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.

Acute intracerebroventricular injection of chemerin-9 increases systemic blood pressure through activating sympathetic nerves via CMKLR1 in brain.

Chemerin is an adipocytokine involved in inflammation and lipid metabolism via G protein-coupled receptor, chemokine-like receptor (CMKLR)1. Since the important nuclei regulating pressure (BP) exist in the brain, we examined the effects of acute intracerebroventricular (i.c.v.) injection of chemerin-9 on systemic BP and explored underlying mechanisms. We examined the effects of acute i.c.v. injection of chemerin-9 (10 nmol/head) on systemic BP by a carotid cannulation method in the control or CMKLR1 small interfering (si) RNA-treated Wistar rats (0.04 nmol, 3 days, i.c.v.). We examined protein expression of CMKLR1 around brain ventricles by Western blotting. We examined the effects of acute i.c.v. injection of chemerin-9 on serum adrenaline by a high performance liquid chromatography. In the control siRNA-treated rats, chemerin-9 significantly increased mean BP, which reached a peak at 2 to 4 min after injection. On the other hand, in the CMKLR1 siRNA-treated rats, chemerin-9 did not affect the mean BP. Protein expression of CMKLR1 specifically in subfornical organ (SFO) and paraventricular nucleus (PVN) from the CMKLR1 siRNA-treated rats decreased compared with the control siRNA-treated rats. In the control siRNA-treated rats, chemerin-9 increased serum adrenaline level. On the other hand, in the CMKLR1 siRNA-treated rats, chemerin-9 did not affect the serum adrenaline level. Further, pretreatment with prazosin, an α-adrenaline receptor blocker, significantly prevented the pressor responses induced by chemerin-9. In summary, we for the first time demonstrated that chemerin-9 stimulates the sympathetic nerves via CMKLR1 perhaps expressed in SFO and PVN, which leads to an increase in systemic BP.

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.

Eukaryotic elongation factor 2 kinase inhibitor, A484954 lowered blood pressure in spontaneously hypertensive rats via inducing vasorelaxation.

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) suppresses protein translation. We previously reported eEF2K expression was upregulated in mesenteric arteries (MA) from spontaneously hypertensive rats (SHR). We have recently revealed A484954, an eEF2K inhibitor, acutely suppressed vasopressor agonists-induced increase of blood pressure (BP) in normal Wistar rats. In this study, we examined the acute effects of A484954 on BP in SHR and explored underlying mechanisms. BP was measured by a carotid cannulation method in SHR. Isometric contraction in MA from SHR was measured. Endothelial nitric oxide synthase (eNOS) dimerization was measured by low-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. A484954 lowered BP in 15-week-old SHR. A484954 induced relaxation in MA from both 4- and 7-9-week-old SHR. In MA from 4-week-old SHR, A484954-induced relaxation was inhibited almost completely by a NOS inhibitor, NG-nitro-l-arginine methyl ester (l-NAME) and significantly by a ß blocker, propranolol. In MA from 7-9-week-old SHR, on the other hand, A484954-induced relaxation was inhibited partly either by l-NAME, indomethacin, a cyclooxygenase inhibitor, or l-NAME + indomethacin. A484954 promoted the dimerization of eNOS in human endothelial cells. In summary, we have revealed A484954 lowers BP in SHR perhaps through the vasorelaxation via the production of endothelium-derived relaxing factors.

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.

Eukaryotic elongation factor 2 (eEF2) kinase/eEF2 plays protective roles against glucose deprivation-induced cell death in H9c2 cardiomyoblasts.

During the development of cardiac hypertrophy, glucose deprivation (GD) associated with coronary microvascular rarefaction is caused, leading to cardiomyocyte death. Phosphorylation (inactivation) of eukaryotic elongation factor 2 (eEF2) by eEF2 kinase (eEF2K) inhibits protein translation, a highly energy consuming process, which plays protective roles against nutrient deprivation-induced cell death. We previously showed that eEF2 phosphorylation was increased in isolated heart from several cardiac hypertrophy models. In this study, we investigated whether eEF2K/eEF2 mediates the inhibition of cardiomyocyte death under GD condition. In H9c2 rat cardiomyoblasts cultured with serum-free medium, GD significantly augmented eEF2 phosphorylation and signals related to autophagy [increase of microtubule-associated protein 1 light chain 3 (LC3)-II to LC3-I ratio] and apoptosis (cleavage of caspase-3) as determined by Western blotting. GD induced cell death, which was augmented by eEF2K gene knockdown using a small interfering RNA. eEF2K gene knockdown significantly augmented GD-induced cleavage of caspase-3 and apoptotic nuclear condensation as determined by 4', 6-diamidino-2-phenylindole staining. In contrast, eEF2K gene knockdown significantly inhibited GD-induced increase of LC3-II to LC3-I ratio and autophagosome formation as determined by an immunofluorescence staining. An inhibitor of autophagy, 3-methyladenine or bafilomycin A1 significantly augmented GD-induced cleavage of caspase-3. Further, eEF2K gene knockdown significantly inhibited GD-induced phosphorylation of adenosine monophosphate-activated protein kinase (AMPK)α and its downstream substrate, unc-51 like autophagy activating kinase (ULK)1. An inhibitor of AMPK, dorsomorphin significantly inhibited GD-induced increase of LC3-II to LC3-I ratio. In conclusion, we for the first time revealed that eEF2K/eEF2 axis under GD condition mediates the inhibition of apoptotic H9c2 cell death at least in part via promotion of autophagy through AMPKα/ULK1 signaling pathway.

A current perspective of canstatin, a fragment of type IV collagen alpha 2 chain.

Type IV collagen is a main component of basement membrane extracellular matrix. Canstatin, a non-collagenous C-terminal fragment of type IV collagen α2 chain, was firstly identified as an endogenous anti-angiogenic and anti-tumor factor, which also has an anti-lymphangiogenic effect. Then, canstatin has been widely investigated as a novel target molecule for cancer therapy. The anti-angiogenic effect of canstatin may be also useful for the treatment of ocular neovascularization. Recently, we have demonstrated that canstatin, which is abundantly expressed in the heart tissue, exerts various biological activities in cardiac cells. In rat H9c2 cardiomyoblasts, canstatin inhibits isoproterenol- or hypoxia-induced apoptosis. Canstatin plays an important role in modulating voltage-dependent calcium channel activity in rat cardiomyocytes. Canstatin also regulates various biological functions in rat cardiac fibroblasts and myofibroblasts. The expression of canstatin decreases in the infarcted area after myocardial infarction. This review focuses on a current perspective for the roles of canstatin in tumorigenesis, ocular neovascularization and cardiac pathology.

Protective effect of T3 peptide, an active fragment of tumstatin, against ischemia/reperfusion injury in rat heart.

Ischemia/reperfusion (I/R)-induced oxidative stress is a serious clinical problem in the reperfusion therapy for ischemic diseases. Tumstatin is an endogenous bioactive peptide cleaved from type IV collagen α3 chain. We previously reported that T3 peptide, an active subfragment of tumstatin, exerts cytoprotective effects on H2O2-induced apoptosis through the inhibition of intracellular reactive oxygen species (ROS) production in H9c2 cardiomyoblasts. In this study, we investigated whether T3 peptide has cardioprotective effects against I/R injury by using in vitro and ex vivo experimental models. H9c2 cardiomyoblasts were stimulated with oxygen and glucose deprivation (OGD) for 12 h followed by reoxygenation for 1-8 h (OGD/R; in vitro model). The cells were treated with T3 peptide (30-1000 ng/ml) during OGD. Ten minutes after the pre-perfusion of T3 peptide (300 ng/ml), Langendorff perfused rat hearts were exposed to ischemia for 30 min followed by reperfusion for 1 h (ex vivo model). T3 peptide inhibited OGD/R-induced apoptosis through the inhibition of mitochondrial ROS production and dysfunction in H9c2 cardiomyoblasts. T3 peptide also prevented I/R-induced cardiac dysfunction, arrhythmia and myocardial infarction in the perfused rat heart. In conclusion, we for the first time demonstrated that T3 peptide exerts cardioprotective effects against I/R injury.

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.

Characterization of fibroblasts from hypertrophied right ventricle of pulmonary hypertensive rats.

Pulmonary arterial hypertension (PAH), which is characterized by an elevation of pulmonary arterial resistance, leads to a lethal right heart failure. It is an urgent issue to clarify the pathogenesis of PAH-induced right heart failure. The present study aimed to elucidate the characteristics of cardiac fibroblasts (CFs) isolated from hypertrophied right ventricles of monocrotaline (MCT)-induced PAH model rats. CFs were isolated from the right ventricles of MCT-injected rats (MCT-CFs) and saline-injected control rats (CONT-CFs). Expression of α-smooth muscle actin and collagen type I in MCT-CFs was lower than that in CONT-CFs. On the other hand, proliferation, migration, and matrix metalloproteinase (MMP)-9 production were significantly enhanced in MCT-CFs. In MCT-CFs, phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK), and Ca2+/calmodulin-dependent protein kinase (CaMK) II was significantly enhanced. In addition to mRNA expression of Orai1, a Ca2+ release-activated Ca2+ channel, and stromal interaction molecules (STIM) 1, an endoplasmic reticulum Ca2+ sensor, the associated store-operated Ca2+ entry (SOCE) was significantly higher in MCT-CFs than CONT-CFs. Pharmacological inhibition of ERK1/2 pathway prevented the enhanced proliferation of MCT-CFs. The enhanced migration of MCT-CFs was prevented by a pharmacological inhibition of ERK1/2, JNK, CaMKII, or SOCE pathway. The enhanced MMP-9 production in MCT-CFs was prevented by a pharmacological inhibition of ERK1/2, CaMKII, or SOCE pathway but not JNK. The present results suggested that MCT-CFs exhibit proliferative and migratory phenotypes perhaps through multiple signaling pathways. This study for the first time determined the characteristics of CFs isolated from hypertrophied right ventricles of MCT-induced PAH model rats.

Canstatin modulates L-type calcium channel activity in rat ventricular cardiomyocytes.

Excessive increase of cytosolic Ca2+ through the activation of L-type Ca2+ channels (LTCCs) via ß adrenergic receptor induces apoptosis of cardiomyocytes. Canstatin, a cleaved fragment of collagen type IV α2 chain, is abundantly expressed in normal heart tissue. We previously reported that canstatin inhibits ß adrenergic receptor-stimulated apoptosis in cardiomyoblasts. Here, we tested the hypothesis that canstatin regulates LTCCs activity in ventricular cardiomyocytes. Collagen type IV α2 chain (COL4A2) small interfering (si) RNA (for canstatin suppression) or control siRNA was injected via jugular vein in Wistar rats. Two days after the injection, electrocardiogram (ECG) was recorded and the left ventricular tissue was isolated using Langendorff apparatus. Immunofluorescence staining was performed to clarify the distribution of canstatin in cardiomyocytes. The knockdown efficiency was confirmed by Western blotting. The L-type Ca2+ channel current (ICaL) of ventricular cardiomyocyte was measured by a whole-cell patch clamp technique. In immunofluorescence staining, colocalization of canstatin and αv integrin was observed in the isolated ventricular cardiomyocytes. The ICaL of ventricular cardiomyocyte isolated from COL4A2 siRNA-injected rats was significantly enhanced compared with control siRNA-injected rats. Recombinant canstatin (250 ng/ml) significantly reversed it. ECG analysis showed that QT interval tended to be shortened and amplitude of T wave was significantly increased in the COL4A2 siRNA-injected rats. In summary, we for the first time clarified that suppressing canstatin expression increases the basal ICaL in ventricular cardiomyocytes. It is proposed that canstatin might play a role in the stabilization of cardiac function through the modulation of LTCC activity in cardiomyocytes.

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.

Mechanisms underlying the relaxation by A484954, a eukaryotic elongation factor 2 kinase inhibitor, in rat isolated mesenteric artery.

Eukaryotic elongation factor 2 kinase (eEF2K) is a calmodulin-related protein kinase which regulates protein translation. A484954 is an inhibitor of eEF2K. In the present study, we investigated the acute effects of A484954 on contractility of isolated blood vessels. Isometric contraction of rat isolated aorta and main branch of superior mesenteric artery (MA) was measured. Expression of an inward rectifier K+ (Kir) channel subtype mRNA and protein was examined. A484954 caused relaxation in endothelium-intact [E (+)] and -denuded [E (-)] aorta or MA precontracted with noradrenaline (NA). The relaxation was higher in MA than aorta. The relaxation was partially inhibited by a nitric oxide (NO) synthase inhibitor, NG-nitro-l-arginine methyl ester (300 µM) in E (+) MA. The relaxation was significantly smaller in MA precontracted with high K+ than NA. The A484954-induced relaxation was significantly inhibited by a Kir channel blocker, BaCl2 (1 mM) compared with vehicle control in E (-) MA. Expression of Kir2.2 mRNA and protein was significantly higher in MA than aorta. We for the first time revealed that A484954 induces relaxation through opening smooth muscle Kir (Kir2.2) channel and through endothelium-derived NO in MA.

Periostin Mediates Right Ventricular Failure through Induction of Inducible Nitric Oxide Synthase Expression in Right Ventricular Fibroblasts from Monocrotaline-Induced Pulmonary Arterial Hypertensive Rats.

Pulmonary arterial hypertension (PAH) leads to lethal right ventricular failure (RVF). Periostin (POSTN) mRNA expression is increased in right ventricles (RVs) of monocrotaline (MCT)-induced PAH model rats. However, the pathophysiological role of POSTN in RVF has not been clarified. We investigated the effects of POSTN on inducible nitric oxide (NO) synthase (iNOS) expression and NO production, which causes cardiac dysfunction, in right ventricular fibroblasts (RVFbs). Male Wistar rats were intraperitoneally injected with MCT (60 mg/kg) or saline. Three weeks after injection, RVFbs were isolated from RVs of MCT- or saline-injected rats (MCT-RVFb or CONT-RVFb). In MCT-RVFb, iNOS expression and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and nuclear factor-kappa B (NF-κB) were higher than those in CONT-RVFb. Recombinant POSTN increased iNOS expression and NO production, which were prevented by a pharmacological inhibition of ERK1/2, JNK or NF-κB in RVFbs isolated from normal rats. Culture medium of POSTN-stimulated RVFbs suppressed Ca2+ inflow through l-type Ca2+ channel (LTCC) in H9c2 cardiomyoblasts. We demonstrated that POSTN enhances iNOS expression and subsequent NO production via ERK1/2, JNK, and NF-κB signaling pathways in RVFbs. POSTN might mediate RVF through the suppression of LTCC activity of cardiomyocytes by producing NO from RVFbs in PAH model rats.

Endostatin Stimulates Proliferation and Migration of Myofibroblasts Isolated from Myocardial Infarction Model Rats.

Myofibroblasts contribute to the healing of infarcted areas after myocardial infarction through proliferation, migration, and production of extracellular matrix (ECM). Expression of endostatin, a cleaved fragment of type XVIII collagen, increases in the heart tissue of an experimental myocardial infarction model. In the present study, we examined the effect of endostatin on the function of myofibroblasts derived from an infarcted area. The myocardial infarction model was created by ligating the left anterior descending artery in rats. Two weeks after the operation, α-smooth muscle actin (α-SMA)-positive myofibroblasts were isolated from the infarcted area. Endostatin significantly increased the proliferation and migration of myofibroblasts in vitro. On the other hand, endostatin had no effect on the production of type I collagen, a major ECM protein produced by myofibroblasts. Endostatin activated Akt and extracellular signal-regulated kinase (ERK), and the pharmacological inhibition of these signaling pathways suppressed the endostatin-induced proliferation and migration. A knockdown of the COL18A1 gene in the myocardial infarction model rats using small interference RNA (siRNA) worsened the cardiac function concomitant with wall thinning and decreased the α-SMA-positive myofibroblasts and scar formation compared with that of control siRNA-injected rats. In summary, we demonstrated for the first time that endostatin might be an important factor in the healing process after myocardial infarction through the activation of myofibroblasts.

Canstatin stimulates migration of rat cardiac fibroblasts via secretion of matrix metalloproteinase-2.

Type IV collagen, a nonfibrillar type, is ubiquitously expressed in the basement membrane around cardiomyocytes. Canstatin, a cleaved product of α2 chain of type IV collagen, is an antiangiogenic factor. Because it has not been clarified whether canstatin exerts other biological activities in heart, we investigated the effects of canstatin on adult rat cardiac fibroblasts. Cell migration was determined by Boyden chamber assay. Western blotting was performed to detect secretion of matrix metalloproteinase-2 (MMP-2) and MMP-9 and phosphorylation of extracellular signal-regulated kinase (ERK). Localization of MMP-2 was detected by immunofluorescence staining. Canstatin (250 ng/ml) significantly increased migration, secretion, and activity of MMP-2 but not MMP-9. CTTHWGFTLC peptide, an MMP inhibitor and small interfering RNA (siRNA) against MMP-2 suppressed the canstatin-induced (250 ng/ml, 24 h) migration. Canstatin (250 ng/ml, 30 min) significantly increased phosphorylation of ERK. PD98059, a MEK inhibitor, significantly suppressed the canstatin-induced (250 ng/ml, 24 h) migration but not secretion of MMP-2. An increase in MMP-2 expression was observed in cytoplasm of the canstatin-treated (250 ng/ml) cardiac fibroblasts (within 30 min). Canstatin induced actin stress fiber formation, which was inhibited by Y-27632, a Rho-associated kinase inhibitor. Y-27632 also suppressed the canstatin-induced (250 ng/ml, 24 h) MMP-2 secretion. Canstatin (250 ng/ml, 30 min) failed to induce ERK phosphorylation in MMP-2 siRNA-treated cardiac fibroblasts. In conclusion, this study revealed a novel function of canstatin for inducing cell migration of adult rat cardiac fibroblasts at least in part by ERK phosphorylation through MMP-2 secretion, possibly via actin cytoskeletal change.