doi: 10.1161/CIRCULATIONAHA.104.524827
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(Circulation. 2005;112:I-73 – I-80.)
© 2005 American Heart Association, Inc.
Cell Transplantation and Tissue Engineering |
Prevention of Left Ventricular Remodeling With Granulocyte Colony-Stimulating Factor After Acute Myocardial Infarction
Final 1-year Results of the Front-Integrated Revascularization and Stem Cell Liberation in Evolving Acute Myocardial Infarction by Granulocyte Colony-Stimulating Factor (FIRSTLINE-AMI) Trial
From the Department of Medicine, Divisions of Cardiology (H.I., M.P., H.E., T.R., D.B., S.K., C.A.N.) and Hematology (H.D.K., M.F.), the University Hospital Rostock, Rostock School of Medicine, Rostock, Germany.
Correspondence to Christoph A. Nienaber, MD, FACC, FESC, Division of Cardiology, University Hospital Rostock, Rostock School of Medicine, Ernst-Heydemann-Str. 6, 18057 Rostock, Germany. E-mail christoph.nienaber{at}med.uni-rostock.de
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Abstract |
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Background— Experimental and clinical evidence has recently shown that pluripotent stem cells can be mobilized by granulocyte colony-stimulating factor (G-CSF) and may enhance myocardial regeneration early after primary percutaneous coronary intervention (PCI) management of acute myocardial infarction. Sustained or long-term effects of mobilized CD34-positive mononuclear stem cells, however, are unknown.
Methods and Results— Thirty consecutive patients with ST-elevation myocardial infarction undergoing primary PCI with stenting and abciximab were selected for the study 85±30 minutes after PCI; 15 patients were randomly assigned to receive subcutaneous G-CSF at 10 µg/kg body weight for 6 days in addition to standard care including aspirin, clopidogrel, an angiotensin-converting enzyme inhibitor, ß-blocking agents, and statins. In patients with comparable demographics and clinical and infarct-related characteristics, G-CSF stimulation led to sustained mobilization of CD34 positive mononuclear cells (MNCCD34+), with a 20-fold increase (from 3±2 at baseline to 66±54 MNCCD34+/µL on day 6; P<0.001); there was no evidence of leukocytoclastic effects, accelerated restenosis rate, or any late adverse events. Within 4 months, G-CSF–induced MNCCD34+ mobilization led to enhanced resting wall thickening in the infarct zone of 1.16±0.29 mm (P<0.05 versus control), which was sustained at 1.20±0.28 after 12 months (P<0.001 versus control). Similarly, left ventricular ejection fraction improved from 48±4% at baseline to 54±8% at 4 months (P<0.005 versus control) and 56±9% at 12 months (P<0.003 versus control and paralleled by sustained improvement of wall-motion score index from 1.70±0.22 to 1.42±0.26 and 1.33±0.21 at 4 and 12 months, respectively), after G-CSF (P<0.05 versus baseline and P<0.03 versus controls). Accordingly, left ventricular end-diastolic diameter showed no remodeling and stable left ventricular dimensions after G-CSF stimulation, whereas left ventricular end-diastolic diameter in controls revealed enlargement from 55±4 mm at baseline to 58±4 mm (P<0.05 versus baseline) at 12 months after infarction and no improvement in diastolic function.
Conclusion— Mobilization of MNCCD34+ by G-CSF after primary PCI may offer a pragmatic strategy for improvement in ventricular function and prevention of left ventricular remodeling 1 year after acute myocardial infarction.
Key Words: myocardial infarction • remodeling • cells • stents
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Introduction |
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Left ventricular (LV) remodeling after myocardial infarction is thought to set the stage for heart failure and premature death through transformation of both necrotic and peri-infarct tissue.1 The dogma of irreversible loss of tissue was challenged with the observation of replicating human cardiac muscle cells in a viable syncytium.2,3 Although animal experiments using cell transplantation techniques (eg, fetal cardiomyocytes or skeletal myoblasts) have succeeded in reconstituting heart muscle, these cells fail to completely integrate structurally and to display characteristic physiological function.4 Conversely, bone marrow-derived pluripotent adult stem cells are not only capable of tissue differentiation, but are likely to regenerate myocardium by myogenesis, angiogenesis, or paracrine effects demonstrated by improved cardiac function in animals and preliminary human studies.5–11
Interestingly, there is mounting evidence that pluripotent cells enhance myocardial restoration regardless of their route of administration, whether by intramyocardial injection, intracoronary or intravenous infusion, or even mobilization with cytokines, with unknown long-term effects. Mobilization of a critical number of pluripotent cells is an interesting concept,6 given that 5x106 circulating stem cells can be removed from the peripheral blood by apheresis of normal human donor blood after several days of granulocyte colony-stimulating factor (G-CSF) stimulation.12To examine the impact of G-CSF on bone marrow, we analyzed the impact of stem cell mobilization in reperfused acute myocardial infarction on long-term changes of LV function and remodeling after 4 and 12 months.
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Methods |
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Patients
The Front-Integrated Revascularization and Stem Cell Liberation in Evolving Acute Myocardial Infarction by Granulocyte Colony-Stimulating Factor (FIRSTLINE-AMI) Trial was initiated in April 2003 to recruit 30 consecutive patients with acute ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PCI) with stenting and adjunctive intravenous abciximab administration according to recent guidelines13; after successful PCI, patients were randomized to 10 µg/kg G-CSF over 6 days in addition to standard care or to standard post-interventional treatment.
Patients between 18 and 65 years of age with first STEMI comprising 
3 of 12 ECG leads were eligible; cardiogenic shock (defined as systolic blood pressure <80 mm Hg requiring intravenous pressors or intra-aortic balloon counterpulsation), major bleeding requiring blood transfusion, history of leukopenia, thrombocytopenia, hepatic or renal dysfunction, evidence of malignant disease, or unwillingness to participate were criteria for exclusion.
After successful recanalization of the infarct-related artery by primary PCI and stent implantation, randomization by the closed-envelope method was initiated. Both G-CSF recipients and controls were continuously monitored for arrhythmogenic or hemodynamic events over 6 days and clinically followed over 12 months.
The FIRSTLINE-AMI trial protocol was approved by the Institutional Ethics Committee and the Ethics Review Board of the University of Rostock, Germany, and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from each patient.
Stem Cell Mobilization
Within 85±30 minutes of successful PCI, patients were randomly assigned to subcutaneous G-CSF (Amgen) at a dose of 10 µg/kg of body weight over 6 days in addition to standard care or standard care alone including aspirin, clopidogrel, angiotensin-converting enzyme inhibitors, ß-blockers, and statins in appropriate doses. Quantification of total white blood cells (WBC) and CD34-positive mononuclear cells (MNCCD34+) by flow cytometry were monitored from whole blood directly sampled before PCI and at days 4, 5. and 6 after PCI. Interleukin-6 and tumor necrosis factor-
as markers of inflammation were measured by use of ELISA (Biosource Diagnostics).
Flow Cytometry (MNCCD34+ Enumeration)
Stem cell concentration in peripheral blood was obtained using the Interdisziplinäre Gruppe für Labor und Durchflusszytometrie protocol14; 100 µL of peripheral blood (anticoagulant K3-EDTA) were incubated for 15 minutes on ice with both pretitered CD45-fluorescein conjugated (clone 2D1) and CD34-phycoerythrin conjugated (clone 8G12) monoclonal antibody (10 µL each). Similarly, control samples were incubated with fluorochrome-conjugated isotypic control antibodies (clone X40), all purchased from Becton-Dickinson. After incubation, erythrocytes were lysed by resuspension of the samples in 2 mL FACS lysing solution for 8 minutes at room temperature. Samples were centrifuged for 10 minutes with 200g at 4°C; cells were washed twice with phosphate buffered saline (pH 7.2) and then measured in a FACSort flow cytometer (Becton Dickinson).
Using the CellQuest Software package, at least 50 000 cells were measured in samples incubated with CD45 and CD34, and 10 000 cells were counted in isotypic control samples. Stem cells were counted by a gating strategy on the basis of a combination of specific CD45 and side scatter pattern of CD34+ cells, allowing exact calculation of the percentage of CD34+ leukocytes down to 0.1%. MNCCD34+ per µL was derived from the relation of CD34+ cells and WBCs in the blood sample.
Angiography and Quantitative Coronary Angiography
Coronary and biplane LV angiograms were obtained and quantitated for left ventricular ejection fraction (LVEF) according to international standards (by observers blinded to the protocol) both with initial PCI and at 6 months. Coronary angiograms were evaluated for binary restenosis of the target lesion. Off-line quantitative coronary angiography was performed from digital cineangiograms of identical coronary projections using a MEDOS workstation connected to SIEMENS HICOR system. Restenosis at 6 months was defined as a diameter stenosis of >50% within the stent ±5 mm.
LV Echocardiography
Resting echocardiography was performed by experts blinded to treatment allocation at baseline and 4 and 12 months after PCI. Global function and regional LV wall motion were digitized using a ATL HDI 5000 ultrasound system and analyzed according to the Standards of the American Society of Echocardiography15 in 4 standard views (parasternal long-axis and short-axis views and apical 4- and 2-chamber views). For quantitation of wall motion, the standardized 16-segment model was used integrating segmental wall motion scores of 1=normal, 2=hypokinesis, 3=akinesis, and 4=dyskinesis16; wall motion score index (WMSI) was calculated as the sum of the scores of the segments divided by the number of segments evaluated. Segmental wall thickening in the infarct territory encompassing the central area of dysfunction by wall motion analysis was assessed from the relation of average end-diastolic and -systolic wall thickness in identical segments over time. Early diastolic mitral flow velocity deceleration time was measured to assess diastolic function.
Clinical Follow-Up Visits
Clinical data were prospectively collected during follow-up visits scheduled at 4 and 12 months. Specific attention was paid to potential signs or symptoms of coronary events (for revascularization) or arrhythmias. Adherence to 6-months follow-up coronary angiography and 1 year adherence to medication per protocol were complete.
Statistical Analysis
Continuous variables are presented as mean±SD. Categoric variables were compared by use of 
2 or Fisher’s exact test. Statistical comparisons within treatment groups were made by paired Student’s t test if data were distributed normally; other comparisons were made by nonparametric Wilcoxon 2-sample test, and Bonferroni-corrected ANOVA was used for longitudinal comparison between baseline and follow-up measurements at 4 and 12 months. Statistical significance was assumed at a value of P<0.05. Statistics were computed using SPSS (Version 11.0, SPSS Inc).
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Results |
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Demographics, clinical characteristics, and comorbidity profiles were homogeneous and revealed a typical distribution of risk factors and medication according to modern guidelines (Table 1). Angiographic and infarction-related characteristics showed a balanced distribution of infarct-related characteristics, location of infarction, enzyme release, and baseline LV function in both groups (Table 2). Moreover, time from onset of symptoms to PCI with stent placement was not different between groups, with 299±101 minutes in the G-CSF group and 304±111 minutes in controls (P=NS). There were no complications associated with acute PCI, and all occluded infarct-related arteries were recanalized and stented under adjunctive abciximab infusion, followed by clopidogrel loading. TIMI III flow was documented in all 30 patients after PCI. Subcutaneous G-CSF injection was begun 85±30 minutes after reperfusion. Postinfarction medication was identical in both groups, including oral continuation of 100 mg aspirin, 75 mg clopidogrel, statins, tailored angiotensin-converting enzyme inhibitors, and up-titrated ß-blocker medication. Two cases of binary restenosis (13.3%) without resting flow limitation (TIMI III flow) were detected in both groups after 6 months, and 3 patients in each group had target vessel revascularization (TVR) within 1 year (20%) (Table 2).
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Table 3 summarizes all in-hospital and 1-year follow-up cumulative events in both the G-CSF and control groups. There were no in-hospital adverse events in either group, and there was a low incidence of events within 1 year with, no significant differences between groups. TVR was achieved in 3 cases in each group, and congestive heart failure was documented in 1 control patient under standard care. As demonstrated in Figure 1, MNCCD34+ and WBCs were increased markedly during G-CSF administration; parameters of rheology such as serum fibrinogen and blood viscosity were similar at baseline, after 4 months, and at 1 year in both groups (data not shown). As markers of inflammation, interleukin-6, C-reactive protein, and tumor necrosis- revealed a similar pattern at baseline, day 6, after 4 months, and at 1 year in both groups. There was no evidence of transiently elevated body temperature, relevant bone pain, or any adverse (or leukocytoclastic) events. In addition, during 6 days of continuous ECG recording, there was no documentation of increased ventricular arrhythmias or any ventricular tachycardia/fibrillation in either group; 24-hour Holter monitoring at day 9±2, 4 months, and 1 year was uneventful, with stable sinus rhythm in all. Morphological and functional parameters at baseline and 4 and 12 months are summarized in Table 4.
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Parameters of Systolic and Diastolic LV Function
At baseline, all parameters of LV function were similar in both the G-CSF-treated and control groups. Interestingly, with G-CSF, mean segmental wall thickening (WT) showed significant recovery to 1.16±0.29 at 4 month, which was sustained at 1.20±0.28 after 1 year (P<0.05 versus baseline), whereas the controls revealed only a borderline intragroup improvement of mean WT within 1 year (P=0.048). Intergroup comparison documented a G-CSF–associated improved mean WT both at 4 months (P<0.005) and 1 year (P<0.001). Recovery of LVEF with G-CSF was documented at 4 months and eventually measured 54±8% (P<0.005 versus control), and was sustained at 56±9% at 1 year (P<0.05 by Bonferroni-corrected ANOVA and P<0.003 versus controls). Conversely, there were no significant longitudinal changes present in controls (Figure 2A and 2B).
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Similar to LVEF, resting WMSI revealed partial recovery with G-CSF to 1.42±0.26 after 4 months (P<0.05 by ANOVA) and to 1.33±0.21 after 1 year (P<0.05 by ANOVA), whereas no recovery was seen with standard treatment (Figure 3A and 3B). Interestingly, with G-CSF, left ventricular end-diastolic diameter showed no enlargement as evidence of LV remodeling of the index infarction, with a stable left ventricular end-diastolic diameter of 54±5 mm over 1 year after postreperfusion G-CSF treatment, whereas control patients revealed progressive LV enlargement to 59±4 mm at 4 months and 58±4 mm after 1 year (P<0.05 by Bonferroni-corrected ANOVA) and evidence of remodeling compared with early G-CSF administration (P<0.03). Finally, diastolic mitral flow velocity deceleration time revealed improvement of diastolic function over 4 months and 1 year after G-CSF (P<0.05 by ANOVA) to an extent not found in controls.
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Discussion |
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This is the first randomized study with completed 1-year follow-up of G-CSF–induced MNCCD34+ mobilization after acute myocardial infarction. The strategy was safe, with no evidence of early or late adverse events, including electrical instability and accelerated restenosis (Table 3). Postreperfusion G-CSF administration over 6 days exposed postischemic human myocardium to approximately 2.8x1010 mobilized MNCCD34+ with potential for homing to necrotic areas, and documented improvement of both regional and global myocardial function with sustained functional benefit over 1 year. Conversely, control patients did not show improving LVEF or WMSI at 4 and 12 months, as is occasionally seen after early PCI,17–19 an observation likely the result of both remote compensatory hyperkinesis at baseline and lack of ischemic preconditioning. Previous studies reported a 4% increase of LVEF at 6 months after PCI,17 especially if PCI was performed within 4 to 6 hours of coronary occlusion18; for instance, the Abciximab before Direct Angioplasty and Stenting in Myocardial Infarction Regarding Acute and Long-term Follow-up study revealed a 4.1% increase in LVEF after stenting of acute myocardial infarction.19 Patients in all 3 studies, however, were older and had more multivessel disease and higher likelihood of ischemic preconditioning,17–19 whereas FIRSTLINE-AMI patients were younger and had single vessel involvement and thus larger infarcts with lower baseline LVEF (Tables 2 and Table 4).
Our findings of sustained long-term improvement of cardiac performance in humans is supported by recent experimental evidence of cytokine-mediated translocation of bone marrow cells repopulating the heart to enhance myocardial regeneration.6 Although both G-CSF and stem cell factor were given before coronary occlusion, cytokine-induced cardiac repair decreased mortality in mice by 68%, infarct size by 40%, cavity remodeling by 26%, and diastolic stress by 70%; even without reperfusion, LVEF and hemodynamics improved significantly as a consequence of 15x106 new myocytes connected with arterioles and capillaries in the circulation of unaffected myocardium.6 Similarly, human MNCCD34+ mobilized by G-CSF led to stem cell infiltration exclusively in injured myocardium 2 days after intravenous injection in rats; at 15 weeks, new blood vessel formation in the infarct bed and proliferation of preexisting vasculature were observed.7 Moreover, apoptotic cells and infarct size were reduced from 36% to 12%, with corresponding enhancement of cardiac output. Although both studies suggested a beneficial impact of G-CSF in the prevention of remodeling, cytokine treatment was either started before infarction6 or given in an non-reperfusion setting; both studies are unlikely to reflect the reperfusion scenario in humans. The results of the FIRSTLINE-AMI trial, however, are corroborated by recent experimental findings in an occlusion-reperfusion experiment20; G-CSF after infarction in rabbits increased LVEF and decreased remodeling in the long term, supporting the notion that G-CSF may enhance regeneration and prevent remodeling after infarction. At present, it is not entirely clear whether acutely infarcted myocardium may trigger spontaneous mobilization of pluripotent MNCCD34+21 or whether release of cytokines and upregulation of G-CSF may set the stage for homing of circulating CD34/CXCR4+ stem cells to the target tissue.22,23 Cardiac tissue-derived G-CSF may in fact have a role for recruitment of stem/progenitor cells and may help regenerate cardiomyocytes,24 supporting the idea of therapeutic utilization of G-CSF.
An important underlying mechanism beyond mobilization of pluripotent cells, at least in experimental settings, is the upregulation of Akt protein by G-CSF coupled with decreased rate of apoptosis and followed by improved cardiac function.25 Activated Akt is likely to protect the heart against damage in vivo by enhancing glucose transport and cell survival and promoting signaling cascades to inhibit cardiomyocyte apoptosis.26,27 Other potential mechanisms may be the parallel mobilization of both mesenchymal stem cells with G-CSF28 and side population cells invading the heart within 14 days.29 Moreover, even resident stem cells may migrate to the site of injury under cytokine stimulation.4 Finally, G-CSF was identified to upregulate expression of CXCR4 and thus enhance the stromal cell-derived factor-1/CXCR4 interaction required for stem cell homing30; similar to experimental myocardial infarction in mice, plasticity of neural structures and brain vascularization was impressively shown with G-CSF in a rat stroke model.31
The above-mentioned experimental data are likely to corroborate our findings of contractile improvement at 4 months and sustained recovery 1 year after G-CSF–induced MNCCD34+ mobilization. A critical issue for successful target delivery of mobilized stem cells, however, is the number of circulating stem cells.5–7 A mild increase of circulating MNCCD34+ to twice the baseline level has been observed in conjunction with acute myocardial infarction and reperfusion in humans.21 Such an archaic natural defense reflex was not seen outside the setting of acute infarction, but appears too subtle for measurable repair in humans, considering the mild spontaneous increase in MNCCD34+ in control patients (without G-CSF stimulation). Moreover, stromal cell-derived factor-1 required for homing is upregulated within 24 hours of myocardial infarction.22 With G-CSF, however, bone marrow mobilization enhanced exposure of MNCCD34+ to injured reperfused myocardium by 1000% over several days, accounting for greater chances of effective translocation and homing than one-time intracoronary infusion of multipotent bone marrow cells. While intracoronary infusion of cells may cause sludge and microemboly in animals,32 no evidence of impaired blood rheology or any microcirculatory disturbance (by elevated serum lactate, liver enzymes, creatine kinase-BB or creatinine) was documented in the FIRSTLINE-AMI trial after G-CSF administration (data not shown).
Whereas intracoronary stem cell delivery was enacted not before 5 to 9 days9 or 4.3±1.5 days after onset of necrosis10 and required bone marrow aspiration, G-CSF induced mobilization of MNCCD34+ was initiated within 2 hours of PCI. Moreover, Strauer et al9 delivered only 5.9x105 CD34+ cells or 2.1±0.28% of all 2.8x107 MNCs harvested after overnight culture, whereas Assmus et al10 infused 7.35±7.31x106 CD34/CD45+ cells per patient. Kocher et al7 transplanted 2x106 freshly isolated 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled human CD34+ cells/100g rat with beneficial effects; for the human setting, this translates into 3x108 to 6x108 cells on a weight-adjusted basis.33 Assuming an average blood flow of 0.8 mL · min–1 · g–1, 100 g of injured myocardium were exposed to approximately 2.8x1010 MNCCD34+ with G-CSF stimulation over several days; blood flow and tissue perfusion in the range of 0.8 mL · min–1 · g–1 were previously demonstrated after stenting with abciximab in the clinical setting of acute myocardial infarction.34
Although our longitudinal functional assessment revealed sustained recovery of both regional and global myocardial function 1 year after G-CSF treatment, the long-term impact of intracoronary infusion of bone marrow-derived stem cells is less homogeneous. Early results by Strauer et al9 were not followed-up long-term; functional data on significant improvement of LVEF in Bone Marrow Transfer to Enhance ST-Elevation Infarct Regeneration35 were recently followed by a regression to the mean at 18-months follow-up.36 Only the 1-year assessment of Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction patients revealed sustained favorable effects on LV remodeling, with a 9.3±8% enhancement of LVEF between 4 and 12 months and late reduction of infarct volume after intracoronary progenitor cell treatment with only 7x106 cells.37 With G-CSF administration, the heart is exposed to vastly more circulating MNCCD34+, although there are at present no convincing data on body distribution. In a non-splenectomized rat model, systemic delivery of technetium Tc 99m-labeled bone marrow-derived stem cells was mainly limited by lung entrapment; a small fraction between <1% and 2% of circulating cells, however, migrate and colonize the infarcted heart.38,39 After a conservative assumption, G-CSF would deliver 2.8x108 MNCCD34+ over several days, eg, 100- to 1000-fold more MNCCD34+ than one-time intracoronary delivery. Recent experiments in nude mice, however, suggest that up to 23% of CD34+ cells migrate to the ischemic heart within 24 hours of systemic delivery.40
Similar to MNCCD34+, circulating leukocytes increase after G-CSF and could destabilize coronary plaque with potentially adverse outcomes in acute myocardial infarction.41 The recent Myocardial Regeneration and Angiogenesis in Myocardial Infarction with G-CSF and Intra-Coronary Stem Cell Infusion trial,42 for instance, reported an unexpectedly high rate of in-stent restenosis in the infarct-related vessel after G-CSF; the controversial impact of G-CSF on in-stent restenosis, however, was deduced from only 3 patients with angiographic follow-up and should be interpreted with great caution, considering the expected 20% (critical) restenosis rate or TVR for bare metal stents in the FIRSTLINE-AMI trial after G-CSF administration. Thus, G-CSF administration did not promote intimal hyperplasia as suggested in MAGIC42 and failed to demonstrate an accelerated restenotic process; our finding are shared by recent reports of the Stem Cells in Myocardial Infarction trial43 and by Franz et al,44 with similar protocols of G-CSF in acute coronary syndromes and no evidence of an unexpected increase in restenosis rate. Moreover, in a model of apolipoprotein E-deficient mice, G-CSF was recently shown to reduce even atherosclerotic deposits and coronary lesions by lowering low-density lipoprotein cholesterol and decreasing plaque burden.45 Thus, G-CSF is safe in selected patients after complete revascularization of single vessel disease with mild LV dysfunction.
Limitations
At present, it is too early to claim that a beneficial effect over 1 year will be sustained at long term. In addition, the small sample size with strong male preponderance (93.3%) characterizes the pilot nature of the study, with limited power to generalize 1-year findings of functional recovery. Moreover, echocardiographic assessment of function may have inherent limitations because of 2-dimensional imaging as compared with both radionuclide blood pool imaging (with a 3-dimensional component) and volumetric magnetic resonance imaging. With focus on safety and feasibility, however, radiation burden had to be avoided in the pilot phase; an upcoming multicenter trial will implement sophisticated magnetic resonance imaging for yearly follow-up evaluation. Finally, this concept study was not blinded by design, although randomization ensured a balanced distribution; future trials should implement a double-blinded approach in addition to blinded evaluation.
Conclusion
Mobilization of MNCCD34+ by G-CSF after reperfusion of infarcted myocardium seems safe and feasible and could offer a pragmatic strategy for sustained 1-year recovery of myocardial function, with no risk of accelerated post-PCI restenosis. This concept warrants further investigation of developmental potential of stem cells, longer follow-up surveillance, and the scrutiny of multicenter, placebo-controlled trials.
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