Yu Misao

Acceleration of the Healing Process and Myocardial Regeneration May Be Important as a Mechanism of Improvement of Cardiac Function and Remodeling by Postinfarction Granulocyte Colony–Stimulating Factor Treatment

Background—We investigated whether the improvement of cardiac function and remodeling after myocardial infarction (MI) by granulocyte colony–stimulating factor (G-CSF) relates to acceleration of the healing process, in addition to myocardial regeneration. Methods and Results—In a 30-minute coronary occlusion and reperfusion rabbit model, saline (S) or 10 μg · kg−1 · d−1 of human recombinant G-CSF (G) was injected subcutaneously from 1 to 5 days after MI. Smaller left ventricular (LV) dimension, increased LV ejection fraction, and thicker infarct-LV wall were seen in G at 3 months after MI. At 2, 7, and 14 days and 3 months after MI, necrotic tissue areas were 14.2±1.5/13.4±1.1, 0.4±0.1/1.8±0.5*, 0/0, and 0/0 mm2 ·· slice−1 · kg−1, granulation areas 0/0, 4.0±0.7/8.5±1.0*, 3.9±0.8/5.7±0.7,* and 0/0 mm2 · slice−1 kg−1, and scar areas 0/0, 0/0, 0/0, and 4.2±0.5/7.9±0.9* mm2 slice−1 kg−1 in G and S, respectively (*P <0.05, G versus S). Clear increases of macrophages and of matrix metalloproteinases (MMP) 1 and 9 were seen in G at 7 days after MI. This suggests that G accelerates absorption of necrotic tissues via increase of macrophages and reduces granulation and scar tissues via expression of MMPs. Meanwhile, surviving myocardial tissue areas within the risk areas were significantly increased in G despite there being no difference in LV weight, LV wall area, or cardiomyocyte size between G and S. Confocal microscopy revealed significant increases of cardiomyocytes with positive 3,3,3′3′-tetramethylindocarbocyanine perchlorate and positive troponin I in G, suggesting enhanced myocardial regeneration by G. Conclusions—The acceleration of the healing process and myocardial regeneration may play an important role for the beneficial effect of post-MI G-CSF treatment.

Granulocyte Colony-Stimulating Factor

Background The purpose of this study was to determine whether treatment with granulocyte colony-stimulating factor (G-CSF), which mobilizes endothelial progenitor cells from bone marrow, can safely improve the clinical outcomes of patients with atherosclerotic peripheral artery disease (PAD). Methods and Results Thirty-nine patients with intractable PAD were randomly assigned to 3 groups: a negative control group (n=12) treated with conventional drug therapy; a positive control group (n=13) treated with conventional drug therapy plus bone marrow transplantation (BMT); and a G-CSF group (n=14) treated with conventional therapy plus subcutaneous injection of 2-5 μg/kg of recombinant human G-CSF once daily for 10 days. One month after treatment, subjective symptoms improved significantly in the G-CSF and BMT groups. Ankle-brachial pressure index and transcutaneous oxygen pressure increased significantly in the BMT and G-CSF groups, but no such improvements were seen in the group receiving conventional therapy alone. Conclusions G-CSF improves the clinical signs and symptoms of patients with intractable PAD to the same degree as BMT does. This noninvasive treatment may thus represent a useful new approach to managing the disease. (Circ J 2006; 70: 1093 - 1098)

Treatment with granulocyte colony-stimulating factor ameliorates chronic heart failure

Chronic heart failure remains a leading cause of mortality. Although granulocyte colony-stimulating factor (G-CSF) is reported to have a beneficial affect on postinfarction cardiac remodeling and dysfunction when administered before the onset of or at the acute stage of myocardial infarction (MI), its effect on established heart failure is unknown. We show here that subcutaneous administration of G-CSF greatly improves the function of murine hearts failing due to a large, healed MI. G-CSF changed the geometry of the infarct scar from elongated and thin to short and thick, induced hypertrophy among surviving cardiomyocytes, and reduced myocardial fibrosis. Expression of G-CSF receptor was confirmed in failing hearts and was upregulated by G-CSF treatment. G-CSF treatment also led to activation of signal transducer and activator of transcription-3 and induction of GATA-4 and various sarcomeric proteins such as myosin heavy chain, troponin I and desmin. Expression of metalloproteinase-2 and -9 was also increased in G-CSF-treated hearts, while that of tumor necrosis factor-α, angiotensin II type 1 receptor (AT1) and transforming growth factor-β1 was reduced. Although activation of Akt was noted in G-CSF-treated hearts, vessel density was unchanged, and apoptosis was too rare to exert a meaningful effect. No bone marrow-derived cardiomyocytes or vascular cells were detected in the failing hearts of green fluorescent protein chimeric mice. Finally, beneficial effects of G-CSF on cardiac function were found persisting long after discontinuing the treatment (2 weeks). Collectively, these findings suggest G-CSF administration could be an effective approach to treating chronic heart failure following a large MI.

Post-infarct treatment with an erythropoietin-gelatin hydrogel drug delivery system for cardiac repair.

AIMS We investigated the effect of an erythropoietin (EPO)-gelatin hydrogel drug delivery system (DDS) applied to the heart on myocardial infarct (MI) size, left ventricular (LV) remodelling and function. METHODS AND RESULTS Experiments were performed in a rabbit model of MI. The infarct size was reduced, and LV remodelling and function were improved 14 days and 2 months after MI but not at 2 days after MI in the EPO-DDS group. The number of cluster of differentiation 31(CD31)-positive microvessels and the expression of erythropoietin receptor (EPO-R), phosphorylated-Akt (p-Akt), phosphorylated glycogen synthase kinase 3beta (p-GSK-3beta), phosphorylated extracellular signal-regulated protein kinase (p-ERK), phosphorylated signal transducer and activator of transcription 3 (p-Stat3), vascular endothelial growth factor (VEGF), and matrix metalloproteinase-1 (MMP-1) were significantly increased in the myocardium of the EPO-DDS group. CONCLUSION Post-MI treatment with an EPO-DDS improves LV remodelling and function by activating prosurvival signalling, antifibrosis, and angiogenesis without causing any side effect.

Cilnidipine, an N+L-Type Dihydropyridine Ca Channel Blocker, Suppresses the Occurrence of Ischemia/Reperfusion Arrhythmia in a Rabbit Model of Myocardial Infarction

Dihydropyridine Ca channel blockers are widely prescribed for the treatment of hypertension and coronary artery diseases, but it remains unknown whether these agents protect against arrhythmias. We investigated whether cilnidipine, an N+L-type Ca channel blocker, reduces the incidences of ventricular premature beats (VPBs) and, if so, via what mechanisms. Japanese white rabbits underwent 30 min of ischemia and 48 h of reperfusion. Cilnidipine (0.5 or 1.0 μg/kg/min, i.v.) or saline (i.v.) was administered from 30 min before ischemia to 30 min after reperfusion. Electrocardiogram and blood pressure were monitored and the incidences of VPBs were measured. At 48 h after reperfusion, myocardial infarct was measured. Myocardial interstitial noradrenaline levels were determined before, during and after 30 min of ischemia with cilnidipine (0.5 and 1.0 μg/kg/min) or saline. The incidences of VPBs during ischemia and reperfusion were significantly attenuated in the cilnidipine 0.5 group (15.6±3.1 and 6.8±1.9 beats/30 min) and in the cilnidipine 1.0 group (10.4±4.9 and 3.5±1.0 beats/30 min) compared to the control group (27.2±4.5 and 24.2±3.1 beats/30 min), respectively. Myocardial interstitial noradrenaline levels were significantly reduced in the cilnidipine 0.5 and 1.0 groups compared to the control group during ischemia and reperfusion. The antiarrhythmic effect of cilnidipine may be related to the attenuation of cardiac sympathetic nerve activity. This finding may provide new insight into therapeutic strategies for hypertensive patients with ventricular arrhythmias.

Benidipine reduces myocardial infarct size involving reduction of hydroxyl radicals and production of protein kinase C-dependent nitric oxide in rabbits.

Japanese white rabbits underwent 30 minutes of ischemia and 48 hours of reperfusion. Benidipine (3 or 10 μg/kg, i.v.) was administered 10 minutes before ischemia with and without pretreatment with L-NAME (10 mg/kg, i.v., a NOS inhibitor), chelerythrine (5 mg/kg, i.v., a PKC blocker) or 5-HD (5 mg/kg, i.v. a mitochondrial KATP channel blocker), genistein (5 mg/kg, i.v. a protein tyrosin kinase blocker). SNAP (2.5 mg/kg/min × 70 minutes, i.v., an NO donor) was also administered 10 minutes before ischemia. Benidipine significantly reduced the infarct size in a dose-dependent manner (3 μg/kg: 29.0 ± 2.7%, n = 8, 10 μg/kg: 23.0 ± 2.4%, n = 10) compared with the control (41.6 ± 3.3%, n = 10). This effect was completely blocked by L-NAME (39.9 ± 3.6%, n = 8) and chelerythrine (35.5 ± 2.4%, n = 8) but not by 5-HD (23.0 ± 2.4%, n = 10) or genistein (24.6 ± 3.1%, n = 10). SNAP also reduced the infarct size (24.6 ± 3.1%, n = 8). Benidipine significantly increased the expression of eNOS mRNA at 30 minutes after reperfusion and significantly increased the expression of eNOS protein at 3 hours after reperfusion in the ischemic area of the left ventricle. Benidipine and SNAP significantly decreased myocardial interstitial 2,5-DHBA levels, an indicator of hydroxyl radicals, during ischemia and reperfusion. Benidipine increased myocardial interstitial NOx levels, which effect was blocked by chelerythrine, during 0 to 30 minutes and 150 to 180 minutes after reperfusion. Benidipine reduces the infarct size through PKC-dependent production of nitric oxide and decreasing hydroxyl radicals but not through involving protein tyrosine kinase or mitochondrial KATP channels in rabbits.

Edaravone reduces myocardial infarct size and improves cardiac function and remodelling in rabbits

1 In the present study, we investigated the effect of 3‐methyl‐1‐phenyl‐2‐pyrazolin‐5‐one (edaravone), a free radical scavenger, on myocardial infarct (MI) size and cardiac function in an in vivo model of MI in rabbits. We further investigated the contribution of hydroxyl radicals, superoxide and nitric oxide (NO) to its effects. 2 Anaesthetized open‐chest Japanese white male rabbits were subjected to 30 min coronary occlusion and 48 h reperfusion. The control group (n = 10) was injected with saline 10 min before reperfusion. The edaravone group (n = 10) was injected with a bolus of 3 mg/kg edaravone 10 min before reperfusion. The edaravone + NG‐nitro‐l‐arginine methyl ester (l‐NAME) group (n = 5) was given 10 mg/kg, i.v., l‐NAME 10 min before the administration of 3 mg/kg edaravone. The l‐NAME group (n = 5) was given 10 mg/kg, i.v., l‐NAME 20 min before reperfusion. Infarct size was measured using the triphenyl tetrazolium chloride method and is expressed as a percentage of area at risk. Cardiac function was assessed by echocardiography 14 days after infarction. 3 In another series of experiments, rabbits were subjected to 30 min coronary occlusion and 30 min reperfusion and myocardial interstitial 2,3‐dihydroxybenzoic acid (DHBA) and 2,5‐DHBA levels, indicators of hydroxyl radical, were measured using a microdialysis technique. 4 Infarct size in the edaravone group was significantly reduced compared with that in the control group (27.4 ± 6.8 vs 43.4 ± 6.8%, respectively; P < 0.05). The edaravone‐induced reduction of infarct size was abolished by pretreatment with l‐NAME. Myocardial interstitial levels of 2,3‐DHBA and 2,5‐DHBA increased 20 and 30 min after ischaemia and peaked at 10 min reperfusion in the control group. Edaravone significantly inhibited the increase in 2,3‐DHBA and 2,5‐DHBA levels seen during reperfusion. Dihydroethidium staining showing in situ detectoion of superoxide was less intense in ischaemic myocardium in the edaravone‐treated group compared with the control group. Edaravone improved cardiac function and left ventricular remodelling 14 days after infarction. 5 In conclusion, edaravone significantly reduces MI size and improves cardiac function and LV remodelling by decreasing hydroxyl radicals and superoxide in the myocardium and increasing the production of NO during reperfusion in rabbits.

Autologous bone marrow cell transplantation improves left ventricular function in rabbit hearts with cardiomyopathy via myocardial regeneration-unrelated mechanisms

Recent studies suggest transplanted bone marrow cells (BMCs) can be used to reconstitute coronary vessels and myocardium following acute myocardial infarction, thereby improving cardiac function. We sought to investigate the therapeutic potential of BMC transplantation in the treatment of nonischemic cardiomyopathy. Experimental cardiomyopathy was produced by treating rabbits for 8 weeks with doxorubicin (2 mg/kg per week). Two weeks after the treatment was finished, freshly aspirated BMCs or an equivalent volume of phosphate-buffered saline was injected directly into the left ventricular free wall. Four weeks later, heart function was examined during perfusion on a Langendorff apparatus. Left ventricular developed pressure and ±dp/dt were significantly better in the transplantation group, among which echocardiography also showed significantly better ejection fractions. In addition, left ventricular weights as a fraction of body weight and left ventricular wall thicknesses were both lower in rabbits transplanted with BMCs than in controls. Immunohistochemical analyses carried out 2 weeks after transplantation showed no new myocardium and a very small number of endothelial cells originating from BMCs. On the other hand, immunoblotting revealed upregulated expression of transforming growth factor-β1 and downregulated expression of matrix metalloproteinase-1 and tumor necrosis factor-α following BMC transplantation. In conclusion, autologous BMC transplantation into cardiomyopathic rabbit hearts ameliorates the decline in ventricular function without regenerating cardiomyocytes, most likely by altering expression of various cytokines.

Quinaprilat reduces myocardial infarct size involving nitric oxide production and mitochondrial KATP channel in rabbits.

This study examined whether quinaprilat, an angiotensin-converting enzyme inhibitor, reduces the infarct size, and investigated the mechanisms for its infarct size–reducing effect, in rabbits. Japanese white rabbits underwent 30 min of ischemia and 48 h of reperfusion. Quinaprilat (100 &mgr;g/kg/h or 300 &mgr;g/kg/h for 70 min, IV) was administered 20 min before ischemia with or without pretreatment with N ω-nitro-l-arginine methyl ester (l-NAME) (10 mg/kg, IV, a nitric oxide synthase inhibitor), 5-hydroxydecanoic acid sodium salt (5-HD) or posttreatment with 5-HD (5 mg/kg, IV, a mitochondrial KATP channel blocker). The area at risk as a percentage of the left ventricle was determined by Evans blue dye and the infarct size was determined as a percent of the area at risk by triphenyl tetrazolium chloride staining. Using a microdialysis technique, myocardial interstitial levels of 2,5-dihydroxybenzoic acid (2,5-DHBA), an indicator of hydroxyl radicals, and NOx, an indicator of nitric oxide, were measured before, during, and after 30 min of ischemia. Quinaprilat significantly reduced the infarct size in a dose-dependent manner (30.1 ± 3%, n = 10, and 27.6 ± 2%, n = 7, respectively) compared with the control (46.5 ± 4%, n = 10). The infarct size–reducing effect of quinaprilat was completely blocked by pretreatment with l-NAME (43.8 ± 2%, n = 8) and 5-HD (50.1 ± 3%, n = 8) and posttreatment with 5-HD (50.3 ± 2%, n = 8), respectively. Quinaprilat did not affect the myocardial interstitial 2,5-DHBA level but significantly increased the NOx level during ischemia and reperfusion. Quinaprilat reduces myocardial infarct size involving NO production and mitochondrial KATP channels in rabbits without collateral circulation.