Vascular- and hepato-protective effects of passion fruit seed extract containing piceatannol in chronic high-fat diet-fed rats.

The effects of chronic administration of piceatannol-enriched (9.5% w/w) passion fruit seed extract (PFSE) on the cardiovascular damage induced in a high-fat (HF) diet-fed model of Fischer 344 rats were evaluated. Rats were fed the control, HF, or HF diets containing PFSE (0.5% w/w) for 16 weeks, and the effects of the various diets on the tissue weight, serum lipid profile, hepatic fibrosis, hepatic ductular reaction, cardiac function and aortic ring reactivity were examined. HF diet-fed rats developed signs of cardiovascular disease with abnormal serum profiles compared to control diet-fed rats. PFSE supplementation improved the liver hypertrophy and hepatic histology of the HF diet-fed rats. In addition, the triglyceride and cholesterol levels, platelet aggregation, cardiac function, and acetylcholine-mediated relaxation of the aortic ring were improved. These results suggest that the chronic intake of PFSE containing piceatannol prevents HF diet-induced cardiovascular disease in rats.

The role of hypertriglyceridemia in the development of atherosclerosis and endothelial dysfunction.

A hereditary postprandial hypertriglyceridemic rabbit (PHT rabbit) is a new dyslipidemic model showing remarkably high plasma triglycerides with only limited elevation of plasma total cholesterol. In PHT rabbits, plasma triglyceride was markedly elevated postprandially compared with healthy Japanese white (JW) rabbits. In physiological experiments, the ring preparation of the thoracic aorta was suspended in an organ bath filled with modified Krebs-Henseleit solution, and the developed tension was recorded. Endothelial function was evaluated by acetylcholine-induced vasorelaxation in each preparation with intact endothelium. The acetylcholine-induced endothelium-dependent relaxation was diminished in PHT compared with JW rabbits, suggesting endothelial dysfunction in PHT rabbits. Histological examination was carried out in adipose tissue, liver and aorta. They were fixed in formaldehyde and embedded in paraffin. The tissues were sliced (4 µm) and stained using hematoxylin-eosin solution. In the adipose tissue, the visceral fat accumulated, and the size of adipose cells was enlarged in PHT rabbits. The liver of the PHT rabbit was fatty and degenerated. In aorta, increased intimal thickness was observed, suggesting the progression of atherosclerosis in the PHT rabbit. This study suggests the important role of postprandial hypertriglyceridemia in atherosclerosis. By using PHT rabbits, the effects of hypertriglyceridemia on health and diseases could be evaluated precisely.

Identification of the strong vasorelaxing substance scirpusin B, a dimer of piceatannol, from passion fruit (Passiflora edulis) seeds.

Piceatannol is present in passion fruit (Passiflora edulis) seeds in high amounts. In this study, we isolated the second major polyphenolic compound of passion fruit seeds and identified it as scirpusin B, which is a dimer of piceatannol. We investigated the antioxidant activities and vasorelaxing effects of these polyphenols. Their antioxidant effects were measured using an in vitro 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, and their vasorelaxant effects were determined ex vivo in rat thoracic aorta. Both polyphenolic compounds exhibited potent antioxidant activities and significant vasorelaxant effects in endothelium-intact aortas. More specifically, scirpusin B exerted a greater antioxidant activity and vasorelaxant effect compared with that of piceatannol. Additionally, the vasorelaxation effects of the compounds were induced via the NO derived from the endothelium. This study provides the possibility that polyphenols in passion fruit seeds are effective against cardiovascular diseases (CVDs).

Investigation of differentially expressed genes in the ventricular myocardium of senescent rats.

Aging alters a variety of physiological functions of the heart. The molecular basis of the age-related functional changes has not been fully understood. Differential gene expression provides the basis for many fundamental cellular processes associated with development and aging. The identification and cloning of genes whose expression is modulated by aging can be of importance for our better understanding of these age-related phenomena. In order to isolate and characterize gene products differentially expressed in senescent hearts, we applied a differential display method for screening those genes in rat ventricular myocardium. Total RNAs were isolated from 2-month-old (young) and 24-month-old rat (senescent) ventricles by the acid-guanidium-phenol-chloroform method. The first-strand synthesis of the cDNAs from each RNA was carried out with oligo-d(T) primers. The differential display screening was performed with three arbitrary primers and eight anchor primers, and the products were isolated on a 6% denaturing polyacrylamide gel. The bands showing differential expression were excised and subcloned into T-vector. We selected 19 upregulated clones and 66 downregulated clones in aged rat hearts. The differential expression of those candidate genes was confirmed by reverse Northern blot analysis. The selected genes were sequenced by dye-terminator methods. Among 31 clones, 15 clones were unknown. The known products included alpha-myosin heavy chain, cytochrome oxidase subunit, H(+)-transporting ATP synthase F0 complex subunit c isoform 3 (ATP5G3), and Na(+)-K(+)-Cl(-) cotransporter. The RT-PCR differential display method effectively identified genes differentially expressed in senescent hearts, and may be a useful tool for investigating factors responsible for age-related physiological changes.

Role of angiotensin II and endothelin-1 receptors in aging-related functional changes in rat cardiovascular system.

Angiotensin II (AII) and endothelin-1 (ET-1) are regarded as key players in the age-related changes in cardiovascular function. They are known to be involved in the pathogenesis of cardiac fibrosis and coronary vascular atherosclerosis. AII- and ET-induced vasoconstriction was augmented in coronary arteries of Langendorff-perfused heart from aged rats. In papillary muscles, ET-1-induced positive inotropic effect (PIE) was diminished by aging. On the other hand, both ET-1 and AII caused greater vasoconstriction in aged rat coronary arteries compared to those in the young rat. To further elucidate the mechanism of these age-dependent changes in cardiovascular effects of ET-1 and AII, we examined the expression of AII and ET-1 receptors in young (2-month-old) and aged (24-month-old) rats. Total RNA was isolated from left ventricles. For determination of the gene expression of AT(1) receptor and ET(A)/ET(B) receptor mRNA, competitive RT-PCR and Northern blot analysis were performed, respectively. [(125)I]ET-1 receptor assay was carried out in left ventricular membrane fraction. AT(1)-receptor, ET(A)-, and ET(B)-receptor mRNA were upregulated in the left ventricles of senescent rats compared with young ones. The affinity of ET-1-receptor was not changed, but receptor density was significantly increased in aged rats. Although the precise mechanism for the upregulation of AT(1) receptor and ET-1 receptor in the aged rat heart has not been clarified yet, these findings suggest that the activation of the renin-angiotensin system as well as ET receptor may be important for the physiological changes in aged hearts.

Progression of severe atherosclerosis and increased arterial pulse pressure in the newly developed heritable mixed hyperlipidaemic rabbits.

We have recently segregated a new line of rabbit, named TGH, with severely high levels of plasma triglyceride and cholesterol. The aim of the present study was to investigate the progression of atherosclerosis and haemodynamic parameters in TGH rabbits. 2. Japanese white (JW) and TGH rabbits (24-27 months old) were anaesthetized with ketamine and xylazine. Plasma concentrations of triglyceride were 63.1 8.0 and 446.0 35.2 mg/dL in JW and TGH rabbits, respectively. Blood pressure was measured by a catheter implanted in the femoral artery. Histological examinations were performed using haematoxylin-eosin and elastica-Masson trichrome staining to detect atherosclerotic lesions. 3. The JW rabbits had no atherosclerotic lesions. In TGH rabbits, severe atherosclerotic lesions were observed throughout the aorta, especially in the aortic arch. Basal femoral arterial pressure was not significantly different between JW and TGH rabbits. However, the basal pulse pressure in TGH rabbits (48.3 4.5 mmHg) was significantly greater than that of JW rabbits (28.0 5.6 mmHg). Intravenous infusion of N(G)-nitro-L-arginine methyl ester (L-NAME; 26.9 mg/kg) increased the blood pressure of TGH and JW rabbits. There was no significant difference in the response to L-NAME between the two rabbit strains. 4. The present study shows that severe atherosclerotic changes develop in TGH rabbits and suggests that the hyperlipidaemia combined with hypercholesterolaemia and hypertriglyceridaemia is an important factor for promoting atherosclerosis in TGH rabbits. The greater pulse pressure in TGH rabbits may be due to the increased vascular stiffness with atherosclerosis. 5. This newly developed TGH rabbit line of heritable hypertriglyceridaemia with hypercholesterolaemia will become a useful animal model for studies on the role of hyperlipidaemia in the progression of atherosclerosis and in many atherosclerosis-related diseases.

Vascular contractile effect of urotensin II in young and aged rats: influence of aging and contribution of endothelial nitric oxide.

To elucidate whether aging influences the vascular contractile effect of urotensin II in rat thoracic aorta, and to evaluate the contribution of endothelial vasodilating substances in mediating the effect of urotensin II, the effect of urotensin II was examined in the vessels of young (2-3-month-old) and aged rat. Isolated rat aortic rings incubated in Krebs-Henseleit solution gassed with 95% O2/5% CO2 were stimulated with urotensin II, and the developed tension was measured. Urotensin II increased the developed tension, which was decreased by aging. In 2-3-months-old young aorta without endothelium, urotensin II (10(-10) to 10(-7)) elicited a concentration-dependent aortic contraction to the maximal response almost equivalent to high KCl-induced contraction (79.4+/-11.3% of KCl(max)). In the presence of endothelium, the urotensin II-induced vasoconstriction in young aorta was significantly attenuated to 33.3+/-4.6% of KCl(max). However, the contractile response was greater in the pretreatment with N(G)-nitro-L-arginine (L-NNA) (100 microM) (50.3+/-8.4% of KCl(max) in endothelial denuded aorta), suggesting the vasorelaxing role of endothelial nitric oxide. In 25-27-months-old aged rat aorta, the urotensin II-mediated contraction was remarkably decreased, both in the presence (6.3+/-2.0% of KCl(max)) and absence (11.7+/-3.0% of KCl(max)) of endothelium. A cyclooxygenase inhibitor, diclofenac (10 microM), did not have any effect on the urotensin II-induced contraction. These results suggest that urotensin II can induce vascular smooth muscle contraction in rat aorta, and there was an aging-related decline in the urotensin II-induced contraction. Endothelial production of nitric oxide in response to urotensin II but not cyclooxygenase metabolites such as prostacyclin may play a role in reducing the vascular constriction especially in young aorta.

Role of prostaglandins in urotensin II-induced vasodilatation in the coronary arteries of aged rats.

Endothelial function is modulated by aging. The objective of this study was to elucidate whether aging influences urotensin II-induced coronary vasodilatation, and whether aging influences the production of endothelial factors in response to urotensin II. We examined the effects of urotensin II on coronary flow in Langendorff-perfused rat hearts. The production of nitric oxide (NO), prostacyclin and prostaglandin (PG)E2 were determined in the coronary effluent of both young and aged rats. Urotensin II increased coronary flow in Langendorff-perfused hearts in both young and aged rats and vasodilation did not differ between young and aged rats. Pretreatment with a NO synthase inhibitor, NG-nitro-L-arginine (L-NNA), significantly inhibited urotensin II-induced vasodilatation in young rats, but not in aged rats. In addition, urotensin II increased the production of NO only in young rats. On the other hand, the cyclooxygenase inhibitor diclofenac significantly attenuated the urotensin II-induced coronary vasodilatation in both young and aged rats. Urotensin II markedly increased the release of the vasodilating prostacyclin and PGE2 into the coronary effluent. Production of these prostanoids was maintained even in the aged coronary arteries. These results indicate that the production of NO in the endothelium of coronary arteries is impaired in aged rats, and that prostacyclin and PGE2 may play an important role in regulating urotensin II-induced coronary vasodilatation.

Aging-related alterations in the contractile responses to acetylcholine, muscarinic cholinoceptors and cholinesterase activities in jejunum and colon of the male Fischer 344 rats.

In an attempt to examine whether the muscarinic receptor-activated intestinal function is altered by aging, we studied the changes in (1) contractile responses to acetylcholine (Ach), (2) muscarinic cholinoceptors and (3) cholinesterase (ChE) activities, in jejunum and colon of the young (2-3 months) and aged (24-28 months) Fischer 344 rats. In the physiological contraction experiments of jejunum and colon, Ach concentration-dependently increased the force of contraction, and the contractile responses to Ach were not affected by aging. In addition, the true- and pseudo-ChE activities were not significantly changed by aging. The Ach-induced contraction was competitively inhibited by muscarinic M3-selective antagonist hexahydro-sila-difenidolhydrochloride p-fluoroanalog (p-F-HHSiD), suggesting that the contractile responses in the rat jejunum and colon were mediated through M3-cholinoceptor. Age-related changes in muscarinic cholinoceptors of jejunum and colon were determined with the use of specific muscarinic radioligand [3H]-quinuclidinylbenzilate (QNB). The [3H]QNB saturation binding experiments revealed that the maximal binding (B(max)) was increased only in aged jejunum without changes in K(D) values. These results suggest that aging may not attenuate the Ach-induced intestinal contraction via muscarinic M3 receptor, although the expression of muscarinic cholinoceptor is differentially modulated in jejunum and colon.

Involvement of p44/42 mitogen-activated protein kinases in regulating angiotensin II- and endothelin-1-induced contraction of rat thoracic aorta.

In order to elucidate the signal transduction pathway of vascular smooth muscle contraction induced by the activation of receptors for angiotensin II and endothelin-1, we examined whether tyrosine kinases and mitogen-activated protein (MAP) kinases are involved in the development of force of contraction in the rat aorta. Isolated aortic smooth muscles without endothelium were incubated in a modified Krebs-Henseleit solution and stimulated with angiotensin II (100 nM) or endothelin-1 (10 nM). A tyrosine kinase inhibitor genistein (10 microM) reduced the angiotensin II- and endothelin-1-induced aortic contraction, while 10 microM of daidzein (an inactive analogue of genistein) did not. The K(+) depolarization-induced contraction was not attenuated by 10 microM of genistein. Selective inhibitors of MAP kinase/extracellular signal-regulated kinase (Erk) kinase (MEK) such as PD98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one] and U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] inhibited the angiotensin II- and endothelin-1-induced vasocontraction. The p44/42 MAP kinases were phosphorylated in cultured aortic smooth muscle cells and in physiologically contracted aortic vessels stimulated with angiotensin II and endothelin-1 for 5 min. The angiotensin II- and endothelin-1-induced phosphorylations of p44/42 MAP kinases were inhibited by PD98059 as well as U0126 in the intact aorta. These results suggest that the activation of genistein-sensitive tyrosine kinases and p44/42 MAP kinases is involved in the angiotensin II- and endothelin-1-induced rat aortic contraction.