Erg is a crucial regulator of endocardial-mesenchymal transformation during cardiac valve morphogenesis.

During murine embryogenesis, the Ets factor Erg is highly expressed in endothelial cells of the developing vasculature and in articular chondrocytes of developing bone. We identified seven isoforms for the mouse Erg gene. Four share a common translational start site encoded by exon 3 (Ex3) and are enriched in chondrocytes. The other three have a separate translational start site encoded by Ex4 and are enriched in endothelial cells. Homozygous Erg(ΔEx3/ΔEx3) knockout mice are viable, fertile and do not display any overt phenotype. By contrast, homozygous Erg(ΔEx4/ΔEx4) knockout mice are embryonic lethal, which is associated with a marked reduction in endocardial-mesenchymal transformation (EnMT) during cardiac valve morphogenesis. We show that Erg is required for the maintenance of the core EnMT regulatory factors that include Snail1 and Snail2 by binding to their promoter and intronic regions.

Ascorbic acid uptake and regulation of type I collagen synthesis in cultured vascular smooth muscle cells.

BACKGROUND/AIMS: Vascular smooth muscle cells contribute both to the structure and function of arteries, but are also involved in pathologic changes that accompany inflammatory diseases such as atherosclerosis. Since inflammation is associated with oxidant stress, we examined the uptake and cellular effects of the antioxidant vitamin ascorbic acid in cultured A10 vascular smooth muscle cells. METHODS/RESULTS: A10 cells concentrated ascorbate against a gradient in a sodium-dependent manner, most likely on the sodium-dependent vitamin C transporter type 2 (SVCT2) ascorbate transporter, which was present in immunoblots of cell extracts. Although ascorbate did not affect A10 cell proliferation, it stimulated radiolabeled proline incorporation and type I collagen synthesis. The latter was evident in the cells as increases in proalpha1(I) collagen and conversion of proalpha1(I) and proalpha2(I) collagen to mature forms that were released from the cells and deposited as extracellular matrix. Intracellular type I procollagen maturation was optimal at intracellular ascorbate concentrations of 200 microM and below, which were readily achieved by culture of the cells at plasma physiologic ascorbate concentrations. CONCLUSION: These results show that the SVCT2 facilitates ascorbate uptake by vascular smooth muscle cells, which in turn increases both the synthesis and maturation of type I collagen.

Comparison of fresh osteochondral autografts and allografts: a canine model.

BACKGROUND: Osteochondral autografts and allografts have been widely used in the treatment of isolated grade IV articular cartilage lesions of the knee. However, the authors are not aware of any study that has prospectively compared fresh osteochondral autografts to fresh allografts with regard to imaging, biomechanical testing, and histology. HYPOTHESIS: The imaging, biomechanical properties, and histologic appearance of fresh osteochondral autograft and fresh allograft are similar with respect to bony incorporation into host bone, articular cartilage composition, and biomechanical properties. STUDY DESIGN: Controlled laboratory study. METHODS: Eighteen adult dogs underwent bilateral knee osteochondral graft implantation after creation of an Outerbridge grade IV cartilage defect. One knee received an autograft, and the contralateral knee received a fresh allograft. Nine dogs were sacrificed at 3 months, and 9 dogs were sacrificed at 6 months. Graft analysis included gross examination, radiographs, magnetic resonance imaging, biomechanical testing, and histology. RESULTS: Magnetic resonance imaging demonstrated excellent bony incorporation of both autografts and allografts. Biomechanical testing demonstrated no significant difference between autografts versus allografts versus control at 3 or 6 months (P = .36-.91). A post hoc calculation showed 80% power to detect a 30% difference between allograft and control. Histologic examination showed normal cartilage structure for both autografts and allografts. CONCLUSION: Fresh osteochondral autograft and fresh allograft tissues are not statistically different with respect to bony incorporation, articular cartilage composition, or biomechanical properties up to 6 months after implantation. CLINICAL RELEVANCE: The use of fresh allograft tissue to treat osteochondral defects eliminates morbidity associated with harvesting autograft tissue without compromising the results of the surgical procedure.

Ascorbic acid decreases oxidant stress in endothelial cells caused by the nitroxide tempol.

Stable nitroxide radicals have been considered as therapeutic antioxidants because they can scavenge more toxic radicals in biologic systems. However, as radicals they also have the potential to increase oxidant stress in cells and tissues. We studied the extent to which this occurs in cultured EA.hy926 endothelial cells exposed to the nitroxide Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl). Tempol was rapidly reduced by the cells, as manifest by an increase in the ability of the cells to reduce extracellular ferricyanide and by disappearance of the Tempol EPR signal. Cells loaded with ascorbic acid, which directly reacts with Tempol, showed increased rates of Tempol-dependent ferricyanide reduction, and a more rapid loss of the Tempol EPR signal than cells not containing ascorbate. In this process, intracellular ascorbate was oxidized, and was depleted at lower Tempol concentrations than was GSH, another important intracellular low molecular weight antioxidant. Further evidence that Tempol concentrations of 100-1000 microM induced an oxidant stress was that it caused an increase in the oxidation of dihydrofluorescein in cells and inhibited ascorbate transport at concentrations as low as 50-100 microM. The presence of intracellular ascorbate both prevented dihydrofluorescein oxidation and spared GSH from oxidation by Tempol. Such sparing was not observed when GSH was depleted by other mechanisms, indicating that it was likely due to protection against oxidant stress. These results show that whereas Tempol may scavenge other more toxic radicals, care must be taken to ensure that it does not itself induce an oxidant stress, especially with regard to depletion of ascorbic acid.

Galectin-3: a biologic marker and diagnostic aid for chordoma.

Galectin-3 is a beta-galactoside binding protein. Its expression is quantitatively and qualitatively altered during self-proliferation, malignant transformation, and tumor progression. Galectin-3 is a lectin-related molecule. Lectins are proteins that bind specific carbohydrate structures. Although their precise biologic function is unclear, the general idea is that these molecules operate in modulating cell-to-cell and cell-to-matrix interactions. Galectins have been implicated in cell growth and differentiation and seem to play a role in malignant transformation and metastasis. Galectin-3 is expressed in primitive notochord. The purpose of the current investigation was to identify an immunohistochemical marker to help distinguish the pathologically overlapping entities of chordoma from myxoid low-grade chondrosarcoma. Twelve of 16 (75%) chordomas stained positive for Galectin-3 whereas only one of 12 low-grade myxoid chondrosarcomas stained positive. Galectin-3 chordoma staining is 75% sensitive and 92% specific.