Genetic Induction of a Releasable Pool of Factor VIII in Human Endothelial Cells

Rosenberg, Jonathan B.; Greengard, Judith S.; Montgomery, Robert R.

doi:10.1161/01.atv.20.12.2689

Cited by 72 publications

(63 citation statements)

References 43 publications

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“…3 Moreover, transplantation of LSEC offers therapeutic potential in coagulation disorders, including hemophilia A. 18,47 Besides the possibility of transducing cultured LSEC via retroviral and lentiviral vectors, 48,49 the feasibility of lentiviral gene transfer in freshly isolated LSEC, as shown here, will offer additional opportunities for cell and gene therapy.…”

Section: Discussionmentioning

confidence: 92%

Hepatic targeting of transplanted liver sinusoidal endothelial cells in intact mice

et al. 2005

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Targeting of cells to specific tissues is critical for cell therapy. To study endothelial cell targeting, we isolated mouse liver sinusoidal endothelial cells (LSEC) and examined cell biodistributions in animals. To identify transplanted LSEC in tissues, we labeled cells metabolically with DiIconjugated acetylated low density lipoprotein particles (DiI-Ac-LDL) or 111 Indium-oxine, used LSEC from Rosa26 donors expressing ␤-galactosidase or Tie-2-GFP donors with green fluorescent protein (GFP) expression, and tranduced LSEC with a GFP-lentiviral vector. LSEC efficiently incorporated 111 Indium and DiI-Ac-LDL and expressed GFP introduced by the lentiviral vector. Use of radiolabeled LSEC showed differences in cell biodistributions in relation to the cell transplantation route. After intraportal injection, LSEC were largely in the liver (60 ؎ 13%) and, after systemic intravenous injection, in lungs (67 ؎ 9%); however, after intrasplenic injection, only some LSEC remained in the spleen (29 ؎ 10%; P < .01), whereas most LSEC migrated to the liver or lungs. Transplanted LSEC were found in the liver, lungs, and spleen shortly after transplantation, whereas longer-term cell survival was observed only in the liver. Transplanted LSEC were distinct from Kupffer cells with expression of Tie-2 promoter-driven GFP and of CD31, without F4/80 reactivity. In further studies using radiolabeled LSEC, we established that the manipulation of receptor-mediated cell adhesion in liver sinusoids or the manipulation of blood flow-dependent cell exit from sinusoids improved intrahepatic retention of LSEC to 89 ؎ 7% and 89 ؎ 5%, respectively (P < .01). In conclusion, the targeting of LSEC to the liver and other organs is directed by vascular bed-specific mechanisms, including blood flow-related processes, and cell-specific factors. These findings may facilitate analysis of LSEC for cell and gene therapy applications. (HEPATOLOGY 2005;42:140-148.)

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Section: Discussionmentioning

confidence: 92%

Hepatic targeting of transplanted liver sinusoidal endothelial cells in intact mice

et al. 2005

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“…3,21 Transfected AtT-20 cells were grown on 35-mm culture plates (Fisher Scientific, Itasca, IL) in Dulbecco modified Eagle medium/G418 media for 72 hours. Negative controls (nontransfected and mock-transfected cells) were processed in parallel with each immunofluorescence labeling experiment.…”

Section: Immunofluorescence Stainingmentioning

confidence: 99%

The role of the D1 domain of the von Willebrand factor propeptide in multimerization of VWF

Rosenberg

Haberichter

Jozwiak

et al. 2002

Blood

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While studying patient plasma containing an unusual pattern of von Willebrand factor (VWF) multimers, we discovered a previously unreported phenomenon: heavy predominance of dimeric VWF. Genomic analysis revealed a new congenital mutation (Tyr87Ser) that altered the final stages of VWF biosynthesis. This mutation in the propeptide (VWFpp) resulted in synthesis of dimeric VWF with an almost complete loss of N-terminal multimerization. The multimer pattern in patient plasma appears to result from separate alleles' synthesizing wild-type or mutant (dimeric) VWF, with homodimers composing the predominant protomeric species. We have expressed VWF protein containing the Tyr87Ser mutation and analyzed the intracellular processing and resulting VWF biological functions. The expressed dimeric VWF displayed a loss of several specific functions: collagen binding, factor VIII binding, and ristocetin-induced platelet binding. However, granular storage of dimeric VWF was normal, demonstrating that the lack of multimerization does not preclude granular storage. Although the tertiary structure of the VWFpp remains unknown, the mutant amino acid is located in a region that is highly conserved across several species and may play a major role in the multimerization of VWF. Our data suggest that one function of the highly cysteine-rich VWFpp is to align the adjacent subunits of VWF into the correct configuration, serving as an intramolecular chaperone. The integrity of the VWFpp is essential to maintain the proper spacing and alignment of the multiple cysteines in the VWFpp and N-terminus of the mature

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“…Recent studies have suggested that it may be beneficial to express FVIII in cells that also express its natural carrier protein von Willebrand factor (VWF). [5][6][7][8][9][10][11] In the circulation, VWF protects FVIII from premature clearance and proteolytic degradation by virtue of its ability to bind to it with high affinity. [12][13][14] VWF is expressed in megakaryocytes and vascular endothelial cells.…”

Section: Introductionmentioning

confidence: 99%

“…[15][16][17][18] Expression of FVIII in endothelial cells results in storage of FVIII in WPBs. 6,19,20 Co-storage of the VWF/FVIII complex in secretory granules and subsequent release of the VWF/FVIII complex upon agonist-induced stimulation has the potential of secreting large amounts of FVIII at sites of vascular injury as well as directly increasing FVIII half-life by protecting FVIII from premature clearance and proteolytic degradation.…”

Section: Introductionmentioning

confidence: 99%

Storage and regulated secretion of factor VIII in blood outgrowth endothelial cells

Biggelaar¹,

Bouwens²,

Kootstra³

et al. 2009

Haematologica

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BackgroundGene therapy provides an attractive alternative for protein replacement therapy in hemophilia A patients. Recent studies have shown the potential benefit of directing factor (F)VIII gene delivery to cells that also express its natural carrier protein von Willebrand factor (VWF). In this study, we explored the feasibility of blood outgrowth endothelial cells as a cellular FVIII delivery device with particular reference to long-term production levels, intracellular storage in Weibel-Palade bodies and agonist-induced regulated secretion. Design and MethodsHuman blood outgrowth endothelial cells were isolated from peripheral blood collected from healthy donors, transduced at passage 5 using a lentiviral vector encoding human Bdomain deleted FVIII-GFP and characterized by flow cytometry and confocal microscopy. ResultsBlood outgrowth endothelial cells displayed typical endothelial morphology and expressed the endothelial-specific marker VWF. Following transduction with a lentivirus encoding FVIII-GFP, 80% of transduced blood outgrowth endothelial cells expressed FVIII-GFP. Levels of FVIII-GFP positive cells declined slowly upon prolonged culturing. Transduced blood outgrowth endothelial cells expressed 1.6±1.0 pmol/1×10 6 cells/24h FVIII. Morphological analysis demonstrated that FVIII-GFP was stored in Weibel-Palade bodies together with VWF and P-selectin. FVIII levels were only slightly increased following agonist-induced stimulation, whereas a 6-to 8-fold increase of VWF levels was observed. Subcellular fractionation revealed that 15-22% of FVIII antigen was present within the dense fraction containing Weibel-Palade bodies. ConclusionsWe conclude that blood outgrowth endothelial cells, by virtue of their ability to store a significant portion of synthesized FVIII-GFP in Weibel-Palade bodies, provide an attractive cellular on-demand delivery device for gene therapy of hemophilia A.

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Genetic Induction of a Releasable Pool of Factor VIII in Human Endothelial Cells

Abstract: Abstract-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.

Cited by 72 publications

References 43 publications

Hepatic targeting of transplanted liver sinusoidal endothelial cells in intact mice

Hepatic targeting of transplanted liver sinusoidal endothelial cells in intact mice

The role of the D1 domain of the von Willebrand factor propeptide in multimerization of VWF

Storage and regulated secretion of factor VIII in blood outgrowth endothelial cells

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