ABSTRACT
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.
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.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Authorship Revised
Nonstandard Abbreviations and Acronyms
- AAV8
- adeno-associated virus-8
- ABCG1
- ATP Binding Cassette Subfamily G Member 1
- ACAT
- acyl-CoA cholesterol acyltransferase
- AMI
- acute myocardial infarction
- BCA
- brachiocephalic artery
- CAD
- coronary artery disease
- CEC
- cholesterol efflux capacity
- DKO
- double knockout
- ENTH
- epsin N-terminal homology
- GEM
- gel beads-in-emulsion
- GO
- gene ontology
- GSEA
- gene set enrichment analysis
- HDL
- high density lipoprotein
- ICAM-1
- intercellular adhesion molecule-1
- IF
- immunofluorescence staining
- IP
- immunoprecipitation
- LXR
- liver X receptor
- M-CSF
- macrophage colony stimulating factor
- NP
- nanoparticle
- ORO
- oil red O
- oxLDL
- oxidized low-density lipoprotein
- PCSK9
- proprotein convertase subtilisin/kexin type 9 serine protease
- RCT
- reverse cholesterol transport
- scRNA-seq
- single-cell RNA sequencing
- siRNA
- small interfering RNA
- SR-A
- scavenger receptor-A
- S2PNP-siEpsin1/2
- S2P-conjugated Epsins 1 and 2 siRNA nanoparticle S2PNP-siCtrl S2P-conjugated Control nanoparticle
- TG
- thioglycolate
- UIM
- ubiquitin-interacting motif
- VCAM-1
- vascular cell adhesion molecule-1
- VSMCs
- vascular smooth muscle cells
- WB
- western blot
- WD
- western diet