Autologous skeletal myoblasts transplanted to infarcted human myocardium: histological analysis of cell survival and differentiation

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Abstracts

The Journal of Heart and Lung Transplantation January 2003

apr. in comparison, 56 of the 96 patients (58%) of the il-10 int/low genotype had apr (p⫽0.024). for the il-10 haplotype gcc/acc versus gcc/ata, which were the genotypes associated with int il-10 production, gcc/acc had 31 out of 43 patients (72%) with apr in comparison with gcc/ata in which there were 10 out of 22 patients (45%) with apr (p⫽0.035). Conclusion: In LT patients, the IL-10 high genotype is protective against APR in comparison to int/low IL-10 genotypes. The IL-10 intermediate genotype in LT patients can be differentiated into two haplotype responses with the GCC/ACC having more APR. The homozygous high IL-10 genotype may be protective against lung rejection by reducing inflammatory cytokines and downregulating APCs. Future pharmacogenomic models may incorporate these associations with APR in LT patients to formulate individualized therapeutic regimens.

13 BRONCHIOLITIS OBLITERANS SYNDROME/OBLITERATIVE BRONCHIOLITIS AMELIORATED BY AZITHROMYCIN S.G. Gerhardt,1 J.S. McDyer,1 R.E. Girgis,1 J.V. Conte,2 S.C. Yang,3 M. Khalid,4 J.B. Orens,1 1Division of Pulmonary and Critical Care Medicine, The Johns Hopkins Hospital; 2Division of Cardiac Surgery; 3 Division of Thoracic Surgery, The Johns Hopkins Hospital, Baltimore, MD; 4Division of Pulmonary Medicine, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia Background: Bronchiolitis obliterans syndrome(BOS) remains the leading cause of morbidity and mortality for lung transplant recipients. Current treatment strategies are ineffective, costly, and associated with significant side effects. Previous studies have shown the effectiveness of azithromycin in the treatment of panbronchiolitis and its value as adjunct therapy in Cystic Fibrosis(CF). Study Objectives: To determine the effect of azithromycin on pulmonary function in patients with BOS and OB. Methods: History, clinical course, and pulmonary function data were gathered on all patients who had received at least 6 weeks of azithromycin (250mg daily for 5 days, followed by 250mg tiw) for treatment of BOS (ISHLT criteria) or obliterative bronchiolitis (OB), including idiopathic OB and post bone marrow transplant OB. Results: Subjects included: 6 pulmonary allograft recipients (2 transplanted for CF, 2 for bronchiectasis, 1 for IPF, and 1 retransplanted for OB) and 3 non-lung transplant patients (2 with idiopathic OB and 1 post bone marrow transplant). 8 of the 9 patients showed marked improvement in FEV1 and FVC. Change in spirometry measurements before and after azithromycin therapy

N ⌬FEV1 (liters) ⌬Percent predicted FEV1 ⌬Percent post transplant maximum FEV1 ⌬FVC (liters) ⌬Percent predicted FVC ⌬Percent post transplant maximum FVC

Transplanted patients

Nontransplanted patients

All patients

6 0.50 17.1* 14.0

3 0.40 14.0* –

9 0.46* 12.7 –

0.40 11.3 12.9

0.84 21.5 –

0.55* 14.7* –

*P ⱕ 0.05.

Conclusions: Azithromycin led to a statistically significant improvement in pulmonary function in 6 of 7 patients with BOS and 3 patients with non-transplant related OB. A randomized trial of this promising, inexpensive, and low-risk treatment for BOS is warranted.

14 THE PREDICTIVE VALUE OF BRONCHIOLITIS OBLITERANS SYNDROME STAGE 0-P R.R. Hachem, G.A. Patterson, E.P. Trulock, Lung Transplant Program, Washington University School of Medicine, St Louis, MO Introduction: New staging criteria for bronchiolitis obliterans syndrome (BOS) were recently proposed. Stage 0-p was added to detect early deterioration in allograft function that might presage BOS 1. The purpose of this study was to determine the predictive value of stage 0-p. Methods: A retrospective analysis of spirometric data for 251 adult lung transplant recipients [203 bilateral (BLTx), 48 single (SLTx)] was performed to determine the onset of BOS 0-p and the onset of BOS 1. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of BOS 0-p were then determined and stratified among BLTx and SLTx recipients. Freedom from BOS 1 was determined after the onset of BOS 0-p. Results: The sensitivity, specificity, and predictive values of each BOS 0-p criterion are shown below: BLTx Recipients FEV1 0-p FEF25-75 0-p

Sensitivity

Specificity

PPV

NPV

74% 78%

86% 44%

79% 51%

82% 72%

SLTx Recipients FEV1 0-p FEF25-75 0-p

Sensitivity

Specificity

PPV

NPV

58% 77%

78% 41%

79% 67%

56% 54%

The following table displays freedom from BOS 1 after the onset of BOS 0-p by each criterion, for BLTx and SLTx recipients: Freedom from BOS 1 after the onset of BOS 0-p Time (years)

BLTx FEV1 BLTx FEF25-75 SLTx FEV1 0p 0p 0p

SLTx FEF25-75 0p

1 2 3 4 5

43% 37% 18% 9% 0%

69% 47% 26% 18% 18%

63% 58% 49% 42% 37%

41% 25% 0% 0% 0%

Time is from the onset of BOS 0-p; percentages represent the percent free from BOS 1.

Conclusion: When defined by FEV1 criterion, BOS 0-p is a reasonable predictor of BOS 1 after BLTx; however, its predictive value is poor after SLTx. Likewise, when defined by FEF25-75 criterion, BOS 0-p is not predictive of developing BOS 1 after BLTx or SLTx.

15 AUTOLOGOUS SKELETAL MYOBLASTS TRANSPLANTED TO INFARCTED HUMAN MYOCARDIUM: HISTOLOGICAL ANALYSIS OF CELL SURVIVAL AND DIFFERENTIATION F.D. Pagani,1 H. DerSimonian,2 A. Zawadzka,2 D.B. Jacoby,2 J. Dinsmore,2 S. Wright,1 T.H. Aretz,3 K. Wetzel,2 K.D. Aaronson,4 1Section of Cardiac Surgery, University of Michigan, Ann Arbor, MI; 2Department of Cell Transplantation, Diacrin, Inc, Charlestown, MA; 3Department of Pathology, Massachusetts General Hopsital, Boston, MA; 4Division of Cardiology, University of Michigan, Ann Arbor, MI

The Journal of Heart and Lung Transplantation Volume 22, Number 1S Background: Autologous skeletal myoblast transplantation is under investigation as a means to repair infarcted myocardium. We report the histological analysis of human hearts in patients with ischemic cardiomyopathy who were transplanted with autologous skeletal muscle (SM) concurrent with LVAD implant as a bridge to transplant. Methods: Three pts underwent SM biopsy from the right quadriceps muscle. The SM from biopsy was subjected to enzymatic digestion to release satellite cells. Skeletal myoblast cultures were expanded according to a modified Ham’s method. Cells were grown for 14 doublings to achieve a final yield of 300 million cells. At the time of LVAD implant, injections of autologous skeletal myoblasts were made into the anterior wall of the LV. At the time of transplant and LVAD explant, the recipient heart was excised for the purpose of assessing SM cell survival and differentiation using immunohistochemistry. Results: Evidence of SM cell survival and differentiation into mature myofibers was demonstrated in heavily scarred myocardium using an antibody against SM-specific myosin heavy chain (MY-32 mAb). The presence of vascular endothelium in grafted and non-grafted myocardium was assessed using an anti-CD-31 mAb. A significant (p⬍0.05) increase in small vessel formation was observed at the site of surviving myotubes, but not in adjacent tissue devoid of engrafted cells (228⫾24 vs. 72⫾17 cells/field). There was no evidence of immune reaction or lymphocyte infiltration in either grafted or non-grafted areas using a T-cell specific anti-CD3 polyclonal antibody. Conclusion: These findings represent the first histological evidence of autologous myoblast cell survival in human hearts. The implanted skeletal myoblasts formed viable grafts in heavily scarred human myocardial tissue. These results demonstrate and establish the feasibility of myoblast transplants for myocardial repair. 16 BONE MARROW CELL ENHANCES THE EFFICACY OF SKELETAL MYOBLAST TRANSPLANTATION IN RATS WITH ISCHEMIC CARDIOMYOPATHY G. Sakaguchi, Y. Sakakibara, K. Tambara, G. Premaratne, X. Lin, K. Nishimura, M. Komeda, Cardiovascular Surgery, Kyoto University, Kyoto, Japan Introduction: Previous studies have shown autologous cell ( skeletal myoblast, SM and bone marrow cell, BMC) transplantation can prevent LV remodeling and maintain LV function after myocardial infarction. However, the efficacy of adult SM transplantation is limited. Hypothesis: We hypothesized that BMC could enhance the efficacy of adult SM transplantation by promoting transplanted SM survival or angiogenesis. Methods: Myocardial infarction was induced in syngeneic Lewis rats by ligating the left coronary artery. Four weeks after myocardial infarction, the animals were randomized into three groups: 8 rats had an intramyocardial injection of culture medium alone (control group), 8 rats had an intramyocardial injection of SM (5x106)(SM group), and 6 rats had an intramyocardial injection of both adult SM (5x106 ) and adult BMC (1x106 )(SM-BM group). LV function was assessed by echocardiography for 4 weeks. Results: There was no death in any groups. There was no significant difference in LV end-diastolic dimension (LVDD) among the 3 groups at baseline(control, SM, SM-BM; 9.47 ⫾0.13, 8.97 ⫾0.28, 9.18 ⫾0.35mm, respectively). Whereas LVDD gradually increased in the control group, that in the SM and SM-BM groups unchanged at 4 weeks(9.99 ⫾0.29, 8.31 ⫾0.36, 8.18 ⫾0.48mm, p⬍0.01 by ANOVA). There was no significant difference in fractional area change (FAC) among the 3 groups at baseline(control, SM, SM-BM; 36 ⫾3, 32 ⫾5, 32 ⫾3%, respectively). While FAC unchanged in the control and SM groups, that in the SM-BM group increased (30 ⫾2, 35 ⫾4, 46 ⫾5% at

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4 weeks, respectively, p⬍0.05). The survival of the transplanted SM was similar between the SM and SM-BMC groups. The hearts in the SM-BM group showed angiogenesis around the injection site. Conclusion: BMC transplantation enhanced the efficacy of SM transplantation in the improvement of LV function by angiogenesis. 17 ELECTROPHYSIOLOGICAL PROPERTIES OF MYOBLASTS INJECTED INTO THE INFARCTED MYOCARDIUM OF RATS H.C. Ott,1 S.A. Berjukow,2 R. Marksteiner,2 E. Margreiter,2 B. Podesser,4 G. Laufer,3 S. Hering,2 1Cardiology, University of Innsbruck, Innsbruck, Austria; 2Biochemical Pharmacology, University of Innsbruck, Innsbruck, Austria; 3Cardiac Surgery, University of Innsbruck, Innsbruck, Austria; 4Ludwig Boltsman Institute for Cardiovascular Research, University of Vienna, Vienna, Austria Aim: Transplantation of skeletal myoblasts (SMB) into an infracted myocardium is a promising therapeutic approach to improve the outcome of patients with ischemic cardiomyopathy. We analyse the functional properties of SMB after different times of engraftment. The expression of ion channels was investigated before and 1, 3 and 6 days after transplantation of the cells into the infracted myocardium. Methods: In male Fisher rats the left ventricle was infarcted by coronary ligation for 30 minutes. Permanently GFP-transfected SMB (107 cells) were injected into the infarct and peri-infarct region. The hearts were removed after 1, 3 and 6 days and single cells isolated by Langendorf perfusion with an collagenase containing solution. Sodium (Na), calcium (Ca) and potassium (K) channels were studied in the freshly isolated GFP-positive SMB by means of the patch clamp technique. Results: Before implantation 68% of the GFP-positive SMB expressed low voltage activated T-type Ca channels and 64% expressed fast Na channels. Voltage-gated outward K channels were observed in 90% of the cells. After 1 day in the myocardial environment the cells did no longer express T-type channels, only 42% expressed Na and 83% K channels. After 3 days in only 13% of the cells expression of Na but in 50% expression of K channels were observed. After 6 days neither Na nor Ca currents were recorded. However, 31% of the cells still expressed voltage-gated K channels. Conclusions: Single SMB that were transplanted into the region of an acute myocardial infarction can be enzymatically isolated up to 6 days after grafting. Implanted cell can be identified by GFP-expression for subsequent patch clamp studies. We observed a down-regulation in Na, K and T-type Ca channel expression progressing with time of engraftment. We conclude that SMB undergo severe functional changes after implantation into the freshly infarcted myocardium. 18 A NOVEL SUB-POPULATION OF BONE MARROW DERIVED MYOCARDIAL STEM CELLS: POTENTIAL AUTOLOGUS CELL THERAPY IN MYOCARDIAL INFARCTION H.J. Soukiasian, L.S.C. Czer, I. Avital, T. Aoki, Y. KIM, Y. Umehara, J. Pass, R. Tabrizi, K. Magliato, G.P. Fontana, W. Cheng, A.A. Demetriou, A. Trento, Cardiothoracic Surgery, Cedars-Sinai Medical Center, Los Angeles, CA Several studies have identified beta-2-microglobulin negative (B2M-) cells as a potential stem cell fraction in the bone marrow of rats and humans. Purpose: we studied the ability of bone marrow derived b2m- cells to differentiate into cardiomyocytes and reconstitute the myocardium in a model of myocardial infarction.