Targeting Epsins by nanotherapy regulates lipid metabolism and promotes ABCG1-mediated cholesterol efflux to fortify atheroma regression
Authors: Cui Kui , Gao Xinlei , Wang Beibei , Wu Hao , Dong Yunzhou , Xiao Yuling , Jiang Xingya , Malovichko Marina V. , Li Kathryn , Peng Qianman , Lu Yaowei , Zhu Bo , Zheng Rongbin , Wong Scott , Cowan Douglas B. , Linton MacRae , Srivastava Sanjay , Shi Jinjun , Chen Kaifu , Chen Hong 
Published: August 2022

BACKGROUND

Excess cholesterol accumulation in lesional macrophages elicits complex responses in atherosclerosis. Epsins, a family of endocytic adaptors, fuel the progression of atherosclerosis; however, the underlying mechanism and therapeutic potential of targeting Epsins remains unknown. In this study, we determined the role of Epsins in macrophage-mediated metabolic regulation. We then developed an innovative method to therapeutically-target macrophage Epsins with specially-designed S2P-conjugated lipid nanoparticles (NPs), which encapsulate small interfering RNAs to suppress Epsins.

METHODS

We used single cell RNA sequencing (scRNA-seq) with our newly developed algorithm MEBOCOST to study cell-cell communications mediated by metabolites from sender cells and sensor proteins on receiver cells. Biomedical, cellular and molecular approaches were utilized to investigate the role of macrophage Epsins in regulating lipid metabolism and transport. We performed this study using myeloid-specific Epsin double knockout (LysM-DKO) mice and mice with a genetic reduction of ABCG1 (LysM-DKO-ABCG1fl/+). The NPs targeting lesional macrophages were developed to encapsulate interfering RNAs to treat atherosclerosis.

RESULTS

We revealed that Epsins regulate lipid metabolism and transport in atherosclerotic macrophages. Inhibiting Epsins by nanotherapy halts inflammation and accelerates atheroma resolution. Harnessing lesional macrophage-specific NP delivery of Epsin siRNAs, we showed that silencing of macrophage Epsins markedly diminished atherosclerotic plaque size and promoted plaque regression. Mechanistically, we demonstrated that Epsins bound to CD36 to facilitate lipid uptake by enhancing CD36 endocytosis and recycling. Conversely, Epsins promoted ABCG1 degradation via lysosomes and hampered ABCG1-mediated cholesterol efflux and reverse cholesterol transport. In a LysM-DKO-ABCG1fl/+ mouse model, enhanced cholesterol efflux and reverse transport due to Epsin deficiency was suppressed by the reduction of ABCG1.

CONCLUSIONS

Our findings suggest that targeting Epsins in lesional macrophages may offer therapeutic benefits for advanced atherosclerosis by reducing CD36-mediated lipid uptake and increasing ABCG1-mediated cholesterol efflux.

Novelty and Significance

WHAT IS KNOWN?

Epsin endocytic adaptor proteins are upregulated in human and mouse atherosclerotic lesions

Lesional macrophages internalize lipids primarily through scavenger receptor-mediated endocytosis such as CD36 and SR-A

Macrophage-mediated cholesterol efflux and reverse cholesterol transport is crucial to atheroma resolution

WHAT NEW INFORMATION DOES THIS ARTICLE CONTRIBUTE?

ScRNA-seq combined with the newly-developed algorithm MEBOCOST reveals that Epsins are involved in macrophage-mediated lipid metabolic regulation

Macrophage epsins promote lipid uptake by targeting CD36 endocytosis and membrane recycling via the Epsin ENTH domain

Epsins bind ubiquitinated ABCG1—resulting in the endocytosis and lysosomal degradation of this cholesterol transporter, which reduces cholesterol efflux.

Macrophage-specific, nanoparticle-mediated RNAi delivery exhibits a therapeutic benefit for the treatment of atherosclerosis

Atherosclerotic plaque regression using this nanoparticle delivery platform represents a clinically-relevant approach for the treatment of advance atherosclerosis

BRIEF SUMMARY

Despite the development of new cholesterol-lowering therapies, including the recently approved PCSK9 small interfering RNA (siRNA) antagonists, patients still face a tremendous risk of developing major acute cardiovascular events resulting from chronic inflammation in the plaque. We employed a novel nanomedicine platform containing a stabilin-2 targeting peptide (S2P) to deliver Epsin-specific siRNAs to lesional macrophages. We discovered that inhibition of these adaptor proteins in lesional macrophages significantly diminished plaque size and necrotic core area, increased fibrous cap thickness, and promoted plaque regression.