Satoko Sasaki

Effect of Shear Stress on Asymmetric Dimethylarginine Release From Vascular Endothelial Cells

Abstract—We demonstrated recently that plasma concentrations of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase, are increased by high salt intake concomitantly with a decrease in plasma levels of NO in human hypertension. We investigated the effect of shear stress on ADMA release in 2 types of cells: transformed human umbilical vein endothelial cells (HUVECs; cell line ECV-304) and HUVECs. Exposure of ECV-304 cells and HUVECs to shear stress with the use of a cone-plate viscometer enhanced gene expression of protein arginine methyltransferase (PRMT-1), ADMA synthase. In HUVECs, the ratio of PRMT-1 to glyceraldehyde 3-phosphate dehydrogenase mRNA was increased by 2-fold by a shear stress of ≥15 dyne/cm2. A dominant-negative mutant of I&kgr;B kinase &agr; and troglitazone at 8 &mgr;mol/L, an activator of peroxisome proliferator–activated receptor &ggr;, abolished the shear stress–induced increase in PRMT-1 gene expression in parallel with the blockade of nuclear factor (NF)-&kgr;B translocation into the nucleus. The activity of dimethylarginine dimethylaminohydrolase, the degradation enzyme of ADMA, was unchanged after shear stress ≤15 dyne/cm2 and was enhanced by 1.48±0.06-fold (P <0.05) by shear stress at 25 dyne/cm2. The release of ADMA was increased by 1.64±0.10-fold (P <0.05) by shear stress at 15 dyne/cm2 but was not affected by shear stress at 25 dyne/cm2. These results indicate that shear stress enhances gene expression of PRMT-1 and ADMA release via activation of the NF-&kgr;B pathway. Shear stress at higher magnitudes facilitates the degradation of ADMA, thus returning ADMA release levels to baseline.

Characteristics of Intracerebral Hemorrhage During Rivaroxaban Treatment Comparison With Those During Warfarin

Background and Purpose— Neuroradiological characteristics and functional outcomes of patients with intracerebral hemorrhage (ICH) during novel oral anticoagulant treatment were not well defined. We examined these in comparison with those during warfarin treatment. Methods— The consecutive 585 patients with ICH admitted from April 2011 through October 2013 were retrospectively studied. Of all, 5 patients (1%) had ICH during rivaroxaban treatment, 56 (10%) during warfarin, and the other 524 (89%) during no anticoagulants. We focused on ICH during rivaroxaban and warfarin treatments and compared the clinical characteristics, neuroradiological findings, and functional outcomes. Results— Patients in the rivaroxaban group were all at high risk for major bleeding with hypertension, abnormal renal/liver function, stroke, bleeding history or predisposition, labile international normalized ratio, elderly, drugs/alcohol concomitantly (HAS-BLED) score of 3 and higher rate of past history of ICH. Moreover, multiple cerebral microbleeds (≥4) were detected more frequently in rivaroxaban group than in warfarin (80% versus 29%; P=0.04). Hematoma volume in rivaroxaban group was markedly smaller than that in warfarin (median: 4 versus 11 mL; P=0.03). No patient in the rivaroxaban group had expansion of hematoma and surgical treatment. Rivaroxaban group showed lower modified Rankin Scale at discharge relative to warfarin, and the difference between modified Rankin Scale before admission and at discharge was smaller in rivaroxaban than in warfarin (median: 1 versus 3; P=0.047). No patient in the rivaroxaban group died during hospitalization, whereas 10 (18%) warfarin patients died. Conclusions— Rivaroxaban-associated ICH occurs in patients at high risk for major bleeding. However, they had a relatively small hematoma, no expansion of hematoma, and favorable functional and vital outcomes compared with warfarin-associated ICH.

Intracellular Signaling for Vasoconstrictor Coupling Factor 6. Novel Function of β-Subunit of ATP Synthase as Receptor

Coupling factor 6 (CF6), a component of adenosine triphosphate (ATP) synthase, is circulating and functions as an endogenous vasoconstrictor by inhibiting cytosolic phospholipase A2. We showed a high plasma level of CF6 in human hypertension. The present study focused on the identification and characterization of a receptor for CF6 and its post-receptor signaling pathway. Incubation of human umbilical vein endothelial cells (HUVECs) with an excess of free CF6 reduced by 50% the immunoreactivity for the antibody to &bgr;-subunit of ATP synthase at the cell surface, but unaffected that for the &agr;-subunit antibody. A significant displacement of radioligand was observed at 3×10−9 through 10−7 M unlabeled CF6, and the Kd was 7.6 nM. Adenosine diphosphate (ADP) at 10−7 M and &bgr;-subunit antibody suppressed the binding of 125I-CF6 by 81.3±9.7% and 32.0±2.0%, respectively, whereas the &agr;-subunit antibody unaffected it. The hydrolysis activity of ATP to ADP was increased by 1.6-fold by CF6 at 10−7 M, and efrapeptin at 10−5 M, an inhibitor of ATP synthase, blocked it. CF6 at 10−7 M decreased intracellular pH in 2′,7′-bis(carboxyethyl-5 (6))-carboxyfluorescein-loaded HUVEC. Amyloride at 10−4 M augmented the pH decrease in response to CF6, whereas efrapeptin at 10−5 M blocked it. Arachidonic acid release was suppressed by CF6, and it was reversed by efrapeptin at 10−5 M or &bgr;-subunit antibody or ADP at 10−7 M. The &bgr;-subunit antibody suppressed coupling factor 6–induced increase in blood pressure. These indicate that membrane-bound ATP synthase functions as a receptor for CF6 and may have a previously unsuspected role in the genesis of hypertension by modulating the concentration of intracellular hydrogen.

Relationship between Salt Intake, Nitric Oxide and Asymmetric Dimethylarginine and Its Relevance to Patients with End-Stage Renal Disease

Patients with essential hypertension (n = 24) were administered a low-salt diet (2 g NaCl/day), a high-salt diet (20–23 g) and then a low-salt diet for 7 days, and plasma levels of nitrate and nitrite (NOx) and asymmetric dimethylarginine (ADMA) were examined. There was a negative correlation between the percent changes in mean blood pressure and the plasma NOx concentration after salt loading and restriction. The percent change in plasma ADMA concentration was negatively correlated with that in the plasma NOx concentration after salt loading and restriction. In patients with end-stage renal disease (n = 51), the plasma ADMA concentration was positively correlated with the duration of dialysis treatment. The frequency of cardiovascular events was greater in patients with a plasma ADMA level of ≥3 µM than in those with a plasma AMDA level of <3 µM. The results indicate that ADMA is not only a modulator of salt sensitivity in hypertension but also a cardiovascular risk factor in end-stage renal disease.

Effect of vasoconstrictor coupling factor 6 on gene expression profile in human vascular endothelial cells: enhanced release of asymmetric dimethylarginine.

Background Coupling factor 6 (CF6), a component of ATP synthase, inhibits phospholipase A2 and induces vasoconstriction. However, because arachidonic acid acts in the widespread fields of vascular biology, CF6 might exert profound effects in addition to vasoconstriction. We investigated the effect of CF6 on the gene expression profile in human umbilical vein endothelial cells. Methods and results The increased gene expression after 24-h exposure to CF6 at 10−7 mol/l, assessed by cDNA microarray (n = 3), included neuregulin-1 (1.84 ± 0.07 fold compared with control, P < 0.05) and relaxin-1 (1.74 ± 0.20, P < 0.05), both relating to congestive heart failure, urokinase type plasminogen activator receptor (1.77 ± 0.24, P = 0.06) and estrogen receptor β (1.74 ± 0.36, P = 0.08), both relating to vascular inflammation and cell infiltration, and protein arginine methyltransferase (PRMT-1; 1.73 ± 0.20, P < 0.05). Out of these genes, the enzyme relating to the synthesis (PRMT-1) of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), was further examined concomitantly with the degradation enzyme, dimethylarginine dimethylaminohydrolase 2 (DDAH-2). The ratio of PRMT-1 to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA, measured by real-time quantitative reverse transcription-polymerase chain reaction, was increased by 9 ± 2% (n = 10, P < 0.01) at 48 h after CF6 at 10−7 mol/l, whereas the ratio of DDAH-2 to GAPDH was decreased by 12 ± 2% (n = 8, P < 0.01). DDAH-2 protein and activity were decreased by 28 ± 5% (n = 5, P < 0.01) and 19 ± 2% (n = 6, P < 0.01) by CF6, respectively. ADMA release was enhanced by 20 ± 8% and NOS activity was decreased by 13 ± 1% (both n = 8, P < 0.05) by CF6. Conclusions CF6 changes the gene expression profile to be proatherogenic and functions as a novel stimulator for ADMA release by enhancing its synthesis and suppressing its degradation.

Impact of Sex Difference on Severity and Functional Outcome in Patients with Cardioembolic Stroke.

INTRODUCTION Female sex is a risk factor for thromboembolic events in Caucasian, but not in Japanese, patients with nonvalvular atrial fibrillation. However, it remains unclear whether the female sex is also a risk factor for severe stroke and unfavorable functional outcome in patients with cardioembolic (CE) stroke. METHODS Three hundred fifty-five consecutive patients with CE stroke within 48 hours after onset and with a modified Rankin Scale (mRS) score of 1 or lower before onset were studied. We compared basic characteristics, stroke severity, and functional outcome between female (n = 157) and male (n = 198) patients. RESULTS The mean age was higher in female than in male patients (80 ± 8 versus 75 ± 9 years, P < .00001). The congestive heart failure, hypertension, age [≥ 75 years], diabetes, stroke/transient ischemic attack [TIA] (CHADS2) score before onset was similar between the two groups (median, 3 [2-4] in both groups). Stroke severity on admission, assessed by the National Institutes of Health Stroke Scale (NIHSS), was higher in female than in male patients (13 [5-20] versus 8 [3-16], P = .0009). Functional outcome at discharge, assessed by mRS, was unfavorable in female than in male patients (3 [1-5] versus 2 [1-4], P = .005). An mRS score of 3 or higher at discharge was found more in female than in male patients (59% versus 39%, P = .0001). Multivariate analyses confirmed that female sex was a significant determinant of severe stroke (NIHSS ≥ 8) on admission (odds ratio [OR] to male = 1.97; 95% confidence interval [CI]; 1.24-3.15, P = .004) and for the mRS score of 3 or higher at discharge (OR = 1.83; 95% CI, 1.16-2.89; P = .01). Similar results were obtained by propensity-score matching analysis. CONCLUSIONS Female sex is a risk factor for severe stroke on admission and unfavorable functional outcome at discharge in Japanese patients with CE stroke.

Novel pro-atherogenic molecule coupling factor 6 is elevated in patients with stroke: a possible linkage to homocysteine.

Abstract Background and purpose. Homocysteine (Hcy) is an independent predictor of stroke. Coupling factor 6 (CF6) is regulated by nuclear factor kappa B (NF-κB) signaling which is activated by Hcy. We tested the hypothesis that CF6 is elevated with Hcy in stroke. We also examined the effect of vitamin treatment on CF6 and Hcy levels. Methods and results. The 59 Japanese patients with a recent history of stroke were randomly assigned to a group without vitamin treatment (Group 1, n = 29) and to a group with treatment with both folic acid and vitamin B12 for 2 months (Group 2, n = 30). The CF6 level was elevated in the patients with stroke compared with that in controls (n = 64) at admission. In a multiple regression model, the plasma CF6 level was weakly correlated to the total Hcy (tHcy) level. In Group 1, the plasma tHcy and CF6 levels were unchanged. In Group 2, however, they were both decreased, and there was a weak positive correlation between the decreases in plasma levels of CF6 and tHcy. Conclusion. CF6 is elevated in patients with stroke independently of risk factors. Since Hcy and vitamin treatment affect CF6 levels in stroke, CF6 appears to be a novel molecule for the pathogenesis and treatment of stroke.

p122 Protein Enhances Intracellular Calcium Increase to Acetylcholine: Its Possible Role in the Pathogenesis of Coronary Spastic Angina

Objective—Phospholipase C-&dgr;1 activity is enhanced in patients with coronary artery spasm, and a p122 protein was recently cloned to potentiate phospholipase C-&dgr;1 activity. To investigate the role of p122 in enhanced vasomotility, we examined p122 expression in the cultured skin fibroblasts obtained from patients with and without coronary spasm, intracellular Ca2+ concentration ([Ca2+]i) at baseline and after stimulation with acetylcholine in the cells transfected with p122, and promoter in genomic DNA. Methods and Results—p122 protein and gene expression levels in patients with coronary spasm (n=11) were enhanced compared with levels in control subjects (n=9) (P<0.01 for both). [Ca2+]i at baseline and the peak increase in [Ca2+]i in response to acetylcholine were both 2 times higher in cells transfected with p122 than in those without p122. Conversely, knockdown of p122 resulted in diminished [Ca2+]i response. In the p122 promoter analysis, the −228G/A and −1466C/T variants revealed the increase in luciferase activity. Although the −1466C/T variant was similar between 144 patients with coronary spasm and 148 controls, the −228G/A variant was more frequent in male patients than in male controls (P<0.05). Conclusion—The p122 protein is upregulated in patients with coronary spasm, causing increased [Ca2+]i to acetylcholine, and thereby seems to be related to enhanced coronary vasomotility.

Impact of Atrial Natriuretic Peptide Value for Predicting Paroxysmal Atrial Fibrillation in Ischemic Stroke Patients.

INTRODUCTION The impact of atrial natriuretic peptide (ANP) value for predicting paroxysmal atrial fibrillation (pAF) in ischemic stroke patients remains uncertain. METHODS The consecutive 222 ischemic stroke patients (median 77 [IQR 68-83] years old, 93 females) within 48 hours after onset were retrospectively studied. Plasma ANP and brain natriuretic peptide (BNP) levels were simultaneously measured at admission. Of all, 158 patients had no evidence of atrial fibrillation (AF) (sinus rhythm [SR] group), 25 patients had pAF (pAF group), and the other 39 patients had chronic AF (cAF group). We investigated predicting factors for pAF, with focus on ANP, BNP, and ANP/BNP ratio. RESULTS ANP value was significantly higher in the pAF than in the SR group (97 [50-157] mg/dL versus 42 [26-72] mg/dL, P < .05) and further increased in the cAF group (228 [120-392], P < .05 versus pAF and SR groups). Similarly, the BNP value was higher in the pAF than in the SR group (116 [70-238] mg/dL versus 34 [14-72] mg/dL, P < .05) and further increased in the cAF group (269 [199-423], P < .05 versus pAF and SR groups). ANP/BNP ratio was lower in the pAF and cAF groups than in the SR group (.6 [.5-1.2] and .7 [.5-1.0] versus 1.3 [.8-2.4], both P < .05]. Multivariate analysis in the SR and pAF groups (n = 183) demonstrated that age, congestive heart failure, ANP, and BNP, but not ANP/BNP ratio, were independent predictors for detecting pAF. Receiver operating characteristic curve analysis further showed that area under the curve was similar between ANP and BNP (.76 and .80). CONCLUSIONS ANPmay be clinically useful for detecting pAF in ischemic stroke patients as well as BNP.

Effect of rivaroxaban on prothrombin fragment 1+2 compared with warfarin in patients with acute cardioembolic stroke: Insight from its serial measurement.

INTRODUCTION Patients with intracerebral hemorrhage during rivaroxaban treatment have small hematoma and favorable outcomes compared with those with warfarin. We investigated its possible mechanism, focusing on prothrombin fragment 1+2 (F1+2), a marker of thrombin generation. MATERIALS AND METHODS In 65 patients with acute cardioembolic stroke (median 77years), rivaroxaban was initiated at 5days after the onset. Plasma F1+2 level (normal range, 69-229pmol/L), prothrombin time (PT), and rivaroxaban concentration evaluated by anti-Xa activity were serially measured. RESULTS Median plasma F1+2 was 276 (IQR, 195-454) pmol/L before starting rivaroxaban, and significantly decreased to 196 (141-267) and 192 (151-248) on 7 and 28days after rivaroxaban, respectively (both p<0.05). Serial measurements of PT and rivaroxaban concentration at trough, 2, 4, and 6h after taking rivaroxaban showed a positive correlation (R2=0.69, p<0.01). PT at 4h after rivaroxaban was significantly prolonged compared with trough (16.6 versus 11.5s, p<0.0001). F1+2 at 4h was also decreased compared with trough (160 (123-245.5) versus 196 (141-266.5), p=0.04), but no patients showed F1+2 below the normal range at 4h. In other 34 patients with warfarin treatment (77years), median PT-INR and F1+2 were 2.06 (1.75-2.50) and 75 (48-111) (p<0.0001 versus 4h after rivaroxaban). Notably, of those with PT-INR≥2.0 (18/34), 12 (12/18, 67%) showed F1+2 below the normal range. CONCLUSIONS Rivaroxaban retains a normal thrombin generation even at its peak level with prolonged PT, whereas warfarin at therapeutic levels inhibits thrombin generation. This may partly explain different outcomes in patients complicated with bleeding events.