Abstract
In patients with stable coronary artery disease (CAD) and refractory angina, we performed direct intramyocardial injections of autologous mesenchymal stromal cells (MSC) and followed the safety and efficacy of the treatment for 12 months. A total of 31 patients with stable CAD, moderate to severe angina, normal left ventricular ejection fraction, and no further revascularization options were included. Bone marrow MSCs were isolated and culture expanded for 6–8 weeks and then stimulated with vascular endothelial growth factor (VEGF) for 1 week. The 12-month follow-up demonstrated that it was safe to culture expand MSCs and use the cells for clinical treatment. The patients' maximal metabolic equivalent (MET) during exercise increased from 4.23 MET at baseline to 4.72 MET at 12-month follow-up (p < 0.001), Canadian Cardiovascular Society Class (CCS) was reduced from 3.0 to 0.8 (p < 0.001), angina attacks per week from 13.8 to 3.2 (p < 0.001), and nitroglycerin consumption from 10.7 to 3.4 per week (p < 0.001). In addition, Seattle Angina Questionnaire (SAQ) evaluations demonstrated highly significant improvements in physical limitation, angina stability, angina frequency, and quality of life (p < 0.001 for all). It is safe in the intermediate/long term to treat patients with stable CAD using autologous culture expanded MSCs. Previously reported, early and highly significant improvements in exercise capacity and clinical symptoms persist after 12 months. The results are encouraging, and a larger controlled study is warranted.
Keywords
Introduction
Stem cell therapy in patients with severe coronary artery disease (CAD) and refractory angina is a treatment modality, which has been investigated in several small clinical trials within the last years (3,4,10,12,13,18,20, 26,27,38,39). The aim of these trials has been to increase the myocardial perfusion by initiating the development of new blood vessels in the ischemic myocardium (21,30). Several different stem cell lines from the bone marrow have been used in clinical trials, and presently there is no consensus on whether one cell line is superior to the other (9,21). The mesenchymal stromal cells (MSCs) could be a promising cell line in patients with CAD, since it has the ability to differentiate towards endothelial and cardiac cell types and hereby regenerate the consequences of CAD (30).
The results in the different stem cell trials have been conflicting in patients with acute and chronic myocardial ischemia (2,5,14,19,29,31,34,35,37,42). Some but not all studies have suggested a beneficial effect on myocardial perfusion, function, and symptoms in patients with chronic myocardial ischemia (13,27,38,39). Due to safety concerns, many of these initial pivotal pilot studies are nonrandomized and without a control group. Moreover, the patient's follow-up is, in most trials, 3–6 months. Therefore, some of the diverging results could be due to a short-term placebo effect, which would disappear with prolonged follow-up.
We have recently, in a nonrandomized unblinded clinical trial, demonstrated that direct intramyocardial injection of autologous MSCs in patients with CAD, refractory angina, and normal left ventricular function (LVEF) was safe in a 6-month follow-up period (12). Moreover, the patients had significant improvements in left ventricular function and exercise capacity, in addition to an improvement in clinical symptoms and Seattle Angina Questionnaire scores (SAQ) evaluations. There is undoubtedly a risk for a placebo effect in the outcome, firstly due to the open design of the study and secondly due to the many follow-up meetings in the first 6 months.
The aim of the present study was to investigate further the safety and efficacy of the MSC therapy in patients with CAD and refractory angina in the following 6-month period with no clinical follow-up to eliminate the influence of study visits on the outcome.
Materials and Methods
In this prospective first-in-man open study with autologous ex vivo culture expanded MSCs, we treated 31 patients with severe CAD and refractory angina (12). Inclusion criteria were reversible ischemia at an adenosine stress single-photon emission computerized tomography (SPECT), aged above 18 years, and Canadian Cardiovascular Society (CCS) angina classes II–III in spite of maximal tolerable medication. All patients were evaluated by an independent thoracic surgeon and cardiologist to be ineligible for further surgical and percutaneous revascularization.
Excluded were patients with LVEF below 40%, unstable angina pectoris, acute myocardial infarction (MI) within the last 3 months, diabetes mellitus with proliferative retinopathy, diagnosed or suspected cancer disease, chronic inflammatory disease, and fertile women. After baseline assessments and stem cell treatment, the patients were followed with clinical controls after 1, 3, 6, and 12 months.
This study protocol complied with the Declaration of Helsinki and was approved by the National Ethical Committee (02-268856) and Danish Medicines Agency (2612–2867). All patients received oral and written information about the study and signed an informed consent.
End Points
Safety of culture MSC expansion was evaluated by testing the culture media for bacteria, yeast, and mycoplasma 1 week before and on the day of treatment. Safety of injection of and treatment with MSCs were evaluated by registration of adverse and serious adverse events in the follow-up period. The study primary end point was changes in perfusion defects with an adenosine single-photon emission computerized tomography from baseline to 6-month follow-up as previously described (12). The prespecified 12-month secondary efficacy end points were change in exercise capacity, CCS angina class, Seattle Angina Questionnaire (SAQ) scores, frequency of angina attacks, and nitroglycerin consumption.
Bone Marrow Cell Preparation and Culturing
The isolation and culture expansion of the MSCs from the bone marrow (BM) has previously been described in details (11,12,15,16). Under local anesthesia, BM cells were obtained from the iliac crest by needle aspiration of 50 ml of bone marrow. MNCs were isolated by gradient centrifugation on Lymphoprep (density, 1,077 g/cm3) (Medinor, Copenhagen, Denmark). During the last week in culture, the cells were stimulated with human recombinant vascular endothelial growth factor A-165 (rhVEGF) (R&D Systems, Minneapolis, MN, USA) (50 ng/ml) to facilitate differentiation towards endothelial precursor cells as described and evaluated previously by qPCR, immunocytochemical, and ECMatrix gel analyses (11,12,15,16). One culture flask was saved for cell characterization by flow cytometry. MSC characteristics were documented by analyzing for CD45, CD34, CD105, CD73, CD13, and CD90 expression. No functional tests were applied due to the small number of cells available. The patients were treated with the full amount of cells reached after two cell passages.
NOGAXP®—Electromechanical Mapping of Left Ventricle and Intramyocardial Injections
Electromechanical evaluation of the left ventricle was performed with the NOGA-XP® system and the intramyocardial injections with the 8-french-sized Myostar® mapping injection catheter (Biological Delivery System, Cordis, Johnson & Johnson, USA) (1,12,22,32). Approximately 12–15 injections of 0.2-ml stem cell solution were given around and within the area with reversible ischemia.
Exercise Test
The bicycle exercise test was performed as a maximal symptom limited test with a start workload on 25 W and with increments of 25 W every 2 min. The test was terminated if normal physiological maximal work level was achieved or if the patient develops limiting symptoms on a submaximal work level. The maximal exercise time was registered, and corresponding metabolic equivalent of task (MET) was calculated.
Statistical Analysis
All statistical analyses were done using PASW 18 (SPSS, Inc., Chicago, IL, USA) and SAS 9.2 (SAS Institute, Inc., Cary, NC, USA). Wilcoxon signed-rank test was used to compare baseline and follow-up data. Due to three comparisons for each variable (3, 6, and 12 months with baseline), a Bonferroni correction was applied and a value of p < 0.05/3 = 0.016 was considered significant. All values are presented as mean ± SD. All graphs were made using R 2.10.1 (http://www.rproject.org).
Results
Patient Characteristics
The demographic characteristics of the 31 patients are presented in Table 1. They were all treated with more than one antiangina drug. The medication was constant at 12-month follow-up in all patients, except for two patients who stopped with long-lasting nitroglycerine, two patient who increased both β-blocker and long-lasting nitroglycerine, and one who initiated calcium antagonist treatment.
Baseline Characteristics of the 31 Patients Treated With Mesenchymal Stromal Cells
Values are expressed as mean ± SD or n (%). LVEF, left ventricular ejection fraction; CCS, Canadian Cardiovascular Society functional classification; CABG, coronary artery bypass grafting; PCI, percutaneous coronary intervention; STEMI, ST elevation Myocardial Infarction > 1 year before inclusion.
Mesenchymal Stromal Cells
MSCs were culture expanded under Good Manufactory Practice (GMP) conditions for 40 ± 9 days (mean ± SD), and the patients were treated with an average of 21.5 × 106 MSCs (range: 3–62 × 106). There was no detectable association between the number of cells reached after cultivation and the patients' baseline characteristics. The flow cytometry characterization of the MSCs at the time of treatment were 1.0% CD45+, 4.0% CD34+ 92.9% CD13+, 92.4% CD73, 92.5% CD90+, and 91.8% CD105+. There were no signs of contaminations with bacteria, yeast, or mycoplasma during culture expansion.
Safety
The direct intramyocardial injection of the cells was without any procedural complications or severe cardiac arrhythmias recorded during in-hospital monitoring or clinically after the injections. Serious adverse events for the entire 12 months of follow-up period were pneumonia in one patient after 1 month and unstable angina pectoris and fever in the same patient 4 months later, and unstable angina in another patient. In one patient, atrial fibrillation recurred. This patient was previously treated with radio frequency ablation.
Exercise Test
Exercise time increased gradually in the follow-up period. The improvement was significant after 6 months (p = 0.007), but after 12 months, the increase from baseline 6:23 ± 1:43 min to 7:15 ± 1:54 min was only borderline significant (p = 0.07) (Fig. 1B). There was a continuous significant improvement in the maximal metabolic equivalent during exercise after the treatment from 4.23 MET at baseline to 4.72 MET at 12-month follow-up (p = 0.001).
The baseline, 3-month, 6-month, and 12-month follow-up of CCS class (A), bicycle exercise time (B), angina pectoris attacks (C), and nitroglycerin consumption (D) in patients with stable chronic coronary artery disease treated with mesenchymal stromal cells.
Clinical Evaluation
The clinical estimation of the angina level demonstrated a stepwise significant reduction in CCS classification for the initial 6 months (p < 0.001), which was even further reduced at the 12-month follow-up (p < 0.001) (Fig. 1A). The same tendency was seen in the weekly number of angina attacks in the follow-up period. The number of attacks decreased gradually from the pretreatment level at 13.8 ± 13.7 attacks per week to 3.2 ± 5.7 attacks per week at 12-month follow-up (p < 0.001) (Fig. 1C). Also the weekly consumption of nitroglycerin showed the same trend. It was reduced from 10.7 ± 10.0 nitroglycerin consumption per week to 3.4 ± 4.9 (p < 0.001) 12 months after treatment (Fig. 1D).
The SAQ evaluation demonstrated a constant increasing improvement in physical limitation score (p < 0.001), angina stability (p < 0.001), angina frequency score (p < 0.001), and quality of life score (p < 0.001) at the 12-month follow-up registration (Fig. 2A–D). The treatment satisfaction score was already high at baseline (90%) and improved slightly at 6 months (93%, p = 0.03) but was unchanged 92% at 12-month follow-up.
Seattle Angina Questionnaire (SAQ) quality of life evaluation of treatment effect in mesenchymal stromal cell-treated patients with stable chronic coronary artery disease at baseline, 3-month, 6-month, and 12-month follow-up. A higher SAQ score denotes improved symptoms or functioning.
The regression analyses demonstrated a positive relation between the number of cells and improvement in angina stability score (r = 0.37, p = 0.05) and a tendency for improvement of CCS class (r = 0.35, p = 0.07) at 12-month follow-up.
Discussion
Several studies have evaluated whether injections of stem cell solutions in patients with CAD, refractory angina, and normal left ventricle ejection fraction could improve myocardial perfusion and hereby reduce patient's symptoms and improve exercise capacity (3,4,10,12,13, 18,20,26,27,38,39). The majority of these clinical stem cell studies have only followed the patients 3–6 months after the treatment. The present study demonstrates 12-month safety and efficacy follow-up data of autologous MSC-derived endothelial precursor cell treatment in patients with stable CAD and refractory angina. The study extended the previous 6-month safety data demonstrating that there was not seen any serious adverse events in a 12-month follow-up period, which could be related to the culture expansion of MSC-derived endothelial precursor cells or intramyocardial injection of cells (12). In the 12-month follow-up period, there were no deaths, cardiac arrhythmias, or other serious adverse events.
It is always a problem, in unblinded treatment study, that the knowledge of a potential active treatment and the extra contact with the study and hospital staff can induce a placebo effect in the evaluation of the patients and in the patient's experience of a treatment effect. This was also a problem in the present study, where the patients had regular visits in the project in the initial 6-month period after the treatment. However, in the next period from 6–12 months control, there was no contact with the patient. It is therefore likely that a placebo effect would diminish in this period without any study contact. However, the improvements demonstrated at 6-month follow-up persisted and even tended to improve further for some parameters at the 12-month evaluation. This could be an indication for an MSC treatment effect, although it needs confirmation in a larger randomized double-blind placebo-controlled trial.
The improvement in the patient's symptoms demonstrated for up to 6-month follow-up was still highly significant at the 12 months evaluation for CCS classification, weekly angina attacks, and nitroglycerin consumption and for the SAQ parameters. In a few clinical trials with a control or placebo group included, the changes in CCS class in these groups were from baseline to 6-month follow-up 3.1 to 2.3 (38) and 2.9 to 2.5 (39) compared to the improvements in the mononuclear cell treated patients from 3.3 to 2 and 3.0 to 2.2, respectively. The present improvement in CCS class from pretreatment 3.0 to 1.3 and 0.8 at 6- and 12-month follow-up is more pronounced, indicating that it is not entirely caused by a placebo effect. The reduction in angina attacks and nitroglycerin consumption is not regularly registered in most trials, but two uncontrolled studies have also demonstrated a reduction in these parameters in patients treated with cell solutions (3,27).
The maximal exercise time was now only borderline significant at 12-month follow-up compared to the previous significant increased level. However, the maximal physiological metabolic equivalent during bicycle exercise was still significant and even slightly further increased at 12 months compared to baseline, 3-month and 6-month improvements. The exercise time has not been significantly improved in most clinical stem cell trials, but there has been a tendency in some of the trials (3,13,27,38,39). Therefore, the demonstration of a significant improvement in exercise time and metabolic equivalent at 3- and 6-month evaluation and the borderline significance at 12-month follow-up is rather interesting and encouraging.
Myocardial perfusion and left ventricular function was not evaluated at 12-month follow-up. However, we have previously demonstrated that, at 6-month follow-up, there was a significant improvement in LVEF, end-systolic wall thickness, and wall thickening determined by MRI, indicating an MSC-induced improvement in myocardial function (12). In agreement with these results, several studies have, in an identical patient population, demonstrated an approximately 3% improvement in LVEF in the cell-treated patients (3,4,38,39).
The mechanisms behind the clinical and objective improvements is uncertain, since we could not detect any changes in SPECT-measured global myocardial perfusion at the 6-month follow-up (12). This is in agreement with other studies, which also could not demonstrate significant changes in myocardial perfusion (13,27,38). The study by van Ramshorst et al (39) could demonstrate a small and modest improvement in SPECT perfusion after mononuclear cell treatment. These observed disappointing results of no changes in the perfusion in many studies could be due to known limitations of SPECT to measure regional myocardial perfusion in multivessel CAD. The influence of local immunomodulation, secretion of cytokines, or retention of injected MSC on the clinical outcome can only be speculative in the present clinical study (8,40,41,43).
It can be discussed whether MSCs obtained from elderly ischemic patients are actually functionally useful for clinical regenerative therapy. To investigate this question, we have compared the presently used culture expansion of MSC in young healthy controls (mean age: 26 years) and older patients with stable chronic CAD and reversible perfusion defects on SPECT (mean age: 64 years). The study demonstrated identical surface markers and proliferation capacity. In addition VEGF stimulation identically significantly increased the expression of the endothelial genes thrombospondin 1, Tie-2, and von Willebrand factor and induced endothelial ring structures on extracellular matrix (11). This finding is supported by a recent study demonstrating that MSCs from persons aged 8 months to 58 years could be expanded without alterations in expansive properties and changes in parameters associated with senescence (28). That age seems to be without any importance on MSC function is in opposition to findings with mononuclear cell (MNC) solutions used in many clinical trials, where MNC functionality decrease with age (25,36). Therefore, it is most likely that the present used MSCs are of a sufficient quality to be used in regenerative clinical trials and maybe are superior to MNCs in older patients. This can be of importance in cost–potency comparisons between different cell populations in future clinical trials.
Our study was not a dose titration study. However, there was a small relationship between the cell number and improvements in CCS classification and angina stability. This is supported by a recent study demonstrating an MSC dose-depending improvement in infarct size in pigs with myocardial infarcts (34).
The MSCs are a potential cell line for regenerative therapies in patients with ischemic heart disease. However, only a few small phase I/II safety and efficacy clinical studies on the therapeutic potency of human in vitro culture expanded autologous MSCs in patients with CAD without and with heart failure have been published (6,7,17,23,24). The present study is the first larger study with direct intramyocardial delivery of MSCs in patients with chronic CAD. In agreement with previous studies, we found that MSC treatment was safe.
The study has several limitations, which is of importance for the interpretation of the results. It is an open nonrandomized study without any control group including a limited number of patients. The 6-month hard end points myocardial perfusion and left ventricular ejection fraction are not included in the 12-month follow-up.
In conclusion, the 12-month follow-up of patients with stable CAD and refractory angina treated with locally in-hospital culture expanded autologous MSCs was safe. The improvements in clinical symptoms and exercise capacity demonstrated at 6-month follow-up still persisted at the 12-month evaluation. To confirm these encouraging results, a double-blind placebo-controlled study is needed, which should aim for injection of a higher number of MSCs and measurement of myocardial perfusion with PET.
Footnotes
Acknowledgments
This study was supported by grants from the Research Foundation at Rigshospitalet, Lundbeck Foundation, Aase og Ejnar Danielsens Foundation, Toyota Foundation, Søren and Helene Hempel Foundation, Brd. Hartmans Foundation and The Danish Heart foundation. The authors declare no conflict of interest.