Structure modeling hints at a granular organization of the Golgi ribbon.

BACKGROUND: In vertebrate cells, the Golgi functional subunits, mini-stacks, are linked into a tri-dimensional network. How this "ribbon" architecture relates to Golgi functions remains unclear. Are all connections between mini-stacks equal? Is the local structure of the ribbon of functional importance? These are difficult questions to address, without a quantifiable readout of the output of ribbon-embedded mini-stacks. Endothelial cells produce secretory granules, the Weibel-Palade bodies (WPB), whose von Willebrand Factor (VWF) cargo is central to hemostasis. The Golgi apparatus controls WPB size at both mini-stack and ribbon levels. Mini-stack dimensions delimit the size of VWF "boluses" whilst the ribbon architecture allows their linear co-packaging, thereby generating WPBs of different lengths. This Golgi/WPB size relationship suits mathematical analysis. RESULTS: WPB lengths were quantized as multiples of the bolus size and mathematical modeling simulated the effects of different Golgi ribbon organizations on WPB size, to be compared with the ground truth of experimental data. An initial simple model, with the Golgi as a single long ribbon composed of linearly interlinked mini-stacks, was refined to a collection of mini-ribbons and then to a mixture of mini-stack dimers plus long ribbon segments. Complementing these models with cell culture experiments led to novel findings. Firstly, one-bolus sized WPBs are secreted faster than larger secretory granules. Secondly, microtubule depolymerization unlinks the Golgi into equal proportions of mini-stack monomers and dimers. Kinetics of binding/unbinding of mini-stack monomers underpinning the presence of stable dimers was then simulated. Assuming that stable mini-stack dimers and monomers persist within the ribbon resulted in a final model that predicts a "breathing" arrangement of the Golgi, where monomer and dimer mini-stacks within longer structures undergo continuous linking/unlinking, consistent with experimentally observed WPB size distributions. CONCLUSIONS: Hypothetical Golgi organizations were validated against a quantifiable secretory output. The best-fitting Golgi model, accounting for stable mini-stack dimers, is consistent with a highly dynamic ribbon structure, capable of rapid rearrangement. Our modeling exercise therefore predicts that at the fine-grained level the Golgi ribbon is more complex than generally thought. Future experiments will confirm whether such a ribbon organization is endothelial-specific or a general feature of vertebrate cells.

V-ATPase V0a1 promotes Weibel-Palade body biogenesis through the regulation of membrane fission.

Membrane fission, the division of a membrane-bound structure into two discrete compartments, is essential for diverse cellular events, such as endocytosis and vesicle/granule biogenesis; however, the process remains unclear. The hemostatic protein von Willebrand factor is produced in vascular endothelial cells and packaged into specialized secretory granules, Weibel-Palade bodies (WPBs) at the trans-Golgi network (TGN). Here, we reported that V0a1, a V-ATPase component, is required for the membrane fission of WPBs. We identified two V0a isoforms in distinct populations of WPBs in cultured endothelial cells, V0a1 and V0a2, on mature and nascent WPBs, respectively. Although WPB buds were formed, WPBs could not separate from the TGN in the absence of V0a1. Screening using dominant-negative forms of known membrane fission regulators revealed protein kinase D (PKD) as an essential factor in biogenesis of WPBs. Further, we showed that the induction of wild-type PKDs in V0a1-depleted cells does not support the segregation of WPBs from the TGN; suggesting a primary role of V0a1 in the membrane fission of WPBs. The identification of V0a1 as a new membrane fission regulator should facilitate the understanding of molecular events that enable membrane fission.

Arf GTPase-activating proteins SMAP1 and AGFG2 regulate the size of Weibel-Palade bodies and exocytosis of von Willebrand factor.

Arf GTPase-Activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors (Arfs), which are critical to form transport intermediates. ArfGAPs have been thought to be negative regulators of Arfs; however, accumulating evidence indicates that ArfGAPs are important for cargo sorting and promote membrane traffic. Weibel-Palade bodies (WPBs) are cigar-shaped secretory granules in endothelial cells that contain von Willebrand factor (vWF) as their main cargo. WPB biogenesis at the Golgi was reported to be regulated by Arf and their regulators, but the role of ArfGAPs has been unknown. In this study, we performed siRNA screening of ArfGAPs to investigate the role of ArfGAPs in the biogenesis of WPBs. We found two ArfGAPs, SMAP1 and AGFG2, to be involved in WPB size and vWF exocytosis, respectively. SMAP1 depletion resulted in small-sized WPBs, and the lysosomal inhibitor leupeptin recovered the size of WPBs. The results indicate that SMAP1 functions in preventing the degradation of cigar-shaped WPBs. On the other hand, AGFG2 downregulation resulted in the inhibition of vWF secretion upon Phorbol 12-myristate 13-acetate (PMA) or histamine stimulation, suggesting that AGFG2 plays a role in vWF exocytosis. Our study revealed unexpected roles of ArfGAPs in vWF transport.

Estrogen activates endothelial exocytosis.

Estrogen therapy is used to treat patients with post-menopausal symptoms, such as hot flashes and dyspareunia. Estrogen therapy also decreases the risk of fractures from osteoporosis in post-menopausal women. However, estrogen increases the risk of venous thromboembolic events, such as pulmonary embolism, but the pathways through which estrogen increase the risk of thromboembolism is unknown. Here, we show that estrogen elicits endothelial exocytosis, the key step in vascular thrombosis and inflammation. Exogenous 17ß-estradiol (E2) stimulated endothelial exocytosis of Weibel-Palade bodies (WPBs), releasing von Willebrand factor (vWF) and interleukin-8 (IL-8). Conversely, the estrogen antagonist ICI-182,780 interfered with E2-induced endothelial exocytosis. The ERα agonist propyl pyrazole triol (PPT) but not the ERß agonist diarylpropionitrile (DPN) induced vWF release, while ERα silencing counteracted vWF release by E2, suggesting that ERα mediates this effect. Exocytosis triggered by E2 occurred rapidly within 15 min and was not inhibited by either actinomycin D or cycloheximide. On the contrary, it was inhibited by the pre-treatment of U0126 or SB203580, an ERK or a p38 inhibitor, respectively, suggesting that E2-induced endothelial exocytosis is non-genomically mediated by the MAP kinase pathway. Finally, E2 treatment enhanced platelet adhesion to endothelial cells ex vivo, which was interfered with the pre-treatment of ICI-182,780 or U0126. Taken together, our data show that estrogen activates endothelial exocytosis non-genomically through the ERα-MAP kinase pathway. Our data suggest that adverse cardiovascular effects such as vascular inflammation and thrombosis should be considered in patients before menopausal hormone treatment.

Plasma membrane phosphatidylinositol (4,5)-bisphosphate promotes Weibel-Palade body exocytosis.

Weibel-Palade bodies (WPB) are specialized secretory organelles of endothelial cells that control vascular hemostasis by regulated, Ca2+-dependent exocytosis of the coagulation-promoting von-Willebrand factor. Some proteins of the WPB docking and fusion machinery have been identified but a role of membrane lipids in regulated WPB exocytosis has so far remained elusive. We show here that the plasma membrane phospholipid composition affects Ca2+-dependent WPB exocytosis and von-Willebrand factor release. Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] becomes enriched at WPB-plasma membrane contact sites at the time of fusion, most likely downstream of phospholipase D1-mediated production of phosphatidic acid (PA) that activates phosphatidylinositol 4-phosphate (PI4P) 5-kinase γ. Depletion of plasma membrane PI(4,5)P2 or down-regulation of PI4P 5-kinase γ interferes with histamine-evoked and Ca2+-dependent WPB exocytosis and a mutant PI4P 5-kinase γ incapable of binding PA affects WPB exocytosis in a dominant-negative manner. This indicates that a unique PI(4,5)P2-rich environment in the plasma membrane governs WPB fusion possibly by providing interaction sites for WPB-associated docking factors.

Orchestration of Primary Hemostasis by Platelet and Endothelial Lysosome-Related Organelles.

Megakaryocyte-derived platelets and endothelial cells store their hemostatic cargo in α- and δ-granules and Weibel-Palade bodies, respectively. These storage granules belong to the lysosome-related organelles (LROs), a heterogeneous group of organelles that are rapidly released following agonist-induced triggering of intracellular signaling pathways. Following vascular injury, endothelial Weibel-Palade bodies release their content into the vascular lumen and promote the formation of long VWF (von Willebrand factor) strings that form an adhesive platform for platelets. Binding to VWF strings as well as exposed subendothelial collagen activates platelets resulting in the release of α- and δ-granules, which are crucial events in formation of a primary hemostatic plug. Biogenesis and secretion of these LROs are pivotal for the maintenance of proper hemostasis. Several bleeding disorders have been linked to abnormal generation of LROs in megakaryocytes and endothelial cells. Recent reviews have emphasized common pathways in the biogenesis and biological properties of LROs, focusing mainly on melanosomes. Despite many similarities, LROs in platelet and endothelial cells clearly possess distinct properties that allow them to provide a highly coordinated and synergistic contribution to primary hemostasis by sequentially releasing hemostatic cargo. In this brief review, we discuss in depth the known regulators of α- and δ-granules in megakaryocytes/platelets and Weibel-Palade bodies in endothelial cells, starting from transcription factors that have been associated with granule formation to protein complexes that promote granule maturation. In addition, we provide a detailed view on the interplay between platelet and endothelial LROs in controlling hemostasis as well as their dysfunction in LRO related bleeding disorders.

Weibel-Palade Bodies Orchestrate Pericytes During Angiogenesis.

Objective Weibel-Palade bodies (WPBs) are endothelial cell (EC)-specific organelles formed by vWF (von Willebrand factor) polymerization and that contain the proangiogenic factor Ang-2 (angiopoietin-2). WPB exocytosis has been shown to be implicated for vascular repair and inflammatory responses. Here, we investigate the role of WPBs during angiogenesis and vessel stabilization. Approach and Results WPB density in ECs decreased at the angiogenic front of retinal vascular network during development and neovascularization compared with stable vessels. In vitro, VEGF (vascular endothelial growth factor) induced a VEGFR-2 (vascular endothelial growth factor receptor-2)-dependent exocytosis of WPBs that contain Ang-2 and consequently the secretion of vWF and Ang-2. Blocking VEGF-dependant WPB exocytosis and Ang-2 secretion promoted pericyte migration toward ECs. Pericyte migration was inhibited by adding recombinant Ang-2 or by silencing Ang-1 (angiopoietin-1) or Tie2 (angiopoietin-1 receptor) in pericytes. Consistently, in vivo anti-VEGF treatment induced accumulation of WPBs in retinal vessels because of the inhibition of WPB exocytosis and promoted the increase of pericyte coverage of retinal vessels during angiogenesis. In tumor angiogenesis, depletion of WPBs in vWF knockout tumor-bearing mice promoted an increase of tumor angiogenesis and a decrease of pericyte coverage of tumor vessels. By another approach, normalized tumor vessels had higher WPB density. Conclusions We demonstrate that WPB exocytosis and Ang-2 secretion are regulated during angiogenesis to limit pericyte coverage of remodeling vessels by disrupting Ang-1/Tie2 autocrine signaling in pericytes.

Apolipoprotein E4 Expression Causes Gain of Toxic Function in Isogenic Human Induced Pluripotent Stem Cell-Derived Endothelial Cells.

OBJECTIVE: The ApoE (apolipoprotein) allele epsilon 4 is a major genetic risk factor for Alzheimer disease, cardiovascular disorders, and stroke, indicating that it significantly impacts cerebral and vascular systems. However, very little is known about how APOE genotype affects brain endothelial cells, which form a network of tight junctions to regulate communication between the brain and circulating blood factors. Approach and Results: Here, we present a novel model of endothelial dysfunction using isogenic human induced pluripotent stem cell-derived cells harboring different alleles of the APOE gene, specifically ApoE 3/3, 3/4, and 4/4. We show for the first time that ApoE4 expression by endothelial cells is sufficient to cause a toxic gain of cellular dysfunction. Using RNAseq, we found significant effects of ApoE4 on signaling pathways involved in blood coagulation and barrier function. These changes were associated with altered cell function, including increased binding of platelets to ECs with the 3/4 or 4/4 genotype. ApoE4-positive cells exhibited a proinflammatory state and prothrombotic state, evidenced by higher secretion of Aß (amyloid-ß) 40 and 42, increased release of cytokines, and overexpression of the platelet-binding protein VWF (vonWillebrand factor). Immunohistochemistry of human brain Alzheimer disease brains also showed increased VWF expression with the ApoE4/4 genotype. Finally, pharmacological inhibition of inflammation in ECs by celastrol rescued overexpression of VWF in cells expressing ApoE4. CONCLUSIONS: These cells provide novel insight into ApoE4-mediated endothelial dysfunction and provide a new platform to test potential therapies for vascular disorders.

Permeability and Weibel-Palade Bodies of the Blood Vessels in the Human Vocal Fold Mucosa.

OBJECTIVES: Transendothelial exchange and permeability of the capillaries in Reinke space (the superficial layer of the lamina propria) of the vocal fold mucosa affect physiological and pathological conditions of the human vocal fold mucosa. The mechanism of permeability and Weibel-Palade bodies of the blood vessels in the human vocal fold mucosa were investigated using electron microscopy. STUDY DESIGN: Histologic analysis of the human vocal fold. METHODS: Six normal human vocal folds (three adults and three newborns) obtained from autopsy cases and three human vocal folds with Reinke edema from surgical specimens were investigated under transmission electron microscopy. RESULTS: There were three possible capillary wall transport systems related to the permeability of the blood vessels in the vocal fold mucosa: 1) Fenestra transport, plasma exuded from the capillaries into surrounding tissue via the fenestration with or without a diaphragm; 2) vesicular transport (transcellular transport via vesicles), the use of vesicles to ferry fluid and solutes across endothelial cells; and 3) junctional transport (intercellular transport), molecules passed through intercellular gaps between endothelial cells. Weibel-Palade bodies were present in the cytoplasm of endothelial cells both in adults and newborns. They were present in high numbers in the cytoplasm of endothelial cells, with intercellular transport in the vocal folds with Reinke edema. CONCLUSION: There were three types of mechanisms for the permeability of the blood vessels in the human vocal fold mucosa. Some physiologically active substances, such as histamine produced by Weibel-Palade bodies, may adversely influence the permeability of the blood vessels. LEVEL OF EVIDENCE: NA. Laryngoscope, 2588-2592, 2018.

Modulation of Angiopoietin 2 release from endothelial cells and angiogenesis by the synaptic protein Neuroligin 2.

The synaptic protein Neuroligin 2, similarly to its isoform Neuroligin 1, is produced by endothelial cells, but its activity in the vascular context remains unknown. This study aimed at verifying the hypothesis that Neuroligin 2, in parallel with its extraneuronal involvement in pancreatic beta cells exocytosis, modulated cytokine release from endothelial cells and consequently angiogenesis. We used in vitro approaches to modulate Neuroligin 2 expression and Neuroligin 2 null mice to test our hypotheses. In vitro, upon VEGF stimulation, Neuroligin 2 silencing strongly reduces Angiopoietin 2 release in the medium and increases the endothelial cell retention of Weibel Palade Bodies, the specialized organelles that store Angiopoietin 2 and various other cytokines. On the contrary, Neuroligin 2 overexpression almost depletes cells of Weibel Palade Bodies, independent of VEGF. In vivo, both the retina and tumor xenografts grown in NLGN2- null mice display an immature vasculature, with lower pericyte coverage and lower Tie2 phosphorylation. At the molecular level NLGN2 colocalizes with its neuronal partner collibystin, a CDC42 guanine nucleotide exchange factor, which is also expressed by endothelial cells and in turn modulates Angiopoietin 2 release. Neuroligin 2, an inhibitory synaptic protein, modulates a peculiar aspect of vascular function and could represent a novel target of therapy in various fields, from tumor angiogenesis to vascular diseases.

Interaction between MyRIP and the actin cytoskeleton regulates Weibel-Palade body trafficking and exocytosis.

Weibel-Palade body (WPB)-actin interactions are essential for the trafficking and secretion of von Willebrand factor; however, the molecular basis for this interaction remains poorly defined. Myosin Va (MyoVa or MYO5A) is recruited to WPBs by a Rab27A-MyRIP complex and is thought to be the prime mediator of actin binding, but direct MyRIP-actin interactions can also occur. To evaluate the specific contribution of MyRIP-actin and MyRIP-MyoVa binding in WPB trafficking and Ca(2+)-driven exocytosis, we used EGFP-MyRIP point mutants with disrupted MyoVa and/or actin binding and high-speed live-cell fluorescence microscopy. We now show that the ability of MyRIP to restrict WPB movement depends upon its actin-binding rather than its MyoVa-binding properties. We also show that, although the role of MyRIP in Ca(2+)-driven exocytosis requires both MyoVa- and actin-binding potential, it is the latter that plays a dominant role. In view of these results and together with the analysis of actin disruption or stabilisation experiments, we propose that the role of MyRIP in regulating WPB trafficking and exocytosis is mediated largely through its interaction with actin rather than with MyoVa.

Towards the imaging of Weibel-Palade body biogenesis by serial block face-scanning electron microscopy.

Electron microscopy is used in biological research to study the ultrastructure at high resolution to obtain information on specific cellular processes. Serial block face-scanning electron microscopy is a relatively novel electron microscopy imaging technique that allows three-dimensional characterization of the ultrastructure in both tissues and cells by measuring volumes of thousands of cubic micrometres yet at nanometre-scale resolution. In the scanning electron microscope, repeatedly an image is acquired followed by the removal of a thin layer resin embedded biological material by either a microtome or a focused ion beam. In this way, each recorded image contains novel structural information which can be used for three-dimensional analysis. Here, we explore focused ion beam facilitated serial block face-scanning electron microscopy to study the endothelial cell-specific storage organelles, the Weibel-Palade bodies, during their biogenesis at the Golgi apparatus. Weibel-Palade bodies predominantly contain the coagulation protein Von Willebrand factor which is secreted by the cell upon vascular damage. Using focused ion beam facilitated serial block face-scanning electron microscopy we show that the technique has the sensitivity to clearly reveal subcellular details like mitochondrial cristae and small vesicles with a diameter of about 50 nm. Also, we reveal numerous associations between Weibel-Palade bodies and Golgi stacks which became conceivable in large-scale three-dimensional data. We demonstrate that serial block face-scanning electron microscopy is a promising tool that offers an alternative for electron tomography to study subcellular organelle interactions in the context of a complete cell.

The Golgi is a measuring cup.

The relation of organelle size to cellular function is a basic question in cell biology about which almost nothing is known. Reporting in this issue of Developmental Cell, Ferraro et al. (2014) show that the size and topology of the Golgi apparatus determines the size and functionality of a medically important secretory granule.

A two-tier Golgi-based control of organelle size underpins the functional plasticity of endothelial cells.

Weibel-Palade bodies (WPBs), endothelial-specific secretory granules that are central to primary hemostasis and inflammation, occur in dimensions ranging between 0.5 and 5 µm. How their size is determined and whether it has a functional relevance are at present unknown. Here, we provide evidence for a dual role of the Golgi apparatus in controlling the size of these secretory carriers. At the ministack level, cisternae constrain the size of nanostructures ("quanta") of von Willebrand factor (vWF), the main WPB cargo. The ribbon architecture of the Golgi then allows copackaging of a variable number of vWF quanta within the continuous lumen of the trans-Golgi network, thereby generating organelles of different sizes. Reducing the WPB size abates endothelial cell hemostatic function by drastically diminishing platelet recruitment, but, strikingly, the inflammatory response (the endothelial capacity to engage leukocytes) is unaltered. Size can thus confer functional plasticity to an organelle by differentially affecting its activities.

Anti-atherogenic effects of statins: Impact on angiopoietin-2 release from endothelial cells.

Beyond lipid lowering, statins are supposed to exert pleiotropic effects positively influencing the progression of atherosclerotic lesions. The development of such lesions is associated with increased release of angiopoietin-2 (Ang-2), an endothelial cell-specific protein growth factor stored in Weibel-Palade bodies (WPBs). The aim of our study was to examine whether statin pretreatment influences the release of Ang-2 from endothelial cells. Stimulation of HUVECs and HMVECs with PMA, thrombin or histamine resulted in significant release of Ang-2, as evidenced by ELISA. Pretreatment with simvastatin and mevastatin suppressed this release to basal level, while pravastatin had no effect. Simvastatin itself increased nitric oxide (NO, EC number 1.14.13.39) synthesis, measured by Griess reaction. Combining the statin pretreatment with the eNOS inhibitor L-NNA as well as bypassing the HMG-CoA reductase (EC number: 1.1.1.34) by adding mevalonic acid or geranyl pyrophosphate restored the exocytotic effect of PMA. Immunofluorescence microscopy showed that depletion of WPBs upon PMA stimulation ceased after pretreatment with simvastatin. This study demonstrates a potent suppressive effect of statins on the release of Ang-2 from endothelial cells. Regarding its harmful effects in the development of atherosclerotic lesions, our data provide further insight into the mechanisms of the anti-atherogenic potential of statins.

Effect of laminar shear stress on the distribution of Weibel-Palade bodies in endothelial cells.

BACKGROUND: Vascular endothelial cells (ECs) provide a highly interactive barrier between blood and the underlying tissues. It is well established that ECs exposed to laminar flow align in the direction of flow and also arrange their actin stress fibers in a parallel manner in the direction of flow. Also the organization of the microtubule network is altered in response to flow with repositioning of the microtubule-organizing centre (MTOC) in the direction of flow. Weibel-Palade bodies (WPBs) are endothelial cell specific storage organelles that contain a number of important homeostatic and inflammatory components. Dynamics of WPBs are controlled by microtubules and the actin cytoskeleton. OBJECTIVES: Here, we monitored flow-induced changes in distribution of WPBs. METHODS: ECs were exposed for five days to laminar shear stress of 10 dyne/cm(2). Subsequently we measured the distance of individual WPBs with respect to the centre of the nucleus using Image Pro Plus. RESULTS: ECs aligned in the direction of flow under these conditions. After 5 days the MTOC was positioned downstream of the nucleus in the direction of the flow. The number of WPBs per cell was slightly reduced as a result of the application of flow. Unexpectedly, only minor differences in the distribution of WPBs in ECs cultured under laminar flow were observed when compared to that of cells grown under static conditions. CONCLUSIONS: Our findings suggest that laminar flow does not induce major changes in number and distribution of WPBs in ECs.

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 angiogenesis.

Thrombin upregulates the angiopoietin-Tie2 Axis: endothelial protein C receptor occupancy prevents the thrombin mobilization of angiopoietin 2 and P-selectin from Weibel-Palade bodies.

SUMMARY BACKGROUND: Activated protein C (APC) in complex with endothelial protein C receptor (EPCR) can reverse the barrier-disruptive and cytotoxic effects of proinflammatory cytokines by cleaving protease-activated receptor 1 (PAR-1). Recently, it was reported that the PAR-1-dependent vascular barrier-protective effect of APC is mediated through transactivation of the angiopoietin (Ang)-Tie2 signaling pathway. The antagonist of this pathway, Ang2, is stored in Weibel-Palade bodies within endothelial cells. OBJECTIVES: To determine whether the occupancy of EPCR by its ligand can switch the PAR-1-dependent signaling specificity of thrombin through the Ang-Tie2 axis. METHODS: We activated endothelial cells with thrombin before and after treating them with the catalytically inactive Ser195-->Ala substitution mutant of protein C. The expression levels of Ang1, Ang2 and Tie2 in response to thrombin were measured by both an enzyme-linked immunosorbent assay and a cell permeability assay in the absence and presence of small interfering RNA and a blocking antibody to Tie2. RESULTS: Thrombin upregulated the expression of both Ang1 and Tie2 but downregulated the expression of Ang2 when EPCR was occupied by its ligand. The Ang1-Tie2-dependent protective effect of thrombin was initiated through protein C inhibiting the rapid mobilization of Ang2 from Weibel-Palade bodies. Interestingly, the protein C mutant also inhibited the thrombin mobilization of P-selectin. CONCLUSIONS: These results suggest a physiologic role for the low concentration of thrombin in maintaining the integrity of the EPCR-containing vasculature through the PAR-1-dependent inhibition of Ang2 and P-selectin release from Weibel-Palade bodies.

[Formation and function of Weibel-Palade bodies].

Weibel-Palade bodies (WPB) are specialized cigar-shaped secretory organelles in endothelial cells, which contain a variety of biologically active molecules. These contents can be released rapidly by stimulation and involved in hemostasis, inflammation and angiogenesis. The main component of WPB is von Willebrand factor (vWF), whose expression and tubulation are necessary for the formation of the unique rod-like WPBs. Different molecules such as vWF, P-selectin, CD63, Rab27A and Rab3D are recruited into WPB mediated by the AP-1, AP-3 or other transport machinery. The underlying mechanism of the formation of WPB remains further investigation, which will gain insights into its function. The molecular mechanism of WPB formation and its function were discussed in this review.