Key Points• Recruitment of STXBP1 by Slp4-a promotes WeibelPalade body exocytosis.• Ex vivo EIEE4 endothelial cells haploinsufficient for STXBP1 have impaired Weibel-Palade body exocytosis.Vascular endothelial cells contain unique rod-shaped secretory organelles, called WeibelPalade bodies (WPBs), which contain the hemostatic protein von Willebrand factor (VWF) and a cocktail of angiogenic and inflammatory mediators. We have shown that the Rab27A effector synaptotagmin-like protein 4-a (Slp4-a) plays a critical role in regulating hormoneevoked WPB exocytosis. Using a nonbiased proteomic screen for targets for Slp4-a, we now identify syntaxin-binding protein 1 (STXBP1) and syntaxin-2 and -3 as endogenous Slp4-a binding partners in endothelial cells. Coimmunoprecipitations showed that STXBP1 interacts with syntaxin-2 and -3, but not with syntaxin-4. Small interfering RNA-mediated silencing of STXBP1 expression impaired histamine-and forskolin-induced VWF secretion.To further substantiate the role of STXBP1, we isolated blood outgrowth endothelial cells (BOECs) from an early infantile epileptic encephalopathy type 4 (EIEE4) patient carrying a de novo mutation in STXBP1. STXBP1-haploinsufficient EIEE4 BOECs contained similar numbers of morphologically normal WPBs compared with control BOECs of healthy donors; however, EIEE4 BOECs displayed significantly impaired histamine-and forskolin-stimulated VWF secretion. Based on these findings, we propose that the Rab27A-Slp4-a complex on WPB promotes exocytosis through an interaction with STXBP1, thereby controlling the release of vaso-active substances in the vasculature. (Blood. 2014;123(20):3185-3194) IntroductionEndothelial cells line the lumen of all blood vessels, providing a highly dynamic barrier that plays a crucial role in maintaining vascular homeostasis. They contain specialized secretory organelles called Weibel-Palade bodies (WPBs) that allow the endothelium to store and release, in a regulated fashion, a presynthesized cocktail of hemostatic, inflammatory, and angiogenic mediators in response to endothelial activation, injury, or stress. [1][2][3] The main component of these organelles is von Willebrand factor (VWF), a multimeric glycoprotein crucial for platelet plug formation and stabilizing coagulation factor VIII. In addition to VWF, several soluble chemokines (eg,, IL-8) as well as the integral membrane proteins CD63 and P-selectin are stored in these organelles. [4][5][6][7][8][9] Coordinated expression of CD63 and P-selectin on the endothelial cell surface after WPB exocytosis is crucial for leukocyte extravasation at sites of inflammation. 10 The presence of angiopoietin-2 and insulin-like growth factor-binding protein 7 in WPBs points toward a critical role for the organelle in regulation of angiogenesis. 11-13The precise composition of mediators stored in WPBs depends crucially on the physical, mechanical, and chemical signals in the local microenvironment; for example, targeting of eotaxin-3, IL-8, and IL-6 has been observed in response to pro-...
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 angiogenesis.
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, such as thrombin, or agonists that increase intracellular levels of cAMP, such as epinephrine. Objective: Previously, we have shown that the exchange protein activated by cAMP, exchange protein activated by cAMP, and the small GTPase Rap1 are involved in cAMP-mediated release of WPBs. In this study, we explored potential downstream effectors of Rap1 in cAMP-mediated WPB release. Methods: Studies were performed in primary human umbilical vein endothelial cells. Activation of the small GTP-binding protein Rac1 was monitored by its ability to bind to the CRIB domain of the serine/threonine kinase P21-activated kinase (PAK)1. Downstream effectors of Rap1 were identified with a proteomic screen using a glutathione-S-transferase fusion of the Ras-binding domain of RalGDS. Functional involvement of candidate proteins in WPB release was determined by RNA interference (RNAi)-mediated knockdown of gene expression. Results: Depletion of Rac1 by RNAi prevented epinephrine-induced VWF secretion. Also, the Rac1 inhibitor EHT1864 reduced epinephrine-induced WPB release. We identified the phosphatidylinositol-3,4,5-triphosphate-dependent Rac exchange factor 1 (PREX1) and the regulatory b-subunit of phosphatidylinositol 3-kinase (PI3K) as downstream targets of Rap1. The PI3K inhibitor LY294002 reduced epinephrine-induced release of VWF. RNAi-mediated downregulation of PREX1 abolished epinephrine-induced but not thrombin-induced release of WPBs. Conclusion: Our findings show that PREX1 regulates epinephrine-induced release of WPBs.
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 S1PR3.
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 ‘Children 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’, 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 0–1-month age group to 33% in the 4–5 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’s awareness of how their friends and family members fed their own babies. The main barriers were a lack of encouragement by husbands, women’s perception that their infants’ 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’ support, and improve knowledge of mothers on breastfeeding are recommended to be incorporated in the design of future health programs.
No abstract available.
Vascular endothelial cells contain unique rod-shaped secretory granules, called Weibel-Palade bodies (WPBs), which contain a number of haemostatic, angiogenic and inflammatory mediators. Several components that are critical for regulated WPB exocytosis have been identified, including the small GTPase Rab27A and its effector synaptotagmin-like protein 4-a (Slp4-a), but the mechanism remains unclear. We have previously identified syntaxin binding protein 1 (STXBP1) as an endogenous Slp4-a binding partner involved in WPB release, along with the SNARE proteins syntaxin-2 and -3. In this study we investigated the possible role of syntaxin-2 and -3 in WPB exocytosis. We characterized the subcellular location of these syntaxins in endothelial cells using immunocytochemistry. Syntaxin-2 was primarily associated with the plasma membrane where it localized at VE-caderin-based adherens junctions and at integrin-based adhesions to the extracellular matrix. Interestingly, the t-SNARE syntaxin-3 was primarily associated with WPBs. To further explore its role in WPB biology we mapped the endothelial interaction partners of syntaxin-3 through an unbiased mass spectrometry approach using pull downs of lentivirally-expressed mEGFP-syntaxin-3 with anti-GFP nanobeads. Among its interaction partners are various SNAREs and associated proteins such as syntaxin binding proteins 2 and 5 (STXBP2/5), N-ethylmaleimide-sensitive factor (NSF), SNAP23 and α-SNAP, suggesting we successfully pulled down a SNARE complex and its regulatory machinery that are involved in exocytosis. We further addressed the role of syntaxin-3 in stimulated WPB exocytosis using siRNA-mediated knockdowns and found that depletion of syntaxin-3 led to a significant potentiation of Ca2+- as well as cAMP-mediated VWF secretion. In contrast, we observed a decrease in basal (unstimulated) VWF secretion after silencing of syntaxin-3, which amounts to a significantly increased intracellular VWF content. The potentiation of VWF secretion after depletion of syntaxin-3 was almost completely attributable to an increased WPB pool size; when corrected for increased WPB content the probability of stimulated release of a WPB was unaltered in the absence of syntaxin-3. Our data position syntaxin-3 as a WPB-linked SNARE-protein that regulates basal secretion of VWF. Disclosures No relevant conflicts of interest to declare.
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