High-Yield Method for Isolation and Culture of Endothelial Cells from Rat Coronary Blood Vessels Suitable for Analysis of Intracellular Calcium and Nitric Oxide Biosynthetic Pathways

We describe here a method for isolating endothelial cells from rat heart blood vessels by means of coronary microperfusion with collagenase. This methods makes it possible to obtain high amounts of endothelial cells in culture which retain the functional properties of their in vivo counterparts, including the ability to uptake fluorescently-labeled acetylated low-density lipoproteins and to respond to vasoactive agents by modulating intracellular calcium and by upregulating intrinsic nitric oxide generation. The main advantages of our technique are: (i) good reproducibility, (ii) accurate sterility that can be maintained throughout the isolation procedure and (iii) high yield of pure endothelial cells, mainly due to microperfusion and temperature-controlled incubation with collagenase which allow an optimal distribution of this enzyme within the coronary vascular bed

Reversible expression of sm α-actin protein and sm α-actin mRNA in cloned cerebral endothelial cells

AbstractThe expression of smooth muscle (sm) α-actin was studied in cloned capillary cerebral endothelial cells of two phenotypes. Type I cells were cultured in medium containing 10% FCS, heparin and ECGS (or α-ECGF) and stained positive for a specific endothelial cell marker (Bandeiraea simplicifolia). Depletion of heparin and ECGS resulted in a smooth muscle-like appearance after 2–3 days. Cells of this phenotype, (type II) stained positive for the endothelial cell marker and for sm α-actin. In contrast to type I cells, type II cells expressed sm α-actin protein and mRNA as evidenced by Immunoblots and Northern blots. This phenotypic switch was shown to be reversible and so was the expression of sm α-actin