Clinical Track

Selected CD133+ Progenitor Cells to Promote Angiogenesis in Patients With Refractory Angina

Final Results of the PROGENITOR Randomized Trial

Pilar Jimenez-Quevedo, Juan Jose Gonzalez-Ferrer, Manel Sabate, Xavier Garcia-Moll, Roberto Delgado-Bolton, Leopoldo Llorente, Esther Bernardo, Aranzazu Ortega-Pozzi, Rosana Hernandez-Antolin, Fernando Alfonso, Nieves Gonzalo, Javier Escaned, Camino Bañuelos, Ander Regueiro, Pedro Marin, Antonio Fernandez-Ortiz, Barbara Das Neves, Maria del Trigo, Cristina Fernandez, Teresa Tejerina, Santiago Redondo, Eulogio Garcia, Carlos Macaya
https://doi.org/10.1161/CIRCRESAHA.115.303463
Circulation Research. 2014;115:950-960
Originally published September 17, 2014

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Abstract

Rationale: Refractory angina 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 refractory angina.

Methods and Results: In this randomized, double-blinded, multicenter controlled trial, eligible patients were treated with granulocyte colony-stimulating factor, underwent an apheresis and electromechanical mapping, and were randomized to receive treatment with CD133+ cells or no treatment. The primary end point was the safety of transendocardial injection of CD133+ cells, as measured by the occurrence of major adverse cardiac and cerebrovascular event at 6 months. Secondary end points analyzed the efficacy. Twenty-eight patients were included (n=19 treatment; n=9 control). At 6 months, 1 patient in each group had 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, −8.5 [95% confidence interval, −15.0 to −4.0]) and in angina functional class in the treatment arm but not in the control group. At 6 months, only 1 simple-photon emission computed tomography (SPECT) parameter: summed score improved significantly in the treatment group at rest and at stress (median absolute difference, −1.0 [95% confidence interval, −1.9 to −0.1]) but not in the control arm.

Conclusions: Our findings support feasibility and safety of transendocardial injection of CD133+ cells in patients with refractory angina. 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.

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

Editorial, see p 904

In This Issue, see p 897

In recent years, 2 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 (PCs) incorporate to the capillary plexus to form collateral circulation in ischemic zones2 and second, the demonstration that adult endothelial cells have a bone marrow (BM) origin.3 Several preclinical studies using transendocardial injection of BM-derived PCs 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 studies,6–13 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 low (≈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 endothelial PROGENITOr cells and Refractory angina (PROGENITOR) 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 on 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, because of 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 Canadian Cardiovascular Society (CCS) class II to 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 simple-photon emission computed tomography (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 past 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 past 5 years, (7) significant aortic valve disease, (8) pregnancy, and (9) hemorrhagic disorders.

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, underwent an apheresis and electromechanical mapping (EMM). However, in the control group, the transendocardial injections were not performed but were simulated to keep the patient and all the investigators except the 2 operators who performed the injections blinded.

Cell Preparation

All patients were treated with granulocyte colony-stimulating factor (Neupogen, Amgen, Thousand Oaks, CA) 5 μg/kg per 12 hours for 4 days. The fifth 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 stain and culture) were performed on the final cell preparation. The target dose was 20 to 30×106. The cells were suspended in normal saline and concentrated in 3 mL for the injection.

Cell Injection Procedure

Although the inmunomagnetic selection was performed, the patient underwent an electromechanical mapping with the NOGA XP platform (BDS, Johnson & Johnson) using the NOGAstar mapping catheter. The target zone was defined as previously described: unipolar voltage >6.9 mV associated with decreased mechanical activity: linear local 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 15. Immediately after the procedure and before discharge 2-dimensional 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 ECG monitoring during 12 hours. Serial creatinine kinase and troponin I was measured each 8 hours ≤3 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, and 1 and 2 years. Clinical history, physical examination, 12-lead ECG, laboratory tests, and 24-hour Holter ECG recording were obtained in all visits. At 6 months all patient underwent a coronary angiogram to rule out new obstructive coronary disease and underwent EMM with NOGA XP (BDS, Johnson & Johnson). Regarding the EMM 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 system) and clearly detectable wall contact (verified by clear detectable bipolar voltage signals). In addition, analysis was performed using NOGA XP 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 3 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. Echocardiogram acquisition and analyses were performed according to the guidelines.15 Treadmill test were performed using the Naughton protocol. Gated SPECT was performed using technetium-99 m sestamibi. Naughton protocol or dipyridamol (0.56 mg/kg in 4 minutes) was used. The LV was divided into 17 segments.15 Summed rest and stress scores were calculated by the summation of the patients’ segmental stress score and with the summation of the patients’ segmental rest score. All the analyses were centralized in an independent core laboratory blinded to the randomization located in the PET Focuscan, Madrid Spain. EMM data were analyzed as previously described.13

End Points and Definitions

The primary end point was the safety of transendocardial injection of circulating CD133+ cells, as measured by the occurrence of major adverse cardiovascular and cerebrovascular event at 6-month follow-up. Major adverse cardiovascular and cerebrovascular event was defined as cardiovascular death, nonfatal MI, ischemic stroke, need for revascularization, and 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 end points 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 (Mann–Whitney U test) for quantitative variables or Fisher 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 [mAD] between 6 months and baseline) 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 major adverse cardiovascular and cerebrovascular event 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 2-sided. Statistical significance was set at P<0.05.

Results

Study Patients

From October 2008 to February 2012, 28 patients were included in the study. Nineteen patients were allocated to the cell group and 9 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 (58.0–73.1) years, 85.7% were men, 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.

View this table:
Table 1.

Baseline Characteristics of the Patients Included in the Study

Figure 1.
Figure 1.

Flow chart of patients in the endothelial PROGENITOr cells and Refractory angina (PROGENITOR) Trial.

Procedural Data and Clinical Outcomes

Granulocyte colony-stimulating factor 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 30×106 CD133+ selected cells, except one who received 24×106 CD133+. After cell injection none of the patients had a significant rise in creatine phosphokinase, symptoms, ECG changes, or echocardiographic abnormalities. Twenty-four hours after the procedure the median troponin levels were 1.3 (1.0–2.0) ng/mL.

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 had ventricular fibrillation 24-hour 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 had cardiac tamponade during mapping. The tamponade was successfully resolved but, eventually, the patient died in cardiogenic shock. Overall, major adverse cardiovascular and cerebrovascular event 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.

View this table:
Table 2.

Clinical Events at 6 Months (Nonhierarchical)

Characterization of CD133+ PC

The CD133+ cell purity was 94.5% with >97% viability (Table 3; Figure 2A). The expression of immature antigens CD133+, CD34+, and aldehyde dehydrogenase activity decreased in cultured cells after 7 and 15 days. However, the expression of endothelial markers (kinase insert domain receptor [KDR], vascular endothelial [VE]-cadherin, P1H12, and tyrosine kinase receptor [TIE-2]) increased significantly in cultured cells after 7 and 15 days (Table 3) An example of 2-month cultured PC is depicted in Figure 2B. In addition, these cells showed capacity of microtubules formation (Figure 2C and 2D).

View this table:
Table 3.

Cell Product Characteristics Before Injection and After Culture

Figure 2.
Figure 2.

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 hematological immature cells consisted of round cell with prominent nucleus and small amount of cytoplasm. B, Two-month cultured PC stained with fluorescent dye AcDilLDL. C, PC (2×105 cell/mL) cocultured with HUVECs (2×105 cell/mL) within extracellular matrix (ECM) gels and 50 ng/mL of VEGF-forming tubule structures (×20 magnification). D, PC (2×105 cell/mL) cocultured with HUVECs (2×105 cell/mL) within ECM gels and 50ng/mL of VEGF stained with lectin Ulexeuropeaus. AcDilLDL indicates acetylated low-density lipoprotein, labeled with 1,1'- dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate; HUVEC, human umbilical vein endothelial cells; and VEGF, vascular endothelial growth factor.

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 (mAD, −8.5 [95% CI, −15.0 to −4.0] and mAD, −3.5 [95% CI, −5.2 to 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 to 0.0], 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.

Figure 3.
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. The y axis represents the 95% confidence interval and the x axis 3 different time points: baseline, 3 and 6 months. Data are expressed in medians. The analyses were performed with paired nonparametric test.

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 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 months, 635 [442.2–706.0]; P=0.046) and the control group (baseline, 466.5 [267.0–673.6]; 6 months, 466.5 [142.5–913.8]; P>0.99).

Myocardial Perfusion, Echocardiography, and EMM Results

Table 4 shows echocardiogram, SPECT and EMM data obtained at baseline and at 6-month 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, all comparisons between groups at 6 months were adjusted for baseline data and age, but remained not significant (Table 4). About 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.

View this table:
Table 4.

Echocardiogram, SPECT, and EMM Results at Baseline and at 6 Months

About 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 with baseline, LLS but not unipolar voltage significantly improved in the injected segments of treated patients. In the control group, no significant changes in LLS and unipolar voltage were observed (Figure 4).

Figure 4.
Figure 4.

An example of 2 patients included in the endothelial PROGENITOr cells and Refractory angina (PROGENITOR) trial. A and B, An example of a patient allocated to the control group. 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 <50% (blue to yellow): it shows inferior and inferolateral reversible defects. Baseline electromechanical maps (EM; bottom) from the same patient: left, the unipolar voltage (UV) map shows the viability of the tissue according to the definition (UV>6.9); right, lineal local shortening map (LLS) shows a decrease in contractile function at the inferolateral wall of the ventricle. B, Follow-up SPECT polar maps at 6 months: they show the persistence of the same perfusion defect in the inferior and inferolateral wall of the ventricle. The NOGA maps show the impairment of contractile function in the same walls. C and D, An example of a patient treated with CD133+ cells. C, Baseline stress and rest SPECT polar maps (upper) showing an inferolateral reversible defect. Baseline EM maps (bottom) viewed from the inferior position shows an area of viability (normal voltage values and decrease local shortening values) at the inferolateral wall of the ventricle (brown dots show the site of cell injections). D, Six-month SPECT shows an improvement in perfusion at inferolateral wall. The EM map shows an improvement of the LLS values at the injection site (right) reflecting improvement in mechanical function.

Discussion

The main findings of this phase I to 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. Second, 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 1 SPECT parameter that significantly improved only in the treatment group: the summed score. Finally, the characterization of CD133+ cells on the basis of aldehyde dehydrogenase 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 was 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 EMM 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. However, CD133+ surface marker is highly expressed on immature stem cell as it is downregulated 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.22 In 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 has 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 CD133+ 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 regard to its primary end points (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 nonenriched cell populations to treat patients with refractory angina. In a phase II study, 50 patients were randomized to receive nonselected 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 (Direct Endomyocardial Implantation of Bone Marrow Cells for Therapeutic Angiogenesis in Coronary Artery Disease) Trial, 28 patients were randomized to different dose of BM mononuclear cells (1×106/0.1 mL and 2× 106/0.1 mL). In this study, a significant increase in the treadmill test, LVEF and New York Heart Association class was reported. However, CCS class was reduced in both groups.8

The only phase II study that has used selected PC to treat patients with refractory angina was the ACT34-CMI study, a phase II study that included 167 patients with severe refractory angina (class III–IV) randomly assigned to receive 2 different doses of selected CD34+ cells: 1×105 (low dose) and 5×105 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 who 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 3-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 under-recognized 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, which has been considered a global perfusion index and has presented correlation with prognosis in previous studies,25 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 angina.6

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 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 in the treatment arm. In addition, LVEF and the finding that standard means of measuring regional function (wall thickening and wall motion) were 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 follow-up.

Another characteristic of the present study were first, all cell processes were injected in the same day avoiding the possible effect of storage and second, in this study, no placebo injections were performed to avoid any potential effect of the needle in the control arm. Regarding the EMM 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 study14 that has used the same criteria (viable myocardium) to inject the cells.

Several considerations have to be made on the safety of transendocardial 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 hypercontractile 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 nonsignificant 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 1 baseline variable between groups (ie, age) probably because of the small number of patients. For this reason all the analyses between groups were adjusted to avoid bias in the results.

Intramyocardial placebo injections were not performed in the control group, and therefore the 2 investigators who performed EMM were unblinded. 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. About the NOGA parameters, LLS values are strongly dependent on the quality of each collected mapping point and the entire electromechanical 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 EMM may present some degree of variability. Thus, larger trials are warranted to corroborate these findings.

Conclusions

This first-in-man study confirms that injection of selected CD133+ cells in ischemic myocardium identified with EMM is feasible and safe. The study is essentially negative from the standpoint of objective measures of ischemia. However, the results derived from the serial analyses in terms of improvement of angina symptoms and some SPECT-derived ischemia parameters in noncandidates for myocardial revascularization are encouraging and should be confirmed in a large randomized trial.

Acknowledgments

We would like to thank 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 a recipient of the ISCIII (Instituto de Salud Carlos III) grant “Fondo de Investigación Sanitaria” (PI11/00299) which relates to the topic of this study.

Disclosures

None.

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 aldehyde dehydrogenase 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 XP 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 1 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.

Footnotes

  • In August, 2014, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.55 days.

  • The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.115.303463/-/DC1.

  • Nonstandard Abbreviations and Acronyms
    BM
    bone marrow
    EMM
    electromechanical mapping
    LLS
    linear local shortening
    LVEF
    left ventricular ejection fraction
    mAD
    median absolute difference
    MI
    myocardial infarction
    PC
    progenitor cell

  • Received January 14, 2014.
  • Revision received September 16, 2014.
  • Accepted September 17, 2014.

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  • Selected CD133+ Progenitor Cells to Promote Angiogenesis in Patients With Refractory Angina
    Pilar Jimenez-Quevedo, Juan Jose Gonzalez-Ferrer, Manel Sabate, Xavier Garcia-Moll, Roberto Delgado-Bolton, Leopoldo Llorente, Esther Bernardo, Aranzazu Ortega-Pozzi, Rosana Hernandez-Antolin, Fernando Alfonso, Nieves Gonzalo, Javier Escaned, Camino Bañuelos, Ander Regueiro, Pedro Marin, Antonio Fernandez-Ortiz, Barbara Das Neves, Maria del Trigo, Cristina Fernandez, Teresa Tejerina, Santiago Redondo, Eulogio Garcia and Carlos Macaya
    Circulation Research. 2014;115:950-960, originally published September 17, 2014
    https://doi.org/10.1161/CIRCRESAHA.115.303463
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