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(Circulation. 2005;112:1798-1805.)
© 2005 American Heart Association, Inc.

Vascular Medicine

Losartan Metabolite EXP3179 Activates Akt and Endothelial Nitric Oxide Synthase via Vascular Endothelial Growth Factor Receptor-2 in Endothelial Cells

Angiotensin II Type 1 Receptor–Independent Effects of EXP3179

Tetsu Watanabe, MD, PhD; Jun Suzuki, MD, PhD^*; Hideyuki Yamawaki, DVM, PhD^*; Virendra K. Sharma, DVM, PhD; Shey-Shing Sheu, PhD; Bradford C. Berk, MD, PhD

From the Cardiovascular Research Institute and Department of Medicine (T.W., J.S., H.Y., B.C.B.) and Department of Pharmacology and Physiology (V.K.S., S.-S.S.), University of Rochester, Rochester, NY.

Correspondence to Bradford C. Berk, MD, PhD, Cardiovascular Research Institute, University of Rochester, Box 679, 601 Elmwood Ave, Rochester, NY 14642. E-mail bradford_berk{at}urmc.rochester.edu

Received September 27, 2004; revision received June 22, 2005; accepted June 24, 2005.

Background— Recent studies suggest that angiotensin type 1 receptor (AT1R) blockers have vascular protective effects beyond blood pressure lowering. Because of the importance of endothelial nitric oxide synthase (eNOS) in vascular and platelet function, we hypothesized that losartan and its metabolites would stimulate eNOS and its upstream activators Akt and phosphatidylinositol 3-kinase (PI3K).

Methods and Results— Losartan is metabolized into EXP3174 (AT1R-blocking metabolite) and EXP3179 (no AT1R-blocking properties). Treatment of endothelial cells (ECs) with losartan and both metabolites stimulated phosphorylation of Akt and eNOS in the absence of angiotensin II. However, the magnitude for EXP3179 was much greater than EXP3174, and the EC₅₀ was significantly lower (–logEC₅₀, 8.2±0.1 versus 5.4±0.2 mol/L), suggesting an AT1R-independent effect. Inhibiting PI3K or vascular endothelial growth factor receptor 2 (VEGFR2) tyrosine phosphorylation abrogated EXP3179-induced eNOS phosphorylation. In endothelium of intact rat aorta, EXP3179 also stimulated Akt and eNOS phosphorylation. VEGFR2 activation was shown to be calcium and Src family kinase dependent by use of specific drug inhibitors and dominant negative kinase transfection. EXP3179 significantly inhibited tumor necrosis factor –induced apoptosis by 60% (from 30.1±5.8% to 12.2±2.0% TUNEL-positive cells), which was abolished by pretreatment with the PI3K inhibitor LY294002. Cleaved caspase-3 was suppressed by 48% with EXP3179.

Conclusions— The losartan metabolite EXP3179 stimulates eNOS phosphorylation and suppresses tumor necrosis factor –induced EC apoptosis by activating the VEGFR2/PI3K/Akt pathway.

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