As classically described, the precardiac mesoderm of the paired heart-forming fields migrate and fuse anteriomedially in the ventral midline to form the first segment of the straight heart tube. This segment ultimately forms the right trabeculated ventricle. Additional segments are added to the caudal end of the first in a sequential fashion from the posteriolateral heart-forming field mesoderm. In this study we report that the final major heart segment, which forms the cardiac outflow tract, does not follow this pattern of embryonic development. The cardiac outlet, consisting of the conus and truncus, does not derive from the paired heart-forming fields, but originates separately from a previously unrecognized source of mesoderm located anterior to the initial primitive heart tube segment. Fate-mapping results show that cells labeled in the mesoderm surrounding the aortic sac and anterior to the primitive right ventricle are incorporated into both the conus and the truncus. Conversely, if cells are labeled in the existing right ventricle no incorporation into the cardiac outlet is observed. Tissue explants microdissected from this anterior mesoderm region are capable of forming beating cardiac muscle in vitro when cocultured with explants of the primitive right ventricle. These findings establish the presence of another heart-forming field. This anterior heart-forming field (AHF) consists of mesoderm surrounding the aortic sac immediately anterior to the existing heart tube. This new concept of the heart outlet's embryonic origin provides a new basis for explaining a variety of gene-expression patterns and cardiac defects described in both transgenic animals and human congenital heart disease.

Bone morphogenetic proteins (BMPs) have been shown to induce ectopic expression of cardiac transcription factors and beating cardiomyocytes in nonprecardiac mesodermal cells in chicks, suggesting that BMPs are inductive signaling molecules that participate in the development of the heart. However, the precise molecular mechanisms by which BMPs regulate cardiac development are largely unknown. In the present study, we examined the molecular mechanisms by which BMPs induce cardiac differentiation by using the P19CL6 in vitro cardiomyocyte differentiation system, a clonal derivative of P19 embryonic teratocarcinoma cells. We established a permanent P19CL6 cell line, P19CL6noggin, which constitutively overexpresses the BMP antagonist noggin. Although almost all parental P19CL6 cells differentiate into beating cardiomyocytes when treated with 1% dimethyl sulfoxide, P19CL6noggin cells did not differentiate into beating cardiomyocytes nor did they express cardiac transcription factors or contractile protein genes. The failure of differentiation was rescued by overexpression of BMP-2 or addition of BMP protein to the culture media, indicating that BMPs were indispensable for cardiomyocyte differentiation in this system. Overexpression of TAK1, a member of the mitogen-activated protein kinase kinase kinase superfamily which transduces BMP signaling, restored the ability of P19CL6noggin cells to differentiate into cardiomyocytes and concomitantly express cardiac genes, whereas overexpression of the dominant negative form of TAK1 in parental P19CL6 cells inhibited cardiomyocyte differentiation. Overexpression of both cardiac transcription factors Csx/Nkx-2.5 and GATA-4 but not of Csx/Nkx-2.5 or GATA-4 alone also induced differentiation of P19CL6noggin cells into cardiomyocytes. These results suggest that TAK1, Csx/Nkx-2.5, and GATA-4 play a pivotal role in the cardiogenic BMP signaling pathway.

Growth factors, cell-surface receptors, adhesion molecules, and extracellular matrix proteins play critical roles in vascular pathophysiology by affecting growth, migration, differentiation, and survival of vascular cells. In a search for secreted and cell-surface molecules expressed in the cardiovascular system, by using a retrovirus-mediated signal sequence trap method, we isolated a cell-surface protein named vasorin. Vasorin is a typical type I membrane protein, containing tandem arrays of a characteristic leucine-rich repeat motif, an epidermal growth factor-like motif, and a fibronectin type III-like motif at the extracellular domain. Expression analyses demonstrated that vasorin is predominantly expressed in vascular smooth muscle cells, and that its expression is developmentally regulated. To clarify biological functions of vasorin, we searched for its binding partners and found that vasorin directly binds to transforming growth factor (TGF)-␤ and attenuates TGF-␤ signaling in vitro. Vasorin expression was downregulated during vessel repair after arterial injury, and reversal of vasorin down-regulation, by using adenovirus-mediated in vivo gene transfer, significantly diminished injury-induced vascular lesion formation, at least in part, by inhibiting TGF-␤ signaling in vivo. These results suggest that down-regulation of vasorin expression contributes to neointimal formation after vascular injury and that vasorin modulates cellular responses to pathological stimuli in the vessel wall. Thus, vasorin is a potential therapeutic target for vascular fibroproliferative disorders. V ascular smooth muscle cells (VSMCs), the major cell type in the vessel wall, show a spectrum of phenotypes, depending on environmental cues. Various injurious stimuli provoke the proliferation of differentiated medial VSMCs, which migrate to the intima and produce extracellular matrix proteins, resulting in the narrowing of the vascular lumen. These processes, called VSMC phenotypic modulation, play a key role in development of atherosclerotic diseases, such as postangioplasty restenosis, vein graft disease, and transplant vasculopathy. Whereas tremendous progress has been made in identifying growth factors and transcription factors that regulate the vascular response to injury, much information is lacking regarding cell-surface molecules that are involved in the pathogenesis of vascular fibroproliferative disorders. The signal sequence trap (SST) is a strategy to identify cDNAs containing signal sequence that encode secreted and type I membrane proteins (1, 2). We recently developed a refined SST system based on retrovirusmediated expression screening (SST-REX) (3). In a search for secreted and cell-surface molecules expressed in the cardiovascular system, by using SST-REX, we identified a TGF-␤ binding protein, vasorin. Vasorin is predominantly expressed in VSMCs and modulates the vascular response to injury, at least in part, by attenuating TGF-␤ signaling in vivo. Here, we describe the molecular and functional characteristics of...

Dnm3os, a gene that is transcribed into a non-coding RNA (ncRNA), contains three micro RNAs (miRNAs), miR-199a, miR-199a*, and miR-214, whose functions remain unknown in mammals. In this study, we introduced the lacZ gene into the Dnm3os locus to recapitulate its expression pattern and disrupt its function. Dnm3os ؉/lacZ heterozygous embryos showed ␤-galactosidase activity, which reflected the authentic expression pattern of Dnm3os RNA. Most of the Dnm3os lacZ/lacZ homozygous pups died within one month of birth. After birth, Dnm3os lacZ/lacZ mice exhibited several skeletal abnormalities, including craniofacial hypoplasia, defects in dorsal neural arches and spinous processes of the vertebrae, and osteopenia. Importantly, the expression of miR-199a, miR-199a*, and miR-214 was significantly downregulated in Dnm3os lacZ/lacZ embryos, supporting the assumption that Dnm3os serves as a precursor of these three miRNAs. Thus, Dnm3os has emerged as an miRNA-encoding gene that is indispensable for normal skeletal development and body growth in mammals.

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Vascular endothelial growth factor (VEGF or vascular permeability factor), a direct-acting, endothelial cell-specific mitogen, has been suggested to be involved in development and maintenance of vasculatures in tumor neovascularization and in normal tissues. To investigate possible roles of VEGF in ischemic hearts, we studied induction of VEGF mRNA by ischemia and hypoxia using coronary artery-ligated hearts in vivo and perfused hearts and cultured myocardial cells in vitro. VEGF mRNA was potently induced by ischemia in the heart in vivo. In perfused hearts, maximum expression was rapidly induced (within 30 min) by transient reversible ischemia (5-10 min of ischemia) and lasted at least 3 h. Induction was also caused by hypoxia, which was confirmed in perfused hearts and cultured myocardial cells. These results suggest that induction of VEGF mRNA is upregulated by oxygen deprivation in the heart and that not only infarction but also chronic ischemia in the clinical setting could induce VEGF as a potent angiogenesis factor to stimulate coronary collateral formation.

Background-In our previous study, adrenomedullin (AM) overexpression could limit the arterial intimal hyperplasia induced by cuff injury in rats. However, it remains to be elucidated whether endogenous AM plays a role against vascular injury. Methods and Results-We used the AM knockout mice to investigate the effect of endogenous AM. Compared with wild-type (AM ϩ/ϩ ) mice, heterozygous AM knockout (AM ϩ/Ϫ ) mice had the increased intimal thickening of the cuff-injured femoral artery, concomitantly with lesser AM staining. In AM ϩ/Ϫ mice, cuff placement increased both the production of superoxide anions (O 2 Ϫ ) measured by coelentarazine chemiluminescence and the immunostaining of p67 phox and gp91 phox , subunits of NAD(P)H oxidase in the adventitia, associated with the increment of CD45-positive leukocytes, suggesting that the stimulated formation of radical oxygen species accompanied chronic adventitial inflammation. Not only the AM gene transfection but also the treatment of NAD(P)H oxidase inhibitor apocynin and membrane-permeable superoxide dismutase mimetic tempol could limit cuff-induced intimal hyperplasia in AM

We conducted a pilot study to assess the feasibility and efficacy of immunotherapy for stage IV malignant melanoma patients resistant to conventional therapies involving vaccination with mature dendritic cells (mDCs) combined with administration of low dose interleukin-2. Autologous monocytes were harvested from a single apheresis and cultured for 7 days with granulocyte-macrophage colony-stimulating factor and interleukin-4, yielding immature dendritic cells (iDCs), which were then cryopreserved until use. For 4 days prior to vaccination, iDCs were exposed to autologous tumour lysate combined with tumour necrosis factor-alpha to induce terminal differentiation into mDCs. Patients were then vaccinated weekly with 107 mDCs for 10 weeks and given 350-700 kIU of interleukin-2 three times per week. Of the 10 patients in the study, one showed stable disease, seven showed progressive disease, and two showed mixed responses, including partial tumour regression, and were therefore given 20 additional injections. Only minimal adverse events were noted, including localized skin reactions and mild fever (NIH-CTC grade 0-1). Median survival from the first vaccination was 240 days (range 31-735 days). In vitro, melanoma patient-derived dendritic cells (DCs) showed reduced cell surface expression of CD1a antigen on iDCs and reduced CD86 and HLA-DR expression on mDCs. In addition, antigen uptake, chemotaxis and antigen presentation were all attenuated in DCs from the patients. In summary, although improvement of clinical efficacy will require further research, autologous tumour lysate-pulsed monocyte-derived mDCs could be safely harvested, cryopreserved and administrated to patients without obvious complications.