Yoshifusa Aizawa

Hydrodynamic-Based Delivery of an Interleukin-22-Ig Fusion Gene Ameliorates Experimental Autoimmune Myocarditis in Rats

IL-22 is one of several cytokines with limited homology to IL-10. However, the biological activities of IL-22 are mostly unknown. The purpose of this study was to evaluate the effect of IL-22 on rat experimental autoimmune myocarditis (EAM) and elucidate an aspect of the biological activities of IL-22. Rats were immunized on day 0; IL-22-Ig-treated rats were injected with pCAGGS-IL-22-Ig and control rats with pCAGGS-Ig using hydrodynamics-based gene delivery on day 1 or day 6. IL-22-Ig gene therapy administered on day 1 or day 6 after immunization was effective in controlling EAM as monitored by the heart weight to body weight ratio, and the myocarditis area in rats was sacrificed on day 17. Examination of the expression of IL-22-related genes in purified cells from EAM hearts suggested that IL-22-Ig acting target cells were noncardiomyocytic (NC) noninflammatory cells such as fibroblasts, smooth muscle cells, and endothelial cells. Therefore, we examined the effect of rIL-22 or serum containing IL-22-Ig on the expression of immune-relevant genes in IL-1-stimulated NC cells cultured from EAM hearts. Results showed that the expression of immunologic molecules (PGE synthase, cyclooxygenase-2, MIP-2, MCP-1, IL-6, and cytokine-induced neutrophil chemoattractant-2) in IL-1-stimulated NC cells was significantly decreased by rIL-22 or serum containing IL-22-Ig. EAM was suppressed by hydrodynamics-based delivery of plasmid DNA encoding IL-22-Ig, and the reason for this effectiveness may be that IL-22 suppressed gene expression of PG synthases, IL-6, and chemokines in activated NC noninflammatory cells.

Blood Pressure and Autonomic Responses to Electrical Stimulation of the Renal Arterial Nerves Before and After Ablation of the Renal Artery

Radiofrequency (RF) catheter ablation of the renal artery is therapeutic in patients with drug-refractory essential hypertension. This study was designed to examine the role of the renal autonomic nerves and of RF application from inside the renal artery in the regulation of blood pressure (BP). An open irrigation catheter was inserted into either the left or right renal artery in 8 dogs. RF current (17±2 watts) was delivered to one renal artery. Electrical autonomic nerve stimulation was applied to each renal artery before and after RF ablation. BP, heart rate, indices of heart rate variability, and serum catecholamines were analyzed. Before RF ablation, electrical autonomic nerve stimulation of either renal artery increased BP from 150±16/92±15 to 173±21/105±16 mm Hg. After RF ablation, BP increased similarly when the nonablated renal artery was electrically stimulated, although the rise in BP was attenuated when the ablated renal artery was stimulated. Serum catecholamines and sympathetic nerve indices of heart rate variability increased when electrical autonomic nerve stimulation was applied before RF ablation and to the nonablated renal artery after RF ablation, although it changed minimally when the ablated renal artery was stimulated, suggesting interconnectivity between afferent renal nerve stimulation and systemic sympathetic activity. Renal artery angiogram showed no apparent injury after RF ablation. In conclusion, electrical stimulation of the renal arterial autonomic nerves increases BP via an increase in central sympathetic nervous activity. This response might be used to determine the target ablation site and end point of renal artery RF ablation.

Expression of the peptide hormone hepcidin increases in cardiomyocytes under myocarditis and myocardial infarction

The micronutrient iron is an essential component that plays a role in many crucial metabolic reactions. The peptide hormone hepcidin is thought to play a central role in iron homeostasis and its expression is induced by iron overloading and inflammation. Recently, hepcidin has been reported to be expressed also in the heart; however, the kinetics of altered hepcidin expression in diseases of the heart remain unknown. In this study, we examined cardiac expression of hepcidin in rat experimental autoimmune myocarditis (EAM), human myocarditis and rat acute myocardial infarction (AMI). In rat EAM and AMI hearts, hepcidin was expressed in cardiomyocytes; ferroportin, which is a cellular iron exporter bound by hepcidin, was also expressed in various cells. Analysis of the time course of the hepcidin to cytochrome oxidase subunit 6a (Cox6a)2 expression ratio showed that it abruptly increased more than 100-fold in hearts in the very early phase of EAM and in infarcted areas 1 day after MI. The hepcidin/Cox6a2 expression ratio correlated significantly with that of interleukin-6/gamma-actin in both EAM and AMI hearts (r=0.781, P<.0001 and r=0.563, P=.0003). In human hearts with histological myocarditis, the ratio was significantly higher than in those without myocarditis (0.0400+/-0.0195 versus 0.0032+/-0.0017, P=.0045). Hepcidin is strongly induced in cardiomyocytes under myocarditis and MI, conditions in which inflammatory cytokine levels increase and may play an important role in iron homeostasis and free radical generation.

Effect of Hydrodynamics-Based Gene Delivery of Plasmid DNA Encoding Interleukin-1 Receptor Antagonist-Ig for Treatment of Rat Autoimmune Myocarditis Possible Mechanism for Lymphocytes and Noncardiac Cells

Background—Interleukin-1 (IL-1) is a powerful and important cytokine in myocarditis. The purpose of this study was to evaluate the effect and possible mechanism of hydrodynamics-based delivery of the IL-1 receptor antagonist (IL-1RA)-immunoglobulin (Ig) gene for treatment of rat experimental autoimmune myocarditis (EAM). Methods and Results—On the day after immunization, rats were transfected with either pCAGGS encoding IL-1RA-Ig or pCAGGS encoding Ig alone. On day 17, IL-1RA-Ig gene therapy was effective in controlling EAM, as monitored by a decreased ratio of heart weight to body weight, reduced myocarditis areas, reduced gene expression of atrial natriuretic peptide in hearts, and improved cardiac function in echocardiographic and hemodynamic parameters. Examination of the expression of IL-1–related genes in purified cells from EAM hearts suggested that ectopic IL-1RA-Ig-acting target cells were &agr;&bgr;T cells and noncardiomyocytic noninflammatory cells such as fibroblasts, smooth muscle cells, and endothelial cells. Therefore, we examined the effect of serum containing IL-1RA-Ig on the expression of immune-relevant genes within noncardiomyocytic cells cultured from EAM hearts or concanavalin A-stimulated lymphocytes derived from lymph nodes in EAM-affected rats. The expression of immunologic molecules (prostaglandin E synthase, cyclooxygenase-2, and IL-1&bgr;) in cultivated noncardiomyocytic cells and Th1 cytokines (IL-2 and IFN-&ggr;) in lymphocytes was significantly decreased by the serum containing IL-1RA-Ig. Conclusions—EAM was suppressed by hydrodynamics-based delivery of plasmid DNA encoding IL-1RA-Ig. In addition, IL-1RA-Ig suppressed gene expression of prostaglandin synthases and IL-1 in noncardiomyocytic cells and Th1 cytokines in lymphocytes.

Effect of antimicrobial periodontal treatment and maintenance on serum adiponectin in type 2 diabetes mellitus.

AIMS The aims of this study were to evaluate the effect of mechanical periodontal treatment with local application of minocycline (APT) on serum adiponectin as a marker of insulin resistance improvement in type 2 diabetes mellitus (T2DM) patients and to investigate if effect of APT on serum adiponectin level was sustained by periodontal maintenance (PM). MATERIAL AND METHODS Twenty-seven T2DM patients were randomly assigned into test or control groups. Test received scaling with ultrasonic devices at baseline and APT biweekly for 2 months while control received scaling at baseline and mechanical tooth cleaning (MPT) at the same interval. At 6 months, all patients received mechanical tooth cleaning as PM. Periodontal examination and blood measurements were performed at baseline, 4 and 9 months. RESULTS Adiponectin concentrations in test had significantly increased by 31.4% after APT (p=0.024) and by 30.4% after PM (p=0.002) compared with baseline. The percentage of >or=4 mm probing depths (PD) had shown 8.3% and 9.3% reduction after APT and PM (p=0.046, 0.02) in test while 5.0% reduction after MPT in control group (p=0.031). CONCLUSIONS Our results suggested that APT and PM not only improve periodontal disease but also increase serum adiponectin in T2DM patients.

The effect of hydrodynamics-based delivery of an IL-13-Ig fusion gene for experimental autoimmune myocarditis in rats and its possible mechanism

Interleukin (IL)‐13 is a pleiotropic cytokine secreted by activated Th2 T lymphocytes. Th1 cytokines are assumed to exacerbate and Th2 cytokines to ameliorate rat experimental autoimmune myocarditis (EAM). Here, we examined the effect of IL‐13 on EAM, using a hydrodynamics‐based delivery of an IL‐13‐Ig fusion gene, as well as the possible mechanism of its effect. Rats were immunized on day 0, and IL‐13‐Ig‐treated rats were injected with pCAGGS‐IL‐13‐Ig, and control rats with pCAGGS‐Ig, on day 1 or 7. On day 17, the IL‐13‐Ig gene therapy was effective in controlling EAM as monitored by a decreased heart weight/body weight ratio, by reduced myocarditis and by reduced atrial natriuretic peptide mRNA in the heart, as a heart failure marker. On the basis of IL‐13 receptor mRNA expression in separated cells from EAM hearts, we proposed that IL‐13‐Ig target cells were CD11b+ cells and non‐cardiomyocytic noninflammatory (NCNI) cells, such as fibroblasts, smooth muscle or endothelial cells. IL‐13‐Ig inhibited expression of the genes for prostaglandin E synthase, cyclooxygenase‐2, inducible nitric oxide synthase, IL‐1β and TNF‐α in cultivated cells from EAM hearts, while it enhanced expression of the IL‐1 receptor antagonist gene. We conclude that IL‐13‐Ig ameliorates EAM and suppose that its effectiveness may be due to the influence on these immunologic molecules in CD11b+ and NCNI cells.

Free heme is a danger signal inducing expression of proinflammatory proteins in cultured cells derived from normal rat hearts.

Endogenous molecules from damaged tissue act as danger signals to trigger or amplify the immune/inflammatory response. In this study, we examined whether free heme induced pro-inflammatory proteins in cultured cells derived from normal hearts and investigated the cells targeted by heme, together with its mechanism of action in these cells. We cultured collagenase-isolated heart-derived cells from normal rats and examined whether free heme induced pro-inflammatory proteins, reactive oxygen species (ROS) production and NF-κB activation, by quantitative RT-PCR, ELISA and flow cytometry. Free heme increased mRNA of various pro-inflammatory proteins in cultured cardiac resident cells (CCRC) (at least 100-fold) and induced intracellular ROS formation. Approximately 85-90% of CCRC are fibroblast/smooth muscle cells and 10-15% are CD11bc-positive macrophages; therefore to examine individual target cells, macrophage-deleted (CD11bc-negative) CCRC, primary cultured cells (cardiac fibroblasts, arterial smooth muscle cells and cardiac microvascular endothelial cells) and macrophage cells lines (NR8383) were similarly treated. Free heme activated NF-κB and induced expression of some pro-inflammatory proteins, including IL-1 and TNF-α in NR8383. On the other hand, macrophage-deleted CCRC strongly increased expression of these proteins on treatment with IL-1 or TNF-α, but not free heme. Induction of expression of pro-inflammatory proteins by free heme was not inhibited by intracellular ROS reduction, but by protease and proteasome inhibitors capable of regulating NF-κB. These data suggest that free heme strongly induces various pro-inflammatory proteins in injured hearts through NF-κB activation in cardiac resident macrophages and through cross-talk between macrophages and fibroblast/smooth muscle cells mediated inter alia by IL-1, TNF-α.

Amiodarone Improves Cardiac Sympathetic Nerve Function to Hold Norepinephrine in the Heart, Prevents Left Ventricular Remodeling, and Improves Cardiac Function in Rat Dilated Cardiomyopathy

Background—It is unclear how amiodarone therapy exerts its effects on left ventricular remodeling and cardiac sympathetic nerve function in chronic heart failure. We investigated long-term effects of amiodarone on rat dilated cardiomyopathy after healing of cardiac myosin–induced autoimmune myocarditis. Methods and Results—Rats were treated with oral amiodarone or vehicle for 6 weeks. We determined cardiac function, left ventricular remodeling, and cardiac sympathetic nerve function with iodine-125–labeled metaiodobenzylguanidine ([I125]MIBG). Amiodarone treatment improved left ventricular pressure, central venous pressure, and rate of isovolumetric contraction and decreased ventricular weight (P<0.005). Expression of cytokine mRNA was unchanged; expression of atrial natriuretic peptide, collagen III, and transforming growth factor-&bgr;1 mRNA was decreased in amiodarone-treated rats (P<0.05). Phenotype of myosin heavy chain was moved toward that of normal rats by amiodarone. Initial myocardial uptake of MIBG decreased by 67% (P<0.001) and washout rate accelerated by 221% in rats with chronic heart failure compared with normal rats. Whereas amiodarone decreased the initial uptake by 71% in normal rats, amiodarone decelerated the early washout and the late washout and improved the late myocardial distribution of MIBG in rats with chronic heart failure (257% compared with vehicle-treated rats with chronic heart failure; P<0.01). In proportion to MIBG distributions, cardiac tissue catecholamines were increased by amiodarone treatment. Conclusions—Long-term amiodarone treatment prevented left ventricular remodeling and improved cardiac function in rat dilated cardiomyopathy. Long-term amiodarone treatment also restored cardiac sympathetic tone to hold norepinephrine in the heart.

Gender differences in correlations among cardiovascular risk factors.

BACKGROUND Although the clustering of a few specific cardiovascular risk factors is known as the metabolic syndrome, sex-specific differences in correlations among risk factors have not been thoroughly examined. OBJECTIVE The analysis was undertaken to detect gender differences in correlations among cardio-vascular risk factors. METHODS Correlations among age, body mass index, systolic and diastolic blood pressures, and serum levels of fasting blood sugar (FBS), triglycerides (TG), high- and low-density lipoprotein cholesterol, high-sensitivity C-reactive protein (CRP), and uric acid were analyzed in apparently healthy Japanese men and women with TG<400 mg/dL and FBS<126 mg/dL. RESULTS Among the 136 men and 136 women examined, the frequency of significant correlations was marginally higher in women than in men: 28/45 correlations versus 17/45 correlations, respectively (P=0.017). Of a total of 45 possible correlations, 5 were marginally or significantly stronger in women, whereas no correlations among these risk factors were marginally or significantly stronger in men (P=0.021). These gender differences were considerably attenuated after adjustment for age. However, a significant sex-specific difference was observed in the correlation between TG and rank transformation of CRP, even after adjustment for age (P<0.01). CONCLUSIONS Correlations among cardiovascular risk factors were marginally stronger in women than in men. These results suggest that the existence of 1 additional risk factor may increase the risk of cardiovascular disease more steeply in women than in men.

Truncated KCNQ1 mutant, A178fs/105, forms hetero-multimer channel with wild-type causing a dominant-negative suppression due to trafficking defect.

We identified a novel mutation Ala178fs/105 missing S3–S6 and C‐terminus portions of KCNQ1 channel. Ala178fs/105‐KCNQ1 expressed in COS‐7 cells demonstrated no current expression. Co‐expression with wild‐type (WT) revealed a dominant‐negative effect, which suggests the formation of hetero‐multimer by mutant and WT. Confocal laser microscopy displayed intracellular retention of Ala178fs/105‐KCNQ1 protein. Co‐expression of the mutant and WT also increased intracellular retention of channel protein compared to WT alone. Our findings suggest a novel mechanism for LQT1 that the truncated S1–S2 KCNQ1 mutant forms hetero‐multimer and cause a dominant‐negative effect due to trafficking defect.