The Nitric Oxide-Donating Pravastatin Derivative, NCX

Statins possess anti-inflammatory effects that may contribute to their ability to slow atherogenesis, whereas nitric oxide (NO) also influences inflammatory cell adhesion. This study aimed to determine whether a novel NO-donating pravastatin derivative, NCX 6550 [(1S-[1 ( S*, S*),2 ,6 ,8 -(R*),8a ]]-1,2,6,7,8,8a-hexahydro, ,6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphthalene-heptanoic acid 4-(nitrooxy)butyl ester)], has greater antiinflammatory properties compared with pravastatin in normal and atherosclerotic apolipoprotein E receptor knockout (ApoE / ) mice. C57BL/6 and ApoE / mice were administered pravastatin (40 mg/kg), NCX 6550 (48.5 mg/kg), or vehicle orally for 5 days. Ex vivo studies assessed splenocyte adhesion to arterial segments and splenocyte reactive oxygen species (ROS) generation. NCX 6550 significantly reduced splenocyte adhesion to artery segments in both C57BL/6 (8.8 1.9% versus 16.6 6.7% adhesion; P 0.05) and ApoE / mice (9.3 2.9% versus 23.4 4.6% adhesion; P 0.05) concomitant with an inhibition of endothelial intercellular adhesion molecule-1 expression. NCX 6550 also significantly reduced phorbol 12-myristate 13-acetate-induced ROS production that was enhanced in isolated ApoE / splenocytes. Conversely, pravastatin had no significant effects on adhesion in normal or ApoE / mice but reduced the enhanced ROS production from ApoE / splenocytes. In separate groups of ApoE / mice, NCX 6550 significantly enhanced endotheliumdependent relaxation to carbachol in aortic segments precontracted with phenylephrine ( logEC50, 6.37 0.37) compared with both vehicle-treated ( logEC50, 5.81 0.15; P 0.001) and pravastatin-treated ( logEC50, 5.57 0.45; P 0.05) mice. NCX 6550 also significantly reduced plasma monocyte chemoattractant protein-1 levels (648.8 pg/ml) compared with both vehicle (1191.1 pg/ml; P 0.001) and pravastatin (847 71.0 pg/ml; P 0.05) treatment. These data show that NCX 6550 exerts superior anti-inflammatory actions compared with pravastatin, possibly through NO-related mechanisms. Atherosclerosis is now generally acknowledged to be an inflammatory disease where inflammation develops at certain predilection sites in response to endothelial injury. Attachment of leukocytes to atherosclerotic blood vessels (Ramos et al., 1999), coupled with up-regulation of the vascular adhesion molecules vascular cell adhesion molecule-1 and ICAM-1 (Nakashima et al., 1998), is a fundamental step in the development of atherosclerosis. Recently, adhesion of cultured murine monocytoid WEH1 78/24 cells to artery segments of ApoE / mice has been demonstrated (Li et al., 2005), supporting the notion that a hyperinflammatory state exists in developing atherosclerosis. This has been attributed to increased levels of a number of cytokines, which act to elevate adhesion molecule expression. Thrombin, in addition to playing a role in the coagulation cascade, is also involved Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.109298. ABBREVIATIONS: ICAM, intercellular adhesion molecule; ApoE / , apolipoprotein E receptor knockout; ROS, reactive oxygen species; NO, nitric oxide; NCX 6550, (1S-[1 ( S*, S*),2 ,6 ,8 -(R*),8a ]]-1,2,6,7,8,8a-hexahydro, ,6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphtalene-heptanoic acid 4-(nitrooxy)butyl ester); MCP, monocyte chemoattractant protein; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; ANOVA, analysis of variance; CL, chemiluminescence. 0022-3565/07/3201-419–426$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 320, No. 1 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 109298/3158998 JPET 320:419–426, 2007 Printed in U.S.A. 419 at A PE T Jornals on M ay 9, 2017 jpet.asjournals.org D ow nladed from in the regulation of inflammation and has been shown to induce monocyte adhesion to endothelial cells (human umbilical vein endothelial cells) through induction of ICAM-1 and increased expression of vascular cell adhesion molecule-1, P-selectin, and E-selectin (Kaplanski et al., 1998) due to an action at protease activated receptor-1. Consequently, thrombin has been implicated in atherogenesis (Coughlin, 2005). However, there has been no direct demonstration of a hyperinflammatory response to thrombin in atherosclerotic blood vessels. The first aim of the current study was therefore to compare adhesion of splenocyte preparations, commonly used as a source of immune cells, with thrombin-challenged arterial tissue from normal (C57BL/6) and atherosclerotic (ApoE / ) mice. The value of introducing the lipid-lowering statins into the management of patients with coronary artery disease has been illustrated through the significant benefit of these drugs in primary (Shepherd et al., 1995) and secondary prevention of symptomatic coronary heart disease [Scandinavian Simvastatin Survival Study (4S), 1994]. Detailed analyses of data from these trials, however, suggest that lipid-lowering by statins does not solely account for the significant clinical outcomes, and that statins possess additional (pleiotropic) effects beyond their lipid-lowering capacity (Downs et al., 1998). Among the reported pleiotropic effects of statins, demonstrations of their anti-inflammatory and antiadhesive effects are abundant (Stalker et al., 2001; Fischetti et al., 2004). Furthermore, fluvastatin (Bandoh et al., 2003), and other statins have been shown to inhibit formation of reactive oxygen species (ROS) by inflammatory cells. However, although these effects are readily demonstrated following acute challenge with supratherapeutic concentrations in vitro, the effects in vivo often require prolonged administration before they are observed. Although nitric oxide (NO) was originally identified as a key mediator in the maintenance of vascular tone, it also exerts anti-inflammatory effects. NO, either generated endogenously or released from NO-donating molecules, inhibits leukocyte adhesion through a reduction in endothelial expression of adhesion molecules such as Pselectin (Davenpeck et al., 1994) and ICAM-1 (Berendji-Grun et al., 2001). Recent studies have reported superior antiinflammatory properties of novel NO-releasing statins (nitrostatins) over the respective native statins in RAW 264.7 murine macrophage cells (Ongini et al., 2004; Rossiello et al., 2005). The NO-donating moiety of nitrostatins is similar to other nitro compounds such as nitro aspirin, which yields NO through metabolic hydrolysis, resulting in relatively longlasting plasma levels of NO (Muscara et al., 2001). Furthermore, studies conducted with the NO-releasing derivative of pravastatin, NCX 6550, showed that the compound given to hypercholesterolemic CD1 mice is equally effective as equivalent doses of the native statin at lowering cholesterol (S. Momi, G. Guglielmini, A. Monopoli, E. Ongini, and P. Gresele, personal communication). Thus, NO released by these molecules may provide a more rapid anti-inflammatory action than can be achieved with a native statin, while still affording a reduction in cholesterol levels. Thus, the aim of the present study was to compare the effects of short-term (5 days) in vivo administration of NCX 6550 and native pravastatin on ex vivo splenocyte adhesion to arterial segments, splenocyte ROS production, and endothelial ICAM-1 expression in tissues from normal (C57BL/6) and atherosclerotic (ApoE / ) mice. In addition, we determined the effects of these interventions on endothelium-dependent vasorelaxant function and plasma MCP-1 levels in ApoE / mice. Although many studies that investigated leukocyte-endothelial adhesion have used in vitro cell models, such as myeloid cell adhesion to human umbilical vein endothelial cell monolayers (McGettrick et al., 2006), we chose to use a more physiological model involving in vivo dosing with statins followed by ex vivo measurement of isolated splenocyte adhesion to arterial tissue. Although this approach has the limitation of being a static model of vascular adhesion, it has the advantage of allowing atherosclerosis-susceptible arteries to be studied, in contrast to intravital microscopy, which is a dynamic model that allows the detection of adhesion in the presence of shear stress but which involves visualizing microvascular beds that are not generally susceptible to atherosclerotic plaque development, such as the mesenteric bed. Materials and Methods Materials. C57BL/6 (Harlan, UK Ltd) and ApoE / (Charles River, Margate, Kent, UK) mice were bred in house at the University of Strathclyde (Glasgow, UK). NCX 6550 was synthesized at Nicox (Bresso, Milan, Italy). All chemicals were purchased from SigmaAldrich (Dorset, Poole, UK), unless otherwise stated. Experimental Design. Forty C57BL/6 (18–24 g) and 52 ApoE / (26–35 g) age-matched mice of either sex were employed in the study, under a project license issued under the UK Animals (Scientific Procedures) Act 1986. The ApoE / mice were fed on an atherogenic diet (1% cholesterol, 5% lard) for 12 weeks postweaning, and control normocholesterolemic C57BL/6 mice were fed normal laboratory chow. The mice were then employed for the following studies. A group of C57BL/6 (n 10) and ApoE / (n 10) mice were used in preliminary experiments to determine any difference between the strains with respect to the adhesive response to thrombin, ICAM-1 expression, and splenocyte ROS generation. Three groups (n 10 per group) of mice of each strain were administered vehicle [dimethyl sulfoxide/castor oil/polyethylene glycol 400/water, 1:2:7:10 (v/v/v/v)], native pravastatin (40 mg/kg), or NCX 6550 (equimolar dose, 48.5 mg/kg) by oral gavage every day (at 10:00 AM) for 5 days. One hour after the final dose, the mice were euthanized by CO2 asphyxiation. Heparinized blood was obtained by cardiac puncture immediately following euthanasia for subsequent measurement of plasma cholesterol levels using standard assay kits (R-Biopharm AG, Darmstadt, Germany). Three groups (n 4 per group) of ApoE / were administered vehicle, pravastatin, or NCX 6550 (all as above) for 5 days. Immediately following euthanasia, blood was collected by cardiac puncture into heparinized tubes for measurement of plasma MCP-1 using an enzyme-linked immunosorbent assay kit (Insight Bioscience, Wembley, UK), and the aorta were harvested for determination of blood vessel function. Splenocyte Isolation and Radiolabeling. Splenocyte suspensions were prepared by disrupting the spleens over a 200m mesh (Cadisch Precision Meshes Limited, London, UK) into 3 ml of RPMI 1640 medium (Dutch modification; Invitrogen, Carlsbad, CA) containing 10% fetal calf serum (Invitrogen). The resulting cell suspensions were passed through a 200m mesh and centrifuged at 125g for 10 min. The supernatants were removed, and the pellets containing the cells were agitated with 4 ml of distilled H2O for 30 s (to lyse erythrocytes), followed by the addition of 4 ml of 1.8% NaCl to restore isotonicity. The splenocyte suspensions were filtered through a 200m mesh, centrifuged, and the resulting cell pellets were resuspended in 2 ml of RPMI. Cell density was determined using a hemocytometer, and the suspension was diluted as necessary to achieve a final density of 1 10 cells/l. The resuspended leukocytes (1 ml) were labeled for 1 h at 37°C in a humidified chamber with 185 420 Dever et al. at A PE T Jornals on M ay 9, 2017 jpet.asjournals.org D ow nladed from kBq of Cr (GE Healthcare, Little Chalfont, Buckinghamshire, UK); the cells were agitated every 15 min to minimize cell sedimentation. The cells were washed twice with RPMI 1640 and resuspended in RPMI 1640 to 1 10 cells/ml. Splenocyte Characterization. Splenocyte preparations were characterized in samples from three separate C57BL/6 and three ApoE / mice using flow cytometry. In brief, cell suspensions were incubated with leukocyte Fc receptor blocking buffer (anti-CD 16/32 hybridoma supernatant, 10% mouse serum, and 0.1% azide) for 5 min to prevent binding of antibody to cells via Fc regions. The cell suspensions were then incubated with a mixture of cell lineage specific antibodies for 40 min at 4°C. B lymphocytes were identified using fluorescein isothiocyanate-conjugated anti-CD45R/B220 (clone RA3–6B2; Pharmingen, Oxford, UK), CD4 T lymphocytes using peridinin chlorophyll protein-cyanin 5.5-conjugated anti-CD4 (clone GK1.5; Pharmingen), and myeloid cells using phycoerythrin-conjugated anti-CD11b (clone M1/70; Pharmingen). The cells were washed in fluorescence-activated cell sorting buffer [phosphate-buffered saline (PBS), 2% fetal calf serum, and 0.1% azide] prior to acquisition using a BD FACSCanto flow cytometer with FACSDiVa software (BD Biosciences, San Jose, CA). FlowJo software (Tree Star Inc., Ashland, OR) was used for three color analysis. To ascertain which cell types were adhering to the artery surface, cells were added to pinned-out segments of aorta for 30 min and adherent cells were harvested by addition of ice-cold PBS solution. The suspensions of adherent cells were incubated with the same three antibodies and run through the flow cytometer. Assessment of Splenocyte Adhesion to Aortic Segments. The method employed was a modification of a method using rabbit tissue developed in our laboratory to determine the effects of vascular injury on inflammatory cell adhesion (Kennedy et al., 2000). Homologous aortic lengths were removed and cut into two segments (aortic arch and thoracic aorta), which were pinned out luminal-side up onto Sylgard blocks (Dow Corning, Midland, MI). The artery segments were incubated with 10 l of 10 U/ml thrombin for 10 min in a humidified chamber (37°C), washed, and then incubated with a 5l aliquot of the labeled leukocytes for a further 30 min. The segments were then washed with RPMI 1640, transferred into microtubes, and assayed for Cr in a gamma counter (Cobra Autogamma; Canberra Industries, Meriden, CT). Aliquots (5 l) of labeled and unlabeled cells were also counted to allow calculation of leukocyte adhesion using the following equation: