Effects of Endogenous Angiotensin II on Abdominal Aortic Aneurysms and Atherosclerosis in Angiotensin II–Infused Mice

Angiotensin II (Ang II) is a major effector of the reninangiotensin system and is important in regulating vascular function. Infusion of Ang II induces abdominal aortic aneurysms (AAA) and exacerbates atherosclerosis in hypercholesterolemic mice. In Ang II– infused normocholesterolemic rats, endogenous Ang II production is maintained in kidney.1 However, the effects of endogenous Ang II on AAA and atherosclerosis during Ang II infusion in hypercholesterolemic mice have not been studied. Detailed methods and results are available in bioRxiv (https://doi.org/10.1101/2020.11.18.377416). All animal experiments were approved by the University of Kentucky Institutional Animal Care and Use Committee. SigmaPlot version 14.0 (Systat Software Inc) was used for statistical analyses. Since either normality (ShapiroWilk test) or homogeneous variation (BrownForsythe test) was not confirmed, all data were analyzed using nonparametric analyses. Statistical tests might be underpowered. Our previous studies revealed that liverspecific deletion of angiotensinogen, the sole precursor of Ang II, reduced atherosclerotic lesion area with decreases of Ang II concentrations in kidney but not plasma.2 In addition, inhibition of angiotensinogen uptake into renal proximal tubular cells ameliorated atherosclerosis development.3 These results indicate an important role of renal Ang II in atherosclerosis formation. In an initial study, either vehicle or murine Ang II (1000 ng/kg per minute) was infused into male C57BL/6J mice for 7 days via osmotic pumps (Alzet model 2001, Durect Corporation). Then, we determined concentrations of angiotensin peptides in plasma and kidney by liquid chromatography– tandem mass spectrometry. Plasma Ang II concentrations were not altered by Ang II infusion (Figure [A]). Consistent with a previous report,1 renal Ang II concentrations were significantly higher in Ang II– infused mice than in vehicleinfused mice (Figure [A]). Since Ang II is metabolized to Ang III and Ang(1– 7) and these peptides may contribute to the pathophysiology of atherosclerosis,4 we assessed Ang III and Ang(1– 7) concentrations in Ang II– infused mice. Ang(1– 7) was not detectable in plasma and kidney from either vehicleor Ang II– infused mice, and Ang III concentrations were not statistically different in plasma and kidney between infusions (Figure [B]). These results indicate that exogenous Ang II does not affect productions of major Ang II metabolites. Since Ang I is the direct substrate of Ang II, we measured plasma and renal Ang I concentrations to evaluate the production of Ang II. Ang II infusion decreased Ang I concentrations in both plasma and kidney (Figure [C]). However, Ang II– induced reduction of Ang I concentrations was modest in kidney