Mice possess a more limited natural antihuman factor VIII antibody repertoire than humans that is produced disproportionately by marginal zone B cells.

BACKGROUND: One-third of patients with severe hemophilia A develop neutralizing antibodies to the factor VIII (FVIII) protein in response to intravenous replacement therapy. Patients may also generate natural, nonneutralizing antibodies to FVIII before FVIII exposure. These patients are at increased risk of developing neutralizing antibodies to FVIII. However, natural anti-FVIII antibodies are also present in healthy human donors. OBJECTIVES: To further characterize the natural antihuman (h) FVIII antibody repertoire in mice and humans. METHODS: An in-house ELISA was developed using a purified polyclonal immunoglobulin (Ig) standard to quantify anti-hFVIII Ig in cell culture supernatant or plasma from mice (wild-type and FVIII-/-) and adult human donors. RESULTS: All naïve wild-type and FVIII-/- mice, as well as healthy human donors, possess natural anti-hFVIII antibodies. Mice only have natural anti-hFVIII IgM, which is present in germ-free mice, suggesting that they are germline encoded. Although murine marginal zone B cells (MZBs) contribute 44% to all circulating natural IgM, they contribute disproportionately to the anti-hFVIII IgM repertoire (82%). This naturally occurring murine MZB-derived IgM is not B-domain specific and is reduced by intravenously administered hFVIII, suggesting that it may form immune complexes immediately upon hFVIII administration. Natural anti-hFVIII antibodies of IgG, IgM, and IgA isotypes can be detected in adult human donors. There were increased levels of B-domain-favoring anti-hFVIII IgG in 14% of healthy donors, which were markedly different from the rest of the "low-titer" population. CONCLUSIONS: There is a preponderance of natural anti-hFVIII antibodies in both mice and healthy adult human donors.

Heterogeneity and reciprocity of FVIII and VWF expression, and the response to shear stress in cultured human endothelial cells.

BACKGROUND: Substantial phenotypic heterogeneity exists in endothelial cells and while much of this heterogeneity results from local microenvironments, epigenetic modifications also contribute. METHODS: Cultured human umbilical vein endothelial cells, human pulmonary microvascular endothelial cells, human hepatic sinusoidal endothelial cells, human lymphatic endothelial cells (hLECs), and two different isolations of endothelial colony forming cells (ECFCs) were assessed for levels of factor VIII (FVIII) and von Willebrand factor (VWF) RNA and protein. The intracellular location and co-localization of both proteins was evaluated with immunofluorescence microscopy and stimulated release toof FVIII and VWF from Weibel-Palade bodies (WPBs) was evaluated. Changes in expression of FVIII and VWF RNA after hLECs and ECFCs were exposed to 2 or 15 dynes/cm2 of laminar shear stress were also assessed. RESULTS: We observed considerable heterogeneity in FVIII and VWF expression among the endothelial cells. With the exception of hLECs, FVIII RNA and protein were barely detectable in any of the endothelial cells and a reciprocal relationship between levels of FVIII and VWF appears to exist. When FVIII and VWF are co-expressed, they do not consistently co-localize in the cytoplasm. However, in hLECs where significantly higher levels of FVIII are expressed, FVIII and VWF co-localize in WPBs and are released together when stimulated. Expression of both FVIII and VWF is markedly reduced when hLECs are exposed to higher or lower levels of laminar shear stress, while in ECFCs there is a minimal response for both proteins. CONCLUSIONS: Variable levels of FVIII and VWF RNA and protein exist in a subset of cultured human endothelial cells. Higher levels of FVIII present in hLECs co-localize with VWF and are released together when exposed to a secretagogue.

Endothelial characteristics in healthy endothelial colony forming cells; generating a robust and valid ex vivo model for vascular disease.

BACKGROUND: Endothelial colony forming cells (ECFCs) derived from peripheral blood can be used to analyze the pathophysiology of vascular diseases ex vivo. However, heterogeneity is observed between ECFC clones and this variability needs to be understood and standardized for ECFCs to be used as a cell model for applications in vascular studies. OBJECTIVE: Determine reference characteristics of healthy control ECFCs to generate a valid ex vivo model for vascular disease. METHODS: Putative ECFCs (n = 47) derived from 21 individual healthy subjects were studied for cell morphology and specific cell characteristics. Clones were analyzed for the production and secretion of von Willebrand factor (VWF), cell proliferation, and the expression of endothelial cell markers. RESULTS: Based on morphology, clones were categorized into three groups. Group 1 consisted of clones with classic endothelial cell morphology, whereas groups 2 and 3 contained less condensed cells with increasing cell sizes. All clones had comparable endothelial cell surface expression profiles, with low levels of non-endothelial markers. However, a decrease in CD31 and a group-related increase in CD309 and CD45 expression, combined with a decrease in cell proliferation and VWF production and secretion, was observed in clones in group 3 and to a lesser extent in group 2. CONCLUSIONS: We observed group-related variations in endothelial cell characteristics when clones lacked the classic endothelial cell morphology. Despite this variation, clones in all groups expressed endothelial cell surface markers. Provided that clones with similar characteristics are compared, we believe ECFCs are a valid ex vivo model to study vascular disease.