Repeated Intracoronary Infusion of Peripheral Blood … X-Circ J. (2011).pdfPeople’s Hospital, Nanjing Medical University, Nanjing (K.C.), China; and Center for Cardiovascular Biology

Circulation Journal Vol.75, April 2011

Circulation JournalOfficial Journal of the Japanese Circulation Societyhttp://www.j-circ.or.jp

ecent studies have suggested that granulocyte-colony stimulating factor (G-CSF) used alone and/or in a combination with intracoronary peripheral blood stem

cells (PBSC) delivery might alleviate cardiac remodeling, improve heart function and myocardium perfusion in post-myocardial infarction (MI) models.1–5 Some clinical studies, however, have generated inadequate evidence showing the efficacy of G-CSF with and without stem cell infusions in post-MI patients.6–9 A correlationship between the number of

cells infused and clinical outcome has been proposed recent-ly.10 Repeated skeletal myoblast implantation reportedly improves left ventricular (LV) function potentially as a more effective cell delivery method.11 Stem cell mobilization by repeated administration of G-CSF together with a single intra-coronary stem cell infusion might reduce angina frequency and increase exercise tolerance, but make no change in car-diac perfusion and function.12

Received September 9, 2010; revised manuscript received October 26, 2010; accepted November 25, 2010; released online February 11, 2011 Time for primary review: 20 days

Department of Cardiology, Northern People’s Hospital, Medical College of Yangzhou University, Yangzhou (X.G., Y.X., J.G., L.S., S.H., R.X., J.D., J.Z., F.H., H.X., M.L.); Department of Cardiology, Jiangsu Institute of Cardiovascular Diseases, Jiangsu Provincial People’s Hospital, Nanjing Medical University, Nanjing (K.C.), China; and Center for Cardiovascular Biology & Atherosclerosis Research, Department of Internal Medicine, School of Medicine, University of Texas Health Science Center at Houston & the Labo-ratory of Heart Failure & Stem Cell Research, Texas Heart Institute, Houston, TX (Y.G.), USA

Grant support: Project supported by the Science Committee (BS2004532) and Health Department of Jiangsu Province (Z200514); Pro-gram for Jiangsu Province Outstanding Medical Talented Leader (JS2006038).

Mailing address: Xiang Gu, MD, PhD, Department of Cardiology, Northern People’s Hospital, Medical College of Yangzhou University, Nantong West Road 98, Yangzhou 225001, China. E-mail: [email protected]

ISSN-1346-9843 doi: 10.1253/circj.CJ-10-0898All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected]

Repeated Intracoronary Infusion of Peripheral Blood Stem Cells With G-CSF in Patients

With Refractory Ischemic Heart Failure– A Pilot Study –

Xiang Gu, MD, PhD; Yong Xie, MD, PhD; Jian Gu, BSc; Lei Sun, BSc; Shenghu He, BSc; Rixin Xu, BSc; Junfei Duan, BSc; Jianye Zhao, BSc; Fei Hang, BSc; Houtian Xu, BSc;

Minghui Li, PhD; Kejiang Cao, MD, PhD; Yongjian Geng, MD, PhD

Background: Recent investigations have suggested the clinical efficacy of granulocyte colony-stimulating factor (G-CSF) infusion alone or in combination with a single dose delivery of peripheral blood stem cells (PBSC) infusion in patients with myocardial infarction (MI) and congestive heart failure (HF). The current study tested the feasibility and effect of repeated intracoronary infusions PBSC and the mobilization of G-CSF in patients with refractory HF after MI.

Methods and Results: Patients with recent large MI and a lower left ventricular ejection fraction (LVEF) were enrolled into one of the following 3 groups: Group R (n=15) received repeated intracoronary infusion of PBSC and one-dose of G-CSF; Group S (n=15) received a single infusion of PBSC and a G-CSF dose; and Group C (n=15) received neither PBSC nor a G-CSF dose. Cardiac performance was evaluated by echocardiography and single photon-emission computed tomography (SPECT). All the patients underwent 12-month follow-up. LVEF in Group R (47.00±4.90%) was significantly higher than that in Group S (44.40±3.87%, P<0.01) and Group C (40.80± 3.41%, P<0.01). Similarly, the improvement of myocardial perfusion assessed by SPECT in Group R was more than that in Group S (P=0.012) and Group C (P<0.01). Neither death nor new MI occurred.

Conclusions: Repeated intracoronary infusions of PBSC plus mobilization of G-CSF might be an optional effective strategy for treating patients with refractory HF after recent large MI. (Circ J 2011; 75: 955 – 963)

Key Words: Granulocyte colony-stimulating factor; Heart failure; Heart performance; Myocardial infarction; Stem cells

R

ORIGINAL ARTICLERegenerative Medicine


956 GU X et al.

Editorial p 789

In this study, we tested the hypothesis that repeated deliv-ery of PBSC might result in better outcome and significant clinical improvement in cardiac function. We evaluated the feasibility and efficacy of repeated intracoronary infusion of PBSC following by mobilization of G-CSF in patients with refractory heart failure (HF) after recent massive MI.

MethodsStudy ProtocolThe current phase-I study was a prospective, open-labeled and controlled trial, designed to investigate the safety, feasi-bility, and effectiveness of repeated intracoronary infusions of PBSC and the mobilization of G-CSF in patients with refractory HF after a recent large MI. Outcomes were deter-mined in a blinded fashion. The protocol was conducted in accordance with the Helsinki declaration and approved by the ethics committee of the Northern People’s Hospital. A written

informed consent was obtained from the patients after having explained the procedure and its risks. Under standard medi-cal and primary percutaneous coronary intervention (PCI) therapy, those patients with refractory HF after a recent large MI were enrolled. To induce proliferation and mobilization of PBSC, we initiated pharmacological stimulation with G-CSF for the cell therapy group. After stimulating the presence of PBSC in the peripheral blood, we performed cell apheresis and the first intracoronary PBSC infusion. The PBSC suspen-sion was injected into the infarct-related region through a balloon catheter. The patients in the repeated infusion group were scheduled to receive the second intracoronary PBSC infusion 6 months later. The PBSC dose was similar for each patient and each timepoint. Patients were followed up to 12 months after the first PBSC transplantation. Change in LV ejection fraction (LVEF) measured by M-mode echocar-diography was the primary endpoint. The second endpoint was the change in LV end-diastolic diameter (LVEDd), LV shortening fraction (LVFS), the myocardial perfusion, exer-cise capacity on a 6-min walking test and New York Heart Association (NYHA) class. All the analyses were performed

Figure 1. CD34+ cell populations from peripheral blood measured by flow cytometry. (A) The representative R2 population in a patient demonstrates significant increase of CD34+ cells at day 5 of treatment with G-CSF (b), compared with baseline (a). A ratio of CD34+ cells in peripheral blood and cell suspension was 0.01% in (a), 0.02% in (b) and 0.7% in (c) respectively. Blue bars=Baseline; Red bars=5 days. (B) Comparison of quantitative CD34+ cells from peripheral blood between cell therapy groups. CD34+ cell populations rose in cell therapy groups (P<0.01), but there was no significant difference between cell therapy groups (P>0.05). Group C, control group; Group S, single infusion group; Group R, repeated infusion group. G-CSF, granulocyte colony-stimulating factor.


957Repeated Infusion of PBSC With G-CSF in HF

by experienced operators who were blinded to the study pro-tocol. Echocardiography was performed at baseline, 6, and 12 months after first transplantation. In the cell therapy group, Tc 99m Sestamibi (MIBI) single-photon emission computed tomography (SPECT) was performed for measuring myocar-dial perfusion. In the repeated infusion group, the coronary angiography was done for evaluating in stent restenosis rate (ISR) at 6-months follow-up. All patients received the same or similar medications, including aspirin, clopidogrel, β-blocker, angiotensin converting enzyme inhibitor or angiotensin-receptor blocker, and a statin unless contraindicated. All data and measured values were obtained prospectively.

Patient PopulationA total of 45 patients with stable HF and with a large (ST elevation ≥4 limb or precordial leads) recent MI (≤3 months from symptom onset) were enrolled in the phase I clinical

study as approved by the ethics committee. Among them, 15 patients who refused cell therapy were assigned to the control group (Group C), 15 patients who were willing to receive repeated cell therapy were assigned to Group R, and the remaining 15 patients were allocated to Group S. Inclusion criteria was symptomatic stable HF with a recent MI and LVEF ≤45%. Exclusion criteria were chronic atrial fibrillation or left bundle-branch block, recent major surgery, known major diseased coronary artery not suitable for cell infusion, apoplexy, malignancies, younger than 18 years or older than 80 years, or any other life-threatening conditions.

PBSC Mobilization, Separation, Collection and PurificationFor patients who received the stem cell therapy, an open-label dose of G-CSF (Lunan Biopharmaceutical Co Ltd) at 10 μg · d–1 · kg–1 of body weight was administered subcutane-ously within a 12 h interval for 4–6 days. The group C did not

Table 1. Baseline Characteristics of Eligible Patients

Characteristic Group C (n=15)

Group S (n=15)

Group R (n=12)

Age, years 58.88±8.90　 59.50±7.96　 59.26±8.37　

Gender, male, n (%) 15 (100)　 14 (93)　 12 (100)　

Hypertension, n (%) 10 (66.7)　 9 (60)　 8 (66.7)

Hyperlipidemia, n (%) 7 (46.7) 8 (53.3) 7 (58.3)

Diabetes, n (%) 6 (40)　 5 (33.3) 5 (41.7)

Previous MI, n (%) 2 (13.3) 2 (13.3) 2 (16.7)

Previous PCI, n (%) 2 (13.3) 2 (13.3) 1 (8.3)　

NYHA class >– II, n (%) 15 (100)　 15 (100)　 12 (100)　

Plasma creatinine >– 170μmol/L, n (%) 1 (6.7)　 1 (6.7)　 2 (16.7)

Peak CK-MB value, u 78.4±46.2 88.7±42.3 90.4±36.8

Time from the onset to the first PBSC infusion, days 16.1±6.9　 17.7±7.5　 16.6±8.2　

Smoking, n (%) 7 (46.7) 8 (53.3) 7 (58.3)

Thrombolysis before PCI, n (%) 8 (53.3) 7 (46.7) 6 (50)　

Infarction extent

Extensive antetheca, n (%) 10 (66.7)　 9 (60)　 9 (75)　

Extensive antetheca and posterior wall, n (%) 2 (13.3) 3 (20)　 2 (16.7)

Extensive antetheca and inferior wall, n (%) 3 (20)　 3 (20)　 1 (8.3)　

Extent of coronary disease and PCI situation

1-vessel, n (%) 8 (53.3) 5 (53.3) 5 (41.7)

2-vessel, n (%) 3 (20)　 3 (20)　 4 (33.3)

3-vessel, n (%) 2 (13.3) 2 (13.3) 3 (25)　

Complete revascularization, n (%) 12 (80)　 11 (73.3)　 9 (75)　

Medication at baseline

ACEI or ARB, n (%) 9 (60)　 10 (66.7)　 8 (66.7)

Nitrates, n (%) 9 (60)　 10 (66.7)　 7 (58.3)

β-blockers, n (%) 8 (53.3) 9 (60)　 7 (58.3)

Diuretics, n (%) 9 (60)　 10 (66.7)　 10 (83.3)　

Mean furosemide dose, mg/d 32.7±8.8　 33.3±7.2　 35.0±5.2　

Medication at 12-month follow up

ACEI or ARB, n (%) 11 (73.3)　 12 (80)　 10 (83.3)　

Nitrates, n (%) 12 (80)　 12 (80)　 11 (91.7)　

β-blockers , n (%) 12 (80)　 12 (80)　 10 (83.3)　

Diuretics , n (%) 7 (46.7) 6 (40)　 3 (25)*　

Mean furosemide dose, mg/d 30.7±8.0　 27.3±7.0*　 23.3±4.9*　

Values expressed as number (percent) or mean ± SD.Group C, control group; Group S, singler intracoronary infusions group; Group R, repeated intracoronary infusions group; MI, myocardial infarction; PCI, percutaneous coronary intervention; NYHA, New York Heart Association; CK, creatine kinase; PBSC, peripheral blood stem cells; ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin-receptor blockers.*P<0.05 vs. baseline.


958 GU X et al.

receive a placebo. Blood tests (including, blood chemistry, C-reactive protein (CRP), troponin I, and creatine kinase – MB (CK-MB)), electrocardiogram (ECG), and clinical review were performed before and after PBSC mobilization. Full blood counts were monitored daily and mononuclear cell CD34+ (MNCCD34+) counts were evaluated by flow cytome-try (Becton Dickinson) with anti-CD34+ antibodies (Beckman Couter Biotechnology Inc) MNCCD34+ per μl was derived from the relation of CD34+ cells and white blood cells in the blood sample. The optimal time for apheresis was established based on the MNCCD34+ ratio in peripheral blood mono-nuclear cells. Circulating mononuclear cells were collected using a blood cell separator (Baxter Fanwal Inc). A total of 4,000 to 10,000 ml blood was processed according to practical required volume. The cytopheresis product was centrifuged using the cell processor to deplete blood platelets. Mononu-clear cells were washed 3 times with phosphate-buffered saline, then measured and adjusted to a final concentration of 7–9×104 CD34+ cells/ml in a flow cytometry (Figure 1). The a single infusion dose of mononuclear cells was 1.5 to 2×108 to guarantee the minimum dose of 1×106 CD34 + cells per patient. Bacterial and fungal cultures of the cell prepara-tions were performed and found negative. The number of surviving cells as assessed by trypan blue staining was more than 95%. Time interval between harvest of stem cells and injection was less than 3 h. The number of the infusion cells in therapy groups was similar. Group R patients had their sur-plus cell suspension kept in freezing medium at –80°C for use in the next transplantation.

Intracoronary Infusion of PBSCTo obtain sufficient coronary blood flow at the infarct-related region, The DES stent was implanted into the infarct-related coronary artery (IRA). Patients were considered not qualified for the protocol if the IRA could not be passed through. An over-the-wire balloon catheter (Maverick, Boston Scientific) was placed at the proximal, middle, and distal end of the IRA. PBSC suspension was injected into the IRA through central lumen of the balloon catheter. The injected volume at each position of the IRA was approximately 5 ml for a total of 15–20 ml per patient. Intracoronary doses of PBSC for the cell therapy groups were similar. There was no intracoronary placebo infusion applied to the control group. A balloon infla-tion, stop-flow technique was not used for injecting the cell suspension. In addition, we performed a contrast medium backflow study to assess whether the cell suspension reflux from the IRA. All angiography images were digitally stored for offline analysis. ECG and routine blood examinations were performed the day after the procedure.

Repeated injection of PBSC was conducted 6 months later after patients received the first cell therapy under a similar procedure and dose of cells. In brief, the frozen monocytes were thawed in the 37°C water. The samples were taken for phenotyping, microbiology testing and living cell assessment. The number of survival cells assessed by trypan blue staining was more than 90%. The remaining steps of the intracoronary infusion were performed as previously described.

Clinical evaluation for the 3 groups included history and physical examination, G-CSF-related complaints, laboratory

Table 2. Comparison of Quantitative Date From Eligible Patients

Group C(n=15)

Group S(n=15)

Group R(n=12)

LVEF (%)

Baseline 39.20±3.05　 39.93±4.17　 38.08±4.34　

6 months 39.87±3.02　 42.80±3.51*,† 42.50±4.85*,†

12 months 40.80±3.41　 44.40±3.87*,† 47.00±4.90*,**,†,††

LVEDd (mm)

Baseline 60.80±2.43　 60.73±2.71　 62.67±3.39　

6 months 60.27±2.34　 56.00±2.88*,† 60.50±3.37†,††

12 months 60.13±2.67　 55.53±3.60*,† 54.92±3.60*,**,†,††

LVFS (%)

Baseline 18.00±1.60　 18.47±1.77　 18.66±2.10　

6 months 19.07±2.05　 23.87±2.97*,† 23.08±3.03*,†

12 months 19.47±3.07*　 24.53±3.56*,† 27.50±2.81*,**,†,††

Myocardial perfusion defect, % (SPECT)

Baseline 41.40±3.50　 43.73±3.20　 43.17±2.98　

6 Months 37.60±3.09*　 37.73±3.03*,† 38.50±3.48*,†

12 Months 36.33±3.13*　 35.73±2.37*,† 32.83±3.83*,**,†,††

6-min walking test distance (m)

Baseline 212.47±15.79　 215.80±17.63　 207.17±14.17　

6 months 219.87±17.76　 247.13±33.25*,† 234.58±16.13*,†,††

12 months 233.27±25.66*　 257.73±33.29*　 264.00±14.01*,**

NYHA class

Baseline 3.27±0.59 3.26±0.52 3.33±0.78

6 months 3.00±0.65 2.53±0.64*,† 2.67±0.65*,†

12 months 2.86±0.74* 2.30±0.83* 1.75±0.45*,**,†,††

Values expressed as mean ± SD.LVEF, left ventricular ejection fraction; LVEDd, left ventricular end-diastolic dimension; LVFS, left ventricular short-ening fraction; SPECT, single photon emission computed tomography. Other abbreviations as in Table 1.*P<0.05 vs. baseline; **P<0.05 vs. 6 months; †P<0.05 vs. control group; ††P<0.05 vs. single group.



evaluation (complete blood count, blood chemistry, CRP, CK-MB and troponin serum concentrations), echocardiogram, 6-min walking test, and 24-h dynamic electrocardiography monitoring scheduled at baseline, 6, and 12 months. Patients were followed by out-patient clinic and telephone. All of patients underwent the above-mentioned evaluations before and after the procedure. In the repeated cell therapy group, CAG evaluated restenosis rate at month 6 follow-up. All images were stored for offline analysis. ISR was defined as a diameter of stenosis >50% within the stented segments includ-ing 5-mm segments from stent margin on CAG. All studies were processed and evaluated by experienced operators who were blinded to the assigned therapy.

Assessment of Cardiac Function and Myocardial PerfusionThe echocardiography was performed by a single senior tech-nician. LVEF, LVED, LVFS and other parameters were ob-tained according to standard protocols. Images were digitally stored and the offline analysis was conducted by a senior reader. If there was an obvious discrepancy, it would be assessed by a third observer. The rest MIBI SPECT (GE Hawkeye Millennium VG5, USA) was performed for measuring myo-cardial perfusion at baseline and at follow-up. Studies were assessed by experienced observers blinded to all patient data. For MIBI SPECT, patients received 300 MBq MIBI IV, and resting tomography was performed >30 min later. SPECT in-volved a Butterworth cut-off frequency of 0.45, with an order of 5 and the reconstructed data created along 3 oblique-axis

planes (short axis, vertical long axis and horizontal long axis) of the heart. Quantitative analysis involved use of Cedars quantitative perfusion SPECT. Perfusion defects were calcu-lated by a scintigraphic “bulls-eye” technique.

Statistical AnalysisContinuous variables were expressed as the mean ± standard deviations (mean ± SD). SPSS 16.0 software was used for sta-tistical analysis. Comparisons among groups were performed by one-way analysis of variance (ANOVA) and least-signifi-cant difference contrast. A value of P<0.05 was considered statistically significant.

ResultsBaseline Clinical Characteristics of PatientsAmong 45 patients enrolled in this study 42 patients completed the protocol. The remaining 3 patients were within Group R who did not complete the stem cell therapy: 2 patients had IRA that could not be crossed; 1 patient was withdrawn due to an accelerating ventricular rhythm after apheresis. The base-line characteristics of all the remaining 42 eligible patients were summarized and well matched in Table 1. The mean time from onset of acute MI (AMI) to first cell transplantation was 16.8±7.5 days. Mean time interval between the 2 cell in-fusions was 6.1±1.2 months. The PBSC infusion doses were not significantly different between the 2 groups receiving their first transplantation (1.30±0.21×106 vs. 1.31 ±0.23×106,

Figure 2. Echocardiographic evaluation of LVEF and LVEDd. (A) Quantification by M-mode echocardiographic analysis of the LVEF. There is significant improve-ment of LVEF (Group R vs. Group S, P= 0.001; Group R vs. Group C, P<0.001, respectively). Blue bars=Baseline; Red bars=6 months; Yellow bars=12 months. (B) Representative 2-D echocardiographic analysis shows decreased gradu-ally LVEDd at baseline (a), and month 6 (b), and month 12 (c) in Group R. LVEF, left ventricular injection fraction; LVEDd, left ventricular end-diastolic diameter. Other abbreviations as in Figure 1.


960 GU X et al.

P=0.115) and the 2 timepoints for Group R (1.30±0.29×106 vs. 1.29±0.35×106, P=0.104). At baseline, there were no sig-nificant differences in medical treatment among the 3 groups. At the 12 month follow up, the proportion of patients of using diuretics in Group R is significantly decreased from 83.3% at baseline to 25% (P=0.006), whereas there were no signifi-cant changes in Group S (from 66.7% to 40%, P=0.136) and Group C (from 60.0 to 46.7%, P=0.136). Meanwhile, the re-duction of mean furosemide dose had a similar tendency (from 35.0±5.2 mg to 23.3±4.9 mg, P≤0.001) in Group R (from 33.3±7.2 mg to 27.3±7.0 mg, P=0.003), in Group S and (from 32.7±8.8 mg to 30.7±8.0 mg, P=0.082) Group C.

Cardiac Function and Myocardial PerfusionThere were no significant differences in the baseline values among the 3 groups. An increase in LVEF measured by quan-titative echocardiography was observed in Group R from baseline to month 6 and month 12 (from 38.08±4.34 to 42.50± 4.85%, P=0.032; from 38.08±4.34 to 47.00±4.90%, P<0.001). Likewise, 6 months and 12 months after the first PBSC trans-plantation, the LVEF increased from 39.93±4.17% at base-line to 42.80±3.51% (P=0.048), and to 44.40±3.87 (P=0.003) in Group S. In Group C, there were no significant changes

during the same period (from 39.20±3.05% to 39.87±3.02%, P=0.567; from 39.20±3.05% to 40.08±3.41%, P=0.173) (Table 2). Moreover, patients in Group S and Group R at month 12 had higher LVEF (P=0.023 and P<0.001) and LVFS (P<0.001 and P<0.001) and lower LVEDd (P<0.001 and P<0.001) respectively when compared with Group C. Furthermore, at 12 months the mean improvement of LVEF was more significant in Group R than in Group S (47.00± 4.90 vs. 44.40±3.87, P=0.001) and in Group C (47.00±4.90 vs. 40.80±3.41, P<0.001). The mean improvement of LVEF from month 6 to month 12 was more significant (4.5%) in Group R than (1.6%) in Group S (P=0.013) and Group C (P= 0.001). Echocardiographic evaluation of LVEF and LVEDd are shown in Figure 2. At month 12, SPECT shows that there was significant difference in quantification of perfusion defect change in Group R and Group S, compared with Group C (P<0.001 and P=0.006). At month 12, the reduced myocardial perfusion in Group R was more obvious than that in Group S (P=0.034). Representative rest images of Group R and quan-titative myocardial perfusion defect changes assessed by SPECT at baseline and at 6, 12 months are shown in Figure 3. Accordingly, a significant change in NYHA classification and 6-min walking distance were observed in all 3 groups at

Figure 3. Myocardial perfusion de-fect assesed by SPECT. (A) At month 12, the difference in quantification of perfusion defect change was sig-nificant in Group R, compared with Group C and Group S. Blue bars= Baseline; Red bars=6 months; Yellow bars=12 months. (B) Representative myocardial perfusion image improve-ment assessed by SPECT at baseline, month 6 and month 12 in Group R. Note myocardial perfusion improved gradually from a to c. SPECT, single photon-emission computed tomog-raphy.



12 months (from 3.27±0.59 to 2.86±0.74 and from 212.47± 15.79 to 233.27±25.66 in Group C; from 3.26±0.52 to 2.30± 0.83 and from 215.80±17.63 to 257.73±33.29 in Group S; and from 3.33±0.78 to 1.75±0.45 and from 207.17±14.17 to 264.00±14.01 in Group R, all P<0.05).However, the improve-ment of NYHA class was more notable in Group R than in Group S (P=0.024) and the Group C (P<0.001) (Table 2).

Safety EvaluationThere were no major peri-procedural complications. Two patients in Group R had 3 transient episodes of angina (last-ing for 3–9 min) with TIMI blood flow slowdown and ST depression during PBSC infusion. The blood flow and ST segment soon returned to normal levels with relieved angina while the coronary artery being washed with heparin water and reinsertion of the balloon catheter being into the distal end of target coronary artery. During G-CSF administration, 4 patients complained of some symptoms: bone pain (n=2), headache (n=1) and dizziness (n=1). The concentrations of CRP indicating inflammatory response at baseline among the 3 groups showed no significant difference (all P>0.05). CRP was not significantly different from the peri-procedural period and follow-up (all P>0.05). Following daily subcutane-ous G-CSF for 5–6 days, a ratio of CD34+ cells from periph-eral blood measured by flow cytometry in the cell therapy group rose but there was no significant difference between cell therapy groups (P>0.05) (Figure 1). There were no sus-tained ventricular arrhythmias by ECG monitoring. The number of premature ventricular contractions was similar in the 3 groups. There was a 50% in stent restenosis in IRA of a patient in Group R at 6 months follow up. There were no increases in troponin T or CK-MB indicating myocardial damage during the procedure. Follow-up results demonstrated no re-infarction, apoplexy, tumor or death in the study. Finally, there was no contrast regurgitation observed in any contrast backflow tests (Figure 4).

DiscussionThe current data provide the first evidence that repeated intra-coronary PBSC infusions plus one-time mobilization of GSF in patients with refractory HF following recent MI is a feasi-ble and safe procedure. The two-time PBSC infusions resulted in more striking improvement in cardiac performance during 12-month follow-up than a single PBSC infusion for patients with refractory HF after recent large MI. It also negates the need for bone marrow aspiration and a two-time mobilization of G-CSF by using cryopreserved PBSC for the second injec-tion.

Some studies have suggested that G-CSF with and without intracoronary PBSC delivery might improve heart function and myocardial perfusion in post-MI patients. Interestingly, there is an inverse correlation between the PBSC with the degree of LV dysfunction: the higher the plasma level of PBSC, the better the ventricular function.13 However, there is still controversy regarding the lack of improvement in cardiac function and relatively few stem cells targeted to the heart.14,15 Recently, a meta-analysis across 8 randomized con-trolled trials6 suggested that administration of G-CSF alone in patients with acute MI fails to improve heart function. An animal model study demonstrated that majority of cells infused into the coronary artery died 72 h after transplanta-tion.16 The BOOST study revealed that the increase in LVEF lasts for only 6 months suggesting that a single intracoronary infusion of PBSC will not provide long-term benefit.17 Simi-

larly, Schots et al concluded in his PBSC intracoronary study that poor clinical effect is likely due to a small number of cells homing to the myocardium after a single intracoronary dose.18 To overcome these effects, optimal routes and times of cell therapy have been the object of several studies. Perin et al was one of the first groups to demonstrate the safety and efficacy of transendocardial BMS injections by electrome-chanical mapping in animal and then clinical studies.19–21 Their study showed that transendocardial delivery was supe-rior to the intracoronary administration and applicable for patients with severe acute or chronic ischemic heart disease. It now seems evident that clinical efficacy is correlated to the number of cells infused.10 Steinwender et al showed that the combination of G-CSF therapy and a single large dose trans-coronary transplantation of CD34+ cells are associated with a significant increase in global LVEF and regional systolic wall motion after 6 months, but seem to imply a high risk for the development of ISR.22 In 1 case of our pilot study, a tran-sient episode of syncope and convulsion but no ECG change might be connected with cellular embolism during intracoro-nary of 1×109 /ml mononuclear cell infusion. It is conceivable that repeated cell transplantation will be necessary for effec-tive cell therapy. Our preliminary study results reinforce this concept. At 12 months, the 8.9% mean improvement of LVEF in Group R was higher than 5.6% in Group S and 1.6% in Group C. In particular, the 4.5% mean improvement of LVEF from month 6 to month 12 follow up in Group R was more significant than the 1.6% in Group S and 1.0% in Group C. Similarly, the improvement of myocardial perfusion in Group R at 12 months was more than that in Group S and Group C. Interestingly, none of the 12 Group R patients required angio-plasty during secondary infusion. These results suggest that repeated intracoronary infusion of PBSC might accelerate LV recovery and induce a larger and continuous improvement compared to a single infusion. It might especially benefit those patients that have not fully recovered after their first cell treatment and might result in a better LVEF improvement

Figure 4. Test of contrast medium flow-back. Representa-tive digital subtraction angiography in a patient showed that contrast medium infused into the coronary artery through Foley’s over-the-wire catheter did not flow back to the proxi-mal end of the coronary artery when the sacculus had not been dilated. Green arrow shows no reflux of contrast me-dium in the proximal balloon. Red arrow appears the flow of contrast agent in its distal.


962 GU X et al.

following the second cell infusion.Recent clinical data, in accordance with our study, de-

scribed that a repeated intracoronary BMC transplantation 3 months after the first infusion in patients with a large AMI can significantly improve cardiac function, exercise capacity, and reduce infarct size with beneficial clinical effects lasting for at least 12 months.23 Another non-randomized clinical study demonstrated no change in LVEF by dobutamine con-trast echocardiogram after repeated intracoronary BMC treat-ment in patients with chronic ischemic HF, but it was asso-ciated with the improvement of LV filling and subjective clinical symptom.24,25 The discrepancies among these studies might be explained by different doses, timing, the type of MI, and duration of intracoronary infusions, and etc.

It is likely that homing signals might be more intense in acute and subacute MI. Additionally, some clinical studies have showed the effects of autologous BMC transplantation after MI usually might last for more than 6 months. Taking all of these into account, we chose recent AMI (less than 3 months) as the inclusion criteria for the first cell infusion and 6 months after the first transplantation as the secondary cells infusion time. Moreover, to reduce the complication of the related to intervention procedure, we simultaneously performed the PCI to IRA and the first PBSC infusion. How-ever, inconsistent with some other studies, we did not use a balloon inflation stop-flow technique to injecting the cells. Several reasons were considered when using continuous intra-coronary cell infusion without balloon inflation in our study. First of all, the initial compensation of failed heart in our enrolled patients with refractory HF after a large recent MI is very poor. The balloon inflation stop-flow could create further ischemia and even induce acute coronary syndrome. Secondly, it is plausible that such a ropiness suspension, fine central lumens and forward flow scarcely could lead to the regurgita-tion of cells suspension. We at least might corroborate little possibility of cell suspension refluxing by the injecting con-trast test. As prescribed previously, Gao et al26 indicated that there were no significant differences regarding clinical effect between the 2 groups using or not sacculus occlusion. So far, it is still not sure whether such a temporary balloon occlu-sion might trigger the homing of injected stem cells to the target myocardium.

Our study can only conclude that the repeated administra-tion of PBSC therapy is safe. We did not observe any death, re-infarction, re-vascularization, or tumorigeneses. The fre-quency and duration of hospitalization in Group R was sig-nificantly reduced which might be associated with an improve-ment of heart function and myocardial perfusion. Indeed, both the dose of diuretics and the number of patients using diuretics were significantly reduced in Group R at 12-month follow up. We observed 2 patients with 3 episodes of tran-sient myocardium ischemia while infusing PBSC in Group R which might be attributed to microemboli following intra-coronary infusion27 according to CAG images. Very recently, bone marrow-derived very small embryonic-like stem cells might have significant implications for cardiac repair after MI. Because its diameter (<6 μm) is too small to cause micro-vascular occlusion, this kind of cells appear to be suitable for intracoronary delivery.28 There was no acceleration of ISR in Group R at 6 months. No further increase in serum troponin, CK-MB, and CRP concentrations was observed which sug-gests that repeated intracoronary PBSC infusion does not sig-nificantly induce severe ischemic damage to the myocardium or induce a systemic inflammatory reaction. Current pub-lished animal and clinical studies have proved the safety of

the repeated intracoronary MSC injections.11,23,25,29 In addi-tion, safe and feasible repeated endomyocardial injections of cryopreserved MSC injections in Yorkshire swine has been demonstrated.29

Our study has several limitations: This is a phase I clini-cal study. The data are still preliminary as each study group has only 15 patients, which might not be sufficient to make a full conclusion on the functional improvement. Furthermore, this study is not randomized and the baseline demographics shown in this study seemed similar among 3 groups. None-theless, our data warrents a phase-II, large scale study taking place in a randomized, double blinded, and placebo controlled manner. There is a clear need to further assess the value of repeated intracoronary PBSC infusions in patients with recent MI, preferably with clinical endpoints such as mortality, mor-bidity, and target vessel revascularization.

In summary, the current clinical study demonstrates that the dual stem cell injection in combination with growth factor injection is a feasible and safe procedure. The repeated stem cell treatment might be potentially beneficial to patients with refractory HF after a recent large MI. Compared to the single dose PBSC infusion, repeated delivery of PBSC plus G-CSF show a better therapeutic potential. The strategy documented in this study offers an option for using celluar therapy with growth factor to better improve post-infarct cardiac function, exercise capacity, and myocardial perfusion.

AcknowledgementsThe authors greatly appreciate the support of nursing and technical staff from the Department of Cardiology, the catheterization laboratory and nuclear medicine department in Northern People’s Hospital. We are grateful to Professor Chaoshu Tang, Professor Fumin Zhang, Professor Zhijian Yang, Dr Yi Zheng, Mr Fred Baimbridge, and Dr Yuqi Liu for their advice and help. This study was supported by the Science Com-mittee (BS2004532), Health Department of Jiangsu Province (Z200514), and Program for Jiangsu Province Outstanding Medical Talented Leader (JS2006038).

References 1. Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B,

et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001; 410: 701 – 705.

2. Hosoda T, Kajstura J, Leri A, Anversa P. Mechanisms of myocar-dial regeneration. Circ J 2010; 74: 13 – 17.

3. Harada M, Qin Y, Takano H, Minamino T, Zou Y, Toko H, et al. G-CSF prevents cardiac remodeling after myocardial infarction by activating the Jak-Stat pathway in cardiomyocytes. Nature Med 2005; 11: 305 – 311.

4. Minatoguchi S, Takemura G, Chen X, Wang N, Uno Y, Koda M, et al. Acceleration of healing process and myocardial regeneration may be important as a mechanism of improvement of cardiac func-tion and remodeling by postinfarction granulocyte colony-stimulat-ing factor treatment. Circulation 2004; 109: 2572 – 2580.

5. Reffelmann T, Könemann S, Kloner RA. Promise of blood-and bone marrow-derived stem cell transplantation for functional cardiac repair: Putting it in perspective with existing therapy. J Am Coll Cardiol 2009; 53: 305 – 308.

6. Abdel-Latif A, Bolli R, Zuba-Surma EK, Tleyjeh IM, Hornung CA, Dawn B. Granulocyte colony-stimulating factor therapy for cardiac repair after acute myocardial infarction: A systematic review and meta-analysis of randomized controlled trials. Am Heart J 2008; 156: 216 – 226.

7. Zohlnhöfer D, Ott I, Mehilli J, Schömig K, Michalk F, Ibrahim T, et al; REVIVAL-2 Investigators. Stem cell mobilization by granu-locyte colony-stimulating factor in patients with acute myocardial infarction: A randomized controlled trial. JAMA 2006; 295: 1003 – 1010.

8. Ripa RS, Jørqensen E, Wang Y, Thune JJ, Nilsson JC, Søndergaard L, et al. Stem cell mobilization induced by subcutaneous granulo-cyte-colony stimulating factor to improve cardiac regeneration after acute ST-elevation myocardial infarction: Result of the double-blind, randomized, placebo-controlled stem cells in myocardial infarction



(STEMMI) trial. Circulation 2006; 113: 1983 – 1992. 9. Zohlnhöfer D, Dibra A, Koppara T, de Waha A, Ripa RS, Kastrup J,

et al. Stem cell mobilization by granulocyte colony-stimulating factor for myocardial recovery after acute myocardial infarction: A meta-analysis. J Am Coll Cardiol 2008; 51: 1429 – 1437.

10. Pouzet B, Vilquin JT, Hagege AA, Scorsin M, Messas E, Fiszman M, et al. Factors affecting functional outcome after autologous skeletal myoblast transplantation. Ann Thorac Surg 2001; 71: 844 – 850.

11. Premaratne GU, Tambara K, Fujita M, Lin X, Kanemitsu N, Tomita S, et al. Repeated implantation is a more effective cell delivery method in skeletal myoblast transplantation for rat myocardial infarction. Circ J 2006; 70: 1184 – 1189.

12. Kovacic JC, Macdonald P, Feneley MP, Muller DW, Freund J, Dodds A, et al. Safety and efficacy of consecutive cycles of granu-locyte-colony stimulating factor, and an intracoronary CD133+ cell infusion in patients with chronic refractory ischemic heart disease: The G-CSF in angina patients with IHD to stimulate neovasculariza-tion (GAIN I) trial. Am Heart J 2008; 156: 954 – 963.

13. Ferrari R, Ceconi C, Campo G, Cangiano E, Cavazza C, Secchiero P, et al. Mechanisms of remodeling: A question of life (stem cell production) and death (myocyte apoptosis). Circ J 2009; 73: 1973 – 1982.

14. Lee SS, Naqvi TZ, Forrester J, Cattley R, Shah A, Frantzen M, et al. The effect of granulocyte colony stimulating factor on regional and global myocardial function in the porcine infarct model. Int J Cardiol 2007; 116: 225 – 230.

15. Kang HJ, Lee HY, Na SH, Chang SA, Park KW, Kim HK, et al. Differential effect of intracoronary infusion of mobilized peripheral blood stem cells by granulocyte colony-stimulating factor on left ventricular function and remodeling in patients with acute myocar-dial infarction versus old myocardial infarction: The MAGIC Cell-3-DES randomized, controlled trial. Circulation 2006; 114(Suppl): I145 – I151.

16. Suzuki K, Murtuza B, Beauchamp JR, Smolenski RT, Varela-Carver A, Fukushima S, et al. Dynamics and mediators of acute graft attri-tion after myoblast transplantation to the heart. FASEB J 2004; 18: 1153 – 1155.

17. Meyer GP, Wollert KC, Lotz J, Pirr J, Rager U, Lippolt P, et al. Intracoronary bone marrow cell transfer after myocardial infarction: 5-year follow-up from the randomized controlled BOOST trial. Eur Heart J 2009; 30: 2978 – 2984.

18. Schots R, De Keulenaer G, Schoors D, Caveliers V, Dujardin M, Verheye S, et al. Evidence that intracoronary-injected CD133+ peripheral blood progenitor cells home to the myocardium in chronic postinfarction heart failure. Exp Hematol 2007; 35: 1884 – 1890.

19. Perin EC, Silva GV, Assad JA, Vela D, Buja LM, Sousa AL, et al. Comparison of intracoronary and transendocardial delivery of allo-

geneic mesenchymal cells in a canine model of acute myocardial infarction. J Mol Cell Cardiol 2008; 44: 473 – 476.

20. Zheng Y, Fernandes MR, Silva GV, Cardoso CO, Canales J, Gahramenpour A, et al. Histopathological validation of electro-mechanical mapping in assessing myocardial viability in a porcine model of chronic ischemia. Exp Clin Cardiol 2008; 13: 198 – 203.

21. Perin EC, Silva GV, Sarmento-Leite R, Sousa AL, Howell M, Muthupillai R, et al. Assessing myocardial viability and infarct transmurality with left ventricular electromechanical mapping in patients with stable coronary artery disease: Validation by delayed-enhancement magnetic resonance imaging. Circulation 2002; 106: 957 – 961.

22. Steinwender C, Hofmann R, Kammler J, Kypta A, Pichler R, Maschek W, et al. Effects of peripheral blood stem cell mobiliza-tion with granulocyte–colony stimulating factor and their transcor-onary transplantation after primary stent implantation for acute myocardial infarction. Am Heart J 2006; 151: 1296.e7 – e13.

23. Yao K, Huang R, Sun A, Qian J, Liu X, Ge L, et al. Repeated autol-ogous bone marrow mononuclear cell therapy in patients with large myocardial infarction. Eur J Heart Fail 2009; 11: 691 – 698.

24. Diederichsen AC, Møller JE, Thayssen P, Junker AB, Videbaek L, Saekmose SG, et al. Effect of repeated intracoronary injection of bone marrow cells in patients with ischaemic heart failure. Eur J Heart Fail 2008; 10: 661 – 667.

25. Diederichsen AC, Møller JE, Thayssen P, Videbaek L, Saekmose SG, Barington T, et al. Changes in left ventricular filling patterns after repeated injection of autologous bone marrow cells in heart failure patients. Scand Cardiovasc J 2010; 6: 139 – 145.

26. Gao LR, Wang ZG, Zhu ZM, Fei YX, He S, Tian HT, et al. Effect of intracoronary transplantation of autologous bone marrow-derived mononuclear cells on outcomes of patients with refractory chronic heart failure secondary to ischemic cardiomyopathy. Am J Cardiol 2006; 98: 597 – 602.

27. Ince H, Petzsch M, Kleine HD, Eckard H, Rehders T, Burska D, et al. 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 Granu-locyte Colony-Stimulating Factor (FIRSTLINE-AMI) Trial. Circu-lation 2005; 112(Suppl): I73 – I80.

28. Tang XL, Rokosh DG, Guo Y, Bolli R. Cardiac progenitor cells and bone marrow-derived very small embryonic-like stem cells for car-diac repair after myocardial infarction. Circ J 2010; 74: 390 – 404.

29. Poh KK, Sperry E, Young RG, Freyman T, Barringhaus KG, Thompson CA. Repeated direct endomyocardial transplantation of allogeneic mesenchymal stem cells: Safety of a high dose, “off-the-shelf”, cellular cardiomyoplasty strategy. Int J Cardiol 2007; 117: 360 – 364.

Documents

Repeated Intracoronary Infusion of Peripheral Blood … X-Circ J. (2011).pdfPeople’s Hospital, Nanjing Medical University, Nanjing (K.C.), China; and Center for Cardiovascular Biology