Shinjiro Sasaki

Increased local angiotensin II formation in aneurysmal aorta.

We investigated the levels and locations of angiotensin II-forming enzymes, angiotensin converting enzyme (ACE) and chymase, in aneurysmal and normal aortas. Aneurysmal aortic specimens (n = 14) were obtained at the time of operative aneurysm repair from 14 patients ranging in age from 57 to 84 y. Normal aortic specimens (n = 16) were obtained from 16 patients (48 to 72 y) who underwent coronary artery bypass surgery. The ACE and chymase activities were determined using each specimen. Sections of each specimen were immunostained with antibodies for ACE and chymase. The ACE activities in the aneurysmal and normal aortas were 0.82 +/- 0.10 and 0.14 +/- 0.05 mU/mg protein, respectively, and this difference was significant. The chymase activities in the aneurysmal and normal aortas were 17.9 +/- 2.40 and 1.02 +/- 0.18 mU/mg protein, respectively, and this difference was also significant. In the aneurysmal aorta, ACE-positive cells were detected with macrophages in the intima and media and chymase-positive cells were detected with mast cells in the media and adventitia, whereas positive ACE and chymase cells in the normal aorta were located only in the endothelium and adventitia, respectively. Angiotensin II-forming enzymes, chymase and ACE, were significantly increased in the aneurysmal aorta, and increased angiotensin II may be associated with the development of aneurysmal formations.

Inhibition of chymase reduces vascular proliferation in dog grafted veins

We investigated the effect of a chymase inhibitor Suc‐Val‐Pro‐PheP(OPh)2 on the proliferation of the grafted vein in dog. By 28 days after the operation, the mean intimal area of the grafted vein in the placebo group was 3.24±0.32 mm2. The intimal area of the grafted vein in the chymase inhibitor‐treated group was reduced to 63.9%. In the placebo group, the activities of chymase and angiotensin‐converting enzyme in grafted vein were significantly increased 15‐ and 2‐fold, respectively. In the chymase inhibitor‐treated group, chymase activity in the grafted veins was decreased significantly. These findings suggest that inhibition of chymase appears useful for preventing vascular proliferation.

ATP regulation of ciliary beat frequency in rat tracheal and distal airway epithelium

Ciliary beat frequency (CBF) was measured by video‐optical microscopy in rat tracheal and distal airway ciliary cells using a slice preparation. In tracheal ciliary cells (tracheal slice), ATP or 2‐methylthio ATP (MeSATP) increased CBF, which was inhibited by suramin (100 μm, an inhibitor of purinergic receptor). Ionomycin (5 μm) or thapsigargin (2 μm) increased CBF similarly. Ca2+‐free solution or addition of Ni2+ (1 mm) decreased CBF gradually by approximately 25% and subsequent stimulation with ATP (10 μm) increased CBF transiently. The purinergic agonist experiments demonstrated that ATP increases CBF in tracheal ciliary cells via both P2X and P2Y receptors. ATP increased the intracellular calcium concentration ([Ca2+]i) in tracheal ciliary cells. However, in distal airway ciliary cells (lung slice), ATP did not increase CBF and [Ca2+]i, although a Ca2+‐free solution decreased CBF, and ionomycin (5 μm) or thapsigargin (2 μm) increased it. Moreover, acetylcholine (100 μm) did not increase CBF in distal airway ciliary cells, although it increased CBF in tracheal ciliary cells. Terbutaline (10 μm), a selective β2‐adrenergic agonist, increased CBF in both tracheal and distal airway ciliary cells. These observations suggest that the Ca2+‐mobilization mechanisms via purinergic or muscarinic receptors of the distal airway ciliary cell may be different from those of the tracheal ciliary cell. In conclusion, the CBF increase is differently regulated in the tracheal and distal airway epithelia of the rat.

Oral administration of a specific chymase inhibitor, NK3201, inhibits vascular proliferation in grafted vein.

Chymase may play an important role in vascular proliferation, as shown by in-vitro experiments, but the role of chymase in vivo has been unclear. In this study, we investigated the effect of a novel chymase inhibitor, NK3201, on this proliferation in dog grafted veins. NK3201 inhibited human and dog chymases, but not rabbit ACE. NK3201 suppressed the Ang I-induced vascular contraction in isolated dog arteries in the presence of an ACE inhibitor, and the IC50 value of chymostatin and NK3201 in dog artery was 320 nM. In dog, the concentration of NK3201 in blood was about 10 microM at 24 h after oral administration of the drug (5 mg/kg). In the group treated with NK3201, each dog was administered orally 5 mg/kg per day from 5 days before to the day before the removal of the grafted veins. Each dog underwent right common carotid artery bypass grafting with the ipsilaterial external jugular vein. By 28 days after grafting, a significant vascular proliferation was observed in the grafted veins and the chymase activity was also increased significantly. Treatment with chymase inhibitor significantly suppressed the proliferation of the grafted veins and the increased chymase activity. In this study, we demonstrate for the first time that oral administration of a specific chymase inhibitor, NK3201, appears useful for preventing vascular proliferation.

Preischemic infusion of alpha-human atrial natriuretic peptide elicits myoprotective effects against ischemia reperfusion in isolated rat hearts.

Carperitide, a synthetic alpha-human atrial natriuretic peptide (ANP) is a newly developed drug for the treatment of heart failure. However, effects of carperitide on susceptibility to ischemia reperfusion injury are left to be determined. Isolated rat hearts were subjected to Langendorff perfusion. Six hearts received 0.1 μM of carperitide for 10 min, 6 hearts received 1 mM of a NO synthetase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) for 5 min before the infusion of carperitide, 6 hearts received 0.02 μM of a PKC synthetase inhibitor chelerythrine chloride for 5 min before the infusion of carperitide, 6 hearts received 100 μM of a selective mitochondrial ATP-sensitive potassium (KATP) channel blocker 5-dehydroxydecanoate (5HD) before the infusion of carperitide, 6 hearts received 10 μM of a soluble guanylate cyclase inhibitor methylene blue for 5 min before the infusion of carperitide, and 6 hearts served as a control with no drug infusion. All hearts were then subjected to 20 min of global ischemia followed by 120 min of reperfusion. Left ventricular pressures and coronary flow were measured throughout the experiment and infarct size was detected at the end of experiment. Both plasma and tissue cGMP levels were also determined. The results showed: (1) Carperitide significantly reduced infarct size compared to control (26.1 ± 2.8 vs. 42.7 ± 2.3%, carperitide vs. control, p < 0.05). This effect was reversed by L-NAME, chelerythrine and 5HD, but not methylene blue. (2) Plasma cGMP levels were increased in carperitide-treated group. This effect was reversed by L-NAME (0.16 ± 0.03 vs. 1.04 ± 0.09* vs. 0.28 ± 0.02 nmol/L, control vs. carperitide vs. L-NAME, *p < 0.01 vs. control). We conclude that preischemic infusion of carperitide exerts cardioprotective effects possibly through NO-PKC dependent pathway followed by mitochondrial KATP channel activation.

Mitochondrial ATP-Sensitive Potassium Channel Plays a Dominant Role in Ischemic Preconditioning of Rabbit Heart

Background: The ATP-sensitive potassium (KATP) channel has been shown to be important in the ischemic preconditioning (IPC) response. Recently, the mitochondrial rather than the sarcolemmal KATP channel has been focused on due to its energy-modulating property. Hence, this study was undertaken to elucidate the role of the mitochondrial KATP channel in IPC by modulating the mitochondrial KATP channel in isolated perfused rabbit hearts. Methods: Seven hearts served as a control with no interventions. Seven hearts underwent IPC consisting of two 5-min cycles of global ischemia followed by 5 min of reperfusion. Seven hearts received the selective mitochondrial KATP channel blocker 5-dehydroxydecanoate (5-HD, 100 µM) for 5 min before IPC, and 7 hearts received the selective mitochondrial KATP channel opener diazoxide (50 µM) for 5 min. Then, all hearts were subjected to 1 h of left anterior descending coronary artery ischemia and 1 h of reperfusion. Left ventricular pressures, monophasic action potentials and coronary flow were measured throughout the experiment and infarct size was detected at the end of experiment. Results: (1) The mitochondria-selective KATP channel opener diazoxide reduced infarct size as compared to control (p < 0.05); (2) IPC reduced infarct size and preserved postischemic diastolic function as compared to control (p < 0.05), and (3) the mitochondria-selective KATP channel blocker 5-HD reversed these effects. Conclusion: The mitochondrial ATP-sensitive potassium channel may be a potential site of cardioprotection.

Intra-Arterial Bone Marrow Cell Transplantation Induces Angiogenesis in Rat Hindlimb Ischemia

Background: Bone marrow (BM) cells have been shown to augment local angiogenesis by differentiating vessels themselves and/or secreting paracrinally angiogenic growth factors. Herein, the angiogenic effects of intra-arterial BM mononuclear cell (BM-MNC) transplantation were evaluated in a rat ischemic hindlimb model. Methods: Unilateral hindlimb ischemia was created by excising the femoral artery and its branch in Lewis rats. BM-MNCs were isolated by centrifugation through a Histopaque density gradient. One week after excision of the unilateral femoral artery, BM-MNCs (5 × 106 cells, Group A, n = 6) or PBS (Group B, n = 7) were injected into the ischemic thigh skeletal muscles at the six points with a gauge needle. Another injection of BM-MNCs (3 × 107 cells, Group C, n = 6) or PBS (Group D, n = 7) was administered via the indwelling catheter in the right common iliac artery. Results: Four weeks after the BM-MNC transplantation, angiographic examination revealed the development of collateral vessels in both BM-MNC-transplanted groups. The difference in skin temperature between right and left hindlimbs was significantly reduced in both BM-MNC-transplanted groups (0.93 ± 0.15 vs. 2.84 ± 0.35 vs. 1.20 ± 0.26 vs. 2.61 ± 0.37°C, Group A vs. Group B vs. Group C vs. Group D, p < 0.05). Moreover, immunohistochemical analysis demonstrated that capillary endothelial cells were increased in both BM-MNC-transplanted groups. Conclusion: BM-MNC implantation was able to induce functional neovascularization in rat ischemic hindlimb. The intra-arterial administration offered similar levels of angiogenic activity as intramuscular injection.

Angiotensin II receptor antagonist, L-158,809, prevents intimal hyperplasia in dog grafted veins.

We investigated the levels of the angiotensin II-forming enzymes, chymase and angiotensin converting enzyme (ACE), in dog grafted veins, and studied the effect of an angiotensin II type 1 receptor antagonist, L-158,809, on vascular proliferation in the grafted veins. The right external jugular vein was grafted to the ipsilaterial carotid artery. In the group treated with L-158,809, the drug (10 mg/kg per day, p.o.) were administered orally from 7 days before the operation to 28 days after it, while the others were administrated placebo. In the placebo-treated group, the chymase activity in the grafted veins was increased about 10-fold and the ACE activity was doubled. The areas of intima and media were significantly increased in the grafted veins in the placebo-treated group. L-158,809 significantly reduced the intimal area of the grafted veins. An angiotensin II receptor antagonist, L-158,809, prevented the vascular proliferation in the grafted veins, and the development of the proliferation may depend on activation of local angiotensin II formation.

The synergistic effects of hyperthermia and anticancer drugs on induction of apoptosis

We studied the synergistic effects of hyperthermia and anticancer drugs on induction of apoptosis in lung cancer cells (LK-2 and LU-65A) using in situ end-labeling of DNA, the DNA fragmentation assay, and transmission electron microscopy. A few apoptotic cells were detected only when both cell lines were heated at relatively high temperature (44°C). Moderate numbers of apoptotic cells were observed when both cell lines were incubated with high concentrations (30 or 40 μM) of anticancer drug. Compared with hyperthermia or anticancer drug alone, the combined treatment induced many apoptotic cells in both cell lines, even in the cells treated with lower concentrations (6 or 8 μM) of anticancer drugs following mild hyperthermia (43°C). In regard to kinetics of apoptotic cells induced by treatment, the maximum induction of apoptosis by the combined treatment was higher than that of hyperthermia or anticancer drug alone in both cell lines, although the time of the peak of apoptotic index differed among the three treatments. Therefore, “hyperthermo-chemotherapy” may reduce the required dosage of anticancer drug and decrease the temperature of hyperthermia on induction of apoptosis.

Descending branch of lateral femoral circumflex artery as a free graft for myocardial revascularization : A case report

The lateral femoral circumflex artery (LFCA) is one of the branches of the deep femoral artery. In the field of plastic and reconstructive surgery, the LFCA has been used to supply composite tissue of skin and/or muscle. 1-3 The LFCA has three major branches: the ascending, transverse, and descending branches. The descending branch of the LFCA passes downward through the intermuscular space between the rectus femoris muscle and the vastus lateralis muscle and finally terminates in the vastus muscle near the knee joint, The descending branches of the LFCA have a very large diameter (2 to 3 ram) and do not taper like those of the radial arteries. 3 The descending branch of the LFCA has an attractive caliber and length. We report the first case in which a descending branch of the LFCA was used as a graft for myocardial revascularization. A 77-year-old woman had a 7-month history of increasing angina on exertion. ElectrocardiOgraphic changes were consistent with effort angina. Cardiac catheterization revealed a 99% stenosis of the proximal right coronary artery and a 75% stenosis of the middle third of the left anterior descending coronary artery. For this patient, we had planned to perform coronary artery bypass grafting (CABG): a left internal thoracic artery (LITA) in situ to the left anterior descending coronary artery and a saphenous vein graft to the right coronary artery. However, the saphenous vein had varices and was unacceptably thin. We also decided to avoid grafting the right internal thoracic artery to the right coronary artery for this elderly woman because devascularization of the sternum suggested a greater risk of sternal wound infections with the use of bilateral internal thoracic artery grafts. We concluded that the descending branch of the LFCA could be used to bypass the right coronary artery. We had evaluated the descending branches of the LFCA bilaterally by means of a femoral arteriogram 5 days before this operation. The arteriogram showed a good qUality vessel Without stenosis (Fig. 1). The descending branch of the left LFCA was harvested