Method of removal of aortic endothelium affects arachidonic acid metabolism and vascular reactivity.

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 [C-14]arachidonic acid and 20-mu-M A23187 for determination of arachidonic acid metabolism, incubated with 20-mu-M 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 < 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 [C-14]12-HETE was markedly increased in these vessels as compared to control or denuded-1 vessels. Specific radioimmunoassays detected enhanced release of 12-HETE in denuded-2 vessels (13 +/- 5 pg/mg) as compared to control (6 +/- 2 pg/mg) or denuded-1 (6 +/- 2 pg/mg) vessels. KCl-induced contractions were significantly less in denuded-2 vessels (2.5 +/- 0.3 g) than in control vessels (3.9 +/- 0.3 g). Both denuded-1 and denuded-2 vessels contracted similarly to a range of concentrations of norepinephrine but significantly less than did control vessels. Control vessels relaxed to methacholine with an EC50 of 2 X 10(-7) M. Neither denuded-1 nor denuded-2 vessels relaxed to methacholine. However, vessels in each of the three groups relaxed similarly to sodium nitroprusside (EC50 = 5 X 10(-7) M). We conclude that some methods of endothelial cell removal may damage smooth muscle cells, increase release of 12-HETE and reduce vascular reactivity.