Effect of exogenous Oct4 overexpression on cardiomyocyte differentiation of human amniotic mesenchymal cells.

Regenerative therapy is a new strategy for the end-stage heart failure; however, the ideal cell source has not yet been established for this therapy. We expected that the amnion might be an ideal cell source for cardiac regenerative therapy and that the differentiation potency of the human amnion mesenchymal cells (hAMCs) could be improved by overexpression of Oct4, a key factor that maintains the undifferentiated state. A plasmid vector was made by insertion of the Oct4 open reading frame (ORF) under control of a cytomegalovirus (CMV) promoter (pCMV-hOct4) and transfected into hAMCs by electroporation. The optimum induction time was investigated by comparing the quantity of stem cell-specific mRNAs, cardiac-specific mRNAs, and cardiac-specific proteins with time. hAMCs already expressed cardiac-specific proteins such as Nkx2.5 and Connexin43. After pCMV-hOct4 transfection, endogenous Oct4 mRNA and other stem cell markers showed a transient increase. With 5-azacytidine treatment, quantities of the cardiac-specific mRNAs, such as GATA4 and myosin light-chain-2v (Mlc-2v), were increased significantly. After Oct4 overexpression, the highest expression of cardiac-specific mRNAs and stem cell makers was seen at almost the same time. Furthermore, more mature myocardial contraction proteins were observed when hAMCs were induced at specific optimal times after gene transfection. In conclusion, hAMCs were activated to an undifferentiated state by overexpression of Oct4, and their cardiac differentiation potency was improved. Thus, the single-time transfection of the Oct4 expression vector may be a useful strategy for effective cell therapy. The use of cryopreserved hAMCs in cell therapy still requires more investigation.

Ventricular septal dysfunction after surgical closure of multiple ventricular septal defects.

BACKGROUND: We assessed the global and regional ventricular septal functions using conventional echocardiography and two-dimensional speckle tracking imaging in children with postoperative multiple ventricular septal defects. METHODS: Thirty-six children were studied: 16 with postoperative multiple ventricular septal defects and 20 normal control subjects. In children with multiple ventricular septal defects, 60 ventricular septal defects were closed using one of three different techniques (patch closure, the sandwich technique, direct closure). Speckle tracking imaging was applied to three short-axis echocardiographic images. RESULTS: The total patch area used in the multiple ventricular septal defects group was correlated with the postoperative ejection fraction (r=0.703) and Tei index (r=0.778). The global septal peak systolic radial displacement and global septal peak systolic radial strain in the multiple ventricular septal defects group were significantly lower than those observed in the control subjects. The peak systolic radial strain in the segments closed with patches and the peak systolic radial displacement in the segments closed with the felt sandwich technique were significantly lower than those observed in the intact septal segments. No significant regional functional depressions were identified in the segments that were closed directly. CONCLUSIONS: The postoperative ventricular global and septal functions were significantly reduced in children with multiple ventricular septal defects, especially in the cases with complex congenital heart disease and that were closed with large prosthetic materials. These results suggest that an effort to minimize the use of patch materials may lead to preserved postoperative ventricular function.

[Current status and prospect of off-pump pulmonary vein isolation for atrial fibrillation].

We reviewed our clinical experience in off-pump pulmonary vein isolation (PVI) using bipolar radiofrequency (RF) device for atrial fibrillation (Af). From August 2004 to July 2007, the combined off-pump PVI and coronary artery bypass grafting (CABG) was performed in 13 of the 22 planned patients. There was no operative mortality or major complication. Sinus rhythmus was established in 69.2% of the patients [paroxysmal Af (PAf), 8/8 patients; chronic Af, 1/5 patients]. Off-pump PVI using bipolar RF may not be indicated to the chronic Af due to the low curability, and bipolar RF is not safe, especially in case with cardiomegaly or low cardiac function. We developed novel technique using RF thermal balloon catheter for off-pump PVI and evaluated the technique in experimental model. Off-pump PVI with RF thermal balloon catheter was considered to be a safe and effective method to ablate the left atrium-pulmonary vein (LA-PV) antrum circumferentially.

Impact of preoperative atrial fibrillation on the late outcome of off-pump coronary artery bypass surgery.

OBJECTIVE: The impact of pre-existing atrial fibrillation on the long-term outcome in patients after off-pump coronary revascularisation is not well known. This study aims to determine the independent effects of preoperative atrial fibrillation on the early and late outcomes of off-pump coronary artery bypass surgery. METHODS: A total of 513 patients undergoing isolated coronary artery bypass surgery using off-pump approach between 2000 and 2005 were studied. Twenty-six of them (5.1%) had preoperative atrial fibrillation (15 had paroxysmal atrial fibrillation and 11 had persistent or permanent atrial fibrillation) and the other 487 patients were in normal sinus rhythm. Early and late outcomes were compared retrospectively between patients with preoperative atrial fibrillation and patients in sinus rhythm. The median follow-up period for the entire study population was 3.3 + or - 2.7 years. RESULTS: The baseline characteristics of the patients with preoperative atrial fibrillation were generally similar to those of patients in sinus rhythm. However, the patients with atrial fibrillation had a significantly lower left ventricular ejection fraction compared with those in sinus rhythm (50 + or - 15 vs 56 + or - 12%, p=0.03). The mean age of the atrial fibrillation group was almost 3 years more than that of the sinus rhythm group. Operative mortality was similar in patients with atrial fibrillation (3.8%) and those in sinus rhythm (1.0%). Ten patients developed cerebral infarction within 7 days after surgery, including one patient (3.8%) from the atrial fibrillation group and nine patients (1.8%) from the sinus rhythm group. Long-term survival was significantly decreased in the atrial fibrillation group (5-year survival: 70 + or - 9.6% vs 87 + or - 1.8%; p=0.0018). Freedom from cerebral complications was also significantly decreased in the atrial fibrillation group (5-year survival: 85 + or - 8.3% vs 95 + or - 1.2%; p=0.0009), but there were no differences in cardiac death and major cardiac adverse events. On Cox proportional hazards regression analysis, preoperative atrial fibrillation was a significant adverse predictor for survival (hazard ratio=3.0, 95% confidence intervals (CIs) 1.3-6.9; p=0.009) and independent predictor of late cerebral infarction (hazard ratio=6.2, 95% CIs 2.0-19.3; p=0.0002). CONCLUSIONS: Uncorrected preoperative atrial fibrillation is strongly associated with poor long-term survival and increased late cerebral complications after off-pump coronary artery bypass surgery. Concomitant atrial fibrillation surgery should be considered to improve the long-term results of surgical revascularisation.

Inducible capillary formation in lymphatic endothelial cells by blocking lipid phosphate phosphatase-3 activity.

Lymphangiogenesis plays critical roles under normal and/or pathological conditions; however, the molecular contributors to this event were unknown until recently. In the present study, we first employed gene chip analysis and confirmed that lipid phosphate phosphatase-3 (LPP3) expression was increased until capillary formation in the conditionally immortalized rat lymphatic endothelial cell line. Signaling responses occur when several lipids induce acute biological functions; further, lipid phosphate phosphatases (LPPs) control their functions via dephosphorylation; however, there is no report on the association between LPP3 and lymphangiogenesis. siRNA-targeted LPP3 significantly increased capillary formation of human lymphatic endothelial cells; in contrast, it decreased cell adhesion to the basement membrane matrix. Furthermore, the inducible effect of the LPP inhibitor on capillary formation was observed. For the first time, we report that LPP3 abolishes accelerated abnormal lymphangiogenesis. Blocking LPP3 activities may aid in the development of novel therapy for lymph vessel defects.

Surgical management of multiple ventricular septal defects: the role of the felt sandwich technique.

OBJECTIVE: Recently, the felt sandwich technique has been widely used to close muscular ventricular septal defects. We evaluated the early and midterm results of our strategy (a combination of the sandwich technique and direct closures) and assessed the role of the sandwich technique in the treatment of multiple ventricular septal defects. METHODS: Twenty-nine consecutive patients underwent an operation for multiple ventricular septal defects and associated cardiac malformations. They included 17 boys and 12 girls with a median age of 6.0 months. Thirteen patients had 4 or more ventricular septal defects (Swiss cheese septum). RESULTS: There was no surgical or follow-up mortality, and no reoperations were required. There were no cases of heart block and no significant residual shunts in the latest follow-up study. Two patients with Swiss cheese septum had postoperative congestive heart failure. Three muscular ventricular septal defects were closed with the sandwich technique in these 2 patients, whereas 1 or fewer ventricular septal defects were closed with the sandwich technique in the other 27 patients. Seven (77.8%) of 9 patients who underwent the sandwich procedure had septal dysfunction, whereas 5 (25.0%) of the other 20 patients showed septal dysfunction (P < .05). CONCLUSIONS: The outcome of the surgical repair of multiple ventricular septal defects was satisfactory. Although the sandwich technique is simple and effective, the use of numerous felt patches disturbed the movement of the interventricular septum. An effort should be made to close the muscular ventricular septal defect directly to avoid postoperative cardiac dysfunction. Large apical ventricular septal defects, especially those located just underneath the moderator band, are considered suitable for the sandwich technique.

An infected pseudoaneurysm following a modified Blalock-Taussig shunt.

A two-month-old male infant with tetralogy of Fallot underwent a right-sided modified Blalock-Taussig shunt using a 4 mm expanded polytetrafluoroethylene graft through a right thoracotomy. Five months later, the patient developed otitis media, followed by repeated relapses of pneumonia and fever of unknown origin. Multidetector-row computed tomography and angiography, performed at 12 months of age, revealed a pseudoaneurysm of the subclavian artery at the insertion of the modified Blalock-Taussig shunt. After 20 days of antibiotic therapy, the pseudoaneurysm and infected graft were successfully resected through a median sternotomy approach. This report describes the treatment strategy of this rare but potentially fatal complication after a modified Blalock-Taussig shunt operation.

A coronary active perfusion system for off-pump coronary artery bypass grafting in an experimental porcine model: the relationship between flow rate and myocardial function.

We developed a coronary active perfusion system (synchronized arterial flow ensuring system [SAFE-System]) to prevent myocardial ischemia during distal anastomosis in off-pump coronary artery bypass grafting (CABG). The purpose of this study was to identify the relationship between the flow rate of the SAFE-System and myocardial function. Studies were performed on pigs, which were divided into five groups: external shunt perfusion group (group A, n = 6); 0.1 mL/beat flow rate for the SAFE-System group (group B, n = 6); 0.2 mL/beat flow rate group (group C, n = 6); 0.3 mL/beat flow rate group (group D, n = 6); and 0.4 mL/beat flow rate group (group E, n = 6). Regional myocardial blood flow and left ventricular function were monitored for 30 min. The regional myocardial blood flow in group A was severely decreased (P < 0.001), and was significantly lower than in the other groups (P < 0.001). The slope of the end-systolic pressure-volume relationship, and the slope of the preload recruitable stroke work relationship in groups A, D, and E were lower than in groups B and C. As compared with the use of a passive external shunt, a coronary active perfusion system provides adequate myocardial blood flow and hemodynamics. It was possible to maintain left ventricular function when using 0.1 or 0.2 mL/beat flow rate. The use of a coronary active perfusion system appears to make the off-pump CABG procedure safer and may increase the application of off-pump bypass grafting.

Experimental mitral valve plasty under the beating heart guided by real-time 3-dimensional echocardiography.

BACKGROUND: This study examines whether real-time 3-dimensional echocardiography can provide an image resolution to serve as a substitute for optical visualization in performing mitral valve plasty. METHODS: Three pigs were used in this study. A 3-dimensional echocardiographic system was evaluated on an epicardial surface. Beating heart mitral valve plasty was performed with a surgical stapler inserted from the apex of the left ventricle using only 3-dimensional echocardiographic visualization. RESULTS: The high-quality images of the mitral valve were obtained with the probe at the epicardial position. However, by inserting the surgical instrument into the left ventricle, an acoustic shadow developed on the images. The images became indistinct because of the acoustic shadow, and operation became difficult. For the mitral valve plasty, an edge-to-edge mitral valve repair was carried out using a stapler (10 mm) under the beating heart. The stapler was confirmed to seize both leaflets evenly in only 1 of the 3 pigs. CONCLUSIONS: Real-time 3-dimensional echocardiography provided clear 3-dimensional images of the mitral valve; however, when a surgical instrument was inserted into the left ventricle, an acoustic shadow appeared on the image and made detailed confirmation difficult. Lessening or eliminating the acoustic shadow would be a key point to improve this procedure.

CCL21 promotes the migration and adhesion of highly lymph node metastatic human non-small cell lung cancer Lu-99 in vitro.

To develop new therapy strategies for lung cancer, we established an animal model, which reflects the clinical features of mediastinal lymph node metastasis of lung cancer. This study was designed to determine whether CCL21 induced biological functions associated with the metastasis of highly lymph node metastatic human non-small cell lung cancer (NSCLC) selected by our model. Orthotopic intrapulmonary implantation of human NSCLC (Lu-99 and A549) was performed to analyze the metastatic characteristics of these cells. The expression of CCR7, which is a receptor of CCL21, was detected using CCL19 [also called EBI1-ligand chemokine (ELC)]-Fc chimera by flow cytometric analysis. The effects of CCL21 on the migration, adhesion and growth of human NSCLC were investigated. After orthotopic implantation of human NSCLC cell lines, Lu-99, but not A549, metastasized to mediastinal lymph nodes, forming large size nodules, and expressed CCR7 on the surface. Accordingly, its ligand CCL21 induced chemotactic migration and alpha4beta1-mediated adhesion to VCAM-1 of Lu-99. The expression of CCR7 and vigorous responses to its ligand CCL21 potentially account for lymph node metastasis of a human NSCLC line Lu-99.

Predictors of early postoperative cerebral infarction after isolated off-pump coronary artery bypass grafting.

BACKGROUND: Risk factors associated with cerebral infarction within 7 days after off-pump coronary artery bypass grafting require further statistical elucidation. METHODS: From January 1997 to July 2006, off-pump coronary artery bypass grafting was performed in 576 patients at Toyama University Hospital. Factors including previous cerebral infarction, lesions in head and neck vessels, preoperative cerebral ischemic symptoms, intraaortic balloon pump use, number of coronary lesions, number of coronary bypasses, sites of coronary bypasses, number of proximal anastomoses in the ascending aorta, use of aortic no-touch procedure, partial aortic clamping, automatic proximal anastomosis devices, and a proximal seal system were compared retrospectively between patients with and without early cerebral infarction. Factors differing between groups were analyzed further by multivariate logistic regression. RESULTS: Ten patients (1.7%) had cerebral infarction within 7 days after surgery. The subjects were divided into two groups, those who had cerebral infarction after surgery (n = 10) and those who did not (n = 566). Factors showing significant intergroup differences were a previous history of cerebral infarction (p < 0.001), preoperative presence of cerebral ischemic symptoms (p < 0.001), vascular lesions in head and neck vessels (p < 0.001), use of partial aortic clamping (p = 0.002), and postoperative atrial fibrillation within 7 days after surgery (p = 0.011). Multiple logistic regression analysis indicated close relationships between previous history of cerebral infarction (odds ratio, 26.6; 95% confidence interval, 2.8 to 251.1; p = 0.004), preoperative presence of cerebral ischemic symptoms plus lesions in head and neck vessels (odds ratio, 22.8; 95% confidence interval 1.8 to 285.7; p = 0.015), and use of partial aortic clamping (odds ratio, 11.1; 95% confidence interval, 1.4 to 85.7; p = 0.021). Postoperative atrial fibrillation within 7 days after surgery (odds ratio, 3.4; 95% confidence interval, 0.7 to 16.5; p = 0.121) was suspected as a risk factor for postoperative cerebral infarction. CONCLUSIONS: Multivariate analysis identified independent factors strongly associated with cerebral infarction after off-pump coronary artery bypass grafting, such as partial aortic clamping, presence of cerebral ischemic symptoms plus head and neck vascular lesions, and previous cerebral infarction.

Open heart operation in a child with congenital heart disease and hereditary spherocytosis.

An 18-month-old girl with hereditary spherocytosis underwent closure of the ventricular septal defect, commissurotomy of the pulmonary valve, and patch angioplasty of the pulmonary trunk without previous splenectomy. No serious complications as a result of hemolysis occurred in the perioperative period. Open heart surgery can therefore be safely performed in young children with congenital heart disease and hereditary spherocytosis who have not previously undergone splenectomy.

Closure of multiple ventricular septal defects by the felt sandwich technique: further analysis of 36 patients.

OBJECTIVES: We previously described a novel technique for closure of trabecular ventricular septal defects by sandwiching the septum with 2 polyester felt patches without requiring left ventriculotomy. We describe the midterm results of this technique and the postoperative cardiac function. METHODS: This is a retrospective study of 36 consecutive patients. The median age and body weight at the time of repair was 15 months (range: 2-115 months) and 7.8 kg (range: 3.9-51.9 kg), respectively. RESULTS: Sixty-three trabecular ventricular septal defects in 36 patients were closed with the felt sandwich technique. In the early postoperative period, 1 patient died of pulmonary hypertensive crisis. There were 2 late deaths. One patient died of pneumonia 6 months after surgery, and another died suddenly of ventricular arrhythmias 2 years after surgery. Three patients required reoperation (closure of major residual ventricular septal defect, cardiac transplantation for dilated cardiomyopathy, and pacemaker implantation for complete atrioventricular block). Postoperative left ventricular fractional shortening in the group with a body surface area less than 0.4 m2 was significantly lower than that in the group with a body surface area of 0.4 m2 or greater (0.22 +/- 0.09 vs 0.31 +/- 0.06, P = .0027). Moreover, there was a strong correlation between postoperative left ventricular ejection fraction and total patch area/body surface area ratio (R = -0.74, P = .0004). CONCLUSION: Multiple trabecular ventricular septal defects can be closed with the felt sandwich technique easily and safely. Although this technique can be used in small infants, the use of numerous felt patches disturbs the movement of ventricular septum, which may cause postoperative cardiac dysfunction.

An echo-contrast agent, Levovist, lowers the ultrasound intensity required to induce apoptosis of human leukemia cells.

To verify the effect of echo-contrast agent (ECA) on apoptosis induced by ultrasound, leukemia cell lines (Jurkat, Molt-4 and U937) were sonicated at intensities previously shown to induce optimal apoptosis with or without Levovist, an ECA. The results showed that loss of viability and apoptosis can be induced in all three cell lines, apoptosis highest with Molt-4, based on viability and DNA fragmentation assay. Such finding was supported by corresponding increase of cells with low mitochondrial membrane potential, high superoxide production, increased intracellular calcium concentration, and phosphorylation of histone H2AX after sonication. Optimal ultrasound condition was 0.3W/cm(2), 1MHz, 10% duty factor pulsed at 100Hz; but in the presence of Levovist, an apparent shift of cell killing induction was observed at 0.2W/cm(2). While these results further confirmed previous findings on ultrasound-induced apoptosis, they also suggest that use of an enhancing factor, such as addition of ECA, may be useful in cancer therapy when a much lower intensity is desired.

Apoptosis induced by the sonomechanical effects of low intensity pulsed ultrasound in a human leukemia cell line.

To obtain an optimal condition for ultrasound (US)-induced apoptosis that could be useful for cancer therapy, we applied low intensity pulsed US to sonicate U937 cells in vitro. Cells were then incubated at different time intervals before measuring apoptosis. The apoptosis was assessed by DNA fragmentation and phosphatidylserine externalization. The pattern of the decrease in mitochondrial membrane potential was determined by flow cytometry. Optimal apoptosis (70.0+/-13.8%) with minimal lysis was attained with 1 MHz ultrasound 0.3 W/cm2, 10% duty factor at 100 Hz for 1 min) at 12 h after sonication. Lack of US-induced free radical detection and absence of Heme oxygenase-1, an intracellular oxidative stress marker, up-regulation in cells, suggest that sonomechanical, not sonochemical, effects are the main mechanism involved.

Ischemic changes in evoked spinal cord potentials during profound hypothermic circulatory arrest in thoracic aortic surgery.

PURPOSE: We examined the changes in evoked spinal cord potentials (ESCP) during profound hypothermic circulatory arrest to estimate the safe ischemic time. METHODS: We monitored ESCPs during surgery for descending thoracic or thoracoabdominal aneurysms in five patients. Evoked spinal cord potential recordings were obtained before cooling (baseline), then every few minutes during circulatory arrest, and at the end of the operation. RESULTS: After circulatory arrest, the amplitude of ESCPs decreased with time. We calculated the simple linear regression between the amplitude of ESCPs and the circulatory arrest time by the least-squares method, and found a highly linear relationship between amplitude and arrest time in all five patients. The time until disappearance of ESCPs was estimated as 50.7 +/- 20.4 min (95% level of confidence). CONCLUSION: When an ESCP disappeared, ischemic spinal cord injury had occurred. This demonstrates the potential value of estimating the time of disappearance of ESCPs to prevent ischemic spinal cord injury during descending thoracic aortic surgery.

Left pulmonary arterioplasty--extended end-to-end anastomosis.

Surgical arterioplasty for left pulmonary branch stenosis often produces unsatisfactory results. We report a new operative approach involving a modification of end-to end anastomosis, providing a widely patent and nonredundant anastomosis.

Surgical repair of coronary sinus orifice atresia.

We report a 10-month-old boy who underwent a bi-directional Glenn procedure and repair of coronary sinus orifice atresia. The left superior vena cava was the only vessel communicating with the coronary sinus in this case. The coronary sinus was allowed to communicate freely with the left atrium by creating a partially unroofed coronary sinus using a left superior vena cava flap.