ET-1 stimulates pulmonary arterial smooth muscle cell proliferation via induction of reactive oxygen species.

Recent studies implicate reactive oxygen species (ROS) such as superoxide anions and H(2)O(2) in the proliferation of systemic vascular smooth muscle cells (SMCs). However, the role of ROS in SMC proliferation within the pulmonary circulation remains unclear. We investigated the effects of endothelin-1 (ET-1), a potential SMC mitogen, on ROS production and proliferation of fetal pulmonary artery SMCs (FPASMCs). Exposure to ET-1 resulted in increases in superoxide production and viable FPASMCs after 72 h. These increases were prevented by pretreatment with PD-156707. Treatment with pertussis toxin blocked the effects of ET-1, whereas cholera toxin stimulated superoxide production and increased viable cell numbers even in the absence of ET-1. Wortmannin, LY-294002, diphenyleneiodonium (DPI), 4-(2-aminoethyl)benzenesulfonyl fluoride, and apocynin also prevented the ET-1-mediated increases in superoxide production and viable cell numbers. Exposure to H(2)O(2) or diethyldithiocarbamate increased viable cell number by 37% and 50%, respectively. Conversely, ascorbic acid and DPI decreased viable cell number, which appeared to be due to an increase in programmed cell death. Our data suggest that ET-1 exerts a mitogenic effect on FPASMCs via an increase in ROS production and that antioxidants can block this effect via induction of apoptosis. Antioxidant treatment may therefore represent a potential therapy for pulmonary vascular diseases.

Positive and negative regulation of epicardial-mesenchymal transformation during avian heart development.

In the developing heart, the epicardium is essential for coronary vasculogenesis as it provides precursor cells that become coronary vascular smooth muscle and perivascular fibroblasts. These precursor cells are derived from the epicardium via epithelial-mesenchymal transformation (EMT). The factors that regulate epicardial EMT are unknown. Using a quantitative in vitro collagen gel assay, we show that serum, FGF-1, -2, and -7, VEGF, and EGF stimulate epicardial EMT. TGFbeta-1 stimulates EMT only weakly, while TGFbeta-2 and -3 do not stimulate EMT. TGFbeta-1, -2, or -3 strongly inhibits transformation of epicardial cells stimulated with FGF-2 or heart-conditioned medium. TGFbeta-3 does not block expression of vimentin, a mesenchymal marker, but appears to inhibit EMT by blocking epithelial cell dissociation and subsequent extracellular matrix invasion. Blocking antisera directed against FGF-1, -2, or -7 substantially inhibit conditioned medium-stimulated EMT in vitro, while antibodies to TGFbeta-1, -2, or -3 increase it. We confirmed FGF stimulation and TGFbeta inhibition of epicardial EMT in organ culture. Immunoblot analysis confirmed the presence of FGF-1, -2, and -7 and TGFbeta-1, -2, and -3 in conditioned medium, and we localized these growth factors to the myocardium and epicardium of stage-appropriate embryos by immunofluorescence. Our results strongly support a model in which myocardially derived FGF-1, -2, or -7 promotes epicardial EMT, while TGFbeta-1, -2, or -3 restrains it. Epicardial EMT appears to be regulated through a different signaling pathway than endocardial EMT.

Embryonic expression of the divergent Drosophila beta3-tubulin isoform is required for larval behavior.

We have sought to define the developmental and cellular roles played by differential expression of distinct beta-tubulins. Drosophila beta3-tubulin (beta3) is a structurally divergent isoform transiently expressed during midembryogenesis. Severe beta3 mutations cause larval lethality resulting from failed gut function and consequent starvation. However, mutant larvae also display behavioral abnormalities consistent with defective sensory perception. We identified embryonic beta3 expression in several previously undefined sites, including different types of sensory organs. We conclude that abnormalities in foraging behavior and photoresponsiveness exhibited by prelethal mutant larvae reflect defective beta3 function in the embryo during development of chordotonal and other mechanosensory organs and of Bolwig's organ and nerve. We show that microtubule organization in the cap cells of chordotonal organs is altered in mutant larvae. Thus transient zygotic beta3 expression has permanent consequences for the architecture of the cap cell microtubule cytoskeleton in the larval sensilla, even when beta3 is no longer present. Our data provide a link between the microtubule cytoskeleton in embryogenesis and the behavioral phenotype manifested as defective proprioreception at the larval stage.

Common epicardial origin of coronary vascular smooth muscle, perivascular fibroblasts, and intermyocardial fibroblasts in the avian heart.

Previous studies have shown that during avian heart development, epicardial and coronary vascular smooth muscle precursors are derived from the proepicardium, a derivative of the developing liver. This finding led to a model of coronary vascular development in which epicardial cells migrate over the postlooped heart, followed by migration of committed endothelial and smooth muscle precursors from the proepicardium through the subepicardial matrix where the coronary arteries develop. Here we show that epicardial cells undergo epithelial-mesenchymal transformation to become coronary vascular smooth muscle, perivascular fibroblasts, and intermyocardial fibroblasts. We began by establishing primary cultures of quail epicardial cells that retain morphologic and antigenic identity to epicardial cells in vivo. Quail epicardial monolayers stimulated with serum or vascular growth factors produced invasive mesenchyme in collagen gels. Chick epicardial cells labeled in ovo with DiI invaded the subepicardial extracellular matrix, demonstrating that mesenchymal transformation of epicardium occurs in vivo. To determine the fates of epicardially derived mesenchymal cells, quail epicardial cells labeled in vitro with LacZ were grafted into the pericardial space of E2 chicks. These cells attached to the heart, formed a chimeric epicardium, invaded the subepicardial matrix and myocardial wall, and became coronary vascular smooth muscle, perivascular fibroblasts, and intermyocardial fibroblasts, demonstrating the common epicardial origin of these cell types. A general model of coronary vascular development should now include epicardial-mesenchymal transformation and direct participation of mesenchyme derived from the epicardium in coronary morphogenesis.

Tenascin-X deficiency is associated with Ehlers-Danlos syndrome.

The tenascins are a family of large extracellular matrix proteins with at least three members: tenascin-X (TNX), tenascin-C (TNC, or cytotactin) and tenascin-R (TN-R, or restrictin). Although the tenascins have been implicated in a number of important cellular processes, no function has been clearly established for any tenascin. We describe a new contiguous-gene syndrome, involving the CYP21B and TNX genes, that results in 21-hydroxylase deficiency and a connective-tissue disorder consisting of skin and joint hyperextensibility, vascular fragility and poor wound healing. The connective tissue findings are typical of the Ehlers-Danlos syndrome (EDS). The abundant expression of TNX in connective tissues is consistent with a role in EDS, and our patient's skin fibroblasts do not synthesize TNX protein in vitro or in vivo. His paternal allele carries a novel deletion arising from recombination between TNX and its partial duplicate gene, XA, which precludes TNX synthesis. Absence of TNX mRNA and protein in the proband, mapping of the TNX gene and HLA typing of this family suggest recessive inheritance of TNX deficiency and connective-tissue disease. Although the precise role of TNX in the pathogenesis of EDS is uncertain, this patient's findings suggest a unique and essential role for TNX in connective-tissue structure and function.

Genetic analysis of the Drosophila beta3-tubulin gene demonstrates that the microtubule cytoskeleton in the cells of the visceral mesoderm is required for morphogenesis of the midgut endoderm.

We have investigated the cellular basis for lethality of mutant alleles of the Drosophila melanogaster beta3-tubulin gene, betaTub60D. Lethal beta3 mutations can be grouped into two classes: the most severe mutations (Class I alleles) cause death during the first larval instar, while weaker alleles (Class II) cause death in later larval stages or in early pupal development. Since beta3 is not expressed during larval development, lethality of the Class I mutations must reflect essential functions of beta3 in embryogenesis. Beta3-tubulin is zygotically expressed during midembryogenesis in the developing mesoderm, and the major site of beta3 accumulation is in the developing muscles during myogenesis. We show that the embryonic pattern of beta3 expression, including accumulation in the developing musculature, is conserved in other Drosophila species. However, we found that loss of beta3 function does not cause discernible defects in either the ultrastructure or function of the larval muscle. Thus beta3-tubulin is dispensable in its highest site of accumulation. Rather, the essential site of function of beta3 in embryos is in cells of the visceral mesoderm. Lethality of Class I alleles is caused by defects in midgut morphogenesis and failure of gut function. Although the folding pattern is irregular and the gut is smaller than normal, a complete folded gut forms in mutant larvae, and the visceral muscle functions normally to move food through the gut. However, mutant larvae cannot absorb nutrients across the gut wall. Thus loss of beta3 function in the mesoderm results in defects in the underlying endodermally derived layer of the gut. Our data provide an assay for cellular interactions between mesoderm and endodermal tissues and reveal a role for the microtubule cytoskeleton of the visceral mesodermal cells in differentiation of the endodermal cell layer of the larval gut.

The beta 3-tubulin gene of Drosophila melanogaster is essential for viability and fertility.

We have previously shown that the beta 3-tubulin gene of Drosophila melanogaster encodes a divergent isoform expressed in a complex developmental pattern. The beta 3 gene is transiently expressed in the embryo and again in the pupa at high levels in the developing musculature, and at lower levels in several different pupal tissues of ectodermal origin. Adult expression is confined to specific somatic cells in the gonads. In some of the cell types in which it is expressed, beta 3 is the sole or predominant beta-tubulin, while in others the beta 3 protein is a minor component of the beta-tubulin pool. The sites and timing of beta 3 expression demonstrated that beta 3-tubulin is utilized primarily in cytoplasmic microtubule arrays involved in changes in cell shape and tissue organization, and suggested to us that this isoform may be functionally specialized. To determine whether the expression of the beta 3 gene is essential for normal development, and to examine the specific functions of this divergent isoform, we have generated mutations within the gene. We determined that the small deficiency Df(2R)Px2, which deletes the 60C5,6-60D9,10 region of chromosome 2, removes all of the beta 3 coding sequences, and that the distal breakpoint of the deficiency is approximately 2 kb upstream from the start of transcription of the beta 3 gene. We have generated a total of 31 ethyl methanesulfonate- or diepoxybutane-induced recessive lethal or visible mutations which map within the deficiency. These mutations define 12 new lethal complementation groups, which together with two previously identified visible mutations, altogether identify 14 genes in this interval of the second chromosome. A lethal complementation group comprising mutations in the beta 3-tubulin gene (beta Tub60D) was identified by rescue of their lethality by a wild-type copy of the gene introduced into the genome via P element-mediated germ line transformation. Analysis of the homozygous and transheterozygous phenotypes of the five beta 3 mutations recovered (alleles designated B3t1-B3t5) demonstrates that beta 3-tubulin is essential for viability and fertility.