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.