Functional Exploration of Conserved Sequences in the Distal Face of Angiotensinogen-Brief Report

BACKGROUND:Angiotensinogen (AGT) is an essential component in the renin-angiotensin system. AGT has highly conserved sequences in the loop and & beta;-sheet regions among species; however, their functions have not been studied. METHODS:Adeno-associated viral vector (AAV) serotype 2/8 encoding mouse AGT with mutations of conserved sequences in the loop (AAV.loop-Mut), & beta;-sheet (AAV.& beta;sheet-Mut), or both regions (AAV.loop/& beta;sheet-Mut) was injected into male hepatocyte-specific AGT-deficient (hepAGT(-/-)) mice in an LDL (low-density lipoprotein) receptor-deficient background. AAV containing mouse wild-type AGT (AAV.mAGT) or a null vector (AAV.null) were used as controls. Two weeks after AAV administration, all mice were fed a western diet for 12 weeks. To determine how AGT secretion is regulated in hepatocytes, AAVs containing the above mutations were transducted into HepG2 cells. RESULTS:In hepAGT(-/-) mice infected with AAV.loop-Mut or & beta;sheet-Mut, plasma AGT concentrations, systolic blood pressure, and atherosclerosis were comparable to those in AAV.mAGT-infected mice. Interestingly, plasma AGT concentrations, systolic blood pressure, and atherosclerotic lesion size in hepAGT(-/-) mice infected with AAV.loop/& beta;sheet-Mut were not different from mice infected with AAV.null. In contrast, hepatic Agt mRNA abundance was elevated to a comparable magnitude as AAV.mAGT-infected mice. Immunostaining showed that AGT protein was accumulated in hepatocytes of mice infected with AAV.loop/& beta;sheet-Mut or HepG2 cells transducted with AAV.loop/& beta;sheet-Mut. Accumulated AGT was not located in the endoplasmic reticulum. CONCLUSIONS:The conserved sequences in either the loop or & beta;-sheet region individually have no effect on AGT regulation, but the conserved sequences in both regions synergistically contribute to the secretion of AGT from hepatocytes.