Neovascularization and vascular markers in a foreign body reaction to subcutaneously implanted degradable biomaterial in mice

To study the spatiotemporal processes of angiogenesis during a foreign body reaction (FBR), biodegradable bovine collagen type-1 (COL-I) discs were implanted in mice for a period up to 28 days. The cellular infiltration (consisting mainly of macrophages, giant cells and fibroblasts), and the extent of neovascularization into the discs were determined. Also the expression levels and/or distribution of the endothelial cell markers von Willebrand factor (vWF), platelet endothelial cell adhesion molecule-1 (PECAM-1)/CD31, MECA-32 antigens and endomucin, and of the basal lamina marker collagen type IV (Coll IV) were analysed. In time, a strong neovascularization of the discs was observed, with frequently occurring vascular sprouting, and intussusceptive growth of vessels. In this model, vWF, MECA-32 and endomucin antibodies often failed to stain neovessels in the COL-I discs. In contrast, staining for collagen IV basal lamina component in combination with CD31 covered the complete range of neo-vessels. We conclude that the model described in this study is a useful model to study FBR induced angiogenesis because of the active neovascularization taking place during prolonged periods of time.</p

Tumor Vascular Morphology Undergoes Dramatic Changes during Outgrowth of B16 Melanoma While Proangiogenic Gene Expression Remains Unchanged

In established tumors, angiogenic endothelial cells (ECs) coexist next to “quiescent” EC in matured vessels. We hypothesized that angio-gene expression of B16.F10 melanoma would differ depending on the growth stage. Unraveling the spatiotemporal nature thereof is essential for drug regimen design aimed to affect multiple neovascularization stages. We determined the angiogenic phenotype—represented by 52 angio-genes—and vascular morphology of small, intermediate, and large s.c. growing mouse B16.F10 tumors and demonstrated that expression of these genes did not differ between the different growth stages. Yet vascular morphology changed dramatically from small vessels without lumen in small to larger vessels with increased lumen size in intermediate/large tumors. Separate analysis of these vascular morphologies revealed a significant difference in αSMA expression in relation to vessel morphology, while no relation with VEGF, HIF-1α, nor Dll4 expression levels was observed. We conclude that the tumor vasculature remains actively engaged in angiogenesis during B16.F10 melanoma outgrowth and that the major change in tumor vascular morphology does not follow molecular concepts generated in other angiogenesis models

A highly efficient synthesis of (R )‐ and (S )‐citramalic acid

An efficient multigram synthesis of optically pure (S )‐ and (R )‐citramalic acid is describe

Determination of enantiomeric purity of hydroxy biaryls using 1H and 31P NMR on their diazaphospholidine derivatives

Chiral biaryl alcohols were readily transformed in a single step, with chiral non-racemic 1,2-diamines, PCl3, and sulfur in CDCl3 - all in a NMR tube - to their diastereomeric diazaphospholidines and their diastereomeric ratios (also ee's) assessed directly

Spatiotemporal distribution of <i>S.</i><i>pneumoniae</i> in the different compartments of the brain.

<p>A. Visualization of <i>S. pneumoniae</i> co-localized with the vasculature in the brain during the entire course of infection, same images as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068408#pone-0068408-g002" target="_blank">Figure 2B</a>; total magnification 50X. Pneumococci that co-localized with the vascular endothelium appear as white, pneumococci not co-localized appear as green. B. Brain slides of mice challenged with <i>S. pneumoniae</i> were stained for vasculature using tomato lectin (red), bacteria (green), and nuclei (blue) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068408#s2" target="_blank">Material and Methods</a>. Total magnification of subarachnoid space, septum, choroid plexus 630X; total magnification of cerebral cortex 1000X. At 14 hours post infection the white arrow indicates the pneumococci forming clusters in the subarachnoid vessels. For each time point of infection, brains from 3 mice were analyzed, and of each mouse 4 brain sections were used for the immunofluorescent detection. These images are representative of the situation in i) each brain compartment during all the time course of infection and ii) each mouse that was analyzed.</p

Interaction of <i>S.</i><i>pneumoniae</i> with endothelial cells does not cause serious disruption of the intercellular junctions <i>in vitro</i>.

<p>Immunofluorescent detection of VE-Cadherin (red) and <i>S. pneumoniae</i> (green) in HBMEC in normal conditions and after 1 hour incubation with 10⁶ CFU pneumococci. Cells were stained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068408#s2" target="_blank">Material and Methods</a>. Panel A shows fluorescence microscope image 630X total magnification; panel B shows confocal microscope image; panels C and D show the YZ and XZ orthogonal view planes.</p

Leukocyte presence in the brain and lungs of mock treated and <i>S.</i><i>pneumoniae</i> infected mice at different time points after infection.

<p>A. Immunofluorescent staining of the leukocyte common antigen CD45 (green) and nuclei (blue) in the subarachnoid space/cerebral cortex, septum and choroid plexus at 14 hours-post infection; total magnification 400X. The CD45 staining showed no influx of leukocytes in all the compartments of the brain in the early stages of infection (data not shown), and even 14 hours after infection the presence of leukocytes was minimal. For each time point of infection, brain sections from 3 mice were analyzed, and of each mouse 3 brain sections were used for the immunofluorescent detection. The images are representative of the situation observed in each mouse that was analyzed. B. Immunofluorescent staining of leukocyte common antigen CD45 (green) and nuclei (blue) in lungs of mock-treated and <i>S. pneumoniae</i>- infected mice; total magnification 630X. For each time point of infection, lungs from 3 mice were analyzed, and of each mouse 3 lung sections were used for the immunofluorescent detection. The images are representative of the situation observed in each mouse that was analyzed.</p

<i>S.</i><i>pneumoniae</i> adheres to the brain vascular endothelium.

<p>Visualization of <i>S. pneumoniae</i> (green) adhering to the brain vascular endothelium (red) using confocal microscopy. The scale of each image is shown by the white scale bar. White arrows points to completely overlapping staining of the bacteria and the lectin resulting in a yellow color, strongly suggesting a co-localization of <i>S. pneumoniae</i> with endothelial cells. For each time point of infection, brains from 3 mice were analyzed, and of each mouse 3 brain sections were used for the confocal detection. The images at the later stages of infection were used because more bacteria were detected at those time points and thus these provide a clear picture of the bacteria anchored to the vasculature. Images of all time points after infection are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068408#pone.0068408.s002" target="_blank">Figure S2</a>.</p

Inflammatory cytokine levels in the brain during pneumococcal infection.

<p>mRNA levels (AU = arbitrary units) of the pro-inflammatory cytokines TNF alpha, IL-1 beta, IL-6 and the housekeeping genes GAPDH and HTR1 in the brains of mock treated mice and mice sacrificed at 14 hours after infection, as quantified by Real Tim ePCR. * indicates p<0.05.</p

Vasculature in the different compartments of the mouse brain.

<p>Immunofluorescent detection of CD31 (488 nm green signal), tomato lectin (594 nm red signal) and nuclei (350 nm blue signal) in the brain of mock infected mice; total magnification 630X. Double staining with anti CD31 antibody and the lectin showed that the tomato lectin provides a more comprehensive detection of the brain vasculature.</p