Hideharu Tomita

Important role of tissue angiotensin-converting enzyme activity in the pathogenesis of coronary vascular and myocardial structural changes induced by long-term blockade of nitric oxide synthesis in rats

The long-term administration of N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthesis, produces coronary vascular remodeling and myocardial hypertrophy in animals. This study used a rat model to investigate the role of angiotensin I converting enzyme (ACE) in the pathogenesis of such changes. We studied the following groups, all of which received drug treatment in their drinking water: untreated controls, and those administered L-NAME, L-NAME, and an ACE inhibitor (ACEI), and L-NAME and hydralazine. Cardiovascular structural changes and tissue ACE activities were evaluated after the first, fourth, and eighth week of treatment. In rats treated with L-NAME alone, vascular remodeling was evident at the fourth and eighth week, and myocardial hypertrophy was present at the eighth week of treatment. The vascular and myocardial remodeling were characterized by increased tissue ACE activities and immunodetectable ACE in those tissues. These changes were markedly reduced by ACEI, but not by hydralazine treatment. Increased local ACE expression may thus be important in the pathogenesis of cardiovascular remodeling in this model.

Early Induction of Transforming Growth Factor-β via Angiotensin II Type 1 Receptors Contributes to Cardiac Fibrosis Induced by Long-term Blockade of Nitric Oxide Synthesis in Rats

We previously reported that the chronic inhibition of nitric oxide (NO) synthesis increases cardiac tissue angiotensin-converting enzyme expression and causes cardiac fibrosis in rats. However, the mechanisms are not known. Transforming growth factor-beta (TGF-beta) is a key molecule that is responsible for tissue fibrosis. The present study investigated the role of TGF-beta in the pathogenesis of cardiac fibrosis. The development of cardiac fibrosis by oral administration of the NO synthesis inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) to normal rats was preceded by increases in mRNA levels of cardiac TGF-beta1 and extracellular matrix (ECM) proteins. TGF-beta immunoreactivity was increased in the areas of fibrosis. Treatment with a specific angiotensin II type 1 receptor antagonist, but not with hydralazine, completely prevented the L-NAME-induced increases in the gene expression of TGF-beta1 and ECM proteins and also prevented cardiac fibrosis. Intraperitoneal injection of neutralizing antibody against TGF-beta did not affect the L-NAME-induced increase in TGF-beta1 mRNA levels but prevented an increase in the mRNA levels of ECM protein. These results suggest that the early induction of TGF-beta1 via the angiotensin II type 1 receptor plays a major role in the development of cardiac fibrosis in this model.

Important Role of Local Angiotensin II Activity Mediated via Type 1 Receptor in the Pathogenesis of Cardiovascular Inflammatory Changes Induced by Chronic Blockade of Nitric Oxide Synthesis in Rats

BACKGROUND The chronic inhibition of NO synthesis by N(omega)-nitro-L-arginine methyl ester (L-NAME) upregulates the cardiovascular tissue angiotensin II (Ang II)-generating system and induces cardiovascular inflammatory changes in rats. METHODS AND RESULTS We used a rat model to investigate the role of local Ang II activity in the pathogenesis of such inflammatory changes. Marked increases in monocyte infiltration into coronary vessels and myocardial interstitial areas, monocyte chemoattractant protein-1 (MCP-1) expression, and nuclear factor-kappaB (NF-kappaB, an important redox-sensitive transcriptional factor that induces MCP-1) activity were observed on day 3 of L-NAME administration. Along with these changes, vascular superoxide anion production was also increased. Treatment with an Ang II type 1 receptor antagonist or with a thiol-containing antioxidant, N-acetylcysteine, prevented all of these changes. CONCLUSIONS Increased Ang II activity mediated via the type 1 receptor may thus be important in the pathogenesis of early cardiovascular inflammatory changes in this model. Endothelium-derived NO may decrease MCP-1 production and oxidative stress-sensitive signals by suppressing localized activity of Ang II.

Inhibition of NO Synthesis Induces Inflammatory Changes and Monocyte Chemoattractant Protein-1 Expression in Rat Hearts and Vessels

We recently showed that chronic inhibition of NO synthesis by N(omega)-nitro-L-arginine methyl ester (L-NAME) causes coronary vascular remodeling (ie, vascular fibrosis and medial thickening) in rats. To test the hypothesis that the inhibition of NO synthesis induces inflammatory changes in the heart, we characterized the inflammatory lesions that occurred during L-NAME administration and determined whether inflammation involved the induction of monocyte chemoattractant protein-1 (MCP-1) in vivo. During the first week of L-NAME administration to Wistar-Kyoto rats, we observed a marked infiltration of mononuclear leukocytes (ED1-positive macrophages) and fibroblast-like cells (alpha-smooth muscle actin-positive myofibroblasts) into the coronary vessels and myocardial interstitial areas. These inflammatory changes were associated with the expression of proliferating cell nuclear antigen and MCP-1 (both mRNA and protein). The areas affected by inflammatory changes, as well as the expression of MCP-1 mRNA, declined after longer (28 days) treatment with L-NAME and were replaced by vascular and myocardial remodeling. Our results support the hypothesis that the inhibition of NO synthesis induces inflammatory changes in coronary vascular and myocardial tissues and involves MCP-1 expression. Results also suggest that the early stages of inflammatory changes are important in the development of later-stage structural changes observed in rat hearts.

Cardiac Angiotensin II Receptors Are Upregulated by Long-Term Inhibition of Nitric Oxide Synthesis in Rats

It has been shown that nitric oxide (NO) may regulate angiotensin II (Ang II) receptors in vitro. To determine whether the chronic inhibition of NO synthesis upregulates cardiac Ang II receptors in a rat model, we evaluated the in vivo effect of Nomega-nitro-L-arginine methyl ester (L-NAME) on several Ang II receptors and on the expression of AT1 receptor mRNA in heart tissue. The chronic administration of L-NAME to normal rats increased the arterial blood pressure. The number of AT1 and AT2 receptors was increased, with no change in affinity, during the first week of L-NAME administration but returned to control levels after 4 weeks of treatment. The AT1 receptor mRNA was changed parallel to AT1 receptor number. Inflammatory changes (monocyte infiltration and myofibroblast formation) in perivascular areas surrounding coronary vessels and myocardial interstitial spaces were observed during the first week. The immunohistochemistry revealed that myofibroblasts expressed AT1 receptor. AT1 receptor blockade or cotreatment with L-arginine, but not cotreatment with hydralazine, prevented the L-NAME-induced increase in Ang II receptors and inflammatory changes. In conclusion, rat cardiac Ang II receptors are upregulated at an early phase of chronic inhibition of NO synthesis. This may contribute to cardiovascular inflammatory changes in an early phase and to remodeling at the later phase, which occurs after inhibition of NO synthesis.

Inhibition of Glycogen Synthase Kinase 3β During Heart Failure Is Protective

Glycogen synthase kinase (GSK)-3, a negative regulator of cardiac hypertrophy, is inactivated in failing hearts. To examine the histopathological and functional consequence of the persistent inhibition of GSK-3&bgr; in the heart in vivo, we generated transgenic mice with cardiac-specific overexpression of dominant negative GSK-3&bgr; (Tg-GSK-3&bgr;-DN) and tetracycline-regulatable wild-type GSK-3&bgr;. GSK-3&bgr;-DN significantly reduced the kinase activity of endogenous GSK-3&bgr;, inhibited phosphorylation of eukaryotic translation initiation factor 2Bϵ, and induced accumulation of &bgr;-catenin and myeloid cell leukemia-1, confirming that GSK-3&bgr;-DN acts as a dominant negative in vivo. Tg-GSK-3&bgr;-DN exhibited concentric hypertrophy at baseline, accompanied by upregulation of the &agr;-myosin heavy chain gene and increases in cardiac function, as evidenced by a significantly greater Emax after dobutamine infusion and percentage of contraction in isolated cardiac myocytes, indicating that inhibition of GSK-3&bgr; induces well-compensated hypertrophy. Although transverse aortic constriction induced a similar increase in hypertrophy in both Tg-GSK-3&bgr;-DN and nontransgenic mice, Tg-GSK-3&bgr;-DN exhibited better left ventricular function and less fibrosis and apoptosis than nontransgenic mice. Induction of the GSK-3&bgr; transgene in tetracycline-regulatable wild-type GSK-3&bgr; mice induced left ventricular dysfunction and premature death, accompanied by increases in apoptosis and fibrosis. Overexpression of GSK-3&bgr;-DN in cardiac myocytes inhibited tumor necrosis factor-&agr;–induced apoptosis, and the antiapoptotic effect of GSK-3&bgr;-DN was abrogated in the absence of myeloid cell leukemia-1. These results suggest that persistent inhibition of GSK-3&bgr; induces compensatory hypertrophy, inhibits apoptosis and fibrosis, and increases cardiac contractility and that the antiapoptotic effect of GSK-3&bgr; inhibition is mediated by myeloid cell leukemia-1. Thus, downregulation of GSK-3&bgr; during heart failure could be compensatory.

Inducible cAMP Early Repressor (ICER) Is a Negative-Feedback Regulator of Cardiac Hypertrophy and an Important Mediator of Cardiac Myocyte Apoptosis in Response to β-Adrenergic Receptor Stimulation

Abstract— Although stimulation of the &bgr;-adrenergic receptor increases levels of cAMP and activation of the cAMP response element (CRE) in cardiac myocytes, the role of the signaling mechanism regulated by cAMP in hypertrophy and apoptosis is not well understood. In this study we show that protein expression of inducible cAMP early repressor (ICER), an endogenous inhibitor of CRE-mediated transcription, is induced by stimulation of isoproterenol (ISO), a &bgr;-adrenergic agonist with a peak at ≈12 hours and persisting for more than 24 hours in neonatal rat cardiac myocytes. ICER is also upregulated by phenylephrine but not by endothelin-1. Continuous infusion of ISO also increased ICER in the rat heart in vivo. Overexpression of ICER significantly attenuated ISO- and phenylephrine-induced cardiac hypertrophy but did not inhibit endothelin-1–induced cardiac hypertrophy. Overexpression of ICER also stimulated cardiac myocyte apoptosis. Antisense inhibition of ICER significantly enhanced &bgr;-adrenergic hypertrophy, whereas it significantly inhibited &bgr;-adrenergic cardiac myocyte apoptosis, suggesting that endogenous ICER works as an important regulator of cardiac hypertrophy and apoptosis. Inhibition of CRE-mediated transcription by dominant-negative CRE binding protein inhibited cardiac hypertrophy, whereas it stimulated cardiac myocyte apoptosis, thereby mimicking the effect of ICER. Both ISO and ICER reduced expression of Bcl-2, an antiapoptotic molecule, whereas antisense ICER prevented ISO-induced downregulation of Bcl-2. These results suggest that ICER is upregulated by cardiac hypertrophic stimuli increasing CRE-mediated transcription in cardiac myocytes and acts as a negative regulator of hypertrophy and a positive mediator of apoptosis, in part through both inhibition of CRE-mediated transcription and downregulation of Bcl-2.

Percutaneous transluminal gene transfer into canine myocardium in vivo by replication-defective adenovirus

OBJECTIVE The aim was to examine the feasibility, efficiency and safety of adenovirus-mediated in vivo gene transfer into the canine myocardium by a percutaneous transluminal method using a needle-catheter. METHODS Either a replication-defective adenovirus (Adex1SRLacZL) or a plasmid (pSRLacZ), both expressing E. coli lacZ coding beta-galactosidase (beta-gal), was directly injected into the left ventricle of dogs through a needle-catheter inserted via a femoral artery. Expression of lacZ was examined by histochemical staining and quantified by measuring beta-gal activity. RESULTS Injections with Adex1SRLacZL induced lacZ expression as a result of 40 out of 41 injections; the expression level was 10 times higher than that obtained with pSRLacZ. Induced beta-gal activity was detected within 24 h, peaked at 7 days and retained for 2 weeks after gene transfer. A repetitive administration of the same adenovirus at 14 days after the first injection also evoked a reduced but significant level of expression despite neutralizing antibodies to adenovirus in serum. Although injection induced an inflammatory response that peaked at 3 days after injection and gradually subsided without a second peak, the temporal change and the extent of inflammation induced by adenovirus injection was not significantly different from those induced by injection with either saline or plasmid. Neither leakage of enzymes such as CPK or LDH nor alteration in the ECG was detected in the 30 days following gene transfer. CONCLUSIONS Our findings demonstrate that a catheter-mediated direct injection with an adenovirus can induce gene expression in the ventricle more efficiently without additional myocardial damage and inflammation compared with injection with a plasmid. A repeat dose of the same adenovirus elicited gene expression at an attenuated but significant level. This method may potentially have clinical applications: in modifying myocardial phenotype and/or improving general circulation under certain circumstances.

Quantitative Analysis of Repeat Adenovirus-Mediated Gene Transfer Into Injured Canine Femoral Arteries

We quantitatively evaluated the effectiveness of a repeat administration of a recombinant adenoviral vector expressing bacterial Escherichia coli lacZ into the same arterial site of a relatively large animal, the dog. The replication-defective adenoviral vector was introduced percutaneously into balloon-injured femoral arteries through a double-balloon catheter. After a single dose of adenoviral vector, up to 90% of surface (73 +/- 16%, n = 7) and smooth muscle cells in multiple layers of the media showed transgene expression as evaluated by 5-bromo-4-chloro-3-indoyl beta-D-galactopyranoside histostaining without extralocal expression, as assessed by polymerase chain reaction. High-level expression (measured as beta-galactosidase activity) peaked 7 days after transfer and was transient, although it was retained for a month. Second does of the same adenovirus to the same arterial site were given 1, 2, 5, or 8 weeks after the first administration. At 1 week the second dose significantly enhanced lacZ expression. At 2, 5, or 8 weeks the second dose reinduced lacZ expression at 25% to 30% of the full expression. lacZ expression was also detected in preimmuned dogs, although the expression levels correlated inversely to the titer of neutralizing antibodies in their serum. These results demonstrate that arterial gene expression can be enhanced by a second administration of the same adenovirus after a short interval and that a repeat dose after a long interval partially but significantly reinduces gene expression despite the presence of an immune response. These data may provide an additional scientific foundation for the use of adenovirus-mediated arterial gene transfer in future clinical practice.

Phosphorylation of Eukaryotic Translation Initiation Factor 2Bε by Glycogen Synthase Kinase-3β Regulates β-Adrenergic Cardiac Myocyte Hypertrophy

Abstract— Glycogen synthase kinase 3β (GSK-3β) negatively regulates cardiac hypertrophy. A potential target mediating the antihypertrophic effect of GSK-3β is eukaryotic translation initiation factor 2B3ε (eIF2Bε). Overexpression of GSK-3β increased the cellular kinase activity toward GST-eIF2Bε in neonatal rat cardiac myocytes, whereas LiCl (10 mmol/L) or isoproterenol (ISO) (10 μmol/L), a treatment known to inhibit GSK-3β, decreased it. Immunoblot analyses using anti-S535 phosphospecific eIF2Bε antibody showed that S535 phosphorylation of endogenous eIF2Bε was decreased by LiCl or ISO, suggesting that GSK-3β is the predominant kinase regulating phosphorylation of eIF2Bε-S535 in cardiac myocytes. Decreases in eIF2Bε-S535 phosphorylation were also observed in a rat model of cardiac hypertrophy in vivo. Overexpression of wild-type eIF2Bε alone moderately increased cell size (+31±11%; P< 0.05 versus control), whereas treatment of eIF2Bε-transduced myocytes with LiCl (+73±22% versus eIF2Bε only; P< 0.05) or ISO (+84±33% versus eIF2Bε only; P< 0.05) enhanced the effect of eIF2Bε. Overexpression of eIF2Bε-S535A, which is not phosphorylated by GSK-3β, increased cell size (+107±35%) as strongly as ISO (+95±25%), and abolished antihypertrophic effects of GSK-3β, indicating that S535 phosphorylation of eIF2Bε critically mediates antihypertrophic effects of GSK-3β. Furthermore, expression of eIF2Bε-F259L, a dominant-negative mutant, inhibited ISO-induced hypertrophy, indicating that eIF2Bε is required for β-adrenergic hypertrophy. Interestingly, expression of eIF2Bε-S535A partially increased cytoskeletal reorganization, whereas it did not increase expression of atrial natriuretic factor gene. These results suggest that GSK-3β is the predominant kinase mediating phosphorylation of eIF2Bε-S535 in cardiac myocytes, which in turn plays an important role in regulating cardiac hypertrophy primarily through protein synthesis.