Atsushi Nakano

Multipotent Embryonic Isl1+ Progenitor Cells Lead to Cardiac, Smooth Muscle, and Endothelial Cell Diversification

Cardiogenesis requires the generation of endothelial, cardiac, and smooth muscle cells, thought to arise from distinct embryonic precursors. We use genetic fate-mapping studies to document that isl1(+) precursors from the second heart field can generate each of these diverse cardiovascular cell types in vivo. Utilizing embryonic stem (ES) cells, we clonally amplified a cellular hierarchy of isl1(+) cardiovascular progenitors, which resemble the developmental precursors in the embryonic heart. The transcriptional signature of isl1(+)/Nkx2.5(+)/flk1(+) defines a multipotent cardiovascular progenitor, which can give rise to cells of all three lineages. These studies document a developmental paradigm for cardiogenesis, where muscle and endothelial lineage diversification arises from a single cell-level decision of a multipotent isl1(+) cardiovascular progenitor cell (MICP). The discovery of ES cell-derived MICPs suggests a strategy for cardiovascular tissue regeneration via their isolation, renewal, and directed differentiation into specific mature cardiac, pacemaker, smooth muscle, and endothelial cell types.

Islet1 cardiovascular progenitors: a single source for heart lineages?

The creation of regenerative stem cell therapies for heart disease requires that we understand the molecular mechanisms that govern the fates and differentiation of the diverse muscle and non-muscle cell lineages of the heart. Recently, different cardiac cell types have been reported to arise from a common, multipotent Islet1 (Isl1)-positive progenitor, suggesting that a clonal model of heart lineage diversification might occur that is analogous to hematopoiesis. The ability to isolate, renew and differentiate Isl1+ precursors from postnatal and embryonic hearts and from embryonic stem cells provides a powerful cell-based system for characterizing the signaling pathways that control cardiovascular progenitor formation, renewal, lineage specification and conversion to specific differentiated progeny.

Pioglitazone, a peroxisome proliferator-activated receptor-γ agonist, attenuates myocardial ischemia/reperfusion injury in a rat model

Thiazolidinediones are insulin-sensitizing drugs, ligands for peroxisome proliferator-activated receptor-γ (PPAR-γ), which play an important role in the modulation of inflammatory responses. Myocardial ischemia/reperfusion (MI/R) injury is associated with inflammation, in which various cells, particularly monocytes and macrophages, are involved. This study examined the effects of the thiazolidinedione peroxisome proliferator-activated receptor-γ ligand, pioglitazone, in a rat model of MI/R injury. Pioglitazone at 3 mg/kg/day or the vehicle was administered for 7 days before rats were subjected to 30 minutes of coronary ligation followed by 24 hours of reperfusion. The mRNA expression [monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1] in the ischemic region, the number of infiltrating macrophages in the ischemic region, and the myocardial infarct size were examined. The inhibitory effects of pioglitazone on activated macrophages were studied in vitro. Phorbol 12-myristate 13-acetate–induced MCP-1 production, in the absence or presence of pioglitazone, were assayed in cultured macrophages. Compared with the control group, (1) mRNA levels of MCP-1 and intercellular adhesion molecule-1 and the number of infiltrating macrophages in the ischemic region were significantly lower in the pioglitazone-treated group; and (2) myocardial infarct size was significantly smaller in the pioglitazone-treated group. Phorbol 12-myristate 13-acetate–stimulated cultured macrophages in the presence of pioglitazone produced significantly lower levels of MCP-1 than the stimulated control in the absence of pioglitazone. These observations demonstrate that pioglitazone has anti-inflammatory effects in MI/R injury that are independent of its insulin-sensitizing effect.

Role of p38 Mitogen-Activated Protein Kinase in Neointimal Hyperplasia After Vascular Injury

Abstract—p38 mitogen-activated protein kinase (MAPK) is involved in intracellular signals that regulate a variety of cellular responses during inflammation. However, the role of p38 MAPK in atherosclerosis, a chronic inflammatory disorder, remains uncertain. The aim of the present study was to examine the role of p38 MAPK in the development of neointimal hyperplasia in balloon-injured rat carotid arteries. Immunohistochemical studies indicated that p38 MAPK was rapidly activated in the majority of medial cells in injured arterial walls. Rats treated with FR167653, a selective inhibitor of p38 MAPK, at a dosage of 10 mg · kg–1 · d–1, had a 29.4% lower intima-to-media ratio than the untreated controls at 14 days after balloon injury (P <0.05). The percentage of proliferating nuclear antigen–positive cells in the media at 48 hours was significantly lower in the FR167653-treated group than in the control group. Quantitative competitive reverse transcription–polymerase chain reaction analysis revealed that interleukin-1&bgr; mRNA expression in arteries was significantly inhibited by FR167653 (to 18.1% of control, P <0.05) at 8 hours after balloon injury. Moreover, p38 MAPK activation and interleukin-1&bgr; production by lipopolysaccharide-stimulated vascular smooth muscle cells were inhibited by FR167653 in a concentration-dependent manner in vitro. These results indicate that p38 MAPK is activated in vascular walls after injury and promotes neointimal formation and suggest that selective inhibition of p38 MAPK may be effective in the prevention of restenosis after percutaneous transluminal coronary angioplasty.

Cytokine gene therapy for myocarditis by in vivo electroporation

Cytokines are important pathophysiologic and pathogenic factors in cardiovascular disorders, including viral myocarditis. We attempted to treat viral myocarditis with cytokine gene therapy by transferring an inhibitory cytokine, IL-1 receptor antagonist (IL-1ra) or viral IL-10 (vIL-10), by in vivo electroporation, a new method for gene transfer into muscle. Four-week-old male DBA/2 mice were inoculated intraperitoneally with 10 PFU of encephalomyocarditis virus. Immediately after virus inoculation, an expression plasmid carrying IL-1ra or vIL-10 was injected into tibialis anterior muscles followed by electroporation. Serum levels of IL1ra and vIL-10 reached 10.5 and 2.3 ng/ml, respectively, on day 5, when gene expression reached its peak. Histopathological examination showed that myocardial cellular infiltration was improved in mice treated with IL-1ra or vIL-10 compared with the control group. On day 14 after the onset of myocarditis, transfer of IL1ra or vIL-10 expression plasmid had significantly improved the survival rates of the animals. The expression of TNF-alpha was decreased to 0.60-fold (p < 0.005) and inducible nitric oxide synthase (iNOS) 0.43-fold (p < 0.005) by IL-1ra treatment, and the expression of IFN-gamma in the heart was decreased to 0.35-fold (p < 0.05), and iNOS 0.21-fold (p < 0.005), by vIL-10 relative to the controls. These results show that gene therapy with IL-1ra or vIL-10 expression plasmid was effective in the treatment of viral myocarditis, and in vivo electroporation may be a useful method by which to deliver cytokine therapy in cardiovascular diseases.

Gene transfer of Fc-fusion cytokine by in vivo electroporation: application to gene therapy for viral myocarditis

Among a number of techniques for gene transfer in vivo, the direct injection of plasmid DNA into muscle is simple, inexpensive and safe. Although combining direct DNA injection with in vivo electroporation increases the efficiency of gene transfer into muscle, applications of this method have remained limited because of the relatively low expression level. To overcome this problem, we developed a plasmid vector that expresses a secretory protein as a fusion protein with the noncytolytic immunoglobulin Fc portion and used it for electroporation-mediated viral interleukin 10 (vIL-10) expression in vivo. The fusion cytokine vIL-10/mutFc was successfully expressed and the peak serum concentration of vIL-10 was almost 100-fold (195 ng/ml) higher than with a non-fusion vIL-10 expression plasmid. The expressed fusion cytokine suppressed the phytohemagglutinin-induced IFN-γ production by human peripheral blood mononuclear cells and decreased the mortality in a mouse viral myocarditis model as effectively as vIL-10 expression. These results demonstrate that the transfer of plasmid DNA expressing a noncytolytic Fc-fusion cytokine is useful to deliver enhanced levels of cytokine without altering general biological activities. This simple and efficient system should provide a new approach to gene therapy for human diseases and prove very useful for investigating the function of newly discovered secretory protein genes.

Multipotent islet-1 cardiovascular progenitors in development and disease.

During the past several years, advances at the intersection of cardiovascular development and heart stem cell biology have begun to reshape our view of the fundamental logic that drives the formation of discrete tissue components in the mammalian heart. Although many of the critical genes that control cardiac myogenesis have been identified, our understanding of how a highly diverse and specialized subset of heart cell lineages arises from mesodermal precursors and is subsequently assembled into distinct muscle chambers, coronary arterial tree and large vessels, valvular tissue, and conduction system/pacemaker cells remains at a relatively primitive stage. Recent studies have uncovered a diverse group of closely related heart progenitors that are central in controlling and coordinating these complex steps of cardiogenesis. Understanding the pathways that control their formation, renewal, and subsequent conversion to specific differentiated progeny forms the underpinning for unraveling the pathways for congenital heart disease and has direct relevance to cardiovascular regenerative medicine. This current brief review highlights the discovery and delineation of the role of Islet-1 cardiovascular progenitors in the generation of diverse heart cell lineages and how the implications of these findings are revising our classification and thinking about congenital heart disease in general.

Long term follow-up in a case of anomalous origin of the left coronary artery from the pulmonary artery.

An adult patient with anomalous origin of the left coronary artery from the pulmonary artery had no serious cardiac event for 19 years after closing the shunt from the aorta to the pulmonary trunk by ligation of the left main coronary artery. Although one-coronary arterial perfusion is considered to have worse prognosis than two-coronary arterial system, this patient proved to have an unexpectedly good long-term prognosis.

Rotational motion effect on sensitivity matrix of MEMS three-axis accelerometer for realization of concurrent calibration using vibration table

The rotational motion effect on the sensitivity matrix of a MEMS three-axis accelerometer was evaluated to realize the concurrent calibration of multiple MEMS accelerometers using a multi-axis shaker. The stage position dependency on the sensitivity matrix of a single-mass capacitive SOI three-axis accelerometer was measured. We found that the rotational motions generated by the resonance of the shaker causes an acceleration error on the accelerometer and can be compensated by measuring it using gyroscopes.