Angiotensinogen Interacts With Megalin And Contributes To Atherosclerosis

Background:Atherosclerosis is the primary origin of numerous cardiovascular diseases as well as the leading cause of death worldwide.The renin-angiotensin system(RAS)plays a pivotal role in the development and progression of atherosclerosis.Angiotensinogen(AGT),predominantly derived from liver,is the unique substrate of this system.Renin cleaves AGT into angiotensin ?(Ang?)and des(Ang?)AGT.Ang? subsequently is catalyzed by angiotensin converting enzyme(ACE)to produce angiotensin ?(Ang?),which promotes atherogenesis.However,it remains ambiguous about how AGT is regulated to accelerate atherosclerosis.Megalin,a member of the low-density lipoprotein receptor family,is present in polarized epithelial cells.Previous studies demonstrated megalin interacted with AGT in proximal tubules of kidney.This study aims to determine whether megalin is involved in the regulation of RAS and atherogenesis.Methods and results:To identify whether AGT promotes atherosclerosis in an Ang?-dependent manner,hepatocyte-specific AGT-deficient(hepAGT-/-)and their wild-type littermates(hepAGT+/+)were bred into a low-density lipoprotein receptor-/-(LDLR-/-)background.hepAGT+/+mice were administered with adeno-associated viral(AAV)vector with a null insert by an intraperitoneal injection.hepAGT-/-mice were injected with AAV containing a null insert,wild-type AGT or des(AngI)AGT,respectively.These mice were fed with Western diet for 12 weeks.AAV-driven expression of wild-type AGT significantly augmented atherosclerotic lesion size in hepAGT-/-mice,while injection of des(AngI)AGT had no effect on atherosclerosis.Plasma Ang?concentrations did not show any difference between hepAGT+/+ and hepAGT-/-mice,but a profound reduction of renal Ang? occurred in hepAGT-/-mice.By using immunofluorescence,we found AGT,renin and ACE were colocalized with megalin in renal proximal tubules of wild-type mice.Inhibition of megalin by using antisense oligonucleotides(ASO)ablated AGT and renin in proximal tubule cells,while strikingly increasing AGT and renin concentrations in urine.Megalin ASO injection also led to profound reduction of renal Ang? concentrations without affecting plasma Ang?.To determine whether megalin contributes to atherosclerosis,both male and female LDLR-/-mice were subcutaneously injected with PBS,control ASO or megalin ASO.After 12 weeks of Western diet,mice with megalin inhibition of both genders displayed a prominent amelioration of atherosclerosis.Bioinformatic analysis revealed highly conserved sequences in the distal region of AGT.To determine whether these sequences mediates AGT binding to megalin,we generated AGT recombinants with mutations in these conserved regions.Surface plasmon resonance analysis recognized a reduced binding affinity between AGT mutants and megalin.To test whether these conserved sequences have influence on atherogenesis,hepAGT-/-mice were injected with AAV containing AGT mutants.hepAGT+/+and hepAGT-/-mice with null AAV injection were used as controls.After 12 weeks of Western diet,hepAGT-/-mice with AGT mutants AAV injection developed atherosclerosis similar to hepAGT+/+ mice.Conclusion:This study unveils megalin as a crucial regulator of intrarenal RAS homeostasis and atherogenesis,positing renal Ang? activation mediated by megalin is an important contributor of atherosclerosis.