Recovery of Regional but Not Global Contractile Function by the Direct Intramyocardial Autologous Bone Marrow Transplantation
Results From a Randomized Controlled Clinical Trial
Background— Recent trials have shown that intracoronary infusion of bone marrow cells (BMCs) improves functional recovery after acute myocardial infarction. However, whether this treatment is effective in heart failure as a consequence of remodeling after organized infarcts remains unclear. In this randomized trial, we assessed the hypothesis that direct intramyocardial injection of autologous mononuclear bone marrow cells during coronary artery bypass graft (CABG) could improve global and regional left ventricular ejection fraction (LVEF) at 4-month follow-up.
Methods and Results— Twenty patients (age 64.8±8.7; 17 male, 3 female) with a postinfarction nonviable scar, as assessed by thallium (Tl) scintigraphy and cardiac magnetic resonance imaging (MRI), scheduled for elective CABG, were included. They were randomized to a control group (n =10, CABG only) or a BMC group (CABG and injection of 60.106±31.106 BMC). Primary end points were global LVEF change and wall thickening changes in the infarct area from baseline to 4-month follow-up, as measured by MRI. Changes in metabolic activity were measured by Tl scintigraphy and expressed as a score with a range from 0 to 4, corresponding to percent of maximal myocardial Tl uptake (4 indicates <50%, nonviable scar; 3, 50% to 60%; 2, 60% to 70%; 1, 70% to 80%; 0>80%). Global LVEF at baseline was 39.5±5.5% in controls and 42.9±10.3% in the BMC group (P=0.38). At 4 months, LVEF had increased to 43.1±10.9% in the control group and to 48.9±9.5% in the BMC group (P=0.23). Systolic thickening had improved from −0.6±1.3 mm at baseline to 1.8±2.6 mm at 4 months in the cell-implanted scars, whereas nontreated scars remained largely akinetic (−0.5±2.0 mm at baseline compared with 0.4±1.7 mm at 4 months, P=0.007 control versus BMC-treated group at 4 months). Defect score decreased from 4 to 3.3±0.9 in the BMC group and to 3.7±0.4 in the control group (P=0.18).
Conclusions— At 4 months, there was no significant difference in global LVEF between both groups, but a recovery of regional contractile function in previously nonviable scar was observed in the BMC group.
Experimental studies have shown that transplantation of bone marrow-derived cells may contribute to the regeneration of infarcted myocardium.1 Based on those data, 2 clinical trials have shown that intracoronary infusion of autologous bone marrow cells improves left ventricular functional recovery after acute myocardial infarction and successful percutaneous coronary intervention.2,3 However, whether this treatment is effective in congestive heart failure as a consequence of remodeling after organized infarcts remains unclear. Loss of viable myocardium initiates a process of adverse remodeling, leading to chamber dilatation and contractile dysfunction. Also, the efficiency of cell transfer in case of intracoronary administration may be hampered by the fact that only a small number of cells remain in the heart.4 Therefore, we performed a randomized controlled trial to assess the functional effect of direct intramyocardial injection of autologous bone marrow cells on left ventricular ejection fraction (LVEF) in patients with a chronic myocardial infarction undergoing coronary artery bypass graft (CABG) surgery.
Materials and Methods
Patients were eligible if they were admitted for elective CABG surgery, had a transmural myocardial infarction on ECG and akinesia or dyskinesia in part of the left ventricle as shown by angiography. Patients needing urgent surgery were excluded, as well as patients in advanced renal or hepatic failure or with documented malignancy. In addition patients carrying a pacemaker were excluded. The randomized controlled trial was approved by the local Ethics Committee. All patients provided written informed consent.
Randomization and Baseline Investigation
Patients were randomly allocated in a 1:1 ratio to either the control, conventional CABG, or bone marrow cell (BMC) group, by means of sequentially numbered sealed envelopes. After randomization, all patients underwent baseline investigations before revascularization, consisting of cardiac magnetic resonance imaging (MRI) and thallium scintigraphy. Mean time between occurrence of infarct and CABG was 217±162 days in the BMC group and 213±145 days in the control group (P=0.95).
Harvest and Transfer of BMCs
In the bone marrow transplant group (n =10), 40 mL of bone marrow was aspirated under local anesthesia from the patients’ iliac crest, the day before surgery. In the control group (n =10), bone marrow (7 mL) was harvested from patients’ sternum under full anesthesia just before surgery, to ascertain that characteristics of the BMC suspension did not significantly differ between both groups. Bone marrow-derived mononuclear cells were immediately isolated by density gradient centrifugation using Lymphoprep (Nycomed). Isolated cells were washed twice with saline and subsequently resuspended in X-Vivo 15 medium (Cambrex) supplemented with 2% autologous serum. This cell suspension was transferred to Teflon Bags (Vuelife CellGenix) at a concentration of &1×106 cells/mL for overnight cultivation. The next day, cells were harvested and washed 3 times before finally suspended in 10 mL heparinized saline. Just before transplantation, cells were filtered by a 70-micron cell strainer (Falcon). Nucleated cell viability was assessed by Trypan blue (Invitrogen) exclusion.
The complete 10 mL was directly injected at the border zone of the infarct scar. This was accomplished using 29-gauge myojector syringes (Terumo) containing 0.5 mL fractions of the cell suspensions. Multiple punctures were performed with a pre-bent needle to make injections parallel to the epicardium and avoid delivery of cells into the ventricular cavity. In the control group, an equivalent volume of heparinized saline was injected. The surgeon was unaware whether cells or only saline was injected. Revascularization of the infarct related area was systematically performed. Complete revascularization was achieved in all patients
Characterization of Transplanted Cells
The absolute counts of CD34 positive cells were determined using the stem kit CD34+HPC Enumeration kit (Beckman Coulter).
As quality ex vivo control, hematopoietic colony-forming cell growth was calculated by standard methylcellulose assay as described previously.
Further characterization of the transplanted cells was performed on a FACSAria (Beckon Dickinson) with 4-color flow cytometry.
Before hospital discharge (9 to 14 days after surgery) and again 4 months after surgery, cardiac MRI and thallium scintigraphy were repeated in all patients.
To assess whether intramyocardial BMC transfer was associated with pro-arrhythmic effects, all patients who accepted underwent programmed ventricular stimulation at 6 to 8 weeks follow-up. Stimulation was performed at the right ventricular apex and the right ventricular outflow tract with single, double, and triple extra stimuli at twice the diastolic threshold and basic cycle lengths of 500 and 400 ms.
Cardiac MRI was performed in a 1.0-T scanner (Gyroscan T 10-NT; Philips Medical Systems) using surface body coil with ECG gating and respiratory triggering.
Sequence parameters were as follows: balanced fast field echo (FFE) gradient echo sequence with dynamic sequences in transversal plane, long axis, short axis, and 4-chamber view; and 3-dimensional T1-weighted FFE gradient echo sequence with multishot and TFE prepulse with variable delay between 200 and 300 ms after intravenous contrast injection of megluminegadoterate (Guerbet, Roissy, France) (“delayed enhancement”).
The balanced FFE gradient echo images were analyzed in cine view to detect dyskinetic or akinetic regions and wall thickness measurement was performed on short axis and long axis views.
Contrast-enhanced MRI was used to assess myocardial injury after infarction and to differentiate between necrosis and viable tissue with scanning after a delay time of 15 minutes after intravenous injection of 15 mL 0.5 mmol/kg megluminegadoterate (delayed enhancement).
All images were analyzed by 1 investigator unaware of treatment assignment (E.B.). Endocardial and epicardial borders were traced in all end-diastolic and end-systolic short axis and long axis slices to determine left ventricular end-diastolic volume (LVEDV) and end-systolic volume (LVESV) for calculation of global left-ventricular ejection fraction, with ejection fraction = ([LVEDV − LVESV] / LVEDV) × 100%.
Regional wall thickening was calculated by determining the short axis slice through the center of the infarct region. A 19-segment model was used and pathologic segments were identified as segments with marked decrease in wall thickness and without thickening during systole. Correlation was made with pathologic regions on delayed enhancement. Wall thickening was calculated by the formula end-systolic thickness minus end-diastolic thickness.
Area length ejection fraction of the left ventricle was calculated mid ventricular on long axis views.
Correlation was made with thallium scintigraphy for evaluation of areas of myocardial scar.
Thallium scintigraphy was performed 10 minutes and 4 hours after the intravenous injection of 111 MBq 201Tl (Tyco Healthcare). Patients were injected at rest, after an overnight fasting period. All studies consisted of SPECT acquisition with a dual-head gamma camera (GE Hawkeye). Images were acquired over a 180° arc with camera heads in rectangular position (60 steps, 50 beats/step, matrix size 642 zoom 1.33). Image reconstruction was performed with filtered back projection (Butterworth filter: cut-off 0.4 to 0.65 cycle/cm, order 5) followed by the reorientation of the tomographic data. Short axis and vertical and horizontal long axis slices of the myocardium were generated on which a 20% background subtraction was performed and a 16-step color scale was applied, yielding a fixed image set used for further analysis.
Each study was evaluated by the consensus of 2 investigators blinded for treatment assignment.
To obtain a semi-quantitative measurement of Tl uptake, the myocardium was divided into 17 segments, each receiving a score with a range from 0 to 4, corresponding to percentage of maximal myocardial Tl uptake (4 indicates <50%, nonviable scar; 3, 50% to 60%; 2, 60% to 70%; 1, 70% to 80%; 0, >80%). The overall decrease in Tl uptake was then expressed as the sum score, obtained by adding up all segmental values.
Continuous variables are reported as mean±SD. The LVEF and volume indices of the 2 groups were statistically compared by means of the Student t test. The analyses were performed for the different measurement occasions and for the changes versus baseline. The nonparametric Wilcoxon-rank test was also used as an alternative test because the data are likely not normally distributed and the sample size of the study is small. ANCOVA was used to compare the changes in LVEF and volume indices in the 2 groups, with treatment as the main factor and baseline EF or volume index as a covariate. For each patient the wall thickening was measured in a number of ventricular segments. These measurements are not independent observations. Therefore, mixed model methodology was used to investigate the changes in wall thickening between groups and alternatively Friedman tests to check the differences with a nonparametric test, taking into account that measurements in ventricular segments are not independent observations.
Categorical variables were compared using the χ2 test and Fishers exact test. Statistical significance was assumed at a value of P<0.05. Computations were performed with SAS 9.1 (SAS Institute, Inc, Cary, NC).
Statement of Responsibility
The authors had full access to the data and take full responsibility for their integrity. All authors have read and agree to the manuscript as written.
Between January 13, 2004 and December 22, 2004, 28 patients were assessed for eligibility. Four patients did not meet the inclusion criteria, based on preoperative MRI and thallium. One patient refused to participate. Twenty-three patients were randomized, 11 to the bone marrow transplant group and 12 to the control group. In the control group one patient died on the fifth postoperative day because of multi-organ failure secondary to low cardiac output syndrome. One patient in the control group was lost to follow-up because of acute psychiatric illness. In the bone marrow group, 1 patient died on postoperative day 7 because of a cardiac unrelated cause (perforated esophageal ulcer complicated by mediastinitis). The final cohort included 10 controls and 10 patients in the BMC group. Table 1 shows baseline characteristics of the treated groups.
The total amount of recovered BMCs used for transplantation was on average 60.25×106±31.35×106 nucleated cells (viability 95.05%±2.54%; recovery 73.0±14.6%). This transplanted cell suspension contained 1.42%±0.99% CD34+ cells and 76.37±44.47 CFU-GM per 105 mononuclear cells. More detailed information about the characteristics of the BMC suspension is described in Table 2.
Global ejection fraction increased in both groups, albeit not significantly. In the control group, it was 39.5±5.5% at baseline, 41.2±10.1% at hospital discharge, and 43.1±10.9% at 4 months (P=0.36 baseline versus 4 months). In the BMC group LVEF was 42.9±10.3% at baseline. Baseline values were not significantly different between groups (P=0.19). LVEF increased to 45.8±13.2% at discharge and 48.9±9.5% at 4 months (P=0.21 baseline versus 4 months). There was no significant difference in improvement between the control and the BMC treated group at 4-month follow-up (P=0.41).
Changes in LVEDV index and LVESV index did not differ significantly between the control and BMC groups (Table 3).
In all patients, thallium scintigraphy was performed before surgery and repeated at hospital discharge and at 4-month follow-up. In the final analysis, only segments in the area with intention to treat with a Tl uptake score of 4 at 10 minutes and 4 hours were considered. In the BMC group, defect score decreased to 3.5±0.9 at discharge and further to 3.3±1.0 at 4 months. In the control group, defect score was 3.7±0.4 at hospital discharge, representing the revascularization effect on Tl uptake. No further decrease in defect score was observed at 4 months (3.7±0.4) (Figure 1). The differences were not statistically significant at hospital discharge (P=0.51) or at 4-month follow-up (P=0.63).
According to MRI, 35 pathologic segments were identified in the BMC group and 39 in the control group. The presence of an infarct was confirmed by thallium uptake <50% in that area. Wall thickening in the affected segments was −0.5±2.0 mm at baseline in the control group and −0.6±1.3 mm in the BMC group. At hospital discharge, wall thickening values did not differ significantly from baseline in both groups: 0.4±1.6 mm in the control group and 0.3±1.6 mm in the treatment group (P=0.18). At 4 months, however, a significant improvement in wall thickening was observed in the BMC group (1.8±2.6 mm), whereas in the control group, wall thickening remained unchanged (0.4±1.7 mm) (P=0.007; Figure 2).
The BMC group could be divided into a subgroup of patients showing a response (n=5), a group of nonresponders (n=4), and 1 patient with discordant findings. The subgroup of responders was defined as those patients showing a significant wall thickening at 4 months postoperatively and a decrease in thallium defect score. In this subset of patients, wall thickening increased from −0.6±1.3 mm to 3.0±2.6 mm. Thallium defect score decreased from 4 to 2.6±1.0. In the group of nonresponders, no wall thickening was observed at 4 months follow-up (from −0.7±1.5 mm preoperatively to 0.0±1.6 mm at 4 months follow-up) and defect score remained 4 in all segments. In the patient with discordant findings, improvement in regional contraction was observed, whereas no change in thallium uptake defect score was noted.
The improvement in the responder group did not correlate with the number of transplanted mononuclear cells (63.8×106±41.6×106 cells in the responder group versus 57.9×106±30.3×106 cells in the nonresponder group, P=0.82). Interestingly, the number and percentage of CD34+ cells in the responder group was significantly higher than in the nonresponder group (3.1±1.97% versus 0.9±0.38%, P=0.03). Table 4 gives an overview of the total number of transplanted cells, the percentage and absolute number of CD34-positive cells, and the Tl defect score and wall thickening changes for each patient in the BMC group. Also, the greater improvement in global LVEF (4 M versus baseline) was observed in patients with higher numbers of engrafted CD34-positive cells. Figure 3 shows LVEF changes (4 M-baseline) for each BMC patient, plotted as a function of absolute numbers of CD34+ cells implanted (r=0.49).
Additional investigations suggest that direct intramyocardial injection of cells does not cause additional damage to the myocardium. Although 1 patient died in the treated group, the cause of death was noncardiac-related. Peak CK-total and CK-MB values were similar in both groups (CK-total 1308±689 versus 824±209U/L in BMC versus control group, P=0.09; CK-MB 48±44 versus 40±29 U/L, P=0.73). Also, peak CTnI values did not differ significantly between both groups (6.6±4.1 U/L versus 9.7±12.8 U/L BMC versus control group, P=0.68).
Five control patients and 9 patients who received bone marrow transfer agreed to undergo an electrophysiological study. In the BMC group, monomorphic ventricular tachycardia could be induced in 5 patients, polymorphic ventricular tachycardia could be induced in 1. In 3 of those 6 patients, an automatic implantable cardioverter defibrillator was implanted. Three patients were treated with amiodarone and closely followed-up. No patient in the control group had inducible ventricular tachycardia.
To investigate a possible confounding effect of amiodarone on the BMC group data, a subgroup analysis was performed. Wall thickening change (4 M baseline) in the amiodarone-treated group was 2.0±3.0 mm versus 3.0±2.1 mm in the nonamiodarone-treated patients (P=0.24), indicating that amiodarone had no significant effect on cell treatment.
This randomized clinical trial addresses the effect of autologous BMC therapy on LVEF in patients with ischemic cardiomyopathy as a consequence of postinfarction myocardial scar. Patient characteristics in both groups were comparable. Complete revascularization was achieved in all patients, but a marginally higher number of bypass grafts were constructed in the BMC group (2.8±0.4) compared with the control group (2.5±0.5, P=0.07). Although these grafts were placed in remote, noninfarcted areas, they might have increased perfusion in the neighboring scar tissue and account for the better recovery of regional function in some of the BMC patients. However, the fact that the number of grafts in the “responder” and “nonresponder” group were similar makes this interpretation unlikely. There was no significant difference in global LVEF between both groups at 4-month follow-up. The increase in LVEF postoperatively was comparable in both groups (41.2±10.1% in controls versus 45.8±13.2% in the BMC group) and reflects the effect of revascularization on the reversal of myocardial stunning. A further increase in LVEF, albeit not significant was observed at 4 months (43.1±10.9% in control and 48.9±9.5% in the BMC group). This could be explained by the presence of hibernating myocardium.5 Although 201thallium uptake <50% relative to the area of maximal tracer uptake, which defines nonviable scar tissue, has a high negative predictive value (90%), comparable to position emission tomography,6 it can be expected that &10% of nonviable segments would improve after revascularization alone. This could account for a further improvement in global LVEF after hospital discharge.
In correspondence to our results, Janssens et al7 reported no significant difference in global LVEF in a double-blind trial of intracoronary transplantation of autologous bone marrow. However, among patients treated within 6 hours of chest pain onset, stem cell therapy was associated with an almost 40% greater reduction in infarct size. No improvement in LVEF was observed in patients with large infarcts treated with intracoronary mononuclear BMC transplantation.8
These findings are in contrast with a number of previous reports, in which improvement in global LVEF by transfer of bone marrow-derived cells was reported.2,3,9 However, in those studies, BMC treatment was administered in the setting of acute myocardial infarction. Furthermore, in those studies baseline LVEF was systematically better preserved than in our patient population.
Our results showed significant improvement of systolic wall thickening between hospital discharge and 4 months follow-up in the BMC treated group. A possible confounding effect of CABG was ruled out because all affected segments in both groups were systematically revascularized. Moreover, Galinanes et al10 have reported that transplantation of autologous bone marrow cells into scar tissue can only enhance cardiac function when used in combination with revascularization. These results are not necessarily in contradiction with the observation that global LVEF did not significantly change. A critical factor of global LVEF recovery is the number of segments with improved wall motion. This number needs to represent &20% of the left ventricle.11 Given the relatively low dose of transplanted cells, it is unlikely that this number was reached in our study. Other trials reporting significant improvement in global LVEF have used higher numbers of transplanted cells (245×106 in the TOPCARE-AMI trial;2 24.6×108 in the BOOST trial3). This observation may warrant an investigation into a possible dose–response effect of BMC transplantation.
LVEDV did not decrease, indicating that BMC treatment did not improve left ventricular remodeling at 4-month follow-up.
Whether bone marrow-derived stem cells can transdifferentiate to cardiomyocytes remains a matter of debate. Adult bone marrow contains a number of multipotential stem cells, such as mesenchymal and hematopoietic stem cells.12 Orlic et al showed that in the mouse model hematopoietic stem cells (Lin− c-kit+) were able to differentiate into cardiac myocytes.1 However, those results could not be reproduced: Murry et al transplanted Lin− c-kit+ cells in MHC-nLAC mice.13 These cells did not differentiate even 4 weeks after transplantation. Also, Balsam et al reported that hematopoietic stem cells could not be found 30 days after injection into the ischemic myocardium of mice.14 Nevertheless, Kajstura et al have recently repeated their previous observation that in the mouse model, in 10 days, nearly 4.5 million biochemically and morphologically differentiated myocytes together with coronary arterioles and capillary structures were generated independently of cell fusion.15
In view of the small size of this trial, subgroup analysis is difficult. However, the “responder” group was transplanted with a cell population, containing a significantly higher percentage and absolute number of CD34+ cells than the nonresponders. Hematopoietic stem cells and endothelial progenitor cells have been described as cells expressing the hematopoietic marker CD34 on their surface. Those cells have the capacity to incorporate in sites of neovascularization and differentiate into endothelial cells in situ.16 Recently, using fluorodeoxyglucose as monitoring, Hofmann et al have shown that 14% to 39% of a CD34-enriched population homed into infarcted myocardium after intracoronary administration, whereas only 1.3% to 2.6% of an unselected BMC population did.4 Those observations taken together suggest that CD34+ cells may play an important role in successful engraftment of BMC in infarcted cardiac tissue. Furthermore, no significant difference could be observed in other surface markers between the responder and nonresponder group (Table 5).
The improvement in wall thickening in the BMC group was observed between hospital discharge and 4 months, suggesting that the time needed for differentiation of stem cells in myocardial infarction may be longer than expected. Dai et al have shown that in the rat model mesenchymal stem cells did not express muscle-specific markers at 2 weeks but expression started at 3 months in some animals and at 6 months in all animals.17 Our group has transplanted mesenchymal (CD34−) cells into the infarct area at 4 hours after coronary occlusion in the sheep model. One month later, immunohistochemical staining for troponin-I and cardiac-specific myosin of transplanted mesenchymal stem cells was negative.18
Our study suggests that direct intramyocardial injection of bone marrow-derived cells did not cause additional damage to the myocardium. The postoperative increase in markers for myocardial damage was not beyond that observed in the conventional CABG group. This may be in contrast to intracoronary administration. Vulliet et al have reported dose-dependent ischemic changes and microinfarctions after intracoronary infusion of mesenchymal cells in noninfarcted dogs.19 Kang et al reported a small increase in markers of myocardial damage after intracoronary infusion of peripheral blood stem cells.20 Other studies using intracoronary administration of bone marrow cells did not observe myocardial injury3 or microvascular dysfunction9 after transient repetitive occlusion and simultaneous infusion of BMCs, or did not report specifically.2
Occurrence of sustained ventricular tachycardia seems the only serious adverse event likely related to the procedure. Comparable to the study by Menasche et al21 performed with skeletal myoblasts, 6 patients had inducible ventricular tachycardia. In the case of skeletal myoblasts, this problem was attributed to expression of different sets of ion channels. Furthermore, although functional gap junction formation between myoblasts/myotubes and neonatal rat cardiomyocytes has been reported in vitro, sustained coupling between engrafted myoblasts/myotubes and myocytes does not occur, creating electrically isolated islands.22 Our findings contrast with at least 1 previous study of bone marrow cell transplantation in association with CABG, in which no serious ventricular arrhythmias were observed up to 14 months.23 However, despite the fact that mesenchymal stem cells do express connexin-specific genes in coculture with cardiomyocytes,24 occurrence of shortened refractory periods and increased slope of restitution were reported and can induce susceptibility to ventricular arrhythmia.25
Our study shows that autologous BMC transplantation promotes regional left ventricular function in chronic heart failure patients. However, this did not translate into an increase in global left ventricular function, as reported in acute myocardial infarction patients. Future studies should be directed toward optimizing restoration of cardiac function.
Presented at the American Heart Association Scientific Sessions, Dallas, Tex, November 13–16, 2005.
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