Abstract P323: Direct Vascular Endothelial Actions Of Angiotensin II Via At ₁ R And No Signaling Revealed By Intravital Multiphoton Microscopy.

The renin-angiotensin system (RAS) controls blood pressure and body fluid balance. The classic vasoactive effects of RAS are mediated by the binding of angiotensin II (ANGII) to ANGII type 1 receptors (AT 1 R) in vascular smooth muscle cells leading to vasoconstriction, or to opposing ANGII type 2 receptors (AT 2 R). However, only few studies investigated the direct effects of ANGII on vascular endothelial cells (VEC) in vivo and the mechanisms how VECs may mitigate ANGII-mediated vasoconstriction. The present study aimed to explore the molecular mechanisms and pathophysiological relevance of the direct actions of ANGII on VEC in the kidney. VEC calcium dynamics and NO synthesis were visualized by using intravital multiphoton microscopy (MPM) of Cdh5-G6 mice that selectively express the ratiometric Ca 2+ reporter GCaMP6f/tdTomato in VECs, or the endothelial uptake of iv injected nitric oxide (NO) sensitive dye DAF-FM, respectively. Bolus injection of ANGII (400 ng/kg, ia.) triggered a >4-fold increase in VEC calcium in afferent (AA) arterioles and glomerular capillaries, but not in peritubular capillaries. These responses were blocked by pretreatment with the AT 1 R inhibitor Losartan (60mg/kg ip.), but not by the AT 2 R inhibitor PD123319 (25mg/kg ip.)(1.2+/-0.4 p<0.001 and 5.6+/-0.9-fold, p=0.3, respectively). VEC responded to acute ANGII by increased NO synthesis as indicated by >1.5-fold increase in DAF-FM fluorescence intensity as compared to baseline (p<0.0001). In mice with unilateral ureteral obstruction (UUO), a well-known high ANGII and vasoconstrictor state associated with endothelial dysfunction, the ANGII induced calcium increase in VECs was significantly reduced (2.8+/-0.3-fold) as compared to control (4.6+/-0.4-fold, p<0.01). These results correlated with the significantly reduced AA diameter in UUO compared to control (8.2+/-0.4 and 10.43+/-0.3 μm, respectively, p<0.001). In summary, ANGII has major direct effects on VECs NO production that counterbalances agonist-induced vasoconstriction. These effects are diminished in conditions with endothelial dysfunction leading to increased vascular contractility. Cell-specific targeting of ANGII actions may represent exciting future therapeutic opportunities.