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Platelet-derived growth factor (PDGF) is a major mediator of neointima formation after arterial injury. We constructed a recombinant adenovirus, Ad/PDGFtr, that expresses the soluble extracellular domain of the murine PDGFbeta receptor (PDGFtr). The expressed PDGFtr was appropriately glycosylated and secreted by chicken vascular smooth muscle cells (SMCs) in vitro. The expressed PDGFtr inhibited human PDGF-BB induced receptor autophosphorylation, and also inhibited PDGF-BB induced cell proliferation without affecting PDGF-AA induced mitogenesis. In vivo transduction of balloon-injured rooster femoral arteries with Ad/PDGFtr resulted in expression and secretion of the glycosylated PDGFtr. The expressed PDGFtr significantly inhibited neointima formation compared with controls. Neointima-associated thrombus was significantly reduced in Ad/PDGFtr transduced arteries compared with controls. Thus, in addition to impacting on SMC proliferation and migration, PDGF-BB plays a role in thrombus formation in response to arterial injury. Growth factor inhibition by localized gene delivery constitutes a powerful approach to intervene in the molecular pathways involved in vascular disease.

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Various genetic conditions produce dysfunctional osteoclasts resulting in osteopetrosis or osteosclerosis. These include human pycnodysostosis, an autosomal recessive syndrome caused by cathepsin K mutation, cathepsin K-deficient mice, and mitf mutant rodent strains. Cathepsin K is a highly expressed cysteine protease in osteoclasts that plays an essential role in the degradation of protein components of bone matrix. Cathepsin K also is expressed in a significant fraction of human breast cancers where it could contribute to tumor invasiveness. Mitf is a member of a helix-loop-helix transcription factor subfamily, which contains the potential dimerization partners TFE3, TFEB, and TFEC. In mice, dominant negative, but not recessive, mutations of mitf, produce osteopetrosis, suggesting a functional requirement for other family members. Mitf also has been found-and TFE3 has been suggested-to modulate age-dependent changes in osteoclast function. This study identifies cathepsin K as a transcriptional target of Mitf and TFE3 via three consensus elements in the cathepsin K promoter. Additionally, cathepsin K mRNA and protein were found to be deficient in mitf mutant osteoclasts, and overexpression of wild-type Mitf dramatically up-regulated expression of endogenous cathepsin K in cultured human osteoclasts. Cathepsin K promoter activity was disrupted by dominant negative, but not recessive, mouse alleles of mitf in a pattern that closely matches their osteopetrotic phenotypes. This relationship between cathepsin K and the Mitf family helps explain the phenotypic overlap of their corresponding deficiencies in pycnodysostosis and osteopetrosis and identifies likely regulators of cathepsin K expression in bone homeostasis and human malignancy.

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Microphthalmia (Mi) is a bHLHZip transcription factor that is essential for melanocyte development and postnatal function. It is thought to regulate both differentiated features of melanocytes such as pigmentation as well as proliferation/survival, based on phenotypes of mutant mouse alleles. Mi activity is controlled by at least two signaling pathways. Melanocyte-stimulating hormone (MSH) promotes transcription of the Mi gene through cAMP elevation, resulting in sustained Mi up-regulation over many hours. c-Kit signaling up-regulates Mi function through MAP kinase phosphorylation of Mi, thereby recruiting the p300 transcriptional coactivator. The current study reveals that c-Kit signaling triggers two phosphorylation events on Mi, which up-regulate transactivation potential yet simultaneously target Mi for ubiquitin-dependent proteolysis. The specific activation/degradation signals derive from MAPK/ERK targeting of serine 73, whereas serine 409 serves as a substrate for p90 Rsk-1. An unphosphorylatable double mutant at these two residues is at once profoundly stable and transcriptionally inert. These c-Kit-induced phosphorylations couple transactivation to proteasome-mediated degradation. c-Kit signaling thus triggers short-lived Mi activation and net Mi degradation, in contrast to the profoundly increased Mi expression after MSH signaling, potentially explaining the functional diversity of this transcription factor in regulating proliferation, survival, and differentiation in melanocytes.

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Avian models of atherosclerosis helped pioneer the study of vascular biology, and offer economic and technical advantages over mammalian models. As an initial step towards investigating important molecular pathways involved in avian atherogenesis and restenosis, we developed a recombinant adenovirus (Ad) which expresses the reporter gene beta-galactosidase (beta-gal), and applied it to cultured chicken vascular smooth muscle cells (SMCs) and a rooster model of acute vascular injury. In cultured chicken SMCs, recombinant gene expression increased as a function of multiplicity of infection (MOI) and incubation time. Maximal expression occurred at an MOI of 10(4) plaque-forming units (pfu)/cell with approximately 50% of quiescent and non-quiescent chicken SMCs expressing beta-gal. Human aorta SMCs had two- to four-fold increased beta-gal expression compared with chicken SMCs at all MOI and incubation times. In vivo instillation of recombinant Ad into uninjured rooster femoral artery segments revealed low efficiency endothelial cell expression of the reporter gene. In contrast, recombinant Ad infection of rooster femoral artery segments 3-21 days after balloon injury revealed up to 60% of luminal surface beta-gal expression, confined predominantly to the neointimal layer. Peak reporter gene expression efficiencies occurred in arterial segments infected 3 days after balloon injury. Uninfected and control Ad infected arteries had no detectable beta-gal expression. Rooster neointimal cells targeted by the recombinant Ad were identified as alpha-smooth muscle actin containing cells by immunohistochemistry. We conclude that Ad-mediated gene transfer is efficient and selective for the neointima in the rooster acute vascular injury model, and offers the potential to efficiently introduce exogenous genes that may impact on the injury response. This model of acute vascular injury may also be manipulated into more established avian models of atherosclerosis, permitting the investigation of acute injury progression to chronic injury.

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Left ventricular wall motion abnormalities, aneurysm formation, and progression to global hypokinesis have been described in patients with myocarditis and in patients with hypertrophic cardiomyopathy. We document a case of reversible aneurysm formation, cardiogenic shock, and complete recovery in a patient with myocarditis and hypertrophic cardiomyopathy. Pathophysiologic mechanisms of myocardial injury and recovery are discussed.

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The amino acid sequences deduced from cDNA clones of human lamin A and lamin C show identity between these two lamins except for an extra 9.0-kDa carboxyl-terminal tail that is present only in lamin A. Both lamins A and C contain an alpha-helical domain of approximately 360 residues that shows striking homology to a corresponding alpha-helical rod domain that is the structural hallmark of all intermediate filament proteins. However, the lamin alpha-helical domain is 14% larger than that of the intermediate filament proteins. In addition to the extensive homology to intermediate filament proteins as reported [McKeon, F., Kirschner, M. & Caput, D. (1986) Nature (London) 319, 463-468], a different 82-amino acid residue stretch at the carboxyl terminus of lamin A has been deduced and verified by amino acid sequencing. This region contains sequence homology to amino- and carboxylterminal domains of type I and type II epidermal keratins. Implications of the presence of these and other domains in lamins A and C for the assembly of the nuclear lamina are discussed.

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