Absence of cardiovascular manifestations in a haploinsufficient Tgfbr1 mouse model

Loeys-Dietz syndrome (LDS) is an autosomal dominant arterial aneurysm disease belonging to the spectrum of transforming growth factor β (TGFβ)-associated vasculopathies. In its most typical form it is characterized by the presence of hypertelorism, bifid uvula/cleft palate and aortic aneurysm and/or arterial tortuosity. LDS is caused by heterozygous loss of function mutations in the genes encoding TGFβ receptor 1 and 2 (TGFBR1 and -2), which lead to a paradoxical increase in TGFβ signaling. To address this apparent paradox and to gain more insight into the pathophysiology of aneurysmal disease, we characterized a new Tgfbr1 mouse model carrying a p.Y378*nonsense mutation. Study of the natural history in this model showed that homozygous mutant mice die during embryonic development due to defective vascularization. Heterozygous mutant mice aged 6 and 12 months were morphologically and (immuno)histochemically indistinguishable from wild-type mice. We show that the mutant allele is degraded by nonsense mediated mRNA decay, expected to result in haploinsufficiency of the mutant allele. Since this haploinsufficiency model does not result in cardiovascular malformations, it does not allow further study of the process of aneurysm formation. In addition to providing a comprehensive method for cardiovascular phenotyping in mice, the results of this study confirm that haploinsuffciency is not the underlying genetic mechanism in human LDS

Morphological and morphometric of the tonsil of the soft palate in sheep

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Improving In Vivo High-Resolution CT Imaging of the Tumour Vasculature in Xenograft Mouse Models through Reduction of Motion and Bone-Streak Artefacts

Preclinical in vivo CT is commonly used to visualise vessels at a macroscopic scale. However, it is prone to many artefacts which can degrade the quality of CT images significantly. Although some artefacts can be partially corrected for during image processing, they are best avoided during acquisition. Here, a novel imaging cradle and tumour holder was designed to maximise CT resolution. This approach was used to improve preclinical in vivo imaging of the tumour vasculature.A custom built cradle containing a tumour holder was developed and fix-mounted to the CT system gantry to avoid artefacts arising from scanner vibrations and out-of-field sample positioning. The tumour holder separated the tumour from bones along the axis of rotation of the CT scanner to avoid bone-streaking. It also kept the tumour stationary and insensitive to respiratory motion. System performance was evaluated in terms of tumour immobilisation and reduction of motion and bone artefacts. Pre- and post-contrast CT followed by sequential DCE-MRI of the tumour vasculature in xenograft transplanted mice was performed to confirm vessel patency and demonstrate the multimodal capacity of the new cradle. Vessel characteristics such as diameter, and branching were quantified.Image artefacts originating from bones and out-of-field sample positioning were avoided whilst those resulting from motions were reduced significantly, thereby maximising the resolution that can be achieved with CT imaging in vivo. Tumour vessels ≥ 77 μm could be resolved and blood flow to the tumour remained functional. The diameter of each tumour vessel was determined and plotted as histograms and vessel branching maps were created. Multimodal imaging using this cradle assembly was preserved and demonstrated.The presented imaging workflow minimised image artefacts arising from scanner induced vibrations, respiratory motion and radiopaque structures and enabled in vivo CT imaging and quantitative analysis of the tumour vasculature at higher resolution than was possible before. Moreover, it can be applied in a multimodal setting, therefore combining anatomical and dynamic information

Expression and Localization of Angiogenic Growth Factors in Developing Porcine Mesonephric Glomeruli

The development and growth of renal glomeruli is regulated by specific angiogenic growth factors, including vascular endothelial growth factor (VEGF) and the angiopoietins (ANGPT1 and ANGPT2). The expression of these factors has already been studied during metanephric glomerulogenesis, but it remains to be elucidated during the development of the embryonic mesonephros, which can function as an interesting model for glomerular development and senescence. In this study, the presence of the angiogenic growth factors was studied in developing porcine mesonephroi, using IHC and real-time RT-qPCR on laser capture microdissected glomeruli. In addition, mesonephric glomerular growth was measured by using stereological methods. ANGPT2 remained upregulated during maturation of glomeruli, which may be explained by the continuous growth of the glomeruli, as observed by stereological examination. The mRNA for VEGFA was expressed in early developing and in maturing glomeruli. The VEGF receptor VEGFR1 was stably expressed during the whole lifespan of mesonephric glomeruli, whereas VEGFR2 mRNA was only upregulated in early glomerulogenesis, suggesting that VEGFR2 is important for the vascular growth but that VEGFR1 is important for the maintenance of endothelial fenestrations. (J Histochem Cytochem 58:1045–1056, 2010

Analysis of tumour vessel diameter.

<p>The diameter of each tumour vessel was determined using a distance map approach and plotted as a histogram. A-D: Vessel diameter histograms for four representative mice, displaying a range in tumour volumes, are presented. The histograms correspond to the same tumours as portrayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128537#pone.0128537.g004" target="_blank">Fig 4</a>. The same annotations were used to identify the tumours; i.e. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128537#pone.0128537.g004" target="_blank">Fig 4</a> column A corresponds to Fig 7A.</p

3D maximum-intensity-projection vessel rendering of a vascular cast for the same animal as Fig 4C.

<p>The box indicates tumour position whilst the arrows indicate the a. femoralis.</p

CT imaging using the proposed method.

<p>A centre slice through the tumour is shown. A: pre-contrast CT, B: post-contrast CT, C: subtraction image (post—pre). The white arrow indicates a 2-pixel discrepancy between the pre- and post-contrast image.</p

Branching maps of the tumour vasculature.

<p>These are based on a skeletonization of the vesselness map segmentations and illustrate the degree of branching and the number of branches for each tumour. A-D: A showcase of branching maps for four representative mice, displaying a range in tumour volumes, is presented. The maps correspond to the same tumours as portrayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128537#pone.0128537.g004" target="_blank">Fig 4</a>. The same annotations were used to identify the tumours; i.e. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128537#pone.0128537.g004" target="_blank">Fig 4</a> column A corresponds to Fig 6A.</p

Tumour vessel image rendering.

<p>Column A-D: A showcase of four representative mice, displaying a range in tumour volumes, is presented. 3D vessel segmentations based on intensity thresholding are portrayed in the top row and are overlaid with the tumour surface. The bottom row presents a composite image of the vessel segmentations with the vesselness map segmentation. Tumour ‘C’ portrays the same animal as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128537#pone.0128537.g005" target="_blank">Fig 5</a>.</p