European Heart Journal (2007) 28, 2998–3005

doi:10.1093/eurheartj/ehm485
Clinical research
Coronary heart disease

Prospective randomized trial of direct endomyocardial

implantation of bone marrow cells for treatment of
severe coronary artery diseases (PROTECT-CAD trial)
Hung-Fat Tse1*†, Sukumaran Thambar3†, Yok-Lam Kwong1, Philip Rowlings4, Greg Bellamy3,
Jane McCrohon4, Paul Thomas5, Bruce Bastian3, John K.F. Chan6, Gladys Lo6, Chi-Lai Ho7,
Wing-Sze Chan1, Raymond Y. Kwong8, Anthony Parker9, Thomas H. Hauser10, Jenny Chan1,

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Daniel Y.T. Fong2, and Chu-Pak Lau1
1
Department of Medicine, University of Hong Kong, Queen Mary Hospital, Hong Kong; 2Department of Nursing Studies
Medicine, University of Hong Kong, Queen Mary Hospital, Hong Kong; 3Cardiovascular Division, Hunter Heart-Lung Research
Guild, John Hunter Hospital, Newcastle, Australia; 4Hunter Area Pathology Service, Newcastle Mater Misericordiae Hospital,
Newcastle, Australia; 5Department of Cardiology, St George Hospital, Sydney, Australia; 6Department of Radiology and
Radiotherapy, Hong Kong Sanatorium and Hospital, Hong Kong; 7Department of Nuclear Medicine and Positron Emission
Tomography, Hong Kong Sanatorium and Hospital, Hong Kong; 8Cardiac Magnetic Resonance Imaging Cardiovascular Division,
Brigham and Women’s Hospital, Harvard Medical School, Boston, USA; 9Nuclear Medicine Division, Beth Israel Deaconess
Medical Center, Harvard Medical School, Boston, USA; and 10Cardiology Division, Beth Israel Deaconess Medical Center,
Harvard Medical School, Boston, USA
Received 25 March 2007; revised 24 August 2007; accepted 26 September 2007; online publish-ahead-of-print 5 November 2007

KEYWORDS Aims Experimental studies have demonstrated that bone marrow (BM) cells can induce angiogenesis in
Angiogenesis; ischaemic myocardium. Recently, several non-randomized pilot studies have also suggested that direct
Bone marrow; BM cells implantation appears to be feasible and safe in patients with severe coronary artery diseases
Coronary artery disease (CAD).
Methods and results We performed a randomized, blinded, and placebo-controlled trial in 28 CAD
patients. After BM harvesting, we assigned patients to receive low dose (1  106 cells/0.1 mL, n ¼ 9),
high dose (2  106 cells/0.1 mL, n ¼ 10) autologous BM cells or control (0.1 mL autologous
plasma/injection, n ¼ 9) catheter-based direct endomyocardial injection as guided by electromechani-
cal mapping. Our primary endpoint was the increase in exercise treadmill time and our secondary end-
points were changes in Canadian Cardiovascular Society (CCS) and New York Heart Association (NYHA)
class, and myocardial perfusion and left ventricular ejection fraction (LVEF) assessed by single-
photon emission computed tomography and magnetic resonance imaging, respectively. A total 422
injections (mean 14.6 + 0.7 per patient) were successfully performed at 41 targeted ischaemic
regions without any acute complication. Baseline exercise treadmill time was 439 + 182 s in controls
and 393 + 136 s in BM-treated patients, and changed after 6 months to 383 + 223s and 464 + 196 s
[BM treatment effect þ0.43 log seconds (þ53%), 95% CI 0.11–0.74, P ¼ 0.014]. Compared with
placebo injection, BM implantation was associated with a significant increase in LVEF (BM treatment
effect þ5.4%, 95% CI 0.4–10.3, P ¼ 0.044) and a lower NYHA class (odds ratio for treatment effect
0.12, 95% CI 0.02–0.73, P ¼ 0.021) after 6 months, but CCS reduced similarly in both groups. We
observed no acute or long-term complications, including ventricular arrhythmia, myocardial damage,
or development of intramyocardial tumour or calcification associated with BM implantation.
Conclusion Direct endomyocardial implantation of autologous BM cells significantly improved exercise
time, LVEF, and NYHA functional class in patients with severe CAD who failed conventional therapy.

* Corresponding author. Tel: þ852 2855 3598; Fax: þ852 2618 6304.
E-mail address: hftse@hkucc.hku.hk
†
The authors contributed equally to the article as first authors.

Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2007.
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PROTECT-CAD trial
Introduction
Coronary artery disease (CAD) remains a major cause of mor-
bidity and mortality worldwide. Recent advances in medical
therapy and coronary revascularization techniques with
either percutaneous or surgical procedures have improved
the prognosis and survival in patients with CAD. However,
with improvement in survival, the number of CAD patients
who have exhausted or failed, or are not suitable candidates
for coronary revascularization, is increasing.1,2 These
patients with severe CAD follow an inexorable downhill
course with disabling symptoms and progressive cardiac
failure, despite maximal medical therapy.3 In recent years,
therapeutic use of bone marrow (BM)-derived or circulating
progenitor cells have been investigated as a potential novel
therapy in patients with CAD, who are not amenable to con-
ventional treatment.4–7 Although these studies indicate
feasibility and safety of direct endomyocardial injection of
autologous BM cells into the chronic ischaemic, non-
infarcted myocardium the clinical efficacy of this therapy
remains unclear. We performed a randomized controlled
trial to investigate the effect of direct endomyocardial
injection of autologous BM cells in patients with severe
CAD who had failed conventional therapy.
Methods
Patient population
This study was a randomized, blinded, placebo-controlled trial per-
formed in Hong Kong and Australia. Eligibility for inclusion was
based on a history of stable Canadian Cardiovascular Society (CCS)
class III or IV angina refractory to medical therapy, no conventional
percutaneous or surgical revascularization option as determined by
interventional cardiologists and cardiothoracic surgeons, ability to
complete .3 min but ,10 min of treadmill exercise using modified
Bruce protocol, and one or two coronary territories of viable,
ischaemic myocardium as documented by dipyridamole single-
photon emission computed tomographic (SPECT) perfusion study.
The main exclusion criteria consisted of unprotected left main
CAD, decompensated heart failure, and/or severe systolic left ven-
tricular (LV) dysfunction, as reflected by an echocardiographic LV
ejection fraction (LVEF) 30%, acute coronary syndrome, or
stroke within the past 3 months, significant renal, liver, or haemato-
logical abnormalities, active systemic infection or malignancy, and
inability to perform percutaneous electromechanical mapping in
the LV, due to atrial fibrillation, LV thrombus, severe peripheral vas-
cular disease, severe aortic stenosis, or an aortic prosthetic valve.
All patients gave informed consent and were willing to comply
with the planned follow-up. The protocol was approved by the insti-
tutional review boards of the two participating centres. This study is
registered with www.hkclinicaltrials.com, number HKCTR-3.
Study protocol
Patients were randomly assigned into low or high BM groups or
control group in 1:1:1 ratio using a randomization table. Randomiz-
ation was constrained, stratified on the study centre, and conducted
via a system of sealed and numbered envelopes provided to each
investigational centre. Two different dosages of BM mononuclear
cells were used: low dose (1  106 cells/0.1 mL injection) and
high dose (2  106 cells/0.1 mL injection), to allow us to explore
the potential dose-dependent treatment effect of BM cell treat-
ment. Patients randomized to the control group received autologous
plasma injection. After randomization, the study processes were
blinded to the patients, study coordinators, and the investigators
who were responsible for patients assessment. Baseline evaluation
2999
in both groups included functional status [New York Heart Associ-
ation (NYHA) and CCS angina classification],8 treadmill exercise
testing (modified Bruce treadmill protocol),9 dipyridamole SPECT
perfusion scanning, and cardiac magnetic resonance imaging (MRI).
On the day of the procedure, BM was harvested from every
patient by an experienced haematologist via posterior iliac crest
puncture under local anaesthesia. A total of 40 mL of BM blood
was aspirated, and an adequate trephine biopsy was performed.
Mononuclear cells were isolated by Ficoll density gradient centrifu-
gation as previously described.6 BM cells were washed twice in
phosphate-buffered saline, re-suspended in phosphate-buffered
saline enriched with 10% autologous plasma to either 1 or 2  107
mononuclear cells/mL and returned directly to cardiac catheteriza-
tion laboratory for use. In the placebo preparation for the control
group, BM cells were not included in the final suspension, which con-
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sisted merely of phosphate-buffered saline with 10% autologous
plasma. BM suspensions were tested by flow cytometry (Elite,
Beckman Coulter, Fullerton, CA, USA) with directly conjugated anti-
bodies against CD34 (Beckman Coulter, Krefeld, Germany).
At an average of 3–4 h after BM harvest, we performed non-
fluoroscopic LV electromechanical mapping (NOGA System,
Biosense-Webster, CA, USA) to identify the foci of ischaemic myo-
cardium as previously described.4–6 During the procedure, systemic
anticoagulation was achieved with intravenous heparin to maintain
an activated clotting time of 250–300 s throughout the procedure.
The targeted injection regions were selected by matching the
area of ischaemic myocardium identified by SPECT. After completion
of the LV electromechanical mapping, the mapping catheter was
replaced by a modified mapping catheter incorporated with a 27 G
needle at the tip (Myostar, Biosense-Webster) that could be used
for direct endomyocardial injection. Depending on the size of the
region, eight to 12 injections of 0.1 mL of BM cell or placebo prep-
aration were delivered evenly in each ischaemic region.
Clinical outcomes
All patients were observed for 24 h after the procedure in the cor-
onary care unit. Serial myocardial isoenzymes (CPK-MB) and electro-
cardiograms were measured for the first 24 h. A standard
transthoracic echocardiogram was also performed within the first
24 h to exclude pericardial effusion post-operatively.
At 3 and 6 months after discharge, patients had follow-up exam-
inations to assess their clinical and functional status by study coor-
dinator in each centre who was blinded to treatment assignment. At
6 months’ follow-up, modified Bruce treadmill testing, dipyridamole
SPECT, and cardiac MRI were repeated in all patients.
Adverse events related to direct endomyocardial injection pro-
cedures and BM cells implantation were carefully evaluated. Specifi-
cally, the occurrence of ventricular arrhythmias was detected by
24 h Holter ECG recording, and the development of myocardial
tumour formation was assessed by MRI at 6 months’ follow-up. Fur-
thermore, computed tomography (CT) of thorax was performed at
12 months after procedure to assess for development of intramyo-
cardial tumor or calcification.
Exercise treadmill testing
At baseline and at 6 months after study enrolment, all patients
underwent standard exercise treadmill testing per the modified
Bruce protocol.9 Each exercise treadmill testing was monitored by
a study coordinator who was not aware of the patients’ treatment
group assignments. Patients were encouraged to perform as much
exercise stress as possible, while their symptoms and ECG signs of
cardiac ischaemia were continuously assessed. Specific indications
for termination of exercise treadmill testing include: patients’
desire to stop the study due to fatigue or symptoms or development
of ECG evidence of myocardial ischaemia/injury by 1 mm ST
deviation.
3000
Dipyridamole single-photon emission computed
tomographic perfusion
Dipyridamole stress and resting SPECT myocardial perfusion imaging
was performed at baseline and 6 months’ follow-up. Gated rest and
stress SPECT imaging of the heart were acquired Helix (General
Electric, Milwaukee, WI, USA) or Marconi Prism 3000 (Philips
Medical Systems, Bothell, WS, USA) gamma camera systems.
SPECT imaging was performed either as a 1 day rest/stress protocol
(sestamibi 10 mCi intravenous injection at rest followed by 30 mCi
post peak pharmacological-induced stress by dipyridamole infusion)
or a 2-day protocol (sestamibi 20 mCi rest and 20 mCi post-stress).
Imaging was performed at least 20 min after the rest injection
and 30 min post-stress injection. Analysis of SPECT myocardial per-
fusion imaging was performed by consensus of two experienced
investigators (J.A.P. and T.H.H.). Pairs of stress and rest images
from baseline and 6 months’ follow-up were interpreted as a
group by investigators blinded to the order of the image pairs.
Defect severity (0 ¼ normal, 1 ¼ equivocal, 2 ¼ moderate, 3 ¼
severe, and 4 ¼ absent) was scored in each of 20 segments from a
computer display of short-axis, vertical long-axis, and horizontal
long-axis images. The sum of stress scores (SSS), sum of rest
scores, and sum of difference scores (SDS) were calculated as
simple sums of the respective scores.
Cardiac magnetic resonance imaging
Cardiac MRI examination was performed at baseline and 6 months’
follow-up using a 1.5 T scanner (Signa CV/i, General Electric, Mil-
waukee, WI, USA; Magnetom Sonata; Siemens, Erlangen, Germany;
Intera, Philips, Eindhoven, The Netherlands) and a four-channel
cardiac phased-array coil. Images were acquired during repeated
breath-holds with the patient in supine position. Cine images cover-
ing the entire LV were obtained using an ECG-gated steady-state
free precession pulse sequence, with the following imaging par-
ameters: TR3.8 ms, TE1.5 ms, flip angle 458, matrix size 192160,
field of view 28–34 cm, slice thickness 8 mm with no gap, views
per segment 16, and number of excitation 1.
All image analyses were performed by two investigators who were
unaware of treatment allocation (W.S.C. and R.Y.K.), using a com-
mercially available off-line software package (CineTool 4.5.2,
General Electric Healthcare). Measurement of LV volume and calcu-
lation of stroke volume and EF were based on previously validated
techniques.10,11 For each slice location, the end-diastolic and end-
systolic cine frames were defined as the ones showing the largest
and smallest LV cavity, respectively. The endocardial border was
manually traced, and the end-diastolic and end-systolic volumes
were the sums of LV cavity sizes across all contiguous slices in the
corresponding phase of the cardiac cycle (Simpson’s rule). LVEF
was calculated as stroke volume divided by end-diastolic volume
and multiplied by 100%. Regional wall thickening was calculated
by the percentage change of end-systolic thickness over end-
diastolic thickness using a 20-segment model.
Statistical analysis
Primary outcome was the change from baseline in total exercise
time on a modified Bruce protocol at 6 months’ follow-up. Second-
ary outcomes were changes in LVEF, NYHA, and CCS angina classifi-
cation and sum of different scores on SPECT. The primary
comparison was performed between the combined BM-treated
groups with two different doses of BM cells vs. control group.
On the basis of the data from our pilot study, we took a standard
deviation of 95 s for the change of total exercise time at 6 months’
follow-up from baseline. In order to have at least 80% power to
detect a difference of 115 s (30 percentage points) between the
control and the combined BM groups, including two different
doses of BM cells with a 5% maximum false positive error rate, we
would need a total 27 patients with 2:1 randomization to BM
(n ¼ 18) and placebo (n ¼ 9) by a two-tailed t-test. The inclusion
H.-F. Tse et al.
of two different doses of BM cells in this study was to explore for
the potential of dose–response effects. The analysis had taken
account of the three-group randomization and was performed in
all randomized subjects according to the intention-to-treat prin-
ciple. The exercise time for the withdrawn patient was available.
For other analysis, the baseline value was used to impute the
missing value.
Continuous variables were presented as mean + standard devi-
ation. Categorical data were presented as frequencies and percen-
tages. Comparison of between groups was performed using
Student’s t-test. The logarithmic transformation was used for the
total exercise time due to the presence of non-constant variance
in residuals. For the analysis of total exercise time (in log), MRI,
and SPECT variables, we did an analysis of covariance (ANCOVA)
analysis to assess the differences between the treatment groups
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at 6 months, after adjusting for baseline values. Specifically,
values at 6 months were taken as the dependent variable and the
associated baseline values and a factor for treatment were taken
as independent variables. Model adequacy was assessed by examin-
ing the standardized residuals. We estimated treatment effects by
computing the differences (combined BM vs. control) between the
adjusted means and their corresponding 95% CIs. Furthermore,
paired t-tests were performed to determine the regional changes
in SPECT scores and percentage of wall thickening measured by
MRI according to the coronary circulation territories in the target
and non-target regions for intramyocardial injection. The relation
between the total number of CD34þ cells injected into the ischae-
mic myocardium and the subsequent total exercise time changes
were assessed with Pearson’s correlation coefficient. Comparisons
of NYHA functional class and CSS angina, taken as ordinal outcomes,
between BM-treated patients and controls at 3 and 6 months were
made by generalized estimating equations with the cumulative
logit link function and adjustment on the baseline values. The
resulting treatment effects were expressed as proportional odds.
All statistical tests were two-sided and used 0.05 as the nominal
level of significance. The statistical analysis was performed in the
Statistical Analysis System version 9.
Results
Between January 2002 and September 2005, 38 patients
were enrolled and 28 patients were randomized into this
study (Figure 1). In this study, 15 and 13 patients were ran-
domized in Hong Kong and Australia, respectively. After ran-
domization, one patient in the control group was withdrawn
because of failure to access LV to perform mapping and
endomyocardial injection due to severe bilateral peripheral
artery disease. The patient was, however, included in the
analysis by the intention-to-treat principle. The baseline
Figure 1 Flow chart of the study design. MNC, mononuclear cell.
PROTECT-CAD trial 3001

Compared with controls, the total exercise time at 6

Table 1 Patients’ characteristics months’ follow-up was significantly greater in BM-treated
patients (þ0.43 in log seconds, 95% CI 0.11–0.74; P ¼
Control group Bone-marrow group
(n ¼ 9) (n ¼ 19) 0.014) (Table 2). This represents a total exercise time
increased by 53% more in BM treated patients (Figure 2).
Age, years 68.9 (6.3) 65.2 (8.3) In the controls, one patient developed dramatically
Men 7 (88) 14 (79) decline in exercise time during follow-up. Further analysis
Diabetes mellitus 5 (63) 8 (42) after omitting this patient revealed that the treatment
Hyperlipidaemia 9 (100) 19 (100) effect of BM cell injection on exercise time remains statisti-
Hypertension 6 (75) 13 (68) cally significant (þ0.32 in log seconds, 95% CI 0.03–0.62;
Current cigarette smoker 4 (50) 8 (42) P ¼ 0.043). Besides, there was one patient in the control
Percutaneous coronary 7 (88) 9 (47)
group who developed myocardial infarction 3 months after
intervention
Coronary artery bypass 5 (63) 13 (68) the procedure. Exclusion of this patient from the analysis
showed again a positive effect of BM cell injection on exer-

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surgery
Medication at baseline cise time (þ0.47 in log seconds, 95% CI ¼ 0.15–0.80; P ¼
ACE-inhibitors or ARB 4 (50) 6 (32) 0.009). Moreover, removal of both patients in the control
Aspirin and/or 9 (100) 19 (100) group resulted in essentially the same conclusion (þ0.36
clopidogrel in log seconds, 95% CI ¼ 0.05–0.67; P ¼ 0.032). Further-
b-Blockers 7 (88) 18 (95) more, there was no difference of BM cell effects on exercise
Calcium channel 4 (50) 8 (42) time between the two centres after adjusting for baseline
blockers
values (P ¼ 0.082). In addition, there was no difference of
Nitrates 4 (50) 8 (42)
BM cell effects on exercise time between the two centres
Statins 9 (100) 19 (100)
Medication at 6 months after adjusting for baseline values (P ¼ 0.082).
ACE-inhibitors or ARB 4 (50) 7 (37) Among those BM-treated patients, the mean total number
Aspirin and/or 9 (100) 19 (100) of mononuclear cells injected in the low-dose group (1.67 +
clopidogrel 0.34  107 cells) was significantly lower than the high-dose
b-Blockers 6 (75) 17 (89) group (4.20 + 2.80  107 cells, P ¼ 0.011). However, there
Calcium channel 4 (50) 7 (37) were no significant differences in the mean number of injec-
blockers tion (16.7 + 3.5 vs. 13.8 + 5.0, P ¼ 0.15) and the mean total
Nitrates 4 (50) 9 (47) number of CD34þ cell injected (0.79 + 0.66  106 vs.
Statins 9 (100) 19 (100)
1.38 + 1.70  106, P ¼ 0.38) between patients treated
Data are means (SD) or n (%) unless otherwise stated.
with low- and high-dose BM mononuclear cells injection.
ACE, angiotensin-converting enzyme; ARB, angiotensin receptor. The change in total exercise time from baseline to 6
months’ follow-up did not differ between patients treated
with low-dose (þ89.0 + 137.9 s) and high-dose (þ51.4 +
characteristics of these patients are shown in Table 1. After 163.9s) BM injections (P ¼ 0.59). Furthermore, the improve-
discharge, all patients were maintained on stable ment in total exercise time in BM-treated patients after 6
cardiovascular medications throughout the study period months was not correlated with the total number of mono-
(Table 1). nuclear cells (r ¼ 20.27, P ¼ 0.30) nor CD34þ BM cells
With respect to BM cell assessment, all the microbiologi- (r ¼ 20.11, P ¼ 0.66) injected into the ischaemic
cal cultures were negative, and BM trephine biopsy revealed myocardium.
no abnormality. Flow cytometry analysis revealed no signifi- The increase in exercise workload during treadmill test as
cant differences in the number of CD34þ cells in the BM measured by metabolic equivalents from baseline to 6
preparation between control (5.4 + 5.8  106 cells) and BM months in BM treated patients was only slightly higher
groups (3.6 + 3.0  106 cells, P ¼ 0.32). than controls (Table 2).
A total of 422 percutaneous catheter-based endomyocar- Paired cardiac MRI was available in 8 controls and 18 BM
dial injections to 41 ischaemic regions (inferior ¼ 16, treated patients (Figure 3). One control patient withdrew
lateral ¼ 9, posterior ¼ 3, and anterior ¼ 13) as guided by from the study due to the procedural failure refused a
the electromechanical mapping were performed in 27 repeated MRI examination and one BM treated patient
patients. On average, 14.6 + 0.7 (range 9–22) injections could not undergo MRI examination due to the presence of
per patient were performed. There was no significant differ- an implanted pacemaker. Compared with controls, LVEF
ence in the number of injections given between controls increased (P ¼ 0.044) and LV end-systolic volume decreased
(12.9 + 4.4) and BM-treated patients (15.3 + 2.7, mean (P ¼ 0.058) in the BM treated patients at 6-months (Table 2).
difference þ2.6, 95% CI 20.55 to 5.82, P ¼ 0.12). Furthermore, post hoc analysis showed that the percentage
The mean procedural time and fluoroscopic time was of regional wall thickening over the target regions was only
152 + 72 min (range 70–370 min) and 33 + 11 min (range significantly increased in BM treated patients at 6 months
15–65 min), respectively. There were no acute procedural- compared with baseline (þ5.33%, 95% CI 2.72–7.94; P ¼
related complications, including stroke, transient ischaemic 0.0032) (Figure 4A). Although the changes in LV end-diastolic
attack, ECG changes, sustained ventricular or atrial arrhyth- volume from baseline to 6 months did not differ significantly
mias, elevation of CPK-MB (more than two times), nor echo- between the two groups, these tended to decrease in the
cardiographic evidence of pericardial effusion within the BM-treated patients (Table 2).
first 24 h after the procedure. Post-procedure, all patients Paired SPECT myocardial perfusion was available in eight
were discharged from the hospital after 24 h of observation. controls and 19 BM-treated patients. One control patient
 Downloaded from https://academic.oup.com/eurheartj/article/28/24/2998/589988 by guest on 19 March 2023
3002 H.-F. Tse et al.
Table 2 Treadmill exercise parameters, SPECT scores, and left ventricular volume and global left ventricular ejection fraction as determined by cardiac magnetic resonance imaging at baseline

P-value

0.014

0.044

0.058
0.14

0.28
0.17

0.16
BM treatment effect at 6 months (estimatea)

29.3 (218.4, 20.2)

211.7 (227.4, 4.2)
0.43 (0.11, 0.74)
0.9 (20.3, 2.2)

22.7 (27.6, 2.1)

22.4 (25.8, 1.0)

5.4 (0.4, 10.4)

Figure 2 Treatment effect of bone marrow cells implantation on total exer-

cise time on treadmill test.
0.14 (0.33)

211.2 (26.8)
28.8 (18.4)
1.0 (1.6)

20.5 (5.0)
21.8 (3.6)

3.7 (5.1)
BM group

20.25 (0.46)

2.0 (22.1)
23.1 (14.4)
0.03 (1.2)

2.4 (6.9)
0.8 (4.9)

20.4 (7.5)
Controls
Change

BM, bone-marrow; LVEDV, left ventricular end-diastolic volume; LVESV, left ventricular end-systolic volume.
6.05 (0.46)

150.3 (29.3)
67.0 (21.5)
5.3 (6.5)
5.1 (1.9)

12.7 (7.0)

55.6 (8.8)
BM group

Treatment effects expressed as differences in least-squares means (ANCOVA model) with 95% CI.
There were no differences between BM group (n ¼ 19) and control group (n ¼ 9) at baseline.

Figure 3 Treatment effect of bone marrow cells implantation on left ventri-

cular ejection fraction as determined by cardiac magnetic resonance
5.74 (0.74)

19.6 (13.5)

160.3 (22.8)
80.8 (18.6)

imaging.
6.6 (5.6)
4.2 (1.8)

45.3 (8.2)
6 months

Controls

withdrew from the study due to the procedural failure

refused a repeated SPECT examination. With respect to
161.4 (41.3)

SPECT, the extent of stress-induced perfusion defects as

5.91 (0.39)

75.9 (33.5)
13.2 (4.8)

51.9 (8.5)
BM group

7.2 (5.6)
4.1 (1.0)

determined by SDS was significantly decreased in BM-treated

n ¼ 19

n ¼ 19

n ¼ 19

patients at 6 months when compared with baseline (P ¼

0.04) (Table 2). Furthermore, post hoc analysis showed
that the SDS over the target regions was only significantly
decreased in BM-treated patients at 6 months compared
158.3 (27.3)
5.99 (0.48)

17.3 (11.0)

83.9 (25.1)

Data are mean (SD) unless otherwise stated.

with baseline (20.49, 95% CI 20.97 to 0.02; P ¼ 0.0047)
45.7 (8.3)
5.9 (5.4)
4.2 (1.3)
Baseline

Controls

(Figure 4B). Although both changes of SSS and SDS from

n¼9

n¼8

n¼8
baseline to 6 months did not differ significantly between
the two groups, they tended to decrease more in the
BM-treated patients (Table 2).

Sum of difference scores

Exercise workload, METs
At 6 months, NYHA functional class was significantly lower
and 6 months’ follow-up

Exercise time, log (s)

Treadmill exercise test
in BM-treated patients than in controls [odds ratio (OR) for

Sum of stress scores

treatment effect 0.12, 95% CI 0.02–0.73, P ¼ 0.021].
However, there was no significant treatment-related differ-

Global LVEF, %
ence on CCS angina class between BM-treated patients and

LVEDV, mL
LVESV, mL
Cardiac MRI
controls (OR for treatment effect 0.43, 95% CI 0.09–2.07,
P ¼ 0.291) (Table 3).

SPECT
After a mean follow-up of 19 + 9months, no patient had

a
sudden cardiac death or was lost to follow-up. During the
PROTECT-CAD trial
Figure 4 (A) cardiac magnetic resonance imaging wall thickening and
regional changes in (B) single-photon emission computed tomography sum
stress score (upper panel) and sum difference score (lower panel) over
target and non-target region in control and bone marrow-treated patients
at baseline and 6 months’ follow-up.
6-month study period, none of them received further coron-
ary revascularization. One patient in the control group with
baseline inferior and lateral wall myocardial ischaemia
developed inferior wall acute myocardial infarction at 3
months after the procedure. Another patient in the control
group died of acute myocardial infarction at 31 months
after the procedure. One patient in the BM group was diag-
nosed to have carcinoma of the urinary bladder during
follow-up. No patients developed symptomatic cardiac
arrhythmias and 24 h Holter recording at 6 months did not
reveal any sustained or non-sustained ventricular tachycar-
dia. In seven controls and 19 BM-treated patients, cardiac
MRI at 6 months did not demonstrate any tumour formation.
Again, in six controls and 18 BM-treated patients who
reached the 12 months’ follow-up after the initial
3003
Table 3 Number of subjects in each class of the New York Heart
Association functional class and Canadian Cardiovascular Society
angina class at baseline and 6 months’ follow-up
Control group (n ¼ 9) Bone marrow group
(n ¼ 19)
Baseline 6 months Baseline 6 months
NYHA class
1 0 0 0 2
2 2 6 7 16
3 7 3 8 1
4 0 0 4 0
CSS class
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1 0 0 0 2
2 0 6 0 15
3 8 3 13 2
4 1 0 6 0
procedure, CT thorax did not show any tumour or intramyo-
cardial calcification.
Discussion
The findings of this randomized controlled trial indicated
that direct endomyocardial injection of autologous BM
cells in patients with severe CAD who failed conventional
therapy resulted in significant but only modest improvement
in total exercise time, NYHA functional class, and LVEF com-
pared with placebo. Nevertheless, the absence of any acute
procedural complications or long-term sequalae, including
ventricular arrhythmia, myocardial damage, or develop-
ment of intramyocardial tumour or calcification, was
encouraging in terms of the safety and feasibility of the
current novel catheter-based cellular transplantation
procedure.
In this study, significant BM treatment effects were
observed on exercise time and LVEF after 6 months. At 3
and 6 months, although both groups reported an improve-
ment in symptoms with respect to NYHA functional class
and CCS angina class when compared with baseline, these
changes tended to disappear in the controls after 6
months. As a result, significant BM treatment effect was
observed on NYHA function class at 6 months.
Recently, BM cell therapy has been investigated as a
means to facilitate myocardial regeneration and/or angio-
genesis.12 Previous experimental studies13 have suggested
that BM-derived cells may have the capacity to regenerate
the myocardium after myocardial infarction. However, the
notion that BM-derived stem cells can trans-differentiate
into cardiomyocytes has been challenged by recent
studies.14,15 With the use of genetic rather than immuno-
fluoresence techniques, these studies have shown that
only a very low level of transplantation of BM cells trans-
differentiation into cardiomyocytes. However, functional
studies after BM cells implantation did demonstrate protec-
tion against ventricular remodelling and preservation of LV
function.15 These functional benefits in the absence of myo-
cardial regeneration after BM cell implantation is likely
attributed to an increased angiogenesis.16,17 There may be
multiple mechanisms for the enhancement of myocardial
3004
angiogenesis by BM cells. Prior studies have demonstrated
that human BM cells contain endothelial progenitor cells
with expression of CD34 surface antigen, which can be
used to induce neovascularization after myocardial infarc-
tion.18 Furthermore, BM cells are a natural source of mul-
tiple growth factors involved in neovascularization,
including vascular endothelial growth factor.19
We did not observe any significant difference in the
improvement in the total exercise time between the low
and high-dose BM-treated patients. Although the mean
total numbers of mononuclear cells injected was signifi-
cantly higher in the high-dose group, the mean total
number of CD34þ cells injected was not different
between the two groups. This may account for the lack of
difference in the change of total exercise time between
them. Recent experimental studies have shown a dose-
dependent effect of CD34þ cells implantation on neovascu-
larization after myocardial infarction.20 However, we also
show no direct relationships between the improvement of
exercise time with neither the total numbers of mono-
nuclear cells nor the total number of CD34þ cells implanted
into the ischaemic myocardium. These findings are consist-
ent with the results of recent studies21,22 which also failed
to demonstrate a relationship between the number of
injected cells and improvement in clinical outcomes. There-
fore, in addition to the numbers of cell implanted, other
factors including the functional impairment of BM cells
associated with ageing and diabetes, cell survival after
transplantation, and optimal composition of types of BM
cells may account for individual variation in clinical
responses.23 Given the large variability in retention, survival
and number of injections, a larger difference (5–10-fold) in
the number of BM cells injected might be needed to show an
incremental benefit of the treatment.
Recent non-randomized studies4–7 have suggested that
direct BM cell implantation into the ischaemic myocardium
improved symptoms and exercise capacity and increased
myocardial perfusion and function in patients with refrac-
tory CAD. However, the impact of placebo effect of treat-
ment in these studies cannot be determined. In this study,
all patients of the BM and control groups underwent identi-
cal procedures and follow-up. Indeed, quite striking placebo
effects on NYHA and CCS class were observed in controls,
but there were no significant improvement on objective
functional parameters. In contrast, total exercise time was
significantly improved in BM-treated patients. This ben-
eficial effect over and beyond the effect noted in controls
suggests a potential therapeutic effect of BM cells on
chronic ischaemic myocardium and highlights the need for
randomized, placebo-controlled group to prove the ben-
eficial effects. Furthermore, serial cardiac MRI also demon-
strated increases in global LVEF and regional wall thickening
over the target regions in BM-treated patients, suggesting
recovery of myocardial function in the ischaemic myocar-
dium. These findings are consistent with the change in myo-
cardial perfusion, as reflected by a modest reduction in the
stress-induced perfusion defects on SPECT in BM-treated
patients. However, this trial was not powered to examine
the treatment effect in these secondary endpoints. There-
fore, it remains unclear that the modest functional ben-
eficial effects observed after BM cells therapy is mediated
via enhancement of angiogenesis as suggested in experimen-
tal studies.16,17
H.-F. Tse et al.
Recent clinical trials have demonstrated the safety and
feasibility of intracoronary route of delivery of BM cells
after successful coronary revascularization after myocardial
infarction.24–28 In our patients with severe CAD, intracoron-
ary injection might not have provided optimal cell transfer,
due to the presence of total occlusion and diffuse distal dis-
eases, and the lack of potent local signals for the homing of
BM cells that might occur during acute myocardial infarc-
tion. Therefore, we performed direct endomyocardial injec-
tion of BM cells as guided by electromechanical mapping.
When compared with intracoronary delivery, direct endo-
myocardial injection provides a higher efficacy of cellular
delivery, but a more uneven distribution of BM cells.29
However, we have not observed any acute or long-term
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adverse effects related to direct endomyocardial implan-
tation of BM cells in chronic ischaemic myocardium. In con-
trast, the requirement of a specialized system and the high
cost of the procedure may limit the use of technique. There-
fore, future development of a more simple and inexpensive
technique for catheter-based direct endomyocardial injec-
tion will increase the feasibility of this treatment strategy.
This study had several limitations. First, although no stat-
istical significant differences in baseline characteristics
between the BM-treated group and placebo group were
observed, there were some imbalances in mean exercise
time, SPECT score, and LVEF between them due to a small
sample size. These might lead to bias in the results.
However, we have made the best effort possible to maintain
the balance between groups by following proper randomiz-
ation procedure. Moreover, adjustment of all possible base-
line imbalances resulted in essentially the same conclusion
of the effect of BM in improvement exercise time. Second,
patients were not trained to perform exercise treadmill
test before the study. As a result, the impact of the training
effects on exercise time cannot be determined. Third, this
trial did not have sufficient power and sample size to allow
us to determine the optimal cell type or dose of BM cell
implantation and to identify any potential clinical predictors
for clinical improvements. Furthermore, no functional tests
such as colony-forming unit and migration capacity and/or
CXCR-4 receptor expression were measured to correlate the
degree of clinical improvement after injection. Forth, our
current results also cannot determine whether the observed
beneficial effects of BM treatment can persist beyond the
6-month period. Therefore, future studies are required to
address the optimal number and phenotype of BM cells for
therapeutic angiogenesis, as well as the long-term clinical
impact of this novel therapeutic approach.
In summary, this randomized, placebo-controlled trial has
shown beneficial effects of direct endomyocardial injection
autologous BM cells on symptoms, functional capacity, and
LVEF in patients with chronic myocardial ischaemia who
failed conventional medical and revascularization pro-
cedures. The documented safety and clinical benefits of
this novel procedure for this patient group prompt future
studies to investigate the therapeutic benefit on survival
and cardiovascular outcomes of this therapy.
Acknowledgement
This study was an academia-initiated study. There was no external
industry sponsor involved in study design, data collection, data
analysis, data interpretation, or writing of the report.
PROTECT-CAD trial
Conflict of interest statement. H.F.T. and S.T. received consultant
fee from Biosense-Webster, CA, USA. All other authors declare that
they have no conflict of interest.
Funding
This study was partially supported by the Sun Chieh Yeh Heart Foun-
dation Fund, S.K. Yee Medical Foundation Grant (Project No.
203217), and The Research Grants Council of Hong Kong(HKU
7357/02M) from Hong Kong.
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