(Pro)renin Receptor Inhibition Reduces Plasma Cholesterol and Triglycerides but Does Not Attenuate Atherosclerosis in Atherosclerotic Mice

Abstract

Objective: Elevated plasma cholesterol concentrations contributes to ischemic cardiovascular diseases. Recently, we showed that inhibiting hepatic (pro)renin receptor [(P)RR] attenuated diet-induced hypercholesterolemia and hypertriglyceridemia in low-density lipoprotein receptor (LDLR) deficient mice. The purpose of this study was to determine whether inhibiting hepatic (P)RR could attenuate atherosclerosis. Approach and Results: Eight-week-old male LDLR^-/- mice were injected with either saline or N-acetylgalactosamine-modified antisense oligonucleotides (G-ASOs) primarily targeting hepatic (P)RR and were fed a western-type diet (WTD) for 16 weeks. (P)RR G-ASOs markedly reduced plasma cholesterol concentrations from 2,211 ± 146 to 1,128 ± 121 mg/dL. Fast protein liquid chromatography (FPLC) analyses revealed that cholesterol in very low-density lipoprotein (VLDL) and intermediate density lipoprotein (IDL)/LDL fraction were potently reduced by (P)RR G-ASOs. Moreover, (P)RR G-ASOs reduced plasma triglyceride concentrations by more than 80%. Strikingly, despite marked reduction in plasma lipid concentrations, atherosclerosis was not reduced but rather increased in these mice. Further testing in ApoE^-/- mice confirmed that (P)RR G-ASOs reduced plasma lipid concentrations but not atherosclerosis. Transcriptomic analysis of the aortas revealed that (P)RR G-ASOs induced the expression of the genes involved in immune responses and inflammation. Further investigation revealed that (P)RR G-ASOs also inhibited (P)RR in macrophages and in enhanced inflammatory responses to exogenous stimuli. Moreover, deleting the (P)RR in macrophages resulted in accelerated atherosclerosis in WTD fed ApoE^-/- mice. Conclusion: (P)RR G-ASOs reduced the plasma lipids in atherosclerotic mice due to hepatic (P)RR deficiency. However, augmented pro-inflammatory responses in macrophages due to (P)RR downregulation counteracted the beneficial effects of lowered plasma lipid concentrations on atherosclerosis. Our study demonstrated that hepatic (P)RR and macrophage (P)RR played a counteracting role in atherosclerosis.

Keywords: (Pro)renin receptor (PRR); V-ATPase = vacuolar H+-adenosine triphosphatase; cholesterol; macrophage; renin- angiotensin system.

Figure 1

(Pro)renin receptor [(P)RR] N-acetylgalactosamine-modified antisense oligonucleotides (G-ASOs) reduced plasma cholesterol and triglyceride concentrations but not atherosclerosis in low-density lipoprotein receptor deficient (LDLR^−/−) mice fed a western-type diet (WTD). Eight-week-old male LDLR^−/− mice were administered with saline (blue) or (P)RR G-ASOs (red) and fed a WTD for 16 weeks. N = 8/group. Total plasma cholesterol (A) and triglycerides concentrations (B). Pooled plasma samples were resolved by FPLC for lipoprotein fractionation analysis, and cholesterol (A) and triglycerides content (B) in each fraction was determined. Representative images of the aorta arch and Oil Red O-stained whole aorta (C). Quantification of lesion areas of the whole aorta (D) and aortic arch region (E). Representative images of cross-sectioned aortic root stained with H&E and ORO and quantification of lesion areas of the aortic root (F). Bar = 1,000 μm.

Figure 2

(P)RR G-ASOs reduced plasma lipid concentrations, but not atherosclerosis in ApoE^−/− mice. Eight-week-old ApoE^−/− mice were administered with either saline (blue) or (P)RR G-ASO (red) and fed a WTD for 16 weeks. N=9 per group. Plasma cholesterol (A) and triglycerides concentrations (B). Representative images of aortic arch and en face whole aorta (C). Quantification of lesions in whole aorta (D) or aortic arch region (E). Representative images showing H&E and ORO-stained sectioned aortic root and quantification of lesion size in aortic root (F). Bar = 1,000 μm.

Figure 3

(P)RR G-ASOs promoted immune responses in aorta by augmenting macrophage inflammatory cytokine production. (A–D) Eight-week-old LDLR^−/− mice were administered with either saline or (P)RR G-ASOs and fed a WTD for 4 weeks, and aortas were isolated for transcriptomic analyses. Aortas from 8-week-old LDLR^−/− mice (0 week) served as control. Experimental procedure (A). Venn graph showing overlapped and non-overlapped DEGs (B). KEGG enrichment analysis (C) and GSVA analysis of curated gene sets (D). (E,F) RAW264.7 cells were incubated with saline (blue) or (P)RR G-ASOs (red) and stimulated with or without LPS. Expression and production of cytokines were determined.

Figure 4

Deleting (P)RR in macrophages accelerated atherosclerosis in ApoE^−/− mice. Eight-week-old Lyz2-Cre^+/0 (P)RR^wt/YApoE^−/− mice (Ctrl) and Lyz2-Cre^+/0 (P)RR^fl/YApoE^−/− mice (CKO) were fed a WTD for 12 weeks. N = 7 per group. Plasma cholesterol and triglycerides concentrations (A,B). Representative images showing aortic arch and ORO-stained whole aorta (C). Quantification of lesion area in whole aorta (D) and aortic arch region (E). Representative images showing H&E and ORO-stained sections of aortic root and quantification of lesion size in aortic root (F). Bar = 1,000 μm.