The Weibel-Palade Body Localized SNARE (Soluble NSF Attachment Protein Receptor) Syntaxin-3 Modulates Von Willebrand Factor Secretion From Endothelial Cells

Objective Endothelial cells store von Willebrand factor (VWF) in rod-shaped secretory organelles, called Weibel-Palade bodies (WPBs). WPB exocytosis is coordinated by a complex network of Rab GTPases, Rab-effectors and SNARE proteins. We have previously identified STXBP1 as the link between the Rab27A-Slp4-a complex on WPBs and the SNARE proteins syntaxin-2 and -3. In this study we investigate the function of syntaxin-3 in VWF secretion. Approach and Results In human umbilical vein endothelial cells (HUVECs) and in blood outgrowth endothelial cells (BOECs) from healthy controls endogenous syntaxin-3 immunolocalized to WPBs. A detailed analysis of BOECs isolated from a patient with variant microvillus inclusion disease (MVID), carrying a homozygous mutation in STX3 (STX3-/-), showed a loss of syntaxin-3 protein and absence of WPB-associated syntaxin-3 immunoreactivity. Ultrastructural analysis revealed no detectable differences in morphology or prevalence of immature or mature WPBs in control versus STX3-/- BOECs. VWF multimer analysis showed normal patterns in plasma of the MVID patient, and media from STX3-/- BOECs, together indicating WPB formation and maturation are unaffected by absence of syntaxin-3. However, a defect in basal as well as Ca2+ - and cAMP-mediated VWF secretion was found in the STX3-/- BOECs. We also show that syntaxin-3 interacts with the WPB-associated SNARE protein VAMP8. Conclusions Our data reveal syntaxin-3 as a novel WPB-associated SNARE protein that controls WPB exocytosis

Phosphatidylinositol-3,4,5-triphosphate-dependent Rac exchange factor 1 regulates epinephrine-induced exocytosis of Weibel-Palade bodies

Thrombosis and Hemostasi

Determinants of breastfeeding practice in Pujehun district, southern Sierra Leone: a mixed-method study

Background: It is well established that exclusive breastfeeding can play a critical role in reducing child morbidity and mortality. Limited research has been done thus far on the practice and perceptions of breastfeeding in Sierra Leone, where more than 10 % of children die before the age of five. This study aimed to gain understanding into and explore both matters in order to develop recommendations for effective strategies to promote breastfeeding practice in Pujehun District, Southern Sierra Leone. Methods: This exploratory mixed-method study included a cross-sectional survey of 194 mothers, semi-structured interviews and focus group discussions. Logistic regression analysis was used calculated odds ratios of factors associated with primarily breastfeeding practice, defined as \u2018Children under six months of age who are fed with breast milk only and children older than six months of age that were exclusively breastfed up to six months\u2019, based on recall from birth. Exclusive breastfeeding rate was based on breastfeeding practice 24 h prior to the survey. Qualitative data was analysed through a deductive approach, using a pre-determined framework on determinants of breastfeeding. Results: This study revealed an exclusive breastfeeding rate of 62.8% (95% CI 53.9, 71.7); dropping from 74% in the 0\u20131-month age group to 33% in the 4\u20135 months group. Triangulation of qualitative and quantitative data revealed enabling factors for primarily breastfeeding practice included mothers receiving support during their first breastfeed, pregnant women being provided with information on the benefits of the practice, counselling by nurses, support from husbands, and women\u2019s awareness of how their friends and family members fed their own babies. The main barriers were a lack of encouragement by husbands, women\u2019s perception that their infants\u2019 stools were abnormal or that they were not producing enough breast milk. Conclusions: Although the exclusive breastfeeding may have risen over recent years, a gap remains compared to World Health Organization recommendations. According to the breastfeeding determinants identified in this study, promotion of counselling by a nurse, encouragement of husbands\u2019 support, and improve knowledge of mothers on breastfeeding are recommended to be incorporated in the design of future health programs

Sphingosine-1-phosphate receptor 3 mediates sphingosine-1-phosphate induced release of weibel-palade bodies from endothelial cells

Sphingosine-1-phosphate (S1P) is an agonist for five distinct G-protein coupled receptors, that is released by platelets, mast cells, erythrocytes and endothelial cells. S1P promotes endothelial cell barrier function and induces release of endothelial cell-specific storage-organelles designated Weibel-Palade bodies (WPBs). S1P-mediated enhancement of endothelial cell barrier function is dependent on S1P receptor 1 (S1PR1) mediated signaling events that result in the activation of the small GTPase Rac1. Recently, we have reported that Rac1 regulates epinephrine-induced WPB exocytosis following its activation by phosphatidylinositol-3,4,5-triphosphate-dependent Rac exchange factor 1 (PREX1). S1P has also been described to induce WPB exocytosis. Here, we confirm that S1P induces release of WPBs using von Willebrand factor (VWF) as a marker. Using siRNA mediated knockdown of gene expression we show that S1PR1 is not involved in S1P-mediated release of WPBs. In contrast depletion of the S1PR3 greatly reduced S1P-induced release of VWF. S1P-mediated enhancement of endothelial barrier function was not affected by S1PR3-depletion whereas it was greatly impaired in cells lacking S1PR1. The Rho kinase inhibitor Y27632 completely abrogated S1P-mediated release of VWF. Also, the calcium chelator BAPTA-AM significantly reduced S1P-induced release of VWF. Our findings indicate that S1P-induced release of haemostatic, inflammatory and angiogenic components stored within WPBs depends on the S1PR

Heteromeric Interactions Required for Abundance and Subcellular Localization of Human CDC50 Proteins and Class 1 P4-ATPases*

Members of the P4 family of P-type ATPases (P4-ATPases) are believed to function as phospholipid flippases in complex with CDC50 proteins. Mutations in the human class 1 P4-ATPase gene ATP8B1 cause a severe syndrome characterized by impaired bile flow (intrahepatic cholestasis), often leading to end-stage liver failure in childhood. In this study, we determined the specificity of human class 1 P4-ATPase interactions with CDC50 proteins and the functional consequences of these interactions on protein abundance and localization of both protein classes. ATP8B1 and ATP8B2 co-immunoprecipitated with CDC50A and CDC50B, whereas ATP8B4, ATP8A1, and ATP8A2 associated only with CDC50A. ATP8B1 shifted from the endoplasmic reticulum (ER) to the plasma membrane upon coexpression of CDC50A or CDC50B. ATP8A1 and ATP8A2 translocated from the ER to the Golgi complex and plasma membrane upon coexpression of CDC50A, but not CDC50B. ATP8B2 and ATP8B4 already displayed partial plasma membrane localization in the absence of CDC50 coexpression but displayed a large increase in plasma membrane abundance upon coexpression of CDC50A. ATP8B3 did not bind CDC50A and CDC50B and was invariably present in the ER. Our data show that interactions between CDC50 proteins and class 1 P4-ATPases are essential for ER exit and stability of both subunits. Furthermore, the subcellular localization of the complex is determined by the P4-ATPase, not the CDC50 protein. The interactions of CDC50A and CDC50B with multiple members of the human P4-ATPase family suggest that these proteins perform broader functions in human physiology than thus far assumed

Proteomic Screen Identifies IGFBP7 as a Novel Component of Endothelial Cell-Specific Weibel-Palade Bodies

Vascular endothelial cells contain unique storage organelles, designated Weibel-Palade bodies (WPBs), that deliver inflammatory and hemostatic mediators to the vascular lumen in response to agonists like thrombin and vasopressin. The main component of WPBs is von Willebrand factor (VWF), a multimeric glycoprotein crucial for platelet plug formation. In addition to VWF, several other components are known to be stored in WPBs, like osteoprotegerin, monocyte chemoattractant protein-1 and angiopoetin-2 (Ang-2). Here, we used an unbiased proteomics approach to identify additional residents of WPBs. Mass spectrometry analysis of purified WPBs revealed the presence of several known components such as VWF, Ang-2, and P-selectin. Thirty-five novel candidate WPB residents were identified that included insulin-like growth factor binding protein-7 (IGFBP7), which has been proposed to regulate angiogenesis. Immunocytochemistry revealed that IGFBP7 is a bona fide WPB component. Cotransfection studies showed that IGFBP7 trafficked to pseudo-WPB in HEK293 cells. Using a series of deletion variants of VWF, we showed that targeting of IGFBP7 to pseudo-WPBs was dependent on the carboxy-terminal D4-C1-C2-C3-CK domains of VWF. IGFBP7 remained attached to ultralarge VWF strings released upon exocytosis of WPBs under flow. The presence of IGFBP7 in WPBs highlights the role of this subcellular compartment in regulation of angiogenesi

Signaling pathways that regulate S1P induced WPB exocytosis.

<p>Stimulation of the S1P receptor 1 (S1PR1) by S1P induces G_i subunit mediated Rac1 activation by PI3K- dependent signaling, resulting in increased barrier function. Stimulation of S1PR3 by S1P results in activation of G_q and G_12/13 subunits. G_q subunit activation results in an increase in intracellular calcium levels. Activation of G_12/13 subunits signals to RhoA and ROCK that promote assembly of actin rings that facilitate WPB exocytosis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091346#pone.0091346-Nightingale1" target="_blank">[48]</a>.</p

S1PR1 is responsible for barrier integrity in S1P stimulated HUVECs.

<p>(A) HUVECs were transfected with a control siRNA SMARTpool (siCTRL) or a siRNA SMARTpool targeting S1PR1 (siS1PR1) or S1PR3 (siS1PR3) and grown to confluence on electrode ECIS-arrays. (B) SiCTRL, siS1PR1 or siS1PR3 transfected HUVECs show no differences in impedance after the indicated time points. (C) HUVECs transfected with a control siRNA SMARTpool (siCTRL) or a siRNA SMARTpool targeting S1PR1 (siS1PR1) or S1PR3 (siS1PR3) were incubated with 1 μM S1P or SF medium alone. The impedance was continuously monitored and was corrected for starting impedance after 1 hr serum starvation. Three independent experiments were performed. Statistical significance was assessed by 2-way ANOVA followed by Bonferroni post-hoc test for selected comparison (***P<0.001; **P<0.01 and *P<0.05).</p

Downregulation of S1PR3 expression reduces WPB release.

<p>(A) HUVECs were incubated for the indicated time points with 1 μM S1P or SF medium. (B) HUVECs were transfected with a control siRNA SMARTpool (siCTRL) or a siRNA SMARTpool targeting S1PR1 (siS1PR1) or S1PR3 (siS1PR3). Western blot analysis at 72 hours post-transfection showed downregulation of S1PR1 expression or S1PR3 expression. Levels of α-tubulin are shown as a protein loading control. (C,E) siCTRL siS1PR1 and siS1PR3 treated HUVECs were incubated for 45 minutes with 1 μM S1P, 10 μM forskolin (FSK) and 100 μM IBMX, 1 U/ml thrombin (T) or SF medium alone (-). (D,F) siCTRL siS1PR1 and siS1PR3 treated HUVECs were incubated for indicated time points with 1 μM S1P. The amount of VWF secreted in the medium was measured by ELISA. Total amount of VWF secreted is displayed in percentages where the amount of VWF secreted by unstimulated cells t = 45 transfected with siCTRL is set to 100%. Four independent experiments were performed.Statistical significance was assessed by 2-way ANOVA followed by Bonferroni post-hoc test for selected comparison (***P<0.001; **P<0.01 and *P<0.05).</p

Pharmacological inhibition of S1P-induced VWF secretion.

<p>CTRL and Y27632 treated HUVECs were incubated for indicated times with 1 μM S1P or SF medium alone (-) (A). (B) siCTRL and siS1PR1 treated HUVECs were incubated for 45 minutes with 1 μM S1P or SF medium alone (-) in the presence of 10 μM Rho kinase inhibitor Y27632 or 100 μM calcium chelator BAPTA-AM. (C) siCTRL and siS1PR3 treated HUVECs were incubated for 45 minutes with 1 μM S1P or SF medium alone (-) in the presence of Rho kinase inhibitor Y27632 or calcium inhibitor BAPTA-AM. The amount of VWF secreted in the medium was measured by ELISA; VWF secreted by unstimulated siCTRL treated cells after 45 minutes was set to 100%. Three independent experiments were performed. Statistical significance was assessed by 2-way ANOVA followed by Bonferroni post-hoc test for selected comparison (***P<0.001; **P<0.01 and *P<0.05).</p