Hirohiko Ise

Human amniotic mesenchymal cells have some characteristics of cardiomyocytes

Background. Cellular cardiomyoplasty (CCM) is a major method for the treatment of heart failure because adult cardiomyocytes do not regenerate after ischemic injury, which results in heart failure. There is a great deal of interest in finding suitable new cell sources for use in CCM. Here, we report that human amniotic mesenchymal cells (hAMC), which are multipotent cells derived from fetal mesoderm, may be a suitable cell source for CCM. Methods. Freshly isolated hAMC were examined to detect the expression of cardiac-specific genes by reverse-transcription polymerase chain reaction and immunocytochemistry. hAMC were cocultivated with neonatal rat heart explants and transplanted into myocardial infarcts in the rat heart. Results. hAMC expressed cardiac-specific transcription factor GATA4, cardiac-specific genes, such as myosin light chain (MLC)-2a, MLC-2v, cTnI, and cTnT, and the &agr;-subunits of the cardiac-specific L-type calcium channel (&agr;1c) and the transient outward potassium channel (Kv4.3). After stimulation with basic fibroblast growth factor (bFGF) or activin A, hAMC expressed Nkx2.5, a specific transcription factor for the cardiomyocyte and cardiac-specific marker atrial natriuretic peptide. In addition, the cardiac-specific gene &agr;-myosin heavy chain was detected after treatment with activin A. Coculture experiments confirmed that hAMC were able to both integrate into cardiac tissues and differentiate into cardiomyocyte-like cells. After transplantation into the myocardial infarcts in rat hearts, hAMC survived in the scar tissue for at least 2 months and differentiated into cardiomyocyte-like cells. Conclusion. The results of the present study suggest that hAMC possess some characteristics of cardiomyocytes.

Galactosylated alginate as a scaffold for hepatocytes entrapment.

Galactose moieties were covalently coupled with alginate through ethylenediamine as the spacer for enhancing the interaction of hepatocytes with alginate. Adhesion of hepatocytes onto the galactosylated alginate (GA)-coated polystyrene (PS) surface showed an 18-fold increase as compared with that of the alginate-coated surface and it increased with an increase in the concentration of GA. The morphologies of attached hepatocytes were observed to spread out at the 0.15 wt% GA-coated PS surface while round cells were observed at the 0.5 wt% GA-coated PS surface. Inhibition of hepatocytes attachment onto the galactose-carrying PS-coated surface occurred with the addition of the GA into the hepatocyte suspension, indicating the binding of GA with hepatocytes via the patch of asialoglycoprotein receptors. Primary hepatocytes were entrapped in the GA/Ca2+ capsules (GAC). Higher cell viability and more spheroid formation of hepatocytes were obtained in the GAC than in the alginate/Ca2+ capsules (AC). Moreover, liver functions of the hepatocytes such as albumin secretion and urea synthesis in the GAC were improved in comparison with those in the AC.

Cardiac Overexpression of Monocyte Chemoattractant Protein-1 in Transgenic Mice Prevents Cardiac Dysfunction and Remodeling After Myocardial Infarction

Myocardial infarction (MI) is accompanied by inflammatory responses that lead to the recruitment of leukocytes and subsequent myocardial damage, healing, and scar formation. Because monocyte chemoattractant protein-1 (MCP-1) (also known as CCL2) regulates monocytic inflammatory responses, we investigated the effect of cardiac MCP-1 overexpression on left ventricular (LV) dysfunction and remodeling in a murine MI model. Transgenic mice expressing the mouse JE-MCP-1 gene under the control of the α-cardiac myosin heavy chain promoter (MHC/MCP-1 mice) were used for this purpose. MHC/MCP-1 mice had reduced infarct area and scar formation and improved LV dysfunction after MI. These mice also showed induction of macrophage infiltration and neovascularization; however, few bone marrow-derived endothelial cells were detected in MHC/MCP-1 mice whose bone marrow was replaced with that of Tie2/LacZ transgenic mice. Flow cytometry analysis showed no increase in endothelial progenitor cells (CD34+/Flk-1+ cells) in MHC/MCP-1 mice. Marked myocardial interleukin (IL)-6 secretion, STAT3 activation, and LV hypertrophy were observed after MI in MHC/MCP-1 mice. Furthermore, cardiac myofibroblasts accumulated after MI in MHC/MCP-1 mice. In vitro experiments revealed that a combination of IL-6 with MCP-1 synergistically stimulated and sustained STAT3 activation in cardiomyocytes. MCP-1, IL-6, and hypoxia directly promoted the differentiation of cardiac fibroblasts into myofibroblasts. Our results suggest that cardiac overexpression of MCP-1 induced macrophage infiltration, neovascularization, myocardial IL-6 secretion, and accumulation of cardiac myofibroblasts, thereby resulting in the prevention of LV dysfunction and remodeling after MI. They also provide a new insight into the role of cardiac MCP-1 in the pathophysiology of MI.

Critical Role of Bone Marrow Apoptosis-Associated Speck-Like Protein, an Inflammasome Adaptor Molecule, in Neointimal Formation After Vascular Injury in Mice

Background— Inflammatory cytokines such as interleukin (IL)-1β and IL-18 play an important role in the development of atherosclerosis and restenosis. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor protein that regulates caspase-1–dependent IL-1β and IL-18 generation; however, the role of ASC in vascular injury remains undefined. Here, we investigated the contribution of ASC to neointimal formation after vascular injury in ASC-deficient (ASC−/−) mice. Methods and Results— Wire-mediated vascular injury was produced in the femoral artery of ASC−/− and wild-type mice. Immunohistochemical analysis revealed that ASC was markedly expressed at the site of vascular injury. Neointimal formation was significantly attenuated in ASC−/− mice after injury. IL-1β and IL-18 were expressed in the neointimal lesion in wild-type mice but showed decreased expression in the lesion of ASC−/− mice. To investigate the contribution of bone marrow–derived cells, we developed bone marrow–transplanted mice and found that neointimal formation was significantly decreased in wild-type mice in which bone marrow was replaced with ASC−/− bone marrow cells. Furthermore, in vitro experiments showed that the proliferation activity of ASC−/− vascular smooth muscle cells was not impaired. Conclusions— These findings suggest that bone marrow–derived ASC is critical for neointimal formation after vascular injury and identify ASC as a novel therapeutic target for atherosclerosis and restenosis.

M-CSF Accelerates Neointimal Formation in the Early Phase After Vascular Injury in Mice. The Critical Role of the SDF-1-CXCR4 System

Objective—Since the macrophage colony-stimulating factor (M-CSF) has been shown to stimulate differentiation and proliferation of monocyte/macrophage lineage and to be involved in the process of neointimal formation after vascular injury, we tested the effects of M-CSF on the recruitment of bone marrow–derived progenitor cells in neointimal formation after vascular injury in mice. Methods and Results—Wire-mediated vascular injury was produced in the femoral artery of C57BL/6 mice. Recombinant human M-CSF [500 &mgr;g/(kg·day)] or saline (control) was administered for 10 consecutive days, starting 4 days before the injury. Treatment with M-CSF accelerated neointimal formation in the early phase after injury, and this neointimal lesion mainly consisted of bone marrow–derived cells. M-CSF treatment had no effect on the mobilization of endothelial progenitor cells (EPCs: CD34+/Flk-1+) and reendothelialization after injury. The stromal cell-derived factor-1 (SDF-1) was markedly expressed in the neointima and media after injury, whereas CXCR4+ cells were observed in the neointima. Further, a novel CXCR4 antagonist, AMD3100, significantly attenuated the M-CSF–induced neointimal formation. Conclusions—These findings suggest that M-CSF accelerated neointimal formation after vascular injury via the SDF-1–CXCR4 system, and the inhibition of this system has therapeutic potential for the treatment of cardiovascular diseases.

MCP-1 induces cardioprotection against ischaemia/reperfusion injury: role of reactive oxygen species

AIMS Monocyte chemoattractant protein-1 (MCP-1: CCL2) has been demonstrated to be involved in the pathophysiology of ischaemic heart disease; however, the precise role of MCP-1 in ischaemia/reperfusion (I/R) injury is controversial. Here, we investigated the role of cardiac MCP-1 expression on left ventricular (LV) dysfunction after global I/R in Langendorff-perfused hearts isolated from transgenic mice expressing the mouse JE-MCP-1 gene under the control of the alpha-cardiac myosin heavy chain promoter (MHC/MCP-1 mice). METHODS AND RESULTS In vitro experiments showed that MCP-1 prevented the apoptosis of murine neonatal cardiomyocytes after hypoxia/reoxygenation. I/R significantly increased the mRNA expression of MCP-1 in the Langendorff-perfused hearts of wild-type mice. Cardiac MCP-1 overexpression in the MHC/MCP-1 mice improved LV dysfunction after I/R without affecting coronary flow; in particular, it ameliorated LV diastolic pressure after reperfusion. This improvement was independent of both sarcolemmal and mitochondrial K(ATP) channels. Cardiac MCP-1 overexpression prevented superoxide generation in the I/R hearts, and these hearts showed decreased expression of the NADPH oxidase family proteins Nox1, gp91phox, and Nox3 compared with the hearts of wild-type mice. Further, superoxide dismutase activity in the hearts of MHC/MCP-1 mice was significantly increased compared with that in the hearts of wild-type mice. CONCLUSION These findings suggest that cardiac MCP-1 prevented LV dysfunction after global I/R through a reactive oxygen species-dependent but K(ATP) channel-independent pathway; this provides new insight into the beneficial role of MCP-1 in the pathophysiology of ischaemic heart diseases.

Vimentin and desmin possess GlcNAc-binding lectin-like properties on cell surfaces

Vimentin and desmin are intermediate filament proteins found in various mesenchymal and skeletal muscle cells, respectively. These proteins play an important role in the stabilization of the cytoplasmic architecture. Here, we found, using artificial biomimicking glycopolymers, that vimentin and desmin possess N-acetylglucosamine (GlcNAc)-binding lectin-like properties on the cell surfaces of various vimentin- and desmin-expressing cells such as cardiomyocytes and vascular smooth muscle cells. The rod II domain of these proteins was demonstrated to be localized to the cell surface and to directly bind to the artificial biomimicking GlcNAc-bearing polymer, by confocal laser microscopy and surface plasmon resonance analysis. These glycopolymers strongly interact with lectins and are useful tools for the analysis of lectin-carbohydrate interactions, since glycopolymers binding to lectins can induce the clustering of lectins due to multivalent glycoside ligand binding. Moreover, immunocytochemistry and pull-down assay with His-tagged vimentin-rod II domain protein showed that the vimentin-rod II domain interacts with O-GlcNAc proteins. These results suggest that O-GlcNAc proteins might be one candidate for physiological GlcNAc-bearing ligands with which vimentin and desmin interact. These findings demonstrate a novel function of vimentin and desmin that does not involve stabilization of the cytoplasmic architecture by which these proteins interact with physiological GlcNAc-bearing ligands such as O-GlcNAc proteins on the cell surface through their GlcNAc-binding lectin-like properties.

Cardiac differentiation of embryonic stem cells by substrate immobilization of insulin-like growth factor binding protein 4 with elastin-like polypeptides

The establishment of cardiomyocyte differentiation of embryonic stem cells (ESCs) is a useful strategy for cardiovascular regenerative medicine. Here, we report a strategy for cardiomyocyte differentiation of ESCs using substrate immobilization of insulin-like growth factor binding protein 4 (IGFBP4) with elastin-like polypeptides. Recently, IGFBP4 was reported to promote cardiomyocyte differentiation of ESCs through inhibition of the Wnt/β-catenin signaling. However, high amounts of IGFBP4 (approximately 1 μg/mL) were required to inhibit the Wnt/β-catenin signaling and induce differentiation to cardiomyocytes. We report herein induction of cardiomyocyte differentiation using IGFBP4-immobilized substrates. IGFBP4-immobilized substrates were created by fusion with elastin-like polypeptides. IGFBP4 was stably immobilized to polystyrene dishes through fusion of elastin-like polypeptides. Cardiomyocyte differentiation of ESCs was effectively promoted by strong and continuous inhibition of Wnt/β-catenin signaling with IGFBP4-immobilized substrates. These results demonstrated that IGFBP4 could be immobilized using fusion of elastin-like polypeptides. Our results also demonstrate that substrate immobilization of IGFBP4 is a powerful tool for differentiation of ESCs into cardiomyocytes. These findings suggest that substrate immobilization of soluble factors is a useful technique for differentiation of ESCs in regenerative medicine and tissue engineering.

Immobilized E-cadherin model can enhance cell attachment and differentiation of primary hepatocytes but not proliferation

E-Cadherin is an intercellular adhesion molecule that regulates cell functions such as differentiation and proliferation of cells. To clarify the potential of E-cadherin-mediated adhesion to induce differentiation, we constructed an adsorbable recombinant E-cadherin molecule by fusing with an immunoglobulin G (IgG) Fc region (E-cad-Fc). Hepatocytes could adhere to the fusion protein-coated surface by a homophilic interaction of E-cadherins and showed differentiated phenotypes such as low DNA synthesizing activity and maintenance of tryptophan oxygenase expression, similar to those of spheroid-formed hepatocytes that are known as a highly differentiated tissue-like cell aggregation. These results suggest that E-cadherin is a key molecule for maintaining differentiation of primary hepatocytes.

Embryonic undifferentiated cells show scattering activity on a surface coated with immobilized E-cadherin

Rearrangement of cell–cell adhesion is a critical event in embryonic development and tissue formation. We investigated the regulatory function of E‐cadherin, a key adhesion protein, in the developmental process by using E‐cadherin/IgG Fc fusion protein as an adhesion matrix in cell culture. F9 embryonal carcinoma cells usually form colonies when cultured on gelatin or fibronectin matrices. However, F9 cells cultured on the E‐cadherin/IgG Fc fusion protein matrix formed a scattered distribution, with a different cytoskeletal organization and E‐cadherin‐rich protrusions that were regulated by Rac1 activity. The same scattering activity was observed in P19 embryonal carcinoma cells. In contrast, three types of differentiated cells, NMuMG mammary gland cells, MDCK kidney epithelial cells, and mouse primary isolated hepatocytes, did not show the scattering activity observed in F9 and P19 cells. These results suggest that migratory behavior on an E‐cadherin‐immobilized surface is only observed in embryonic cells, and that the regulatory mechanisms underlying E‐cadherin‐mediated cell adhesion vary with the state of differentiation. J. Cell. Biochem. 103: 296–310, 2008.