Endothelial cells contain specialized storage organelles called Weibel-Palade bodies (WPBs) that release their content into the vascular lumen in response to specific agonists that raise intracellular Ca2+ or cAMP. We have previously shown that cAMP-mediated WPB release is dependent on protein kinase A (PKA) and involves activation of the small GTPase RalA. Here, we have investigated a possible role for another PKA-independent cAMP-mediated signaling pathway in the regulation of WPB exocytosis, namely the guanine nucleotide exchange factor Epac1 and its substrate, the small GTPase Rap1. Epinephrine stimulation of endothelial cells leads to Rap1 activation in a PKA-independent fashion. siRNA-mediated knockdown of Epac1 abolished epinephrine-induced activation of Rap1 and resulted in decreased epinephrine-induced WPB exocytosis. Down-regulation of Rap1 expression and prevention of Rap1 activation through overexpression of Rap1GAP effectively reduced epinephrine- but not thrombin-induced WPB exocytosis. Taken together, these data uncover a new Epac-Rap1-dependent pathway by which endothelial cells can regulate WPB exocytosis in response to agonists that signal through cAMP.Background: Ca2+- and cAMP-raising agonists promote exocytosis of Weibel-Palade bodies from endothelial cells.Results: cAMP-mediated Weibel-Palade body release depends on Rap1 activation by the exchange protein activated by cAMP (Epac).Conclusion: The Epac-Rap1 pathway is involved in the regulation of cAMP-mediated Weibel-Palade body release.Significance: We unraveled a new signaling cascade that regulates cAMP-mediated Weibel-Palade body exocytosis and systemic VWF levels in plasma. Endothelial cells contain specialized storage organelles called Weibel-Palade bodies (WPBs) that release their content into the vascular lumen in response to specific agonists that raise intracellular Ca2+ or cAMP. We have previously shown that cAMP-mediated WPB release is dependent on protein kinase A (PKA) and involves activation of the small GTPase RalA. Here, we have investigated a possible role for another PKA-independent cAMP-mediated signaling pathway in the regulation of WPB exocytosis, namely the guanine nucleotide exchange factor Epac1 and its substrate, the small GTPase Rap1. Epinephrine stimulation of endothelial cells leads to Rap1 activation in a PKA-independent fashion. siRNA-mediated knockdown of Epac1 abolished epinephrine-induced activation of Rap1 and resulted in decreased epinephrine-induced WPB exocytosis. Down-regulation of Rap1 expression and prevention of Rap1 activation through overexpression of Rap1GAP effectively reduced epinephrine- but not thrombin-induced WPB exocytosis. Taken together, these data uncover a new Epac-Rap1-dependent pathway by which endothelial cells can regulate WPB exocytosis in response to agonists that signal through cAMP. Background: Ca2+- and cAMP-raising agonists promote exocytosis of Weibel-Palade bodies from endothelial cells. Results: cAMP-mediated Weibel-Palade body release depends on Rap1 activation by the exchange protein activated by cAMP (Epac). Conclusion: The Epac-Rap1 pathway is involved in the regulation of cAMP-mediated Weibel-Palade body release. Significance: We unraveled a new signaling cascade that regulates cAMP-mediated Weibel-Palade body exocytosis and systemic VWF levels in plasma.
Vascular endothelial cells contain typical elongated vesicles, known as Weibel-Palade bodies. These organelles serve as a storage compartment for a variety of proteins that play a part in controlling vascular homeostasis, including von Willebrand factor, endothelin, P-selectin and interleukin-8. Upon activation of endothelial cells, Weibel-Palade bodies are translocated to the periphery of the cell and there fuse with the plasma membrane to release their contents into the blood circulation and subendothelial connective tissue. This process provides an adequate means by which endothelial cells can actively participate in controlling the arrest of bleeding upon vascular damage or modulate inflammatory reactions and other physiological and pathophysiological processes at the blood-tissue interface. Weibel-Palade bodies may also move to the nucleus of the cell, thus escaping secretion. This phenomenon may play a part in controlling stimulus-induced exocytosis.
The presence of Factor VIII-related antigen (F VIIIRA) in haemostatic plugs was demonstrated by immunofluorescent techniques. Immunofluorescent studies of intact washed platelets incubated with rabbit antifactor VIII in suspension showed that most of the platelets did not stain, whereas a positive staining was obtained after disruption of the membranes after air-drying of a drop of the same platelet suspension on a glass slide. This suggested that F VIIIRA was localized inside the platelets. F VIIIRA was detected in the supernatant of washed platelet suspensions that had been lysed by freezing and thawing (4 ×). This platelet F VIIIRA could not be distinguished from plasma factor VIII in immuno diffusion studies and cross-immuno-electrophoresis using antinormal factor VIII and the antisera against the low ionic strength subunits of factor VIII. The concentration of F VIIIRA in normal platelets was about 60 times as high as the concentration in plasma. Normal concentrations of F VIIIRA were detected in blood platelets and in 13 out of 15 patients with Von Willebrand’s Disease. No F VIIIRA was detected in the plasma of these patients with Von Willebrand’s Disease. Blood platelet F VIIIRA of normal platelets and of platelets of patients with Von Willebrand’s Disease supported the Ristocetin aggregation of washed platelets.
Leukocyte adhesion to endothelial cells and migration into the subendothelial matrix was studied with a three-dimensional model system, consisting of human endothelial cells cultured on a loose collagen matrix. We developed a new method to separate the endothelial cell monolayer and adhering leukocytes, from the subendothelial matrix, allowing simultaneous analysis of leukocyte adhesion and transendothelial migration. Monocytes adhered more avidly to untreated endothelial cells than did neutrophils (2.5 +/- 0.3 vs. 1.0 +/- 0.2 leukocytes per endothelial cell). Only a small fraction (10%-20%) of these leukocytes migrated into the subendothelium. Pretreatment of endothelial cells with interleukin 1 (IL 1) enhanced adhesion (20%), but not migration of monocytes. In contrast, neutrophil adhesion was markedly and in a time-dependent manner increased by IL 1 treatment (i.e. 200% after 6 h and 110% after 24 h of IL 1 treatment). Moreover, IL 1 pretreatment enhanced neutrophil migration twofold. Activation of leukocytes with formyl-methionyl-leucyl-phenylalanine (fMLP) enhanced both monocyte and neutrophil adhesion, but did not affect leukocyte migration. Under all conditions, monocyte adhesion was only partly (30%-40%) inhibited by monoclonal antibodies (mAb) against the common beta subunit of the leukocyte-cell adhesion molecules (LeuCAM: CD18) and 25%-30% by mAb against the alpha subunit of LFA-1 (CD11a). In contrast, mAb against the alpha subunits of Mac-1 (CD11b) and p150.95 (CD11c) were hardly effective. fMLP-mediated neutrophil adhesion was reduced to below baseline levels by anti-LeuCAM (CD18) mAb, whereas the LeuCAM contribution in IL 1-mediated neutrophil adhesion was less pronounced and varied in time. IL 1-mediated neutrophil migration, however, was completely blocked by anti-LeuCAM mAb. fMLP-mediated neutrophil adhesion was inhibited by mAb against the alpha subunits of Mac, while mAb against the alpha subunits of LFA-1 and Mac-1 both reduced IL 1-mediated adherence. In summary, we describe a novel leukocyte adhesion/migration method and demonstrate that the contribution of the LeuCAM complex in leukocyte-endothelium interaction varies depending on cell type and stimulus used.
