Biomedical subjects

Jan A van Mourik

Publications and source records attributed to Jan A van Mourik.

12 recordsLinked to original sources

PubMed2006

Cav3.1 (alpha1G) controls von Willebrand factor secretion in rat pulmonary microvascular endothelial cells.

The T-type Ca2+ channel Cav3.1 subunit is present in pulmonary microvascular endothelial cells (PMVECs), but not in pulmonary artery endothelial cells (PAECs). The present study sought to assess the role of Cav3.1 in thrombin-induced Weibel-Palade body exocytosis and consequent von Willebrand factor (VWF) release. In PMVECs and PAECs transduced with a green fluorescent protein (GFP)-tagged VWF chimera, we examined the real-time dynamics and secretory process of VWF-GFP-containing vesicles in response to thrombin and the cAMP-elevating agent isoproterenol. Whereas thrombin stimulated a progressive decrease in the number of VWF-GFP-containing vesicles in both cell types, isoproterenol only decreased the number of VWF-GFP-containing vesicles in PAECs. In PMVECs, thrombin-induced decrease in the number of VWF-GFP-containing vesicles was nearly abolished by the T-type Ca2+ channel blocker mibefradil as well as by Cav3.1 gene silencing with small hairpin RNA. Expression of recombinant Cav3.1 subunit in PAECs resulted in pronounced increase in thrombin-stimulated Ca2+ entry, which is sensitive to mibefradil. Together, these data indicate that VWF secretion from lung endothelial cells is regulated by two distinct pathways involving Ca2+ or cAMP, and support the hypothesis that activation of Cav3.1 T-type Ca2+ channels in PMVECs provides a unique cytosolic Ca2+ source important for Gq-linked agonist-induced VWF release.

Animals↗

PubMed2006

Dynamics and plasticity of Weibel-Palade bodies in endothelial cells.

Agonist-induced release of endothelial cell specific storage granules, designated Weibel-Palade bodies (WPBs), provides the endothelium with the ability to rapidly respond to changes in its micro-environment. Originally being defined as an intracellular storage pool for von Willebrand factor (VWF), it has recently been shown that an increasing number of other components, including P-selectin, interleukin (IL)-8, eotaxin-3, endothelin-1, and angiopoietin-2, is present within this subcellular organelle, implicating a role for WPB exocytosis in inflammation, hemostasis, regulation of vascular tone and angiogenesis. Recent studies emphasize that WPBs provide a dynamic storage compartment whose contents can be regulated depending on the presence of inflammatory mediators in the vascular micro-environment. Additionally, release of WPBs is tightly regulated and feedback mechanisms have been identified that prevent excessive release of bioactive components from this subcellular organelle. The ability to regulate both contents and exocytosis of WPBs endows these endothelial cell specific organelles with a remarkable plasticity. This is most likely needed to allow for controlled delivery of bioactive components into the circulation on vascular perturbation.

Animals↗

PubMed2006

von Willebrand factor propeptide in malaria: evidence of acute endothelial cell activation.

The pathogenicity of Plasmodium falciparum is thought to relate to the unique ability of infected erythrocytes to adhere to and subsequently activate the vascular endothelium. To study the state of endothelial activation during falciparum malaria, we measured plasma levels of both von Willebrand factor (VWF) and its propeptide, indices of chronic and acute endothelial cell perturbation, respectively. Results were correlated with clinical and biochemical markers of disease severity, including plasma lactate. Our data show that acute endothelial cell activation is a hallmark of malaria in children, indicated by a significant rise in VWF and VWF propeptide. The highest VWF and propeptide levels were seen in cerebral and non-cerebral severe malaria, and associations found between VWF propeptide level and lactate (P < 0.001). Mean VWF propeptide levels (nmol/l) were in cerebral malaria 33.4, non-cerebral severe malaria 26.3, mild malaria 22.1, non-malaria febrile illness 10.2, and controls 10.1. Differences between patient and control groups were highly significant (P < 0.005). Follow-up of 26 cerebral malaria cases showed that levels of VWF propeptide, but not VWF fell by 24 h, following the clinical course of disease and recovery. These novel findings potentially implicate acute, regulated exocytosis of endothelial cell Weibel-Palade bodies in the pathogenesis of Plasmodium falciparum malaria.

ABO Blood-Group System↗

PubMed2006

Amino acid regions 572-579 and 657-666 of the spacer domain of ADAMTS13 provide a common antigenic core required for binding of antibodies in patients with acquired TTP.

Antibodies directed against ADAMTS13 have been detected in the majority of patients with acquired thrombotic thrombocytopenic purpura (TTP). We have previously localized a major antigenic determinant within the spacer domain of ADAMTS13. To identify the amino acid residues of the spacer domain that are involved in binding of anti-ADAMTS13 antibodies, we constructed a series of fifteen hybrids (designated A-O) in which 5-10 amino acids of the spacer domain were exchanged for the corresponding region of ADAMTS1. Plasma from six patients with antibodies directed against the spacer domain was analyzed for reactivity with the ADAMTS13/ADAMTS1 chimeras. Exchange of amino acid residues 572-579 (hybrid C) and 657-666 (hybrid M) completely abolished the binding of antibodies from all six patients analyzed. Regions 580-587 (D), 602-620 (G, H), 629-638 (J), and 667-767 (N) contributed to binding of antibodies from patients 2, 4, and 5 (epitope present within regions CDGHJMN). Antibodies derived from patient 1 required region 602-620 (G, H) for binding (CGHM-epitope). For antibodies of patient 3, residues 564-571 (B), 580-587 (D), and 629-638 (J) were required (BCDJM-epitope), whereas replacement of residues 602-610 (G) and 629-638 (J) greatly diminished binding of antibodies from patient 6 (CGJM-epitope). Despite the presumably polyclonal origin of the antibodies present in plasma of patients, our results suggest that residues 572-579 (C) and 657-666 (M) comprise a common antigenic core region that is crucial for binding of anti-ADAMTS13 antibodies. Other regions that spatially surround this antigenic core further modulate binding of antibodies to the spacer domain.

ADAM Proteins↗

PubMed2005

Dynein-dynactin complex mediates protein kinase A-dependent clustering of Weibel-Palade bodies in endothelial cells.

OBJECTIVE: Perinuclear clustering is observed for several different organelles and illustrates dynamic regulation of the secretory pathway and organelle distribution. Previously, we observed that a subset of Weibel-Palade bodies (WPBs), endothelial cell-specific storage organelles, undergo centralization when endothelial cells are stimulated with cAMP-raising agonists of von Willebrand factor (vWF) secretion. In this study, we investigated this phenomenon of WPB clustering in more detail. METHODS AND RESULTS: Our results demonstrate that the clustered WPBs are localized at the microtubule organizing center and that cluster formation depends on an intact microtubule network. Disruption of the microtubules by nocodazole completely abolished clustering, whereas treatment with the actin depolymerizing compound cytochalasin B had no effect on WPB clustering. Interfering with the dynein-dynactin interaction by overexpression of the p50 dynamitin subunit or the CC1 domain of the p150glued subunit of the dynactin complex completely inhibited perinuclear clustering of WPBs, suggesting that dynein activity mediates this process. Furthermore, we found that inhibition of dephosphorylation resulted in an increase in clustering, whereas inhibition of protein kinase A (PKA) markedly reduced WPB clustering. CONCLUSIONS: These results suggest that perinuclear clustering of WPBs involves PKA-dependent regulation of the dynein-dynactin complex. Endothelial cell stimulation with epinephrine results in retrograde movement of a subset of WPBs to the microtubule organizing center. This minus-end directed transport requires an intact microtubular network and is mediated by the motor protein dynein. Together, our results suggest that epinephrine-induced clustering of WPBs involves PKA-dependent regulation of the dynein-dynactin complex.

1-Methyl-3-isobutylxanthine↗

PubMed2004

Small GTP-binding protein Ral is involved in cAMP-mediated release of von Willebrand factor from endothelial cells.

OBJECTIVE: von Willebrand factor (vWF) is synthesized by endothelial cells and stored in specialized vesicles called Weibel-Palade bodies (WPBs). Recently, we have shown that the small GTP-binding protein Ral is involved in thrombin-induced exocytosis of WPBs. In addition to Ca2+-elevating secretagogues such as histamine and thrombin, release of WPB is also observed after administration of cAMP-raising substances such as epinephrine and vasopressin. In the present study, we investigated whether Ral is also involved in cAMP-mediated vWF release. METHODS AND RESULTS: Activation of Ral was observed 15 to 20 minutes after stimulation of endothelial cells with epinephrine, forskolin, or dibutyryl-cAMP. A cell-permeable peptide comprising the carboxy-terminal part of the Ral protein reduced both thrombin-induced and epinephrine-induced vWF secretion supporting a crucial role for Ral in this process. Furthermore, inhibition of protein kinase A by H-89 resulted in a marked reduction of vWF release and greatly diminished levels of GTP-Ral on stimulation with epinephrine. Activation of Ral was independent of the activation of Epac, a cAMP-regulated exchange factor for the small GTPases Rap1 and Rap2. CONCLUSIONS: These results suggest that protein kinase A-dependent activation of Ral regulates cAMP-mediated exocytosis of WPB in endothelial cells.

Bucladesine↗

PubMed2004

Factor VIII expression in liver disease.

Liver disease is associated with markedly elevated plasma factor VIII (FVIII) levels, whereas the synthesis of many other coagulation factors and proteins is reduced. In order to define the mechanism of FVIII increase, we have determined the expression levels of FVIII, both at mRNA and protein level, in patients with liver disease who underwent partial liver resection. In addition, the expression of von Willebrand factor (VWF) and low density lipoprotein receptor-related protein (LRP), proteins known for their ability to modulate FVIII plasma levels, were examined. Tissue samples for RNA extraction were obtained from 4 patients with cirrhosis, 9 patients with liver failure without cirrhosis and 6 patients with liver metastasis of a colon or rectum carcinoma (control group). In patients with liver cirrhosis hepatic FVIII and LRP mRNA levels were significantly lower than controls (p < or = 0.010), while VWF mRNA was significantly higher (p < or = 0.050). Immunohistochemical analysis revealed that cellular VWF protein distribution was also increased in cirrhotic livers compared to liver tissue from patients with non-cirrhotic liver disease. In cirrhotic tissue enlarged portal veins appeared to overgrow FVIII producing sinusoidal endothelial cells. Similarly, the number of LRP-producing cells appeared to be lower in cirrhotic tissue than in controls. The plasma concentration of both FVIII and VWF was significantly higher in patients with cirrhosis than control subjects (p = 0.038 and 0.010 respectively). These results demonstrate that elevated plasma FVIII levels in liver cirrhosis are associated with increased hepatic biosynthesis of VWF and decreased expression of LRP, rather than increased FVIII synthesis.

Adult↗

PubMed2003

Von Willebrand factor targets IL-8 to Weibel-Palade bodies in an endothelial cell line.

Vascular endothelial cells are able to store the chemotactic cytokine interleukin-8 (IL-8) in specialized storage vesicles, Weibel-Palade bodies, together with von Willebrand factor (VWF) and P-selectin. We investigated whether VWF plays a role in the sorting of IL-8 into these organelles. We examined the effect of VWF expression on IL-8 targeting in an endothelial cell line (EC-RF24). This cell line has retained the typical phenotypic characteristics of primary endothelial cells but has lost the capacity to produce VWF in appreciable amounts. EC-RF24 cells were retrovirally transduced with a vector encoding a VWF-green fluorescent protein chimera (VWF-GFP). This approach enables direct visualization of the cellular distribution and secretory behavior of the VWF-GFP hybrid. Expression of VWF-GFP resulted in the generation of Weibel-Palade body-like organelles as shown by the colocalization of VWF-GFP and P-selectin. VWF-GFP expressing EC-RF24 cells also showed significant colocalization of VWF-GFP with IL-8 in these storage vesicles. Live cell imaging revealed that the number of VWF-GFP-containing granules decreased upon cell stimulation. These observations indicate that VWF plays an active role in sequestering IL-8 into Weibel-Palade bodies.

Antibodies, Monoclonal↗

PubMed2003

Real-time imaging of the dynamics and secretory behavior of Weibel-Palade bodies.

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.

Calcium↗

PubMed2003

Endothelial cell activation in thrombotic thrombocytopenic purpura (TTP): a prospective analysis.

It is generally assumed that endothelial cell injury is the primary event in the pathogenesis of thrombotic thrombocytopenic purpura (TTP). In this study, we have determined the extent of vascular perturbation during acute episodes of the disease. We performed a prospective, serial study of nine patients with relapsing TTP during hospitalization and treatment, and assessed the degree of endothelial cell involvement at admission, exacerbation and remission by measurement of von Willebrand factor (VWF) and VWF-propeptide levels. Measurement of both VWF and its propeptide enabled discrimination between acute and chronic perturbation of the endothelium. Elevated levels of both VWF and propeptide were found at admission. These levels decreased immediately upon plasma exchange therapy. However, plasma VWF and propeptide concentrations did not change, even at the time of acute exacerbation. These observations suggest that endothelial cell activation is not the primary event leading to TTP. Vascular perturbation seems to be a consequence, rather than a cause, of the disease.

Acute Disease↗

PubMed2002

Phospholipid-binding domain of factor VIII is involved in endothelial cell-mediated activation of factor X by factor IXa.

Apparently quiescent, nonapoptotic endothelial cells mediate the activation of factor X by activated factor IX in the presence of its cofactor, activated factor VIII. In a previous study, we reported that during the activation of factor X, the interaction of the cofactor with the endothelial cell membrane clearly differs from the interaction of the cofactor with artificial lipid membranes. In the present study, we identified the peptide domain of factor VIII involved in the assembly of the enzyme-cofactor complex on the endothelial cell surface. With the use of monoclonal antibodies against different peptide sequences on the factor VIII light chain, it was observed that the lipid-binding region of the C2 domain on the factor VIII light chain mediates the assembly of the factor X-activating complex on the endothelial cell surface. In addition, a synthetic peptide that constitutes region Ala2318-Tyr2332 of the C2 domain and that is known for its ability to inhibit the binding of factor VIII to artificial lipid membranes also showed inhibition of the cofactor activity of factor VIII on endothelial cells. Thus, the carboxy-terminal part of the factor VIII light chain not only contains sites involved in lipid binding but also contains sites involved in complex assembly on the endothelial cell membrane.

Annexin A5↗

PubMed2002

Biogenesis and exocytosis of Weibel-Palade bodies.

Vascular endothelial cells contain typical, elongated vesicles, the so-called Weibel-Palade bodies, which serve as a storage compartment for von Willebrand factor (VWF), a plasma protein that plays an essential role in controlling the adhesion and aggregation of platelets at sites of vascular injury. Upon activation of endothelial cells by agonists such as thrombin, epinephrine or histamine, the Weibel-Palade bodies fuse with the plasma membrane and release their contents into the blood circulation. This process provides an adequate means by which endothelial cells can actively participate in controlling the arrest of bleeding upon vascular damage. Besides VWF, Weibel-Palade bodies contain a subset of other proteins, including interleukin-8 (IL-8), P-selectin and endothelin. Similar to VWF, these proteins are transported to the outside of the cell upon stimulation and may control local or systemic biological effects, including inflammatory and vasoactive responses. Apparently, endothelial cells are able to create a storage pool for a variety of bioactive molecules which can be mobilised upon demand. Endothelial cells that are deficient of VWF synthesis are not only unable to form Weibel-Palade bodies, but also lack the ability to store IL-8 or P-selectin or release these proteins in a regulated manner. It thus appears that VWF not only plays a prominent role in controlling primary haemostasis, but also may modulate inflammatory processes through its ability to target inflammatory mediators to the regulated secretion pathway of the endothelium.

Animals↗