An in vitro system for study of effects of angiotensin I on cultured endothelial cells

Since certain non-vascular angiotensin II (AII) receptors may be activated by angiotensin I (AI), and since sustained increase in AI levels accompanies chronic treatment with converting enzyme inhibitors (CEI) which block conversion of AI to AII, the question of whether AI has significant biological effects is of clinical relevance. We therefore sought to develop an in vitro culture system in which effects of angiotensin I, independently of its conversion to AII, could be studied in cloned aortic vascular endothelial cells (VEC). This was complicated by peptide degradation during the period of observation, both by angiotensin converting enzyme (ACE) on the surface of VEC and by angiotensinases in either the serum component of culture media or associated with the cell monolayer. Accordingly, we examined the half life of AI under relevant cell culture conditions, with and without confluent fetal bovine aortic endothelial cells (FBAEC). Factors assessed included (1) fetal calf serum: commercial source, concentration in culture media, effects of converting enzyme inhibitor (CEI: MK422) and/or heat inactivation (superimposed on the commercially performed process); and (2) effect of FBAEC in monolayer culture, with and without CEI. Results showed that (1) in the absence of cells, loss of AI in culture media, when present, was solely due to the presence of fetal calf serum (FCS) and showed a dose dependent response; (2) FCS from differing sources may vary dramatically in capacity for AI breakdown; and (3) serum related AI disapperance included a heat resistant ACE like component (inhibitable by CEI) and a heat sensitive/CEI resistant component dominant at concentrations of FCS exceeding 5%. FBAEC dependent AI loss was solely due to CEI inhibitable ACE like activity, with no evidence for non-ACE angiotensin activity detectable in confluent monolayer cultures. Cloned FBAEC retained ACE like activity, which is compatible with persistence of a differentiated phenotype. Thus optimum conditions for study of effects of AI on vascular endothelial cells, with sustained AI levels over a 24 h period, can be achieved by inclusion of 10^-6M MK422, provided that the culture medium is either serum free or contains FCS selected for low AI degrading activity.