Masanori Obana

Connective tissue growth factor induces cardiac hypertrophy through Akt signaling

In the process of cardiac remodeling, connective tissue growth factor (CTGF/CCN2) is secreted from cardiac myocytes. Though CTGF is well known to promote fibroblast proliferation, its pathophysiological effects in cardiac myocytes remain to be elucidated. In this study, we examined the biological effects of CTGF in rat neonatal cardiomyocytes. Cardiac myocytes stimulated with full length CTGF and its C-terminal region peptide showed the increase in cell surface area. Similar to hypertrophic ligands for G-protein coupled receptors, such as endothelin-1, CTGF activated amino acid uptake; however, CTGF-induced hypertrophy is not associated with the increased expression of skeletal actin or BNP, analyzed by Northern-blotting. CTGF treatment activated ERK1/2, p38 MAPK, JNK and Akt. The inhibition of Akt by transducing dominant-negative Akt abrogated CTGF-mediated increase in cell size, while the inhibition of MAP kinases did not affect the cardiac hypertrophy. These findings indicate that CTGF is a novel hypertrophic factor in cardiac myocytes.

Therapeutic Activation of Signal Transducer and Activator of Transcription 3 by Interleukin-11 Ameliorates Cardiac Fibrosis After Myocardial Infarction

Background— Glycoprotein 130 is the common receptor subunit for the interleukin (IL)-6 cytokine family. Previously, we reported that pretreatment of IL-11, an IL-6 family cytokine, activates the glycoprotein 130 signaling pathway in cardiomyocytes and prevents ischemia/reperfusion injury in vivo; however, its long-term effects on cardiac remodeling after myocardial infarction (MI) remain to be elucidated. Methods and Results— MI was generated by ligating the left coronary artery in C57BL/6 mice. Real-time reverse transcription polymerase chain reaction analyses showed that IL-11 mRNA was remarkably upregulated in the hearts exposed to MI. Intravenous injection of IL-11 activated signal transducer and activator of transcription 3 (STAT3), a downstream signaling molecule of glycoprotein 130, in cardiomyocytes in vivo, suggesting that cardiac myocytes are target cells of IL-11 in the hearts. Twenty-four hours after coronary ligation, IL-11 was administered intravenously, followed by consecutive administration every 24 hours for 4 days. IL-11 treatment reduced fibrosis area 14 days after MI, attenuating cardiac dysfunction. Consistent with a previous report that STAT3 exhibits antiapoptotic and angiogenic activity in the heart, IL-11 treatment prevented apoptotic cell death of the bordering myocardium adjacent to the infarct zone and increased capillary density at the border zone. Importantly, cardiac-specific ablation of STAT3 abrogated IL-11–mediated attenuation of fibrosis and was associated with left ventricular enlargement. Moreover, with the use of cardiac-specific transgenic mice expressing constitutively active STAT3, cardiac STAT3 activation was shown to be sufficient to prevent adverse cardiac remodeling. Conclusions— IL-11 attenuated cardiac fibrosis after MI through STAT3. Activation of the IL-11/glycoprotein 130/STAT3 axis may be a novel therapeutic strategy against cardiovascular diseases.

Atrogin-1 ubiquitin ligase is upregulated by doxorubicin via p38-MAP kinase in cardiac myocytes

AIMS Doxorubicin (DOX) is one of the most effective anti-neoplastic agents; however, its clinical use is limited by drug-induced cardiomyopathy. The molecular mechanisms responsible for this toxicity remain to be fully addressed. In the present study, we investigated the involvement of atrogin-1, one of the muscle-specific ubiquitin ligases, in DOX-induced cardiotoxicity. METHODS AND RESULTS This method involved intraperitoneal administration of DOX-induced atrogin-1 in the hearts and skeletal muscles of C57BL/6 mice. Consistently, atrogin-1 mRNA was upregulated with DOX treatment in cultured rat neonatal cardiomyocytes. Adenoviral transfer of atrogin-1 induced a reduction in cell size that was ameliorated by the ubiquitin proteasome inhibitor, MG-132. The transduction of constitutively active Akt (caAkt), a serine/threonine protein kinase, inhibited the DOX-mediated induction of atrogin-1. The phosphorylation status of Akt and its downstream target, FOXO, was not affected by DOX. DOX treatment did not activate the atrogin-1 promoter that contains FOXO-binding sites, suggesting that DOX induced atrogin-1 without modulating the Akt/FOXO pathway; importantly, DOX activated p38-mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Furthermore, pharmacological inhibition of p38-MAPK, but not JNK, abrogated DOX-mediated induction of atrogin-1. Finally, adenoviral transfer of caAkt inhibited the DOX-induced p38-MAPK activation. CONCLUSIONS DOX induces atrogin-1 through a p38-MAPK-dependent pathway in cardiac myocytes. Constitutive activation of Akt negatively regulates DOX-mediated atrogin-1 induction by inhibiting p38-MAPK activity as a novel mechanism.

IL-6-mediated Th17 differentiation through RORγt is essential for the initiation of experimental autoimmune myocarditis

AIMS Interleukin (IL)-17-producing helper T (Th17) cells have been proposed to participate in the pathogenesis of chronic inflammation, such as autoimmune myocarditis. IL-6 gene ablation confers the resistance to experimental autoimmune myocarditis (EAM). In this study, we have addressed the pathological roles of IL-6 in the regulation of Th17 cells in EAM. METHODS AND RESULTS To induce EAM, mice were immunized twice with α-myosin heavy chain peptide. Three weeks after the first injection, the cardiac expression of the Th17-specific transcription factor, retinoic acid receptor-related orphan nuclear receptor (ROR γt), was up-regulated. Consistently, Th17 cells were recruited into EAM hearts, as analysed by flow cytometry. Using the mice with enhanced green fluorescence protein (eGFP) gene knocked-in at RORγt locus (RORγt-eGFP mice), we observed Th17 cell infiltration into inflamed lesions. Pre-treatment with IL-6 receptor (IL-6R)-blocking antibody (anti-IL-6R Ab) inhibited EAM induction in terms of disease severity score (3.5 ± 0.8; IgG vs. 0.5 ± 0.8; anti-IL-6R Ab, n = 6, P< 0.01) and suppressed the myocardial expression of IL-17 and RORγt. In contrast, the administration of anti-IL-6R Ab 7 days after the first immunization failed to show the inhibitory effects, suggesting that IL-6 plays important roles in EAM initiation. Finally, by generating RORγt-eGFP homozygous mice, we revealed that RORγt gene ablation conferred the resistance to EAM induction. CONCLUSION IL-6-mediated induction of Th17 cells is critical for the onset of EAM, but not for its progression. IL-6/Th17 signalling could be a promising therapeutic target for the prevention of myocardial inflammation.

STAT3/Pim-1 signaling pathway plays a crucial role in endothelial differentiation of cardiac resident Sca-1+ cells both in vitro and in vivo

Cardiac stem cells potentially differentiate into cardiac cells, including cardiomyocytes and endothelial cells (ECs). Previously we demonstrated that STAT3 activation by IL-6 family cytokines, such as leukemia inhibitory factor (LIF), induces the endothelial differentiation of cardiac Sca-1+ cells. In this study, we addressed molecular mechanisms for EC differentiation of Sca-1+ cells. First, DNA array experiments were performed to search for the molecules induced by LIF. Among 134 genes that LIF upregulated by more than 4 fold, we focused on Pim-1 gene transcript, because Pim-1 is associated with the differentiation of some cell lineages. Real time RT-PCR analyses confirmed that LIF stimulation upregulated Pim-1 expression. Adenoviral transfection of dominant negative (dn) STAT3 inhibited LIF-mediated induction of Pim-1, while the overexpression of constitutively active STAT3 upregulated Pim-1 expression, suggesting that STAT3 activation is necessary and sufficient for Pim-1 induction. Moreover, in STAT3-deficient Sca-1+ cells, LIF failed to induce Pim-1 expression and EC differentiation. Importantly, the overexpression of dnPim-1 abrogated the induction of EC markers, indicating Pim kinase activity is indispensable for STAT3-mediated EC differentiation in vitro. Finally, Sca-1+ cells labeled with LacZ were transplanted into post-infarct myocardium and the transdifferentiation was estimated. The overexpression of wild-type STAT3 by adenovirus vector significantly promoted EC differentiation, while STAT3 gene ablation reduced the frequency of differentiating cells in post-infarct myocardium. Furthermore, transplanted Sca-1+ cells overexpressing dnPim-1 showed the reduced frequency of EC differentiation and capillary density. Collectively, Pim-1 kinase is upregulated by STAT3 activation in cardiac Sca-1+ cells and plays a pivotal role in EC differentiation both in vitro and in vivo.

Signals Through Glycoprotein 130 Regulate the Endothelial Differentiation of Cardiac Stem Cells

Objective—Cardiac Sca-1+ cells were originally identified as multipotent stem cells. To address the regulation of their differentiation, we investigated the effects of the proinflammatory cytokines on their endothelial differentiation. Methods and Results—We examined the effects of the proinflammatory cytokines including tumor necrosis factor-&agr; (TNF-&agr;), interleukin (IL)-1&bgr;, IL-6, IL-11, and cardiotrophin-1 (CT-1) on the cardiac Sca-1+ cell differentiation. IL-11 and CT-1, whose receptor systems use glycoprotein 130 (gp130), induced endothelial-specific genes in Sca-1+ cells, but not TNF-&agr;, IL-1&bgr;, or IL-6, analyzed by RT-PCR and by immunocytochemistry. Immnunoblot analyses showed that IL-11 and CT-1 activated signal transducer and activator of transcription 3 (STAT3), a downstream target of gp130, but not other cytokines. Though IL-6 receptor is not endogenously expressed in Sca-1+ cells, IL-6 exhibited the activity to induce the endothelial markers in the presence of soluble IL-6 receptor, an agonistic receptor, associated with STAT3 phosphorylation. Moreover, the inhibition of STAT3, by its dominant-negative form or siRNA, suppressed the induction of endothelial specific genes by IL-11 and CT-1. Finally, LIF and IL-11 transcripts were upregulated in postinfarct myocardium, accompanied by the induction of Sca-1+/VE-cadherin+ cells. Conclusions—Gp130/STAT3 pathway plays critical roles in the regulation of endothelial differentiation of cardiac Sca-1+ cells.

Therapeutic administration of IL-11 exhibits the postconditioning effects against ischemia-reperfusion injury via STAT3 in the heart.

Activation of cardiac STAT3 by IL-6 cytokine family contributes to cardioprotection. Previously, we demonstrated that IL-11, an IL-6 cytokine family, has the therapeutic potential to prevent adverse cardiac remodeling after myocardial infarction; however, it remains to be elucidated whether IL-11 exhibits postconditioning effects. To address the possibility that IL-11 treatment improves clinical outcome of recanalization therapy against acute myocardial infarction, we examined its postconditioning effects on ischemia/reperfusion (I/R) injury. C57BL/6 mice were exposed to ischemia (30 min) and reperfusion (24 h), and IL-11 was intravenously administered at the start of reperfusion. I/R injury mediated the activation of STAT3, which was enhanced by IL-11 administration. IL-11 treatment reduced I/R injury, analyzed by triphenyl tetrazolium chloride staining [PBS, 46.7 ± 14.4%; IL-11 (20 μg/kg), 28.6 ± 7.5% in the ratio of infarct to risk area]. Moreover, echocardiographic and hemodynamic analyses clarified that IL-11 treatment preserved cardiac function after I/R. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining revealed that IL-11 reduced the frequency of apoptotic cardiomyocytes after I/R. Interestingly, IL-11 reduced superoxide production assessed by in situ dihydroethidium fluorescence analysis, accompanied by the increased expression of metallothionein 1 and 2, reactive oxygen species (ROS) scavengers. Importantly, with the use of cardiac-specific STAT3 conditional knockout (STAT3 CKO) mice, it was revealed that cardiac-specific ablation of STAT3 abrogated IL-11-mediated attenuation of I/R injury. Finally, IL-11 failed to suppress the ROS production after I/R in STAT3 CKO mice. IL-11 administration exhibits the postconditioning effects through cardiac STAT3 activation, suggesting that IL-11 has the clinical therapeutic potential to prevent I/R injury in heart.

Mouse model of Epstein–Barr virus LMP1- and LMP2A-driven germinal center B-cell lymphoproliferative disease

Significance Epstein–Barr virus (EBV) is a major cause of B-cell lymphomas in immunocompromised hosts, where viral oncoproteins cause malignancy with loss of immunosurveillance. The EBV membrane oncoproteins latent membrane protein 1 (LMP1) and LMP2A are coexpressed in EBV-associated immunosuppressed host and Hodgkin lymphomas, where they activate key B-cell growth and survival pathways. We established a transgenic mouse model of LMP1/2A coexpression in germinal center (GC) B cells, the cell type from which most EBV-driven lymphomas arise. Our model highlighted synergistic LMP1/2A effects on this important B-cell population. Upon suppression of T and natural killer cells, LMP1/2A caused B-cell differentiation into rapidly growing plasmablasts that were not observed with LMP1 or LMP2A alone. Our model highlights synergistic LMP1/2A GC B-cell effects and recapitulates key aspects of EBV-driven lymphoproliferative disease. Epstein–Barr virus (EBV) is a major cause of immunosuppression-related B-cell lymphomas and Hodgkin lymphoma (HL). In these malignancies, EBV latent membrane protein 1 (LMP1) and LMP2A provide infected B cells with surrogate CD40 and B-cell receptor growth and survival signals. To gain insights into their synergistic in vivo roles in germinal center (GC) B cells, from which most EBV-driven lymphomas arise, we generated a mouse model with conditional GC B-cell LMP1 and LMP2A coexpression. LMP1 and LMP2A had limited effects in immunocompetent mice. However, upon T- and NK-cell depletion, LMP1/2A caused massive plasmablast outgrowth, organ damage, and death. RNA-sequencing analyses identified EBV oncoprotein effects on GC B-cell target genes, including up-regulation of multiple proinflammatory chemokines and master regulators of plasma cell differentiation. LMP1/2A coexpression also up-regulated key HL markers, including CD30 and mixed hematopoietic lineage markers. Collectively, our results highlight synergistic EBV membrane oncoprotein effects on GC B cells and provide a model for studies of their roles in immunosuppression-related lymphoproliferative diseases.

Myeloid cell-derived LRG attenuates adverse cardiac remodelling after myocardial infarction.

AIMS Leucine-rich α2-glycoprotein (LRG) is considered as a biomarker of the clinical activities of chronic inflammatory diseases, including heart failure. However, its pathophysiological roles in cardiac remodelling after myocardial infarction (MI) remain to be clarified. In this study, we have addressed functional roles of LRG in cardiac remodelling after MI. METHODS AND RESULTS MI was generated by ligating the left coronary artery in mice. Real-time reverse transcription (RT)-PCR and immunoblot analyses revealed that the expressions of LRG transcript and protein were up-regulated in post-infarct myocardium. LRG protein was produced by heart-infiltrating myeloid cells, such as macrophages and neutrophils. To elucidate functional roles of LRG in cardiac remodelling, we generated MI in wild-type (WT) and LRG-deficient (LRG(-/-)) mice and found that LRG gene ablation aggravated myocardial fibrosis with cardiac dysfunction after MI. Immunohistochemical analyses with anti-CD31 antibody revealed that capillary density decreased at border zone in LRG(-/-) mice compared with WT mice. Consistently, the expression of apelin receptor was reduced in LRG(-/-) mice, implying that the impaired angiogenic activity is associated with adverse cardiac remodelling in LRG(-/-) mice. Moreover, LRG gene ablation suppressed the activation of smad1/5/8, a pro-angiogenic signalling pathway. Finally, the transplantation of WT bone marrow cells into LRG(-/-) mice attenuated cardiac fibrosis with functional improvement after MI, accompanied by restoration of capillary density compared with the bone marrow transplantation from LRG(-/-) mice. CONCLUSION LRG, produced by heart-infiltrating myeloid cells, suppresses adverse cardiac remodelling after MI as a novel cardioprotective factor. LRG signalling could be a therapeutic target against cardiovascular diseases.

Cardiac-specific ablation of the STAT3 gene in the subacute phase of myocardial infarction exacerbated cardiac remodeling

STAT3 is a cardioprotective molecule against acute myocardial injury; however, recent studies have suggested that chronic STAT3 activation in genetically modified mice was detrimental after myocardial infarction (MI). In the present study, we assessed the biological significance of STAT3 activity in subacute MI using tamoxifen (TM)-inducible cardiac-specific STAT3 knockout (STAT3 iCKO) mice. After coronary ligation, STAT3 was rapidly activated in hearts, and its activation was sustained to the subacute phase. To make clear the pathophysiological roles of STAT3 activation specifically in subacute MI, MI was generated in STAT3 iCKO mice followed by TM treatment for 14 consecutive days beginning from day 11 after MI, which ablated the STAT3 gene in the subacute phase. Intriguingly, mortality was increased by TM treatment in STAT3 iCKO mice, accompanied by an increased heart weight-to-body weight ratio. Massons trichrome staining demonstrated that cardiac fibrosis was dramatically exacerbated in STAT3 iCKO mice 24 days after MI (fibrotic circumference: 58.3 ± 6.7% in iCKO mice and 40.8 ± 9.3% in control mice), concomitant with increased expressions of fibrosis-related gene transcripts, including matrix metalloproteinase 9, procollagen 1, and procollagen 3. Echocardiography clarified that cardiac function was deteriorated in STAT3 iCKO mice (fractional shortening: 20.6 ± 4.1% in iCKO mice and 29.1 ± 6.0% in control mice). Dihydroethidium fluorescence analysis revealed that superoxide production was increased in STAT3 iCKO mice. Moreover, immunohistochemical analyses revealed that capillary density was decreased in STAT3 iCKO mice. Finally, STAT3 deletion in subacute MI evoked severe cardiac hypertrophy in the border zone. In conclusion, the intrinsic activity of STAT3 in the myocardium confers the resistance to cardiac remodeling in subacute MI.