Differential effects of enzymatic treatments on the storage and secretion of von Willebrand factor by human endothelial cells.
Enzymatic treatment used for passaging of endothelial cells may induce release of von Willebrand factor (vWF). Decreased ability to replenish intracellular stores results in decreased secretion of vWF in later passages of cells. Since both trypsin and pancreatin complex have been used for passaging endothelial cells, we analyzed the effects of successive passaging with these two enzyme preparations on the storage and secretion of vWF by human umbilical vein endothelial cells (HUVECs). Measurements were performed after the second to fifth passages. Cytoplasmic vWF was analyzed by indirect immunofluorescence and secreted vWF was measured in the supernatant of cultured HUVECs by ELISA. In trypsin-passaged cells, secreted vWF decreased progressively from passages 2 to 5. Respective concentrations were 355.0 +/- 30.4, 201.0 +/- 84.5, 150.0 +/- 1.4 and 120.5 +/- 38.9 ng vWF/10(5) cells. Comparatively, pancreatin-passaged cells secreted even less vWF protein (P = .001) at passages 4 and 5 (108.5 +/- 12.0 and 100.0 +/- 4.2 ng/10(5) cells, respectively) and had less vWF-positive cytoplasmic granules per cell. Thus, in experiments involving measurements of endothelial vWF, the use of low passage cells is recommendable and passaging with a pure trypsin preparation appears to be more appropriate.
Lopes
A A
AA
Heart Institute, University of São Paulo, São Paulo, Brazil. aablopes@usp.br
Peranovich
T M
TM
Maeda
N Y
NY
Bydlowski
S P
SP
D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D010194:Pancreatin / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology*; D014841:von Willebrand Factor / Q000378:metabolism*
D014841:von Willebrand Factor; D010194:Pancreatin; D014357:Trypsin