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Electrical competence of infarcted heart following stem cell based regenerative therapy, in a rat model
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ABSTRACTS / Journal of Molecular and Cellular Cardiology 42 (2007) S88–S101 +
Impaired contribution of circulating CD34 in myocardial ischemia-induced neovascularization with age Mohamed Gaballa, Hoang Thai, Steven Goldman. University of Arizona, SHC, Tucson, AZ, USA Impaired angiogenesis is documented in the aged. However, the effects of age on ischemia-induced cardiac vasculogenesis are unknown. To examine changes in ischemia-induced cardiac neovascularization with aging, myocardial infarction (MI) was created by LAD ligation in Brown Norway Fisher F344 cross at 3 ages: 6 weeks, 6 months, and 24 months rats (n = 10 per group). A 3D collagen scaffold transplanted onto the heart immediately after MI and bone marrow derived CD34+ cells or 50 ng/kg G-CSF was injected IV. Aging increased MAP, 90 ± 4, 115 ± 6, 121 ± 6, n = 10, P < 0.05, while MI decreased MAP (115 ± 6 to 85 ± 3 and 121 ± 6 to 90 ± 3, n = 10, P < 0.05) only in the adult and the aged group and decreased (P < 0.05) LVdP/dt in all age groups. All MI groups had higher (P < 0.05) LVEDPs. Basal vascular density was decreased (6 ± 0.2 vs. 9±0.4 1/mm2, n = 7, P < 0.05) with aging. MI increased vascular density in all animal groups. However the percent of vascular density increase after MI is lower (P < 0.05) in the old rats. G-CSF administration after MI had no effect on vascular density. Contribution of CD34+ to neovascularization was decreased (27 ± 2, 13 ± 2, 8 ± 1 1/mm2, n = 5, P < 0.05) in both adult and aged. The number of circulating and BM CD34+ cells after MI is decreased (72±4 vs. 28±4 and 16±1 vs. 12±0.5, n = 6, P < 0.05) in the aged. Aging decreased (P < 0.05) the levels of secreted VEGF2 (315 ± 40, 213 ± 33, and 81 ± 9 pg/mg, n = 6). Thus, myocardial ischemia-induced neovascularization is impaired in the aged at least in part due to decreased number of circulating CD34+ and diminished levels of VEGF-2 released by these cells. Keywords: Myocardial infarction; Stem cells; Angiogenesis doi:10.1016/j.yjmcc.2007.03.213
Improvement of post-infarction cardiac remodeling by grafting stromal stem cells with a hyaluronan-based scaffold Marco Carboni, Matvey Tsivian, Giorgio Arpesella, Laura Foroni, Emanuela Fiumana, Gianandrea Pasquinelli, Bruno Nardo, Claudio Caldarera. INRC (I), Italy Tissue engineering has been proposed as a strategy to regenerate the infarcted myocardium. In this study, rat hearts were explanted, subjected to left coronary descending artery occlusion, and then grafted into the abdomen (aorta–aorta anastomosis) of receiving syngeneic rats. After 2 weeks, a pouch of 3 mm2 was made in the thickness of the ventricular wall at the level of the post-infarction scar. A hyaluronan-benzyl ester nonwoven mesh (Hyaff-11®, Fidia Advanced Biopolymers, Abano Terme, Padua, Italy), previously engineered for 3 weeks with rat bone marrow mesenchymal stem cells (MSCs), was introduced
into the pouch and the myocardial edges sutured with few stitches. Two weeks later we observed that the hyaluronan fibres had not been substantially degraded and most MSCs had migrated to the surrounding infarcted area where they were especially found close to small-sized vessels. Scar tissue was moderate in the engrafted region and the thickness of the corresponding ventricular wall was comparable to that of the non-infarcted remote area. Also, the left ventricular shortening fraction, evaluated by M-Mode echography, was not found decreased when compared to that measured just before construct transplantation. Therefore, this study suggests that postinfarction myocardial remodeling can be favourably affected by the grafting of MSCs delivered through a hyaluronan-based scaffold. Acknowledgment This research was funded by a grant of Compagnia di San Paolo, Turin, Italy. Keywords: Cardiac remodeling; Bone marrow stromal cells; Infarction doi:10.1016/j.yjmcc.2007.03.214
Electrical competence of infarcted heart following stem cell based regenerative therapy, in a rat model Leonardo Bocchi, Monia Savi, Roberta Berni, Gallia Graiani, Costanza Lagrasta, Federico Quaini, Ezio Musso. CISTAC, University of Parma, Italy Electrophysiological consequences of stem cell based cardiac regeneration have not been systematically analysed. We specifically addressed this issue in a rat model of myocardial infarction (MI). In 83 male Wistar rats with 1-month-old MI we measured: (i) the incidence of ventricular arrhythmias (VAs) by telemetry ECG recordings and (ii) basic electrophysiological parameters by epicardial mapping. Then, in 43 rats resident cardiac stem cells were mobilized by intramyocardial injection of IGF-1 + HGF while in the remaining 40 control animals saline was administered. A continuous infusion of BrdU via an osmotic pump was also started. Two weeks later electrophysiological measurements were repeated and, at sacrifice, left ventricular pressure and ± dP/dt were invasively recorded. Eventually, the heart was perfusion fixed for morphometric and immunohistochemical studies. Cytochine injection significantly reduced the incidence of VAs and increased ventricular refractoriness and the liminal length for action potential generation. Amelioration of hemodynamics was observed together with a positive remodelling of the left ventricle and a lower volume fraction of fibrosis. BrdU labelling significantly increased in myocytes of spared as well as infarcted myocardium where we found clusters of small cycling myocytes expressing Cx43 and N-chaderin. A 2- to 3-fold increased expression of Cx43 in the infarcted tissue was also shown by Western blot analysis. In
ABSTRACTS / Journal of Molecular and Cellular Cardiology 42 (2007) S88–S101
conclusion, stem cell based regeneration of the infarcted heart leads to both a better mechanical function and a reduction of cardiac electrical instability. Keywords: Stem cells; Arrhythmias; Infarction doi:10.1016/j.yjmcc.2007.03.215
Mesenchymal stem cells supernatant protects cardiomyocytes from death induced by hypoxia and reoxygenation D. Angoulvant, F. Ivanes, R. Ferrera, V. Guyot, O. Gateau Roesch, M. Ovize. Inserm U886, Université Claude Bernard Lyon1, Lyon, France Objectives: To study the protective effect of mesenchymal stem cells (MSC) on cell death induced by hypoxia followed by reperfusion (H/R) on cultured neonatal rat cardiomyocytes (NRC). This effect will be compared with the protection induced by ciclosporin A (CsA), a potent inhibitor of mitochondrial permeability transition pore (mPTP) opening that reduces cell death during post ischemic reperfusion. Method: MSC and NRC were isolated and cultured. NRC (2.106) were subjected to 4 h hypoxia followed by 16-h reoxygenation. Five groups were used (n = 4): sham with no hypoxia, control subjected to H/R only, CsA with 200 nM of CsA added at the onset of reoxygenation, MSC with 2.105 MSC added at the onset of reoxygenation, and MSCSN with MSC supernatant only (MSCSN) added at the onset of reoxygenation. Cell death was assessed by LDH level in NRC supernatant at the end of reoxygenation. Results: LDH after reoxygenation reached 398 U/l in the control group versus 1 U/l in the sham group. Compared to control group we observed a 95% reduction of LDH in the CsA group (p < 0.01), 83% reduction in the MSC group (p < 0.05) and 94% reduction in the MSCSN group (p < 0.01). There was no significant difference between these 3 groups. Conclusion: MSC added at the onset of reoxygenation reduce cell death induced by H/R. This protection is comparable to that observed after mPTP inhibition by CsA and is also observed with MSCSN only suggesting a paracrine mechanism of this cell protection. Keywords: Cardioprotection; Stem cells; Ischemia reperfusion doi:10.1016/j.yjmcc.2007.03.216
Non-invasive monitoring of in situ pO2 and functional recovery in infarct hearts transplanted with myoblasts Periannan Kuppusamy, Mahmood Khan, Vijay K. Kutala. Davis Heart and Lung Research Institute, Ohio State University, Columbus, OH 43210, USA A major limitation of cell therapy for the treatment of ischemic heart disease is the inability to determine the distribution of
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the transplanted cells and the measurement of local tissue oxygenation (pO2). The goal of this study was to establish a non-invasive method, using oxygen-sensing nanoparticulate probes detectable by EPR spectroscopy, for in vivo monitoring of stem-cell therapy in infarct myocardium. Myocardial infarction was created by permanent ligation of LCA in mice. Mouse skeletal myoblasts (SM; isolated from skeletal muscle) were labeled with oxygen-sensing probes (LiNc-BuO) and injected in the infarcted region. In situ myocardial pO2 was monitored noninvasively and repeatedly for several weeks by using in vivo EPR spectroscopy. Histological and hemodynamic studies were performed at the termination of the pO2 measurements. Differentiation of SM cells into skeletal myocytes was confirmed by MY-32 staining at 1 to 4 weeks after celltransplantation. EPR measurements established the presence of the probe in the infarcted hearts for up to 4 weeks, and possibly beyond. The baseline myocardial pO2 of normal (non-ischemic) tissue was 15.8±1.2 mm Hg (N = 7), which decreased to 1.7 ± 0.7 mm Hg in the ischemic region induced by LCA ligation. The tissue pO2 was significantly enhanced at the site of treatment. The SM-treated mice also showed significant improvements in heart function. For the first time, using an oxygen-sensing paramagnetic spin probe, we have demonstrated the feasibility of in vivo tracking of transplanted stem cells and changes in myocardial tissue pO2 at the site of implantation. Keywords: Cell therapy; Tissue oxygenation; Myocardial infarction doi:10.1016/j.yjmcc.2007.03.217
Role of the stem cell antigen-1 (SCA-1) in the maintenance of resident cardiac precursors N. Rosenblatt-Velin, S. Ogay, A. Felley, A. Domenighetti, M. Lepore, Th. Pedrazzini. Department of Medicine, University of Lausanne, Medical School, 1011 Lausanne, Switzerland In a previous work, we showed that cardiac precursor cells (CPCs) found in the heart of neonatal mice can differentiate in vitro and in vivo into functional cardiomyocytes. CPCs express the stem cell antigen (Sca)-1. Thus, the aim of our project was to determine the importance of Sca-1 in the maintenance and selfrenewal capacity of CPCs. Therefore, we sorted Sca-1+ and Sca-1- cells from wild-type mouse hearts, and induced them to differentiate into cardiomyocytes. Both Sca-1− or Sca-1+ cell populations contained precursors capable of producing differentiated cardiomyocytes in vitro but also in vivo. We next isolated a cardiac stem cell clone that stably expressed Sca-1. Clonal cells expressed markers of mesenchymal stem cells and could differentiate spontaneously into immature cardiomyocytes in vitro. Furthermore, these cells migrated to the heart and differentiated into cardiomyocytes when injected into neonatal mice. Finally, we showed that cardiac function was significantly affected in
ABSTRACTS / Journal of Molecular and Cellular Cardiology 42 (2007) S88–S101 +
Impaired contribution of circulating CD34 in myocardial ischemia-induced neovascularization with age Mohamed Gaballa, Hoang Thai, Steven Goldman. University of Arizona, SHC, Tucson, AZ, USA Impaired angiogenesis is documented in the aged. However, the effects of age on ischemia-induced cardiac vasculogenesis are unknown. To examine changes in ischemia-induced cardiac neovascularization with aging, myocardial infarction (MI) was created by LAD ligation in Brown Norway Fisher F344 cross at 3 ages: 6 weeks, 6 months, and 24 months rats (n = 10 per group). A 3D collagen scaffold transplanted onto the heart immediately after MI and bone marrow derived CD34+ cells or 50 ng/kg G-CSF was injected IV. Aging increased MAP, 90 ± 4, 115 ± 6, 121 ± 6, n = 10, P < 0.05, while MI decreased MAP (115 ± 6 to 85 ± 3 and 121 ± 6 to 90 ± 3, n = 10, P < 0.05) only in the adult and the aged group and decreased (P < 0.05) LVdP/dt in all age groups. All MI groups had higher (P < 0.05) LVEDPs. Basal vascular density was decreased (6 ± 0.2 vs. 9±0.4 1/mm2, n = 7, P < 0.05) with aging. MI increased vascular density in all animal groups. However the percent of vascular density increase after MI is lower (P < 0.05) in the old rats. G-CSF administration after MI had no effect on vascular density. Contribution of CD34+ to neovascularization was decreased (27 ± 2, 13 ± 2, 8 ± 1 1/mm2, n = 5, P < 0.05) in both adult and aged. The number of circulating and BM CD34+ cells after MI is decreased (72±4 vs. 28±4 and 16±1 vs. 12±0.5, n = 6, P < 0.05) in the aged. Aging decreased (P < 0.05) the levels of secreted VEGF2 (315 ± 40, 213 ± 33, and 81 ± 9 pg/mg, n = 6). Thus, myocardial ischemia-induced neovascularization is impaired in the aged at least in part due to decreased number of circulating CD34+ and diminished levels of VEGF-2 released by these cells. Keywords: Myocardial infarction; Stem cells; Angiogenesis doi:10.1016/j.yjmcc.2007.03.213
Improvement of post-infarction cardiac remodeling by grafting stromal stem cells with a hyaluronan-based scaffold Marco Carboni, Matvey Tsivian, Giorgio Arpesella, Laura Foroni, Emanuela Fiumana, Gianandrea Pasquinelli, Bruno Nardo, Claudio Caldarera. INRC (I), Italy Tissue engineering has been proposed as a strategy to regenerate the infarcted myocardium. In this study, rat hearts were explanted, subjected to left coronary descending artery occlusion, and then grafted into the abdomen (aorta–aorta anastomosis) of receiving syngeneic rats. After 2 weeks, a pouch of 3 mm2 was made in the thickness of the ventricular wall at the level of the post-infarction scar. A hyaluronan-benzyl ester nonwoven mesh (Hyaff-11®, Fidia Advanced Biopolymers, Abano Terme, Padua, Italy), previously engineered for 3 weeks with rat bone marrow mesenchymal stem cells (MSCs), was introduced
into the pouch and the myocardial edges sutured with few stitches. Two weeks later we observed that the hyaluronan fibres had not been substantially degraded and most MSCs had migrated to the surrounding infarcted area where they were especially found close to small-sized vessels. Scar tissue was moderate in the engrafted region and the thickness of the corresponding ventricular wall was comparable to that of the non-infarcted remote area. Also, the left ventricular shortening fraction, evaluated by M-Mode echography, was not found decreased when compared to that measured just before construct transplantation. Therefore, this study suggests that postinfarction myocardial remodeling can be favourably affected by the grafting of MSCs delivered through a hyaluronan-based scaffold. Acknowledgment This research was funded by a grant of Compagnia di San Paolo, Turin, Italy. Keywords: Cardiac remodeling; Bone marrow stromal cells; Infarction doi:10.1016/j.yjmcc.2007.03.214
Electrical competence of infarcted heart following stem cell based regenerative therapy, in a rat model Leonardo Bocchi, Monia Savi, Roberta Berni, Gallia Graiani, Costanza Lagrasta, Federico Quaini, Ezio Musso. CISTAC, University of Parma, Italy Electrophysiological consequences of stem cell based cardiac regeneration have not been systematically analysed. We specifically addressed this issue in a rat model of myocardial infarction (MI). In 83 male Wistar rats with 1-month-old MI we measured: (i) the incidence of ventricular arrhythmias (VAs) by telemetry ECG recordings and (ii) basic electrophysiological parameters by epicardial mapping. Then, in 43 rats resident cardiac stem cells were mobilized by intramyocardial injection of IGF-1 + HGF while in the remaining 40 control animals saline was administered. A continuous infusion of BrdU via an osmotic pump was also started. Two weeks later electrophysiological measurements were repeated and, at sacrifice, left ventricular pressure and ± dP/dt were invasively recorded. Eventually, the heart was perfusion fixed for morphometric and immunohistochemical studies. Cytochine injection significantly reduced the incidence of VAs and increased ventricular refractoriness and the liminal length for action potential generation. Amelioration of hemodynamics was observed together with a positive remodelling of the left ventricle and a lower volume fraction of fibrosis. BrdU labelling significantly increased in myocytes of spared as well as infarcted myocardium where we found clusters of small cycling myocytes expressing Cx43 and N-chaderin. A 2- to 3-fold increased expression of Cx43 in the infarcted tissue was also shown by Western blot analysis. In
ABSTRACTS / Journal of Molecular and Cellular Cardiology 42 (2007) S88–S101
conclusion, stem cell based regeneration of the infarcted heart leads to both a better mechanical function and a reduction of cardiac electrical instability. Keywords: Stem cells; Arrhythmias; Infarction doi:10.1016/j.yjmcc.2007.03.215
Mesenchymal stem cells supernatant protects cardiomyocytes from death induced by hypoxia and reoxygenation D. Angoulvant, F. Ivanes, R. Ferrera, V. Guyot, O. Gateau Roesch, M. Ovize. Inserm U886, Université Claude Bernard Lyon1, Lyon, France Objectives: To study the protective effect of mesenchymal stem cells (MSC) on cell death induced by hypoxia followed by reperfusion (H/R) on cultured neonatal rat cardiomyocytes (NRC). This effect will be compared with the protection induced by ciclosporin A (CsA), a potent inhibitor of mitochondrial permeability transition pore (mPTP) opening that reduces cell death during post ischemic reperfusion. Method: MSC and NRC were isolated and cultured. NRC (2.106) were subjected to 4 h hypoxia followed by 16-h reoxygenation. Five groups were used (n = 4): sham with no hypoxia, control subjected to H/R only, CsA with 200 nM of CsA added at the onset of reoxygenation, MSC with 2.105 MSC added at the onset of reoxygenation, and MSCSN with MSC supernatant only (MSCSN) added at the onset of reoxygenation. Cell death was assessed by LDH level in NRC supernatant at the end of reoxygenation. Results: LDH after reoxygenation reached 398 U/l in the control group versus 1 U/l in the sham group. Compared to control group we observed a 95% reduction of LDH in the CsA group (p < 0.01), 83% reduction in the MSC group (p < 0.05) and 94% reduction in the MSCSN group (p < 0.01). There was no significant difference between these 3 groups. Conclusion: MSC added at the onset of reoxygenation reduce cell death induced by H/R. This protection is comparable to that observed after mPTP inhibition by CsA and is also observed with MSCSN only suggesting a paracrine mechanism of this cell protection. Keywords: Cardioprotection; Stem cells; Ischemia reperfusion doi:10.1016/j.yjmcc.2007.03.216
Non-invasive monitoring of in situ pO2 and functional recovery in infarct hearts transplanted with myoblasts Periannan Kuppusamy, Mahmood Khan, Vijay K. Kutala. Davis Heart and Lung Research Institute, Ohio State University, Columbus, OH 43210, USA A major limitation of cell therapy for the treatment of ischemic heart disease is the inability to determine the distribution of
S99
the transplanted cells and the measurement of local tissue oxygenation (pO2). The goal of this study was to establish a non-invasive method, using oxygen-sensing nanoparticulate probes detectable by EPR spectroscopy, for in vivo monitoring of stem-cell therapy in infarct myocardium. Myocardial infarction was created by permanent ligation of LCA in mice. Mouse skeletal myoblasts (SM; isolated from skeletal muscle) were labeled with oxygen-sensing probes (LiNc-BuO) and injected in the infarcted region. In situ myocardial pO2 was monitored noninvasively and repeatedly for several weeks by using in vivo EPR spectroscopy. Histological and hemodynamic studies were performed at the termination of the pO2 measurements. Differentiation of SM cells into skeletal myocytes was confirmed by MY-32 staining at 1 to 4 weeks after celltransplantation. EPR measurements established the presence of the probe in the infarcted hearts for up to 4 weeks, and possibly beyond. The baseline myocardial pO2 of normal (non-ischemic) tissue was 15.8±1.2 mm Hg (N = 7), which decreased to 1.7 ± 0.7 mm Hg in the ischemic region induced by LCA ligation. The tissue pO2 was significantly enhanced at the site of treatment. The SM-treated mice also showed significant improvements in heart function. For the first time, using an oxygen-sensing paramagnetic spin probe, we have demonstrated the feasibility of in vivo tracking of transplanted stem cells and changes in myocardial tissue pO2 at the site of implantation. Keywords: Cell therapy; Tissue oxygenation; Myocardial infarction doi:10.1016/j.yjmcc.2007.03.217
Role of the stem cell antigen-1 (SCA-1) in the maintenance of resident cardiac precursors N. Rosenblatt-Velin, S. Ogay, A. Felley, A. Domenighetti, M. Lepore, Th. Pedrazzini. Department of Medicine, University of Lausanne, Medical School, 1011 Lausanne, Switzerland In a previous work, we showed that cardiac precursor cells (CPCs) found in the heart of neonatal mice can differentiate in vitro and in vivo into functional cardiomyocytes. CPCs express the stem cell antigen (Sca)-1. Thus, the aim of our project was to determine the importance of Sca-1 in the maintenance and selfrenewal capacity of CPCs. Therefore, we sorted Sca-1+ and Sca-1- cells from wild-type mouse hearts, and induced them to differentiate into cardiomyocytes. Both Sca-1− or Sca-1+ cell populations contained precursors capable of producing differentiated cardiomyocytes in vitro but also in vivo. We next isolated a cardiac stem cell clone that stably expressed Sca-1. Clonal cells expressed markers of mesenchymal stem cells and could differentiate spontaneously into immature cardiomyocytes in vitro. Furthermore, these cells migrated to the heart and differentiated into cardiomyocytes when injected into neonatal mice. Finally, we showed that cardiac function was significantly affected in