Teruhiko Toyo-oka

Increased plasma level of endothelin-1 and coronary spasm induction in patients with vasospastic angina pectoris.

To elucidate the pathogenic contribution of a potent vasoconstrictor, endothelin-1, to coronary artery spasm, we provoked spasm with intracoronary administration of acetylcholine or ergonovine and performed sensitive immunoassays of plasma levels of endothelin-1 and atrial natriuretic factor (ANF) in the peripheral vein and coronary sinus of patients with a tentative diagnosis of vasospastic angina (VSA, n = 19). The validity of coronary sinus blood sampling was verified by simultaneous measurement of the ANF level. The plasma endothelin-1 levels in venous and coronary sinus blood of the spasm-provoked patients (n = 12) were 1.71-fold and 2.16-fold higher, respectively, than those of nonprovoked cases (n = 5, p less than 0.01). During left coronary spasm, the endothelin-1 level in coronary sinus transiently decreased from 2.27 +/- 0.14 to 1.76 +/- 0.14 pg/ml (p less than 0.01) and returned to the control level (1.98 +/- 0.20 pg/ml) after the spasm resolved, whereas the change was equivocal during right coronary spasm. In contrast, the patients in whom spasm was not provoked showed no changes and maintained low endothelin-1 levels both before and after the maximal provocation (0.90 +/- 0.13 versus 0.90 +/- 0.13 pg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of Hydroxymethylglutaryl-Coenzyme A Reductase Reduces Th1 Development and Promotes Th2 Development

Abstract— Several prospective clinical studies have indicated that hydroxymethylglutaryl-coenzyme A reductase inhibitors, statins, prevent cardiovascular events in part through their antiinflammatory properties. Because inflammation is positively and negatively regulated by T helper (Th) 1 cells and Th2 cells, respectively, we examined the effects of statins on the Th polarization in vitro and in vivo. Here we demonstrated that the statins tested, ie, cerivastatin, simvastatin, lovastatin, and atorvastatin, promoted Th2 polarization through both inhibition of Th1 development and augmentation of Th2 development of CD4+ T cells primed in vitro with anti-CD3 antibody and splenic antigen-presenting cells. Cerivastatin exerted most potent effect on modulation of Th1/Th2 development, and the effect was completely abrogated by an addition of mevalonate. Consistent with in vitro experiments, cerivastatin treatment decreased IFN-&ggr; production of lymph node cells from mice immunized with ovalbumin emulsified in complete Freund’s adjuvant, indicating that Th1 development is also suppressed in an in vivo proinflammatory environment. In this murine model, cerivastatin significantly reduced mesangial matrix expansion of glomeruli in the kidney and attenuated proteinuria. The decrease of glomerular sclerosis by cerivastatin treatment was positively related to the suppression of interferon (IFN)-&ggr;–producing Th1 response in draining lymph node cells. Hence, these findings strongly suggest that statins’ inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase regulates Th1/Th2 polarization in vivo and such a mechanism possibly plays a pathophysiological role in immune-related glomerular injury.

Inhibition of proteolytic activity of calcium activated neutral protease by leupeptin and antipain

Abstract The calcium activated neutral protease from bovine ventricular muscle requires milli-molar concentration of Ca ions for the activation of the proteolysis of troponin-T, troponin-I and tropomyosin. The exogenous protease inhibitors were examined concerning the blocking action of this enzyme. Both leupeptin and antipain were effective for the inhibition at the nearly same molar concentration as the protease. Lineweaver plot for both the protease alone and protease with leupeptin showed straight lines, and the mode of the inhibition was non-competitive type. Natural actomyosin, pretreated with this protease showed markedly reduced sensitivity to Ca ions. With the addition of leupeptin to the pretreatment, however, the Ca sensitivity was well preserved.

Contribution of Sustained Ca Elevation for Nitric Oxide Production in Endothelial Cells and Subsequent Modulation of Ca Transient in Vascular Smooth Muscle Cells in Coculture

To elucidate the intracellular Ca (Ca) transient responsible for nitric oxide (NO) production in endothelial cells (ECs) and the subsequent Ca reduction in vascular smooth muscle cells (VSMCs), we administrated four agonists with different Ca-mobilizing mechanisms for both cells in iso- or coculture. We monitored the Ca of both cells by two-dimensional fura-2 imaging, simultaneously measuring NO production as NO. The order of potency of the agonists in terms of the peak Ca in ECs was bradykinin (100 nM) > ATP (10 μM) > ionomycin (50 nM) > thapsigargin (1 μM). In contrast, the order in reference to both the extent of Ca reduction in cocultured VSMCs and the elevation in NO production over the level of basal release in ECs completely matched and was ranked as thapsigargin > ionomycin > ATP > bradykinin. Treatment by N-monomethyl-L-arginine monoacetate but not indomethacin or glybenclamide restored the Ca response in cocultured VSMCs to the isoculture level. In ECs, when the Ca influx was blocked by Ni or by chelating extracellular Ca, all four agonists markedly decreased NO production, the half decay time of the Ca degenerating phase, and the area under the Ca curve. The amount of produced NO hyperbolically correlated to the half decay time and the area under the Ca curve but not to the Ca peak level. Thus, the sustained elevation of Ca in ECs, mainly a result of Ca influx, determines the active NO production and subsequent Ca reduction in adjacent VSMCs. Furthermore, L-arginine but not D-arginine or L-lysine at high dose (5 mM) without agonist enhanced the NO production, weakly reduced the Ca in ECs, and markedly decreased the Ca in VSMCs, demonstrating the autocrine and paracrine effects of NO (Shin, W. S., Sasaki, T., Kato, M., Hara, K., Seko, A., Yang, W. D., Shimamoto, N., Sugimoto, T., and Toyo-oka, T.(1992) J. Biol. Chem. 267, 20377-20382).

Rescue of hereditary form of dilated cardiomyopathy by rAAV-mediated somatic gene therapy: amelioration of morphological findings, sarcolemmal permeability, cardiac performances, and the prognosis of TO-2 hamsters.

The hereditary form comprises ≈1/5 of patients with dilated cardiomyopathy (DCM) and is a major cause of advanced heart failure. Medical and socioeconomic settings require novel treatments other than cardiac transplantation. TO-2 strain hamsters with congenital DCM show similar clinical and genetic backgrounds to human cases that have defects in the δ-sarcoglycan (δ-SG) gene. To examine the long-term in vivo supplement of normal δ-SG gene driven by cytomegalovirus promoter, we analyzed the pathophysiologic effects of the transgene expression in TO-2 hearts by using recombinant adeno-associated virus vector. The transgene preserved sarcolemmal permeability detected in situ by mutual exclusivity between cardiomyocytes taking up intravenously administered Evans blue dye and expressing the δ-SG transgene throughout life. The persistent amelioration of sarcolemmal integrity improved wall thickness and the calcification score postmortem. Furthermore, in vivo myocardial contractility and hemodynamics, measured by echocardiography and cardiac catheterization, respectively, were normalized, especially in the diastolic performance. Most importantly, the survival period of the TO-2 hamsters was prolonged after the δ-SG gene transduction, and the animals remained active, exceeding the life expectancy of animals without transduction of the responsible gene. These results provide the first evidence that somatic gene therapy is promising for human DCM treatment, if the rAAV vector can be justified for clinical use.

Long-term infusion of kallikrein attenuates renal injury in Dahl salt-sensitive rats.

We investigated whether long-term infusion of kallikrein would attenuate renal injury in salt-induced hypertension in Dahl salt-sensitive rats. A subdepressor dose of purified rat urinary kallikrein (700 ng/d IV) was infused by osmotic minipump for 4 weeks in male Dahl salt-sensitive rats fed a high salt (2% NaCl) diet. This dose did not affect the time-dependent elevation of blood pressure; however, urinary protein excretion was significantly decreased, and glomerular filtration rate was increased. These beneficial effects were reflected morphologically by an attenuation of glomerulosclerotic lesions and tubular injury seen in the hypertensive Dahl salt-sensitive rats. Kallikrein infusion increased urinary excretion of bradykinin and stimulated excretion of cyclic GMP, suggesting that the kallikrein-kinin-prostaglandin and nitric oxide axes were enhanced by rat urinary kallikrein infusion. The alterations induced by kallikrein infusion were potentiated by the concomitant administration of the angiotensin-converting enzyme inhibitor alacepril. These studies indicated that long-term replacement with rat tissue kallikrein attenuates renal injury in hypertensive Dahl salt-sensitive rats.

Calcium-activated neutral protease from bovine ventricular muscle: Isolation and some of its properties

A new protease has been isolated and purified from bovine ventricular muscle, by a new method which employs affinity chromatography. This protease required both millimolar concentrations of Ca2+ and the SH-group for the activation, and it was active at neutral pH. The molecular weight of this calcium-activated neutral protease was estimated to be 92 000 and 80 000 by gel filtration and sodium dodecylsulfate polyacrylamide gel electrophoresis, respectively. The isoeletric point was determined to be 4.3 by isoelectric focusing. The synthetic substrates including N-benzoyl l-arginine ethylester and acetyltyrosine ethylester were not hydrolysed by this protease. Among the endogenous myofibrillar proteins, the contractile proteins (myosin and actin) were not degraded, while the regulatory proteins, tropomyosin and troponin were degraded only in the presence of Ca2+. Of three components of troponin, both troponin-T and troponin-I were susceptible to the proteolytic action of calcium-activated neutral protease, while troponin-C was well preserved, as verified by the electrophoretic pattern in sodium dodecyl-sulfate-polyacrylamide gel. The breakdown of the regulatory proteins was also functional, as demonstrated in natural actomyosin pretreated by calcium-activated neutral protease by measurement of Ca2+-sensitivity of superprecipitation. Although the physiological feature of calcium-activated neutral protease is still unknown, this enzyme might act as an aggravating factor in the course of myocardial necrosis, when the intracellular concentration of free Ca2+ is considered to be elevated to millimolar levels.

In Vivo Gene Transfection of Human Endothelial Cell Nitric Oxide Synthase in Cardiomyocytes Causes Apoptosis-Like Cell Death Identification Using Sendai Virus–Coated Liposomes

BACKGROUND Nitric oxide (NO) has various actions on the cardiovascular system, although its pathophysiological significance in myocardial cells remains obscure. The aim of the present study was to identify direct NO actions on cardiomyocytes by gene transfection in vivo using a newly developed vector under physiological conditions. METHODS AND RESULTS Liposomes containing the beta-galactosidase (beta-gal) gene alone or with the human endothelial cell nitric oxide synthase (ecNOS) gene were coated with UV-inactivated Sendai virus and injected into the left ventricular wall of rat heart in vivo. Histological examination confirmed that the transfection efficiency was comparable to adenovirus-mediated transfection and that the new vector per se caused no inflammation. beta-Gal expression was confined to cardiomyocytes between two intercalated discs, suggesting that the transfected gene did not permeate the discs. An immunohistochemical study showed that cotransfection of the ecNOS gene induced massive myocardial cell shrinkage in both transfected cells and the adjacent myocytes in a time- and dose-dependent manner. Histochemical findings in shrunk cells coincided with apoptosis as identified by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling. Electron microscopy of the lesion revealed myofibrillar degradation and accumulation of mitochondria but no apoptotic bodies. Pre-treatment with the NOS inhibitor N omega-nitro-L-arginine methyl ester abolished these morphological alterations. CONCLUSIONS The efficient expression of the human ecNOS gene in vivo suggests that NO or its toxic metabolite caused myocardial degradation, a part of which was compatible with apoptosis of the transfected cardiomyocytes themselves and the adjacent cells as a paracrine effect. These morphological features mimicked acute myocarditis or ischemic injury.

Direct action of nitric oxide on osteoblastic differentiation

The effect of nitric oxide (NO) on osteoblastic differentiation was examined in cultured mouse osteoblasts. Interleukin‐1β and tumor necrosis factor‐α expressed inducible NO synthase gene with little effect on constitutive NO synthase gene. These cytokines increased NO production, which was inhibited by l‐NMMA pretreatment, and decreased alkaline phosphatase (AIPase) activity, which was not restored by l‐NMMA. Furthermore, NO donors, sodium nitroprusside and NONOate dose‐dependently elevated AIPase activity and expression of osteocalcin gene. These results suggest that NO directly facilitates osteoblastic differentiation and the cytokine‐induced inhibition of AIPase activity is mediated via mechanism other than NO.

Long-Term Inhibition of Renin-Angiotensin System Sustains Memory Function in Aged Dahl Rats

The Dahl salt-sensitive (DS) rat, a genetic model of salt-induced hypertension in humans, is more likely to develop severe vascular injuries than a rat with spontaneous hypertension. We designed an experiment to scrutinize the effects of renin-angiotensin inhibition on cognitive dysfunction in the aged, normotensive DS with a passive avoidance test. Eighteen months of treatment with a very low dose of the angiotensin-converting enzyme (ACE) inhibitor cilazapril (2.5 microg/mL in drinking water) or the angiotensin II type 1 receptor antagonist E4177 did not reduce blood pressure throughout the experiment, although in the low dose cilazapril group (12.5 microg/mL in drinking water), blood pressure dropped within 6 months after treatment began. The cilazapril treatments dose-dependently improved memory function in the aged, normotensive DS fed a low-salt diet compared with the untreated, control rats. This improvement was associated with significant increases in hippocampal CA1 cells and capillary densities in the CA1 regions compared with those in the untreated DS. Similarly, E4177 slightly improved the memory dysfunction observed in the aged DS. The cells in the hippocampal CA1 region were restored slightly, but the capillary densities were not influenced by the receptor antagonist. On the other hand, the ACE inhibitor and receptor antagonist both attenuated urinary protein excretions with an improvement of glomerular sclerosis. These data suggest that long-term treatment with an ACE inhibitor improves memory dysfunction probably through restoration of capillary and hippocampal cells. The effects are due to the inhibition of the angiotensin II type 1 receptor and probably to the enhancement of the kallikrein-kinin system.