Efficacy and long-term evaluation of intramyocardial injection of autologous CD34-enriched PBMSC in old myocardial infarction

José Luis Aceves, Abel Archundia, Araceli Páez, Rafael Vilchis, Elvira Varela, Emma Rodriguez, Guillermo Diaz, Lourdes Flores-Luna, Martha Alvarado, Manuel Lopez H., Luis Felipe Montaño, Felipe Masso

Center of Population Health Research, National Institute of Public Health, Mexico City, Mexico.
Department of Cardiovascular Surgery, National Medic Center 20 de Noviembre ISSSTE, Mexico City, Mexico.
Department of Cellular Physiology, National Institute of Cardiology “Ignacio Chavez”, Mexico City, Mexico.
Department of Hematology, National Medic Center 20 de Noviembre ISSSTE, Mexico City, Mexico.
Immunobiology Laboratory, Department of Cell and Tissue Biology, School of Medicine UNAM, Mexico City, Mexico.

DOI: 10.4236/wjcd.2012.24044                

Abstract

Aims: We have shown that autologous transplant of CD34⁺-enriched peripheral-blood mononuclear cells (PBMSC) could restore depressed myocardial function, and sustain adequate myocardial function 12 months after surgery in patients with old (>one year-old) myocardial infarction. Our aim is to report the long-term morbidity and mortality efficacy of this procedure. Methods and results: Seventy patients with an old anteroseptal myocardial infarction were followed for 2 to 7 years, 35 had a revascularization procedure and received an intra-myocardial injection of autologous CD34⁺-enriched PBMSC (8 × 108 mononuclear cells/ml including 3 × 107 CD34⁺ cells/ml)(Group A). Group B patients only had the revascularization. Abnormal pre-surgical values of LVEF (33.2% ± 4.8%), LVDV (178 ± 13.7 ml), LVSV (120 ± 16 m), LVDD (58.9 ± 3.84 mm), E and A waves without contractility in infarction area in group A patients improved to approximate normal values (50% ± 3% for LVEF; 90 ± 9.3 ml for LVDV; 80 ± 9.9 ml for LVSV; 55.3 ± 3 mm for LVDD; 5.2 ± 0.5 cm/s for E wave and 4.18 ± 0.3 cm/s for A wave) 1 year after the procedure and have remained unaltered for all the follow-up period. All the patients remain alive. Only seven patients have been readmitted to the hospital for non-myocardial related events. Group B only 11 patients continued alive to 5 years after surgery and LEVF never increased more than 6%, all of them with many hospitalizations (n ≥ 10) by heart failure events. Conclusion: Intramyocardial injection of CD34⁺ highly enriched PBSC represent an encouraging alternative for patients with severely scarred and dysfunctional myocardium.

Keywords

Stem Cells; Myocardial Infarction; CD34⁺ Cells; Revascularization; Heart Failure; Ventricular Function

1. INTRODUCTION

Myocardial infarction is a main cause of mortality and morbidity in Western societies. The intracoronary injection of autologous bone marrow stem progenitor cells in acute or sub-acute lesions with viable tissue improves hemodynamic criteria and cardiac function [1,2]. Intracoronary infusion of peripheral blood mononuclear stem cells (PBMSC) mobilized by Granulocyte-Colony Stimulating Factor (G-CSF) in acute myocardial infarction [3] generates peri-stent tissue growth and a modest increase in cardiac function [4,5]. The beneficial effects, cardiac repair and restraint fibrosis, may be the result of increased angiogenesis and the production of cytokines from endothelial progenitors [6]. The delivery of progenitor cells into ischaemic cardiac muscle improves angiogenesis and revascularization [7].

The potential benefits and final outcome of stem cell transplantation in patients with old (>1 year) myocardial infarction require further evaluation. One trial reports the results of the intracoronary application of unfractionated BMSC, mobilized with G-CSF, in patients with old myocardial infarction [8]. We have previously shown improved ventricular function in patients with old myocardial infarction subjected to the peri-infarction injecttion of unfractionated PBMSC [9]. The aim of this paper was to report the long-term feasibility and safety of our process as well as to determine if depressed myocardial function is restored and sustains an adequate myocardial function for extended periods of time.

2. METHODS

2.1. Patients

Seventy patients (Mean age of 54.3 ± 8.5-year-old) with ischemic cardiomyopathy and more than one-year-old anteroseptal myocardial infarction (43.4 ± 14.7 months) were eligible for inclusion into the study. They all had left ventricular failure as determined by the low left ventricular ejection fraction (LVEF) value, were considered as NYHA-III/IV functional class and were being treated with appropriate doses of angiotensin converting enzyme inhibitors, AT1R blockers, beta blockers and statins before the surgical procedure. The presence and extent of the infarct was determined by ECG, angiography (C type proximal injury in the left anterior descending coronary artery and akinesia in the anterior wall and apical heart zones), transthoracic echocardiogram (akinesia in the apex and the anterior myocardial side) and radionuclide myocardial perfusion imaging (old transmural infarction in the anterior wall and apical zone without evidence of viable tissue according to 201-Thalium rest, stress and 24 hour redistribution images). All the patients were candidates for a revascularization of the anterior descendent artery, and were divided into two groups. Group A individuals gave their informed consent for the revascularization procedure and the injection of their autologous CD34-enriched cell fraction, whereas group B included the individuals who only accepted the revascularization procedure and placebo (Physiologic solution). We only considered individuals with lesion in the anterior descending artery, since the possible inclusion of individuals having occlusion of the other arteries could bias our study. Each patient was evaluated every six months for the first year after surgery and then yearly. The ethics review board of the Hospital approved the protocol in January 2002, and the study was conducted in accordance with the Declaration of Helsinki.

2.2. Surgical Procedure and Follow Up

Our original stem cell protocol [9] was used in every patient. Briefly, patients are treated with G-CSF (Filgistrin, Neupogen^TM; 300 mg/day) subcutaneously for five days. On the fifth day, a Mahurkar catheter is inserted into the subclavian artery and mononuclear cells are harvested with a close circuit in the apheresis machine. The percentage of CD34⁺ cells is then estimated by cytofluorometry before the CD34⁺ enriched with mononuclear cells are cryopreserved at a ratio 1:1 with patient plasma. The revascularization procedure was always performed from the left internal thoracic artery to the anterior descending coronary artery with beating heart, that is, without extracorporeal circulatory support. Group A patients were subjected to the surgical procedure 48 hr. After that mononuclear cells were harvested. Once the revascularization procedure was finished, group A patients received 20 ml of their autologous CD34⁺ enriched mononuclear cell preparation (8 × 10⁸ mononuclear cells/ml including 3 × 10⁷ CD34⁺ cells/ml) distributed in 10 × 2 ml radial injections (2 inches deep) from the center of the scar to the border zone between healthy and infarcted tissue, following the course of the anterior descending coronary artery, thus assuring that the anterior wall and septum receive the CD34-enriched cells. None of the patients developed arrhythmia during or after the surgical procedure. Group B Patients received revascularization procedure and 20 ml of physiological solution (Placebo) in the same manner in which the cells were implanted in group A.

After surgery the patients were monitored at 6 and 12 months, and then, yearly by tissue doppler and standard transthoracic echocardiography. The hemodynamic parameters included heart motility, left ventricular ejection fraction (LVEF), left ventricular end diastolic volume (LVDV), left ventricular end systolic volume (LVSV), left-ventricular end diastolic diameter (LVDD), mitral ring velocity, E and A wave in zone infarction. Tc 99 and 201-Thallium SPECT were made in each post-surgery evaluation.

Statistical analysis. The results were evaluated with non-parametric analysis using Prisma software; survival tables and graphs were done using STATA v.9 software. A p value <0.05 was considered statistically significant.

3. RESULTS

Mean age of group A patients (51.91 ± 8.9-year-old) was lower than those in group B (56.86 ± 8.1-year-old; p = 0.018) but the time interval between the acute infarct event and the first visit to us was significantly higher in group A vs group B patients (48.8 ± 14.2 months vs 38.1 ± 15.2 months, p = 0.004). Body mass index was lower in group A patients (27.14 ± 2.75 vs 29.31 ± 2.81 Kg/m²; p = 0.0018). The follow-up period has been greater in group A patients (61.97 ± 54.65 months vs 40.11 ± 13.89 months in group B, p = 0.015).

Post-surgical complications were not reported in any of the patients. As surgical experience increased, the number of days the patients spent in the intensive care unit has diminished from 7 - 10 days, in the first five patients, to 3 - 5 days. Surgical experience also allowed us to choose patients with lower LVEF basal value (40.2% ± 3.7% for our first eight patients vs 33.4 ± 4.8% for the subsequent; p = 0.036).

The basal value of LVEF in group A and B patients was not significantly different. There was a highly significant difference between the LVEF basal value (35.06 ± 5.44) and the six-months (50.17 ± 7.95, p < 0.0001), one year (51.03 ± 7.57, p < 0.001) and 2 - 7 years follow-up (52.06 ± 8.06; p < 0.0001) values in group A. Only three of group A patients had higher than 40% basal values (41%, 41% and 42%) whereas six had basal values below 30% (25.3 ± 2.8 %); interestingly. Two of the latter increased their LVEF percentage to values above the 40% limit after one year and have remained above that figure during the follow-up. The remaining four did not reach the 40% value but they increased their LVEF percentage by 17%, 32%, 36%, and 71%, compared to their basal value. In group B patients, the LVEF value remained almost unchanged six months after the revascularization (38 ± 4.03, p N.S.). The survival at five years was nil in group B patients, those who remained alive one year after the revascularization only showed a 3% increase in the LVEF value.

Table 1. Echocardiographic values before and after treatment

Table 1 shows the basal and follow-up values of LVDV, LVSV and LVDD in groups A and B. A signifycant improvement was observed in group A patients six months after surgery. Figures 1 and 2 show the preand post-operative SPECT imaging of a representative patient of group A.

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Assmus, B., Schachinger, V., Teupe, C., Britten, M., Leh-mann, R., Dobert, N., Grunwald, F., Aicher, A., Urbich, C., Martin, H., Hoelzer, D., Dimmeler, S. and Zeiher, A.M. (2002) Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI). Circulation, 106, 3009-3017. Hdoi:10.1161/01.CIR.0000043246.74879.CD
[2]	Strauer, B.E., Brehm, M., Zeus, T., Kostering, M., Hernandez, A., Sorg, R.V., Kogler, G. and Wernet, P. (2002) Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation, 106, 1913-1918. Hdoi:10.1161/01.CIR.0000034046.87607.1C
[3]	Kang, H.J., Kim, H.S., Koo, B.K., Park, K.W., Lee, H.Y., Sohn, D.W., Oh, B.H., Park, Y.B. and Kim, H.S. (2008) Effects of stem cell therapy with G-CSF on coronary artery after drug-eluting stent implantation in patients with acute myocardial infarction. Heart, 94, 604-609. Hdoi:10.1136/hrt.2007.128348
[4]	Singh, S., Arora, R., Handa, K., Khraisat, A., Nagajothi, N., Molnar, J. and Khosla, S. (2009) Stem cells improve left ventricular function in acute myocardial infarction. Clinical Cardiology, 32, 176-180. Hdoi:10.1002/clc.20470
[5]	Martin-Rendon, E., Brunskill, S., Hyde, C., Stanworth, S., Mathur, A. and Watt, S. (2008) Autologous bone marrow stem cells to treat acute myocardial infarction: A systematic review. European Heart Journal, 29, 1807-1818. Hdoi:10.1093/eurheartj/ehn220
[6]	Mathur, A. and Martin, J.F. (2004) Stem cells and repair of the heart. Lancet, 364, 183-192. Hdoi:10.1016/S0140-6736(04)16632-4
[7]	Tse, H.F., Kwong, Y.L., Chan, J.K., Lo, G., Ho, C.L. and Lau, C.P. (2003) Angiogenesis in ischaemic myocardium by intramyocardial autologous bone marrow mononuclear cell implantation. Lancet, 361, 47-49. Hdoi:10.1016/S0140-6736(03)12111-3
[8]	Chang, S.A., Kang, H.J., Lee, H.Y., Kim, K.H., Hur, J., Han, K.S., Park, Y.B. and Kim, H.S. (2009) Peripheral blood stem cell mobilization by granulocyte-colony stimulating factor in patients with acute and old myocardial infarction for intracoronary cell infusion. Heart, 95, 1326- 1330. Hdoi:10.1136/hrt.2008.148429
[9]	Archundia, A., Aceves, J.L., Lopez-Hernandez, M., Al- varado, M., Rodriguez, E., Diaz Quiroz, G., Paez, A., Rojas, F.M. and Monta?o, L.F. (2005) Direct cardiac injection of G-CSF mobilized bone-marrow stem-cells improves ventricular function in old myocardial infarction. Life Sciences, 78, 279-283. Hdoi:10.1016/j.lfs.2005.04.080
[10]	Lévesque, J.P., Hendy, J., Winkler, I.G., Takamatsu, Y. and Simmons, P.J. (2003) Granulocyte colony-stimulating factor induces the release in the bone marrow of proteases that cleave c-KIT receptor (CD117) from the surface of hematopoietic progenitor cells. Experimental Hematology, 31, 109-117. Hdoi:10.1016/S0301-472X(02)01028-7
[11]	Mathieu, M., Bartunek, J., El Oumeiri, B., Touibri, K., Hadad, I., Thoma, P., Metens, T., Da Costa, A.M., Mah-moudabady, M., Egrise, D., Blocklet, D., Mazouz, N., Naejje, R., Heyndrickx, G. and McEntee, K. (2009) Cell therapy with autologous bone marrow mononuclear stem cells is associated with superior cardiac recovery compared with use of nonmodified mesenchymal stem cells in a canine model of chronic myocardial infarction. The Journal of Thoracic and Cardiovascular Surgery, 138, 646- 653. Hdoi:10.1016/j.
[12]	Kang, H.J., Lee, H.Y., Na, S.H., Chang, S.A., Park, K.W., Kim, H.K., Kim, S.Y., Chang, H.J., Lee, W., Kang, W.J., Koo, B.K., Kim, Y.J., Lee, D.S., Sohn, D.W., Han, K.S., Oh, B.H., Park, Y.B. and Kim, H.S. (2006) Differential effect of intracoronary infusion of mobilized peripheral blood stem cells by granulocyte colony-stimulating factor on left ventricular function and remodeling in patients with acute myocardial infarction versus old myocardial infarction: The MAGIC Cell-3-DES randomized, controlled trial
[13]	Brunskill, S.J., Hyde, C.J., Doree, C.J., Watt, S.M. and Martin-Rendon, E. (2009) Route of delivery and baseline left ventricular ejection fraction, key factors of bone- marrow-derived cell therapy for ischaemic heart disease. European Journal of Heart Failure, 11, 887-896. Hdoi:10.1093/eurjhf/hfp101
[14]	Suarez de Lezo, J., Herrera, C., Pan, M., Romero, M., Pavlovic, D., Segura, J., Sanchez, J., Ojeda, S. and Torres, A. (2007) Regenerative therapy in patients with a revas- cularized acute anterior myocardial infarction and depressed ventricular function. Revista Espa?ola de Cardiología, 60, 357-365.
[15]	Herrmann, J.L., Wang, Y., Abarbanell, A.M., Well, B.R., Tan, J. and Meldrum, D.R. (2010) Precondiotioning mesen-chymal stem cells with transforming growth factoralpha improves mesenchymal stem cell-mediated cardio-protection. Shock, 33, 24-30. Hdoi:10.1097/SHK.0b013e3181b7d137
[16]	Sanganalmath, S.K., Abdel-Latif, A., Bolli, R., Xuan, Y.T. and Dawn, B. (2011) Hematopoietic cytokines for cardiac repair: Mobilization of bone marrow cells and beyond. Basic Research in Cardiology, 106, 709-733. Hdoi:10.1007/s00395-011-0183-y
[17]	Burchfield, J.S., Iwasaki, M., Koyanagi, M., Urbich, C., Rosenthal, N., Zelher, A.M. and Dimmeler, S. (2008) Interleukin-10 from transplanted bone marrow mononuclear cells contributes to cardiac protection after myocardial infarction. Circulation Research 103, 201-211. doi:10.1161/CIRCRESAHA.108.178475
[18]	Kanellakis, P., Slater, N.J., Du, X.J., Bobik, A. and Curtis, D.J. (2006) Granulocyte colony-stimulating factor and stem cell factor improve endogenous repair after myo- cardial infarction. Cardiovascular Research, 70, 117-125. Hdoi:10.1016/j.cardiores.2006.01.005
[19]	Bartunek, J., Dimmeler, S., Drexler, H., Fernandez-Aviles, F., Galinanes, M., Janssens, S., Martin, J., Mathur, A., Menasche, P., Priori, S., Strauer, B., Tendera, M., Wijns, W. and Zeiher, A. (2006) Task force of the European Society of Cardiology. The consensus of the task force of the European Society of Cardiology concerning the clinical investigation of the use of autologous adult stem cells for repair of the heart. European Heart Journal, 27, 1338-1340. Hdoi:10.1093/eurheartj/ehi793
[20]	Yeh, E.T., Zhang, S., Wu, H.D., Korbling, M., Willerson, J.T. and Estrov, Z. (2003) Transdifferentiation of human peripheral blood CD34-enriched cell population into cardiomyocytes, endothelial cells, and smooth muscle cells in vivo. Circulation, 108, 2070-2073. Hdoi:10.1161/01.CIR.0000099501.52718.70
[21]	Balsam, L.B., Wagers, A.J., Christensen, J.L., Kofidis, T., Weissman, I.L. and Robbins, R.C. (2004) Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature, 428, 668-673. Hdoi:10.1038/nature02460
[22]	Murry, C.E., Soonpaa, M.H., Reinecke, H., Nakajima, H., Nakajima, H.O., Rubart, M., Pasumarthi, K.B., Virag, J.I., Bartelmez, S.H., Poppa, V., Bradford, G., Dowell, J.D., Williams, D.A. and Field, L.J. (2004) Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Nature, 428, 664-668. Hdoi:10.1038/nature02446
[23]	Menasche, P. (2004) Embryonic stem cells pace the heart. Nature Biotechnology, 22, 1237-1238. Hdoi:10.1038/nbt1004-1237
[24]	Jackson, K.A., Majka, S.M., Wang, H., Pocius, J., Hart- ley, C.J., Majesky, M.W., Entman, M.L., Michael, L.H., Hirschi, K.K. and Goodell, M.A. (2001) Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. Journal of Clinical Investigation, 107, 1395-1402. Hdoi:10.1172/JCI12150
[25]	Castello, S., Podesta, M., Menditto, V.G., Ibatici, A., Pitto, A., Figari, O., Scarpati, D., Magrassi, L., Bacigalupo, A., Piaggio, G. and Frassoni, F. (2004) Intra-bone marrow injection of bone marrow and cord blood cells: An alternative way of transplantation associated with a higher seeding efficiency. Experimental Hematology, 32, 782-787. Hdoi:10.1016/j.exphem.2004.05.026
[26]	Kastrup, J., Jorgensen, E., Ruck, A., Tagil, K., Glogar, D., Ruzyllo, W., Botker, H.E., Dudek, D., Drvota, V., Hesse, B., Thuesen, L., Blomberg, P., Gyongyosi, M. and Sylven, C. (2005) Direct intramyocardial plasmid vascular endothelial growth factor-A165 gene therapy in patients with stable severe angina pectoris. Journal of the American College of Cardiology, 45, 982-988. Hdoi:10.1016/j.jacc.2004.12.068
[27]	HMias, CH., HLairez, OH., HTrouche, EH., HRoncalli, JH., HCalise, DH., HSeguelas, M.HH., HOrdener, CH., HPiercecchi-Marti, M.DH., HAuge, NH., HSalvayre, A.NH., HBourin, PH., HParini, AH. and HCussac, DH., (2009) Mesenchymal stem cells promote matrix metal- loproteinase secretion by cardiac fibroblasts and reduce cardiac ventricular fibrosis after myocardial infarction. Stem Cells, 27, 2734-2743. Hdoi:10.1002/stem.169
[28]	Mozid, A.M., Arnous, S., Sammut, E.C. and Mathur, A. (2011) Stem cell therapy for heart diseases. British Medical Bulletin, 98, 143-159. Hdoi:10.1093/bmb/ldr014
[29]	Strauer, B.E., Schannwell, C.M. and Brehm, M. (2009) Therapeutic potentials of stem cells in cardiac diseases. Minerva Cardioangiologica, 57, 249-267.