Expression of Factor VIII by Murine Liver Sinusoidal Endothelial Cells

Abstract

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 ...

Hepatic targeting of transplanted liver sinusoidal endothelial cells in intact mice

“…Previously, murine LSEC were isolated by centrifugal elutriation or magnetic sorting, which are more complex processes. 7,10 The simpler density gradients may be sufficient for some applications. We used Tie-2-GFP cells to exclude the possibility that Kupffer cells would be mistaken for LSEC.…”

“…7,18 Moreover, hepatic endothelial damage is associated with conditions, such as sepsis, impaired liver function, veno-occlusive disease, and graft versus host disease, that could potentially benefit from endothelial reconstitution. [22][23][24][25] In principle, transplanted LSEC could enter the liver through the systemic or portal circulation, because hepatic sinusoids receive blood from both the portal vein (70%) and the hepatic artery (30%).…”

“…[1][2][3][4] Liver sinusoidal endothelial cells (LSEC) exhibit unique properties, such as hyaluronic acid receptors 5,6 and production of coagulation factor VIII, 7 and play a role in the modulation of immune responses. [8][9][10] Angiogenesis requires proliferation of EC and mesenchymal cells as well as complex interactions between regulators of vasculogenesis and blood vessel maturation.…”

Absence of a desmopressin response after therapeutic expression of factor VIII in hemophilia A dogs with liver-directed neonatal gene therapy

“…However, the source of the released FVIII after DDAVP stimulation has not been determined (3). Liver is a major site of FVIII production because liver transplantation completely corrects HA (46), and hepatocytes express FVIII mRNA (47). However, endstage liver disease does not cause a decrease in FVIII, FVIII mRNA is found in other organs, and spleen and lung transplantation can ameliorate HA (3).…”

Gene therapy for hemophilia

“…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). However, the preponderance of recent evidence implicates LSECs and by extension endothelial cells in other tissues as well, which may explain the observations of F.VIII production in extrahepatic vascularized tissues such as the kidney, spleen, and lung (247)(248)(249)(250).…”