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Comparison of intracoronary and transendocardial delivery of allogeneic mesenchymal cells in a canine model of acute myocardial infarction
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Journal of Molecular and Cellular Cardiology 44 (2008) 486 – 495 www.elsevier.com/locate/yjmcc
Original article
Comparison of intracoronary and transendocardial delivery of allogeneic mesenchymal cells in a canine model of acute myocardial infarction Emerson C. Perin a,⁎, Guilherme V. Silva a , Joao A.R. Assad a , Deborah Vela a,b , L. Maximilian Buja a,b , Andre L.S. Sousa a , Silvio Litovsky a , Jing Lin a , William K. Vaughn a , Stephanie Coulter a , Marlos R. Fernandes a,b , James T. Willerson a,b a b
The Texas Heart Institute at St. Luke's Episcopal Hospital, Houston, TX 77030, USA The University of Texas Health Science Center at Houston, Houston, TX 77030, USA
Received 2 July 2007; received in revised form 30 August 2007; accepted 27 September 2007 Available online 4 October 2007
Abstract This study assessed safety of transendocardial (TE) electromechanical-guided delivery of bone marrow mesenchymal stem cells (MSCs) after acute myocardial infarction (AMI) and compared intracoronary (IC) delivery with TE delivery. In a canine acute myocardial ischemia model, 100 × 106 MSCs were delivered 7 days after AMI via IC and TE routes. Functional assessment was performed by 2D echocardiogram, and detailed histopathologic analyses were performed to assess the impact of cell therapy in vascular density and fibrosis. Patterns of cell distribution in both delivery methods were also compared. There was a statistically significant reduction in the amount of myocardial ischemia in the TE group (P = 0.007). Left ventricular ejection fraction (LVEF) increased 13% (mean) in the TE group (21-day follow-up) and was significantly better than that of the controls (P = 0.01), but did not improve in the IC-delivery group. Dissimilar patterns of cell distribution were noted between the IC and TE groups. This study suggests that MSC treatment is probably safe and effective after AMI. In the comparison of TE and IC delivery, the TE group showed higher cell retention (clusters even in the injury center of the infarct) with an increased vascularity and greater functional improvement than did the IC group (no clusters; cells at the border of the infarct). The higher local cell density in the TE group may be important for therapeutic effectiveness. © 2008 Elsevier Inc. All rights reserved. Keywords: Acute myocardial infarction; Stem cell therapy; Transendocardial delivery; Intracoronary delivery; Mesenchymal stem cell; Stem cell injection
1. Introduction Previous human studies have suggested that bone marrowderived mononuclear stem cells (BMNCs) can positively affect postinfarction remodeling processes [1–6]. The safety and therapeutic benefits of intracoronary (IC) infusion of BMNCs through the stop-flow technique have been recently confirmed with the publication of the REPAIR-MI trial [7]. Within the bone marrow, the mesenchymal stem cell (MSC) subfraction exhibits a high degree of plasticity and the potential for differentiating into both mesenchymal and non-mesenchymal lineages; therefore, MSCs have been proposed for use in
⁎ Corresponding author. Tel.: +1 713 791 9400; fax: +1 713 795 5651. E-mail address: [email protected] (E.C. Perin). 0022-2828/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yjmcc.2007.09.012
cardiac repair after myocardial infarction (MI). MSCs, in contrast to BMNCs, are very large cells, and an isolated preclinical study has questioned the safety of delivering MSCs via IC infusion [8]. For the treatment of chronic myocardial ischemia, both experimental and human stem cell studies have been performed either with direct (surgical) delivery or with transendocardial (TE) delivery guided by electromechanical mapping (EMM) [9–12]. The TE delivery route has been shown to be safe in patients with end-stage ischemic heart failure [12], and numerous preclinical studies support its safety [9,13]. TE delivery also adds the potential advantage of targeting viable ischemic myocardium [14] (the border zone of an infarct), which has been postulated as an optimal milieu for bone marrow stem cell engraftment and effect. The role of TE injection after acute myocardial infarction (AMI) in clinical trials, however, has not been established because of potential safety concerns.
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Therefore, we designed a canine study to assess the safety and feasibility of electromechanical-guided TE delivery of MSCs in the setting of AMI. In addition, we compared electromechanicalguided TE delivery of MSCs at the border zone of acute infarction with that of IC delivery of MSCs to the infarct-related artery. 2. Materials and methods This study complies with the Declaration of Helsinki and was reviewed and approved by The University of Texas Health Science Center at Houston's Animal Welfare Committee. The study was conducted at The University's Center for Laboratory Animal Medicine and Care, located at The University of Texas M. D. Anderson Cancer Center's Department of Veterinary Medicine and Surgery (Houston, TX). 2.1. MSC isolation, culture, and labeling Allogeneic canine MSC isolation was performed at Osiris Therapeutics, Inc. (Baltimore, MD), as previously described [15]. Briefly, a purified bone marrow MSC population was expanded in culture. Cells were then harvested, labeled with the cross-linkable membrane dye CM-DiI (Molecular Probes, Inc, Eugene, OR) and the nuclear stain DAPI, and frozen in cryocyte bags. Frozen cells were stored in the vapor phase of liquid nitrogen until the time of implantation. Before injection, the cells were thoroughly washed and resuspended in a 5 ml volume of saline (20 × 106 MSC/ml). At the time of delivery, the viability of the cell suspension was confirmed to be greater than 90%. 2.2. Pilot safety study of IC delivery of MSCs To assess safety of IC delivery, 100 million MSCs were infused in 9 dogs with normal coronary arteries at 3 different velocities: 1 × 106 cells/min (n = 5); 1.5 × 106 cells/min (n = 2), and 3 × 106 cells/min (n = 2).
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2.4. Canine AMI model In brief, a left thoracotomy was performed with the aid of anesthesia induced with Pentothal (17 mg/kg, IV) and maintained with isoflurane (1.5–2.0%). Acute myocardial ischemia was produced by transient occlusion (total of 3 h) of the proximal LAD coronary artery and followed by reperfusion. In addition, ligation of the diagonal branch was performed to decrease collateral flow to the infarct area. 2.5. Coronary angiography The right femoral artery was surgically exposed, and after insertion of a 6F sheath, the animals were systemically anticoagulated with unfractionated heparin. A 5F Amplatz left 1 diagnostic catheter was introduced, and selective coronary angiograms from the right coronary artery and left coronary system were obtained in multiple projections. 2.6. Electromechanical mapping (EMM) acquisition and functional assessment EMM was performed as previously described [16]. After point acquisition was completed, post-processing analysis was performed with a series of filters in the moderate setting to eliminate inner points, points that did not fit the standard stability criteria (location stability b4 mm, loop stability b6 mm, and cycle length variation b10%), points acquired during STsegment elevation, and points not related to the left ventricle (e.g., those on the atrium). Ischemic myocardium was defined as a zone with unipolar voltage greater than 6.9 mV and linear local shortening of less than 6% on the linear local shortening map. EMM was used to quantify the ischemic area before injections and immediately before the animals were humanely killed, as previously described [17]. 2.7. IC and TE delivery of MSCs
2.3. Study design After the pilot safety study (IC delivery of MSCs) was completed, the IC vs. TE delivery study was performed. Twentyone dogs were used for the study. Day 0: All 21 dogs were scheduled to undergo two-dimensional (2D) echocardiography followed by left anterior descending (LAD) occlusion/reperfusion procedures, as described below (canine AMI model). Two dogs died during surgery at the time of occlusion due to ventricular fibrillation and infarction and were not randomized. Therefore, 19 dogs were randomized. Day 7: All 19 dogs underwent coronary angiography, EMM, and 2D echocardiography. The animals were randomly assigned to 3 groups: (1) TE delivery (n = 6), (2) IC delivery (n = 7), and (3) control group (n = 6). The injection procedures are described below. Day 21: Immediately before the animals were humanely killed, repeat coronary angiography, EMM, and 2D echocardiography were performed.
In the IC delivery group (n = 7), after coronary angiography was performed to confirm LAD patency, a 5F Amplatz 1 diagnostic catheter was positioned under fluoroscopic guidance subselectively into the LAD. After the catheter was positioned, a total of 100 × 10 6 allogeneic MSCs were infused at 1 × 106 cells/min. MSCs were infused during 5-min periods (total of 20) with 1-min intervals between infusions to allow for normal coronary flow. Our technique differed from the stopflow technique utilized in the major clinical trials in that it did not involve proximal occlusion of the vessel with a coronary angioplasty balloon. In the TE delivery group (n = 6), allogeneic MSCs (100 × 106 diluted in 5 ml phosphate buffered saline [PBS]) were delivered via 25 EMM-guided TE injections. Each injection was composed of 0.2 cm3 of a solution of 2 million cells per 0.1 cm3. The injection catheter (Myostar™ injection catheter, BiosenseWebster, Diamond Bar, CA) is composed of a 27-gauge needle housed in an 8F catheter that is able to assess endocardial
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voltage and catheter endocardial contact, allowing for targeting of viable ischemic myocardium at the border zone of the anterior infarction. None of the animals received immunosuppressive therapy. 2.8. Control group The animals in the control group (n = 6) underwent the occlusion/reperfusion, coronary angiography, EMM, and 2Dechocardiography in a similar fashion to that of the IC and TE groups. The control group did not receive IC placebo infusion or TE placebo injection. 2.9. Functional assessment by echocardiography Transthoracic rest echocardiography was performed at baseline (before infarction), immediately before cell therapy (7 days after infarction), and 14 days after cell treatment (21 days after infarction). Echocardiography was performed with a commercially available echocardiographic system (Sonos1000, Hewlett Packard Co, Palo Alto, CA) equipped with a 10 MHz linear-array transducer, as described elsewhere [18]. The left ventricular ejection fraction (LVEF), end-diastolic dimension (EDD), and end-systolic dimension (ESD) at rest were obtained from at least 3 consecutive cardiac cycles, according to the American Society of Echocardiography leading-edge method [19]. A blinded, experienced observer performed all measurements (SC). 2.10. Tissue preparation and infarct sizing Dogs were humanely killed 21 days after occlusion/reperfusion (14 days after MSC therapy). Their hearts were exposed by median sternotomy and quickly removed. The heart weight was recorded. The hearts were sliced in a bread-loaf manner into 4 transverse sections from apex to base. Each section was separated into anterior, anterolateral, lateral, posterolateral, and posterior left ventricular free wall; anterior, mid, and posterior interventricular septum; and right ventricular free wall. The sizes of myocardial infarcts and bed at risk (B-A-R) were measured by the tetrazolium method, as previously described [20]. The heart was the only organ examined in this study. 2.11. Immunofluorescence labeling procedure All frozen specimens were cut on positively charged slides at 4 μm and allowed to air dry for 2 h at room temperature. Sections were then fixed in 4 °C acetone for 2 min, allowed to air dry at room temperature for 15 min, and then placed in PBS (pH 7.5) for 5 min. The following antibodies and dilutions were used for antigen detection: anti-α-sarcomeric actinin, 1:20,000 (Sigma, Cat #A7811); troponin I, 1:200 (Chemicon International, Cat #MAB3438); smooth muscle actin, 1:40,000 (Sigma, Cat #A2547); and von Willebrand factor VIII, 1:8000 (Dako, Cat #A0082). All antibodies were diluted with 0.02% bovine serum albumin (BSA)/PBS. Tissue sections were removed from PBS and placed in 1.5% normal horse serum (Vector, Cat #S-2000) for 10 min. The horse
serum was gently tapped off, and the sections were incubated with the primary antibodies for 1 h at room temperature. The sections were then rinsed with PBS for 5 min and placed in fluorescein solution for 10 min in a dark chamber at room temperature. For polyclonal antibody factor VIII, goat anti-rabbit fluorescein (Vector, Cat #Fl-1000) was used. For monoclonal antibodies, smooth muscle actin, anti-α-sarcomeric actinin, troponin I, and horse anti-mouse fluorescein (Vector, Cat #Fl-2000) were used. The sections were thoroughly rinsed in PBS and aqueous mounted with Aquaperm (Thermo-Shandon, Cat #484985). 2.12. Fibrosis quantification Trichrome staining was used to evaluate collagen deposition. Six anterolateral sections from each heart were evaluated in their entirety and quantified. The results were expressed as a percentage of each ventricular section. 2.13. Vascular density assessment The effect of stem cell transplantation on angiogenesis was evaluated by quantitative morphometry, performed with Olympus MicroSuite software on an Olympus BX61 microscope (Olympus America, Inc., Melville, NY). Seven to eight paraffinembedded sections per heart, from mid-level left ventricular anterior and anterolateral walls to apex, were immunostained for the endothelial cell marker, factor VIII (Dako, Carpinteria, CA), and α-smooth muscle actin (Sigma). The number of vessels was counted under a light microscope in 5 random fields (each field measuring 0.58 mm2). Capillary density was expressed as the area of factor VIII positive blood vessels in μm2 per mm2 of each ventricular section. Arteriolar density was obtained by counting all microvessels (arterioles fully coated with a muscular layer positive for α-smooth muscle actin) with a minor axis b 15 μm, and was expressed as arterioles/mm2. 2.14. Mesenchymal cell retention and distribution Histopathologic quantitative analysis (fluorescence microscopy) for the presence of DAPI and/or DiI-positive MSCs was performed in an average of 12 segments, from mid-level to the apex for each heart. Frozen sections were raster-scanned at high-power magnification (× 400) on an Olympus BX61 microscope (Olympus America, Inc., Center Valley, PA) and were then quantified with Olympus MicroSuite Five software. 2.15. Statistical analyses The primary end point was assessment of LVEF by echo at the last follow-up. The study was powered and designed to answer the question of whether cell treatment would lead to a higher LVEF in the treatment groups than in the control group. Importantly, the study was specifically designed to compare the IC and TE delivery routes with the controls as the primary end point. All other analyses were designed to be secondary end points. All values are expressed as mean ± SD. All analyses were performed with appropriate software (Statview; SAS Institute,
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Inc., Cary, NC). A sample size of 6 animals per group (total of 18 dogs) provided a power of 0.8, considering an alpha equal to 0.05 to detect an absolute difference of 10% of LVEF 21 days after MI, considering means of 34 ± 5% and 45 ± 7% for the control group and each treatment arm, respectively. Comparisons of vascular density, LVEF, left ventricular diameter, and the amount of fibrosis were performed utilizing analysis of variance (ANOVA) and Kruskal–Wallis tests for non-normally distributed variables. The Bonferroni test (parametric data) or Kruskal–Wallis Z test (non-parametric data) was used for comparison between pairs (IC vs. TE, IC vs. control, and TE vs. control). Comparisons of MSC engraftment between the IC and TE groups were performed by using an unpaired two-tailed Student's t-test. A value of P b 0.05 was considered statistically significant. 3. Results 3.1. Pilot safety study of IC delivery of MSCs In the study of the safety of IC delivery of 100 million MSCs at 3 different velocities (1 × 106 cells/min [n = 5]; 1.5 × 106 cells/ min [n = 2]; and 3 × 106 cells/min [n = 2]), post-procedural transient TIMI-2 flow was seen in all dogs at the 2 higher velocities. In dogs that received the cell infusion at 1 × 106 cells/min, there was no significant elevation in CK-MB (1.16 ± 1.15 ng/ml at baseline; 1.78 ± 1.67 ng/ml at 24 h [P = 0.51]) or troponin I (0.03 ± 0.01 ng/ml at baseline; 0.45 ± 0.17 ng/ml at 24 h after cell infusion [P = 0.85]). Peak troponin I values were 2 times the normal level in dogs that received 1.5 × 106 cells/min and 7 times the normal level in dogs that received 3 × 106 cells/min. Histopathologic study did not show capillary plugging in animals in the pilot study. 3.2. IC vs. TE delivery study Of the 19 dogs that were randomized, 2 dogs died after randomization and IC infusion of MSCs and did not complete the evaluation at day 21 (described below).
Fig. 1. H & E stain showing microvascular plugging that occurred in the dog that died during intracoronary cell injection.
3.4. Coronary angiography Two animals in the IC group did not complete follow-up angiography (see above). All other animals in the IC group (n = 5) had TIMI-3 flow in the LAD territory at 7- and 21-day follow-up. All animals in the TE and control groups had TIMI-3 flow in the LAD territory at 7- and 21-day follow-up. Coronary collaterals did not develop in any animal, based on visual assessment. 3.5. Functional assessment by electromechanical mapping (EMM) Fig. 2A shows the net change in ischemic area in the 3 groups over time. Overall, there was a statistically significant reduction in the amount of myocardial ischemia in the TE group vs. the IC and control groups (P = 0.007). All other comparisons between groups are shown in Fig. 2A and stated in the legend.
3.3. Procedural safety 3.6. Functional assessment by echocardiography One dog in the IC group had no-reflow of the LAD coronary artery during the injection procedure. ST-elevation and ventricular fibrillation developed, and the dog died despite resuscitative efforts. Histopathologic analysis revealed extensive microvascular “plugging” associated with the presence of MSCs (Fig. 1). Another dog in the IC group died 2 days after cell injection from intestinal ischemia/infarct, which was confirmed at postmortem study. All animals underwent TE injection of MSCs without complication 7 days postinfarction. There were no sustained arrhythmias associated with either the IC or TE injection procedure. In the TE group, no pericardial effusion or cardiac tamponade was observed on immediate post-procedural surveillance or on the 2D echocardiograms performed before the animals were humanely killed.
All groups had similar LVEFs before ischemia/reperfusion (Fig. 2B). Treated and control dogs had similar resting LVEFs immediately before stem cell implantation (Fig. 2B). At 21-day follow-up, the TE-delivery group had a significant improvement in LVEF and ventricular dimensions as compared to the control group (13%, P = 0.01). This improvement was not seen in the IC delivery group (Fig. 2B–D). 3.7. Histopathologic analyses Organs other than the heart were not examined in this protocol. The injected hearts showed no evidence of either gross or microscopic calcification, tumor formation, or unwanted cell differentiation.
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3.7.1. Infarct size and bed at risk There were no significant differences among the 3 groups (Fig. 3A). 3.7.2. Fibrosis quantification There were no significant differences among the 3 groups in terms of total LAD territory and the amount of fibrosis (data not shown).
3.7.3. Enhancement of neovascularization by transplanted MSCs Capillary density was increased in the MSC-TE treated dogs as compared to controls (Fig. 3B, TE vs. control, P = 0.01). Arteriolar density is shown in Fig. 3C (TE vs. IC and control, P = 0.03). 3.7.4. Stem cell retention and distribution patterns Persistence of grafted cells in the myocardium was confirmed in approximately one third of the sections examined (for both TE and IC groups) by identifying MSCs showing DAPI and/or DiI positivity. The TE delivery route achieved a higher MSC concentration per square micron than the IC route in the myocardium (P b 0.01). The results for local cell density can be seen in Fig. 3D. The distribution of DAPI-labeled MSCs within positive regions in dogs in the main study group was studied in a detailed analysis. This analysis showed that certain distribution patterns appear to be predominant and specific for one group or the other (Fig. 4A–D): IC group: IC delivery appears to result in a distribution of vast areas of relatively constant concentration at the positive sites, which are homogeneous in pattern (Fig. 4A). DAPI-labeled cells were observed in both the injured and normal myocardium, although the highest cell density for this group was at the injury border site (Fig. 4B). There was a tendency for cells to be absent from the injury center. The cells seen in the normal myocardium in proximity to the injury diminished in number until they were absent at sites very distant from the healing infarct. TE group: DAPI positive cells were found in approximately one third of the examined segments in each heart. Like the cells in the previous group, these cells were found in both injured and normal myocardium, with a higher concentration at the injury border (as seen in IC dog in Fig. 4B). However, a finding particular only to this group is that areas coinciding with an injection site tended to retain relatively high cell
Fig. 2. (A) Mean change in resting ischemic area within the groups by electromechanical mapping. *P = 0.03 for TE vs. control; P = 0.08 for intracoronary vs. control; P = 0.18 for transendocardial vs. intracoronary. (B) Left ventricular ejection fraction at rest. Assessments were made at baseline, before occlusion/reperfusion (left), 7 days later at the time of cell injection (middle), and 21 days after occlusion/reperfusion (right). *P = 0.01 for TE vs. control after cell delivery; P = 0.83 for intracoronary vs. control after cell delivery; P = 0.63 for TE vs. intracoronary after cell delivery. (C) Left ventricular end diastolic dimension at rest. Assessments were made at baseline, before occlusion/reperfusion (left), 7 days later at the time of cell injection (middle), and 21 days after occlusion/reperfusion (right). *P = 0.04 for TE vs. control after cell delivery; P = 0.87 for intracoronary vs. control after cell delivery; P = 0.36 for TE vs. intracoronary after cell delivery. (D) Left ventricular end systolic dimension at rest. Assessments were made at baseline, before occlusion/reperfusion (left), 7 days later at the time of cell injection (middle), and 21 days after occlusion/reperfusion (right). *P = 0.02, for TE vs. control after cell delivery; P = 0.51, for intracoronary vs. control after cell delivery; and P = 0.16 for TE vs intracoronary after cell delivery.
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Fig. 3. (A) Infarct size and bed at risk (B-A-R). (B) Vascular density was statistically greater in the anterolateral walls of animals that received transendocardial (TE) injections of mesenchymal stem cells as compared to controls; * P = 0.01, vs. control. (C) Arteriolar count (diameter b 15 µm) at the infarct border; * P b 0.05, vs. control. (D) Absolute cell density within normal and injured myocardial areas, at positive sites, in the TE and IC groups. * P b 0.01 for TE-cluster vs. IC.
concentrations. This higher density of DAPI positive cells was observed even in a few rare instances where the injection site was found up to 500–600 μm from the infarct border, toward the direction of the center of the injury. The high-density cell clustering at these latter sites was identical to the clustering pattern of the cells at the injection sites in proximity to the infarct, suggesting that these clusters might not have been a product of cell migration. This pattern contrasts to that of the IC group where the cells tended to remain at the border, and their density gradually decreased or was absent toward the injury center. In the TE group, very high cell concentrations were seen in the normal tissue as well, with true needle tracts and cluster patterns representing the injection sites (Fig. 4C and D). 3.7.5. Stem cell co-localization in ischemic myocardium The survival of engrafted cells was identified by the presence of DiI- and/or DAPI-positive cells in frozen sections as performed with Olympus MicroSuite Five software on an Olympus BX61 microscope. In many instances, DAPI positive nuclei co-localized with mature phenotypical markers such as α–smooth-muscle actin, factor VIII, or α-sarcomeric actinin (Fig. 5A–F).
4. Discussion This study demonstrates the safety and feasibility of TE delivery of MSCs in the post-AMI setting. In this canine model of acute myocardial infarction, significant functional improvement was demonstrated after EMMguided TE delivery of bone marrow-derived MSCs. In addition, TE delivery of MSCs resulted in higher vascular density and a higher density of MSCs at the peri-infarct region. 4.1. Allogeneic MSCs Previous work has demonstrated the functional benefits of bone marrow MSC surgical injection after MI [21]. Within the bone marrow mononuclear cells, the MSC subfraction is capable of secreting numerous cytokines and has a high degree of plasticity, which has led to increasing interest in this cell subtype for possible use in cardiac regeneration. Because MSCs are rare in the bone marrow, ex-vivo expansion (for at least 10 days after harvest) is mandatory for clinical utilization. Bone marrow mononuclear cells have been shown to be functionally impaired with aging and with disease states such as coronary artery disease [22]. Therefore, we used allogeneic MSCs, given the theoretical advantage of clinical availability
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Fig. 4. Various TE and IC delivery distribution patterns in normal myocardium. (A) Homogenous distribution of DAPI+ cells, as can be found in either IC delivery or in non-clustered areas TE delivery, away from injection sites, magnification x 200; (B) DAPI+ cells at infarct border of an IC delivery dog. Note on the left side (infarct side) the higher density of DAPI+ nuclei (arrows) when compared with the right side showing DAPI+ nuclei amidst normal myocardium, magnification x 200; (C) High concentration of DAPI+ cells corresponding to a needle tract of a TE dog, magnification x100; (D) DAPI+ cell cluster, corresponding to an injection site of a TE dog, magnification x 100. (Scale bar = 200 µm for A, B; 100 µm for C, D).
and optimal functional capacity over autologous, ex-vivoexpanded MSCs. Allogeneic MSCs have been suggested to be non-immunogenic stem cells [23]. Dai et al. [24] have demonstrated that transplanted allogeneic MSCs in rats can survive up to 6 months after transplantation and did not induce immunorejection. MSCs have been shown to inhibit T-cell proliferation and also to prevent T-cell maturation to their antigen [23]. This study has once more demonstrated the non-immunogenic profile of allogeneic MSCs. Importantly, the animals were not given any immunosuppressive drugs, which lends a favorable perspective for the clinical use of this cell type. 4.2. Comparison of IC and TE distribution patterns: correlation with functional and histopathologic findings The issues of timing of cell delivery after infarction and persistence of MSCs post MI have been evaluated in previous studies in small animal models. Cell retention after infarction progressively declines after cell injection as shown by MüllerEhmsen et al. [25]. In their study, cell engraftment was quantified, using quantitative real-time PCR with Y-chromosome specific primers, at 0 h, 48 h, 5 days, 3 weeks, and 6 weeks after cell injection. The percentage of MSCs in the heart decreased rapidly from a maximal percentage of 80% of injected cells at 0 h down to a maximal percentage of approximately 3.5% at 6 weeks, follow-up. At a similar follow-up time as the
present study, a maximal percentage of 4.7% of injected MSCs was detected in the heart. Furthermore, the functional benefit of MSC injections post MI seems to depend on the timing of cell delivery. In a preliminary study, Hu et al. [26] observed that the ideal timing of cell delivery is 1 week after infarction (as done in the present study) to achieve optimal functional benefit. In large animal models, previous studies have confirmed the safety and feasibility of TE and surgical intramyocardial delivery of MSCs after AMI [21,27–29]. Amado et al. [27] have focused on the functional aspect of TE-MSC injection after MI. They have shown that when allogeneic MSCs are injected 3 days after MI, cardiac regeneration is stimulated, infarct size is decreased, and cardiac function is near normalized. Other studies have focused on quantifying MSC engraftment after MI. Hou et al. [28] have found higher engraftment rates of stem cells delivered via surgical intramyocardial injection (11.3%) when compared to engraftment rates of cells delivered via the IC route (2.6%). Freyman et al. [29] have compared postMI MSC injection by TE delivery to IV and IC delivery. At 14 days, cell engraftment was higher in the IC group vs. the 2 other groups. TE delivery had higher engraftment rates than did IV infusion. Our study compared IC and TE delivery methods taking both combined functional and engraftment aspects into account. Our study confirms that the functional benefit of MSC
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Fig. 5. (A) Normal myocardium of an IC dog immunostained for α-sarcomeric actinin shows numerous DAPI+ nuclei, some of which appear to colocalize with the myocytes (arrows) while others appear at the cell periphery or in interstitium (arrowheads). (B) Normal myocardium of a control animal immunostained for αsarcomeric actinin, DAPI-negative. (C–E) Healing infarct of a TE dog immunostained for α-smooth-muscle actin (C, green), showing abundant DAPI+ cells (D, blue), many of which appear to contribute to the neovascularization (E, merged imaged of C–D). (F) Healing infarct of a DAPI-negative control dog immunostined for αsmooth-muscle actin. Magnification x 400 A, B; x4 00 C–F. Scale bar = 50 µm A, B; 100 µm C–F.
treatment is preserved when TE injections are extended to 7 days after MI. However, a dramatic reduction in infarct size was not observed. This finding might be explained by the shorter follow-up time after cell injection in our study; previous studies in small [26] and large animals [25] have shown a reduction in infarct size at 4 and 8 weeks post MSC injections, respectively. Our study results conflict with that of Freyman et al. [29] because we had higher engraftment rates with EMM-guided TE than with IC infusion. One possible explanation is the precise targeting offered by EMM-guided TE. Both TE delivery studies cited above differ from the current study in that they utilized fluoroscopy-only guided TE injections. The electromechanicalguided TE delivery unites real-time 3D navigation capabilities with real-time viability assessment. Thus, therapy may be accu-
rately placed at the border zone of an infarct, avoiding myocardial scar [14]. Agbulut et al. [30] have shown that when bone marrow stem cells are injected into scar tissue, engraftment decreases (as does efficacy). Alternatively, the lower engraftment with the IC infusion might have resulted from the technique utilized. There was no proximal occlusion of the vessel before injection and, therefore, it is possible that a significant number of cells were injected retrograde to the proximal part of the coronary artery and to the ascending aorta. In addition, the slow infusion rate of cells might not have been concentrated enough to create a significant gradient to enable more cells to enter the myocardium. Kinnaird et al. [31] have reported that MSCs produce a wide array of arteriogenic cytokines and improve perfusion and remodeling in a mouse model of hind-limb ischemia, possibly
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through neoangiogenesis. Thus, in the present study, higher engraftment rates of MSCs might have contributed to the significantly higher capillary density in the anterolateral wall of TE stem cell–treated animals. The higher capillary density in the TE-delivery group accompanied a substantial improvement in LVEF as compared to the control group. These findings were not present in the animals who received cells via IC delivery. In addition to different engraftment rates, TE and IC delivery modes also differ in cell engraftment patterns. While the MSCs seem to be distributed in a more widespread fashion by IC delivery, cluster areas of MSCs were present in the TE-injected animals, which could indicate that a considerable portion of cells delivered by the TE route will stay at or very near the injection site without migrating. Moreover, TE animals had MSCs distributed not only in the peri-injury area but also inside the injured area. The finding of dissimilar patterns of cell distribution between IC and TE delivery modes might also result from the different targeting capabilities of each delivery method. The importance of cell distribution patterns for therapeutic effectiveness remains to be determined. 4.3. Safety of cell delivery In Freyman's study [29], half of the animals had impaired coronary flow distal to the infusion site after IC infusion. In our study, 1 animal had no-reflow and subsequently died immediately after IC-MSC infusion. The no-reflow phenomenon occurs as a consequence of microvascular flow impairment secondary to distal embolization and disruption of the microvasculature [32,33]. Those findings might relate to the results of the study by Vulliet et al. [8], who described elevated cardiac enzymes and micro infarctions in dogs with normal coronary arteries after IC injection of MSCs. Thus, IC delivery of MSCs might cause microvascular plugging and consequent no-reflow phenomena. Interestingly, Chen et al. [3] did not report reduction in blood flow after IC infusion of larger doses of MSCs (total of 8– 10 × 109 cells). Importantly in the present study, histopathologic study showed that the administration of MSCs did not result in gross or microscopic calcification or tumor formation, as has been reported in a previous study [34]. This issue is particularly critical in the use of the TE delivery route in which cells can reach higher density in cluster patterns. 4.4. Study limitations The major limitation of the present study is the small number of animals in each group, which limits conclusions about efficacy. Still, statistically significant differences between the IC, TE, and control groups were shown in regard to 2D-echocardiographic parameters and capillary density. Because organs other than the heart were not examined in this study, the conclusions regarding safety apply only to the heart. In addition, the IC delivery technique used in our study was different from the one used in the clinical trials. Hence, trans-
lation of our preclinical results regarding safety of IC injections into clinical terms is uncertain. Dogs might develop substantial collateral circulation, which could limit interpretation of cardiac function data. It is possible that a significant number of the injected stem cells did not reach the AMI region. However, coronary angiography was performed both before cell therapy and immediately before animals were humanely killed to ensure patency of the LAD/diagonal coronary arteries and to assess for the development of new collateral vessels. No significant differences in major collateral development were seen between treated and control hearts. The fewer cells present in the IC delivery group could be due either to reduced immediate delivery or early retention. Having a group of animals humanely killed early (24 h) would have answered this question to some extent. However, our study provides the cell therapy field with additional evidence that viability-targeted delivery after MI might enhance therapeutic effectiveness. EMM is known to be capable of detecting and delineating infarcted and ischemic territories. However, no true validation has been made in humans regarding EMM quantification of ischemic area vs. quantification by other modalities, such as SPECT. 5. Conclusions In conclusion, our study suggests that MSC treatment in a canine model of acute myocardial infarction increased vascularity and improved cardiac function. Second, TE injection of MSCs into ischemic myocardium after AMI is probably safe, achieving higher retention rates in a more targeted cell distribution pattern (within the injured area) as compared to IC infusion. Finally, TE delivery of MSCs was associated with a greater functional improvement than was IC infusion. Acknowledgments The authors wish to acknowledge Marianne Mallia, ELS, and Chrissie Chambers, ELS, for their editorial assistance in the preparation of this manuscript. References [1] Assmus B, Schachinger V, Teupe C, Britten M, Lehmann R, Dobert N, et al. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI). Circulation 2002;106: 3009–17. [2] Britten MB, Abolmaali ND, Assmus B, Lehmann R, Honold J, Schmitt J, et al. Infarct remodeling after intracoronary progenitor cell treatment in patients with acute myocardial infarction (TOPCARE-AMI): mechanistic insights from serial contrast-enhanced magnetic resonance imaging. Circulation 2003;108:2212–8. [3] Chen SL, Fang WW, Ye F, Liu YH, Qian J, Shan SJ, et al. Effect on left ventricular function of intracoronary transplantation of autologous bone marrow mesenchymal stem cell in patients with acute myocardial infarction. Am J Cardiol 2004;94:92–5. [4] Janssens S, Dubois C, Bogaert J, Theunissen K, Deroose C, Desmet W, et al. Autologous bone marrow-derived stem-cell transfer in patients with ST-segment elevation myocardial infarction: double-blind, randomised controlled trial. Lancet 2006;367:113–21.
E.C. Perin et al. / Journal of Molecular and Cellular Cardiology 44 (2008) 486–495 [5] Strauer BE, Brehm M, Zeus T, Kostering M, Hernandez A, Sorg RV, et al. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation 2002;106: 1913–8. [6] Wollert KC, Meyer GP, Lotz J, Ringes-Lichtenberg S, Lippolt P, Breidenbach C, et al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet 2004;364:141–8. [7] Schachinger V, Erbs S, Elsasser A, Haberbosch W, Hambrecht R, Holschermann H, et al. Intracoronary bone marrow-derived progenitor cells in acute myocardial infarction. N Engl J Med 2006;355:1210–21. [8] Vulliet PR, Greeley M, Halloran SM, MacDonald KA, Kittleson MD. Intra-coronary arterial injection of mesenchymal stromal cells and microinfarction in dogs. Lancet 2004;363:783–4. [9] Chazaud B, Hittinger L, Sonnet C, Champagne S, Le Corvoisier P, Benhaiem-Sigaux N, et al. Endoventricular porcine autologous myoblast transplantation can be successfully achieved with minor mechanical cell damage. Cardiovasc Res 2003;58:444–50. [10] Forrester JS, Price MJ, Makkar RR. Stem cell repair of infarcted myocardium: an overview for clinicians. Circulation 2003;108:1139–45. [11] Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410: 701–5. [12] Perin EC, Dohmann HF, Borojevic R, Silva SA, Sousa AL, Silva GV, et al. Improved exercise capacity and ischemia 6 and 12 months after transendocardial injection of autologous bone marrow mononuclear cells for ischemic cardiomyopathy. Circulation 2004;110:II213–8. [13] Kornowski R, Fuchs S. Catheter-based transendocardial gene delivery for therapeutic myocardial angiogenesis. Int J Cardiovasc Intervent 2000;3: 67–70. [14] Perin EC, Silva GV, Sarmento-Leite R. Assessing myocardial viability and infarct transmurality with left ventricular electromechanical mapping in patients with stable coronary artery disease: validation by delayedenhancement magnetic resonance imaging. Circulation 2002;106:957–61. [15] Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–7. [16] Vale PR, Losordo DW, Milliken CE, Maysky M, Esakof DD, Symes JF, et al. Left ventricular electromechanical mapping to assess efficacy of phVEGF(165) gene transfer for therapeutic angiogenesis in chronic myocardial ischemia. Circulation 2000;102:965–74. [17] Kawamoto A, Tkebuchava T, Yamaguchi J, Nishimura H, Yoon YS, Milliken C, et al. Intramyocardial transplantation of autologous endothelial progenitor cells for therapeutic neovascularization of myocardial ischemia. Circulation 2003;107:461–8. [18] Morillo CA, Klein GJ, Jones DL, Guiraudon CM. Chronic rapid atrial pacing. Structural, functional, and electrophysiological characteristics of a new model of sustained atrial fibrillation. Circulation 1995;91:1588–95. [19] Sahn DJ, DeMaria A, Kisslo J, Weyman A. Recommendations regarding quantitation in M-mode echocardiography: results of a survey of echocardiographic measurements. Circulation 1978;58:1072–83.
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[20] Bush LR, Buja LM, Samowitz W, Rude RE, Wathen M, Tilton GD, et al. Recovery of left ventricular segmental function after long-term reperfusion following temporary coronary occlusion in conscious dogs. Comparison of 2- and 4-hour occlusions. Circ Res 1983;53:248–63. [21] Shake JG, Gruber PJ, Baumgartner WA, Senechal G, Meyers J, Redmond JM, et al. Mesenchymal stem cell implantation in a swine myocardial infarct model: engraftment and functional effects. Ann Thorac Surg 2002;73: 1919–25 [discussion 1926]. [22] Walter DH, Haendeler J, Reinhold J, Rochwalsky U, Seeger F, Honold J, et al. Impaired CXCR4 signaling contributes to the reduced neovascularization capacity of endothelial progenitor cells from patients with coronary artery disease. Circ Res 2005;97:1142–51. [23] Pittenger MF, Martin BJ. Mesenchymal stem cells and their potential as cardiac therapeutics. Circ Res 2004;95:9–20. [24] Dai W, Hale SL, Martin BJ, Kuang JQ, Dow JS, Wold LE, et al. Allogeneic mesenchymal stem cell transplantation in postinfarcted rat myocardium: short- and long-term effects. Circulation 2005;112:214–23. [25] Müller-Ehmsen J, Krausgrill B, Burst V, Schenk K, Neisen UC, Fries JW, et al. Effective engraftment but poor mid-term persistence of mononuclear and mesenchymal bone marrow cells in acute and chronic rat myocardial infarction. J Mol Cell Cardiol 2006;41:876–84. [26] Hu X, Wang J, Chen J, Luo R, He A, Xie X, et al. Optimal temporal delivery of bone marrow mesenchymal stem cells in rats with myocardial infarction. Eur J Cardiothorac Surg 2007;31:438–43. [27] Amado LC, Saliaris AP, Schuleri KH, St John M, Xie JS, Cattaneo S, et al. Cardiac repair with intramyocardial injection of allogeneic mesenchymal stem cells after myocardial infarction. Proc Natl Acad Sci U S A 2005;102: 11474–9. [28] Hou D, Youssef EA, Brinton TJ, Zhang P, Rogers P, Price ET, et al. Radiolabeled cell distribution after intramyocardial, intracoronary, and interstitial retrograde coronary venous delivery: implications for current clinical trials. Circulation 2005;112:I150–6. [29] Freyman T, Polin G, Osman H, Crary J, Lu M, Cheng L, et al. A quantitative, randomized study evaluating three methods of mesenchymal stem cell delivery following myocardial infarction. Eur Heart J 2006;27: 1114–22. [30] Agbulut O, Vandervelde S, Al Attar N, Larghero J, Ghostine S, Leobon B, et al. Comparison of human skeletal myoblasts and bone marrow-derived CD133+ progenitors for the repair of infarcted myocardium. J Am Coll Cardiol 2004;44:458–63. [31] Kinnaird T, Stabile E, Burnett MS, Shou M, Lee CW, Barr S, et al. Local delivery of marrow-derived stromal cells augments collateral perfusion through paracrine mechanisms. Circulation 2004;109:1543–9. [32] Kelly RV, Cohen MG, Runge MS, Stouffer GA. The no-reflow phenomenon in coronary arteries. J Thromb Haemost 2004;2:1903–7. [33] Willerson JT, Watson JT, Hutton I, Templeton GH, Fixler DE. Reduced myocardial reflow and increased coronary vascular resistance following prolonged myocardial ischemia in the dog. Circ Res 1975;36:771–81. [34] Yoon YS, Park JS, Tkebuchava T, Luedeman C, Losordo DW. Unexpected severe calcification after transplantation of bone marrow cells in acute myocardial infarction. Circulation 2004;109:3154–7.
Journal of Molecular and Cellular Cardiology 44 (2008) 486 – 495 www.elsevier.com/locate/yjmcc
Original article
Comparison of intracoronary and transendocardial delivery of allogeneic mesenchymal cells in a canine model of acute myocardial infarction Emerson C. Perin a,⁎, Guilherme V. Silva a , Joao A.R. Assad a , Deborah Vela a,b , L. Maximilian Buja a,b , Andre L.S. Sousa a , Silvio Litovsky a , Jing Lin a , William K. Vaughn a , Stephanie Coulter a , Marlos R. Fernandes a,b , James T. Willerson a,b a b
The Texas Heart Institute at St. Luke's Episcopal Hospital, Houston, TX 77030, USA The University of Texas Health Science Center at Houston, Houston, TX 77030, USA
Received 2 July 2007; received in revised form 30 August 2007; accepted 27 September 2007 Available online 4 October 2007
Abstract This study assessed safety of transendocardial (TE) electromechanical-guided delivery of bone marrow mesenchymal stem cells (MSCs) after acute myocardial infarction (AMI) and compared intracoronary (IC) delivery with TE delivery. In a canine acute myocardial ischemia model, 100 × 106 MSCs were delivered 7 days after AMI via IC and TE routes. Functional assessment was performed by 2D echocardiogram, and detailed histopathologic analyses were performed to assess the impact of cell therapy in vascular density and fibrosis. Patterns of cell distribution in both delivery methods were also compared. There was a statistically significant reduction in the amount of myocardial ischemia in the TE group (P = 0.007). Left ventricular ejection fraction (LVEF) increased 13% (mean) in the TE group (21-day follow-up) and was significantly better than that of the controls (P = 0.01), but did not improve in the IC-delivery group. Dissimilar patterns of cell distribution were noted between the IC and TE groups. This study suggests that MSC treatment is probably safe and effective after AMI. In the comparison of TE and IC delivery, the TE group showed higher cell retention (clusters even in the injury center of the infarct) with an increased vascularity and greater functional improvement than did the IC group (no clusters; cells at the border of the infarct). The higher local cell density in the TE group may be important for therapeutic effectiveness. © 2008 Elsevier Inc. All rights reserved. Keywords: Acute myocardial infarction; Stem cell therapy; Transendocardial delivery; Intracoronary delivery; Mesenchymal stem cell; Stem cell injection
1. Introduction Previous human studies have suggested that bone marrowderived mononuclear stem cells (BMNCs) can positively affect postinfarction remodeling processes [1–6]. The safety and therapeutic benefits of intracoronary (IC) infusion of BMNCs through the stop-flow technique have been recently confirmed with the publication of the REPAIR-MI trial [7]. Within the bone marrow, the mesenchymal stem cell (MSC) subfraction exhibits a high degree of plasticity and the potential for differentiating into both mesenchymal and non-mesenchymal lineages; therefore, MSCs have been proposed for use in
⁎ Corresponding author. Tel.: +1 713 791 9400; fax: +1 713 795 5651. E-mail address: [email protected] (E.C. Perin). 0022-2828/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yjmcc.2007.09.012
cardiac repair after myocardial infarction (MI). MSCs, in contrast to BMNCs, are very large cells, and an isolated preclinical study has questioned the safety of delivering MSCs via IC infusion [8]. For the treatment of chronic myocardial ischemia, both experimental and human stem cell studies have been performed either with direct (surgical) delivery or with transendocardial (TE) delivery guided by electromechanical mapping (EMM) [9–12]. The TE delivery route has been shown to be safe in patients with end-stage ischemic heart failure [12], and numerous preclinical studies support its safety [9,13]. TE delivery also adds the potential advantage of targeting viable ischemic myocardium [14] (the border zone of an infarct), which has been postulated as an optimal milieu for bone marrow stem cell engraftment and effect. The role of TE injection after acute myocardial infarction (AMI) in clinical trials, however, has not been established because of potential safety concerns.
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Therefore, we designed a canine study to assess the safety and feasibility of electromechanical-guided TE delivery of MSCs in the setting of AMI. In addition, we compared electromechanicalguided TE delivery of MSCs at the border zone of acute infarction with that of IC delivery of MSCs to the infarct-related artery. 2. Materials and methods This study complies with the Declaration of Helsinki and was reviewed and approved by The University of Texas Health Science Center at Houston's Animal Welfare Committee. The study was conducted at The University's Center for Laboratory Animal Medicine and Care, located at The University of Texas M. D. Anderson Cancer Center's Department of Veterinary Medicine and Surgery (Houston, TX). 2.1. MSC isolation, culture, and labeling Allogeneic canine MSC isolation was performed at Osiris Therapeutics, Inc. (Baltimore, MD), as previously described [15]. Briefly, a purified bone marrow MSC population was expanded in culture. Cells were then harvested, labeled with the cross-linkable membrane dye CM-DiI (Molecular Probes, Inc, Eugene, OR) and the nuclear stain DAPI, and frozen in cryocyte bags. Frozen cells were stored in the vapor phase of liquid nitrogen until the time of implantation. Before injection, the cells were thoroughly washed and resuspended in a 5 ml volume of saline (20 × 106 MSC/ml). At the time of delivery, the viability of the cell suspension was confirmed to be greater than 90%. 2.2. Pilot safety study of IC delivery of MSCs To assess safety of IC delivery, 100 million MSCs were infused in 9 dogs with normal coronary arteries at 3 different velocities: 1 × 106 cells/min (n = 5); 1.5 × 106 cells/min (n = 2), and 3 × 106 cells/min (n = 2).
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2.4. Canine AMI model In brief, a left thoracotomy was performed with the aid of anesthesia induced with Pentothal (17 mg/kg, IV) and maintained with isoflurane (1.5–2.0%). Acute myocardial ischemia was produced by transient occlusion (total of 3 h) of the proximal LAD coronary artery and followed by reperfusion. In addition, ligation of the diagonal branch was performed to decrease collateral flow to the infarct area. 2.5. Coronary angiography The right femoral artery was surgically exposed, and after insertion of a 6F sheath, the animals were systemically anticoagulated with unfractionated heparin. A 5F Amplatz left 1 diagnostic catheter was introduced, and selective coronary angiograms from the right coronary artery and left coronary system were obtained in multiple projections. 2.6. Electromechanical mapping (EMM) acquisition and functional assessment EMM was performed as previously described [16]. After point acquisition was completed, post-processing analysis was performed with a series of filters in the moderate setting to eliminate inner points, points that did not fit the standard stability criteria (location stability b4 mm, loop stability b6 mm, and cycle length variation b10%), points acquired during STsegment elevation, and points not related to the left ventricle (e.g., those on the atrium). Ischemic myocardium was defined as a zone with unipolar voltage greater than 6.9 mV and linear local shortening of less than 6% on the linear local shortening map. EMM was used to quantify the ischemic area before injections and immediately before the animals were humanely killed, as previously described [17]. 2.7. IC and TE delivery of MSCs
2.3. Study design After the pilot safety study (IC delivery of MSCs) was completed, the IC vs. TE delivery study was performed. Twentyone dogs were used for the study. Day 0: All 21 dogs were scheduled to undergo two-dimensional (2D) echocardiography followed by left anterior descending (LAD) occlusion/reperfusion procedures, as described below (canine AMI model). Two dogs died during surgery at the time of occlusion due to ventricular fibrillation and infarction and were not randomized. Therefore, 19 dogs were randomized. Day 7: All 19 dogs underwent coronary angiography, EMM, and 2D echocardiography. The animals were randomly assigned to 3 groups: (1) TE delivery (n = 6), (2) IC delivery (n = 7), and (3) control group (n = 6). The injection procedures are described below. Day 21: Immediately before the animals were humanely killed, repeat coronary angiography, EMM, and 2D echocardiography were performed.
In the IC delivery group (n = 7), after coronary angiography was performed to confirm LAD patency, a 5F Amplatz 1 diagnostic catheter was positioned under fluoroscopic guidance subselectively into the LAD. After the catheter was positioned, a total of 100 × 10 6 allogeneic MSCs were infused at 1 × 106 cells/min. MSCs were infused during 5-min periods (total of 20) with 1-min intervals between infusions to allow for normal coronary flow. Our technique differed from the stopflow technique utilized in the major clinical trials in that it did not involve proximal occlusion of the vessel with a coronary angioplasty balloon. In the TE delivery group (n = 6), allogeneic MSCs (100 × 106 diluted in 5 ml phosphate buffered saline [PBS]) were delivered via 25 EMM-guided TE injections. Each injection was composed of 0.2 cm3 of a solution of 2 million cells per 0.1 cm3. The injection catheter (Myostar™ injection catheter, BiosenseWebster, Diamond Bar, CA) is composed of a 27-gauge needle housed in an 8F catheter that is able to assess endocardial
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voltage and catheter endocardial contact, allowing for targeting of viable ischemic myocardium at the border zone of the anterior infarction. None of the animals received immunosuppressive therapy. 2.8. Control group The animals in the control group (n = 6) underwent the occlusion/reperfusion, coronary angiography, EMM, and 2Dechocardiography in a similar fashion to that of the IC and TE groups. The control group did not receive IC placebo infusion or TE placebo injection. 2.9. Functional assessment by echocardiography Transthoracic rest echocardiography was performed at baseline (before infarction), immediately before cell therapy (7 days after infarction), and 14 days after cell treatment (21 days after infarction). Echocardiography was performed with a commercially available echocardiographic system (Sonos1000, Hewlett Packard Co, Palo Alto, CA) equipped with a 10 MHz linear-array transducer, as described elsewhere [18]. The left ventricular ejection fraction (LVEF), end-diastolic dimension (EDD), and end-systolic dimension (ESD) at rest were obtained from at least 3 consecutive cardiac cycles, according to the American Society of Echocardiography leading-edge method [19]. A blinded, experienced observer performed all measurements (SC). 2.10. Tissue preparation and infarct sizing Dogs were humanely killed 21 days after occlusion/reperfusion (14 days after MSC therapy). Their hearts were exposed by median sternotomy and quickly removed. The heart weight was recorded. The hearts were sliced in a bread-loaf manner into 4 transverse sections from apex to base. Each section was separated into anterior, anterolateral, lateral, posterolateral, and posterior left ventricular free wall; anterior, mid, and posterior interventricular septum; and right ventricular free wall. The sizes of myocardial infarcts and bed at risk (B-A-R) were measured by the tetrazolium method, as previously described [20]. The heart was the only organ examined in this study. 2.11. Immunofluorescence labeling procedure All frozen specimens were cut on positively charged slides at 4 μm and allowed to air dry for 2 h at room temperature. Sections were then fixed in 4 °C acetone for 2 min, allowed to air dry at room temperature for 15 min, and then placed in PBS (pH 7.5) for 5 min. The following antibodies and dilutions were used for antigen detection: anti-α-sarcomeric actinin, 1:20,000 (Sigma, Cat #A7811); troponin I, 1:200 (Chemicon International, Cat #MAB3438); smooth muscle actin, 1:40,000 (Sigma, Cat #A2547); and von Willebrand factor VIII, 1:8000 (Dako, Cat #A0082). All antibodies were diluted with 0.02% bovine serum albumin (BSA)/PBS. Tissue sections were removed from PBS and placed in 1.5% normal horse serum (Vector, Cat #S-2000) for 10 min. The horse
serum was gently tapped off, and the sections were incubated with the primary antibodies for 1 h at room temperature. The sections were then rinsed with PBS for 5 min and placed in fluorescein solution for 10 min in a dark chamber at room temperature. For polyclonal antibody factor VIII, goat anti-rabbit fluorescein (Vector, Cat #Fl-1000) was used. For monoclonal antibodies, smooth muscle actin, anti-α-sarcomeric actinin, troponin I, and horse anti-mouse fluorescein (Vector, Cat #Fl-2000) were used. The sections were thoroughly rinsed in PBS and aqueous mounted with Aquaperm (Thermo-Shandon, Cat #484985). 2.12. Fibrosis quantification Trichrome staining was used to evaluate collagen deposition. Six anterolateral sections from each heart were evaluated in their entirety and quantified. The results were expressed as a percentage of each ventricular section. 2.13. Vascular density assessment The effect of stem cell transplantation on angiogenesis was evaluated by quantitative morphometry, performed with Olympus MicroSuite software on an Olympus BX61 microscope (Olympus America, Inc., Melville, NY). Seven to eight paraffinembedded sections per heart, from mid-level left ventricular anterior and anterolateral walls to apex, were immunostained for the endothelial cell marker, factor VIII (Dako, Carpinteria, CA), and α-smooth muscle actin (Sigma). The number of vessels was counted under a light microscope in 5 random fields (each field measuring 0.58 mm2). Capillary density was expressed as the area of factor VIII positive blood vessels in μm2 per mm2 of each ventricular section. Arteriolar density was obtained by counting all microvessels (arterioles fully coated with a muscular layer positive for α-smooth muscle actin) with a minor axis b 15 μm, and was expressed as arterioles/mm2. 2.14. Mesenchymal cell retention and distribution Histopathologic quantitative analysis (fluorescence microscopy) for the presence of DAPI and/or DiI-positive MSCs was performed in an average of 12 segments, from mid-level to the apex for each heart. Frozen sections were raster-scanned at high-power magnification (× 400) on an Olympus BX61 microscope (Olympus America, Inc., Center Valley, PA) and were then quantified with Olympus MicroSuite Five software. 2.15. Statistical analyses The primary end point was assessment of LVEF by echo at the last follow-up. The study was powered and designed to answer the question of whether cell treatment would lead to a higher LVEF in the treatment groups than in the control group. Importantly, the study was specifically designed to compare the IC and TE delivery routes with the controls as the primary end point. All other analyses were designed to be secondary end points. All values are expressed as mean ± SD. All analyses were performed with appropriate software (Statview; SAS Institute,
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Inc., Cary, NC). A sample size of 6 animals per group (total of 18 dogs) provided a power of 0.8, considering an alpha equal to 0.05 to detect an absolute difference of 10% of LVEF 21 days after MI, considering means of 34 ± 5% and 45 ± 7% for the control group and each treatment arm, respectively. Comparisons of vascular density, LVEF, left ventricular diameter, and the amount of fibrosis were performed utilizing analysis of variance (ANOVA) and Kruskal–Wallis tests for non-normally distributed variables. The Bonferroni test (parametric data) or Kruskal–Wallis Z test (non-parametric data) was used for comparison between pairs (IC vs. TE, IC vs. control, and TE vs. control). Comparisons of MSC engraftment between the IC and TE groups were performed by using an unpaired two-tailed Student's t-test. A value of P b 0.05 was considered statistically significant. 3. Results 3.1. Pilot safety study of IC delivery of MSCs In the study of the safety of IC delivery of 100 million MSCs at 3 different velocities (1 × 106 cells/min [n = 5]; 1.5 × 106 cells/ min [n = 2]; and 3 × 106 cells/min [n = 2]), post-procedural transient TIMI-2 flow was seen in all dogs at the 2 higher velocities. In dogs that received the cell infusion at 1 × 106 cells/min, there was no significant elevation in CK-MB (1.16 ± 1.15 ng/ml at baseline; 1.78 ± 1.67 ng/ml at 24 h [P = 0.51]) or troponin I (0.03 ± 0.01 ng/ml at baseline; 0.45 ± 0.17 ng/ml at 24 h after cell infusion [P = 0.85]). Peak troponin I values were 2 times the normal level in dogs that received 1.5 × 106 cells/min and 7 times the normal level in dogs that received 3 × 106 cells/min. Histopathologic study did not show capillary plugging in animals in the pilot study. 3.2. IC vs. TE delivery study Of the 19 dogs that were randomized, 2 dogs died after randomization and IC infusion of MSCs and did not complete the evaluation at day 21 (described below).
Fig. 1. H & E stain showing microvascular plugging that occurred in the dog that died during intracoronary cell injection.
3.4. Coronary angiography Two animals in the IC group did not complete follow-up angiography (see above). All other animals in the IC group (n = 5) had TIMI-3 flow in the LAD territory at 7- and 21-day follow-up. All animals in the TE and control groups had TIMI-3 flow in the LAD territory at 7- and 21-day follow-up. Coronary collaterals did not develop in any animal, based on visual assessment. 3.5. Functional assessment by electromechanical mapping (EMM) Fig. 2A shows the net change in ischemic area in the 3 groups over time. Overall, there was a statistically significant reduction in the amount of myocardial ischemia in the TE group vs. the IC and control groups (P = 0.007). All other comparisons between groups are shown in Fig. 2A and stated in the legend.
3.3. Procedural safety 3.6. Functional assessment by echocardiography One dog in the IC group had no-reflow of the LAD coronary artery during the injection procedure. ST-elevation and ventricular fibrillation developed, and the dog died despite resuscitative efforts. Histopathologic analysis revealed extensive microvascular “plugging” associated with the presence of MSCs (Fig. 1). Another dog in the IC group died 2 days after cell injection from intestinal ischemia/infarct, which was confirmed at postmortem study. All animals underwent TE injection of MSCs without complication 7 days postinfarction. There were no sustained arrhythmias associated with either the IC or TE injection procedure. In the TE group, no pericardial effusion or cardiac tamponade was observed on immediate post-procedural surveillance or on the 2D echocardiograms performed before the animals were humanely killed.
All groups had similar LVEFs before ischemia/reperfusion (Fig. 2B). Treated and control dogs had similar resting LVEFs immediately before stem cell implantation (Fig. 2B). At 21-day follow-up, the TE-delivery group had a significant improvement in LVEF and ventricular dimensions as compared to the control group (13%, P = 0.01). This improvement was not seen in the IC delivery group (Fig. 2B–D). 3.7. Histopathologic analyses Organs other than the heart were not examined in this protocol. The injected hearts showed no evidence of either gross or microscopic calcification, tumor formation, or unwanted cell differentiation.
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3.7.1. Infarct size and bed at risk There were no significant differences among the 3 groups (Fig. 3A). 3.7.2. Fibrosis quantification There were no significant differences among the 3 groups in terms of total LAD territory and the amount of fibrosis (data not shown).
3.7.3. Enhancement of neovascularization by transplanted MSCs Capillary density was increased in the MSC-TE treated dogs as compared to controls (Fig. 3B, TE vs. control, P = 0.01). Arteriolar density is shown in Fig. 3C (TE vs. IC and control, P = 0.03). 3.7.4. Stem cell retention and distribution patterns Persistence of grafted cells in the myocardium was confirmed in approximately one third of the sections examined (for both TE and IC groups) by identifying MSCs showing DAPI and/or DiI positivity. The TE delivery route achieved a higher MSC concentration per square micron than the IC route in the myocardium (P b 0.01). The results for local cell density can be seen in Fig. 3D. The distribution of DAPI-labeled MSCs within positive regions in dogs in the main study group was studied in a detailed analysis. This analysis showed that certain distribution patterns appear to be predominant and specific for one group or the other (Fig. 4A–D): IC group: IC delivery appears to result in a distribution of vast areas of relatively constant concentration at the positive sites, which are homogeneous in pattern (Fig. 4A). DAPI-labeled cells were observed in both the injured and normal myocardium, although the highest cell density for this group was at the injury border site (Fig. 4B). There was a tendency for cells to be absent from the injury center. The cells seen in the normal myocardium in proximity to the injury diminished in number until they were absent at sites very distant from the healing infarct. TE group: DAPI positive cells were found in approximately one third of the examined segments in each heart. Like the cells in the previous group, these cells were found in both injured and normal myocardium, with a higher concentration at the injury border (as seen in IC dog in Fig. 4B). However, a finding particular only to this group is that areas coinciding with an injection site tended to retain relatively high cell
Fig. 2. (A) Mean change in resting ischemic area within the groups by electromechanical mapping. *P = 0.03 for TE vs. control; P = 0.08 for intracoronary vs. control; P = 0.18 for transendocardial vs. intracoronary. (B) Left ventricular ejection fraction at rest. Assessments were made at baseline, before occlusion/reperfusion (left), 7 days later at the time of cell injection (middle), and 21 days after occlusion/reperfusion (right). *P = 0.01 for TE vs. control after cell delivery; P = 0.83 for intracoronary vs. control after cell delivery; P = 0.63 for TE vs. intracoronary after cell delivery. (C) Left ventricular end diastolic dimension at rest. Assessments were made at baseline, before occlusion/reperfusion (left), 7 days later at the time of cell injection (middle), and 21 days after occlusion/reperfusion (right). *P = 0.04 for TE vs. control after cell delivery; P = 0.87 for intracoronary vs. control after cell delivery; P = 0.36 for TE vs. intracoronary after cell delivery. (D) Left ventricular end systolic dimension at rest. Assessments were made at baseline, before occlusion/reperfusion (left), 7 days later at the time of cell injection (middle), and 21 days after occlusion/reperfusion (right). *P = 0.02, for TE vs. control after cell delivery; P = 0.51, for intracoronary vs. control after cell delivery; and P = 0.16 for TE vs intracoronary after cell delivery.
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Fig. 3. (A) Infarct size and bed at risk (B-A-R). (B) Vascular density was statistically greater in the anterolateral walls of animals that received transendocardial (TE) injections of mesenchymal stem cells as compared to controls; * P = 0.01, vs. control. (C) Arteriolar count (diameter b 15 µm) at the infarct border; * P b 0.05, vs. control. (D) Absolute cell density within normal and injured myocardial areas, at positive sites, in the TE and IC groups. * P b 0.01 for TE-cluster vs. IC.
concentrations. This higher density of DAPI positive cells was observed even in a few rare instances where the injection site was found up to 500–600 μm from the infarct border, toward the direction of the center of the injury. The high-density cell clustering at these latter sites was identical to the clustering pattern of the cells at the injection sites in proximity to the infarct, suggesting that these clusters might not have been a product of cell migration. This pattern contrasts to that of the IC group where the cells tended to remain at the border, and their density gradually decreased or was absent toward the injury center. In the TE group, very high cell concentrations were seen in the normal tissue as well, with true needle tracts and cluster patterns representing the injection sites (Fig. 4C and D). 3.7.5. Stem cell co-localization in ischemic myocardium The survival of engrafted cells was identified by the presence of DiI- and/or DAPI-positive cells in frozen sections as performed with Olympus MicroSuite Five software on an Olympus BX61 microscope. In many instances, DAPI positive nuclei co-localized with mature phenotypical markers such as α–smooth-muscle actin, factor VIII, or α-sarcomeric actinin (Fig. 5A–F).
4. Discussion This study demonstrates the safety and feasibility of TE delivery of MSCs in the post-AMI setting. In this canine model of acute myocardial infarction, significant functional improvement was demonstrated after EMMguided TE delivery of bone marrow-derived MSCs. In addition, TE delivery of MSCs resulted in higher vascular density and a higher density of MSCs at the peri-infarct region. 4.1. Allogeneic MSCs Previous work has demonstrated the functional benefits of bone marrow MSC surgical injection after MI [21]. Within the bone marrow mononuclear cells, the MSC subfraction is capable of secreting numerous cytokines and has a high degree of plasticity, which has led to increasing interest in this cell subtype for possible use in cardiac regeneration. Because MSCs are rare in the bone marrow, ex-vivo expansion (for at least 10 days after harvest) is mandatory for clinical utilization. Bone marrow mononuclear cells have been shown to be functionally impaired with aging and with disease states such as coronary artery disease [22]. Therefore, we used allogeneic MSCs, given the theoretical advantage of clinical availability
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Fig. 4. Various TE and IC delivery distribution patterns in normal myocardium. (A) Homogenous distribution of DAPI+ cells, as can be found in either IC delivery or in non-clustered areas TE delivery, away from injection sites, magnification x 200; (B) DAPI+ cells at infarct border of an IC delivery dog. Note on the left side (infarct side) the higher density of DAPI+ nuclei (arrows) when compared with the right side showing DAPI+ nuclei amidst normal myocardium, magnification x 200; (C) High concentration of DAPI+ cells corresponding to a needle tract of a TE dog, magnification x100; (D) DAPI+ cell cluster, corresponding to an injection site of a TE dog, magnification x 100. (Scale bar = 200 µm for A, B; 100 µm for C, D).
and optimal functional capacity over autologous, ex-vivoexpanded MSCs. Allogeneic MSCs have been suggested to be non-immunogenic stem cells [23]. Dai et al. [24] have demonstrated that transplanted allogeneic MSCs in rats can survive up to 6 months after transplantation and did not induce immunorejection. MSCs have been shown to inhibit T-cell proliferation and also to prevent T-cell maturation to their antigen [23]. This study has once more demonstrated the non-immunogenic profile of allogeneic MSCs. Importantly, the animals were not given any immunosuppressive drugs, which lends a favorable perspective for the clinical use of this cell type. 4.2. Comparison of IC and TE distribution patterns: correlation with functional and histopathologic findings The issues of timing of cell delivery after infarction and persistence of MSCs post MI have been evaluated in previous studies in small animal models. Cell retention after infarction progressively declines after cell injection as shown by MüllerEhmsen et al. [25]. In their study, cell engraftment was quantified, using quantitative real-time PCR with Y-chromosome specific primers, at 0 h, 48 h, 5 days, 3 weeks, and 6 weeks after cell injection. The percentage of MSCs in the heart decreased rapidly from a maximal percentage of 80% of injected cells at 0 h down to a maximal percentage of approximately 3.5% at 6 weeks, follow-up. At a similar follow-up time as the
present study, a maximal percentage of 4.7% of injected MSCs was detected in the heart. Furthermore, the functional benefit of MSC injections post MI seems to depend on the timing of cell delivery. In a preliminary study, Hu et al. [26] observed that the ideal timing of cell delivery is 1 week after infarction (as done in the present study) to achieve optimal functional benefit. In large animal models, previous studies have confirmed the safety and feasibility of TE and surgical intramyocardial delivery of MSCs after AMI [21,27–29]. Amado et al. [27] have focused on the functional aspect of TE-MSC injection after MI. They have shown that when allogeneic MSCs are injected 3 days after MI, cardiac regeneration is stimulated, infarct size is decreased, and cardiac function is near normalized. Other studies have focused on quantifying MSC engraftment after MI. Hou et al. [28] have found higher engraftment rates of stem cells delivered via surgical intramyocardial injection (11.3%) when compared to engraftment rates of cells delivered via the IC route (2.6%). Freyman et al. [29] have compared postMI MSC injection by TE delivery to IV and IC delivery. At 14 days, cell engraftment was higher in the IC group vs. the 2 other groups. TE delivery had higher engraftment rates than did IV infusion. Our study compared IC and TE delivery methods taking both combined functional and engraftment aspects into account. Our study confirms that the functional benefit of MSC
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Fig. 5. (A) Normal myocardium of an IC dog immunostained for α-sarcomeric actinin shows numerous DAPI+ nuclei, some of which appear to colocalize with the myocytes (arrows) while others appear at the cell periphery or in interstitium (arrowheads). (B) Normal myocardium of a control animal immunostained for αsarcomeric actinin, DAPI-negative. (C–E) Healing infarct of a TE dog immunostained for α-smooth-muscle actin (C, green), showing abundant DAPI+ cells (D, blue), many of which appear to contribute to the neovascularization (E, merged imaged of C–D). (F) Healing infarct of a DAPI-negative control dog immunostined for αsmooth-muscle actin. Magnification x 400 A, B; x4 00 C–F. Scale bar = 50 µm A, B; 100 µm C–F.
treatment is preserved when TE injections are extended to 7 days after MI. However, a dramatic reduction in infarct size was not observed. This finding might be explained by the shorter follow-up time after cell injection in our study; previous studies in small [26] and large animals [25] have shown a reduction in infarct size at 4 and 8 weeks post MSC injections, respectively. Our study results conflict with that of Freyman et al. [29] because we had higher engraftment rates with EMM-guided TE than with IC infusion. One possible explanation is the precise targeting offered by EMM-guided TE. Both TE delivery studies cited above differ from the current study in that they utilized fluoroscopy-only guided TE injections. The electromechanicalguided TE delivery unites real-time 3D navigation capabilities with real-time viability assessment. Thus, therapy may be accu-
rately placed at the border zone of an infarct, avoiding myocardial scar [14]. Agbulut et al. [30] have shown that when bone marrow stem cells are injected into scar tissue, engraftment decreases (as does efficacy). Alternatively, the lower engraftment with the IC infusion might have resulted from the technique utilized. There was no proximal occlusion of the vessel before injection and, therefore, it is possible that a significant number of cells were injected retrograde to the proximal part of the coronary artery and to the ascending aorta. In addition, the slow infusion rate of cells might not have been concentrated enough to create a significant gradient to enable more cells to enter the myocardium. Kinnaird et al. [31] have reported that MSCs produce a wide array of arteriogenic cytokines and improve perfusion and remodeling in a mouse model of hind-limb ischemia, possibly
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through neoangiogenesis. Thus, in the present study, higher engraftment rates of MSCs might have contributed to the significantly higher capillary density in the anterolateral wall of TE stem cell–treated animals. The higher capillary density in the TE-delivery group accompanied a substantial improvement in LVEF as compared to the control group. These findings were not present in the animals who received cells via IC delivery. In addition to different engraftment rates, TE and IC delivery modes also differ in cell engraftment patterns. While the MSCs seem to be distributed in a more widespread fashion by IC delivery, cluster areas of MSCs were present in the TE-injected animals, which could indicate that a considerable portion of cells delivered by the TE route will stay at or very near the injection site without migrating. Moreover, TE animals had MSCs distributed not only in the peri-injury area but also inside the injured area. The finding of dissimilar patterns of cell distribution between IC and TE delivery modes might also result from the different targeting capabilities of each delivery method. The importance of cell distribution patterns for therapeutic effectiveness remains to be determined. 4.3. Safety of cell delivery In Freyman's study [29], half of the animals had impaired coronary flow distal to the infusion site after IC infusion. In our study, 1 animal had no-reflow and subsequently died immediately after IC-MSC infusion. The no-reflow phenomenon occurs as a consequence of microvascular flow impairment secondary to distal embolization and disruption of the microvasculature [32,33]. Those findings might relate to the results of the study by Vulliet et al. [8], who described elevated cardiac enzymes and micro infarctions in dogs with normal coronary arteries after IC injection of MSCs. Thus, IC delivery of MSCs might cause microvascular plugging and consequent no-reflow phenomena. Interestingly, Chen et al. [3] did not report reduction in blood flow after IC infusion of larger doses of MSCs (total of 8– 10 × 109 cells). Importantly in the present study, histopathologic study showed that the administration of MSCs did not result in gross or microscopic calcification or tumor formation, as has been reported in a previous study [34]. This issue is particularly critical in the use of the TE delivery route in which cells can reach higher density in cluster patterns. 4.4. Study limitations The major limitation of the present study is the small number of animals in each group, which limits conclusions about efficacy. Still, statistically significant differences between the IC, TE, and control groups were shown in regard to 2D-echocardiographic parameters and capillary density. Because organs other than the heart were not examined in this study, the conclusions regarding safety apply only to the heart. In addition, the IC delivery technique used in our study was different from the one used in the clinical trials. Hence, trans-
lation of our preclinical results regarding safety of IC injections into clinical terms is uncertain. Dogs might develop substantial collateral circulation, which could limit interpretation of cardiac function data. It is possible that a significant number of the injected stem cells did not reach the AMI region. However, coronary angiography was performed both before cell therapy and immediately before animals were humanely killed to ensure patency of the LAD/diagonal coronary arteries and to assess for the development of new collateral vessels. No significant differences in major collateral development were seen between treated and control hearts. The fewer cells present in the IC delivery group could be due either to reduced immediate delivery or early retention. Having a group of animals humanely killed early (24 h) would have answered this question to some extent. However, our study provides the cell therapy field with additional evidence that viability-targeted delivery after MI might enhance therapeutic effectiveness. EMM is known to be capable of detecting and delineating infarcted and ischemic territories. However, no true validation has been made in humans regarding EMM quantification of ischemic area vs. quantification by other modalities, such as SPECT. 5. Conclusions In conclusion, our study suggests that MSC treatment in a canine model of acute myocardial infarction increased vascularity and improved cardiac function. Second, TE injection of MSCs into ischemic myocardium after AMI is probably safe, achieving higher retention rates in a more targeted cell distribution pattern (within the injured area) as compared to IC infusion. Finally, TE delivery of MSCs was associated with a greater functional improvement than was IC infusion. Acknowledgments The authors wish to acknowledge Marianne Mallia, ELS, and Chrissie Chambers, ELS, for their editorial assistance in the preparation of this manuscript. References [1] Assmus B, Schachinger V, Teupe C, Britten M, Lehmann R, Dobert N, et al. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI). Circulation 2002;106: 3009–17. [2] Britten MB, Abolmaali ND, Assmus B, Lehmann R, Honold J, Schmitt J, et al. Infarct remodeling after intracoronary progenitor cell treatment in patients with acute myocardial infarction (TOPCARE-AMI): mechanistic insights from serial contrast-enhanced magnetic resonance imaging. Circulation 2003;108:2212–8. [3] Chen SL, Fang WW, Ye F, Liu YH, Qian J, Shan SJ, et al. Effect on left ventricular function of intracoronary transplantation of autologous bone marrow mesenchymal stem cell in patients with acute myocardial infarction. Am J Cardiol 2004;94:92–5. [4] Janssens S, Dubois C, Bogaert J, Theunissen K, Deroose C, Desmet W, et al. Autologous bone marrow-derived stem-cell transfer in patients with ST-segment elevation myocardial infarction: double-blind, randomised controlled trial. Lancet 2006;367:113–21.
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