Human liver sinusoidal endothelial cells but not hepatocytes contain factor VIII

Abstract

Summary. Background: Although the liver is the major site of coagulation factor VIII (FVIII) synthesis, the type of cells producing FVIII within the liver is still unclear. Objectives: To measure FVIII in extracts of primary liver sinusoidal endothelial cells (LSECs) and hepatocytes, thereby preventing potential bias resulting from the modifications of the cell phenotype that can take place during in vitro culture. Methods: LSECs were purified by flow cytometry cell sorting on the basis of their coexpression of Tie2 and CD32b. The purity of the cells was controlled by RNA sequencing. FVIII activity (FVIII:C) in extracts of purified cells was measured with a sensitive FVIII chromogenic assay, in which the specificity of the reaction is controlled by neutralization of FVIII activity with specific inhibitor antibodies. Results: The FVIII:C concentration in purified LSECs ranged from 0.3 to 2.8 nU per cell. In contrast, FVIII:C was undetectable in hepatocytes. The intracellular FVIII:C concentrations are therefore at least 10-100-fold higher in LSECs than in hepatocytes. Conclusions: Our data demonstrate that LSECs, but not hepatocytes, contain measurable amounts of FVIII:C, and suggest that the former are the main cells producing FVIII in the human liver.

The search for the origin of factor VIII synthesis and its impact on therapeutic strategies for hemophilia A

“…19,20 However, this strategy may be limited by the intrinsic inability of the human hepatocyte to fully synthesize and secrete factor VIII, despite the presence of mRNA. 6 This may explain, at least in part, the higher dose of vectors required for expression of factor VIII compared to factor IX. On the other hand, despite the limiting effect on the efficacy of factor VIII secretion, the use of hepatocyte-specific expression is a favorable strategy in terms of immune tolerance induction to factor VIII, which is the most serious and common complication (~20%) of hemophilia A treatment.…”

“…These include acute myeloid leukemia and acute lymphoblastic leukemias caused by translocations of the Mixed Lineage Leukemia (MLL) gene, [2][3][4] fusions of the HOXA9 gene that produce a novel HOXA9-NUP98 fusion protein in acute myeloid leukemia, 5 and T-cell acute lymphoblastic leukemias that have translocations between the TCRβ and HOXA9/A10 loci. 6 Interestingly however, despite this seemingly central role in a subset of acute leukemias, Hoxa9 expression alone is only weakly oncogenic in mouse leukemia models and usually requires a second "hit" via overexpression of Meis1, 7,8 or in some cases Pbx3. 9 Much work has been done trying to understand the molecular function of the HOXA9 protein.…”

“…[4][5][6] The overall conclusion was that liver sinusoid endothelial cells (LSEC), not hepatocytes, are the main cellular source of hepatic factor VIII. Here, in a unique set of experiments, the authors demonstrated that transplant of non-parenchymal cells (a mix of Küpffer cells and LSEC), improved circulating factor VIII levels, but infusion of isolated hepatocyte did not.…”

“…[4][5][6] In this issue of the journal, Zanolini and colleagues provide new insight into both the hepatic and hematopoietic-derived cellular origin of circulating factor VIII. 7 Using multiple sources of hematopoietic cells (bonemarrow derived or cord blood CD34 + cells) from humans, they identified (by both mRNA and immunofluorescence staining of protein) that monocytes and monocyte-derived macrophages are the main cells of the hematopoietic system responsible for factor VIII.…”

Replacing bad (F)actors: hemophilia

“…They observed that endothelial cells, but not hepatocytes, contained measurable levels of FVIII coagulant activity. 8 Before these studies, there existed conflicting reports and significant speculation that endothelial cells, hepatocytes, and hematopoietic and mesenchymal cells were important contributors to the circulating pool of FVIII. Without clinical evidence of nonendothelial FVIII biosynthesis and nonhepatocyte FIX biosynthesis and with no existing source/transplantation protocols for endothelial or hepatocyte stem cells, allogeneic transplantation does not appear to be a viable near-term option.…”