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Hepatocyte-specific knockout of YAP protects against atherosclerosis via inhibition of ANGPTL3 in mice

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE316646 | GEO | 2026/01/20

REPOSITORIES: GEO

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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 showed increased LDL particle clearance and enhanced LDLR-cholesterol interactions under WD treatment in ApoE-/-/Liver-DKO mice compared with ApoE-/- controls, correlating 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 an increased epsin1 and epsin2 expression in atherosclerotic cardiovascular disease patients. 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 in the liver.

Project description:Scavenger receptors Stabilin-1 (Stab1) and Stabilin-2 (Stab2) are preferentially expressed by liver sinusoidal endothelial cells. They mediate the clearance of circulating plasma molecules controlling distant organ homeostasis. Studies suggest that Stab1 and Stab2 may impact atherosclerosis. Although subsets of tissue macrophages also express Stab1, hematopoietic Stab1 deficiency does not modulate atherogenesis. Here, we comprehensively studied how targeting Stab1 and Stab2 affects atherosclerosis. Methods: ApoE-KO mice were interbred with Stab1-KO and Stab2-KO mice and fed a Western diet (WD). For antibody targeting, Ldlr-KO mice were also used. Plasma proteomics were performed in Stabilin-deficient mice and candidates were independently confirmed. Ligand binding studies comprised GST-pull down and endocytosis assays. Plasma proteome effects on monocytes were studied by single cell RNA sequencing in vivo, and by gene expression analyses of Stabilin-ligand-stimulated and plasma-stimulated bone marrow-derived monocytes/macrophages in vitro. Results: Spontaneous and WD-associated atherogenesis was significantly reduced in ApoE-Stab1- and ApoE-Stab2-KO. Similarly, inhibition of Stab1 or Stab2 by monoclonal antibodies significantly reduced WD-associated atherosclerosis in ApoE-KO and Ldlr-KO. While neither plasma lipid levels nor circulating immune cell numbers were decisively altered, plasma proteomics revealed a switch in the plasma proteome, consisting of 231 dysregulated proteins comparing Wildtype with Stab1/2 single and Stab1/2-double KO, and of 43 proteins comparing ApoE-, ApoE-Stab1- and ApoE-Stab2-KO. Among this broad spectrum of common, but also disparate scavenger receptor ligands, Periostin, Reelin and TGFBi, known to modulate atherosclerosis, were independently confirmed as novel circulating ligands of Stab1/2. scRNA-Seq of circulating myeloid cells of ApoE-, ApoE-Stab1- and ApoE-Stab2-KO showed transcriptomic alterations in patrolling (Ccr2-/Cx3cr1++/Ly6Clo) and inflammatory (Ccr2+/Cx3cr1+/Ly6Chi) monocytes including downregulation of pro-atherogenic transcription factor Egr1. Ligand exposure did not alter Egr1 expression in vitro, but exposure of Wildtype bone marrow-derived monocytes/macrophages to plasma from ApoE-Stab1- and ApoE-Stab2-KO mice showed a reverted pro-atherogenic macrophage activation as compared to ApoE-KO plasma including downregulation of Egr1. Conclusions: Inhibition of Stab1/Stab2 mediates an anti-inflammatory switch in the plasma proteome which includes direct Stabilin ligands. The altered plasma proteome suppresses both patrolling and inflammatory macrophages and, thus, systemically protects against atherogenesis. Altogether, anti-Stab1- and anti-Stab2-targeted therapies provide a novel approach for the future treatment of atherosclerosis.

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Hepatocyte-specific knockout of YAP protects against atherosclerosis via inhibition of ANGPTL3 in mice

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