Selective isolation of rat aortic wall layers and their cell types in culture--application to converting enzyme activity measurement

Abstract

The rat aorta, whose three wall layers can be separated by microdissection offers the rare possibility of comparing physiological characteristics of in vivo tissular cell components and corresponding cells after culture. We developed a technique allowing the dissociation of the three tunicae (intima, media and adventitia) of the rat aorta and the culture of their main cell types, i.e.: endothelial cells (EC) from intima, smooth muscle cells (SMC) from media and fibroblasts (Fib) from adventitia. Comparison between selected tunicae in vivo and their corresponding cells in vitro was performed via arterial angiotensin converting enzyme (ACE) activity measurements in Wistar rats. In vivo microsomial ACE activity for each tunica was as follows: 368.9 +/- 34.3 (endothelium), 10.5 +/- 1.9 (media) and 10.2 +/- 4.9 (adventitia) pmol/mg protein/min. Corresponding cell primary culture values were 1.2 +/- 0.1 (EC), 0.06 +/- 0.02 (SMC) and 0.24 +/- 0.01 (Fib) pmol/mg protein/min. Incubation of serum-deprived cells with Dexamethasone (10(-7) M) over 48 hr induced a statistically significant shift of total ACE activity from controls to stimulated cells of 2.9 +/- 0.3 to 9.7 +/- 1.0 in EC, 0.8 +/- 0.1 to 32.1 +/- 4.9 in SMC and 1.03 +/- 0.65 to 57.2 +/- 2.1 pmol/mg prot/min in fibroblasts. In the rat aorta, ACE was present not only in the intimal endothelial cell lining, but also in the media and the adventitia. ACE activity levels in primary cultured vascular cells were about 100-fold less than those found in the ex vivo tissues. Nevertheless, ACE expression seems to be more constitutive in endothelial cells and more inducible in smooth muscle cells and fibroblasts. This methodological approach should be of interest in studying environmental or genetic regulation of protein expression in the three layers/three cell types of the vascular wall.