Method of removal of aortic endothelium affects arachidonic acid metabolism and vascular reactivity.
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abstract
To assess endothelium-dependent responses of blood vessels in vitro, endothelial cells are removed by a variety of mechanical means. We sought to determine if the method of removal of the endothelium affected arachidonic acid metabolism and vascular reactivity of isolated strips of rabbit aorta. Thoracic aorta of New Zealand White rabbits were excised and sectioned into strips with a sharp razor blade. The luminal surface of the vessel was then gently stroked (denuded-1) or forcefully rubbed (denuded-2) with a moist cotton swab. Vessels were then either fixed in 3% glutaraldehyde and processed for electron microscopy, incubated with [14C]arachidonic acid and 20 microM A23187 for determination of arachidonic acid metabolism, incubated with 20 microM A23187 for measurement of endogenous release of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and 12-hydroxyeicosatetraenoic acid (12-HETE) by specific radioimmunoassays, or suspended in an organ chamber filled with Krebs bicarbonate solution for vascular reactivity experiments. Electron micrographs showed that denuded-1 vessels lacked an endothelial cell layer and had slight degeneration of the smooth muscle cells. Additionally, these vessels had a diminished capacity to produce 6-keto-PGF1 alpha as compared to control vessels (214 +/- 25 vs. 360 +/- 36 pg/mg of tissue, P less than 0.05). Denuded-2 vessels contained severe degeneration and rupture of smooth muscle cells in addition to the loss of the endothelial cell layer. While the 6-keto-PGF1 alpha concentration (168 +/- 23 pg/mg) was less in denuded-2 vessels, HPLC indicated that the production of [14C]12-HETE was markedly increased in these vessels as compared to control or denuded-1 vessels.(ABSTRACT TRUNCATED AT 250 WORDS)
