FUNCTIONAL IMPACT OF ANGIOTENSINOGEN SPECIFIC DOMAINS ON ANGIOTENSIN II-MEDIATED FUNCTIONS

OF DISSERTATION FUNCTIONAL IMPACT OF ANGIOTENSINOGEN SPECIFIC DOMAINS ON ANGIOTENSIN II-MEDIATED FUNCTIONS Angiotensinogen (AGT) is the only substrate for all angiotensin peptides in the renin angiotensin system (RAS). Cleavage of AGT by renin is a rate-limiting step of angiotensin peptides productions in the RAS and regulates angiotensin peptides-associated pathophysiological functions. Only ten N-terminal residues are cleaved by renin and the functions of remaining part of AGT protein, which is called des(angiotensin I)AGT, remain unclear. Despite of pivotal roles of AGT in the RAS, studies related to how AGT is metabolized and how des(angiotensin I)AGT regulates AGT functions or AngII-mediated functions are limited. Renin cleavage of AGT shows species specificity. It has been reported that residues in N-terminus of AGT surrounding renin cleavage site play important roles of affecting renin activity in vitro by using synthetic peptides. However, there is no in vivo evidence showing the effects of N-terminal residues in renin cleavage of AGT and consequently AngII-mediated functions, such as blood pressure and atherosclerosis. In this study, we compared expressions of AGT proteins by adeno-associated viral (AAV) vectors encoding human AGT or mouse AGT with Leu11Val and Tyr12Ile mutations which mimics two residues at the same position in human AGT to determine whether substitution of these residues regulated AngII-mediated functions. All mice were LDL receptor mice. Hepatocyte-specific AGT deficient (hepAGT) mice were injected with AAVs encoding null AAV, human AGT AAV or mutated mouse AGT (L11V;Y12I). hepAGT littermates injected with null AAV as positive controls. Expression of human AGT did not affect endogenous mouse AGT expression, blood pressure and atherosclerosis in hepAGT mice. In a subsequent study, expression of L1V;Y12I AGT significantly increased plasma AGT concentration, blood pressure and atherosclerosis, showing that replacement of L11 and Y12 to V11 and I12, respectively, in mouse AGT does not affect renin cleavage, blood pressure, and atherosclerosis in LDL receptor deficient mice. AGT has been reported to have both AngII-dependent and -independent functions. In our previous publication, we found two highly conserved regions, the β-sheet and loop region, on the surface of AGT which are distal to renin interacting face, and showed that these two highly conserved regions might have renin-independent roles. Moreover, we demonstrated that AGT and megalin interactions predispose animals to atherosclerosis. Thus, in this study, we investigated whether these two highly conserved regions affect AngII-mediated functions thorough influencing AGT interaction with megalin. Firstly, we determined the roles of the loop region in the interaction of AGT with megalin in vitro. All mice were LDL receptor background. However, repopulation of mouse AGT with single nucleotide mutation on the loop region, triple nucleotide mutations on the β-sheet or the loop region, and replacement of whole conserved sequences in the loop region with a GA linker did not change blood pressure and atherosclerosis in hepAGT mice, which is associated with plasma AGT concentration. Interestingly, mutations on both the β-sheet and loop region inhibited plasmas AGT level, blood pressure and atherosclerosis, showing the β-sheet and loop region synergistically regulate AGT production and consequently affect AngII-mediated functions in hepAGT mice. Taken together, residues in AGT have different biological effects which regulates angiotensin-associated functions.