Akima Harada

Cell-sheet Therapy With Omentopexy Promotes Arteriogenesis and Improves Coronary Circulation Physiology in Failing Heart

Cell-sheet transplantation induces angiogenesis for chronic myocardial infarction (MI), though insufficient capillary maturation and paucity of arteriogenesis may limit its therapeutic effects. Omentum has been used clinically to promote revascularization and healing of ischemic tissues. We hypothesized that cell-sheet transplantation covered with an omentum-flap would effectively establish mature blood vessels and improve coronary microcirculation physiology, enhancing the therapeutic effects of cell-sheet therapy. Rats were divided into four groups after coronary ligation; skeletal myoblast cell-sheet plus omentum-flap (combined), cell-sheet only, omentum-flap only, and sham operation. At 4 weeks after the treatment, the combined group showed attenuated cardiac hypertrophy and fibrosis, and a greater amount of functionally (CD31(+)/lectin(+)) and structurally (CD31(+)/α-SMA(+)) mature blood vessels, along with myocardial upregulation of relevant genes. Synchrotron-based microangiography revealed that the combined procedure increased vascularization in resistance arterial vessels with better dilatory responses to endothelium-dependent agents. Serial (13)N-ammonia PET showed better global coronary flow reserve in the combined group, mainly attributed to improvement in the basal left ventricle. Consequently, the combined group had sustained improvements in cardiac function parameters and better functional capacity. Cell-sheet transplantation with an omentum-flap better promoted arteriogenesis and improved coronary microcirculation physiology in ischemic myocardium, leading to potent functional recovery in the failing heart.

Improvement of Cardiac Stem Cell-Sheet Therapy for Chronic Ischemic Injury by Adding Endothelial Progenitor Cell Transplantation : Analysis of Layer-Specific Regional Cardiac Function

The transplantation of cardiac stem cell sheets (CSC sheets) is a promising therapeutic strategy for ischemic cardiomyopathy, although potential ischemia in the transplanted area remains a problem. Injected endothelial progenitor cells (EPCs) can reportedly induce angiogenesis in the injected area. We hypothesized that concomitant CSC sheet transplantation and EPC injection might show better therapeutic effects for chronic ischemic injury model than the transplantation of CSC sheets alone. Scaffold-free CSC sheets were generated from human c-kit-positive heart-derived cells. A porcine chronic ischemic injury model was generated by placing an ameroid constrictor around the left coronary artery for 4 weeks. The animals then underwent a sham operation, epicardial transplantation of CSC sheet over the ischemic area, intramyocardial injection of EPCs into the ischemic and peri-ischemic area, or CSC sheet transplantation plus EPC injection. The efficacy of each treatment was then assessed for 2 months. Speckle-tracking echocardiography was used to dissect the layer-specific regional systolic function by measuring the radial strain (RS). The epicardial RS in the ischemic area was similarly greater after treatment with the CSC-derived cell sheets alone (19 ± 5%) or in combination with EPC injection (20 ± 5%) compared with the EPC only (9 ± 4%) or sham (7 ± 1%) treatment. The endocardial RS in the ischemic area was greatest after the combined treatment (14 ± 1%), followed by EPC only (12 ± 1%), compared to the CSC only (11 ± 1%) and sham (9 ± 1%) treatments. Consistently, either epicardial CSC sheet implantation or intramyocardial EPC injection yielded increased capillary number and reduced cardiac fibrosis in the ischemic epicardium or endocardium, respectively. Concomitant EPC injection induced the migration of transplanted CSCs into the host myocardium, leading to further neovascularization and reduced fibrosis in the ischemic endocardium, compared to the CSC sole therapy. Transplantation of CSC sheets induced significant functional recovery of the ischemic epicardium, and concomitant EPC transplantation elicited transmural improvement in chronic ischemic injury.

Development of PET Imaging to Visualize Activated Macrophages Accumulated in the Transplanted iPSc-Derived Cardiac Myocytes of Allogeneic Origin for Detecting the Immune Rejection of Allogeneic Cell Transplants in Mice.

Allogeneic transplantation (Tx) of induced pluripotent stem cells (iPSCs) is a promising tissue regeneration therapy. However, this inevitably induces macrophage-mediated immune response against the graft, limiting its therapeutic efficacy. Monitoring the magnitude of the immune response using imaging tools would be useful for prolonging graft survival and increasing the therapy longevity. Minimally invasive quantitative detection of activated macrophages by medical imaging technologies such as positron emission tomography (PET) imaging targets translocator protein (TSPO), which is highly expressed on mitochondrial membrane, especially in activated macrophage. N,N-diethyl-2-[4-(2-fluoroethoxy) phenyl]-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-acetamide (DPA-714) is known as a TSPO ligand used in clinical settings. We herein hypothesized that immune rejection of the transplanted iPSC-derived cardiomyocytes (iPSC-CMs) of allogeneic origin may be quantitated using 18F-DPA-714-PET imaging study. iPSC-CM cell-sheets of C57BL/6 mice origin were transplanted on the surface of the left ventricle (LV) of C57BL/6 mice as a syngeneic cell-transplant model (syngeneic Tx group), or Balb/c mice as an allogeneic model (allogeneic Tx group). 18F-DPA-714-PET was used to determine the uptake ratio, calculated as the maximum standardized uptake value in the anterior and septal wall of the LV. The uptake ratio was significantly higher in the allogeneic Tx group than in the syngeneic group or the sham group at days 7 and day 10 after the cell transplantation. In addition, the immunochemistry showed significant presence of CD68 and CD3-positive cells at day 7 and 10 in the transplanted graft of the allogeneic Tx group. The expression of TSPO, CD68, IL-1 beta, and MCP-1 was significantly higher in the allogeneic Tx group than in the syngeneic Tx and the sham groups at day 7. The 18F-DPA-714-PET imaging study enabled quantitative visualization of the macrophages-mediated immune rejection of the allogeneic iPSC-cardiac. This imaging tool may enable the understanding and monitoring host-immune response of the host, allogeneic cell transplantation therapy.

Immunologic targeting of CD30 eliminates tumourigenic human pluripotent stem cells, allowing safer clinical application of hiPSC-based cell therapy

Induced pluripotent stem cells (iPSCs) are promising candidate cells for cardiomyogenesis in the failing heart. However, teratoma/tumour formation originating from undifferentiated iPSCs contaminating the graft is a critical concern for clinical application. Here, we hypothesized that brentuximab vedotin, which targets CD30, induces apoptosis in tumourigenic cells, thus increasing the safety of iPSC therapy for heart failure. Flow cytometry analysis identified consistent expression of CD30 in undifferentiated human iPSCs. Addition of brentuximab vedotin in vitro for 72 h efficiently induced cell death in human iPSCs, associated with a significant increase in G2/M phase cells. Brentuximab vedotin significantly reduced Lin28 expression in cardiomyogenically differentiated human iPSCs. Transplantation of human iPSC-derived cardiomyocytes (CMs) without treatment into NOG mice consistently induced teratoma/tumour formation, with a substantial number of Ki-67–positive cells in the graft at 4 months post-transplant, whereas iPSC-derived CMs treated with brentuximab vedotin prior to the transplantation did not show teratoma/tumour formation, which was associated with absence of Ki-67–positive cells in the graft over the same period. These findings suggest that in vitro treatment with brentuximab vedotin, targeting the CD30-positive iPSC fraction, reduced tumourigenicity in human iPSC-derived CMs, potentially providing enhanced safety for iPSC-based cardiomyogenesis therapy in clinical scenarios.

A prostacyclin agonist and an omental flap increased myocardial blood flow in a porcine chronic ischemia model

Objective: We hypothesized that therapeutic efficacy may be augmented by a combination of placing a sheet immersed in ONO‐1301SR, a slow‐release synthetic prostacyclin agonist‐inducing multiproangiogenic cytokines, over the left ventricle and a pedicled omental flap in a chronic myocardial infarct heart. Methods: A minipig chronic myocardial infarction was generated by placing an ameroid constrictor ring around the left anterior descending artery for 4 weeks. The minipigs were then assigned into 4 groups of 6 each: sham, omental flap only, ONO‐1301SR only, and ONO‐1301SR combined with an omental flap (combined). Four weeks after treatment, therapeutic efficacy was evaluated histologically and via several modalities used in the clinical setting. Results: In an angiogram and pressure wire study, the combined group induced development of collateral arteries to decrease the resistance and increase the flow reserve of microvasculature in the left circumflex territory. In a 13N‐ammonia positron emission tomography study, the combined group displayed a prominent increase in myocardial blood flow and myocardial flow reserve in the left circumflex territory, particularly at the infarct‐border region. Consequently, the combined group showed greater regional cardiac function in the left circumflex territory particularly at the infarct‐border region, contributing to a greater global ejection fraction with a smaller left ventricular endosystolic volume. Pathologically, attenuated fibrosis, nonswollen myocytes, and upgraded capillary density and proangiogenic cytokines were prominent in the combined group. Conclusions: ONO‐1301SR combined with a pedicled omental flap synergistically promoted myocardial angiogenesis, leading to function recovery in a porcine chronic myocardial infarction model.

Histone Modification Is Correlated With Reverse Left Ventricular Remodeling in Nonischemic Dilated Cardiomyopathy

BACKGROUND Although implantation of a left ventricular assist device (LVAD) induces reverse remodeling of the left ventricle in end-stage nonischemic dilated cardiomyopathy (DCM), the underlying mechanism is not fully understood. It has been shown that epigenetic modification, such as methylation or acetylation of the histone, is one of the most important upstream signals in cardiac failure. This study hypothesized that histone profiles may be modified by LVAD implantation for end-stage nonischemic DCM, in association with reverse left ventricular remodeling. METHODS Hemodynamic changes associated with histone modification profiles in the left ventricle were comprehensively assessed in 14 patients with a diagnosis of end-stage nonischemic DCM. These patients underwent LVAD implantation and subsequent cardiac transplantation in our institution (Osaka University Hospital, Osaka, Japan). Samples of normal left ventricle from 3 different people were used as a control. RESULTS After LVAD support for 2.5 ± 1.2 years, the study cohort showed a significant reverse remodeling of left ventricular function associated with histopathologic changes in the left ventricle, such as reduction of myocyte size. Although the left ventricle of the cohort histologically expressed less 3 histone methylation-related molecules (eg, H3 lysine 4 trimethylation [H3K4me3], H3 lysine 9 dimethylation [H3K9me2], and H3 lysine 9 trimethylation [H3K9me3]) compared with normal left ventricle, LVAD support reversed expression of these molecules, associated with up-regulation of H3 lysine 9 [H3K9] methyltransferase and suppressor of variegation 3-9 homologue 1 [SUV39H1] and with down-regulation of H3K9 demethylase and jumonji domains [JMJDs] in the LVAD-supported left ventricle. Moreover, expression of atrial natriuretic peptide and brain natriuretic peptide (ANP and BNP) was negatively correlated with that of H3K9me2 and H3K9me3. CONCLUSIONS The epigenetic state of cardiac myocytes (eg, as histone methylation) was substantially modulated in end-stage nonischemic DCM. LVAD support partially reversed the epigenetic state and its upstream signals, in association with pathologic and functional reverse remodeling.

Intravital imaging with two-photon microscopy reveals cellular dynamics in the ischeamia-reperfused rat heart

Recent advances in intravital microscopy have provided insight into dynamic biological events at the cellular level in both healthy and pathological tissue. However, real-time in vivo cellular imaging of the beating heart has not been fully established, mainly due to the difficulty of obtaining clear images through cycles of cardiac and respiratory motion. Here we report the successful recording of clear in vivo moving images of the beating rat heart by two-photon microscopy facilitated by cardiothoracic surgery and a novel cardiac stabiliser. Subcellular dynamics of the major cardiac components including the myocardium and its subcellular structures (i.e., nuclei and myofibrils) and mitochondrial distribution in cardiac myocytes were visualised for 4–5 h in green fluorescent protein-expressing transgenic Lewis rats at 15 frames/s. We also observed ischaemia/reperfusion (I/R) injury-induced suppression of the contraction/relaxation cycle and the consequent increase in cell permeability and leukocyte accumulation in cardiac tissue. I/R injury was induced in other transgenic mouse lines to further clarify the biological events in cardiac tissue. This imaging system can serve as an alternative modality for real time monitoring in animal models and cardiological drug screening, and can contribute to the development of more effective treatments for cardiac diseases.

Influence of coronary architecture on the variability in myocardial infarction induced by coronary ligation in rats

It has been shown that the size of myocardial infarction in rats created by coronary ligation technique is not uniform, varying from 4% to 65%. We hypothesized that infarct size variability induced by coronary artery ligation might be caused by coronary artery branching pattern. Coronary artery angiography was performed in 50 normal Lewis rats and in chronic myocardial infarction models in which coronary artery was ligated immediately below the left atrial appendage or 2mm distal to the left atrial appendage (n = 25 for each), followed by histological analysis. Unlike the human, the rats had a single major septal artery arising from the proximal part of the left coronary artery (n = 30) or right coronary artery (n = 20). There were three branching patterns of left circumflex artery (LCX): 33 (66%) had LCX branching peripherally from a long left main coronary artery (LMCA), while the remainder 17 (34%) had the LCX branching from the proximal part of the septal artery or a short LMCA. The rats with distal coronary ligation presented myocardial infarction localized to an anterior territory irrespective of LCX branching pattern. In the rats with proximal coronary ligation, 64% (n = 16) had broad myocardial infarction involving the anterior and lateral territories, while the remainder (36%, n = 9) had myocardial infarction localized to an anterior territory with the intact LCX arising proximally from a short LMCA. The interventricular septum was spared from infarction in all rats because of its anatomical location. Infarct size variations were caused not only by ligation site but also by varying LCX branching patterns. There are potential risks to create different sizes of myocardial infarction, particularly when targeting a broad range of myocardial infarction. The territory of the septal artery always appears to be spared from myocardial infarction induced by the coronary ligation technique.

172 A Slow-Releasing Synthetic Prostacyclin Agonist “Ono 1301-sr†Combined with Omental Flap Increases Myocardial Blood Flow and Reduces Microvascular Resistance, Associated with Functional Recovery, in a Porcine Chronic Myocardial Infarction Model

Introduction Coronary microvascular dysfunction (MVD) has been shown to be the major cause of persistent myocardial ischaemia and progressive left ventricular (LV) remodelling in the chronic myocardial infarction (MI) heart. ONO-1301SR is a slow-releasing synthetic prostacyclin agonist, being developed to induce angiogenesis by upregulating a variety of proangiogenic cytokines in the targeted region. In addition, pedicle omental flap covering over the LV surface reportedly yields proangiogenic effects on chronic MI heart. We herein hypothesised pedicle omental flap covering, combined with ONO-1301SR placement. over the LV surface may be effective on the MVD and related functional deterioration in a chronic MI heart. To test this hypothesis, clinically latest imaging studies were applied in a porcine chronic MI model. Methods A mini-pig chronic MI model was generated by placing an ameroid constrictor around the left anterior descending artery for 4 weeks. The mini-pigs were then randomly assigned into the following 4 groups; sham group, omental flap alone (OM group), ONO-1301SR alone (ONO group), ONO-1301SR plus omental flap (ONO+OM group, n = 6 each). Results At 4 weeks after the treatment, 13N-ammonia PET study showed that the ONO+OM group, but not the other groups, displayed a significantly increase in myocardial blood flow (189 ± 89% increase, P < 0.05) and coronary flow reserve (179 ± 121% increase, P < 0.05) under the stress compared to those under the rest. This trend was predominated in the left circumflex territory. In addition, coronary angiogram and pressure-temperature sensor wire study showed that the ONO+OM and the ONO groups, but not the OM group, showed a significantly greater number of collateral arteries and smaller indexed of microvascular resistance, in particular, in the left circumflex territory, compared to the sham group. Moreover, echocardiographical EF was significantly greater in the ONO+OM group (50 ± 7%) and the ONO group (48 ± 3%), compared to the OM group (41 ± 3%) and the sham group (38 ± 5%) at 4 weeks. Furthermore, histological density of CD31-positive capillaries and CD31SMA-double positive arterioles were significantly greater in the ONO+OM group than the sham group or the OM group, in association with a significantly smaller myocyte size and interstitial fibrous area in the ONO+OM and the ONO group compared to those in the other groups. However, expression of proangiogenic cytokines, such as vascular endothelial growth factor, hepatocyte growth factor or stem cell-derived factor-1, were not significantly different among the groups, indicating that angiogenic process might have been completed at the 4 weeks after the treatment. Conclusions ONO-1301SR combined with omental flap therapy promoted myocardial angiogenesis with collateral artery growth, leading to recovery of the cardiac function in a porcine ICM model. This combined therapy may be useful in regenerating myocardial microvasculature.