Seibu Mochizuki

RETRACTED: Valsartan in a Japanese population with hypertension and other cardiovascular disease (Jikei Heart Study): a randomised, open-label, blinded endpoint morbidity-mortality study

BACKGROUND Drugs that inhibit the renin-angiotensin-aldosterone system benefit patients at risk for or with existing cardiovascular disease. However, evidence for this effect in Asian populations is scarce. We aimed to investigate whether addition of an angiotensin receptor blocker, valsartan, to conventional cardiovascular treatment was effective in Japanese patients with cardiovascular disease. METHODS We initiated a multicentre, prospective, randomised controlled trial of 3081 Japanese patients, aged 20-79 years, (mean 65 [SD 10] years) who were undergoing conventional treatment for hypertension, coronary heart disease, heart failure, or a combination of these disorders. In addition to conventional treatment, patients were assigned either to valsartan (40-160 mg per day) or to other treatment without angiotensin receptor blockers. Our primary endpoint was a composite of cardiovascular morbidity and mortality. Analysis was by intention to treat. The study was registered at clintrials.gov with the identifier NCT00133328. FINDINGS After a median follow-up of 3.1 years (range 1-3.9) the primary endpoint was recorded in fewer individuals given valsartan than in controls (92 vs 149; absolute risk 21.3 vs 34.5 per 1000 patient years; hazard ratio 0.61, 95% CI 0.47-0.79, p=0.0002). This difference was mainly attributable to fewer incidences of stroke and transient ischaemic attack (29 vs 48; 0.60, 0.38-0.95, p=0.028), angina pectoris (19 vs 53; 0.35, 0.20-0.58, p<0.0001), and heart failure (19 vs 36; 0.53, 0.31-0.94, p=0.029) in those given valsartan than in the control group. Mortality or tolerability did not differ between groups. INTERPRETATION The addition of valsartan to conventional treatment prevented more cardiovascular events than supplementary conventional treatment. These benefits cannot be entirely explained by a difference in blood pressure control.

Effect of 17β‐oestradiol on contraction, Ca2+ current and intracellular free Ca2+ in guinea‐pig isolated cardiac myocytes

1 The effect of 17β‐oestradiol on cardiac cell contraction, inward Ca2+ current and intracellular free Ca2+ ([free Ca2+]i) was investigated in guinea‐pig single, isolated ventricular myocytes. The changes of cell length were measured by use of a photodiode array, the voltage‐clamp experiments were performed with a switch clamp system and [free Ca2+]i was measured with the Ca2+ indicator, Fura‐2. 2 17β‐Oestradiol (10, 30 μm) caused a decrease in cell shortening at both 22 and 35°C. This negative inotropic effect was accompanied by a decrease in action potential duration mainly brought about by a shortening of the plateau region of the action potential. 17β‐Oestradiol (10, 30 μm) induced a similar decrease in cell shortening in voltage‐clamped and current‐clamped cells. 3 In Fura‐2 loaded cells, 17β‐oestradiol (10 and 30 μm) decreased systolic Fura‐2 fluorescence to 72 ± 7% and 47 ± 4% (n = 6, P < 0.001) of control respectively. 17β‐Oestradiol (10 μm) had no significant effect on diastolic Fura‐2 fluorescence, but at higher concentration (30 μm) induced a slight decrease in resting Fura‐2 fluorescence. The effect of 17β‐oestradiol was reversible after 1–2 min of washout of the steroid. 4 17β‐Oestradiol (10 and 30 μm) decreased the peak inward Ca2+ current (ICa), which was sensitive to [Ca2+]o, dihydropyridines and isoprenaline, to 59 ± 3% and 39 ± 5% (n = 7∼9, P < 0.01) respectively, without producing any significant change in the shape of the current‐voltage relationship. 5 The recovery time of ICa from inactivation was delayed by 17β‐oestradiol (10 μm). The inhibitory effect of 17β‐oestradiol on ICa was less at a holding potential of −80 mV than at −40mV. 6 We conclude that 17β‐oestradiol has a negative inotropic effect on guinea‐pig single ventricular myocytes by inhibiting ICa and so reducing systolic [Ca2+]i. 17β‐Oestradiol may therefore have a Ca2+ channel blocking property in guinea‐pig isolated ventricular myocytes.

Alterations in sarcoplasmic reticulum function and gene expression in ischemic-reperfused rat heart.

In view of the critical role of sarcoplasmic reticular (SR) Ca(2+) release and the Ca(2+) pump in cardiac contraction-relaxation, this study was undertaken to assess the status of SR function, protein content, and gene expression in isolated rat hearts subjected to global ischemia for 30 min followed by 60 min of reperfusion (I/R). Attenuated recovery of contractile function in the I/R hearts was associated with reduced SR Ca(2+) uptake, Ca(2+) release, and ryanodine-binding activities. mRNA levels and protein contents for SR Ca(2+) pump ATPase and Ca(2+) release channels were markedly depressed in the I/R hearts. Perfusion of hearts with superoxide dismutase plus catalase, well-known scavengers of oxyradicals, prevented the I/R-induced alterations in cardiac function and partially prevented SR Ca(2+) transport activities and mRNA abundance. In hearts perfused with xanthine plus xanthine oxidase or H(2)O(2), changes similar to those in the I/R hearts were observed. These results indicate that oxyradicals may participate in depressing the SR Ca(2+) handling and gene expression in the I/R heart. It is suggested that treatment of hearts with antioxidants may improve the recovery of cardiac function by preserving the SR function and partially protecting the SR gene expression.In view of the critical role of sarcoplasmic reticular (SR) Ca2+ release and the Ca2+ pump in cardiac contraction-relaxation, this study was undertaken to assess the status of SR function, protein content, and gene expression in isolated rat hearts subjected to global ischemia for 30 min followed by 60 min of reperfusion (I/R). Attenuated recovery of contractile function in the I/R hearts was associated with reduced SR Ca2+ uptake, Ca2+ release, and ryanodine-binding activities. mRNA levels and protein contents for SR Ca2+ pump ATPase and Ca2+ release channels were markedly depressed in the I/R hearts. Perfusion of hearts with superoxide dismutase plus catalase, well-known scavengers of oxyradicals, prevented the I/R-induced alterations in cardiac function and partially prevented SR Ca2+transport activities and mRNA abundance. In hearts perfused with xanthine plus xanthine oxidase or H2O2, changes similar to those in the I/R hearts were observed. These results indicate that oxyradicals may participate in depressing the SR Ca2+ handling and gene expression in the I/R heart. It is suggested that treatment of hearts with antioxidants may improve the recovery of cardiac function by preserving the SR function and partially protecting the SR gene expression.

Blockade of the natriuretic peptide receptor guanylyl cyclase-A inhibits NF-κB activation and alleviates myocardial ischemia/reperfusion injury

Acute myocardial infarction (AMI) remains the leading cause of death in developed countries. Although reperfusion of coronary arteries reduces mortality, it is associated with tissue injury. Endothelial P-selectin-mediated infiltration of neutrophils plays a key role in reperfusion injury. However, the mechanism of the P-selectin induction is not known. Here we show that infarct size after ischemia/reperfusion was significantly smaller in mice lacking guanylyl cyclase-A (GC-A), a natriuretic peptide receptor. The decrease was accompanied by decreases in neutrophil infiltration in coronary endothelial P-selectin expression. Pretreatment with HS-142-1, a GC-A antagonist, also decreased infarct size and P-selectin induction in wild-type mice. In cultured endothelial cells, activation of GC-A augmented H2O2-induced P-selectin expression. Furthermore, ischemia/reperfusion-induced activation of NF-kappaB, a transcription factor that is known to promote P-selectin expression, is suppressed in GC-A-deficient mice. These results suggest that inhibition of GC-A alleviates ischemia/reperfusion injury through suppression of NF-kappaB-mediated P-selectin induction. This novel, GC-A-mediated mechanism of ischemia/reperfusion injury may provide the basis for applying GC-A blockade in the clinical treatment of reperfusion injury.

Sphingosine 1-Phosphate Induces Membrane Ruffling and Increases Motility of Human Umbilical Vein Endothelial Cells via Vascular Endothelial Growth Factor Receptor and CrkII

Sphingosine 1-phosphate (S1P), a ligand for endothelial differentiation gene family proteins, is one of the most potent signal mediators released from activated platelets. Here, we report that S1P induces membrane ruffling of human umbilical vein endothelial cells (HUVECs) via the vascular endothelial growth factor receptor (VEGFR), Src family tyrosine kinase(s), and the CrkII adaptor protein. S1P induced prominent phosphorylation of CrkII in HUVECs, indicating that CrkII was involved in the S1P-induced signaling pathway. S1P-induced CrkII phosphorylation was blocked by pertussis toxin and overexpression of the carboxyl terminus of β-adrenergic receptor kinase, indicating that the βγ subunit of Gi was required for the phosphorylation. Notably, the S1P-induced CrkII phosphorylation was also abolished by inhibitors of VEGFR or Src family tyrosine kinases. By using Picchu, a real time monitoring protein for CrkII phosphorylation, we found that S1P induced rapid CrkII phosphorylation at membrane ruffles. Finally, we observed that expression of a dominant negative mutant of CrkII inhibited the S1P-induced membrane ruffling and cell migration. These results delineated a novel S1P signaling pathway that involves sequential activation of Gi-coupled receptor(s), VEGFR, Src family tyrosine kinase(s), and the CrkII adaptor protein, and which is responsible for both the induction of membrane ruffling and the increase in cell motility.

Complete Deficiency of the Low-Density Lipoprotein Receptor Is Associated With Increased Apolipoprotein B-100 Production

Objective—We addressed the role of the low-density lipoprotein (LDL) receptor in determining clearance rates and production rate (PR) of apolipoprotein B (apoB) in humans. Methods and Results—Kinetic studies using endogenous labeling of apoB with deuterated leucine were performed in 7 genetically defined patients with homozygous familial hypercholesterolemia (FH) and compared with 4 controls. The fractional catabolic rates (FCR) and PRs for apoB were determined by multicompartmental modeling. The FCRs of very-low-density lipoprotein 1 (VLDL1), VLDL2, intermediate-density lipoprotein (IDL), and LDL apoB were lower in FH than in controls, with the LDL apoB FCR being significantly lower (0.148±0.049 versus 0.499±0.099 pools · d−1; P=0.008). Whereas receptor-defective FH patients had a total apoB PR similar to controls, receptor-null FH patients had a significantly greater total apoB PR than controls (35.97±10.51 versus 21.32±4.21 mg · kg−1 · d−1, respectively; P=0.02). Conclusions—This first study of apoB metabolism in homozygous FH using endogenous labeling with stable isotopes demonstrates that the LDL receptor contributes significantly to the clearance of LDL from plasma but plays a lesser role in the clearance of larger apoB-containing lipoproteins. Furthermore, these data also indicate that absence of a LDL receptor in humans substantially influences the apoB PR in vivo.

Pioglitazone enhances cholesterol efflux from macrophages by increasing ABCA1/ABCG1 expressions via PPARγ/LXRα pathway: Findings from in vitro and ex vivo studies

OBJECTIVE Pioglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist, reportedly reduces cardiovascular events in diabetic patients. ATP cassette binding transporters (ABC) A1 and G1 are pivotal molecules for cholesterol efflux (ChE) from macrophages and high density-lipoprotein biogenesis, and the A1 transporter is regulated by a PPARγ-liver receptor X (LXR) pathway. Also, pioglitazone induces ABCG1 expression, though the exact mechanism remains unclear. We therefore investigated the effects of pioglitazone on ABCA1/G1 expression in vitro and ex vivo. METHODS The effects of pioglitazone on ChE and ABCA1/G1 expressions in macrophages were assessed. Then, mRNA was quantified in macrophages when PPARγ/LXR inhibition by siRNA or overexpression of oxysterol sulfotransferase was performed. ABCA1/G1 promoter activity with mutated LXR-responsive elements was also measured. As an ex vivo study, 15 type 2 diabetic patients were administered pioglitazone or placebo, and ChE assays and protein expressions were determined using macrophages cultured with the corresponding sera. RESULTS Pioglitazone increased LXRα/ABCA1/G1 expressions, which enhanced ChE from macrophages. Inhibition of PPARγ/LXR pathways revealed that LXR was primarily involved in pioglitazones transactivation of ABCA1 but only partially involved for ABCG1. Promoter assays showed that ABCG1 was regulated more by the promoter in intron 4 than that upstream of exon 1 but both promoters were responsive to LXR activation. Sera obtained after pioglitazone treatment promoted ChE and ABCA1/G1 expressions in macrophages. CONCLUSION Pioglitazone enhanced ChE from macrophages by increasing ABCA1/G1 in LXR-dependent and -independent manners. Our comparable in vitro and ex vivo results shed new light on pioglitazones novel anti-atherogenic property.

Delayed In Vivo Catabolism of Intermediate-Density Lipoprotein and Low-Density Lipoprotein in Hemodialysis Patients as Potential Cause of Premature Atherosclerosis

Objective—Premature cardiovascular disease is the leading cause of death in patients with end-stage renal disease treated by hemodialysis (HD). Low-density lipoprotein (LDL) levels are not generally increased in HD patients, but their LDL metabolism is still poorly understood. We therefore investigated the in vivo metabolism of apoB-containing lipoproteins in two different ethnic populations of HD patients and controls. Methods and Results—We performed stable isotope kinetic studies using a primed constant infusion of deuterated leucine in 12 HD patients and 13 healthy controls. Tracer/tracee ratio of apoB was determined by means of gas chromatography/mass spectrometry, and the modeling program SAAMII was used to estimate the fractional catabolic rate (FCR) of apoB. Mean LDL-apoB plasma concentrations were almost identical in both groups (HD: 95±30 mg/dL, controls: 91±40 mg/dL), whereas LDL-apoB FCR was 50% lower in HD patients as compared with controls (0.22±0.12 days−1 versus 0.46±0.20 days−1, P=0.001) with concomitantly decreased production rates of LDL. Compared with controls, intermediate-density lipoprotein (IDL)-apoB FCR was 65% lower (2.87±1.02 days−1 versus 8.89±4.94 days−1, P=0.014), accompanied by 1.5-fold higher IDL-apoB levels in HD. Very low-density lipoprotein metabolism was similar in both study groups. Conclusions—In vivo catabolism of LDL and IDL is severely impaired in HD patients but misleadingly masked by normal plasma cholesterol levels. The resulting markedly prolonged residence times of both IDL and LDL particles might thus significantly contribute to the well-documented high risk for premature cardiovascular disease in HD patients.

Down-regulation of sarcolipin mRNA expression in chronic atrial fibrillation

Background  Abnormal intracellular Ca2+ homeostasis is an important modulator of chronic atrial fibrillation. Sarcolipin, a homologue of phospholamban, is specifically expressed in the atria, and may play an important role in modulating intracellular Ca2+ homeostasis in the atria. The aim of this study was to investigate the expression of sarcolipin mRNA in the atrial myocardium of patients with chronic atrial fibrillation.

Abnormal HDL Apolipoprotein A-I and A-II Kinetics in Hemodialysis Patients: A Stable Isotope Study

Low levels of HDL cholesterol and its major apoprotein constituents apoA-I and apoA-II are common in patients who have ESRD and are undergoing hemodialysis (HD), but the metabolic basis for the low HDL is poorly understood. This study aimed to investigate in vivo metabolism of apoA-I and apoA-II in five normotriglyceridemic ESRD-HD patients and compared it with five control subjects using endogenous stable isotope labeling methods coupled with a multicompartmental modeling. HDL cholesterol, apoA-I, and apoA-II levels were markedly decreased in the ESRD-HD patients by 39, 30, and 44%, respectively, in comparison with the control subjects. Fractional catabolic rate of apoA-I was found to be significantly increased by 59% to 0.360 +/- 0.084/d in ESRD-HD patients as compared with control subjects of 0.227 +/- 0.076/d (P = 0.028), whereas the production rates remained unchanged. Conversely, the apoA-II production rate significantly decreased by 31% to 1.50 +/- 0.61 mg/kg per d in the ESRD-HD patients in comparison with control subjects of 2.17 +/- 0.40 mg/kg per d (P = 0.047) with apoA-II fractional catabolic rate unchanged. These results revealed that the decreased levels of apoA-I are due solely to the increased rate of catabolism, whereas the reduced apoA-II levels are due primarily to the decreased rate of production in ESRD-HD patients. This differential regulation of apoA-I and apoA-II further supports the concept that apoA-I and apoA-II have distinct metabolic pathways.