79 research outputs found

    Flow velocity mapping using contrast enhanced high-frame-rate plane wave ultrasound and image tracking: methods and initial in vitro and in vivo evaluation

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    Ultrasound imaging is the most widely used method for visualising and quantifying blood flow in medical practice, but existing techniques have various limitations in terms of imaging sensitivity, field of view, flow angle dependence, and imaging depth. In this study, we developed an ultrasound imaging velocimetry approach capable of visualising and quantifying dynamic flow, by combining high-frame-rate plane wave ultrasound imaging, microbubble contrast agents, pulse inversion contrast imaging and speckle image tracking algorithms. The system was initially evaluated in vitro on both straight and carotid-mimicking vessels with steady and pulsatile flows and in vivo in the rabbit aorta. Colour and spectral Doppler measurements were also made. Initial flow mapping results were compared with theoretical prediction and reference Doppler measurements and indicate the potential of the new system as a highly sensitive, accurate, angle-independent and full field-of-view velocity mapping tool capable of tracking and quantifying fast and dynamic flows

    Mass Transport Properties of the Rabbit Aortic Wall

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    Uptake of circulating macromolecules by the arterial wall may be a critical step in atherogenesis. Here we investigate the age-related changes in patterns of uptake that occur in the rabbit. In immature aortas, uptake was elevated in a triangle downstream of branch ostia, a region prone to disease in immature rabbits and children. By 16-22 months, uptake was high lateral to ostia, as is lesion prevalence in mature rabbits and young adults. In older rabbits there was a more upstream pattern, similar to the disease distribution in older people. These variations were predominantly caused by the branches themselves, rather than reflecting larger patterns within which the branches happened to be situated (as may occur with patterns of haemodynamic wall shear stress). The narrow streaks of high uptake reported in some previous studies were shown to be post mortem artefacts. Finally, heparin (which interferes with the NO pathway) had no effect on the difference in uptake between regions upstream and downstream of branches in immature rabbits but reversed the difference in older rabbits, as does inhibiting NO synthesis directly. Nevertheless, examination of uptake all around the branch showed that changes occurred at both ages and that they were quite subtle, potentially explaining why inhibiting NO has only minor effects on lesion patterns in mature rabbits and contradicting the earlier conclusion that mechanotransduction pathways change with age. We suggest that recently-established changes in the patterns of haemodynamic forces themselves are more likely to account for the age-dependence of uptake patterns

    Novel Characterization of Lymphatic Valve Formation during Corneal Inflammation

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    Lymphatic research has progressed rapidly in recent years. Though lymphatic dysfunction has been found in a wide array of disorders from transplant rejection to cancer metastasis, to date, there is still little effective treatment for lymphatic diseases. The cornea offers an optimal site for lymphatic research due to its accessible location, transparent nature, and lymphatic-free but inducible features. However, it still remains unknown whether lymphatic valves exist in newly formed lymphatic vessels in the cornea, and how this relates to an inflammatory response. In this study, we provide the first evidence showing that lymphatic valves were formed in mouse cornea during suture-induced inflammation with the up-regulation of integrin alpha 9. The number of corneal valves increased with the progression of inflammatory lymphangiogenesis. Moreover, we have detected lymphatic valves at various developmental stages, from incomplete to more developed ones. In addition to defining the average diameter of lymphatic vessels equipped with lymphatic valves, we also report that lymphatic valves were more often located near the branching points. Taken together, these novel findings not only provide new insights into corneal lymphatic formation and maturation, but also identify a new model for future investigation on lymphatic valve formation and possibly therapeutic intervention

    High-resolution 3D analysis of mouse small-intestinal stroma.

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    Here we detail a protocol for whole-mount immunostaining of mouse small-intestinal villi that can be used to generate high-resolution 3D images of all gut cell types, including blood and lymphatic vessel cells, neurons, smooth muscle cells, fibroblasts and immune cells. The procedure describes perfusion, fixation, dissection, immunostaining, mounting, clearing, confocal imaging and quantification, using intestinal vasculature as an example. As intestinal epithelial cells prevent visualization with some antibodies, we also provide an optional protocol to remove these cells before fixation. In contrast to alternative current techniques, our protocol enables the entire villus to be visualized with increased spatial resolution of cell location, morphology and cell-cell interactions, thus allowing for easy quantification of phenotypes. The technique, which takes 7 d from mouse dissection to microscopic examination, will be useful for researchers who are interested in most aspects of intestinal biology, including mucosal immunology, infection, nutrition, cancer biology and intestinal microbiota

    Genome-Wide Gene Expression Analysis Implicates the Immune Response and Lymphangiogenesis in the Pathogenesis of Fetal Chylothorax

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    Fetal chylothorax (FC) is a rare condition characterized by lymphocyte-rich pleural effusion. Although its pathogenesis remains elusive, it may involve inflammation, since there are increased concentrations of proinflammatory mediators in pleural fluids. Only a few hereditary lymphedema-associated gene loci, e.g. VEGFR3, ITGA9 and PTPN11, were detected in human fetuses with this condition; these cases had a poorer prognosis, due to defective lymphangiogenesis. In the present study, genome-wide gene expression analysis was conducted, comparing pleural and ascitic fluids in three hydropic fetuses, one with and two without the ITGA9 mutation. One fetus (the index case), from a dizygotic pregnancy (the cotwin was unaffected), received antenatal OK-432 pleurodesis and survived beyond the neonatal stage, despite having the ITGA9 mutation. Genes and pathways involved in the immune response were universally up-regulated in fetal pleural fluids compared to those in ascitic fluids. Furthermore, genes involved in the lymphangiogenesis pathway were down-regulated in fetal pleural fluids (compared to ascitic fluid), but following OK-432 pleurodesis, they were up-regulated. Expression of ITGA9 was concordant with overall trends of lymphangiogenesis. In conclusion, we inferred that both the immune response and lymphangiogenesis were implicated in the pathogenesis of fetal chylothorax. Furthermore, genome-wide gene expression microarray analysis may facilitate personalized medicine by selecting the most appropriate treatment, according to the specific circumstances of the patient, for this rare, but heterogeneous disease

    The Receptor Tyrosine Kinase Alk Controls Neurofibromin Functions in Drosophila Growth and Learning

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    Anaplastic Lymphoma Kinase (Alk) is a Receptor Tyrosine Kinase (RTK) activated in several cancers, but with largely unknown physiological functions. We report two unexpected roles for the Drosophila ortholog dAlk, in body size determination and associative learning. Remarkably, reducing neuronal dAlk activity increased body size and enhanced associative learning, suggesting that its activation is inhibitory in both processes. Consistently, dAlk activation reduced body size and caused learning deficits resembling phenotypes of null mutations in dNf1, the Ras GTPase Activating Protein-encoding conserved ortholog of the Neurofibromatosis type 1 (NF1) disease gene. We show that dAlk and dNf1 co-localize extensively and interact functionally in the nervous system. Importantly, genetic or pharmacological inhibition of dAlk rescued the reduced body size, adult learning deficits, and Extracellular-Regulated-Kinase (ERK) overactivation dNf1 mutant phenotypes. These results identify dAlk as an upstream activator of dNf1-regulated Ras signaling responsible for several dNf1 defects, and they implicate human Alk as a potential therapeutic target in NF1

    An Evolutionary Conserved Role for Anaplastic Lymphoma Kinase in Behavioral Responses to Ethanol

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    Anaplastic lymphoma kinase (Alk) is a gene expressed in the nervous system that encodes a receptor tyrosine kinase commonly known for its oncogenic function in various human cancers. We have determined that Alk is associated with altered behavioral responses to ethanol in the fruit fly Drosophila melanogaster, in mice, and in humans. Mutant flies containing transposon insertions in dAlk demonstrate increased resistance to the sedating effect of ethanol. Database analyses revealed that Alk expression levels in the brains of recombinant inbred mice are negatively correlated with ethanol-induced ataxia and ethanol consumption. We therefore tested Alk gene knockout mice and found that they sedate longer in response to high doses of ethanol and consume more ethanol than wild-type mice. Finally, sequencing of human ALK led to the discovery of four polymorphisms associated with a low level of response to ethanol, an intermediate phenotype that is predictive of future alcohol use disorders (AUDs). These results suggest that Alk plays an evolutionary conserved role in ethanol-related behaviors. Moreover, ALK may be a novel candidate gene conferring risk for AUDs as well as a potential target for pharmacological intervention

    Leukocyte Tyrosine Kinase Functions in Pigment Cell Development

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    A fundamental problem in developmental biology concerns how multipotent precursors choose specific fates. Neural crest cells (NCCs) are multipotent, yet the mechanisms driving specific fate choices remain incompletely understood. Sox10 is required for specification of neural cells and melanocytes from NCCs. Like sox10 mutants, zebrafish shady mutants lack iridophores; we have proposed that sox10 and shady are required for iridophore specification from NCCs. We show using diverse approaches that shady encodes zebrafish leukocyte tyrosine kinase (Ltk). Cell transplantation studies show that Ltk acts cell-autonomously within the iridophore lineage. Consistent with this, ltk is expressed in a subset of NCCs, before becoming restricted to the iridophore lineage. Marker analysis reveals a primary defect in iridophore specification in ltk mutants. We saw no evidence for a fate-shift of neural crest cells into other pigment cell fates and some NCCs were subsequently lost by apoptosis. These features are also characteristic of the neural crest cell phenotype in sox10 mutants, leading us to examine iridophores in sox10 mutants. As expected, sox10 mutants largely lacked iridophore markers at late stages. In addition, sox10 mutants unexpectedly showed more ltk-expressing cells than wild-type siblings. These cells remained in a premigratory position and expressed sox10 but not the earliest neural crest markers and may represent multipotent, but partially-restricted, progenitors. In summary, we have discovered a novel signalling pathway in NCC development and demonstrate fate specification of iridophores as the first identified role for Ltk

    HHEX is a transcriptional regulator of the VEGFC/FLT4/PROX1 signaling axis during vascular development.

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    Formation of the lymphatic system requires the coordinated expression of several key regulators: vascular endothelial growth factor C (VEGFC), its receptor FLT4, and a key transcriptional effector, PROX1. Yet, how expression of these signaling components is regulated remains poorly understood. Here, using a combination of genetic and molecular approaches, we identify the transcription factor hematopoietically expressed homeobox (HHEX) as an upstream regulator of VEGFC, FLT4, and PROX1 during angiogenic sprouting and lymphatic formation in vertebrates. By analyzing zebrafish mutants, we found that hhex is necessary for sprouting angiogenesis from the posterior cardinal vein, a process required for lymphangiogenesis. Furthermore, studies of mammalian HHEX using tissue-specific genetic deletions in mouse and knockdowns in cultured human endothelial cells reveal its highly conserved function during vascular and lymphatic development. Our findings that HHEX is essential for the regulation of the VEGFC/FLT4/PROX1 axis provide insights into the molecular regulation of lymphangiogenesis

    Plasma and cellular fibronectin: distinct and independent functions during tissue repair

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    Fibronectin (FN) is a ubiquitous extracellular matrix (ECM) glycoprotein that plays vital roles during tissue repair. The plasma form of FN circulates in the blood, and upon tissue injury, is incorporated into fibrin clots to exert effects on platelet function and to mediate hemostasis. Cellular FN is then synthesized and assembled by cells as they migrate into the clot to reconstitute damaged tissue. The assembly of FN into a complex three-dimensional matrix during physiological repair plays a key role not only as a structural scaffold, but also as a regulator of cell function during this stage of tissue repair. FN fibrillogenesis is a complex, stepwise process that is strictly regulated by a multitude of factors. During fibrosis, there is excessive deposition of ECM, of which FN is one of the major components. Aberrant FN-matrix assembly is a major contributing factor to the switch from normal tissue repair to misregulated fibrosis. Understanding the mechanisms involved in FN assembly and how these interplay with cellular, fibrotic and immune responses may reveal targets for the future development of therapies to regulate aberrant tissue-repair processes
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