Tomoka Morita

HDAC4 mediates development of hypertension via vascular inflammation in spontaneous hypertensive rats

Histone deacetylases (HDACs) are transcriptional corepressors. Our recent study demonstrated that HDAC4 protein specifically increases in mesenteric artery from spontaneous hypertensive rats (SHR) compared with Wistar Kyoto rats (WKY). Vascular inflammation is important for pathogenesis of hypertension. We examined whether HDAC4 affects vascular inflammatory responses and promotes hypertension. In vivo, blood pressure, reactive oxygen species (ROS) production, and VCAM-1 expression in isolated mesenteric artery were elevated in young SHR (7 wk old) compared with age-matched WKY, which were prevented by long-term treatment of SHR with an HDACs inhibitor, trichostatin A (TSA; 500 μg·kg(-1)·day(-1) for 3 wk). In isolated mesenteric artery, the increased angiotensin II-induced contraction in SHR was reversed by TSA. The endothelium-dependent relaxation induced by ACh in SHR was augmented by TSA. In cultured rat mesenteric arterial smooth muscle cells (SMCs), expression of HDAC4 mRNA and protein was increased by TNF-α (10 ng/ml). TSA (10 μM, pretreatment for 30 min) inhibited VCAM-1 expression and NF-κB phosphorylation induced by TNF (10 ng/ml, 24 h or 20 min) in SMCs. HDAC4 small interfering RNA inhibited TNF-induced monocyte adhesion, VCAM-1 expression, transcriptional activity of NF-κB, and ROS production in SMCs. The present results demonstrated that proinflammatory effects of HDACs may mediate the further development of hypertension in SHR. It is also suggested in cultured vascular SMCs that TNF-induced HDAC4 mediates vascular inflammation likely via VCAM-1 induction through ROS-dependent NF-κB activation.

Histone Deacetylase 4 Controls Neointimal Hyperplasia via Stimulating Proliferation and Migration of Vascular Smooth Muscle Cells

Histone deacetylases (HDACs) are transcriptional coregulators. Recently, we demonstrated that HDAC4, one of class IIa family members, promotes reactive oxygen species–dependent vascular smooth muscle inflammation and mediates development of hypertension in spontaneously hypertensive rats. Pathogenesis of hypertension is, in part, modulated by vascular structural remodeling via proliferation and migration of vascular smooth muscle cells (SMCs). Thus, we examined whether HDAC4 controls SMC proliferation and migration. In rat mesenteric arterial SMCs, small interfering RNA against HDAC4 inhibited platelet-derived growth factor (PDGF)-BB–induced SMC proliferation as determined by a cell counting and bromodeoxyuridine incorporation assay as well as migration as determined by Boyden chamber assay. Expression and activity of HDAC4 were increased by PDGF-BB. HDAC4 small interfering RNA inhibited phosphorylation of p38 mitogen–activated protein kinase and heat shock protein 27 and expression of cyclin D1 as measured by Western blotting. HDAC4 small interfering RNA also inhibited PDGF-BB–induced reactive oxygen species production as measured fluorometrically using 2′, 7′-dichlorofluorescein diacetate and nicotinamide adenine dinucleotide phosphate oxidase activity as measured by lucigenin assay. A Ca2+/calmodulin-dependent protein kinase II inhibitor, KN93, inhibited PDGF-BB–induced SMC proliferation and migration as well as phosphorylation of HDAC4. In vivo, a class IIa HDACs inhibitor, MC1568 prevented neointimal hyperplasia in mice carotid ligation model. MC1568 also prevented increased activation of HDAC4 in the neointimal lesions. The present results for the first time demonstrate that HDAC4 controls PDGF-BB–induced SMC proliferation and migration through activation of p38 mitogen–activated protein kinase/heat shock protein 27 signals via reactive oxygen species generation in a Ca2+/calmodulin-dependent protein kinase-dependent manner, which may lead to the neointimal hyperplasia in vivo.

A novel adipocytokine, nesfatin-1 modulates peripheral arterial contractility and blood pressure in rats

Nesfatin-1 is a novel adipocytokine which exerts not only anorexigenic but also hypertensive roles through acting on hypothalamus melanocortin-3/4 receptors. Although it is logical to hypothesize that nesfatin-1 could also affect the contractile reactivity of peripheral blood vessels, it still remains to be examined. The present study was performed to test the hypothesis. In both endothelium-intact and -denuded mesenteric artery of rats, acute treatment with nesfatin-1 (10nM, 30min pretreatment) had no influence on the noradrenaline- and 5-hydroxytryptamine-induced concentration-dependent contractions. Chronic treatment of mesenteric artery with nesfatin-1 (10nM, 3days) using organ-culture method had also no influence on the agonists-induced contractions. In contrast, nesfatin-1 (10nM, 30min) significantly inhibited the sodium nitroprusside (SNP)-induced relaxations of smooth muscle in mesenteric artery. A membrane permeable cyclic GMP (cGMP) analog, 8-bromo-cGMP-induced relaxations were not affected by nesfatin-1. Consistently, the SNP-induced cGMP production in smooth muscle was impaired by nesfatin-1. Intravenous application of nesfatin-1 to rats not only increased blood pressure but also impaired the SNP-induced decreases in blood pressure. The present study for the first time reveals that nesfatin-1 affects peripheral arterial blood vessel and inhibits the nitric oxide donor-induced smooth muscle relaxations via impairing the cGMP production. The results are the first to demonstrate that nesfatin-1 modulates blood pressure through directly acting on peripheral arterial resistance.

Mechanisms underlying impairment of endothelium-dependent relaxation by fetal bovine serum in organ-cultured rat mesenteric artery

Organ culture of blood vessels provides a useful technique to investigate long-term effects of drugs because tissue architecture and function are well preserved. Various growth factors are responsible for structural and functional changes during vascular diseases. We investigated long-term effects of fetal bovine serum (FBS) which contains such factors on endothelium-dependent relaxation using organ-culture method. Rat isolated mesenteric arteries with endothelium were cultured for 3 days without or with 10% FBS (FBS). Acetylcholine- and bradykinin-induced endothelium-dependent relaxations were significantly impaired in FBS, whereas sodium nitroprusside-induced relaxation of endothelium-removed artery was unchanged. Morphological examination revealed that endothelium was intact in FBS. Acetylcholine-induced nitric oxide (NO) release as detected by 4, 5-diaminofluorescein significantly decreased in FBS, whereas endothelial NO synthase expression was unchanged. A Ca(2+) ionophore, A23187-induced relaxation was unchanged in FBS. A phospholipase C activator, m-3M3FBS-induced relaxation of FBS was unchanged in either Ca(2+)-containing or -free solution. Total expressions of transient receptor potential canonical channels (TRPCs: TRPC-1, -4, -5) were similar in FBS. These data suggest that FBS impairs endothelium-dependent relaxation by inhibiting events upstream of phospholipase C activation including phospholipase C, G-protein, and receptors in endothelium.

Long-term methylglyoxal treatment impairs smooth muscle contractility in organ-cultured rat mesenteric artery

Methylglyoxal (MGO), a metabolite of glucose accumulates in vascular tissues of hypertensive rats. We recently showed that short-term (30min) treatment with MGO inhibits noradrenaline (NA)-induced smooth muscle contraction in rat aorta and mesenteric artery. In the present study, long-term effect of MGO was examined using organ culture method. The contractility, morphology, and protein expression of rat mesenteric artery after organ culture with MGO for 3 days were examined. MGO (4 and 42μM) inhibited NA (0.1nM to 3μM) or KCl (72.7mM)-induced contraction. The inhibitory effect was higher in endothelium-denuded than endothelium-intact artery. An anti-oxidant drug, N-acetyl-l-cysteine (NAC; 1mM) or an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), gp91ds-tat (0.1μM) prevented the inhibitory effect of MGO. MGO increased superoxide production as detected by lucigenin assay. In the medial layer of the arteries cultured with MGO, apoptotic morphological change was observed, and NAC or gp91ds-tat prevented it. MGO significantly increased expression of a homolog of gp91(phox), NOX1 but not gp91(phox) as determined by Western blotting. An NF-κB inhibitor, pyrrolidine dithiocarbamate prevented the MGO-induced NOX1 expression. MGO had no effect on protein expression of p22(phox), p67(phox), p47(phox), as well as superoxide dismutase (SOD)-1, SOD-2 and SOD-3. Present results indicate that long-term MGO treatment has an inhibitory effect on contractility of isolated blood vessel, which is likely mediated via increased NOX1-derived superoxide production and subsequent apoptosis.

Visceral adipose tissue-derived serine protease inhibitor augments acetylcholine-induced relaxation via the inhibition of acetylcholine esterase activity in rat isolated mesenteric artery

Visceral adipose tissue‐derived serine protease inhibitor (vaspin) is an adipocytokine with insulin‐sensitizing activity originally identified in visceral adipose tissues of obesity‐related type II diabetic rats. We previously showed that vaspin inhibits vascular cell migration and apoptosis as well as inflammatory responses, which are crucial for the development of hypertension. However, little is known about the effects of vaspin on vascular reactivity. The aim of this study was thus to explore the effects of vaspin on contraction and relaxation of isolated blood vessel.

Mechanisms Underlying a Decrease in KCl-Induced Contraction after Long-Term Serum-Free Organ Culture of Rat Isolated Mesenteric Artery

ABSTRACT Organ culture of blood vessel is a better technique to investigate the long-term effects of drugs. However, some functional changes may occur from freshly isolated vessel (Fresh). Mammalian/mechanistic target of rapamycin (mTOR) regulates smooth muscle differentiation and Ca2+ mobilization. We thus investigated mechanisms of alteration in smooth muscle contractility after serum-free organ culture focusing on mTOR. Rat isolated mesenteric arteries were cultured for 5 days without (0% serum) or with rapamycin. In 0% serum, absolute contraction by KCl significantly decreased from Fresh, which was significantly rescued by rapamycin. In 0% serum, mTOR expression significantly increased from Fresh, which was significantly rescued by rapamycin. In 0% serum, expression of myocardin, a key regulator of smooth muscle differentiation markers, significantly decreased from Fresh, which was significantly rescued by rapamycin. However, the decrease in expression of contractile proteins, including SM22α and calponin, was not changed by rapamycin. Basal phosphorylation of calmodulin-dependent protein kinase II significantly increased in 0% serum, which was significantly rescued by rapamycin. In 0% serum, absolute contraction by caffeine significantly decreased from Fresh, which was significantly rescued by rapamycin. In conclusion, expression of mTOR increased during serum-free organ culture of rat isolated mesenteric artery for 5 days, which may be at least partly responsible for the decreased smooth muscle contractility perhaps due to the decrease in the stored Ca2+ in smooth muscle.