Cultured murine cerebral microvascular endothelial cells contain von Willebrand factor-positive Weibel-Palade bodies and support rapid cytokine-induced neutrophil adhesion.

Abstract

To obtain cultures of rodent brain microvascular endothelium (BMEC) that retain endothelial cell-specific markers and functions for two purposes: investigating whether these cultures contain endothelial cell-specific storage granules or Weibel-Palade bodies and have the ability to rapidly bind neutrophils upon cytokine induction; and setting the groundwork for future studies examining endothelium derived from mice strains with targeted deficiencies in endothelial adhesion molecules. Capillaries were obtained by collagenase/dispase digestion and subsequent density centrifugation of either rodent brain or meninges. The yield was then plated onto fibronectin-coated dishes. For some studies, pure murine endothelial cultures were obtained by flow-cytometric sorting, using uptake of fluorescently labeled diI-acetylated low-density lipoprotein as a marker for endothelium. Endothelial cell-specific markers were analyzed via immunofluorescence, immunoprecipitation and light microscopy. Cytokine-induced neutrophil adhesion and associated upregulation of leukocyte adhesion molecules were measured as described previously for human umbilical vein endothelial cells. BMEC possess numerous von Willebrand factor-containing Weibel-Palade bodies and synthesize and secrete all multimeric forms of von Willebrand factor. They take up diI-acetylated low-density lipoproteins, contain platelet-endothelial cell adhesion molecules and form capillary-like structures on three-dimensional extracellular matrix substrates. Sorted murine brain microvascular endothelial cells treated with IL-1 beta or TNF-alpha for 4 h show an increase in surface expression of the cytokine-inducible leukocyte adhesion molecules E-selectin, VCAM-1, and ICAM-1, and they support rapid neutrophil adhesion, which is, on average, three times greater than that of nonstimulated cells. The brain microvascular endothelial cultures described here exhibit many of the markers of endothelial cells including the presence of Weibel-Palade bodies. The localization of von Willebrand factor almost exclusively to Weibel-Palade bodies indicates that murine cerebral endothelium has evolved an efficient mechanism for storage of this platelet adhesion protein, which plays an important role in hemostasis. In addition, this is the first demonstration of rapid neutrophil adhesion to murine brain microvascular endothelial cells. Finally, the reproducible culture and the characterization of murine BMEC makes feasible future studies on endothelium isolated from gene-targeted mice.