Wangde Dai

Allogeneic Mesenchymal Stem Cell Transplantation in Postinfarcted Rat Myocardium Short- and Long-Term Effects

Background—Mesenchymal stem cells (MSCs) have the potential to replace infarct scar, but the long-term effects are unknown. We studied short- and long-term effects of MSC transplantation on left ventricular (LV) function in a rat myocardial infarction model. Methods and Results—Saline (n=46) or MSCs labeled with 1,1′-dioctadecyl-3,3,3′3′-testramethylindocarbocyanine perchlorate (DiI; n=49, 2×106 cells each) were injected into the scar of a 1-week-old myocardial infarction in Fischer rats. The presence and differentiation of engrafted cells and their effect on LV ejection fraction was assessed. At 4 weeks, LV stroke volume was significantly greater in the MSC-treated group (145±9 &mgr;L) than in the saline group (122±3 &mgr;L, P=0.032), and LV ejection fraction was significantly greater in MSC-treated animals (43.8±1.0%) than in the saline group (38.8±1.1%, P=0.0027). However, at 6 months, these benefits of MSC treatment were lost. DiI-positive cells were observed in the MSC group at 2 weeks and at 3 and 6 months. Expression of the muscle-specific markers α-actinin, myosin heavy chain, phospholamban, and tropomyosin was not observed at 2 weeks in DiI-positive cells. At 3 and 6 months, the DiI-positive cells were observed to express the above muscle-specific markers, but they did not fully evolve into an adult cardiac phenotype. Some of the DiI-positive cells expressed von Willebrand factor. Conclusions—Allogeneic MSCs survive in infarcted myocardium as long as 6 months and express markers that suggest muscle and endothelium phenotypes. MSCs improved global LV function at 4 weeks; however, this benefit was transient, which suggests a possible early paracrine effect.

Reduction of Ischemia/Reperfusion Injury With Bendavia, a Mitochondria-Targeting Cytoprotective Peptide

Background Manifestations of reperfusion injury include myocyte death leading to infarction, contractile dysfunction, and vascular injury characterized by the “no-reflow” phenomenon. Mitochondria-produced reactive oxygen species are believed to be centrally involved in each of these aspects of reperfusion injury, although currently no therapies reduce reperfusion injury by targeting mitochondria specifically. Methods and Results We investigated the cardioprotective effects of a mitochondria-targeted peptide, Bendavia (Stealth Peptides), across a spectrum of experimental cardiac ischemia/reperfusion models. Postischemic administration of Bendavia reduced infarct size in an in vivo sheep model by 15% (P=0.02) and in an ex vivo guinea pig model by 38% to 42% (P<0.05). In an in vivo rabbit model, the extent of coronary no-reflow was assessed with Thioflavin S staining and was significantly smaller in the Bendavia group for any given ischemic risk area than in the control group (P=0.0085). Myocardial uptake of Bendavia was ≈25% per minute, and uptake remained consistent throughout reperfusion. Postischemic recovery of cardiac hemodynamics was not influenced by Bendavia in any of the models studied. Isolated myocytes exposed to hypoxia/reoxygenation showed improved survival when treated with Bendavia. This protection appeared to be mediated by lowered reactive oxygen species–mediated cell death during reoxygenation, associated with sustainment of mitochondrial membrane potential in Bendavia-treated myocytes. Conclusions Postischemic administration of Bendavia protected against reperfusion injury in several distinct models of injury. These data suggest that Bendavia is a mitochondria-targeted therapy that reduces reperfusion injury by maintaining mitochondrial energetics and suppressing cellular reactive oxygen species levels. (J Am Heart Assoc. 2012;1:e001644 doi: 10.1161/JAHA.112.001644.)

Delivering stem cells to the heart in a collagen matrix reduces relocation of cells to other organs as assessed by nanoparticle technology

AIM A limitation of cell therapy for heart disease is the fact that stem cells injected directly into the myocardium are capable of entering the vasculature and migrating to remote organs. We determined whether retention of mesenchymal stem cells (MSCs) in the infarcted myocardium could be improved by implanting the cells in a collagen matrix. METHODS A myocardial infarction was induced by ligation of the left anterior descending coronary artery in Fischer rats. A total of 7 days after myocardial infarction, saline (n = 12), saline plus 2 million bone marrow-derived rat MSCs labeled with isotopic colloidal nanoparticles containing europium (n = 13), collagen (n = 13) or collagen plus 2 million labeled MSCs (n = 13) were directly injected into the infarcted myocardium. Tissues from the infarcted myocardium, noninfarcted myocardium, lung, liver, spleen and kidney were sampled 4 weeks later. Distribution of grafted MSCs was quantitatively analyzed by measuring the nanoparticle radioactivity in these tissues. Cardiac function was assessed by left ventriculography. RESULTS There were zero nanoparticles detected in the tissues that received saline or collagen alone into the heart. Nanoparticles were detected in the heart and remote organs in the saline plus MSC group. Labeled cells (expressed as cell number/g tissue weight) were present in three out of 13 lungs (mean of 12,724 +/- 7060 cells/g), four out of 13 livers (12,301 +/- 5924 cells/g), 11 out of 13 spleens (57,228 +/- 11,483 cells/g), zero out of 13 kidneys, 13 out of 13 infarcted myocardium (8,006,835 +/- 1,846,462 cells/g) and nine out of 13 noninfarcted myocardium (167,331 +/- 47,007 cells/g). However, compared with the saline plus MSC group, nanoparticles were detected to a lesser extent in remote organs in collagen plus MSC group. Nanoparticles were detected in two out of 13 lungs (4631 +/- 3176 cells/g; p = NS), zero out of 13 livers (0 cells/g; p <0.05 vs saline plus MSC), four out of 13 spleens (24,060 +/- 17,373 cells/g; p <0.05), zero out of 13 kidneys (p = NS) and five out of 13 noninfarcted myocardium (51,522 +/- 21,548 cells/g; p <0.05). In the collagen plus MSC group, nanoparticles were detected in 12 out of 13 infarcted myocardium (4,830,050 +/- 592,215 cells/g), which did not significantly differ from that in the saline plus MSC group (p = NS). Both saline plus MSCs and collagen alone improved left ventricular ejection fraction compared with saline treatment. However, collagen plus MSCs failed to improve cardiac function. CONCLUSIONS Collagen matrix as a delivery vehicle significantly reduced the relocation of transplanted MSCs to remote organs and noninfarcted myocardium.

Role of a paracrine action of mesenchymal stem cells in the improvement of left ventricular function after coronary artery occlusion in rats.

PURPOSE We aimed to determine whether soluble factors released by cultured mesenchymal stem cells (MSCs) improved cardiac function in an experimental model of myocardial infarction. METHODS MSCs were cultured in fresh medium. The conditioned medium, which contained factors secreted by MSCs, was collected after 4 days of culture. Fischer rats with 1-week-old myocardial infarction were divided into four groups that received: saline (n = 12); fresh medium (n = 10); conditioned medium (n = 8); or 2 million MSCs in fresh medium (n = 10) by direct intramyocardial injection. A total of 4 weeks later, left ventricular (LV) function was assessed by LV angiogram and by LV catheterization. Hearts were processed for histology. RESULTS Before treatment, LV angiogram assessment demonstrated that the baseline LV function was comparable among the four groups. At 4 weeks after treatment, LV angiogram and LV catheterization showed that LV ejection fraction was better in the fresh medium (49.5 +/- 1.0%), conditioned medium (48.5 +/- 2.1%) and MSCs groups (49.9 +/- 4.2%) than in the saline group (43.7 +/- 1.2%; p < 0.05). There were no significant differences in heart rate, blood pressure, postmortem LV volume, infarct size or septum thickness among the groups. The scar thickness was similar in the saline (395 +/- 31 microm), fresh medium (404 +/- 30 microm) and conditioned medium (397 +/- 34 microm) groups, but significantly thicker in the MSCs group (560 +/- 51 microm; p < 0.05). CONCLUSION Fresh medium, conditioned medium and MSC injection all improved LV function at 4 weeks after treatment compared with saline treatment in a rat myocardial infarct model; only MSCs increased wall thickness. Since the culture medium contains nutrients and bovine serum, the roles of the soluble factors released by MSCs might be masked. The effect of these nutrients needs further investigation.

Transplantation of neonatal cardiomyocytes after permanent coronary artery occlusion increases regional blood flow of infarcted myocardium.

BACKGROUND Cellular cardiomyoplasty is a promising approach for rebuilding scar tissue after acute myocardial infarction. However, the angiogenic potential of transplanted immature cardiomyocytes and their effect on regional myocardial blood flow (RMBF) after coronary artery occlusion remain to be evaluated. METHODS AND RESULTS Intramyocardial injection of cultured neonatal cardiomyocytes (4 x 10(6) cells/50-70 microliter) into the scar 1 week after permanent coronary occlusion in rats resulted in improved RMBF in the infarct 4 weeks after transplantation (radioactive microspheres, 0.97 +/- 0.18 ml/min/g) in comparison to medium-injected hearts (0.61 +/- 0.11 ml/min/g, P < 0.047). The macroscopic perfusion defect after in vivo staining with the blue dye 50% Uniperse blue was significantly smaller in the cell transplantation group (1.5 +/- 0.3% of the heart) compared to the medium group (3.0 +/- 0.6%, P < 0.017). Clusters of engrafted cells within the scar demonstrated a high capillary density (1217 +/- 114 perfused (blue) capillaries/mm(2)); however, in the scar tissue itself capillary density in the cell group (156 +/- 62/mm(2)) did not significantly differ from the medium group (125 +/- 10/mm(2)), suggesting that neo-angiogenesis was confined to regions of successful engraftment (non-infarcted tissue: 1924 +/- 114 perfused capillaries/mm(2)). The transplantation group was characterized by smaller diastolic and systolic left ventricular volumes, as assessed by intravenous ventriculography, along with thickened infarcts (0.93 +/- 0.07 vs. 0.75 +/- 0.04 mm, P < 0.020) and lower infarct expansion indices (0.64 +/- 0.07 vs. 0.83 +/- 0.06, P < 0.023), as determined by post-mortem morphometry of histologic slides. CONCLUSIONS Transplantation of neonatal cardiomyocytes induced neo-angiogenesis in zones of successful cell engraftment within the scar, which effectively enhanced tissue perfusion.

Bendavia, a mitochondria-targeting peptide, improves postinfarction cardiac function, prevents adverse left ventricular remodeling, and restores mitochondria-related gene expression in rats.

Abstract: We evaluated the post-myocardial infarction (MI) therapeutic effects of Bendavia. Two hours after coronary artery ligation, rats were randomized to receive chronic Bendavia treatment (n = 28) or water (n = 26). Six weeks later, Bendavia significantly reduced scar circumference (39.7% ± 2.2%) compared with water treatment (47.4% ± 0.03%, P = 0.024) and reduced left ventricular (LV) volume by 8.9% (P = 0.019). LV fractional shortening was significantly improved by Bendavia (28.8% ± 1.7%) compared with water treatment (23.8% ± 1.8%, P = 0.047). LV ejection fraction was higher with Bendavia (55.3% ± 1.4%) than water treatment (49.3% ± 1.4%, P = 0.005). Apoptosis, within the MI border zone, was significantly less in the Bendavia group (32% ± 3%, n = 12) compared with the water group (41% ± 2%, n = 12; P = 0.029). Bendavia reversed mitochondrial function–related gene expression in the MI border, which was largely reduced in water-treated rats. Bendavia improved complex-I and -IV activity, and reduced production of reactive oxygen species and cytosolic cytochrome c level in the peri-infarcted region. Bendavia improved post-MI cardiac function, prevented infarct expansion and adverse LV remodeling, and restored mitochondria-related gene expression, complex-I and -IV activity, and reduced reactive oxygen species and cardiomyocyte apoptosis in the noninfarcted MI border.

Bendavia restores mitochondrial energy metabolism gene expression and suppresses cardiac fibrosis in the border zone of the infarcted heart

AIMS We have observed that Bendavia, a mitochondrial-targeting peptide that binds the phospholipid cardiolipin and stabilizes the components of electron transport and ATP generation, improves cardiac function and prevents left ventricular remodeling in a 6week rat myocardial infarction (MI) model. We hypothesized that Bendavia restores mitochondrial biogenesis and gene expression, suppresses cardiac fibrosis, and preserves sarco/endoplasmic reticulum (SERCA2a) level in the noninfarcted border zone of infarcted hearts. MAIN METHODS Starting 2h after left coronary artery ligation, rats were randomized to receive Bendavia (3mg/kg/day), water or sham operation. At 6weeks, PCR array and qRT-PCR was performed to detect gene expression. Picrosirius red staining was used to analyze collagen deposition. KEY FINDINGS There was decreased expression of 70 out of 84 genes related to mitochondrial energy metabolism in the border zone of untreated hearts. This down-regulation was largely reversed by Bendavia treatment. Downregulated mitochondrial biogenesis and glucose & fatty acid (FA) oxidation related genes were restored by administration of Bendavia. Matrix metalloproteinase (MMP9) and tissue inhibitor of metalloproteinase (TIMP1) gene expression were significantly increased in the border zone of untreated hearts. Bendavia completely prevented up-regulation of MMP9, but maintained TIMP1 gene expression. Picrosirius red staining demonstrated that Bendavia suppressed collagen deposition within border zone. In addition, Bendavia showed a trend toward restoring SERCA2a expression. SIGNIFICANCE Bendavia restored expression of mitochondrial energy metabolism related genes, prevented myocardial matrix remodeling and preserved SERCA2a expression in the noninfarcted border, which may have contributed to the preservation of cardiac structure and function.

Ischemic preconditioning maintains cardioprotection in aging normotensive and spontaneously hypertensive rats.

We determined whether ischemic preconditioning could reduce infarct size and improve cardiac function in both aging normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). The left anterior descending coronary artery was occluded for 1h followed by 3 h reperfusion in aging ( approximately 16 months old) SHR rats and age-matched WKY rats. Hearts were either preconditioned or not (control group) prior to 1h of coronary artery occlusion. The preconditioning regimen consisted of three cycles of 3 min occlusion followed by 5 min reperfusion applied prior to the subsequent 1h occlusion. In WKY (n=12 each group), the risk zone was similar in the control (51+/-2%) and preconditioned group (46+/-2%; p=0.1). Preconditioning significantly reduced infarct size (as a percentage of the ischemic risk zone) (24+/-6%) compared to controls (51+/-5%; p=0.0026). In SHR rats (n=9 each group), the risk zone was smaller in the preconditioning group (41+/-3%) than in the control group (51+/-3%; p=0.035). Infarct size (as % of ischemic risk zone) was also significantly reduced in the preconditioned group (13+/-4%) compared to controls (62+/-5%; p<0.0001). For both WKY and SHR rats, for any sized risk zone the infarct size was smaller in preconditioned hearts compared with the control hearts. Preconditioning improved aspects of LV function during ischemia and reperfusion phase in SHR rats, but these benefits were not observed in the WKY rats. Preconditioning maintains powerful cardioprotection in aging normotensive hearts as well as aging hypertrophied hearts.

Functional and Histological Assessment of an Experimental Model of Takotsubo's Cardiomyopathy

Background Our objectives were to characterize functional and structural features of an experimental model of Takotsubo cardiomyopathy, and its response to beta‐blockers. Methods and Results In protocol 1, a dose‐finding study: 69 rats received various doses of isoproterenol (ISO) and echocardiographic and histologic parameters were measured on days 2 to 3 or day 8. There were no dose‐dependent effects and, out of 69 ISO‐treated rats, 40 (58.0%) survived and 29 (42.0%) died within 24 hours. Of survivors, 30 had apical akinesis averaging 12.1±1.6% of the long axis LV circumference. Out of the 40 survivors, 32.5% showed apical akinesis ≥10%, 42.5% showed akinesis<10% and 25% showed no apical akinesis. The basal portion of the LV was always preserved. At 24 hours, histology and ultrastructure showed necrosis, vacuolization, lipid droplets, mononuclear cell infiltration, damaged mitochondria, and edema. On day 8, apical akinesis fully resolved but histologic abnormalities were still present. In protocol 2, rats were randomized to Control; ISO100 mg/kg; propranolol+ISO; and metoprolol+ISO groups. Pretreatment with propranolol and metoprolol improved survival to 90% and 100% respectively, compared with 60% in the ISO group, but did not reduce the incidence and extent of akinesis or the structural damage. Conclusion TC can be mimicked in a rat model of ISO exposure that demonstrates apical akinesis on days 2 to 3 with full recovery of systolic regional wall motion abnormality despite the presence of persistent foci of necrosis and fibrosis on day 8. Pretreatment with beta‐blockers improved survival but did not affect structural and functional alterations.

An apoptosis signal-regulating kinase 1 inhibitor reduces cardiomyocyte apoptosis and infarct size in a rat ischemia-reperfusion model.

Purposes: We determined whether a small molecule inhibitor of apoptosis signal–regulating kinase 1 (ASK1-i) could reduce myocardial infarct size in a rat ischemia/reperfusion model. Methods and Results: Sprague–Dawley rats were randomized to 3 groups: ASK1-i infusion (n = 16), vehicle infusion (n = 16), or ischemic preconditioning (IPC; n = 15). Infusion of ASK1-i (10 mg/kg, iv) or vehicle commenced 45 minutes before myocardial ischemia. IPC consisted of 3 cycles of 3 minutes of coronary occlusion followed by 5 minutes of reperfusion immediately before index myocardial ischemia, which consisted of 30-minute left coronary occlusion followed by 180 minutes of reperfusion. Pathologic analysis revealed no significant difference in the ischemic risk size among the 3 groups. ASK1-I and IPC significantly reduced myocardial infarct size (27.7% ± 3.3%, 16.5% ± 3.4%, and 41.5% ± 4.8% in the ASK1-i group, the IPC group, and the vehicle group, respectively; P = 0.0002) and apoptosis (the percentage of apoptotic nuclei averaged 11.6% ± 1.0%, 10.2% ± 1.7%, and 17.7% ± 2.0% in the ASK1-i group, IPC group, and vehicle group, respectively, P = 0.0055). Conclusions: A small molecule inhibitor of ASK1 was shown for the first time to reduce apoptosis and myocardial infarct size in a rat model of ischemia/reperfusion.