CD133+ Progenitor Cells to Promote Angiogenesis p 950

Volume 115, Number 11, November 7, 2014
ISSN 0009-7330
http://circres.ahajournals.org
American
Heart
Association®
Circulation
Research
CD133+ Progenitor Cells to Promote Angiogenesis p 950
Nox1 Phosphorylation Mediates NoxA1 Association p 911
Ankyrin-G Signaling Platform p 929
MIF–CXCR7 Interaction and Platelet Survival p 939
Selected CD133+ Progenitor Cells to Promote Angiogenesis in Patients with Refractory Angina:
The Final Results of the PROGENITOR Randomized Trial.
Pilar Jimenez-Quevedo1, Juan Jose Gonzalez-Ferrer1, Manel Sabate2, Xavier Garcia-Moll3, Roberto
Delgado-Bolton4 , Leopoldo Llorente1, Esther Bernardo1, Aranzazu Ortega-Pozzi1, Rosana HernandezAntolin1, Fernando Alfonso1, Nieves Gonzalo1, Javier Escaned1, Camino Bañuelos1, Ander Regueiro2,
Pedro Marin2,Antonio Fernandez-Ortiz1, Barbara Das Neves1, Maria del Trigo1, Cristina Fernandez5,
Teresa Tejerina6, Santiago Redondo6, Eulogio Garcia1, Carlos Macaya1
1
Cardiology and Hematology Department ,Hospital Clínico San Carlos, Madrid, Spain IdISSC;
Cardiology and Hematology Department, Hospital Clinic, IDIBAPS; Barcelona, Spain; 3Cardiology
Department, Hospital Sant Pau, Barcelona, Spain; 4Instituto PET Focuscan; 5Statistic Department
Hospital Clínico San Carlos, and; 6Pharmacology Department, Complutense University, Madrid,
Spain.
2
Running title: CD133+ Progenitor Cells to Promote Angiogenesis
Subject codes:
[7] Chronic ischemic heart disease
[129] Angiogenesis
Address correspondence to:
Dr. Pilar Jiménez-Quevedo
Interventional Cardiology Department.
Hospital Clínico San Carlos. IdISSC
c/ Martín Lagos s/n, 28040, Madrid.
Tel: +34913303283.
Fax: +34913303290
[email protected]
In August, 2014, the average time from submission to first decision for all original research papers submitted
to Circulation Research was 13.55 days.
DOI: 10.1161/CIRCRESAHA.115.303463
Copyright by American Heart Association, Inc. All rights reserved.
1
ABSTRACT
Rationale: Refractory angina (RA) constitutes a clinical problem.
Objective: The aim of this study was to assess the safety and the feasibility of transendocardial injection
of CD133+cells to foster angiogenesis in patients with RA.
Methods and Results: in this randomized, double-blinded, multicenter controlled trial, eligible patients
were treated with granulocyte-colony-stimulating factor, underwent an apheresis and NOGA mapping and
were randomized to receive treatment with CD133+cells or no treatment. The primary endpoint was the
safety of transendocardial injection of CD133+cells, as measured by the occurrence of major adverse
cardiac and cerebrovascular event at 6-month. Secondary endpoints analyzed the efficacy. Twenty-eight
patients were included (n=19 treatment; n=9 control). At 6-month, 1 patient in each group suffered
ventricular fibrillation and 1 patient in each group died. One patient (treatment group) had a cardiac
tamponade during mapping. There were no significant differences between groups with respect to
efficacy parameters, however, the comparison within groups showed: a significant improvement in the
number of angina episodes/per month (median-absolute difference (mAD):-8.5(95%CI, -15.0;-4-0) and in
angina functional class in the treatment arm but not in the control group. At 6-months, only one SPECT
parameter: Summed Score improved significantly in the treatment group: at rest and at stress:(mAD:1.0(95%CI,-1.9;-0.1) but not in the control arm.
Conclusions: Our findings support feasibility and safety of transendocardial injection of CD133+cells in
patients with RA. The promising clinical results and favorable data observed in SPECT summed score
may set up the basis to test the efficacy of cell therapy in a larger randomized trial.
Keywords:
Angiogenesis, refractory angina, stem cell, chronic ischemia.
Nonstandard Abbreviations and Acronyms:
PC
progenitor cell
BM
bone marrow
G-CSF
granulocyte-colony-stimulating factor
VV
unipolar voltage
LLS
linear local shortening
MACCE
major adverse cardiovascular and cerebrovascular events
MI
myocardial infarction
LVEF
left ventricular ejection fraction
DOI: 10.1161/CIRCRESAHA.115.303463
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2
INTRODUCTION
Refractory angina in patients with ischemic heart disease who are not candidates for coronary
revascularization constitutes a major cause of disablement, impairs patient´s quality of life, and remains a
major clinical challenge.1
In recent years two pivotal areas of research opened new possibilities for the development of new
treatment strategies in these patients: first, the finding that circulating endothelial progenitor cells
incorporate to the capillary plexus to form collateral circulation in ischemic zones;2and second, the
demonstration that adult endothelial cells have a bone marrow (BM) origin.3Several preclinical studies
using transendocardial injection of BM-derived progenitor cells (PC) to treat chronic myocardial ischemia,
have demonstrated the safety and feasibility of this type of treatment and have showed positive results in
terms of increasing capillary density4, and in improving left ventricular ejection fraction (LVEF) and
myocardial perfusion5 in the treated segments. The results of further clinical studies6-13, focused on
enhancing collateral circulation in patients with chronic angina and a coronary anatomy not amenable for
revascularization, were consistent with the results of preclinical research. Most of these clinical studies
used the mononuclear fraction of BM, demonstrated a significant decrease in angina severity, and
improvements in quality of life, myocardial perfusion and exercise capacity. 6-13
Importantly, available evidence suggests that the biological activity of BM derived cells varies
substantially, and that the CD34+/CD133+ PCs fraction might be the most active in promoting
angiogenesis in the treated myocardium.14 In previous studies the percentage of these CD34+/CD133+PCs
in the mononuclear fraction of BM used was very low (around 3%), and no clinical studies using the
enriched CD133+PC to treat patients with chronic myocardial ischemia have been reported. Therefore, we
investigated the safety and feasibility of transendocardial injection of peripheral isolated CD133+PC with
the aim to improve myocardial ischemia and angina symptoms in patients not amenable for coronary
revascularization. Besides, we sought to characterize phenotypically the injected CD133+PC and to
determine their angiogenic capacity.
METHODS
Study design and eligibility.
The PROGENITOR (endothelial PROGENITOr cells and Refractory angina) trial is a Phase I/II,
multicenter, prospective, single-blinded and randomized clinical trial (ClinicalTrials.gov
identifier:NCT00694642).The study was performed at Hospital Clínico San Carlos, Madrid, Spain;
Hospital Clinic, Barcelona, Spain; and Hospital de Sant Pau, Barcelona, Spain. The study complied with
the provisions of the Declaration of Helsinki regarding investigation in humans and was approved by the
corresponding institutional review boards at all 3 investigational sites.The pilot study was originally
designed to enrol 30 patients however, due to slow enrolment, the study was stopped after the inclusion
of 28 patients.
Inclusion criteria in the PROGENITOR trial were: 1) patients with refractory angina with functional
CCS class II- IV for angina on optimal medical therapy with coronary anatomy not amenable for surgical
or percutaneous revascularization, as judged during a heart team discussion at the recruiting center; 2)
myocardial ischemia/viability demonstrated by a reversible perfusion defect by SPECT; and 3) signed
informed consent before randomization. Exclusion criteria were: 1) age <18 or >80 years; 2) permanent
atrial fibrillation; 3) acute myocardial infarction (MI) in the last 3 months; 4) presence of LV thrombus;
5) LV wall thickness <8 mm at the target site for cell injection; 6) history of malignancy in the last 5
years; 7) significant aortic valve disease; 8) pregnancy and 9) hemorrhagic disorders.
DOI: 10.1161/CIRCRESAHA.115.303463
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3
After obtaining the informed consent a centralized telephonic randomization was performed using a
computer-generated code before the index procedure. Patients were randomized to receive treatment with
CD133+PC or no treatment in a 2:1 ratio. Both groups were treated with granulocyte colony-stimulating
factor (G-CSF), underwent an apheresis and a NOGA mapping. However, in the control group, the
transendocardial injections were not performed but were simulated to keep the patient and all the
investigators except the two operators that performed the injections blinded.
Cell preparation.
All patients were treated with G-CSF (Neupogen ®, Amgen, Thousand Oaks, CA) 5µg/kg/12 hours for 4
days. The 5th day all patients underwent leukapheresis to isolate the mononuclear fraction from the
peripheral blood. Only those patients allocated to the cell group CD133+PC were isolated by
immunomagnetic selection with CliniMacs®cell separation system (MiltenyiBiotec, Bergisch-Gladback,
Germany). Sterility tests (Gram´s stain and culture) were performed on the final cell preparation. The
target dose was 20-30x10 6. The cells were suspended in normal saline and concentrated in 3 ml for the
injection.
Cell injection procedure.
While the inmunomagnetic selection was performed the patient underwent an electromechanical mapping
with the NOGA XPTMplatform (BDS, Johnson & Johnson) using the NOGAstarTM mapping catheter. The
target zone was defined as previously described: unipolar voltage (UV)>6.9 mV associated with
decreased mechanical activity: local linear shortening (LLS)<12. 14 Immediately after the positive
selection was performed, the cells were delivered into the ventricle with the Myostar injection catheter
(BDS, Johnson & Johnson) on the same day. The number of injections recommended per protocol was
fifteen. Immediately after the procedure and before discharge 2-D echocardiogram was performed.
The methodology of Characterization of the injected cells is explained in Online Appendix I.
Clinical and invasive follow-up.
After myocardial cell injection the patients were admitted to the coronary care unit for continuous
electrocardiogram monitoring during 12hours. Serial creatinine kinase and troponin I was measured each
8 hours up to three times. In the absence of complications patients were discharged the following day
after myocardial cell injection. Clinical follow-up was scheduled at 1 week, 1, 3, 6 months, 1 and 2 years.
Clinical history, physical examination, 12-lead electrocardiogram, laboratory tests and 24-hour holter
electrocardiogram recording were obtained in all visits. At 6-months all patient underwent a coronary
angiogram to rule out new obstructive coronary disease and underwent electromechanical mapping with
NOGA XPTM (BDS, Johnson & Johnson). Regarding the NOGA mapping in order to assure the quality of
the maps in our study, special care was taken to only accept points with a stable (measured by loop
stability, cycle length, local activation time stability parameters in NOGA) and clearly detectable wall
contact (verified by clear detectable bipolar voltage signals). In addition, analysis was performed using
NOGA Bulls eye segmental analysis, where a homogenous point distribution with a minimum of 3 to 5
edited points per segment (17-segment bulleye) was anticipated.
Functional and imaging studies.
Patients underwent perfusion evaluation with SPECT, echocardiograms and treadmill test preprocedurally, at 6-months and at 1-year. LVEF was measured by three methods: echocardiogram, gated
SPECT and ventriculography. LV angiograms were obtained in the same projections at the time of the
baseline procedure and at 6-months. A blinded investigator analyzed the LV angiograms with the use of a
computer-based system (CASS). Echocardiogram acquisition and analyses were performed according to
the guidelines.15Treadmill test were performed using the Naughton protocol. Gated-SPECT was
performed using technetium-99m sestamibi. Naughton protocol or dipyridamol (0.56mg/kg in 4 minutes)
was used. The LV was divided into 17 segments.15 Rest and stress Summed stress score was calculated by
the summation of the patients` segmental stress score and with the summation of the patients` segmental
DOI: 10.1161/CIRCRESAHA.115.303463
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4
rest score. All the analyses were centralized in an independent core lab blinded to the randomization
located in the PET Focuscan, Madrid Spain. NOGA data was analyzed as previously described.13
End points and definitions.
The primary endpoint was the safety of transendocardial injection of circulating CD133+ cells, as
measured by the occurrence of major adverse cardiac and cerebrovascular event (MACCE) at 6-month
follow-up. MACCE was defined as cardiovascular death, non-fatal MI, ischemic stroke, need for
revascularization, procedure-related complication: pericardial effusion/cardiac tamponade, vascular
complications and sustained ventricular arrhythmias. MI was defined according to the third universal
definition of MI.16
Secondary endpoints included the efficacy of the transendocardial injection of PC CD133+ assessed by
means of the following variables: the change in the myocardial perfusion defect as measured by SPECT,
symptom-limited treadmill test, Quality of life, CCS angina classification and antianginal medication
requirement.
Statistical analyses.
Continuous variables were expressed as median (interquartile range) and categorical variables as
percentages. Baseline comparisons between groups were performed by unpaired nonparametric test (U of
Mann-Whitney) for quantitative variables or Fisher's exact test for categorical variables. All comparisons
within groups were performed by nonparametric-paired test for quantitative variables (Wilcoxon paired
test), The results were expressed by absolute difference (median absolute difference between 6-monthsbaseline) and interquartile range. To assess the treatment effect between groups the covariance analysis
was used and was adjusted by baseline value and age (ANCOVA). The results were expressed by
absolute difference (average absolute difference) and 95% interval confidence. To contrast the difference
in the probability of MACCE between groups, the probability of an event was estimated using the
probability of the control group as a reference under a binomial distribution. This is a pilot study and
therefore no specific sample size was calculated. All statistical analyses were performed according to the
intention to treat principle using SPSS (version 15.0) or STATA (version 9.0) software, and all reported
P-values were two-sided. Statistical significance was set at P<.05.
RESULTS
Study patients.
From October 2008 to February 2012 twenty-eight patients were included in the study. Nineteen
patients were allocated to the cell group and nine to the control group. The flow chart of the study is
depicted in figure 1. Baseline characteristics are described in Table 1. Overall, median age was 64.4(58.073.1) years, 85.7% were male, 71.4% had CCS angina ≥class III. Both groups were well balanced except
that patients from the treatment arm were significantly older than those of the control group.
Procedural data and clinical outcomes.
G-CSF treatment was well tolerated, all patients presented bone pain as the only symptom that
was relieved with analgesics. In the treated arm all patients received a fixed dose of 30x106CD133+
selected cells, except one who received 24x106CD133+. After cell injection none of the patients had a
significant rise in CPK, symptoms, ECG changes or echocardiographic abnormalities. Twenty-four hour
after the procedure the median troponin levels was 1.3 (1.0-2.0) ng/mL.
DOI: 10.1161/CIRCRESAHA.115.303463
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5
Six-month clinical follow-up was performed in 100% of patients. Clinical events at 6-months are
summarized in Table 2. In the control group, 1 patient with low LVEF suffered from ventricular
fibrillation 24-hours after the baseline procedure and required an implantable cardioverter defibrillator.
This patient died from a fatal MI 3.5 months after his inclusion, no autopsy was performed. In the
treatment group, 1 patient presented ventricular fibrillation during the injection procedure that was
successfully cardioverted. This patient had normal LVEF and his clinical outcome during follow-up was
uneventful. Another patient in the treatment group suffered from cardiac tamponade during mapping. The
tamponade was successfully resolved but, eventually, the patient died in cardiogenic shock. Overall,
MACCE rate was comparable between groups based on the binomial probability (0.20). None of the
patients showed progression of atherosclerosis disease at the 6-month coronary angiogram
Characterization of CD133+PC.
The CD133+ cell purity was 94.5% with >97% viability (Table 3, Figure 2-panel A). The
expression of immature antigens CD133+, CD34+ and ALDH activity decreased in cultured cells after 7
and 15 days. However, the expression of endothelial markers (KDR, VE-Cadherin, P1H12, TIE-2)
increased significantly in cultured cells after 7 and 15 days (Table3) An example of 2-month cultured PC
is depicted in Figure 2: panel B. In addition these cells showed capacity of microtubules formation
(Figure 2; panel C-D).
Clinical results and exercise capacity.
The (adjusted) comparison between groups in clinical parameters and Seattle Angina
questionnaire showed no significant differences of any of them. (All comparisons are provided in Online
Appendix II).
With respect to the comparison within groups of clinical parameters: after 6-months the number
of angina episodes per month and the number of nitroglycerin-tablet consumption per month decreased
significantly in the treatment group [median absolute difference (mAD) -8.5 (95%CI, -15.0;-4.0) and
mAD -3.5 (95%-5.2;0.0), respectively]. Alternatively, no significant changes were observed in the control
group [mAD: -1.5, (95%CI, -8.7 to 15.0), and mAD: -0.5 (95%CI, -6.5 to0.0), respectively]. Likewise,
the CCS class significantly improved in the cell group [mAD -1.0 (95% CI, 2.0 to 0.0)] whereas no
significant changes were observed in the control group [mAD 0.0 (95%CI, -1.0 to 0.0)] Figure 3. The
analysis within groups of Seattle Angina questionnaires is described in Online Appendix III.
Exercise capacity measure by treadmill test was not performed in 6 patients (4 patients in the
treatment group and 2 patients in the control group) who were unable to perform adequate exercise
because of physical limitation or lack of motivation. The change in the median time of exercise and
metabolic equivalents (METS) were not different in the 2 groups at 6-months: Treatment: [from 515
(487.5-708.5) to 652 (471.0-709.5); p=0.39], and (from 4.6 (4.4-5.7) to 5.7 (4.4-6.5); p=0.72); control
group: [from 604.8(438.0-865.2); to 609.0 (372.0-935.4);p=0.80] and (from 6.0 (5.2-8.7) to 7.0 (5.5-8.2);
p=0.13). However, the median time to the onset of angina was significantly greater at 6 months in the
treatment group without significant changes in the control group. Treatment group: [baseline:
428.4(357.1-542.8); 6-month: 635 (442.2-706.0); p=0.046]. In the control group: [baseline: 466.5(267.0673.6); 6-month: 466.5 (142.5-913.8); p>0.99].
Myocardial perfusion, echocardiography and NOGA results.
Table 4 shows echocardiogram, SPECT and NOGA data obtained at baseline and at 6 months
follow-up. The percentage of baseline reversible defect was similar between groups. In contrast an
increase in the percentage in baseline fixed defect was observed in the treatment group. For this reason,
DOI: 10.1161/CIRCRESAHA.115.303463
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all comparisons between groups at 6 months were adjusted for baseline data and age, but remained not
significant (Table 4). Regarding the comparison within groups no significant change over time in the
percentage of reversible ischemic segments in SPECT was noted. Of note, the only SPECT parameters
that were associated with significant improvements in the treatment group were the summed rest and
stress scores that were not documented in the control group.
Regarding the echocardiographic findings no significant changes were observed in the ventricular
diameters in either group. LVEF measured by different methods (echo, Gated SPECT and
ventriculography) was also similar.
At 6 months, NOGA mapping was performed in all patients except 2 (1 in each group) who
refused to undergo this invasive diagnostic study. At 6-months compared to baseline LLS but not UV
significantly improved in the injected segments of treated patients. In the control group, no significant
changes in LLS and UV were observed. Figure 4.
DISCUSSION
The main findings of this phase I-II randomized controlled study were as follows. First,
transendocardial injection of selected CD133+PC in patients with refractory angina is feasible and safe.
No differences in safety events were observed between groups. Secondly, the comparison between groups
was not significant and therefore the study is essentially negative from the standpoint of objective
measures of ischemia. However, the serial comparison showed that the treatment with CD133+ cells
injection was associated with a significant improvement in clinical status, quality of life, time to angina in
the treadmill test at 6-months, whereas these improvements were not observed in the control group. In
addition, there was only one SPECT parameter that significantly improved only in the treatment group:
the summed score. Finally, the characterization of CD133+ cells on the basis of ALDH levels measured
after their isolation suggested an important proliferate and reparative capacity. In addition, these cells
expressed endothelial markers and showed “in vitro” angiogenic capacity after culture.
Selected CD33+ cells have been used previously to treat patients with acute or chronic MI 17-19 In
these studies the delivery method were the intracoronary injection or transmyocardial injection during
surgery. To the best to our knowledge the current trial represents the first study using transendocardial
injection guided by NOGA of enriched selected CD133+ cells to treat patient with refractory angina.
Previous studies have demonstrated the presence of mature endothelial cells and PC in the peripheral
circulation. Both populations may express the CD34+ surface marker and therefore, this marker is not
useful to select only the more immature cells. On the other hand, CD133+ surface marker is highly
expressed on immature stem cell as it is down-regulated as the cells differentiate to mature endothelial
cell.20 Therefore, the population of cells obtained after CD133+ positive selection, although the majority
of them are CD34+, constitutes an immature population of PC able to differentiate into mature endothelial
cells.21 Importantly, within peripheral CD133+PC there is a subpopulation of cells defined as CD34-/133+
which are functionally more potent than CD34+/CD133+ cells. 22In the present study the rationale to select
this marker was to obtain an immature population of PC, however, the clinical effect of this selection
compared with others selection of PC is unknown and have to be explored.
The potential relevance of using enriched selected CD133+or CD34+ cells find support in a
previous randomized study in which a relationship between the magnitude of the effect of transendocardial cell injection and the percentage of CD 133+ cells was found during the analysis of study data.
In this regard, an interesting randomized study included 92 no-option patients, defined as LVEF ≤45%
with a demonstrated myocardial ischemia and limiting heart failure or angina symptoms, allocated to 100
million BM mononuclear cells or placebo (2:1 ratio). The study was negative with regards to its primary
DOI: 10.1161/CIRCRESAHA.115.303463
Copyright by American Heart Association, Inc. All rights reserved.
7
endpoints (change in LVESV, maximal oxygen consumption and defect size), but during the analysis of
data a proportional relationship between the percentages of CD34+ or CD133+ cells injected and increase
in LVEF improvement was found. This is the first time that a study evaluates this interaction and suggests
that selecting these cells may improve the efficacy after treatment.14
Leaving aside the different type of cells used in the PROGENITOR trial, its overall favorable
results in terms of reducing angina frequency and improving quality of life are in agreement with other
randomized studies most of them that used non-enriched cell populations to treat patients with refractory
angina. In a phase II study, 50 patients were randomized to receive non-selected mononuclear fraction of
the BM or placebo.7 At 6-months the CCS, exercise capacity and LVEF measured by MRI improved
significantly in the treatment group, but not in the control group. Quality of life and perfusion
measurement by SPECT (summed stress score and the mean number of ischemic segments) significantly
improved in both study groups; however, the documented improvement was greater in the treatment
group. The authors posed the hypothesis that improvement in the control group might reflect angiogenesis
triggered by intramyocardial injections performed to deliver placebo. In the PROTECT-CAD TRIAL, 28
patients were randomized to different dose of BM mononuclear cells (1X106/0.1mL and 2x106/0.1mL). In
this study a significant increase in the treadmill test, LVEF and NYHA class was reported. However, CCS
class was reduced in both groups. 8
The only phase II study that have used selected PC to treat patients with refractory angina was
theACT34-CMI study, a phase II study that included 167 patients with severe refractory angina (class IIIIV) randomly assigned to receive two different doses of selected CD34+ cells: 1x105(low dose) and 5x105
cells/Kg (high dose). In this study only the low dose was associated with a significantly lower weekly
angina frequency and a significantly improvement in exercise tolerance. In addition, total severity score
stress significantly improved compared with the control group, the remaining standard SPECT imaging
parameters revealed no significant differences between treated and control groups. Although stem cell
therapy using BM derived stem cell have shown controversial results in other clinical scenarios such as
acute or chronic MI, in patients with refractory angina all studies demonstrated consistent results.23-24
SPECT is an important imaging modality in the management of patients with cardiovascular
disease. However, SPECT has some limitations especially in patients with refractory angina that usually
have multivessel disease. One of the main limitations of SPECT is the fact that it measures only relative
uptake, and therefore, in patients with three-vessel disease; the decreased perfusion to all walls may not
be recognized and this may lead to an underestimation of the extent of ischemia. In our study all patient
had multivessel disease, and for this reason small changes in perfusion within a segment or several
segments may be underrecognized by this technique. This may explain the lack of significant changes in
the percentage of ventricle reversible segments in each group. However, the Summed score, that has been
considered a global perfusion index and has presented correlation with prognosis in previous studies25,
improved significantly in the treatment group but not in the control arm. This particular situation
involving lack of significant changes in the number of reversible defects but with significant changes in
the summed score has been seen in other clinical trials involving patients with refractory angina6.
At a difference with the above-mentioned studies, in our study the exercise capacity did not
experience a significant increased in the treatment group. We consider that this may be as a result of a
small sample size. However, the median time to angina onset improved significantly in the treatment
group and was unchanged in the control group. This result is in accordance with the clinical improvement
observed the treatment arm. In addition, LVEF and the finding that standard means of measuring regional
function (wall thickening and wall motion) was also similar at 6 months in both groups. These results
were initially expected as baseline LVEF was almost preserved in both groups, especially in the control
arm. This may account for the lack of difference in the change of LVEF and regional function at followup.
DOI: 10.1161/CIRCRESAHA.115.303463
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Another characteristic of the present study were: first, all cell processes were injected in the same
day avoiding the possible effect of storage and secondly, in this study no placebo injections were
performed in order to avoid any potential effect of the needle in the control arm. Regarding the NOGA
parameters, the LLS is a parameter that has been previously validated in previous studies 26. In our study
the significant improvement in the LLS observed in the treatment group was in agreement with a previous
study 14 that has used the same criteria (viable myocardium) to inject the cells.
Several considerations have to be made regarding the safety of trans-endocardial cell injections.
This constitute an invasive modality of treatment with obvious potential for complications, and for which
estimating the net benefit in the future should take into account not only the potential to treat a large
number of patients with high risk profile that currently do not have a therapeutic option for a disabling
condition, but also the foreseeable developments in dedicated hardware and operator expertise that might
contribute to procedural safety. This might decrease, for example, the risk of LV wall perforation and
cardiac tamponade during electromechanical endocardial mapping or LV wall injections that have been
described in previous studies6,27 and that in the PROGENITOR study occurred in 1 patient. In our case,
the patient with a complication had a small body surface area, small ventricular cavity and normal
ejection fraction with a hyper-contractile ventricle. In view of the occurrence of LV perforation, we
propose that electromechanical mapping in patients similar to this one should be performed with the
smaller curve of the catheter (B), at a difference with the one that was actually used (D curve).
Limitations.
This is a pilot study that was not designed to assess efficacy. Only the comparisons that resulted
significant were those performed within groups. The comparisons between groups were no significant.
Therefore any claims of efficacy should be taken with caution given the size of the study and the potential
presence of Type II error given the multiplicity of parameter evaluated. Further and larger studies are
warranted to confirm these initial positive results. Although the 2:1 randomization may have the tendency
to reach non-significant when there is a big difference in the sample sizes, in this study we have used 2:1
randomization to maximize the probability to be treated and to increase patient acceptance of the trial.
Despite being a randomized trial, a significant difference was observed in one baseline variable
between groups (i.e. age) probably due to the small number of patients. For this reason all the analyses
between groups were adjusted in order to avoid bias in the results.
Intramyocardial placebo injections were no performed in the control group, and therefore the two
investigators who performed NOGA mapping were un-blinded. However, these investigators do not have
access to the patient data. The remaining investigators involved in the clinical follow-up, the analyses of
data and the patients were blinded to the randomization. Although this may constitute a limitation of the
study, we consider that the possible stimulation of angiogenesis mediated by the needle itself was ruled
out in the control group. Regarding the NOGA parameters, LLS values are strongly dependent on the
quality of each collected mapping point and the entire NOGA map for this reason special care was
taken to build a good quality map. However, we cannot completely rule out that serial measurement of
LLS by NOGA may present some degree of variability. Thus larger trials are warranted to corroborate
these findings
Conclusions.
In conclusion, this first-in-man study confirms that injection of selected CD133+ cells in
ischemic myocardium identified with EM mapping is feasible and safe. The study is essentially negative
from the standpoint of objective measures of ischemia. However, the results derived from the serial
DOI: 10.1161/CIRCRESAHA.115.303463
Copyright by American Heart Association, Inc. All rights reserved.
9
analyses in terms of improvement of angina symptoms and some SPECT-derived ischemia parameters in
non-candidates for myocardial revascularization are encouraging and should be confirmed in a large
randomized trial.
ACKNOWLEDGMENT
We would like to thank to Emerson Perin for performing the transendocardial injections of the first
patient included in the study.
SOURCES OF FUNDING
This study trial was a noncommercial trial. It was funded by an independent research grant from the
Spanish National Ministry of Health and Social Policy (Direccion general de Terapias Avanzadas y
Transplante (TRA-019) and an unrestricted Grant from Mutua Madrileña Foundation (FMM08). Dr.
Jimenez-Quevedo is recipient of the ISCIII grant “Fondo de Investigación Sanitaria (PI11/00299) which
relates to the topic of this study.
DISCLOSURES
None of the authors have any conflict of interest
REFERENCES
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Lootens N, Heyndrickx G, WijnsW. Intracoronary injection of CD133-positive enriched bone marrow
progenitor cells promotes cardiac recovery after recent myocardial infarction: feasibility and safety.
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Am Coll Cardiol. 2005;45:982-988
DOI: 10.1161/CIRCRESAHA.115.303463
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12
TABLES
Table 1: Baseline characteristics of the patients included in the study
Treatment Group
Control Group (n=9)
(n= 19)
P value
Age
70.0 (61-73.3)
58.2 (47.8-65.7)
0.02
Male, n (%)
15 (78.9)
9 (100.0)
0.27
Current smoker, n (%)
3 (15)
1 (11)
0.80
Diabetes Mellitus, n (%)
10 (52)
5 (55)
1
Hypertension, n (%)
16 (84)
9 (100)
0.50
Hypercholesterolemia, n(%)
17 (89)
6 (66)
0.30
Body mass index
29.0 (26.0-31.9)
29.3 (28.4-30.4)
0.60
Left ventricular ejection
fraction, %
Prior myocardial infarction, n
(%)
Prior revascularization, n (%)
51.0 (40.0-63.0)
55.0 (51.2-63.5)
0.40
13(68.4)
6(66.7)
>0.99
17(89.5)
7(77.8)
>0.99
Peripheral vascular disease, n
(%)
ACE inhibitors, n (%)
2(10.5)
1(11.1)
>0.99
17(89.5)
7(77.8)
0.57
Β-Blockers, n (%)
18(94.7)
7(77.8)
0.23
Nitrates, n (%)
16(84.2)
8(88.9)
>0.99
Statins, n (%)
19 (100)
9(100)
-
Calcium-antagonists, n (%)
8(42.1)
6(66.7)
.042
Aspirin, n (%)
18(94.7)
8(88.9)
>0.99
Clopidogrel, n (%)
15(78.9)
5(55.6)
0.37
Nº angina epidodes/month
12.0 (6.0-28.0)
10.0 (5.0-11.0)
0.26
Nº nitroglicerin/month
4.0 (2.0-12.0)
5.0 (2.0-9.0)
0.88
CCS angina class
3.0 (2.0-3.0)
3.0 (2.0-3.0)
0.78
Data are expressed as median (interquartile range) or percentage.
CCS: Canadian Cardiovascular Society, ACE inhibitors: angiotensin-converting-enzyme inhibitors
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13
Table 2: Clinical events at 6-months (non-hierarchical)
Treatment
Group
n=19
Major Cardiac and cerebrovascular events*, 2 (10.5)
n(%)
Cardiac death, n (%)
1 (5.3)
Control
Group
n=9
1 (11.1)
P value
>0.99
1 (11.1)
>0.99
Non-fatal myocardial infarction, n (%)
0
0
>0.99
Sustained VT/VF, n(%)
1(5.3)
1(11.1)
>0.99
Pericardial effusion/ Cardiac tamponade,
n(%)
1(5.3)
0
>0.99
Vascular complications, n(%)
0
0
-
Neurological complications, n(%)
0
0
-
2 (25)
>0.99
Repeat hospitalization for cardiac cause,
2 (11.1)
n(%)
VF: ventricular fibrillation, VT: ventricular tachycardia.
*Hierarchical events
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14
Table 3: Cell product characteristics before injection and after culture
Injected cells
7 days of culture 14 days of
(N= 19)
culture
Viability, (7 7Aminoactinomycin D) %
Nº CD 133 cells X106
P
value
97.3 (95.9-98.4)
Cell dose X106
65.2 (37.9100.8)
30 (24-30)
CD45+low/Cellproduct, %
98.7(96.5-99.2)
94.1 (88.8-96.7)
94.0 (85.6-96.3)
0.006
CD133+/Cellproduct, %
94.5 (91.2-98.1)
40.6 (13.8-83.1)
7.8 (2.8-26.2)
<0.001
CD34+/Cellproduct, %
93.7 (86.3-97.2)
30.2 (7.4-68.0)
2.2 (0.6-4.2)
<0.001
CD14+/Cellproduct, %
0.7 (0.1-2.4)
0.2 (0.2-3.1)
0.8(0.0-13.1)
0.35
CD31+/Cellproduct, %
63.5 (31.0-86.9)
63.1 (47.7-78.3)
73.4 (67.3-74.7)
0.91
CD117+/Cellproduct, %
27.9 (13.1-48.3)
41.1 (18.5-48.8)
36.3 (26.5-44.5)
0.67
KDR/Cellproduct, %
0.3 (0.2-2.4)
3.3 (1.9-8.3)
5.7 (2.0-10.7)
0.006
VEcadherina/Cellproduct, %
P1H12/Cellproduct, %
0.6 (0.2-2.1)
25.8 (13.4-49.7)
43.4 (36.5-64.6)
<0.001
0.2 (0.1-0.4)
4.5 (2.6-13.9)
13.3 (6.4-27.5)
<0.001
Tie+/Cellproduct, %
2.4 (0.8-3.7)
11.5 (8.9-24.0)
21.3 (14.9-50.9)
<0.001
11.2 (4.5-24.8)
2.2 (1.6-14.0)
<0.001
84.0 (75.5-95.7)
91.2 (83.4-96.5)
0.18
Functional analyses
(n=11)
Colony forming units
ALDH bright
3.2 (1.1-18.1)
88.3 (80.8-92-6)
Dil-AcLDL
Microtubules formation
100%
Data are expressed as median (interquartile range) or percentage.
ALDHbright: aldehyde dehydrogenase
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15
Table 4: Echocardiogram, SPECT and NOGA results at baseline and at 6-months
Treatment
Control
Inter-group
Inter-group
Absolute
Absolute
difference
Difference
(95%CI) *
(95%CI)†
n=17
n=7
Echocardiogram
Left ventricular
ejection fraction (%)
Baseline
6-months
Intra-groupP value
End Diastolic
diameter (mm)
Baseline
6-months
Intra-group P value
End Systolic diameter
(mm)
Baseline
6-months
Intra-group P value
Wall motion index
Baseline
6-months
Intra-group P value
Wall Thickening (%)
Baseline
6-months
Intra-group P value
SPECT
Gated SPECT
Ejection fraction (%)
Baseline
6-months
Intra-group P value
Percentage of
ventricle reversible
segments
Baseline
6-months
Intra-group P value
Percentage of
ventricle fixed
segments
Baseline
6-months
Intra-group P value
Summed rest score
51.0 (40.0-63.0)
61.1 (48.2-70.2)
0.20
55.0 (51.2-63.5)
55.0 (55.0-64.0)
0.34
1.6(-8.9;12.1)
5.0(-17.4;7.4)
52.0 (46.0-56.0)
53.0 (45.5-58.5)
50.0 (47.0-53.0)
51.5 (46.2-54.7)
1.9(-2.7;6.4)
2.7(-3.2;8.5)
0.11
0.93
38 (30.0-43.7)
37.5 (21.2-44.5)
34.0(29.0-37.5)
31.0 (30.0-38.0)
2.9(-2.6;8.5)
2.9(-2.6;8.5)
0.23
0.83
1.2 (1.1-1.8)
1.3 (1.0-1.7)
0.52
1.1 (1.1-1.4)
1.0 (1.0-1.1)
0.20
0.1(-0.1;0.4)
0.03(-0.2;0.3)
87.0 (85.0-90.0)
86.0 (85.0-88.0)
0.22
n=17
86.0 (86.0-89.0)
86.0 (85.0-87.5)
0.26
n=7
0.4 (-0.6;1.5)
0.2 (-1.0;1.5)
53.5 (32.2-66.2)
55.0 (31.5-64.0)
0.22
57.0 (50.5-71.0)
57.0 (54.0-64.0)
0.20
0.83(-9.2;10.9)
-0.5(13.1;12.0)
11.8 (11.8-23.5)
11.8 (5.9-19.1)
17.6 (11.8-26.4)
12.0 (3.0-20.6)
2.2(-3.6;8.1)
5.1 (-1.4;11.7)
0.07
0.11
14.7 (0-19.1)
8.8 (0-17.6)
0.0 (0.0-10.3)
0.0 (0.0-10.3)
0.2 (-4.4;4.7)
-1.6 (-6.9;3.7)
0.21
>0.99
DOI: 10.1161/CIRCRESAHA.115.303463
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16
Baseline
21.0 (17.0-26.0) 18.0 (17.0-21.0) -0.4(-1.2;0.4)
-0.7(-1.6;0.1)
19.5 (17.7-23.0) 17.5 (17.0-20.5)
6-months
Intra-group P value
0.03
0.32
Summed stress score
Baseline
24.0 (21.0-26.0) 20.0 (19.5-25.0) -0.9(-2.3;0.3)
-0.7(-2.3;0.9)
6-months
23.5 (19.7-27.0) 20.0 (18.5-23.7)
Intra-group P value
0.003
0.20
NOGA
n=16
n=6
Unipolar Voltage‡
Baseline
10.4 (7.4-14.2)
12.5 (8.1-15.0)
-0.4(-1.3;2.0)
1.0(-1.0;3.1)
6-months
11.4 (7.4-16.6)
10.1 (7.7-13.9)
Intra-group P value
0.55
0.57
Linear Local
Shortening
Baseline
10.6 (5.2-13.6)
10.7 (3.8-17.4)
-4.1(-7.2;-0.9) -2.5(-6.4;1.3)
6-months
12.3 (6.8-18.5)
8.9 (3.5-12.3)
Intra-group P value
0.01
0.30
Ventriculography
Ejection fraction
Baseline
50.0 (38.5-64.5) 59.0 (51.2-66.2) -0.4(-8.2;7.4)
-1.6(-11.9;8.6)
6-months
52.0 (40.0-65.5) 63.0 (57.5-65.5)
Intra-group P value
0.37
0.78
Data expressed as median (interquartile range)
Absolute difference refers to the absolute effect between groups at 6-months analyzed by ANCOVA and
adjusted by baseline data
† Absolute difference refers to the absolute effect between groups at 6-months analyzed by ANCOVA
and adjusted by baseline data and age
Intragroup p-value was calculated by paired non-parametric test
‡ Injected segments
DOI: 10.1161/CIRCRESAHA.115.303463
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17
FIGURE LEGENDS
Figure 1: Flow chart of patients in the PROGENITOR Trial.
Figure 2: Panel A: Hematoxylin-eosin stain of progenitor cells (PC) immediately after positive selection
of CD133+ cells. Note the uniformity of the cell shape with a characteristic morphology of haematological
immature cells consisted of round cell with prominent nucleus and very small amount of cytoplasm,
Panel B: 2-months cultured PC stained with fluorescent dye AcDilLDL. Panel C: PC (2x105 cell/ml)
cocultured with HUVECs (2x105 cell/ml) within extracellular matrix (ECM) gels and 50ng/ml of VEGF
forming tubule structures 20x magnification. Panel D: PC (2x105 cell/ml) cocultured with HUVECs
(2x105 cell/ml) within extracellular matrix (ECM) gels and 50ng/ml of VEGF stained with lectin
Ulexeuropeaus.
Figure 3: Change (from 6 months to baseline) in the number of angina episodes, the number of
nitroglycerin-tablet consumption and Canadian Cardiovascular Society class (CCS). The Y-axis
represents the 95% confidence interval and the X-axis three different time points: Baseline, 3 and 6
months. Data are expressed in medians. The analyses were performed with paired non-parametric test.
Figure 4: An example of two patients included in the PROGENITOR trial. Panel A and B show an
example of a patient allocated to the control group. Panel A: Baseline stress and rest SPECT polar maps
(upper), where perfusion is color-coded according to a linear semicontinuous scale located at the right, in
which each color represents a 10% interval and reduced perfusion is considered when below 50% (blue to
yellow): it shows inferior and infero-lateral reversible defects. Baseline NOGA maps (botton) from the
same patient: on the left the unipolar voltage (UV) map shows the viability of the tissue according to the
definition (UV>6.9), on the right side lineal local shortening map (LLS) shows a decrease in contractile
function at the infero-lateral wall of the ventricle. Panel B: Follow up SPECT polar maps at 6-month:
they show the persistence of the same perfusion defect in the inferior and infero-lateral wall of the
ventricle. The NOGA maps show the impairment of contractile function in the same walls. Panel C: and
D an example of a patient treated with CD133+ cells. Panel C: Baseline stress and rest SPECT polar
maps (upper) showing an infero-lateral reversible defect. Baseline NOGA maps (bottom) viewed from the
inferior position shows an area of viability (normal voltage values and decrease local shortening values)
at the infero-lateral wall of the ventricle (brown dots show the site of cell injections): Panel D: 6-months
SPECT shows an improvement in perfusion at infero-lateral wall. The NOGA map shows an
improvement of the LLS values at the injection site (on the right) reflecting improvement in mechanical
function.
DOI: 10.1161/CIRCRESAHA.115.303463
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18
Novelty and Significance
What Is Known?
 Refractory angina is caused by myocardial ischemia and currently without an effective treatment.
 Stem cell therapy has emerged as a promising treatment for patients with refractory angina.
 Several preclinical and clinical studies have demonstrated the safety and the feasibility of stem
cell transplantation in ischemic myocardium, with some positive results in terms of efficacy.
 Most stem cell trials have used bone marrow derived mononuclear cells or CD34+ cells .
What New Information Does This Article Contribute?

This phase I/II, multicenter, prospective, single‐blinded and randomized clinical trial used
CD133+ cells that constitute a more immature bone marrow-derived progenitor cell population.

High levels of ALDH activity in freshly isolated CD133+ cells suggest a high proliferative and
reparative capacity.

After culture in an endothelial medium, CD133+ cells expressed endothelial markers and showed
“in vitro” angiogenic capacity.
In this study, we used a selection of an immature progenitor population (CD133+ cells) to treat ischemic
myocardium using a percutaneous injection catheter (NOGA technology) to infuse cells directly to the
ischemic tissue. The results of the study show that transendocardial injection of CD133+cells is safe and
feasible in patients with refractory angina. However, with a small number of patients, the pilot study
showed no significant differences between the control and the treatment groups. Interestingly,
improvement in symptoms, quality of life, and one SPECT parameter (summed score) were observed only
in the treatment group during follow-up. These findings provide the basis for testing the efficacy of
CD133+ cell therapy in larger randomized trials.
DOI: 10.1161/CIRCRESAHA.115.303463
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19
Figure 1
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Figure 2
Copyright by American Heart Association, Inc. All rights reserved.
Figure 3
Copyright by American Heart Association, Inc. All rights reserved.
Figure 4
Copyright by American Heart Association, Inc. All rights reserved.