Hiroshi Akimaru

Sonic Hedgehog-induced activation of the Gli1 promoter is mediated by GLI3.

Drosophila transcription factorcubitus interruptus (Ci) and its co-activator CRE (cAMP response element)-binding protein (CBP) activate a group of target genes on the anterior-posterior border in response to hedgehog protein (Hh) signaling. In the anterior region, in contrast, the carboxyl-truncated form of Ci generated by protein processing represses Hh expression. In vertebrates, three Ci-related transcription factors (glioblastoma gene products (GLIs) 1, 2, and 3) were identified, but their functional difference in Hh signal transduction is unknown. Here, we report distinct roles for GLI1 and GLI3 in Sonic hedgehog (Shh) signaling. GLI3 containing both repression and activation domains acts both as an activator and a repressor, as does Ci, whereas GLI1 contains only the activation domain. Consistent with this, GLI3, but not GLI1, is processed to generate the repressor form. Transcriptional co-activator CBP binds to GLI3, but not to GLI1. Thetrans-activating capacity of GLI3 is positively and negatively regulated by Shh and cAMP-dependent protein kinase, respectively, through a specific region of GLI3, which contains the CBP-binding domain and the phosphorylation sites of cAMP-dependent protein kinase. GLI3 directly binds to theGli1 promoter and induces Gli1 transcription in response to Shh. Thus, GLI3 may act as a mediator of Shh signaling in the activation of the target gene Gli1.

Methodological Development of a Clonogenic Assay to Determine Endothelial Progenitor Cell Potential

The precise and conceptual insight of circulating endothelial progenitor cell (EPC) kinetics is hampered by the absence of an assay system capable of evaluating the EPC differentiation cascade. An assay system for EPC colony formation was developed to delineate circulating EPC differentiation. EPC colony-forming assay using semisolid medium and single or bulk CD133+ cells from umbilical cord blood exhibited the formation of two types of attaching cell colonies made of small or large cells featuring endothelial lineage potential and properties, termed small EPC colony-forming units and large EPC colony-forming units, respectively. In vitro and in vivo assays of each EPC colony-forming unit cell revealed a differentiation hierarchy from small EPC to large EPC colonies, indicating a primitive EPC stage with highly proliferative activity and a definitive EPC stage with vasculogenic properties, respectively. Experimental comparison with a conventional EPC culture assay system disclosed EPC colony-forming unit cells differentiate into noncolony-forming early EPC. The fate analysis of single CD133+ cells into the endothelial and hematopoietic lineage was achieved by combining this assay system with a hematopoietic progenitor assay and demonstrated the development of colony-forming EPC and hematopoietic progenitor cells from a single hematopoietic stem cell. EPC colony-forming assay permits the determination of circulating EPC kinetics from single or bulk cells, based on the evaluation of hierarchical EPC colony formation. This assay further enables a proper exploration of possible links between the origin of EPC and hematopoietic stem cells, representing a novel and powerful tool to investigate the molecular signaling pathways involved in EPC biology.

CD34+ Cells Represent Highly Functional Endothelial Progenitor Cells in Murine Bone Marrow

Background Endothelial progenitor cells (EPCs) were shown to have angiogenic potential contributing to neovascularization. However, a clear definition of mouse EPCs by cell surface markers still remains elusive. We hypothesized that CD34 could be used for identification and isolation of functional EPCs from mouse bone marrow. Methodology/Principal Findings CD34+ cells, c-Kit+/Sca-1+/Lin− (KSL) cells, c-Kit+/Lin− (KL) cells and Sca-1+/Lin− (SL) cells were isolated from mouse bone marrow mononuclear cells (BMMNCs) using fluorescent activated cell sorting. EPC colony forming capacity and differentiation capacity into endothelial lineage were examined in the cells. Although CD34+ cells showed the lowest EPC colony forming activity, CD34+ cells exhibited under endothelial culture conditions a more adherent phenotype compared with the others, demonstrating the highest mRNA expression levels of endothelial markers vWF, VE-cadherin, and Flk-1. Furthermore, a dramatic increase in immediate recruitment of cells to the myocardium following myocardial infarction and systemic cell injection was observed for CD34+ cells comparing with others, which could be explained by the highest mRNA expression levels of key homing-related molecules Integrin β2 and CXCR4 in CD34+ cells. Cell retention and incorporation into the vasculature of the ischemic myocardium was also markedly increased in the CD34+ cell-injected group, giving a possible explanation for significant reduction in fibrosis area, significant increase in neovascularization and the best cardiac functional recovery in this group in comparison with the others. Conclusion These findings suggest that mouse CD34+ cells may represent a functional EPC population in bone marrow, which could benefit the investigation of therapeutic EPC biology.

Myb controls G2/M progression by inducing cyclin B expression in the Drosophila eye imaginal disc

The c‐myb proto‐oncogene product (c‐Myb) is a transcriptional activator. Vertebrate c‐Myb is a key regulator of the G1/S transition in cell cycle, while Drosophila Myb (dMyb) is important for the G2/M transition. Here we report that dMyb induces expression of cyclin B, a critical regulator of the G2/M transition, in Drosophila eye imaginal disc. In the wild‐type eye disc, dmyb mRNA was expressed in the stripes both anterior and posterior to the morphogenetic furrow. Ectopic expression of C‐terminal‐truncated dMyb in the eye disc caused ectopic expression of cyclin B and the rough eye phenotype. This rough eye phenotype correlated with prolonged M phase, caused by overexpression of cyclin B. Cyclin B expression was lost in dmyb‐deficient clones. In Schneider cells, the activity of the cyclin B promoter was dramatically reduced by loss of dMyb using the RNA interference method. Mutations of the multiple AACNG sequences in the cyclin B promoter also abolished the promoter activity. These results indicate that dMyb regulates the G2/M transition by inducing cyclin B expression via binding to its promoter.

Dissecting the Molecular Hierarchy for Mesendoderm Differentiation Through a Combination of Embryonic Stem Cell Culture and RNA Interference

Although there is a criticism that embryonic stem (ES) cell differentiation does not always reflect the differentiation process involved in mouse development, it is a suitable model system to dissect the specific differentiation pathway. We established the culture conditions that selectively differentiated mouse ES cells into three germ layers containing mesendoderm, definitive endoderm (DE), visceral endoderm (VE), mesoderm, and neuroectoderm. However, the molecular mechanisms of differentiation under each specific condition still remain unclear. Here, in combination with the RNA interference‐mediated gene knockdown (KD) method, we show that Eomesodermin (Eomes), Mixl1, Brachyury (T), and GATA6 are major molecular determinants in the differentiation of mesendoderm, DE, VE, and mesoderm. Eomes plays a pivotal role in an early stage of mesendoderm differentiation, whereas Mixl1 does the same in the later stage where mesendoderm differentiates into DE. Further analyses of quantitative reverse transcription polymerase chain reaction and overexpression of Mixl1 demonstrated that Mixl1 is genetically a downstream molecule of Eomes. In addition, both Eomes and Mixl1 act as negative regulators of T expression. This strategy also reveals that Eomes and T play cell‐autonomous roles in platelet‐derived growth factor receptor α (PDGFRα)+ vascular endothelial growth factor receptor 2 (VEGFR2)+ and PDGFRα+ mesoderm generations, respectively. Our results obtained from this study are fully consistent with previous knockout studies of those genes. The present study, therefore, demonstrates that the major molecular mechanism underlying in vitro ES cell differentiation largely recapitulates that in actual embryogenesis, and the combination of our culture system and RNAi‐mediated gene KD is an useful tool to elucidate the molecular hierarchy in in vitro ES cell differentiation.

Therapeutic effect of local administration of low-dose simvastatin-conjugated gelatin hydrogel for fracture healing

Several reports have shown the therapeutic effect of statins on bone formation and neovascularization. However, the effect of the systemic administration of statins is limited due to its metabolism in the liver and clearance in the digestive system. In addition, high‐dose administration may cause adverse side effects. To avoid low‐efficacy/frequent side effects of high‐dose statin treatment, we utilized biodegradable gelatin hydrogel as a drug delivery system of statin for fracture healing. A femoral fracture was created in rats with periosteum cauterization leading to nonunion at 8 weeks postfracture. Rats received local administration of either simvastatin‐conjugated gelatin hydrogel (ST‐Gel group) or gelatin hydrogel alone (Gel group). Approximately 70% of animals in the ST‐Gel group achieved fracture union radiographically and histologically, while only 7% of animals achieved fracture healing in the Gel group. Functional bone healing was also significantly greater with increased angiogenesis‐ and osteogenesis‐related growth factor expressions in periosteal granulation tissue in the ST‐Gel group than in the Gel group. Simvastatin locally applied with gelatin hydrogel to fracture sites at a dose similar to that used in clinical settings successfully induced fracture union in a rat unhealing bone fracture model via its effect on both angiogenesis and osteogenesis.

Development of Serum-Free Quality and Quantity Control Culture of Colony-Forming Endothelial Progenitor Cell for Vasculogenesis

Quantitative and qualitative impairment of endothelial progenitor cells (EPCs) limits the efficacy of autologous cell therapy in patients with cardiovascular diseases. Here, we developed a serum‐free quality and quantity control culture system for colony‐forming EPCs to enhance their regenerative potential. A culture with serum‐free medium containing stem cell factor, thrombopoietin, vascular endothelial growth factor, interleukin‐6, and Flt‐3 ligand was determined as optimal quality and quantity culture (QQc) in terms of the most vasculogenic colony‐forming EPC expansion, evaluated by the newly established EPC colony formation assay. The QQc of umbilical cord blood‐CD133+ cells for 7 days produced a 52.9‐fold increase in total cell number and 3.28‐fold frequency in definitive EPC colony development, resulting in a 203.9‐fold increase in estimated total definitive EPC colony number in vitro. Pre‐ or post‐QQc cells were intramyocardially transplanted into nude rats with myocardial infarction (MI). Echocardiographic and micromanometer‐tipped conductance catheter examinations 28 days post‐MI revealed significant preservation of left ventricular (LV) function in rats receiving pre‐ or post‐QQc cells compared with those receiving phosphate‐buffered saline. Assessments of global LV contractility indicated a dose‐dependent effect of pre‐ or post‐QQc cells and the superior potency of post‐QQc cells over pre‐QQc cells. Furthermore, immunohistochemistry showed more abundant formation of both human and rat endothelial cells and cardiomyocytes in the infarcted myocardium following transplantation of post‐QQc cells compared with pre‐QQc cells. Our optimal serum‐free quality and quantity culture may enhance the therapeutic potential of EPCs in both quantitative and qualitative aspects for cardiovascular regeneration.

A Hedgehog-Responsive Region in the Drosophila Wing Disc Is Defined by Debra-Mediated Ubiquitination and Lysosomal Degradation of Ci

Transcription factor Ci mediates Hedgehog (Hh) signaling to determine the anterior/posterior (A/P) compartment of Drosophila wing disc. While Hh-inducible genes are expressed in A compartment cells abutting the A/P border, it is unclear how the boundaries of this region are established. Here, we have identified a Ci binding protein, Debra, that is expressed at relatively high levels in the band abutting the border of the Hh-responsive A compartment region. Debra mediates the polyubiquitination of full-length Ci, which then leads to its lysosomal degradation. Debra is localized in the multivesicular body, suggesting that the polyubiquitination of Ci directs its sorting into lysosome. Thus, Debra defines the border of the Hh-responsive region in the A compartment by inducing the lysosomal degradation of Ci.

Lnk Deletion Reinforces the Function of Bone Marrow Progenitors in Promoting Neovascularization and Astrogliosis Following Spinal Cord Injury

Lnk is an intracellular adaptor protein reported as a negative regulator of proliferation in c‐Kit positive, Sca‐1 positive, lineage marker‐negative (KSL) bone marrow cells. The KSL fraction in mouse bone marrow is believed to represent a population of hematopoietic and endothelial progenitor cells (EPCs). We report here that, in vitro, Lnk−/− KSL cells form more EPC colonies than Lnk+/+ KSL cells and show higher expression levels of endothelial marker genes, including CD105, CD144, Tie‐1, and Tie2, than their wild‐type counterparts. In vivo, the administration of Lnk+/+ KSL cells to a mouse spinal cord injury model promoted angiogenesis, astrogliosis, axon growth, and functional recovery following injury, with Lnk−/− KSL being significantly more effective in inducing and promoting these regenerative events. At day 3 following injury, large vessels could be observed in spinal cords treated with KSL cells, and reactive astrocytes were found to have migrated along these large vessels. We could further show that the enhancement of astrogliosis appears to be caused in conjunction with the acceleration of angiogenesis. These findings suggest that Lnk deletion reinforces the commitment of KSL cells to EPCs, promoting subsequent repair of injured spinal cord through the acceleration of angiogenesis and astrogliosis. STEM CELLS 2010;28:365–375

SDF‐1/CXCR4 Axis in Tie2‐Lineage Cells Including Endothelial Progenitor Cells Contributes to Bone Fracture Healing

CXC chemokine receptor 4 (CXCR4) is a specific receptor for stromal‐derived‐factor 1 (SDF‐1). SDF‐1/CXCR4 interaction is reported to play an important role in vascular development. On the other hand, the therapeutic potential of endothelial progenitor cells (EPCs) in fracture healing has been demonstrated with mechanistic insight of vasculogenesis/angiogenesis and osteogenesis enhancement at sites of fracture. The purpose of this study was to investigate the influence of the SDF‐1/CXCR4 pathway in Tie2‐lineage cells (including EPCs) in bone formation. We created CXCR4 gene conditional knockout mice using the Cre/loxP system and set two groups of mice: Tie2‐CreER CXCR4 knockout mice (CXCR4−/−) and wild‐type mice (WT). We report here that in vitro, EPCs derived from of CXCR4−/− mouse bone marrow demonstrated severe reduction of migration activity and EPC colony‐forming activity when compared with those derived from WT mouse bone marrow. In vivo, radiological and morphological examinations showed fracture healing delayed in the CXCR4−/− group and the relative callus area at weeks 2 and 3 was significantly smaller in CXCR4−/− group mice. Quantitative analysis of capillary density at perifracture sites also showed a significant decrease in the CXCR4−/− group. Especially, CXCR4−/−group mice demonstrated significant early reduction of blood flow recovery at fracture sites compared with the WT group in laser Doppler perfusion imaging analysis. Real‐time RT‐PCR analysis showed that the gene expressions of angiogenic markers (CD31, VE‐cadherin, vascular endothelial growth factor [VEGF]) and osteogenic markers (osteocalcin, collagen 1A1, bone morphogenetic protein 2 [BMP2]) were lower in the CXCR4−/− group. In the gain‐of‐function study, the fracture in the SDF‐1 intraperitoneally injected WT group healed significantly faster with enough callus formation compared with the SDF‐1 injected CXCR4−/− group. We demonstrated that an EPC SDF‐1/CXCR4 axis plays an important role in bone fracture healing using Tie2‐CreER CXCR4 conditional knockout mice.