Venular endothelium in vitro: isolation and characterization

The structural and functional properties of the endothelium vary in relation to anatomic site and position along the vascular tree. Cultures of endothelial cells have been obtained so far from large arteries, large veins and capillaries, but not from venules. We now report techniques for culturing not only rat arterial and venous endothelium, but also a special method for obtaining and culturing venular endothelium. The technique is based on the principle of "vascular labeling," whereby an insoluble pigment can be permanently deposited in the wall of the venules, making them easily visible by light microscopy. The venules of a rat cremaster muscle are labeled with a local injection of histamine followed by Monastral blue B intravenously (i.v.); 24 hours later selected venules are isolated by microdissection and either enzymatically dispersed or placed directly into tissue culture wells. The wells are coated with fibronectin and laminin and supplemented with DMEM, 20% fetal calf serum, and endothelial cell growth factor. Polygonal and spindly endothelial cells begin as clusters, grow in sheets, and sometimes form tubes. The cells stain variably for Factor VIII-related antigen, Ulex Europeus I lectin, and non-muscle specific actin. They synthesize angiotensin-converting enzyme, but do not metabolize acetylated LDL. Ultrastructurally, they display pinocytic vesicles, microtendons, and tight junctions, but not Weibel-Palade bodies. We believe that this method will be important for studying the pathophysiology of venules, which are the preferential target of inflammatory mediators and the typical site of inflammatory cell diapedesis.