docROSUVASTATIN FOR REDUCTION OF MYOCARDIAL DAMAGE
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ROSUVASTATIN FOR REDUCTION OF MYOCARDIAL DAMAGE
Briguori C. et al.Online Supplemental Material, page 1 ROSUVASTATIN FOR REDUCTION OF MYOCARDIAL DAMAGE DURING CORONARY ANGIOPLASTY - THE REMEDY TRIAL Carlo Briguori, M.D, PhD1, Rosalinda Madonna, MD, PhD2, Marco Zimarino, MD, PhD2, Paolo Calabrò, MD, PhD3, Cristina Quintavalle, PhD4, Maria Salomone, MD, DPharm5, Gerolama Condorelli, MD, PhD4, Raffaele De Caterina, MD, PhD2 From: 1 Clinica Mediterranea, Naples; 2 Instiute of Cardiology and Center of Excellence on Aging, G. d’Annunzio” University, Chieti; 3 Department of Cardiothoracic Sciences, Second University of Naples, Monaldi Hospital, Naples; 4 Department of Molecular Medicine and Medical Biotechnologies "Federico II” University of Naples; 5 Dimensione Ricerca, Milan, and ES Health Science Foundation, Lugo, Italy Short title: rosuvastin and periprocedural myocardial infarction ONLINE SUPPLEMENTAL MATERIAL Correspondence: Raffaele De Caterina, MD, PhD Institute of Cardiology, “G. d’Annunzio” University – Chieti C/o Ospedale SS. Annunziata Via dei Vestini 66013 Chieti, Italy. Tel. +39-0871-41512 FAX: +39-0871-402817 E-mail: [email protected] Briguori C. et al.Online Supplemental Material, page 2 LEGEND TO ONLINE SUPPLEMENTAL FIGURES OS Figure 1. CFU-EC and late ECFCs in patients randomized to standard or intensive lipid-lowering treatments. Representative images of colony forming units-endothelial cells or CFU-Hill (A) and late ECFCs (B) of patients at time of treatment reload. OS Figure 2. Panel A, B and C: Sequential gating strategy used in sample 3 to select live CD34+ cells. Panel D: Q1, Q2, Q3 and Q4 quadrants imposition to establish a cut-off for PE-positive cells OS Figure 3. Panels A, B and C: Sequential gating strategy used in sample 1 and 2 to select live CD34+ cells. Panel D: Q1, Q2, Q3 and Q4 quadrants imposition in sample 2 to establish a cut-off for APC-positive cells. Panel E: in sample 1, no quadrants modification respect to sample 2 to obtain the percentage of CD34+CD309+ (Q1+Q2 events) and CD34+CD13++CD309+ cells (Q2 events). Briguori C. et al.Online Supplemental Material, page 3 Appendix 1 REMEDY Study Structure Steering Committee (Clinical Study): Marco Tubaro, Maddalena Lettino, Paolo Golino, Alberto Corsini, Alberico Catapano, Pericle Di Napoli, Rosalinda Madonna, Marco Zimarino Participating Centers Centre Prof. Raffaele De Caterina Ospedale Clinicizzato SS. Annunziata U.O. di Cardiologia Universitaria Via dei Vestini - 66100 Chieti Dr. Maddalena Lettino 2 Dipartimento Cardiotoracovascolare U.O. Complessa di Cardiologia Fondazione I.R.C.C.S. Policlinico S. Matteo Via Golgi 2 - 27100 Pavia 3 Prof. Paolo Golino U.O.C. Cardiologia A.O. S. Anna e S. Sebastiano – II Università di Napoli Via Tescione - 81100 Caserta 4 Dr. Paolo Calabrò U.O. Complessa Cardiologia Suni Azienda Ospedaliera Monaldi Via Leonardo Bianchi 1 - 80131 Napoli 5 Dr. Enrico Magagnini U.O. Emodinamica Azienda ASL 6 – P. Ospedaliero Livorno Viale Alfieri, 36- 57100 Livorno 6 Prof. Massimo Santini U.O. Complessa di Cardiologia Azienda Complesso Ospedaliero S. Filippo Neri Via Martinetti, 20 - 00135 Roma 7 Prof. Angelo Branzi U.O. Cardiologia Azienda Ospedaliero-Universitaria di Bologna – Policlinico S. Orsola-Malpighi Via Massarenti - 40138 Bologna 1 Ethic Commitee COMITATO DI ETICA PER LA RICERCA BIOMEDICA DELL'UNIVERSITA' DEGLI STUDI GABRIELE D'ANNUNZIO E DELLA ASL DI CHIETI Via dei Vestini, 31 - 66013 CHIETI COMITATO DI BIOETICA DELLA FONDAZIONE I.R.C.C.S. POLICLINICO S. MATTEO Viale Golgi, 19 - 27100 PAVIA COMITATO ETICO DELL’AZIENDA OSPEDALIERA S. ANNA E S. SEBASTIANO DI CASERTA Via Tescione trav. Palasciano 81100 CASERTA COMITATO ETICO DELL´AZIENDA OSPEDALIERA VINCENZO MONALDI DI NAPOLI Via Leonardo Bianchi – 80131 NAPOLI SOTTOCOMITATO ETICO PER LA SPERIMENTAZIONE CLINICA DEI FARMACI DELLA AUSL 6 DI LIVORNO Via di Monterotondo, 49 - 57128 LIVORNO COMITATO ETICO PER LA SPERIMENTAZIONE CLINICA DELL'AZIENDA COMPLESSO OSPEDALIERO S. FILIPPO NERI DI ROMA Piazza S. Maria della Pietà, 5 - 00135 ROMA COMITATO ETICO INDIPENDENTE DELL´AZIENDA OSPEDALIERO-UNIVERSITARIA POLICLINICO S. ORSOLA-MALPIGHI DI BOLOGNA Via Albertoni, 15 - 40138 BOLOGNA Briguori C. et al.Online Supplemental Material, page 4 8 9 10 11 12 13 14 15 16 17 Dr. Giancarlo Piovaccari U.O. Cardiologia Ospedale Infermi Via Settembrini, 2 47900 Rimini Dr. Silva Severi U.O. Cardiologia UTIC Ospedale della Misericordia Via Senese,161 - 58100 Grosseto Prof. Corrado Tamburino U.O di Cardiologia Ospedale Ferrarotto Via S. Citelli, 1 95124- Catania Dr. Luciano Moretti U.O. Cardiologia Ospedale Generale Provinciale C.G. Mazzoni Via degli Iris, 6 - 63100 Ascoli Piceno Prof. Gian Franco Gensini SOD Cardiologia Generale Azienda Ospedaliero-Universitaria Careggi Viale Morgagni 85 - 50134 Firenze Dr. Giuseppe Mariani (De Servi) U.O. Cardiologia Ospedale Civile G. Fornaroli Via Al Donatore di Sangue 50 - 20013 Magenta Prof. Germano Di Sciascio U.O. Cardiologia Policlinico Universitario Campus Bio-Medico di Roma Via Alvaro del Portillo, 200 - 00128 Roma Dr. Maria Grazia Bongiorni U.O. Malattie Cardiovascolari 2 Aziena Ospedaliero-Universitaria Pisana Stabilimento Ospedaliero di santa Chiara Via Roma, 67 - 56126 Pisa Dr. Franco Prosperi U.O. Cardiologia 2 Emodinamica ASL 106 Teramo - P.O. G. Mazzini Piazza Italia - 64100 Teramo Prof. Filippo Crea UTIC e Subintensiva Policlinico Agostino Gemelli Largo Gemelli, 8 - 00168 Roma COMITATO ETICO DI AREA VASTA ROMAGNA DI CESENA E ISTITUTO SCIENTIFICO ROMAGNOLO PER LO STUDIO E LA CURA DEI TUMORI DI MELDOLA (FO) Piazza L. Sciascia n. 111/2 - 47023 CESENA COMITATO ETICO PER LA SPERIMENTAZIONE DEI FARMACI DELLA AUSL 9 DI GROSSETO Via Genova 6 d - 58100 GROSSETO COMITATO ETICO DELL'AZIENDA OSPEDALIERA-UNIVERSITARIA VITTORIO EMANUELE, FERRAROTTO, SANTO BAMBINO DI CATANIA Via Gesualdo Clementi, 36 - 95124 CATANIA COMITATO ETICO DELLA ASUR ZONA TERRITORIALE 13 DI ASCOLI PICENO Via Degli Iris Snc 63100 ASCOLI PICENO COMITATO ETICO PER LA SPERIMENTAZIONE CLINICA DEI MEDICINALI DELL´AZIENDA OSPEDALIERO-UNIVERSITARIA CAREGGI DI FIRENZE Largo Palagi 1 c/o C.T.O. - 50139 FIRENZE COMITATO ETICO DELL´AZIENDA OSPEDALIERA OSPEDALE CIVILE DI LEGNANO Via Candiani, 2 - 20025 LEGNANO (MI) COMITATO ETICO DELL´UNIVERSITA´ CAMPUS BIO-MEDICO DI ROMA Via Alvaro del Portilllo, 21 - 00128 ROMA COMITATO PER LA SPERIMENTAZIONE CLINICA DEI MEDICINALI DELL´AZIENDA OSPEDALIERO UNIVERSITARIA PISANA DI PISA Via Roma, 67 - 56126 PISA COMITATO ETICO DELLA ASL 106 DI TERAMO Circonvallazione Ragusa, 1 - 64100 TERAMO COMITATO ETICO DELL'UNIVERSITÀ' CATTOLICA DEL SACRO CUORE - POLICLINICO UNIVERSITARIO AGOSTINO GEMELLI Largo A. Gemelli, 8 - 00168 ROMA Briguori C. et al.Online Supplemental Material, page 5 18 19 20 21 22 23 Prof. Gianfranco Parati U.O. Cardiologia IRCCS Istituto Auxologico-Ospedale S.Luca Via Spagnolettio 3 - 20149 Milano Prof. Sabino Iliceto U.O.C. Cardiologia Azienda Ospedaliera di Padova Via Giustiniani, 2 - 35128 Padova Dr. Sergio Berti U.O. Cardiologia Adulto Fondazione <toscana Gabriele Monasterio Ospedale Pediatrico Apuano - O.P.A. Pasquinucci Via Aurelia Sud- 54100 Massa Prof. Federico Lombardi Struttura Complessa di Cardiologia e UTIC Azienda Ospedaliera – Ospedale San Paolo Via A. di Rudinì, 8 - 20142 Milano Dr. Flavio Airoldi Servizio di Cardiologia Emodinamica IRCCS Sesto San Giovanni Via Milanese, 300 – Sesto San Giovanni Dr. Carlo Briguori Unità Operativa di Cardiologia Interventistica Clinica Mediterranea Via Orazio, 2, 80122 Napoli COMITATO ETICO DELL´IRCCS ISTITUTO AUXOLOGICO ITALIANO DI MILANO Via Ariosto 13 – 20145 MILANO COMITATO ETICO PER LA SPERIMENTAZIONE DELL´AZIENDA OSPEDALIERA DI PADOVA Via Giustiniani, 1 – 35128 PADOVA COMITATO ETICO PER LA SPERIMENTAZIONE CLINICA DEI MEDICINALI DELLA ASL 1 DI MASSA E CARRARA Viale Risorgimento, 18 ( c/o Medicina Legale) 54100 MASSA COMITATO ETICO DELL'AZIENDA OSPEDALIERA S. PAOLO DI MILANO Via A. di Rudinì, 8 - 20142 MILANO COMITATO ETICO DELL´IRCCS MULTIMEDICA DI SESTO S. GIOVANNI Via Milanese, 300 20099 SESTO SAN GIOVANNI COMITATO ETICO ASL-NAPOLI 1 Via Comunale del Principe, 13/a 80145 Napoli Briguori C. et al.Online Supplemental Material, page 6 REMEDY EPC substudies Steering Commitee Raffaele De Caterina, MD, PhD Carlo Briguori, MD, PhD Rosalinda Madonna, MD, PhD Gerolama Condorelli, MD, PhD Data Monitoring and Safety Commitee Maria Salomone, D. Pharm. Clinical Events Commitee Flavio Airoldi, MD, PhD, Departmenr of Cardiology, IRCCS Multimedica, Milan, Italy Davide D’Andrea, MD, Department of Cardiology, AORN Cardarelli, Naples, Italy Clinical Trials and Evaluation Unit Member Statistician; Giuseppe Signoriello, PhD, Department Medicine, Second University of Naples, Italy Mental Health and Preventive Partecipating Centers Center Investigators Study Coordinators “G. D’Annunzio” University - Chieti Raffaele De Caterina Raffaele De Caterina Rosalinda Madonna Rosalinda Madonna Carlo Briguori Francesca De Micco Clinica Mediterranea - Naples Michael Donahue "Federico II” University - Naples Luigi Del Vecchio Francesca D’Alessio "Federico II” University - Naples Gerolama Condorelli Cristina Quintavalle, Giuseppina Roscigno Briguori C. et al.Online Supplemental Material, page 7 Appendix 2- The REMEDY EPC substudies The REMEDY early-EPC substudy Collection and processing of peripheral blood EPC will be evaluated by both cytometric analysis and culture assays. To monitor changes in the EPC levels, 12 ml peripheral blood (PB, which represents the minimal amount of blood required to obtained a visible three-layer stratification for mononuclear cell isolation) will be collected in ethylenediaminetetraacetic acid (EDTA)-treated tubes (about 4 tubes/patient) at the time of randomization to placebo or lipid-lowering treatments (12 hours before PCI, sample R), and 1 hour before the diagnostic angiography and PCI at the time of treatment reload (placebo or lipid-lowering treatments, sample T0). The blood samples will be maintained in EDTA-treated tubes at +4 °C and used within 5 hours for mononuclear cell (MNC) isolation and assays of EPC functional activities. Peripheral blood mononuclear cells (PBMCs, which include monocytes and lymphocytes) will be isolated from 12 ml of PB by gradient centrifugation using Ficoll-Paque PLUS (Amersham). 12 ml of PB (contained in 4 EDTA-vacutainers) will be mixed with one part of phosphate buffer saline (PBS). An equal volume of Ficoll will be placed in 50 ml falcon tube and blood-PBS mixture will be carefully stratified onto Ficoll. The tube will be centrifuged at 400 g or 1600 rpm at 20 °C for 35 min. Three layers will be obtained at the end of centrifugation: a. upper layer, containing Plasma + PBS; b. middle layer, containing monocytes and lymphocytes; c. lower layer, containing Ficoll, neutrophils and erythrocytes. The middle layer will be withdrawn and placed in 50 ml Falcon tube. 25 ml cold PBS will be added and centrifuged at 1500 rpm for 5 min. The upper layer will be frozen at -80 °C in aliquots in 3 ml criovials for further biochemical determinations (adhesion molecules, growth factors, cytokines). The pellet will be Briguori C. et al.Online Supplemental Material, page 8 resuspended in 30 ml PBS/5% fetal calf serum (FCS), centrifuged at 1500 rpm for 5 min and washed again. The final pellet will be resuspended in Roswell Park Memorial Institute (RPMI) 1640 (Invitrogen), then used for colony forming units-endothelial cells (CFU-EC) and late outgrowth colonies isolations. Detailed procedure for flow cytometry analysis of whole peripheral blood PB will be drawn (BD Vacutainer Eclipse, 21 G x 1-1/4”, 0.8 x 32 mm), in EDTA (2 mg/ml) tubes (BD K2E EDTA, Becton Dickinson Biosciences - BD, San Jose, CA, USA). The first 3 ml of blood sampling will be discarded and not included in the flow cytometry analysis, in order to avoid the effects of the vascular damage caused by venipuncture on EPC numbers. Instead, every first ml of blood sampling will be used to determine sample leukocyte count, in order to assess double platform counting. Instrument Setting: Once set the instrument, flow cytometer performance, stability, and data reproducibility will be daily checked in real time by using the CS&T quality control Module (BD Biosciences) and further validated by the acquisition of Spherotech 8 peaks Rainbow Beads (Spherotec. Lake Forest, IL, USA), as well as Cytometer Setup and Tracking (CS&T) bright beads (1). Afterwards, the stabilization of the laser lamp will be obtained for a period of thirty minutes . Panel of reagents/antibodies: EPC will be identified by using an already established panel of reagents (48). In order to achieve a high level of standardization, liquid reagents for the panel and the respective control tube (Online Supplement (OS) Tables 1 and 2) will be lyophilized as already described (1). A single lyophilized-reagent tube lot will be used along the study. Sample staining: For each sample, 20 x 106 leukocytes will be processed within 4 hours from blood collection, as already described (2). Briefly, the sample volume containing 20 x 106 leukocytes will undergo an erythrocyte-lysis step, by adding 45 ml of Pharm Lyse Briguori C. et al.Online Supplemental Material, page 9 solution (BD Biosciences) for 15 minutes at room temperature under gentle agitation, as suggested by the manufacturer’s instructions. Samples will be then centrifuged (400 g, 10 min, room temperature) and washed by adding 2 ml of Stain Buffer, containing bovine serum albumin (BD Biosciences). Surface staining will be carried out by adding the pellet of each sample to the re-hydrated lyophilized cocktail of reagents. One µM Syto-16 (Thermo Fisher Scientific, Eisai, Medipost - USA) and anti-CD144 V450-conjugated will be added as liquid drop-in to each panel tube (OS Tables 1 and 2). Samples will be incubated in the dark for 30 minutes at 4 °C, washed (2 ml of Stain Buffer with BSA, BD Biosciences) and resuspended in 1.5 ml of FACSFlow (BD Biosciences). Data acquisition: a minimum of 2 x 106 and a maximum of 4 x 106 events/sample with lymphmonocyte morphology will be acquired by flow cytometry (FACSCanto, BD Biosciences) at “medium” flow rate mode. A threshold combination will be used on forward-scatter (FSC) and Fluorescein isothiocyanate (FITC) (Syto16) channels to get rid of very small and nonnucleated events. The specificity of anti-CD144, anti-CD146 and anti-vascular endothelial growth factor receptor (VEGFR)2 binding will be assessed by the use of isotype matched controls at the same concentration and from the same manufacturers of the respective specific antibody. Compensations will be calculated using CompBeads (BD Biosciences) and single stained fluorescent cells. Carryover between samples will be prevented by appropriate instrument cleaning at the end of each sample acquisition. Data analysis: Data will be centrally collected and analyzed by a single operator by using FACSuite v1.04 (BD Biosciences) software. To ensure correct identification of negative and positive populations, cells will be plotted using dot-plot bi-exponential display, as suggested. In order to assess non-specific fluorescence, both fluorescence minus one (FMO) and isotype controls in combination with all the remaining surface reagents present in the panels will be used (3, 4). Briguori C. et al.Online Supplemental Material, page 10 Counting formula CEC and hematopoietic stem cell (HSC) numbers will be calculated by a dual-platform counting method and absolute numbers will be obtained by using the already reported formula (2). CFU-EC isolation and quantification CFU-EC (colony forming units-endothelial cells or CFU-Hill (5)) will be cultured using the EndoCultTM Liquid Medium kit (Stem Cells Inc.), according to the manufacturer’s instructions. Briefly, 5 x106 PBMCs per well will be plated onto fibronectin-coated six-well plate in duplicate and incubated in EndoCult TM medium for two days at 37 °C, 5% CO2 with 95% humidity. After 48 h, non-adherent cells will be collected and transferred into individual 5 ml tubes. 1 x 106 cells of non-aherent cells will be re-plated in each well of fibronectincoated 24-well plates and cultured in EndoCult TM medium for additional 5 days. These cells organize in small clusters of central rounded cells with radiating spindle-shaped cells that disappear from the 10-14 day on (OS Figure 1 panel A). At day 5 after plating in fibronectin-coated 24-well plates clusters will be counted in 8 randomly selected high-power fields. Late outgrowth colonies isolation and quantification Late outgrowth colonies, named also endothelial colony forming cells (ECFCs), will be isolated as described elsewhere (6, 7). Briefly, 1.0 × 106 PBMCs will be plated onto fibronectin-coated six-well plates, cultured in endothelial basal medium (EBM)-2 (Cambrex Bio Science Walkersville, Inc., Walkersville, MD, USA) with endothelial growth medium (EGM)-2 supplement (Cambrex) and grown for 28 days. Culture medium will be changed first on day 4 and then every 2 days. With this culture system, attaching cells rapidly assume an endothelial-like shape, and starting from days 3–6 of culture, cells proliferate in clusters Briguori C. et al.Online Supplemental Material, page 11 or small colonies made up of a central core of rounded cells surrounded by radiating spindleshaped cells. Starting from days 10–14, cells also organize in large colonies of cobblestone cells, which are considered endothelial colonies and become confluent starting from days 21 (OS Figure 1 panel B). Clusters will be visualized under an inverted microscope every 3 days starting from days 7–14, and counted at days 14 and 21 after plating in 8 randomly selected high-power fields. Tube formation assay Tube formation assay will be performed on late ECFCs collected at days 21, with a minimal volume of Matrigel in 96-well plates (BD Biosciences), which allows the formation of both tubules and a vascular network. After being detached from the plates at day 21 by 0.25% trypsin, 1×104 late ECFCs will be placed on matrix solution with EGM-2 medium, and incubated at 37 °C for 16 h. Tube formation will be monitored by an inverted phase contrast microscopy (Leica, Wetzlar, Germany) and picture will be taken by an attached digital output Olympus camera. Several tube formation indices (tube areas, tube length, and tube number) will be quantified with National Institutes of Health (NIH) Image software. Statistical analyses 1) acute (<24 hours) changes of EPCs levels and functional activity (in vitro angiogenesis and clonogenic activity) before PCI according to pre-treatment with rosuvastatin or atorvastatin or placebo. Wilcoxon matched-pairs signed ranks test will be used to evaluate whether the various parameters significantly will change after treatments. Kruskal-Wallis test will be then used to assess whether the difference between post and pre treatments in each parameter is influenced by treatment group, gender, smoking, history of cardiovascular diseases (CVD), hypercholesterolemia, dyslipidemia, diabetes, hypertension, and coronary diagnosis. As a secondary analysis, Pearson's correlation coefficients will be used to explore Briguori C. et al.Online Supplemental Material, page 12 the relationships among each parameter at baseline, and among each parameter difference between pre and post treatments. Statistical significance will be defined as a two-sided pvalue <0.05 for all analyses, which will be carried out using Stata version 13.1 (Stata Corp., College Station, Texas, USA, 2013). Briguori C. et al.Online Supplemental Material, page 13 The REMEDY late-EPC substudy Collection and processing of peripheral blood EPC will be evaluated by both cytometric analysis and culture assays. To monitor changes in the EPC levels, 12 ml peripheral blood will be collected in EDTA-treated tubes (about 4 tubes/patient) at the time of randomization to placebo or statins (12 hours before PCI, sample R), at 24 (sample T24) and at 3-month (sample T3M) after PCI. PBMCs will be isolated from freshly collected whole blood using Ficoll-Paque PLUS (Amersham). 12 ml of PB (contained in 4 EDTA-vacutainers) will be pooled and mixed with one part of PBS. An equal volume of Ficoll (12 ml) will be placed in 50 ml Falcon tube and blood-PBS mixture will be carefully stratified onto Ficoll. The tube will be centrifuged at 400 g or 1600 rpm at 20 °C for 35 min. Three layers will be obtained at the end of centrifugation: a. upper layer containing Plasma + PBS; b. middle layer containing monocytes and lymphocytes; c. lower layer containing Ficoll, neutrophils and erythrocytes. The upper layer will be frozen at -80 °C in 3 ml aliquots in criovials for future biochemical determinations (adhesion molecules, growth factors, cytokines). The middle layer will be withdrawn and placed in 50 ml Falcon tube. 25 ml cold PBS will be added and centrifuged at 1500 rpm for 5 min. The pellet will be resuspend in 30 ml PBS/5% FCS, centrifuged at 1500 rpm for 5 min and washed again. The final pellet will be resuspended in RPMI1640 (Invitrogen), then used for CFU-EC isolations. Detailed procedure for flow cytometry analysis of whole peripheral blood For each patient, 3 tubes of whole blood will be created and labeled as follows: a) sample 1: “EPC sample” (10 ml); sample 2: “Control sample CD309” (10 ml); and sample 3: “Control sample CD133” (200 µl). All samples will be incubated with Red Blood Cell Lysis Solution for 10 min at room temperature and then centrifuged at 300xg for 10 min. As to samples 1 and 2, the pellet will be resuspended in 300 µl of AutoMACS Running buffer (Miltenyi Biotec Briguori C. et al.Online Supplemental Material, page 14 S.r.l; Calderara di Reno; Italy) and 100 µl of FcR blocking reagent and 100 µl EPC Enrichment Cocktail will be added to each of the two pellets. After 30 min of incubation in the dark at 4 °C, 50 µl of EPC staining Cocktail [CD34-FITC, CD133/2-Phycoerythrin (PE), CD309-allophycocyanin (APC) and CD14-PE-Cy5)] and 50 µL of EPC Control Cocktail CD309 (CD34-FITC, CD133/2-PE, Mouse IgG1-APC and CD14-PE-Cy5) will be added to the samples 1 and 2, respectively. After 10 min of incubation in the dark at 4 °C, cells will be washed and resuspended in 500 µl of AutoMACS Running buffer (Miltenyi Biotec S.r.l; Calderara di Reno, Italy) and then separated on MS columns (Miltenyi). As to sample 3, after red blood cell lysis, the pellet will be resuspended in 70 µl of AutoMACS Running buffer (Miltenyi). Twenty µl of FcR blocking reagent and 10 µl of EPC Control Cocktail CD133 (CD34-FITC, Mouse IgG2b-PE and CD14-pe-Cy5) will be added to the sample 3 and incubated for 10 min in the dark at 4 °C. After incubation, cells will be washed and resuspended in 500 µl of AutoMACS Running buffer (Miltenyi). Before flow cytometric acquisition, 10 µl of Propidium Iodide (PI) solution will be added to samples 1 and 2; and 5 µl to sample 3. Flow cytometric analysis will be performed on a FACSCanto II flow cytometer (BD Biosciences). Calibration and compensation will be carried out using CaliBRITE beads (BD catalog no. 340486) as quality controls across the study (54, 55). Appropriate amount of beads will be used following the manufacturer’s instructions. Daily control of CaliBRITE intensity will be performed in order to assess changes in instrument sensitivity throughout the study. The relative voltage range for each detector will be assessed una tantum by the “eight-peak” technology (Rainbow Calibration Particles, BD catalog number 559123) at the beginning of the study. Compensation will be set in FACS-DiVa (BD) software, and samples will be analyzed compensated using the same software. Then samples will be acquired within 30 min on the BDFacsCanto II and, for each patient, a minimum of 100,000 events will be recorded for samples 1 and 2, and 1x106 events for sample 3. Briguori C. et al.Online Supplemental Material, page 15 The gating strategy starts from sample 3, which is useful to identify both CD34+ and CD133+ cells. In order to select CD34+ cells in a side scatter (SSC) versus forward-scatter (FSC) dot-plot, a P1 gate will be created to select leukocytes and to exclude debris and platelets (OS Figure 2, panel A). Then P1 events will be displayed in a CD14/PI versus Mouse IgG2b dot-plot and a P2 gate will be imposed on CD14/PIneg cells to select live cells (OS Figure S, panel B). Then, we will create P3 region in a CD34-FITC vs SSC dot-plot to select CD34+ cells (OS Figure 2, panel C). The percentage of CD34+ cells, identified in sample 3 using the aforementioned gating strategy along with the white blood cell count in 10 ml of whole blood, is necessary to calculate the absolute number of CD34+ cells, by which we can obtain the absolute number of EPC. The following formula will be applied: Absolute number of CD34+ cells = (% viable CD34+ cells X absolute number of leukocytes) /100. We will use sample 3 also to identify CD133 specificity. In details, P3 events will be displayed in an APC vs Mouse-IgG2b dot-plot, and we will impose quadrants Q1, Q2, Q3 and Q4 to establish the cutoff for CD133-PE-positive cells (OS Figure 2, panel D). In order to identify the EPC population, we will use sample 1 and 2 to select live cells by the same gating strategy of the sample 3 (P1 and P2 regions in OS Figure 3, panels A-B). Then, in sample 2, we will selected CD34+ cells imposing a P3 gate in a CD34 vs. CD133 dot-plot (OS Figure 3, panel C). In sample 2, CD34+ cells will be displayed in a Mouse IgG-APC vs CD133-PE dot-plot imposing the same quadrants Q1, Q2, Q3 and Q4 of the sample 3. In sample 2, we will use these quadrants in position also to determine a cutoff for CD309-EPCpositive cells (OS Figure 3, panel D), such as including in Q3 and Q4 all cellular events that will be APC negative. Finally, in sample 1, CD34+ cells (P3 events) will be displayed in a CD309-APC versus CD133-PE dot-plot (OS Figure 3, panel E) without quadrants modification. We will calculate the absolute percentages of: Briguori C. et al.Online Supplemental Material, page 16 1. CD34+/CD133+/CD309+ cells as Q2 cellular events percentage of sample 1 (OS Figure 3 panel E) minus Q2 events percentage of sample 2 (OS Figure 3 panel D); 2. CD34+CD309+ cells as Q1+Q2 cellular events percentage of sample 1 (OS Figure 3 panel E) minus Q1+Q2 events percentage of sample 2 (OS Figure 3 panel D). Absolute number of EPC will be calculated as follow: (absolute % of CD34+CD133+CD309+ cells X absolute number of CD34+ cells) /100. Absolute number of EC will be calculated as follow: (absolute % of CD34+CD309+ cells X absolute number of CD34+ cells)/100. CFU-EC isolation and quantification CFU-EC (colony forming units-endothelial cells or CFU-Hill (5)) will be cultured using the EndoCultTM Liquid Medium kit (Stem Cells Inc.), according to the manufacturer’s instructions. Briefly, 5 × 106 PBMCs will be resuspended in CFU-Hill EndoCultTM Liquid Medium Kit (Human) (Stem Cell Technologies) and cultured on fibronectin-coated 6 well plates (BD Falcon). After 48 hours, non adherent cell will be collected and 4x106 cells will be plated on fibronectin-coated 24 well for additional 3 days. At the end (i.e. at day 5 after plating in fibronectin-coated 24-well plates), adherent cells will be fixed in 4% paraformaldehide, stored at 4 °C, and clusters will be counted in 8 randomly selected highpower fields. Optical coherence tomography Optical coherence tomography (OCT) offers 10 times higher resolution (12 to 15 µm vs 150 µm) and 40 times faster imaging acquisition compared with intravascular ultrasound, and to date represents the gold standard in evaluating vessel healing after PCI and in detecting Briguori C. et al.Online Supplemental Material, page 17 incomplete apposition of stents to the arterial wall (8, 9). Enrolled patients will undergo coronary angiography and OCT imaging at 3-month post-procedure. Imaging of the target DES will be performed using a Fourier-domain OCT system (Ilumien imaging system, LightLab Imaging, Inc., St. Jude Medical, St. Paul, MN) and the corresponding 6F guide catheter compatible Dragonfly Intravascular Imaging Catheter. The catheter will be introduced into the coronary artery via a standard 0.014′′ angioplasty wire after the administration of an unfractionated heparin bolus of 70 IU/kg. OCT cross-sectional images are generated at a rotational speed of 100 frames/s while the fiber is withdrawn at a speed of 20 mm/s within the stationary imaging sheath, using a non-occlusive technique. All cross sectional images will be initially screened for quality assessment and excluded from analysis if the image has poor quality caused by artifacts or inadequate blood clearance, as defined by the inability to visualize lumen contour in more than 45° (1 quadrant). OCT analysis will be performed by an independent investigator blinded to patient and procedural information using proprietary offline software (LightLab Imaging, St. Jude Medical). Cross-sectional OCT images of the stented segments will be analyzed at 1 mm intervals. Stent and luminal crosssectional areas (CSA) will be measured; neointimal hyperplasia (NIH) CSA will be calculated as the stent minus luminal CSA, while the mean NI area stenosis is defined as the NI area divided by the stent area multiplied by 100. NIH thickness will be measured as the distance between the endoluminal surface of the neointima and the strut, and an uncovered strut was defined as having a NIH thickness of 0 μm (10). The NI volume will be calculated as difference between stent volume and lumen volume and the NI volume obstruction will be defined as the NI volume (mm3) divided by the stent volume (mm3) multiplied by 100. For totally occluded vessels not associated with stent thrombosis, it will be estimated that the entire length of the stent is filled with neointima. A malapposed strut is defined as a strut that had detached from the vessel wall by ≥100 μm (that is, strut thickness 80 µm + OCT Briguori C. et al.Online Supplemental Material, page 18 resolution limit 20 µm) (11). Cross-sections with major side branches (diameter ≥2 mm) and overlapping stents will be excluded from this analysis. Statistical analysis and sample calculation 1) acute (<24 hours) changes of EPCs levels after PCI according to pre-treatment with rosuvastatin or atorvastatin or placebo. The independent t-test will be used to test for differences between the placebo and three groups on the means of continuous variables. One-way ANOVA will be performed to compare differences between groups and the serial changes in cell counts to baseline. Proportions will be compared by the Chi-square test. Data will be expressed as mean ± SD. The power of the sample size calculation will be set at 90%. This will result in a required sample size of 30 patients per each group. Accounting for an additional 10 to 15% drop out rate, a sample size of 33 patients is anticipated as needed. All statistical analyses will be performed using the SPSS 14.0 (SPSS Inc., Chicago, Illinois) and P values <0.05 will be considered significant. 2) 3-month changes of EPC levels after PCI according to pre-treatment with high (80 mg) versus low (20 mg) atorvastatin dose in a subgroup of diabetic patients. Previous studies have reported 0.0038 per 100 PMNCs as the EPCs level threshold predicting future cardiovascular events (60). Low (<0.0038/100 PMNCs) EPCs levels have been observed in approximately 30% of diabetic patients (8). We hypothesize that at 3-month follow-up, due to a larger increase of EPC levels in the High Atorvastatin Dose group, this proportion would remain stable in Low Atorvastatin Dose group, and, on the contrary, would decrease in the High Atorvastatin Dose group. Therefore, expecting a reduction of at least 10% in the High Atorvastatin Dose group, we have calculated an overall sample size of 80 subjects (40 per group) needed to achieve 80% power (95% confidence interval) to detect statistical significant difference (p <0.05) in the proportion of low (<0.0038/100 PMNCs) EPC level at 3-month follow-up in the 2 groups of treatment (12-17), Briguori C. et al.Online Supplemental Material, page 19 ONLINE SUPPLEMENTAL REFERENCES 1. Orru V, Steri M, Sole G, et al. Genetic variants regulating immune cell levels in health and disease. Cell 2013; 155: 242-56. 2. Lanuti P, Rotta G, Almici C, et al. Endothelial progenitor cells, defined by the simultaneous surface expression of VEGFR2 and CD133, are not detectable in healthy peripheral and cord blood. Cytometry A 2015. 3. Lachmann R, Lanuti P, Miscia S. OMIP-011: Characterization of circulating endothelial cells (CECs) in peripheral blood. Cytometry A 2012; 81: 549-51. 4. Lanuti P, Santilli F, Marchisio M, et al. A novel flow cytometric approach to distinguish circulating endothelial cells from endothelial microparticles: relevance for the evaluation of endothelial dysfunction. J Immunol Methods 2012; 380: 16-22. 5. Hill JM, Zalos G, Halcox JP, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med 2003; 348: 593-600. 6. Ingram DA, Mead LE, Tanaka H, et al. Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood. Blood 2004; 104: 2752-60. 7. Yoder MC, Mead LE, Prater D, et al. Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals. Blood 2007; 109: 1801-9. 8. Bezerra HG, Attizzani GF, Sirbu V, et al. Optical coherence tomography versus intravascular ultrasound to evaluate coronary artery disease and percutaneous coronary intervention. JACC Cardiovasc Interv 2013; 6: 228-36. 9. Guagliumi G, Sirbu V. Optical coherence tomography: high resolution intravascular imaging to evaluate vascular healing after coronary stenting. Catheter Cardiovasc Interv 2008; 72: 237-47. 10. Takano M, Inami S, Jang IK, et al. Evaluation by optical coherence tomography of neointimal coverage of sirolimus-eluting stent three months after implantation. Am J Cardiol 2007; 99: 1033-8. 11. Guagliumi G, Ikejima H, Sirbu V, et al. Impact of drug release kinetics on vascular response to different zotarolimus-eluting stents implanted in patients with long coronary stenoses: the LongOCT study (Optical Coherence Tomography in Long Lesions). JACC Cardiovasc Interv 2011; 4: 778-85. Briguori C. et al.Online Supplemental Material, page 20 12. Patti G, Pasceri V, Colonna G, et al. Atorvastatin pretreatment improves outcomes in patients with acute coronary syndromes undergoing early percutaneous coronary intervention: results of the ARMYDA-ACS randomized trial. J Am Coll Cardiol 2007; 49: 1272-8. 13. Field KM. Effect of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors on high-sensitivity C-reactive protein levels. Pharmacotherapy 2005; 25: 1365-77. 14. Notarbartolo A, Davi G, Averna M, et al. Inhibition of thromboxane biosynthesis and platelet function by simvastatin in type IIa hypercholesterolemia. Arterioscler Thromb Vasc Biol 1995; 15: 247-51. 15. Schmidt-Lucke C, Rossig L, Fichtlscherer S, et al. Reduced number of circulating endothelial progenitor cells predicts future cardiovascular events: proof of concept for the clinical importance of endogenous vascular repair. Circulation 2005; 111: 29817. 16. Werner N, Kosiol S, Schiegl T, et al. Circulating endothelial progenitor cells and cardiovascular outcomes. N Engl J Med 2005; 353: 999-1007. 17. Vasa M, Fichtlscherer S, Aicher A, et al. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res 2001; 89: E1-7. Briguori C. et al.Online Supplemental Material, page 21 OS Table 1. Flow cytometry specificities and reagents Detection Fluorochrome/ Vendor Ab Clone Catalog Number Amount per Test - S-7578 1 µM P1H12 623920* 40 µL - 623920* 10 µL 8G12 623920* 10 µL 2D1 623920* 10 µL 89106 623920* 20 µL 55-7H1 561569 5 µL Reagent DNA CD146 Viability CD34 CD45 VEGFR2 CD144 ThermoFisher Scientific BD PE Biosciences BD 7-AAD Biosciences BD PE-Cy7 Biosciences BD APC-H7 Biosciences AlexaFluor64 BD 7 Biosciences V450 BD Biosciences Syto16 Reagents composing the lyophilized panel are evidenced in bold face. Other reagents added to the basic panel are listed in plain font. *Catalog number of the lyophilized combination. Legend: R-phycoerythrin (PE); 7-AminoActinomycin D (7-AAD), PE-Cyanine 7 (Cy7), Allophycocyanin-Hilite®7 (APC-H7). Becton Dickinson (BD) Biosciences (San Jose, CA, USA); Life technologies (Monza, Italy). Briguori C. et al.Online Supplemental Material, page 22 OS Table 2. Composition of the flow cytometry panel and control tubes CONTROL TUBE PANEL TUBE Tube Antibody/Reagent Syto16 CD146 PE 7-AAD CD34 PE-Cy7 CD45 APC-H7 VEGFR2 (CD309) Alexa 647 CD144 V450 Syto16 Isotype PE 7-AAD CD34 PE-Cy7 CD45 APC-H7 Isotype Alexa Fluor 647 Isotype V450 Ab Clone P1H12 8G12 2D1 89106 55-7H1 MOPC-21 8G12 2D1 MOPC-21 MOPC-21 Purpose DNA probe Endothelial marker Viablity probe Endothelial marker Leukocyte marker Endothelial marker Endothelial marker DNA probe Isotype Control Viablity probe Endothelial marker Leukocyte marker Isotype Control Isotype Control Reagents composing the lyophilized panel are evidenced in bold face. Other reagents added as liquid drop in to the basic panel are listed in plain font. Legend: R-phycoerythrin (PE); 7-AminoActinomycin D (7-AAD), PE-Cyanine 7 (Cy7), Allophycocyanin-Hilite®7 (APC-H7), Allophycocyanin (APC). B Late ECFCs CFU-EC A 5d 20 x 7d 20 x 14 d 20 x 7d 20 x 14 d 20 x 21 d 20 x OS Figure 1 OS Figure 2 OS Figure 3
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