Differential regulation of thrombospondin-1 and fibronectin by angiotensin II receptor subtypes in cultured endothelial cells

Objectives: Angiotensin II (ANG II) can modulate cellular proliferation in various cell types via AT(1) and AT(2), receptors. In the present study, we investigated the effect of the angiotensin AT(1) and AT(2) receptors on DNA-synthesis as well as on the expression of the extracellular matrix (ECM) components, thrombospondin-1 (TSP-1) and fibronectin (FN) in endothelial cells (EC). Methods: The experiments were performed in microvascular EC derived from rat heart (CEC) and macrovascular EC derived from bovine aorta (BAEC). The experiments were performed in cells of the second and third passage and the expression of AT(1) and AT(2) receptors was verified by binding studies, Northern analysis or RT-PCR. Quiescent rat CEC and BAEC were stimulated to proliferate by the addition of 25 ng/ml bFGF, while ANG II (10(-7) M) and the selective ANG II receptor antagonists, Losartan (10(-5) M) and PD123177 (10(-6) M) or the AT, agonist, CGP42112A (10(-7) M) were added 16 h later. Results: ANG II induced a dose-dependent decrease of DNA-synthesis in BAEC measured by [H-3]-thymidine incorporation. This inhibitory effect of ANG II was prevented by the addition of the AT(2) receptor antagonist PD123177 (10(-6) M), demonstrating, that the inhibition of DNA synthesis is mediated by the AT(2) receptor. In the presence of Losartan, stimulation of both, CEC and BAEC, with ANG II resulted in a marked increase of TSP-1 mRNA levels, which was maximal between 3 and 6 h in rat CEC and after 9 h in BAEC. In addition, TSP-1 was clearly induced by the AT(2) agonist CGP42112A. In contrast, blockade of the AT(2) receptor by the selective AT(2) antagonist, PD123177 (10(-6) M), resulted in a pronounced down regulation of FN mRNA 9 h after the stimulation. Conclusions: The present results suggest that the ANG II receptor subtype AT(2) mediates growth inhibition in macrovascular EC similar to what has been shown before in microvascular rat EC and that AT(2) receptors mediates remodeling of the endothelial ECM by upregulation of TSP-1 expression in both macro- and micro-vascular endothelial cells. (C) 2001 Elsevier Science B.V. All rights reserved.