Kazuhiko Seya

Tumor Necrosis Factor-α Accelerates the Calcification of Human Aortic Valve Interstitial Cells Obtained from Patients with Calcific Aortic Valve Stenosis via the BMP2-Dlx5 Pathway

Calcific aortic valve stenosis (CAS) is the most frequent heart valve disease in the elderly, accompanied by valve calcification. Tumor necrosis factor-α (TNF-α), a pleiotropic cytokine secreted mainly from macrophages, has been detected in human calcified valves. However, the role of TNF-α in valve calcification remains unclear. To clarify whether TNF-α accelerates the calcification of aortic valves, we investigated the effect of TNF-α on human aortic valve interstitial cells (HAVICs) obtained from patients with CAS (CAS group) and with aortic regurgitation or aortic dissection having a noncalcified aortic valve (control group). HAVICs (2 × 104) were cultured in a 12-well dish in Dulbeccos modified Eagles medium with 10% fetal bovine serum. The medium containing TNF-α (30 ng/ml) was replenished every 3 days after the cells reached confluence. TNF-α significantly accelerated the calcification and alkaline phosphatase (ALP) activity of HAVICs from CAS but not the control group after 12 days of culture. Furthermore, gene expression of calcigenic markers, ALP, bone morphogenetic protein 2 (BMP2), and distal-less homeobox 5 (Dlx5) were significantly increased after 6 days of TNF-α treatment in the CAS group but not the control group. Dorsomorphin, an inhibitor of mothers against decapentaplegic homologs (Smads) 1/5/8 phosphorylation, significantly inhibited the enhancement of TNF-α-induced calcification, ALP activity, Smad phosphorylation, and Dlx5 gene expression of HAVICs from the CAS group. These results suggest that HAVICs from the CAS group have greater sensitivity to TNF-α, which accelerates the calcification of aortic valves via the BMP2-Dlx5 pathway.

Biogenesis-like transformation of salidroside to rengyol and its related cyclohexyletanoids of forsythia suspensa

Abstract Photooxygenation of salidroside (8) in methanol in presence of Rose Bengal afforded cornoside (9) , which, on high pressure hydrogenation with 5 % palladium on activated carbon, yielded rengyoside B (6) Reduction of 6 with sodium borohydride gave rengyoside A (5) stereoselectively. By enzymatic hydrolysis, 9 , 6 and 5 furnished rengyolone (4) , rengyoxide (3) and rengyol (1) , respectively. Similarly, p -hydroxyphenylethanol (10) , the aglycone part of salidroside (8) , was oxygenated photochemically to a dienone alcohol, which cyclized spontaneously to rengyolone (4) . Hallerone (17) was obtained by the photooxygenation of p -hydroxyphenylethyl acetate (10b) . Thus the plausible biosynthetic routes from salidroside (8) to rengyol (1) and the related natural cyclohexylethanoids were simulated chemically.

Structures of rengyosides A, B and C, three glucosides of Forsythia suspensa fruits

Abstract Three new glucosides, rengyosides A, B and C, having as aglycones the reduced forms of phenylethanol were isolated from Forsythia suspensa fruits. Chemical and spectroscopic studies established the structures of these natural products to be 2-(1,4-dihydroxycyclohexyl)ethyl β- d -glucopyranoside, 2-(1-hydroxy-4-ketocyclohexyl)ethyl β- d -glucopyranoside and 2-(1,4-dihydroxycyclohexyl)ethyl β- d -6-O-[2-(4-hydroxyphenyl)acetyl]glucopyranoside, respectively. Salidroside, a possible biogenetic precursor of these glucosides, was also isolated.

Fragility of the electron transport chain and superoxide generation in mitochondria of the liver graft after cold ischemia

BACKGROUND After cold ischemia, electrons transferred in the electron transport chain may leak out of the mitochondria in proportion to the deterioration of mitochondrial oxidative phosphorylation. This seems to be one major cause of the lipid peroxidation that occurs mainly in the hepatocytes at reperfusion in liver transplantation. To examine this hypothesis, we investigated superoxide generation and the amount of oxidative phosphorylation in the mitochondria isolated from rat livers after cold preservation. METHODS Rat liver was preserved in University of Wisconsin solution at 4 degrees C for 24 hr. The mitochondrial fraction was prepared, and the amount of ATP synthesis and superoxide generation was investigated. Superoxide generation in the electron transport chain of submitochondrial particles was also measured by a chemiluminescence recorder. RESULTS The amount of ATP synthesis was significantly decreased after 12 hr of cold preservation. In the whole mitochondria, superoxide production in the presence of succinate was approximately 1/2000 to 1/3000 less than that observed in the submitochondrial particles at any determination point, and superoxide production was not affected by cold preservation. In the presence of antimycin A, superoxide production in the mitochondria after 18 hr of preservation increased significantly. CONCLUSION These results indicate that the electron transfer in the complex III of the mitochondrial membrane becomes leaky after long periods of cold ischemia, but that leakage of superoxide anion did not increase, although the mitochondrial respiratory phosphorylation was deteriorated. We conclude that superoxide through the mitochondrial membrane cannot cause lipid peroxidation in hepatocytes at reperfusion even after a long period of cold ischemia.

Opposite Effects of Two Resveratrol (trans-3,5,4′-Trihydroxystilbene) Tetramers, Vitisin A and Hopeaphenol, on Apoptosis of Myocytes Isolated from Adult Rat Heart

It has been reported that resveratrol (trans-3,5,4′-trihydroxystilbene) from Vitis plants has various cardioprotective effects. Vitis plants also include various resveratrol tetramers. The aim of our study is to clarify the pharmacological properties of resveratrol tetramers. We isolated two resveratrol tetramers as major products of Vitis plants. One is vitisin A, a complex of two resveratrol dimers, (+)-ϵ-viniferin and ampelopsin B, and the other is hopeaphenol, composed of 2 mol ampelopsin B. Vitisin A (30–300 nM) unexpectedly dose-dependently facilitated swelling and depolarization of mitochondria and cytochrome c release from mitochondria, which are indices of cardiomyocyte apoptosis. Furthermore, vitisin A induced apoptosis in the primary culture of adult rat ventricular myocytes. On the other hand, hopeaphenol (1–10 μM) dose-dependently inhibited Ca2+ (30 μM)-induced mitochondrial depolarization and cytochrome c release from mitochondria but had not affected mitochondrial swelling. Moreover, hopeaphenol inhibited vitisin A-induced apoptosis. In structural and functional studies, we further confirmed that vitisin B, one of the resveratrol tetramers having (+)-ϵ-viniferin unit, induces mitochondrial swelling and cytochrome c release from mitochondria like vitisin A and that vitisifuran A, one of the resveratrol tetramers having the ampelopsin B unit, inhibits Ca2+-induced cytochrome c release from mitochondria like hopeaphenol. These results show that resveratrol tetramers have at least two opposite effects on cardiomyocytes; the one having the (+)-ϵ-viniferin unit induces cardiomyocyte apoptosis, and the other having ampelopsin B but not (+)-ϵ-viniferin unit inhibits it.

Gene expression and functional activity of sodium/calcium exchanger enhanced in vascular smooth muscle cells of spontaneously hypertensive rats.

Abstract: Effects of hypertension on the function of the Na+/Ca2+ exchanger (NCX) were investigated by analyzing vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats. Angiotensin II-induced 45Ca2+ efflux from VSMCs mediated by NCX was enhanced by up to 3-fold in SHR compared with WKY, whereas ionomycin-induced 45Ca2+ efflux mediated by NCX was not different between SHR and WKY. The decline rate from the peak value of intracellular Ca2+ concentration ([Ca2+]i) mobilized by angiotensin II was decelerated by removal of extracellular sodium (Na+o) in SHR but not in WKY. Gene expressions of NCX subtype 1 and angiotensin II receptor type1A assessed by quantitative RT-PCR were increased by 1.3- and 1.5-fold, respectively in SHR compared with WKY. NCX protein was also increased 1.6-fold in SHR compared with WKY. MEK inhibitor, PD98059, partly blocked the Na+o-dependent acceleration of the [Ca2+]i recovery rate and tyrosine kinase inhibitor, genistein, diminished it in SHR. Genistein decreased angiotensin II-induced Na+o- dependent 45Ca2+ efflux. However, angiotensin II did not enhance the tyrosine phosphorylation of NCX. These results suggest that acceleration of Ca2+ efflux from VSMCs of SHR was at least partly due to the enhancement of functional activity of NCX via increased gene expression and tyrosine phosphorylation in connection with hypertension.

Stereostructure of Rengyol and Isocengyol, Phenylethanoids of Forsythiasuspensa

Abstract Determination of the stereostructure of rengyol (1), a novel nonaromatic phenylethanoid natural product isolated from Forsythia suspensa , by synthetic means has been described. The Reformatsky reaction of 4-acetoxycyclohexanone with ethyl bromoaoetate afforded two isomeric acetoxy esters (5, 6) and the one (5) which has an equatorial acetoxy1 group yielded on IAH reduction a triol identified as rengyol (1). The isomer (7), obtained similarly from the other isomeric acetoxy ester (6), has also been isolated from the natural source and is named isorengyol. Further, dehydration of the esters (5,6) and subsequent pyrolytic deacetoxylation afforded a 1,3-cyclohexadiene derivative (12), which on photosensitized cis -dioxygenation, followed by reduction, yielded rengyol (1) establishing its stereostructure to have 1,4- cis -cyclohexanediol system. These results supported the previous conclusion based on the 1H and 13C NMR spectral data.

Photo-oxidation of L-tyrosine, an efficient 1,4-chirality transfer reaction

Dye-sensitized oxidation of L-tyrosine with Rose Bengal yielded the optically pure ketolactam (2) stereoselectively in one step.

Endothelium-dependent vasodilatory effect of vitisin C, a novel plant oligostilbene from Vitis plants (Vitaceae), in rabbit aorta.

We investigated the pharmacological properties of vitisin C, a novel plant oligostilbene from Vitis plants. Vitisin C (1-10 microM) dose-dependently inhibited the contractile responses of endothelium-intact rabbit thoracic aorta induced by phenylephrine (1 microM). These inhibitory effects were abolished in the presence of N (G)-nitro-L-arginine methyl ester (L-NAME; 300 microM), a potent inhibitor of nitric oxide synthase, but not atropine (1 microM), a non-selective muscarinic cholinoceptor antagonist. In endothelium-denuded rabbit aorta, vitisin C was ineffective in attenuating phenylephrine-induced contraction. Moreover, vitisin C (10 microM) increased cGMP production in endothelium-intact, but not endothelium-denuded, aorta, and this increase was abolished in the presence of L-NAME (300 microM). To assess Ca(2+) movement across the endothelial cell membrane induced by vitisin C, we further investigated (45)Ca(2+) influx into cultured rabbit aortic endothelial cells in the presence of vitisin C (3 microM), carbachol (1 microM) or A23187 (10 nM). Vitisin C and carbachol significantly enhanced (45)Ca(2+) influx, which was inhibited by nifedipine (10 microM), a blocker of L-type Ca(2+) channels. In the presence of SK&F96365, a blocker of receptor-operated Ca(2+) channels, (45)Ca(2+) influx induced by carbachol was significantly inhibited, whereas that induced by vitisin C was not affected. On the other hand, A23187 enhanced (45)Ca(2+) influx in the presence and absence of nifedipine and SK&F96365. These results suggest that vitisin C evokes endothelium-dependent vasorelaxation through enhancing nitric oxide release, which is facilitated by Ca(2+) influx into endothelial cells via nifedipine-sensitive Ca(2+) channels.

8-Methyltryptanthrin-induced differentiation of P19CL6 embryonal carcinoma cells into spontaneously beating cardiomyocyte-like cells.

Enhancement of cardiac differentiation is critical to stem cell transplantation therapy for severe ischemic heart disease. The aim of this study was to investigate whether several derivatives of tryptanthrin (1), extracted from the medicinal plant Polygonum tinctorium, induce the differentiation of P19CL6 mouse embryonal carcinoma cells into beating cardiomyocyte-like cells. P19CL6 cells were cultured in α-MEM supplemented with 10% FBS including a test compound or vehicle. Drug-induced differentiation was assessed by measuring the number of beating and nonbeating aggregates and the area of beating aggregates, and the expression of genes involved in cardiac differentiation was evaluated by real-time PCR. A 1 μM concentration of 8-methyltryptanthrin (2) induced the differentiation of P19CL6 cells into cardiomyocyte-like cells to a significantly greater degree than 1% dimethyl sulfoxide (DMSO), a conventional differentiation inducer of P19CL6 cells. Furthermore, 2 strongly increased both the number and the area of spontaneously beating aggregates in comparison with DMSO. Two distinct genes of the calcium channel family, Cav1.2 and Cav3.1, underlying cardiac automaticity were significantly expressed in the presence of 2. Gap junction genes GJA1 and GJA5 contributing to the synchronized contraction of the myocardium were also induced significantly by 2. These results suggest that 2 successfully differentiated P19CL6 cells into spontaneously beating cardiomyocyte-like cells by activating the gene expression of pacemaker channels and gap junctions.