Satoshi Taketani

Myocardial Regeneration Therapy for Heart Failure Hepatocyte Growth Factor Enhances the Effect of Cellular Cardiomyoplasty

Background—We hypothesized that transfection of the gene for human hepatocyte growth factor (hHGF) combined with cellular cardiomyoplasty might regenerate the impaired myocardium. Methods and Results—We used a ligation model of proximal left anterior descending coronary artery (LAD) of Lewis rats. Two weeks after LAD ligation, 3 different treatments were conducted: (1) neonatal rat cardiomyocytes group (106 cells, T group, n=11), (2) HVJ-liposomes bearing the hHGF gene group (H group, n=10), and (3) combined (T-H group, n=10). The injection site was the scar area of myocardial infarction. For control, culture medium was injected (C group, n=13). Echocardiography demonstrated that cardiac performance was significantly ameliorated in the T-H group 4 and 8 weeks after injection. Contrast echocardiography also showed a marked increase in myocardial perfusion in the T-H group but not in the other groups. In the T-H group, neovascularization and a marked reduction of fibrosis were observed histologically. In an immunohistochemical study, strong staining for &bgr;1-integrin, &agr;-, and &bgr;-dystroglycan were found principally in the basement membrane of myocytes in the T-H group 8 weeks after transplantation, although there was weak immunoreactivity in the T group. Conclusions—hHGF gene transfection enhanced the cellular cardiomyoplasty possibly by stimulating angiogenesis, restoring the impaired ECM, and promoting the integration of the dissociated grafted myocytes. The combined effects might have lead to the improved cardiac performance. Thus, combined therapy may be a promising strategy for the treatment of heart failure caused by myocardial infarction.

Tissue cardiomyoplasty using bioengineered contractile cardiomyocyte sheets to repair damaged myocardium: their integration with recipient myocardium.

Background. We hypothesized that tissue-engineered contractile cardiomyocyte sheets without a scaffold would show histological and electrical integration with impaired myocardium, leading to the regeneration of infarcted myocardium. Methods. Neonatal rat cardiomyocytes were cultured on Poly(N-isopropylacrylamide)-grafted polystyrene dishes and detached as a square cell sheet at 20°C. Two sheets were stacked to make thicker contractile cardiac sheets. In cross-section, the stacked sheets looked like homogeneous heart-like tissue. Two weeks after rats were subjected to left anterior descending (LAD) ligation, two treatments were conducted: 1) cardiomyocyte sheet implantation (T group, n=10), and 2) fibroblast sheet implantation (F group, n=10). The control group underwent no additional treatment (C group, n=10). Results. Echocardiography demonstrated that cardiac performance was significantly ameliorated in the T group 2, 4, and 8 weeks after implantation. The cardiomyocyte sheets became attached to the infarcted myocardium, showed angiogenesis, expressed connexin-43, and appeared as homogeneous tissue in the myocardium Electrophysiological experiments showed a QRS complex with one peak in the treated scar area in the T group, but two peaks, indicative of branch block, in that of the other groups. Furthermore, the threshold for pacing of the recipient heart was lower in the T group than in the other groups. Conclusions. Cardiomyocyte sheets integrated with the impaired myocardium and improved cardiac performance in a model of ischemic myocardium. Techniques using such tissue-engineered cell sheets are introducing the promising concept of tissue cardiomyoplasty to the field of regenerative medicine.

Reduction in myocardial apoptosis associated with overexpression of heat shock protein 70.

Abstract It is reported that ischemia-reperfusion induces apoptotic cell death in myocardium. It is also demonstrated that heat shock protein 70 (HSP70) enhances myocardial tolerance. Therefore, it is hypothesized that HSP70 may play a role in the attenuation of myocardial apoptosis. To elucidate this goal, HSP70-overexpressing and control-transfected rat hearts were prepared using gene transfection by intra-coronary infusion of the hemagglutinating virus of Japan-liposome. In vivo experiment Hearts fo both groups were subjected to global ischemia, followed by reperfusion in situ. Shorter recovery time to spontaneous beating (HSP70-transfected vs control-transfected; 46.7±4.6 vs 67.5±7.0 s, p = 0.033) and lower serum CPK levels (415±27 vs 533±36 IU, p = 0.027) were observed in the HSP70-transfected group. The HSP70-transfected group also showed a lower percentage of cardiac myocytes positively stained by nick end labeling after ischemia-reperfusion (17.5±4.9 vs 40.0±5.1%, p = 0.010). In vitro experiment Cardiac myocytes isolated from the hearts of both groups (prepared separately from the in vivo eperiment) were subjected to hypoxia-reoxygenation. Flow cytometry was used to identify the cells that showed sub-G1 DNA content as apoptotic cells. Apoptotic cels as a percentage of viable cells increased more in the control-transfected group after hypoxia-reoxygenation (13.0±0.77 vs 21.9±1.18%, p < 0.0001).In conclusion, we demonstrated that apoptosis after ischemia-reperfusion was decreased in the HSP70-overexpressing heart in vivo and in vitro, leading to the suggestions that HSP70 could be associated with the reduction in myocardial apoptosis.

Analysis of sympathetic nerve activity in end-stage cardiomyopathy patients receiving left ventricular support

BACKGROUND The left ventricular assist system (LVAS) has been used increasingly for patients with end-stage heart failure who are awaiting transplantation. Sympathetic nerve activity is known to correlate with cardiac function in chronic heart failure patients, but little is known about sympathetic nerve activity during LVAS support. In this study, we examined the status of sympathetic nerve activity in relation to mechanical support. METHODS In this study, we included 10 consecutive patients with end-stage cardiomyopathy who were on LVAS support for at least 2 months (duration, 222 +/- 59 days). None of these patients achieved enough functional recovery to be taken off LVAS. In these patients, we used iodine-125-metaiodobenzylguanidine (125I-MIBG) scintigraphy to examine the change of sympathetic nerve activity after LVAS implantation, and compared the results with the change of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) levels as well as with histologic optional findings. Samples for ANP and BNP measurement were obtained before and 30 days after LVAS implantation. Specimens for histologic analysis were obtained at the time of LVAS implantation and at the time of cardiac transplantation or autopsy. RESULTS We observed marked decrease in serum levels of ANP and BNP 1 month after LVAS implantation. But myocardial sympathetic nerve function, which was evaluated with 125I-MIBG scintigraphy and expressed as the heart-to-mediastinum activity ratio, remained below normal even 2 months after the LVAS implantation (1.57 +/- 0.19; normal, 2.34 +/- 0.36). Serial histologic analysis in these 10 patients showed continuous increase in percentage of fibrosis and cell diameter despite ventricular unloading by the LVAS. CONCLUSIONS Sympathetic nerve function, which was evaluated on 125I-MIBG scintigraphy, did not improve during left ventricular support. Because none of the patients included in our study showed improvement in cardiac function or histologic findings, the recovery of myocardial sympathetic nerve function may be an important factor in myocardial recovery for cardiomyopathy patients on LVAS support.

Late true aneurysm after bypass grafting for long aortic coarctation

Two adolescent patients who underwent a repair of long aortic coarctation using bypass grafting with subsequent late true aneurysm formation are reported. To our knowledge, only 1 case of late true aneurysm formation after bypass grafting has been reported in the English-language literature.

Angiogenic gene cell therapy using suicide gene system regulates the effect of angiogenesis in infarcted rat heart

Background. Although angiogenic gene therapy has been reported to be effective in restoring ischemic heart function, there are several obstacles to its clinical application, such as unreliable efficiency of transfection and uncontrollable expression. We developed human HGF (hHGF)-producing cells that regulated hHGF production using the thymidine kinase gene of Herpes Simplex Virus (TK) and the Ganciclovir (GCV) system. We tested whether these cells induced and regulated angiogenic effects in infarcted myocardium. Methods. NIH3T3 cells were stably transfected with an hHGF cDNA expression plasmid (NIH/HGF). Next, the NIH/HGF cells were stably transfected with TK (NIH/HGF/TK). The left anterior descending artery was ligated in the heart of severe combined immunodeficiency rats, and four materials were transplanted: 1) NIH/HGF (n=10), 2) NIH/HGF/TK, with orally administered GCV (n=10), 3) NIH3T3 (n=10), and 4) culture medium (n=10). Results. In vitro, the proliferation of NIH/HGF/TK cells was suppressed by GCV. In vivo, significant increases in cardiac performance and angiogenesis were observed in the NIH/HGF and NIH/HGF/TK groups 4 weeks after transplantation. Although tumorous lesions were detected in the NIH/HGF group, their growth was completely controlled in the NIH/HGF/TK group. Conclusions. Angiogenic gene cell therapy using the TK-GCV suicide gene system induces and regulates angiogenesis under the control of cell growth, suggesting it as a promising system for therapeutic angiogenesis.

Myocardial histological changes in dilated cardiomyopathy during a long-term left ventricular assist device support

SummaryAs the myocardium in patients with dilated cardiomyopathy (DCM) is deteriorating progressively, resulting in a decrease in left ventricular function, patients with end-stage DCM may require implantation of a left ventricular assist device (LVAD) unless they undergo heart transplantation. Although LVAD has been reported to provide excellent hemodynamic support, no data are currently available about the effects of long-term LVAD support on the myocardium in patients with DCM. We describe two patients with end-stage DCM who underwent LVAD implantation and were supported with LVAD for 524 and 245 days, respectively. Serial myocardial biopsies showed increases in myocardial cell diameter and intercellular fibrosis, despite excellent hemodynamic support by LVAD. These data suggest that the myocardium in patients with end-stage DCM deteriorates progressively, even if the preload of the left ventricle is reduced by LVAD.

Impact of synovial membrane-derived stem cell transplantation in a rat model of myocardial infarction

To explore a new source of cell therapy for myocardial infarction (MI), we assessed the usefulness of mesenchymal stem cells derived from synovial membrane samples (SM MSCs). We developed a model of MI by ligation of the proximal left anterior descending coronary artery (LAD) in Lewis rats. Two weeks after ligation, 5 × 106 SM MSCs were injected into the MI scar area (T group, n = 9), while buffer was injected into the control group (C group, n = 9). Cardiac performances measured by echocardiography at 4 weeks after transplantation were significantly increased in the T group as compared with the C group. Masson’s trichrome staining showed that SM MSC transplantation decreased collagen volume in the myocardium. Engrafted SM MSCs were found in the border zone of the infarct area. Immunohistological analysis showed that these cells were positive for the sarcomeric markers alpha-actinin and titin, and negative for desmin, troponin T, and connexin 43. SM MSC transplantation improved cardiac performance in a rat model of MI in the subacute phase, possibly through transdifferentiation of the engrafted cells into a myogenic lineage, which led to inhibition of myocardial fibrosis. Our results suggest that SM MSCs are a potential new regeneration therapy candidate for heart failure.

Change of c-Myc expression and cardiac hypertrophy in patients with aortic valve replacement

BACKGROUND Long-term volume overload to the left ventricle (LV) due to aortic regurgitation (AR) tends to cause severe impairment in LV function that cannot be reversed even with aortic valve replacement (AVR). Recently, we reported that the protooncogene c-myc is related to the onset of the cardiac hypertrophy and LV dysfunction in patients with chronic AR. However, it is still unclear whether c-myc is related to reversibility of the cardiac hypertrophy or LV dysfunction after AVR. METHODS AND RESULTS Twenty patients with isolated chronic AR who underwent AVR were included in this study. LV function was calculated before and after AVR. After AVR, end-systolic volume index (ESVI) and enddiastolic volume index (EDVI) were improved, but not mass index (LVMI). However, normalization of ESVI and EDVI was observed only in 12 and 9 patients, respectively. Preoperatively, c-Myc protein was expressed in the myocardium of 16 out of 20 patients with an average point count of 35+/-30%. After AVR, c-Myc protein was observed only in 2 patients. Preoperative ejection fraction (EF), ESVI, and postoperative end-systolic stress (ESS)/ESVI had significant correlation to postoperative cell diameter (CD). Percent c-Myc protein expression before the operation was significantly correlated to postoperative CD, ESVI, and ESS/ESVI. Average c-Myc expression was higher in patients who showed normalization of CD and ESS/ESVI after AVR than the patients who did not. CONCLUSIONS These data suggest that preoperative expression of c-Myc can be indicative of the reversibility of myocardial cellular hypertrophy and LV dysfunction.

NITRIC OXIDE GAS INFUSION TO THE OXYGENATOR ENHANCES THE BIOCOMPATIBILITY OF HEPARIN COATED EXTRACORPOREAL BYPASS CIRCUITS

Heparin coated bypass circuits have been reported to improve the biocompatibility of extracorporeal circulation, although it is still insufficient and improvable. Nitric oxide (NO) is known to inhibit platelet activation and inflammatory reactions. In this study, the authors evaluated exogenous NO infusion in enhancing the effect of a heparin coated bypass circuit on the biocompatibility of an extracorporeal circuit, especially in view of the attenuation of the inflammatory response. A miniature closed bypass circuit, including an oxygenator (BioActive surface; Carmeda, Stockholm, Sweden) was primed with fresh human heparinized blood and perfused with a centrifugal pump. Either pure N2 gas (control group: n = 7) or NO gas (NO group [100 ppm in N2]: n = 7) was infused to the oxygenator. NO metabolites (nitrite and nitrate), platelet count, thrombin-antithrombin III complex (TAT), alpha2-plasmin-plasminogen inhibitor complex (PIC), beta-thromboglobulin (beta-TG), platelet factor 4 (PF4), serotonin, complement 3 activation products (C3a), granulocyte elastase, and bradykinin were measured at 0, 30, 60, 120, and 180 min after starting perfusion. At every sampling point, platelet counts were significantly higher, and TAT, beta-TG, and bradykinin were lower in the NO group than in the control group. PF4, C3a, and granulocyte elastase were significantly lower in the NO group at 60, 120, and 180 min. These results suggest that NO gas infusion to the oxygenator enhances the biocompatibility of heparin coated extracorporeal circuits.