Hiroaki Obata

Nonviral delivery of siRNA into mesenchymal stem cells by a combination of ultrasound and microbubbles

Cell therapy is a promising therapeutic strategy for regenerative medicine. However, its current efficacy is insufficient, because of the short lifetime and low engraftment of transplanted cells. Transplantation of genetically modified stem cells has been reported to improve the efficacy of cell therapy. The aim of this study was to elucidate the feasibility of a combination of ultrasound and microbubbles (US-MB) for delivery of small interfering RNA (siRNA) into mesenchymal stem cells (MSC). Although cell damage was observed after US-MB treatment, the transfection efficiency determined using fluorescent-labeled siRNA was significantly increased after US-MB. Furthermore, the intracellular delivery of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) siRNA by US-MB resulted in significant knockdown of PTEN mRNA expression and activation of Akt, a mediator of a survival signaling pathway. Our results indicate that US-MB could serve as a nonviral delivery method of siRNA into MSC. The transplantation of genetically modified MSC by US-MB could be a useful strategy for regenerative medicine in the future.

Transplantation of mesenchymal stem cells improves atrioventricular conduction in a rat model of complete atrioventricular block.

Mesenchymal stem cells (MSCs) are multipotent cells that differentiate into a variety of lineages including myocytes and vascular endothelial cells. However, little information is available regarding the therapeutic potential of MSCs in patients with atrioventricular block (AVB). We investigated whether local implantation of MSCs improves AV conduction in a rat model of complete AVB. Complete AVB was achieved by injection of ethanol into the AV nodal region of Lewis rats. Five days after ethanol injection, 2 × 106 of MSCs (MSC group) or vehicle (Control group) were injected into the AV nodal region. Animals were monitored by electrocardiograms for 14 days, and physiological and histological examinations were performed. The 1:1 AV conduction was recovered in 5 of 15 rats (33%) in the MSC group during the follow-up period, whereas no improvement was observed in the control group. MSC transplantation significantly decreased collagen deposition in the AV node, which was associated with a marked decrease in transforming growth factor-β1 expression. In vitro experiments demonstrated that MSCs secreted a large amount of antifibrotic factors such as hepatocyte growth factor and interleukin-10, and MSC conditioned medium inhibited the growth of adult cardiac fibroblasts. In addition, local injection of MSC conditioned medium recovered AV conduction in 2 of 15 rats (13%). MSC transplantation improved AV conduction in a rat model of complete AVB, at least in part through antifibrotic paracrine effects.

Role of mineralocorticoid receptor on atrial structural remodeling and inducibility of atrial fibrillation in hypertensive rats

Hypertension is well known to increase atrial fibrillation (AF) and the development of AF is associated with atrial chamber remodeling. Although mineralocorticoid receptor (MR) inhibition provides cardiovascular protection, the role of MR on atrial structural remodeling and inducibility of AF in hypertension remains unclear. Here, we investigated roles of the MR on atrial structural remodeling and inducibility of AF in hypertensive rats by using MR antagonist eplerenone (EPL). Dahl salt-sensitive (DS) rats were fed a normal-salt or a high-salt (HS) diet from 7 weeks, and a non-antihypertensive dose of EPL or vehicle was administrated from 13 weeks, at which time myocytes hypertrophy, interstitial fibrosis in the atrium and AF inducibility had increased, until 20 weeks. There was no significant difference in systolic blood pressure between DS+HS (186±4 mm Hg) and DS+HS+EPL (184±5 mm Hg) at 20 weeks. Burst atrial pacing demonstrated decreased AF inducibility in DS+HS+EPL (0 of 10) compared with DS+HS (7 of 10). Fibrosis and myocytes hypertrophy in the atrium were decreased in DS+HS+EPL with the reduction of atrial inflammatory cytokines. These beneficial effects of EPL were associated with less atrial oxidative stress, as assessed by 4-hydroxy-2-nonenal staining, and reduced activation of the Rho GTPase Rac1 in the atrium. Thus, MR has important roles in atrial structural remodeling and AF inducibility in Dahl rats. The effects of MR are associated, at least in part, with activation of Rac1-oxidative stress/inflammatory axis.

Metabolic syndrome and risk of development of chronic kidney disease: the Niigata preventive medicine study.

The metabolic syndrome consists of a cluster of cardiovascular risk factors, many of which have also been implicated in the genesis of chronic kidney disease. We studied the impact of the metabolic syndrome on chronic kidney disease in general population.

Inappropriate expression of hepcidin by liver congestion contributes to anemia and relative iron deficiency.

BACKGROUND Anemia and relative iron deficiency (RID) are prevalent in patients with heart failure (HF). The etiology of anemia and RID in HF patients is unclear. Hepcidin expression may be closely related to anemia and RID in HF patients. Although hepcidin is produced mainly by the liver, and the most frequent histologic appearance of liver in HF patients is congestion, the influence of liver congestion (LC) on hepcidin production has not yet been investigated. We investigated whether hepcidin contributed to anemia and RID in rats with LC. METHODS AND RESULTS LC was induced in rats by ligating the inferior vena cava and compared with bleeding anemia (BA) model induced by phlebotomy and hemolytic anemia (HA) model induced by injection of phenylhydrazine. BA and HA strongly suppressed expression of hepcidin in liver and so did not cause decrease in serum iron and transferrin saturation. However, hepcidin expression did not decrease in LC rats, which resulted in anemia and lower transferrin saturation. In addition, many cells with hemosiderin deposits were observed in the liver and spleen and not in the bone marrow, and this appeared to be related to suppression of hepcidin expression. Iron accumulated in hepatocytes, and bone morphogenetic protein 6, which induces hepcidin, increased. Inflammation was observed in the congestive liver, and there was an increase in interleukin-6, which also induced hepcidin and was induced by free heme and hemoglobin via Toll-like receptor 4. CONCLUSIONS We conclude that LC contributes to RID and anemia, and it does so via inappropriate expression of hepcidin.

Mechanical alternans in human idiopathic dilated cardiomyopathy is caused with impaired force-frequency relationship and enhanced poststimulation potentiation.

Mechanical alternans (MA) is frequently observed in patients with heart failure, and is a predictor of cardiac events. However, there have been controversies regarding the conditions and mechanisms of MA. To clarify heart rate-dependent contractile properties related to MA, we performed incremental right atrial pacing in 17 idiopathic dilated cardiomyopathy (DCM) patients and in six control patients. The maximal increase in left ventricular dP/dt during pacing-induced tachycardia was assessed as the force gain in the force–frequency relationship (FG-FFR), and the maximal increase in left ventricular dP/dt of the first post-pacing beats was examined as the force gain in poststimulation potentiation (FG-PSP). As a result, MA was induced in 9 DCM patients (DCM MA(+)) but not in the other 8 DCM patients (DCM MA(−)), and not in any of the control patients. DCM MA(+) had significantly lower FG-FFR (34.7 ± 40.9 vs 159.4 ± 103.9 mmHg/s, P = 0.0091) and higher FG-PSP (500.0 ± 96.8 vs 321.9 ± 94.9 mmHg/s, P = 0.0017), and accordingly a wider gap between FG-PSP and FG-FFR (465.3 ± 119.4 vs 162.5 ± 123.6 mmHg/s, P = 0.0001) than DCM MA(−) patients. These characteristics of DCM MA(+) showed clear contrasts to those of the control patients. In conclusion, MA is caused with an impaired force–frequency relationship despite significant poststimulation potentiation, suggesting that MA reflects ineffective utilization of the potentiated intrinsic force during tachycardia.

Improvement of anemia with decreasing hepcidin levels following valve replacement for severe tricuspid regurgitation

To the Editor: Severe right heart failure is occasionally accompanied by anemia (1, 2), but the underlying mechanisms for this association remain unknown. Hepcidin, a circulating hormone that is synthesized mainly by the liver, has emerged as a key regulator of systemic iron homeostasis (3). Hepcidin suppresses the expression of the iron transporter, ferroportin-1, thereby inhibiting the absorption of iron from the duodenum and the release of iron from the reticuloendothelial system. Therefore, the excessive production of hepcidin decreases serum iron levels, consequently causing and consequently causes anemia (3). Here, we report on a sixty-two-year-old woman of severe tricuspid regurgitation with refractory anemia in a patient with a very high serum hepcidin level who recovered from anemia after valve replacement, which was accompanied by a decrease in the hepcidin level. She had undergone mitral valve replacement for mitral stenosis 24 yrs before this episode. An echocardiogram revealed severe tricuspid regurgitation and dilatation of the right atrium. Right heart catheterization was performed after admission. Her right atrial pressure (RAP) was high (mean RAP, 15 mmHg; prominent V wave, 21 mmHg). Abdominal CT revealed hepatic vein dilatation and massive ascites. She received drug therapy for right heart failure, but this failed to reduce the massive ascites. She also had normocytic, normochromic anemia, but fecal occult blood tests were negative, and no bleeding source was found. She received oral iron preparation and red blood cell transfusions; however, her anemia did not resolve. As the massive ascites and progressive abdominal distension were believed to be life threatening, we decided to perform valve replacement surgery. The patient’s preoperative hemoglobin (Hb) level was 7.9 g ⁄dL, red blood cell (RBC) count was 271 million ⁄L, hematocrit (Ht) was 25.0%, serum iron level was low (26 lg ⁄dL), and serum ferritin level was high (724 ng ⁄mL; Table 1). Moreover, using a hepcidin EIA kit (Bachem Americans Inc., San Carlos, CA, USA), we found that her serum hepcidin levels (828 ng ⁄mL) were very high compared with the normal range (normal range, 1.8– 48.7 ng ⁄mL). Her preoperative serum C-reactive protein (CRP) level (15.91 mg ⁄dL) and interleukin-6 (IL-6) level (695 pg ⁄mL) were very high, but infectious disease was not found in any region of her body. Tricuspid valve replacement for severe tricuspid valve regurgitation and mitral valve replacement for the old ball valve were performed on July 1, 2010. Three months after the operation, the massive ascites completely disappeared. Postoperative right heart catheterization showed a decrease in RAP (mean RAP, 7 mmHg; V wave, 9 mmHg). Serum

Comparison of Patients With Pulmonary Arterial Hypertension With Versus Without Right-Sided Mechanical Alternans

The clinical implications of mechanical alternans in patients with pulmonary arterial hypertension (PAH) remain unknown. In this study, the prevalence, characteristics, and prognostic implications of mechanical alternans in patients with PAH were investigated. Thirty-two consecutive patients with PAH confirmed by cardiac catheterization from 2000 to 2010 were included in this cohort study. During cardiac catheterization, 8 patients (25%) showed mechanical alternans at rest. All alternans were detected in the right ventricle and pulmonary trunk. Serum level of brain natriuretic peptide (584 ± 177 vs 238 ± 252 pg/ml, p = 0.001), World Health Organization functional class (3.5 ± 0.5 vs 2.9 ± 0.4, p = 0.02), mean pulmonary arterial pressure (59 ± 10 vs 47 ± 18 mm Hg, p = 0.03), mean right atrial pressure (10 ± 4 vs 5 ± 4 mm Hg, p = 0.01), right ventricular end-diastolic pressure (15 ± 5 vs 9 ± 5 mm Hg, p = 0.01), and heart rate at catheterization (96 ± 17 vs 70 ± 11 beats/min, p = 0.003) were significantly higher in patients with alternans than in those without. Twelve-month mortality of patients with alternans was higher than in patients without alternans (p = 0.03): the 12-month survival rate after cardiac catheterization was 37% for the alternans group and 75% for the group without alternans. In conclusion, isolated right-sided mechanical alternans is not an uncommon event in patients with PAH. The existence of alternans is associated with the severity of PAH and right ventricular dysfunction and implies a poor prognosis in the short term.

Contrast Sonography Enables Noninvasive and Quantitative Assessment of Neovascularization After Stem Cell Transplantation

Stem cell transplantation is one of the attractive therapeutic strategies for the treatment of hindlimb ischemia. However, few studies have quantitatively assessed perfusion noninvasively in deep tissues after cell transplantation. In this study, we examined the feasibility of contrast sonography for the assessment of perfusion after bone marrow-derived mesenchymal stem cell (MSC) transplantation by using a rat unilateral hindlimb ischemia model. The quantitative parameters derived from contrast sonography were compared with the colored microspheres-derived blood flow and the capillary density. Nine rats were assigned each to a control (saline injection) or a treated (MSC transplantation) group. Video intensity vs. pulsing interval plots were acquired with ultraharmonic imaging of SONOS5500 during IV infusion of Levovist. The left-to-right ratio of hindlimb blood volume (A-ratio), microbubble velocity (beta-ratio) and hindlimb blood flow (Abeta-ratio) were calculated. The MS-ratio, the ratio of the left to the right hindlimb blood flow determined using colored microspheres, was also calculated. Although A-ratio did not change, beta- and Abeta-ratio in the treated group were significantly higher than those in the control group. In addition, MS-ratio and capillary density in the treated group were significantly higher than those in the control group. Compared with A- and Abeta-ratio, beta-ratio had the highest correlation with MS-ratio and capillary density (vs. MS-ratio: r = 0.66, p < 0.01; vs. capillary density: r = 0.52, p < 0.05). The results of our study imply that the contrast sonography-derived beta-ratio is a useful parameter that reflects the perfusion after cell transplantation in ischemic hindlimb.

Intravascular Ultrasonic Imaging of Vulnerable Plaque in a Bare Metal Stent 10 Years After Implantation

A 77-year-old woman was admitted to our hospital with unstable angina pectoris. She was previously diagnosed as having both mitral valve regurgitation and aortic valve stenosis and underwent surgery for replacement of both valves when she was 61 years of age. At 66 years of age, she was admitted to our hospital for angina pectoris and underwent implantation of a bare metal stent (BMS; 3.5×15 mm, Multilink, Guidant, Santa Clara, Calif) at the proximal left anterior descending coronary artery. Her …