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Hepatocyte-Specific Nrf2 Deficiency Alters Cholesterol Metabolism, Leading to Mitigated Atherosclerosis in ApoE-Knockout Mice

ABSTRACT: Hypercholesterolemia and hyperlipidemia are major contributors to the pathogenesis and progression of atherosclerosis and cardiovascular diseases (CVDs). Nuclear factor erythroid-derived 2-like 2 (NRF2) exerts antioxidant and anti-inflammatory effects while regulating glucose and lipid metabolism. However, the precise role and molecular mechanisms of NRF2 in hepatic cholesterol metabolism remain incompletely understood. Approach and Results: ApoE-knockout (ApoE-KO) mice were crossed with hepatocyte-specific Nrf2-knockout (Nrf2(H)-KO) mice to explore how NRF2 modulated cholesterol metabolism and the progression of atherosclerosis in vivo. Atherosclerotic lesion areas in Nrf2(H)-KO;ApoE-KO mice were significantly reduced compared with control counterparts (Nrf2-LoxP;ApoE-KO). Total cholesterol and LDL-cholesterol levels in plasma of Nrf2(H)-KO;ApoE-KO mice were significantly decreased, consistent with atherosclerotic phenotypes. Furthermore, hepatic triglyceride levels and cholesterol contents were increased in Nrf2(H)-KO;ApoE-KO mice. The hepatic transcriptomic analysis highlighted lipid metabolism pathway alterations in Nrf2(H)-KO;ApoE-KO mice, and carboxylesterase 1 (Ces1s) and lipocalin gene family member major urinary proteins (Mups) clusters markedly reduced expression in Nrf2(H)-KO;ApoE-KO livers. Liver proteomics analysis corroborated the transcriptomic findings. The current findings suggest that NRF2-modulated CES1s and MUPs may regulate cholesterol metabolism in the ApoE-KO model. Conclusions: Hepatocyte-specific Nrf2 deficiency significantly reduced plasma cholesterol levels, thereby attenuating atherosclerotic plaque formation and progression in the ApoE-KO background. This study established NRF2 in hepatocytes as a potential therapeutic target for controlling hypercholesterolemia and preventing atherosclerosis development.

ORGANISM(S): Mus musculus

PROVIDER: GSE346814 | GEO | 2026/09/15

REPOSITORIES: GEO

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Project description:Background and Aims Lipid-lowering therapy is a cornerstone in the treatment of atherosclerotic cardiovascular disease (ASCVD). Although some lipid-lowering drugs have demonstrated positive effects in patients with ASCVD, their effects are limited in those with homozygous familial hypercholesterolemia (HoFH). It is essential to seek new lipid-lowering targets. Yes-associated protein (YAP) may be involved in lipid metabolism in liver; therefore, we investigated the function of hepatocyte YAP in hyperlipidemia and atherosclerosis. Methods Hyperlipidemia models were generated in apoE-/- mice or mice injected with AAV8-D377Y-mPCSK9, which degrades and deletes low density lipoprotein receptor (LDLR), by being fed a high cholesterol diet (HCD) for 12 weeks. We measured the expression level of hepatic YAP in these apoE-/- mice. Next, we created YAPΔHep apoE-/- mice to further determine the role of YAP in hyperlipidemia and atherosclerosis. AML12 cells and mice injected with AAV8-D377Y-mPCSK9 or YAPΔHepapoE-/- mice were used to elucidate its mechanism. Finally, apoE-/- or LDLR-/- mice were used to observe the therapeutic efficacy of AAV8-Alb-shYAP for hyperlipidemia and atherosclerosis. Results HCD-fed apoE-/- mice showed increased levels of YAP in the liver. Further investigation indicated that YAPΔHepapoE-/- mice exhibited lighter hyperlipidemia and atherosclerosis than YAPflox/floxapoE-/- mice fed with HCD. Conversely, hepatocyte-specific overexpression of YAP (5S) deteriorated hyperlipidemia and atherosclerosis in HCD-fed apoE-/- mice. Furthermore, the lipid-lowering effect of YAP deficiency in hepatocytes was independent of LDLR. Hepatocyte-specific overexpression of angiopoietin-like-3 (ANGPTL3) aggravated hyperlipidemia and atherosclerosis in YAPΔHepapoE-/- mice, indicating that ANGPTL3 is responsible for the function of YAP in hyperlipidemia. Mechanistically, YAP upregulated ANGPTL3 via TEAD4 in hepatocytes independent of LDLR. Notably, AAV8-Alb-shYAP lowered lipid levels in apoE-/- or LDLR-/- mice. Conclusion Taken together, our findings revealed a novel role for the YAP-TEAD4-ANGPTL3 axis in lipid metabolism independent of LDLR. Inhibition of hepatocyte YAP may be an effective lipid-lowering strategy for HoFH.

Project description:Background: The low-density lipoprotein receptor (LDLR) in the liver plays a crucial role in clearing low-density lipoprotein cholesterol (LDL-C) from the bloodstream. This process takes place mainly in the liver. Under atherogenic conditions, Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9), secreted by the liver, binds to LDLR on hepatocytes, preventing its recycling and enhancing its lysosomal degradation. This process reduces LDL-C clearance, promoting hypercholesterolemia. Epsins, a family of ubiquitin-binding endocytic adaptors, are key regulators of atherogenesis in lesional cells, including endothelial cells and macrophages. Given epsins' canonical role in regulating endocytosis of cell-surface receptors, we aimed to determine whether and how liver epsins contribute to PCSK9-mediated LDLR endocytosis and degradation, thereby impairing LDL-C clearance and accelerating atherosclerosis. Methods: Liver-specific epsin knockout (Liver-DKO) atherosclerotic models were generated in ApoE-/- and PCSK9-AAV8-induced atheroprone mice fed on a Western diet. We utilized single-cell RNA sequencing, along with molecular, cellular, and biochemical analyses, to investigate the physiological role of liver epsins in PCSK9-mediated LDLR degradation. Additionally, we explored the therapeutic potential of nanoparticle-encapsulated siRNAs targeting epsins 1 and 2 in ApoE-/- mice with established atherosclerosis. Results: Western diet (WD)-induced atherosclerosis was significantly attenuated in ApoE-/-/Liver-DKO mice compared with ApoE-/- controls, as well as in PCSK9-AAV8-induced Liver-DKO mice compared with PCSK9-AAV8-induced wild-type (WT) mice, accompanied by reductions in blood cholesterol and triglyceride levels. Mechanistically, single-cell RNA sequencing of hepatocytes and aortas isolated from atherosclerotic ApoE-/- and ApoE-/-/Liver-DKO mice revealed epsin-deficient Ldlrhi hepatocytes with diminished lipogenic potential. Notably, pathway analysis of hepatocytes revealed increased LDL particle clearance and enhanced LDLR-cholesterol interactions under WD treatment in ApoE-/-/Liver-DKO mice compared with ApoE-/- controls, which correlated with decreased plasma LDL-C levels. Furthermore, pathway analysis of the aortas showed attenuated inflammation and endothelial activation, coupled with reduced lipid uptake, and enhanced cholesterol efflux under WD treatment in ApoE-/-/Liver-DKO mice compared with ApoE-/- controls. Moreover, the absence of liver epsins led to an upregulation of LDLR protein expression in hepatocytes. We further demonstrated that epsins bind LDLR via the ubiquitin-interacting motif (UIM), enabling PCSK9-mediated LDLR degradation. Depleting epsins abolished this degradation, thereby preventing atheroma progression. Lastly, targeting liver epsins with nanoparticle-encapsulated epsins siRNAs effectively ameliorates dyslipidemia and inhibits atherosclerosis progression. These results are consistent with findings showing increased expression of epsin1 and epsin2 in patients with atherosclerotic cardiovascular disease. Conclusions: Liver epsins drive atherogenesis by promoting PCSK9-mediated LDLR degradation, thereby elevating circulating LDL-C levels and heightening lesional inflammation. As such, targeting epsins in the liver represents a promising therapeutic strategy to mitigate atherosclerosis by preserving LDLR and enhancing LDL-C clearance.

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Hepatocyte-Specific Nrf2 Deficiency Alters Cholesterol Metabolism, Leading to Mitigated Atherosclerosis in ApoE-Knockout Mice

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