The Safety of Autologous Intracoronary Stem Cell Injections
in a Porcine Model of Chronic Myocardial Ischemia

Intracoronary mononuclear cell therapy may lead to angiogenesis in chronic myocardial ischemia. A total of four cell therapy trials in patients with myocardial ischemia not amenable to revascularization have been performed.¹ Tse et al. studied transendocardial bone marrow cell transplantation guided by electromechanical mapping in a pilot study of 8 patients with severe ischemic heart disease via a percutaneous approach. All patients had an ejection fraction > 30 percent. Marrow (40 mL) was processed to isolate mononuclear cells prior to injection. At 3-month follow up, the mean number of weekly anginal episodes decreased, as did the number of nitroglycerin tablets consumed. Cardiac magnetic resonance imaging demonstrated significant increases in target wall thickening and motion. Intramyocardial injection was safe, as there were no increases in serologic markers of myocardial necrosis, and ambulatory electrocardiographic monitoring did not demonstrate arrhythmias.² Perin et al. studied transendocardial bone marrow cell transplantation in 21 patients with severe heart failure secondary to ischemic heart disease with an ejection fraction 3 Hamano et al. treated 5 patients during concomitant surgical revascularization with epicardial autologous bone marrow cell transplantation in myocardium for which the native diseased vessel was not amenable to surgical revascularization. They transplanted 5 x 10^7 to 1 x 10^8 cells/0.1 mL injection in injections spaced 1 cm apart. One-month and 1-year follow up demonstrated no adverse events and improvements in coronary perfusion by nuclear scintigraphy.⁴ Fuchs et al. transplanted autologous bone marrow cells in 10 patients with severe angina who were not candidates for revascularization via twelve 0.2 mL injections guided by electromechanical mapping. Cell therapy was safe, as there were no occurrences of arrhythmia, infection, myocarditis or inflammation, or scar expansion. Cell therapy improved angina and decreased ischemic burden on perfusion imaging.⁵ We proposed a novel approach of delivering autologous stem cells via an epicardial coronary artery supplying collateral vessels distal to an occluded coronary artery not amenable to revascularization. No clinical studies to date have studied the safety and efficacy of intracoronary cell injection into an epicardial coronary artery supplying collateral vessels to myocardium distal to a chronically, totally occluded coronary artery. This novel approach may alleviate ischemia via cell-based vasculogenesis in myocardium not amenable to percutaneous or surgical revascularization. The purpose of this study was to evaluate the safety of intracoronary injections of autologous bone marrow mononuclear cells in a porcine chronic myocardial ischemia model. Methods Animal procedures. All animal procedures were approved by the Case Western Reserve University Institutional Animal Care and Use Committee. Animals were housed in the Animal Resource Center Health Science Animal Facility. Three animals were selected to test the safety of intracoronary mononuclear cell injection in patent coronary arteries without hemodynamically significant stenoses. Five animals were then chosen to study this procedure in a chronic myocardial ischemia model, as it was agreed that injecting a cell dose at least 5 times that to be used in clinical studies in at least 5 animals would be adequate for safety studies (V. Pompili, personal communication). Anesthesia was induced with intravenous ketamine (100 mg/kg), acepromazine (10 mg/kg) (both from Phoenix), and 2.5% pentothol (Abbott Laboratories). Anesthesia was maintained with mechanical ventilation (Narkovet VCII Series One) using inhaled isoflurane (1–2%) with 1–2 L/hour of oxygen. All animals received cefazolin 1 g intravenously prior to all procedures. Coronary angiography and contrast ventriculography. The right groin was shaved, then prepped and draped in a sterile fashion. A sagittal incision was made in the right groin for direct exposure of the femoral artery. An 8 Fr sheath was introduced into the artery, aspirated and flushed with sterile saline. All animals received intravenous unfractionated heparin (50 units/kg, Baxter, Deerfield, Illinois), magnesium sulfate (1–2 g, American Reagent, Mirus Bio Corp. Madison, Wisconsin), and lidocaine (50–100 mg 2% solution, Abbott Laboratories, Abbott Park, Illinois) at the start of the procedure. All cardiac catheterization procedures were performed using a Philips 6028 Cath Lab Fluoroscopy Unit (Philips, Andover, Massachusetts). All animals underwent quantitative coronary angiography [Camtronics Vericis Cardiac Workstation, Rev. 2.2.1, (Camtronics, Hartland, Wisconsin)], and contrast ventriculography both at baseline and following surgical placement of ameroid constrictor on the left circumflex artery. A 7 Fr AL2 catheter (Boston Scientific, Natick, Massachusetts) was advanced over a 0.035 inch guidewire to selectively engage the left and right coronary arteries. Digital cineangiograms were taken in standard right anterior oblique (RAO) and left anterior oblique (LAO) views. A 7 Fr pigtail catheter (SciMed, Maple Grove, Minnesota) was used to cross the aortic valve and enter the left ventricle. Contrast ventriculography was performed using a power contrast injector (Medrad Medmark V, 514V, Indianola, Pennsylvania) in standard RAO and LAO views. Following all coronary diagnostic and cell injection procedures, the sheath site was repaired in multiple layers. All animals received 0.5% bupivicaine (Abbott Laboratories) at incisional sites at the conclusion of all procedures. Surgical placement of left circumflex artery ameroid constrictor placement. Following coronary angiography and ventriculography, animals were repositioned, and the chest was prepped and draped in a sterile fashion. A left lateral thoracotomy was performed through the fourth intercostal space. Blunt dissection was used to make a 20 cm parascapular incision in the animal’s side. The ribs were minimally retracted for sufficient visualization of the heart. Pericardial fat was removed by blunt and sharp dissection, and an 8-cm incision was made in the pericardium, which was retracted to create a cradle to expose the heart. The left circumflex artery was isolated and an appropriately-sized ameroid constrictor (1.75–2.5 mm in diameter) was placed, usually proximal to the first obtuse marginal branch, through an 8–10 mm arteriotomy. Silicone elastomer vascular loops were passed inferior to and looped over the left circumflex artery at the proximal and distal ends of the arteriotomy site. Papaverine (30 mg/ml diluted 1:10 in normal saline) was administered topically to treat arterial spasm. Heparin (100 IU/kg intravenously) was given to achieve an activated clotting time of at least 300 seconds before ameroid constrictor placement. The constrictor loops were tightened so that the arterial segment would be pushed through the constrictor slot. The constrictor was rotated so that the slot would face away from the heart surface. Following successful positioning, the constrictor loops were released to allow antegrade flow down the left circumflex artery. Coronary angiography was repeated immediately postoperatively to confirm successful ameroid constrictor placement and decreased coronary blood flow (Figure 1). Pericardium was closed loosely with 3–0 silk suture. A small puncture in the sixth intercostal space distal to the thoracotomy incision was made, and a 21-Fr thoracostomy tube with Heimlich valve was placed prior to reapposition of ribs and soft tissues using polyamide (Nylon) sutures. Unless excessive drainage was present, all chest tubes were removed within 15 minutes postoperatively. All animals received buprenorphine (0.3 mL subcutaneously) 30 minutes prior to closure of the chest cavity and femoral arteriotomy site using 3.0 Vicryl (Ethicon, Somerville, New Jersey) suture. All animals received 5 mL 0.5% bupivicaine (Abbott Labs) at the incisional sites and 75 µg/hour transdermal fentanyl patch (Janssen, Titusville, New Jersey) for analgesia. Repeat coronary angiography and ventriculography 3 weeks later was performed to confirm severe (> 80%) stenosis at the site of ameroid constrictor placement, TIMI grades 0 or 1 (Thrombolysis in Myocardial Infarction) blood flow and collateral vessels to the circumflex distribution distal to the stenosis. Bone marrow aspiration and mononuclear cell isolation. Animals were anesthetized as described above. The ventral pelvic region was shaved, prepped and draped in a sterile fashion. The distal portion of the femur just superior to the knee was located. A 5–10 mm incision in the skin directly over the selected harvest was made. A bone biopsy needle was advanced perpendicularly to the femur. Marrow (40–60 ml) was withdrawn while advancing the biopsy needle. Heparin (400 IU per 20 ml marrow aspirate) was added, after which the biopsy needle was removed. Mononuclear cells were isolated by Ficoll density gradient centrifugation and labeled with CM-dioctadecyl tetramethylindocarbocyanine (CM-DiI) (Molecular Probes™, Invitrogen Detection Technologies, Carlsbad, California), a lipophilic dye known to be nontoxic and which allows for cell tracking in vivo. Intracoronary mononuclear cell injection. An over-the-wire catheter (Maverick 2.5 x 12-mm, REF 20620-1225, Boston Scientific) was selectively engaged into the coronary artery supplying the majority of the collateral vessels to the myocardium distal to the occluded circumflex artery and advanced over a 0.14 inch guidewire (Choice Standard, Scimed). In the animals used in the dose-escalation study, cell injections were performed in the first or second diagonal branch from the left anterior descending artery, depending on vessel size and ease of intubating the vessel with the perfusion balloon catheter. The balloon-tipped catheter was inflated to prevent retrograde flow of delivered cells. Cells (in 3 mL phosphate-buffered saline containing 2% autologous plasma) were injected through the guidewire lumen at a rate of 1 mL/minute, followed by flush with 1 mL 2% autologous plasma in heparinized phosphate-buffered saline. Coronary angiography and ventriculography were repeated immediately following cell delivery to rule out acute occlusion of injected vessels. Nitroglycerin (100 mcg/ml, Abbott Laboratories) was injected intracoronary after follow-up coronary angiography. Animals were monitored for hemodynamic and electrocardiographic changes following cell delivery. Follow-up care and post-mortem protocol. Animals were monitored clinically for 7 days, after which all animals underwent repeat coronary angiography and ventriculography (Figure 2). Animals used in the chronic ischemia model also underwent bone marrow aspiration from the femur opposite to that from which autologous mononuclear cells were harvested. Animals then were euthanized by intravenous injection of 200 mg/kg sodium pentobarbital (Fatal-Plus, Vortech Pharmaceuticals, Dearborn, Michigan). Heart and vital organs (lung, liver, spleen, kidney) were harvested for post-mortem examination and histopathology. Hearts were removed and pressure-perfused with normal saline followed by 10% formalin. Sections of heart and visceral organs were immersed in 10% buffered formalin for 2–3 days, sectioned, placed in cassettes, and fixed for an additional 2–3 days before embedding. Alternate sections were either stained with hematoxylin and eosin (H&E) or left unstained and covered with coverslips for immunofluorescence microscopy. Three of the 5 animals that underwent ameroid constrictor placement were selected randomly and underwent bone marrow aspiration, which was analyzed for the presence of CM-DiI-labeled cells. Peripheral blood was sampled for CK, CK-MB, and troponin I prior to initial surgery as well as prior to, and 1 and 7 days after, cell delivery. Results Intracoronary cell injections in increasing doses in patent coronary arteries did not result in ischemia or infarction. The 3 animals received 1 of 3 increasing doses of cells, ranging from 4.5 x 10^6 to 15.0 x 10^6 cells, similar to the dose range of 0.5 x 10^6 to 5.0 x 10^6 cells to be used in anticipated clinical trials. Vessel intubation using the perfusion balloon catheter was safe and feasible. All 3 animals tolerated cell injection clinically without evidence of periprocedural ischemia or infarction. However, injection of the highest cell dose — 15 x 10^6 cells — resulted in reduced TIMI grade flow that resolved spontaneously after 10 minutes. Gross and histopathologic examination of hearts harvested post-mortem did not show evidence of ischemia or infarction (data not shown). Ameroid constrictor placement resulted in myocardial ischemia but not myocardial infarction. Coronary angiography performed 3 weeks following ameroid constrictor placement and immediately before intracoronary cell delivery demonstrated TIMI grade 0 flow in 3 animals and TIMI grade 1 flow in the other 2, thus simulating a chronically, totally occluded artery, a common etiology of chronic myocardial ischemia. All animals demonstrated collateral vessels from a patent artery supplying myocardium supplied by the target artery distal to the ameroid constrictor. To assess whether surgical induction of myocardial ischemia resulted in myocardial infarction, we measured serum levels of CK-MB and troponin I immediately after ameroid constrictor placement (timepoint -21 d in Table 1), and found no abnormal elevations. To rule out the possibility of subclinical infarction secondary to ameroid constrictor placement, below the detection limits of laboratory assays for biomarkers of myocardial necrosis, we prepared H&E-stained sections of myocardium supplied by the left circumflex artery and distal to ameroid constrictor placement. Representative sections showed evidence of myocardial ischemia (Figure 3), but no evidence of myocardial infarction, as expected with this preclinical model. Intracoronary cell injection into the target epicardial artery resulted in vasospasm that resolved 10 minutes following intracoronary nitroglycerin in all animals. Hemodynamic monitoring revealed no evidence of cardiovascular compromise in any of the animals (data not shown). To evaluate the possibility that intracoronary cell delivery would result in periprocedural infarction, we sampled peripheral blood both immediately before (day 0, baseline), 1 (day 1), and 7 (day 7) days after cell therapy and demonstrated no abnormal elevations in CK-MB or troponin I (Table 1). CM-DiI-labeled mononuclear cells localized in perivascular structures in ischemic myocardium and in spleen. We investigated homing and localization of labeled cells in myocardium seven days following intracoronary delivery. Comparison of consecutive histologic sections either stained with H&E or left unstained and visualized by immunofluorescence microscopy demonstrated cell localization in the perivascular tissues in myocardium supplied by the distal branches of the epicardial artery receiving injected cells (Figure 4). We also investigated extracardiac homing and migration of injected cells. H&E-stained sections of vital organs (lung, liver, spleen, kidney) demonstrated no significant cell localization in any animal, with the exception of the spleen in 1 animal (Figure 5). Immunofluorescence microscopy of aspirated bone marrow detected labeled cells. Given the lack of significant homing and migration of labeled cells to vital organs, we tested the hypothesis that progenitor cells delivered by intracoronary injection during myocardial ischemia migrate to bone marrow. Bone marrow can then serve as a reservoir of progenitor cells that can, subsequently, participate in angiogenesis to alleviate ischemia. Bone marrow from the 3 animals that underwent bone marrow aspiration demonstrated the presence of CM-DiI-labeled cells in all 3 animals (Figure 6). Although the method of bone marrow harvest (aspiration vs. biopsy) precludes quantification of bone marrow engraftment by labeled cells, these results suggest bone marrow as a major destination of labeled progenitor cells following intracoronary delivery. Discussion Ameroid constrictor placement reproducibly resulted in total occlusion, defined as having TIMI grades 0 or 1 flow for at least 2 weeks’ duration,⁶ and present in a significant percentage of patients with chronic stable angina. Our model resulted in chronic myocardial ischemia without infarction, as demonstrated both serologically and histologically. Hence, ameroid constrictor placement around the left circumflex artery in pigs is a valid and reproducible model in which to study cell-based therapies for chronic myocardial ischemia. Results of the dose-escalating experiments in 3 normal animals without surgically-induced myocardial ischemia indicate that cannulation of coronary arteries with a balloon-tipped perfusion catheter is safe and feasible. These data also indicate that intracoronary delivery of mononuclear cells in doses several-fold higher than those likely to be used in future clinical trials is safe and feasible and does not induce periprocedural ischemia or infarction. While the highest cell dose resulted in decreased TIMI grade flow, secondary most likely to microvascular dysfunction distally, this resolved spontaneously and did not result in myocardial necrosis, determined by serum cardiac biomarkers and post-mortem analysis. Based on the results of our report, intracoronary injection of autologous mononuclear cells in chronic myocardial ischemia appears safe. Although intracoronary cell delivery in the epicardial artery supplying collateral vessels resulted in coronary vasospasm, this resolved after 10 minutes following intracoronary nitroglycerin injection and did not result in abnormal elevations of serum biomarkers of myocardial necrosis. The lack of adverse effects secondary to intracoronary cell delivery is consistent with the findings reported in the seven clinical studies of intracoronary autologous cell delivery following myocardial infarction.^7–13 Although two studies used selected cells — one studied mesenchymal stem cells, the other studied hematopoetic stem cells selected for CD133 expression — most studies injected mononuclear cells, analogous to our preclinical study. Bone marrow mononuclear cells are able to circulate freely in peripheral capillaries, so there is less of a concern of vascular obstruction as may exist with other bone marrow stem cells, such as mesenchymal stem cells, which are larger in size, and may occlude the microvasculature.¹⁴ Our study differs from the four clinical studies in chronic myocardial ischemia mentioned earlier^2–5 in that those studies injected cells directly — either percutaneously or surgically — into the myocardium, whereas we delivered cells via intracoronary injection. One potential advantage of direct intramyocardial cell delivery over intracoronary delivery is that it obviates the risks of vasospasm, acute vessel occlusion, and periprocedural infarction that may occur during intracoronary cell delivery as well as other, non-cellular percutaneous coronary interventions. However, catheter-based transendocardial cell therapy is more invasive and carries the small but serious risk of myocardial perforation. Electromechanical mapping used during transendocardial cell delivery is labor-intensive and technically demanding, requiring prolonged procedure times and considerable experience to be used safely and effectively. Although other surgical studies,¹⁵ in addition to the one by Fuchs et al.⁵ demonstrated improvements in ventricular function, dimensions and perfusion defect following intramyocardial cell injection during bypass surgery, such an approach is invasive and not a treatment option in patients who otherwise are not candidates for open-heart surgery. Whereas the four prior studies utilized direct myocardial injection, Erbs et al.¹⁶ studied intracoronary delivery of peripheral blood progenitor cells following successful percutaneous revascularization of a chronically occluded artery. This study randomized 26 patients who underwent successful revascularization of a chronically occluded artery to placebo or cell therapy. Cell therapy resulted in a 43% increase in coronary flow reserve in the target vessel that corresponded to a decreased amount of hibernating myocardium, a 16% relative decrease in infarct size and a 14% relative increase in ejection fraction. Intracoronary cell injection resulted in cell localization in the perivascular areas of myocardium supplied by the injected vessel, and, as stated earlier, did not produce clinical, laboratory, or histologic evidence of periprocedural infarction. Dohmann et al.¹⁷ published a unique follow up to the study by Perin et al.³ in which they performed post-mortem examination on a patient who died from a noncardiovascular cause 11 months after receiving transendocardial cell therapy. Examination of the anterolateral left ventricular wall that received cell injections compared to the infarcted inferoposterior wall that did not demonstrate significantly greater capillary density accompanied by proliferation of pericytes, mural cells, and adventitial fibroblasts. Some injected cells also demonstrated immunohistochemical characteristics of cardiomyocytes, suggesting transdifferentiation. While follow-up coronary angiography after cell therapy did not demonstrate increased collateral vessels, the prior clinical studies suggest that 7 days following cell therapy is an insufficient period of time to expect significant cell-based vasculogenesis. The report by Dohmann et al. observed benefits of cell therapy almost one year after initial cell therapy, suggesting that preclinical models of chronic myocardial ischemia such as ours may require several weeks or months of observation before demonstrating clinical and histologic evidence of vasculogenesis. Cell localization was observed outside the heart only in the spleen of only one animal. This was not unexpected, given the selective homing and migration to ischemic myocardium that intracoronary cell delivery through a balloon-occlusion catheter may yield. However, labeled cells were identified in bone marrow aspirates from all 3 animals that underwent repeat bone marrow aspiration following injection. Although we were unable to quantitate bone marrow engraftment with labeled cells, these findings suggest a role for bone marrow engraftment and repopulation as a possible mechanism for progenitor cell localization in myocardium. We plan to study further the molecular and cellular mechanisms of mononuclear cells in angiogenesis and alleviating ischemia. Broader applications of our model include the study of selected mononuclear cells, such as CD133+ hematopoetic stem cells, in small and large animal models of both acute infarction and chronic myocardial ischemia. Bone marrow CD133+ mononuclear cells are early mononuclear cells with high angiogenic potential.¹⁸ We recently obtained approval from both the U.S. Food and Drug Administration and our health system’s Institutional Review Board to inject autologous bone marrow-derived CD133+ mononuclear cells in a phase I trial in patients analogous to our animal model, i.e., with a chronically occluded artery supplied by collateral vessels from another epicardial artery. The results from these future studies will guide future studies into the mechanisms of cell-based therapy for ischemic heart disease, as well as larger clinical studies of stem cells for therapeutic uses in cardiovascular disease. Acknowledgement. We wish to thank the Animal Resource Center and Health Sciences Animal Facility of Case Western Reserve University.

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