Coronary development is regulated by ATP-dependent SWI/SNF chromatin remodeling component BAF180.

Dissecting the molecular mechanisms that guide the proper development of epicardial cell lineages is critical for understanding the etiology of both congenital and adult forms of human cardiovascular disease. In this study, we describe the function of BAF180, a polybromo protein in ATP-dependent SWI/SNF chromatin remodeling complexes, in coronary development. Ablation of BAF180 leads to impaired epithelial-to-mesenchymal-transition (EMT) and arrested maturation of epicardium around E11.5. Three-dimensional collagen gel assays revealed that the BAF180 mutant epicardial cells indeed possess significantly compromised migrating and EMT potentials. Consequently, the mutant hearts form abnormal surface nodules and fail to develop the fine and continuous plexus of coronary vessels that cover the entire ventricle around E14. PECAM and *-SMA staining assays indicate that these nodules are defective structures resulting from the failure of endothelial and smooth muscle cells within them to form coronary vessels. PECAM staining also reveal that there are very few coronary vessels inside the myocardium of mutant hearts. Consistent with this, quantitative RT-PCR analysis indicate that the expression of genes involved in FGF, TGF, and VEGF pathways essential for coronary development are down-regulated in mutant hearts. Together, these data reveal for the first time that BAF180 is critical for coronary vessel formation.

Critical role for the Ets transcription factor ELF-1 in the development of tumor angiogenesis.

The Ets transcription factors regulate a wide variety of biologic processes. Several members have been shown to play a role in regulating angiogenesis and vascular development. For example, the Ets factor ELF-1 is enriched in the developing vasculature of the embryo, where it regulates the expression of the Tie2 gene. We have determined that ELF-1 and Tie2 expression is also enriched in tumor blood vessels, and have identified a short peptide, 34 amino acids in length, corresponding to the terminal portion of the highly conserved ETS domain that potently blocks the function of ELF-1. A tailored ELF-1 blocking peptide, containing a 12-amino acid HIV-1 TAT protein, readily crosses the cell membrane and enters into the nucleus of endothelial cells, leading to a marked reduction in the expression of ELF-1 gene targets including Tie2 and endothelial nitric oxide synthase. Furthermore, the ELF-1 blocking peptide potently inhibits angiopoietin-1-mediated endothelial cell migration. Systemic administration of this peptide markedly attenuates B16 melanoma tumor growth and tumor-associated angiogenesis in nude mice. These results support the function of ELF-1 in the regulation of Tie2 gene expression during the development of tumor angiogenesis.

Homing of intravenously infused embryonic stem cell-derived cells to injured hearts after myocardial infarction.

OBJECTIVE: The present study was designed to test whether intravenously infused embryonic stem cell-derived cells could translocate to injured myocardium after myocardial infarction and improve cardiac function. METHODS: Cultured embryonic stem cell-derived cells were transfected with green fluorescent protein. Embryonic stem cell-derived cells were administered through the tail vein (approximately 10(7) cells in 1 mL of medium for each rat) every other day for 6 days in 45 rats after myocardial infarction. Six weeks after myocardial infarction and cell infusion, cardiac function, blood flow, and the numeric density of arterioles were measured to test the benefits of cell therapy. An in vitro Transwell assay was performed to evaluate the embryonic stem cell migration. RESULTS: Ventricular function, regional blood flow, and arteriole density were significantly increased in rats receiving intravenously infused embryonic stem cell-derived cells compared with control rats after myocardial infarction. Histologic analysis demonstrated that infused embryonic stem cell-derived cells formed green fluorescent protein-positive grafts in infarcted myocardium. Additionally, positive immunostaining for cardiac troponin I was found in hearts after myocardial infarction receiving embryonic stem cell-derived cell infusion that corresponded to the green fluorescent protein-positive staining. The Transwell migration assay indicated that cultured neonatal rat cardiomyocytes with overexpression of tumor necrosis factor alpha induced greater migration of embryonic stem cells compared with cardiomyocytes without tumor necrosis factor alpha expression. CONCLUSIONS: Our data demonstrate that intravenously infused embryonic stem cell-derived cells homed to the infarcted heart, improved cardiac function, and enhanced regional blood flow at 6 weeks after myocardial infarction. The in vitro migration assay suggested that such a homing mechanism could be associated with locally released cytokines, such as tumor necrosis factor alpha, that are upregulated in the setting of acute myocardial infarction and heart failure.

A novel role for GADD45beta as a mediator of MMP-13 gene expression during chondrocyte terminal differentiation.

The growth arrest and DNA damage-inducible 45beta (GADD45beta) gene product has been implicated in the stress response, cell cycle arrest, and apoptosis. Here we demonstrated the unexpected expression of GADD45beta in the embryonic growth plate and uncovered its novel role as an essential mediator of matrix metalloproteinase-13 (MMP-13) expression during terminal chondrocyte differentiation. We identified GADD45beta as a prominent early response gene induced by bone morphogenetic protein-2 (BMP-2) through a Smad1/Runx2-dependent pathway. Because this pathway is involved in skeletal development, we examined mouse embryonic growth plates, and we observed expression of Gadd45beta mRNA coincident with Runx2 protein in pre-hypertrophic chondrocytes, whereas GADD45beta protein was localized prominently in the nucleus in late stage hypertrophic chondrocytes where Mmp-13 mRNA was expressed. In Gadd45beta(-/-) mouse embryos, defective mineralization and decreased bone growth accompanied deficient Mmp-13 and Col10a1 gene expression in the hypertrophic zone. Transduction of small interfering RNA-GADD45beta in epiphyseal chondrocytes in vitro blocked terminal differentiation and the associated expression of Mmp-13 and Col10a1 mRNA in vitro. Finally, GADD45beta stimulated MMP-13 promoter activity in chondrocytes through the JNK-mediated phosphorylation of JunD, partnered with Fra2, in synergy with Runx2. These observations indicated that GADD45beta plays an essential role during chondrocyte terminal differentiation.

In vivo MRI of embryonic stem cells in a mouse model of myocardial infarction.

The therapeutic potential of administering stem cells to promote angiogenesis and myocardial tissue regeneration after infarction has recently been demonstrated. Given the advantages of using embryonic stem cells and mouse models of myocardial infarction for furthering the development of this therapeutic approach, the purpose of this study was to determine if embryonic stem cells could be loaded with superparamagnetic iron oxide (SPIO) particles and imaged in a mouse model of myocardial infarction over time using MRI. Mouse embryonic stem cells were labeled with SPIO particles. When incubated with 11.2, 22.4, and 44.8 microg Fe/ml of SPIO particles, cells took up increasing amounts of iron oxide. Embryonic stem cells loaded with SPIO compared to unlabeled cells had similar viability and proliferation profiles for up to 14 days. Free SPIO injected into infarcted myocardium was not observable within 12 hr after injection. After injection of three 10-microl aliquots of 10(7) SPIO-loaded cells/ml into infarcted myocardium, MRI demonstrated that the mouse embryonic stem cells were observable and could be seen for at least 5 weeks after injection. These findings support the ability of MRI to test the long-term therapeutic potential of embryonic stem cells in small animals in the setting of myocardial infarction.