Functional Exploration of Conserved Sequences in the Distal Face of Angiotensinogen

Abstract

ABSTRACTBackgroundAngiotensinogen (AGT) is an essential component in the renin-angiotensin system. AGT has highly conserved sequences in the loop and β-sheet regions among species; however, their functions have not been studied.MethodsAdeno-associated viral vector serotype 8 (AAV8) encoding mouse AGT with mutations of conserved sequences in the loop (AAV8.loop-Mut), β-sheet (AAV8.βsheet-Mut), or both regions (AAV8.loop/βsheet-Mut) were injected into male hepatocyte-specific AGT deficient (hepAGT-/-) mice in an LDL receptor -/- background. AAV8 containing mouse wild-type AGT (AAV8.mAGT) or a null vector (AAV8.null) were used as controls. Two weeks after AAV administration, all mice were fed Western diet for 12 weeks.ResultsIn hepAGT-/- mice infected with AAV8.loop-Mut or βsheet-Mut, plasma AGT concentrations, systolic blood pressure, and atherosclerosis were comparable to those in AAV8.mAGT-infected mice. To determine the synergistic effects of conserved sequences in the loop and β-sheet regions, hepAGT-/- mice were infected with AAV8 encoding AGT with mutations in both regions. Surprisingly, plasma AGT concentrations, systolic blood pressure, and atherosclerotic lesion size in mice infected with AAV8.loop/βsheet-Mut were not different from mice infected with AAV8.null. However, hepaticAgtmRNA abundance was elevated to a comparable magnitude as AAV8.mAGT-infected mice. Immunostaining showed that AGT protein was accumulated in hepatocytes of mice infected with AAV8.loop/βsheet-Mut, while AGT protein was hardly detectable in hepatocytes of hepAGT-/- mice infected with AAV8.mAGT.ConclusionsThe conserved sequences in either the loop or β-sheet region individually have no effect on AGT regulation; the conserved sequences in both regions synergistically contribute to the secretion of AGT from hepatocytes.HIGHLIGHTSThe loop and β-sheet regions in the distal face of angiotensinogen (AGT) have highly conserved sequences across species.Mutations on either the loop or β-sheet regions do not affect plasma AGT concentrations, blood pressure, and atherosclerosis in hypercholesterolemic mice.The conserved sequences in the loop and β-sheet regions regulate the secretion of AGT from hepatocytes synergistically.