To investigate potential interactions between angiotensin II (AII) and the insulin signaling system in the vasculature, insulin and AII regulation of insulin receptor substrate-1 (IRS-1) phosphorylation and phosphatidylinositol (PI) 3-kinase activation were examined in rat aortic smooth muscle cells. Pretreatment of cells with AII inhibited insulin-stimulated PI 3-kinase activity associated with IRS-1 by 60%. While AII did not impair insulin-stimulated tyrosine phosphorylation of the insulin receptor (IR) ␤ -subunit, it decreased insulin-stimulated tyrosine phosphorylation of IRS-1 by 50%. AII inhibited the insulin-stimulated association between IRS-1 and the p85 subunit of PI 3-kinase by 30-50% in a dose-dependent manner. This inhibitory effect of AII on IRS-1/PI 3-kinase association was blocked by the AII receptor antagonist saralasin, but not by AT 1 antagonist losartan or AT 2 antagonist PD123319. AII increased the serine phosphorylation of both the IR ␤ -subunit and IRS-1. In vitro binding experiments showed that autophosphorylation increased IR binding to IRS-1 from control cells by 2.5-fold versus 1.2-fold for IRS-1 from AII-stimulated cells, suggesting that AII stimulation reduces IRS-1's ability to associate with activated IR. In addition, AII increased p85 serine phosphorylation, inhibited the total pool of p85 associated PI 3-kinase activity, and decreased levels of the p50/ p55 regulatory subunit of PI 3-kinase. These results suggest that activation of the renin-angiotensin system may lead to insulin resistance in the vasculature. (

Abstract-Increased expression of plasminogen activator inhibitor-1 (PAI-1) has been reported in atherosclerotic and balloon-injured vessels. Little is known regarding the factors and mechanisms that may negatively regulate PAI-1 expression. In this report, the effect of cGMP-coupled vasoactive hormones, including natriuretic factors and nitric oxide, on the regulation of PAI-1 expression in vascular smooth muscle cells was examined. Atrial natriuretic factor 1-28 (ANF) and C-type natriuretic factor-22 (CNP) reduced angiotensin II (Ang II)-and platelet-derived growth factor-stimulated PAI-1 mRNA expression in rat aortic smooth muscle cells by 50% to 70%, with corresponding reductions in PAI-1 protein release. Treatment of human aortic smooth muscle cells with CNP similarly inhibited both platelet-derived growth factor-induced PAI-1 mRNA expression and PAI-1 protein release by 50%. Dose-response studies revealed that the inhibitory effects of CNP and ANF on PAI-1 expression were concentration dependent, with IC 50 s of Ϸ1 nmol/L for both natriuretic peptides. Ang II-stimulated PAI-1 expression was also inhibited by the nitric oxide donor S-nitroso-N-acetylpenicillamine.

Abstract-Although the renin-angiotensin system has been implicated in increasing plasminogen activator inhibitor-1 (PAI-1) expression, the role of the angiotensin type 1 (AT 1 ) receptor is controversial. This report examines the effects of angiotensin peptides, angiotensin-converting enzyme inhibition, and AT 1 antagonism on rat aortic and cardiac PAI-1 gene expression. In vitro, angiotensin (Ang) I, Ang II, and angiotensin Arg 2 -Phe 8 (Ang III) were potent agonists of PAI-1 mRNA expression in rat aortic smooth muscle cells (RASMCs), and stimulation of PAI-1 by these peptides was blocked by the AT 1 antagonist candesartan. Angiotensin Val 3 -Phe 8 (Ang IV) and angiotensin Asp 1 -Pro 7 (Ang [1-7]) did not affect PAI-1 expression in RASMCs. In neonatal rat cardiomyocytes, Ang II increased PAI-1 mRNA expression by 4-fold (PϽ0.01), and this response was completely blocked by AT 1 receptor antagonism. Continuous intrajugular infusion of Ang II into Sprague-Dawley rats for 3 hours increased aortic and cardiac PAI-1 mRNA expression by 17-and 9 fold, respectively, and these Ang II responses were completely blocked by coinfusion with candesartan. Aortic and cardiac PAI-1 expressions were compared in spontaneously hypertensive rats and Wistar-Kyoto rats. PAI-1 expression in the aorta and heart from spontaneously hypertensive rats was 5.8-fold and 2-fold higher, respectively, than in control Wistar-Kyoto rats (PϽ0.05). Candesartan treatment for 1 week reduced aortic and cardiac PAI-1 expression in spontaneously hypertensive rats by 94% and 72%, respectively (PϽ0.05), but did not affect vascular PAI-1 levels in Wistar-Kyoto rats. These results demonstrate a role for the AT 1 receptor in mediating the effects of Ang II on aortic and cardiac PAI-1 gene expression. Key Words: angiotensin II Ⅲ plasminogen activator inhibitor Ⅲ hypertension Ⅲ aorta Ⅲ vascular smooth muscle cells P lasminogen activator inhibitor-1 (PAI-1) is the major inhibitor of tissue and urokinase plasminogen activators and thereby reduces the conversion of plasminogen to plasmin, an extracellular protease that mediates fibrinolysis and activates matrix metalloproteinases. 1-3 An elevated level of PAI-1, which occurs in diabetes, insulin resistance, obesity, and hypertension, has been implicated as a contributing risk factor for cardiovascular disease. 4 -7 Recent studies suggest that the renin-angiotensin system (RAS) may exert an important role in the regulation of circulating and vascular PAI-1 expression and may thereby affect the fibrinolytic balance. Reports from our laboratory and others have demonstrated that angiotensin II (Ang II) is a potent stimulator of PAI-1 mRNA and protein expression in both cultured endothelial and vascular smooth muscle cells. 8 -11 The physiological importance of the RAS in modulating PAI-1 levels is supported by in vivo studies, which have demonstrated that treatment of rats with the angiotensin-converting enzyme (ACE) inhibitor captopril suppresses the induction of PAI-1 expression in the aortic neointima induced by b...