Isolation of rabbit pulmonary microvascular endothelial cells and characterization of their angiotensin converting...

An in vitro model using cultured rabbit pulmonary endothelial cells of microvascular origin was developed to define the luminal surface membrane characteristics of microvascular endothelium. Endothelial cells were isolated from peripheral lung segments and sorted after preferential uptake of a fluorescent derivative, diiodoindocarbo cyanine acetylated-LDL. Cells were further characterized by demonstrating angiotensin converting enzyme (ACE) on their surface by means of indirect immunofluorescence. ACE activity and its pharmacologic modification were then studied as functional assays of cell activity. Hydrolysis of Benz-phe-ala-pro (BPAP), a synthetic substrate for ACE was saturable over a concentration range of 1 to 100 microns. Thus, BPAP hydrolysis in cultured microvascular endothelial cells behaves overall in a manner similar to that seen in large resistance vessels except that a portion of the hydrolysis is not inhibited by captopril, an ACE-specific inhibitor, indicating the presence of another protease capable of BPAP hydrolysis. Accordingly, this system can be used to compare ACE and other protease kinetics in microvessel cells with those of large vessel endothelium or perfused lungs.