Keiichi Tambara

Adrenomedullin Infusion Attenuates Myocardial Ischemia/Reperfusion Injury Through the Phosphatidylinositol 3-Kinase/Akt-Dependent Pathway

Background—Infusion of adrenomedullin (AM) has beneficial hemodynamic effects in patients with heart failure. However, the effect of AM on myocardial ischemia/reperfusion remains unknown. Methods and Results—Male Sprague-Dawley rats were exposed to a 30-minute period of ischemia induced by ligation of the left coronary artery. They were randomized to receive AM, AM plus wortmannin (a phosphatidylinositol 3-kinase [PI3K] inhibitor), or saline for 60 minutes after coronary ligation. Hemodynamics and infarct size were examined 24 hours after reperfusion. Myocardial apoptosis was also examined 6 hours after reperfusion. The effect of AM on Akt phosphorylation in cardiac tissues was examined by Western blotting. Intravenous administration of AM significantly reduced myocardial infarct size (28±4% to 16±1%, P <0.01), left ventricular end-diastolic pressure (19±2 to 8±2 mm Hg, P <0.05), and myocardial apoptotic death (19±2% to 9±4%, P <0.05). Western blot analysis showed that AM infusion accelerated Akt phosphorylation in cardiac tissues and that pretreatment with wortmannin significantly attenuated AM-induced Akt phosphorylation. Moreover, pretreatment with wortmannin abolished the beneficial effects of AM: a reduction of infarct size, a decrease in left ventricular end-diastolic pressure, and inhibition of myocardial apoptosis after ischemia/reperfusion. Conclusions—Short-term infusion of AM significantly attenuated myocardial ischemia/reperfusion injury. These cardioprotective effects are attributed mainly to antiapoptotic effects of AM via a PI3K/Akt-dependent pathway.

Intramyocardial sustained delivery of basic fibroblast growth factor improves angiogenesis and ventricular function in a rat infarct model

Abstract Recently we have demonstrated that the release of basic fibroblast growth factor (bFGF) from a biodegradable gelatin hydrogel carrier depends on the degradation of hydrogel in vivo. The purpose of our study was to assess whether bFGF-incorporating gelatin hydrogels induce myocardial angiogenesis and improve left ventricular function in the infarcted myocardium of rats. Studies were conducted in 22 Lewis rats after a 4-week ligation of the proximal left anterior descending coronary artery. The rats were randomized into the following two groups: the control group (n = 11) had an intramyocardial injection of saline alone, and the FGF group (n = 11) had gelatin hydrogel microspheres containing 100 μg of bFGF injected into the border zone of the infarct area after the repeat left thoracotomy. For visualization of the regional myocardial blood flow in the rat heart, 201Tl images were taken just before and 4 weeks after the treatment using a 4-head single photon emission computed tomography scanner with pinhole collimators. Left ventricular function was also assessed with echocardiography and a micromanometer-tipped catheter. Finally, the extent of myocardial angiogenesis was evaluated quantitatively in the postmortem analysis. The 201Tl defect score in the control group remained unchanged before and after the treatment, whereas it decreased significantly in the FGF group. Both regional and global left ventricular function was significantly better in the FGF group compared with the control group. The vascular density in the border zone of the infarct in the FGF group was significantly higher than that in the control group. In conclusion, intramyocardial injection of bFGF-impregnated gelatin hydrogels induces functionally significant angiogenesis and improves left ventricular systolic and diastolic function in the infarcted myocardium of rats.

Transplanted Skeletal Myoblasts Can Fully Replace the Infarcted Myocardium When They Survive in the Host in Large Numbers

Background—It is not clear how many skeletal myoblsts (SM) can survive and exert beneficial effects in the host myocardial infarction (MI) area. We assessed the hypothesis that a large number of SM can replace the MI area with reverse left ventricular (LV) remodeling. Methods and Results—MI was created by left coronary artery ligation in male Lewis rats. Four weeks after ligation, 45 rats had skeletal myoblast transplantation in the MI area. They were randomized into 3 groups according to the number of SM: group I (n=15), 5×107; group II (n=15), 5×106; and group III (n=15), 5×105 cells. Donor SM were obtained from neonatal Lewis rats and directly used without expansion. Another four weeks later, all rats were sacrificed following hemodynamic assessment. All heart sections were stained with anti-fast skeletal myosin heavy chain (FSMHC) antibody to determine the spacial extent of donor myocytes. Results—Four weeks after transplantation, LV diastolic dimension was decreased, fractional area change was increased, and MI size was decreased maximally in group I. Histological study showed that donor cells positive for FSMHC occupied the MI area with nearly normal wall thickness in group I, in which estimated volume of donor-derived muscle tissue was 40 mm3. In the other groups, FSMHC-positive cells were found only partly in the MI area. Conclusions—A large number of freshly isolated neonatal SM can survive in the host and fully replace the infarcted myocardium with reverse LV remodeling in rats with MI.

Toward surgical angiogenesis using slow-released basic fibroblast growth factor

OBJECTIVE Therapeutic angiogenesis using basic fibroblast growth factor (bFGF) in coronary artery disease has been documented in a number of papers. However, the effectiveness is discrepant among documents. In this study, we evaluated the distribution of bFGF in the rat heart by different administration methods, and investigated the efficacy of slow-released administration of bFGF using biodegradable hydrogel microspheres (bFGF microspheres) in a pig infarction model toward an enhanced coronary bypass surgery. METHODS Heart failure due to myocardial infarction was induced in rats and pigs. In the rat study, free form of bFGF (central venous injection, intracoronary injection, and intramyocardial administration) and bFGF microspheres (intramyocardial administration) were given 4 weeks later. The remaining radioactivity of bFGF in the hearts was estimated 1, 24, and 72 h later. On the other hand, the pigs were randomized into two groups 4 weeks after myocardial infarction. While the control group (n=8) had gelatin hydrogel microspheres with saline, the FGF group (n=8) received bFGF microspheres in the left ventricular (LV) wall. RESULTS In the rat study, after intramyocardial administration of bFGF microspheres, more bFGF remained in the rat heart 72 h later compared with the other methods (P<0.0001). In the pig study, 4 weeks after the treatment, the FGF group had smaller LV diastolic diameter (48.7+/-5.3 vs. 56.7+/-5.2 mm, P<0.01) than the control group. LV end-systolic elastance was higher in the FGF group (2.96+/-1.2 vs. 1.06+/-0.3 mmHg/ml, P<0.01). In microscopic examinations, many neovessels were found in and around the scar tissue, and the vascular density in the FGF group was significantly higher (61.5+/-18.3 vs. 153.0+/-29.0/mm2, P<0.01). In addition, the infarcted LV walls were less expanded and more thickened in the FGF group. CONCLUSIONS Biodegradable hydrogel microspheres with bFGF improved LV function and inhibited LV remodeling by angiogenesis in pigs with chronic myocardial infarction. bFGF microspheres into ischemic myocardium may revascularize small ungraftable vessels and may potentially increase distal run-off when applied in coronary bypass surgery.

Composite arterial Y graft has less coronary flow reserve than independent grafts.

BACKGROUND It is not known whether a composite Y graft of the left internal thoracic artery can provide sufficient blood flow to the whole left coronary system. The aim of this study was to compare regional myocardial blood flow (MBF) and coronary flow reserve after coronary artery bypass grafting using arterial composite Y graft or independent arterial grafts. METHODS Positron emission tomography was performed at rest and after dipyridamole infusion using oxygen-15-labeled water 2 weeks after coronary artery bypass grafting. Regional MBF was calculated in seven segments of the left ventricle. Coronary flow reserve was defined as the ratio of MBF after dipyridamole infusion to MBF at rest. In the Y graft group (n = 22), a free arterial graft to obtuse marginal arteries was anastomosed to the proximal side of in situ left internal thoracic artery, which was anastomosed to the left anterior descending artery. In the independent graft group (n = 13), left anterior descending and obtuse marginal arteries were independently revascularized using in situ left internal thoracic artery and a free arterial graft. RESULTS There was no difference between the groups in MBF at rest. Coronary flow reserve in the Y graft group was lower than that in the independent group in the anterobasal (1.43 +/- 0.07 versus 1.90 +/- 0.13, p = 0.038), apical (1.24 +/- 0.06 versus 1.64 +/- 0.12, p = 0.003), septal (1.34 +/- 0.05 versus 1.75 +/- 0.13, p = 0.023), and lateral regions (1.19 +/- 0.04 versus 1.66 +/- 0.09, p = 0.001). CONCLUSIONS Although arterial composite Y graft improved MBF at rest, it was not as effective as independent grafts for improving coronary flow reserve soon after coronary artery bypass grafting.

Coronary angiography by 64-detector row computed tomography using low dose of contrast material with saline chaser: influence of total injection volume on vessel attenuation.

Objective: To assess the influence of total injection volume on thoracic great vessels and coronary arteries enhancement in 64-detecter row computed tomography (CT) coronary angiography using low dose of contrast material. Methods: Sixty patients underwent cardiac CT (64 × 0.5 mm, 0.4 rot/s) using 40 mL of contrast material (350 mg of Iodine per milliliter) in 30 patients and 50 mL in 30 patients. Computed tomography densities (Hounsfield units) in ascending aorta, descending aorta, and main pulmonary artery were measured at every second with the time of CT data acquisition recorded in each reconstructed image. Computed tomography densities of proximal and distal coronary arteries were also measured. Differences in CT densities between 40 and 50 mL contrast material were assessed with the Student t test. In addition, the relation between the injection volume (mL) of contrast material per kilogram body weight and contrast enhancement in coronary arteries was studied. Results: The average attenuations in the ascending and descending aorta and coronary arteries were significantly lower in 40-mL group than in 50-mL group (<0.05). In addition, the average attenuations in the pulmonary artery were significantly lower in 40-mL group than 50-mL group (<0.01). Every patient with the total injection volume of more than 0.9 mL/kg body weight showed a contrast enhancement more than 250 Hounsfield units. Conclusions: The reduction of total injection volume lowered the enhancement of thoracic great vessels and coronary arteries in 64-detector row cardiac CT. The injection volume of at least 0.9 mL/kg body weight was necessary for a steady contrast enhancement in coronary arteries.

Administration of Control-Released Hepatocyte Growth Factor Enhances the Efficacy of Skeletal Myoblast Transplantation in Rat Infarcted Hearts by Greatly Increasing Both Quantity and Quality of the Graft

Background—We investigated whether simultaneous administration of control-released hepatocyte growth factor (HGF) enhances the efficacy of skeletal myoblast (SM) transplantation (Tx) through its antiapoptotic, angiogenic, and antifibrotic effects in myocardial infarction (MI). Methods and Results—Forty-eight Lewis rats with chronic MI were divided into 4 groups. In Group I (n=14), neonatal SMs (5×106) were transplanted in the MI area with a gelatin sheet incorporating 40 &mgr;g (1 g/L) of HGF applied. Group II (n=14) had SM Tx and placement of a saline sheet. Groups III (n=10) and IV (n=10) had culture medium injection plus HGF and saline sheet application, respectively. Four rats each from Groups I and II were sacrificed at day 1 for TUNEL assay on donor SMs. The percentage of TUNEL-positive donor cells was much lower in Group I than in Group II (P<0.05). At 4 weeks, in Group I, left ventricular diastolic dimension was smallest in echocardiography, end-systolic elastance was highest, and &tgr; was the lowest (both P<0.0005 in ANOVA) in cardiac catheterization. Vascular density inside the graft was higher in Group I than in Group II (P<0.0001). The percentage of fibrotic area inside the graft was smaller in Group I than in Group II (P<0.001). The graft volume as estimated by fast skeletal myosin heavy chain-positive areas was ≈7-fold larger in Group I than in Group II (P<0.0001). Conclusions—In SM Tx, HGF can greatly increase the graft volume and vascularity and reduce fibrosis inside the graft, which enhances the efficacy of SM Tx to infarcted hearts.

Therapeutic angiogenesis by the controlled release of basic fibroblast growth factor for ischemic limb and heart injury: toward safety and minimal invasiveness.

We review our studies on therapeutic angiogenesis using basic fibroblast growth factor (bFGF) released in a controlled manner from biodegradable gelatin hydrogel (GH). The bFGF-GH was intramuscularly injected in rabbits with limb ischemia. The group treated with bFGF showed an increase in tissue blood flow under laser Doppler imaging and histology showed a greater vascular density compared with controls. Also, bFGF-GH was subepicardially injected into old heart infarcts in rats. In the group treated with bFGF, improved left ventricular function was shown by echocardiography and cardiac catheterization, increased regional blood flow in the peri-infarct area was detected by pinhole single-photon emission computed tomography using 201Tl, and increased vascular density was demonstrated by histology. In rabbits with acute myocardial infarction, the heart was wrapped with the omentum (including the gastroepiploic artery) and a bFGF-GH sheet was applied. Postoperative assessment revealed rich communication from the gastroepiploic artery to the coronary artery and improved cardiac function. The controlled release of bFGF was effective for both limb and heart ischemia and is considered to be suitable for clinical use because its application in animals was feasible and safe with minimal invasiveness.

Recent insights into human coronary collateral development

Enhancement of coronary collateral function is an intriguing approach to the preservation of ischaemic myocardium. Coronary collateral development consists of collateral recruitment and collateral growth. Collateral growth encompasses proliferation of capillaries in the ischaemic area (angiogenesis) and maturation of pre-existing collateral vessels (arteriogenesis), with the latter being more relevant in humans. Therefore, treatment intended directly for arteriogenesis of collateral vessels appears to be more effective. Promotion of coronary collateral growth has many attractive features, particularly in patients with angina who are not indicated for percutaneous coronary intervention or coronary artery bypass grafting surgery. A complete elucidation of the remaining practical and mechanistic questions of arteriogenesis may lead to a new remedy capable of developing collateral vessels more effectively.

Cardiomyocyte transplantation does not reverse cardiac remodeling in rats with chronic myocardial infarction

BACKGROUND Several reports have documented the potential benefits of cell transplantation as an alternative to cardiac transplantation. This study was designed to investigate whether cardiomyocyte transplantation is effective in rats with chronic myocardial infarction. METHODS Syngeneic Lewis rats were used in this study. Chronic myocardial infarction was induced in rats by ligating the left anterior descending artery. Four weeks later, after left ventricular (LV) dysfunction with akinetic regions was confirmed by echocardiography, the rats were randomized into two groups: a group that received fetal cardiomyocyte transplantation (TX group; n = 11); and a group that received an intramyocardial injection of culture medium only (control group; n = 12). RESULTS Four weeks after treatment, the TX group had smaller end-systolic dimension (LVDs) (7.5 +/- 0.9 vs 8.9 +/- 0.8 mm, p < 0.01) and better fractional shortening (FS) (26.2 +/- 5.9 vs 17.7% +/- 5.1%, p < 0.01) than the control group. However, there were no differences in LV end-diastolic dimension, LVDs, and FS between baseline and post-treatment values in the TX group. In addition, plasma levels of atrial natriuretic peptide were not significantly different between the two groups 4 weeks after treatment. In microscopic examination, small amounts of transplanted cardiomyocytes were found only in the periinfarct area, not in the center of scar area, and a thicker ventricular wall in the infarct area was detected in the TX group. CONCLUSIONS Fetal cardiomyocyte transplantation prevented, but did not reverse, cardiac remodeling that was accompanied with heart failure in myocardial infarction rats. Further investigation is warranted for optimal clinical application to the failing heart.