Oxysterol-induced endothelial cell dysfunction in culture

Abstract

Cholesterol oxidation products (oxysterols), such as cholestan-3 beta,5 alpha,6 beta-triol (Triol), may be atherogenic by altering the barrier function of the vascular endothelium. We have shown that incubation of endothelial cell monolayers with Triol increased transendothelial albumin transfer (i.e., decreased barrier function) in a concentration- and time-dependent manner. Such dysfunction of endothelium could result from alterations in membrane characteristics, including changes in membrane-associated enzyme activities. To test this hypothesis, endothelial monolayers were treated with 20 microM Triol and the activities of selected membrane enzymes were measured at 0, 2, 4, 6, 12 and 24 hours. Calcium-adenosine triphosphatase (Ca(++)-ATPase) and sodium, potassium, magnesium-adenosine triphosphatase (Na+, K+, Mg(++)-ATPase) activities were significantly increased after 4 or 2 hours incubation with 20 microM Triol, respectively. 5'-nucleotidase activity was significantly elevated only after a 24-hour exposure to Triol, whereas there was no change in angiotensin-converting enzyme (ACE) activity in response to 20 microM Triol treatment at any time studied. Compared with all concentrations tested 40 microM Triol increased Ca(++)-ATPase activity most markedly, with a significant increase already after a 2-hour exposure. No major morphological changes were noted until 12 hours of exposure to 20 microM Triol; obvious cellular damage was observed by 24 hours. Cultures treated with Triol for 24 hours showed significant signs of toxicity, measured by an elevated [3H]adenine release, compared with control cultures. These data demonstrate that Triol alters the activity of certain membrane-bound enzymes, particularly Na+, K+, Mg(++)-ATPase and Ca(++)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)