Atai Watanabe

Increased Connective Tissue Growth Factor Relative to Brain Natriuretic Peptide as a Determinant of Myocardial Fibrosis

Excessive fibrosis contributes to an increase in left ventricular stiffness. The goal of the present study was to investigate the role of connective tissue growth factor (CCN2/CTGF), a profibrotic cytokine of the CCN (Cyr61, CTGF, and Nov) family, and its functional interactions with brain natriuretic peptide (BNP), an antifibrotic peptide, in the development of myocardial fibrosis and diastolic heart failure. Histological examination on endomyocardial biopsy samples from patients without systolic dysfunction revealed that the abundance of CTGF-immunopositive cardiac myocytes was correlated with the excessive interstitial fibrosis and a clinical history of acute pulmonary congestion. In a rat pressure overload cardiac hypertrophy model, CTGF mRNA levels and BNP mRNA were increased in proportion to one another in the myocardium. Interestingly, relative abundance of mRNA for CTGF compared with BNP was positively correlated with diastolic dysfunction, myocardial fibrosis area, and procollagen type 1 mRNA expression. Investigation with conditioned medium and subsequent neutralization experiments using primary cultured cells demonstrated that CTGF secreted by cardiac myocytes induced collagen production in cardiac fibroblasts. Further, G protein-coupled receptor ligands induced expression of the CTGF and BNP genes in cardiac myocytes, whereas aldosterone and transforming growth factor-&bgr; preferentially induced expression of the CTGF gene. Finally, exogenous BNP prevented the production of CTGF in cardiac myocytes. These data suggest that a disproportionate increase in CTGF relative to BNP in cardiac myocytes plays a central role in the induction of excessive myocardial fibrosis and diastolic heart failure.

Plasma connective tissue growth factor is a novel potential biomarker of cardiac dysfunction in patients with chronic heart failure.

Connective tissue growth factor (CTGF) has been recently reported as a mediator of myocardial fibrosis; however, the significance of plasma CTGF concentration has not been evaluated in patients with heart failure. The aim of this study was to investigate the clinical utility of plasma CTGF concentration for the diagnosis of heart failure.

Lentiviral Vector–mediated SERCA2 Gene Transfer Protects Against Heart Failure and Left Ventricular Remodeling After Myocardial Infarction in Rats

Reduced expression of the SERCA2 gene impairs the calcium-handling and contractile functions of the heart. We developed an SERCA2 gene transfer system using lentiviral vectors, and examined the long-term effect of SERCA2 gene transfer in the rat ischemic heart failure model. A lentiviral vector containing the SERCA2 gene was infused into a rat heart by hypothermic intracoronary delivery 2 weeks after myocardial infarction (MI). The transduction efficiency was approximately 40%. Six months after transduction, echocardiogram and pressure-volume measurements revealed that the SERCA2 gene transfer had significantly protected against left ventricular (LV) dilation, and had improved systolic and diastolic function, resulting in reduction in mortality rates. The brain natriuretic peptide mRNA level showed a significantly decrease and the phosphorylation level of serine residue of phospholamban (PLN) showed an increase in the Lenti-SERCA2-transduced heart. Further, DNA microarray analysis disclosed that SERCA2 gene transfer had increased cardioprotective gene expression and lowered the expression of genes that are known to exacerbate heart failure. The SERCA2 gene was successfully integrated into the host heart, induced favorable molecular remodeling, prevented LV geometrical remodeling, and improved the survival rate. These results suggest that a strategy to compensate for reduced SERCA2 gene expression by lentiviral vectors serves as a positive inotropic, lucitropic, and cardioprotective therapy for post-MI heart failure.

Integral Role of RhoA Activation in Monocyte Adhesion–Triggered Tissue Factor Expression in Endothelial Cells

Objective—The role of Rho activation in the regulation of tissue factor (TF) is not clear. This study was undertaken to investigate this in endothelial cells induced by monocyte adhesion. Methods and Results—Isolated human peripheral blood monocytes were added to cultured human coronary endothelial cells. Monocyte adhesion to endothelial cells increased the levels of TF antigen in the endothelial cells. The results of transient transfection of the human TF promoter/luciferase gene into endothelial cells indicated that the increase in endothelial expression of the TF gene caused by monocyte adhesion occurred at the transcriptional level. The upregulation of TF was inhibited by statins, and the suppressive effect of statins was reversed by geranylgeranylpyrophosphate. Monocyte adhesion rapidly upregulated the membrane translocation and GTP/GDP exchange of RhoA, but not of Cdc42 or Rac, in endothelial cells. Rho inhibition by C3 exoenzyme or adenovirus-mediated expression of N19RhoA prevented the endothelial upregulation of TF caused by monocyte adhesion, and this was mimicked by Rho-kinase inhibitors. Moreover, monocyte adhesion increased the phosphorylation of nuclear factor-&kgr;B p65 in endothelial cells, and this was prevented by statins and Rho inhibition. Conclusions—Our study shows that RhoA activation plays an integral role in TF expression in endothelial cells.

Regulation of the human tumor necrosis factor-α promoter by angiotensin II and lipopolysaccharide in cardiac fibroblasts: different cis-acting promoter sequences and transcriptional factors

We recently showed that angiotensin (ANG) II as well as mechanical stretch stimulated production of tumor necrosis factor (TNF) in cardiac fibroblasts. Presently, we examined the molecular mechanisms by which ANGII and lipopolysaccharide (LPS) upregulate TNF-alpha gene expression. In neonatal rat cardiac fibroblasts, increased transcription of TNF-alpha mRNA was detected as luciferase activity associated with activity of the TNF-alpha promoter. Progressive deletion from this promoter located the LPS-responsive region between -200 and -120 bp from the transcription initiation site, while the sequence between -120 and -70 bp was required for ANGII-induced expression. Next, we examined which cis-acting sequences in the TNF-alpha promoter region were essential for induction of TNF-alpha transcription. Competition analysis by electrophoretic mobility shift assay with and without specific antibodies showed that LPS increased binding of Sp1 and Sp3 to the Sp1-binding site, while Egr-1 was unimportant. With ANGII, binding of ATF-2/c-jun to the CRE site was required for TNF-alpha gene induction; neither Ets nor NF-kappaB was essential. Mutation analysis confirmed that response to LPS relied upon the Sp1 site in the TNF-alpha promoter, while the CRE-binding site was essential for stimulation by ANGII. We concluded that since TNF-alpha gene expression is transcriptionally activated by ANGII or LPS in cardiac fibroblasts via different cis-acting sequences in the TNF-alpha promoter and different transcriptional factors, mechanisms inducing TNF production differ between heart failure or cardiac hypertrophy and infectious disease.

Competitive Binding of CREB and ATF2 to cAMP/ATF Responsive Element Regulates eNOS Gene Expression in Endothelial Cells

Objective—Expression of endothelial nitric oxide synthase (eNOS) is a critical determinant for vascular homeostasis. We examined the effects of Beraprost sodium (BPS), a stable analogue of prostacyclin, on the eNOS gene expression in the presence of inflammatory cytokine interleukin (IL)-1β in cultured endothelial cells. Method and Results—Exposure of human and bovine endothelial cells to IL-1β decreased eNOS expression. Western blot analysis using phospho-specific antibodies showed that IL-1β stimulated p38 MAP kinase and phosphorylated ATF2. BPS inhibited these effects via protein kinase A (PKA)/cAMP-responsive element binding protein (CREB) activation. Transfection assays using site-specific mutation constructs showed that CRE/ATF elements located at −733 and −603 within the human eNOS promoter are necessary for full IL-1β responsiveness. BPS attenuated the IL-1β–mediated decrease in eNOS promoter activity and the expression of eNOS gene through PKA pathway. Electrophoretic gel mobility shift assays showed that IL-1β increased the binding of phosphorylated ATF2 to CRE/ATF. On treatment with BPS, phosphorylated CREB predominantly bound to CRE/ATF. Conclusions—These results indicate that IL-1β and BPS antagonistically regulates the eNOS expression through the activation of p38 and PKA, respectively. Furthermore, the ability to bind both CREB and ATF2 implicates the CRE/ATF sequence as a potential target for multiple signaling pathways in the regulation of the eNOS gene transcription.

Mitochondrial transcription factors TFAM and TFB2M regulate Serca2 gene transcription.

AIMS Sarco(endo)plasmic reticulum Ca²(+)-ATPase 2a (SERCA2a) transports Ca²(+) by consuming ATP produced by mitochondrial respiratory chain enzymes. Messenger RNA (mRNA) for these enzymes is transcribed by mitochondrial transcription factors A (TFAM) and B2 (TFB2M). This study examined whether TFAM and TFB2M coordinately regulate the transcription of the Serca2 gene and mitochondrial genes. METHODS AND RESULTS Nuclear localization of TFAM and TFB2M was demonstrated by immunostaining in rat neonatal cardiac myocytes. Chromatin immunoprecipitation assay and fluorescence correlation spectroscopy revealed that TFAM and TFB2M bind to the -122 to -114 nt and -122 to -117 nt regions of the rat Serca2 gene promoter, respectively. Mutation of these sites resulted in decreased Serca2 gene transcription. In a rat myocardial infarction model, Serca2a mRNA levels significantly correlated with those of Tfam (r = 0.54, P < 0.001) and Tfb2m (r = 0.73, P < 0.001). Overexpression of TFAM and TFB2M blocked hydrogen peroxide- and norepinephrine-induced decreases in Serca2a mRNA levels. In addition, overexpression of TFAM and TFB2M increased the mitochondrial DNA (mtDNA) copy number and mRNA levels of mitochondrial enzymes. CONCLUSION Although TFAM and TFB2M are recognized as mtDNA-specific transcription factors, they also regulate transcription of nuclear DNA, i.e. the Serca2 gene. Our findings suggest a novel paradigm in which the transcription of genes for mitochondrial enzymes that produce ATP and the gene for SERCA2a that consumes ATP is coordinately regulated by the same transcription factors. This mechanism may contribute to maintaining proper cardiac function.

Retinoids induce the PAI-1 gene expression through tyrosine kinase-dependent pathways in vascular smooth muscle cells.

Retinoids exert their pleiotropic effects on several pathophysiologic processes, including neointima formation after experimental vascular injury. Plasminogen activator inhibitor-1 (PAI-1) has been proposed to play an inhibitory role in arterial neointima formation after injury. We examined whether retinoids regulate PAI-1 expression in cultured vascular smooth muscle cells (SMCs). Northern blot analysis showed that all-trans retinoic acid (atRA) and 9-cis retinoic acid (9cRA) increased PAI-1 mRNA levels in a dose-dependent manner. These responses were completely inhibited by tyrosine kinase inhibitors. The half-life of PAI-1 was not affected by atRA, suggesting that induction of PAI-1 mRNA was mainly regulated at the transcriptional levels. Stable and transient transfection assays of the human PAI-1 promoter-luciferase constructs indicate that DNA sequence responsive to either ligand-stimulated or overexpressed retinoic acid receptor-alpha expression vector lies downstream of −363 relative to the transcription start site, where no putative retinoic acid response element is found. These results indicate that atRA and 9cRA increase PAI-1 gene transcription through pathways involving tyrosine kinases in SMCs. Because PAI-1 inhibits the production of fibrinolytic protein plasmin that facilitates SMC migration, induction of the PAI-1 gene expression by atRA may at least partly account for the role of atRA as an important inhibitor of neointima formation.