Epsin Nanotherapy Regulates Cholesterol Transport to Fortify Atheroma Regression

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, which encapsulate small-interfering RNAs to suppress Epsins.

Methods: We used single-cell RNA sequencing with our newly developed algorithm MEBOCOST (Metabolite-mediated Cell Communication Modeling by Single Cell Transcriptome) 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 (ATP-binding cassette subfamily G member 1; LysM-DKO-ABCG1^fl/+). The nanoparticles 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 nanoparticle delivery of Epsin small-interfering RNAs, we showed that silencing of macrophage Epsins 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-ABCG1^fl/+ 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.

Keywords: Epsin; atherosclerosis; chronic; lipid metabolism; nanoparticle.

Figure 1.. scRNA-seq data revealed down-regulation of cholesterol-related cell-cell communications mediated by metabolites and their sensor proteins in macrophage subpopulations in LysM-DKO aorta compared to WT.

Aortas from WT and LysM-DKO mice (n=3 mice/each group) were isolated, digested and mixed for scRNA sequencing. (A) UMAP plot of cell clusters in aortas from WT and LysM-DKO mice on normal diet. Macrophage populations are indicated in the red circle. (B) UMAP of subclusters of major macrophage populations in (A). Trajectory inferred by Monocle3 was displayed. (C) A bar plot showing the number of communication events in cell groups in WT and LysM-DKO. (D) A circle plot showing the differential communications between cell groups in LysM-DKO compared to WT. The arrows indicate directions of communication from sender cells to receiver cells. The size of nodes positively correlates with the number of connected nodes. Line colors indicate the difference in communication scores of LysM-DKO compared to WT. (E) Violin plots of the abundance of representative metabolites and sensors across cell types in WT and LysM-DKO. Metabolite abundance (upper), sensor abundance (below). (F) ScRNA-seq identified the different macrophage subpopulations in aortas from WT/ApoE^−/− and LysM-DKO/ApoE^−/− mice on Western diet for 16 weeks. UMAP plot of each macrophage subpopulation in WT/ApoE^−/− and LysM-DKO/ApoE^−/−. (G-H) Feature plots (G) and Violin plots (H) showing the global expression level of marker genes in the macrophage sub types (e.g., Foamy Trem2 macrophage).

Figure 2.. Epsin facilitates CD36-mediated lipid uptake by promoting CD36 endocytosis and recycling.

(A-C) Thioglycolate (TG) induced peritoneal macrophages from WT/ApoE^−/− (n=5) and LysM-DKO/ApoE^−/− (n=5) mice on normal diet (ND) were incubated in lipid-deficient medium for 24h and treated with or without 100μg/mL oxLDL for 1h at 37⁰C. qRT-PCR analysis of CD36 expression (A), western blot (WB) analysis for total protein level of CD36 (B) and flow cytometry for surface level of CD36 (C). (D-E) Elicited TG-induced peritoneal macrophages from WT/ApoE^−/− and LysM-DKO/ApoE^−/− mice on ND were incubated in lipid-deficient medium for 24h and treated with or without 100μg/mL oxLDL for 15min (D) or 30min (E) at 37⁰C. Macrophages were co-stained with CD36 (green), the early endosome marker EEA1 (red) or the recycling endosome marker Rab11 (Red) and DAPI (bule), and imaged using confocal microscope. White arrows indicate the endocytic vesicles, scale bar=5μm, n=8/group. (F) BODIPY staining of peritoneal macrophages from WT and LysM-DKO mice on normal diet were pre-incubated with 25μg/mL oxLDL for 24h in lipid-deficient medium, n=6/group, scale bar=10μm. (G) Cholesterol and triglycerides levels in WT and LysM-DKO macrophages treated with 25μg/mL oxLDL for 24h in lipid-deficient medium (n=6). (H) Peritoneal macrophages isolated from WT and LysM-DKO mice on normal diet were incubated in lipid-deficient medium for 24h followed by the treatment with DiI-oxLDL for 2h at 37⁰C to assess the lipoprotein uptake by flow cytometry, n=6/group. Data from A-H are presented as mean ± SD. Mann-Whitney U test was utilized in A-C. Two-way ANOVA followed by Sidak post hoc multiple comparisons test was conducted in D-E and G. Unpaired t test was conducted in F and H.

Figure 3.. Epsin interacts with CD36 via Epsin-ENTH domain which is critical for CD36-mediated lipid uptake and foam cell formation.

(A) Bone marrow derived macrophages (BMDM) isolated from WT and LysM-DKO mice on normal diet were incubated in lipid-deficient medium for 24h followed by the treatment with 100μg/mL oxLDL for 1h, IP and WB analysis of Epsin1 and CD36 (n=4). (B) CD36 plasmids and full length (FLAG-Epsin1^WT) or domain-deletion constructs (FLAG-Epsin1^ΔENTH or FLAG-Epsin1^ΔUIM) in the pcDNA3 vector were transfected into HEK 293T cells for 48 h and then treated with 100μg/mL oxLDL for 1h, followed by immunoprecipitation (IP) and WB analysis using antibodies against FLAG tags and CD36 (n=4). (C) Peritoneal macrophages isolated from WT/ApoE^−/− mice on normal diet were incubated in lipid-deficient medium for 24h followed by treatment with 5 μM MG132 for 3h. Cells were subsequently treated with 100 μg/mL oxLDL for 1h followed by IP and WB for ubiquitin and CD36. (D) FLAG-Epsin1^WT, FLAG-Epsin1^ΔENTH, and FLAG-Epsin1^ΔUIM constructs were transfected into LysM-DKO/ApoE^−/− macrophages for 48 h and treated with 100μg/mL oxLDL for 1 h, followed by staining with F4/80 (red), BODIPY (green) and DAPI (blue). Scale bar= 200μm. (E) Statistics for (D and Figure S10), n=6. Data from A-D are presented as mean ± SD. Mann-Whitney U test was utilized in A. Kruskal-Wallis test followed by Dunn’s post hoc multiple comparisons test was conducted in B. One-way ANOVA followed by Tukey post hoc multiple comparisons test was conducted in E.

Figure 4.. RNA-seq analyses of WT, DKO and ABCG1 knockout (ABCG1KO) peritoneal macrophages indicate inverse regulation of lipid metabolism and inflammatory response.

(A-C) The Gene Set Enrichment Analysis (GSEA) (top panels) indicates the tendency of individual pathways to be up or down regulated in ABCG1KO macrophages compared to wild type. Genes associated with inflammatory response (HALLMAKR) (A), TNFα signaling via NFκB (HALLMARK) (B), and cholesterol homeostasis (HALLMARK) (C) are analyzed. The bar plots (bottom panels) showing log2 fold changes of expression of altered genes. (D) GO enrichment analysis for up- and down- regulated genes in ABCG1KO relative to WT. (E) Genome browser tracks to show expression of Itgb3 in individual samples. (F) The GO enrichment analysis revealed pathways reversely regulated in ABCG1 knock out and Epsin deficient peritoneal macrophages, compared to wild type. n=3/each group, * Adjusted P<0.05, ** adjusted P<1×10⁻⁵.

Figure 5.. Epsins deficiency in macrophages shows increased cholesterol efflux in vitro and reverse cholesterol transport (RCT) in vivo.

(A-B) Peritoneal macrophages were isolated from WT, LysM-DKO and LysM-DKO/ABCG1^fl/+ mice on normal diet. In vitro [³H]-cholesterol labeled WT, LysM-DKO or LysM-DKO/ABCG1^fl/+ macrophages were incubated in the presence or absence of HDL (25μg/mL) and ApoA-1 (10μg/mL) in the presence of 3μmol/L LXR agonist (T0901317) (n=9). (C) Schematic of [³H]-cholesterol loaded macrophage-RCT pathway. WT, LysM-DKO or LysM-DKO/ABCG1^fl/+ peritoneal macrophages were treated with 4μCi/mL [³H]-cholesterol and 50μg/mL ac-LDL for 48h followed by the injection of radiolabeled foam cells to C57BL/6/WT mice. After 48h, blood, liver and feces were collected and measured using a scintillation counter. (D-I) Distribution of [³H]-radioactivity counts in serum, HDL, liver feces and intestinal contents were determined by scintillation counter (n=6). (J) LysM-DKO mice were crossed with ABCG1^fl/+ mice to generate LysM-DKO-ABCG1^fl/+ mice. (K-L) Bone marrow derived macrophages isolated from WT, LysM-DKO and LysM-DKO/ABCG1^fl/+ mice on normal diet (n=6) were analyzed by qRT-PCR (K) and WB (L) (n≥3). Data from A-L are presented as mean ± SD. One-way ANOVA followed by Tukey post hoc multiple comparisons test was conducted in A, B, and D-I; Kruskal-Wallis test followed by Dunn’s post hoc multiple comparisons test was conducted in K and L.

Figure 6.. Epsins bind to ABCG1 and facilitate the internalization and degradation of ABCG1 via lysosomes.

(A) Peritoneal macrophages from WT/ApoE^−/− and LysM-DKO/ApoE^−/− mice on normal diet (ND) were lysed for WB analysis (n=4). (B) Peritoneal macrophages from WT/ApoE^−/− and LysM-DKO/ApoE^−/− mice on ND were pretreated with a liver X receptor (LXR) activator and followed by a cell surface biotinylation assay to evaluate the cell surface ABCG1 levels (n=4). (C) LXR agonist activated WT and LysM-DKO peritoneal macrophages were incubated in lipid-deficient medium for 24h and treated with or without 100μg/mL oxLDL for 5, 15 and 45 min followed by staining with anti-ABCG1 and analyzed by flow cytometry. (D) LXR agonist pretreated peritoneal macrophages isolated from WT and LysM-DKO mice on ND were treated with 100μg/mL oxLDL for 1h followed by IP and WB for Epsin1 and ABCG1 or ABCA1 (n=4). (E) ABCG1 plasmids and full length (FLAG-Epsin1^WT) or domain-deletion constructs (FLAG-Epsin1^ΔENTH or FLAG-Epsin1^ΔUIM) in the pcDNA3 vector were transfected into HEK 293T cells for 24 h in the presence of LXR agonist. Cells were then treated with 100μg/mL oxLDL for 1h, followed by IP and WB analysis using antibodies against FLAG tags and ABCG1 (n=4). (F) Peritoneal macrophages isolated from WT mice on ND were cultured in serum-free medium for 24h followed by treatment with 5 μM MG132 for 3h. Cells were then treated with 100μg/mL oxLDL for 1h followed by IP and WB for ubiquitin and ABCG1. (G-H) WT and LysM-DKO peritoneal macrophages were incubated in lipid-deficient medium for 24h followed by incubation with or without 100μg/mL oxLDL for 15min (G) or 45min (H) at 37⁰C. Macrophages were stained with ABCG1 (green), early endosome marker EEA1 (red) or lysosome marker Lamp1 (red) and DAPI (blue), then assessed by confocal microscopy. White arrows indicate the endocytic vesicles, scale bar=5μm, n=8/group. Data from A-H are presented as mean ± SD. Unpaired non-parametric Mann-Whitney U test was conducted in A, B and D; Kruskal-Wallis test followed by Dunn’s post hoc multiple comparisons test was conducted in C, E and F. Two-way ANOVA followed by Sidak post hoc multiple comparisons test was conducted in G and H.

Figure 7.. S2PNP-siEpsin1/2 delivery inhibits atheroma progression, decreases necrotic core content, and increases smooth muscle cell content in advanced stage of atherosclerosis.

(A) Male ApoE^−/− mice fed a Western Diet (WD) for 17 weeks followed by treatment of S2PNP-siCtrl or S2PNP-siEpsin1/2 for 3 weeks (2 doses per week). (B) En face ORO staining of aortas from baseline, control siRNA NP-treated ApoE^−/− or Epsin1/2 siRNA NP treated male ApoE^−/− mice fed a WD, lesion areas were analyzed with NIH ImageJ, scale bar=5mm. (C) ORO staining of brachiocephalic artery (BCA) and aortic root sections of above three groups. Lesional area (black dash line outlined) were analyzed by NIH ImageJ. (D) Aortic roots from S2PNP-siCtrl-treated ApoE^−/− or S2PNP-siEpsin1/2 treated male ApoE^−/− mice were stained with the macrophage marker CD68 (dashed white line outlined) and αSMA (white arrow). (E) H&E staining of BCA and aortic root sections of the above three groups. Necrotic areas (black dash line outlined) were analyzed by NIH ImageJ. Data from B-E (n=6) are presented as mean ± SD. Scale bar: B=5mm; C, D, E=500μm. One-way ANOVA followed by Tukey’s post hoc multiple comparisons test was conducted in B-E.

Figure 8.. S2PNP-siEpsin1/2 promotes the resolution of atheroma, reduces inflammation, and stabilizes atherosclerotic plaques.

(A) Male C57BL/6 WT mice were injected twice with PCSK9-AAV8 (D377Y) virus and fed a WD for 16 weeks and followed by normal diet feeding with the treatment of S2PNP-siCtrl or S2PNP-siEpsin1/2 for 4 weeks (2 doses per week). (B) En face ORO staining of aortas from above mice. Lesion area was analyzed using NIH ImageJ, scale bar=5mm. (C) Immunostaining of CD68 (green) and αSMA (red) in the above groups. White arrows indicate the luminal αSMA. For (B) and (C), n=6/group, scale bar: B (aorta)=5mm, B (aortic root) =500μm, C=500μm. (D) Statistics for ORO staining (B) and IF staining (C). (E-H) Co-staining of CD31 (endothelial cell marker, green) with vascular cell adhesion molecule-1 (VCAM-1, red, E), intercellular adhesion molecule-1 (ICAM-1, red, F), P-selectin (red, G) or E-selectin (red, H) and DAPI on the luminal surface of aortic root sections. White arrows indicate CD31/ICAM-1, CD31/VCAM-1, CD31/E-selectin or CD31/P-selectin colocalization. Scale bars=20μm. (I) Statistical analysis of E-H, n=6/group. (J) Isolated macrophages from PCSK9-mice were treated with S2PNP-siCtrl or S2PNP-siEpsin1/2 for 24h, RNA was isolated, and qRT-PCR was performed (n=4). B-J are presented as mean ± SD. and were analyzed using one-way ANOVA (B and C) and the unpaired Student’s t-test (E-J). One-way ANOVA followed by Tukey’s post hoc multiple comparisons test was conducted in D; Unpaired t test was conducted in I; Mann-Whitney U test was conducted in J.