Effect Of Receptor Associated Protein On The Angiotensin Ⅱ-induced Atherosclerosis And Abdominal Aortic Aneurysms In Mice

Receptor associated protein(RAP) is an endoplasmic reticulum resident protein and can tightly bind to multiple moleculars including low density lipoprotein receptor-related protein(LRP1),lipoprotein lipase,very low density lipoprotein receptor and glucoprotein 330/megalin.It may serve as molecular chaperone of the protein and prevent from premature binding during protein synthesis and secret pathway. LRP1 deficiency in smooth muscle cells,bone marrow-derived cells or hepatocytes promotes atherosclerosis in hypercholesterolemia mice,However,LPL deficiency in bone marrow-derived cells attenuates atherosclerosis.Although the effect of LRP1 and LPL on the vascular diseases is widely researched,the role of RAP is poorly understood. The purpose of the project is to determine whether RAP deficiency affect the AngiotensinⅡ-induced atherosclerosis and abdominal aortic aneurysms(AAAs) in mice.RAP deficiency mouse was purchased from Jackson Lab.All the mice were back crossed to C57BL6 for more than 10 times.Chronic AngⅡinfusion(28 days) was achieved by osmotic mini pumps.Plasma cholesterol concentration and lipoprotein distribution were measured by enzymatic assay kit and size exclusion chromatography. Systolic blood pressure was measured on conscious,restrained mice using the Kent 8 tail cuff system..Atherosclerosis was quantified on the aortic arch by en face measurement.The maximum width of the abdominal aorta was measured using computerized morphometry(Image-Pro).Firstly,strain and age(6-12 weeks) matched male mice(RAP+/+,n=11,RAP-/-, n=10) were fed normal chow and infused with AngⅡ(1000ng/kg/min) by osmotic mini pumps.RAP deficiency had no effect on the body weight and plasma cholesterol.AngⅡinfusion increased systolic blood pressure similarly in two groups(RAP+/+, 177±7mmHg,RAP-/-,170±5mmHg,P=NS).RAP deficiency did not augment AngⅡ-induced supra-renal aortic diameter and the incidence of AAAs formation.Secondly,Strain and age matched low density lipoprotein receptor knockout(LDL receptor-/-) mice that are either RAP+/+(n=20) or RAP-/-(n=17) male mice(6-12 week old) were fed fat-enriched diets.Mice were infused with AngⅡ(500 ng/kg/min) for 28 days by osmotic pumps.There was no difference of body weight between the two group.AngⅡinfusion led to a comparable increase of systolic blood pressure in two groups(RAP+/+:171±6 mmHg;RAP-/-:165±5 mmHg;P=NS).RAP deficiency increased plasma total cholesterol concentrations(RAP+/+:1122±40;RAP-/-:1500±56 mg/dl;P＜0.001).FPLC and nonlinear fitting analysis showed that RAP deficiency led to an increase of VLDL(RAP+/+:484±22;RAP-/-:713±55 mg/dl;P=0.001) and IDL/LDL(RAP+/+:566±25;RAP-/-:757±42 mg/dl;P＜0.001) concentration, while decrease the HDL(RAP+/+:75±3;RAP -/-:63±4 mg/dl;P=0.028) concentration.Although the pro-atherosclerosis changes of lipoprotein distribution in RAP-/- mice,RAP deficiency decreased the arch atherosclerosis lesion percentage (RAP +/+:18.28±2.24%;RAP -/-:11.74±0.99 mg/dl;P=0.041).While AngⅡinfusion led to a comparable dilation in RAP +/+ and RAP -/- mice(1.48±0.30 versus 1.25±0.15 mm,P=NS).Finally,LRLR -/- mice were irradiated and repopulated with bone marrow- derived cells that were either RAP+/+(n=24) or RAP-/-(n=25).Mice were fed fat-enriched diets and infused with AngⅡ(500 ng/kg/min) for 28 days.Bone marrow-derived cells specific RAP deficiency had no effect on the body weight and plasma cholesterol. Systolic blood pressure was comparably increased after AngⅡinfusion(RAP +/+:168±3 mmHg;RAP -/-:173±3 mmHg;P=NS).Bone marrow-derived cells specific RAP deficiency did not reduce the aortic arch atherosclerosis lesion percentage(RAP +/+: 24.07±2.14%;RAP -/-:19.24±2.21 mg/dl;P=0.124).However,RAP deficiency in bone marrow-derived cells led to significant increase of maximal suprarenal aortic diameter(RAP+/+:0.99±0.04mm;RAP-/-:1.28±0.12mm,P=0.01) and AAAs formation incidence(RAP+/+:4%;RAP-/-:41%,P=0.001).Western blotting showed that RAP deficiency totally blocked the expression of LRP1 in macrophages,but had no effect in SMCs.Immunostaining indicated that RAP deficiency reduced the expression of LRP1 in hepatocytes.RAP in resident cells promoted atherosclerosis by the mechanisms independent of LRP1 in mice;the role of RAP in AngⅡ-induced AAAs formation need to be further investigated.The results firstly demonstrated the role of RAP in vascular diseases;these results supported the ideal that atherosclerosis and abdominal aneurysms are two independent diseases.