Alteration of reactivity in isolated mesenteric artery from Zucker fatty diabetes mellitus rats.

Obesity and diabetes are major risk factors for cardiovascular diseases. Zucker fatty diabetes mellitus (ZFDM) rats are novel animal model of obesity and type 2 diabetes. We have recently reported that blood pressure in ZFDM-Leprfa/fa (Homo) rats was normal, while blood adrenaline level and heart rate were lower than those in control ZFDM-Leprfa/+ (Hetero) rats. Here, we compared the reactivity in isolated mesenteric artery between Hetero and Homo rats. Contraction induced by phenylephrine was increased, while relaxation induced by isoprenaline was decreased in Homo rats at 21-23 weeks old compared with those in Hetero rats. The mRNA expression for α1A but not ß2 adrenoreceptor in Homo rats was increased. Nitric oxide (NO)-mediated relaxation induced by acetylcholine was decreased, while the mRNA expression for endothelial NO synthase (eNOS) was rather increased in mesenteric artery from Homo rats. These findings for the first time revealed that in Homo rats with reduced plasma adrenaline, blood pressure could be maintained by enhancing vascular contractility induced by adrenaline through the increased α1 adrenoceptor expression and the attenuated ß2 adrenoceptor signaling. Additionally, NO-mediated endothelium-dependent relaxation is impaired perhaps due to eNOS dysfunction, which might also contribute to maintain the blood pressure in Homo rats.

Human Omentin-1 Administration Ameliorates Hypertensive Complications without Affecting Hypertension in Spontaneously Hypertensive Rats.

Hypertension is one of the major risk factors for cardiovascular diseases and is caused by various abnormalities including the contractility of blood vessels. Spontaneously hypertensive rats (SHR), whose systemic blood pressure increases with aging, are a frequently used animal model for investigating essential hypertension and related complications in humans due to the damage of several organs. Human omentin-1 is an adipocytokine consisting of 313 amino acids. Serum omentin-1 levels decreased in hypertensive patients compared with normotensive controls. Furthermore, omentin-1 knockout mice showed elevated blood pressure and impaired endothelial vasodilation. Taken together, we hypothesized that adipocytokine, human omentin-1 may improve the hypertension and its complications including heart and renal failure in the aged SHR (65-68-weeks-old). SHR were subcutaneously administered with human omentin-1 (18 µg/kg/day, 2 weeks). Human omentin-1 had no effect on body weight, heart rate, and systolic blood pressure in SHR. The measurement of isometric contraction revealed that human omentin-1 had no influence on the enhanced vasocontractile or impaired vasodilator responses in the isolated thoracic aorta from SHR. On the other hand, human omentin-1 tended to improve left ventricular diastolic failure and renal failure in SHR. In summary, human omentin-1 tended to improve hypertensive complications (heart and renal failure), while it had no influence on the severe hypertension in the aged SHR. The further study of human omentin-1 may lead to the development of therapeutic agents for hypertensive complications.

Cardiovascular Characteristics of Zucker Fatty Diabetes Mellitus Rats, an Animal Model for Obesity and Type 2 Diabetes.

Zucker fatty diabetes mellitus (ZFDM) rats harboring the missense mutation (fa) in a leptin receptor gene have been recently established as a novel animal model of obesity and type 2 diabetes (T2D). Here, we explored changes in cardiovascular dynamics including blood pressure and heart rate (HR) associated with the progression of obesity and T2D, as well as pathological changes in adipose tissue and kidney. There was no significant difference in systolic blood pressure (SBP) in ZFDM-Leprfa/fa (Homo) compared with ZFDM-Leprfa/+ (Hetero) rats, while HR and plasma adrenaline in Homo were significantly lower than Hetero. The mRNA expression of monocyte chemotactic protein-1 in perirenal white adipose tissue (WAT) from Homo was significantly higher than Hetero. Interscapular brown adipose tissue (BAT) in Homo was degenerated and whitened. The plasma blood urea nitrogen in Homo was significantly higher than Hetero. In summary, we demonstrated for the first time that HR and plasma adrenaline concentration but not SBP in Homo decrease with obesity and T2D. In addition, inflammation occurs in WAT from Homo, while whitening occurs in BAT. Further, renal function is impaired in Homo. In the future, ZFDM rats will be useful for investigating metabolic changes associated with the progression of obesity and T2D.

Chemokine-like Receptor 1 in Brain of Spontaneously Hypertensive Rats Mediates Systemic Hypertension.

Adipocytokine chemerin is a biologically active molecule secreted from adipose tissue. Chemerin elicits a variety of functions via chemokine-like receptor 1 (CMKLR1). The cardiovascular center in brain that regulates blood pressure (BP) is involved in pathophysiology of systemic hypertension. Thus, we explored the roles of brain chemerin/CMKLR1 on regulation of BP in spontaneously hypertensive rats (SHR). For this aim, we examined effects of intracerebroventricular (i.c.v.) injection of CMKLR1 small interfering (si)RNA on both systemic BP as measured by tail cuff system and protein expression in paraventricular nucleus (PVN) of SHR as determined by Western blotting. We also examined both central and peripheral protein expression of chemerin by Western blotting. Systolic BP of SHR but not normotensive Wistar Kyoto rats (WKY) was decreased by CMKLR1 siRNA. The decrease of BP by CMKLR1 siRNA persisted for 3 days. Protein expression of CMKLR1 in PVN of SHR tended to be increased compared with WKY, which was suppressed by CMKLR1 siRNA. Protein expression of chemerin in brain, peripheral plasma, and adipose tissue was not different between WKY and SHR. In summary, we for the first time revealed that the increased protein expression of CMKLR1 in PVN is at least partly responsible for systemic hypertension in SHR.

Preventive Effect of Canstatin against Ventricular Arrhythmia Induced by Ischemia/Reperfusion Injury: A Pilot Study.

Ventricular arrhythmia induced by ischemia/reperfusion (I/R) injury is a clinical problem in reperfusion therapies for acute myocardial infarction. Ca2+ overload through reactive oxygen species (ROS) production is a major cause for I/R-induced arrhythmia. We previously demonstrated that canstatin, a C-terminal fragment of type IV collagen α2 chain, regulated Ca2+ handling in rat heart. In this study, we aimed to clarify the effects of canstatin on I/R-induced ventricular arrhythmia in rats. Male Wistar rats were subjected to I/R injury by ligating the left anterior descending artery followed by reperfusion. Ventricular arrhythmia (ventricular tachycardia and ventricular fibrillation) was recorded by electrocardiogram. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) activity and ROS production in neonatal rat cardiomyocytes (NRCMs) stimulated with oxygen glucose deprivation/reperfusion (OGD/R) were measured by lucigenin assay and 2',7'-dichlorodihydrofluorescein diacetate staining, respectively. The H2O2-induced intracellular Ca2+ ([Ca2+]i) rise in NRCMs was measured by a fluorescent Ca2+ indicator. Canstatin (20 µg/kg) inhibited I/R-induced ventricular arrhythmia in rats. Canstatin (250 ng/mL) inhibited OGD/R-induced NOX activation and ROS production and suppressed the H2O2-induced [Ca2+]i rise in NRCMs. We for the first time demonstrated that canstatin exerts a preventive effect against I/R-induced ventricular arrhythmia, perhaps in part through the suppression of ROS production and the subsequent [Ca2+]i rise.

Chemerin-9-induced contraction was enhanced through the upregulation of smooth muscle chemokine-like receptor 1 in isolated pulmonary artery of pulmonary arterial hypertensive rats.

Chemerin is an adipocytokine having cardiovascular effects. Chemokine-like receptor 1 (CMKLR1) and chemokine (CC motif) receptor-like 2 (CCRL2) are chemerin receptors. Chemerin-9, an active fragment, causes contraction via smooth muscle CMKLR1 in isolated blood vessels. Pulmonary arterial hypertension (PAH) is a fatal disease resulting ultimately in right heart failure. To test the hypothesis that chemerin affects pulmonary artery (PA) resistance, we examined the effects of chemerin-9 on contractility of isolated PA from PAH rats. Wistar rats were injected with monocrotaline (MCT) for 2 weeks to make PAH rats (MCT rats). Control (Cont) rats received a saline injection. Chemerin-9-induced contraction of isolated intrapulmonary artery (IPA) from left lung was isometrically measured. Protein expression of CMKLR1 and CCRL2 in isolated left lung was determined by Western blotting. Localization of CMKLR1 in IPA of left lung was examined immunohistochemically. Chemerin-9-induced contraction was significantly enhanced in IPA from MCT compared with Cont rats. Protein expression of CMKLR1 was significantly elevated in isolated left lung from MCT compared with Cont rats, while protein expression of CCRL2, a decoy receptor, was significantly decreased. CMKLR1 was localized mainly in endothelium of IPA in Cont rats. The CMKLR1 expression was significantly decreased in endothelium of IPA in MCT rats, while it was significantly elevated in smooth muscle. The present study for the first time demonstrated that the enhanced chemerin-9-induced contraction of isolated IPA from MCT rats was at least partly caused by the increase of CMKLR1 in smooth muscle.

Decreased Expression of Canstatin in Rat Model of Monocrotaline-Induced Pulmonary Arterial Hypertension: Protective Effect of Canstatin on Right Ventricular Remodeling.

Pulmonary arterial hypertension (PAH) is a progressive disease which causes right ventricular (RV) failure. Canstatin, a C-terminal fragment of type IV collagen α2 chain, is expressed in various rat organs. However, the expression level of canstatin in plasma and organs during PAH is still unclear. We aimed to clarify it and further investigated the protective effects of canstatin in a rat model of monocrotaline-induced PAH. Cardiac functions were assessed by echocardiography. Expression levels of canstatin in plasma and organs were evaluated by enzyme-linked immunosorbent assay and Western blotting, respectively. PAH was evaluated by catheterization. RV remodeling was evaluated by histological analyses. Real-time polymerase chain reaction was performed to evaluate RV remodeling-related genes. The plasma concentration of canstatin in PAH rats was decreased, which was correlated with a reduction in acceleration time/ejection time ratio and an increase in RV weight/body weight ratio. The protein expression of canstatin in RV, lung and kidney was decreased in PAH rats. While recombinant canstatin had no effect on PAH, it significantly improved RV remodeling, including hypertrophy and fibrosis, and prevented the increase in RV remodeling-related genes. We demonstrated that plasma canstatin is decreased in PAH rats and that administration of canstatin exerts cardioprotective effects.

Optimal Isolation Method of Small Extracellular Vesicles from Rat Plasma.

Small extracellular vesicles (sEVs) mediate cell-to-cell communication. We recently reported that circulating sEVs regulate systolic blood pressure in an animal model of human systemic hypertension. However, the underlying mechanisms still remain to be elucidated. As the first step for detailed analyses, we sought to increase the yield and purity of sEVs isolated from rat plasma. We compared the concentration and size distribution of sEVs as well as protein expression of the sEV marker and contaminants among plasma sEVs isolated by the ultracentrifugation (UC) method, the precipitation with polyethylene-glycol and ultracentrifugation (PEG-UC) method, or the precipitation with polyethylene-glycol (PEG) method. Effects of anticoagulants were also examined. The total concentration of plasma sEVs isolated by the PEG or PEG-UC method was much higher than that of the UC method. In the plasma sEVs isolated by the PEG-UC method, contaminating proteins were lower, while the protein expression of certain sEV markers was higher than that of the PEG method. There was no significant difference in total concentration or protein expression of sEV markers in sEVs isolated from rat plasma treated with three different anticoagulants (heparin, ethylenediaminetetraacetic acid, or acid citrate dextrose buffer) by the PEG-UC method. We, for the first time, determined that the PEG-UC method was optimal for sEV isolation from rat plasma.

Plasma exosomes regulate systemic blood pressure in rats.

Exosomes, the smallest extracellular vesicle, contain various molecules and mediate cell-cell communication. A number of studies demonstrate exosomes are involved in important physiological and pathological processes. Moreover, microRNA (miRNA) regulating hypertension development through the suppression of certain translation was recently reported. However, roles of exosomes containing various molecules including miRNA on development of essential hypertension have not been examined. We tested the hypothesis that plasma exosomes regulate systemic blood pressure in normotensive and spontaneously hypertensive rats (SHR). Normotensive Wistar Kyoto rats (WKY) and SHR (5-10-week-old) were intraperitoneally administrated with exosomes derived from plasma in WKY or SHR weekly for 6 weeks. Exosomes were isolated by an ultracentrifuge method. SHR-derived exosomes significantly increased systolic blood pressure in WKY, while WKY-derived exosomes decreased it in SHR. In WKY, SHR-derived exosomes induced modest structural changes of thoracic aorta, such as wall thickening and decreased abundance of collagen, which were similar to the changes observed in SHR. On the contrary, WKY-derived exosomes tended to reverse the changes in SHR. WKY-derived exosomes significantly suppressed the increased prostaglandin F2α-induced contraction of mesenteric arterial smooth muscle in SHR. In addition, wet weight and perivascular fibrosis of left ventricles in WKY were significantly increased by SHR-derived exosomes, while the fibrosis but not ventricular weight was significantly decreased by WKY-derived exosomes in SHR. We for the first time demonstrated that plasma exosomes can modulate systemic blood pressure as well as structure and function of cardiovascular tissues in both normotensive and hypertensive rats.

Vasculo-protective effect of BMS-309403 is independent of its specific inhibition of fatty acid-binding protein 4.

Fatty acid-binding protein (FABP) 4 is an adipocytokine mainly expressed in adipocyte and macrophage. Blood FABP4 is related not only to metabolic disorders including insulin resistance and atherosclerosis but also increased blood pressure. We tested the hypothesis that FABP4 plays roles in pathogenesis of hypertension development including proliferation, migration, and inflammation of vascular smooth muscle cells (SMCs) as well as contractile reactivity. FABP4 alone had no influence on proliferation, migration, and inflammation of rat mesenteric arterial SMCs, while it significantly enhanced smooth muscle contraction and increases of systolic blood pressure (SBP) induced by noradrenaline (NA). BMS-309403, an FABP4 inhibitor, significantly inhibited platelet-derived growth factor-BB-induced DNA synthesis and migration via preventing p38 and HSP27 activation. Further, BMS-309403 significantly inhibited tumor necrosis factor-α-induced expression of vascular cell adhesion molecule-1 and monocyte chemotactic protein-1 as well as monocyte adhesion via preventing NF-κB activation. Interestingly, SMCs do not express FABP4. Long-term treatment of spontaneously hypertensive rats (SHR) with BMS-309403 significantly inhibited impaired relaxation in isolated mesenteric arteries and left ventricular hypertrophy, while it had no influence on SBP. We for the first time showed that FABP4 acutely enhances NA-induced increases of SBP possibly through the enhancement of peripheral arterial contractility. BMS-309403 prevents proliferation, migration, and inflammatory responses of SMCs, although exogenous application of FABP4 has no influence on the cellular responses. Furthermore, we demonstrated that long-term treatment with BMS-309403 partially improves the pathological conditions of SHR. These results indicate that BMS-309403 would be useful for developing a new pharmacotherapeutic agent against obesity-associated hypertension and complications.

Expression pattern and function of tyrosine receptor kinase B isoforms in rat mesenteric arterial smooth muscle cells.

Tyrosine receptor kinaseB (TrkB) is a high affinity receptor for brain-derived neurotrophic factor (BDNF). TrkB isoforms involve full length TrkB (TrkB FL) and truncated TrkB type1 (TrkB T1) and type 2 (TrkB T2) in rats. The aim of present study was to explore their expression pattern and function in mesenteric arterial smooth muscle cells (MASMCs). The expression of TrkB isoform protein and mRNA was examined by Western blotting, immunofluorescence and quantitative RT-PCR analyses. Cell proliferation was measured by a bromodeoxyuridine (BrdU) incorporation assay. Cell migration was measured by a Boyden chamber assay. Cell morphology was observed with a phase-contrast microscope. Protein and mRNA expression of BDNF and TrkB isoforms was confirmed in MASMCs. Expression level of TrkB FL was less, while that of TrkB T1 was the highest in MASMCs. Although BDNF increased phosphorylation of ERK, it had no influence on migration and proliferation of MASMCs. TrkB T1 gene knockdown by a RNA interference induced morphological changes and reduced expression level of α-smooth muscle actin (α-SMA) in MASMCs. Similar morphological changes and reduced α-SMA expression were induced in MASMCs by a Rho kinase inhibitor, Y-27632. In conclusion, we for the first time demonstrate that TrkB T1 expressed highly in MASMCs contributes to maintain normal cell morphology possibly via regulation of Rho activity. This study firstly defined expression level of TrkB isoforms and partly revealed their functions in peripheral vascular cells.

Death-associated protein kinase 3 mediates vascular structural remodelling via stimulating smooth muscle cell proliferation and migration.

Death-associated protein kinase 3 (DAPK3) also known as zipper-interacting kinase is a serine/threonine kinase that mainly regulates cell death and smooth muscle contraction. We have previously found that protein expression of DAPK3 increases in the mesenteric artery from spontaneously hypertensive rats (SHRs) and that DAPK3 mediates the development of hypertension in SHRs partly through promoting reactive oxygen species-dependent vascular inflammation. However, it remains to be clarified how DAPK3 controls smooth muscle cell (SMC) proliferation and migration, which are also important processes for hypertension development. We, therefore, sought to investigate whether DAPK3 affects SMC proliferation and migration. siRNA against DAPK3 significantly inhibited platelet-derived growth factor (PDGF)-BB-induced SMC proliferation and migration as determined by bromodeoxyuridine (BrdU) incorporation and a cell counting assay as well as a Boyden chamber assay respectively. DAPK3 siRNA or a pharmacological inhibitor of DAPK3 inhibited PDGF-BB-induced lamellipodia formation as determined by rhodamine-phalloidin staining. DAPK3 siRNA or the DAPK inhibitor significantly reduced PDGF-BB-induced activation of p38 and heat-shock protein 27 (HSP27) as determined by Western blotting. In ex vivo studies, PDGF-BB-induced SMC out-growth was significantly inhibited by the DAPK inhibitor. In vivo, the DAPK inhibitor significantly prevented carotid neointimal hyperplasia in a mouse ligation model. The present results, for the first time, revealed that DAPK3 mediates PDGF-BB-induced SMC proliferation and migration through activation of p38/HSP27 signals, which may lead to vascular structural remodelling including neointimal hyperplasia. The present study suggests DAPK3 as a novel pharmaceutical target for the prevention of hypertensive cardiovascular diseases.

Eukaryotic elongation factor 2 kinase inhibitor, A484954 induced diuresis via nitric oxide production in spontaneously hypertensive rats.

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) is a protein kinase that inactivates eEF2, a protein that mediates a peptidyl-tRNA translocation during an elongation step of protein synthesis. We have previously shown that eEF2K was involved in pathogenesis of essential and pulmonary hypertension. A484954 (7-amino-1-cyclopropyl-3-ethyl-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d] pyrimidine-6-carboxamide), a selective eEF2K inhibitor, is a membrane permeable small molecule. We have previously shown that A484954 lowered blood pressure and induced diuretic effects in spontaneously hypertensive rats (SHR) due to an increase in renal blood flow. Here we aimed to reveal mechanisms underlying the diuretic effects of A484954 in SHR. A484954-induced diuresis and increase in urinary Na+ excretion were inhibited by N (G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase inhibitor. A484954 increased mRNA expression of angiotensin type 2 receptor (AT2R) and nuclear factor-erythroid 2-related factor 2 (Nrf2). In summary, we for the first time revealed that A484954 induces diuresis in SHR at least partly via the activation of NO/Nrf2/AT2R pathway.

Autocrine role of senescent cardiac fibroblasts-derived extracellular vesicles.

Cellular senescence is a highly stable state associated with cell cycle arrest, that is elicited in response to various stresses. The accumulation of senescent cells in tissues drives age-related diseases. Recent studies have shown that the cellular senescence enhances an extracellular vesicles (EV) secretion. EV are lipid-bilayer-capsuled particles released by various cells mediating cell-to-cell communication. It was recently reported that EV secreted by the senescent cells had several functions such as cancer cell proliferation and immune cell activation. In the present study, we investigated whether senescent cardiac fibroblasts-derived EV play an autocrine/paracrine role in the heart cells. Neonatal rat cardiac fibroblasts (NRCFs) were treated with doxorubicin (DOX) to induce cellular senescence. EV were isolated from NRCFs culture media. The vehicle-treated NRCFs-derived EV (D0-EV, 72 hr) increased a living cell number in NRCFs, which was attenuated by DOX (1,000 nM)-treated NRCFs-derived EV (D103-EV, 72 hr). While D0-EV did not affect protein concentration in NRCFs, D103-EV decreased it. Furthermore, D103-EV significantly increased a ratio of microtubule-associated protein 1 light chain 3 (LC3)-II to LC3-I in NRCFs, indicating an induction of autophagy. In addition, D103-EV increased phosphorylation of adenosine monophosphate-activated kinase (AMPK) α in NRCFs. In neonatal rat cardiomyocytes, however, NRCFs-derived EV (72 hr) had no effect on the living cell number, protein concentration, and ratio of LC3-II to LC3-I. In conclusion, we for the first time revealed that DOX-induced senescent NRCFs-derived EV induce autophagy in NRCFs perhaps partly through the activation of AMPKα.

Human omentin-1 reduces vascular insulin resistance and hypertension in Otsuka Long-Evans Tokushima Fatty rats.

PURPOSE: Hypertension is one of the major risk factors for renal failure and cardiovascular diseases, and is caused by various abnormalities including the contractility of blood vessels. Otsuka Long-Evans Tokushima Fatty (OLETF) rats, which mimic human type 2 diabetes, are frequently used to study obesity-induced insulin resistance (IR) and hypertension. Human omentin-1 is one of the recently identified adipocytokines. We previously demonstrated that human omentin-1 not only caused vasodilation in rat isolated blood vessels, but also prevented inflammatory responses, a possible mechanism relating IR, in human vascular endothelial cells. Taken together, we hypothesized that human omentin-1 may reduce obesity-induced IR and hypertension in OLETF rats. METHODS: OLETF rats were intraperitoneally administered with human omentin-1 for 7 days. RESULTS: Human omentin-1 had no influence on overweight, hyperglycemia, urinary glucose extraction, hyperinsulinemia, and systemic IR in OLETF rats. Human omentin-1 decreased systolic blood pressure in OLETF rats. The measurement of isometric contraction revealed that human omentin-1 had no influence on the agonist-induced contractile and relaxant responses in isolated thoracic aorta from OLETF rats. However, the relaxant response mediated by human insulin was converted into the contractile response in thoracic aorta from OLETF rats, which was prevented by human omentin-1. The Western blotting revealed that human omentin-1 improved the decrease in endothelial nitric oxide synthase activation in isolated thoracic aorta from OLETF rats. CONCLUSION: In summary, we for the first time revealed that human omentin-1 partly reduces vascular IR and thereby inhibits hypertension in OLETF rats.

Eukaryotic elongation factor 2 kinase inhibitor, A484954 inhibits perivascular sympathetic nerve stimulation-induced vasoconstriction in isolated renal artery.

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) repressively regulates protein translation through phosphorylating eEF2. We previously showed that expression and activity of eEF2K are increased in isolated mesenteric arteries from spontaneously hypertensive rats (SHR) contributing to development of essential hypertension. Furthermore, we have recently shown that 7-Amino-1-cyclopropyl-3-ethyl-1,2,3,4-tetrahydro-2,4-dioxopyrido[2,3-d]pyrimidine-6-carboxamide (A484954), a selective eEF2K inhibitor, induces endothelium-dependent relaxation in isolated mesenteric arteries from SHR inducing an antihypertensive effect. In order to test the hypothesis that inhibition of eEF2K activity induces vasodilatation by suppressing sympathetic nerve activity, we examined the effects of A484954 on perivascular sympathetic nerve stimulation-induced contraction in isolated renal artery from normotensive and hypertensive rats. Electrodes were placed near the isolated renal arteries that were applied with transmural nerve stimulation (TNS). Then, contraction of the arteries was isometrically measured. A484954 inhibited TNS-induced contraction. The A484954-mediated inhibition of TNS-induced contraction was significantly prevented by NG-nitro-L-arginine methyl ester. In SHR isolated renal artery, TNS-induced contraction was enhanced compared with normotensive Wistar rats. Furthermore, A484954-mediated inhibition of TNS-induced contraction in SHR was enhanced compared with Wistar rats. In conclusion, this study demonstrates for the first time that A484954 inhibits perivascular sympathetic nerve stimulation-induced vasoconstriction at least in part perhaps through nitric oxide (NO) release from NO-operating nerve.

Chemerin fragments show different effects on systemic blood pressure dependent on carboxyl-terminal cleavage site.

Chemerin is an adipocytokine whose concentration in blood correlates positively with blood pressure (BP). We have recently revealed that acute intracerebroventricular (i.c.v.) injection of chemerin-9, an active fragment of human chemerin, increased systemic BP in normal Wistar rats, suggesting that chemerin is involved in the central nervous control of peripheral BP. After secreted as an inactive form as prochemerin, a mature form of active chemerin is produced through the cleavage of its carboxyl (C)-terminus by proteases. Although the activity of cleaved products of chemerin has been examined in vitro, in vivo effects remained to be elusive. In order to explore them, we performed acute i.c.v. injection of mouse chemerin-9 (mChemerin-9; 148F-156S), mouse chemerin-8 (mChemerin-8; 148F-155F), and mouse chemerin-7 (mChemerin-7; 148F-154A) into Wistar rats, and examined the effects on systemic BP. After chemerin fragment (1-30 nmol/head, i.c.v.) was cumulatively administered, systemic BP was measured by a cannulation method under an isoflurane anesthesia. mChemerin-9 but not mChemerin-8 and -7 induced a pressor response, which was concentration-dependent. In conclusion, we for the first time demonstrated that mChemerin-9 that corresponds to the C-terminal nine amino acids of active mouse chemerin156S increased systemic BP in rats, and also that chemerin fragments showed different effects on systemic BP dependent on how their C-terminus was cleaved.

Age-dependent increase in activity of eukaryotic elongation factor 2 kinase in mesenteric arteries from spontaneously hypertensive rats.

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) negatively regulates protein translation through the phosphorylation of its specific substrate, eEF2. We previously found that expression of eEF2K was increased in arteries from 13-15-week-old spontaneously hypertensive rats (SHR) as well as in left ventricles of cardiac hypertrophy models. Furthermore, we demonstrated that eEF2K mediates the development of essential hypertension and pulmonary arterial hypertension in animal models. Protein expression changes with age during development of hypertension in SHR. In the present study, we examined whether activity and expression of eEF2K change in isolated mesenteric arteries dependent on the age. After superior mesenteric arteries were isolated from 4-10-week-old Wistar Kyoto rats (WKY) and SHR, Western blotting was performed. The phosphorylation of eEF2K at Ser500, an activating phosphorylation site, was increased in the arteries from 10-week-old SHR, whereas the phosphorylation of eEF2K at Ser366, an inactivating phosphorylation site, was increased in the arteries from 4-5-week-old SHR compared with WKY. The expression of eEF2K was increased in the arteries from 10-week-old SHR compared with WKY. The phosphorylation of eEF2 at Thr56 was decreased in the arteries from 4-5-week-old SHR, whereas it was increased in the arteries from 10-week-old SHR compared with WKY. We for the first time revealed that eEF2K activity is lower in prehypertensive stage but higher in hypertensive stage in SHR, suggesting that an inhibition of eEF2K activity may be a potential therapeutic strategy for the treatment of essential hypertension.

[Development of basic research toward clinical application of cleaved fragment of type IV collagen].

Basement membrane is a dense sheet-like extracellular matrix (ECM), which separates cells from surrounding interstitium. Type IV collagen is a major component of basement membrane and three of six α chains (namely α1-α6 chains) form a triple-helix structure. Recently, endogenous bioactive factors called "matricryptins" or "matrikines", which are produced by degrading and cleaving C-terminal domain of type IV collagen, attract attentions as a novel therapeutic target or a candidate for biomarkers. In all type IV collagens, matricryptins called arresten (α1 chain), canstatin (α2), tumstatin (α3), tetrastatin (α4), pentastatin (α5), and hexastatin (α6), have been identified. The type IV collagen-derived matricryptins have been previously studied as new therapeutic targets for neoplastic diseases since they exert anti-angiogenic and/or anti-tumor effects. On the other hand, we have recently demonstrated the cardioprotective effects of matricryptins in addition to the altered expression levels in cardiac diseases. In this review, we introduce the results of fundamental studies for the type IV collagen-derived matricryptins in various diseases, such as neoplastic diseases and cardiac diseases, and discuss the potential clinical application as novel therapeutic agents and biomarkers.

Eukaryotic elongation factor 2 kinase inhibitor, A484954 induces diuretic effect via renal vasorelaxation in spontaneously hypertensive rats.

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K), alternatively known as calmodulin-dependent protein kinase III, inhibits protein translation via phosphorylating its sole substrate, eEF2. We previously demonstrated that expression and activity of eEF2K change in mesenteric artery from spontaneously hypertensive rats (SHR) with aging and that eEF2K is involved in pathogenesis of essential hypertension. In addition, we have recently revealed that acute intravenous injection with A484954, a selective eEF2K inhibitor, lowers blood pressure specifically in SHR partly via inducing vasorelaxation. In this study, we examined whether A484954 induces diuretic effect. After male SHR and normotensive Wistar Kyoto rats (WKY) were given a single intraperitoneal injection of A484954 (2.5 mg/kg, 0.5-9 h), urine was collected using metabolic cage. Contraction of isolated renal arteries form SHR was isometrically measured. While A484954 did not induce diuretic effect in WKY, it increased urine output, water intake, and urinary sodium excretion in SHR. A484954 (10 µM) induced vasorelaxation in isolated renal arteries, which was inhibited by a ß-adrenergic receptor antagonist, propranolol. It was confirmed that A484954 increased renal blood flow in SHR as measured by renal ultrasonography. In summary, it was for the first time revealed that A484954 induces diuretic effect in SHR at least partly via renal vasorelaxation through ß-adrenergic receptor.