat, opened longitudinally, and placed in 10% formalin for up to 10 days until the time of Sudan IV staining. To prepare for the Sudan IV staining, the aortas were rinsed in PBS for 5 min and in 1 ml of 70% ethanol for 3 min, followed by gentle mixing in 70% ethanol two more times. The aortas were then immersed in 1 ml of 0.5% Sudan IV in a solution of ethanol-acetone-water (35:50:15) for 8 min, followed by rocking. The aortas were destained in 1 ml of 80% ethanol for 3 min twice and washed with PBS twice. The Sudan IV-stained aorta image was captured with a digital camera and analyzed with Image-Pro Plus software (Media Cybernetics, Inc.; Bethesa, MD) to quantify the Sudan IV-stained surface area. Imaging and analysis were performed by a blinded operator.

Aortic root histomorphometric analysis
After removal of the aortas as described above, the intact heart was removed and transected through a plane perpendicular to the aorta. The section containing the aortic root was placed in a standard cryomold, covered with OCT cryo-embedding media, and immediately frozen. The whole specimen was cut into 8 µm sections, and every 4th section was processed for hematoxylin and eosin staining. The area (in µm 2 ) of atherosclerotic lesions was then measured on digital images of the slides using computer-based image analysis. Concurrent with image acquisition, the position of the distal end of the aortic valves was determined by a human operator on the basis of direct observation. This point was arbitrarily defi ned as zero microns. Lesion volume in the 300 µm of aorta distal to the zero point was calculated as the summation of the lesion area times the section thickness for all the sections in 300 µm.

Statistical methods
Comparisons between treatment groups were made using two-sample t -tests, using the Welch-Satterthwaite method to avoid assuming equal variances. A two-sided P value of less than 0.05  ( Fig. 4 ) and female Ldlr Ϫ / Ϫ mice (data not shown). As a control, the use of SQ-29,548 indicated that the vasoconstriction induced by U46619 could be prevented by TP antagonism.
These results indicate that in Ldlr Ϫ / Ϫ mice, L-655 at doses у 100 ppm can completely block PGD 2 -induced vasodilation ( Fig. 4 ), without measureable off-target effects on TP. Thus, the dose selected for our atherosclerosis studies in Ldlr Ϫ / Ϫ mice (male and female) was 100 ppm.

Effects of L-655 ± nicotinic acid in ApoE
In three independent studies, 4-wk-old ApoE Ϫ / Ϫ mice were fed a HFWD for 16 weeks followed by 8 weeks of treatment with L-655 alone or in combination with nicotinic acid. Two additional studies included a dose of 1,000 ppm L-655, which was considered too high because of potential nonspecifi c effects, and are therefore not reported here. However, results at 30, 100, and 300 ppm of L-655 indicated that plasma levels at these doses were suffi cient to inhibit vasodilation but not high enough to affect vasoconstriction ( Fig. 3 ; supplementary Table I); therefore, results from these studies are presented here and summarized in Table 2 . Note that the presence of nicotinic acid did not alter the plasma levels of L-655 (see supplementary Table I), which were comparable to levels obtained in the dose-fi nding study ( Fig. 3 ). Two studies used only male mice, whereas the third included both males and females ( Table 2 ; see supplementary  Table II). Nicotinic acid alone reduced aortic CE and total aortic cholesterol ( Fig. 5 ), compared with vehicle; the reduction was statistically signifi cant in two of three studies than to TP and murine DP2 ( Table 1 ). L-655 also inhibited PGD 2 -induced cAMP formation in human platelet-rich plasma (PRP) with an IC 50 value of 2.51 ± 0.76 nM ( n = 13). In comparison, in human PRP, L-655 inhibited U46619 (1.36 µM)-induced platelet aggregation in a dosedependent manner, with a mean IC 50 ± SD of 19.4 ± 8.6 µM, indicating a high degree of selectivity for DP1 over TP ( 19 ). In addition, U46619 demonstrated >400× selectivity of binding over EP1, EP2, EP3, EP4, FP, and IP receptors ( 19 ).
Similarly, L-655 exhibited a high degree of selectivity in vivo between functional DP1 and TP antagonism in ApoE Ϫ / Ϫ mice ( Fig. 3 ). At doses of L-655 у 100 ppm, trough afternoon plasma levels completely blocked PGD 2 -induced vasodilation, whereas morning peak levels had no off-target effects in blocking induction of cutaneous vasoconstriction by the TP agonist U44619 ( Figs. 3, 4 ) . In comparison, a 2 mg/kg dose of the TP antagonist SQ-29548 completely blocked the vasoconstriction in this model. The fi nding that vasodilation was blocked ‫ف‬ 7 h after drug-containing chow had been removed suggests that DP1 antagonism occurs throughout the day using this route of administration. Conversely, no evidence for TP antagonism was observed at any dose of L-655 tested ( Figs. 3, 4 ), including a dose of 1,000 ppm that resulted in high plasma levels of this compound (5.06 ± 1.14 µM, data not shown). Given that L-655 had high selectivity between functional DP1 and TP antagonism in ApoE Ϫ / Ϫ mice, doses of 30, 100, and 300 ppm admixed in high-fat chow were chosen for subsequent atherosclerosis studies. PGD 2 at 2 mg/kg induced a robust vasodilatory response in both male Ldlr Ϫ / Ϫ mice ( Fig. 4 )    group ( Ϫ 20%, P = 0.045) or the niacin-only group ( Ϫ 22%, P = 0.021).
In the same study (Study 3), L-655, with or without nicotinic acid, caused statistically signifi cant increases of 17% to 25% in plasma TG levels in female mice but not in males. Conversely, in male mice, there was a signifi cant 16% increase in plasma cholesterol when L-655 was combined with nicotinic acid, compared with nicotinic acid alone, but it was not signifi cant in females. In females, there was also a signifi cant decrease in plasma cholesterol with L-655 alone (see supplementary Table II).

Effects of L-655 ± nicotinic acid in Ldlr ؊ / ؊ mice
Based on the results from the dose-fi nding study shown in Fig. 4 , a dose of 100 ppm of L-655 was chosen for the large study carried out in Ldlr Ϫ / Ϫ mice. The plasma levels of L-655 were suffi cient to see inhibition of vasodilation ( Table 2 ). L-655 alone produced statistically signifi cant reductions in aortic CE ( Table 2 ), TC, and FC ( Table 2 and  supplementary Table II) in female mice at a dose of 100 ppm. An analogous benefi cial effect of L-655 was not induced in male mice, suggesting a gender-specifi c effect. In two studies, CE was similar or signifi cantly less in the nicotinic acid/L-655 combination group, compared with the nicotinic acid-only group ( Table 2 ). In summary, L-655 produces a neutral to benefi cial effect on aortic CE and does not attenuate the anti-athero effects of nicotinic acid ( Table 2 ). Similar patterns were observed for aortic TC and FC (see supplementary Table II).
In the one study that included histologic assessments of aortic lesions (supplementary Table II), there were no signifi cant differences between treatment groups for male mice, but female mice receiving L-655 plus nicotinic acid had signifi cantly smaller lesions than the vehicle-only  In Part 2 of the Ldlr Ϫ / Ϫ mouse study, examining 8 weeks of drug treatment that occurred between 16 and 24 weeks of age, similar decreases of plasma TC and TGs were observed with nicotinic acid alone and with the nicotinic acid/L-655 combination in both males and females when compared with vehicle ( Fig. 8A and supplementary Table V). Aortic CE, FC, and TC signifi cantly decreased both with nicotinic acid treatment alone and with the nicotinic acid/L-655 combination in males, but only with the nicotinic acid/L-655 combination in females ( Fig. 8B and supplementary Table V). Statistically signifi cant reductions in percent aortic lesion area, as measured en face, were observed with treatment by nicotinic acid alone or in combination with L-655 in female but not male mice ( Fig. 8C , without affecting vasoconstriction ( Fig. 4 and supplementary Table III). In Ldlr Ϫ / Ϫ mice, there were sporadic but no consistent or sustained increases in atherosclerotic measures across the 24 week period tested in both males and females ( Figs. 6 and 7 ; supplementary Table IV). In female mice, L-655 treatment resulted in 1.2-to 1.3-fold increases compared with vehicle in aortic lipids, and plasma cholesterol was signifi cantly higher, by 7%. In males, aortic lesions were signifi cantly higher with L-655 than with vehicle, by 1.5-to 5.1-fold. However, after 16 and 24 weeks of treatment, there were no signifi cant differences between vehicle and L-655 in any of the measures for either males or females, except for a decrease in plasma TGs for females after 16 weeks ( Fig. 7 and supplementary Table IV).   24 weeks of age), but not at 32 weeks. These early but modest increases in aortic lipids are consistent with results obtained by another group in double-defi cient Ptgdr Ϫ / Ϫ Ldlr Ϫ / Ϫ mice ( 22 ). In comparison, pharmacological inhibition with the DP1 antagonist L-655 did not increase aortic  Table V). Moreover, the nicotinic acid/L-655 combination, although not nicotinic acid alone, signifi cantly decreased lesion area of the aortic root, as measured by histology, in females only (see supplementary Table V). In Part 3 of the Ldlr Ϫ / Ϫ mouse study, examining 8 weeks of drug treatment that occurred between 24 and 32 weeks of age (see supplementary Table VI), there were statistically signifi cant reductions in plasma TC in both males and females, with nicotinic acid alone and with the nicotinic acid/L-655 combination. Overall, very similar trends were observed with plasma TGs, with statistically significant lower TGs measured with nicotinic acid alone in both males and females and with the nicotinic acid/L-655 combination in males. Consistent with the reductions in plasma TGs and TGs, percent aortic lesion area was signifi cantly reduced by L-655 and the nicotinic acid/L-655 combination in males. Furthermore, aortic TC and FC were significantly reduced by nicotinic acid. There were some sporadic and gender-specifi c increases in atherosclerotic measures; specifi cally, L-655 alone signifi cantly increased aortic CE in male mice only, and plasma TC in females only (see supplementary Table VI)

DISCUSSION
In the present study, genetic ablation of the DP1 receptor in ApoE Ϫ / Ϫ mice fed a high-fat diet resulted in a modest increase in aortic cholesterol at early time points (16 and

DP1 antagonism in ApoE
؊ / ؊ and Ldlr ؊ / ؊ mice 185 Early stages of plaque formation are similar between these mouse models and humans; however, the size, composition, and histology of the plaques are quite dissimilar. Importantly, unlike humans with advanced coronary plaque and elevated plasma lipids, ApoE Ϫ / Ϫ and Ldlr Ϫ / Ϫ mice do not generate end-stage ischemic lesions or coronary heart disease ( 23,24  Mouse models have been used to study the effects of lipid-lowering and anti-atherosclerotic agents, and provide practical tools for examining the biology of several targets and the pharmacological effects of drugs. As with all animal studies, there are inherent limitations with respect to extrapolating the fi ndings to human disease and important distinctions between the ApoE Ϫ / Ϫ and Ldlr Ϫ / Ϫ mice that are noteworthy. For example, high-fat feeding of ApoE Ϫ / Ϫ mice results in accumulation of lipids mostly in VLDL particles, unlike in humans, whereas high-fat feeding of Ldlr Ϫ / Ϫ mice results in accumulation of cholesterol in LDL particles, similar to humans ( 23 ). The ApoE Ϫ / Ϫ mouse has elevated circulating cholesterol and develops plaque even when fed a chow diet, whereas the Ldlr Ϫ / Ϫ mouse model develops substantive dyslipidemia and plaque only after exposure to a Western-style, high-fat, cholesterol-containing diet. In the context of studying plaque, Ldlr Ϫ / Ϫ and ApoE Ϫ / Ϫ models tend to accumulate plaque in the aorta, unlike humans who localize plaque predominantly to the coronary arteries ( 24 ). and has no effect on aortic lesions as measured en face (C). L-655 alone has no effect and does not alter the effects of nicotinic acid. P < 0.05 vs. vehicle. NA, nicotinic acid. and not the other (males), in general, the directionality of changes was similar for both genders. There were some anomalous gender-specifi c effects of L-655 on TGs and plasma cholesterol levels in female and male ApoE Ϫ / Ϫ mice; however, similar effects were not seen in Ldlr Ϫ / Ϫ mice. Given that the mice were prone to developing skin lesions, as discussed in the Methods section, despite our efforts to eliminate animals with lesions, there could have been immune responses from mice with transient lesions that could have subsequently led to sporadic effects on decreased plaque volume. There has been no reported direct link between DP1 biology and cardiovascular risk; nevertheless, in vitro data have indirectly suggested that DP1 antagonism in platelets could promote platelet aggregation and could be pro-atherogenic ( 22 ). Specifi cally, in vitro studies have shown that PGD 2 inhibits platelet aggregation, and platelet activation results in the release of PGD 2 . It has been speculated that antagonism of DP1 could block the ability of endogenous PGD 2 to inhibit further platelet aggregation and thereby may indirectly enhance the reactivity of platelets. However, several clinical studies have indicated that treatment with the DP1 antagonist laropiprant has no platelet-aggregatory effect; in fact, a transient and mild increase in bleeding time has been reported (32)(33)(34). One caveat in interpreting this observation is that laropiprant has been shown to antagonize both DP1 and TP in humans (32)(33)(34), unlike the DP1specifi c antagonist L-655 used in this mouse work. Therefore, it is likely that the mild anti-platelet effects in humans are a result of TP antagonism and theoretically represent a net effect of antagonizing both DP1 and TP on platelets. As such, the DP1-and TP-specifi c effects on platelets cannot be teased out from cited clinical studies of laropiprant.
In this work, in vivo dose selection of the DP1 antagonist L-655 (that was 20-fold more selective for DP1 over TP binding in vitro) was made such that trough afternoon plasma levels were suffi cient to completely block PGD 2induced vasodilation (a measure of DP1-specifi c activity), whereas morning peak levels had no off-target effects in blocking TP-induced vasoconstriction. Assuming that vasodilation and vasoconstriction were reasonable measures of all DP1-specifi c and TP-specifi c activity, the DP1 antagonist had no consistent or sustained effects on multiple measures of atherosclerosis. In agreement with fi ndings of others ( 22 ), we have observed that mouse platelets do not respond to PGD 2 (as measured by cAMP production) but do respond to forskolin and iloprost treatment, suggesting very low or undetectable levels of murine DP1 expression in mouse platelets. Therefore, the overall lack of effects of L-655 on atherosclerotic measures in mice reported in this work does not necessarily apply to DP1 antagonism of human platelets and the potential effect on atherosclerosis in the clinic. However, clinical treatment with large amounts of PGD 2 that engage the DP1 receptor did not reduce bleeding times, supporting the hypothesis that engagement of the DP1 receptor does not promote pro-aggregatory platelet effects in humans (35)(36)(37).

Ldlr
Ϫ / Ϫ , and other mouse models do not provide consistent responses for several endpoints, relative to one another and to humans ( 23,25,26 ). Given these differences, caution must be exercised in the interpretation of data from murine models of disease. Nevertheless, these studies provide important mechanistic insight into atherosclerosis.
Whereas the effects of nicotinic acid on circulating lipids and lipoprotein particles have been well studied both clinically and in multiple preclinical species ( 1,11,12,27 ), few studies in preclinical species address the potential effect of nicotinic acid on plaque. In an 8 week study of male rabbits on a cholesterol-enriched (2% w/w) diet, the addition of nicotinic acid (0.4% w/w) reduced serum, liver, and aortic cholesterol (by 60%, 77%, and 64%, respectively) compared with a cholesterol diet alone ( 28 ). Nicotinic acid has also been studied in high-fat-fed ApoE Ϫ / Ϫ mice ( 29,30 ). In that study, nicotinic acid admixed in chow (0.5% w/w) had no effect on plasma TG or HDL-C, nor did it affect atherosclerosis as measured by histologic and morphometric analysis of aortic lesions. Given that relatively high doses of nicotinic acid were required to alter plasma lipids in other studies [e.g., 1% in the study employing CETP-transgenic mice above ( 31 ) and 3% in this work], it is very likely that a nicotinic acid dose of 0.5% w/w is not suffi cient to impact plasma or aortic lipids.
In our studies in ApoE Ϫ / Ϫ and Ldlr Ϫ / Ϫ mice, treatment with nicotinic acid (3% w/w), in the presence or absence of L-655, generally signifi cantly lowered both plasma and aortic lipid levels by approximately 20% to 30%. Also notable was that both strains of mice, particularly on a Western diet, were prone to developing skin lesions, which were exacerbated by fi ghting of cage mates. We eliminated animals with large or unhealed lesions because we have found that the presence of these skin lesions is associated with decreased plaque volume (K. Cheng, unpublished observations), presumably because of the immune responses generated. Mice with small skin lesions that had healed over may have been included; however, an attempt was made to closely monitor the mice across the duration of the experiments to minimize this possible variable. However, in both models, this dose of nicotinic acid tended to have smaller and less-statistically signifi cant effects on aortic lesions as compared with aortic lipids. A caveat to this comparison is the use of measurement techniques, specifi cally, a greater number of samples were used when studying aortic lipids by the biochemical method as compared with aortic lesions, which were measured by en face staining or histology. Historically, en face analysis has been most commonly used in the fi eld to visually assess aortic lesions. Although en face analysis provides detailed spatial information about the lesion location, it cannot delineate the three-dimensional structure of the lesion as provided by histological assessment. Variability in these studies may arise from multiple sources, including the relatively short half-life of the nicotinic acid that is administered in the feed. Although niacin effects were comparable across males and females, there may be a slight gender effect with L-655 treatment alone. However, although statistical signifi cance was met in one gender (females)