Genetic induction of a releasable pool of factor VIII in human endothelial cells.
Although it is known that factor VIII (FVIII) plasma levels increase rapidly in response to a number of stimuli, the biological stimuli behind this release is less clear. Previously, we showed that FVIII can traffic together with von Willebrand factor (vWF) into storage granules in a pituitary tumor cell line, AtT-20; however, AtT-20 cells could not be used to address the release or functional activity of released FVIII. To investigate the regulated secretion of stored FVIII, endothelial cells with intact agonist-stimulated release pathways were used. Human umbilical vein endothelial cells (HUVECs) were transduced with retroviral FVIII construct [hFVIII(V)] to create a FVIII/vWF storage pool. Immunofluorescent staining of transduced cells demonstrated FVIII in Weibel-Palade bodies. In contrast, the transduction of hFVIII(V) into HT-1080 and HepG2 cells displayed FVIII only in the cytoplasm. We studied the regulated release of both FVIII and vWF from endothelial cells after agonist-induced stimulation and demonstrated a parallel release of FVIII and vWF proteins. This released FVIII was functionally active. Hence, endothelial cells transduced with hFVIII(V) store FVIII together with vWF in Weibel-Palade bodies, creating a releasable storage pool of both proteins. Because FVIII secretion can be physiologically regulated by agonists in culture, this may explain the pharmacological agonist-induced release of FVIII by drugs such as desmopressin in vivo and suggests vascular endothelium as a reasonable target of gene therapy of hemophilia A.
Rosenberg
J B
JB
Blood Research Institute, The Blood Center of Southeastern Wisconsin, Milwaukee, WI, USA.
Greengard
J S
JS
Montgomery
R R
RR
D002460:Cell Line; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D003894:Deamino Arginine Vasopressin / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000378:metabolism*; D005169:Factor VIII / Q000096:biosynthesis / Q000235:genetics*; D019084:Fluorescent Antibody Technique, Indirect; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006467:Hemophilia A / Q000628:therapy; D006490:Hemostatics / Q000494:pharmacology; D006801:Humans; D014161:Transduction, Genetic; D014841:von Willebrand Factor / Q000096:biosynthesis / Q000235:genetics
D006490:Hemostatics; D014841:von Willebrand Factor; D005169:Factor VIII; D003894:Deamino Arginine Vasopressin