Flow cytometric determination of cell proliferation in hypertensive blood vessels

Background: Measurement of vascular cell proliferation in animal models of hypertension is currently accomplished by demonstrating [H-3]-thymidine ([H -3]-dT) incorporation into DNA using autoradiography. This method, however, is labor intensive, requires radioactivity, and is Limited by the inherent difficulty in discriminating labeled and unlabeled cells. To address these limitations, a flow cytometric-based method is described utilizing incorpo ration of 5-bromo-2'-deoxyuridine (BrdU) into DNA of nuclei isolated from b lood vessels. Methods: Pulmonary hypertension was induced in rats by exposure to 10% O-2 (hypoxia) for varying periods of time. Pulmonary arteries and aorta from ra ts injected with BrdU prior to sacrifice were isolated, fixed with 10% form alin, and digested with Protease XIV. The intact nuclei liberated by this t reatment were successively treated with HCl/Triton X-100 and sodium berate. Processed nuclei were probed with a BrdU-specific fluorescein-conjugated a ntibody, and the percentage of BrdU staining cells was determined using now cytometry. Results: An approximate to 20-fold increase in BrdU-positive cells at 3 day s of hypoxia in pulmonary arteries (relative to control) with no change in aorta was observed. These results were similar to previous studies using [H -3]-dT labeling. Conclusions: Flow cytometric determination of cell proliferation in blood v essels is a simple, objective technique that may facilitate measurement of cell proliferation in animal models of vascular disease. (C) 1999 Wiley-Lis s, Inc.