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.

JAK-STAT signaling in cardiomyogenesis of cardiac stem cells.

Recently various kinds of cardiac stem/progenitor cells have been identified and suggested to be involved in cardiac repair and regeneration in injured myocardium. In this review, we focus on the roles of JAK-STAT signaling in cardiac stem/progenitor cells in cardiomyogenesis. JAK-STAT signaling plays important roles in the differentiation of stem cells into cardiac lineage cells. The activation of JAK-STAT signal elicits the mobilization of mesenchymal stem cells as well, contributing to the maintenance of cardiac function. Thus we propose that JAK-STAT could be a target signaling pathway in cardiac regenerative therapy.

Glycoprotein 130 cytokine signal as a therapeutic target against cardiovascular diseases.

Postnatal cardiomyocytes have only limited capacity of proliferation. Therefore, the myocardium is intrinsically equipped with cardioprotective machineries and protects itself from pathological stresses. One of the most important cardioprotective systems is the signal network of autocrine/paracrine factors, including neurohumoral factors, growth factors, and cytokines. In this review, we focus on the roles of interleukin-6 (IL-6) family cytokines, also known as glycoprotein 130 (gp130) cytokines, in cardioprotection. These cytokines make a complex with their specific cytokine receptor α-subunits. The cytokine-receptor α-subunit complex binds to gp130, a common receptor of the IL-6 family, followed by the activation of JAK/STAT, ERK, and PI3 kinase/Akt pathways. In cardiomyocytes, signals through gp130 promote cell survival and angiogenesis through the JAK/STAT pathway. Activation of gp130 in cardiac stem cells induces their endothelial transdifferentiation, leading to neovascularization. Recently, accumulating evidence has revealed that altered JAK/STAT activity is associated with heart failure, suggesting that the JAK/STAT pathway is a therapeutic target against cardiovascular diseases. Interestingly, activation of the JAK/STAT pathway with interleukin-11 (IL-11) exhibits preconditioning effects in ischemia/reperfusion model. Moreover, IL-11 treatment after coronary ligation prevents cardiac remodeling through the JAK/STAT pathway. Since IL-11 is used for patients with thrombocytopenia, we propose that IL-11 is a candidate cytokine clinically available for cardioprotection therapy.

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-alpha (TNF-alpha), interleukin (IL)-1beta, 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-alpha, IL-1beta, 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.

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.

Leukemia inhibitory factor induces endothelial differentiation in cardiac stem cells.

The importance of interleukin 6 (IL-6)-related cytokines in cardiac homeostasis has been studied extensively; however, little is known about their biological significance in cardiac stem cells. Here we describe that leukemia inhibitory factor (LIF), a member of IL-6-related cytokines, activated STAT3 and ERK1/2 in cardiac Sca-1+ stem cells. LIF stimulation resulted in the induction of endothelial cell-specific genes, including VE-cadherin, Flk-1, and CD31, whereas neither smooth muscle nor cardiac muscle marker genes such as GATA4, GATA6, Nkx-2.5, and calponin were up-regulated. Immunocytochemical examination showed that about 25% of total cells were positively stained with anti-CD31 antibody 14 days after LIF stimulation. Immunofluorescent microscopic analyses identified the Sca-1+ cells that were also positively stained with anti-von Willebrand factor antibody, indicating the differentiating process of Sca-1+ cells into the endothelial cells. IL-6, which did not activate STAT3 and ERK1/2, failed to induce the differentiation of cardiac stem cells into the endothelial cells. In cardiac stem cells, the transduction with dominant negative STAT3 abrogated the LIF-induced endothelial differentiation. And the inhibition of ERK1/2 with the MEK1/2 inhibitor U0126 also prevented the differentiation of Sca-1+ cells into endothelial cells. Thus, both STAT3 and ERK1/2 are required for LIF-mediated endothelial differentiation in cardiac stem cells. Collectively, it is proposed that LIF regulates the commitment of cardiac stem cells into the endothelial cell lineage, contributing to neovascularization in the process of tissue remodeling and/or regeneration.