Abstract
Aim
To assess the safety and efficacy of the intracoronary infusion of CD133+ hematopoietic stem cells to improve ventricular function and quality of life in candidates for heart transplantation due to post-infarct chronic heart failure.
Methods
We selected seven candidates for heart transplantation (six males/one female, age range 44–65 years) in whom all treatment alternatives were exhausted (angioplasty/stent and bypass surgery). These subjects had a symptomatic New York Heart Association (NYHA) scale of at least II and ejection fractions (EFs) below 35%. After obtaining informed consent, CD133+ cells were obtained by stimulation with granulocyte-colony stimulating factor, apheresis, and separation with magnetic beads. Stem cells were implanted in the infarcted zone via intracoronary percutaneous angiography. Evaluations (NYHA scale classification, plasma concentration of pro-B-natriuretic-peptide and the risk of sudden death, echocardiography, cardiac magnetic resonance, and gated-SPECT with MIBI) were performed at baseline and at 3, 6, 12, and 24 months after cell infusion.
Results
Stem cell isolation was efficient and safe (around 10 7 cells/patient and >92% CD133+ viable cells). Two patients died during observation due to noncardiac conditions. In the five remaining subjects, the NYHA scale improved and no accounts of hospital admissions for heart failure were documented. Plasma concentrations of pro-B-natriuretic peptide and the risk of sudden death clearly decreased, while the EF increased significantly to 35% and 40% by echocardiography and cardiac MRI, respectively ( P =.013 and .009, respectively) 24 months after treatment. No other major adverse events were noticed.
Conclusions
The intracoronary inoculation of CD133+ stem cells was safe and effective to improve ventricular contraction and symptomatic class function in patients with refractory post-infarct heart failure.
1
Introduction
Post-infarct heart failure (PIHF) is a frequent adverse event that severely restricts quality of life. Heart failure accounts for almost 1 million hospital admissions and for approximately 50,000 annual deaths in the US . Although there are no data regarding the prevalence and impact of PIHF in Mexico, the National Institute for Statistics, Geography, and Informatics (INEGI) reveals that 48,573 people died due to ischemic heart disease in the year 2003 and that 12,444 patients were admitted to hospitals due to infarct of the myocardium . The aims of standard PIHF treatment are to control the symptoms, reduce the handicap, and delay the progress of the heart failure . Pharmacologic agents such as inotropic drugs, diuretics, vasodilators, and neuro-hormonal inhibitors are useful to control some symptoms and improve heart function and survival. Nevertheless, these treatments are not very effective in moderate or severe cases of PIHF; therefore these limitations justify exploring new therapeutic approaches. Several preclinical and clinical trials with adult endothelial precursor cells (EPCs) have demonstrated that these cells are able to stimulate angiogenesis in the post-infarct viable tissue and that this modality of cellular therapy is safe and effective. CD133+ cells are committed EPCs that have been successfully used in previous clinical trials for ischemic cardiomyopathy . The goal of the present study was to analyze the effectiveness of the intracoronary infusion of CD133+ (a particular set of EPCs) in patients with moderate to severe PIHF to improve heart function and quality of life, and to contribute with additional data on the safety of the procedure.
2
Materials and methods
2.1
Human subjects
Seven patients who were candidates for heart transplantation (six males/one female, age range 44–65 years) and in whom all treatment alternatives were exhausted (angioplasty/stent and bypass surgery) were recruited for the trial between September 2005 and February 2006. The protocol was submitted and approved by the hospital’s IRB and by the Federal Commission for the Protection Against Sanitary Risks (COFEPRIS) of the Mexican Secretariat of Health, and in accordance with the 1975 Declaration of Helsinki. All patients were verbally informed about the risks and possible clinical outcomes, and voluntary signatures of the informed consent were collected in all cases. Fig. 1 describes the flow of procedures and evaluations for study subjects included in this clinical trial. After admission, all subjects were clinically evaluated by an examining cardiologist who classified their condition and cardiac function according to New York Heart Association (NYHA) classification, and cardiologic and laboratory tests were performed to assess heart function and baseline condition. Electrocardiography was performed to evaluate ventricular arrhythmia. The plasma concentrations of pro-B-type natriuretic peptide (proBNP) were measured in the Triage Meter Plus instrument (Biosite, Inc., San Diego, CA, USA), following manufacturer instructions.
Inclusion criteria were as follows: (1) patients with PIHF of at least 4 weeks of evolution, (2) age between 18 and 75 years, (3) NYHA class of at least II, (4) ventricular ejection fraction (EF) below 35%, and (5) severe concurrent organic dysfunction involving the brain, liver, or kidneys. Patients with the following criteria were excluded from the study: (1) eligible candidates for revascularization, (2) unstable angina, (3) proliferative retinopathy, (4) thrombocytopenia or leucopenia, (5) history or evidence of concurrent malignancy, (6) drug addiction, and (7) impediments to accomplish the scheduled evaluations.
2.2
Cardiac function
2.2.1
Echocardiography
Cardiac transthoracic ultrasonographic studies were performed for measurement of LV contractility and volumes, using a Philips iE33 echocardiography system (Philips, The Netherlands) equipped with an S3-1 xMATRIX Array transducer (3 MHz). For the study, patients were positioned supine left lateral, with the head slightly elevated. The standard approach included the parasternal long- and short-axis views and the apical two and four-chamber views. Two-dimensional techniques were used to provide visual assessment of LV systolic function, both regional and global. LVEF was calculated with the Simpson method . Images and loops were digitally stored for offline analysis. The studies were analyzed by two experienced echocardiographers, one nonblinded (RFR) and one blinded to the cell-implant intervention (PGF).
2.2.2
Cardiac MRI
This study was done with ECG-gated sequences in a 1.5-T Intera System (Philips). To determine LVEF, LV end-diastolic and end-systolic volumes were determined for calculation of LVEF with breath-hold gradient echo sequences. Sequence parameters were as follows: TR 40.05 ms, TE 1.3 ms, flip angle 80° to 65°, matrix 192×156, slice thickness 8 mm, and field of view 34 to 40 cm. The LV was covered by a continuous stack of short-axis slices. An end-diastolic, end-expiratory four-chamber view served as a reference to plan the short-axis slices. Image analysis was done blinded, without knowledge of the echocardiographic data.
2.3
Cell preparation
After 6 days of stimulation with 5 mg/kg per day of granulocyte colony stimulating factor (G-CSF), patients underwent apheresis. The total volume of collected cell suspension was transferred between blood bags under Good Manufacturing Procedure conditions. The CD133+ cells were processed and separated using the CliniMACS instrument (Miltenyi Biotec, Auburn, CA, USA) as recommended by the manufacturer. Aliquots from the apheresis product and from the injected cell fraction were collected to measure composition and purity by a fluorescence-activated cell sorter. Samples from the positive cell injection were collected immediately before intracoronary infusion and sent for microbiological screening to exclude contamination. Viability was tested by dye exclusion staining with trypan blue.
2.3.1
Cell transplantation
After collected cells were analyzed and purified, patients were taken to the catheter laboratory for intracoronary application. Inguinal punction was performed by the Seldinger technique and a coronary angiogram was performed to identify the culprit artery for cell infusion. A guide catheter (Viking C-4) was placed in the culprit artery (left anterior descending artery in all patients) and an extra-support guide wire of 0.14×300 mm was used as guide for the perfusion catheter (Open-Sail 3×10 mm). Collected cells were infused with a perfusion system (Perfusor Compact, B Braun, Germany) at a rate of 10 ml/min, using a balloon inflated at 4 atm with a deflated lapse of 2 min between every infusion, until total volume was infused. No complications were observed during the procedure.
2.3.2
Statistical analyses
All data are presented as mean±S.E.M. Parametric values were analyzed by the Student’s t -coupled test, while nonparametric values of the NYHA scale were studied by the Wilcoxon test. In both cases, comparisons were established between the data at any observation point against the baseline registered data. P values <.05 were considered significant in the study.
2
Materials and methods
2.1
Human subjects
Seven patients who were candidates for heart transplantation (six males/one female, age range 44–65 years) and in whom all treatment alternatives were exhausted (angioplasty/stent and bypass surgery) were recruited for the trial between September 2005 and February 2006. The protocol was submitted and approved by the hospital’s IRB and by the Federal Commission for the Protection Against Sanitary Risks (COFEPRIS) of the Mexican Secretariat of Health, and in accordance with the 1975 Declaration of Helsinki. All patients were verbally informed about the risks and possible clinical outcomes, and voluntary signatures of the informed consent were collected in all cases. Fig. 1 describes the flow of procedures and evaluations for study subjects included in this clinical trial. After admission, all subjects were clinically evaluated by an examining cardiologist who classified their condition and cardiac function according to New York Heart Association (NYHA) classification, and cardiologic and laboratory tests were performed to assess heart function and baseline condition. Electrocardiography was performed to evaluate ventricular arrhythmia. The plasma concentrations of pro-B-type natriuretic peptide (proBNP) were measured in the Triage Meter Plus instrument (Biosite, Inc., San Diego, CA, USA), following manufacturer instructions.
Inclusion criteria were as follows: (1) patients with PIHF of at least 4 weeks of evolution, (2) age between 18 and 75 years, (3) NYHA class of at least II, (4) ventricular ejection fraction (EF) below 35%, and (5) severe concurrent organic dysfunction involving the brain, liver, or kidneys. Patients with the following criteria were excluded from the study: (1) eligible candidates for revascularization, (2) unstable angina, (3) proliferative retinopathy, (4) thrombocytopenia or leucopenia, (5) history or evidence of concurrent malignancy, (6) drug addiction, and (7) impediments to accomplish the scheduled evaluations.
2.2
Cardiac function
2.2.1
Echocardiography
Cardiac transthoracic ultrasonographic studies were performed for measurement of LV contractility and volumes, using a Philips iE33 echocardiography system (Philips, The Netherlands) equipped with an S3-1 xMATRIX Array transducer (3 MHz). For the study, patients were positioned supine left lateral, with the head slightly elevated. The standard approach included the parasternal long- and short-axis views and the apical two and four-chamber views. Two-dimensional techniques were used to provide visual assessment of LV systolic function, both regional and global. LVEF was calculated with the Simpson method . Images and loops were digitally stored for offline analysis. The studies were analyzed by two experienced echocardiographers, one nonblinded (RFR) and one blinded to the cell-implant intervention (PGF).
2.2.2
Cardiac MRI
This study was done with ECG-gated sequences in a 1.5-T Intera System (Philips). To determine LVEF, LV end-diastolic and end-systolic volumes were determined for calculation of LVEF with breath-hold gradient echo sequences. Sequence parameters were as follows: TR 40.05 ms, TE 1.3 ms, flip angle 80° to 65°, matrix 192×156, slice thickness 8 mm, and field of view 34 to 40 cm. The LV was covered by a continuous stack of short-axis slices. An end-diastolic, end-expiratory four-chamber view served as a reference to plan the short-axis slices. Image analysis was done blinded, without knowledge of the echocardiographic data.
2.3
Cell preparation
After 6 days of stimulation with 5 mg/kg per day of granulocyte colony stimulating factor (G-CSF), patients underwent apheresis. The total volume of collected cell suspension was transferred between blood bags under Good Manufacturing Procedure conditions. The CD133+ cells were processed and separated using the CliniMACS instrument (Miltenyi Biotec, Auburn, CA, USA) as recommended by the manufacturer. Aliquots from the apheresis product and from the injected cell fraction were collected to measure composition and purity by a fluorescence-activated cell sorter. Samples from the positive cell injection were collected immediately before intracoronary infusion and sent for microbiological screening to exclude contamination. Viability was tested by dye exclusion staining with trypan blue.
2.3.1
Cell transplantation
After collected cells were analyzed and purified, patients were taken to the catheter laboratory for intracoronary application. Inguinal punction was performed by the Seldinger technique and a coronary angiogram was performed to identify the culprit artery for cell infusion. A guide catheter (Viking C-4) was placed in the culprit artery (left anterior descending artery in all patients) and an extra-support guide wire of 0.14×300 mm was used as guide for the perfusion catheter (Open-Sail 3×10 mm). Collected cells were infused with a perfusion system (Perfusor Compact, B Braun, Germany) at a rate of 10 ml/min, using a balloon inflated at 4 atm with a deflated lapse of 2 min between every infusion, until total volume was infused. No complications were observed during the procedure.
2.3.2
Statistical analyses
All data are presented as mean±S.E.M. Parametric values were analyzed by the Student’s t -coupled test, while nonparametric values of the NYHA scale were studied by the Wilcoxon test. In both cases, comparisons were established between the data at any observation point against the baseline registered data. P values <.05 were considered significant in the study.
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