Autologous bone marrow mononuclear cell transplantation in patients undergoing coronary artery bypass grafting

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1 Surgery Autologous bone marrow mononuclear cell transplantation in patients undergoing coronary artery bypass grafting David Mocini, MD, a Mario Staibano, MD, b Luca Mele, MD, PhD, c Paride Giannantoni, MD, a Giacomo Menichella, MD, PhD, c Furio Colivicchi, MD, FESC, a Paolo Sordini, MD, b Paola Salera, MD, c Marco Tubaro, MD, a and Massimo Santini, MD, FACC, FESC a Rome, Italy Background Recent studies have shown that autologous bone marrow mononuclear cell (abm-mnc) transplantation can be effectively performed in human beings either by the coronary route or by endoventricular injections. However, scanty data are available for patients undergoing coronary artery bypass grafting (CABG). Accordingly, the aim of this study was to evaluate the feasibility and safety of abm-mnc transplantation in patients with recent myocardial infarction undergoing CABG. Methods and Results The study population included 36 consecutive patients with recent myocardial infarction (b6 months) undergoing CABG. Eighteen patients (17 men, mean age 64 years) underwent CABG plus abm-mnc transplantation, whereas 18 subjects undergoing conventional CABG (17 men, mean age 67 years) served as control subjects. Cell transplantation was performed by direct injections in the border zone of the recently infarcted area. An average number of 292 F abm-mncs was injected in each patient. When compared with control subjects, transplanted patients showed higher values of troponin I peak after CABG (median values of 1.65 ng/ml vs 0.64 ng/ml, P b.001). No major transplant-related adverse event could be detected. During follow-up, transplanted patients had an improvement in left ventricular ejection fraction (from 0.46 to 0.51, P b.05) and wall motion score index (from 1.71 to 1.42, P b.01). The incidence of arrhythmias immediately after CABG and during follow-up was similar in the 2 groups. Conclusions Our data support the idea that direct injection of abm-mncs in the myocardium during CABG is feasible and safe. Larger studies are needed to assess the efficacy of such an approach in patients undergoing CABG. (Am Heart J 2006;151:192-7.) Stem cell transplantation may represent an effective therapeutic option to restore tissue viability after myocardial damage. Actually, stem cells from the bone marrow or the peripheral blood have been used with this specific purpose and with promising results Besides, stem cell transplantation has been performed either by the coronary route 1-5 or by endoventricular injections. 6-8 However, to date, only scanty data are available for patients undergoing coronary artery bypass grafting (CABG), 9-15 even if such surgical setting may provide the opportunity for direct delivery of stem cells in the diseased myocardium. From the a Division of Cardiology, Cardiovascular Department, b Division of Cardiac Surgery, Cardiovascular Department, and c Laboratory Medicine Department, S. Filippo Neri Hospital, Rome, Italy. Submitted August 27, 2004; accepted February 2, Reprint requests: David Mocini, MD, Giuseppe Gioeni 30, Rome, Italy. david.mocini@fastwebnet.it /$ - see front matter n 2005, Mosby, Inc. All rights reserved. doi: /j.ahj This study was designed and undertaken to evaluate the feasibility and safety of autologous bone marrow mononuclear cell (abm-mnc) transplantation as performed by direct myocardial injection during CABG in patients with recent myocardial infarction (MI). Methods Selection criteria All patients undergoing CABG at our institution were prospectively screened for inclusion in the investigation. Consecutive patients with the following characteristics were selected for inclusion in the study: 1. recent MI (N4 weeks but b6 months); 2. planned CABG for at least 2 vessels, 3. no evidence of myocardial viability in the infarct area, as shown by a preoperative low-dose dobutamine echocardiography. Patients were excluded in the following instances: 1. age N80 years; 2. left ventricular ejection fraction b35%;

2 Volume 151, Number 1 Mocini et al myocardial revascularization in the previous 6 months; 4. left ventricular aneurysm; 5. permanent or persistent atrial fibrillation; 6. severe valvular disease; 7. history of any hematological disease, including leukopenia (b3.000 white cells/al), thrombocytopenia (b platelets/al), or thrombocytosis (N platelets/al); 8. hepatic or renal dysfunction (serum creatinine N2 mg/ml, ALT N2 times of normal value); 9. evidence of malignant diseases or unwillingness to participate. Among 231 consecutive patients undergoing CABG at our institution between November 15, 2003, and March 15, 2004, all patients matching inclusion criteria and without exclusion criteria accepted to take part in the investigation and were enrolled in the study. Study protocol The study was designed to include 6 consecutive blocks of 6 subjects. Such consecutive blocks of patients were alternatively allocated to conventional CABG plus cell transplantation (transplant group) or to conventional CABG alone (control group). The initial block was allocated to cell transplantation. Patients were not aware of which treatment they would receive in case of participation to the study, whereas the enrolling medical personnel (DM) knew what treatment each of them would in case of enrollment. The final study sample included 36 consecutive subjects who were divided in 2 groups of 18 patients each. The study protocol was approved by the ethical committee of our institution. Written informed consent was obtained from all patients. Echocardiography All echocardiographic examinations (Vivid 7, GE Medical Systems, Solingen, Germany) were performed by an experienced cardiologist (PG), who was blinded to clinical details and group allocation, both during the preoperative baseline evaluation and during the follow-up period. All echocardiographic measurements and calculations were made in accordance with the recommendations of the American Society of Echocardiography. 16 Moreover, the low-dose dobutamine echocardiography necessary for inclusion in the study was performed as elsewhere described. 17 Transplantation procedure Coronary artery bypass grafting was done in all cases during cardiopulmonary bypass and cold-blood cardioplegic arrest. After the beginning of anesthesia, 50 ml of bone marrow was collected from the posterior iliac crest and transferred into a sterile bag. Cells were filtrated with a 140-Am filter and seeded in 7%/vol hydroxyethyl starch (Plasmasteril 7%, Fresenius Kabi, Bad Homburg, Germany) for 90 minutes. After 2 phosphatebuffered saline washing steps and a centrifugation for 10 minutes at 3000 rpm, a final volume of 4 to 6 ml of bone marrow cell solution was obtained for injection into the myocardial tissue. All the procedures from harvesting to cell injection were performed in a closed-circuit system into sterile bags. Table I. Patient characteristics Transplanted patients Control subjects Subjects (n) Age (y) 64.4 F F 4.5 NS Men NS Inferior or posterior 67 (12) 67 (12) NS MI Revascularization 0 (0) 0 (0) NS of the infarct area Anterior, lateral, 33 (6) 33 (6) NS apex MI Revascularization of 33 (6) 33 (6) NS the infarct area Hypertension 78 (14) 72 (13) NS Diabetes mellitus 56 (10) 50 (9) NS Current smoking 33 (6) 38 (7) NS Hypercholesterolemia 61 (11) 61 (11) NS Current medications Aspirin 83 (15) 89 (16) NS h-blockers 50 (9) 38 (7) NS ACE inhibitors 38 (7) 33 (6) NS Insulin 28 (5) 33 (6) NS Statin 72 (13) 78 (14) NS Nitrates 78 (14) 72 (13) NS Diseased coronary arteries (median) 3 3 NS ACE, angiotensin-converting enzyme; NS, not significant. All counts of total cells and abm-mncs were performed by a Beckman Coulter device. Determinations of specific subpopulations of abm-mncs were performed after washing by flow cytometric analysis (FacScan, Becton Dickinson, Franklin Lakes, NJ [BD]) using a phycoerythrin-labeled murine CD34 monoclonal antibody (clone 8G12, BD Procount, BD), peridin chlorophyll protein labeled murine CD45 (CD45-perCP) monoclonal antibody (clone 2D1, BD Procount, BD), and phycoerythrinlabeled murine CD133 monoclonal antibody (clone AC133, Miltenyi Biotec, Bergisch Gladbach, Germany). Cell viability was determined by 7-amin-actinomycin D staining test. 18 The CD45 +, CD34 +, and CD133 + cells were counted as elsewhere described. 19 Briefly, 50 AL of the cell solution was incubated with a nucleic acid dye, CD45-perCP, and phycoerythrin-labeled murine CD34 in bead-containing TruCount tubes (BD). The control reagent (nucleic acid dye, g1-pe, and CD45- percp) was used to assess the amount of nonspecific antibody binding. After red cell lysis with a diluted FACS Lysing Solution (BD), the samples were measured with a FACScan flow cytometer (BD). The absolute number of CD34 + cells in the sample was determined by dividing the number of CD34 + cellular events by the number of fluorescent bead events and then multiplying with the bead concentration. The ProCOUNT software system (BD) was used to acquire and analyze data. For CD45 + /CD133 + quantification, 50 AL of the cell solution was labeled with CD45-perCP and PE-CD133. After red cell lysis, flow cytometric analyses were performed on a FACScan analytical flow cytometer (BD). P

3 194 Mocini et al January 2006 Table II. Cell subpopulations in the injected solution Total cells BM-MNCs CD45 + CD34 + CD133 + Mean SD Median Minimum Maximum Interquartile range During surgery, before the beginning of extracorporeal circulation, epicardial echocardiography with a 6-MHz probe was used to identify the infarct area and its border zone (Sonos 2000, Hewlett Packard, Palo Alto, CA). The area was immediately marked for subsequent identification. Injections of cell solutions were performed in the previously marked area at the end of cardiopulmonary bypass, starting from the border zone toward the center of the infarct area, using a 27- gauge, 4-mm needle. The cell solution volume/injection was 0.2 ml. Surgeons paid particular attention to deliver the cell solution in a gentle and slow way in the subepicardial myocardium. Autologous fibrin glue was used to immediately close the hole made by the needle, to avoid cell solution regurgitation and abm-mnc loss. Perioperative care and follow-up Continuous electrocardiographic monitoring with automatic arrhythmia detection was performed for 3 days after surgery. In the same period, troponin I was dosed on a daily basis. A 24-hour Holter recording and an echocardiographic examination were performed before discharge in all patients. All patients underwent a clinical follow-up visit on a monthly basis. Besides, after 90 days from surgery, all patients underwent 48-hour Holter recording, echocardiography, and cardiac nuclear magnetic resonance imaging. Statistical analysis Mean (F SD) and median values were calculated for continuous variables, whereas frequencies were measured for categorical variables. Distributions of continuous variables were determined by the Kolmogorov-Smirnov test. Group differences for continuous data were then examined by the Student t test or the Mann-Whitney U test as appropriate. In case of categorical variables, group differences were examined by m 2 analysis or the Fisher exact test as appropriate. Correlations were assessed by the Spearman correlation coefficient. All statistical tests were 2 tailed. A P value of b.05 was considered significant. Statistical analysis was performed with commercially available software (SPSS 12.0, Chicago, IL). Results Baseline clinical features of the study population are shown in Table I. Patients with inferior or posterior MI did not undergo revascularization of the infarct-related vessel, whereas patients with anterior or apical MI did. Table III. Safety and efficacy data Transplanted patients Controls subjects Graft (n), median (IQR) 2.5 (2-3) 3 (2-3) NS CPB (min), mean F SD 113 F F 38 NS Aortic cross clamp time 59 F F 22 NS (min), mean F SD Troponin I after CABG 1.65 ( ) 0.64 ( ) b.001 (ng/ml), median (IQR) Pre-CABG echocardiography LVEF, mean F SD 0.46 F F 0.08 NS WMSI, mean F SD 1.71 F F 0.45 NS Follow-up echocardiography LVEF, mean F SD 0.51 F 0.09T 0.49 F 0.09 NS WMSI, mean F SD 1.42 F 0.25y 1.52 F 0.40 NS Arrhythmias predischarge Atrial fibrillation 6 5 NS (patients) VPBs (number/patient), 513 ( ) 763 ( ) NS median (IQR) Couplets (number/ 6 (0-42) 3 (0-7) NS patient), median (IQR) NSVT (patients) 5 6 NS SVT (patients) 1 0 NS Follow-up arrhythmias Atrial fibrillation 1 1 (patients) VPBs (number/patient), 276 ( ) 133 (27-635) NS median (IQR) Couplets (number/ 0.5 (0-6) 3 ( ) NS patient), median (IQR) NSVT (patients) 1 3 NS SVT (patients) 0 0 NS IQR, interquartile range; CPB, cardiopulmonary bypass; LVEF, left ventricular ejection fraction; WMSI, wall motion score index; VPBs, ventricular premature beats; NSVT, nonsustained ventricular tachycardia; SVT, sustained ventricular tachycardia. TP =.048 vs pre-cabg LVEF. yp =.003 vs pre-cabg WMSI. Feasibility We injected a cell suspension including a mean number of 640 F total cells/patient (median , interquartile range ). The cell solution consisted of heterogeneous cell populations. Cellular marker results are presented in Table II. P

4 Volume 151, Number 1 Mocini et al 195 Figure 1 Echocardiographic left ventricular ejection fraction in transplanted patients (A) and in control subjects (B) at baseline and after 3 months. Dashed lines represent variations in mean values of left ventricular ejection fraction. Small dots show data for individual patients; large dots show mean values. Vertical bars show SD. Table IV. Stem cell transplantation in patients undergoing CABG First author Reference Patients (n) Control group Cell source Ficoll use BM-MNCs injected (number) CD34 + injected CD133 + injected Stamm [9] 6 No BM Yes NA 1.16 Hamano [10] 5 No BM No NA NA Galinanes [11] 14 No BM No T NA Li [12] 6 No BM No NA NA Ozbaran [13] 6 No PB No NA Pompilio [14] 4 No PB No NA Stamm [15] 12 No BM Yes NA NA 1.5 NA, not available; BM, bone marrow; PB, peripheral blood. TCD34 + /CD Cell viability was at least 94% as determined by the 7-amin-actinomycin D staining test. An average of 26 F 10 injections/patient (range 10-35) was done. The mean injected volume/patient was 5.2 F 1.7 ml. Safety No major surgical complications were noted in any patient. Moreover, no patient reported pain or showed any complication at the bone marrow harvesting site. There were no significant differences between the 2 groups in cardiopulmonary bypass time, aortic cross clamp time, or the number of coronary artery grafts performed (Table III). The incidence of atrial fibrillation and that of ventricular arrhythmias were similar in the 2 groups (Table III). Median peak troponin I values within 3 days of surgery were higher in transplanted patients than in control subjects (Table III). In transplanted patients, peak troponin I values were significantly correlated both with the total number of injected cells and with the number of abm-mncs (r = 0.54, P =.021 and r = 0.57, P =.014, respectively). No other significant correlation between peak troponin I values and the number of any other cell subpopulation was found. Furthermore, no significant correlation was noted between peak troponin I values and the injected volume of cell solution. Follow-up All patients were discharged alive and in good clinical condition and were followed up for a median period of 354 days (median 356, minimum 294, maximum 422,

5 196 Mocini et al January 2006 interquartile range days). No patient was lost to follow-up. Besides, clinical, echocardiographic, and Holter follow-up data were available for all patients. During follow-up, the incidence of arrhythmias in 48-hour Holter monitoring was similar in the 2 study groups (Table III). Transplanted patients showed a significant improvement in left ventricular ejection fraction and wall motion score index, whereas control subjects did not show any significant modification of such parameters (Table III, Figure 1). Cardiac nuclear magnetic resonance imaging did not show any unexpected image in myocardial tissue receiving cell therapy. In particular, differently from animal studies, 20 no calcifications within the myocardium were revealed by such methodology after 90 days from surgery and cell transplantation. Discussion It has been accepted for a long time that cardiac cell death results in an irreparable damage to the structure of the adult heart. However, recent research has challenged the dogmatic notion that the heart represents a terminally differentiated postmitotic organ incapable of self-renewal. 21 As a matter of fact, both preclinical studies in animal models and preliminary clinical investigations have suggested that stem cell therapy may promote cardiac angiogenesis and cell regeneration, thereby improving cardiac function. In human beings, cell transplantation has been performed either by the coronary route 1-5 or by endoventricular injections 6-8 ; however, scanty data are available as to the direct injection of stem cells into the myocardial wall during CABG In addition, all studies performed in the specific setting of coronary surgery showed major inherent limitations because they were not controlled and included small and heterogeneous study populations Moreover, those investigations dealt with small amounts of transplanted cells or were only focused on selected stem cell subpopulations. 9,14,15 Main data from previous studies performed in the specific setting of coronary surgery are presented in Table IV. When compared with available research and clinical data, our study shows some relevant features. In fact, to the best of our knowledge, this is the first controlled investigation performed in the specific setting of stem cell transplantation during coronary surgery, while also including the largest study population ever considered. As to the methodology, intraoperative epicardial echocardiography was used for the first time to identify precisely the infarct area and its border zone, thereby improving stem cell delivery. Furthermore, in this study, stem cell manipulations were performed only with materials approved for clinical practice. In particular, differently from previous studies, 9,15 cells were not seeded on Ficoll, which is a polymer of sucrose not intended for clinical use but only for in vitro applications. Overall, this study clearly shows that the direct injection of abm-mnc in patients with recent MI is feasible. The procedure is not associated with an increase in circulatory extracorporeal or aortic cross clamp time, while the preparation and transplantation of abm-mncs can be performed with minimal bone marrow manipulations, lasting b3 hours. Another point of interest is that our study provides evidence that the procedure is reasonably safe. In our series, the injection of up to 1.72 billion total cells, billion abm-mncs, and 15 million CD34 + cells was associated with only a mild rise in troponin I values, whereas no major adverse clinical events were noted either in the early postoperative period or during followup. In our opinion, this observation retains particular clinical relevance. In fact, although direct stem cell injection seems to be associated with a limited myocardial damage, the functional outcome of the left ventricle may critically depend on the total number of cells delivered. 25 Furthermore, our data clearly show that, despite reported concerns, 26 stem cell transplantation by direct injection in the myocardial wall is not associated with an increased incidence of life-threatening arrhythmias. Finally, even if this study was not aimed at assessing the efficacy of the procedure, a significant improvement in left ventricular function was noted in transplanted patients during a relatively short follow-up period. Limitations of the study We did not transplant a selected cell population. The use of a selected CD133 + or CD117 + population can be used to reduce the number of cells delivered, thereby reducing potential myocardial damage. However, current knowledge of cell traffic is largely incomplete. Consequently, cell selection might exclude important elements in the differentiation process. For this reason, we preferred an unselected bone marrow mononuclear cell population. In this study, which is not properly randomized, 6 consecutive patients received stem cell therapy, followed by 6 control subjects. This pattern was repeated 3 times to achieve 18 patients in each group. This kind of patient allocation has been chosen for merely internal practical reasons. However, we are conscious that such methodology is superior to historical controls but inferior to proper randomization. We therefore recognize this point as a limitation of our study. Conclusions Autologous bone marrow mononuclear cell transplantation by direct myocardial injection in patients with recent MI undergoing CABG is feasible and safe. The

6 Volume 151, Number 1 Mocini et al 197 procedure neither implies any significant alteration in the current surgical practice of CABG nor is associated with any major clinical complication. Consequently, our approach could represent a fairly practical method of stem cell therapy during CABG, which could be adopted by many surgical programs. However, larger randomized and blinded trials are needed to confirm safety and to test effectiveness in this specific surgical setting. References 1. Assmus B, Schächinger V, Teupe C, et al. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI). Circulation 2002;106: Strauer BE, Brehm M, Zeus T, et al. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation 2002;106: Kang H-J, Kim H-S, Zhang S-Y, et al. 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