Preservation From Left Ventricular Remodeling by Front-Integrated Revascularization and Stem Cell Liberation in Evolving Acute Myocardial In
http://www.100md.com
《循环学杂志》
the Department of Internal Medicine, Divisions of Cardiology and Hematology (H.D.K., M.F.), and the Department of Nuclear Medicine (C.S.) at the University Hospital Rostock, Rostock School of Medicine, Rostock, Germany.
Abstract
Background— Considering experimental evidence that stem cells enhance myocardial regeneration and granulocyte colony–stimulating factor (G-CSF) mediates mobilization of CD34+ mononuclear blood stem cells (MNCCD34+), we tested the impact of G-CSF integrated into primary percutaneous coronary intervention (PCI) management of acute myocardial infarction in man.
Methods and Results— Fifty consecutive patients with ST-segment elevation myocardial infarction were subjected to primary PCI stenting with abciximab and followed up for 6 months; 89±35 minutes after successful PCI, 25 patients were randomly assigned in this pilot study (PROBE design) to receive subcutaneous G-CSF at 10 μg/kg body weight for 6 days in addition to standard care, including aspirin, clopidogrel, an ACE inhibitor, -blocking agents, and statins. By use of CellQuest software on peripheral blood samples incubated with CD45 and CD34, mobilized MNCCD34+ were quantified on a daily basis. With homogeneous demographics and clinical and infarct-related characteristics, G-CSF stimulation led to mobilization of MNCCD34+ to between 3.17±2.93 MNCCD34+/μL at baseline and 64.55±37.11 MNCCD34+/μL on day 6 (P<0.001 versus control); there was no indication of leukocytoclastic effects, significant pain, impaired rheology, inflammatory reactions, or accelerated restenosis at 6 months. Within 35 days, G-CSF and MNCCD34+ liberation led to enhanced resting wall thickening in the infarct zone of between 0.29±0.22 and 0.99±0.32 mm versus 0.49±0.29 mm in control subjects (P<0.001); under inotropic challenge with dobutamine (10 μg · kg–1 · min–1), wall motion score index showed improvement from 1.66±0.23 to 1.41±0.21 (P<0.004 versus control) and to 1.35±0.24 after 4 months (P<0.001 versus control), respectively, coupled with sustained recovery of wall thickening to 1.24±0.31 mm (P<0.001 versus control) at 4 months. Accordingly, resting wall motion score index improved with G-CSF to 1.41±0.25 (P<0.001 versus control), left ventricular end-diastolic diameter to 55±5 mm (P<0.002 versus control), and ejection fraction to 54±8% (P<0.001 versus control) after 4 months. Morphological and functional improvement with G-CSF was corroborated by enhanced metabolic activity and 18F-deoxyglucose uptake in the infarct zone (P<0.001 versus control).
Conclusions— G-CSF and mobilization of MNCCD34+ after reperfusion of infarcted myocardium may offer a pragmatic strategy for preservation of myocardium and prevention of remodeling without evidence of aggravated restenosis.
Key Words: myocardial infarction remodeling cells angioplasty glucose
Introduction
Left ventricular (LV) remodeling after myocardial infarction is considered to set the stage for heart failure and premature death by 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,
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fetal cardiomyocytes or skeletal myoblasts) have succeeded in reconstituting heart muscle, these cells fail both to completely integrate structurally and to display characteristic physiological function.4–6 Conversely, bone marrow–derived pluripotent adult stem cells not only are capable of tissue differentiation7–9 but also are likely to regenerate myocardium by inducing myogenesis and angiogenesis, as shown by improved cardiac function in animals and preliminary human studies.10–16
Interestingly, there is mounting evidence that pluripotent cells enhance myocardial restoration regardless of their route of administration either by intramyocardial injection, by intracoronary and intravenous infusion, or even by mobilization with cytokines. Mobilization of a critical number of pluripotent cells is an interesting concept,11 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.17 Recent observations, however, have challenged myocardial regeneration in favor of direct cytokine effects.
In this context, we tested both the safety and impact of G-CSF in the setting of human myocardial infarction in conjunction with primary percutaneous coronary intervention (PCI) and abciximab18 in a randomized study with serial assessment of LV function and metabolism after 1 and 4 months and coronary morphology at 6 months.
Methods
Patients and PCI
FIRSTLINE-AMI was initiated in April 2003 and set up to recruit 50 consecutive patients with acute ST-elevation myocardial infarction (STEMI) subjected to primary PCI with stenting and abciximab administration according to recent guidelines18; after successful reperfusion, patients were randomized by use of the closed-envelope method in 1:1 allocation (with 25 subjects per group) to 10 μg/kg G-CSF over a period of 6 days in addition to standard care or to standard postinterventional care alone.
Patients between 18 and 65 years of age and 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, a 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 facilitated PCI using abciximab and modern bare-metal stents, randomization by the closed-envelope method was initiated. Both G-CSF recipients and control subjects were monitored continuously for arrhythmogenic or hemodynamic events over a period of 6 days and followed up for 6 months. The pilot study, using PROBE design with open-label application of G-CSF but blinded evaluation by expert readers unaware of patient group assignment (flow chart in online-only Appendix; see http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.105.541433/DC1), was approved by the Institutional Ethics Committee and the Ethics Review Board of the University of Rostock, Germany, and was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from each patient.
Stem Cell Mobilization
Within 89±35 minutes of successful PCI, patients were randomly assigned to subcutaneous G-CSF (Amgen GmbH) at a dose of 10 μg/kg body wt over a period of 6 days in addition to standard care or to standard care alone, including aspirin, clopidogrel, ACE inhibitors, -blockers, and statins in appropriate doses. Quantification of total white blood cells (WBCs) and CD34+ mononuclear blood stem cells (MNCCD34+) by flow cytometry was monitored from whole blood directly sampled before PCI and at days 4, 5, and 6 after PCI; parameters of blood viscosity were determined at baseline and at 6 and 35 days.19
Flow Cytometry (MNCCD34+ Enumeration)
Stem cell concentration in peripheral blood was obtained according to the IGLD protocol20; 100 μL of peripheral blood (anticoagulant K3-EDTA) was incubated for 15 minutes on ice with both pretitered CD45-fluorescein–conjugated (clone 2D1) and CD34-phycoerythrin–conjugated (clone 8G12) monoclonal antibody of 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 at 200g and 4°C; cells were washed 2 times with PBS at pH 7.2 and then measured in a FACSort flow cytometer (Becton Dickinson).
By use of 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 based on a combination of specific CD45 and side-scatter patterns of CD34+ cells, allowing exact calculation of the percentage of CD34-positive leukocytes down to 0.1%. The number of MNCCD34+ per μL was derived from the relation of CD34+ cells and WBCs of the blood sample (Figure 1).
Angiography and Quantitative Coronary Arteriography
Coronary and biplane LV angiograms were obtained according to standard acquisition guidelines both with initial PCI and at follow-up. Baseline ejection fraction (LVEF) and volumes were calculated by use of the area-length method21; coronary angiograms were evaluated for binary restenosis, (in-stent) late lumen loss, and minimal lumen diameter of the target lesion. Offline quantitative coronary arteriography was performed from digital cineangiograms by use of a MEDOS workstation and a validated edge-detection algorithm.22 Identical unforeshortened coronary projections were used and analyzed at baseline and at 6 months. Restenosis at follow-up was defined as a diameter stenosis of >50% within the stent ±5 mm.
Echocardiography and Dobutamine Challenge
Four days, 35±2 days, and 121±7 days (4 months) after facilitated PCI, resting and low-dose dobutamine echocardiography was performed to assess viable myocardium. In brief, dobutamine was infused at doses of 5 and 10 μg · kg–1 · min–1 in 5-minute stages on days 4±1 and 35±2 and after 4 months of PCI in conjunction with 2D echocardiographic imaging using a phased-array electronic ultrasound system in 4 standard views (parasternal long-axis and short-axis views and apical 4- and 2-chamber views) while 12-lead ECG and blood pressure were recorded continuously. Global function and regional LV wall motion were digitized by use of an ATL HDI 5000 ultrasound system and analyzed according to the Standards of the American Society of Echocardiography23; both intraobserver and interobserver variability of independent expert readers blinded to treatment allocation was low, with correlation coefficients of 0.96 and 0.94, respectively. For quantification of wall motion, the standardized 16-segment model was used, integrating segmental wall motion scores of 1=normal, 2=hypokinesis, 3=akinesis, and 4=dyskinesis23; wall motion score index (WMSI) was calculated as the sum of the scores of the segments divided by the number of the 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 thicknesses. Any possible effort was made to compare identical segments over time by adherence to recent expert recommendations.24 Early diastolic mitral flow velocity deceleration time was measured as a parameter of diastolic function correlating closely with LV end-diastolic pressure.25
18F-Deoxyglucose–Positron Emission Tomography
Baseline and follow-up 18F-deoxyglucose–positron emission tomography (18FDG-PET) imaging was performed in all 50 patients 5±1 and 128±6 days (4 months) after index STEMI by use of a whole-body scanner (Philips, IRIX Gamma PET III). Two hours after oral administration of 250 mg acipimox and 30 minutes after 50 g oral glucose, 350 to 470 MBq 18FDG was administered26,27; 2 diabetic patients were subjected to hyperinsulinemic euglycemic clamping.26 Image acquisition was started 40 minutes after 18FDG administration, and standardized quantitative image analysis was performed in a polar map display of the mean 18FDG uptake signal intensity in the 3 respective myocardial perfusion territories.
Data Collection and Follow-Up Examination
Clinical and laboratory data were collected prospectively, and follow-up visits were scheduled at 35 days and 4 months; standard C-reactive protein and fibrinogen measurements were accompanied by ELISA for interleukin-6 and transforming growth factor- (Biosource Diagnostics). Specific attention was paid to potential signs or symptoms of coronary events or arrhythmia. Low-dose dobutamine echocardiography was repeated on day 35 and after 4 months in all patients; 18FDG-PET studies were executed at 5±1 days and 4 months after index STEMI. Adherence to medication and 6-month coronary angiography according to the study flow chart were complete (see online-only Appendix).
Statistical Analysis
Continuous variables are presented as mean±SD; categoric variables were compared by use of 2 or Fisher’s exact test. Statistical comparisons between treatment groups were made by repeated-measures ANOVA or by paired or unpaired Student’s t test if appropriate. Repeated measurements were analyzed by 2-factor ANOVA to evaluate differences across time and between groups encompassing serological and hematological variables as well as morphological and functional echocardiographic parameters; reporting of the trial results followed established CONSORT guidelines.28,29 Statistical computing was performed by use of SPSS (Version 11.5, SPSS Inc.); for all tests, a probability level of P<0.05 was considered significant.
Results
Demographics, clinical characteristics, and comorbidity profiles were homogeneous and revealed a typical distribution of risk factors and evidence-based medication (Table 1). Angiographic and infarction-related characteristics showed a balanced distribution, including enzyme release and baseline LV function (Table 2). Moreover, time from onset of symptoms to PCI with stent placement was similar, at 299±101 minutes in the G-CSF group and 304±111 minutes in control subjects (P=0.86). No complications were associated with acute PCI, and all occluded infarct-related arteries were recanalized and stented with adjunctive abciximab infusion and subsequent clopidogrel loading; TIMI III flow was documented in all patients after PCI. Subcutaneous G-CSF injection was begun 89±35 minutes after reperfusion. Postinfarction medication was identical in both groups, with an oral intake of 100 mg aspirin, 75 mg clopidogrel, statins, and tailored ACE inhibitors and uptitrated -blocker medication. Four and 5 cases of binary restenosis at 6 months without resting flow limitation (TIMI III flow) were detected in the G-CSF and control groups, respectively; both late lumen loss and minimal lumen diameter were not different between groups (Table 2).
Parameters of Systolic and Diastolic LV Function
At baseline, all parameters of LV function were similar in both the G-CSF–treated and the control groups (Table 4). Interestingly, with G-CSF, LV end-diastolic diameter (LVEDD) showed no enlargement over a period of 4 months, whereas LVEDD increased to 58±4 mm in control subjects and was larger than with G-CSF (P=0.002). Moreover, with G-CSF, mean wall thickness in the infarct territory revealed enhancement by 0.7 mm at day 35 (P<0.01), a finding sustained after 4 months (P<0.01). Recovery of LVEF with G-CSF was documented at 35 days and over 4 months (P<0.01) and eventually measured 54±8% (P<0.001 versus control), whereas no longitudinal improvement was present in control subjects.
Similar to LVEF, resting WMSI revealed partial recovery with G-CSF, from 1.71±0.22 at baseline to 1.41±0.25 after 4 months (P<0.001), but no change in control subjects (Figure 2, A and B). Finally, deceleration time revealed improvement of diastolic function in both groups over 4 months; however, it was more pronounced after G-CSF (P<0.01).
Response to Inotropic Challenge
As in resting studies, the response of all LV functional parameters to dobutamine was similar in both groups at baseline (Table 4). After 35 days of follow-up, low-dose inotropic challenge enhanced both wall thickness and WMSI in patients treated with G-CSF to 1.18±0.33 mm wall thickness (P<0.001 versus control) and to 1.41±0.21 WMSI (P<0.004 versus control), respectively; improved response to inotropic challenge was sustained both for wall thickness and for WMSI at 4 months (Figure 2, C and D). Parameters such as LVEDD and LVEF failed to respond to inotropic challenge at 35 days; however, at 4 months, LVEDD was 53±5 mm with G-CSF and without any sign of progressive enlargement as found in control subjects (P<0.001). Similarly, with G-CSF, LVEF, which was unresponsive to inotropic challenge at 35 days, improved by 4% at 4 months to 59±7% (P<0.001 versus control). Deceleration time was not assessed under inotropic stimulation.
Myocardial Viability
In all patients myocardial 18FDG uptake was measured by PET both at baseline and at 4 months for longitudinal comparison (Table 5). Patients randomized to G-CSF revealed enhanced mean 18FDG uptake in the infarct territory after 4 months and greater 18FDG uptake differential than control subjects (Table 5). The G-CSF group was associated with myocardial 18FDG uptake enhanced by 11% after 4 months (P<0.001 versus baseline) versus none in control subjects (P<0.001). The noninfarct territories show no significant changes in either group.
Discussion
This is the first randomized study to use G-CSF to liberate MNCCD34+ over the initial 6 days of acute myocardial infarction. The strategy was safe, with no evidence of such adverse events as enhanced inflammation, thrombogenicity, electrical instability, or accelerated restenosis. G-CSF administration with reperfusion over 6 days exposes postischemic human myocardium to approximately 2.8x1010 mobilized MNCCD34+ within 8 days, enhances the chance of populating necrotic areas of the myocardium, and improves regional myocardial function with adrenergic challenge at day 35; with G-CSF, regional improvement was sustained after 4 months and was accompanied by both global and regional functional recovery and enhanced myocardial uptake of 18FDG in the infarct-related territory. Conversely, control patients failed to show any mild increase in LVEF at follow-up, as expected with PCI after 5 hours,30–32 an observation likely to result from both remote hyperkinesis at baseline and uptitrated oral -blockade at follow-up. Previous studies reported a 4% increase of LVEF at 6 months after PCI30 and a 3.7±11% increase with PCI within 4 to 6 hours of coronary occlusion31; similarly, the ADMIRAL trial revealed a 4.1% increase in LVEF after stenting of acute myocardial infarction.32 None of these studies, however, reported the fraction of patients on -blockers, which was probably low30–32; conversely, all FIRSTLINE-AMI patients were subject to uptitrated -blockade, were 10 years younger, had single-vessel disease without any chance for ischemic preconditioning, and thus were prone to adverse remodeling (Table 2), which may in turn explain the lower LVEF without G-CSF at follow-up (Table 3); the latter is corroborated by the lack of viability in the infarct-related region of the control group (Table 5).
Interestingly, experimental data in splenectomized mice exposed to G-CSF and stem cell factor have shown enhanced myocardial regeneration after translocation of bone marrow cells.11 However, despite potential differentiation into cardiomyocytes and vascular cells,12,33–35 recent observations have challenged the potential of bone marrow cells36,37 in favor of direct cytokine effects.38,39 Another critical issue seems to be the number of circulating stem cells.10–12 Although a mild increase of circulating MNCCD34+ has been observed with myocardial reperfusion,40 such an archaic natural defense reflex was not seen outside the setting of acute infarction, as evidenced by measurements in 10 age-matched healthy volunteers with 2.11±2.09 MNCCD34+/μL. The mild spontaneous increase in MNCCD34+ in control patients (without G-CSF stimulation; Table 4) is supported by additional findings of twice the normal CXCR4+ and CD34+ cells in human STEMI.41 Moreover, stromal cell–derived factor 1, required for cell engraftment, is upregulated within 24 hours of myocardial infarction but is not expressed later than 7 days.42 Nevertheless, it is not understood whether infarction may trigger mobilization of MNCCD34+ or whether some cytokines may set the stage for engraftment of circulating CD34/CXCR4+ stem cells in target tissue. With G-CSF, however, bone marrow mobilization enhanced exposure of MNCCD34+ to injured reperfused myocardium more than 10-fold over several days. Although intracoronary cell infusion may cause sludge and microemboli in animals,43 no evidence of impaired rheology or microcirculatory plugging by leukocytes (by elevated serum lactate, liver enzymes, creatine kinase-BB, or creatinine) was documented after G-CSF.
Another potentially beneficial effect may be derived from G-CSF–induced mobilization of leukocytes that are known to play an important role for infarct repair by regulating phagocytosis of necrotic tissue, fibroblast proliferation, and angiogenesis in the experimental reperfusion setting.38 Accordingly, in FIRSTLINE, the postischemic vascular bed was exposed to approximately 3.1x1013 total leukocytes over a period of 8 days, enabling an increased number of migrated neutrophils and macrophages to accelerate the healing process of human infarction. Moreover, there is experimental evidence of a G-CSF–dependent protection of cultured cardiomyocytes from apoptotic cell death through upregulation of Bcl-2 and Bcl-xL expression via the G-CSF receptor and the Jak-Stat pathway. In vivo experiments have shown that the G-CSF led to upregulation of G-CSF receptor and activation of the Stat-3 pathway, thereby preventing both cardiomyocyte apoptosis and remodeling after myocardial infarction; favorable effects of G-CSF were documented after 7 days of treatment, without any signs of enhanced cardiac engraftment of mobilized bone marrow cells.39 In contrast to intracoronary infusion of autologous bone marrow cells, MNCCD34+ mobilization by G-CSF differs in various ways: first, MNCCD34+ mobilization is noninvasive and requires only subcutaneous injections; second, bone marrow aspiration and preparation is not required (potentially difficult in acute patients); third, repeat catheterization with intracoronary infusion or intramyocardial injections are avoided; fourth, exposure to both G-CSF and to mobilized MNCCD34+ begins early after reperfusion in the susceptible phase39; and fifth, exposure of postischemic injured myocardium to mobilized MNCCD34+ and leukocytes is sustained over a period of 1 week at concentrations markedly exceeding natural cell mobilization in acute infarction. Whereas intracoronary delivery was enacted as early as 5 to 9 days14 or 4.3±1.5 days after the onset of necrosis,15 G-CSF–induced liberation of MNCCD34+ was initiated within 89 minutes of PCI. Moreover, Strauer et al14 delivered only 5.9x105 CD34+ cells, or 2.1±0.28% of all 2.8x107 MNCs harvested after overnight culture, whereas Schchinger et al15 infused 7.35±7.31x106 CD34/CD45+ cells per patient. Kocher et al12 transplanted 2x106 freshly isolated DiI-labeled human CD34+ cells/100 g rat; for the human setting, this translates into 3x108 to 6x108 cells on a weight-adjusted basis. Assuming an average blood flow of 0.8 mL · min–1 · g–1, 100 g of injured myocardium was exposed to approximately 2.8x1010 MNCCD34+ and 3.1x1013 leukocytes with G-CSF stimulation over 8 days (online-only Appendix); blood flow and tissue perfusion in the range of 0.8 mL · min–1 · g–1 was previously demonstrated after stenting with abciximab in the clinical setting of acute myocardial infarction.44
At present, there are no convincing data on body distribution of mobilized stem cells in human infarction. In a nonsplenectomized rat model, systemic delivery of 99mTc-labeled bone marrow–derived stem cells was limited by lung entrapment; a small fraction of less than 2% migrate and colonize infarcted heart.45,46 With this conservative (hypothetic) assumption, G-CSF would deliver 2.8x108 MNCCD34+ over several days, eg, 100- to 1000-fold more MNCCD34+ than one-time intracoronary delivery. Nevertheless, this calculation should be used with caution, because the quantity of engrafting MNCCD34+ in extracardiac organs is unknown without visualization of stem cells. In the aggregate, the present evidence is too scarce to conclude which method of administration and what number or nature of cells have relevance for potential improvement of cardiac performance.
Limitations
A G-CSF–induced mobilization strategy may have limitations, because no animal model resembles human coronary arteries with multiple unstable coronary plaque in the setting of acute myocardial infarction.47,48 Unstable coronary plaque could destabilize from mobilized leukocytes after G-CSF, and elevated WBCs may predict adverse outcomes.49 The recent MAGIC trial,50 for instance, reported an unexpectedly high rate of in-stent restenosis in the infarct-related vessel, although accelerated restenosis was also found after intracoronary bone marrow-derived stem cells. The controversial impact of G-CSF with (n=7) or without cell infusion (n=3) on in-stent restenosis, however, was deduced from 10 patients with angiographic follow-up and should be interpreted with caution, considering an expected binary restenosis rate of 16% in 25 FIRSTLINE-AMI patients and a clinical restenosis rate of 19% in the Danish STEMMI trial.51 Interestingly, patients enrolled in STEMMI and FIRSTLINE-AMI were between 50 and 55 years of age and had single-vessel obstruction, whereas MAGIC included heterogeneous patients even with chronic infarction and incomplete follow-up; moreover, G-CSF was initiated before plaque removal, potentially allowing cytokines and mobilized cells to destabilize a culprit lesion and promote accelerated restenosis.50 However, with G-CSF given after successful stenting in 65 patients, restenosis was found to be within the expected range.51,52 Finally, with abciximab given to all patients, neither impaired coronary microcirculation nor impaired rheology was seen, corroborating the safety profile of G-CSF in hematological patients.53,54 Similar to recent experience,50 inflammatory markers were not enhanced by G-CSF in the setting of infarction, confirming experimental findings without aggravated inflammation in murine infarcted heart.55 Conversely, enhanced myocardial uptake of 18FDG over time (versus control subjects) argues not only against potential deleterious effects of elevated MNCCD34+ or leukocytes in target tissue but for colonization of metabolically active cells. Although stem cell–derived cardiomyocytes, studied in vitro by patch-clamp techniques, show spontaneous activity, low dV/dT, prolonged action potential duration, and easily inducible triggered arrhythmias,56 no such arrhythmogenic phenomena were observed during continuous monitoring over 6 days and additional Holter recording on days 9±2 and 35±2 and at 4 months, probably because of lateral seeding of pluripotent MNCCD34+ rather than formation of islands of cells. In vivo imaging of labeled mobilized MNCCD34+, however, is not feasible, and mechanisms such as transdifferentiation of MNCCD34+ or aborted apoptosis of cardiomyocytes are difficult to elucidate in the human setting. Moreover, although transdifferentiation of MNCCD34+ has recently been challenged,36,37 G-CSF may have direct antiapoptotic potential,39 may activate prosurvival gene Akt 1 encoding for the Akt protein required to repair infarcted myocardium,57,58 or may accelerate the process of healing.38 At present, however, both the issue of plasticity and direct effects in humans are unresolved and should be subject to further investigation; in fact, MNCCD34+ may act via paracrine effects, with delivery of growth factors, which could in turn activate resident progenitor cells or other stem cells (c-kit+), eventually promoting tissue repair.
Although all 4 patients with restenosis in the G-CSF group revealed TIMI III flow at 6 months, a potentially inhomogeneous occurrence of in-stent hyperplasia with negative impact on functional recovery may not be fully excluded. In addition, the small trial size, with a strong male preponderance (92%), characterizes the pilot nature with limited power to generalize preliminary findings of functional preservation with G-CSF.
Conclusion
G-CSF after reperfusion of infarcted myocardium seems to be safe and feasible, was correlated with better preservation of ventricular function and less remodeling, and was not associated with aggravated post-PCI restenosis rate. This concept of myocardial preservation warrants further investigation, longer follow-up surveillance, and the scrutiny of multicenter, placebo-controlled trials.
Moreover, echocardiographic assessment of function may have inherent limitations as a result of 2D imaging compared with either radionuclide blood pool imaging or volumetric MRI. With a focus on safety and feasibility, a radiation burden had to be avoided in the pilot phase; however, an upcoming multicenter trial will implement sophisticated MRI. Finally, although open by design, this concept trial used randomization and blinded evaluation; additional trials should implement a double-blinded approach and independent evaluation, considering that the predominant findings are that G-CSF tended to blunt deteriorating cardiac function as seen in control subjects. Thus, the present beneficial effects of G-CSF should not be overemphasized.
Footnotes
The first 2 authors contributed equally to this work.
The online-only Data Supplement can be found with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.105.541433/DC1.
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Abstract
Background— Considering experimental evidence that stem cells enhance myocardial regeneration and granulocyte colony–stimulating factor (G-CSF) mediates mobilization of CD34+ mononuclear blood stem cells (MNCCD34+), we tested the impact of G-CSF integrated into primary percutaneous coronary intervention (PCI) management of acute myocardial infarction in man.
Methods and Results— Fifty consecutive patients with ST-segment elevation myocardial infarction were subjected to primary PCI stenting with abciximab and followed up for 6 months; 89±35 minutes after successful PCI, 25 patients were randomly assigned in this pilot study (PROBE design) to receive subcutaneous G-CSF at 10 μg/kg body weight for 6 days in addition to standard care, including aspirin, clopidogrel, an ACE inhibitor, -blocking agents, and statins. By use of CellQuest software on peripheral blood samples incubated with CD45 and CD34, mobilized MNCCD34+ were quantified on a daily basis. With homogeneous demographics and clinical and infarct-related characteristics, G-CSF stimulation led to mobilization of MNCCD34+ to between 3.17±2.93 MNCCD34+/μL at baseline and 64.55±37.11 MNCCD34+/μL on day 6 (P<0.001 versus control); there was no indication of leukocytoclastic effects, significant pain, impaired rheology, inflammatory reactions, or accelerated restenosis at 6 months. Within 35 days, G-CSF and MNCCD34+ liberation led to enhanced resting wall thickening in the infarct zone of between 0.29±0.22 and 0.99±0.32 mm versus 0.49±0.29 mm in control subjects (P<0.001); under inotropic challenge with dobutamine (10 μg · kg–1 · min–1), wall motion score index showed improvement from 1.66±0.23 to 1.41±0.21 (P<0.004 versus control) and to 1.35±0.24 after 4 months (P<0.001 versus control), respectively, coupled with sustained recovery of wall thickening to 1.24±0.31 mm (P<0.001 versus control) at 4 months. Accordingly, resting wall motion score index improved with G-CSF to 1.41±0.25 (P<0.001 versus control), left ventricular end-diastolic diameter to 55±5 mm (P<0.002 versus control), and ejection fraction to 54±8% (P<0.001 versus control) after 4 months. Morphological and functional improvement with G-CSF was corroborated by enhanced metabolic activity and 18F-deoxyglucose uptake in the infarct zone (P<0.001 versus control).
Conclusions— G-CSF and mobilization of MNCCD34+ after reperfusion of infarcted myocardium may offer a pragmatic strategy for preservation of myocardium and prevention of remodeling without evidence of aggravated restenosis.
Key Words: myocardial infarction remodeling cells angioplasty glucose
Introduction
Left ventricular (LV) remodeling after myocardial infarction is considered to set the stage for heart failure and premature death by 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,
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fetal cardiomyocytes or skeletal myoblasts) have succeeded in reconstituting heart muscle, these cells fail both to completely integrate structurally and to display characteristic physiological function.4–6 Conversely, bone marrow–derived pluripotent adult stem cells not only are capable of tissue differentiation7–9 but also are likely to regenerate myocardium by inducing myogenesis and angiogenesis, as shown by improved cardiac function in animals and preliminary human studies.10–16
Interestingly, there is mounting evidence that pluripotent cells enhance myocardial restoration regardless of their route of administration either by intramyocardial injection, by intracoronary and intravenous infusion, or even by mobilization with cytokines. Mobilization of a critical number of pluripotent cells is an interesting concept,11 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.17 Recent observations, however, have challenged myocardial regeneration in favor of direct cytokine effects.
In this context, we tested both the safety and impact of G-CSF in the setting of human myocardial infarction in conjunction with primary percutaneous coronary intervention (PCI) and abciximab18 in a randomized study with serial assessment of LV function and metabolism after 1 and 4 months and coronary morphology at 6 months.
Methods
Patients and PCI
FIRSTLINE-AMI was initiated in April 2003 and set up to recruit 50 consecutive patients with acute ST-elevation myocardial infarction (STEMI) subjected to primary PCI with stenting and abciximab administration according to recent guidelines18; after successful reperfusion, patients were randomized by use of the closed-envelope method in 1:1 allocation (with 25 subjects per group) to 10 μg/kg G-CSF over a period of 6 days in addition to standard care or to standard postinterventional care alone.
Patients between 18 and 65 years of age and 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, a 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 facilitated PCI using abciximab and modern bare-metal stents, randomization by the closed-envelope method was initiated. Both G-CSF recipients and control subjects were monitored continuously for arrhythmogenic or hemodynamic events over a period of 6 days and followed up for 6 months. The pilot study, using PROBE design with open-label application of G-CSF but blinded evaluation by expert readers unaware of patient group assignment (flow chart in online-only Appendix; see http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.105.541433/DC1), was approved by the Institutional Ethics Committee and the Ethics Review Board of the University of Rostock, Germany, and was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from each patient.
Stem Cell Mobilization
Within 89±35 minutes of successful PCI, patients were randomly assigned to subcutaneous G-CSF (Amgen GmbH) at a dose of 10 μg/kg body wt over a period of 6 days in addition to standard care or to standard care alone, including aspirin, clopidogrel, ACE inhibitors, -blockers, and statins in appropriate doses. Quantification of total white blood cells (WBCs) and CD34+ mononuclear blood stem cells (MNCCD34+) by flow cytometry was monitored from whole blood directly sampled before PCI and at days 4, 5, and 6 after PCI; parameters of blood viscosity were determined at baseline and at 6 and 35 days.19
Flow Cytometry (MNCCD34+ Enumeration)
Stem cell concentration in peripheral blood was obtained according to the IGLD protocol20; 100 μL of peripheral blood (anticoagulant K3-EDTA) was incubated for 15 minutes on ice with both pretitered CD45-fluorescein–conjugated (clone 2D1) and CD34-phycoerythrin–conjugated (clone 8G12) monoclonal antibody of 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 at 200g and 4°C; cells were washed 2 times with PBS at pH 7.2 and then measured in a FACSort flow cytometer (Becton Dickinson).
By use of 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 based on a combination of specific CD45 and side-scatter patterns of CD34+ cells, allowing exact calculation of the percentage of CD34-positive leukocytes down to 0.1%. The number of MNCCD34+ per μL was derived from the relation of CD34+ cells and WBCs of the blood sample (Figure 1).
Angiography and Quantitative Coronary Arteriography
Coronary and biplane LV angiograms were obtained according to standard acquisition guidelines both with initial PCI and at follow-up. Baseline ejection fraction (LVEF) and volumes were calculated by use of the area-length method21; coronary angiograms were evaluated for binary restenosis, (in-stent) late lumen loss, and minimal lumen diameter of the target lesion. Offline quantitative coronary arteriography was performed from digital cineangiograms by use of a MEDOS workstation and a validated edge-detection algorithm.22 Identical unforeshortened coronary projections were used and analyzed at baseline and at 6 months. Restenosis at follow-up was defined as a diameter stenosis of >50% within the stent ±5 mm.
Echocardiography and Dobutamine Challenge
Four days, 35±2 days, and 121±7 days (4 months) after facilitated PCI, resting and low-dose dobutamine echocardiography was performed to assess viable myocardium. In brief, dobutamine was infused at doses of 5 and 10 μg · kg–1 · min–1 in 5-minute stages on days 4±1 and 35±2 and after 4 months of PCI in conjunction with 2D echocardiographic imaging using a phased-array electronic ultrasound system in 4 standard views (parasternal long-axis and short-axis views and apical 4- and 2-chamber views) while 12-lead ECG and blood pressure were recorded continuously. Global function and regional LV wall motion were digitized by use of an ATL HDI 5000 ultrasound system and analyzed according to the Standards of the American Society of Echocardiography23; both intraobserver and interobserver variability of independent expert readers blinded to treatment allocation was low, with correlation coefficients of 0.96 and 0.94, respectively. For quantification of wall motion, the standardized 16-segment model was used, integrating segmental wall motion scores of 1=normal, 2=hypokinesis, 3=akinesis, and 4=dyskinesis23; wall motion score index (WMSI) was calculated as the sum of the scores of the segments divided by the number of the 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 thicknesses. Any possible effort was made to compare identical segments over time by adherence to recent expert recommendations.24 Early diastolic mitral flow velocity deceleration time was measured as a parameter of diastolic function correlating closely with LV end-diastolic pressure.25
18F-Deoxyglucose–Positron Emission Tomography
Baseline and follow-up 18F-deoxyglucose–positron emission tomography (18FDG-PET) imaging was performed in all 50 patients 5±1 and 128±6 days (4 months) after index STEMI by use of a whole-body scanner (Philips, IRIX Gamma PET III). Two hours after oral administration of 250 mg acipimox and 30 minutes after 50 g oral glucose, 350 to 470 MBq 18FDG was administered26,27; 2 diabetic patients were subjected to hyperinsulinemic euglycemic clamping.26 Image acquisition was started 40 minutes after 18FDG administration, and standardized quantitative image analysis was performed in a polar map display of the mean 18FDG uptake signal intensity in the 3 respective myocardial perfusion territories.
Data Collection and Follow-Up Examination
Clinical and laboratory data were collected prospectively, and follow-up visits were scheduled at 35 days and 4 months; standard C-reactive protein and fibrinogen measurements were accompanied by ELISA for interleukin-6 and transforming growth factor- (Biosource Diagnostics). Specific attention was paid to potential signs or symptoms of coronary events or arrhythmia. Low-dose dobutamine echocardiography was repeated on day 35 and after 4 months in all patients; 18FDG-PET studies were executed at 5±1 days and 4 months after index STEMI. Adherence to medication and 6-month coronary angiography according to the study flow chart were complete (see online-only Appendix).
Statistical Analysis
Continuous variables are presented as mean±SD; categoric variables were compared by use of 2 or Fisher’s exact test. Statistical comparisons between treatment groups were made by repeated-measures ANOVA or by paired or unpaired Student’s t test if appropriate. Repeated measurements were analyzed by 2-factor ANOVA to evaluate differences across time and between groups encompassing serological and hematological variables as well as morphological and functional echocardiographic parameters; reporting of the trial results followed established CONSORT guidelines.28,29 Statistical computing was performed by use of SPSS (Version 11.5, SPSS Inc.); for all tests, a probability level of P<0.05 was considered significant.
Results
Demographics, clinical characteristics, and comorbidity profiles were homogeneous and revealed a typical distribution of risk factors and evidence-based medication (Table 1). Angiographic and infarction-related characteristics showed a balanced distribution, including enzyme release and baseline LV function (Table 2). Moreover, time from onset of symptoms to PCI with stent placement was similar, at 299±101 minutes in the G-CSF group and 304±111 minutes in control subjects (P=0.86). No complications were associated with acute PCI, and all occluded infarct-related arteries were recanalized and stented with adjunctive abciximab infusion and subsequent clopidogrel loading; TIMI III flow was documented in all patients after PCI. Subcutaneous G-CSF injection was begun 89±35 minutes after reperfusion. Postinfarction medication was identical in both groups, with an oral intake of 100 mg aspirin, 75 mg clopidogrel, statins, and tailored ACE inhibitors and uptitrated -blocker medication. Four and 5 cases of binary restenosis at 6 months without resting flow limitation (TIMI III flow) were detected in the G-CSF and control groups, respectively; both late lumen loss and minimal lumen diameter were not different between groups (Table 2).
Parameters of Systolic and Diastolic LV Function
At baseline, all parameters of LV function were similar in both the G-CSF–treated and the control groups (Table 4). Interestingly, with G-CSF, LV end-diastolic diameter (LVEDD) showed no enlargement over a period of 4 months, whereas LVEDD increased to 58±4 mm in control subjects and was larger than with G-CSF (P=0.002). Moreover, with G-CSF, mean wall thickness in the infarct territory revealed enhancement by 0.7 mm at day 35 (P<0.01), a finding sustained after 4 months (P<0.01). Recovery of LVEF with G-CSF was documented at 35 days and over 4 months (P<0.01) and eventually measured 54±8% (P<0.001 versus control), whereas no longitudinal improvement was present in control subjects.
Similar to LVEF, resting WMSI revealed partial recovery with G-CSF, from 1.71±0.22 at baseline to 1.41±0.25 after 4 months (P<0.001), but no change in control subjects (Figure 2, A and B). Finally, deceleration time revealed improvement of diastolic function in both groups over 4 months; however, it was more pronounced after G-CSF (P<0.01).
Response to Inotropic Challenge
As in resting studies, the response of all LV functional parameters to dobutamine was similar in both groups at baseline (Table 4). After 35 days of follow-up, low-dose inotropic challenge enhanced both wall thickness and WMSI in patients treated with G-CSF to 1.18±0.33 mm wall thickness (P<0.001 versus control) and to 1.41±0.21 WMSI (P<0.004 versus control), respectively; improved response to inotropic challenge was sustained both for wall thickness and for WMSI at 4 months (Figure 2, C and D). Parameters such as LVEDD and LVEF failed to respond to inotropic challenge at 35 days; however, at 4 months, LVEDD was 53±5 mm with G-CSF and without any sign of progressive enlargement as found in control subjects (P<0.001). Similarly, with G-CSF, LVEF, which was unresponsive to inotropic challenge at 35 days, improved by 4% at 4 months to 59±7% (P<0.001 versus control). Deceleration time was not assessed under inotropic stimulation.
Myocardial Viability
In all patients myocardial 18FDG uptake was measured by PET both at baseline and at 4 months for longitudinal comparison (Table 5). Patients randomized to G-CSF revealed enhanced mean 18FDG uptake in the infarct territory after 4 months and greater 18FDG uptake differential than control subjects (Table 5). The G-CSF group was associated with myocardial 18FDG uptake enhanced by 11% after 4 months (P<0.001 versus baseline) versus none in control subjects (P<0.001). The noninfarct territories show no significant changes in either group.
Discussion
This is the first randomized study to use G-CSF to liberate MNCCD34+ over the initial 6 days of acute myocardial infarction. The strategy was safe, with no evidence of such adverse events as enhanced inflammation, thrombogenicity, electrical instability, or accelerated restenosis. G-CSF administration with reperfusion over 6 days exposes postischemic human myocardium to approximately 2.8x1010 mobilized MNCCD34+ within 8 days, enhances the chance of populating necrotic areas of the myocardium, and improves regional myocardial function with adrenergic challenge at day 35; with G-CSF, regional improvement was sustained after 4 months and was accompanied by both global and regional functional recovery and enhanced myocardial uptake of 18FDG in the infarct-related territory. Conversely, control patients failed to show any mild increase in LVEF at follow-up, as expected with PCI after 5 hours,30–32 an observation likely to result from both remote hyperkinesis at baseline and uptitrated oral -blockade at follow-up. Previous studies reported a 4% increase of LVEF at 6 months after PCI30 and a 3.7±11% increase with PCI within 4 to 6 hours of coronary occlusion31; similarly, the ADMIRAL trial revealed a 4.1% increase in LVEF after stenting of acute myocardial infarction.32 None of these studies, however, reported the fraction of patients on -blockers, which was probably low30–32; conversely, all FIRSTLINE-AMI patients were subject to uptitrated -blockade, were 10 years younger, had single-vessel disease without any chance for ischemic preconditioning, and thus were prone to adverse remodeling (Table 2), which may in turn explain the lower LVEF without G-CSF at follow-up (Table 3); the latter is corroborated by the lack of viability in the infarct-related region of the control group (Table 5).
Interestingly, experimental data in splenectomized mice exposed to G-CSF and stem cell factor have shown enhanced myocardial regeneration after translocation of bone marrow cells.11 However, despite potential differentiation into cardiomyocytes and vascular cells,12,33–35 recent observations have challenged the potential of bone marrow cells36,37 in favor of direct cytokine effects.38,39 Another critical issue seems to be the number of circulating stem cells.10–12 Although a mild increase of circulating MNCCD34+ has been observed with myocardial reperfusion,40 such an archaic natural defense reflex was not seen outside the setting of acute infarction, as evidenced by measurements in 10 age-matched healthy volunteers with 2.11±2.09 MNCCD34+/μL. The mild spontaneous increase in MNCCD34+ in control patients (without G-CSF stimulation; Table 4) is supported by additional findings of twice the normal CXCR4+ and CD34+ cells in human STEMI.41 Moreover, stromal cell–derived factor 1, required for cell engraftment, is upregulated within 24 hours of myocardial infarction but is not expressed later than 7 days.42 Nevertheless, it is not understood whether infarction may trigger mobilization of MNCCD34+ or whether some cytokines may set the stage for engraftment of circulating CD34/CXCR4+ stem cells in target tissue. With G-CSF, however, bone marrow mobilization enhanced exposure of MNCCD34+ to injured reperfused myocardium more than 10-fold over several days. Although intracoronary cell infusion may cause sludge and microemboli in animals,43 no evidence of impaired rheology or microcirculatory plugging by leukocytes (by elevated serum lactate, liver enzymes, creatine kinase-BB, or creatinine) was documented after G-CSF.
Another potentially beneficial effect may be derived from G-CSF–induced mobilization of leukocytes that are known to play an important role for infarct repair by regulating phagocytosis of necrotic tissue, fibroblast proliferation, and angiogenesis in the experimental reperfusion setting.38 Accordingly, in FIRSTLINE, the postischemic vascular bed was exposed to approximately 3.1x1013 total leukocytes over a period of 8 days, enabling an increased number of migrated neutrophils and macrophages to accelerate the healing process of human infarction. Moreover, there is experimental evidence of a G-CSF–dependent protection of cultured cardiomyocytes from apoptotic cell death through upregulation of Bcl-2 and Bcl-xL expression via the G-CSF receptor and the Jak-Stat pathway. In vivo experiments have shown that the G-CSF led to upregulation of G-CSF receptor and activation of the Stat-3 pathway, thereby preventing both cardiomyocyte apoptosis and remodeling after myocardial infarction; favorable effects of G-CSF were documented after 7 days of treatment, without any signs of enhanced cardiac engraftment of mobilized bone marrow cells.39 In contrast to intracoronary infusion of autologous bone marrow cells, MNCCD34+ mobilization by G-CSF differs in various ways: first, MNCCD34+ mobilization is noninvasive and requires only subcutaneous injections; second, bone marrow aspiration and preparation is not required (potentially difficult in acute patients); third, repeat catheterization with intracoronary infusion or intramyocardial injections are avoided; fourth, exposure to both G-CSF and to mobilized MNCCD34+ begins early after reperfusion in the susceptible phase39; and fifth, exposure of postischemic injured myocardium to mobilized MNCCD34+ and leukocytes is sustained over a period of 1 week at concentrations markedly exceeding natural cell mobilization in acute infarction. Whereas intracoronary delivery was enacted as early as 5 to 9 days14 or 4.3±1.5 days after the onset of necrosis,15 G-CSF–induced liberation of MNCCD34+ was initiated within 89 minutes of PCI. Moreover, Strauer et al14 delivered only 5.9x105 CD34+ cells, or 2.1±0.28% of all 2.8x107 MNCs harvested after overnight culture, whereas Schchinger et al15 infused 7.35±7.31x106 CD34/CD45+ cells per patient. Kocher et al12 transplanted 2x106 freshly isolated DiI-labeled human CD34+ cells/100 g rat; for the human setting, this translates into 3x108 to 6x108 cells on a weight-adjusted basis. Assuming an average blood flow of 0.8 mL · min–1 · g–1, 100 g of injured myocardium was exposed to approximately 2.8x1010 MNCCD34+ and 3.1x1013 leukocytes with G-CSF stimulation over 8 days (online-only Appendix); blood flow and tissue perfusion in the range of 0.8 mL · min–1 · g–1 was previously demonstrated after stenting with abciximab in the clinical setting of acute myocardial infarction.44
At present, there are no convincing data on body distribution of mobilized stem cells in human infarction. In a nonsplenectomized rat model, systemic delivery of 99mTc-labeled bone marrow–derived stem cells was limited by lung entrapment; a small fraction of less than 2% migrate and colonize infarcted heart.45,46 With this conservative (hypothetic) assumption, G-CSF would deliver 2.8x108 MNCCD34+ over several days, eg, 100- to 1000-fold more MNCCD34+ than one-time intracoronary delivery. Nevertheless, this calculation should be used with caution, because the quantity of engrafting MNCCD34+ in extracardiac organs is unknown without visualization of stem cells. In the aggregate, the present evidence is too scarce to conclude which method of administration and what number or nature of cells have relevance for potential improvement of cardiac performance.
Limitations
A G-CSF–induced mobilization strategy may have limitations, because no animal model resembles human coronary arteries with multiple unstable coronary plaque in the setting of acute myocardial infarction.47,48 Unstable coronary plaque could destabilize from mobilized leukocytes after G-CSF, and elevated WBCs may predict adverse outcomes.49 The recent MAGIC trial,50 for instance, reported an unexpectedly high rate of in-stent restenosis in the infarct-related vessel, although accelerated restenosis was also found after intracoronary bone marrow-derived stem cells. The controversial impact of G-CSF with (n=7) or without cell infusion (n=3) on in-stent restenosis, however, was deduced from 10 patients with angiographic follow-up and should be interpreted with caution, considering an expected binary restenosis rate of 16% in 25 FIRSTLINE-AMI patients and a clinical restenosis rate of 19% in the Danish STEMMI trial.51 Interestingly, patients enrolled in STEMMI and FIRSTLINE-AMI were between 50 and 55 years of age and had single-vessel obstruction, whereas MAGIC included heterogeneous patients even with chronic infarction and incomplete follow-up; moreover, G-CSF was initiated before plaque removal, potentially allowing cytokines and mobilized cells to destabilize a culprit lesion and promote accelerated restenosis.50 However, with G-CSF given after successful stenting in 65 patients, restenosis was found to be within the expected range.51,52 Finally, with abciximab given to all patients, neither impaired coronary microcirculation nor impaired rheology was seen, corroborating the safety profile of G-CSF in hematological patients.53,54 Similar to recent experience,50 inflammatory markers were not enhanced by G-CSF in the setting of infarction, confirming experimental findings without aggravated inflammation in murine infarcted heart.55 Conversely, enhanced myocardial uptake of 18FDG over time (versus control subjects) argues not only against potential deleterious effects of elevated MNCCD34+ or leukocytes in target tissue but for colonization of metabolically active cells. Although stem cell–derived cardiomyocytes, studied in vitro by patch-clamp techniques, show spontaneous activity, low dV/dT, prolonged action potential duration, and easily inducible triggered arrhythmias,56 no such arrhythmogenic phenomena were observed during continuous monitoring over 6 days and additional Holter recording on days 9±2 and 35±2 and at 4 months, probably because of lateral seeding of pluripotent MNCCD34+ rather than formation of islands of cells. In vivo imaging of labeled mobilized MNCCD34+, however, is not feasible, and mechanisms such as transdifferentiation of MNCCD34+ or aborted apoptosis of cardiomyocytes are difficult to elucidate in the human setting. Moreover, although transdifferentiation of MNCCD34+ has recently been challenged,36,37 G-CSF may have direct antiapoptotic potential,39 may activate prosurvival gene Akt 1 encoding for the Akt protein required to repair infarcted myocardium,57,58 or may accelerate the process of healing.38 At present, however, both the issue of plasticity and direct effects in humans are unresolved and should be subject to further investigation; in fact, MNCCD34+ may act via paracrine effects, with delivery of growth factors, which could in turn activate resident progenitor cells or other stem cells (c-kit+), eventually promoting tissue repair.
Although all 4 patients with restenosis in the G-CSF group revealed TIMI III flow at 6 months, a potentially inhomogeneous occurrence of in-stent hyperplasia with negative impact on functional recovery may not be fully excluded. In addition, the small trial size, with a strong male preponderance (92%), characterizes the pilot nature with limited power to generalize preliminary findings of functional preservation with G-CSF.
Conclusion
G-CSF after reperfusion of infarcted myocardium seems to be safe and feasible, was correlated with better preservation of ventricular function and less remodeling, and was not associated with aggravated post-PCI restenosis rate. This concept of myocardial preservation warrants further investigation, longer follow-up surveillance, and the scrutiny of multicenter, placebo-controlled trials.
Moreover, echocardiographic assessment of function may have inherent limitations as a result of 2D imaging compared with either radionuclide blood pool imaging or volumetric MRI. With a focus on safety and feasibility, a radiation burden had to be avoided in the pilot phase; however, an upcoming multicenter trial will implement sophisticated MRI. Finally, although open by design, this concept trial used randomization and blinded evaluation; additional trials should implement a double-blinded approach and independent evaluation, considering that the predominant findings are that G-CSF tended to blunt deteriorating cardiac function as seen in control subjects. Thus, the present beneficial effects of G-CSF should not be overemphasized.
Footnotes
The first 2 authors contributed equally to this work.
The online-only Data Supplement can be found with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.105.541433/DC1.
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