Angiotensin-converting Enzyme: II. Pulmonary Endothelial Cells in Culture

Direct studies of the function of a given cell type often require that the cell type be obtained in pure culture. A number of different specific metabolic activities have been attributed to pulmonary endothelial cells, yet with few exceptions the conclusions were based on indirect evidence. Thus, to improve our ability to examine directly for specific metabolic activities, we began a program to obtain pulmonary endothelial cells in culture. Two methods have been developed: (1) cells can be obtained from pulmonary artery and vein of large animals (cow, pig), and (2) cells can be obtained from the microvasculature of small animals (rat, guinea pig, and rabbit). The latter technique can also be used to obtain cells from a lobe of lung from large animals and may be adaptable for use with human tissue. In the first technique, pulmonary arteries, free of blood, are filled with collagenase (0.25%, 500 units) in Puck's saline for 25 min. The collagenase mixture containing cells is removed and centrifuged. The pellet is resuspended and seeded into culture flasks. In the second method, lungs are perfused (artery to vein) with Krebs-Henseleit solution until the effluent is blood-free. Collagenase (0.25%, 500 units) is introduced, and the lungs are then perfused in the opposite direction (vein to artery) until the flow stops spontaneously (ca. 15 min). The detached cells are collected and seeded as before. The endothelial cells attach as small clumps (10-50 cells). Those flasks which contain more than 95% endothelial cells (by phase microscopy) are retained for culture and the lines are purified usng differential adherence procedures. The cells grow as monolayers with a cobblestone appearance. They contain Weibel-Palade bodies. They possess converting enzyme activity and are reactive with antibodies to converting enzyme, Factor VIII and alpha 2-macroglobulin. The cells synthesize prostaglandins and related substances. In addition, they possess ADPase and synthesize angiotensin-converting enzyme.