Published online before print April 3, 2003, doi: 10.1161/01.ATV.0000069847.72001.E8
© 2003 American Heart Association, Inc.
Vascular Biology |
Real-Time Imaging of the Dynamics and Secretory Behavior of Weibel-Palade Bodies
From the Departments of Plasma Proteins (T.R.d.W., M.G.R., J.V., J.A.v.M.) and Experimental Immunohaematology (P.L.H.), Sanquin Research at CLB, Amsterdam, and the Department of Vascular Medicine (J.A.v.M.), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
Correspondence to J.A. van Mourik, PhD, Department of Plasma Proteins, Sanquin Research at CLB, Plesmanlaan 125, 1066 CX Amsterdam, the Netherlands. E-mail J_van_Mourik{at}clb.nl
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Abstract |
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Objective— Weibel-Palade bodies (WPBs) are specialized secretory granules found in endothelial cells. These vesicles store hormones, enzymes, and receptors and exhibit regulated exocytosis on cellular stimulation. Here we have directly visualized intracellular trafficking and the secretory behavior of WPBs in living cells by using a hybrid protein consisting of von Willebrand factor (vWF), a prominent WPB constituent, and green fluorescent protein (GFP).
Methods and Results— Immunofluorescence microscopy demonstrated that this chimera was targeted into WPBs. In resting cells, some WPBs seemed motionless, whereas others moved at low speed in a stochastic manner. On stimulation of cells with [Ca2+]i- or cAMP-raising secretagogues, membrane-apposed patches were formed, suggesting fusion of WPBs with the plasma membrane. Patches remained visible for >20 minutes. This sustained, membrane-associated retention of vWF might play a role in focal adhesion of blood constituents to the endothelium after vascular injury. In addition, stimulation with cAMP-raising agonists resulted in clustering of a subset of WPBs in the perinuclear region of the cell. Apparently, these WPBs escaped secretion. This feature might provide a mechanism to control regulated exocytosis.
Conclusions— In conclusion, the fusion protein vWF-GFP provides a powerful tool to study, in real time, signal-mediated trafficking of WPBs.
Key Words: von Willebrand factor • Weibel-Palade bodies • endothelial cells • real-time imaging • green fluorescent protein
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Introduction |
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Vascular endothelial cells are equipped with a machinery that, on perturbation, allows prompt delivery of a number of bioactive substances, including hormones, receptors, and adhesive molecules, to the surface of the cell. A distinct subset of proteins destined to be released on stimulation of the endothelium stems from Weibel-Palade bodies (WPBs), typical and morphologically highly organized storage vesicles that release their contents by regulated exocytosis. WPBs are endothelial cell–specific elongated organelles, enclosed by a limiting membrane, which are
0.1 µm wide and up to 4 µm long1 and originate from the trans-Golgi network.2,3 They serve as storage vesicles for a variety of proteins with different biologic functions, such as the leukocyte adhesion receptor P-selectin4,5 and the chemokine interleukin-8 (IL-8).6,7 Effective translocation of P-selectin from WPBs to the cell surface is critical for the binding and rolling of leukocytes on the endothelium at sites of inflammation.8,9 Similarly, regulated exocytosis of IL-8 provides an effective means for controlling local leukocyte extravasation.10 One of the most prominent WPB residents is von Willebrand factor (vWF), an adhesive multimeric glycoprotein that contributes to platelet adhesion and hemostatic plug formation at sites of vascular injury (reviewed in Sadler11 and Ruggeri12). Regulated secretion of vWF provides an adequate means for endothelial cells to actively participate in controlling the arrest of bleeding after vascular damage. Thus, it seems likely that regulated exocytosis of WPBs serves several physiological functions, including inflammatory and hemostatic responses.
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Regulated exocytosis of vWF and other WPB residents involves the translocation of WPBs from the cytoplasm toward the cell surface and the fusion of these vesicles with the plasma membrane. Increased concentrations of cytosolic Ca2+ have been implicated in the mechanism of exocytosis of a number of agonists, including thrombin and histamine.13,14 Release of WPBs can also be induced by secretagogues, such as epinephrine or forskolin, agents known for their ability to activate cAMP-dependent signaling.15,16 The cellular responses to increased cytosolic Ca2+ are most likely mediated by calmodulin and small GTP-binding proteins.17–19 The molecular mechanisms associated with cAMP-dependent exocytosis of WPBs remain to be identified. It has been shown that cAMP-mediated responses differ from regulated secretion elicited by a rise in cytosolic Ca2+ in that secretion induced by Ca2+-raising agents involves the release of both peripheral and central granules, whereas cAMP-mediated secretion primarily involves vesicles located in the periphery of the cell.18
Although studies performed so far have clearly provided the basis for understanding the molecular machinery responsible for exocytotic trafficking of WPBs, little is known about the dynamics of this secretory process. To date, exocytosis of WPBs has been studied only morphologically, by monitoring defined stages of this process in fixed cells. These conditions do not necessarily reflect the dynamics found in intact, living cells. The aim of this study was to investigate the intracellular trafficking of WPBs in living, wild-type endothelial cells in real time. For this purpose, we introduced, by retroviral transduction, a green fluorescent protein (GFP)-tagged vWF into primary human umbilical vein endothelial cells (HUVECs). The vWF-GFP chimera was correctly targeted to WPBs, together with endogenous vWF and P-selectin. This approach, exploiting the intrinsic fluorescence of vWF-GFP, allows direct visualization of the routing and fate of WPBs on stimulation of the cell. Our data reveal novel features of the dynamics and secretory behavior of WPBs, including perinuclear redistribution and membrane-apposed accumulation of GFP-containing granules.
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Methods |
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Please see http://atvb.ahajournals.org for details of the methods used for this study.
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Results |
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vWF-GFP Expression and Distribution in HUVECs
HUVECs were retrovirally transfected with either the vWF-GFP hybrid protein alone or together with hTERT-NGFR (hTERT, human telomerase reverse transcriptase; NGFR, neural growth factor). The transduction efficiency by either method was
20%, as determined by measuring GFP expression by flow cytometry. GFP-positive cells revealed a vesicular distribution of the green fluorescence (Figure IB, available at http://atvb.ahajournals.org). Three-dimensional analysis of the cells showed rod-shaped vesicles that were distributed throughout the cytoplasm. Closer examination of the position of the GFP-containing organelles within the cell, visualized with a color scale (Figure IB), revealed that some vesicles were located at the bottom of the cell, whereas the majority was positioned in the middle, and some others were located at the top of the cell. Thus, transfection of HUVECs with vWF-GFP results in the formation of GFP-labeled vesicles, typical of WPBs, which were heterogeneously distributed throughout the cell. We next confirmed whether vWF-GFP was indeed targeted to genuine WPBs. Staining of vWF-GFP–infected HUVECs with anti-vWF (CLB-RAg 35, directed against the A1 domain) revealed rod-shaped granules that were always colocalized with the GFP signal, consistent with targeting of vWF-GFP to WPBs (Figure IIa through IIc, available at http://atvb.ahajournals.org). Similarly, vWF-GFP was colocalized with P-selectin, a typical marker of WPBs (Figure IIg through IIi). GFP-positive vesicles were distinct from lysosomes, because GFP-associated vesicles were not colocalized with granules that stained with an antibody to cathepsin D, a lysosomal marker (Figure IIj through IIl). These data demonstrate that vWF-GFP is targeted to WPBs, together with endogenous, wild-type vWF. To discriminate between endogenous vWF and vWF-GFP, cells were stained with an anti-vWF antibody (CLB-RAg 50) directed against the A2 domain of vWF. All GFP-positive vesicles were stained with this antibody (Figure IId through IIf). This indicates that WPBs of vWF-GFP–infected HUVECs contained both endogenous, wild-type vWF and the vWF-GFP hybrid protein.
vWF-GFP Multimerization and Secretion
Polymerization of vWF is one of the most characteristic events that occurs during its posttranslational maturation. Only vWF in its multimeric form is stored in WPBs.11 To verify whether vWF-GFP was also able to multimerize, wild-type HUVECs and vWF-GFP–transfected HUVECs were stimulated with thrombin, and the respective media were subjected to multimer analysis. vWF-GFP–transfected cells produced vWF multimers that were similar to wild-type vWF multimers (Figure III, available at http://atvb.ahajournals.org). As expected, vWF multimers produced by wild-type HUVECs did not stain with an antibody against GFP. However, the distribution of multimers secreted by vWF-GFP HUVECs, revealed by staining with anti-GFP, was identical to the pattern observed after staining with polyclonal antibodies to vWF. Thus, replacement of the A2 domain by GFP did not affect the ability of vWF to multimerize.
We also quantified the secreted vWF-GFP. Stimulation with phorbol myristate acetate (PMA) for 60 minutes resulted in a 1.5-fold increase of vWF (P<0.05) and GFP antigen (P<0.01) secreted into the medium compared with the amounts secreted by unstimulated cells (Figure IV, available at http://atvb.ahajournals.org). Similarly, after incubation with thrombin, PMA, or forskolin for 60 minutes, we observed a significant decrease in the number of WPBs compared with the number secreted by untreated cells (Table I, available at http://atvb.ahajournals.org). Collectively, these data indicate that WPBs containing vWF-GFP retained their ability to secrete their "cargo" in a regulated manner.
Dynamics of WPBs in Resting vWF-GFP–Transfected HUVECs
Having established that vWF-GFP properly accumulated in WPBs, was releasable, and had retained its ability to multimerize, we next examined the dynamics of WPBs in living, resting cells. To follow individual vesicles, we selected cells with relatively few WPBs. Preliminary observations revealed that the secretory behavior of WPBs was not affected by the WPB density of the cell. These vesicles were monitored for 60 minutes at intervals of 2 minutes. Figure 1 shows individual frames of the first 40 minutes of a real-time movie of resting vWF-GFP–transfected cells (Video I, available at http://atvb.ahajournals.org). We observed vesicle traffic in an apparently random and uncoordinated fashion throughout the cell body. Some granules seemed motionless during the entire recording period, as if they were "tethered" (Figure 1; WPB 1, 3), whereas others were continuously moving in a stochastic manner (Figure 1; WPB 2 and 4 through 7). Some vesicles seemed to travel longer distances, notably toward the periphery (Figure 1; WPB 4, 7), whereas others returned to their starting point (Figure 1; WPB 2, 5). We also observed WPBs that rotated along their longitudinal axes and sometimes appeared as round vesicles (Figure 1; WPB 6).
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Dynamics of WPBs in PMA-Stimulated vWF-GFP–Infected HUVECs
We next investigated WPB trafficking induced by different agonists. Cells were monitored during 60 minutes of stimulation at intervals of 1 minute. Figure 2 shows a typical time-lapse sequence of vWF-GFP–infected HUVECs stimulated with PMA (Video II, available at http://atvb.ahajournals.org). The apparent random movement of WPBs changed radically on stimulation. In each real-time stimulation experiment performed (n=8), we observed the same succession of events. Cells contracted slightly, an event which coincided with slight movement of the WPBs toward the center of the cell. Vesicles were not seen to clearly move to the periphery of the cell. However, 15 minutes after stimulation of the cells, the rod-shaped WPBs transformed into bright, stationary patches (Figure 2A and 2B; WPB 1 through 3). The lag phase probably reflects diffusion of the agonist after addition to the medium. Typically, individual patches remained visible for 20 minutes. Finally, these patches completely disappeared, and the cells became depleted of fluorescent material. Interestingly, disappearance of a patch often coincided with the appearance of a diffuse "cloud" (Figure 2; WPB 2, 30 minutes and WPB 3, 40 minutes) at the surface of the cell. This feature most likely reflects gradual dispersion of vWF-GFP into the extracellular environment. The formation of patches within a single cell was not synchronized. The first patches appeared, on average, 15 minutes after stimulation; others appeared only after 60 minutes. Similarly, patches disappeared at apparently different rates. Cells were totally depleted of WPBs between 60 and 120 minutes after stimulation.
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Figure 3 shows a 3-dimensional analysis of the transformation of a single WPB into a patch (arrow). To facilitate this analysis, we selected a cell with few vesicles. The x-y view of this cell (Figure 3A) shows that during the first 20 minutes of stimulation with PMA, the WPB appeared as a thin, rod-shaped vesicle. Its depth-coding staining signal suggested that the vesicle was located in the middle of the cell (green). A y-z view of this WPB (Figure 3B) confirmed its tubular morphology and central localization. About 20 minutes after stimulation, this vesicle transformed into a bright, round patch located at the apical side (blue, Figure 3A; 20 minutes). Examination of the cell on the y-z plane indicated that the patch represented the same vesicle (Figure 3B, arrow). However, it had rotated 45° and was positioned perpendicularly to the focal plane. The vesicle was apposed to the cell membrane at the apical part of the cell (Figure 3B). The patch remained visible in the x-y view for 20 minutes (Figure 3A, 50 minutes). The y-z view of the vesicle showed that its size diminished in time (Figure 3B, 22 to 40 minutes). Finally, 50 minutes after stimulation, the vesicle had completely disappeared. On stimulation, WPBs moved at a similar speed as in untreated cells (<10 nm/s). The entire process of patch formation, disappearance of vesicles, and appearance of the vWF-GFP clouds (see Figure 2) most likely reflects fusion of WPBs with the plasma membrane and secretion of vWF-GFP at the fusion site into the extracellular milieu. No preferential movement of WPBs to either the basal or the apical side of the cell was observed. However, vesicles that were primarily located at the bottom of the cell tended to form patches at the basal side of the cell, whereas vesicles residing at the upper part of the cell tended to dock at the apical side.
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Dynamics of WPBs in Thrombin-Stimulated vWF-GFP–Infected HUVECs
When vWF-GFP–infected HUVECs were stimulated with thrombin, we often observed an immediate contraction of the cells. This somewhat obscured visualization of the dynamics of WPB secretion. This process started a few minutes after addition of the stimulus. Thrombin also induced more rapid formation of patches than did PMA. Most patches were formed within the first 5 minutes of stimulation (Figure V, available at http://atvb.ahajournals.org) and disappeared faster (after 10 minutes) than did PMA-induced patches. In some cases, concomitant formation of clouds was observed. Fewer patches were formed with thrombin than with PMA. As during PMA-induced stimulation, patch formation and fading were observed at the apical as well as the basal part of the cells.
WPB Dynamics Induced by cAMP Agonists
Unlike PMA and thrombin, neither forskolin (not shown) nor epinephrine induced contraction of the cell. However, we observed that these secretagogues induced a vectorial migration of WPBs toward the nucleus (Figure 4). Perinuclear redistribution was much more pronounced than that induced by either PMA or thrombin. After 30 minutes, most of the vesicles clustered around the nucleus in a starlike structure (Figure 4A). Also, y-z analysis of the cell demonstrated that the initial random distribution of WPBs changed completely on stimulation with epinephrine (Figure 4B). We clearly observed that on stimulation, vesicles moved to the center of the cell, in particular, to the luminal side. Furthermore, vesicles moved up to 10 times faster (<100 nm/s) than in resting cells.
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During migration, secretion was suggested to occur by means of the formation of patches that started to appear within 20 minutes of stimulation. However, they disappeared faster than after PMA stimulation. Indeed, patches lasted for 5 minutes (Video III, available at http://atvb. ahajournals.org). Patches were seen both at the periphery of the cell and around the nucleus. Furthermore, patch formation was observed at the basal (Figure 4, WPB 1) as well as the apical (WPB 2) part of the cell. Thus, cAMP agonists induced distinct migration of WPBs to a specific site directly above the nucleus and concomitant formation of a starlike clustering of vesicles. Tentative fusion events started within the same time interval as observed on stimulation with PMA, but the event itself was as fast as after stimulation with thrombin.
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Discussion |
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In the past, biogenesis and regulated exocytosis of WPBs have been extensively studied by biochemical and morphological analyses of endothelial cells. So far, the dynamics of this unique endothelial cell–specific storage device could not be addressed. In this study, the first evidence is presented for the complexity of the dynamics of WPBs. We constructed a vWF-GFP chimera that was able to multimerize and was properly targeted to WPBs and secreted on stimulation. This allowed direct visualization of WPB trafficking in living, primary HUVECs.
We observed that in resting cells, these vesicles are not static but consist of pools with different motilities (Figure 1). Some vesicles barely moved, as if they were tethered; others seemed to travel in a stochastic manner and frequently reversed their direction. All WPBs moved with remarkably low speed, not exceeding 10 nm/s. Previous studies on other cell types have shown that secretory vesicles may travel in different directions and move with different speeds. Newly formed, microtubule-associated vesicles travel at high speed (1 µm/s) from the Golgi to the plasma membrane.20–23 On arrival at the plasma membrane, these vesicles are trapped and mature in the dense meshwork of the actin cortex, which restricts their motility (50 nm/s).23,24 On the basis of these data and on the similar dynamics observed in WPBs, we speculate that the tethered WPBs seen in our study (eg, Figure 1; WPB 1, 3) are "docked" at the plasma membrane or trapped in the actin cortex of endothelial cells. WPBs that moved in all directions are reminiscent of microtubule-associated granules.20 Whether the WPBs observed here are immature or mature granules cannot be concluded from this study.
Stimulation with secretagogues dramatically changes the dynamics of WPBs. We observed 2 characteristic features of this traffic: (1) formation of patches observed on stimulation with both Ca2+ and cAMP agonists and (2) perinuclear clustering of WPBs only induced by cAMP secretagogues.
Formation of Membrane-Associated Patches
Previously, in studies with fixed endothelial cells, the presence of large, extracellular patches of vWF was noticed on stimulation of the cells.25,26 Our study clearly documents the generation of this typical feature. This is most apparent in the real-time movie of PMA-stimulated cells (Video I). These observations suggest that patch formation reflects fusion of a single WPB with the plasma membrane rather than the clustering of vesicles and subsequent bulk extrusion.26 Changes in fluorescence signal could be due to changes in the pH of the microenvironment and/or changes in fluorescence quenching because of dispersion of vWF-GFP. Striking was the rather long life span of WPB patches.22 The rapid (seconds) increase of the fluorescence signal due to fusion with the plasma membrane was followed by a slow (minutes) decay of the fluorescence (Figure 2B) as vWF-GFP diffused into the extracellular space. The apparent slow release of vWF-GFP from the cell could be due to the compact, crystalloid structure of vWF, which may hamper its dissolution. The possible interaction of vWF with other WPB constituents or with proteins localized at the plasma membrane might also affect the rate of vWF dispersion.
These long-lasting patches could have physiological significance. Patches at the cell surface might not only reflect diffusion of vWF-GFP but might also provide focal sites with a high concentration of vWF. These sites could play a role in adequately recruiting and binding plasma proteins, blood cells, or matrix components to sites of vascular injury. Pertinent to this point is the observation that platelets adhered rapidly, though transiently, to vWF secreted at the luminal face of endothelial cells on triggering of the cell with Ca2+-agonists.27,28 The observation that adherence of platelets to the endothelium was transient (minutes) corresponds with the time course of the fading of patches. Although this has not been demonstrated, it is possible that vWF-containing patches also present IL-8, P-selectin, or other WPB residents at high concentrations at the cell surface. Focal sites expressing these proteins at high levels could contribute to the rapid recruitment of leukocytes to endothelial cells after simulation. Indeed, the time frame of P-selectin–mediated rolling of leukocytes on the endothelium is on the same order of magnitude as patch fading.29
The vectorial movement of WPBs induced by stimulation with either Ca2+- or cAMP-raising agents was less prominent than expected on the basis of previous observations. Basolateral as well apical secretion of vWF after simulation of endothelial cells has been observed.30,31 In this study, we observed that WPBs that are recruited toward the nucleus tended to secrete their contents at the apical side of the cell, whereas vesicles that resided at the periphery of the cell secreted at the basal side. The location of the microtubule-organizing center (MTOC, see next paragraph) at the apical side of the cell might contribute to preferential luminal secretion associated with exocytosis of vesicles located in the vicinity of the nucleus. However, we did not observe any preferential accumulation of WPBs or a distinct vectorial movement. Quantitative analysis of the dynamics of WPBs in real time of multiple cells might provide more insight into a possible vectorial behavior of vesicle trafficking.
Perinuclear Clustering of WPBs
Another prominent feature of the dynamics of WPBs was the perinuclear clustering of WPBs. Only when cells were exposed to cAMP-raising agonists, such as forskolin or epinephrine, were WPBs docked at a distinct site proximal to the nucleus and spatially organized in a starlike structure (Figure 4). WPBs migrated to that site at velocities 10 times higher than in resting cells. On the basis of the observed localization and the morphology of these clusters, we assume that the site toward which WPBs migrated is associated with the MTOC. This observation suggests that microtubules play a role in the cAMP-induced migration of WPBs. Although not studied here, the cAMP-dependent mechanism responsible for the perinuclear recruitment of WPBs is most likely caused by protein kinase A–dependent modulation of the activity of microtubule-associated motor proteins.32,33 In addition to perinuclear clustering, patch formation was also observed (Figure 4; WPB 1, 2). Notably, tethered WPBs escaped clustering and fused directly with the plasma membrane. On stimulation with thrombin or PMA, WPBs did not accumulate around the nucleus (Figure 2 and Figure V). Apparently, WPBs are directly translocated to the plasma membrane under these conditions. Both actin filaments and microtubules are most likely involved in the Ca2+-dependent trafficking of WPBs.18,34,35
Taken together, although both cAMP- and Ca2+-raising agents induce patch formation, WPB dynamics triggered by these agonists are clearly different. cAMP-dependent perinuclear recruitment of WPBs might provide a means to limit excessive release of prothrombotic and proinflammatory mediators stored in WPBs under physiological conditions that raise intracellular levels of cAMP, such as physical exercise or other stress situations.18 On the other hand, Ca2+-mediated secretion, eg, in response to vascular damage, most likely reflects mobilization of the entire WPB population to accomplish adequate release of bioactive molecules at sites of vascular injury.
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Acknowledgments |
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This work was supported by a grant from the Netherlands Heart Foundation (grant No. 2000.97). We kindly thank Drs C.P. Engelfriet and A. Sonnenberg for critically reading the manuscript. We also gratefully acknowledge Erik Mul for his technical assistance.
Received January 19, 2003; accepted March 12, 2003.
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Y. Suzuki, H. Mogami, H. Ihara, and T. Urano Unique secretory dynamics of tissue plasminogen activator and its modulation by plasminogen activator inhibitor-1 in vascular endothelial cells Blood, January 8, 2009; 113(2): 470 - 478. [Abstract] [Full Text] [PDF]
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 A. Moldobaeva, J. Jenkins, and E. Wagner Effects of distension on airway inflammation and venular P-selectin expression Am J Physiol Lung Cell Mol Physiol, November 1, 2008; 295(5): L941 - L948. [Abstract] [Full Text] [PDF]
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 R. Noubade, R. del Rio, B. McElvany, J. F. Zachary, J. M. Millward, D. D. Wagner, H. Offner, E. P. Blankenhorn, and C. Teuscher Von-Willebrand Factor Influences Blood Brain Barrier Permeability and Brain Inflammation in Experimental Allergic Encephalomyelitis Am. J. Pathol., September 1, 2008; 173(3): 892 - 900. [Abstract] [Full Text] [PDF]
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C. J. van Schooten, S. Shahbazi, E. Groot, B. D. Oortwijn, H. M. van den Berg, C. V. Denis, and P. J. Lenting Macrophages contribute to the cellular uptake of von Willebrand factor and factor VIII in vivo Blood, September 1, 2008; 112(5): 1704 - 1712. [Abstract] [Full Text] [PDF]
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J. Johnsen and J. A. Lopez VWF secretion: what's in a name? Blood, August 15, 2008; 112(4): 926 - 927. [Full Text] [PDF]
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M. G. Rondaij, R. Bierings, E. L. van Agtmaal, K. A. Gijzen, E. Sellink, A. Kragt, S. S. G. Ferguson, K. Mertens, M. J. Hannah, J. A. van Mourik, et al. Guanine exchange factor RalGDS mediates exocytosis of Weibel-Palade bodies from endothelial cells Blood, July 1, 2008; 112(1): 56 - 63. [Abstract] [Full Text] [PDF]
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 D. J. Metcalf, T. D. Nightingale, H. L. Zenner, W. W. Lui-Roberts, and D. F. Cutler Formation and function of Weibel-Palade bodies J. Cell Sci., January 1, 2008; 121(1): 19 - 27. [Abstract] [Full Text] [PDF]
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 M. Erent, A. Meli, N. Moisoi, V. Babich, M. J. Hannah, P. Skehel, L. Knipe, G. Zupancic, D. Ogden, and T. Carter Rate, extent and concentration dependence of histamine-evoked Weibel Palade body exocytosis determined from individual fusion events in human endothelial cells J. Physiol., August 15, 2007; 583(1): 195 - 212. [Abstract] [Full Text] [PDF]
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H. L. Zenner, L. M. Collinson, G. Michaux, and D. F. Cutler High-pressure freezing provides insights into Weibel-Palade body biogenesis J. Cell Sci., June 15, 2007; 120(12): 2117 - 2125. [Abstract] [Full Text] [PDF]
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 C. D. Anfuso, G. Lupo, L. Romeo, G. Giurdanella, C. Motta, A. Pascale, C. Tirolo, B. Marchetti, and M. Alberghina Endothelial cell-pericyte cocultures induce PLA2 protein expression through activation of PKC{alpha} and the MAPK/ERK cascade J. Lipid Res., April 1, 2007; 48(4): 782 - 793. [Abstract] [Full Text] [PDF]
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C. Zhou, H. Chen, F. Lu, H. Sellak, J. A. Daigle, M. F. Alexeyev, Y. Xi, J. Ju, J. A. van Mourik, and S. Wu Cav3.1 ({alpha}1G) controls von Willebrand factor secretion in rat pulmonary microvascular endothelial cells Am J Physiol Lung Cell Mol Physiol, April 1, 2007; 292(4): L833 - L844. [Abstract] [Full Text] [PDF]
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R. J. Dekker, R. A. Boon, M. G. Rondaij, A. Kragt, O. L. Volger, Y. W. Elderkamp, J. C. M. Meijers, J. Voorberg, H. Pannekoek, and A. J. G. Horrevoets KLF2 provokes a gene expression pattern that establishes functional quiescent differentiation of the endothelium Blood, June 1, 2006; 107(11): 4354 - 4363. [Abstract] [Full Text] [PDF]
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G. Michaux, T. J. Pullen, S. L. Haberichter, and D. F. Cutler P-selectin binds to the D'-D3 domains of von Willebrand factor in Weibel-Palade bodies Blood, May 15, 2006; 107(10): 3922 - 3924. [Abstract] [Full Text] [PDF]
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 M. G. Rondaij, R. Bierings, A. Kragt, J. A. van Mourik, and J. Voorberg Dynamics and Plasticity of Weibel-Palade Bodies in Endothelial Cells Arterioscler Thromb Vasc Biol, May 1, 2006; 26(5): 1002 - 1007. [Abstract] [Full Text] [PDF]
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J. H. Cleator, W. Q. Zhu, D. E. Vaughan, and H. E. Hamm Differential regulation of endothelial exocytosis of P-selectin and von Willebrand factor by protease-activated receptors and cAMP Blood, April 1, 2006; 107(7): 2736 - 2744. [Abstract] [Full Text] [PDF]
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M. G. Rondaij, R. Bierings, A. Kragt, K. A. Gijzen, E. Sellink, J. A. van Mourik, M. Fernandez-Borja, and J. Voorberg Dynein-Dynactin Complex Mediates Protein Kinase A-Dependent Clustering of Weibel-Palade Bodies in Endothelial Cells Arterioscler Thromb Vasc Biol, January 1, 2006; 26(1): 49 - 55. [Abstract] [Full Text] [PDF]
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 I. Oynebraten, N. Barois, K. Hagelsteen, F.-E. Johansen, O. Bakke, and G. Haraldsen Characterization of a Novel Chemokine-Containing Storage Granule in Endothelial Cells: Evidence for Preferential Exocytosis Mediated by Protein Kinase A and Diacylglycerol J. Immunol., October 15, 2005; 175(8): 5358 - 5369. [Abstract] [Full Text] [PDF]
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 M. J. Hannah, P. Skehel, M. Erent, L. Knipe, D. Ogden, and T. Carter Differential Kinetics of Cell Surface Loss of von Willebrand Factor and Its Propolypeptide after Secretion from Weibel-Palade Bodies in Living Human Endothelial Cells J. Biol. Chem., June 17, 2005; 280(24): 22827 - 22830. [Abstract] [Full Text] [PDF]
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M. G. Rondaij, E. Sellink, K. A. Gijzen, J. P. ten Klooster, P. L. Hordijk, J. A. van Mourik, and J. Voorberg Small GTP-Binding Protein Ral Is Involved in cAMP-Mediated Release of von Willebrand Factor From Endothelial Cells Arterioscler Thromb Vasc Biol, July 1, 2004; 24(7): 1315 - 1320. [Abstract] [Full Text] [PDF]
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U. Fiedler, M. Scharpfenecker, S. Koidl, A. Hegen, V. Grunow, J. M. Schmidt, W. Kriz, G. Thurston, and H. G. Augustin The Tie-2 ligand Angiopoietin-2 is stored in and rapidly released upon stimulation from endothelial cell Weibel-Palade bodies Blood, June 1, 2004; 103(11): 4150 - 4156. [Abstract] [Full Text] [PDF]
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