Hiroaki Kodama

Cardiomyocytes can be generated from marrow stromal cells in vitro

We have isolated a cardiomyogenic cell line (CMG) from murine bone marrow stromal cells. Stromal cells were immortalized, treated with 5-azacytidine, and spontaneously beating cells were repeatedly screened. The cells showed a fibroblast-like morphology, but the morphology changed after 5-azacytidine treatment in approximately 30% of the cells; they connected with adjoining cells after one week, formed myotube-like structures, began spontaneously beating after two weeks, and beat synchronously after three weeks. They expressed atrial natriuretic peptide and brain natriuretic peptide and were stained with anti-myosin, anti-desmin, and anti-actinin antibodies. Electron microscopy revealed a cardiomyocyte-like ultrastructure, including typical sarcomeres, a centrally positioned nucleus, and atrial granules. These cells had several types of action potentials, such as sinus node-like and ventricular cell-like action potentials. All cells had a long action potential duration or plateau, a relatively shallow resting membrane potential, and a pacemaker-like late diastolic slow depolarization. Analysis of the isoform of contractile protein genes, such as myosin heavy chain, myosin light chain, and alpha-actin, indicated that their muscle phenotype was similar to that of fetal ventricular cardiomyocytes. These cells expressed Nkx2.5/Csx, GATA4, TEF-1, and MEF-2C mRNA before 5-azacytidine treatment and expressed MEF-2A and MEF-2D after treatment. This new cell line provides a powerful model for the study of cardiomyocyte differentiation.

Mechanical Stretch Activates the JAK/STAT Pathway in Rat Cardiomyocytes

This study was designed to determine whether mechanical stretch activates the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway in cardiomyocytes and, if so, by what mechanism. Neonatal rat/murine cardiomyocytes were cultured on malleable silicone dishes and were stretched by 20%. Mechanical stretch induced rapid phosphorylation of JAK1, JAK2, Tyk2, STAT1, STAT3, and glycoprotein 130 as early as 2 minutes and peaked at 5 to 15 minutes. It also caused gel mobility shift of sis-inducing element, which was supershifted by preincubation with anti-STAT3 antibody. Preincubation with CV11974 (AT1 blocker) partially inhibited the phosphorylation of STAT1, but not that of STAT3. Preincubation with TAK044 (endothelin-1-type A/B-receptor blocker) did not attenuate this pathway. RX435 (anti-glycoprotein 130 blocking antibody) inhibited the phosphorylation of STAT3 and partially inhibited that of STAT1. Phosphorylation of STAT1 and STAT3 was strongly inhibited by HOE642 (Na+/H+ exchanger inhibitor) and BAPTA-AM (intracellular calcium chelator), but not by gadolinium (stretch-activated ion channel inhibitor), EGTA (extracellular Ca2+ chelator), or KN62 (Ca2+/calmodulin kinase II inhibitor). Chelerythrine (protein kinase C inhibitor) partially inhibited the phosphorylation of STAT1 and STAT3. Mechanical stretch also augmented the mRNA expression of cardiotrophin-1, interleukin-6, and leukemia inhibitory factor at 60 to 120 minutes. These results indicated that the JAK/STAT pathway was activated by mechanical stretch, and that this activation was partially dependent on autocrine/paracrine-secreted angiotensin II and was mainly dependent on the interleukin-6 family of cytokines but was independent of endothelin-1. Moreover, certain levels of intracellular Ca2+ were necessary for stretch-induced activation of this pathway, and protein kinase C was also partially involved in this activation.

Human circulating CD14+ monocytes as a source of progenitors that exhibit mesenchymal cell differentiation

Circulating CD14+ monocytes are precursors of phagocytes, such as macrophages and dendritic cells. Here we report primitive cells with a fibroblast‐like morphology derived from human peripheral blood CD14+ monocytes that can differentiate into several distinct mesenchymal cell lineages. We named this cell population monocyte‐derived mesenchymal progenitor (MOMP). MOMPs were obtained in vitro from human peripheral blood mononuclear cells cultured on fibronectin in the presence of fetal bovine serum alone as a source of growth factors. MOMPs had a unique molecular phenotype–CD14+CD45+CD34+ type I collagen+–and showed mixed morphologic and molecular features of monocytes and endothelial and mesenchymal cells. MOMPs were found to be derived from a subset of circulating CD14+ monocytes, and their differentiation required that they bind fibronectin and be exposed to one or more soluble factors derived from peripheral blood CD14− cells. MOMPs could be expanded in culture without losing their original phenotype for up to five passages. The induction of MOMPs to differentiate along multiple limb‐bud mesodermal lineages resulted in the expression of genes and proteins specific for osteoblasts, skeletal myoblasts, chondrocytes, and adipocytes. Our findings represent the first evidence that human circulating CD14+ monocytes are a source of progenitors that exhibit mesenchymal cell differentiation.

Interleukin-6 Family of Cytokines Mediate Angiotensin II-induced Cardiac Hypertrophy in Rodent Cardiomyocytes

This study was designed to investigate whether angiotensin II induces the interleukin (IL)-6 family of cytokines in cardiac fibroblasts and, if so, whether these cytokines can augment cardiac hypertrophy. Angiotensin II increased IL-6, leukemia inhibitory factor (LIF) and cardiotrophin-1 mRNA by 6.5-, 10.2-, and 2.0-fold, respectively, but did not affect IL-11, ciliary neurotrophic factor, or oncostatin M in cardiac fibroblasts. Enzyme-linked immunosorbent assay revealed that angiotensin II-stimulated conditioned medium from cardiac fibroblasts contained 9.3 ng/ml IL-6 at 24 h, which was 24-fold higher than the control. It phosphorylated gp130 and STAT3 in cardiomyocytes, which was reduced with RX435 (anti-gp130 blocking antibody). It increased [3H]phenylalanine uptake and cell area by 44% and 86% in cardiomyocytes compared with mock medium. RX435 suppressed these increases by 26% and 38%, while TAK044 (endothelin-A/B-R blocker) suppressed them by 52% and 52%, respectively. Antisense oligonucleotides against LIF and cardiotrophin-1 blocked their up-regulation, and attenuated the conditioned medium-induced increase in [3H]phenylalanine uptake by 21% and 13%, respectively. The combination of antisense oligonucleotides to LIF and cardiotrophin-1 decreased their uptake by 33%. These results indicated that angiotensin II induced IL-6, LIF, and cardiotrophin-1 in cardiac fibroblasts, and that these cytokines, particularly LIF and cardiotrophin-1, activated gp130-linked signaling and contributed to angiotensin II-induced cardiomyocyte hypertrophy.

Leukemia Inhibitory Factor, a Potent Cardiac Hypertrophic Cytokine, Activates the JAK/STAT Pathway in Rat Cardiomyocytes

Leukemia inhibitory factor (LIF) is a member of the interleukin-6 family of cytokines, which induces a wide range of responses in a variety of cells. The aim of this study was to investigate whether LIF induces cardiomyocyte hypertrophy and transmits signals through the JAK/STAT (indicating just another kinase/signal transducer and activator of transcription) pathway in primary cultured neonatal rat cardiomyocytes. LIF increased protein content and [3H]phenylalanine uptake in cardiomyocytes in a dose-dependent manner. LIF (10(3) U/mL) induced rapid tyrosine phosphorylation of gp130, JAK1, JAK2, STAT1, and STAT3 but not Tyk2 or STAT2. LIF also induced autokinase activity of JAK1 in a time-dependent manner. Gel shift assays for interferon gamma activation site/interferon-stimulated responsive element and sis-inducible element (SIE) revealed that LIF induced dimerization of STAT1 and STAT3 and formation of sis-inducing factor complexes, which subsequently interacted with SIE in the promoter. Preincubation with anti-STAT1 and anti-STAT3 antibodies inhibited the binding of SIF complexes. In conclusion, LIF induces cardiac hypertrophy and directly stimulates the JAK/STAT pathway in cardiomyocytes.

Role of Angiotensin II in Activation of the JAK/STAT Pathway Induced by Acute Pressure Overload in the Rat Heart

This study was designed to determine whether the JAK/STAT (indicating just another kinase/signal transducer and activator of transcription) pathway is activated in cardiac hypertrophy induced in vivo by pressure overload in rats and to demonstrate whether angiotensin II is involved in the activation of the JAK/STAT pathway. Acute pressure overload was produced by constricting the abdominal aorta of Wistar rats. Immunoprecipitation-Western blot analysis revealed that pressure overload activated JAK1, JAK2, and Tyk2 as early as 5 minutes and that STAT1, STAT2, and STAT3 were tyrosine-phosphorylated rapidly after exposure to the pressure overload. Phosphorylation of STAT1 and STAT2 peaked in the early stage at 5 to 15 minutes, whereas that of STAT3 peaked in the late stage at 60 minutes. Gel mobility shift of the interferon gamma activation site/interferon alpha-stimulating response element was observed immediately after the aortic banding, whereas the band of sis-inducing element was shifted in the late stage at 60 minutes. Both cilazapril (angiotensin II-converting enzyme inhibitor) and E4177 (angiotensin II type 1 [AT1] receptor antagonist) significantly suppressed the phosphorylation of Tyk2 and partially inhibited the phosphorylation of JAK2, but neither affected JAK1. Coimmunoprecipitation of the AT1 receptor with JAK2 or Tyk2 was clearly observed at 5 minutes and peaked at 15 minutes (20-fold the control value). These results indicate that the JAK/STAT pathway is activated by acute pressure overload in rats and that angiotensin II is involved in activating Tyk2, and partially activating JAK2, via the AT1 receptor. Both angiotensin II-dependent and -independent pathways take part in activating the JAK/STAT pathway in the pressure-overloaded rat heart.

Biphasic Activation of the JAK/STAT Pathway by Angiotensin II in Rat Cardiomyocytes

This study was designed to demonstrate the characteristic pattern of angiotensin II-induced JAK/STAT (indicating just another kinase/signal transducer and activator of transcription) activation in cultured rat cardiomyocytes by comparing it with leukemia inhibitory factor (LIF)-induced activation. Angiotensin II (10(-7) mol/L) induced rapid phosphorylation of JAK2 and Tyk2, but not JAK1, and phosphorylated STAT1 and STAT2, but not STAT3, in the early stage up to 30 minutes. The time course of JAK/STAT activation by angiotensin II was apparently slower than that by LIF. Interestingly, angiotensin II phosphorylated STAT3 and rephosphorylated STAT1 in the late stage at 120 minutes. We also found that angiotensin II induced the formation of interferon-stimulating gene factor (ISGF) complexes biphasically, in the early stage at 15 to 30 minutes and in the late stage at 120 minutes, and that angiotensin II induced delayed activation of the sis-inducing factor (SIF) complex at 120 minutes. Formation of ISGF and SIF complexes in response to angiotensin II paralleled the phosphorylation pattern of STAT1 and STAT3 and was quite different from those obtained in response to LIF. The phosphorylation of STAT1 was suppressed by pretreatment with the angiotensin II type-1 (AT1) receptor antagonist CV11974, but the delayed addition of CV11974 failed to suppress phosphorylation of STAT3 at 120 minutes. In conclusion, angiotensin II-induced JAK/STAT activation in rat cardiomyocytes is biphasic and entirely different from LIF-induced activation.

Endothelial Differentiation Potential of Human Monocyte-Derived Multipotential Cells

We previously reported a unique CD14+CD45+CD34+ type I collagen+ cell fraction derived from human circulating CD14+ monocytes, named monocyte‐derived multipotential cells (MOMCs). This primitive cell population contains progenitors capable of differentiating along the mesenchymal and neuronal lineages. Here, we investigated whether MOMCs can also differentiate along the endothelial lineage. MOMCs treated with angiogenic growth factors for 7 days changed morphologically and adopted a caudate appearance with rod‐shaped microtubulated structures resembling Weibel‐Palade bodies. Almost every cell expressed CD31, CD144, vascular endothelial growth factor (VEGF) type 1 and 2 receptors, Tie‐2, von Willebrand factor (vWF), endothelial nitric‐oxide synthase, and CD146, but CD14/CD45 expression was markedly downregulated. Under these culture conditions, the MOMCs continued to proliferate for up to 7 days. Functional characteristics, including vWF release upon histamine stimulation and upregulated expression of VEGF and VEGF type 1 receptor in response to hypoxia, were indistinguishable between the MOMC‐derived endothelial‐like cells and cultured mature endothelial cells. The MOMCs responded to angiogenic stimuli and promoted the formation of mature endothelial cell tubules in Matrigel cultures. Finally, in xenogenic transplantation studies using a severe combined immunodeficient mouse model, syngeneic colon carcinoma cells were injected subcutaneously with or without human MOMCs. Cotransplantation of the MOMCs promoted the formation of blood vessels, and more than 40% of the tumor vessel sections incorporated human endothelial cells derived from MOMCs. These findings indicate that human MOMCs can proliferate and differentiate along the endothelial lineage in a specific permissive environment and thus represent an autologous transplantable cell source for therapeutic neovasculogenesis.

Involvement of gp130-mediated signaling in pressure overload-induced activation of the JAK/STAT pathway in rodent heart

SummaryPreviously, we showed that the JAK/STAT pathway was activated in pressure-overloaded rat heart, and that angiotensin II was partially involved in this activation. The present study was designed to investigate whether gp130-mediated signaling is involved in this activation, and if so, which interleukin (IL)-6 family cytokine is involved. Pressure overload was produced by ligation of the abdominal aorta of Wistar rats or ICR mice. IP-Western blot was performed to detect tyrosine phosphorylation of STATs, gp130, and the association of gp130 with JAK kinases. The serum concentration of IL-6 was measured by enzyme-linked immunosorbent assay. Expression of IL-6, IL-11, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), oncostatin M (OSM), and cardiotrophin-1 (CT-1) mRNA was quantitated. After pressure overload, rapid phosphorylation of STAT1 and STAT3 was observed at 5min, STAT1 was rephosphorylated at 60min, and intense phosphorylation of STAT3 was observed at 60min. Both the phosphorylation of gp130 and the association of gp130 with JAK1 and JAK2 were increased after pressure overload. IL-6 was significantly increased by two-fold in the pressure-overloaded rats. Only CT-1 mRNA expression could be detected by Northern blot, and it increased after pressure overload. Reverse transcription-polymerase chain reaction revealed that IL-6 mRNA expression was increased 9.5-fold. IL-11, LIF, CNTF, and OSM expression were unaffected by pressure overload. These results suggested that gp130mediated signaling was involved in the pressure overload-induced activation of the JAK/STAT pathway, and that IL-6 and CT-1 might be involved in this activation.

Characterization of Insulin-Like Growth Factor-1–Induced Activation of the JAK/STAT Pathway in Rat Cardiomyocytes

This study was designed to investigate whether insulin-like growth factor-1 (IGF-1) transduces signaling through the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway in cardiomyocytes and to assess the upstream signals of serine and tyrosine phosphorylation of STAT family proteins. Primary cultured neonatal rat cardiomyocytes were stimulated with IGF-1 (10(-8) mol/L). JAK1, but not JAK2 or Tyk2, was phosphorylated by IGF-1 as early as 2 minutes and peaked at 5 minutes. IGF-1 induced both tyrosine and serine phosphorylation of STAT1 and STAT3. Tyrosine phosphorylation of STAT1 peaked at 15 minutes and correlated with that of JAK1, whereas that of STAT3 was sustained up to 120 minutes and was dissociated from the activation of JAK1. Tyrosine phosphorylation of STAT3 was unaffected by the preincubation with CV11974 (AT(1) blocker), TAK044 (endothelin-1 receptor blocker), RX435 (anti-gp130 blocking antibody), PD98058, wortmannin, EDTA, or KN62 but was significantly attenuated by BAPTA-AM and chelerythrine. The time course of a gel mobility shift of SIE (sis-inducing element) coincided with the phosphorylation of STAT3. Serine phosphorylation of STAT1 peaked at 30 minutes and that of STAT3 was observed from 5 to 60 minutes. These results indicated that (1) IGF-1 activated JAK1 but not JAK2 or Tyk2 in rat cardiomyocytes; (2) IGF-1 induced both tyrosine and serine phosphorylation of STAT1 and STAT3; and (3) the tyrosine phosphorylation of STAT3 was not caused by JAK1 alone, and protein kinase C and intracellular Ca(2+) were required for phosphorylation.