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 [³H]-thymidine ([³H]-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 incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA of nuclei isolated from blood vessels. Methods: Pulmonary hypertension was induced in rats by exposure to 10% O₂ (hypoxia) for varying periods of time. Pulmonary arteries and aorta from rats injected with BrdU prior to sacrifice were isolated, fixed with 10% formalin, and digested with Protease XIV. The intact nuclei liberated by this treatment were successively treated with HCl/Triton X-100 and sodium borate. Processed nuclei were probed with a BrdU-specific fluorescein-conjugated antibody, and the percentage of BrdU staining cells was determined using flow cytometry. Results: An ≃20-fold increase in BrdU- positive cells at 3 days of hypoxia in pulmonary arteries (relative to control) with no change in aorta was observed. These results were similar to previous studies using [³H]-dT labeling. Conclusions: Flow cytometric determination of cell proliferation in blood vessels is a simple, objective technique that may facilitate measurement of cell proliferation in animal models of vascular disease.