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 [(3)H]-thymidine ([(3)H]-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% O(2) (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 approximately 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 [(3)H]-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.
Hirsch
A
A
Department of Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey, USA.
Tozzi
C A
CA
Das
A K
AK
Thakker-Varia
S
S
Poiani
G J
GJ
Riley
D J
DJ
Yurkow
E J
EJ
D000818:Animals; D001011:Aorta / Q000503:physiopathology; D001808:Blood Vessels / Q000503:physiopathology*; D001973:Bromodeoxyuridine; D002455:Cell Division / Q000502:physiology*; D002467:Cell Nucleus; D005434:Flow Cytometry / Q000379:methods*; D006976:Hypertension, Pulmonary / Q000139:chemically induced / Q000503:physiopathology*; D000860:Hypoxia / Q000139:chemically induced; D011651:Pulmonary Artery / Q000503:physiopathology; D051381:Rats; D017207:Rats, Sprague-Dawley
D001973:Bromodeoxyuridine