Hitoshi Yaku

Controlled Delivery of Basic Fibroblast Growth Factor Promotes Human Cardiosphere-Derived Cell Engraftment to Enhance Cardiac Repair for Chronic Myocardial Infarction

OBJECTIVES This study was designed to determine whether controlled release of basic fibroblast growth factor (bFGF) might improve human cardiosphere-derived cell (hCDC) therapy in a pig model of chronic myocardial infarction. BACKGROUND Current cell therapies for cardiac repair are limited by loss of the transplanted cells and poor differentiation. METHODS We conducted 2 randomized, placebo-controlled studies in immunosuppressed pigs with anterior myocardial infarctions. Four weeks after coronary reperfusion, 14 pigs were randomly assigned to receive an intramyocardial injection of placebo medium with or without bFGF-incorporating hydrogel implantation. As a second study, 26 pigs were randomized to receive controlled release of bFGF combined with or without hCDCs or bone marrow-derived mesenchymal stem cell transplantation 4 weeks after reperfusion. RESULTS Controlled release of bFGF in ischemic myocardium significantly augmented the formation of microvascular networks to enhance myocardial perfusion and contractile function. When combined with cell transplantation, the additive effects of bFGF were confined to hCDC-injected animals, but were not observed in animals receiving human bone marrow-derived mesenchymal stem cell transplantation. This was shown by increased donor-cell engraftment and enhanced cardiomyocyte differentiation in the transplanted hearts, resulting in synergistically improved ventricular function and regional wall motion and reduced infarct size. CONCLUSIONS Controlled delivery of bFGF modulates the post-ischemic microenvironment to enhance hCDC engraftment and differentiation. This novel strategy demonstrates significant functional improvements after myocardial infarction and may potentially represent a therapeutic approach to be studied in a clinical trial in human heart failure.

Early Outcome of a Randomized Comparison of Off-Pump and On-Pump Multiple Arterial Coronary Revascularization

Background—Previous randomized comparisons of off-pump and on-pump coronary artery bypass grafting (CABG) have yielded controversial results about the cardiac and neurological events and graft patency. In addition, these randomized studies were composed of CABG with a few arterial grafts. We performed a prospective randomized controlled study to compare off-pump and on-pump CABG with multiple arterial grafts. Methods and Results—Between July, 2002, and September, 2004, 167 consecutive unselected patients referred for elective primary CABG were randomly assigned to undergo multiple arterial off-pump CABG (n=81) or on-pump CABG (n=86). The clinical outcomes and S-100 protein, neuron-specific enolase, and maximum creatine kinase-MB levels were compared. Early graft patency was examined within 3 weeks after the operation by angiography. The number of grafts performed per patient (3.5±1.0 for off-pump CABG and 3.6±0.9 for on-pump CABG) and the number of arterial grafts performed per patient (3.3±1.0 for off-pump CABG and 3.4±0.9 for on-pump CABG) were similar. Completeness of revascularization (completed grafts/planned grafts) was 98% in both procedures. There were no hospital deaths in either group. The operation time was significantly (P<0.001) shorter in the off-pump group than in the on-pump group (267±60 minutes versus 307±59 minutes). The incidence of perioperative complications was similar. The frequency of no need for transfusion was higher in the off-pump group than in the on-pump group (80% versus 55%, P<0.001). The S-100 protein levels at the admission into the intensive care unit were significantly (P<0.001) lower in the off-pump group than in the on-pump group (0.20±0.11 ng/mL versus 0.34±0.22 ng/mL). The neuron-specific enolase levels at the intensive care unit admission were significantly (P<0.001) lower in the off-pump group than in the on-pump group (10.4±9.0 ng/mL versus 16.9±6.9 ng/mL). Maximum creatine kinase-MB levels were significantly (P=0.046) lower in the off-pump group than in the on-pump group (17.1±16.7 IU/L versus 21.5±10.6 IU/L). The overall early graft patency rate with or without stenosis was the same (98%) in both groups, but the rate without stenosis was slightly worse in the off-pump group (93%) than in the on-pump group (96%) (P=0.093). The stenosis-free patency rate in the right coronary area was significantly (P=0.028) worse in the off-pump CABG group (90%) than in the on-pump group (99%). Conclusions—Off-pump CABG with multiple arterial grafts was as safe as the conventional on-pump CABG, with similar completeness of revascularization and early graft patency.

Differentiation from embryonic stem cells to vascular wall cells under in vitro pulsatile flow loading

This study evaluated the possibility of differentiation from embryonic stem (ES) cells to vascular wall cells by physical (mechanical) stress loading in vitro. A cell mixture containing Flk1-positive cells (ca. 30%) derived from murine ES cells was added to a compliant microporous tube made of segmented polyurethane. The compliance of the tube was close to that of the human artery [the stiffness parameter (β) = 57.2 (n = 5, SD < 5%)]. The luminal surface of the tube was fully covered with the cells by preincubation for two days in the presence of vascular endothelial growth factor (VEGF). After 2 days of additional incubation without VEGF under static conditions, layering of the grown cells, mostly smooth muscle actin (SMA)-positive cells, was observed only on the luminal surface of the tube. The cells were flat, polygonal, and randomly oriented. On the other hand, after a 2-day incubation under a weak pulsatile flow simulating the human venous systems [wall shear stress (WSS) from −0.98 to 2.2 dyn/cm2; circumferential strain (CS) 4.6–9.6 × 104 dyn/cm2] without VEGF, cells in the superficial layer were regularly oriented in the direction of the pulsatial flow. The oriented cells exhibited endothelial-like appearance, indicating that they were platelet endothelial cell adhesion molecule 1 (PECAM1)-positive. In addition, the cells growing into the interstices in the deeper layer showed smooth muscle-like appearance, indicating that they were SMA-positive. Differentiation to two different cell types and segregation of incorporated ES cells may be simultaneously encouraged by the combination of WSS and CS. It is expected that the monobloc building of hierarchically structured hybrid vascular prostheses composed of several vascular wall cell types is possible by physically synchronized differentiation of ES cells.

Label-free biochemical imaging of heart tissue with high-speed spontaneous Raman microscopy

Label-free imaging is desirable for elucidating morphological and biochemical changes of heart tissue in vivo. Spontaneous Raman microscopy (SRM) provides high chemical contrast without labeling, but presents disadvantage in acquiring images due to low sensitivity and consequent long imaging time. Here, we report a novel technique for label-free imaging of rat heart tissues with high-speed SRM combined with resonance Raman effect of heme proteins. We found that individual cardiomyocytes were identified with resonance Raman signal arising mainly from reduced b- and c-type cytochromes, and that cardiomyocytes and blood vessels were imaged by distinguishing cytochromes from oxy- and deoxy-hemoglobin in intact hearts, while cardiomyocytes and fibrotic tissue were imaged by distinguishing cytochromes from collagen type-I in infarct hearts with principal component analysis. These results suggest the potential of SRM as a label-free high-contrast imaging technique, providing a new approach for studying biochemical changes, based on the molecular composition, in the heart.

Expanded polytetrafluoroethylene conduits and patches with bulging sinuses and fan-shaped valves in right ventricular outflow tract reconstruction: Multicenter study in Japan

OBJECTIVE There is no optimal substitute for right ventricular outflow tract (RVOT) reconstruction in congenital heart defects. Expanded polytetrafluoroethylene (ePTFE) valved conduits and patches may be a good alternative to homografts and bovine jugular veins. We have developed a fan-shaped ePTFE valve and an ePTFE valved conduit and patch with bulging sinuses with the aim of enhancing the long-term valve function. METHOD Bulging sinuses were constructed on ePTFE conduits and patches as described previously (J Thorac Cardiovasc Surg. 2007;134:327-32). Between February 2001 and January 2011, 794 patients (aged 14 days to 56.8 years old; median, 2.0 years old) had ePTFE valves implanted for RVOT reconstruction at 52 Japanese institutes. Conduits with a fan-shaped ePTFE valve were implanted in 325 patients and a patch with a fan-shaped ePTFE valve was implanted in 469 patients. Valve function was assessed by a series of echocardiograms postoperatively. RESULTS The mean follow-up was 3.6 years (1.1 months to 10.0 years). Freedom from reoperation at 10 years was 95.4% in patients with conduits and 92.3% in those with patches. Pulmonary insufficiency was mild or nonexistent in 95.0% of patients with conduits and 79.6% of patients with patches. The pressure gradient between the right ventricle and the pulmonary artery was 14.0 ± 13.2 mm Hg in patients with conduits and 11.6 ± 11.6 mm Hg in patients with patches. CONCLUSIONS Fan-shaped ePTFE valved conduits and patches with bulging sinuses have a high freedom from reoperation and prevent pulmonary insufficiency. They represent a promising material for RVOT reconstruction.

Development of a Completely Autologous Valved Conduit With the Sinus of Valsalva Using In-Body Tissue Architecture Technology A Pilot Study in Pulmonary Valve Replacement in a Beagle Model

Background— We developed autologous prosthetic implants by simple and safe in-body tissue architecture technology. We present the first report on the development of autologous valved conduit with the sinus of Valsalva (BIOVALVE) by using this unique technology and its subsequent implantation in the pulmonary valves in a beagle model. Methods and Results— A mold of BIOVALVE organization was assembled using 2 types of specially designed silicone rods with a small aperture in a trileaflet shape between them. The concave rods had 3 projections that resembled the protrusions of the sinus of Valsalva. The molds were placed in the dorsal subcutaneous spaces of beagle dogs for 4 weeks. The molds were covered with autologous connective tissues. BIOVALVEs with 3 leaflets in the inner side of the conduit with the sinus of Valsalva were obtained after removing the molds. These valves had adequate burst strength, similar to that of native valves. Tight valvular coaptation and sufficient open orifice area were observed in vitro. These BIOVALVEs were implanted to the main pulmonary arteries as allogenic conduit valves (n=3). Postoperative echocardiography demonstrated smooth movement of the leaflets with trivial regurgitation. Histological examination of specimens obtained at 84 days showed that the surface of the leaflet was covered by endothelial cells and neointima, including an elastin fiber network, and was formed at the anastomosis sides on the luminal surface of the conduit. Conclusion— We developed the first completely autologous BIOVALVE and successfully implanted these BIOVALVEs in a beagle model in a pilot study.

Dynamic cardiac compression improves contractile efficiency of the heart

The effect of dynamic cardiac compression on left ventricular contractile efficiency was assessed in terms of the pressure-volume relationship and myocardial oxygen consumption. In 11 excised cross-circulated dog hearts, the ventricle was directly compressed during systole (dynamic cardiac compression). Measurements for pressure-volume area (a measure of total mechanical energy), external work, and myocardial oxygen consumption were done before and during dynamic cardiac compression. Dynamic cardiac compression increased pressure-volume area by 28% +/- 17% (mean plus or minus the standard deviation) and external work by 24% +/- 20% (p = 0.0000185 and 0.0000212, respectively) at given end-diastolic and stroke volumes without affecting myocardial oxygen consumption. As a result, the oxygen cost of pressure-volume area, that is, the slope of the myocardial oxygen consumption-pressure-volume area relationship, significantly decreased by 16% +/- 13% (p = 0.0000135) whereas the pressure-volume area-independent myocardial oxygen consumption was unchanged. Then, contractile efficiency, that is, the reciprocal of the slope of the myocardial oxygen consumption-pressure-volume area relationship in joules significantly improved from 45% +/- 8% to 53% +/- 13% (p = 0.0000437). When the native myocardial oxygen consumption-pressure-volume area relationship was assessed by subtracting the dynamic cardiac compression pressure applied to the heart, the slope of the myocardial oxygen comsumption-pressure-volume area relationship returned to the control level. This indicates that the contractile efficiency of the native heart was not affected by dynamic cardiac compression. We conclude that dynamic cardiac compression enhances left ventricular pump function by improving the contractile efficiency of the overall heart leaving the energetics of the native heart unchanged.

Local elasticity imaging of vascular tissues using a tactile mapping system

This study aimed to map the elasticity of a natural artery at the micron level by using a tactile mapping system (TMS) that was recently developed for characterization of the stiffness of tissue slices. The sample used was a circumferential section (thickness, approximately 1 mm) of a small-caliber porcine artery (diameter, approximately 3 mm). Elasticity was measured with a probe of diameter 1 μm and a spatial resolution of 2 μm at a rate of 0.3 s per point, without significant sample invasion. Topographical measurements were also performed simultaneously. Wavy regions of high elasticity, layered in the circumferential direction, were measured at the tunica media, which was identified as an elastin-rich region. The Young’s modulus of the elastin-rich region in the media was 50.8 ± 13.8 kPa, and that of the elastin-rich region of the lamina elastica interna was 69.0 ± 12.8 kPa. Both these values were higher than the Young’s modulus of the other regions in the media, including smooth muscle cells and collagen fibrils (17.0 ± 9.0 kPa). TMS is simple and inexpensive to perform and allows observation of the distribution of the surface elastic modulus at the extracellular matrix level in vascular tissue. TMS is expected to be a powerful tool in evaluation of the maturation and degree of reconstruction in the development of tissue-engineered or artificial tissues and organs.

Mechanoenergetic effects of pimobendan in canine left ventricles. Comparison with dobutamine.

BackgroundWe hypothesized that the effect of pimobendan (UD-CG 115 BS) to increase calcium sensitivity of contractile protein might result in less myocardial oxygen consumption (Vo2) in comparison with dobutamine when they enhance ventricular contractility to the same extent. To examine this hypothesis, we compared the effects of pimobendan and dobutamine on left ventricular contractility and energetics using the frameworks of Emax (contractility index) and the relation between Vo2 and PVA (systolic pressure-volume area, a measure of left ventricular total mechanical energy) Methods and ResultsWe measured Vo2, Emax, PVA, and force-time integral (FVI) in excised, cross-circulated, nonfailing dog hearts. The slope of the Vo2-PVA relation reciprocally indicates the efficiency from PVA-dependent Vo2 to the total mechanical energy (contractile efficiency). The Vo2 intercept of the Vo2-PVA relation, i.e., PVA-independent Vo2, reflects energy utilization for excitationcontraction coupling. The ratio of FTI to PVA-dependent Vo2 can be called contractile economy. Both drugs comparably enhanced Emax. Although the contractile economy was greater by 14±19% (p<0.05>) for pimobendan than for dobutamine, the contractile efficiency was similar between the two drugs. Oxygen cost of contractility, defined as the slope of the relation between the PVA-independent Vo2 and Emax, was the same between the two drugs. Other mechanoenergetic effects of both drugs were similar except for a greater coronary vasodilating effect of pimobendan ConclusionsPimobendan has almost the same mechanoenergetic effects as dobutamine but slightly greater contractile economy and coronary vasodilation. The calcium-sensitizing effect of pimobendan did not save the oxygen cost of contractility.

Continuous Thrombus in the Right and Left Atria Penetrating the Patent Foramen Ovalis

A 34-year-old man with protein S deficiency and deep vein thrombosis was admitted to our hospital because of chest discomfort. Transthoracic echocardiography demonstrated mobile hyperechoic masses in the right and left atria (Figure 1). Contrast-enhanced, ECG-gated, multidetector-16 row CT (Aquilion 16, Toshiba) revealed homogeneous lesions located on the atrial septum in both the right and left atria (Figure …