Hisataka Yasui

Extracorporeal Cardiac Shock Wave Therapy Markedly Ameliorates Ischemia-Induced Myocardial Dysfunction in Pigs in Vivo

Background—Prognosis of ischemic cardiomyopathy still remains poor because of the lack of effective treatments. To develop a noninvasive therapy for the disorder, we examined the in vitro and vivo effects of extracorporeal shock wave (SW) that could enhance angiogenesis. Methods and Results—SW treatment applied to cultured human umbilical vein endothelial cells significantly upregulated mRNA expression of vascular endothelial growth factor and its receptor Flt-1 in vitro. A porcine model of chronic myocardial ischemia was made by placing an ameroid constrictor at the proximal segment of the left circumflex coronary artery, which gradually induced a total occlusion of the artery with sustained myocardial dysfunction but without myocardial infarction in 4 weeks. Thereafter, extracorporeal SW therapy to the ischemic myocardial region (200 shots/spot for 9 spots at 0.09 mJ/mm2) was performed (n=8), which induced a complete recovery of left ventricular ejection fraction (51±2% to 62±2%), wall thickening fraction (13±3% to 30±3%), and regional myocardial blood flow (1.0±0.2 to 1.4±0.3 mL · min−1 · g−1) of the ischemic region in 4 weeks (all P<0.01). By contrast, animals that did not receive the therapy (n=8) had sustained myocardial dysfunction (left ventricular ejection fraction, 48±3% to 48±1%; wall thickening fraction, 13±2% to 9±2%) and regional myocardial blood flow (1.0±0.3 to 0.6±0.1 mL · min−1 · g−1). Neither arrhythmias nor other complications were observed during or after the treatment. SW treatment of the ischemic myocardium significantly upregulated vascular endothelial growth factor expression in vivo. Conclusions—These results suggest that extracorporeal cardiac SW therapy is an effective and noninvasive therapeutic strategy for ischemic heart disease.

Human Endothelial Progenitor Cell-Seeded Hybrid Graft: Proliferative and Antithrombogenic Potentials in Vitro and Fabrication Processing

In this article, we show that human endothelial progenitor cells (EPCs) in circulating peripheral blood may be a novel cell source for a cell-incorporated engineered vascular graft. Cultures of human peripheral blood mononuclear cells collected by the density gradient technique developed highly proliferative EPC colonies, which expanded with culture time. The production rates of antiplatelet substances such as endothelial-type nitric oxide synthase and 6-keto-prostaglandin-F(1)-alpha were approximately one-third and approximately one-half of those of mature endothelial cells (ECs), respectively. On the other hand, the tissue-type plasminogen activator production rate of EPCs was almost the same as that of ECs. EPCs were seeded and cultured on a small-diameter compliant graft (inner diameter, 1.5 mm) made of microporous segmented polyurethane film coated with a photo-reactive gelatin layer, and subsequently subjected to hydrodynamic shear stress by ex vivo circulation. EPCs fully covering the graft elongated and aligned themselves with the direction of the flow, resulting in the production of an integrated EPC-engineered graft. These results indicate that EPCs, which have high proliferative potential and high antithrombogenic potential, comparable to those of ECs, are a suitable cell source for cardiovascular tissue engineering.

Fabrication of endothelial progenitor cell (EPC)-seeded intravascular stent devices and in vitro endothelialization on hybrid vascular tissue

Rapid re-endothelialization at an atherosclerotic lesion after balloon inflation or stent deployment may be essential for reducing or preventing local thrombus formation and restenosis. In order to prevent these complications via enhanced rapid re-endothelialization, we fabricated two types of endothelial progenitor cell (EPC)-seeded intravascular stent devices. One was a photocured gelatin-coated metallic stent, and the other was a microporous thin segmented polyurethane (SPU) film-covered stent on which photocured gelatin was coated. Both devices were seeded with ex vivo expanded EPCs obtained from canine peripheral blood. Seeded EPCs formed confluent monolayers onto surfaces of both photocured gelatin-coated stent struts and SPU film, and a majority of cells remained on surfaces of stents after stent expansion. The EPC-seeded stent was expanded in a tubular hybrid vascular medial tissue composed of vascular smooth muscle cells and collagen as an arterial media mimic. After 7-day culture, EPCs, which migrated from the stent struts, proliferated and endothelialized the luminal surfaces of the hybrid vascular medial tissue. This in vitro pilot study prior to in vivo experiments suggests that on-stent cell delivery of EPCs may be novel therapeutic devices for re-endothelialization or endothelium lining or paving at an atherosclerotic arterial wall, resulting in the prevention of on-stent thrombus formation and in-stent restenosis, as well as the rapid formation of normal tissue architecture.

Canine endothelial progenitor cell-lined hybrid vascular graft with nonthrombogenic potential

OBJECTIVE We sought to fabricate a compliant engineered vascular graft (inner diameter of approximately 4.5 mm and length of 6 cm) lined with endothelial progenitor cells derived from circulating peripheral canine blood and to verify its nonthrombogenicity potential in vivo. METHODS Autologous circulating endothelial progenitor cells derived from the peripheral veins of 6 adult mongrel dogs were isolated by using a density gradient method. The cells were proliferated in vitro in EGM-2 culture medium, prelined on the luminal surface of in situ-formed collagen type I meshes as an extracellular matrix, and wrapped with a segmented polyurethane thin film with multiple micropores as a compliant scaffold. After canine carotid arteries were bilaterally implanted with these grafts for 1 and 3 months, microscopic observation, histologic staining, and immunochemical staining were performed to evaluate morphogenesis. RESULTS After 33.3 +/- 10.5 days of culture in vitro, 4.2 +/- 1.2 x 10(6) endothelial progenitor cells were obtained. Eleven of the 12 engineered vascular grafts were patent. The grafts possessed smooth, glistening, and ivory-colored luminal surfaces at the predetermined observation period up to 3 months. The intimal layer was covered with confluent, cobblestone-like monolayered cells that were positively stained with factor VIIIB-related antigen. The thickness of the neoarterial walls was approximately 300 microm at 3 months after implantation. A few smooth muscle cells were observed in the medial tissue, and fibroblasts dominated the adventitial tissue. CONCLUSION Circulating endothelial progenitor cells could be a substitute source of endothelial cells for endothelialization on small-diameter-vessel prostheses to ensure nonthrombogenicity.

Adenovirus-Mediated Local Expression of Human Tissue Factor Pathway Inhibitor Eliminates Shear Stress–Induced Recurrent Thrombosis in the Injured Carotid Artery of the Rabbit

The main cause of acute coronary syndrome may be recurrent thrombosis, which is initiated by the activation of the extrinsic coagulation pathway. Tissue factor (TF) pathway inhibitor (TFPI) efficiently inhibits an early step in this pathway by the formation of a complex with factor VIIa, TF, and factor Xa. We determined whether local TFPI gene transfer can inhibit thrombosis in an injured artery without inducing systemic side effects. Balloon-injured rabbit carotid arteries were infected with an adenoviral vector that expressed either human TFPI (AdCATFPI) or bacterial beta-galactosidase (AdCALacZ). Two to 6 days after gene transfer, thrombosis was induced by the production of constant stenosis of the artery, and blood flow was measured continuously with an electromagnetic flow probe. A cyclic flow variation, which is thought to reflect the recurrent formation and dislodgment of mural thrombi, was observed in all AdCALacZ-infected arteries as well as in saline-infused arteries. In contrast, no cyclic flow variation was detectable in AdCATFPI-transfected arteries, even in the presence of epinephrine (1 microg. kg-1. min-1 infusion). Prothrombin time, activated partial thromboplastin time, and the ex vivo platelet aggregation induced by either adenosine diphosphate or collagen were unaltered in AdCATFPI-infected rabbits. We found that in vivo TFPI gene transfer into an injured artery completely inhibits the recurrent thrombosis induced by shear stress even in the presence of catecholamine, without affecting systemic coagulation status. Adenovirus-mediated local expression of TFPI may have the potential for the treatment of human thrombosis.

The effects of pulsatile and nonpulsatile systemic perfusion on renal sympathetic nerve activity in anesthetized dogs.

It is still controversial whether to pulse or not to pulse for the establishment of ideal extracorporeal circulation. We directly measured the renal sympathetic nerve activity in mongrel dogs (n = 10, weighing from 13 to 21 kg) to determine the effects of pulsatile and nonpulsatile systemic perfusion on the control of the sympathetic nerve activity during left ventricular assistance. Pulsatile perfusion was generated with an air-driven, diaphragm-type blood pump, and nonpulsatile perfusion was generated with a centrifugal pump. Renal sympathetic nerve activity and the blood flow of the descending aorta were then recorded during pulsatile and nonpulsatile systemic perfusion. Other variables, such as mean arterial pressure, central venous pressure, left atrial pressure, and blood gas levels, were kept constant. At the same mean arterial pressure, renal sympathetic nerve activity during pulsatile perfusion decreased significantly to 80% of renal sympathetic nerve activity during nonpulsatile perfusion (26.8 +/- 2.4 vs 33.4 +/- 2.9 spikes/sec, p < 0.01). Total systemic vascular resistance during pulsatile perfusion decreased significantly to 85% of that during nonpulsatile perfusion (5700 +/- 580 vs 6667 +/- 709 dynes.sec.cm-5, p < 0.05). These results suggest that pulsatile systemic perfusion, compared with nonpulsatile systemic perfusion, reduces sympathetic nerve activity and peripheral vascular resistance and thus may improve both microcirculation and organ function.

Total cavopulmonary connection with an extracardiac conduit: experience with 100 patients

BACKGROUND In the Fontan procedures total cavopulmonary connection with an extracardiac conduit is a concern. The potential benefits of an extracardiac conduit may be the avoidance of postoperative supraventricular arrhythmias over the long-term, hemodynamic benefits due to laminar flow, possibility of completion without anoxic arrest, and applicability to anomalous systemic or pulmonary venous return, or both anomalous systemic and pulmonary venous return. We demonstrate early to midterm results of total cavopulmonary connection with an extracardiac conduit. METHODS Between March 1994 and February 2000, a total of 100 patients underwent total cavopulmonary connection with an extracardiac conduit. In 27 patients, who underwent a single stage total cavopulmonary connection operation, 7 were done without palliation. Seventy-three patients had undergone a bidirectional Glenn shunt before completion of the total cavopulmonary connection. We used an expanded polytetrafluoroethylene tube graft as the extracardiac conduit. RESULTS Cardiopulmonary bypass time was 133.2+/-55.2 minutes. Myocardial ischemic time was 38.5+/-23.2 minutes in 40 patients who needed cardioplegic cardiac arrest for intracardiac procedures. Intraoperative fenestration was done in only 1 patient. There were no operative deaths. During follow-up of 37.3 months, there were 5 late deaths. When compared with the patients treated by the lateral tunnel technique in our institute, there was no significant difference in actuarial survival rate, but the event free rate of the extracardiac conduit group was significantly superior to the lateral tunnel group. CONCLUSIONS Total cavopulmonary connection with the extracardiac conduit produced good results in short to midterm follow-up.

Induction of Permanent Mixed Chimerism and Skin Allograft Tolerance Across Fully MHC-Mismatched Barriers by the Additional Myelosuppressive Treatments in Mice Primed with Allogeneic Spleen Cells Followed by Cyclophosphamide

A pure method of drug (cyclophosphamide plus busulfan)-induced skin allograft tolerance in mice that can regularly overcome fully H-2-mismatched barriers in mice has been established. The components of the method are i.v. administration of 1 × 108 allogeneic spleen cells on day 0, i.p. injection of 200 mg/kg CP and 25 mg/kg busulfan on day 2, and i.v. injection of T cell-depleted 1 × 107 bone marrow cells from the same donor on day 3. Recipient B10 (H-2b; IE−) mice prepared with this conditioning developed donor-specific tolerance, and long-lasting survival of skin allografts was shown in almost of the recipient mice. In the tolerant B10 mice prepared with new conditioning, stable multilineage mixed chimerism was observed permanently, and IE-reactive Vβ11+ T cells were reduced in periphery as seen in untreated B10.D2 (H-2d; IE+) mice. The specific tolerant state was confirmed by the specific abrogation against donor Ag in the assays of CTL activity and MLR and donor-specific acceptance in the second skin grafting. These results demonstrated that the limitation of standard protocol of cyclophosphamide-induced tolerance, which have been reported by us since 1984, can be overcome by the additional treatments with the myelosuppressive drug busulfan, followed by 1 × 107 T cell-depleted bone marrow cells. To our knowledge, this is the first report to induce allograft tolerance with a short course of the Ag plus immunosuppressive drug treatment without any kind of mAbs (pure drug-induced tolerance).

Coenzyme Q10: The Prophylactic Effect on Low Cardiac Output Following Cardiac Valve Replacement

A randomized, prospective study of the effectiveness of preoperative administration of coenzyme Q10 on the prophylaxis of postoperative low cardiac output state was performed in 50 patients with acquired valvular diseases necessitating valve replacement. There were 25 patients in the treatment group and 25 in the control group. Patients in the treatment group received 30 to 60 mg of coenzyme Q10 orally for six days before operation. Preoperative clinical variables, operative procedures, total cardiopulmonary bypass time, and aortic cross-clamping time were similar for the two groups. Postoperatively, mild to severe low cardiac output state developed in 28 of 50 patients (56%) and necessitated the administration of considerable amounts of inotropic agent. The treatment group showed a significantly lower incidence of low cardiac output state during the recovery period than the control group (p less than 0.05). These results suggest that preoperative administration of coenzyme Q10 will increase the tolerance of human hearts to ischemia during aortic cross-clamping.

Comparative study of retrograde and selective cerebral perfusion with transcranial Doppler

BACKGROUND Retrograde cerebral perfusion (RCP) is a simple technique and is expected to provide cerebral protection. However, its optimum management and limitations remain unclear. Transcranial Doppler has been used to monitor cerebral perfusion. Using this Doppler technique, we compared cerebral blood flow for RCP with that for selective cerebral perfusion. METHODS Thirty-two consecutive patients underwent elective surgical repair of an aortic aneurysm involving the aortic arch at Kyushu University Hospital. Retrograde cerebral perfusion was used in 15 patients and selective cerebral perfusion, in 17 patients. Continuous measurement of middle cerebral artery blood flow velocities was performed by transcranial Doppler technique. RESULTS Retrograde middle cerebral artery blood flow velocities during RCP could be measured in only 3 patients, whereas middle cerebral artery blood flow velocities during selective cerebral perfusion could be measured in all but 1 woman. The increase in middle cerebral artery blood flow velocities after RCP was significantly greater than that after selective cerebral perfusion. CONCLUSIONS The measurement of middle cerebral artery blood flow velocities with transcranial Doppler technique is practicable during selective cerebral perfusion but difficult during RCP. The increase in middle cerebral artery blood flow velocities after RCP indicates reactive hyperemia and reflects the critical decrease in cerebral blood flow during this type of perfusion.