Tokumitsu Watanabe

Estrogen Prevents Oxidative Stress–Induced Endothelial Cell Apoptosis in Rats

Background —Estrogen replacement attenuates the increased risk of cardiovascular disease in postmenopausal women. Recent studies using an in vitro culture system have shown that estrogen inhibits endothelial cell (EC) apoptosis. The in vivo relevance of this finding, however, is not defined. To do so, we have developed a rat vascular injury model in which EC apoptosis induced by hydrogen peroxide plays a role. Methods and Results —Intracarotid arterial administration of 0.01 mmol/L hydrogen peroxide for 5 minutes evoked EC apoptosis after 6 to 24 hours, determined by nuclear staining with Hoechst 33342, terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling, and electron microscopy. Apoptosis was associated with EC loss and was followed by EC regeneration at 72 hours and neointima formation at 1 to 2 weeks. Estradiol replacement in ovariectomized female Wistar rats decreased the rate of apoptotic ECs by ≈50%, assayed by nuclear morphology of en face specimens, resulting in increased remaining ECs and decreased neointima formation. Progesterone did not influence the effects of estradiol on EC apoptosis. Conclusions —These results provide new insight into the cardioprotective action of estrogen as well as a paradigm of the response-to-injury hypothesis.

Dnm3os, a non‐coding RNA, is required for normal growth and skeletal development in mice

Dnm3os, a gene that is transcribed into a non‐coding RNA (ncRNA), contains three micro RNAs (miRNAs), miR‐199a, miR‐199a*, and miR‐214, whose functions remain unknown in mammals. In this study, we introduced the lacZ gene into the Dnm3os locus to recapitulate its expression pattern and disrupt its function. Dnm3os+/lacZ heterozygous embryos showed β‐galactosidase activity, which reflected the authentic expression pattern of Dnm3os RNA. Most of the Dnm3oslacZ/lacZ homozygous pups died within one month of birth. After birth, Dnm3oslacZ/lacZ mice exhibited several skeletal abnormalities, including craniofacial hypoplasia, defects in dorsal neural arches and spinous processes of the vertebrae, and osteopenia. Importantly, the expression of miR‐199a, miR‐199a*, and miR‐214 was significantly down‐regulated in Dnm3oslacZ/lacZ embryos, supporting the assumption that Dnm3os serves as a precursor of these three miRNAs. Thus, Dnm3os has emerged as an miRNA‐encoding gene that is indispensable for normal skeletal development and body growth in mammals. Developmental Dynamics 237:3738–3748, 2008.

Impact of Blood Pressure Variability on Cardiovascular Events in Elderly Patients with Hypertension

Blood pressure variability is one of the characteristic features of hypertension in the elderly. However, its clinical significance remains to be determined. We therefore examined the impact of blood pressure variability on the development of cardiovascular events in elderly hypertensive patients. A total of 106 consecutive hypertensive patients aged more than 60 years old (mean age, 73.9±8.1 years old; male, 54%), all of whom underwent 24-h ambulatory blood pressure monitoring, were followed up (median, 34 months; range, 3−60 months). During the follow-up period, 39 cardiovascular events were observed, including 14 cases of cerebral infarction and 7 cases of acute myocardial infarction. The coefficient of variation (CV) of 24-h systolic blood pressure (SBP) values was used as an index of blood pressure variability. The patients showed a mean CV value of 10.6%, and were divided into two groups according to this mean value as a cut-off point: a high CV group (n=46) and a low CV group (n=60). Although baseline clinical characteristics were similar in the two groups, Kaplan-Meier plots for event-free survival revealed that the rate of cardiovascular events was significantly higher in high CV group than in low CV group (p<0.05). Coxs proportional hazards analysis showed that increased blood pressure variability (a high CV value of 24-h SBP) was an independent predictive variable for cardiovascular events. The CV value of daytime SBP and the SD value of both 24-h SBP and daytime SBP also had positive correlations with the onset of cardiovascular events. These results suggest that increased blood pressure variability may be an independent risk factor for cardiovascular events in elderly hypertensive patients.

Estrogen receptor β mediates the inhibitory effect of estradiol on vascular smooth muscle cell proliferation

Objectives: It has been demonstrated that 17β-estradiol (E2) has an inhibitory effect on the proliferation of vascular smooth muscle cells (VSMCs) through an estrogen receptor (ER)-dependent pathway. Both ER subtypes, classical ER (ERα) and the newly identified ER subtype (ERβ), are expressed in VSMCs. However, it remains unknown which receptor plays the critical role in the inhibitory effect on VSMC proliferation. Methods and results: We constructed replication-deficient adenoviruses bearing the coding region of human ERα, ERβ, and the dominant-negative form of ERβ (designated AxCAERα, AxCAERβ, and AxCADNERβ, respectively). Prior to infection with the adenoviruses, 100 nmol/l E2 attenuated DNA synthesis by up to 14% and transactivated the estrogen-induced expression of the desired mRNA in rat VSMCs. This was accompanied by increased transcriptional activity of estrogen responsive element in response to E2, and the increase was comparable between AxCAERα and AxCAERβ. When VSMCs were infected with AxCAERβ at a multiplicity of infection of 5 or higher, DNA synthesis as well as cell number decreased by 50% in response to E2, and the effect was abolished by co-infection with AxCADNERβ. In contrast, when VSMCs were infected with AxCAERα, the reduction in DNA synthesis was minimal. Conclusions: Our results indicate that ERβ is more potent than ERα in the inhibitory effect on VSMC proliferation.

Torsades de Pointes Ventricular Tachycardia Induced by Clarithromycin and Disopyramide in the Presence of Hypokalemia

We report a 76‐year‐Old woman who developed TdP ventricular tachycardia induced by combined use of clarithromycin and disopyramide. She had a history of myocardial infarction 5 years earlier and has taken disopyramide for supraventricular arrhythmias. In addition, she had taken clarithromycin for upper respiratory tract infection. On admission, an ECG showed prolongation of QTc interval to 0.71 seconds and self‐terminating TdP occurred several times. Disopyramide was metabolized by the cytochrome enzyme CYP3A4 and clarithromycin competitively inhibits this enzyme, probably resulting in an increase in plasma concentration of disopyramide. We should consider this possibility when prescribing clarithromycin in combination with antiarrhythmic agent disopyramide.

Renin-Angiotensin System Modulates Oxidative Stress-Induced Endothelial Cell Apoptosis in Rats

The role of the renin-angiotensin system in oxidative stress–induced apoptosis of endothelial cells (ECs) was investigated using a rat model and cultured ECs. EC apoptosis was induced by 5-minute intra-arterial treatment of a rat carotid artery with 0.01 mmol/L H2O2 and was evaluated at 24 hours by chromatin staining of en face specimens with Hoechst 33342. Although activity of angiotensin-converting enzyme in arterial homogenates was not increased, administration of an angiotensin-converting enzyme inhibitor temocapril for 3 days before H2O2 treatment inhibited EC apoptosis, followed by reduced neointimal formation 2 weeks later. Also, an angiotensin II type 1 (AT1) receptor blocker (olmesartan) inhibited EC apoptosis, whereas angiotensin II administration accelerated apoptosis independently of blood pressure. Next, cultured ECs derived from a bovine carotid artery were treated with H2O2 to induce apoptosis, as evaluated by DNA fragmentation. Combination of angiotensin II and H2O2 dose-dependently increased EC apoptosis and 8-isoprostane formation, a marker of oxidative stress. Conversely, temocapril and olmesartan reduced apoptosis and 8-isoprostane formation induced by H2O2, suggesting that endogenous angiotensin II interacts with H2O2 to elevate oxidative stress levels and EC apoptosis. Neither an AT2 receptor blocker, PD123319, affected H2O2-induced apoptosis, nor a NO synthase inhibitor, NG-nitro-l-arginine methyl ester, influenced the effect of temocapril on apoptosis in cell culture experiments. These results suggest that AT1 receptor signaling augments EC apoptosis in the process of oxidative stress–induced vascular injury.

Influence of sex and age on serum nitrite/nitrate concentration in healthy subjects.

Measurement of serum nitrite/nitrate (NOx) concentrations has been considered useful to estimate nitric oxide production in humans. However little is known about the physiologic range and the factors affecting serum NOx levels. The aim of this study was, thus, to investigate the influence of sex and age on serum NOx levels in healthy subjects. We selected 263 healthy subjects (118 women and 145 men, 20-69 y) from 505 consecutive subjects who received annual medical checkups at our hospital. Serum NOx levels were determined using an analyzer employing the Griess method. The linear regression analysis showed that NOx increased significantly according to age in women (r=0.22, P<0.05), but did not in men (P=NS). Women of the younger age group (<40 y) showed significantly lower NOx levels than men of the same age group (P<0.05), whereas there was no significant difference between men and women of the older age (> or =40 y). Then, to investigate whether menopause affects serum NOx levels, middle aged women (46-55 y) were selected. Multiple logistic regression analysis showed that menopause was an independent factor for increased serum NOx levels in middle aged women (r=0.4, P<0.05). These results suggest that the serum NOx concentration is affected by age in healthy women, possibly depending on menopausal state.

Prostanoids regulate proliferation of vascular smooth muscle cells induced by arginine vasopressin

The aim of the present study was to investigate the effect of arginine [Arg(8)]vasopressin (vasopressin) on proliferation of vascular smooth muscle cells and the mechanisms underlying the action of vasopressin. To clarify these issues, we used two different types of vascular smooth muscle cells, cultured adult rat aortic smooth muscle cells and A10 cells, a cell line derived from fetal rat aorta. Vasopressin (10(-8) to 10(-6) M) significantly stimulated the proliferation of rat aortic smooth muscle cells in a dose-dependent manner. In contrast, vasopressin significantly inhibited the proliferation of A10 cells. This inhibition was abolished when A10 cells were treated with indomethacin. Vasopressin stimulated the production of prostanoids several-fold in A10 cells but not in rat aortic smooth muscle cells. These effects were completely blocked by the vasopressin V(1) receptor antagonist, 1-¿1-[4-(3-acetylamino-propoxy)benzoyl]4-piperidyl¿-3, 4-dihydro-2(1H)-quinolinone (OPC21268), but not by the vasopressin V(2) receptor antagonist, (+/-)-5-dimethylamino-1-[4-(2-methylbenzoylamino)benzol]-2, 3,4,5-tetrahydro-1H-benzazepine hydrochloride (OPC31260). These results indicate that vasopressin has diverse effect on proliferation of vascular smooth muscle cells through the vasopressin V(1) receptor, depending on the production of growth regulatory prostanoids.

A case of severe hypoglycemia due to pimobendan

Several drugs are reported to cause severe hypo142 mmol / l, potassium 4.6 mmol / l, creatinine 1.6 glycemia. We report here a case of severe hypomg/dl (141 mmol / l), calcium 9.3 mg/dl (2.3 mmol / glycemia during treatment of heart failure, which was l). Plasma glucose was only 9 mg/dl (0.49 mmol / l). presumably caused by a novel inotropic agent, His consciousness became clear soon after intravenpimobendan. ous infusion of glucose. He had no episode of A 74-year-old man had been diagnosed as having hypoglycemia after pimobendan was discontinued. severe congestive heart failure due to aortic insufSevere hypoglycemia due to medications has been ficiency with severe left ventricular dysfunction. The reported. The causative agents include cardiovascular chest X-ray showed mild pulmonary congestion with drugs, such as a-blockers, b-blockers, disopyramide, a cardio-thoracic ratio of 71%. The echocardiography angiotensin converting enzyme (ACE) inhibitors [1]. demonstrated severe aortic regurgitation and severely Pimobendan belongs to a novel class of non-digitalis impaired left ventricular function (LVDd 71 mm, oral inotropic drug and is clinically effective for the LVDs 60 mm, ejection fraction 33%). He was not treatment of heart failure [2]. The agent inhibits diabetic. He was receiving 2.5 mg enalapril, 30 mg mainly phosphodiesterase III activity and elevates 21 furosemide, and 0.125 mg digoxin. After the worsenintracellular cAMP concentration. It also has a Ca ing of heart failure, pimobendan 2.5 mg daily was sensitizing effect, increasing myocardial contractility initiated and increased to a dose of 5 mg 4 weeks by generating more force per given amount of 21 21 later. No other medication was added. His symptoms cytoplasmic Ca . Intracellular Ca plays an imof heart failure were significantly improved after the portant role as a key second messenger in various initiation of pimobendan. organs including the heart and the pancreas. 21 Five weeks after pimobendan was increased to a Pimobendan, as a Ca sensitizer, not only acts on dose of 5 mg, he was urgently transferred to our cardiac myocytes but also acts on pancreatic b-cells. hospital. He was totally unconscious, blood pressure It is reported to enhance insulin release from pan21 was 126/80 mmHg and pulse rate was 88 beats /min. creatic b-cells by directly sensitizing the Ca -sensiAdmission laboratory tests were as follows: sodium tive exocytotic mechanism [3]. Enalapril causes severe hypoglycemia in diabetic patients treated with sulfonylurea, presumably via improving insulin sen*Corresponding author. Tel.: 181-3-5800-8652; fax: 181-3-5800sitivity [4]. Enalapril alone, however, is not reported 6530. E-mail address: ako-ger@h.u-tokyo.ac.jp (J. Ako). to cause severe hypoglycemia in non diabetic pa-

Senile cardiac amyloidosis

A case of senile cardiac amyloidosis with recurrent episodes of congestive heart failure is reported here. An 83‐year‐old woman with a history of hypertension was admitted for the treatment of worsening dyspnea. The echocardiography revealed diffuse mild hypokinesis of the left ventricle with an ejection fraction of 46%. An elevated E wave and shortened deceleration time in the left ventricular inflow waveform was demonstrated with Doppler echocardiography, which suggested that the nature of the heart failure was restrictive. The patient required hospitalization for the treatment of congestive heart failure four times in 2 years. An autopsy revealed deposition of prealbumin‐positive amyloid substance in the atrial myocardium and the intramyocardial arterioles of the left ventricle. Recurrent congestive heart failure was presumed to be a result of diastolic dysfunction due to senile cardiac amyloidosis.