- Email: [email protected]
Genetic studies, cell therapy and angiogenesis II
MONDAY 9/15/03 9:00 –11:00
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TCT-235 Experimental Myometrial Cell-Patch Cardiomyoplasty. S.A. Taheri1, H. Ashraf2, Y.V. Fang1, J. Naughton3, B. Nemes1, A. Orlick1. 1Kaleida Health, Buffalo, New York, USA; 2Buffalo Thoracic Associates, Buffalo, New York, USA; 3State University of New York at Buffalo, Buffalo, New York, USA.
Monday, September 15, 2003 9:00 –11:00 AM 2:00 – 4:00 PM Exhibit Hall B on the Lower Level (Abstract nos. 234 –245)
TCT-234 Allogeneic Myoblasts Rejuvenate Degenerative Hearts: First Human Study. P.K. Law1, G. Fang1, F. Chua1, J. Weinstein1, T. Kakuchaya2, V.S. Repin2, L.A. Bockeria2. 1Cell Transplants International, Memphis, Tennessee, USA; 2Bakoulev Scientific Center for Cardiovascular Surgery, Russian Academy of Medical Science, Moscow, Russia.
A B S T R A C T S
Background: Endomyocardial myoblast injections transferred myoblast nuclei into host cardiomyocytes. Some myoblasts transdifferentiated into cardiomyocytes. Others formed myofibers with regenerative satellite cells. Newly deposited myofilaments augmented contractility. We report here the world’s first myoblast allografts into two myocardial infarct patients. Methods: Quadriceps biopsy (2.18 g) from a 20-year-old, pathogen-free male was cultured, in compliance with cGMP/ISO 9001, to yield 3.64 ⫻ 109 myoblasts that were 98.3% pure (desmin immunopositive), 91.5% viable, potent in myotube formation, Gram-negative, and negative for endotoxin, mycoplasma, and sterility. The patients were male subjects, aged 63 and 49, who had developed atherosclerosis, coronary artery disease, chronic myocardial infarction, stable angina (Canadian Cardiovascular Society functional class IV), and arterial hypertension II (risk 4). Positron emission tomography scanning with 18 FDG revealed scars with hibernated myocardium in the left ventricular (LV) septal, apical, and anterior and posterior wall regions. Echocardiography showed regional akinesis and hypokinesis, with LV ejection fraction (LVEF) measurements of 0.41 and 0.38, respectively. On January 17, 2003, the patients received 18/19 injections and 1.1/1.2 billion myoblasts (108/mL), respectively. The 0.5-mL injections were placed between infarcted and viable myocardium after coronary artery bypass graft on ice-chilled, nonbeating hearts. Subjects took oral cyclosporine (5 to 7 mg/kg body weight per day) for 8 weeks. Doses were adjusted to maintain whole blood trough level at about 250 ng/mL. In addition, 24-hour Holter electrocardiogram was monitored. Results: The subjects recovered with no rash or high fever. Both developed arrhythmia and extrasystoles, which were eliminated with amiodarone. Rejection symptoms were not observed, despite cyclosporine discontinuation. At 3-month follow-up, both were in stable condition with angina at class I-II. Echocardiography showed 14.6% and 10.5%, respectively, increases in LVEF, with no local hypokinesis. Single-photon computed tomography with 30 mCi 99mTc-tetrophosphomin demonstrated positive dynamics, LVEF increases, and reduction in perfusion defect areas during exercise and rest. 18FDG accumulation was equable throughout the LV myocardium; glucose metabolism was sustained.
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Conclusion: The feasibility and preliminary safety and efficacy demonstrated in these myoblast allografts provide a new potential therapy for heart failure and its prevention, with virtually unlimited cell availability and only 2-month immunosuppression.
Genetic Studies, Cell Therapy and Angiogenesis II
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Background: The purpose of this study was to evaluate the viability and function of patches of autologous myometrium to infarcted myocardium of rabbits. Methods: Seven rabbits weighing between 5 and 6 pounds were anesthetized and intubated. The heart was exposed via midline sternotomy. Myocardial infarction was induced by ligating the left anterior descending coronary artery, which was verified by ST segment changes. Then, via laparotomy incision, a small segment of uterus was removed and applied to the area of infarcted myocardium. This was reinforced by greater omentum. Results: Positron emission tomography scanning of 3 rabbits revealed viable myometrium with normal perfusion. Ventriculography on 4 rabbits revealed slight dyskinesis of the posterior wall, with an ejection fraction of 0.42. Histology revealed viable myometrium, firmly adhered to the infarcted left ventricle, demonstrating enhanced angiogenesis and estrogen and progesterone receptors. Conclusion: Experimental myometrial cell-patch cardiomyoplasty to infarcted myocardium revealed viable myometrium well adhered to infarcted myocardium. The viability was also further demonstrated by positive stain for smooth muscle actin, estrogen, or progesterone receptors. The transplanted myometrium contracted synchronously, with an average ejection fraction of 0.42.
TCT-236 Intracoronary Angiogenic Gene Therapy Is Well Tolerated in Patients with Coronary Artery Disease. M. Watkins1, N. Kleiman2, G. Helmer3, W. Penny4, B. Tao5, H. Morales-Ballejo5, P. Marrott5, C.L. Grines6; for Angiogenic Gene Therapy (AGENT) and AGENT2 investigators. 1University of Vermont, Burlington, Vermont, USA; 2Methodist Hospital, Houston, Texas, USA; 3Minnesota Heart Clinic, Minneapolis, Minnesota, USA; 4University of California, San Diego, California, USA; 5Berlex, Montville, New Jersey, USA; 6 William Beaumont Hospital, Royal Oak, Michigan, USA. Background: Safety data from early angiogenesis trials are encouraging. We report experience with adenovirus 5 mediated human fibroblast growth factor (Ad5FGF-4), a replication-deficient adenovirus, serotype 5, encoding the fibroblast growth factor (FGF)-4 gene. Methods: The Angiogenic Gene Therapy (AGENT) and AGENT2 trials enrolled patients with stable angina (Canadian Cardiovascular Society [CCS] class II-III), left ventricular ejection fraction (LVEF) ⱖ30%, and New York Heart Association (NYHA) heart failure class ⱕIII, who neither required immediate coronary bypass graft surgery (CABG) nor percutaneous transluminal coronary angioplasty (PTCA) (AGENT), or were not optimal candidates (AGENT2). Patients were randomized, double-blind, to one-time intracoronary Ad5FGF-4 or placebo (AGENT: placebo n ⫽ 19, Ad5FGF-4 3.2 x 108 to 3.2 ⫻ 1010 viral particles (vp), n ⫽ 60; AGENT2: placebo n ⫽ 17, Ad5FGF-4 1010 vp, n ⫽ 35) and followed for 12 months. Anti-ischemic effects were evaluated by exercise testing (AGENT) and perfusion radionuclide imaging (AGENT2).
SEPTEMBER 15–17, 2003
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Results: First-pass cardiac vector uptake was high (median 87%). For doses ⱖ3.2 x 109 vp, vector was found in venous blood at 1 hour (36% patients, highest dose) but not seen in urine over 6 hours. No Ad5FGF-4 DNA was seen in semen (n ⫽ 12) at 2 months. Neutralizing Ad5 antibody titer increased ⱖ4 x baseline in 56/60 patients. Safety response to Ad5FGF-4 (n ⫽ 95) was favorable, with no procedural complications. Transient dose-related fever was seen (n ⫽ 8). Mild inflammatory response to Ad5FGF-4 included transiently increased (⬎2 ⫻ normal) liver enzymes (n ⫽ 3), increased uric acid (n ⫽ 18), and decreased (⬍100,000/ /L) platelet count (n ⫽ 1). Remote angiogenesis, including retinal vessels, was absent. Two patients were diagnosed with cancer (judged present prior to treatment, tumor negative for Ad5FGF-4 DNA). One death occurred from unrelated cardiac arrest. There was a trend toward less worsening and unstable angina (13% vs 22%) and CABG or PTCA (8% vs 17%) with Ad5FGF-4 versus placebo. A further 174 patients have received Ad5FGF-4 in ongoing trials. An independent data safety monitoring board found no safety concerns. Conclusion: Intracoronary Ad5FGF-4 appears well tolerated in stable angina patients, but further evaluation in a larger population is warranted.
TCT-237 Infarct Size Reduction and Functional Recovery with Transcoronary Venous, Direct Myocardial Injection of Bone Marrow–Derived Cells. C.A. Thompson1, V. Reddy, M. Hayase, E. Pomerantsev, A. Srinivasan, A D’Avila, S. Houser, J. McGregor, J. Makower, T. Lamson, J.P. Vacanti, H.K. Gold. 1Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA; Massachusetts Institute of Technology, Cambridge, Massachusetts, USA; TransVascular, Inc., Menlo Park, California. Background: The purpose of this study was to determine if a subpopulation of bone marrow– derived cells can improve myocardial viability and function in chronic myocardial infarction. Methods: Ten Yorkshire swine had anterior myocardial infarction induced by coil embolization of the left anterior descending coronary artery. At 5 weeks following myocardial infarction, the animals were allocated to cell therapy versus saline control injections. Bone marrow was harvested and a mononuclear cell subpopulation acquired and tagged with a rhodamine-conjugated cell membrane label. Transcoronary venous direct myocardial injections (⬃50/animal) were performed in the infarct and peri-infarct areas using the CrossPoint TransAccess catheter (TransVascular, Inc., Menlo Park, California). The animals were killed at about 8 weeks (5 cell therapy, 3 saline controls) post-transplant procedure, and histologic examination was performed. Coronary angiography and biplane left ventriculography were performed at all timepoints. Endo- and epicardial voltage mapping (CARTO, Biosense Webster, Diamond Bar, California, USA) and right- and left-ventricular stimulation (2-cycle length up to S4) were performed immediately before killing the animals. Results: One animal died in each arm prior to killing. Viable cell grafts in infarct and peri-infarct myocardium were recovered up to 10 weeks post-implantation in cell-treated animals. The mean left ventricular ejection fraction by quantitative angiography improved from 38.1% to 48.5% in the cell therapy arm, but declined from 38% to 34.5% in the control arm (p ⫽ 0.04). Percentage of scar area with hypokinesis and akinesis was reduced in the cell-treated animals from 52.1% to 42.9% and 24.8% to 17.7%, respectively. Epicardial scar size appeared reduced in the treatment group compared to controls. Provocation of ventricular arrhythmia was related to epicardial scar size, but not treatment assignment. Quantitative measures of electrophysiologic data will be presented. Conclusion: Catheter-based, transcoronary venous implantation of bone marrow– derived progenitor cells may reduce infarct size and rejuvenate lost left ventricular systolic function in chronic myocardial infarction.
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TCT-238 Segmental Occlusion of the Anterior Intraventricular Vein Significantly Improves Regional Delivery After High-Pressure Retroperfusion. S. Gnanashanmugam, K. Wei, E.T. Price, F. Ikeno, J.K. Lyons, A.C. Yeung, P.G. Yock, M. Rezaee. Stanford University Medical Center, Stanford, California, USA. Background: Percutaneous coronary venous delivery (PCVD) through high-pressure venous retroperfusion offers advantages in targeted regional delivery and delivery efficiency of growth factors and cell-based therapies. Delivery through the anterior intraventricular vein (AIV) after segmental occlusion was compared to the conventional proximal occlusion approach. Methods: Distribution patterns were compared between proximal occlusion delivery and both proximal and distal occlusion delivery (two-balloon system) ex vivo (n ⫽ 10) using tracer dyes, and in vivo (n ⫽ 6) using radiolabeled albumin (two radiolabels, BioPhysics Assay Laboratory, Inc., Worcester, Massachusetts, USA). These were performed by placing a percutaneous transluminal coronary angioplasty (PTCA) balloon 3.5 ⫻ 10 to 20 mm in size in the mid to distal segment of the AIV, and a 6.0-x-20-mm balloon at the junction of the AIV with the great cardiac vein. Sequential deliveries after occlusion through either system were performed at a rate of 1 mL/sec to achieve a delivery pressure of 100 to 200 mm Hg, as determined through the injection gauge (external) and intravascular monitoring using a pressure wire (RADI Medical Systems AB, Uppsala, Sweden). Animals were killed within 1 hour; blood samples and tissue samples were analyzed for radioactivity to determine delivery distributions. Results: Ex vivo studies demonstrated that the two-balloon system resulted in more localized tracer delivery within the left anterior descending coronary artery (LAD) territory under high-pressure retroinjection. These results were corroborated in vivo as the two-balloon system demonstrated decreased collateral backflow into the coronary sinus and improved targeted delivery of albumin within the target LAD region (restricted to the region between the two balloons). All deliveries were accomplished without any complications. The two-balloon system was more efficient than proximal occlusion (4.81 ⫻ 105 ⫾ 4.14 ⫻ 105 mean disintegrations per minute (dpms) delivered vs 3.28 ⫻ 105 ⫾ 2.22 ⫻ 105 mean dpms delivered), although, due to the small number of cases, significance was not established. Conclusion: Combining proximal and distal occlusion improves delivery effectiveness by nearly 100%. Proximal balloon occlusion results in a broad, uniform distribution from base to apex of the LAD region, whereas proximal and distal occlusion results in a more targeted, concentrated distribution into the area corresponding to the interballoon space.
TCT-239 Endothelial Cells Within Implantable Scaffolds As a Vector for Angiogenesis. N.J. Goswami1, N. Joshi1, J. Polan1, L.D. Waggoner1, O. Munoz2, J. Elliot1, C.M. Agrawal1, S.R. Bailey1. 1 University of Texas Health Science Center, San Antonio, Texas, USA Background: The ischemic rabbit hind limb is an established animal model for angiogenesis. We have previously documented angiogenic cord formation in both in vitro and in vivo models using threedimensional scaffolds (SCA) seeded with human aortic endothelial cells (HAEC). Gas-plasma sterilization (GP) of the implants resulted in enhanced angiogenic cord formation in a nonischemic, nude mouse model. This increase in angiogenesis appeared to be the result of increased growth factor release. In this study, we evaluated the ability of these GP and nontreated control (NT) SCA seeded with HAEC to produce collateral formation in an ischemic rabbit hind limb model.
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Methods: New Zealand White rabbits were divided into three groups: (1) NT implants (n ⫽ 5); (2) GP implants (n ⫽ 6); and (3) sham (n ⫽ 2). The femoral artery was dissected free, ligated, and excised. After 10 days, the animals were implanted with the HAEC-seeded SCA. Angiography was performed at the baseline and repeated at 28 days post-implant. A quantitative angiographic score (QAS) was determined using a technique previously described by Isner et al. The QASs were then compared between the three groups. Paired t tests were performed when comparing the QASs. Results: The primary end point of this study was angiographic collateral vessel formation at 28 days, as determined by the QAS. All animals receiving SCA displayed an increased QAS at 28 days compared with the baseline. The NT SCA group had a 57% increase in the QAS (p ⬍0.005), while the GP SCA group had a 30% increase in the QAS (p ⫽ 0.012). At 28 days, the QAS was not significantly different in the sham group (p ⫽ NS). The NT SCA group displayed higher collateral formation compared with the GP SCA group (QAS ⫹ 0.33 vs ⫹ 0.18; p ⫽ 0.02). Conclusion: Implantation of SCA seeded with HAEC increased angiogenesis in ischemic rabbit hind limb. The NT SCA group exhibited more collateral formation than the GP SCA group in this model.
TCT-240 Autologous Stem Cell Injection in Patients After Acute Myocardial Infarction. G. Beran1, H. Sochor1, A. Kocher2, M. Dettke3, M. Gyo¨ ngyosi1, D. Glogar1, I.M. Lang1. 1Department of Cardiology; 2 Department of Cardiac Thoracic Surgery; 3 Department of Transfusion Medicine, University of Vienna, Vienna, Austria.
P O S T E R A B S T R A C T S
Background: Experimental data suggest that injection of adult bone marrow (BM) stem cells into the border zones of infarcted heart muscle may reduce myocardial infarction (MI) size by affecting myocardial remodeling. Methods: Four patients (55.3 ⫾ 5.1 yrs; 3 men, 1 woman) with anterior MI were treated 8 weeks post-infarction by a combined intramyocardial and intracoronary injection of BM mononuclear cells (BMMNC). Bone marrow (500 ⫾ 125 mL) was aspirated from the iliac crest, and mononuclear cells were concentrated by automated leucopheresis. The cell suspension (4.1 ⫾ 0.8 mL containing 39.5 ⫾ 5.4 ⫻ 106/mL mononuclear cells [CD45/34⫹ cells, 1.6 ⫾ 0.2%]) was implanted intramyocardially by NOGA (Biosense Webster, Diamond Bar, California, USA) injection needle at 12 sites into the transition zone between scar and viable myocardium, guided by NOGA electromechanical mapping. In addition, an intracoronary injection of 11.5 ⫾ 2.6 mL was administered to the infarct-related artery. Myocardial perfusion imaging single photon emission computed tomography (SPECT) and NOGA mapping were performed before and 6 months after stem cell implantation. Results: There were no peri- and postprocedural complications; in particular, no bleeding or sustained arrhythmias were observed. Noreflow/slow flow after intracoronary BMMNC injection occurred in two patients. At 6-months’ follow-up (FUP), the infarct area (perfusion defect at rest) measured by SPECT was significantly reduced (pre-33 ⫾ 3.4% vs 27.2 ⫾ 1.7% at FUP; p ⬍0.04) compared to the defect immediately before mononuclear cell implantation. In addition, the left ventricular ejection fraction obtained by gated SPECT improved from 35.5 ⫾ 8.9% to 42.3 ⫾ 12.7%. Cardiac output assessed by cardiac stroke volume improved significantly, from 43.2 ⫾ 5.7 to 64.7 ⫾ 5.5 mL at FUP (p ⬍0.01). The Canadian Cardiovascular Society score decreased from 2.8 ⫾ 0.5 pre-implantation to 1.3 ⫾ 0.6 at FUP (p ⬍0.02). Conclusion: Intramyocardial and intracoronary injection of BM stem cells was feasible and safe in patients with recent acute MI. Our preliminary results were encouraging and indicated improvement in
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perfusion, cardiac function, and clinical symptoms 6 months after BM stem cell implantation.
TCT-241 A Comparison Between Adult Bone Marrow and Umbilical Cord Blood–Derived Endothelial Precursor Cells: Immunohistochemical Properties and Mediation of Murine Ischemic Hind Limb Neovascularization. J.M. Martin, M. Finney, J. Swan, M. Joseph, S. Kadereit, M. Kozik, P. Fu, H. Meyerson, M.J. Laughlin, V.J. Pompili. University Hospitals of Cleveland/Case Western Reserve University, Cleveland, Ohio, USA. Background: Although several studies have independently demonstrated augmentation of neovascularization in murine ischemic models, no experiments have directly compared umbilical cord blood (UCB) and adult bone marrow (BM)– derived endothelial precursor cells (EPCs). Our goals were to conduct this comparison in the murine ischemic hind limb model as well as to examine the surface phenotype of both EPC populations in vitro. Methods: Using standard techniques (Kalka, C. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci U S A. 2000 Mar 28;97[7]:3422–27), EPCs were cultured and harvested by trypsinization on day 7. Endothelial properties were confirmed by acetylated low density lipoprotein (acLDL) uptake, and Ulex Europaeus adherence by fluorescence microscopy. Flow cytometry quantified populations of CXCR-4, CD14, Stro-1, P1H12, vascular endothelial (VE)-cadherin, and CD31⫹ cells. Unilateral femoral artery ligation was performed on irradiated Fox Chase SCID/NOD (Taconic M&B, Ry, Denmark) mice, followed by intracardiac injection of 1 million EPCs from either BM (n ⫽ 14) or UCB (n ⫽ 21) versus saline controls (n ⫽ 20). Laser Doppler flow ratios were obtained at day 14. On day 28, samples were harvested from the muscle of the ischemic lower limb. Alkaline phosphatase staining was utilized for capillary density measurements. Results: Flow cytometric analysis on day 7 in culture revealed greater expression of CXCR-4 on UCB EPCs (59.5 ⫾ 13.7%) versus BM-derived EPCs (19.7 ⫾ 7.7%). Conversely, CD14⫹ and Stro-1⫹ populations were more abundant in BM-derived EPC cultures. No difference was noted in the expression of other markers. At day 14 post–femoral artery ligation, laser Doppler flow ratios were statistically higher in both EPC study groups, versus saline (NS) controls (BM 0.48 ⫾ 0.04, UCB 0.41 ⫾ 0.03, NS 0.22 ⫾ 0.03; p ⬍0.0001). Capillary density was greater in the samples harvested from mice injected with EPC from either BM or UCB versus controls. Larger numbers of CD31⫹ human EPCs were localized in the ischemic versus the uninjured limb. Conclusion: Although possessing remarkably different in vitro properties, BM and UCB-derived EPCs are equipotent in their capacity to mediate neovascularization.
TCT-242 CD34ⴙ Cells from Patients with Clinical Evidence of Ischemic Syndromes Induce Therapeutic Neovascularization in Animal Model of Hind Limb Ischemia. L.J. Diaz-Sandoval, T. Murayama, O. Tepper, K. Kusano, M. Kearney, A. Kawamoto, T. Asahara, J.M. Isner, D.W. Losordo. St. Elizabeth’s Medical Center, Boston, Massachusetts, USA. Background: Adult human CD34⫹ cells contain an enriched fraction of endothelial progenitor cells, and their autologous transplantation has been shown to promote neovascularization. However, whether preexisting clinical evidence of ischemia alters the function of these cells is unknown. We assessed the hypothesis that administration of human
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CD34⫹ cells from subjects with peripheral or myocardial ischemia could promote neovascularization in peripheral ischemic tissues. Methods: Mononuclear cells (MNCs) were isolated by density gradient centrifugation from peripheral blood samples of patients with either peripheral or myocardial ischemia (documented by positive stress test and ankle– brachial index; n ⫽ 7; 59 ⫾ 10 years old). CD34⫹ cells were selected by magnetic beads. After surgical induction of hind limb ischemia, 3 groups of 5 athymic nude rats received an intramuscular injection of: (1) 100,000 CD34⫹ cells; (2) 500,000 CD34⫹ cells; and (3) 500,000 nonselected MNCs (control group). Physiologic assessment was performed by serial blood flow measurements using laser Doppler perfusion imaging (LDPI), and recorded in a blinded fashion for 4 weeks postoperatively. Blood flow recovery was assessed by LDPI ratio (mean ⫾ SEM ischemic/control limb). Capillary density in ischemic muscles was measured by endothelial cell staining. A subset of CD34⫾ cells was fluorescently labeled (DiI) and administered in the same fashion to track their fate. Results: Postoperatively, limb perfusion was severely reduced in all three groups. On day 28, group 2 demonstrated the greatest blood flow recovery versus group 3 (0.609 ⫾ 0.095 vs 0.427 ⫾ 0.048; p ⬍0.05), as well as significantly increased capillary density (712 ⫾ 75/mm2 vs 467 ⫾ 27/mm2; p ⬍0.01). DiI–labeled CD34⫹ cells were incorporated into the vasculature and stained positive for endothelial cell marker. Conclusion: Circulating CD34⫹ cells from patients with evidence of ischemia are capable of endothelial differentiation in vivo and can induce therapeutic neovascularization in ischemic tissues. Furthermore, our findings support the possible use of autologous cell therapy for therapeutic angiogenesis in the treatment of patients with peripheral ischemia.
TCT-243 A New Antisense–Based Vaccine Significantly Ameliorated Morphological Changes in the Myocardium of Mice Infected with Coxsackie Viruses. P. Iversen1, M. Rogava2, M. Tsapenko3, N.N. Kipshidze2, N. Kipshidze3. 1AVI-Biopharma, Corvallis, Oregon, USA; 2National Center of Therapy, Tbilisi, Republic of Georgia; 3 Lenox Hill Hospital/Cardiovascular Research Foundation, New York, New York, USA. Background: We examined the possibility of antisense inhibition of Coxsackie viruses in mice in different periods of viral infection and myocarditis. Methods: One hundred clean-linear mice were subdivided into 5 equal groups: (1) treated with antisense phosphorodiamidate morpholino oligomer (PMO) Coxsackie B1 and infected with Coxsackie B1; (2) treated with antisense PMO Coxsackie B3 and infected with Coxsackie B3; (3) and (4) infected with Coxsackie B1 and Coxsackie B3, respectively; and (5) control (buffer solution). Antisense treatment was performed by intraperitoneal injection in a dose of 120 g per injection throughout the first 28 days. Animals were infected (0.2 mL of axenic virus culture with 100 LD50) at day 38 of the experiment and killed at days 3, 5, 7, 9, 15, and 30 days post–viral infection for histological and ultasructural examination. Complement-fixation reaction was used to determine the antiserum titer. Results: Accumulation of antibodies in groups 1 and 2 reached a titer of 1:512 and 1:256, respectively, 3 days post-infection. In nontreated mice (Groups 3 and 4), the antiserum titer never rose above 1:16 for up to 15 days post-infection. Morphologic changes were consistent with viral myocarditis (focal necrosis with mononuclear infiltration and interstitial edema) in all 4 experimental groups, although they were more prominent and widespread in groups 3 and 4. Subsequent morphological changes in the course of infection became more pronounced in nontreated animals. By day 30 post-infection, mice in groups 3 and 4 showed diffuse spreading of fibrosis throughout the walls of the left
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ventricle and retained mononuclear infiltration. In contrast, treated animals had only focal changes and diffuse reaction in the interstitium on a background of diffuse myocardial dystrophy. Conclusion: Pretreatment with antisense designed to inhibit Coxsackie B1 and B3 viruses did not protect the groups from acute viral myocarditis, but significantly ameliorated the course of the disease and morphological changes associated with pathology in the myocardium.
TCT-244 Granulocyte-Colony Stimulating Factor–Induced Mobilization and Intracoronary Infusion of Mobilized Whole Leukocytes Is a Feasible and Safe Method of Stem Cell Transplantation in Patients with Myocardial Infarction. K. Hyun-Jae, K. Hyo-Soo, C. Hyun-Jae, K. Bon-Kwon, K. Yong-Jin, O. Byung-Hee, L. Myoungmook, P. Young-Bae. Department of Internal Medicine, Seoul National University Hospital, Seoul, Republic of Korea. Background: Recent studies demonstrated the beneficial effect of transplantation of bone marrow– derived stem cells in patients with myocardial infarction (MI). However, bone marrow aspiration is too invasive a procedure to be applied to patients with MI. The safety and feasibility of granulocyte-colony stimulating factor (G-CSF)–induced stem cell mobilization and intracoronary infusion of the mobilized peripheral blood stem cells are unknown. We tested the feasibility and safety of G-CSF–induced stem cell mobilization and intracoronary infusion of the mobilized cells in patients with MI. Methods: We randomly allocated 14 patients (57.4 ⫾ 11.3 years old) with MI to G-CSF–induced mobilization only (G-CSF group; n ⫽ 7) or intracoronary infusion of G-CSF–mobilized peripheral blood stem cells (infusion group; n ⫽ 7). The G-CSF and infusion groups included 3 and 4 patients with AMI, respectively. G-CSF with 10 g/kg/day for 4.2 ⫾ 0.8 days was administrated to all patients, then percutaneous coronary intervention (PCI) for culprit lesion were performed. Results: No adverse reaction was observed, except for one case of mild headache with G-CSF administration. Patients in the infusion group underwent apheresis before PCI, and 1.3 ⫾ 0.4 ⫻ 1010 leukocytes (7.6 ⫾ 7.6 ⫻ 108 CD34⫹ cells) were infused after PCI without complication. C-Reactive protein (before and 1 day after infusion: 2.0 ⫾ 3.2 vs 2.2 ⫾ 1.9 mg/dL; p ⫽ 0.91), creatine kinase-MB (before and 12 hours after infusion: 3.0 ⫾ 3.3 vs 4.7 ⫾ 4.9 /L; p ⫽ 0.07), symptoms, and electrocardiogram had not significantly changed after cell infusion. No-reflow phenomenon was not observed on coronary angiography, which was confirmed by measuring coronary flow reserve (CRF) by intracoronary Doppler flow wire. CFR did not worsen with cell infusion (before and 10 minutes after infusion: 1.5 ⫾ 0.2 vs 1.7 ⫾ 0.5; p ⫽ 0.27). No morbidity and mortality were observed until the 1-month clinical follow-up visit. Conclusion: G-CSF is a feasible and safe stem cell mobilizer in both acute and old MI. Apheresis and intracoronary infusion of whole leukocytes after G-CSF mobilization is a practical method of stem cell transplantation.
TCT-245 Anti-angiogenesis May Be a Novel Antirestenosis Effect of Paclitaxel-Eluting Stents. A.G. Touchard, M. Ungs, B.C. Poff, J.J. Barry, M.E. Russell, G.J. Wilson, R.S. Schwartz. Minneapolis Heart Institute Foundation, Minneapolis, Minnesota, USA. Background: Neointimal hyperplasia is a cellular repair process following arterial injury. Paclitaxel-eluting stents reduce restenosis by limiting neointimal hyperplasia. Proposed mechanisms include blocking cell migration and proliferation. Coronary stenting is a known, potent stimulator of adventitial angiogenesis, and post-stent angiogenesis may help neointimal growth and maintenance. Although paclitaxel is a known angiogenesis inhibitor, its effect on coronary arteries has not
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been studied. We thus examined adventitial neovascularization in porcine coronary arteries following paclitaxel-eluting stent implant and compared them with control stents. Methods: Forty-three stented arteries, 26 paclitaxel stents (1 g/ mm2), and 17 controls were examined blindly, without knowledge of stent type (paclitaxel or control). Adventitial vessels (vasa vasorum) were identified as arterial structures with endothelial cells surrounding a lumen, with or without erythrocytes. Adventitial vessel density was determined by manual counting using a curved geometric shape 400 ⫻ 50 mm in size, placed in the adventitia, bordering the external elastic lamina. Intimal thickness was measured from the back of each strut to the lumen, and an average was calculated for each artery.
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Results: Paclitaxel-stented arteries showed a marked decrease in adventitial vessels compared with control stented vessels (1.5 ⫾ 1.2 vs 3.1 ⫾ 2.2 vessels/20,000 mm2; p ⬍0.003). There was no statistical difference in minimum intima thickness between the treated and control groups in this mild injury model (paclitaxel: 308.9 ⫹ 57.3 mm, controls: 284.9 ⫹ 59.0 mm; p ⬎0.5). Conclusion: Paclitaxel elution from stents is associated with markedly decreased adventitial angiogenesis. In addition to its antimigratory and antiproliferative effects, paclitaxel may prevent neointimal growth by preventing adventitial vasa vasorum, limiting oxygen and nutrient supply to the neointima. If true, this opens novel therapeutic strategies for limiting neointimal hyperplasia.
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TCT-235 Experimental Myometrial Cell-Patch Cardiomyoplasty. S.A. Taheri1, H. Ashraf2, Y.V. Fang1, J. Naughton3, B. Nemes1, A. Orlick1. 1Kaleida Health, Buffalo, New York, USA; 2Buffalo Thoracic Associates, Buffalo, New York, USA; 3State University of New York at Buffalo, Buffalo, New York, USA.
Monday, September 15, 2003 9:00 –11:00 AM 2:00 – 4:00 PM Exhibit Hall B on the Lower Level (Abstract nos. 234 –245)
TCT-234 Allogeneic Myoblasts Rejuvenate Degenerative Hearts: First Human Study. P.K. Law1, G. Fang1, F. Chua1, J. Weinstein1, T. Kakuchaya2, V.S. Repin2, L.A. Bockeria2. 1Cell Transplants International, Memphis, Tennessee, USA; 2Bakoulev Scientific Center for Cardiovascular Surgery, Russian Academy of Medical Science, Moscow, Russia.
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Background: Endomyocardial myoblast injections transferred myoblast nuclei into host cardiomyocytes. Some myoblasts transdifferentiated into cardiomyocytes. Others formed myofibers with regenerative satellite cells. Newly deposited myofilaments augmented contractility. We report here the world’s first myoblast allografts into two myocardial infarct patients. Methods: Quadriceps biopsy (2.18 g) from a 20-year-old, pathogen-free male was cultured, in compliance with cGMP/ISO 9001, to yield 3.64 ⫻ 109 myoblasts that were 98.3% pure (desmin immunopositive), 91.5% viable, potent in myotube formation, Gram-negative, and negative for endotoxin, mycoplasma, and sterility. The patients were male subjects, aged 63 and 49, who had developed atherosclerosis, coronary artery disease, chronic myocardial infarction, stable angina (Canadian Cardiovascular Society functional class IV), and arterial hypertension II (risk 4). Positron emission tomography scanning with 18 FDG revealed scars with hibernated myocardium in the left ventricular (LV) septal, apical, and anterior and posterior wall regions. Echocardiography showed regional akinesis and hypokinesis, with LV ejection fraction (LVEF) measurements of 0.41 and 0.38, respectively. On January 17, 2003, the patients received 18/19 injections and 1.1/1.2 billion myoblasts (108/mL), respectively. The 0.5-mL injections were placed between infarcted and viable myocardium after coronary artery bypass graft on ice-chilled, nonbeating hearts. Subjects took oral cyclosporine (5 to 7 mg/kg body weight per day) for 8 weeks. Doses were adjusted to maintain whole blood trough level at about 250 ng/mL. In addition, 24-hour Holter electrocardiogram was monitored. Results: The subjects recovered with no rash or high fever. Both developed arrhythmia and extrasystoles, which were eliminated with amiodarone. Rejection symptoms were not observed, despite cyclosporine discontinuation. At 3-month follow-up, both were in stable condition with angina at class I-II. Echocardiography showed 14.6% and 10.5%, respectively, increases in LVEF, with no local hypokinesis. Single-photon computed tomography with 30 mCi 99mTc-tetrophosphomin demonstrated positive dynamics, LVEF increases, and reduction in perfusion defect areas during exercise and rest. 18FDG accumulation was equable throughout the LV myocardium; glucose metabolism was sustained.
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Conclusion: The feasibility and preliminary safety and efficacy demonstrated in these myoblast allografts provide a new potential therapy for heart failure and its prevention, with virtually unlimited cell availability and only 2-month immunosuppression.
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Background: The purpose of this study was to evaluate the viability and function of patches of autologous myometrium to infarcted myocardium of rabbits. Methods: Seven rabbits weighing between 5 and 6 pounds were anesthetized and intubated. The heart was exposed via midline sternotomy. Myocardial infarction was induced by ligating the left anterior descending coronary artery, which was verified by ST segment changes. Then, via laparotomy incision, a small segment of uterus was removed and applied to the area of infarcted myocardium. This was reinforced by greater omentum. Results: Positron emission tomography scanning of 3 rabbits revealed viable myometrium with normal perfusion. Ventriculography on 4 rabbits revealed slight dyskinesis of the posterior wall, with an ejection fraction of 0.42. Histology revealed viable myometrium, firmly adhered to the infarcted left ventricle, demonstrating enhanced angiogenesis and estrogen and progesterone receptors. Conclusion: Experimental myometrial cell-patch cardiomyoplasty to infarcted myocardium revealed viable myometrium well adhered to infarcted myocardium. The viability was also further demonstrated by positive stain for smooth muscle actin, estrogen, or progesterone receptors. The transplanted myometrium contracted synchronously, with an average ejection fraction of 0.42.
TCT-236 Intracoronary Angiogenic Gene Therapy Is Well Tolerated in Patients with Coronary Artery Disease. M. Watkins1, N. Kleiman2, G. Helmer3, W. Penny4, B. Tao5, H. Morales-Ballejo5, P. Marrott5, C.L. Grines6; for Angiogenic Gene Therapy (AGENT) and AGENT2 investigators. 1University of Vermont, Burlington, Vermont, USA; 2Methodist Hospital, Houston, Texas, USA; 3Minnesota Heart Clinic, Minneapolis, Minnesota, USA; 4University of California, San Diego, California, USA; 5Berlex, Montville, New Jersey, USA; 6 William Beaumont Hospital, Royal Oak, Michigan, USA. Background: Safety data from early angiogenesis trials are encouraging. We report experience with adenovirus 5 mediated human fibroblast growth factor (Ad5FGF-4), a replication-deficient adenovirus, serotype 5, encoding the fibroblast growth factor (FGF)-4 gene. Methods: The Angiogenic Gene Therapy (AGENT) and AGENT2 trials enrolled patients with stable angina (Canadian Cardiovascular Society [CCS] class II-III), left ventricular ejection fraction (LVEF) ⱖ30%, and New York Heart Association (NYHA) heart failure class ⱕIII, who neither required immediate coronary bypass graft surgery (CABG) nor percutaneous transluminal coronary angioplasty (PTCA) (AGENT), or were not optimal candidates (AGENT2). Patients were randomized, double-blind, to one-time intracoronary Ad5FGF-4 or placebo (AGENT: placebo n ⫽ 19, Ad5FGF-4 3.2 x 108 to 3.2 ⫻ 1010 viral particles (vp), n ⫽ 60; AGENT2: placebo n ⫽ 17, Ad5FGF-4 1010 vp, n ⫽ 35) and followed for 12 months. Anti-ischemic effects were evaluated by exercise testing (AGENT) and perfusion radionuclide imaging (AGENT2).
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Results: First-pass cardiac vector uptake was high (median 87%). For doses ⱖ3.2 x 109 vp, vector was found in venous blood at 1 hour (36% patients, highest dose) but not seen in urine over 6 hours. No Ad5FGF-4 DNA was seen in semen (n ⫽ 12) at 2 months. Neutralizing Ad5 antibody titer increased ⱖ4 x baseline in 56/60 patients. Safety response to Ad5FGF-4 (n ⫽ 95) was favorable, with no procedural complications. Transient dose-related fever was seen (n ⫽ 8). Mild inflammatory response to Ad5FGF-4 included transiently increased (⬎2 ⫻ normal) liver enzymes (n ⫽ 3), increased uric acid (n ⫽ 18), and decreased (⬍100,000/ /L) platelet count (n ⫽ 1). Remote angiogenesis, including retinal vessels, was absent. Two patients were diagnosed with cancer (judged present prior to treatment, tumor negative for Ad5FGF-4 DNA). One death occurred from unrelated cardiac arrest. There was a trend toward less worsening and unstable angina (13% vs 22%) and CABG or PTCA (8% vs 17%) with Ad5FGF-4 versus placebo. A further 174 patients have received Ad5FGF-4 in ongoing trials. An independent data safety monitoring board found no safety concerns. Conclusion: Intracoronary Ad5FGF-4 appears well tolerated in stable angina patients, but further evaluation in a larger population is warranted.
TCT-237 Infarct Size Reduction and Functional Recovery with Transcoronary Venous, Direct Myocardial Injection of Bone Marrow–Derived Cells. C.A. Thompson1, V. Reddy, M. Hayase, E. Pomerantsev, A. Srinivasan, A D’Avila, S. Houser, J. McGregor, J. Makower, T. Lamson, J.P. Vacanti, H.K. Gold. 1Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA; Massachusetts Institute of Technology, Cambridge, Massachusetts, USA; TransVascular, Inc., Menlo Park, California. Background: The purpose of this study was to determine if a subpopulation of bone marrow– derived cells can improve myocardial viability and function in chronic myocardial infarction. Methods: Ten Yorkshire swine had anterior myocardial infarction induced by coil embolization of the left anterior descending coronary artery. At 5 weeks following myocardial infarction, the animals were allocated to cell therapy versus saline control injections. Bone marrow was harvested and a mononuclear cell subpopulation acquired and tagged with a rhodamine-conjugated cell membrane label. Transcoronary venous direct myocardial injections (⬃50/animal) were performed in the infarct and peri-infarct areas using the CrossPoint TransAccess catheter (TransVascular, Inc., Menlo Park, California). The animals were killed at about 8 weeks (5 cell therapy, 3 saline controls) post-transplant procedure, and histologic examination was performed. Coronary angiography and biplane left ventriculography were performed at all timepoints. Endo- and epicardial voltage mapping (CARTO, Biosense Webster, Diamond Bar, California, USA) and right- and left-ventricular stimulation (2-cycle length up to S4) were performed immediately before killing the animals. Results: One animal died in each arm prior to killing. Viable cell grafts in infarct and peri-infarct myocardium were recovered up to 10 weeks post-implantation in cell-treated animals. The mean left ventricular ejection fraction by quantitative angiography improved from 38.1% to 48.5% in the cell therapy arm, but declined from 38% to 34.5% in the control arm (p ⫽ 0.04). Percentage of scar area with hypokinesis and akinesis was reduced in the cell-treated animals from 52.1% to 42.9% and 24.8% to 17.7%, respectively. Epicardial scar size appeared reduced in the treatment group compared to controls. Provocation of ventricular arrhythmia was related to epicardial scar size, but not treatment assignment. Quantitative measures of electrophysiologic data will be presented. Conclusion: Catheter-based, transcoronary venous implantation of bone marrow– derived progenitor cells may reduce infarct size and rejuvenate lost left ventricular systolic function in chronic myocardial infarction.
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TCT-238 Segmental Occlusion of the Anterior Intraventricular Vein Significantly Improves Regional Delivery After High-Pressure Retroperfusion. S. Gnanashanmugam, K. Wei, E.T. Price, F. Ikeno, J.K. Lyons, A.C. Yeung, P.G. Yock, M. Rezaee. Stanford University Medical Center, Stanford, California, USA. Background: Percutaneous coronary venous delivery (PCVD) through high-pressure venous retroperfusion offers advantages in targeted regional delivery and delivery efficiency of growth factors and cell-based therapies. Delivery through the anterior intraventricular vein (AIV) after segmental occlusion was compared to the conventional proximal occlusion approach. Methods: Distribution patterns were compared between proximal occlusion delivery and both proximal and distal occlusion delivery (two-balloon system) ex vivo (n ⫽ 10) using tracer dyes, and in vivo (n ⫽ 6) using radiolabeled albumin (two radiolabels, BioPhysics Assay Laboratory, Inc., Worcester, Massachusetts, USA). These were performed by placing a percutaneous transluminal coronary angioplasty (PTCA) balloon 3.5 ⫻ 10 to 20 mm in size in the mid to distal segment of the AIV, and a 6.0-x-20-mm balloon at the junction of the AIV with the great cardiac vein. Sequential deliveries after occlusion through either system were performed at a rate of 1 mL/sec to achieve a delivery pressure of 100 to 200 mm Hg, as determined through the injection gauge (external) and intravascular monitoring using a pressure wire (RADI Medical Systems AB, Uppsala, Sweden). Animals were killed within 1 hour; blood samples and tissue samples were analyzed for radioactivity to determine delivery distributions. Results: Ex vivo studies demonstrated that the two-balloon system resulted in more localized tracer delivery within the left anterior descending coronary artery (LAD) territory under high-pressure retroinjection. These results were corroborated in vivo as the two-balloon system demonstrated decreased collateral backflow into the coronary sinus and improved targeted delivery of albumin within the target LAD region (restricted to the region between the two balloons). All deliveries were accomplished without any complications. The two-balloon system was more efficient than proximal occlusion (4.81 ⫻ 105 ⫾ 4.14 ⫻ 105 mean disintegrations per minute (dpms) delivered vs 3.28 ⫻ 105 ⫾ 2.22 ⫻ 105 mean dpms delivered), although, due to the small number of cases, significance was not established. Conclusion: Combining proximal and distal occlusion improves delivery effectiveness by nearly 100%. Proximal balloon occlusion results in a broad, uniform distribution from base to apex of the LAD region, whereas proximal and distal occlusion results in a more targeted, concentrated distribution into the area corresponding to the interballoon space.
TCT-239 Endothelial Cells Within Implantable Scaffolds As a Vector for Angiogenesis. N.J. Goswami1, N. Joshi1, J. Polan1, L.D. Waggoner1, O. Munoz2, J. Elliot1, C.M. Agrawal1, S.R. Bailey1. 1 University of Texas Health Science Center, San Antonio, Texas, USA Background: The ischemic rabbit hind limb is an established animal model for angiogenesis. We have previously documented angiogenic cord formation in both in vitro and in vivo models using threedimensional scaffolds (SCA) seeded with human aortic endothelial cells (HAEC). Gas-plasma sterilization (GP) of the implants resulted in enhanced angiogenic cord formation in a nonischemic, nude mouse model. This increase in angiogenesis appeared to be the result of increased growth factor release. In this study, we evaluated the ability of these GP and nontreated control (NT) SCA seeded with HAEC to produce collateral formation in an ischemic rabbit hind limb model.
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Methods: New Zealand White rabbits were divided into three groups: (1) NT implants (n ⫽ 5); (2) GP implants (n ⫽ 6); and (3) sham (n ⫽ 2). The femoral artery was dissected free, ligated, and excised. After 10 days, the animals were implanted with the HAEC-seeded SCA. Angiography was performed at the baseline and repeated at 28 days post-implant. A quantitative angiographic score (QAS) was determined using a technique previously described by Isner et al. The QASs were then compared between the three groups. Paired t tests were performed when comparing the QASs. Results: The primary end point of this study was angiographic collateral vessel formation at 28 days, as determined by the QAS. All animals receiving SCA displayed an increased QAS at 28 days compared with the baseline. The NT SCA group had a 57% increase in the QAS (p ⬍0.005), while the GP SCA group had a 30% increase in the QAS (p ⫽ 0.012). At 28 days, the QAS was not significantly different in the sham group (p ⫽ NS). The NT SCA group displayed higher collateral formation compared with the GP SCA group (QAS ⫹ 0.33 vs ⫹ 0.18; p ⫽ 0.02). Conclusion: Implantation of SCA seeded with HAEC increased angiogenesis in ischemic rabbit hind limb. The NT SCA group exhibited more collateral formation than the GP SCA group in this model.
TCT-240 Autologous Stem Cell Injection in Patients After Acute Myocardial Infarction. G. Beran1, H. Sochor1, A. Kocher2, M. Dettke3, M. Gyo¨ ngyosi1, D. Glogar1, I.M. Lang1. 1Department of Cardiology; 2 Department of Cardiac Thoracic Surgery; 3 Department of Transfusion Medicine, University of Vienna, Vienna, Austria.
P O S T E R A B S T R A C T S
Background: Experimental data suggest that injection of adult bone marrow (BM) stem cells into the border zones of infarcted heart muscle may reduce myocardial infarction (MI) size by affecting myocardial remodeling. Methods: Four patients (55.3 ⫾ 5.1 yrs; 3 men, 1 woman) with anterior MI were treated 8 weeks post-infarction by a combined intramyocardial and intracoronary injection of BM mononuclear cells (BMMNC). Bone marrow (500 ⫾ 125 mL) was aspirated from the iliac crest, and mononuclear cells were concentrated by automated leucopheresis. The cell suspension (4.1 ⫾ 0.8 mL containing 39.5 ⫾ 5.4 ⫻ 106/mL mononuclear cells [CD45/34⫹ cells, 1.6 ⫾ 0.2%]) was implanted intramyocardially by NOGA (Biosense Webster, Diamond Bar, California, USA) injection needle at 12 sites into the transition zone between scar and viable myocardium, guided by NOGA electromechanical mapping. In addition, an intracoronary injection of 11.5 ⫾ 2.6 mL was administered to the infarct-related artery. Myocardial perfusion imaging single photon emission computed tomography (SPECT) and NOGA mapping were performed before and 6 months after stem cell implantation. Results: There were no peri- and postprocedural complications; in particular, no bleeding or sustained arrhythmias were observed. Noreflow/slow flow after intracoronary BMMNC injection occurred in two patients. At 6-months’ follow-up (FUP), the infarct area (perfusion defect at rest) measured by SPECT was significantly reduced (pre-33 ⫾ 3.4% vs 27.2 ⫾ 1.7% at FUP; p ⬍0.04) compared to the defect immediately before mononuclear cell implantation. In addition, the left ventricular ejection fraction obtained by gated SPECT improved from 35.5 ⫾ 8.9% to 42.3 ⫾ 12.7%. Cardiac output assessed by cardiac stroke volume improved significantly, from 43.2 ⫾ 5.7 to 64.7 ⫾ 5.5 mL at FUP (p ⬍0.01). The Canadian Cardiovascular Society score decreased from 2.8 ⫾ 0.5 pre-implantation to 1.3 ⫾ 0.6 at FUP (p ⬍0.02). Conclusion: Intramyocardial and intracoronary injection of BM stem cells was feasible and safe in patients with recent acute MI. Our preliminary results were encouraging and indicated improvement in
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perfusion, cardiac function, and clinical symptoms 6 months after BM stem cell implantation.
TCT-241 A Comparison Between Adult Bone Marrow and Umbilical Cord Blood–Derived Endothelial Precursor Cells: Immunohistochemical Properties and Mediation of Murine Ischemic Hind Limb Neovascularization. J.M. Martin, M. Finney, J. Swan, M. Joseph, S. Kadereit, M. Kozik, P. Fu, H. Meyerson, M.J. Laughlin, V.J. Pompili. University Hospitals of Cleveland/Case Western Reserve University, Cleveland, Ohio, USA. Background: Although several studies have independently demonstrated augmentation of neovascularization in murine ischemic models, no experiments have directly compared umbilical cord blood (UCB) and adult bone marrow (BM)– derived endothelial precursor cells (EPCs). Our goals were to conduct this comparison in the murine ischemic hind limb model as well as to examine the surface phenotype of both EPC populations in vitro. Methods: Using standard techniques (Kalka, C. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci U S A. 2000 Mar 28;97[7]:3422–27), EPCs were cultured and harvested by trypsinization on day 7. Endothelial properties were confirmed by acetylated low density lipoprotein (acLDL) uptake, and Ulex Europaeus adherence by fluorescence microscopy. Flow cytometry quantified populations of CXCR-4, CD14, Stro-1, P1H12, vascular endothelial (VE)-cadherin, and CD31⫹ cells. Unilateral femoral artery ligation was performed on irradiated Fox Chase SCID/NOD (Taconic M&B, Ry, Denmark) mice, followed by intracardiac injection of 1 million EPCs from either BM (n ⫽ 14) or UCB (n ⫽ 21) versus saline controls (n ⫽ 20). Laser Doppler flow ratios were obtained at day 14. On day 28, samples were harvested from the muscle of the ischemic lower limb. Alkaline phosphatase staining was utilized for capillary density measurements. Results: Flow cytometric analysis on day 7 in culture revealed greater expression of CXCR-4 on UCB EPCs (59.5 ⫾ 13.7%) versus BM-derived EPCs (19.7 ⫾ 7.7%). Conversely, CD14⫹ and Stro-1⫹ populations were more abundant in BM-derived EPC cultures. No difference was noted in the expression of other markers. At day 14 post–femoral artery ligation, laser Doppler flow ratios were statistically higher in both EPC study groups, versus saline (NS) controls (BM 0.48 ⫾ 0.04, UCB 0.41 ⫾ 0.03, NS 0.22 ⫾ 0.03; p ⬍0.0001). Capillary density was greater in the samples harvested from mice injected with EPC from either BM or UCB versus controls. Larger numbers of CD31⫹ human EPCs were localized in the ischemic versus the uninjured limb. Conclusion: Although possessing remarkably different in vitro properties, BM and UCB-derived EPCs are equipotent in their capacity to mediate neovascularization.
TCT-242 CD34ⴙ Cells from Patients with Clinical Evidence of Ischemic Syndromes Induce Therapeutic Neovascularization in Animal Model of Hind Limb Ischemia. L.J. Diaz-Sandoval, T. Murayama, O. Tepper, K. Kusano, M. Kearney, A. Kawamoto, T. Asahara, J.M. Isner, D.W. Losordo. St. Elizabeth’s Medical Center, Boston, Massachusetts, USA. Background: Adult human CD34⫹ cells contain an enriched fraction of endothelial progenitor cells, and their autologous transplantation has been shown to promote neovascularization. However, whether preexisting clinical evidence of ischemia alters the function of these cells is unknown. We assessed the hypothesis that administration of human
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CD34⫹ cells from subjects with peripheral or myocardial ischemia could promote neovascularization in peripheral ischemic tissues. Methods: Mononuclear cells (MNCs) were isolated by density gradient centrifugation from peripheral blood samples of patients with either peripheral or myocardial ischemia (documented by positive stress test and ankle– brachial index; n ⫽ 7; 59 ⫾ 10 years old). CD34⫹ cells were selected by magnetic beads. After surgical induction of hind limb ischemia, 3 groups of 5 athymic nude rats received an intramuscular injection of: (1) 100,000 CD34⫹ cells; (2) 500,000 CD34⫹ cells; and (3) 500,000 nonselected MNCs (control group). Physiologic assessment was performed by serial blood flow measurements using laser Doppler perfusion imaging (LDPI), and recorded in a blinded fashion for 4 weeks postoperatively. Blood flow recovery was assessed by LDPI ratio (mean ⫾ SEM ischemic/control limb). Capillary density in ischemic muscles was measured by endothelial cell staining. A subset of CD34⫾ cells was fluorescently labeled (DiI) and administered in the same fashion to track their fate. Results: Postoperatively, limb perfusion was severely reduced in all three groups. On day 28, group 2 demonstrated the greatest blood flow recovery versus group 3 (0.609 ⫾ 0.095 vs 0.427 ⫾ 0.048; p ⬍0.05), as well as significantly increased capillary density (712 ⫾ 75/mm2 vs 467 ⫾ 27/mm2; p ⬍0.01). DiI–labeled CD34⫹ cells were incorporated into the vasculature and stained positive for endothelial cell marker. Conclusion: Circulating CD34⫹ cells from patients with evidence of ischemia are capable of endothelial differentiation in vivo and can induce therapeutic neovascularization in ischemic tissues. Furthermore, our findings support the possible use of autologous cell therapy for therapeutic angiogenesis in the treatment of patients with peripheral ischemia.
TCT-243 A New Antisense–Based Vaccine Significantly Ameliorated Morphological Changes in the Myocardium of Mice Infected with Coxsackie Viruses. P. Iversen1, M. Rogava2, M. Tsapenko3, N.N. Kipshidze2, N. Kipshidze3. 1AVI-Biopharma, Corvallis, Oregon, USA; 2National Center of Therapy, Tbilisi, Republic of Georgia; 3 Lenox Hill Hospital/Cardiovascular Research Foundation, New York, New York, USA. Background: We examined the possibility of antisense inhibition of Coxsackie viruses in mice in different periods of viral infection and myocarditis. Methods: One hundred clean-linear mice were subdivided into 5 equal groups: (1) treated with antisense phosphorodiamidate morpholino oligomer (PMO) Coxsackie B1 and infected with Coxsackie B1; (2) treated with antisense PMO Coxsackie B3 and infected with Coxsackie B3; (3) and (4) infected with Coxsackie B1 and Coxsackie B3, respectively; and (5) control (buffer solution). Antisense treatment was performed by intraperitoneal injection in a dose of 120 g per injection throughout the first 28 days. Animals were infected (0.2 mL of axenic virus culture with 100 LD50) at day 38 of the experiment and killed at days 3, 5, 7, 9, 15, and 30 days post–viral infection for histological and ultasructural examination. Complement-fixation reaction was used to determine the antiserum titer. Results: Accumulation of antibodies in groups 1 and 2 reached a titer of 1:512 and 1:256, respectively, 3 days post-infection. In nontreated mice (Groups 3 and 4), the antiserum titer never rose above 1:16 for up to 15 days post-infection. Morphologic changes were consistent with viral myocarditis (focal necrosis with mononuclear infiltration and interstitial edema) in all 4 experimental groups, although they were more prominent and widespread in groups 3 and 4. Subsequent morphological changes in the course of infection became more pronounced in nontreated animals. By day 30 post-infection, mice in groups 3 and 4 showed diffuse spreading of fibrosis throughout the walls of the left
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ventricle and retained mononuclear infiltration. In contrast, treated animals had only focal changes and diffuse reaction in the interstitium on a background of diffuse myocardial dystrophy. Conclusion: Pretreatment with antisense designed to inhibit Coxsackie B1 and B3 viruses did not protect the groups from acute viral myocarditis, but significantly ameliorated the course of the disease and morphological changes associated with pathology in the myocardium.
TCT-244 Granulocyte-Colony Stimulating Factor–Induced Mobilization and Intracoronary Infusion of Mobilized Whole Leukocytes Is a Feasible and Safe Method of Stem Cell Transplantation in Patients with Myocardial Infarction. K. Hyun-Jae, K. Hyo-Soo, C. Hyun-Jae, K. Bon-Kwon, K. Yong-Jin, O. Byung-Hee, L. Myoungmook, P. Young-Bae. Department of Internal Medicine, Seoul National University Hospital, Seoul, Republic of Korea. Background: Recent studies demonstrated the beneficial effect of transplantation of bone marrow– derived stem cells in patients with myocardial infarction (MI). However, bone marrow aspiration is too invasive a procedure to be applied to patients with MI. The safety and feasibility of granulocyte-colony stimulating factor (G-CSF)–induced stem cell mobilization and intracoronary infusion of the mobilized peripheral blood stem cells are unknown. We tested the feasibility and safety of G-CSF–induced stem cell mobilization and intracoronary infusion of the mobilized cells in patients with MI. Methods: We randomly allocated 14 patients (57.4 ⫾ 11.3 years old) with MI to G-CSF–induced mobilization only (G-CSF group; n ⫽ 7) or intracoronary infusion of G-CSF–mobilized peripheral blood stem cells (infusion group; n ⫽ 7). The G-CSF and infusion groups included 3 and 4 patients with AMI, respectively. G-CSF with 10 g/kg/day for 4.2 ⫾ 0.8 days was administrated to all patients, then percutaneous coronary intervention (PCI) for culprit lesion were performed. Results: No adverse reaction was observed, except for one case of mild headache with G-CSF administration. Patients in the infusion group underwent apheresis before PCI, and 1.3 ⫾ 0.4 ⫻ 1010 leukocytes (7.6 ⫾ 7.6 ⫻ 108 CD34⫹ cells) were infused after PCI without complication. C-Reactive protein (before and 1 day after infusion: 2.0 ⫾ 3.2 vs 2.2 ⫾ 1.9 mg/dL; p ⫽ 0.91), creatine kinase-MB (before and 12 hours after infusion: 3.0 ⫾ 3.3 vs 4.7 ⫾ 4.9 /L; p ⫽ 0.07), symptoms, and electrocardiogram had not significantly changed after cell infusion. No-reflow phenomenon was not observed on coronary angiography, which was confirmed by measuring coronary flow reserve (CRF) by intracoronary Doppler flow wire. CFR did not worsen with cell infusion (before and 10 minutes after infusion: 1.5 ⫾ 0.2 vs 1.7 ⫾ 0.5; p ⫽ 0.27). No morbidity and mortality were observed until the 1-month clinical follow-up visit. Conclusion: G-CSF is a feasible and safe stem cell mobilizer in both acute and old MI. Apheresis and intracoronary infusion of whole leukocytes after G-CSF mobilization is a practical method of stem cell transplantation.
TCT-245 Anti-angiogenesis May Be a Novel Antirestenosis Effect of Paclitaxel-Eluting Stents. A.G. Touchard, M. Ungs, B.C. Poff, J.J. Barry, M.E. Russell, G.J. Wilson, R.S. Schwartz. Minneapolis Heart Institute Foundation, Minneapolis, Minnesota, USA. Background: Neointimal hyperplasia is a cellular repair process following arterial injury. Paclitaxel-eluting stents reduce restenosis by limiting neointimal hyperplasia. Proposed mechanisms include blocking cell migration and proliferation. Coronary stenting is a known, potent stimulator of adventitial angiogenesis, and post-stent angiogenesis may help neointimal growth and maintenance. Although paclitaxel is a known angiogenesis inhibitor, its effect on coronary arteries has not
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been studied. We thus examined adventitial neovascularization in porcine coronary arteries following paclitaxel-eluting stent implant and compared them with control stents. Methods: Forty-three stented arteries, 26 paclitaxel stents (1 g/ mm2), and 17 controls were examined blindly, without knowledge of stent type (paclitaxel or control). Adventitial vessels (vasa vasorum) were identified as arterial structures with endothelial cells surrounding a lumen, with or without erythrocytes. Adventitial vessel density was determined by manual counting using a curved geometric shape 400 ⫻ 50 mm in size, placed in the adventitia, bordering the external elastic lamina. Intimal thickness was measured from the back of each strut to the lumen, and an average was calculated for each artery.
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Results: Paclitaxel-stented arteries showed a marked decrease in adventitial vessels compared with control stented vessels (1.5 ⫾ 1.2 vs 3.1 ⫾ 2.2 vessels/20,000 mm2; p ⬍0.003). There was no statistical difference in minimum intima thickness between the treated and control groups in this mild injury model (paclitaxel: 308.9 ⫹ 57.3 mm, controls: 284.9 ⫹ 59.0 mm; p ⬎0.5). Conclusion: Paclitaxel elution from stents is associated with markedly decreased adventitial angiogenesis. In addition to its antimigratory and antiproliferative effects, paclitaxel may prevent neointimal growth by preventing adventitial vasa vasorum, limiting oxygen and nutrient supply to the neointima. If true, this opens novel therapeutic strategies for limiting neointimal hyperplasia.
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