Unique Cell Type-Specific Junctional Complexes in Vascular Endothelium of Human and Rat Liver Sinusoids

Liver sinusoidal endothelium is strategically positioned to control access of fluids, macromolecules and cells to the liver parenchyma and to serve clearance functions upstream of the hepatocytes. While clearance of macromolecular debris from the peripheral blood is performed by liver sinusoidal endothelial cells (LSECs) using a delicate endocytic receptor system featuring stabilin-1 and -2, the mannose receptor and CD32b, vascular permeability and cell trafficking are controlled by transcellular pores, i.e. the fenestrae, and by intercellular junctional complexes. In contrast to blood vascular and lymphatic endothelial cells in other organs, the junctional complexes of LSECs have not yet been consistently characterized in molecular terms. In a comprehensive analysis, we here show that LSECs express the typical proteins found in endothelial adherens junctions (AJ), i.e. VE-cadherin as well as α-, β-, p120-catenin and plakoglobin. Tight junction (TJ) transmembrane proteins typical of endothelial cells, i.e. claudin-5 and occludin, were not expressed by rat LSECs while heterogenous immunreactivity for claudin-5 was detected in human LSECs. In contrast, junctional molecules preferentially associating with TJ such as JAM-A, B and C and zonula occludens proteins ZO-1 and ZO-2 were readily detected in LSECs. Remarkably, among the JAMs JAM-C was considerably over-expressed in LSECs as compared to lung microvascular endothelial cells. In conclusion, we show here that LSECs form a special kind of mixed-type intercellular junctions characterized by co-occurrence of endothelial AJ proteins, and of ZO-1 and -2, and JAMs. The distinct molecular architecture of the intercellular junctional complexes of LSECs corroborates previous ultrastructural findings and provides the molecular basis for further analyses of the endothelial barrier function of liver sinusoids under pathologic conditions ranging from hepatic inflammation to formation of liver metastasis

Factors influencing the consumption of seafood among young children in Perth: a qualitative study

<p>Abstract</p> <p>Background</p> <p>This formative study sought to explore the factors that influence the consumption of fish and seafood among 4–6 year old children in the Perth metropolitan area. Focus groups were conducted with mothers of young children to gain insights into the enablers and barriers to regular seafood consumption in children, and the knowledge, attitudes and perceptions of their mothers to including seafood as a regular part of their children's diet.</p> <p>Methods</p> <p>Purposive sampling techniques were used to select and recruit mothers of children aged between four and six years from within the Perth metropolitan area. A total of seven focus groups were conducted. Thematic content analysis was employed to code data generated and to extract major themes.</p> <p>Results</p> <p>Findings indicated that all children of study participants had tried fish and seafood products, with some being exposed to a wide variety from an early age. Across focus groups, several dominant factors were apparent in influencing the frequency and type of seafood purchased and consumed. Perceived cost, freshness, availability/accessibility, and the level of confidence to prepare a meal to suit all family members were significant determinants of whether seafood featured regularly on the household menu. The influence of others in the family (particularly the husband or partner) also tended to impact upon the likelihood of serving fish and seafood, and the types of products mothers were willing to serve.</p> <p>Conclusion</p> <p>Findings from this qualitative study indicate that interventions seeking to promote seafood (particularly fish) as an integral part of a healthy diet should address existing negative attitudes and beliefs around the storage and preparation of seafood. The influence of dominant male influences within the family unit should also be considered. Strategies directed at parents and children should include experimental 'hands-on' components to encourage experimentation, particularly focussing on ease of preparation and the variety of lower cost seafood available.</p

Effect of Glycoamphiphiles on the Solubilization and Dendritic Cell Uptake of a Lipopeptide: A Preliminary Study

International audienc

Wnt2 acts as a cell type-specific, autocrine growth factor in rat hepatic sinusoidal endothelial cells cross-stimulating the VEGF pathway

The mechanisms regulating the growth and differentiation of hepatic sinusoidal endothelial cells (HSECs) are not well defined. Because Wnt signaling has become increasingly important in developmental processes such as vascular and hepatic differentiation, we analyzed HSEC-specific Wnt signaling in detail. Using highly pure HSECs isolated by a newly developed protocol selecting against nonsinusoidal hepatic endothelial cells, we comparatively screened the multiple components of the Wnt pathway for differential expression in HSECs and lung microvascular endothelial cells (LMECs) via reverse-transcription polymerase chain reaction (RT-PCR). As confirmed via quantitative RTPCR and northern and western blotting experiments, Wnt2 (and less so Wnt transporter wls/evi) and Wnt coreceptor Ryk were overexpressed by HSECs, whereas Wnt inhibitory factor (WIF) was strongly overexpressed by LMECs. Exogenous Wnt2 superinduced proliferation of HSECs (P <0.05). The Wnt inhibitor secreted frizzled-related protein 1 (sFRPI) (P <0.005) and transfection of HSECs with Wnt2 small interfering RNA (siRNA) reduced proliferation of HSECs. These effects were rescued by exogenous Wnt2. Tube formation of HSECs on matrigel was strongly inhibited by Wnt inhibitors sFRP I and WIF (P <0.0005). Wnt signaling in HSECs activated the canonical pathway inducing nuclear translocation of beta-catenin. GST (glutathione transferase) pull-down and co-immunoprecipitation assays showed Fzd4 to be a novel Wnt2 receptor in HSECs. Gene profiling identified vascular endothelial growth factor receptor-2 (VEGFR-2) as a target of Wnt2 signaling in HSECs. Inhibition of Wnt signaling down-regulated VEGFR-2 messenger RNA and protein. Wnt2 siRNA knock-down confirmed Wnt2 specificity of VEGFR-2 regulation in HSECs. Conclusion: Wnt2 is an autocrine growth and differentiation factor specific for HSECs that synergizes with the VEGF signaling pathway to exert its effects

Physiological Impact of a Synthetic Elastic Protein in Arterial Diseases Related to Alterations of Elastic Fibers: Effect on the Aorta of Elastin-Haploinsufficient Male and Female Mice

International audienceElastic fibers, made of elastin (90%) and fibrillin-rich microfibrils (10%), are the key extracellular components, which endow the arteries with elasticity. The alteration of elastic fibers leads to cardiovascular dysfunctions, as observed in elastin haploinsufficiency in mice (Eln+/-) or humans (supravalvular aortic stenosis or Williams–Beuren syndrome). In Eln+/+ and Eln+/- mice, we evaluated (arteriography, histology, qPCR, Western blots and cell cultures) the beneficial impact of treatment with a synthetic elastic protein (SEP), mimicking several domains of tropoelastin, the precursor of elastin, including hydrophobic elasticity-related domains and binding sites for elastin receptors. In the aorta or cultured aortic smooth muscle cells from these animals, SEP treatment induced a synthesis of elastin and fibrillin-1, a thickening of the aortic elastic lamellae, a decrease in wall stiffness and/or a strong trend toward a reduction in the elastic lamella disruptions in Eln+/- mice. SEP also modified collagen conformation and transcript expressions, enhanced the aorta constrictive response to phenylephrine in several animal groups, and, in female Eln+/- mice, it restored the normal vasodilatory response to acetylcholine. SEP should now be considered as a biomimetic molecule with an interesting potential for future treatments of elastin-deficient patients with altered arterial structure/function

E- and N-cadherin are absent in liver sinusoidal endothelial cells.

<p>(A, B) Immunofluorescent co-staining of rat liver cryosections with anti-E-cadherin (A, green) or anti-N-cadherin (B, green) and anti-LYVE-1 (A, B, red) antibodies. (C, D) Immunofluorescent co-staining of human liver cryosections with anti-E-cadherin (C, green) or anti-N-cadherin (D, green) and anti-VE-cadherin (C, D, red) antibodies. Images were acquired using laser scanning confocal microscopy. Bars 14.14 µm (A, B, D), 11.9 µm (C). (E) Reverse transcriptase-PCR with mRNA of freshly isolated rat LSECs. Primers specific for VE-cadherin (1), E-cadherin (2), N-cadherin (3) or β-actin (4) were used.</p

α-catenin, β-catenin, p120-catenin, and plakoglobin co-localize with VE-cadherin in rat liver sinusoidal endothelial cells.

<p>(A-D) Immunofluorescent co-staining of rat liver cryosections with anti-α-Catenin (A, green), anti-β-Catenin (B, green), anti-p120-Catenin (C, green), anti-Plakoglobin (D, green), anti-VE -cadherin (A-D, red), anti-Stabilin-2 (A, blue), and anti-LYVE-1 (B-D, blue) antibodies. Images were acquired using laser scanning confocal microscopy. Bars 11.9 µm.</p

VE-cadherin is expressed in liver sinusoidal endothelial cells in rats and humans.

<p>(A) Immunofluorescent co-staining of human liver cryosections with anti-VE-cadherin (green) and anti-CD32b (red) antibodies. (B) Immunofluorescent co-staining of rat liver cryosections with anti-VE-cadherin (green) and anti-LYVE-1 (red) antibodies. (C) Immunofluorescent co-staining of isolated rat LSECs with anti-VE-cadherin (green) and anti-Stabilin-2 (red) antibodies. Toto3 (blue) was used to counterstain the cell nuclei. Images were acquired using laser scanning confocal microscopy. Bars 11.9 µm (A, B), 14.14 µm (C). (D) Reverse transcriptase-PCR with mRNA isolated from rat hepatoma McA-RH7777 cell line (1), freshly isolated rat LMECs (2), and freshly isolated rat LSECs (3). Primers specific for VE-cadherin or β-actin were used.</p