Ultrastructural localization of factor VIII procoagulant antigen in human liver hepatocytes

Zelechowska, M; Mourik, Jan A. van; Brodniewicz-Proba, Teresa

doi:10.1038/317729a0

Cited by 105 publications

(55 citation statements)

References 11 publications

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“…The data represent the mean and standard deviation from cultures obtained from three separate preparations. (12). In contrast, fVIII was localized immunohistologically to LSECs but not hepatocytes (9,10,25,26).…”

Section: Fig 4 Quantitation Of Fviii From Cultured Lsecsmentioning

confidence: 85%

Expression of Factor VIII by Murine Liver Sinusoidal Endothelial Cells

Healey

Waller

et al. 1999

Journal of Biological Chemistry

183

168

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Factor VIII (fVIII) is the procoagulant plasma glycoprotein that is missing or decreased in hemophilia A. The cellular origin of fVIII synthesis is controversial. Liver transplantation cures hemophilia A, demonstrating that the liver is a major site of fVIII synthesis. We detected fVIII mRNA in purified populations of murine liver sinusoidal endothelial cells (LSECs) and hepatocytes, but not Kupffer cells. LSECs and hepatocytes contained comparable numbers of fVIII mRNA (40 and 70 transcripts per cell, respectively) by quantitative competitive reverse transcriptase-polymerase chain reaction analysis. There was not detectable mRNA for factor IX, a hepatocyte marker, in the LSEC preparation, nor was there detectable mRNA for von Willebrand factor, an endothelial cell marker, in the hepatocyte preparation. This excludes the possibility that detectable fVIII mRNA is due to cross-contamination in the hepatocyte or LSEC preparations. Primary cultures of LSECs were established in which fVIII mRNA levels were indistinguishable from purified LSECs. LSECs secreted active fVIII into the culture medium. This finding represents the first demonstration of homologous expression of fVIII mRNA and protein in cell culture and should facilitate studies of fVIII gene regulation. Additionally, LSECs potentially are targets for a fVIII transgene during gene therapy of hemophilia A.The site of the cellular origin for the biosynthesis of fVIII 1 remains controversial despite studies that date back nearly 50 years (see Refs. 1 and 2, for reviews). Human and canine hemophilia A are cured by liver transplantation (3-6), which demonstrates that the liver contributes significantly to fVIII synthesis. Hepatocytes (7,8), liver sinusoidal endothelial cells (LSECs) (9 -11), or both (12), have been proposed as sites of fVIII synthesis. In this study, we isolated hepatocytes, LSECs, and Kupffer cells and measured steady-state levels of fVIII mRNA in these preparations. Our results indicate that both LSECs and hepatocytes synthesize fVIII mRNA. Additionally, LSECs in culture secrete active fVIII, providing a model for studies of the regulation of homologous fVIII gene expression. EXPERIMENTAL PROCEDURESMaterials-Gey's balanced salt solution, Hank's balanced salt solution (HBSS), Dulbecco's phosphate-buffered saline (PBS), Liver Digest Medium, DMEM/F-12 medium, and AIM-V medium were purchased from Life Technologies, Inc. (Gaithersburg, MD). Penicillin (50 units/ ml) and streptomycin (50 g/ml) were added to DMEM/F-12 medium. Collagenase (type IV), gelatin, and dibutyryl cAMP were purchased from Sigma. DNase I was purchased from Roche Molecular Biochemicals (Indianapolis, IN). The following murine monoclonal IgG 1 antibodies were purchased from Pharmigen (San Diego, CA): FITC-conjugated anti-PECAM-1 (anti-CD31), FITC-conjugated anti-VCAM-1 (anti-CD106), PE-conjugated anti-ICAM-1 (anti-CD54), the corresponding FITC-conjugated-and PE-conjugated isotype-specific control antibodies, and biotinylated anti-ICAM-1. FITC-conjugated wheat germ agglutinin was ...

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Section: Fig 4 Quantitation Of Fviii From Cultured Lsecsmentioning

confidence: 85%

Expression of Factor VIII by Murine Liver Sinusoidal Endothelial Cells

Healey

Waller

et al. 1999

Journal of Biological Chemistry

183

168

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“…Early studies suggested that FVIII was expressed mostly in hepatocytes, 40 but other studies found that FVIII was expressed in LSEC and macrophages as well, whereas virtually no signal for FVIII was detected in hepatocytes. 20,41 More recently, we and others established that FVIII is largely produced and secreted by LSEC.…”

Section: Discussionmentioning

confidence: 99%

Extrahepatic sources of factor VIII potentially contribute to the coagulation cascade correcting the bleeding phenotype of mice with hemophilia A

et al. 2015

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“…The liver has been implicated as a major site of F.VIII synthesis due to the ability of liver transplantation to cure hemophilia A in canine models as well as human patients (232)(233)(234). A number of studies have reported the presence of F.VIII mRNA and protein in hepatocytes in vivo, F.VIII production by hepatocytes cultured in vitro, and even the ability to restore clotting activity through hepatocyte transplantation (235)(236)(237)(238)(239). Conversely, others have reported F.VIII synthesis in liver sinusoidal epithelial cells (LSECs) but not hepatocytes, or even in both cell types (240)(241)(242)(243)(244)(245)(246).…”

Section: Additional Challenges In Hemophilia Amentioning

confidence: 99%

Gene therapy for hemophilia

2001

Indian J Pediatr

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Hemophilia is an X-linked inherited bleeding disorder consisting of two classifications, hemophilia A and hemophilia B, depending on the underlying mutation. Although the disease is currently treatable with intravenous delivery of replacement recombinant clotting factor, this approach represents a significant cost both monetarily and in terms of quality of life. Gene therapy is an attractive alternative approach to the treatment of hemophilia that would ideally provide lifelong correction of clotting activity with a single injection. In this review, we will discuss the multitude of approaches that have been explored for the treatment of both hemophilia A and B, including both in vivo and ex vivo approaches with viral and nonviral delivery vectors.

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Ultrastructural localization of factor VIII procoagulant antigen in human liver hepatocytes

Cited by 105 publications

References 11 publications

Expression of Factor VIII by Murine Liver Sinusoidal Endothelial Cells

Expression of Factor VIII by Murine Liver Sinusoidal Endothelial Cells

Extrahepatic sources of factor VIII potentially contribute to the coagulation cascade correcting the bleeding phenotype of mice with hemophilia A

Gene therapy for hemophilia

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