Nitric Oxide-Donating Atorvastatin Attenuates Neutrophil Recruitment ...

Cardiovasc Drugs Ther (2013) 27:211–219 DOI 10.1007/s10557-013-6445-1

ORIGINAL ARTICLE

Nitric Oxide-Donating Atorvastatin Attenuates Neutrophil Recruitment During Vascular Inflammation Independent of Changes in Plasma Cholesterol Roberta Baetta & Agnese Granata & Daniela Miglietta & Francesca Oliva & Lorenzo Arnaboldi & Alessandra Bonomo & Nicola Ferri & Ennio Ongini & Stefano Bellosta & Alberto Corsini

Published online: 12 February 2013 # Springer Science+Business Media New York 2013

Abstract Purpose Polymorphonuclear neutrophils, the first leukocytes to infiltrate the inflamed tissue, can make important contributions to vascular inflammatory processes driving the development of atherosclerosis. We herein investigated the effects of atorvastatin and NCX 6560 (a nitric oxide (NO)donating atorvastatin derivative that has completed a successful phase 1b study) on neutrophilic inflammation in carotid arteries of normocholesterolemic rabbits subjected to perivascular collar placement. Methods Atorvastatin or NCX 6560 were administered orally (5 mg/kg/day or equimolar dose) to New Zealand White

Electronic supplementary material The online version of this article (doi:10.1007/s10557-013-6445-1) contains supplementary material, which is available to authorized users. R. Baetta : A. Granata : L. Arnaboldi : A. Bonomo : N. Ferri : S. Bellosta : A. Corsini Dipartimento di Scienze Farmacologiche e Biomolecolari, University of Milan, Via Balzaretti 9, 20133 Milan, Italy D. Miglietta : F. Oliva : E. Ongini Nicox Research Institute, Via Ludovico Ariosto 21, 20091 Bresso, Milan, Italy R. Baetta (*) Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Via Balzaretti 9, 20133 Milan, Italy e-mail: [email protected]

rabbits for 6 days, followed by collar implantation 1 h after the last dose. Twenty-four hours later carotids were harvested for neutrophil quantification by immunostaining. Results Treatment with NCX 6560 was associated with a lower neutrophil infiltration (−39.5 %), while atorvastatin did not affect neutrophil content. The result was independent of effects on plasma cholesterol or differences in atorvastatin bioavailability, which suggests an important role of NO-related mechanisms in mediating this effect. Consistent with these in vivo findings, in vitro studies showed that NCX 6560, as compared to atorvastatin, had greater inhibitory activity on processes involved in neutrophil recruitment, such as migration in response to IL-8 and IL-8 release by endothelial cells and by neutrophils themselves. Pretreatment with NCX 6560, but not with atorvastatin, reduced the ability of neutrophil supernatants to promote monocyte chemotaxis, a well-known pro-inflammatory activity of neutrophils. Conclusion Experimental data suggest a potential role of NO-releasing statins in the control of the vascular inflammatory process mediated by polymorphonuclear neutrophils. Keywords Atherosclerosis . Inflammation . Neutrophil (polymorphonuclear leukocyte [PMN]) . Leukocytes . Nitric oxide . Statins . NO-statins . NCX-6560

Introduction Present Address: R. Baetta Laboratorio di Biologia Cellulare e Biochimica dell’Aterotrombosi, Centro Cardiologico Monzino, Via C. Parea 4, 20138 Milan, Italy

Atherosclerosis is the main underlying cause of cardiovascular disease, which remains the number one killer and a major cause of illness and disability in populations worldwide [1, 2].

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Currently, low-density lipoprotein (LDL)-cholesterol lowering by statin therapy is the most successful approach in reducing the atherosclerosis-associated cardiovascular diseases [3]. Although elevated LDL-cholesterol plasma levels remain the most important risk factor for atherosclerosis, the growing understanding of the importance of immune and inflammatory mechanisms in all stages of atherosclerotic disease has uncovered an intriguing diversity of targetable mechanisms that can be exploited to complement lipidlowering therapies [4–6]. In this regard, besides the wellknown role of monocyte and T cells in the pathogenesis of atherosclerosis, evidence revealing a previously unappreciated role of polymorphonuclear neutrophils is being accumulating, and neutrophilic inflammation is increasingly regarded as a possible future target for atheroprotection [4, 7–10]. Until now, however, only few experimental investigations, using recombinant HDL and HDL apolipoproteins or niacin, have addressed the effects of pharmacological interventions on neutrophil-mediated arterial inflammation [11–13]. A potential target for neutrophil-directed therapy is the nitric oxide (NO)–cGMP signal transduction pathway, which has a critical role in the pathophysiology of the vasculature and is involved in the regulation of a variety of pathophysiological processes, including leukocyte-vessel wall interactions [14–16]. A dysregulation of this pathway has been implicated in the pathogenesis of cardiovascular diseases, and a restoration of NO function has consistently been found to be associated with vascular protective effects in experimental models of atherosclerosis and restenosis [14]. To have a therapeutic benefit from NO properties, an approach has been designed coupling well-known existing drugs with moieties able to slowly release NO following enzymatic metabolism, leading to hybrid nitrates, in which activities of both the native drug and NO are present [17]. Application of this approach to upgrade the pharmacological profile of statins has led to the synthesis of NO-donating statins, new molecular entities that, compared to their parent compounds, have shown enhanced anti-proliferative, antiatherothrombotic, and vascular reparative properties in several in vitro and animal models [18–25]. In addition, NCX 6560, a derivative of atorvastatin and the most studied among NO-donating statins, has successfully completed a proof-of-principle clinical phase 1b study, showing favourable pharmacodynamic and pharmacokinetic profiles, as well as good safety and tolerability [26]. Here we investigated the effect of a short-term treatment with NCX 6560, as compared to atorvastatin, in carotid arteries of normocholesterolemic rabbits subjected to perivascular insertion of a silicone collar, a valuable experimental model of acute arterial inflammation as related to atherogenesis [11, 27].

Cardiovasc Drugs Ther (2013) 27:211–219

Methods Compounds NO-donating atorvastatin derivative NCX 6560 ((3R,5R)4-(nitrooxy)butyl 7-(2-(4-fluorophenyl)-5-isopropyl-3-phenyl-4-(phenylcarbamoyl)-1H-pyrrol-1-yl)-3,5-dihydroxyheptanoate; Fig. 1) was synthesized atNicOx Research Institute (Milan, Italy) starting from atorvastatin according to a synthetic procedure used for other NO-releasing agents [18]. For in vitro experiments, compounds were dissolved at 10−2 M in DMSO. For in vivo study, dosing suspensions were prepared fresh daily by dissolving the compounds in DMSO and diluting the solution in PEG400/H2O (70:30 v:v). The suspensions were administered by oral gavage in a volume of 2.5 ml/kg body weight. Control group animals received an equal volume of vehicle. The concentration of DMSO in the administered suspensions was always below 1 %. The doses of NCX 6560 and atorvastatin used were equivalent and calculated on the basis of their different molecular weights (MW NCX 6560/MW atorvastatin: ratio 1.17). In Vivo Study in the Perivascular Carotid Collar Model of Acute Vascular Inflammation Animals, Surgical Procedures and Experimental Protocol The study conformed the principles stated in the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and the research protocol was approved by the responsible public authorities as required by the Italian Law in accordance with EU regulations. A total of 63 New Zealand White rabbits (male, 2.75–3 kg body weight; Charles River Laboratories) were utilized for the investigations. In order to define the design for the main study, a preliminary dose-finding experiment was performed in 25 male New Zealand White rabbits (male, 2.75–3 kg body weight; Charles River Laboratories). In this study, rabbits were randomly divided into 5 groups (N=5/group), receiving atorvastatin at doses of 1 or 5 mg/kg/day, equimolar doses

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Fig. 1 Chemical structure of atorvastatin and NCX 6560 ((3R,5R)-4(nitrooxy)butyl 7-(2-(4-fluorophenyl)-5-isopropyl-3-phenyl-4-(phenylcarbamoyl)-1H-pyrrol-1-yl)-3,5-dihydroxyheptanoate)


of NCX 6560 (1.17 or 5.85 mg/kg/day) or vehicle, given by oral gavage for 6 consecutive days. Quantification of carotid artery neutrophil accumulation induced by perivascular carotid collar, performed as described below, identified the higher dose as more promising for subsequent investigation (percent neutrophil content vs. control: atorvastatin 1 mg/kg/day, 112±39; atorvastatin 5 mg/kg/day, 89±24; NCX 6560 1 mg/kg/day, 110±21; NCX 6560 5 mg/kg/day, 41±13; one-way ANOVA p value>0.05). In the main study, 48 rabbits were randomized to treatment with 5 mg/kg/day atorvastatin, the equimolar dose of NCX 6560 or vehicle (N=16/group) for the same duration. Animals were provided ad libitum with standard rabbit chow (Laboratorio Dottori Piccioni, Milan, Italy) and water. The weight of the animals was controlled daily to adjust the dose of drug; behaviour and food intake were monitored regularly. Pericarotid collar placement was performed 1 h after the last dose of treatment. Rabbits were anaesthetized by intramuscular injection of xylazine (5 mg/kg, Rompun Bayer, Milan, Italy) and ketamine (35 mg/kg, Ketavet, Farmaceutici Gellini Spa, Aprilia, Italy). A midline neck incision was made to surgically expose both carotid arteries, and a nonocclusive silastic collar was positioned around the right common carotid artery, while the contralateral carotid artery was sham-operated as previously described [28]. At this time, blood samples were drawn from the ear central artery for plasma cholesterol measurement by standard colorimetric assay (cholesterol oxidase method) [28] and for assessment of plasma drug levels (main study only, see below). Animals were sacrificed 24 h later, coincident with the peak of neutrophil infiltration [27]. Euthanasia was performed under general anaesthesia by intravenous administration of Tanax (Hoechst Roussel Vet, Milan, Italy); carotids were harvested, rinsed in normal saline, and immersion-fixed overnight in 3 % formalin. Samples were then embedded in paraffin and cut transversally into 5-μm sections. Immunohistochemical Analysis of Arterial Wall Infiltration by Polymorphonuclear Neutrophils Tissue sections were rehydrated, deparaffinized, and blocked for endogenous peroxidase activity and nonspecific staining. Immunohistochemical staining was performed with a monoclonal antibody specific for rabbit neutrophils (MCA805G, AbDSerotec; working dilution 1:100) according to the ABC method (Vector) by use of an avidin-biotin-peroxidase kit (Vectastain ABC Elite), developed with 3,3′-diaminobenzidine (Sigma), and counterstained with hematoxylin. For negative control the primary antibody was omitted and sections were incubated with normal horse serum. MCA805G-positive regions within the artery were measured by computer-assisted

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colour image analysis (OPTIMAS 6.2, Media Cybernetics) by 2 independent observers that were blinded to treatment allocation, and expressed as percentage of the area comprised between the internal elastic lamina and the external elastic lamina. A threshold for immunostaining was defined by sampling. A minimum of 8 sections (at least 250 μm apart) were analysed per each rabbits. Plasma Drug Levels Plasma concentrations of atorvastatin and its active metabolites (2- and 4-OH atorvastatin) were quantified by a sensitive and specific LC/MS/MS method. Detection was performed using a Waters Quattro Micro API mass spectrometer operated in ESI + mode. The mobile phase was A, 0.1 % formic acid in water; and B, 0.1 % formic acid in methanol, with a flow rate of 0.500 ml/min. The method was linear over the concentration range of 2–200 nM for atorvastatin, 6–20,000 nM for 2-OH atorvastatin, and 2–600 nM for 4-OH atorvastatin. The analytical data were processed by Masslynx software. For each analysis, data below the limit of quantification (LOQ) were replaced with ½ LOQ. In Vitro Studies Isolation of Human Neutrophils and Monocytes Human neutrophils and mononuclear cells were isolated from buffy coats obtained from the blood of healthy donors by standard techniques of dextran sedimentation and centrifugation on a discontinuous density gradient (formed by sterile Histopaque-1077 and Histopaque-1119, SigmaAldrich; 700 xg, 30 min, room temperature). Neutrophils collected at the interface between the Histopaque-1119 and the Histopaque-1077 were purified by hypotonic lysis of erythrocytes, washed twice in PBS, resuspended at a density of 107 cells/ml in serum-free DMEM containing 0.2 % albumin, and immediately used for experiments. Mononuclear cells were collected at the interface between the Histopaque-1077 and the plasma, washed twice in PBS and resuspended in serum-free DMEM containing 0.2 % albumin. Monocytes were then separated from other mononuclear cells by plastic adherence. To this end, cells were plated at 37 °C at a density of 4×106 cells in 35-mm tissue culture dishes and allowed to adhere for 1 h. Non-adhered cells were then removed and dishes were washed twice with PBS, replenished with DMEM containing 10 % FCS, and used within 24 h. Human Umbilical Vein Endothelial Cells (HUVEC) Culture HUVEC were isolated according to established procedures [29], cultured under standard conditions in medium M-199

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containing 20 % FCS, heparin (15 U/ml) and ECGF (endothelial cell growth factor, 20 μg/ml) (Roche, Italy) and used within the 4th passage as subconfluent cultures. Chemotaxis Assays The effect of atorvastatin and NCX 6560 on neutrophil chemotaxis was assayed by using a 48-well microchemotaxis Boyden chamber. Cells were pre-incubated for 90 min in serum-free DMEM containing 0.2 % albumin in the absence or the presence of increasing concentrations of the experimental compounds. Triplicate wells of the base of the chamber were filled with DMEM containing 0.2 % albumin ± human recombinant interleukin 8 (IL-8; Sigma) at a concentration of 10 ng/ml. A 5-μmpore-diameter polycarbonate membrane (Nucleopore) coated with type I collagen was placed on the top of the chamber. Aliquots of cell suspensions containing 105 cells, with or without the experimental compounds at the indicated concentrations, were added to the upper wells. The chamber was incubated for 1 h at 37 °C to allow cell migration. The membrane was then removed, adherent cells on the top were eliminated, and the membrane was stained with Diff-Quik reagents. Four fields/well were photographed under high magnification (x400) using a Zeiss microscope equipped with a Nikon Coolpix990 digital camera. The numbers of transmigrated cells/field were then counted by Optimas 6.2 image software. To assess the effect of atorvastatin and NCX 6560 on the monocyte-chemotactic activity of neutrophil secretion products, neutrophils were treated with experimental compounds (10−6 M) for 90 min with addition of fMLP (formyl-MetLeu-Phe) (Sigma; 10−6 M) or vehicle (DMSO) during the last 30 min of incubation. Cells were next centrifuged at 1200 rpm, resuspended in fresh serum-free medium and incubated for additional 30 min, after which cell-free supernatants were collected for the evaluation of monocytechemotactic activity. Microchemotaxis assays were performed as described for neutrophils, except that triplicate wells of the base of the chamber were filled with neutrophil supernatants (or appropriate controls) and untreated monocytes were added to the upper wells. The chamber was incubated for 3 h at 37 °C and processed as described above. IL-8 Release by Neutrophils and Endothelial Cells Neutrophils were resuspended at a density of 107 cells/ml in serum-free DMEM containing 0.2 % albumin with or without of atorvastatin or NCX 6560 (10−6 M) for 90 min and stimulated with fMLP 10−6 M during the last 30 min of incubation. HUVECs were incubated for 24 h in 1 % FCS medium in the absence or presence of atorvastatin, NCX


6560 (10 −6 M) or vehicle, and stimulated with TNFα (10 ng/ml) during the last 6 h of incubation. IL-8 release in cell free supernatants was assessed by a specific commercially available ELISA kit (Immunotools, Friesoythe, Germany) according to manufacturer’s instructions. Cytotoxicity Assays To assess the effect of atorvastatin and NCX 6560 on neutrophil and endothelial cell viability, cellfree supernatants from neutrophils and HUVEC cultures prepared and treated as described above were assayed for release of lactate dehydrogenase (LDH). LDH was detected according to the protocol provided with the LDH Cytotoxicity Detection KitPLUS (Roche Applied Science). Statistical Analysis The data were analysed using GraphPad Prism software and are expressed as mean ± SEM. Statistical comparisons were performed using 1-way ANOVA followed by Dunnet’s Multiple Comparison post-hoc test. P values