Noboru Murakami

Periostin promotes the generation of fibrous membranes in proliferative vitreoretinopathy

Proliferative vitreoretinopathy (PVR) is a severe, vision‐threatening disorder characterized by the fibrous membrane formation that leads to trac‐tional retinal detachment. There has been no effective therapeutic approach other than vitreoretinal surgery. In this study, DNA microarray analysis of the fibrous membranes revealed significant up‐regulation of periostin. We also found increased periostin expression in the vitreous and retinal pigment epithelial (RPE) cells from fibrous membranes of PVR patients. In vitro, periostin increased proliferation, adhesion, migration, and collagen production in RPE cells through integrin αVmediated FAK and AKT phosphorylation. Periostin blockade suppressed migration and adhesion induced by TGFβ2 and PVR vitreous. In vivo, periostin inhibition had the inhibitory effect on progression of experimental PVR in rabbit eyes without affecting the viability of retinal cells. These results identified periostin as a pivotal molecule for fibrous membrane formation as well as a promising therapeutic target for PVR.—Ishikawa, K., Yoshida, S., Nakao, S., Nakama, T., Kita, T., Asato, R., Sassa, Y., Arita, R., Miyazaki, M., Enaida, H., Oshima, Y., Murakami, N., Niiro, H., Ono, J., Matsuda, A., Goto, Y., Akashi, K., Izuhara, K., Kudo, A., Kono, T., Hafezi‐Moghadam, A., Ishibashi, T. Periostin promotes the generation of fibrous membranes in proliferative vitreoretinopathy. FASEB J. 28, 131–142 (2014). www.fasebj.org

Mice Heterozygous for the Xanthine Oxidoreductase Gene Facilitate Lipid Accumulation in Adipocytes

Objective— Xanthine oxidoreductase (XOR) catalyzes the production of uric acid with concomitant generation of reactive oxygen species. XOR has been shown to regulate adipogenesis through the control of peroxisome proliferator–activated receptor &ggr;, but its role in adipose tissue remains unclear. The aim of this study was to examine the role of XOR in adipose tissue using XOR genetically modified mice. Approach and Results— Experiments were performed using 2-, 4-, and 18-month-old XOR heterozygous mice (XOR+/−) and their wild-type littermates to evaluate the physiological role of XOR as the mice aged. Stromal vascular fraction cells were prepared from epididymal white adipose tissue in 2-month-old XOR mice to assess adipogenesis. At 18 months, XOR+/− mice had significantly higher body weight, higher systolic blood pressure, and higher incidence of insulin resistance compared with wild-type mice. At 4 months, blood glucose and the expressions of CCAAT enhancer–binding protein &bgr;, peroxisome proliferator–activated receptor &ggr;, monocyte chemoattractant protein-1, and tumor necrosis factor &agr; mRNA in epididymal white adipose tissue were significantly higher in XOR+/− than in wild-type mice. Furthermore, histological analysis of epididymal white adipose tissue in XOR+/− mice revealed that adipocyte size and the F4/80-positive macrophage count were increased. Experiments with a high-fat diet exhibited that body weight gain was also significantly higher in XOR+/− than in wild-type mice. In stromal vascular fraction cells derived from XOR+/− mice, the levels of peroxisome proliferator–activated receptor &ggr;, fatty acid–binding protein 4, and CCAAT enhancer–binding protein &agr; mRNA were upregulated, and oxidative stress levels were elevated during differentiation into adipocytes. Conclusions— These results suggest that the reduction in XOR gene expression in mice augments lipid accumulation in adipocytes, accompanied by an increase in oxidative stress, and induces obesity with insulin resistance in older age.

Downregulation of Endothelial Transient Receptor Potential Vanilloid Type 4 Channel and Small-Conductance of Ca2+-Activated K+ Channels Underpins Impaired Endothelium-Dependent Hyperpolarization in Hypertension

Endothelium-dependent hyperpolarization (EDH)–mediated responses are impaired in hypertension, but the underlying mechanisms have not yet been determined. The activation of small- and intermediate-conductance of Ca2+-activated K+ channels (SKCa and IKCa) underpins EDH-mediated responses. It was recently reported that Ca2+ influx through endothelial transient receptor potential vanilloid type 4 channel (TRPV4) is a prerequisite for the activation of SKCa/IKCa in endothelial cells in specific beds. Here, we attempted to determine whether the impairment of EDH in hypertension is attributable to the dysfunction of TRPV4 and S/IKCa, using isolated superior mesenteric arteries of 20-week-old stroke-prone spontaneously hypertensive rats (SHRSP) and age-matched Wistar–Kyoto (WKY) rats. In the WKY arteries, EDH-mediated responses were reduced by a combination of SKCa/IKCa blockers (apamin plus TRAM-34; 1-[(2-chlorophenyl)diphenylmethl]-1H-pyrazole) and by the blockade of TRPV4 with the selective antagonist RN-1734 or HC-067047. In the SHRSP arteries, EDH-mediated hyperpolarization and relaxation were significantly impaired when compared with WKY. GSK1016790A, a selective TRPV4 activator, evoked robust hyperpolarization and relaxation in WKY arteries. In contrast, in SHRSP arteries, the GSK1016790A-evoked hyperpolarization was small and relaxation was absent. Hyperpolarization and relaxation to cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine, a selective SKCa activator, were marginally decreased in SHRSP arteries compared with WKY arteries. The expression of endothelial TRPV4 and SKCa protein was significantly decreased in the SHRSP mesenteric arteries compared with those of WKY, whereas function and expression of IKCa were preserved in SHRSP arteries. These findings suggest that EDH-mediated responses are impaired in superior mesenteric arteries of SHRSP because of a reduction in both TRPV4 and SKCa input to EDH.

Effects of the Superoxide Dismutase Mimetic Tempol on Impaired Endothelium-Dependent and Endothelium-Independent Relaxations in Type II Diabetic Rats

Endothelium-derived hyperpolarizing factor (EDHF)-mediated hyperpolarization and relaxation, and endothelium-independent relaxations to the nitric oxide donor sodium nitroprusside and the adenosine 5′-triphosphate (ATP)-sensitive K+-channel opener levcromakalim were both impaired in mesenteric arteries of type II diabetic Goto–Kakizaki rats. The treatment with the superoxide dismutase mimetic tempol or its combination with the angiotensin II type 1 receptor blocker candesartan failed to improve EDHF-mediated responses, although both treatments partially improved endothelium-independent relaxations. These findings suggest that increased oxidative stress may in part account for the impaired endothelium-independent relaxations in diabetes, while it does not play a major role in the impaired EDHF-mediated responses.

Facilitation of sympathetic neurotransmission by phosphatidylinositol-4,5- bisphosphate-dependent regulation of KCNQ channels in rat mesenteric arteries

Sympathetic nerves regulate vascular tone by releasing neurotransmitters into the vasculature. We previously demonstrated that bradykinin facilitates sympathetic neurotransmission in rat mesenteric arteries. Although little is known about the intracellular mechanism modulating this neurotransmission, recent cell line experiments have shown that the KCNQ channel, which is inhibited by the depletion of membrane phosphatidylinositol-4,5-bisphosphate (PIP2), participates in the control of neurotransmission by bradykinin. In the present study, we examined the mechanism regulating neurotransmitter release from rat perivascular sympathetic nerves. Excitatory junction potentials (EJPs) elicited by repetitive nerve stimulation (1 Hz, 11 pulses, 20 μs, 20–50 V), a measure of sympathetic purinergic neurotransmission, were recorded with a conventional microelectrode technique in rat mesenteric arteries. Bradykinin (10−7 mol l−1) significantly enhanced the amplitude of EJPs (n=22, P<0.05). This enhancing effect was abolished by N-type calcium-channel inhibition with ω-conotoxin GVIA (2 × 10−9 mol l−1, n=8). The blockade of phospholipase C with U-73122 (10−6 mol l−1, n=17) also eliminated the facilitatory effect of bradykinin. In addition, the effects of bradykinin were diminished by the prevention of PIP2 resynthesis with wortmannin (10−5 mol l−1 n=7) or KCNQ channel inhibition with XE-991 (10−5 mol l−1, n=7). On the other hand, depletion of intracellular calcium stores with cyclopiazonic acid (3 × 10−6 mol l−1, n=6) or the inhibition of protein kinase C with bisindolylmaleimide-I (10−6 mol l−1, n=9) did not alter the action of bradykinin. These data demonstrate that the hydrolysis of PIP2 by phospholipase C, which is activated by Gq/11-coupled receptors, and subsequent KCNQ channel inhibition enhance sympathetic purinergic neurotransmission presumably via the activation of N-type calcium channels in rat mesenteric arteries.

Disruption of xanthine oxidoreductase gene attenuates renal ischemia reperfusion injury in mice

Aims: We examined the roles of xanthine oxidoreductase (XOR) in renal ischemia reperfusion (IR) injury. Main methods: XOR +/+ and XOR +/− mice were subjected to 24‐h reperfusion after a 45‐min bilateral renal artery occlusion or sham operation. We evaluated the renal damage based on the concentrations of blood urea nitrogen (BUN) and serum creatinine (Cr), and histological changes were detected by PAS staining. Xanthine dehydrogenase, oxidase (XO) and XOR activities, amounts of blood and urine 8‐OHdG, and expressions of TNF‐&agr; and MCP‐1 mRNA were examined. F4/80 and nitrotyrosine‐positive cells were assessed by immunohistochemical staining. Key findings: The BUN and Cr concentrations in the XOR +/+IR mice were increased significantly compared to those in XOR +/−IR and allopurinol‐treated XOR +/+IR mice. XO and XOR activity, which were increased in IR mice, were reduced in the allopurinol‐treated XOR +/+IR and XOR +/−IR mice compared to the XOR +/+IR mice. The concentrations of blood and urine 8‐OHdG, and the expressions of MCP‐1 and TNF‐&agr; mRNA were increased significantly in the XOR +/+IR mice compared to those in the XOR +/−IR mice. The histological analysis revealed that the XOR +/−IR and allopurinol‐treated XOR +/+IR mice showed less tubular injury than the XOR +/+IR mice in the cortex regions, with the reduction of inflammation and oxidative stress assessed by the immunohistological staining for F4/80 and nitrotyrosine. Significance: Both the disruption of XOR gene in XOR +/− mice and the reduction of XOR activity in allopurinol‐treated XOR +/+IR mice attenuated renal tissue injury in this IR model. Reduced XOR activity during renal IR could be a beneficial treatment target.

Low diastolic blood pressure was one of the independent predictors of ischemia-like findings of electrocardiogram in patients who underwent coronary angiography

BACKGROUND The underlying cause of a high cardiovascular event rate in the population with low diastolic blood pressure (DBP) has not been fully elucidated. METHODS AND RESULTS The relationship between DBP and ischemia-like findings on electrocardiography (ECG) was investigated in 187 patients who underwent coronary angiography. Patients with conditions affecting ECG (e.g. patients taking digitalis or those with old myocardial infarction, complete right bundle branch block, or hypokalemia) were excluded from the analyses. Ischemia-like ECG was defined as having one or more of the following: borderline Q wave [Minnesota code (MC) I 3], ST depression (MC IV 1-3), negative T wave (MC V 1-3), and complete left bundle branch block (MC VII 1). Based on this definition, 70 of 187 patients (37%) had ischemia-like ECG. Compared with the group without it, the group with ischemia-like ECG included more females (p<0.01), and had lower values of body mass index (p = 0.01), DBP (p<0.01), estimated glomerular filtration rate (p<0.01), left ventricular ejection fraction (LVEF; p<0.01), and higher values of age (p<0.01) and left ventricular mass index (LVMI; p<0.01). The severity of coronary artery disease did not differ between the groups. Receiver operating characteristics curve analysis revealed that 74.5 mmHg was the optimal cut-off point of DBP to predict ischemia-like ECG (area under curve, 0.63; 95% confidence interval, 0.55-0.71, p = 0.003). There were no significant relationships between systolic blood pressure and ischemia-like ECG. A multivariate analysis showed that female sex, low DBP (≤ 74.5 mmHg), LVMI, and LVEF were the significant factors for the ischemia-like ECG. The odds ratio of low DBP was 2.53 (95% confidence interval, 1.19-5.40; p = 0.02). CONCLUSIONS Low DBP was one of the significant predictors of the ischemia-like ECG in the present study. Myocardial ischemia may be a part of the cause of high cardiovascular morbidity in the population with low DBP.

ISH NIA PS 01-06 Downregulation of endothelial TRPV4 channel contributes to the reduced endothelium-dependent hyperpolarization in genetically hypertensive rats

Objective: Endothelium-dependent hyperpolarization (EDH)-mediated responses are decreased in hypertension. However, the underlying mechanisms have not yet been determined. Several recent studies have suggested that Ca2+ influx through endothelial transient receptor potential vanilloid 4 channel (TRPV4) plays a role in EDH-mediated hyperpolarization via the downstream activation of small- and intermediate-conductance of Ca2+-activated K+ channels (SKCa and IKCa). The present study was designed to elucidate whether the impairment of EDH-mediated responses in hypertension is attributable to the dysfunction of TRPV4 and/or KCa. Design and Method: Conventional electrophysiological and molecular biology techniques were used in isolated superior mesenteric arteries of 20-week-old stroke-prone spontaneously hypertensive rats (SHRSP) and age-matched Wistar-Kyoto (WKY) rats. Results: In the WKY mesenteric arteries, acetylcholine (ACh)-induced, EDH-mediated relaxations were reduced by a combination of KCa blockers (apamin plus TRAM-34), and by the blockade of TRPV4 with the selective antagonist RN-1734 or HC-067047. In the SHRSP mesenteric arteries, the ACh-induced, EDH-mediated hyperpolarization and relaxation were significantly smaller compared with those of WKY. GSK1016790A, a selective TRPV4 activator, evoked robust hyperpolarization and relaxation in WKY arteries. In contrast, in SHRSP arteries, the GSK1016790A-evoked hyperpolarization was small and relaxation was absent. Hyperpolarization and relaxation in response to CyPPA, a selective SKCa activator, were marginally decreased in SHRSP arteries compared with those of WKY. On the other hand, hyperpolarization and relaxation in response to 1-EBIO, a selective IKCa activator, and to levcromakalim, an ATP-sensitive K+ channel opener, were comparable between groups. The expression of endothelial TRPV4 and SKCa protein were significantly decreased in the SHRSP mesenteric arteries compared to those of WKY. Conclusions: These findings suggest that downregulation of endothelial TRPV4 predominantly underpins the reduced ACh-induced, EDH-mediated responses in mesenteric arteries of SHRSP.

310 CLINICAL CHARCTERISTICS OF RESISTANT HYPERTENSION EVALUATED BY AMBULATORY BLOOD PRESSURE MONITORING

Objectives: It has been shown that strict control of blood pressure is important to prevent cardiovascular disease, although it is sometimes difficult to decrease blood pressure to target blood pressure levels. The aim of the present study was to investigate the clinical characteristics of resistant hypertension using ambulatory blood pressure monitoring. Methods: One hundred in hospital patients, whose 24-hour average blood pressure was higher than 130/80 mmHg even after treatment with more than three antihypertensive drugs, were included in the present analysis. Circadian variation of blood pressure was evaluated by the nighttime decrease in systolic blood pressure (&Dgr;SBP); i.e. dipper (10%⩽&Dgr;SBP<20%), non-dipper (0%⩽&Dgr;SBP<10%), riser (&Dgr;SBP<0%), and extreme-dipper (&Dgr;SBP≥20%). An estimated glomerular filtration rate (eGFR) was calculated by using modified MDRD equation for Japanese. Results: Average blood pressures were both high in daytime and nighttime, 150.0/82.3 and 143.8/78.2 mmHg, respectively. Twenty patients had been treated with hemodialysis or peritoneal dialysis. In 63 patients out of the other 80 patients (79%), eGFR was also decreased (< 60 ml/min/1.73m2). The patients classified into dipper, non-dipper, riser and extreme-dipper were 20%, 43%, 34% and 3%, respectively. In addition, in 17 patients whose eGFR was preserved, 12 patients were non-dipper or riser, suggesting that it seemed difficult to account for this altered circadian blood pressure variation only by renal dysfunction. Conclusions: A large number of the patients with resistant hypertension suffered from renal dysfunction, although altered circadian blood pressure variation was difficult to be explained only by renal dysfunction.

219 HETEROZYGOUS DELETION OF XANTHINE OXIDOREDUCTASE AUGMENTS ADIPOGENESIS IN WHITE ADIPOSE TISSUE AND INDUCES OBESITY WITH INSULIN RESISTANCE

Background: Xanthine oxidoreductase (XOR) produces uric acid with concomitant generation of reactive oxygen species. In addition, it has been found that XOR is a regulator of adipogenesis and PPAR&ggr; activity. The aim of this study was to elucidate a role of XOR in adiposity using XOR disrupted mice. Methods: Body weight, blood pressure, insulin resistance and expression of mRNA and protein in epididymal adipose tissue (EAT) were investigated in 2-, 4- and 18-month-old XOR+/+ and XOR+/- mice. Primary stromal vascular fraction (SVF) was prepared from EAT of 2-month-old XOR+/+ and XOR+/- mice. Results: At 2 months, there were no significant differences between XOR+/+ and XOR+/- mice. At 4 months, blood glucose concentrations measured before and after intraperitoneal glucose infusion were siginificantly higher in XOR+/- mice. Expression of MCP-1, TNF&agr; and PPAR&ggr; mRNA in EAT of XOR+/- mice were significantly increased. Furthermore, histological analysis revealed that the size of adipocyte and the number of F4/80 positive cells were increased in EAT of XOR+/- mice. At 18 months, the body weight was significantly increased in XOR+/- mice compared with XOR+/+ mice (46.2±1.0 g vs 39.0±0.9 g, P<0.0005). Expression of PPAR&ggr;, FABP4 and C/EBP&agr; mRNA and production of hydrogen peroxide were significantly increased in SVF from XOR+/- mice during adipocyte differentiation. Conclusions: These results suggest that decreased XOR expression in white adipose tissue augments adipogenesis with increasing oxidative stress and induces insulin resistance and visceral fat deposition with aging.